SECONDARY METABOLITE PRODUCTION IN IN

Оригинален научен труд Original Scientific Article

SECONDARY METABOLITE PRODUCTION IN IN VITRO SHOOTS OF MARIGOLD (Calendula officinalis L.) Sonja GADZOVSKA1, Valentina PAVLOVA2, Maja NASTESKA3, Aleksandra NESKOSKA3 & Mirko SPASENOSKI1 Department of Plant Physiology, Institute of Biology, Faculty of Natural Sciences and Mathematics, University “Ss. Cyril and Methodius”, P.O. Box 162, 1000 Skopje, Macedonia. 2 Department of Analytical Chemistry, Institute of Chemistry, Faculty of Natural Sciences and Mathematics, University “Ss. Cyril and Methodius”, P.O. Box 162, 1000 Skopje, Macedonia. 3 Biology Students Research Society (BSRS), Institute of Biology, Faculty of Natural Sciences and Mathematics, University “Ss. Cyril and Methodius”, P.O. Box 162, 1000 Skopje, Macedonia. 1

ABSTRACT Gadzovska S���������������������������������������������������������������������������������������� ., Pavlova V., Nasteska M., Neskoska A., Spasenoski M. (2008): Secondary metabolite pro� duction in in vitro shoots of marigold (Calendula officinalis L.). Proceedings of the III Congress of Ecologists of the Republic of Macedonia with International Participation, 06-09.10.2007, Struga. Special issues of Macedonian Ecological Society, Vol. 8, Skopje. The aim of the present work was to determine whether secondary metabolite production could be enhanced in Calendula in vitro cultures by exogenous application of phytohormones. This study has been focused on two ar� eas: (a) consequences of phytohormonal treatments on biomass production of in vitro cultures; (b) relationships be� tween growth regulator treatments and in vitro culture ability to produce phenylpropanoids (phenolic compounds, flavonoid and anthocyanins). Multiplication of apical segments from sterile germinated seedlings was obtained on solid MS/B5 culture medium in the presence of cytokinin N6-benzyladenine (0.5 mg⋅L-1 BA). ��������������������� Regenerative po� tential of shoots was assessed on medium supplemented with auxins: 2,4-dichlorophenoxyacetic acid (1.0 mg⋅L-1 2,4-D) and α-naphthaleneacetic acid (0.1 mg⋅L-1 NAA). Phytohormones stimulated growth after 7 days in Calendula shoots. Shoot cultures did not give clear cut answer to exogenously applied phytohormones. C. officinalis in vitro shoots could be proposed as a source for rapid production of pharmacological compounds of interest.

ИЗВОД Гаџовска С., Павлова В., Настеска М., Нескоска А., Спасеноски М. (2008): Secondary metabolite production in in vitro shoots of marigold (Calendula officinalis L.). Зборник на трудови од III Конгрес на еко� лозите на Македонија со меѓународно учество, 06-09.10.2007, Струга. Посебни изданија на Македонското еколошко друштво, Кн. 8, Скопје. Во оваа студија беше испитано влијанието на егзогено аплицираните фитохормони врз продукција� та на секундарни метаболити кај in vitro културите од невен (C. officinalis L). Во текот на истражувањата беа поставени две цели: (а) да се испита влијанието на фитохормоните врз продукцијата на биомаса кај in vitro културите; (б) да се испита корелацијата помеѓу концентрацијата на фитохормоните и продукцијата на фе� нилпропаноиди. За иницијација на мултиплицирани изданоци, од стерилно из‘ртеното семе беа изолира� ни апикални сегменти кои беа култивирани на MS/B5 медиум во присуство на цитокиниот N6-бензиладенин (0.5 mg⋅L-1 BA). Регенеративниот потенцијал на изданоците беше испитан во присуство на ауксините: 2,4дихлорофеноксиоцетна киселина (1.0 mg⋅L-1 2,4-D) и α-нафтилоцетна киселина (0.1 mg⋅L-1 NAA). Егзогено аплицираните фитохормони го стимулираа растот и развитокот на изданоците во in vitro услови. Мултип� лицираните изданоци не покажаа значајни промени во продукцијата на секундарни метаболити зависно од концентрацијата на фитохормони. In vitro културите од C. officinalis би можеле да се користат во производ� ството на секундарни метаболити кои имаат примена во фармацевтската индустрија.

al and homeopathic medicine it has been used for skin complaints, wounds and burns, conjunctivi� tis and poor eyesight, menstrual irregularities, var� icose veins, hemorrhoids, duodenal ulcers, etc. Ca-

Introduction Marigold (Calendula officinalis L.) is an an� nual plant of ancient medicinal repute. In tradition�

54

Secondary metabolite production in in vitro shoots of marigold (Calendula officinalis L.) lendula has a flower consists of orange-yellow pet� als which are used for medicinal purposes to re� duce inflammation and control bleeding. Herbal preparations are used to treat stomach ulcers, liver complaints, conjunctivitis and wounds (Newall et al., 1996). Marigold grows as a wild and common garden plant throughout Europe and North Amer� ica. The yellow or golden-orange flowers of mari� gold are used as spice, tea and medicine. They may be used as fresh or dried, and can be made into tea, tinctures, ointments and creams. Marigold is a com� mon flower, well known for its potential against skin problems. The plant and its extracts present sever� al properties important to phytotherapy as well as to dermatology and cosmetology. The extract obtained from the flowers can stimulate bile production, pres� ents anti-fungi activity and is also used for digest in� fection and for gastric and duodenal ulcer treatment. The therapeutic characteristic of the marigold ex� tract is partially due to the terpene content, especial� ly glycol triterpenoids, with sedative action against ulcer (Bako et al., 2002). It has been indicated that the marigold extract does not affect the cardiovas� cular system, the liver or kidney functions and has been shown anti-HIV activity (Campos et al., 2005). The most important components of the extract are the triterpenoids, especially the faradiol with anti-in� flammatory activity (Kalvatchev et al., 1997). Oth� er components in the chemical composition of the marigold extract are flavonoids, essential oil and sesquiterpenes. The pharmacological activity of marigold is related to the content of several classes of second� ary metabolites such as essential oils, flavonoids, sterols, carotenoids, tannins, saponins, triterpene al� cohols, polysaccharides, a bitter principle, mucilage, and resin (Vidal-Ollivier et al., 1989). Bilia et al., (2001) found that marigold flowers contain rutin, isoquercitrin, quercetin-3-O-rutinosylrhamnoside, isorhamnetin-3-Orutinosylrhamnoside, isorhamne� tin-3-O-glucosylglucoside, and isorhamnetin-3-Oglucoside. Recently, in vitro culture systems have be� come worth studying as a useful alternative because of increased demand by pharmaceutical industry and unequal quality of products caused by environmen� tal factors. Moreover, in vitro cultures play an im� portant role in the studies of plant secondary metab� olism. Phytohormone concentrations are often a cru� cial factor of secondary metabolite accumulation in in vitro cultures. The type and concentration of aux� in or cytokinin or the auxin/cytokinin ratio could al� ter growth and secondary metabolite production in cultured plant cells. A large number of in vitro cul� tures cultivated on medium with phytohormones produced specific medicinal compounds at a rate similar or superior to that of intact plants (Vanisree et al., 2004). The response of cells, tissues, and or�

gans in vitro to exogenously applied phytohormones can vary with cultural conditions, the type of ex� plant, and the genotype. Therefore, it is necessary to find an optimal concentration of phytohormones for growth and secondary metabolite production of dif� ferent in vitro cultures. The objective of our research was to develop an efficient protocol for micropropagation to improve secondary metabolite productions in C. officinalis in vitro cultures. The consequences of growth regulator concentrations according to the developmental stages of in vitro cultures have been examined. To determine whether secondary metabolite productions could be enhanced by phytohormone supplementation, in vitro cultures have been exposed to exogenous appli� cation of these chemicals. The effects of cytokinins on secondary metabolite productions in Calendula in vitro shoots cultures have been examined. The main goal of the research was to summarize the influence of cytokinin N6-benzyladenine on ��������������������� particular second� ary metabolites as phenolic compounds, flavonoids and anthocyanins.

Material and methods Plant material Seeds of C. officinalis L. were washed over night, air dried and surface sterilized with 1 % NaO� Cl for 20 minutes, rinsed 3 times in sterile deionized water and germinated on the MS/B5 medium (Mu� rashige and Skoog, 1962; Gamborg, 1968) without growth regulators. Nutrition medium contained MS salt mixture, B5 vitamin solution, 3 % sucrose and 0.7 % agar. The medium was adjusted to pH 5.6 be� fore autoclaving (20 minutes at 120°C). Apical segments, cotyledons and hypocotyls were isolated from 2 weeks old in vitro grown plants. They were used as a source of explants for establish� ment of tissue cultures. The explants were cultured on MS/B5 medium with 100 mg·L-1 myo-inositol, 200 mg·L-1 casein enzymatic hydrolisate and supple� mented with different concentrations of cytokinin N6-benzyladenine (0.5 mg⋅L-1 BA) or auxins: 2,4dichlorophenoxyacetic acid (1.0 mg⋅L-1 2,4-D) and α-naphthaleneacetic acid (0.1 mg⋅L-1 NAA). Mul� tiple shoots formation was developed on medium with 0.5 mg·L-1 BA. Tissue cultures were maintained in a growth chamber at 26±1°C under a photoperi� od of 16-h light and 8 h dark, irradiance of 50 µmol m-2 s-1 and 60% relative humidity. The in vitro cul� tures were harvested by vacuum filtration on Days 7, 14, 21, 28 and 35 of culture, weighted for growth analysis, frozen in liquid nitrogen or lyophilized and stored at -80°C, until analysis. Phenolic compounds assay Phenolic quantification was performed as de� scribed by Torti et al., (1995). The method used to de�

Proceedings of the III Congress of Ecologists of Macedonia

55

Sonja GADZOVSKA et al. termine total soluble phenolic content was based on the reduction of phospho-molybdene/phospho-tung� state present in the Folin–Ciocalteau reagent. Phe� nolics were extracted from air dried powdered plant material (0.2-0.5 g) with 80 % (v/v) methanol for 1 hour at 4ºC. After centrifugation (10 min at 3000 rpm), supernatant was used for quantification of phenolic compounds. Total phenol content was de� termined as follows: methanolic extract was mixed with Folin-Ciocalteau reagent (Carlo Erba Reagenti, Rodano, Italy) and 0.7 M Na2CO3. Samples were in� cubated for 5 min at 50ºC and then cooled 5 min at room temperature. Absorbance was measured spec� ����� trophotometrically at 765 nm. The concentration of total phenolic compounds was calculated using (+)-catechin (0-10 mg⋅mL-1) as a standard.

anidin-3-glucoside (ε530=34300 M-1⋅cm-1) in acidic methanol.

Flavonoids assay Flavonoid contents were determined in methanolic extracts with the method described by Markham (1993). The extracts were filtered through Sep-pack C18 cartridges to exclude chlorophyll and carotenoid pigments. S�������������������������� pectrophotometric��������� measure� ments of the absorbance were made at 360 nm����� .���� Mo� lar extinction coefficient of quercetin (ε360=13.6 mM1 ⋅cm-1) was used to determine total flavonoid con� tents.

In vitro culture of C. officinalis L. Surface sterilized C. officinalis seeds pro� duced normal seedlings with almost 100 % germi� nation on MS/B5 medium without hormones. After 2-3 weeks, in vitro grown seedlings produced 1012 nodes (Fig. 1). Apical segments with 2-4 leaves were isolated as primary explants and cultivated on MS/B5 medium supplemented with 0.1 to 5.0 mg·L-1 BA (Fig. 2). After 20-30 days of culture, on medium with 0.5 mg·L-1 BA, multiple shoots were obtained (Fig. 3).

Anthocyanin assay Anthocyanin determination was performed as described by Giusti et al., (1999). Anthocyanins were extracted from freeze-dried lyophilized and powdered plant material (0.2-0.5 g) with 2 mL solu� tion of 1% HCl/CH3OH (15/85, v/v) ultrasonicated for 60 min at 4°C and than centrifugated at 20 000 g for 30 min. The absorbance of supernatant was mea� sured at 530 nm. The anthocyanin content was cal� culated using the molar extinction coefficient of cy�

Statistical analyses The experiments were independently repeat� ed twice under the same conditions and all analy� ses were performed in triplicate. Error bars of graphs show the standard error of mean value (±SE). The statistical analyses were performed with the SPSS statistical software program (SPSS version 11.0.1 PC, USA, IL). Means were expressed with their standard error and compared by one-way ANOVA (GML procedure). All statistical tests were consid� ered significant at P ≤ 0.05.

Results

Secondary metabolite production in C. officinalis L. shoots The objective of the experiment was to ex� amine the influence of cytokinin N6-benzyladenine (0.5 mg⋅L-1 BA) on secondary metabolite production in marigold in vitro shoots. Cytokinin BA did not in� fluence phenolic contents in in vitro shoots (Fig. 4). Contents of phenols were not significantly different compared with those in control shoots. From Day 7 to

Fig. 1. Germinated seedlings from C. officinalis L. on medium without phytohormones.

56

Зборник на трудови од III Конгрес на еколозите од Македонија

Secondary metabolite production in in vitro shoots of marigold (Calendula officinalis L.)

Fig. 2. Isolated apical segments from C. officinalis L. sterile germinated seedlings.

Fig. 3. Shoot formations and multiplication of C. officinalis L. apical segments. Day 28, phenolic contents in in vitro shoots were from 8.5 to 9.8 mg·g-1. During the treatment non-significant positive correlation (r = 0.32)���������������������� between ��������������������� phenolic con� tents and days of cultivation were noted (Fig. 4). Flavonoid contents were higher on Day 7

and Day 14 but after decrease compared with con� trol levels (Fig. 5). Flavonoid production in in vitro shoots from Day 21 to Day 35 (2.1-2.5 mol·g-1) was lower compared with Day 7 and Day 14 (6.26.4 mol·g-1). Linear negative correlation (r=-0.86,

Proceedings of the III Congress of Ecologists of Macedonia

57

Sonja GADZOVSKA et al.

Phenolic contents [mg·g -1]

p