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CHEMISTRY RESEARCH AND APPLICATIONS

NEW DEVELOPMENTS IN TERPENES RESEARCH

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CHEMISTRY RESEARCH AND APPLICATIONS

NEW DEVELOPMENTS IN TERPENES RESEARCH

JINNAN HU EDITOR

New York


Copyright © 2014 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers‘ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Additional color graphics may be available in the e-book version of this book.

Library of Congress Cataloging-in-Publication Data New developments in terpenes research / [edited by] Jinnan Hu (Department of Plant Pathology, The Ohio State University, Columbus, Ohio). pages cm Includes bibliographical references and index. ISBN: (eBook)

1. Terpenes. I. Hu, Jinnan, editor of compilation. QP752.T47N49 2014 547'.71--dc23 2013046309

Published by Nova Science Publishers, Inc. † New York


CONTENTS Preface Chapter 1

vii Production of Natural Flavor Compounds Using Monoterpenes As Substrates Gustavo Molina, Marina G. Pêssoa, Mariana R. Pimentel, Franciele M. Pelissari, Juliano L. Bicas and Gláucia M. Pastore

1

Chapter 2

Diterpenes As Cancer Therapy Elena González-Burgos and M. Pilar Gómez-Serranillos

Chapter 3

Phenolic Diterpenes from Rosemary As Enhancement Agents of Usual Chemotherapeutic Drugs for Colorectal Cancer Therapy Tiziana Fornari, Ana Ramírez de Molina and Guillermo Reglero

47

The Possible Use of Terpene Compounds in DC Immunotherapy against Cancer Masao Takei, Akemi Umeyama and Je-Jung Lee

79

Chapter 4

Chapter 5

Essential Oil Compositions and In Vitro Biological Activities of Three Szyzgium Species from Nigeria Oladipupo A. Lawal, Isiaka A. Ogunwande, Christiana A Bullem, Olayinka T. Taiwo and Andy R. Opoku

Chapter 6

From Terpenoids to Amines: A Critical Review Arno Behr and Andreas Wintzer

Chapter 7

Illudane-Type Sesquiterpenes: Challenges and Opportunities for Toxicology and Chemotherapy Rui M. Gil da Costa, Carlos Lopes, Paula A. Oliveira and Margarida M. S. M. Bastos

Chapter 8

Palladium-Catalysed Monoterpenes Oxidation: Environmentally Benign Synthesis of Valuable Oxigenate Derivatives Márcio José da Silva, Abiney Lemos Cardoso, Ligia Maria Mendonca Vieira and Danieli Marcolan Carari


25

93

113

135

185

vi Chapter 9

Chapter 10

Chapter 11

Contents Theoretical Properties of Terpenes and their Relationship with Biological Activities S. Andrade-Ochoa, A. A. Camacho-Dávila, L. M. Rodríguez-Valdez, M. Villanueva-García and G. V. Nevárez-Moorillón Configurational and Conformational Analyses by Theoretical Methods of Pentacyclic Triterpenes Isolated from Myricaria elegans A. G. Pacheco, G. Salgado-Morán and A. F. C. Alcântara Applications and Advances in the Extraction and Analysis of Monoterpenes and Sesquiterpenes in Plants J. Omar, M. Olivares, A. Vallejo, A. Delgado, P. Navarro, O. Aizpurua and N. Etxebarria

Index

213

237

249

271


PREFACE As the largest class of nature products which have high potential in chemical and pharmaceutical usage, terpenes have been studied and utilized by human society for centuries. Terpenes are derived biosynthetically from units of isoprene, and it‘s surprising that many phylogenetically distant organisms have evolved to use the very similar structures for common purposes. Despite the fact that over 22000 types of terpenes have been identified and reported, undoubtedly many novel structures are yet undiscovered and the functional activities of the identified are remaining unclear. This is the rational of is book, that is to give a comprehensive update to all the important discovers and application that have been achieved in this field in recent years. There are many topics covered by this book, which include but not is limited to: the evaluation of different terpenes used as cancer treatment or flavor enrichment; review of advanced approach in terpenes extraction and processing; structure analysis of terpenes molecules. This book is useful for both researchers in various fields of chemistry, biochemistry, materials, pharmacology, and other related fields, as well as by graduate students and scientists would like to apply the experimental and analysis procedures in their specific areas. The chapters are relatively independent of each other and can be read separately.

October, 2013



In: New Developments in Terpenes Research Editor: Jinnan Hu

ISBN: 978-1-62948-760-1 © 2014 Nova Science Publishers, Inc.

Chapter 1

PRODUCTION OF NATURAL FLAVOR COMPOUNDS USING MONOTERPENES AS SUBSTRATES Gustavo Molina1,2, Marina G. Pêssoa1, Mariana R. Pimentel1, Franciele M. Pelissari2, Juliano L. Bicas3 and Gláucia M. Pastore1 1

Laboratory of Bioflavors, Department of Food Science, Faculty of Food Engineering, University of Campinas, Campinas, SP, Brazil 2 Institute of Science and Technology, Food Engineering, University of Jequitinhonha and Mucuri, Diamantina, MG, Brazil 3 Department of Chemistry, Biotechnology and Bioprocess Engineering, University of São João del-Rei. Ouro Branco, MG, Brazil

ABSTRACT The biocatalytic bioconversion or biotransformation of monoterpenes allows significantly enhanced accumulation of a desired flavor product, since they are a structurally related precursor molecule. As a prerequisite for this strategy, the precursor must be present in nature and its isolation in sufficient amounts from the natural source must be easily feasible in an economically viable fashion (e.g., the monoterpenes limonene and α-pinene). This chapter is intended to review some monoterpenes with potential to be used as susbtrates to obtain new natural flavor compounds with economic and commercial interest. This chapter also describes the properties and uses of the most promising monoterpenes used as substrates, which could be important for an industrial standpoint increasing their importance as starting materials to obtain new molecules for biotechnological and biological applications.

Keywords: Terpenes, Biotransformation, Bioconversion, Aroma compounds, Bioflavors



Corresponding author: Gustavo Molina. E-mail: [email protected].


2

Gustavo Molina, Marina G. Pêssoa, Mariana R. Pimentel et al.

INTRODUCTION The methods for obtaining flavor compounds include the direct extraction from nature, chemical transformations and biotechnological transformations (which include microbial and enzymatic biotransformations, de novo synthesis and the use of genetic engineering tools) [1]. The scientific literature contains many examples of reviews dealing with the chemical reactions of terpenes to produce flavors [2] and the biotransformation of volatile terpenes for aroma production [3-5]. Despite the great industrial application of aroma compounds produced via chemical synthesis (still responsible for a large portion of the market due to the satisfactory yields), the bioprocesses possess a number of inherent advantages when compared with the classical chemical processing, since it occurs at mild conditions, presents high regio- and enantioselectivity, does not generate toxic wastes and the products obtained may be labeled as ―natural‖ [3, 6]. Also, biotechnological processes usually involve less damaging process conditions for the environment and yield desirable enantiomeric flavor compounds. Thus, bioflavors appeal to many sectors and represent a high market value [7, 8]. Therefore, the biocatalytic conversion of a structurally related precursor molecule (bioconversion or biotransformation processes) is often a more adequate strategy which allows significantly enhanced accumulation of a desired flavor product. As a prerequisite for this strategy, the precursor must be present in nature and its isolation in sufficient amounts from the natural source must be easily feasible in an economically viable fashion (e.g., the monoterpenes limonene and α-pinene). Among the most targeted substrates for biotransformation/bioconversion approaches are the monoterpenes [3, 8]. Thus, this chapter is intended to review some monoterpenes with potential to be used as susbtrates to obtain new natural flavor compounds with economic and commercial interest. This chapter also describes the properties and uses of the most promising monoterpenes used as substrates, which could be important for an industrial standpoint increasing their importance as starting materials to obtain new molecules for biotechnological and biological applications.

MONOTERPENE BIOSYNTHESIS Terpenes are secondary metabolites of plants produced in part for defense against microorganisms and insects. Previously, it was believed that these compounds were derived solely from the mevalonate pathway. However, some inconsistencies were observed, leading to the discovery of new biosynthetic pathways independent of the mevalonate pathway [9, 10]. Anyway, all terpenes are from common intermediates - the isopentyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) - whose synthesis is based on the path of mevalonate or towards deoxixilulose phosphate [11]. In other words, by having the same basic biochemistry these terpenes presents structural features and are grouped into a single class. The common feature of all terpenoids refers to


Production of Natural Flavor Compounds Using Monoterpenes as Substrates

3

the fact that they have a structure that can be decomposed into isoprene units (C5H10) and, therefore, have the general formula (C5H10)n. The volatile terpenes, i.e., mono (n = 2), and sesquiterpenes (n = 3) are major constituents of a variety of essential oils (Table 1), due to their organoleptic properties, are widely used in the flavor industry, is as fragrances or as ingredients in foods and cosmetics. Table 1. Major components of some essential oils (Rowe, 2005) Essential oil

Main components*

Caraway

S-(+)-Carvone, limonene, myrcene, α-phelandrene, α-pinene, β-pinene

Dill

S-(+)-Carvone, α-phelandrene, limonene

Eucalyptus

1,8-Cineol, α-pinene, p-cimene, limonene

Ginger

Zingiberene, α-curcumene, β-sesquiphelandrene, bisabolene, camphene, βphelandrene, 1,8-cineol

Orange peel

Limonene, myrcene, linalool, citronelal, neral, geranial,valencene, α- and βsinensal

Rose

Citronellol, geraniol, nerol, eugenol, geranilacetate, rose oxide

Mint

R-(–)-Carvone, limonene, myrcene, 1,8-cineol, dihydrocarveol

*

The compounds listed are terpenes or their derivatives.

MONOTERPENES AS SUBSTRATES FOR BIOTRANSFORMATION PROCESSES Limonene (1-Methyl-4-(1-Methylethenyl)-Cyclohexene) Limonene is the most abundant naturally occurring monoterpene and represents up to 90% of orange peel oil, an interesting and inexpensive citrus byproduct [12]. The expansion of the orange juice industry in Florida between 1945 and 1960 increased the percentage of processed oranges from 1% to 80%, and consequently the availability of large amounts of low-cost D-limonene from the peel oil [13]. Therefore, limonene has become one of the most studied precursors in bioconversion processes for production of high-value derivatives, which may be a good strategy for enrich the commercial value of agroindustrial residues, since the bulk price of this compound is around US$ 1-2/Kg, while its oxygenated couterparts, such as menthol and carvone, cost around US$ 30-60/Kg [14, 15]. Figure 1 shows the structure of limonene and the other monoterpenes selected in this chapter, with potential to be used as substrate for the bio-production of new compounds. Research and development on the degradation of limonene continued to draw much attention on a wide variety of conversion products such as perillic compounds, carveol, carvone at significant amounts, which could be more valuable in the fields of cosmetics, food ingredients, drug, and chemical synthesis. A number of reports in the literature concerned


4


with the biotransformation of limonene leading to many oxygenated derivatives were reviewed by Maróstica and Pastore [14] and Duetz et al. [16]. More recently Bicas et al. [17] reported the capability of P. fluorescens to metabolize limonene in α-terpineol at high concentrations of about 11 g/L, besides other products like limonene-1,2-oxide. Pseudomonas putida GS1 was able to convert limonene to perillic acid (up to 11 g/L) when the bacteria was cultivated in fed-batch culture with non-limiting amounts of glycerol, ammonium, and limonene [18].

Figure 1. Structure of monoterpenes with potential to be used in bioprocess as starting material.

Later on, an efficient integrated bioprocess was developed using a method for in situ product recovery (ISPR) to overcome the inhibition of bioconversion activity, leading to cumulative perilic acid concentration of 31 g/L after 7 days by Pseudomonas putida DSM 12264 [19]. More recently, other studies reported fungi as potential biocatalysts for limonene degradation leading to the formation of α-terpineol at concentrations of about 3.45 g/L by Penicillium sp. [20] and 2.4 g/L by Fusarium oxysporum 152b [21] which was further optimized to almost 4 g/L after 48 h of fermentation [22]. Carvone and carveol, high value-added compounds, were also reported as a conversion products from limonene by several microorganisms such as Pseudomonas aeruginosa, yielding about 0.63 g carvone/L [23], basidiomycete Pleurotus sapidus achieving a sum of carvone and carveol yield of up to 0.1 g/L [24], and Rhodococcus opacus obtained transcarveol with 94 to 97% conversion rate [25]. It is expected that these advances will soon result in viable processes from an industrial point of view.

Citronellol (3,7-Dimethyl-6-Octen-1-Ol) Citronellol is a linear monoterpene alcohol naturally occurring in various citrus plants. The R-(+)-isomer is commonly found in essential oils of plants of Rutaceae, whereas S-(-)-



5

isomer is found in geranium and citronella oil and is much less common [26]. Citronellol is an interesting flavor compound for food and flavor industries due to the presence of floral notes [27], but is also used as flavor agent in cosmetics, detergents, and ―biosafe‖ insect repellents. It may also be applicable as a precursor to produce other aroma compounds such as rose oxide, one of the most interesting citronellol derivatives [28, 29]. First studies on microbial transformation of terpenes were carried out by Mayer and Neuberg as early as 1915, who reported the synthesis of (+)-citronellol from (+)-citronellal mediated by yeast cells. Further investigations performed by Seubert [30] were based on the use of linear terpenes as sole carbon source by micro-organisms, thus a soil pseudomonad named Pseudomonas citronellolis was reported with the ability to use citronellol and related compounds as the sole source of carbon and energy. Subsequent studies elucidated probable enzymatic steps of the degradation pathway of citronellol in P. citronellolis [31 34]. The presence of β-methyl groups makes the oxidation of linear terpenes harder, thus the first steps of the catabolic pathway of citronellol are the oxidation of primary alcohols to respective aldehydes and acids, and subsequent conversion to the corresponding CoA esters citronellyl-CoA [35]. Then, βoxidation is activated by a key enzyme (genaryl-CoA carboxylase) that converts the β-methyl group to acetate, via carboxylation. The carboxymethyl group is removed by another enzyme, 3-hydroxy-3-isohexenylglutaryl-CoA lyase, and the product is able to be degraded by the leucine pathway [36, 37]. Other strains, P. mendocina and P. aeruginosa, were also reported to degrade citronellol [36, 38]. Investigations on the gene level through molecular biological tools also contributed for the understanding of the acyclic terpene catabolism by Pseudomonas species [39, 40]. Botrytis cinerea is an important example of fungal biocatalyst used in the bioconversion of citronellol, citral and its analogous alcohols nerol and geraniol. The infection of this fungus is especially interesting in fully ripe grapes where it delivers a desirable sweet flavor for winemaking [41]. Brunerie et al. [42, 43] published one of the first studies on the bioconversion of citronellol by the fungus Botrytis cinerea using grape must as medium culture. The main conversion products were the hydroxylation product (E)-2,6-dimethyl-2octen-1,8-diol and its reduction product 2,6-dimethyl-1,8-octanediol. When using a small amount of grape must in a synthetic medium, other products were additionally found such as 2-methyl-2-hepten-6-one, 2-methyl-2-hepten-6-ol and citronellic acid. Later, another research group reported the formation of 8-hydroxy citronellol as a conversion product of citronellol by a strain of Aspergillus niger in the presence of NADPH and O2. Rapp and Mandery [44] also detected hydroxylation products as the main metabolites in the biotransformation of citronellol by B. cinerea. The biotransformation of citronellol into 3,7-dimethyl-1,6,7-octanetriol in an aeratedmembrane bioreactor by the basidiomycete Cystoderma carcharias was described by Onken and Berger [45]. The increase of aeration promoted greater rates of microbial growth and biotransformation. They also reported the formation of an important side-product, 3,7dimethyl- 6,7-epoxy-1-octanol, and minor products such as 2,6-dimethyl-2-octene-1,8-diol, 3,7-dimethyl-5-octene-1,7-diol and 3,7-dimethyl-7-octene-1,6-diol. In minor amounts, microbial formation of rose oxide was observed for the first time in biotransformation of citronellol. The diol products are generated by the photooxygenation reaction of citronellol, from which the 3,7- dimethyl-5-octene-1,7-diol leads directly to cis/trans rose oxide upon dehydration and cyclisation [45]. Another group found the (S)-3,7-dimethyl-5-octene-1,7-


6


diol, a monoterpene from petals of Rose damascene Mill., to be the major genuine precursor for isomeric rose oxides through acid-catalyzed conversion [46]. Rose oxide (4-methyl-2-(2-methyl-1-propenyl)-tetrahydropyran) is a high value flavor compound, which occurs in trace amounts in essential plant oils such as Bulgarian rose oil and geranium oil [45, 47]. Thus, the biotechnological production of rose-oxide is especially interesting to flavor industry, since it is one of the most important components in creating rosy notes in perfumery. One of the last studies in the production of rose oxides by bioconversion of citronellol were published by Maróstica and Pastore [48]. They performed the biotransformation procedure by using agro-industrial residues in the process in order to provide alternative substrate, cost reduction, and minimize pollution problems. Liquid cassava waste is originated from pressing cassava roots and contains ―harmful‖ pollutants substances. Nevertheless, it was used as an interesting alternative for nutritive medium in bioconversion of citronellol, since the production of rose oxide reached yields of more than 70 and 30 mg/L of cis and trans-forms, respectively. Fungal strains of Aspergillus niger were found to bioconvert citronellol into cis- and trans-rose oxides and nerol oxide, by using solid-phase microextraction (SPME) as a fast monitoring technique for microbial screening. The bioconversion products cis- and trans-rose oxide and nerol oxide presented relative contents up to 54, 21 and 12% in the headspace SPME extracts, respectively [49].

Α- and Β-Pinenes ((1S,5S)-2,6,6-Trimethylbicyclo[3.1.1]Hept-2-Ene) Pinenes are naturally occurring bicyclic monoterpenes, found in major amounts in turpentine oils from most plants of the Pinaceae family [50]. Turpentine is a fluid obtained by the distillation of pine resin arising from the pulp and paper industries, where is collected as a by-product [50]. It is composed by terpenes, mainly the monoterpenes α- and β-pinene and other monocyclic terpenes such as limonene and 3-carene, however the ratio between the different terpenes in turpentine varies according to origin and this greatly influences its value and end use [52]. Likewise, the pulp and paper industry generates hundreds of thousands of tons of turpentine byproduct whose composition has high amounts of α-pinene [53] a bicyclic monoterpene very important as a starting substrate in industrial syntheses [54], with an annual output of 160.000t [55]. In Brazil, for example, the annual production of turpentine should reach the scale of a few tens of thousands of tons (estimates based on data from the Brazilian participation in the production of turpentine presented in Karthikeyan and Mahalakshmi [56]. Considering that turpentine is an abundant and inexpensive source of α- and β-pinenes, it would be interesting to develop methods for converting these monoterpenes into more valuable compounds in order to increase the commercial value of the turpentine oil. Therefore, efforts have been done to select biocatalysts with the ability of selective oxidation of pinenes into value-added compounds, such as verbenone and verbenol. This isfeasible, for example, by hydroxylating α-pinene to produce verbenol, which can be further converted to verbenone through dehydrogenation [57, 58]. Verbenol is a highly valued food-flavoring compound that is widely used in soft drink, soups, meats, sausages and ice cream [59]. Verbenone is the major constituent of the strawberry, raspberry, and spearmint flavor complexes and is in great demand in the food



7

industry due to its flavor notes of camphor and menthol [58, 60, 61], and can also be used as a precursor for the synthesis of Taxol® [62], a pharmaceutical drug used in chemotherapy. Moreover, verbenone and verbenol have also application in agriculture as antiaggregation and aggregation pheromones, respectively, for the control of southern pine beetle infestations [6365]. In fact, α-pinene has been widely studied in the bio-oxidation of terpenes using microbial cell cultures. Since 1960, selective oxidation of α-pinene to verbenol, verbenone, and trans-sobrerol by a strain of Aspergillus niger was described, and the process was later optimized by the same research group [57, 66]. In this approach, the potential to biotransform α-pinene was recognized using the yeast Hormonema sp., where the two biotransformation products formed were trans-verbenol and verbenone, reaching concentrations of 0.4 and 0.3 g/L after 96 h, respectively [67]. Lindmark-Henriksson [68] studied the same pathway to obtain these compounds using as biocatalyst a cell suspension of Picea abies. Another interesting report achieved a 15-fold increase in the biotransformation efficiency of α-pinene into verbenol by an Aspergillus niger and Penicillium sp., when these strains were subjected to mutation in UV light [59]. The ability of Pseudomonas fluorescens NCIMB 11671 to utilize α-pinene as sole carbon and energy sources was described by Best et al. [69]. The microorganism attacked the molecule through epoxidation reaction followed by two rings cleavage by α-pinene oxide lyase to form the isonovalal and novalal. Further reports described another Pseudomonas strain, P. rhodesiae CIP 107491 capable of catalyzing the cleavage of both rings of α-pinene oxide to form isonovalal [70]. Later, the same research group optimized the process to convert α-pinene oxide into isonovalal, with recovery of 400 g/L after only 2.5 h reaction [71]. Afterwards, the microorganism P. rhodesiae CIP 107491 was investigated for the bioconversion of α- and β-pinene by using different pinene sources. The results demonstrated the substitution of α-pinene by turpentine have given almost the same products profile, with isonovalal as mains product (80 g/L after 4 h) [17]. Other oxidative compounds derived from α-pinene, such as trans-sobrerol [66, 72], myrtenol [50, 73], α-pinene oxide [74], pinocarveol and pinocarvone, pinocamphone and others have also been reported [3]. Likewise, β-pinene also yields commercially attarctive compounds after its oxyfunctionalization. Toniazzo et al. [75] reported the conversion of β-pinene to α-terpineol by using Aspergillus niger ATCC 9462. The same product could also be obtained through the oxidation of α-pinene by Candida tropicalis [76] or Serratia marcescens [77]. Another study investigated the biotransformation of the substrates (+)- and (–)-limonene, α- and β-pinene and camphor by Aspergillus niger IOC-3913, and identified verbenone and α-terpineol as main derivative products from α- and β-pinene, while no bioconversion products from limonene and camphor were observed [78]. The same monterpenes were applied as substrates for bioconversion procedures by yeast isolated from pine forest litter, identified as Hormonema sp. UOFS Y-0067, resulting in trans-isopiperitenol from limonene, a mixture of verbenone and trans-verbenol from αpinene, and pinocamphone from β-pinene, which was latter hydroxylated to 3-hydroxypinocamphone [67]. Table 2 summarizes some products obtained from these substrates and the others discussed in this chapter, including the biocatalysts used in each process. Over the past few decades, a large number of biotransformations of α-pinene has been reported by fungi [79], bacteria [50], and plant cell culture [80, 81].


8


Linalool (3,7-Dimethylocta-1,6-Dien-3-Ol) Linalool is a very important molecule to flavor and fragrance industry due to its flowerlike odor. This compound has two isoforms ((S)-(+)-linalool and (R)-(-)-linalool), both of them are used in decorative cosmetics, shampoos, soaps, household cleaners and detergents [82]. Table 2. Monoterpenes used as substrates in bioprocesses for the production of natural flavor compounds Terpene Biotransformation substrate product Acyclic monoterpenes Citronellol Rose Oxide Geraniol α-Terpineol Linalool Linalool oxides Dihydrolinalool β-Myrcene and others Citral

Thymol

γ-Terpinene p-Cymene-9-ol Monocyclic monoterpenes α-Terpineol Carveol, carvone Limonene Perillic acid α-Pinene, sabinene and others Bicyclic monoterpenes α-Pinene Verbenol Menthol

β-Pinene

α-Terpineol

Example of Biocatalyst

Reference

Aspergillus niger Aspergillus niger Corynespora cassiicola

Demyttenaere et al., 2004 Demyttenaere et al., 2000 Mirata et al., 2008

Pseudomonas putida

Esmaeili et al., 2011

Penicillium digitatum Stemphyluim botryosum

Esmaeili and Tavassoli 2010 Krings et al., 2005

Pseudomonas fluorescens Rhodococcus opacus Pseudomonas putida DSM 12264

Bicas et al., 2008 Duetz et al., 2001

Penicillium sp

Esmaeili et al., 2009a,b

Aspergillus niger Aspergillus niger ATCC 9462

Agrawal et al., 1998

Mirata et al., 2009

Toniazzo et al., 2005

This acyclic monoterpene can be found in many plants like Cananga dorata,Citrus aurantium, Citrus bergamia, Coriandrum sativum seed oil, Melissa officinalis, Pelargonium roseum and Salvia sclarea [83] and in a variety of essential oils, as recently described in ―Nutmeg Geranium‖ (Pelargonium x fragrans Willd.) [84], honeybush (Cyclopia subternata) [85], Longjing tea (Camellia sinensis) [86] and in Cinnamomum osmophloeum ct. linalool essential oil, in which linalool found as pure S-(+)- isomer [83]. This monoterpene is also a potential therapeutic candidate for treatment of various diseases, because of its antiinflammatory and antinociceptive properties [87], it presents bactericidal effect on some microorganisms [88] and its ability of inducing apoptosis of human leukemia cells [89], besides other bioactive properties. When used as substrate in biotransformation procedures, linalool can result in a variety of high–value compounds, depending of the microorganism used. In general, the metabolites obtained have a floral, creamy odor and are used in perfume industry [90]. The degradation of



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linalool by Pseudomonas sp. was accomplished by reactions of oxidation and hydroxylation, leading to compounds such as furanoid linalool oxide, 2-vinyl-2-methyl-tetrahydrofuran-5one, 8-hydroxylinalool (2,6-dimethyl-2,7-octadiene-1,6-diol), 8-carboxylinalool, oleuropeic acid, α-terpineol and perillic acid [91]. When the fungus Botrytis cinerea was grown in grape must, linalool was converted mainly in (E)-2,6-dimethyl-2,7-octadiene-1,6-diol by a direct enzymatic hydroxylation of this substrate. In lower concentrations, 2-vinil-2-methyl-tetrahydrofuran-5-one, four furanoid and pyranoid forms of (E)- and (Z)- linalool oxides and (E)- and (Z)- acetates of pyranoid linalool oxides were found [92]. Despite the diversity of the compounds obtained, there is no information on their flavor potential. In other experiment, Mirata et al. [93] screened 19 types of fungi, of which, four strains were capable of bioconverting linalool. These researchers were the first to observe lilac aldehydes and lilac alcohols as by-product of fungal biotransformation of linalool. A. niger and B. cinerea were able to produce isomers of lilac aldehyde and lilac alcohol via 8hidroxylinalool, but linalool oxides and 8-hidroxylinalool were the major products of this biotransfomation. The major productivity was obtained by Corynespora cassiicola, reaching 120mg/L.day of linalool oxides. This fungus was identified as a highly stereoselective linalool biotransforming biocatalyst by the authors [93]. This last strain was further evaluated in a biotransformation process, where bioreactors and a system for substrate feeding and product removal were used in order to avoid toxic effects [94]. In another fungal screening, Molina et al. [95] showed that among the 36 strains isolated from Brazilian fruits, few strains were able to use this substrate and accumulate derivative compounds. Authors found the formation of products such as linalool oxide, geraniol, and α-terpineol. To confirm the enantioselective biotransformation of linalool by A. niger, a racemic mixture of linalool and pure (R)-(-)-linalool were used as substrate in liquid cultures. The results showed that the mixture of linalool isoforms were converted into a mixture of cis- and trans-furanoid and pyranoid linalool oxide. When pure (R)-(-)-linalool was used, only transfuranoid and trans-pyranoid oxide were found. It was also shown that the growing conditions were not the reasons of these differences [91]. Changes in culture conditions were performed by Demyttenaere et al. [90] in order to improve linalool biotransformation rates. They found that (S)-(+)-linalool was much better metabolized than the (R)-(-)-isomer and that the cosolvent applied affected the bioconversion rate.

Geraniol ((Trans)-3,7-Dimethyl-2,6-Octadien-1-Ol) Geraniol is an acyclic terpene alcohol, obtained from natural oils of several plants, such as rose (Rosa damascena), geranium (Pelargonium graveolens), citronella (Cymbopogun winterianus) and palmarosa (Cymbopogun martini). This rose-like flavor compound is mainly used as substrate in biotransformation, allowing the production of a variety of other terpenes, like citronellol, linalool, nerol, hidroxygeraniol, α-terpineol, cineol and 6-methyl-5-hepten-2one (MHO) [96, 97]. Geraniol has also been described with anthelmintic [96] and nematicidal activity [98] besides inhibiting Salmonella sp growth when combined with others food antimicrobials [99]. Moreover, some studies relate the efficacy of geraniol as pontential chemopreventive agent against renal carcinogenesis, since itsuppress renal oxidative stress


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and tumor incidence [100]. Geraniol also inhibits prostate cancer growth [101] and proliferation of hepatocarcinoma cells, because it modulates the expression of cell cycle regulators and induces apoptosis [102]. The biotransformation of geraniol to form value-added compounds such as citronellol, linalool, hidroxygeraniol, α-terpineol and 6-methyl-5-hepten-2-one has been the focus of many researchers. Changes in growth conditions and search for enzymes and microorganisms to perform the biotransformation have been made aiming improve yields. In order to find new microorganisms that are able to modify geraniol as substrate, the filamentous fungi Bipolaris sorokiniana was identified with a potential application in monoterpenes biotransformation due to the presence of a sesquiterpene phytotoxin that suggests an active terpenoid metabolism. After 5 days incubation in potassium phosphate buffer, the culture showed an oxidative profile with 74.6% yield of conversion of geraniol to 6-methyl-5-hepten-2-one, showing that B. sorokiniana is also capable of bioconverting geraniol into higher-value compounds, as already described in Penicillium digitatum, P. italicium and Pseudomonas incognita [97]. Studies on biotransformation of geraniol by cultures of Aspergillus niger and Penicillium sp. were made by Demyttenaere et al. [103]. The authors described that in liquid cultures, geraniol was converted mainly into linalool and α-terpineol, whereas in sporulated surface cultures, linalool was predominantly produced form geraniol, resulting in higher yields [103]. One of the biggest problems of biotransformation is that the product frequently is very toxic for living cells and has inhibitory effects on microorganisms. Fisher et al. [104] described that variations on pH affects the conversion of geraniol into linalool and nerol. They monitored this chemical reaction over a 65-day period and reported that at pH 3.4, 20.6% geraniol was converted to linalool, while at pH 7 no linalool formation was observed. For the biotransformation of geraniol into nerol, the major conversion rate was observed at pH 7.0 (14%). At lower pH (3.4), only 1.7% geraniol was converted [104]. Arifin et al. [105] described a new way of growing Saccharomyces cerevisiae for biotransformation of geraniol into citronellol in a gas-phase system, avoiding direct contact between the reaction medium, geraniol and citronellol. Previous experiments used S. cerevisiae in a resting cells system that also separates the cell growth and biotransformation process, but in the continuous-closed-gas-loop bioreactor (CCGLB) system, they obtained a maximum concentration of 1.18 g/L of citronellol [105]. In the same approach, Bluemke et al. [106] developed and integrated bioprocess where as possible to remove the products from the cells, consisting in a bioreactor and a downstream unit with a pervaporation membrane module. In this sense, a fast removal of the products was possible, minimizing the interaction cell-product and increasing microbial growth rates and reducing the product loss during biotransformation.

Citral (3,7-Dimethyl-2,6-Octadienal or Lemonal) Citral is a mixture of stereoisomers where the E-form is known as geranial and the Zform is known as neral. This mixture of aldehydes is readily available terpenoid that can be found in large amounts at reasonable costs from several herbs such as lemon grass, ginger, and some varieties of sweet basil [40, 107]. Citral is considered a valuable flavor component for the perfumery and food industries due to its ―lemony‖ scent odor characteristics [107].



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Several previous investigations have reported the bioconversion of citral into more valuable compounds with interesting properties to the flavor and food industries. For instance, thymol is an important natural preservative that has been used in food products such as cheese to prevent fungal growth [108]. The production of thymol (21.5%) as the major derivative from the biotransformation of citral by Penicillium digitatum was reported by Esmaeili and Tavassoli [108]. The authors also emphasized the effectiveness of the sporulated surface cultures method (SSCM) for biotransformation processes. Afterwards, the same research group reported the bioconversion of citral into citronellol by spores of cells of Saccharomyces cerevisiae [109]. Fungal transformation of citral using SSCM approach also resulted in the production of 6-methylhept-5-en-2-one by spores of Penicillium digitatum; similar results were obtained in the bioconversion of nerol [110]. The enantiospecific reduction of citral to produce citronellal by filamentous fungi, yeast and bacteria strains was reported by several authors [111]. According to Hall et al. [111], the reductase activity may compete with an alcohol dehydrogenases leading to the formation of nerol/geraniol and citronellol, depending on the microorganism. Strains of Pseudomonas were also reported as potent biocatalysts in bioconversion of citral resulting, for instance, in the production of geranic acid by P. convexa [112]. Likewise, citral was converted to geranic acid (62%) as the main conversion product followed by other compounds such as 1-hydroxy-3,7-dimethyl-6-octen-2-one (0.75%), 6methyl-5-heptenoic acid (0.5%), and 3-methyl-2-butenoic acid (1%) by Pseudomonas aeruginosa [113].

Menthol ((1R,2S,5R)-2-Isopropyl-5-Methylcyclohexanol) Menthol is one of the most important monoterpene alcohol worldwide. It is mainly applied in cigarettes, cosmetics, toothpastes, chewing gum, candies, and medicines [54, 114, 115]. Chemically, menthol is a cyclic monoterpene alcohol with three asymmetric carbon atoms and consequently four pairs of optical isomers are possible: (+)-and (–)-menthol, (+)and (–)-neomenthol, (+)- and (–)-isomenthol and (+)- and (–)-neoisomenthol, being (–)menthol the isomer that occurs most widely in nature (peppermint and other mint oils). This compound imparts a mint-like odor and exerts a cooling sensation when in contact to skin and mucosal surfaces, which is one of its most attracting attribute for industry [116]. The oil of plants from the genus Mentha in the family Lamiaceae is one of the main sources of menthol, that is generally found in the free state [115]. Besides its importance in the flavor industry, menthol has other applications. It acts as a topical analgesic and helps the percutaneous penetration of others anesthetic agents in the skin [117]; it is capable of enhancing ocular drug delivery [118] and inhibiting bone absorption, suggesting a protective effect against osteoporosis [119]; it also attenuates respiratory irritation responses to multiple cigarette smoke irritants [120] and decreases the viability of cancer cell lines [121, 122]. Menthol can be also used as substrate in biotransformation generating higher-value compounds. Hydroxilation products were obtainded for biotransformation of (+)- and (-)menthol by A. niger. In this case, (-)-menthol was converted into 1-,2-,6-,7-,8- and 9hydroxymenthols, whereas (+)-menthol was mainly biotransformed into 7-hydroxymenthol, with 1-, 6-, 8- and 9-hydroxymenthol as minor products [123]. When the fungus A. niger is


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grown in a sporulated surface culture, it was possible to observe the formation of cis-pmenthan-7-ol, whereas in sporulates surface cultures of Penicillium sp., the products obtained were limonene, p-cymene and γ-terpinene [124]. The products α-pinene, sabinene, trans-pmenthan-1-ol, p-menth-1-ene, 1,8-cineole and limenone were also identified in a subsequent study with Penicillium sp. [125]. The fungal biotransformation of menthol was also performed by Cephalosporium aphidicola and Macrophomina phaseolina. In a 12-day incubation of C. aphidicola with (1R, 2S, 5R)-(-) menthol, yielded six products: 10-acetoxymenthol, 4α-hydroxymenthol, 3α-hydroxymenthol, 7-, 9-, and 10hydroxymenthol [126]. The biotransformation of (+)-menthol with M. phaseolina generated 8,9-, 6R-, 1R-hydroxymenthol and others, with the C-8 position of menthol preferentially oxidized [127]. This terpene is an interesting example of the importance of these substrates and the products arising from bioprocesses, considering that the current demand of menthol exceeds the supply from natural sources, in this sense it is needed efforts to obtain menthol by natural means and from other more readly availabe raw materials, in addition to synthetic and semi-synthetic routes [115].

Myrcenes β-myrcene (7-methyl-3-methylene-1,6-octadiene) is an acyclic monoterpene found in essential oils of several plants. It was identified as major constituent in Artemisia scoparia oil [128] and present in lemongrass (Cymbopogon citratus), hop, bay and verbena [129]. This monoterpene can be used in food flavor additives, cosmetics, soaps, and detergents, but it has also been described as analgesic, anti-mutagenic, and as a tyrosinase inhibitor [130]. Some studies show that β-myrcene can induce liver isoenzymes [131], interfere in the xenobiotics‘ metabolism by inhibition of CYP2B1 monooxygenase [129] and it is a potential bioherbicide since this compound has phytotoxic character and inhibits the growth of Avena fatua, Cyperus rotundus and Phalaris minor through generation of ROS-induced oxidative damage [128]. The products of biotransformation of myrcenes have great importance for flavor and fragrance industries due to their lilaceous fragrance. The biotransformation of myrcenes by Pseudomonas putida generate dihydrolinalool, cis-β-dihydroterpineol, linalool and cisocimene-8-oxo as major products, varying according to the incubation time [132]. In the same approach, a similar study was made using Pseudomonas aeruginosa for the biotransformation of this monoterpene where the time of incubation was also observed as an important parameter. Different products were obtained in high yields and selectivity, such as dihydrolinalool (79.5%) and 2,6-dimethyloctane (9.3%) after 1.5 days of incubation, and αterpineol (7.7%) and 2,6-dimethyloctane (90.0%) after 3 days of process [133]. Thompson et al. [134] used a strain of Rhodococcus erythropolis for the biotransformation of β-myrcene into geraniol. They observed that at least four proteins were overproduced when R. erythropolis was growth on β-myrcene. Three of them were identified: an aldehyde dehydrogenase, an acyl-CoA dehydrogenase and a chaperone-like protein, involved in β-myrcene degradation pathway. Other studies were performed in order to find new strains capable of biotransformation of β-myrcene and the enzymes and pathways involved in this process. Iurescia et al. [135] isolated a β-myrcene-utilizing strain, identified as Pseudomonas sp. M1. This strain is able to grow on β-myrcene as the sole carbon source.



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They also obtained a β-myrcene-negative mutant, which only accumulates myrcen-8-ol as product of biotransformation of β-myrcene. The analyses of the genome of these microorganisms, lead to identification and sequencing of the β-myrcene catabolism genes. They found four open reading frames named myrA, myrB, myrC and myrD, that potentially code for an aldehyde dehydrogenase, an alcohol dehydrogenase, an acyl-coenzyme A (CoA) shynthetase and an enoyl-CoA hydratase, repectively, and a relationship between these gene and β-myrcene catabolism is suggested by the authors [135]. The bioconversion of β-myrcene was also shown when using solubilized enzyme fraction from mycelium lyophilisates of Pleurotus ostreatus. Perillene and rosefuran were the products of this reaction, the first one with a fresh citrus-flowery odor and the second with a flowery rose-like note. An intracellular pathway of perillene formation by Pleurotus ostreatus starts with the epoxidation of β-myrcene at conjugated double bound, but another pathway is described using soluble enzymes. In this pathway, β-myrcene and its derivatives were transformed into perillene, 6,7epoxyperillene, 7-hydroxyperillene and rosefuran through the corresponding endoperoxides, concluding that a dioxygenase-type enzyme introduces O2 at the 1,3-diene moieties of the monoterpene precursors [136].

Terpinenes α-Terpinene (1-isopropyl-4-methylcyclohexa-1,3diene) and γ-terpinene (1,4-pmenthadiene) are monocyclic terpenes used in pharmaceutical and perfume industries. They are the major compounds of tea tree (Melaleuca alternifolia) oil [137] and of others different essential oils, like from Artemisia annua L. and Senecio graveolens, to which they confer antioxidant properties [138]. Beside the antioxidant capacity, α–terpinene and γ–terpinene have been described as antibacterial, antifungal, anti-inflammatory, anticancer [139] and acaricidal activity against Hyalomma marginatum [140], but the use of these substances in medicine and cosmetics requires care. α-Terpinene, for example, autoxidizes to form allergens [141], while γterpinene is instable and presents cytotoxicity properties [142]. Some biotransformation assays using α–terpinene or γ–terpinene as substrate have been made. In a fermentation process with Corynesporium cassiicola, these terpenes were converted into (1R,2R)-p-menth-3-ene-1,2-diol and (1R,2R)-p-menth-4-ene-1,2-diol [143]. In another study, Krings et al. [142] identified a wild strain of the fungus Stemphyluim botryosum that was able to grown in high concentrations of γ–terpinene. The authores used this strain in a γ–terpinene biotransformation process and found that the two major products were identified as p-cymene-9-ol and p-mentha-1,4-dien-9-ol. The last one has never been described as a biotransformation product, due its chemical instability. The authors showed that the enzymatic process involved is the addition of oxygen atom at non activated carbon.

Others Besides the monoterpenes commonly used, several other have a great potential to be used as the substrate in biotransformation processes, but were little used and investigated. One


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example is camphor (C10H16O), a dicyclic terpenic ketone widely distributed in nature. The main source of d-camphor ((+)-camphor) is the wood of the tree Cinnamomum camphora Ness, l-camphor ((-)-camphor) can be found in the essential oil from Blumea balsamifera, Artemisa tridentate and Lavandula pedunculata, whilst dl-camphor is mainly present in the oil of Chrysanthemum sinense var. japonicum [144-147]. Due to its minty and diffusive aroma, camphor is used in perfuming industrial products, beverages, condiments, baked goods and frozen dairy [12, 114] and it was also described as substrate for biotransformation. Besides its importance in the flavor industry, camphor acts on bone metabolism inhibiting bone reabsorption not due to toxic effects [148], has strong antifungal and antibacterial activity, and may be useful in the clinical treatment of fungal diseases, particularly dermatophytosis [147, 149]. On the same approach, fenchone (C10H16O) is an irregular bicyclic monoterpene ketone that occurs in many fennel (Foeniculum vulgare Mill.) and thuja (Thuja orientalis, Cupressaceae) oils [150-152]. This compound resembles camphor very closely on its properties, as it has a camphoraceous odor, threshold of 510 ppb, and is used to prepare artificial fennel oils, to perfume household products and as a flavor additive in some food products, having an annual consumption of about 10.00lb [12, 114]. Besides its importance in the industry, fenchone has other applications such as repellent activity against Aedes aegypti (Diptera: Culicidae) females, moderate antifungal activity and acute local anesthetic activity [147, 153, 154]. These properties highlight the importance of fenchone and the need for more researches to obtain new derivatives from this terpene. Few biothecnological studies have been conducted for the application of these and other compounds as substrate, which could be an interesting field of study in order to obtain the derivatives of commercial interest.

CONCLUSION The use of monoterpenes as the substrate for obtaining new flavor compounds presents a great potential field of research and development. Besides the recognized importance of these substrates, directed both by their sensory profile as well by their economic interest, these bioprocesses can lead to obtaining new natural molecules with high added value and biological or biotechnological potential. Despite this great potential, many efforts should be directed towards the identification of new compounds, new metabolic pathways, suitable microorganisms with potential for this area and also new strategies to overcome the problems related with these processes. In fact, there are yet some challenges related to the biotransformation of terpenes, such as the chemical instability of both substrate and product, the low water solubility of the substrate, the high volatility of both substrate and product, the high toxicity of both substrate and product, the low yields and the high costs related to fermentation processes. Overall, this chapter showed the importance of some monoterpenes as substrates for the production of new metabolites. It is important to note that many of them remain without any study aiming to evaluate their biological and biotechnological potential, becoming a research field that requires further efforts directed to obtain new natural flavor compounds and



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improve their biotechnological production for the food and flavor industry, cosmetics and pharmaceuticals.

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[134] Thompson, M. L., Marriott, R., Dowle, A., Grogan, G., (2010). Biotransformation of beta-myrcene to geraniol by a strain of Rhodococcus erythropolis isolated by selective enrichment from hop plants. Appl. Microbiol. Biotechnol. 85, 721–730. [135] Iurescia, S., Marconi, A. M., Tofani, D., Gambacorta, A., Paternò, A., Devirgiliis, C., van der Werf, M. J., Zennaro, E., (1999). Identification and sequencing of beta-myrcene catabolism genes from Pseudomonas sp. strain M1. Appl. Environ. Microbiol. 65, 2871–2876. [136] Krügener, S., Schaper, C., Krings, U., Berger, R. G., (2009). Pleurotus species convert monoterpenes to furanoterpenoids through 1,4-endoperoxides. Bioresource Technol. 100, 2855–60. [137] Gomes-Carneiro, M. R., Viana, M. E. S., Felzenszwalb, I., Paumgartten, F. J. R., (2005). Evaluation of beta-myrcene, alpha-terpinene and (+)- and (-)-alpha-pinene in the Salmonella/microsome assay. Food Chem. Toxicol. 43, 247–252. [138] Pyka, A., Bober, K., (2002). On the importance of topological indices in research of αand γ-terpinene as well as α- and β-pinene separated by TLC. J. Liq. Chromatogr. R. T. 25, 1301–1315. [139] Marzec, K. M., Reva, I., Fausto, R., Malek, K., Proniewicz, L. M., (2010). Conformational space and photochemistry of alpha-terpinene. J. Phys. Chem. A 114, 5526–5236. [140] Cetin, H., Cilek, J. E., Oz, E., Aydin, L., Deveci, O., Yanikoglu. A., (2010). Acaricidal activity of Satureja thymbra L. essential oil and its major components, carvacrol and gamma-terpinene against adult Hyalomma marginatum (Acari: Ixodidae). Vet. Parasitol. 170, 287–290. [141] Rudbäck, J., Bergström, M. A., Börje, A., Nilsson, U., Karlberg, A.-T., (2012). αTerpinene, an antioxidant in tea tree oil, autoxidizes rapidly to skin allergens on air exposure. Chem. Res. Toxicol. 25, 713–721. [142] Krings, U., Brauer, B., Kaspera, R., Berger, R. G., (2005). Biotransformation of γ terpinene using Stemphylium botryosum (Wallroth) yields p-mentha-1,4-dien-9-ol, a novel odorous monoterpenol. Biocatal. Biotransfor. 23, 457–463. [143] Farooq, A., Atta-ur-Rahman, B. S. P., Choudhary, M., (2004). Fungal transformation of monoterpenes. Curr. Org. Chem. 8, 353–366. [144] Chizzola, R., Hochsteiner, W., Hajek, S., (2004). GC analysis of essential oils in the rumen fluid after incubation of Thuja orientalis twigs in the Rusitec system. Res. Vet. Sci. 76(1), 77–82. [145] Tabanca, N., Demirci, B., Baser, K. H., Aytac, Z., Ekici, M., Khan, S. I., Jacob, M. R., Wedge, D. E., (2006). Chemical composition and antifungal activity of Salvia macrochlamys and Salvia recognita essential oils. J. Agric. Food Chem. 54, 6593– 6597. [146] Mighri, H. El-jeni, H., Zaidi, S., Tomi, F., Casanova, J., Neffati, M., (2010). Composition and intraspecific chemical variability of the essential oil from Artemisia herba-alba growing wild in a Tunisian arid zone. Chem. Biodivers. 7, 2709–2717. [147] Zuzarte, M., Gonçalves, M. J., Cavaleiro, C., Dinis, A. M., Canhoto, J. M., Salgueiro, L. R., (2009). Chemical composition and antifungal activity of the essential oils of Lavandula pedunculata (Miller) Cav. Chem. Biodivers. 6, 1283–1292.


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[148] Mühlbauer, R., Lozano, A., Palacio, S., Reinli, A., Felix, R., (2003). Common herbs, essential oils, and monoterpenes potently modulate bone metabolism. Bone 32, 372– 380. [149] Tabanca, N., Kirimer, N., Demirci, B., Demirci, F., Başer, K. H., (2001). Composition and antimicrobial activity of the essential oils of Micromeria cristata subsp. phrygia and the enantiomeric distribution of borneol. J. Agric. Food Chem. 49, 4300-4303. [150] Croteau, R., Felton, M., Ronald, C. R., (1980). Biosynthesis of monoterpenes: Conversion of the acyclic precursors geranyl pyrophosphate and neryl pyrophosphate to the rearrangerd monoterpenes fenchol and fenchone by a soluble enzyme preparation from fennel (Foeniculum vulgare). Arch. Biochem. Biophys., 200, 524–533. [151] Díaz-Maroto, M. C., Díaz-Maroto Hidalgo, I. J., Sánchez-Palomo, E., Pérez-Coello, M. S., (2005). Volatile components and key odorants of fennel (Foeniculum vulgare Mill.) and thyme (Thymus vulgaris L.) oil extracts obtained by simultaneous distillationextraction and supercritical fluid extraction. J. Agric. Food Chem. 53, 5385–5389. [152] Raal, A., Orav, A., Arak, E., (2012). Essential oil composition of Foeniculum vulgare Mill. fruits from pharmacies in different countries. Nat. Prod. Res. 26, 1173-1178. [153] Mimica-Dukić, N., Kujundzić, S., Soković, M., Couladis, M., (2003). Essential oil composition and antifungal activity of Foeniculum vulgare Mill. obtained by different distillation conditions. Phytother. Res. 17, 368-371. [154] Zalachoras, I., Kagiava, A., Vokou, D., Theophilidis, G., (2010). Assessing the local anesthetic effect of five essential oil constituents. Planta Med. 76, 1647–1653. [155] Rowe, D. J., (2005). Chemistry and Technology of Flavors and Fragrances. Blackwell Publishing.




Chapter 2

DITERPENES AS CANCER THERAPY Elena González-Burgos and M. Pilar Gómez-Serranillos* Department of Pharmacology, Faculty of Pharmacy, University Complutense of Madrid, Madrid, Spain

ABSTRACT Cancer causes the death of about 7.6 million people each year. According to the World Health Organization (WHO), it is estimated that this number will be increased to 11.5 million of deaths around 2030, which implies an over the incidence of 75%. There is still a need for effective therapies for cancer. Diterpenes are secondary metabolites with a large diversity of structures that are widely distributed throughout the plant kingdom, particularly in Lamiaceae and Euphorbiaceae families. Recent evidence supports the beneficial effects of diterpenes occurring in the nature against human diseases, including different types of cancers. This chapter reviews current knowledge (in vitro, in vivo and clinical studies) on the antitumor potential of diterpenes in those relevant cancers, including breast, colon, lung, and stomach. Biomedical bibliographic databases from January 2000 to July 2013 and the keywords ―diterpene(s)‖, and ―cancer‖ have been employed for the preparation of this chapter. No restrictions were placed upon the language of publication, although only English language publications or papers with an English abstract were taken into account. The data reviewed suggest that some of these natural compounds may be promising candidates for the treatment of cancer.

INTRODUCTION Cancer term includes more than 200 different malignant types of diseases in which abnormal cells grow and disseminate overall in the body in an uncontrolled manner. Carcinogenesis (also called as oncogenesis and tumorigenesis) is a multistep and multifactorial process that initiates and proceeds after the exposition of cells to carcinogens (cancer-causing agents) (Table 1). The repeated interaction of these agents with DNA causes *

Corresponding author: E-mail address: [email protected].


26

Elena González-Burgos and M. Pilar Gómez-Serranillos

damage and mutations. As a consequence of the accumulated and constant cellular damage, normal cells are transformed into cancer cells that grow uncontrollably, and invade and destroy adjacent tissues (Figure 1) [1]. The majority of the cases of cancers (more than 65%) are thought to be caused by environmental factors and occupational exposures, including the alcohol, the smoking, the type of diet, the lack of exercise, some chemical products and the excessive exposure to sunlight, among others. For the rest of cases of cancers, about 5-10% has a hereditary tendency and, for a 20-25%, the cause is unknown and has not been yet identified [2-6]. Tobacco smoke contains multiple carcinogens, including polycyclic aromatic hydrocarbons, acetaldehyde, 1,3-butadiene, hydrazine and isoprene, among others, that are directly involved in the etiology of lung and breast cancer [7, 8]. Alcohol intake (frequency, duration, cumulative lifetime consumption, type of alcoholic beverage) can also increase the risk to develop breast and gastrointestinal tract cancers [9]. Eating a diet poor in fruits and vegetables and rich in fat is strongly associated with getting many cancers, including stomach, colon and breast [10 ,11]. Moreover, many epidemiological studies evidence that doing physical exercise may reduce the risk and development of different cancers such as breast and colon types [12-14]. Furthermore, exposure to environmental pollutants and chemical products in a workplace increase the risk of lung and breast cancers [15, 16]. Table 1. Classification of carcinogens

physical carcinogens Radiation:  ionizing radiation (e.g., gamma rays and X-ray).  - non-ionizing radiation (e.g., ultraviolet [UV])

Classification of carcinogens biologic carcinogens chemical carcinogens  Virus (i.e., papilloma  Inorganic compounds (i.e., viruses, Epstein-Barr arsenic, cadmium, chromium). virus, hepatitis viruses  Aromatic amines (i.e., 2B and C, adenoviruses naphthylamine, benzidine, 2cytomegalovirus). aminofluorene).  Bacteria (i.e.,  Aromatic nitro compounds (i.e., Helicobacter pylori). 4-nitrobiphenyl, 4,4'dinitrobiphenyl).  Dyes (4-dimethylaminoazobenzene, o-aminoazotoluene).  Alkylating agents (i.e., bis(2chloroethyl) sulphide (mustard gas), bis(chloromethyl) ether (BCME), methyl fluorosulphonate).  N-Nitroso compounds and hydrazines (i.e., Nnitrosodimethylamine, Nnitrosodiethylamine).  Naturally occurring carcinogens (i.e., aflatoxin B1 and less active analogues (from an Aspergillus), sterigmatocystin (from an Aspergillus).


Diterpenes As Cancer Therapy

27

Figure 1. Carcinogenesis.

In addition to external factors, hereditary factors are also involved in developing cancers. As example, about 5% to 10% of breast cancers are associated with mutations of over twenty genes such as BRCA1, BRCA2, TP53, CHEK2, and BARD1, among others [17]. Over 2030% of all colorectal cancers are linked to mutations within the mismatch repair genes (MMR) such as (MMR)-MSH2, MLH1, MSH6, and PMS2 [18]. Every year, 12.7 million of new cases of cancer are diagnosed, of which 7.6 million derived in death (which represents about 13% of all human deaths in the world). For the year 2030, it is estimated that the number of cancer deaths will be increased by a 75% [Data from the International Agency for Research on Cancer (IARC)] [19]. The most common types of cancer who are diagnosed in men are lung, stomach, liver, colorectal and oesophagy. Among women, they are breast, lung, stomach, colorectal and cervical [(data from the Agency for Research on Cancer GLOBOCAN database (version 1.2)]. In the recent years, the number of natural diterpenes and synthetic compounds derived from diterpenes has significantly increased as promising agents to prevent and suppress the development of cancers. Among mechanisms of cancer chemoprevention are included the scavenging of free radicals, the antioxidant activity, the induction of apoptosis, the alteration in gene expression, the modulation of signal transduction and the reparation of DNA, among others [20]. The efficiency of diterpenes as anticancer compounds has been tested in in vitro, in vivo and clinical trials. The taxane classes, which are anti-mitotic chemotherapeutic agents, are the only diterpenes-derived compounds available commercially (Table 2). Paclitaxel, isolated from the bark of Taxus brevifolia, was the first diterpene commercialized in 1992 for the treatment of cancers, including ovarian carcinoma, breast carcinoma, advanced forms of Kaposi´s sarcoma associated with AIDS, and non-small cell lung carcinoma [21]. Years later, in 1996, docetaxel, a semisynthetic analogue, was approved for the treatment of breast carcinoma, non-small cell lung carcinoma, prostate cancer and stomach cancer [22]. Cabazitaxel, was the last taxane approved by the US Food and Drug Administration (FDA) for hormone-refractory metastatic prostate cancer. This synthetic diterpene has the advantage, compared to paclitaxel and docetaxel, which has low affinity for P-glycoprotein. P-glycoprotein is an ATP-


28


dependent pump which can efflux several compounds, including anticancer agents, reducing its activity and efficiency [23]. This chapter reviews the main promising diterpenes for the prevention and treatment of breast, colon, lung and stomach cancers.

DITERPENES Diterpenes are secondary metabolites made up of four isoprene units (20 carbons atoms), and they are derived from geranylgeranyl pyrophosphate (GGPP) (Figure 2). In plants, previous studies using stable isotope markers suggest that biosynthesis of diterpene takes place in the plasmids via glyceraldehyde phosphate / pyruvate [also named as DOXP / MEP (1-deoxi-D-xilulosa-5-fosfato/2-C-metil-D-eritritol-4 phosphate)] through GGPP group [24, 25]. Diterpenes are naturally found in plants, including species of the botanical families Euphorbiaceae, Labiateae, Pinaceae and Leguminoseae, in fungus (i.e., Myrothecium verrucaria, Diplodia cupressi and Periconia sp.) and in animals such as soft corals and sea fans [26]. There have been identified more than 5000 different diterpenes, being classified in function of its structure into one of 200 different skeletons (i.e., kaurane, pimarane, labdane, etc.) [26]. Diterpenes are in continuous research for their therapeutically used as antioxidant, antiinflammatory, antimicrobial and anticancer agents [27]. In this chapter, we will focus upon the role of diterpenes as promising candidates against cancer (Figure 3).

Figure 2. Geranylgeranyl pyrophosphate (GGPP) structure.

Figure 3. Diterpenes as potential agents against breast, colon, lung and stomach cancers.


Table 2. Commercially available taxanes TAXANES Generic name Paclitaxel

Trade names Taxol® Abraxane®

Docetaxel

Taxotere®

Cabazitaxel

Jevtana®

Chemical name (2α,4α,5β,7β,10β,13α)-4,10-bis(acetyloxy)-13{[(2R,3S)- 3-(benzoylamino)-2-hydroxy-3phenylpropanoyl]oxy}- 1,7-dihydroxy-9-oxo5,20-epoxytax-11-en-2-yl benzoate 1,7β,10β-trihydroxy-9-oxo-5β,20-epoxytax-11ene-2α,4,13α-triyl 4-acetate 2-benzoate 13{(2R,3S)-3-[(tert-butoxycarbonyl) amino]-2hydroxy-3-phenylpropanoate} (1S,2S,3R,4S,7R,9S,10S,12R,15S)-4(Acetyloxy)-15-{[(2R,3S)-3-{[(tertbutoxy)carbonyl]amino}-2-hydroxy-3phenylpropa-noyl]oxy}-1-hydroxy-9,12dimethoxy-10,14,17,17-tetramethyl-11-oxo-6oxatetracyclo [11.3.1.03, 10.04,7]heptadec-13ene-2-yl benzoate

Action mechanism Disruption of microtubule function, leading to mitotic inhibition Suppression of microtubule dynamics (assembly and dis-assembly) Binding to tubulin and promotion of its assembly.

Therapeutic indications

References

Ovarian carcinoma, breast carcinoma, advanced forms of Kaposi´s sarcoma associated with AIDS, and non-small cell lung carcinoma Breast carcinoma, non-small cell lung carcinoma, prostate cancer and stomach cancer

[21]

Hormone-refractory metastatic prostate cancer

[23]


[22]

30


CANCER AND DITERPENES Colon Cancer and Diterpenes The diterpenoids quinones horminone and acetylhorminone have been reported to be promising therapeutic agents for colon cancer by inducing strans breaks in DNA in malignant cells [28]. Andrographolide, the main component of Andrographis paniculata, has been found to suppress in vitro colon cancer metastasis in the range of concentrations from 0.3 µM to 3 µM by inhibiting the activity of the enzyme matrix metalloproteinase 2 (MMP2). This zincdependent enzyme degrades components of the extracellular matrix, and it is directly implicated in the cancer metastasis process [32]. Jada et al. (2007) synthesized several andrographolide analogues with the aim to identify new potent anticancer compounds. The in vitro cell line screening revealed that 3,19-isopropyl-idene-andrographolide suppressed notably the proliferation and growth of tumoral cells, including the colon and breast types [50]. The briaranes diterpenes brialalepolides B and C, isolated from a soft coral (Briareum sp.), reduced the levels of the enzyme cyclooxygenase-2 (COX-2), which is increased in patients suffer from colon cancer, claimed to have beneficial effects in this type of cancer [36]. Another diterpene isolated from the sea (1S,2S,3E,7E,11E)-3,7,11,15-cembratetraen17,2-olide (LS-1) has been reported to have anti-colorectal cancer action through the production of reactive oxygen species that leads to growth suppression and apoptosis promotion [37]. In an experimental model with rats of colon cancer induced by azoxymethane, the oral administration with the dietary columbin from the crude drug Calumbae Radix significantly protected against the occurrence and expanding of colon cancer cells [38]. Ojo-Amaize et al. (2007) reported that the diterpene hypoestoxide possessed significant antitumor activity against colon cancer [47]. Several kaurane diterpenes have been identified in the treatment for colon cancer. The kaurane diterpene inflexinol from the aerial parts Isodon excisus has been identified as a promising anti-colon cancer agent by inducing the apoptosis in tumoral cells through the regulation of NF-κB target factor [48]. Gao et al. (2012) demonstrated that oridonin (kaurane type diterpene) increases significantly the levels of hydrogen peroxide (the main reactive oxygen specie in the human body) and causes the depletion of glutathione (the main antioxidant endogenous compound), resulted in the death of colorectal cancer cells via apoptosis mechanisms [52]. In 2008, Serova and coworkers observed that the diterpene ingenol-3-angelate, isolated from the aerial parts of Euphorbia peplus has a remarkable anti-cancer activity against colon tumoral cells via apoptosis pathway and inhibition of S phase of cell cycle [49]. The semisynthetic spiramine 15-oxospiramilactone suppressed the growth of colon cancer cells via inhibition of Wnt/β-catenin pathways [53]. The diterpene acid pseudolaric acid B, from the root bark of Pseudolarix kaempferi, exerted a potent in vitro anti-colon cancer effect by inducing apoptosis in tumoral cells [55].


Diterpenes as Cancer Therapy

Figure 4. (Continued)


31

32


Figure 4. Structures of diterpenes with anticancer properties.

In vitro and in vivo studies have also reported the antitumoral action against colon cancer that exhibits the labdane diterpene sclareol. This compound acts as an anticancer agent through different mechanisms, including induction of apoptosis, inhibition of cell growth and damaging DNA [56, 57]. Tanshinone IIA (abietane type), from the roots of Salvia miltiorrhiza, has been reported to be an effective inhibitor of colon cancer metastasis [59]. The diterpene lactone triptolide obtained from the roots of Tripterygium wilfordii has demonstrated to be a potential therapeutic candidate against colon cancer, a process mediated by the inhibition of inflammatory factors (COX-2 and INOS) and the induction of apoptotic factors [61-64].


Diterpenes as Cancer Therapy

33

Breast Cancer and Diterpenes Andrographolide, a labdane-type diterpene isolated from the Chinese official herbal of Andrographis paniculata, has been reported to be a promising effective agent against breast cancer. Different in vitro mechanistic studies have determined that andrographolide exerts its anticancer action through apoptosis activation, cell proliferation inhibition and induction of phase II enzymes (heme oxygenase-1) [29-31]. Apart from andrographolide, its semisynthetic analog named as DRF3188 has been identified as a potent anti-breast cancer via blockage of the G0-G1 phase cell cycle [39]. The semisynthetic diterpene ent-kaur-16-ene-13,19-diol 19-O-4',4',4'-trifluorocrotonate, derived from stevioside, has been demonstrated in an in vitro cancer cell model to be a promising therapeutic candidate against stomach, lung and breast cancers. The functional 19OH group contained in this diterpene has been found responsible for its potent cytotoxic activity [45]. Other kaurane diterpene, oridonin obtained from the Chinese herbal plant Rabdosia rubescens, has shown to inhibit the proliferation of breast cancer cells and induce its death via the apoptotic pathway [51]. Besides, the kaurane diterpene glycoside stevioside resulted to be effective in in vitro studies as an apoptotic inductor in breast cancer cell lines [58]. The diterpene13-epi-sclareol, found on the roots of the Indian herbal medicine Coleus forskohlii, has also demonstrated beneficial effect as an anticancer agent in an in vitro model using breast cancer cell line [46]. Several clinical trials have been performed to evaluate the efficacy and tolerability dose response of paclitaxel and docetaxel combined with other anticancer drugs. For docetaxel, as an example, in a phase II study over three years of duration, docetaxel combined with bevacizumab and cisplatin, increased overall survival of patients suffered from breast cancer [65]. Paclitaxel has been the main anticancer agent for breast cancer patients in many clinical trials. Favorable survival benefits with controllable tolerance have been reported on the combination of paclitaxel with neratinib [77], gemcitabine [78], lapatinib [79], trastuzumab [81], capecitabine [82], bevacizumab [84, 85] and sorafenib [86].

Stomach Cancer and Diterpenes Andrographolide from the aerial parts of Andrographis paniculata has exhibited in vitro antitumor effects against stomach cancer. Particularly, Jiang et al. (2007) demonstrated that this labdane-type diterpene can block the adhesion of gastric cells by inhibiting the expression of E-selectin encoded by the SELE gene [33]. In vitro studies using human stomach cell lines have determined that the growth inhibition properties of docetaxel may be potentiated when it is combined with the potent matrix metalloproteinase inhibitor batismatat. This drug combination could be effective in the treatment of stomach cancers [40-42]. Studies aimed to evaluate the anticancer effects of ent-11alpha-hydroxy-15-oxo-kaur-16en-19-oic-acid (5F) obtained from Pteris semipinnata L. has reported that this diterpene induces apoptosis via production of intracellular reactive oxygen species [43].


Table 3. In vitro and in vivo studies of promising diterpenes as anticancer agents Compound Acetyl horminone Andrographolide

Andrographolide

Type of compound diterpenoid quinones labdane

labdane

Brialalepolide B

briaranes

Brialalepolide C

briaranes

(1S,2S,3E,7E,11E)3,7,11,15-Cembratetraen-17, 2-olide

cembrane

Origin Roots of Salvia officinalis L. Aerial parts of Andrographis paniculata

Aerial parts of Andrographis paniculata

Soft coral (Briareum sp.) Soft coral (Briareum sp.) Vietnamese marine soft coral Lobophytum species

Anticancer activity Colon cancer

Type of study In vitro

Breast cancer

In vitro

Colon cancer

In vitro

Stomach cancer

In vitro

Lung cancer

In vivo In vitro

Colon cancer

In vitro

Colon cancer

In vitro

Colon cancer

In vitro

Effect

 Induction of DNA damage in cancer cells  Inhibition of cell proliferation.  Arrest cell cycle at G2/M phase.  Induction of apoptosis via caspase independent pathway  Induction of heme oxygenase-1 and inhibition of TPA-induced matrix metalloproteinase-9 expression  Induction of apoptosis  Anti-invasive activity via inhibition of MMP2 activity.  Suppression of the adhesion of gastric cancer cells by blocking Eselect in expression  Modulation of signal transduction  Inhibition of migration and invasion of cancer cells via down-regulation of PI3K/ Akt signaling pathway  Reduction of the expression of COX-2  Reduction of the expression of COX-2  Anti-proliferative and anti-apoptotic cell death via ROS generation.


References [28] [29]

[30]

[31] [32] [33]

[34] [35]

[36] [36] [37]

Compound Columbin

Type of compound diterpenoid furanolactone

DRF 3188

labdane

Docetaxel

taxane

ENT-11ALPHAHYDROXY-15-OXOKAUR-16-EN-19-OICACID (5F) Ent-3b-hydroxytrachyloban-18-al Ent-kaur-16-ene13,19-diol 19-O-4',4',4'trifluorocrotonate Ent-trachyloban3b,18-diol 13-Epi-sclareol

Horminone

Origin From the crude drug Calumbae Radix (the root of Jateorhiza columba) semi-synthetic analog of andrographolide A semisynthetic analogue of paclitaxel


Type of study In vivo

Breast cancer Stomach cancer

Effect

References

 Chemopreventive ability during the initiation phase

[38]

In vitro

 Arrest cell cycle at G0-G1 phase.

[39]

In vivo

[40, 41]

In vitro In vitro

 Tumor growth inhibition.  Batimastat (BB-94) potentiates the antitumor activity of docetaxel.  Tumor growth inhibition.  Cell death via ROS generation.

[42] [43]

kaurane

Pteris semipinnata

Stomach cancer

enttrachylobane diterpenes kaurane

Branches of Mitrephora alba

Lung cancer

In vitro

 Moderate cytotoxicity

[44]

Semysinthetic

In vitro

 Apoptosis-inducing activity

[45]

enttrachylobane diterpenes labdane


Stomach, lung and breast cancers Lung cancer

In vitro


[44]

Roots of Coleus forskohlii

Breast cancer

In vitro

[46]

diterpenoid quinones

Roots of Salvia officinalis L.

Colon cancer

In vitro

 Antiproliferative activity  Induction of apoptosis in tumor cells  Induction of DNA damage in cancer cells.


[28]

Table 3. (Continued)

Inflexinol

Type of compound Miscellaneous diterpenes kaurane

Ingenol-3-angelate

ingenane

3,19-Isopropylidene-andrographolide 3,19-IsopropylIDENE- ANDRO-

labdane

Hypoestes rosea dried leaf Aerial parts Isodon excisus Aerial parts of Euphorbia peplus Semysinthesis

labdane

Semysinthesis

Breast cancer

In vitro

 Tumor growth inhibition

[50]

enttrachylobane diterpenes ent-kaurane


Lung cancer

In vitro


[44]

Aerial parts of Rabdosia rubescens

Breast cancer

In vitro

[51]

Colon cancer

In vitro

 Antiproliferative activity  Induction of apoptosis in tumor cells  Induction of apoptosis and senescence in tumor cells  Tumor growth inhibition

Compound Hypoestoxide

Origin


Type of study In vivo

Colon cancer Colon cancer

In vivo In vitro In vitro

Colon cancer

Effect

References

 Tumor growth inhibition

[47] [48]

In vitro

 Induction of apoptosis through inactivation of NF-kappaB  Arrest cell cycle at S phase and induction of apoptosis.  Tumor growth inhibition

[49] [50]

GRAPHOLIDE

Methyl ent-3b-hydroxy trachyloban-18-oate. Oridonin

[52]

15-Oxo spiramilactone (NC043) Paclitaxel

spiramines

semisynthesis

Colon cancer

In vitro

taxane

Bark from Taxus brevifolia

In vitro

 Induction of apoptosis in tumor cells

[54]

Pseudolaric acid B

diterpene acid

Root bark of Pseudolarix kaempferi

Colon and stomach cancers Colon cancer

In vitro

 Induction of apoptosis in tumor cells.  Arrest cell cycle at G(2)/M phase.

[55]


[53]

Compound Royleanone Sclareol

Stevioside

Tanshinone I

Type of compound diterpenoid quinones labdane

Kaurene diterpenoid glycoside abietane

Origin Roots of Salvia officinalis L. Essential oil of Salvia sclarea


Type of study In vitro

Colon cancer

Effect

References [28]

In vivo In vitro

 Induction of DNA damage in cancer cells.  Antiproliferative activity  Induction of apoptosis in tumor cells  Tumor growth inhibition  Arrest cell cycle at G(1) phase.  Activation od caspases  Induction of DNA fragmentation.  Induction of apoptosis in tumor cells

[57]

 Inhibition of tumor growth  Down-regulation of cell cycle at S and G2/M phases  Inhibition of invasion and metastasis of cancer cells.  Antiproliferative activity  Induction of apoptosis in tumor cells

[34]

 Arrest cell cycle at G(1) phase.  Antiproliferative activity  Induction of apoptosis in tumor cells  Antiproliferative activity  Inhibition of inflamma-tory factor COX-2 and iNOS activity

[61, 62] [63]

Leaf of Stevia rebaudiana

Breast cancer

In vitro

Roots of Salvia miltiorrhiza

Lung cancer

In vivo In vitro

Tanshinone II-A

abietane

Roots of Salvia miltiorrhiza

Colon cancer Stomach cancer

Triptolide

Diterpene lactone

Root extracts Tripterygium wilfordii Hook F

Colon cancer

In vivo In vitro In vivo In vitro In vitro In vivo In vitro In vitro


[56]

[58]

[59] [60]

[64]

Table 4. Clinical trials of the taxane-tyoe diterpenes docetaxel and paclitaxel against breast, colon, stomach and lung cancers Compound Docetaxel

Drug combination Cisplatin bevacizumab

Breast cancer

Phase II study

Cisplatin S-1 (DCS)

Stomach cancer

S-1

Duration

Results

References

20 patients

Between 2005 and 2008

[65]

-

59 patients

Stomach cancer Stomach cancer Stomach cancer

-

113 patients

February 2009 to January 2011 -

The combinations of these drugs increase the median time-to-progression and overall survival The combinations of these drugs increase overall survival

[67]

Phase II study

43 patients

-

Phase II study

90 patients

-

Gemcitabine

Lung cancer

Phase II multicenter trial

50 patients

-

Favorable survival benefits with controllable tolerance Favorable survival benefits with controllable tolerance The combinations of these drugs have promising efficacy and may be an alternative treatment option that avoids cisplatin. Favorable survival benefits with controllable tolerance

Cisplatin

Stomach cancer

-

43 patients

-

Favorable survival benefits with controllable tolerance.

[76]

neratinib

Breast cancer

Phase I/II, open-label, two-part study

102 patients

The overall median treatment duration was 47.9 weeks

High rate of response

[77]

cisplatin Docetaxel

Paclitaxel

Fluorouracil

Cancer

Type of study

Patients


[66]

[68] [74]

[75]

Compound

Drug combination gemcitabine

lapatinib cisplatin

Paclitaxel

Cancer Breast cancer

Breast cancer Stomach cancer

Type of study

Patients

Duration

231 patients

7.5 months

The combinations of these drugs increase overall survival

[78]

-

-

[79]

52 patients

1, 8, and 15 of a 4-week regimen January 2008 and January 2011 Every 28 days until disease progression or unacceptable toxicity

The combinations of these drugs increase overall survival Favorable survival benefits with controllable tolerance Favorable survival benefits with controllable tolerance

[81]

Effective and well-tolerated

[82]

Favorable survival benefits with controllable tolerance

[83]

Effective and well-tolerated

[84]

Improved disease in control but did not significantly improve in breast cancer patients

[85]

Breast cancer

-

40 patients

capecitabine

Breast cancer

-

44 patients

bevacizumab

Breast cancer

Randomised phase 3 trials open-label, noninferiority, phase 3 trial.

564 patients

September 2008 to August 2010

bevacizumab

Breast cancer

62 patients

Between February 2009 and August 2011

Breast cancer

References

Prospective, randomized, multicenter, phase III trial Phase III, randomized, double-blind study Phase II trial

trastuzumab

sorafenib

Results

-

Phase II, randomised, double-blind, placebocontrolled

170 patients from India, 52 patients from the United States and

-


[80]

Table 4. (Continued) Compound

Paclitaxel

Drug combination

Cancer

Type of study

S-1

Stomach cancer

Randomised, openlabel, phase II study

doxifluridine

Stomach cancer

phase I clinical trial

Stomach cancer

Case report

doxifluridine

Patients 15 from Brazil 83 patients

Duration

January 2006 and November 2010

28 patients -

A 50-year-old female

Five days per week on a 28day cycle

Results

References

Favorable survival benefits with controllable tolerance

[86]

Recommended dose 80 mg/m2 of paclitaxel (days 1 and 8) and 800 mg/m2) of doxifluridine (days 1-14) every 3 weeks. Effective and well-tolerated gastric cancer patients.

[87]


[88]


41

In stomach cancer models, the abietane type tanshinone IIA inhibited the proliferation of tumoral cells by inducing apoptosis [60]. Important clinical trials results have been obtained for docetaxel in combination with cisplatin in the treatment of stomach cancer [66, 67, 70, 72, 73, 76]. As example, Park et al. (2004) concluded that docetaxel plus cisplatin could constitute second-line chemotherapy for stomach cancer [76]. In addition of cisplatin, docetaxel has been investigated combined to other anticancer drugs, including S1 [66, 67], capecitabine [71] and fluorouracil [74], highlight as alternative option that avoids cisplatin. Besides docetaxel, clinical data have demonstrated the effectivity of paclitaxel in combination with the anticancer drugs doxifluridine, S-1 and cisplatin [80, 86, 87, 88] for the treatment of stomach cancer.

Lung Cancer and Diterpenes Andrographolide has also shown cancer chemopreventive ability against lung cancer through the modulation of signal transduction [34] and the inhibition of PI3K/Akt/AP-1 signaling pathway [35]. In a recent study, Rayail et al. (2013) analyzed the potential in vitro antitumoral action through a series of ent-pimarane and ent-trachylobane diterpenoids from Mitrephora alba against several cancer types. The findings from this research revealed that three diterpenes assayed to belong ent-trachylobane structure (ent-3b-hydroxy-trachyloban-18-al, enttrachyloban-3b,18-diol and methyl ent-3b-hydroxytrachyloban-18-oate) were active in lung cancer [44]. The abietane diterpene tanshinone I, isolated from the roots of Salvia miltiorrhiza, inhibited the tumor growth and the cell cycle at S and G2/M phases in in vitro and in vivo lung cancer models [34]. As a highlight clinical trial, in 2005 it has been assayed the efficacy of the combination of docetaxel plus gemcitabine against lung cancer, being the results satisfactory as evidenced in the rate of survival [75].

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Chapter 3

PHENOLIC DITERPENES FROM ROSEMARY AS ENHANCEMENT AGENTS OF USUAL CHEMOTHERAPEUTIC DRUGS FOR COLORECTAL CANCER THERAPY Tiziana Fornari1*, Ana Ramírez de Molina2 and Guillermo Reglero1,2 1

Instituto de Investigación en Ciencias de la Alimentación CEI UAM+CSIC, Madrid, Spain 2 IMDEA-Food Institute, CEI UAM + CSIC, Madrid, Spain

ABSTRACT Rosemary (Rosmarinus officinalis) is a perennial herb from Lamiaceae family, typical of the Mediterranean region, which has been recognized to have numerous and important biological properties, such as antioxidant, antiviral, antimicrobial, hepatoprotective, antidiabetic, antiproliferative, and antidepressant, among others. Some of these activities point to a favorable effect of rosemary in controlling cancer development. Particularly, the large antioxidant power of rosemary extracts is well related with the presence of phenolic compounds, particularly carnosic acid, carnosol, methyl carnosate, rosmanol and rosmarinic acid. Moreover, carnosic acid and carnosol, two phenolic diterpenes, are the most abundant antioxidants of rosemary. Among the several investigations connected with the anticarcinogenic effects of rosemary extracts, the authors have recently reported the beneficial activity of supercritical rosemary extracts in colorectal cancer. This is the second most frequently diagnosed cancer in females and the third in males. Although the chemotherapy currently used in colorectal cancer is mostly based on the antimetabolite 5-fluorouracil (5-FU), only 10–15% of patients with advanced colorectal cancer respond to the administration of 5-FU alone, and response rates modestly increase to near 50% in combination with other antitumor agents. *

C/Nicolás Cabrera 9, Campus de la Universidad Autónoma de Madrid, 28049 Madrid, Spain. Email: [email protected].


48

Tiziana Fornari, Ana Ramírez de Molina and Guillermo Reglero This chapter provides a comprehensive and updated discussion about the production of rosemary extracts with high concentration of phenolic diterpenes, their effectiveness in cancer therapy to inhibit the proliferation of human colon cancer cells, and their role in the promotion of the antitumoral effect of 5-FU.

Keywords: Phenolic diterpenes; Rosemary; Carnosic acid; Supercritical extraction; Colon cancer

INTRODUCTION Recent studies exposed that certain substances contained in many plants and herbs reveal capacity to prevent, reverse and/or inhibit certain processes of carcinogenesis before the development of invasive cancer [1, 2]. Scientific studies are currently in progress, in order to identify those phytochemicals and prove their specific functional activities. Principally, catechins present in green tea leaves [3], resveratrol of grapes, berries and peanuts [4], lycopene of tomato [5] and the natural phenol antioxidant found in numerous fruits and vegetables, namelly ellagic acid [6], have been reported to show good capability to prevent the development of certain types of cancer. Particularly, rosemary (Rosmarinus officinalis L.) has been recognized to have several and important biological properties, such as hepatoprotective [7], antidiabetic [8], antioxidant [9], antiproliferative [10], antiviral [9], antimicrobial [12], antinociceptive [13] and antidepressant [14], among others. Some of these activities are related with controlling cancer development and hence, studies have been reported showing that rosemary extracts and certain specific substances present in these extracts possess inhibitory effects on the growth of breast, liver, prostate, lung and leukemia cancer cells [14] and represses the initiation and promotion of tumorogenesis of melanoma and glioma in animal models [15-17]. Unquestionably, the antioxidant activity of rosemary extracts is the biological property most widely studied. Rosemary antioxidant capacity was attributed to the presence of phenolic diterpenes, such as carnosol, rosmanol, carnosic acid, methyl carnosate, and phenolic acids, such as rosmarinic and caffeic acids [18-20]. Carnosic acid, followed by carnosol, is the most abundant antioxidant of rosemary. In vitro investigations have shown that carnosic acid has an antioxidant activity three times higher than that of carnosol [21]. Furthermore, different authors [23, 24] established the superior antioxidant activity of supercritical rosemary extracts when compared with those obtained by hydro-distillation or using liquid solvent extraction. Supercritical fluid extraction (SFE) using carbon dioxide (CO2) is an innovative, clean and environmental friendly technology currently applied for the extraction of bioactive substances from plants and herbs. Extraction temperatures are low, times are moderate, extraction is high selective, and there is no associated waste treatment of toxic solvents. CO2 is particularly suitable for the extraction of lipophilic substances, such as terpenes, and thus, the SFE of rosemary leaves to produce natural antioxidant extracts has been extensively investigated and reported [23-31]. In this respect, an important advantage of SFE is the possibility of making an easy fractionation of the extract, and separate the volatile oil, comprised mainly of monoterpenes and oxygenized derivatives, from the antioxidant diterpene phenolic compounds. For example, this fractionation can be accomplished by


Phenolic Diterpenes from Rosemary As Enhancement Agents

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applying two sequential extractions, at different extraction conditions each, or by producing a cascade decompression in two or more separator vessels. In this chapter, the supercritical production of rosemary antioxidant extracts is reviewed, elucidating about the concentration and isolation of the most abundant phenolic diterpene of rosemary, namely carnosic acid. The effect of pressure, temperature, amount of ethanol cosolvent and extraction time is discussed in terms of yield, composition and antioxidant activity of the extracts produced. Fractionation of the supercritical extract is employed to concentrate carnosic acid, by attaining its separation from the plant volatile oil. Additionally, it is explained the isolation of carnosic acid (95% purity) by coupling SFE with preparative supercritical fluid chromatography (SFC). Also, supercritical fluid technology is compared with other extraction techniques, such as pressurized solvent extraction (PLE) and ultrasound assisted extraction (UAE). The antioxidant power and antitumor activity of rosemary extracts is discussed, and the relationship between the content of phenolic diterpenes and the ability of rosemary to inhibit the proliferation of human colon cancer cells is analyzed, focusing on dose-dependency and the effects on both citoestaticity and cytotoxicity. In addition, the antitumor properties of rosemary extract further than antiprolefative effect on colon cancer cells are reported, and analyzed in combination with the most widely used chemotherapeutic agent in colorectal cancer, 5-fluoaracil (5-FU). Furthermore, the effect of rosemary extract as a natural agent useful to overcome resistance to this drug is shown, focusing on the putative molecular mechanism of action mediating this effect. Finally, the potential of supercritical-fluid rosemary extract as a personalized adjuvant agent for colorectal cancer patients is discussed.

ROSEMARY PLANT Rosemary (Rosmarinus officinalis) is a perennial herb, member of family Lamiaceae, genus Rosmarinus. It is an aromatic shrub with needle-like leaves and white, pink, purple, or blue flowers. Rosemary is native to the Mediterranean and Asia, but is reasonably hardy in cool climates. It can withstand droughts, surviving a severe lack of water for lengthy periods. The plant can reach 1.5 m tall, rarely 2 m. The leaves are evergreen, 2–4 cm long and 2–5 mm broad, of green color above and white below. The plant flowers in spring and summer in temperate climates, but can be in constant bloom in warm climates. The leaves, both fresh and dried, are used in traditional Mediterranean cuisine. They have a bitter, astringent taste and are highly aromatic, which complements a wide variety of foods. Also, tisanes can be made from the leaves. Rosemary extract has been shown to improve the shelf life and heat stability of omega 3rich oils, which are prone to rancidity [32]. Recently, the European Commission published Directive 2010/67/EU of 20 October 2010 [33] informed on the safety of rosemary extracts when used as an antioxidant in foodstuffs. Such document establishes appropriate specifications to authorize rosemary extracts as a new food additive for use in foodstuffs, and assigned E392 as its E number. Moreover, several types of production process are described, using solvent (ethanol, acetone and hexane) extraction and also supercritical CO2 extraction. According to Directive 2010/67/EU, rosemary extracts for use as food additive should contain


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Tiziana Fornari, Ana Ramírez de Molina and Guillermo Reglero

more than 13 % mass of antioxidant compounds (carnosic acid + carnosol) and the antioxidant/volatiles ratio should be greater than 15. Besides the cuisine uses of rosemary, many medicinal properties has been attributed to this plant since ancient times [34] and it has been used as a tonic, a digestive aid, headaches, muscle spasms, expectorant, promoter of menstrual flow, and stimulant for production of bile. Rosemary has also been related with enhancing memory. Shakespeare's Ophelia petitions Hamlet with, "There's rosemary, that's for remembrance, pray you love, remember." Scholars of ancient Greece carried wreaths of rosemary about the brow to improve memory during exams. Nowadays, several biological activities of rosemary were methodically confirmed and hence, this plant has been taken as a reference for the scientific community to conduct important studies in the field of biomedicine, seeking beneficial results for human health. The phytochemical composition of the different parts of the plant can vary significantly depending on the source, the chemo-type concerned and their development stage at the time of collection. A large number of terpenes and oxygenated derivatives, which can represent up to 2.5 % of the composition of rosemary leaves, are part of what is called the essential oil of the plant, a colorless or slightly greenish yellow dye, camphor odor, primarily responsible for its characteristic aroma and flavor. The main terpenes found in rosemary essential oil are αpinene and limonene, and main terpenoids are 1,8 cineol, linalool, camphor, borneol, terpineol, verbenone and α-caryophyllene (see Figure 1). Rosemary essential oil is used for purposes of fragrant bodily perfumes or to emit an aroma into a room. It is also burnt as incense, and used in shampoos and cleaning products. Another large group of compounds that are an important part of the plant due to their demonstrated high antioxidant activity are the phenolic compounds such as carnosic acid, a lipophilic terpene-type substance, rosmarinic acid, which is a phenolic hydrophilic acid, and derivatives of the foregoing such as carnosol, rosmanol, rosmadial and methyl carnosate (see Figure 2). As mentioned before, some of these compounds were associated to control cancer development, showing inhibitory effects on the growth several types of cancer cells and repressing tumorogenesis in animal models. However, the potential synergism among components, as well as the putative mechanism of action by which it exerts this biological activity has not been clearly addressed to date.

Figure 1. Main components of rosemary essential oil.



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Figure 2. Main antioxidant phenolic compounds of rosemary.

EXTRACTION TECHNOLOGIES APPLIED TO THE PRODUCTION OF ROSEMARY EXTRACTS Solvent extraction at ambient pressure is the most custom technology exploited to extract phytochemicals from plant matrix. Suitable solvents should have low boiling points, in order to be easily removed from the extract and re-utilized. Hence, the main drawback of this technique is the occurrence of solvent residues in the extracted product. Solvents employed to extract lipophilic phytochemicals are non-polar paraffin-type (hexane, pentane, petroleum ether) or moderate polar solvents (tetrahydrofuran, dichloromethane). On the other side, high polar compounds are usually extracted using water, alcohols or their mixtures. Although high


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temperatures favor the solubility of the solute in the solvent, extraction temperature is limited by solvent boiling point. In this respect, pressurized liquid extraction (PLE) is a new technology that allows employing higher extraction temperatures, providing that pressure be high enough so as to maintain the solvent in liquid state. Moreover, due to the raised pressure employed, compression is made on the vegetal particle contributing to improve extraction. Thus, lower amounts of solvent are required, extraction is faster, higher yields are attained and the loss of volatiles is minimized [35]. Nevertheless, PLE also requires a post-extraction procedure to separate the solvent from the extract. Traditional solvent extraction assisted by ultrasound (UAE) is another very used technique for the extraction of phytochemicals [36, 37]. The use of ultrasound enhances extraction rate and yield, and allows reduction of extraction temperature [38] what is crucial in the case of thermolabile substances. Ultrasound produces turbulence, particle agglomeration and cell disruption, physical effects that arise principally from cavitation, i.e., the formation, growth and violent collapse of microbubbles owing to pressure fluctuations. Supercritical fluid extraction (SFE) is indeed the most widely studied application to extract and isolate bioactive substances from plant matrix [18, 19, 39, 40]. The commercial production of supercritical plant extracts has brought a wide variety of products that are actually in the market. The general agreement is that supercritical extracts proved to be of superior quality, i.e., better functional activity, in comparison with extracts produced using liquid solvents [23, 41-43]. Carbon dioxide (CO2) is the most employed supercritical solvent, due to its moderately low critical pressure (7.4 MPa) and temperature (31.1C). Furthermore, is non-toxic, non-flammable and available in high purity at relatively low cost. In SFE, selectivity is mainly determined by CO2 density, which could be varied by modification of the extraction conditions (temperature and pressure). SFE with pure CO2 has the advantage of recovering the extract with high purity, completely free of solvent, and thus it is considered the most effective and environmentally benign technological approach to recover phytochemicals from plant matrix. Supercritical CO2 has a polarity similar to liquid pentane and thus is suitable for extraction of lipophilic compounds. Polar cosolvents, such as alcohols, are employed to improve the extraction of polar substances. Yet, if a cosolvent is employed, the separation of the liquid solvent from the product is unavoidable. Mechanical stirring is difficult to be accomplished in SFE and thus, the most practical application is assisting the extraction using ultrasound. This combination usually produces important benefits to improve mass transfer processes [44, 45].

PRESSURIZED LIQUID EXTRACTION (PLE) A scheme of typical equipment is shown in Figure 3. The extraction cell is loaded with the solid sample and placed into an oven. Then, the cell is filled with the corresponding solvent up to a pressure that ensures the liquid state of the solvent and is heated-up to the desired extraction temperature. Then, a static extraction continued, while all the system valves are closed. In order to prevent over-pressurization of the cell, a static valve is pulsed open and closed automatically when the cell pressure exceeded the set point. After extraction


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the cell is washed with the solvent and subsequently the solvent is purged from the cell using N2 until complete depressurization is accomplished.

Figure 3. Scheme of typical PLE equipment.

Table 1. Presurized liquid extraction (PLE) and solid-liquid extraction (SLE) of rosemary using hexane and ethanol. CA: carnosic acid; CAR: carnosol

solvent solvent/plant ratio (ml/g) temperature pressure time yield (%) % mass CA % mass CAR CA recovery (mg / g plant)

PLE hexane 20 150C 115 bar 10 min 13.7 13.41 0.96 18.37

SLE hexane 100 50C 1 bar 4h 7.4 24.6 18.20

PLE ethanol 20 150C 115 bar 10 min 63.8 4.63 0.25 29.54


SLE ethanol 100 50C 1 bar 4h 20.7 12.36 1.29 25.56

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PLE of rosemary was accomplished in an Accelerated Solvent Extraction system ASE 350 from Dionex Corporation (Sunnyvale, CA, USA), similar to equipment of Figure 3. The extraction of key antioxidant compounds is reported in Table 1, and compared with traditional solid-liquid extraction at ambient pressure (SLE). Taking into account each type of solvent, it is obvious from Table 1 that PLE can produce the same recovery of carnosic acid from rosemary leaves than that obtained using solvent extraction at ambient pressure. Yet, considerable lower solvent/plant ratio and shorter extraction time are required. Additionally, PLE result in higher extraction yields due to the higher extraction temperature and pressure applied. According to Table 1 PLE yield is around two and three times higher than SLE yield when, respectively, hexane and ethanol are used. Nevertheless, these increased yields are a consequence of the extraction of substances other than lipophilic antioxidants, since the concentration of these compounds in the PLE extracts are reduced almost in the same magnitude that yield increase. Indeed, selectivity of hexane is better than that of ethanol to extract rosemary phenolic diterpenes. Nevertheless, residues of toxic solvents in the extracts limit the application of paraffin-type solvents in the production of rosemary extracts.

ULTRASOUND ASSISTED EXTRACTION (UAE) Table 2 shows the UAE of rosemary leaves using the green solvent ethanol and different sonication times. As can be observed in the table, sonication considerably increases the extraction of phenolic diterpenes from rosemary: after very short extraction time (15 min) the recovery of carnosic acid is higher than that produced by PLE. Furthermore, as in PLE, low solvent/plant ratio is required but, on the contrary of PLE, high extraction temperatures are not mandatory. Nevertheless, high carnosic acid recovery demand long sonication times what produce lower concentrations of phenolic diterpenes. Table 2. UAE of rosemary at ambient pressure and using ethanol as extractive solvent. Extraction temperature was maintained around 30C. CA: carnosic acid; CAR: carnosol Sonication time (min) solvent / plant ratio yield (%) % mass CA % mass CAR CA recovery (mg / g plant)

5 20 9.0 20.44 2.20 18.40

10 20 12.8 18.52 1.76 23.71

15 20 25.6 12.94 0.59 33.13

Very important differences between SLE, PLE and UAE could be established taking into account the amount of antioxidants extracted and the amount of solvent employed. In this respect, and considering the consumption of ethanol, SLE is the most inefficient procedure since only 0.3 mg of carnosic acid was recovered per ml of ethanol employed. In the PLE, this value is increased up to 1.5 mg carnosic acid / ml ethanol, which is of the same order of



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those obtained in the UAE assays (0.9, 1.2 and 1.7 for, respectively, 5, 10 and 15 min of sonication).

SUPERCRITICAL FLUID EXTRACTION (SFE) A basic extraction scheme of semi-continuous SFE equipment for processing solid materials is shown in Figure 4. The extraction vessel is charged with the raw material (vegetal matter dried and grinded) and supercritical CO2 is continuously loaded from the bottom of the extraction vessel. At the exit of the extractor the supercritical CO2 with the solutes extracted flows through a depressurization valve (back pressure regulator, BPR) to a separator (S1) in which, due to the lower pressure, the extracts are separated from the gaseous solvent and collected. Some SFE devices contain two or more separators, as is the case of the scheme shown in Figure 4. In these cases, it is possible to fractionate the extract in two or more fractions (on-line fractionation) by setting suitable temperatures and pressures in the separators. In the last separator, CO2 reaches the pressure of the recirculation system (generally around 4-6 MPa). After flowing through a filter, the solvent is liquefied in a heat exchanger (HE1) and stored in a tank. CO2 is withdrawn from this tank, and is pumped and heated up to the desired extraction pressure and temperature. Before pumping, precooling of the solvent is generally required (HE3) in order to avoid pump cavitation. If a cosolvent is employed an additional pump is required, and the cosolvent is mixed with the CO2 previously of heating (HE2) as is depicted in Figure 4.

Figure 4. Scheme of a SFE plant for processing solid materials.


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As mentioned before, supercritical CO2 is a good solvent for lipophilic (non-polar) compounds. Additional, to increase the extraction of phenolic compounds and get more antioxidant power, small amounts of a polar cosolvent are applied. Despite the commercial and scientific interest on the antioxidant compounds present in rosemary, data about the solubility of such substances in supercritical CO2 is scarce [46]. Certainly, the antioxidant power of supercritical rosemary extracts depends not only on process conditions, but also on the origin of the raw material employed. Carvalho et al. [47] studied the SFE of organic cultivated rosemary (Sao Paulo, Brazil) using cells of different size. Extraction conditions studied were in the range of 303 and 313 K and pressures of 10 30 MPa. The higher content of carnosic acid was 20 % mass, and was obtained using pure CO2 at 30 MPa and 313 K. At these extraction conditions, the overall extraction yield was near 0.05 g of extract per g of plant material loaded to the extraction cell (5 %). In general, lower yields and concentrations of carnosic acid were obtained by other authors. For example, Celiktas et al. [48] studied the extraction of rosemary leaves collected from different locations of Turkey at different harvesting time intervals. Even employing the same extraction conditions (35 MPa, 373 K and 5 % of methanol co-solvent) the concentration of carnosic acid in the extracts varied from 0.5 to 11.6 % mass. Chang et al. [49] studied rosemary plants grown in experimental fields of Taiwan. They investigate pressures in the range of 20-35 MPa and temperatures of 313-343 K; the best antioxidant extract was obtained at 35 MPa and 343 K, with 4.3% overall yield and just 3.5 % mass of carnosic acid. García-Risco et al. [50] studied the kinetic behavior of the SFE of rosemary leaves from Murcia (Spain) in a pilot-scale plant at 30 MPa, 313 K and without cosolvents, and obtained around 10 % mass of carnosic acid and 4.74 % yield. Figure 5 depicts the corresponding overall extraction curve as a function of the amount of supercritical solvent employed. Table 3 report the yield, concentration of carnosic acid and antioxidant activity obtained in the fraction collected at the different intervals of time. The antioxidant activities reported in Table 3 were evaluated in terms of the DPPH (2, 2- diphenil-1-pycril hydrazyl hydrate) radical test, determining the amount of rosemary extract necessary to decrease the initial DPPH concentration by 50% (EC50 value). As can be observed from Table 3 the mass collected in successive periods of 2 h of extraction is considerably reduced, while the concentration of carnosic acid in these samples increase from 7.8 to 28.0 % mass. Accordingly, the antioxidant capacity of the successive samples collected increase, as deduced from the EC50 values (the lower the EC50 value, the higher the antioxidant activity). This effect could be explained due to a decrease of the amount extracted of volatile oil compounds (mainly monoterpenes and oxygenated monoterpenes) as extraction time increases. Reverchon et al. [51] reported that extraction time proved to be one of the main parameters that determine the composition of the fraction extracted. Decreasing percentages of lighter compounds (monoterpenes and oxygenated monoterpenes) were found as extraction time increase, while higher molecular weight compounds showed a continuous percentage increase at increasing extraction times. In comparison with SLE, PLE and UAE, supercritical fluid extraction using pure CO2 provided a considerably lower recovery of antioxidant compounds. Considering the data provided in Table 3 it could be estimated that the recovery of carnosic acid is c.a. 6 mg / g plant. Yet, the mean concentration of carnosic acid in the overall extract is around 13 % mass (Table 3) indicating high selectivity of CO2 in the extraction of this antioxidant compound.



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Table 3. Kinetic behavior of rosemary CO2-SFE at 30 MPa and 313 K time (h) 2 4 6 8

yield (%) 2.69 0.87 0.79 0.40

carnosic acid (% mass) 7.8 14.7 18.0 28.0

antioxidant activity (EC50, µg·ml-1) 21.8 9.9 7.2 6.0

Thus, SFE using pure CO2 could be rather competitive in comparison with liquid solvent techniques, since evaporation steps for the elimination of liquid solvents are unnecessary. Nevertheless, the use of polar cosolvents in CO2-SFE has a pronounced impact in the recovery of carnosic acid from rosemary leaves. The effect of employing ethanol as cosolvent in the SFE of rosemary leaves is explained in Table 4. Extraction I was carried out using pure CO2, while 5 % of ethanol was employed as cosolvent in Extraction II. Table 4. Effect of ethanol cosolvent in the CO2-SFE of rosemary leaves (30 MPa, 40C) 

1,8 cineole camphor borneol bornyl acetate carnosic acid carnosol

Extraction I Extraction II (mg compound / g leaves) 3.86 4.44 1.32 2.27 0.49 0.70 0.11 0.18 4.92 24.32 0.47 2.77

II / I 1.15 1.72 1.43 1.61 4.94 5.83

The recovery (mg compound / g leaves) of the main substances comprising rosemary volatile oil, together with the recovery of phenolic diterpenes (carnosic acid and carnosol) are reported and compared. It is observed that the recovery of essential oil compounds is not significantly increased when ethanol cosolvent is added to supercritical CO2, while around 5 and 6 fold increases in the recovery of, respectively, carnosic acid and carnosol is observed. That is, the most important effect of using ethanol as polar cosolvent in the supercritical CO2 extraction of rosemary is noticed in the recovery of phenolic antioxidant diterpenes but not in the volatile oil substances. The recovery of carnosic acid was increased form 4.92 mg / g plant to 24.32 mg / g plant (values similar to those attained with PLE and UAE). Furthermore, high concentration of carnosic acid was obtained in the extracts (close to 30 % mass) and the consumption of ethanol was considerably low in comparison with PLE or UAE (12 mg of carnosic acid were recovered per g of plant).

SUPERCRITICAL FRACTIONATION OF ROSEMARY EXTRACTS SFE fractionation techniques take advantage of the fact that the supercritical solvent power can be sensitively varied with pressure and temperature. Two different alternatives are


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possible: an extraction accomplished by combining successive steps and fractionation of the extract in a cascade decompression system. In the case of multi-step extraction, the conditions applied in the extraction vessel are varied step by step, increasing CO2 density. Thus, the most soluble solutes are recovered in the first fraction, while substances with decreasing solubility in supercritical CO2 are extracted in the successive steps. Several works were reported in the literature [52, 53] in which two successive steps were applied in rosemary SFE. The first step was accomplished at 40C and pressures around 10 MPa (CO2 density = 630 kg/m3) resulting in a low-antioxidant but essential oil rich extract. In the second step (40-60C, pressures close to 40 MPa, CO2 density  900 kg/m3) a high-antioxidant fraction was recovered. A two-step SFE was also employed by Vicente [54] to produce the complete elimination of the essential oil compounds, using pure CO2 in a first step, and a fraction containing high concentration of phenolic diterpenes in the second step in which CO2 with ethanol as cosolvent was employed. Extraction conditions applied in the first step were 30 MPa and 40C (CO2 density = 911 kg/m3) and thus some antioxidants were also co-extracted, but the high CO2 density applied guaranteed the exhaustion of volatile oil compounds from plant matrix. Then, a second step with ethanol as cosolvent was applied at lower pressure (15 MPa and 40C, CO2 density = 781 kg/m3) producing an extract with 5% yield, and containing 33 % mass of carnosic acid plus carnosol and less than 2.5 % mass of volatile oil compounds. This extract satisfied Directive 2010/67/EU to authorize it as a food additive, i.e., the carnosic acid plus carnosol content in the extract was greater than 13 % mass and the ratio between key antioxidants defined by the Directive (carnosic acid and carnosol) and the key volatile compounds (borneol, bornyl acetate, camphor, 1,8-cineol and verbenone) was greater than 15. On-line fractionation is an alternative which permits keeping the same extraction temperature and pressure during the whole extraction time, while several separators placed on-line (setting different temperatures and decreasing pressures) allow the selective precipitation of different compound families, as a function of their solubility in the supercritical solvent. On-line fractionation in a two-step depressurization system was studied by Cavero et al. [55] using pure CO2 and CO2 with ethanol cosolvent. In the first separator the antioxidant fraction was isolated, while a fraction with high concentration of the volatile oil compounds was recovered in the second separator. Nevertheless, when using ethanol as cosolvent, the differences in the recovery of carnosic acid between the fractions collected in each separator were smaller, showing a decrease in selectivity. The kinetic behavior of rosemary supercritical on-line fractionation is analyzed in Table 5 for and experiment carried out at the same extraction conditions of experiment reported in Table 3 (30 MPa and 313 K) but accomplishing fractionation of the extract. As can be observed in the table, the fractions recovered in S1 (first separator) have higher concentration of carnosic acid than S2 fractions. Accordingly, considering the same period of extraction, the antioxidant activity of S1 samples are higher than those of S2 fraction. Yet, the fraction collected in S2 during the third period of time (3.0-4.5 h) has almost the same carnosic acid concentration and antioxidant activity than the first fraction (0-1.5 h) collected in S1. This observation motivated a novel on-line fractionation scheme to improve the isolation of phenolic diterpenes from supercritical rosemary extracts, as presented by Vicente et al. [56].



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Table 5. Kinetic behavior of rosemary supercritical fractionation at 30 MPa and 313 K time (h) 0-1.5 1.5-3.0 3.0-4.5

yield (%) S1 S2 1.26 1.12 0.60 0.75 0.44 0.43

carnosic acid (% mass) S1 S2 12.0 1.8 15.5 7.5 19.0 12.3

antioxidant activity (EC50, g·ml-1) S1 S2 22.3 39.8 14.2 22.1 12.6 18.0

In this novel fractionation scheme, the extraction temperature and pressure were kept constant (30 MPa and 40C) during the whole extraction time (5 h) but the depressurization procedure implemented to fractionate the extracted material was varied. In the first period (01.0 h) on-line fractionation of the extract was accomplished and thus, due to the lower solubility of the phenolic diterpenes in comparison to the essential oil substances, the antioxidants were precipitated in S1 and the essential oil was mainly recovered in S2. Then, a second period continued, in which S1 pressure is lowered down to CO2 recirculation pressure and all the substances extracted are conducted to precipitate in S1, and mix with the material that had been recovered in this separator during the first period. In this way, a product is obtained in S1 separator with a 2-fold increase of phenolic diterpenes in comparison with a SFE carried out without fractionation, and attaining an extraction yield five times higher than that obtained when on-line fractionation is accomplished during the entire extraction time.

ISOLATION OF PHENOLIC DITERPENES BY SUPERCRITICAL FLUID CHROMATOGRAPHY Supercritical chromatography of rosemary was reported for the first time by Ramirez et al. [57-59]. The authors evaluated the separation of carnosic acid from rosemary by SFC employing specially designed capillary chromatographic columns, that is commercial octadecylsilica and silica particles coated with a stationary phase commonly used in gas chromatography, such as Carbowax 20M (polyethylenglycol) of high polarity. Using high pressures (pressure gradient from 15 to 37 MPa) and temperatures (100C) the elution of carnosic acid was achieved with high selectivity and using pure CO2 (no cosolvent). Later [58] a preparative-SFC system was employed to fractionate a supercritical rosemary extract, using a LC-Diol packed column at 13 MPa, 80C and using 10 % of ethanol as CO2 cosolvent. At similar conditions of pressure and temperature, rosemary supercritical extracts were also fractionated using a home-made column [59], which was prepared using a new method for packing commercial silica particles coated with a stationary phase (5% phenyl-, 95% methylsilicone). In all cases a slight increase (1.2-1.4 fold) of carnosic acid was obtained. Recently, different chromatographic packed columns and process conditions were studied in order to attain SCF purification of carnosic acid [54]. The packed columns tested were (PC1) Kromasil 60-DIOL, (PC2) ACE 5 silica, (PC3) Viridis SFC silica and (PC4) Viridis SFC 2-Ethylpyridine. The SFC conditions tested were pressures in the range of 10-20 MPa, temperatures of 313-333 K and 5-20 % mass of ethanol cosolvent. For all packed columns tested, and in the range of temperatures and pressures employed, the elution of standard


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carnosic acid was achieved only when using ethanol as CO2 cosolvent, being the amount of cosolvent the most important factor in this elution. As expected, for increasing pressure decreasing retention times were observed. Furthermore, as also reported by Lesellier [60], the effect of temperature becomes less significant as increasing the amount of cosolvent. The resolution of the different columns was tested by observing the separation of a carnosol + carnosic acid mixture (70 % mass carnosic acid) as depicted in Figure 6. These two compounds were selected as model mixture to separate in the chromatographic system, because they are the most abundant phenolic antioxidants of rosemary and have very similar chemical structure.

Figure 5. Overall extraction curve of rosemary SFE at 30 MPa and 40C.

Figure 6. Elution of a mixture of (1) carnosic acid and (2) carnosol (70 % mass carnosic acid) at 10 MPa and 313 K: (a) PC2 and (b) PC3 (5% ethanol); (c) PC4 (20% ethanol).

Figure 6 compares the chromatograms attained with the PC2, PC3 and PC4 columns at 10 MPa and 313 K (poor resolution was attained with PC1column). The separation of carnosol and carnosic acid attained with PC4 column is considerably enhanced in comparison to that obtained with the PC2 and PC3 columns.



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When injecting rosemary extracts in the SFC system, the concentration of carnosic acid was significantly increased, producing a fraction with a 1.4-1.9 fold increase when using PC2 and PC3 columns. Further, most of the carnosic acid injected in the SFC was recovered in this fraction (carnosic acid recovery > 70 %). In this way, fractions of rosemary supercritical extracts were produced with concentrations ranging from 30 to 60 % mass of carnosic acid. SFC semi-preparative fractionation using PC4 (Viridis SFC 2-Ethylpyridine) produced much better isolation of carnosic acid, as reported in Table 6 and illustrated in Figure 7.

Figure 7. SFC fractionation of rosemary supercritical extract using PC4 (Viridis SFC 2-Ethylpyridine) column at 10 MPa, 313 K and 20 % mass ethanol cosolvent. F1, F2 and F3: intervals of time employed.

Table 6. Semi-preparative SFC fractionation of rosemary extracts using 2-ethylpyridine (PC4) packed column at 10 MPa, 313 K and 20 % ethanol % mass carnosic acid

carnosic acid recovery (%)

concentration factor (CF)

rosemary F1 F2 F3 F1 F2 F3 F2 extract (raw material) (fractions collected) 22.83 0.53 92.57 N.I. 1.78 98.21 N.I. 4.46 22.83 0.82 96.30 N.I. 2.67 97.32 N.I. 4.64 CF: % mass carnosic acid in F2 / % mass carnosic acid in extract. F1, F2 and F3: fractions collected in the intervals 0-9.3min, 9.3-12.5 min and 12.5-13.5 min. N.I.: non-identified.

Figure 7 show the separation attained in the PC4-SFC systems using a rosemary supercritical extract with an initial content of carnosic acid of 22.83 % mass. Fractionation conditions were 10 MPa, 313 K and 20 % mass ethanol cosolvent. The concentration factors attained with this column were considerably higher, in the range of 4.4 to 4.6. Furthermore,


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the carnosic acid purity obtained was close to 92 % mass, and its recovery was higher than 97 % (see Table 6).

BIOACTIVE PROPERTIES OF ROSEMARY DITERPENES: IMPLICATIONS IN CANCER THERAPY Proverbially, rosemary has been reported to be of potential benefit in the treatment and/or prevention of health conditions including renal colic, respiratory diseases, spasmogenic disorders, peptic ulcer, inflammatory diseases, poor sperm motility [61, 62]. The biological activities of the plant have been mainly attributed to phenolic diterpenes such as carnosic acid, and have been traditionally related to their antioxidant activity. Thus, rosemary diterpenes have demonstrated to exhibit antibacterial effects against various Gram-positive and Gram-negative bacteria, as well as anti-inflammatory effects in animal models [63], which have been suggested to be mainly mediated through the activation of the nuclear transcription factor kappaB and attenuation of the formation of reactive oxygen species (ROS) [64]. However, currently scientific interest is moving towards the specific effects of rosemary extracts, finding additional mechanisms of action involved in its biological activity. In this sense, the effects of phenolic diterpenes of rosemary are being analyzed within the frame of chronic diseases currently considered the main causes of morbidity and mortality worldwide in industrialized countries such as cardiovascular disease, obesity, diabetes or cancer. Thus, the role of rosemary extract as an antiatherosclerotic agent has been recently supported and attributed to the modulation of cell migration through the regulation of matrix metalloproteinase-9 (MMP-9) and monocyte chemoattractant protein-1(MCP-1) activities [65]. Regarding diabetes, rosemary has been shown to reduce blood glucose levels and improve lipid profile in several studies [66, 67]. In this sense, it has been recently suggested that these effects might be mediated by the induction of liver glycolysis and fatty acid oxidation by rosemary through the activation of the AMP-activated protein kinase (AMPK) and the peroxisome proliferator-activated receptors (PPAR) pathways [68]. A potential role of rosemary in obesity has also been recently reinforced, demonstrating a mitigation of weight gain by this agent without affecting food intake [69, 70], in part explained by a significant inhibition of gastric lipase and consequent modulation of fat absorption [70]. PPAR pathway together with the C/EBP, CCAAT/Enhancer Binding Protein, has been also proposed as essential partners in the anti-adipogenic activity of carnosic acid of rosemary extract [71]. Moreover, the bioavailability of carnosic acid and derived diterpenes of rosemary has been evaluated in a rat model of obesity with the aim of identifying a bioactive metabolic profile in this system [72]. Since oxidative damage to cells has been associated to cancer development, antioxidants have been extensively proposed as promising agents for cancer treatment and/or prevention. Extensive evidence show that rosemary, and in particular, its phenolic diterpenes, potentially exert beneficial effects on the inhibition of cancer development and progression [73]. First evidences were related to the activity of rosemary extract and its individual antioxidative constituents as chemopreventive agents in mammary tumorogenesis [74], based on the first observation that rosemary extract inhibits 7,12-dimethylbenz[a]anthracene (DMBA)-induced



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tumor occurrence in animal models decreasing tumor incidence and burden. These effects were afterwards demonstrated also in induced skin mouse tumorogenesis models, and the activation of detoxifying enzymes was implicated in the mechanism of action [75]. Moreover, beyond their interest as chemopreventive agents, the antioxidant and antiinflammatory properties of phenolic diterpenes of rosemary suggested also a potential interesting activity of these components in cancer therapy. Supporting this hypothesis, several studies have demonstrated the anti-proliferative activity of rosemary extract and main constituents on several human cancer cell systems including leukemia, breast, liver, prostate, lung and colon cancer cells [10, 76-78]. In addition, rosemary extracts and/or its main antioxidant constitutents have been reported to show antiproliferative effects on, and to repress the initiation and promotion of melanoma and glioma in vivo in animal models [79]. Moreover, when combined with 1 alpha, 25-dihydroxyvitamin D(3) (1,25D(3)), a carnosic acid rich-rosemary extract has been shown to significantly increase the survival of mice with acute myeloid leukemia [80]. As a relevant process in tumor progression, the role of two of the main diterpenes of rosemary, carnosol and carnosic acid, as potential antiangiogenic components have been also recently evaluated [81]. These authors have shown that the mentioned diterpenes inhibit differentiation, proliferation, migration and proteolytic capability on endothelial cells, resulting in the inhibition of angiogensis in vivo, and suggests their potential use in the treatment of angiogenesis-related malignancies. In addition, a greater activity of rosemary as an antitumor agent has been suggested for the extract obtained by supercritical fluid technology [78], joining this circumstance to the group of advantages observed for this extraction technology. However, though evidence demonstrates an anticancer effect of rosemary extracts and main antioxidant components, and both activities have been traditionally linked, the relationship between antioxidant activity and antitumor effect has not been demonstrated, as a matter of fact, it has been recently questioned [82]. Thus, much research has still to be developed on one hand focused on identifying the most active composition of rosemary extract as an anticancer agent, and on the other hand on elucidating the real dependency on its antioxidant activity for their antitumor action. In this sense, only two studies have been reported, Kontogianni et al. [83] that have shown the anti-proliferative activity of rosemary extracts against cancer cells with respect to their phytochemical profiles, and Vicente et al. [82] that have recently correlated the antioxidant activity of different supercritical rosemary extracts with their effect on hepatic tumor cell proliferation.

ANTIOXIDANT VS. ANTIPROLIFERATIVE ACTIVITY OF SUPERCRITICAL ROSEMARY EXTRACTS Table 7 report the concentration of carnosic acid of different rosemary extracts produced by SFE and combining SFE with semi-preparative SFC. Additionally, the antioxidant activity of these samples is given in terms of the DPPH test and their corresponding EC50 value. As can be observed the antioxidant power of rosemary extract increases with increasing content of the main antioxidant phenolic diterpene identified in rosemary. A mathematical relationship between the EC50 values of the rosemary supercritical extracts and the concentration of carnosic acid in the sample can be established:


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EC50 (g / mL)  11.6641 ln (%mass carnosic acid )  52.9284 The high regression coefficient obtained (R² = 0.912, see Figure 8) is indicating that carnosic acid is the most important antioxidant present in rosemary supercritical extracts, strongly determining the antioxidant activity of rosemary supercritical extracts. Table 8 shows the antioxidant activity and the antiproliferative effect of the different rosemary extracts produced by SFE on hepatoma cancer cells HepG2. As can be observed, a 2-fold increase in the antioxidant power of the rosemary extract (M4-1 vs M5-1) only increases its effect on the inhibition of tumor cell growth by 1.18. By contrast, a slight increase on the antioxidant activity when comparing R4 and R5 (1.07 fold increase) is observed, but antiproliferative potential is significantily higher (more than 2-fold increase). Finally, though R6 presents higher antioxidant activity than R5, the anti-proliferative effect of the latter is not increased, resulting even lower. Similar lack of correlation has been found in colorectal and breast cancer cells, where in addition to inhibition of cell proliferation, induction of tumor cell death has been found independent of their antioxidant power (Gonzalez-Vallinas et al., manuscript under preparation).

Figure 8. Correlation between the EC50 values obtained for different supercritical rosemary extracts and the concentration (% mass) of carnosic acid.

First observations on breast and colon cancer cells further support these results. Table 9 shows the lack of correlation between antioxidant activity and antiproliferative activity against human colon cancer cells. As it is shown, the differences between the antioxidant activity of the different supercritical rosemary extracts are not associated with the differences found in the cytoestatic nor cytotoxic effect mediated by rosemary on human colon cancer cells. Furthermore, the lack of correlation in rosemary-induced cytotoxicity is even more noticeable since the same grade of antioxidant activity displays a significant gap on inducing cell death (R4 and R5 showing medium-range of antioxidant activity but with low and high grade of induced cytotoxic effect).


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Table 7. Rosemary extracts produced by different SFE procedures and combining SFE with semi-preparative SFC rosemary extract R1

R2

R3 R4

R5 R6

R7 R8 R9 R10

supercritical procedure 30 MPa, 40C, no cosolvent. Fractionation during first hour. Then, extraction continued for 5 h without fractionation. First step: 30 MPa, 40C, no cosolvent, 6 h. Second step: 15 MPa, 40C, 10% ethanol, 3 h. 15 MPa, 40C, 10% cosolvent, 3h. No fractionation. 30 MPa, 40C, no cosolvent. Fractionation during first hour. Then, extraction continued for 5 h without fractionation. 15 MPa, 40C, 5% cosolvent, 3h. No fractionation. First step: 30 MPa, 40C, 6 h, no cosolvent. Second step: 15 MPa, 40C, 3 h, 10% ethanol. SFE-SFC (ACE 5 silica column) SFE-SFC (Viridis SFC silica packed column) SFE-SFC (Viridis SFC silica column) SFE-SFC (Viridis SFC 2Ethylpyridine column)

sample collected mass precipitated in second separator mass precipitated in first step all mass extracted mass precipitated in first separator

yield (%) 1.5

carnosic acid (% mass) 3.1

EC50 (μg/mL) 40.0

5.2

10.9

32.9

13.4

14.2

18.1

2.83

16.9

15.9

all mass extracted mass precipitated in second step (E392) second fraction second fraction

7.26

25.7

14.8

4.93

30.7

9.8

38.6 45.4

9.5 9.3

54.6 92.6

7.1 2.5

second fraction second fraction

Table 8. Comparative biological effect of rosemary extracts produced by different SFE procedures determined as the concentration to inhibit 50% of tumoral hepatoma cell proliferation rosemary extract R2 R4 R5 R6

supercritical procedure First step: 30 MPa, 40C, no cosolvent, 6 h. Second step: 15 MPa, 40C, 10% ethanol, 3 h. 30 MPa, 40C, no cosolvent. Fractionation during first hour. Then, extraction continued for 5 h without fractionation. 15 MPa, 40C, 5% cosolvent, 3h. No fractionation. First step: 30 MPa, 40C, 6 h, no cosolvent. Second step: 15 MPa, 40C, 3 h, 10% ethanol.

EC50 (μg/mL) 32.9 15.9 14.8 9.8


IC50 (μg/mL) 110.7 ± 18.7 93.26 ± 22.1 42.16 ± 5.9 48.01 ± 3.2

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Figure 9. Molecular mechanism involved in the synergistic effect of 5-FU and rosemary extract in colon cancer cells.

Table 9. Antioxidant vs antitumor activity of supercritical rosemary extracts determined as the concentration to inhibit 50% of cell proliferation (inhibitory concentration IC50, indicative of cell sensitivity), the concentration to induce Tumor Growth Inhibition (TGI, cytoestatic effect) and the concentration to induce 50% of cell death (lethal concentration LC5O, cytotoxic effect) against human colon cancer cells rosemary EC50 (μg/mL) Cell sensitivity Cytoestatic effect Cytotoxic effect extract R2 L M M L R4 M M M L R5 M H H H R6 H H H H Legend: L (low), M (medium), H (high). For antioxidant activity: Low activity: EC50 ≥ 20 µg/mL; Medium range: 20 µg/mL  EC50  10 µg/mL; High activity: EC50 ≤ 10 µg/mL. For biological effect: cell sensitivity and citoestaticity; Low activity: IC50 /TGI ≥ 75 µg/mL; Medium range: 35 µg/mL  IC50 /TGI  75 µg/mL; High activity: IC50 /TGI ≤ 35 µg/mL. For cytotoxicity: Low activity: LC50 ≥ 75 µg/mL; Medium range: 55 µg/mL  LC50  75 µg/mL; High activity: LC50 ≤ 50 µg/mL.

Thus, though the concentration of the most important antioxidant compound carnosic acid has demonstrated to display a key role on the antitumoral potential of rosemary including growth inhibition and induction of cell death, the putative antitumoral activity of supercritical rosemary extracts might not be exclusively attributed to carnosic acid antioxidant content but



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might be mediated by a specific mechanism. Furthermore, evidences suggest that reaching a significant content of the main bioactive phenolic diterpenes, the presence of additional compounds do not interfere with its antitumor activity, but by might be synergizing in this effect [82], suggesting a potential advantage of the use of a selected whole extract as an anticancer agent more than individual phenolic diterpenes. This constitutes a promising observation regarding the potential application of rosemary extracts for cancer patients since it is now well-known that high concentration of antioxidants may interfere with therapeutic agents (i.e., alkylating agents, anthracyclines, or platinumbased compounds), which cytotoxic effect partially depend on the oxidative damage to tumor cells. Moreover, specific alternative antitumor mechanism of action of antioxidants further than this activity might explain the controversial results found assaying these compounds in cancer patients [84, 85]. In addition, since supercritical rosemary extract has been recognized as a healthy component by EFSA (European Food Safety Authority) [86], being currently used as food additive, a properly conducted research in this area migh result in the putative evaluation of a selected rosemary extract as co-adjuvant agent in cancer therapy.

PHENOLIC DITERPENES FROM ROSEMARY AS ENHANCERS OF CHEMOTHERAPEUTIC AGENTS Many of the commonly used drugs in cancer therapy (i.e., amptothecin from Camptotheca acuminate Decne or paclitaxel from Taxus brevifolia) are derived from natural compounds, directly extracted from natural sources or chemically derived from selected naturally occurring estructures [87], further supporting a relevant role of the use of natural extracts in cancer therapy. However, it is well-established that response rates to chemotherapeutic drugs are usually increased when treated in combination with additional antitumor agents, in part due to cooperative effects modulating drug resistance, main cause of anticancer treatment failure. Therefore, the potential use of rosemary or its main phenolic diterpenes in the clinical setting might be considered in combination with currently used chemotherapeutic agents. In this sense, it has been reported that multidrug-resistant P-gp over-expressing KB-C2 cells cells were sensitized to vinblastine cytotoxicity by the rosemary diterpene carnosic acid [88]. Thus, it has been suggested that rosemary might increase the intracellular accumulation of commonly used chemotherapeutic agents such as doxorubicin and vinblastine in Pglycoprotein-expressing cancer cells, potentiating their effects [88, 89]. Strong evidence has been recently generated in colorectal cancer cells regarding 5-Fluorouracil (5-FU), the most used chemotherapeutic agent in this type of cancer, among others. Supercritical rosemary extracts have been assayed alone and in combination with 5-FU in different colon cancerderived cells in terms of cell viability, cytotoxicity, and cell transformation, finding a synergistic antitumor effect of the combination [78]. Moreover, supercritical rosemary extract was able to overcome acquired resistance to the drug, sensitizing 5-FU-resistant colon cancer cells to the antitumor action of the compound. A genetic approach was followed to provide a hypothesis of the potential molecular mechanism underlying this effect, showing that besides general molecular mechanisms associated to drug resistance, rosemary exerted a specific modulation of key enzymes involved in 5-FU metabolization. Thus, rosemary extract induces


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the downregulation of thymidylate synthetase (TYMS) [78], essential for the synthesis of deoxythymidine monophosphate (dTMP), which constitutes the main mechanism of action of 5-FU [90]. In addition, rosemary also induces the downregulation of thymidine kinase (TK1), main pathway of cell recovery to the action of 5-FU, constituting one of the most important mechanisms of 5-FU resistance (see Figure 9). These observations suggest that rosemary extract might synergize with 5-FU by having an impact in TYMS, that on one hand will be postranslationally inhibited by 5-FU, and on the other will be transcriptionally repressed by rosemary, avoiding the expression of active TYMS not blocked by 5-FU. In addition, rosemary sensitizes colon cancer cells to the effect of 5-FU modulating resistance to this drug by downregulating the main enzymes involved in its savage pathway (Figure 9).

MOLECULAR TARGETS OF PHENOLIC DITERPENES FROM ROSEMARY EXTRACTS The target of efficient chemotherapy is the interference for cellular pathways crucial for tumor cell growth and survival. To that end, a deep knowledge of molecular effects of antitumor agents is essential for their proper application and therefore to achieve the higher efficacy. Whereas regarding chemotherapeutic drugs most of the studies in the past decades have been focused on the identification of molecular targets for personalized prescription, this approach has not been followed regarding natural phytochemicals [87]. Thus, based on promising results obtained in cell systems, no mechanism-based preclinical studies were been performed with natural agents, resulting in controversial results. Intracellular 5-FU is metabolized to three main active metabolites, 5-Fluorodeoxyuridine Monophosphate (FdUMP), which irreversible binds to thymidylate synthase (TYMS/TS) inhibiting DNA synthesis consituting its main mechanism of action; fluorodeoxyuridine triphosphate (FdUTP) and fluorouridine triphosphate (FUTP), which respectively incur in DNA and RNA damage. Conversion to fluorouridine monophosphate (FUMP) might be either directly by orotate phosphoribosyltransferase (OPRT) or indirectly via fluorouridine (FUR) through the sequential action of uridine phosphorylase (UP) and uridine kinase (UK). FUMP is then phosphorylated to fluorouridine diphosphate (FUDP), which can be either converted to the active metabolite FUTP, or to fluorodeoxyuridine diphosphate (FdUDP) by ribonucleotide reductase (RR). In turn, FdUDP can either be phosphorylated or dephosphorylated to the active metabolites FdUTP and FdUMP respectively. TYMS catalyzes the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) for DNA synthesis. 5-FU active metabolite FdUMP binds to the nucleotide-binding site of TYMS and forms a stable complex blocking access of dUMP and inhibiting dTMP synthesis and therefore, inhibiting DNA synthesis. dTMP can be salvaged through the action of thymidine kinase (TK). Rosemary induces the transcriptional downregulation of TYMS and TK. However, in order to properly apply natural compounds as supplements or adjuvant therapy for cancer patients, research strategy must be conducted as that of targeted antitumor drugs, including the selection of the most active composition and the identification of the molecular targets of the candidates in order to identify the conditions and patients that might



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benefit most from these agents. Following this approach, success expectancy in the evaluation of phytochemicals such as phenolic diterpenes from rosemary as antitumor agents will be significantly increased. Regarding rosemary and its constituents, anticancer effects might be exerted through a variety of mechanisms, as it has been previously reported for different phytochemicals [87]. Phenolic diterpenes from rosemary carnosol and carnosic acid have been the most studied components concerning molecular effects. Carnosol inhibits beta-catenin tyrosine phosphorylation and suppresses metalloproteinase-9 through down-regulation of nuclear factor-kappa B and c-Jun [91, 92]. In addition, in prostate cancer cells it has been shown a carnosol-mediated increase of cell cycle regulatory proteins p21 and p27, decrease of cyclins and cyclin-dependent kinases, Bcl-2 downregulation and inhibition of the PI3K/Akt pathway [93]. It has been shown that PI3K pathway is also modulated by carnosic acid, suggesting that modulation of Akt/IKK/NF-κB pathway through activation of PP2A is the main mechanism of action for carnosic acid-induced apoptosis [94]. However, though apoptotic induction of cell death mediated by rosemary bioactive compounds has been suggested, results indicate that other signaling pathways are also contributing to tumor cell death induced by this agent [78]. A more efficient antitumor effect of supercritical rosemary extract than its main phenolic diterpenes treated separately has been previously suggested [82]. Regarding rosemary, it has been shown that extracts of this plant induce glutathione-S-transferase and quinine reductase activity and cells and animal models [94]. In addition, transcriptomic and metabolomic profiles have been recently analyzed after treatment of colon cancer cells with rosemary [95, 96] in order to provide clues of its molecular action. However, the potential molecular targets mediating the antitumor action of rosemary have not been properly defined yet, and further studies are needed in order to identify its molecular targets to bring up the application of this agent to the clinical setting.

PRESENT LIMITS AND FUTURE PROSPECTS OF THE POTENTIAL USE OF PHENOLIC DITERPENES OF ROSEMARY EXTRACTS IN ANTICANCER THERAPY Plants provide a broad spectrum of potential anticancer agents with possible low associated toxicity and a variety of biological effects conducted by the affection of multiple targets. Phenolic diterpenes of rosemary constitute a group of promising molecules in this regard. Their antiproliferative effects have been confirmed in a series of cancer cell lines from different origins, including hematopoietic, colon, breast or prostate. In addition, several studies have further verified that the antiproliferative action might result in antitumorogenic effect in vivo in animal models. Moreover, despite the activity of individual diterpenes present in rosemary which point at them as promising structures for the development of anticancer drugs, the combination of components present in the extract seems to be synergistic, with the additional advantage of the previous approval of rosemary extracts by FDA for its nutritional use in humans. The challenge now is to conduct this knowledge from the bench to the clinical setting. This will be possible by following a similar approach to that of the development of new


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antitumor drugs. This approach includes the identification of the most appropriate extraction technology and final composition which will allow the standardization of the process. Then, the identification of the optimal doses and the balance between efficacy and safety is essential. In addition, a deeper knowledge of the molecular basis underlying its effects is needed, in order to identify the specific population and conditions in which they must be applied to expect the highest efficacy. To that end, the application of the –omics technologies together with an epigenetic analysis might be of greater impact. Finally, high-quality human trials investigating putative rosemary effects and its possible applications, not only in cancer therapy, are required. There is an extended common practice of using botanical products with the wrong assumption that ―plant‖ means either ―almost inactive‖ or ―safe‖, considering these compounds as ‗‗alternative‘‘ therapies that might be used with little or no scientific basis. Thus, plants as rosemary, known for their phenolic components or basal antioxidant activity, are used despite a lack of knowledge of effectiveness or safety. However, it is important to take into account that a significant proportion of the current used drugs are derived from naturally occurring compounds, and display deep impact in human health. Indeed, several traditional Chinese medicinal herbs have been associated to kidney failure, and popular plant extracts containing phytoestrogens have been associated to carcinogenesis [62]. Based on lack of information and absence of a specific regulation, there is a common practice by the population of self-administering natural products without taking into account both direct and side effects, as well as pharmacokinetics. This situation turns into a dangerous condition when cancer patients combine these products with chemotherapeutic agents without a targeted management, conducting even to a detrimental impact on clinical outcome due to well-demonstrated phytochemical-drug interactions. Therefore, a mechanism-based personalized application is needed before integrating phytochemicals as adjuvant supplements for cancer patients. In this sense, recognizing the potential impact of phytochemicals in human health and their role as main source of anticancer compounds, the NCI established a repository for crude natural product materials, the Natural Products Branch (NPB). However, patient, clinicians and companies awareness of the potential impact of the application of these compounds in cancer treatment is still needed. Moreover, deep efforts regarding preclinical and clinical evaluation of these compounds are still required to include them into an evidencebased integrative cancer care.

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CV OF CHAPTER AUTHORS Dr. Guillermo J. Reglero Rada. PhD in Chemical Sciences, Chairman of Food Sciences at the Autonomous University of Madrid and researcher of The Spanish National Research Council-CSIC (on leave). He directs a research team in Food Science and Technology that is focused on obtaining functional ingredients for specific health used. The team takes part of the Spanish programme CONSOLIDER FUN-C-FOOD in Health and Food, and different consortia of industrial research such as CENIT programme, European projects and R&D National Plan. Between 2002 and 2006, he has been Manager of the Area of Science and Food technology of the R&D National Plan. He is author of more than 180 scientific publications with a high international impact and several patents. His research highlights include the development of processes based on novel and clean technologies to obtain functional food ingredients, the identification of substances with interesting biological activities and determination of their potential beneficial/detrimental functionality on human health. In 2001 he received the ―Archer Daniels" award bestowed by the American Oil Chemists Society. Dr. Ana Ramírez de Molina. Extraordinary awarded as an outstanding PhD Thesis in Biochemistry and Molecular Biology (Universidad Autónoma de Madrid, Spain, 2002), Dr. Ramírez de Molina has developed her scientific career in the identification of new biomarkers and molecular targets for anticancer treatment within lipid metabolism. As a postdoctoral researcher she worked at the Traslational Oncology Unit CSIC-UAM-La Paz Hospital, developing postdoctoral projects in collaboration with the Cancer Research UK Centre for Cancer Therapeutics (Prof. Paul Workman), and at the Molecular Pathology Department at Sloan Kettering Cancer Center of New York (Prof. Carlos Cordón). In this period, she published more than 25 articles in prestigious international journals of her research field including lancet oncology, cancer research, journal of medicinal chemistry, etc., and presented 5 different patents, from which one of them, accepted and validated in 18 different countries, promoted the creation of a spin-off company focused on the development of new therapies in cancer, TCD Pharma, in which she was the Director of Research, Development and Innovation for more than 3 years. In 2009 she was awarded by The National Ministry of


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Science and Innovation with a Ramón y Cajal position, and in the frame of this programme she joined IMDEA Food Institute, in which she is currently the Coordinator of Research, Development and Transfer, and leads the Molecular Oncology and Nutritional Genomics of Cancer group. Dr. Tiziana Fornari. Full professor at Universidad Autónoma de Madrid (UAM) and member of the research staff at the Food Science Research Institute (CIAL) from UAM University and from the Spanish Council of Scientific Research (CSIC), in Madrid, Spain. She starts her scientific career in Chemical Engineering in the Universidad Nacional del Sur (UNS) in Argentine, and joined the Pilot Plant of Chemical Engineering (PLAPIQUI) during 15 years, research institute which depends on UNS University and the Argentinian National Council of Scientific and Technological Research (CONICET). In 2004, she was awarded by The National Ministry of Science and Innovation of Spain with a Ramón y Cajal contract in the area of Food Technology at UAM. Her research highlights include the production of functional food ingredients (antioxidants, essential oils, prebiotic carbohydrates) by applying innovative technologies, the thermodynamic fundamentals of separation processes and its engineering design, simulation and optimization. She has co-authored 70 publications in high impact journals and presented 6 patents in the field of novel foods and healthy food ingredients.




Chapter 4

THE POSSIBLE USE OF TERPENE COMPOUNDS IN DC IMMUNOTHERAPY AGAINST CANCER Masao Takei1, Akemi Umeyama2 and Je-Jung Lee3 1

Research center for Cancer Immunotherapy, Chonnam National University Hwasun Hospital, Jeonnam, South Korea 2 Faculty of Pharmaceutical Sciences, Tokushima University, Tokushima, Japan 3 Department of Hematology-Oncology, Chonnam National University Medical School, Gwangju, South Korea

ABSTRACT The immune system is confronted with antigens and proteins that have not been encountered previously. Dendritic cells (DC) play a pivotal role in the initiation of T-cellmediated immune responses, making them an attractive cellular adjuvant for use in cancer vaccines. The interaction of T cell with DC is crucial for directing T cell differentiation towards the Th1, Th2 or Th17 type, and several factors determining the direction of the T cell polarization. Cytokines, secreted by DC at the time of initial T cell stimulation, play an important role in the subsequent differentiation of effector T cells. Th1 cells, through interferon gamma (IFN-) production, regulate antigen presentation and immunity against intracellular pathogens. Although different DC subsets may have some intrinsic potential to preferentially induce Th1, Th2 or Th17 cells, DC also display considerable functional plasticity in response to signals from microbes and the local microenvironment. Multiple reagents have been reported to induce DC maturation and the known DC maturation stimuli include the CD40 ligand (CD40-L), IFN-, TNF-, oligo CpG nucleotides or Toll-like receptor ligands (TLRs). Toll-like receptors are expressed mainly on macrophages and DC, triggering results in the development of effector DC that promote Th1 responses. Recently, several studies proposed the significance of TLR signaling in the induction of anti-cancer immunity. The hooks of Uncaria sp. are contained in Choto-san as the main component herb. Choto-san has been used for hypertension and dementia, and well used as an important of many Chinese prescriptions in China, Korea and Japan. Uncarinic acid and Ursolic acid 

Corresponding author: Chonnam National University Hwasun Hospital, Jeonnam, 519-809 South Korea. Email:[email protected]; [email protected].


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Masao Takei, Akemi Umeyama and Je-Jung Lee are isolated from Uncaria rhynchophylla and phytochemically classified as triterpene. Uncarinic acid showed potent inhibitory activity against phospholipase C1 (PLC1) and inhibited the growth of cancer cells at high dose. Ursolic acid augments the inhibitory effects of anticancer drugs on growth human tumor cells and triggers apoptosis in cancer cells. Triterpene have been identified as a unique class of natural products possessing diverse biological activities and Terpenes contain pharmacologically active substance. A number of alkaloids have been reported as antihypertensive principles from the genus Uncaris. We have reported that numerous terpenes induce the differentiation of DC from human monocytes, and drive Th1, Th2 and IL-10-producing Treg cells. For immunotherapeutic applications, it appears crucial identify factors that might affect the differentiation and function of DC. Although various terpene compounds have pharmacological activity, relatively little is known in regards to the influences Uncarinic acid and Ursolic acid exert on the initiation of specific immune response at the level of DC. Therefore, to further understand the cellular basis of immunological abnormalities associated with Uncarinic acid and Ursolic acid exposure, we have investigated ability of Uncarinic acid and Ursolic acid on human DC differentiation (surface molecule upregulation), function (cytokine productions) and their activation (NF- translocation to the nucleus) in detail. Uncarinic acid and Ursolic acid activate human DC via TLR2 and/or TLR4 and induce the production of IFN- by CD4+ naïve T cells. Triterpene compounds such as Uncarinic acid and Ursolic acid, which may lead to the development of effective immunotherapy for cancer, are discussed in this chapter.

Keywords: Terpene compounds, cancer immunotherapy, dendritic cells, IL-12, Toll-like receptor (TLR)

INTRODUCTION Dendritic cells (DC) are antigen-presenting cells (APC) which play a critical role in the regulation of the adaptive immune response. DC are unique APC and have been referred to as ―professional‖ APC, since the principal function of DC is to present antigens. DC have the ability to induce a primary immune response in resting naïve T lymphocytes. To perform this function, DC are capable of capturing antigens, processing them, and presenting them on the cell surface along with appropriate costimulation molecules. DC also play a role in the maintenance of B cell function and recall responses. DC are critical in the establishment of immunological memory [1-3]. Clinical application of DC has been initiated as a cellular immunotherapy against cancer [4]. DC are generated from either myeloid bone marrow progenitors through intermediate DC precursors that home to sites of potential antigen entry, where they differentiate locally into immature DC. After antigen capture in the presence of maturation signals associated with inflammation or infection, immature DC are activated by Toll-like receptor (TLR) ligands, interferons (IFNs such as IFN-α and IFN-γ,). This process in vitro is paralleled by migration of DC to T-cell-rich areas of lymphoid organs, where they present antigen-derived peptides to antigen-specific T-cells and direct their differentiation into T effector or memory cells [5]. Recently, it has reported that after binding its natural ligand cluster of differentiation CD70, CD27, a tumor necrosis factor receptor (TNFR)associated factor-binding member of the TNFR family, regulates cellular activity in subsets of T, B, and NK cells as well as hematopoietic progenitor cells [6]. Optimal tuning of CD27CD70 interaction is crucial for the regulation of the cellular immune responses. The


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interaction of T cells with DC is crucial for directing T cell differentiation towards the Th1, Th2 or Th17 type and several factors determine the direction of T cell polarization [7, 8]. Th1 responses predominate in organ-specific autoimmune disorders, acute allograft rejection and in some chronic inflammatory disorders [9]. Human-monocytes-derived DC matured by either poly I:C, dsRNA, interferon-alpha (IFN-α), tumor necrosis factor-alpha (TNF-α), LPS, TLR ligands (R848, dsRNA) or cytokine cocktail in vitro produce high levels of IL-12 and induce Th1 cells [10-12]. Cytokines are most important in the environment responses and IL12 is made by cell types that can process and present antigen to T cells, such as DC and macrophages, and additionally by neutrophils. IL-12 is essential for inducing Th1 polarization, and a key cytokine in anti-tumor responses [13]. One of the most important goal of DC research is the development of DC-based strategies for enhancing immune responses against tumors and infectious agents. A variety of preparations of DC can stimulate antitumor immunity, including DC loaded with proteins, DC fused with tumor cells and DC transduced with tumor-derived DNA or viral vectors. Analyses of the clinical results suggest that the maturation status of DC used in such protocols greatly affects the immune response that follows the treatments [14]. Thus, for immunotherapeutic applications, it appears crucial to identify factors that might affect the differentiation, maturation and function of DC. Therefore, it is important to identify factors that might produce IL-12 by monocytes-derived DC, and affect the differentiation, maturation and function of DC.

Role of Cytokine Production by DC against Cancer Certain cytokines are produced rapidly in response to pathogens and can profoundly affect the function of DC, by inducing differentiation, activation, maturation and migration of these types of cells. Cytokine are most important in the environment response and the major factors driving the differentiation of Th1, Th2 or Th17 cells. The best known DC-derived soluble factor mediating the effects of cytokines and other immune stimulating agents is IL12, a key regulator of the induction of adaptive Th1 type immunity [13]. Recently, new knowledge on the immune regulatory activities of IL-12 has been acquired, indicating this cytokine as a critical third signal for CD8+ T cell differentiation [15, 16], and as an important factor that, by promoting the reactivation and survival of memory CD4+T cells [17], can contribute to the repolarization of dysfunctional antitumor Th2 CD4+T cells into Th1 cells [18]. Many researchers have demonstrated that the level of IL-12p70 production by human DC correlated with their ability to activated T cell to produce IFN-γ. IL-12 plays a central role in the immune system, not only by augmenting the cytotoxic activity of T cells and NK cells and regulating IFN-γ production, but also by the capacity of IL-12 to promote the development of Th1 [18]. The use of IL-12 for the treatment of cancer patients has been promoted by the promising results obtained in a number of animal tumor models on the strong anti-angiogeneic and immune-mediated antitumor activities of IL-12 [19]. IL-12 has been used for the treatment of patients with advanced solid tumors and hematologic malignancies, either as a signal agent or in combination with other therapies [13]. Braugher et al., has recently invented a method of producing dendritic cells in vivo. These engineered dendritic cells express interleukin-12 and help in the treatment of tumor [20]. The Th1 cells that produce IFN-γ have shown to exert a powerful anti-tumor effect. Therefore, it is an important to identify factors that might produce IL-12 by monocytes-derived DC and affect


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the differentiation of DC. It should be kept into consideration that the effects induced by some cytokines may be due to the induction of other cytokines. For example, part of the IFN-γinduced effects on differentiation/activation of DC and expansion/survival of CD8+T cells may be mediated by IL-15 [21]. IFN-γ mandatory in Th1 polarization and endowed with regulatory properties, is currently used to condition monocytes-derived DC in cancer therapy and in clinical trials to treat chronic infectious diseases. IL-27 arms naïve T cells naïve T cells to responds to IL-12p70 through up-regulation of the specific receptor. Frasca et al., [22] reported that IFN-γ up-regulated IL-27 and IL-12 production and is a modulator of multiple DC effector functions that can be helpful in monocytes-derived DC-based vaccination protocol. Similar to IL-12, IL-23 is a heterodimeric cytokine composed of two subunits named p40, which is shared with IL-12, with a specific subunit called p19 [23]. IL-12 is important in Th1 development and subsequent exposure of IL-23 might be required for the maturation of effector cells. IL-23 strongly linked to autoimmune pathology and several recent reviews have focused on its role in Th17 biology and its suitability as a therapeutic target [24-26]. It remains unclear whether IL-23 is essential for the survival of Th17 cells, but it has an important role in their functional capacity. The cells that can produce both IL-17 and IFN-γ, as well as the flexibility of human TH17 clones to produce IFN-γ in addition to IL-17 in response to IL-12, suggests that there may be a developmental relationship between Th17 and Th1 cells, at least in human.

NF- Activation and Toll-like Receptor (TLR) Stimulation is Good Candidate for Cancer using DC Immunotherapy DC activation and maturation driven by TLR agonist such as LPS has been clearly associated to NF-B activation. TLR agonists are potent activators of innate immune responses, activating DC maturation and inflammatory cytokine secretion by innate immune cells. Several populations of DC have characterized. Isolated human plasmacytoid DC (pDC) express TLR7 and TLR9, whereas human myeloid DC expresses TLR1, TLR2, TLR3, TLR4, TLR6 and TLR8. Myeloid DC produce sIL-12 upon TLR engagement both in the human and in the mice. Recent studies have demonstrated that the signaling via TLR which are newly identified receptor molecules recognizing many pathogens, are involved in the induction of anti-cancer immunity [27]. An important immunomodulatory property of TLR agonists is their capacity to enhance IL-12 production from DC and other innate immune cells and consequently their ability to promote Th1-type responses, which play a key protective role in immunity to tumors [28]. Recently studies have focused on the molecular mechanisms underlying Th1/Th2 development and eleven members of the TLR family (TLR1 to TLR10 and PR105), co-factors (CD14, MD-1 and MD-2) and their ligands have been reported. TLR signaling promotes activation of an innate immune response, and then triggers antigenspecific adaptive immunity [29-31]. Several studies have been recently proposed the significance of TLR signaling in the induction of anti-cancer immunity. Recent studies suggest that TLR ligands are useful applications of immunotherapy for cancer patients. TLR2 is expressed by most CD11c DC and TLR2 ligands might induce various types of response. TLR2 recognizes peptidoglycan and lipopeptides. A synthetic lipopetide agonist of TLR2 has been shown to stimulate IFN-γ and IL-10 production, which had a regulatory role against Th2



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type responses [32]. Immature monocytes-derived DC expresses TLR3 and ligation by dsRNA results in increased secretion of IL-12 and type 1 IFNs [33]. The unclear correlation between DC-derived cytokine levels and their Th1-cell-polarizing capacity is underlined by the finding that dsRNA-primed mature monocytes-derived DC no longer produce high levels of IL-12 after with CD40L binding, but nevertheless still promote the development of Th1 cells partly through residual IL-12, type I IFNs and ICAM-1, and partly by as-yetunidentified factors. LPS is TLR4 agonist and induces IL-12 production by mice DC and human DC. LPS drive the development of Th1 polarization both in mice and humans. TLR4 agonist, including monophosphoryl lipid A (MPL) enhanced Th1 polarization from naïve T cells via the activation of IL-12p70 and dependent on TLR4, have been used as adjuvant for vaccines against HBV and other pathogens [34]. TLR7 is expressed by plasmacytoid DC (pDC) and monocytes-derived DC. TLR7 is induced during LPS-maturation of DC in a type I IFN--dependent manner [22]. TLR7 ligands are imiquimod (R-837, S-26308) and R-848, a synthetic TLR7 agonist, in mice and humans. Imidazoquinolines-activated DC drives the development of Th1 polarization from naïve T cells dependent on IL-12 secretion [35]. The TLR7 agonist imiquimod has been used to treat superficial basal cell carcinoma. Successful topical treatment of basal cell carcinoma with imiquimod was associated with infiltration of myeloid and plasmacytoid DC in the peritumoral infiltrate and it appears that these cells may mediate direct antitumor cytotoxicity [36]. TLR8 is expressed in myeloid DC (mDC) and dsRNA is agonist of TLR8. TLR8-acitavated DC leads to IL-12 and then drives the development of Th1 polarization from naïve T cells. Several mechanisms have been proposed to explain the apparent adjuvant effects of TLR agonists on antitumor immunity. TLR trigger the secretion of critical cytokines such as IL-1, IL-6 and IL-12 by DC, and TLR can stimulate the proliferation of CD4+T cells and CD8+ T cells. Moreover, TLR signaling frequently enhances the production of IL-12 on DC. Thus, it is strongly suggested that the DC matured by TLR stimulation may induce T cell differentiation toward Th1 by presenting antigens to the T cells while promoting a Th1-leading situation in the local environment. Several TLR agonists have been developed as anticancer drugs. Therefore, it is possible that the ligands of TLR are able to be effective immunoadjuvants for cancer therapy. Brasel et al., recently disclosed a technique for increasing the number of dendritic cell by administering FLT 3 ligands [37].

Feasibility of Cancer Immunotherapy Using Terpene Compounds Terpenes have biological activities including anti-tumor and immunosuppressant [38]. US20070259056 describes the administration of terpenes or its derivatives for the treatment of cancer [39]. Some terpene compounds have properties related to the regulation of apoptosis and cytotoxicity of cells and exhibit potent anti-tumor effects against a variety of tumor cells. Triterpene isolated from Acacia victoriae have been applied for patents [39]. We have reported that diterpene compounds and sesquiterpene compounds induce DC from human monocytes and drive Th1 polarization [40-42]. Sandaracopimaric acid, Sandaracopimaradiene-3β-ol and 16-phyllocladanol are diterpenes isolated from the heatwood of Cryptomeria japonica.


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Figure 1. Structure of terpene compounds.

Figure 2. Uncarinic acid C induces NFulates TLR2 and TLR4. (A) DC were stimulated with Uncarinic acid C (0.5 M), LPS (100 ng/ml) or medium for 30 min, 1 h. and 2 h., and nuclear extracts were analyzed for their NF-B binding activity using EMSA. 1: Competition (x 100 cold) negative control 2: immature dendritic cells (0 min), 3: immature dendritic cells (30 min) 4: immature dendritic cells (1 h) 5: immature dendritic cells (2 h) 6: LPS (0 min) 7: LPS (30 min) 8: LPS (1 h) 9: LPS (2 h) 10: Uncarinic acid C (0 min) 11: Uncarinic acid C (30 min) 12: Uncarinic acid C (1 h) 13: Uncarinic acid C (2 h). (B) Relative TLR expression during the differentiation of dendritic cells. Monocytes were cultured with GM-CSF and IL-4 to differentiate to immature dendritic cells for 8 days. Maturation of dendritic cells was induced by URC (0.1 M), Ursolic acid (1 M) or LPS (1 g/ml). Cells were collected at indicated time; mRNA were extracted and converted to cDNA. The cDNA were subjected to Realtime SYBR Green quantitative PCR using gene-specific primers pair for TLR2, TLR4 and -actin. Relative gene expression was calculated using 2-ΔΔCt method. Left panel: Relative TLR4 expression during differentiation of dendritic cells by LPS in comparison to stimulation of dendritic cells with Uncarinic acid C or Ursolic acid. Right panel: Relative TLR2 expression during differentiation of DC by LPS in comparison to stimulation of dendritic cells with Uncarinic acid C or Ursolic acid. Data are the mean  S.E.M. of three independent experiments. *P< 0.05 compared with immature dendritic cells (iDC).



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These terpene compounds regulated differentiation of DC from human monocytes in combination with GM-CSF and IL-4. DC differentiated with these terpene compounds enhanced the differentiation of naïve T cells towards the Th1 type, which is dependent on IL12 p70 secretion. Moreover, T-cadinol and calamenene are sesquiterpenes isolated from the heatwood of Cryptomeria japonica and also induced DC from human monocytes and drive Th1 polarization via the activation of IL-12p70 Figure 1. However, Th1 polarization induced by these terpenes-primed DC independent on TLR2 and TLR4. The mechanism of matured DC from human monocytes by these terpene compounds requires further investigation. DC activation and maturation derive by TLR agonist such as LPS has been clearly associated to NF-κB activation. Recently, we have studied that Uncarinic acid C, triterpene, isolated from Uncaria rhynchophylla activate human DC and promoted the skew of T cells towards Th1depending on IL-12 secretion [44]. We found that DC activated with Uncarinic acid C and Ursolic acid express significant levels of mRNA coding for both TLR2 and TLR4 (Figure 2). To determine whether Uncarinic acid C and Ursolic acid uses similar activation pathways, we monitored their ability to activate of the NF-B translocation into the nucleus. EMES was performed with the Gel Shift assay system (Promega, El, USA). DC was cultured in the presence of Uncarinic acid C and Ursolic acid for 30 min, 1 h. and 2 h., and nuclear extracts were analyzed for NF-κB content (Figure 2). Uncarinic acid C -primed DC was able to induce NF-κB translocation and activation. Similar results were obtained with Ursolic acid-primed DC and LPS-primed DC. Thus, Uncarinic acid C and Ursolic acid were able to induce NF-κB translocation and activation. Anti-cancer agent Taxol, a plant-derived diterpene, recognized TLR4 in mice and induced NF-κB activation [43]. However, the effects of Taxol on human monocytes-derived DC are not known yet. Anti-tumor immunity has classically been measured by the quantity of tumor-antigen-specific CD8+T cells. Immunotherapy is based on this mechanism. DC play a central role in various immunotherapy protocols through generation of cytotoxic T lymphocytes (CTL). DC-based vaccines have become the most attractive tool for cancer immunotherapy and have been used in the treatment of more than 20 malignancies. In this context, it is important whether Uncarinic acid C-primed DC enhanced specific CTL responses. As expected from their Th1-polarizing effect, the DC differentiated with Uncarinic acid C induced a stronger CTL response (Figure 3A and B). Similar results were obtained with Ursolic acid-primed DC. The effects of diterpene compounds or sesquiterpene compounds in the CTL response are not known yet. However, we expect to obtain similar results with Uncarinic acid C, because diterpenes- or sesquiterpenes-primed DC enhanced the differentiation of naïve T cells towards the Th1 type. The schema of terpene compoundsinduced anti-cancer immunity is shown in Figure 4. TLR-stimulated DC enhanced the producing ability of cytokines such as IL-12, potential Th1-inducung cytokines. Therefore, TLR-stimulated DC may effectively induce tumor-antigen specific Th1 and CTL by presenting antigen to CD4+T cells and CD8+T cells while promoting a Th1-leading situation. The molecular events leading to the effects of diterpene compounds and sesquiterpene compounds on DC function remain to be resolved. However, it is possible that terpenes or some ligands for TLRs are able to be effective immunotherapeutic agents for patients against cancer. Some terpene compounds and/or ligands of TLR should be used in immunotherapy for cancer. Hasumi recently patented a cancer vaccine consisting immature DCs, LCM adjuvant and antigen. This vaccine serves as a predecessor step of cancer treatment along with chemotherapy and radiation [45]. It seems that clinical trials are expected in the near future.


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Figure 3. Autologus CD8+T cells incubated with URC-primed dendritic cells showed higher cytolytic activity against T2 target cells loaded with WT-1 peptide at a high effector-to-target ratio thanagainst T2 target cell without WT-1 peptide. (A) The CTL were labeled with [3H]-methylthymidine and served as the target for tumor cell lines. T2 cells induced more DNA fragmentation of CTL that were generated with Uncarinic acid C -primed dendritic cells pulsed with WT-1 peptides than those that were generated with immature dendritic cells or TNF--primed DC pulsed with WT-1 peptides. (B) Specific lysis was measured by 51Cr release assay. Data are the mean  S.E.M. of three independent experiments. *P< 0.05 compared with T2.



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Figure 4. Sequential stages of terpene compounds mediated Th1 response.

Uncarinic Acid C –P rimed DC and Ursolic Acid-Primed DC Are Capable of Migration In Vitro The ability of DC to migrate to local lymph nodes and their subsequent presentation of antigen to T cells play an essential role in the initiation of adaptive immunity. In tissues, mature DC must be responsive to lymph node derived signals, but must also be able to downregulate tissue anchoring proteins including E-cadherin that would otherwise detrimental DC migration and antigen presentation to naïve T cells. Migration to the secondary lymphoid organs and subsequent antigen presentation requires DC maturation, a process that is associated with up-regulation of co-stimulatory molecules. We investigated the migratory capacity of Uncarinic acid C-primed DC and Ursolic acid-primed DC toward CCL19 and CCL21. Uncarinic acid-primed DC and Ursolic acid-primed DC had migration in response to CCL19 and CCL21, and slightly up-regulated the expression of CCR7 and CD38 on Uncarinic acid C-primed DC and Ursolic acid-primed DC. Expression of CCR7 seems also to be important for other aspects of dendritic cells biology, in particular in enhancing chemotaxis and trans-endothelial passage in response to CCL19 and CCL21. Recently, it has been reported that expression of CD38 on DC is essential for their coordinated migration to the T cell area of draining lymph node and increase dendritic cells function [46]. These results suggest that Uncarinic acid C-primed DC and Ursolic acidprimed DC migrate in vivo and is a promising approach for the treatment of cancer. In most clinical trials using DC-based immunotherapy, immature monocyte-derived dendritic cells pulsed with tumor antigen peptides were used. Recent studies showed that mature DC could be a better antitumor adjuvant. However, there is no clear answer yet.


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CURRENT AND FUTURE DEVELOPMENTS DC are an attractive target for therapeutic manipulation of the immune system to increase otherwise insufficient immune responses to tumor antigens. However, the complexity of the DC system requires rational manipulation of DC to achieve protective or therapeutic immunity. More than 200 clinical studies have been reported using DC vaccines in patients with various hematological and solid malignancies, including multiple myeloma, leukemia, melanoma, renal cell carcinoma, breast and prostate cancer. Although surrogate immunological responses can be detected in many patients after DC vaccination, overall clinical response rates are much lower. However, DC vaccines are safe but require careful design and validation. And an understanding of DC biology will allow conventional DC vaccines to be improved. Their activation status will be enhanced with combination of novel molecular adjuvant. TLR agonists can prove useful in cancer vaccines for the in vitro maturation of Antigen presenting cells (APC). As described above, some terpene compounds are ligands of TLR and induce efficient maturation and activation of DC. Therefore, some terpene compounds may be used in DC-based immunotherapy against cancer in the near future. Because of their pleiotropic effects, activating several immune cells simultaneously, Uncarinic acid C are also able to boost the immune system. The combination of some terpene compounds with other factors amplifies their capacity to enhance immune responses. Ongoing and future preclinical and clinical studies will clarify whether or not terpene compounds can fulfill their promise as effective antitumor treatments and/or vaccine adjuvant.

ACKNOWLEDGMENT We thank Drs. N. Shoji and T. Hashimoto for critical reading of this chapter.

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Chapter 5

ESSENTIAL OIL COMPOSITIONS AND IN VITRO BIOLOGICAL ACTIVITIES OF THREE SZYZGIUM SPECIES FROM NIGERIA Oladipupo A. Lawal1, Isiaka A. Ogunwande1,, Christiana A Bullem1, Olayinka T. Taiwo1 and Andy R. Opoku2 1

Department of Chemistry, Lagos State University, PMB LASU Post Office, Ojo, Lagos, Nigeria 2 Department of Biochemistry and Microbiology, University of Zululand, KwaDlangezwa, South Africa

ABSTRACT The in vitro antibacterial, antioxidant, phytotoxic and insecticidal activities of essential oils from the leaves of Syzygium malaccense and Syzygium samarangense, and the buds of Syzygium aromaticum have been investigated. The hydrodistilled essential oils from the three Syzygium species were analyzed by Gas Chromatography (GC) and Gas chromatography coupled with Mass spectrometry (GC/MS). Eight, twenty-three and forty constituents representing 94.7%, 97.0% and 90.3% of the total oil contents, were identified, respectively in S. aromaticum, S. malaccense and S. samarangense essential oils. Eugenol (78.5%) was the most significant compound of S. aromaticum oil. Limonene (48.8%) and γ-terpinene (26.2%) are the main compounds of S. malaccense oil, while the oil of S. samarangense had α-cadinol (12.7%), juniper camphor (12.5%), caryophyllene oxide (8.2%) and -cadinene (5.7%) as the major constituents. The biological activities of the oils were assayed using different in vitro methods. The oils exhibited moderate antibacterial activity against the tested microorganism except Kiebsiella spp, Proteus spp, Pseudomonas spp and Mucor mucedo. The mean zones of inhibition (MZI) and minimum inhibitory concentrations (MIC) of the oils ranged between 6.3-27.7 mm and 0.16 - 10.0 mg/mL. In the antioxidant assay, the oils exhibited significant scavenging activity for DPPH, nitric oxide and superoxide anion radicals. The oils also showed notable phytotoxic effects on the seeds of Zea mays, with percentage 

Corresponding author: [email protected]; [email protected]; Tel.: +234 8059929304.


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Oladipupo A. Lawal, Isiaka A. Ogunwande, Christiana A Bullem et al. seed germination, root and shoot elongations from 85.0% to 96.6 %, 12.3 to 20.9 mm and 2.9 to 5.3 mm, respectively. Furthermore, the results of the insecticidal activity showed that the oils were significantly and concentration dependent.

Keywords: Syzygium aromaticum, Syzygium malaccense, Syzygium samarangense, essential oil composition, biological activities

INTRODUCTION Syzygium is a genus of flowering plants that belongs to the myrtle family, Myrtaceae. The genus comprises about 1100 species, and has a native range that extends from Africa and Madagascar through southern Asia to the Pacific. Most species are evergreen trees and shrubs, which are grown as ornamental plants for their attractive glossy foliage, and a few produce edible fruits that are eaten fresh or used in jams and jellies. At times Syzygium was confused taxonomically with the genus Eugenia (ca. 1000 species), but the latter genus has its highest specific diversity in the neotropics. Many species formerly classed as Eugenia are now included in the genus Syzygium, although the former name may persist in horticulture [1]. Syzygium malaccense L. (Merr.) & Perry, is a species of flowering tree native to Malaysia, Indonesia Sumatra and Java and Vietnam. It has been introduced into several parts of the world including tropical Africa. It grows up to 16m long. The evergreen leaves are opposite, soft leathery and dark green: the flowers are purplish - red and form a carpet after falling under the tree. The fruit is oblong - to pear shaped with a dark red skin and white flesh; sometimes it is seedless. A decoction of the bark is used against vaginal infection, while the root is used to treat itching. The root is also effective against dysentery and as a diuretic. The methanolic extract exhibited strong antioxidant activity and contains a higher amount of phenolics and flavonoids when compared to aqueous extract [2]. The preliminary ichthyotoxic test on all parts of S. malaccens [3] revealed that the leaves fraction was the most ichthyotoxic against tilapia-fish (Tilapia oreochromis). The extracts of S. malaccense with their beneficial effects on blood sugar and hyperlipidemia associated with diabetes could serve as good adjuvant to other oral hypoglycemic agents [4]. The non-volatile components include ursolic acid, β-sitosterol and sitost-4-en-3-one [3]. The hydrodistilled essential oil from the fresh leaves of S. malaccense grown in Nigeria was largely composed of monoterpenes (61.1%) characterized mainly by α-pinene, β-pinene, p-cymene and αterpineol. The sesquiterpenes constituted 30.8% of the oil with β-caryophyllene as the major component [5]. Syzygium samarangense Merr. & Perry, commonly known as wax jambu, is an evergreen tree with origins in Asia. It produces a pink fleshy fruit which is eaten fresh. The fruit is oblong, pear-shaped, and 5 to 12 cm in length, with four fleshy calyx lobes and 1 to 4 seeds (1 to 2 cm in diameter). The green fruits of wax jambu are eaten raw with salt or cooked as a sauce. The flowers, which contain tannins, desmethoxymatteucinol, 5-O-methyl-4′desmethoxymatteucinol, oleanic acid, and β-sitosterol, are used in Taiwan to treat fever and halt diarrhea [6]. Previous phytochemical studies have shown the presence of ellagitannins, flavanones, flavonol glycosides, proanthocyanidins, anthocyanidins, triterpenoids, cytotoxic and anti-oxidant compounds [7-13] and volatile terpenoids [14]. Five compounds are


Essential Oil Compositions nd In Vitro Biological Activities …

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considered as the main components of its essential oil, including caryophyllene (7.9%), decahydro-4‘-methyl-1-methylene-7-(1-methylethenyl)-naphthalene (7.6%), 1,2,3,4,4‘,5,6,8‘Octahydro-4‘,8-dimethyl-2-(1-methylethenyl)- naphthalene (7.3%), 1,2,4,5-tetramethyl benzene (7.2%), and caryophyllene oxide (6.3%). The essential oil exhibits moderate antioxidant ability in vitro [15]. Clove is the dried flower bud of Syzygium aromaticum (L.) Merr. & Perry. It is an evergreen tree that grows up to 20 m high. The plant is indigenous to India, Indonesia, Zanzibar, Mauritius and Sri Lanka [16]. Clove is reported as aphrodisiac, stomachic, carminative, antispasmodic [17]. It is reported to be useful in conceiving in high doses and act as a contraceptive in low doses and useful in cataract [18]. Clove is also reported to have anticarcinogenic [19], anticonvulsant [20] and antioxidant [21, 22] properties. It improves learning and memory in mice [23]. Extracts of the plant have been shown to possess antiviral activity against Herpes simplex [24]. Phytochemical studies indicate that the clove contains free eugenol, eugenol acetate, caryophyllene, sesquetrepene ester, phenyl propanoid, β caryophyllene, eugenol and acetyle eugenol, with potential biological activities [24-28]. Eugenol, the major constituent, inhibits lipid peroxidation and maintains activities of enzyme superoxide dismutase, catalase, glutathione peroxidase-6 phosphate dehydrogenase, and has also been reported to have vasodilatory, and smooth muscle relaxant property [26-28].

MATERIALS AND METHODS Chemicals 2,2-Diphenyl-1-picryl-hydrazyl (DPPH), sodium nitroprusside, trichloro acetic acid, thiobarbituric acid, naphthylethylenediamine dihydrochloride, xanthine oxidase, xanthine, nitroblue tetrazolium, naphthylethylenediamine dihydrochloride, sulphanilic acid, ethylenediaminetetraacetic acid, dimethylsulfoxide, bovine serum albumin, butyl hydroxyl anisole and were obtained from Sigma-Aldrich Co., Ltd (Steinheim, Germany). All other chemicals and solvents are of analytical grade.

Plant Materials Fresh leaf materials of S. malaccense and S. samarangense were collected from private gardens in Ikotun Town, while the dry buds of S. aromaticum were purchased from Iyana-Iba Central market, in Ikotun/Igando Local Government Development Area and Ojo Local Government Area, Lagos State, Nigeria, respectively. Botanical identification of the plant materials were carried out by Mr. T. K Odewo of the Department of Botany, University of Lagos, Akoka-Yaba, Lagos, Nigeria. Voucher specimens LUH 5783, LUH 1507A and LUH 5783A respectively for S. aromaticum, S. malaccense and S. samarangense were deposited in the Herbarium of the University.


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Oladipupo A. Lawal, Isiaka A. Ogunwande, Christiana A Bullem et al.

Isolation of Essential Oils Aliquots of air-dried and pulverized leaves of S. malaccense (300 g), S. samarangense (300 g) and S. aromaticum (500 g) were separately subjected to hydrodistillation using Clevenger-type apparatus for 3 h in accordance with the standard procedure [29]. The distillated oils were preserved in a sealed sample tube and stored under refrigeration until analysis.

Gas Chromatography (GC) Analyses GC analyses of the oils were carried out on a Hewlett Packard HP 6820 Gas Chromatograph equipped with a FID detector and DB-5 column (60 m x 0.25 mm id, film thickness was 0.25 μm) and the split ratio was 1:25. The oven temperature was programmed from 50 °C (after 2 min) to 240 °C at 5 °C/min and the final temperature was held for 10 min. Injection and detector temperatures were maintained at 200 °C and 240 °C, respectively. Hydrogen was the carrier gas at a flow rate of 1 mL/min. 0.5 µL of the individual oil was injected into the GC. Peaks were measured by electronic integration. A homologous series of n-alkanes were run under the same conditions for determination of retention indices.

Gas Chromatography-Mass Spectrometry (GC/MS) Analyses GC-MS analyses of the oils were performed on a Hewlett Packard Gas Chromatograph HP 6890 interfaced with a Hewlett Packard 5973 Mass Spectrometer system equipped with a DB-5 capillary column (30 m x 0.25 mm id, film thickness 0.25 μm). The GC conditions were as described above. The ion source was set at 240 oC and electron ionization at 70 eV. Helium was used as the carrier gas at a flow rate of 1 mL/min. The scanning range was 35 to 425 amu. Diluted oil in n-hexane (1.0 μL) was injected into the GC/MS.

Identification of Compounds The identification of constituents was performed on the basis of retention indices (RI) determined by co-injection with reference to a homologous series of n-alkanes, under identical experimental conditions. Further identification was performed by comparison of their mass spectra with those from NIST 08 and Wiley 9th Version and the home-made MS library built up from pure substances and components of known essential oils, as well as by comparison of their retention indices with literature values [30, 31].



97

Antibacterial Activity Microbial strains The essential oils were tested against eleven local isolates (two Gram-positive, seven Gram-negative strains and two fungal) and one reference strain obtained from the Department of Microbiology, Lagos State University, Ojo, Lagos and Nigerian Institute of Medical Research (NIMR), Yaba, Lagos, Nigeria, respectively. These microbes were Bacillus subtilis, Staphylococcus aureus, Citrobacter youagae, Escherichia coli, Escherichia coli (ATCC 34523), Kiebsiella spp, Micrococcus spp, Proteus spp, Pseudomonas spp, Salmonella spp, Mucor mucedo and Rhizopus stolonifer. The stock cultures were maintained at 4 oC in Müeller-Hinton agar (Oxoid, Germany).

Disc Diffusion Assay The oils were tested for antibacterial activity by the agar disk diffusion method according to standard procedure [32]. The microorganisms were grown overnight at 37 oC in 20 mL of Mueller-Hinton broth (MHB) (Oxoid, Germany). The cultures were adjusted with sterile saline solution to obtain turbidity comparable to that of McFarland no. 5 standard (1.0 x 108) CFU/mL. Petri dishes, 90 mm (Merck, Germany) containing 12 mL of sterilized Mueller-Hinton agar were inoculated with these microbial suspensions. Sterile Whatman No.1 (6mm) discs papers were individually placed on the surface of the seeded agar plates and 10 µL of each in 1% DMSO was applied to the filter paper disk. The plates were incubated at 37 oC for 24 h and the diameter of the resulting zones of inhibition (mm) of growth was measured. All tests were performed in triplicates. Gentamycin (25 µg) and 1% DMSO were used as controls.

Determination of Minimum Inhibitory Concentrations The minimum inhibitory concentrations (MICs) of the oils were determined using 96well microtitre dilution method as earlier described [33]. Bacterial cultures were incubated in Müller-Hinton broth overnight at 37 oC and a 1:1 dilution of each culture in fresh MH broth was prepared prior to use in the micro dilution assay. Serial dilutions were made to obtain concentrations ranging from 10 mg/mL to 0.078 mg/mL. 100 μL of bacterial culture of an approximate inoculum size of 1.0 x 108 CFU/mL was added to all well and incubated at 37 oC for 24 h. After incubation, 40 μL of 0.2 mg/mL p-iodo-nitotetrazolium (INT) solution was added to each well and incubated at 37 oC. Plates were examined after about 30-60 min of incubation. MIC is defined as the lowest concentration that produces an almost complete inhibition of visible micro-organism growth. Solvent control (1% DMSO) and standard antibiotics (Gentamycin) were included in the assay.


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Antioxidant Activity 1,1-Diphenylpicryl-Hydrazyl Radical Scavenging Activity The free radical scavenging (DPPH) ability of the essential oils was evaluated following a standard method [34, 35]. About 0.2 mL of different concentrations (19-115 µg/mL) of each oil in methanol was mixed with 2.7 mL of 1.0 x 10-4M methanol solution of 2,2-diphenyl-1picrylhydrazyl (DPPH). The absorbance at 517 nm was measured using UV-Visible Genesys 20 spectrophotometer after the solution had been allowed to stand in the dark for 60 min. The absorbance of the oil samples, the control and the blank were measured in comparison with methanol. Synthetic antioxidant: butyl hydroxyl anisole (BHA) and butyl hydroxyl toluene (BHT) were used as standards. Nitric Oxide Radical (NO·) Scavenging Activity The scavenger activity of S. aromaticum, S. malaccense and S. samarangense essential oils to compete with oxygen and reduces the production nitric oxide was determined using Griess Illosvoy reaction Garrat [36]. The reaction mixture (3 mL) containing 2 mL of 10 mM sodium nitroprusside, 0.5 mL of phosphate buffer saline (pH 7.4, 0.01 M) and 0.5 mL of different concentrations of essential oils were incubated at 25 °C for 150 min. Thereafter, 0.5 mL of the reaction mixture containing nitrite was pipette and mixed with 1 mL of sulphanilic acid reagent (0.33% in 20% glacial acetic acid) and allowed to stand for 5 min for completing diazotisation. Then, 1 mL of naphthylethylenediamine dihydrochloride (0.1%) was added, and allowed to stand for 30 min in diffused light. The absorbance of the pink coloured chromophore was measured at 540 nm against the corresponding blank solution. BHT and BHA were used as standards. The inhibitory effect of the oils was calculated by: % Inhibition = {(A0 – A1)/A0 x 100} where A0 is the absorbance of the control and A1 is the absorbance in the presence of the essential oils. The concentration providing 50% inhibition (IC50) was calculated from the graph of percentage inhibition against oil concentrations.

Superoxide Anion (O2 -) Radical Scavenging The ability of S. aromaticum, S. malaccense and S. samarangense essential oils to inhibit photochemical reduction of nitroblue tetrazolium (NBT) in the riboflavin–light–NBT system was assessed according to the modified method [37]. The reaction mixture containing 0.02 mL each of (50.0 mM, pH 10.5) sodium carbonate buffer, 0.15% bovine serum albumin, 3 mM xanthine, 3 mM ethylenediaminetetraacetic acid (EDTA), 0.75 mM NBT and 0.02 mL of different concentrations (20 - 120 mg/L) of each oil was incubated for 20 min at 25°C. Then, 0.02 mL of xanthine oxidase (XOD) was added to initiate the reaction. The production of blue formazan was monitored for 20 min at 25°C, after adding 0.02 mL of 6 mM copper (II) chloride. The absorbance of the mixture was measured at 560 nm. The inhibitory effect of the essential oils was calculated by:



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% Inhibition = {(A0 – A1)/A0 x 100} where A0 is the absorbance of the control and A1 is the absorbance in the presence of the essential oils. The concentration providing 50% inhibition (IC50) was calculated from the graph of percentage inhibition against oil concentrations.

Allelopathic Activity A bioassay based on germination and consequent seedling growth (root length) was used to study the phytotoxic effects of essential oils of S. aromaticum, S. malaccense and S. samarangense on seeds of maize (Zea mays L.) according to the method described earlier [38]. Seeds of Z. mays were purchased from Iyana-Iba Market, Ojo Local Government Area, Lagos, Nigeria. The seeds were surface-sterilized in 95% ethanol for 15 s and sown in Petri dishes (Ø = 90 mm), containing two layers of Whatman filter paper No.1, impregnated with 10 mL of test- solutions of each oil at the different concentrations (20 - 120) mg/ml, 10 mL of 1% DMSO and distilled water as controls, respectively. Each bioassay was repeated three times with 10 seeds for each determination at 27 ± 1 °C with natural photoperiod. The percentage germination and (root and shoot) growth for each experiment was measured after 7 days of incubation period.

Insecticidal Activity The fumigant toxicity of S. aromaticum, S. malaccense and S. samarangense essential oils was assay according to a standard method [39]. Adult insects of mixed sex, 7-14 days old of Sitophilus zeamais reared on maize and at 25 ± 2 o C and 65% ± 5% relative humidity (R.H.) was used for the bioassay. Filter paper (Whatman No. 1, cut into 2-cm diameter pieces) was impregnated with the oils at doses calculated to give equivalent fumigant concentrations of (20 - 120) mg/L air. The impregnated filter paper was placed in the Petri dishes (90 mm) containing 10 adults of S. zeamais to different concentrations of each essential oil. Each concentration and the control were replicated three times. Mortality was determined after 24, 48, 72 and 96 h from the commencement of exposure. When no leg movement was observed, insects were considered dead. The percentage insect mortality was calculated using Abbott‘s formula for natural mortality in untreated controls [40]. Probit analysis was used to estimate LC50 value.

Statistical Analysis The mean and standard deviation of three experiments were determined. Statistical analysis of the differences between mean values obtained for experimental groups were calculated as means  standard deviation (SD) of three independent measurements using Microsoft excel program, 2003 and Origin 6.0 for IC50. Data were subjected to one way analysis of variance (ANOVA). P values ≤ 0.05 were regarded as significant and P values ≤


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0.01 as very significant. The percentage of mortality and lethal concentrations (LC50) values for insecticidal and larvicidal activities were determined using Abbott‘s formula [40] and probit analysis program, version 1.5, respectively and reported as LC50 with 95% confidence intervals, representing the concentrations in μg/mL with 50 % mortality rate in 72 H.

RESULTS AND DISCUSSION Table 1 indicates the percentage compositions of compounds identified from the studied oil samples. Eight, twenty-three and forty constituents representing 95.0%, 97.0% and 93.4% of the total oil contents were identified respectively from the essential oils of S. aromaticum, S. malaccense and S. samarangense. Table 1. Percentage composition of essential oils of three Syzygium species Compounds a

RI b

RI c

α-Thujene α-Pinene β-Pinene 1-Octen-3-ol Myrcene 2-Pentyl furan n-Octanal α-Phellandrene δ-3-Carene α-Terpinene p-Cymene Limonene γ-Terpinene n-Octanol Terpinolene Linalool n-Nonanal Fenchol d (-)-Isopulegol Borneol Terpinen-4-ol α-Terpineol Dihydrocarveol endo-Fenchyl acetate Citronellol Thymol α-Cubebene Citronellyl acetate Eugenol Neryl acetate α-Copaene n-Tetradecane

935 940 979 982 991 995 1005 1007 1010 1017 1021 1029 1061 1079 1087 1099 1106 1109 1160 1169 1187 1189 1201 1221 1226 1296 1354 1362 1364 1369 1378 1400

924 932 974 974 988 984 998 1002 1008 1014 1020 1024 1054 1063 1086 1095 1100 1167 1165 1174 1186 1192 1218 1223 1289 1345 1350 1356 1359 1374 1400

Percent composition (%) S. aromaticum S. malaccense 0.3 1.3 1.0 0.6 3.0 0.3 0.2 0.7 48.8 26.2 0.7 1.1 1.6 1.2 1.1 0.6 0.8 78.5 0.3 -

S. samarangense 0.2 0.3 1.8 0.8 2.8 0.7 0.9 0.8 0.4 2.1 3.3 0.2 0.4 0.7 1.3 0.1 0.3 0.6 -


Essential Oil Compositions nd In Vitro Biological Activities … Compounds a

RI b

β-Caryophyllene 1427 (E)-α-Ionene 1431 Neryl acetone 1434 α-Cedrene 1437 (E)-Isoeugenol 1460 α-Humulene 1461 γ-Muurolene 1478 (E)-β-ionene 1500 α-Selinene 1492 α-Muurolene 1497 (E,E)-α-farnesene 1499 α-Bulnesene 1505 epi-α-Selinene 1515 δ-Cadinene 1523 Eugenyl acetate 1533 Palustrol 1566 Caryophyllene oxide 1589 Ledol 1608 Guaiol 1610 α-Cadinol 1652 Patchouli alcohol 1656 Juniper camphor 1689 Palmitic acid 1917 Ethyl hexadecanoate 1993 Methyl linoleate 2090 Phytol 2113 Oleic acid ethyl ester 2182 Stearic acid ethyl 2189 ester Total Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Diterpenoids Phenyl propanoids Fatty acids Non-terpenes

101

1417 1428 1434 1410 1448 1452 1478 1487 1498 1500 1505 1509 1520 1522 1521 1567 1582 1602 1600 1652 1656 1680 1920 1992 1095 1942 2180

Percent composition (%) S. aromaticum S. malaccense 1.9 1.5 0.2 1.4 0.9 13.3 0.3 0.6 2.6 0.2 0.3 0.5 -

S. samarangense 3.4 0.6 4.8 0.7 2.0 0.1 1.2 2.7 4.6 2.0 5.7 3.5 8.2 1.7 12.7 1.6 12.5 1.0 4.5 0.7 1.0

2191

-

-

1.1

95.0 0.2 1.9 0.3 91.8 0.8 -

97.0 79.9 2.4 2.4 0.6 0.5 2.6 3.5

93.4 6.6 11.1 27.2 40.2 0.7 7.6 -

RI c

a

Elution order on DB-5 column; Retention Indices relative to C9-C24 n-alkanes on the DB-5 column; c Literature retention indices (see Experimental); d correct isomer not identified; - Not identified and not found in Literature b

The compositional pattern differed from each other. Monoterpene hydrocarbons (79.9%) represented mainly by limonene (48.8%) and -terpiene (26.2%) was the most important oil fraction of S. malaccense. The chemical pattern resembles those of previous study from Nigeria [5], only in the general prevalence of monoterpene compounds, but the identities of all the prominent compounds differ from each other. However, p-cymene and β-


102


caryophyllene, the prominent compounds in the result of previous analysis [5] are conspicuously absent in the present investigation. In addition, compounds such as 2phenylethanol and its esters (2-phenylethyl acetate, 2-phenylethyl isopentanoate, 2phenylethyl benzoate and 2-phenylethyl phenylacetate) which are the major constituents of its fruit volatiles of Cuban sample [41] were not identified in this study. However, sesquiterpene hydrocarbons (27.2%) and their oxygenated derivatives (40.2%) as well as the oxygenated monoterpenes (11.1%) were the main oil fractions of S. samarangense. The major compounds were identified as α-cedrol (12.7%), juniper camphor (12.5%), caryophyllene oxide (8.2%) and -cadinene (5.7%). Aldehydes and alcohols compounds were less common in this study in contrast to previous study from Malaysia [14], while benzenoid compounds such as decahydro-4‘-methyl-1-methylene-7-(1-methylethenyl)naphthalene, 1,2,3,4,4‘,5,6,8‘-octahydro-4‘,8-dimethyl-2-(1-methylethenyl)-naphthalene and 1,2,4,5-tetramethyl benzene which are the major compounds in the Chinese sample [15] could not be detected in our study. Eugenol (78.5%) and eugenyl acetate (13.3%), two phenyl propanoids, are the abundant constituents of S. aromaticum. None of the monoterpene hydrocarbons was detected in the oil. All other compounds except β-caryophyllene (1.9%) were identified in amount less than 1%. This compositional pattern was a contrast to a previous report from Nigeria [42] which comprised mainly of methyl salicylate (46.6%), β-caryophyllene (33.3%) and 3, 4-dimethoxy styrene (9.1%). Table 2 indicates a summary of the chemical constituents and biological activities of previously studied of clove oils [43-67]. A review of literature indicated that there is homogeneity in its oil content irrespective of the plant parts or the region of production, with minimal variations. Samples contained eugenol as the major compound, with significant proportions of either eugenyl actate, β-caryophyllene or caryophyllene oxide. The in vitro biological activities of oils were summarized in Table 3. The antimicrobial test indicated that S. samarangense displayed very weak activity with zones of inhibition (6.3 - 19.9 mm) and MIC (2.5 - 10.0 ug/mL). Both S. aromaticum and S. malaccense displayed mild to moderate activities against all the tested microorganisms, with S. aromaticum being more active. The weakest activities were exhibited against Kiebsiella spp, Proteus spp and Pseudomonas spp, and M. mecudo. This is notiveable when compared with the standard antibiotic. The ability of S. aromaticum, S. malaccense and S. samarangense essential oils to scavenge DPPH radical, nitric oxide and superoxide anion radical scavenging activities were evaluated as seen in Table 3. DPPH is a free radical compound that has been widely used to test the free radical scavenging ability of essential oils. DPPH is considered to be a model for lipophilic radical in which a chain reaction is initiated by the lipid autoxidation. The standard antioxidant BHA demonstrated higher inhibitory activity, when compared to the essential oils of Syzygium species (IC50 = 43.6 - 58.4 mg/mL). However, the essential oils also showed a remarkable inhibition activity. In addition, the scavenging effects of the essential oil samples were concentration dependent. The concentration of the essential oils resulting in a 50% inhibition of the DPPH radical scavenging (IC50) was greater than of ascorbic acid except for S. malaccense essential oil with IC50 value of 58.4 mg/ml.



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Table 2. Summary of known chemical constituents and biological activities of S. aromaticum essential oil Main constituents eugenol (78%) and β-caryophyllene (13%) eugenol (24.371 mg/g) and eugenyl acetate (2.354 mg/g) eugenol (49.0%) and caryophyllene (7.5%) eugenol (87.0%), eugenyl acetate (8.0%) and β-caryophyllene (3.6%) 2-methoxy-4-(2-propenyl)-phenol (83%) or eugenol and transcaryophyllene (12%) eugenol (94.4%) and β-caryophyllene (2.9%) eugenol a myrtenone (49.1%), eugenol (27.1%), β-caryophyllene (8.7%), palmitic acid (4.3%) and pulegone (4.1%) eugenol (82.95 %), eugenyl acetate (5.01 %), β-caryophyllène (3.14 %) eugenol (69.8%), β-caryophyllene (13.0%) and eugenyl acetate (16.1%) b eugenol (78.1%) and β-caryophyllene (20.5%) c eugenol

a

eugenol (75.8%) and eugenyl acetate (18.98 %) eugenol (76.8%) eugenol (77.37%) and trans-caryophyllene (13.66%) eugenol (88.6%), eugenyl acetate (5.6%), eugenol (88.6%) eugenol , eugenol acetate, β-caryophyllene, α-humulene and caryophyllene oxide a eugenol (70%) and β-caryophyllene (15%) eugenol (76.8%), β-caryophyllene (17.4%), α-humulene (2.1%) eugenol acetyleugenol, isoeugenol, and methyleugenol a acetyleugenol, β-caryophyllene, eugenol, α-humulene, and methyl salicylate a eugenol (86.2%) eugenol (74.3%), eucalyptol (5.8%) b eugenol (49.7%), caryophyllene (18.9%), benzene,1-ethyl-3nitro (11.1%) and benzoic acid,3-(1-methylethyl) (8.9%) c eugenol (71.56 %) and eugenol acetate (8.99 %) d

Activities cytotoxic antioxidant antimicrobial

References 43 44 45

-

46

insecticidal

47

anti-giardial

48 49

-

50

antimcorbial

51

-

52

free radical scavenging antioxidant, antimicrobial antimicrobial larvicidal antioxidant and antifungal

52

-

58

-

59

-

60

acaricidal activity ovicidal and adulticidal antioxidant -

61

-

―

antioxidant and hepatoprotective

65

eugenol (75.04 - 83.58%). β-caryophyllene (11.65 - 19.53%) and α-humulene (1.38 - 2.17%) b d eugenol (87.52 - 96.65%), β-caryophyllene (1.66 - 9.7%) eugenol (75–88%), acetyl eugenol (4–15%), β-caryophyllene (5-14%) eugenol (82.3-91.4%) and trans-β-caryophyllene (6.3-12.7%) a Quantitative data not available; b leaf sample; c bud sample; d stem sample.


53 54 55 56 57

62 63 64

― ― 66 67

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Table 3. Antibacterial , antioxidant and insecticidal activities of essential oils of three Syzygium speciesa S. aromaticum IZa MICb B. subtilis 19.7 ± 2.1 1.25 S. aureus 27.7 ± 0.6 0.16 C. youagae 12.3 ± 2.1 5 E. coli 20.0 ± 1.0 0.63 E. coli (ATCC) 23.3 ± 2.1 0.31 Kiebsiella spp 7.3 ± 0.6 10 Micrococcus spp 13.0 ± 1.7 2.5 Proteus spp 6.7 ± 1.2 10 Pseudomonas spp 7.3 ± 0.6 10 Salmonella spp 16.0 ± 1.0 1.25 M. mucedo 7.3 ± 0.6 10 R. stolonifer 14.0 ± 1.0 2.5 Antioxidant S. aromaticum DPPH 43.6 Nitric oxide 34.9 Superoxide anion 54.5 Insecticidale S. aromaticum

S. malaccense S. samarangense Gentamycin IZ MIC IZ MIC IZ MIC 13.7 ± 1.5 2.5 14.0 ± 2.7 5 20.7 ± 1.2 0.31 20.3 ± 2.5 1.25 19.0 ± 1.0 2.5 28.3 ± 2.1 0.63 10.7 ± 0.6 10 12.0 ± 1.0 5 ND ND 18.3 ± 1.5 0.63 14.3 ± 0.6 5 24.7 ± 1.2 0.31 24.0 ± 0.0 0.16 15.7 ± 1.5 2.5 32.3 ± 1.7 0.08 6.3 ± 0.6 > 10 8.3 ± 0.6 10 10.7 ± 1.5 2.5 12.3 ± 2.5 5 10.7 ± 2.1 10 14.3 ± 1.3 1.25 7.0 ± 1.0 10 6.3 ± 0.6 ND 12.3 ± 2.5 1.25 6.7 ± 0.6 > 10 6.3 ± 0.6 ND 15.7 ± 2.1 2.5 17.0 ± 0.0 2.5 12.3 ± 1.5 10 22.0 ± 2.0 0.63 8.3 ± 0.6 10 6.3 ± 0.6 ND 10.3 ± 0.6 5 13.7 ± 1.2 5 12.3 ± 1.5 10 14.3 ± 1.3 1.25 S. malaccense S. samarangense BHA AA 58.4 53.3 36.1 56.4 64.6 40.9 36.8 25.2 103.1 88.2 64.9 S. malaccense S. samarangense Permethrin 39.17 (21.2327.11 (18.34LC50 (95% CI) 7.68 (6.11-16.03) 11.10 (6.03-23.19) 51.66) 38.12) a IZ: Inhibition zones diameter (mm) including diameter of sterile disc (6 mm), with values given as mean ± SD (3 replicates); ATCC = American Type Culture Collection; aMIC values are given as (mg/mL); b Methanolic solutions of Gentamycine - 5µg/mL; ND = Not Determined. Microorganisms

Nitric oxide or reactive nitrogen species, formed during their reaction with oxygen or superoxide are very reactive. These compounds/radicals are liable for the changes in the structural and functional responses of many cellular components, causing serious toxic reactions with proteins, lipids, nucleic acids among others. The percentage inhibition generated from the essential oils and the standard antioxidants (BHA and ascorbic acid) at different concentrations by nitric oxide radical from sodium nitropruside at the physiological pH are given in Table 3. The results are inversely proportional to concentration. The IC50 of the oils, BHA and ascorbic acid were found to range from very active to moderately active. Although, the IC50 of BHA and ascorbic acid (25.2 - 36.8 mg/mL) shows more inhibitory activity than the essential oils (IC50 = 34.9 - 64.6 mg/mL). However, S. aromaticum essential oil (IC50 = 34.9 mg/ml) displayed very effective scavenger of nitric oxide, when compared with BHA (IC50 = 36.8 mg/ml). Superoxide anion radical generated by the xanthine oxidase (a dehydrogenase enzyme) that transfers electrons to nicotinamide adenine dinucleotide (NAD+), reducing it to NADH



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and oxidizes xanthine or hypoxanthine to uric acid was spectrophotometrically by monitoring its ability to reduce nitroblue tetrazolium to formazan. The superoxide anion scavenging activity of the oils was evaluated by the enzymatic hypoxanthine/xanthine oxidase system. The results show the scavenging activities of the oils and ascorbic acid to be dose dependent. S. aromaticum essential oil was found to be very effective scavenger of superoxide anion radical with IC50 of 34.9 mg/mL when compared to ascorbic acid (IC50 = 64.9 mg/mL). Based on these results, it is apparent that S. aromaticum essential oil showed highest antioxidant activity. The phytotoxic effects of the essential oils on seed germination and elongations (roots and shoots) of Z. mays at different concentrations are shown in Figures 1 and 2. The results showed that the oils exerted allelopathic effects on Z. mays seeds. Although, the oils did promote the germination and radical elongations of Z. mays seeds, however, germination percentages were significantly decreased (P < 0.05) as the concentrations of the oils increased. The oil of S. aromaticum had the highest allelopathic potential compared to the oils of S. malaccense and S. samarangense. After 7 days of incubation period, the inhibitory effects of the oils on germination of Z. mays seeds ranged between 85.3 to 96.7 %. In addition, the radicle elongations (root and shoot) of Z. mays seeds were affected by the oils, and the radicles were significantly increased, although, root and shoot length increases from (12.3 ± 0.7 to 20.9 ± 2.0 and 2.9 ± 1.8 to 5.3 ± 1.6) mm, (0.8 ± 0.4 to 4.3 ± 0.6 and 0.2 ± 2.2 to 2.2 ± 0.6) mm and (2.6 ± 0.7 to 4.9 ± 1.2 and 0.3 ± 0.2 to 2.0 ± 1.0) mm for the oils of S. aromaticum, S. malaccense and S. samarangense, respectively. But, at 50 % concentration, growth was retarded and radicles of seeds were reduced as concentration of the essential oils increases (Figure 2).

Figure 1. Allelopathic potential of S. aromaticum, S. malaccense and S. samarangense essential oils on seed germination of Zea mays after 7 days.


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Figure 2. Phytotoxic effects of S. aromaticum, S. malaccense and S. samarangense essential oils on root and shoot length of Zea mays after 7 days.

The insecticidal activity of Syzygium species essential oils was determined using fumigant toxicity against Sitophilus zeamais. Table 3 shows the results of the insecticidal potential of the oils and the control (Permethrin) after 96 h, and was found to be significant and concentration dependent for the different concentrations of the oils. The lethal concentration (LC50) of oils against S. zeamais were 7.68, 39.17 and 27.11 mg/L air, respectively. When compared to Permethrin (LC50 = 7.45) mg/L air. It is perceptible that S. aromaticum, S. malaccense and S. samarangense essential oils has strong toxic activity against S. zeamais, and may be use as fumigant in protecting stored products. However, further studies ought to be performed with other storage weevils in order to ascertain the insecticidal effects of these plants (Figure 1). In addition, the findings of this study are in agreement with other previous reports on biological activities of S. aromaticum essential oils (Table 2).

ACKNOWLEDGMENTS Authors are grateful to Mrs. Musilimat Ogunwande for typesetting of the manuscript.



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CV OF CHAPTER AUTHORS Name: OGUNWANDE ISIAKA AJANI Affiliation: DEPARTMENT OF CHEMISTRY, FACULTY OF SCIENCE, LAGOS STATE UNIVERSITY, PMB 0001, LASU POST OFFICE, OJO, LAGOS, NIGERIA Education: (i) Obafemi Awolowo University, Ile-ife, Nigeria- B.Sc Chemistry (ii) University of Ibadan, Nigeria, M.Sc and Ph.D Organic Chemistry Address: Natural Products Research Unit, Department of Chemistry, Faculty of Science, Lagos State University, PMB 0001, Ojo, Lagos, Nigeria. E-mail: isiaka.ogunwande@ lasu.edu.ng; Tel. + 234 8059929304 Research and Professional Experience:   

Associate Editor, Journal of Medicinal Plant Research, 2010 till date Member, Editorial Board, Der Chemica Sinica, 2010 till date Reviewer-(to)

Journal of Essential Oil-Bearing Plants (JEOBP), Journal of Herbs, Spices and Medicinal Plant (JHSMP), Environmental Pollution (EP), Flavour and Fragrance Journal (FFJ), African Journal of Environmental Science and Technology (AJEST), African Journal of Traditional and Complementary Medicine (AJTCAM), African Journal of Pure and Applied Chemistry


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(AJPPC, Kenya), African Journal of Biotechnology (AJB), African Journal of Pure and Applied Chemistry (AJPPC), African Journal of Pharmacy and Pharmacology (AJPP), South African Journal of Botany (SAJB), International Journal of Biological and Chemical Sciences (IJBCS), Journal of Medicinal Plant Research (JMPR), Journal of Brazilian Chemical Society (JBCS), International Journal of Medicinal and Medical Sciences (IJMMS), Food and Chemical Toxicology (FCT), European Journal of Chemistry (EUJC), European Journal of Medicinal Plants (EJMP), Natural Product Research (NPR), Record of Natural Products (RNP), Natural Product Communications (NPC) and Journal of the Serbian Chemical Society (JSCS), African Journal of Agricultural Research (AJGR) and International Journal of Microbiology Research and Review (IJMRR). Professional Appointments: External Examiner- PhD Thesis Evaluation, Department of Chemistry and Department of Biochemistry, University of Karachi, Pakistan Honors: 1) Conference of All Muslim Organizations (CAMO, Ibadan) Scholarship for Doctoral Studies, 1998-2000 2) Third World Academy of Sciences (TWAS) South-South Fellowship, International Centre for Chemical Sciences, University of Karachi, Pakistan, January-March 2002 3) Leverhulme Postdoctoral Fellow, Department of Chemistry, University of Coventry, UK, 2004. 4) Japan Society for Promotion of Science (JSPS) Invitational Fellowship for Foreign Scholars, Institute of Biotechnology, Graduate School of Kyushu University, Fukuoka, Japan, November 2004- November 2006. 5) Chinese Academy of Sciences and Third World Academy of Sciences (CAS-TWAS) Visiting Scholars Fellowship award to the Kunming Institute of Botany, State Key Laboratory of Phytochemistry and Plant Resources, Yunnan, China, 2010




Chapter 6

FROM TERPENOIDS TO AMINES: A CRITICAL REVIEW Arno Behr and Andreas Wintzer Technical University of Dortmund, Emil-Figge-Straße, Dortmund, Germany

ABSTRACT Aliphatic amines are of great significance in the chemical industry. Many ways for synthesising different types of amines are currently known. The production of amine compounds from renewable resources has great potential for commercial use. In this chapter, the authors describe homogeneously catalysed amination reactions of terpenes and terpenoids. The authors illustrate the actual impact of terpene aminations on industrial processes and productions of fine chemicals. At the same time, the authors provide insight into their research on atom economic functionalisations of terpenoids with transition metal catalysis. The structural properties of terpenes and terpenoids and the broad variety of potential starting compounds in combination with sustainable production methods of amines are among the great advantages of terpene chemistry.

INTRODUCTION The importance of organic primary, secondary and tertiary amines for the global supply chain is great. They have endless uses for products used in daily life, and present a plethora of different synthesis routes for creating more specific and valuable products. The significance of these chemicals for mankind is best reflected by ammonia, the most important amine. 170 million tons of ammonia are produced annually. Thereof 70 % is used for fertilizer production, 10 % for nylon manufacture, 7 % for the production of explosives and the last 13 % for different applications like refrigeration. 5-7 million tonnes of ammonia are used for



Corresponding author: [email protected].


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Arno Behr and Andreas Wintzer

the production of amines (excluding the two most important diamines, caprolactam and hexamethylene diamine). [1] The products of amination reactions with terpenes are among the aliphatic amines due to their typical aliphatic structure. Aliphatic amines are devided into four different classes: short alkylamines (C1 to C7 chain), fatty amines (C8 to C24 chain), di- and polyamines, as well as cyclic compounds. [2] Amines such as hydrazine or chloramine are inorganic examples. However, the authors‘ observations focus on the amination of monoterpenes and terpenoids, which usually have a carbon number of ten to thirteen. In combination with different amine substrates with varying substitutions, ranging from carbon number C0 (NH3) to C8 (di-nbutylamine), it is clear that aliphatic fatty amines are relevant end products. The significance of aliphatic fatty amines is strongly reflected in their possible applications, and the subsequent chemistry. To demonstrate their crucial importance, the most common applications for industrially produced amines are listed here: plant growth control [3], flotation agents [4], corrosion inhibitors, use in pharmaceuticals, anti-cancer-agents, cosmetics and sanitary articles, azine or azo dyes, in the form of hydrazines in agrochemicals (about 5∙105 t) or blowing agents (about 4∙105 t), in water treatment, or as rocket fuel [1], as asphalt emulsifier, fabric softener or lubricant [5]. Amines with no direct uses are further processed to create valuable products that often belong to the group of fine chemicals. The possible chemistry of aliphatic amines is manifold: aside from their reactions with oxo compounds such as carboxylic acids and their derivatives, carbonyls, carbon dioxide, carbon disulfide and epoxides, the formation of isocyanates and ureas is important in the production of herbizides and polyurethanes. The reaction with acrylonitrile is also important for the synthesis of polyamines. Aliphatic amines can be oxidised by hydrogen peroxide or nitrous acid, and dealkylated when heated. [5] In addition, the growing interest in ionic liquids causes increased capacities of quaternary ammonium compounds, built by protonation or the alkylation of aliphatic amines. [2]

Figure 1. An overview of the various synthesis methods of aliphatic amines.


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From Terpenoids to Amines: A Critical Review

The two most important examples for cycloaliphatic amines and diamines are caprolactam and hexamethylene diamine, which are used for polymer synthesis and are therefore produced in high amounts. Up to now, commercial processes for caprolactam synthesis are based on toluene or benzene, 90 % of which are produced by the well-known cyclohexanone route. New methods of producing caprolactam have been investigated with the goal of maximising atom economy and cost reduction. [6] Hexamethylene diamine, the precursor for nylon 6.6, is based on the hydrocyanation of butadiene (USA, Europe) or the dimerisation of acrylonitrile (Japan, China). The United States, South America, Western Europe, Japan and China produced a combined total of 1,58∙106 t of it in 2010. [7] Typically, the following preparation methods are used for the synthesis of aliphatic amines: synthesis from alcohols via amino-dehydroxylation, synthesis from carbonyl groups, synthesis from nitriles, the Ritter reaction, synthesis from alkyl halides, synthesis from nitroalkanes, amination of alkenes, synthesis from amides, via rearrangement (Curtius reaction), synthesis from isocyanates and the synthesis from allylic or styrenic bonds (Figure 1). [1] Table 1. General processes for the synthesis of amines [2] Process Amination of lower alcohols C1 Amination of lower alcohols C2C6 Amination of higher alcohols C8C15 Amination of alkanol amines Reductive amination of aldehydes and ketones Hydrogenation of nitriles

Procedure Gas phase without H2

T [°C] 250-450

p [bar] 10-40

Catalyst Mixed oxides from Si, Al, Ti, W, Zeolites On the base of Ni, Co, Fe, Cu, Re, Ru

Product(s) Methylamines

Gas or liquid phase with H2

150-250

30-80

Liquid phase with H2

180-250

180-300

On the base of Ni, Co, Fe, Cu, Re, Ru

Fatty amines


140-230

150-250

Ethyleneamines


50-150

10-200

On the base of Ni, Co, Fe, Cu, Re, Ru On the base of Ni, Co, Cu, Pd, Pt


50-150

20-250

On the base of Ni, Co, Fe, Rh, Ru

Hydroamination of alkenes

Supercritical

250-350

200-300

Zeolites

Alkylamines C2C6

Fatty amines, special amines, dimethylcyclohexylamine Fatty amines, hexamethylene diamine, dimethylaminopropylamine, isophorone diamine tert-Butylamine


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In terms of the terpenoid structure, the amination of alkenes and synthesis from carbonyl groups are of special interest in this chapter and research report. There are many examples for industrial amine synthesis from molecules that have already been functionalised. The goal was to achieve a more sustainable process by combining the reaction steps in one pot, which would entail the synthesis of useful amines from unfunctionalised natural resources such as terpenes. Typical commercial-scale production methods with reaction conditions and catalysts used are listed in Table 1. [2] These examples show the importance of process alternatives, using natural resources as starting materials. Although the importance of terpenes in the chemical industry compared to fats and oils is relatively low [8], they can be used and are already used for the commercial synthesis of important industrial products today. The authors will provide examples for the commercial use of terpenes in existing processes and attempt to forecast their use in the future by describing atom economic syntheses in terms of the current research of the amination of terpenes. The homogeneously catalysed hydroamination (HA), hydroaminomethylation (HAM) and reductive amination (RA) of terpenoids will be presented in detail.

Hydroamination of Terpenoids The hydroamination (HA, Figure 2) is the most important step for terpenoid functionalisation. This reaction is known for various monoterpenes, but it should be mentioned that the hydroamination of myrcene, an acyclic monoterpene, is of great significance in terms of the industrial chemistry of monoterpenes in general. There are a plenty of product molecules that are crucially important for the chemical industry that can be produced from the hydroamination product of myrcene: (-)-menthol, (+)-citronellal, (+)-citronellol, geraniol, hydroxycitronellol, linalool, myrcenol and nerol (Figure 3). [9] In terms of its chemical meaning, the hydroamination reaction is a nucleophilic addition reaction. As a consequence, the double bonds of the olefinic terpene molecules usually undergo a 1,2-addition with an amine substrate. In the case of a 1,3-diene such as myrcene as a starting material, a 1,4-addition is the favoured reaction path, which is demonstrated in Figure 4 with diethylamine as substrate. The most-used catalyst systems for hydroamination consist of alkali metals or transition metals in combination with a chiral ligand. Photoinduced procedures have been described as well. [10] Diethylamine and morpholine are typical amine substrates in metal catalysed hydroaminations.

Figure 2. Reaction scheme for a typical alkene hydroamination.



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Figure 3. Chemistry of N,N-diethylnerylamine, the product from the hydroamination of myrcene with diethylamine.

Figure 4. Reaction scheme for the hydroamination of myrcene with diethylamine.

The hydroamination of myrcene was first reported in 1974, when Fujita et al. transformed myrcene with diethylamine in presence of 33.3 mol% of sodium and 16.7 mol% of naphthalene at room temperature. [11] Within one hour, an amine yield of 53 % and 80 % selectivity of the geranylamine was reached. Chalk and Magennis also used sodium as an active catalyst, but in a far lower concentration of 1.7 mol%. [12] With different dialkylamines and at a reaction temperature of 50 °C, this resulted in amine yields of up to 83 % within four hours. Aside from sodium, lithium is another good hydroamination catalyst. Different research has been published using lithium in form of butyl lithium [13], or elementary [12, 13a, 14]. In all cases, the activity was similar to sodium catalysis, reaching a maximum turnover number (TON) of 49. The typical model substrate is diethylamine, but piperidine or morpholine have also been tested. In comparison to the alkali metal catalysis, transition metal catalysis clearly showed higher activation. Using rhodium as catalyst in an aqueous biphasic solvent system, myrcene was hydroaminated with morpholine with less yield (59 %) and selectivity (53 % geranylamine) but higher TON (590), demonstrating the


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improved activity of this system. [15] In addition to rhodium, palladium catalysis is used for myrcene hydroamination, which will be discussed in detail later on. [16] To the best of our knowledge, these are the only known transition metal catalysed hydroaminations of myrcene. The importance of the resulting amines is associated with the various means of further functionalisation. In amine function, the molecule provides access to reaction pathways, which lead to basic flavor and fragrance compounds. For example, isomerisation with following hydrolysis results in (+)-citronellal, a fragrance compound which is used in perfumes or insect repellent. Further functionalisation in form of hydrogenation results in (+)citronellol, which is found in many deodorants. But the main application of (+)-citronellal is the industrial synthesis of (-)-menthol via the Takasago process (Figure 5). [9] The applications of (-)-menthol are manifold, but mostly it is used as an ingredient in fragrances or in medicine and pharmacology due to its cooling and anesthetic properties. The important isomerisation step of diethylamine with rhodium(I) and (+)- or (-)-2,2‘bis(diphenylphosphino)-1,1‘-binaphthyl (BINAP) in order to create the stereocentre was developed by Noyori et al. [17] Further steps include the cyclisation of (+)-citronellal with ZnBr2 to (-)-isopulegol and hydrogenation to (-)-menthol. Further monoterpene hydroaminations are known from limonene, -pinene and citronellyl acetate. The photoinduced synthesis method of Beauchemin et al. used methyl benzoate as a sensitizer. [10] Only the internal double bond of limonene reacts with imidazole under irradiation with 254 nm and trifluoric acid to the cyclic amine product (30 % yield). As a side reaction, the isomerisation of the internal double bond occurs, resulting in an exocyclic double bond. Isomerisations and rearrangements are known to play a great role in terpene funtionalisations. Nájera and Giner have described the silver-catalysed hydroamination of pinene with 4-toluenesulfonamide (TsNH2). [18] Compared to the other substrates that have been investigated, the hydroamination of -pinene is peculiar. Under the given reaction conditions, hydroamination only occured in neat -pinene without a solvent, and led to a rearrangemant of the scaffold, resulting in N-tosyl-isobornylamine (Figure 6). Within 14 hours at 85 °C and with a molar ratio of alkene/amine of 4:1, 98 % of the product was formed using 5 mol% of silver trifluoromethanesulfonate (silver triflate, AgOTf). With 1 mol% of trifluoromethanesulfonic acid (triflic acid, HOTf), the yield was 66 % under the same reaction conditions. The yields are based on TsNH2 and demonstrate the potential of silver-catalysis compared to gold-catalysed hydroaminations which had been used up to then. [19] In this case, citronellyl acetate was used as starting material. Surprisingly, the internal double bond was aminated with 4-toluenesulfonamide (Figure 6). The hydroamination of myrcene is very well known as a component of the Takasago process and as means of starting functionalisation for a broad variety of fine chemicals. In the industrial process, the hydroamination with diethylamine is catalysed by lithium, which is cheap but also offers relatively low activities, in other words turnover frequencies (TOF) up to 12 h-1. [20] Our group investigated a palladium catalysed alternative with the hydroamination of myrcene with morpholine (Figure 7). [16c] The advantage of transition metal catalysis compared to alkali metals is the high activity, whereas the cost effectiveness is very low due to the high cost of noble metals. Therefore, the reaction was not only realised under single phase conditions, but catalyst recycling concepts were applied and investigated as well.



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Figure 5. Reaction scheme of the Takasago process [9].

Figure 6. Silver and gold catalysed hydroaminations of -pinene and citronellyl acetate with 4toluenesulfonamide.

Figure 7. Reaction scheme of the hydroamination of myrcene with morpholine.

A very promising alternative is the concept of thermomorphic multiphase solvent systems (TMS). This temperature-regulated solvent system was developed by Behr and has already been applied in other homogeneously catalysed reactions [21] and also reviewed [22].


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Figure 8. Principle of TMS systems for the separation of the catalyst from the product.

Figure 9. Impact of catalyst and substrate concentration of the hydroamination selectivity.

The principle of TMS is simple: A selected combination of two or three solvents is biphasic at room temperature (Figure 8). The catalyst is solved in the polar phase, and the starting material in the apolar phase. At reaction temperature, the system becomes a homogeneous phase, providing a good interaction between the substrates and the catalyst. At the end of the reaction the system is cooled down and shifts back to a biphasic system. The catalyst remains in the polar phase while the product is found in the apolar phase. The catalyst system for single phase hydroamination consists of 0.2 mol% of Pd catalyst and a phosphine catalyst at a ratio of Pd/P = 1:32. The variable parameters are the precursor, phosphine ligand, Pd/P ratio, temperature, solvent and substrate concentration. An important side reaction in this hydroamination reaction was the telomerisation of myrcene with morpholine. The telomerisation can be described as the dimerisation of the starting material with the subsequent addition of the substrate. The selectivity control between these two reactions was a crucial part of this work. In conclusion, the best catalyst system identified for hydroamination consists of palladium (II) trifluoroacetate [Pd(CF3CO2)2] as the precursor and the bidentate phosphine ligand bis(diphenylphosphino)butane (DPPB). The proper Pd/P ratio



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was the most decisive factor in terms of selectivity (Table 2). The ligand sphere around the active metal centre has a great impact on the coordination possibilities of the reacting components. With high Pd/P ratios such as 1:32 and 1:16, there is no coordination sphere for two myrcene molecules and one morpholine molecule. By decreasing the ratio to 1:4, in this case for the system Pd(hfacac)2/DPPB, more space on the catalytic centre resulted, facilitating the formation of telomers (59 %). Further factors that result in higher telomerisation can be found in the substrate concentration of the solvent (Figure 9) and in the catalyst concentration (Figure 10). However, with decreasing catalyst concentration telomerisation occurs. This is achieved by raising the substrate amount when the amount of catalyst remains constant, and by increasing the substrate concentration at the same time. With 0.05 mol% of catalyst, a substrate concentration of 0.68 mol∙l-1, 73 % of telomer products was observed. Due to the low catalyst amount in combination with the higher substrate amount of the same volume, the reaction of two myrcene molecules and one morpholine molecule is favorised, even at Pd/P ratio 1:8. At a constant catalyst concentration of 0.2 mol% (Figure 10) the variation of the substrate concentration had no effect, due to the relatively high amount of active catalyst. This indicated that when catalyst concentration remains at a constant level, very high space-time yields are achieved. Table 2. Comparison of different catalyst systems in the palladium catalysed hydroamination of myrcene Hydroamination Telomerisation yield yield Pd(CF3CO2)2/DPPB 1:32 43 % 43 % 0% Pd(CF3CO2)2/DPPB 1:8 96 % 92 % 4% Pd(hfacac)2/DPPB 1:16 39 % 39 % 0% Pd(hfacac)2/DPPB 1:4 77 % 18 % 59 % Conditions: 0.2 mol% precursor, T = 100 °C, t = 5 h, 500 rpm, c (myrcene) = 0.17 mol·L -1, myrcene:morpholine 1:1, 30 mL toluene, 5 bar argon. Catalyst system

Pd/P ratio

Conversion

Table 3. Comparison of two different TMS systems in the hydroamination of myrcene with morpholine Molar Hydroamination Telomerisation Leaching solvent Conversion yield yield Pd/P [ppm] ratio ACN/heptane 40:60 75 % 71 % 4% 4/