Volatile metabolite profiling of ten Hedychium species

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Industrial Crops & Products 126 (2018) 135–142

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Volatile metabolite profiling of ten Hedychium species by gas chromatography mass spectrometry coupled to chemometrics

T

Asit Raya, Sudipta Jenab, Basudeba Karb, Ambika Sahoob, Pratap Chandra Pandac, ⁎ ⁎ Sanghamitra Nayakb, , Namita Mahapatraa, a

Regional Medical Research Centre (Indian Council of Medical Research), Chandrasekharpur, Bhubaneswar, Odisha, India Centre for Biotechnology, Siksha O Anusandhan (Deemed to be University), Bhubaneswar, Odisha, India c Taxonomy and Conservation Division, Regional Plant Resource Centre, Bhubaneswar, Odisha, India b

ARTICLE INFO

ABSTRACT

Keywords: Antioxidant Essential oil Gas chromatography Hedychium

The Hedychium genus (Zingiberaceae) produces a plethora of secondary metabolites that include terpenoids, some of which are known for their aromatic properties. The volatile compositions of several members of this genus have not been chemically defined, as many species are endemic to remote ecosystems. In the present research, the essential oil composition of ten Hedychium species (H. gardnerianum, H. flavescens, H. thyrisiforme, H. flavum, H. ellipticum, H. aurantiacum, H. gracile, H. greenii, H. spicatum, and H. coronarium) was studied by gas chromatography flame ionization detector (GC-FID) and gas chromatography mass spectrometry (GCeMS). The essential oil yield obtained by hydrodistillation varied from 0.05 (%v/w) in H. greenii to 0.75 (%v/w) in H. ellipticum and H. thyrsiforme, respectively. A total of 56 constituents representing 75.93–99.66% of volatile constituents were characterized in the essential oil of 10 Hedychium species. The predominant components were β-pinene in H. gardnerianum, H. gracile, H. thyrsiforme, H. flavum and H. flavescens; bornyl acetate in H. greenii; humulene epoxide I in H. aurantiacum; 1,8-cineole in H. spicatum and H. coronarium. Agglomerative Hierarchical Clustering (AHC) and Principal component analysis (PCA) based on essential oil compositions grouped Hedychium species into three different clusters. In view of the ambiguity existing among different Hedychium species, the developed chemical fingerprint in the present study would assume great significance in identification and classification of Hedychium species.

1. Introduction

documented pharmacological activities such as antimicrobial, antioxidant, analgesic, anti-inflammatory, cercaricidal, and pediculicidal activity (Warren and Peters, 1968; Jadhav et al., 2007; Joshi et al., 2008; Joy et al., 2007; Sabulal et al., 2007; Shrotriya et al., 2007; Ray et al., 2018). Some species are grown for their edible flowers (He, 2000). The essential oil composition varies with inter and intraspecific variation, ontogenic variation, topography, soil, maturity, harvest time, storage and extraction methods (Raut and Karuppayil, 2014). Hence, it is required that comparative study of essential oil constituent among diff ;erent species should be carried out under identical conditions. Similar comparative analysis of essential oil components among different Curcuma species collected from one place has been carried out under similar conditions studies previously by many researchers (Angel et al., 2014; Zhang et al., 2017). Species level delineation of vegetatively propagating Hedychium species is complex due to the short-lived flowers and owing to the fact that flowering occurs in monsoon season

The genus Hedychium (Zingiberaceae) comprises approximately 80 species and is widely distributed in India, China and South East Asia (Ray et al., 2017). The genus Hedychium is represented by 44 taxa, of which 17 are endemic to North Eastern India (Jain and Prakash, 1995). Hedychium species are commonly known as ginger lilies and are herbs with thick, fleshy and aromatic rhizomes. Hedychium species are extensively cultivated due to the perfumery fragrances of their essential oil (Hartati et al., 2014). The aerial stems of Hedychium species are used as a raw material in manufacturing paper (Gao et al., 2008). The extract made from the rhizomes of H. coronarium is used as an eye drops in the event of an infection (Lim, 2014). The Hedychium species are used in the traditional medicine system for treating asthma, gastric and bronchitis (Hartati et al., 2014). The therapeutic properties of the Hedychium species are mainly because of the presence of essential oil in the rhizomes. Essential oil of Hedychium species possess several well



Corresponding authors. E-mail addresses: [email protected] (S. Nayak), [email protected] (N. Mahapatra).

https://doi.org/10.1016/j.indcrop.2018.10.012 Received 17 June 2018; Received in revised form 1 October 2018; Accepted 4 October 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.

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Table 1 Flower description, common name, rhizome color and color of essential oil of 10 Hedychium species used in the study. S.No

Hedychium species

Common name

Flower description

Oil color

Rhizome color

1

ellipticum Buch.-Ham. ex

Shaving Brush Ginger

White transparent

Brown

gardnerianum Wall. ex

Kahili ginger

Cream flowers arranged in a umbelliforme cyme inflorescence with orange stamens Soft yellow flowers with showy red stamens

Dark yellow

Light brown

3 4 5 6 7 8

Hedychium Smith. Hedychium Spreng. Hedychium Hedychium Hedychium Hedychium Hedychium Hedychium

greenii W. W. Smith. flavum Roxb. gracile Roxb. thyrisiforme Smith. flavescens Carey ex Rosc. aurantiacum Wall.

Brown Pale yellow Light yellow Light brown Pale yellow Brown

Hedychium spicatum Smith. Hedychium coronarium J. Koenig

Scarlet red butterfly shaped flowers Cream yellow flowers with yellow stamens Greenish-white flowers with long red stamens White flowers from a green pincushion-like bract Sulfur yellow flowers with yellow stamens Bright orange-red flowers, many in a long upright spike up with long-projecting stamens White flowers with an orange-red base White butterfly like flower in dense elliptical racemes

Pale yellow Light yellow White transparent White transparent Dark brown Pale yellow

9 10

Red ginger Yellow Butterfly ginger Dainty Ginger Lily Pincushion ginger Yellow ginger Orange bottlebrush ginger Spiked ginger lily White ginger lily

White transparent Pale yellow

Light brown Light yellow

2

determined to analyze the variation in chemical composition among ten Hedychium species (Hedychium gardnerianum, Hedychium flavescens, Hedychium thyrisiforme, Hedychium flavum, Hedychium ellipticum, Hedychium aurantiacum, Hedychium gracile, Hedychium greenii, Hedychium spicatum, and Hedychium coronarium) cultivated under similar environmental conditions. In order to classify the relationship between Hedychium species, Agglomerative hierarchical clustering and principal component analysis were carried out based on the essential oil compositions of GC–MS. The data thus obtained were used to determine the Indian Hedychium chemotypes. 2. Material and methodology 2.1. Plant material and isolation of essential oil

Fig. 1. Essential oil yield (%v/w) of different Hedychium species. Oil yield values with different superscript letters (a–f) shows significant difference at p < 0.05 (Tukey test).

Rhizome parts of ten Hedychium species were collected from Pradhan’s Nursery, Kalimpong, West Bengal, India and were raised in the greenhouse of Centre of Biotechnology, Siksha O Anusandhan University, Bhubaneswar, India (location 20°17′3.45″ N, 85°46′31.14″ E, 75 m above sea level). Rhizomes were harvested for 2 years after planting under the same conditions in the greenhouse for essential oil extraction. Plants authentication and identification was done by a Principal Scientist, Dr. P.C. Panda, and the voucher specimens of these ten Hedychium species were deposited at the Herbarium of Regional Plant Resource Centre, Bhubaneswar, India. Voucher specimen numbers were as follows: Hedychium gardnerianum (No: 10630), Hedychium flavescens (No: 10631), Hedychium thyrisiforme (No: 10632), Hedychium flavum (No: 10633), Hedychium ellipticum (No: 10634), Hedychium aurantiacum (No: 10635), Hedychium gracile (No: 10636), Hedychium greenii (No: 10637), Hedychium spicatum (No: 10638), Hedychium coronarium (No: 10639). Essential oils from freshly crushed rhizomes were extracted by hydrodistillation using a Clevenger-type apparatus for 6 h. Essential oil yield was measured from the ratio of volume of the essential oil to the weight of fresh rhizomes (%v/w). All volatile oils were kept at 4 °C in the refrigerator for further use.

(Basak et al., 2014). Morphological characters like the arrangement of bract, flowers per bract, and color of petals are relative and would not assess the degree of similarity between species (Gao et al., 2008). Selfsterility is a major problem in Hedychium species, as they readily hybridize, thereby leading to the taxonomic confusion (Wood et al., 2000). Cole et al. (2007) recommended that dissimilarity in essential oils constituents can be an important criterion for elucidating phylogenetic relationships in plants species that are not easy to distinguish. Hierarchical clustering constructed on the composition of essential oil constituents can clarify the ambiguity in the classification of Hedychium species. The essential oils of Hedychium species are a complex mixture of volatile compounds mainly composed of terpenes. These include monoterpenes (hydrocarbons and oxygenated) and sesquiterpene (hydrocarbons and oxygenated) compounds (Joshi et al., 2008). Sakhanokho et al. (2013) analyzed the volatile constituents and reported β-pinene, α-pinene, 1,8-cineole, linalool and E-nerolidol as the characteristic constituents of Hedychium species. Most of the reports on Indian Hedychium species are confined to profiling of essential oil of few Hedychium species such as H. coronarium (Joy et al., 2007; Sabulal et al., 2007; Joshi et al., 2008), H. spicatum (Sabulal et al., 2007; Joshi et al., 2008; Verma and Padalia, 2010), H. larsenii (Dan et al., 2005), H. acuminatum (Weyerstahl et al., 1998), H. gardenerianum (Weyerstahl et al., 1995), lacking any comprehensive and comparative study on the diversity of chemical constituents of Hedychium species grown under identical environmental conditions. Thus, the present research was

2.2. Analysis of the essential oil Gas chromatography mass spectrometry (GC–MS) analysis was performed in a Perkin Elmer Gas Chromatograph (Model: Clarus 580) coupled with a SQ8 mass spectrometric detector. The volume of sample injection was 0.1 μl. Elite-5 column (30 m length x 0.25 mm i.d., film thickness 0.25 μm) was used for analysis. The condition used was as follows: 50 °C for 1 min; 50 °C–230 °C at 5 °C/min and held at 230 °C for 5 min, increased to 260 °C at 15 °C/min with a 1 min hold, carrier gas:

136

RIexpi

923 925 934 948 969 976 980 988 1005 1009 1016 1023 1027 1030 1032 1043 1056 1070 1083 1098 1100 1122 1140 1144 1168 1175 1191 1194 1207 1220 1247 1289 1342 1413 1449 1475 1488 1495 1506 1512 1541 1551 1558 1566 1574 1582 1593 1603 1613 1620 1635 1637

S.No

H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 H15 H16 H17 H18 H19 H20 H21 H22 H23 H24 H25 H26 H27 H28 H29 H30 H31 H32 H33 H34 H35 H36 H37 H38 H39 H40 H41 H42 H43 H44 H45 H46 H47 H48 H49 H50 H51 H52

921 924 932 946 969 974 981 988 1002 1008 1014 1022 1024 1026 1032 1044 1054 1068 1086 1095 1098 1122 1141 1145 1165 1174 1186 1194 1204 1215 1249 1287 1346 1408 1452 1475 1489 1492 1505 1513 1546 1548 1559 1561 1577 1582 1595 1608 1618 1622 1630 1638

RIlitii

Tricyclene α-Thujene α-Pinene Camphene Sabinene β-Pinene Methyl heptenone Myrcene α-Phellandrene δ-3-Carene α-Terpinene p-Cymene Limonene 1,8-Cineole cis-β-Ocimene trans-β-Ocimene γ-Terpinene p-Mentha-3,8-diene α-Terpinolene Linalool trans-Sabinene hydrate α-Campholenal Camphor Camphene hydrate Borneol Terpinen-4-ol α-Terpineol Myrtenol p-Cymen-9-ol trans-Carveol Geraniol Bornyl acetate α-Terpinyl acetate β-Caryophyllene α-Humulene γ-Gurjunene β-Selinene δ -Selinene (E,E)-α-Farnesene γ-Cadinene Hedycaryol Elemol Germacrene-B E-Nerolidol Spathulenol Caryophyllene oxide Humulene epoxide I Humulene epoxide II 1,10-di-epi-Cubebol 10-epi-γ-Eudesmol γ-Eudesmol epi-α-Cadinol

Compoundiii

– 2.82 ± 0.01a 1.84 ± 0.02i 0.04 ± 0.01f – 18.46 ± 0.02g 3.33 ± 0.01b 1.61 ± 0.04b 0.88 ± 0.01c – 6.19 ± 0.03a 3.49 ± 0.06d 0.89 ± 0.01f 4.63 ± 0.04e – 0.11 ± 0.01d 35.18 ± 0.03a 0.57 ± 0.01c 1.10 ± 0.02b 0.59 ± 0.02e – 0.81 ± 0.01b 0.53 ± 0.02a 0.03 ± 0.01b 0.06 ± 0.01f 14.89 ± 0.05b 0.87 ± 0.01f 0.11 ± 0.02b 0.27 ± 0.01b 0.08 ± 0.01b – – – 0.23 ± 0.01ef 0.03 ± 0.01f – – – – – – – – – – – – – – – – –

H.el

% Peak area

Table 2 Chemical composition of essential oil of 10 Hedychium species.

– 0.39 ± 0.02de 21.15 ± 0.04a 0.74 ± 0.01d 0.56 ± 0.01d 28.03 ± 0.03d – 0.58 ± 0.01g – – – 11.91 ± 0.03a 1.72 ± 0.02c 0.42 ± 0.01g – – 1.18 ± 0.01g – 0.54 ± 0.01f – – – 0.33 ± 0.01d – 0.82 ± 0.02b 1.05 ± 0.02g 1.30 ± 0.01e – – – – – – 0.82 ± 0.01d 0.33 ± 0.01b 0.70 ± 0.02a – – 0.53 ± 0.02b 1.42 ± 0.03a – 0.53 ± 0.01a – 0.88 ± 0.01a – – – – – – – –

H. ga b

0.96 ± 0.01 – 14.49 ± 0.03b 12.81 ± 0.04a 0.86 ± 0.01c 5.44 ± 0.02h 0.71 ± 0.01c 1.85 ± 0.01a – – 0.08 ± 0.01g 0.44 ± 0.02g 10.55 ± 0.01a 3.36 ± 0.05f 0.78 ± 0.01a 1.05 ± 0.02a 0.37 ± 0.01i – 0.33 ± 0.02g – 0.11 ± 0.02c – – – 1.00 ± 0.04a 0.36 ± 0.01h 0.18 ± 0.01g – – – – 31.32 ± 0.02a – – – 0.66 ± 0.02a 0.80 ± 0.03b 1.39 ± 0.01a – – – – – 0.54 ± 0.01c 0.16 ± 0.01b – – 0.14 ± 0.01c 1.59 ± 0.02a 0.19 ± 0.02d – –

H.gre – 0.34 ± 0.02e 11.17 ± 0.04e 0.61 ± 0.01e – 24.77 ± 0.05f – 0.85 ± 0.02f 2.42 ± 0.04b 0.47 ± 0.01b 0.61 ± 0.01e 5.52 ± 0.05c 2.40 ± 0.01b 8.90 ± 0.09d – – 4.47 ± 0.01e – 0.76 ± 0.01e 6.29 ± 0.02b – – – 0.35 ± 0.01a – 2.47 ± 0.01d 1.54 ± 0.02d – – – – – – 1.18 ± 0.03c 0.21 ± 0.02d – 0.19 ± 0.03c – – 0.22 ± 0.02c – 0.18 ± 0.01c – 0.21 ± 0.01d – 0.16 ± 0.01c 0.11 ± 0.01b 0.26 ± 0.03b – 0.44 ± 0.02c 0.72 ± 0.03a –

H.f a

1.87 ± 0.02 – 1.57 ± 0.01j – – 25.24 ± 0.03e 4.24 ± 0.02a 1.24 ± 0.02d 0.48 ± 0.01d – 4.09 ± 0.03b 2.08 ± 0.02e 0.68 ± 0.02g 7.51 ± 0.03d 0.01 ± 0.01b – 24.62 ± 0.02b 1.43 ± 0.02a 0.86 ± 0.01d 1.34 ± 0.02d – 0.84 ± 0.02b 0.59 ± 0.02b – 0.18 ± 0.02e 14.87 ± 0.03b 1.36 ± 0.02e 0.12 ± 0.02b 0.31 ± 0.02ab 0.29 ± 0.02a – – – 0.22 ± 0.02ef 0.11 ± 0.02e – – 0.08 ± 0.01c – – – – – – – – – – 0.08 ± 0.01d – – 0.27 ± 0.02c

H. gr – 0.72 ± 0.02c 5.85 ± 0.04h – – 41.97 ± 0.05a – 1.56 ± 0.02b – – 3.89 ± 0.03c 0.37 ± 0.01g 1.20 ± 0.02e 3.21 ± 0.02ef – 0.38 ± 0.01c 5.80 ± 0.03c 0.82 ± 0.02b 1.37 ± 0.05a 1.55 ± 0.03c 0.56 ± 0.01a 1.05 ± 0.01a 0.66 ± 0.02a – – 16.60 ± 0.08a 1.30 ± 0.02e 0.17 ± 0.01a 0.33 ± 0.02a – – – – 3.40 ± 0.04a 0.55 ± 0.01a – – – – 0.57 ± 0.03b – – – 0.24 ± 0.02d 0.53 ± 0.05a 0.30 ± 0.02b – – – – – 0.61 ± 0.02a

H.th – 0.44 ± 0.02d 13.17 ± 0.05c 0.80 ± 0.02d – 29.76 ± 0.08c – 1.19 ± 0.02d 2.90 ± 0.01a 0.55 ± 0.01a 0.74 ± 0.02d 6.40 ± 0.07b – 12.80 ± 0.08c – – 5.12 ± 0.06d – 0.95 ± 0.02c 7.30 ± 0.09a – 0.19 ± 0.02c – – 0.40 ± 0.02d 3.05 ± 0.02c 2.03 ± 0.02c – – – – – – 1.59 ± 0.02b 0.25 ± 0.01c – 0.18 ± 0.01c – – – – – – 0.21 ± 0.02d 0.19 ± 0.02b 0.21 ± 0.01c – 0.20 ± 0.02bc 0.16 ± 0.01c 0.61 ± 0.02a 0.17 ± 0.01c 0.27 ± 0.01c

H. fl c

0.33 ± 0.02 – 9.55 ± 0.02f 8.25 ± 0.06b – 4.80 ± 0.02i – 0.91 ± 0.02e – – – 0.63 ± 0.02f – 2.62 ± 0.02f – 0.82 ± 0.02b 0.83 ± 0.03h – – 0.32 ± 0.01g – – 0.29 ± 0.01d – – – – – – – – 11.38 ± 0.03b – – – 0.30 ± 0.02b 1.10 ± 0.03a – 1.93 ± 0.01a – 0.39 ± 0.03a 0.35 ± 0.01b 1.89 ± 0.05 0.82 ± 0.02b – 7.28 ± 0.06a 20.74 ± 0.07a 14.23 ± 0.04a 0.40 ± 0.01b 0.54 ± 0.03b 0.36 ± 0.02b 0.45 ± 0.03b

H. au

137

– 0.93 ± 0.02b 12.54 ± 0.03d 0.97 ± 0.01c 1.38 ± 0.01a 30.22 ± 0.08b – 1.43 ± 0.01c 2.47 ± 0.02b 0.45 ± 0.01b 0.69 ± 0.03d – 1.48 ± 0.02d 33.13 ± 0.11b – 0.06 ± 0.01e 1.72 ± 0.03f 0.15 ± 0.02d 0.47 ± 0.03f 0.43 ± 0.02f 0.17 ± 0.01b 0.12 ± 0.02d 0.08 ± 0.01e – 0.99 ± 0.02a 2.34 ± 0.04e 5.07 ± 0.06a – – – – 0.13 ± 0.02c 0.24 ± 0.04 0.21 ± 0.02f – – – – – – – – – – 0.17 ± 0.02b – – – – 0.09 ± 0.01e – –

H. co

(continued on next page)

– 0.18 ± 0.02f 6.51 ± 0.09g 0.06 ± 0.01f 0.97 ± 0.02b 3.18 ± 0.01j – 0.50 ± 0.01h – – 0.23 ± 0.01f 0.03 ± 0.01h – 79.05 ± 0.02a – 0.04 ± 0.01e 0.44 ± 0.01i 0.04 ± 0.01e 0.09 ± 0.01h 0.36 ± 0.02fg 0.03 ± 0.01d 0.07 ± 0.01e 0.08 ± 0.01e – 0.62 ± 0.02c 1.81 ± 0.04f 3.90 ± 0.05b – – – 0.10 ± 0.01 0.03 ± 0.01d – 0.28 ± 0.02e – – – 0.11 ± 0.01b – – 0.03 ± 0.02b – – – – – – – 0.05 ± 0.01d – – 0.05 ± 0.01d

H. sp

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H.el

Data is expressed as mean ± S.D (n=3), Means followed by different superscript letters in the same row are significantly different according to Tukey test (p < 0.05). -, not detected. Abbreviations: H.el - Hedychium ellipticum, H. ga -Hedychium gardnerianum, H. gre - Hedychium greenii, H. f - Hedychium flavum, H. gr – Hedychium gracile, H. th – Hedychium thyrisiforme, H. fl – Hedychium flavescens, H. au – Hedychium aurantiacum, H. sp – Hedychium spicatum, H. co – Hedychium corornarium. i Retention indices (RI) calculated against C8-C20 n-alkane series on Elite-5 MS column. ii Retention indices from literature (Adams, 2007). iii Compound listed in the order of elution on Elite-5 MS capillary column (30 m x 0.25 mm, 0.25 μm film thickness).

– 0.09 ± 0.02c – – 98.22 ± 0.75 54.96 ± 0.33 42.7 ± 0.35 0.21 ± 0.02 0.35 ± 0.05 0.20 ± 0.02 – – 0.06 ± 0.01 99.1 ± 0.52 12.27 ± 0.22 86.05 ± 0.20 0.39 ± 0.03 0.39 ± 0.07 – 0.77 ± 0.01b 0.55 ± 0.02 – 92.83 ± 0.74 26.12 ± 0.21 14.61 ± 0.07 5.22 ± 0.11 46.88 ± 0.35 1.85 ± 0.03 – – – 97.41 ± 0.74 63.93 ± 0.27 25.43 ± 0.25 4.52 ± 0.08 3.53 ± 0.14 0.16 ± 0.01 – – – 96.74 ± 0.56 68.41 ± 0.26 27.41 ± 0.21 0.41 ± 0.05 0.51 ± 0.04 0.23 ± 0.01 – – – 78.05 ± 0.65 54.39 ± 0.36 19.55 ± 0.16 1.80 ± 0.10 2.31 ± 0.13 0.74 ± 0.01 6.26 ± 0.03a – – 99.52 ± 0.58 50.72 ± 0.26 36.33 ± 0.15 2.85 ± 0.06 9.62 ± 0.11 – – – – 75.93 ± 0.37 66.8 ± 0.19 3.92 ± 0.07 3.80 ± 0.09 1.41 ± 0.02 0.05 ± 0.01 – – – 99.66 ± 0.52 76.48 ± 0.29 22.87 ± 0.20 0.26 ± 0.02 0.05 ± 0.01 H53 1649 1649 β-Eudesmol H54 1666 1662 7-epi-α-Eudesmol H55 1675 1674 β-Bisabolol H56 1788 1783 γ-Eudesmol acetate Total Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes

RIlitii RIexpi S.No

Table 2 (continued)

Compoundiii

% Peak area

f

H. ga

H.gre

c

H.f

d

H. gr

e

H.th

a

H. fl

b

0.94 ± 0.02 – – – 92.77 ± 0.81 62.02 ± 0.38 25.77 ± 0.25 2.02 ± 0.04 2.96 ± 0.14

H. sp H. au

de

H. co

A. Ray et al.

helium at a flow rate of 1 ml/min. Injector and detector temperature were set at 250 °C and 260 °C, respectively. The ionization energy was set at 70 eV. Scanning was carried out in the range of 50–600 amu. Gas chromatography (GC) analysis was carried out in a Perkin Elmer Clarus Gas Chromatograph (Model: Clarus 580) coupled with a Flame ionization detector (FID). GC column and conditions were the same as in GC–MS. The percentage composition of the identified compounds was computed from GC-FID peak areas. Retention Index (RI) was calculated against the homologous n-alkanes series (C8-C20) run under identical GC conditions as that of the sample. Identification of compounds was done by comparing the mass spectra with the library (NIST 08) and by comparing retention indices values with those published in the literature (Adams, 2007). 2.3. Statistical analysis All data are expressed as mean ± S.D (n = 3). Data of essential oil compositions were subjected to analysis of variance (ANOVA) and the significant differences among constituents were measured by Tukey HSD test (p < 0.05). Agglomerative Hierarchical Clustering analysis (AHC) and principal component analysis (PCA) was carried out on the compositions of essential oils of 10 species to determine the relationship and similarity among diff ;erent species using Minitab 17 statistical software (Minitab Inc., State College, PA, USA). Data matrix constructed for AHC and PCA analysis consists of 10 species x 56 constituents. Euclidean distance was used to measure the dissimilarity between samples and hierarchical clustering was performed according to the Wards variance method. Principal component analysis was accomplished by diagonalization of the correlation matrix of the data, which transforms the variables into uncorrelated (orthogonal) ones called principal components (PCs). The participation of the original variables in the principal components is given by the loadings, and the individual transformed observations are called scores. Statistical analysis was performed using SPSS 13.0. 3. Results and discussion 3.1. Essential oil yield The flower description, common name, the color of rhizome and the essential oil color of Hedychium genus are listed in Table 1. The essential oils yields of different Hedychium species ranged from 0.05% to 0.75% (v/w, based on the fresh weight) (Fig. 1). Statistically significant differences existed between the oil of different Hedychium species. H. ellipticum and H. thyrsiforme had the highest yield (0.75%) followed by H. gracile (0.65%), H. spicatum (0.30%), H. flavescens (0.20%), H. gardnerianum (0.18%), H. aurantiacum (0.17%), H. coronarium (0.15%), while H. flavum and H. greenii, had the lowest oil content of 0.10% and 0.05%, respectively (Fig. 1). The rhizome color of Hedychium species was either light brown, brown, pale yellow or light yellow, while the color of the essential oil were either dark yellow, light yellow, pale yellow, dark brown or white transparent. It was apparent that the rhizome color was not related to the color of the essential oil. All the Hedychium species used in the current study were grown in the same conditions so as to rule out environmental variations. Sakhanokho et al. (2013) reported oils from 19 Hedychium species and cultivars to have an oil yield in the range of 0.08% to 0.53% calculated on a moisture-free basis. Our study was in agreement with the report of Prakash et al. (2010) who described an oil yield of 0.30% in H. spicatum. 3.2. Essential oil composition of Hedychium species A total of 56 constituents representing 75.93–99.66% of the total volatiles were identified in the essential oil of 10 Hedychium species by gas chromatography (Table 2). Essential oil of 10 Hedychium species consists of mainly monoterpenes, except Hedychium aurantiacum whose 138

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Fig. 2. GC–MS total ion chromatogram (TIC) of essential oil of ten (A–J) different Hedychium species. (A) H. gardnerianum, (B) H. flavescens, (C) H. thyrsiforme, (D) H. flavum, (E) H. ellipticum, (F) H. aurantiacum, (G) H. gracile, (H) H. greenii, (I) H. spicatum and (J) H. coronarium. 1: α-pinene; 2: camphene; 3: β-pinene; 4: α-terpinene; 5: p-cymene; 6: limonene; 7: 1,8-cineole; 8: γ-terpinene; 9: linalool; 10: terpinen-4-ol; 11: α-terpineol; 12: bornyl acetate; 13: β-caryophyllene; 14: caryophyllene oxide; 15: humulene epoxide I; 16: humulene epoxide II and 17: 7-epi-α-eudesmol.

Fig. 3. Percentage composition of 4 common constituents (α-pinene, β-pinene, 1,8-cineole, and γ-terpinene) in ten Hedychium species. H. au – H. aurantiacum, H. co – H. corornarium, H. fl – H. flavescens, H. ga – H. gardnerianum, H. gr – H. gracile, H. sp – H. spicatum, H.el – H. ellipticum, H. f - H. flavum, H. gre – H. greenii, and H. th – H. thyrisiforme.

major fraction was oxygenated sesquiterpene. The content of monoterpene hydrocarbons was higher in the essential oil of all Hedychium species as compared with oxygenated monoterpenes except Hedychium spicatum. The major components present in H. ellipticum essential oil are γ-terpinene (35.18%), β-pinene (18.46%), terpinen-4-ol (14.89%), and

α-terpinene (6.19%) (Fig. 2). The predominant compounds of H. gardnerianum oil are β-pinene (28.03%), α-pinene (21.15%), and p-cymene (11.91%). The major compounds of H. greenii are bornyl acetate (31.32%), α-pinene (14.49%), camphene (12.81%), and limonene (10.55%). The essential oil of H. flavum has β-pinene (24.77%), α-

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H. gracile and H. thyrsiforme. 3.3. Relation between Hedychium species The principal component analysis (PCA) was employed to assess similarity and relationship among Hedychium essential oils. A total of nine principal components (PC) could explain 100% of total variation. As the first two principal components (PC1 and PC2) had the highest share of the total variance (46%) among Hedychium species, they were displayed as scatter plot to determine the phytochemical distance. The first PC (PC1) analyzed 28% of the variation and had positive contribution from β-pinene, γ-terpinene, p-mentha-3,8-diene, α-terpinolene, α-campholenal, camphor, myrtenol, and p-cymen-9-ol and negative contribution from α-pinene, camphene, trans-β-ocimene, bornyl acetate, γ-gurjunene, (E,E)-α-farnesene, hedycaryol, elemol, germacrene-B, E-nerolidol, caryophyllene oxide, humulene epoxide I, humulene epoxide II, 1,10-di-epi-cubebol, and 7-epi-α-eudesmol. Similarly, the second PC (PC2) analyzed 18% of the total variation and had positive contributions from trans-β-ocimene, p-mentha-3,8-diene, α-campholenal, camphor, myrtenol, p-cymen-9-ol, germacrene-B, caryophyllene oxide, humulene epoxide I, humulene epoxide II, and epi-αcadinol and negative contributions from sabinene, α-phellandrene, δ-3carene, 1,8-cineole, α-terpineol, and borneol (Fig. 4). The remainder principal components (PC3-PC9) were shown in Table 3. The PCA scatter plots showed classification among different species in the plot that reflect their relationships. H. gardnerianum, H. flavum, H. flavescens, H. spicatum and H. coronarium belonged to one group and shared 11 common compounds such as α-thujene, α-pinene, camphene, β-pinene, myrcene, 1,8-cineole, γ-terpinene, α-terpinolene, terpinen-4ol, α-terpineol, and β-caryophyllene. H. aurantiacum and H. greenii formed an individual group separating from other Hedychium species, is characterized by high content of bornyl acetate and α-pinene. Another group contained H. thyrsiforme, H. ellipticum, and H. gracile and contains a high amount of β-pinene, terpinen-4-ol, and γ-terpinene (Fig. 5). A dendrogram was created from the statistical analysis performed on the identified constituents (56 compounds) of all Hedychium species, as represented in Fig. 6. It comprises of 3 clusters. Cluster I consists of H. aurantiacum and H. greenii. Cluster II consists of H. coronarium, H. spicatum, H. flavescens, H. flavum, and H. gardenarium Cluster III included H. thyrsiforme, H. gracile, and H. ellipticum. The genetic relationship of Hedychium species based on SRAP primer combinations also showed H. flavescens, H. flavum, and H. coronarium to fall in one cluster group (Gao et al., 2008). Similarly, inter-species genetic relationship based on AFLP marker data of different Hedychium species showed H. spicatum, H. flavum, H. gardenarium, and H. coronarium to fall under single cluster in accordance to our results (Basak et al., 2014). Botanically, H. flavum and H. flavescens are close and are often confused due to similar morphological characteristics. The two latter species have a similar yellow flower. But in the present study from Fig. 6, it was evident that the H. flavum was an independent species closely related to H. flavescens. The cluster classification was similar to the PCA analysis which also categorized different Hedychium species into three major groups. Since morphological and phenotypic characteristics are inadequate for species delineation, therefore the present chemical fingerprint of essential oil will assume a great significance in identification and classification of Hedychium species.

Fig. 4. PCA loading plot of chemical constituents (H1-H56) of essential oil of 10 Hedychium species.

pinene (11.17%), 1,8-cineole (8.90%), and linalool (6.29%) as the predominant constituents. Analysis of essential oil of H. gracile revealed β-pinene (25.24%), γ-terpinene (24.62%), terpinen-4-ol (14.87%), and 1,8-cineole (7.51%) as the dominant constituents. The essential oil of H. thyrsiforme had β-pinene (41.97%) and terpinen-4-ol (16.60%) as the major compounds. β-pinene (29.76%), α-pinene (13.17%), and 1,8-cineole (12.80%) are the principal compounds of H. flavescens. H. aurantiacum essential oil contains humulene epoxide I (20.74%) and humulene epoxide II (14.23%) as the major constituents. 1,8-cineole (79.05%) is the predominant compound of H. spicatum oil. Analysis of H. coronarium oil revealed 1,8-cineole (33.13%), β-pinene (30.22%), and α-pinene (12.54%) as the major compounds. The data in Table 2 also showed that α-pinene, β-pinene, 1,8-cineole, and γ-terpinene were present in the essential oils of all Hedychium species. The concentration of these four compounds varied from 0.42% to 79.05% and could be used as the marker for differentiating these ten Hedychium species (Fig. 3). These results proved that essential oils of Hedychium species could be considered as a good source of α-pinene, camphene, β-pinene, p-cymene, 1,8-cineole, γ-terpinene, αterpinolene, linalool, α-terpinene, terpinen-4-ol, and β-caryophyllene which possess outstanding biological activity and have enormous applications in pharmaceutical and perfumery industry. These compounds are known to possess analgesic, antimicrobial, anti-inflammatory, anticancer, and insecticidal activity (Bakkali et al., 2008; Afzal et al., 2013). Previous authors have explained diversity in the composition of essential oil of different Hedychium species. Van Thanh et al. (2014) reported the presence of α-pinene, α-humulene, and β-caryophyllene as the major constituents in the rhizome of H. coronarium from Vietnam. Joshi et al. (2008) reported trans-meta-mentha-2,8-diene and linalool as the predominant constituents of H. coronarium. Sakhanokho et al. (2013) identified linalool and 1,8-cineole as the major constituents in H. flavum and H. flavescens, respectively. In H. flavum, α-pinene and linalool were reported to be the major constituents in rhizomes from Vietnam (Sakhanokho et al., 2013). Prakash et al. (2010) have reported the presence of 1,8-cineole, α-eudesmol, and 10-epi-γ-eudesmol in rhizomes of H. spicatum and linalool, limonene, trans-meta-mentha,2,8diene, and γ-terpinene in rhizomes of H. coronarium, respectively. Raina and Negi, (2015) analyzed H. spicatum oils from seven different locations of Central Himalayas and found 1,8-cineole to range from 15.5% to 58.2%. Hardly any reports are available on essential oil of H. greenii,

4. Conclusions The present study showed high interspecies variation in chemical constituents among ten different Hedychium species. The predominant constituents in the essential oils were 1,8-cineole (0.42–79.05%), γ-

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Table 3 Principal components data of essential oil constituents in Hedychium species essential oils. Constituents

PC1

PC2

PC3

PC4

PC5

PC6

PC7

PC8

PC9

Tricyclene α-Thujene α-Pinene Camphene Sabinene β-Pinene Methyl heptenone Myrcene α-Phellandrene δ-3-Carene α-Terpinene p-Cymene Limonene 1,8-Cineole cis-β-Ocimene trans-β-Ocimene γ-Terpinene p-Mentha-3,8-diene α-Terpinolene Linalool trans-Sabinene hydrate α-Campholenal Camphor Camphene hydrate Borneol Terpinen-4-ol α-Terpineol Myrtenol p-Cymen-9-ol trans-Carveol Geraniol Bornyl acetate α-Terpinyl acetate β-Caryophyllene α-Humulene γ-Gurjunene β-Selinene δ –Selinene (E,E)-α-Farnesene γ-Cadinene Hedycaryol Elemol Germacrene-B E-Nerolidol Spathulenol Caryophyllene oxide Humulene epoxide I Humulene epoxide II 1,10-di-epi-Cubebol 10-epi-γ-Eudesmol γ-eudesmol epi-α-Cadinol β-Eudesmol 7-epi-α-Eudesmol β-bisabolol γ-Eudesmol acetate Eigen value Variability (%) Cumulative (%)

0.01 0.13 −0.15 −0.20 −0.08 0.17 0.13 0.06 0.04 0.01 0.21 0.01 −0.10 −0.01 −0.12 −0.16 0.17 0.19 0.22 0.04 0.10 0.22 0.16 0.00 −0.10 0.22 0.05 0.20 0.20 0.12 −0.02 −0.17 0.00 0.13 0.12 −0.16 −0.21 −0.12 −0.16 0.01 −0.15 −0.12 −0.15 −0.17 0.09 −0.14 −0.15 −0.15 −0.15 −0.12 −0.07 0.05 0.08 −0.14 −0.15 −0.02 15.77 28 28

0.15 0.01 −0.14 0.13 −0.18 −0.04 0.14 0.13 −0.18 −0.20 0.14 −0.10 0.03 −0.19 0.06 0.20 0.12 0.16 0.05 −0.11 0.07 0.16 0.21 −0.09 −0.16 0.15 −0.25 0.18 0.18 0.12 −0.13 0.12 −0.14 0.01 0.02 0.05 0.16 0.06 0.15 −0.04 0.16 0.01 0.17 0.10 0.05 0.17 0.17 0.17 0.10 0.00 −0.03 0.20 0.07 0.08 0.17 −0.13 9.83 18 46

0.13 0.00 0.01 0.14 0.17 −0.05 0.07 0.21 −0.07 −0.08 0.03 −0.10 0.27 0.01 0.28 0.12 0.01 0.04 0.01 −0.12 0.07 0.03 −0.04 −0.09 0.20 0.02 0.00 0.03 0.03 0.03 0.01 0.21 0.03 −0.06 −0.07 0.11 −0.01 0.28 −0.19 −0.05 −0.18 −0.17 −0.18 −0.06 0.08 −0.19 −0.18 −0.18 0.23 −0.15 −0.19 −0.12 0.08 0.26 −0.18 0.01 8.86 16 62

−0.12 −0.06 0.21 0.04 −0.10 0.23 −0.20 0.08 0.08 0.12 −0.07 0.15 0.10 −0.22 0.06 0.06 −0.15 −0.09 0.17 0.17 0.19 −0.01 −0.03 0.08 −0.01 −0.02 −0.12 0.01 −0.03 −0.18 −0.21 0.04 −0.05 0.28 0.30 0.11 0.03 0.04 −0.03 0.19 −0.07 0.09 −0.06 0.16 0.25 −0.04 −0.06 −0.05 0.05 0.12 0.09 0.12 0.26 0.06 −0.06 −0.21 6.65 12 73

0.10 0.06 −0.10 0.07 −0.15 −0.02 0.13 0.17 0.31 0.27 0.06 −0.04 0.08 −0.12 0.07 0.00 0.14 0.00 0.08 0.29 −0.17 −0.03 −0.16 0.24 −0.13 0.00 −0.07 −0.06 −0.03 0.09 −0.18 0.07 0.01 −0.09 −0.15 −0.16 0.10 0.06 −0.08 −0.30 −0.02 −0.18 −0.01 −0.16 −0.09 0.00 −0.01 0.00 0.08 0.27 0.25 −0.08 −0.05 0.07 −0.01 −0.18 4.75 9 82

−0.11 −0.06 −0.14 0.03 0.09 −0.01 −0.19 0.10 0.05 0.09 −0.04 −0.36 −0.06 0.21 −0.01 0.11 −0.16 −0.03 −0.03 0.03 0.26 0.05 −0.07 −0.08 −0.04 0.01 0.18 0.04 0.00 −0.16 0.16 0.02 0.14 0.15 −0.01 −0.23 0.07 −0.01 −0.01 −0.25 0.10 −0.26 0.08 −0.16 0.27 0.09 0.08 0.08 0.02 0.08 −0.01 0.22 0.23 0.00 0.08 0.16 4.58 8 90

−0.07 0.23 0.11 0.02 0.21 0.18 0.02 0.23 0.17 0.13 0.05 −0.04 −0.04 −0.15 −0.06 0.01 0.05 0.00 0.01 −0.19 0.07 0.01 0.06 −0.20 0.19 0.00 0.17 −0.02 −0.01 −0.03 −0.31 −0.03 0.48 −0.12 −0.11 0.03 −0.01 −0.09 0.10 0.00 0.06 0.04 0.08 0.02 0.07 0.08 0.08 0.08 −0.06 −0.05 −0.17 −0.05 −0.16 −0.05 0.08 −0.31 2.72 5 95

0.48 −0.54 0.07 −0.02 0.04 0.16 0.07 −0.11 0.06 0.11 −0.19 −0.02 −0.05 −0.01 −0.02 −0.09 −0.17 0.26 −0.08 0.08 −0.02 −0.02 0.00 −0.12 0.12 −0.04 0.12 −0.03 0.00 0.37 −0.08 −0.03 0.11 −0.01 0.07 0.00 −0.02 −0.01 0.01 0.00 −0.01 0.00 0.00 0.00 0.03 0.00 0.00 0.00 0.00 0.05 −0.04 0.20 0.02 −0.03 0.00 −0.08 1.81 3 98

0.05 −0.11 −0.08 0.00 0.13 0.05 −0.06 −0.05 −0.10 −0.06 −0.02 −0.22 0.19 −0.02 0.01 0.06 −0.11 0.10 −0.08 −0.19 0.24 −0.02 0.07 0.59 −0.13 0.03 0.06 0.09 0.07 0.01 −0.06 0.01 0.22 0.00 0.01 −0.04 −0.01 0.01 −0.02 0.10 0.00 0.13 0.00 −0.04 −0.04 0.00 0.00 0.00 −0.07 −0.24 0.40 −0.13 −0.15 0.01 0.00 −0.06 1.03 2 100

PC-1 to PC-9 represents the nine major principal components of the principal component analysis.

terpinene (0.37–35.18%), β-pinene (3.18–41.97%), bornyl acetate (0.03–31.32%), α-pinene (1.57–21.15%), humulene epoxide I (0.11–20.74%), and terpinen-4-ol (0.36–16.60%). Further, the developed chemical fingerprint will help in their identification and utilization. Most of the Hedychium species have not been exploited phytochemically as well as pharmacologically, therefore the present study would be of enough significance.

Funding This research did not receive any specific grant from any funding agencies in the public, commercial, or not-for-profit sectors. Conflict of interest None

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Fig. 5. Principal component analysis score plot (PC1 vs. PC2) of different Hedychium species. H.el – H. ellipticum, H. ga – H. gardnerianum, H. gre – H. greenii, H. f - H. flavum, H. gr – H. gracile, H. th – H. thyrisiforme, H. fl – H. flavescens, H. au – H. aurantiacum, H. sp – H. spicatum, H. co – H. corornarium.

Fig. 6. Dendrogram constructed on essential oil compositions of 10 Hedychium species using Euclidean distance and Ward linkage method by Minitab version 17 software. The numerical scale represents the distance between the clusters.

Acknowledgements The authors acknowledge the Director, ICMR-Regional Medical Research Centre and Dean, School of Pharmaceutical Sciences, SOA University for providing necessary infrastructure facilities. References Adams, R.P., 2007. Identification of Essential Oil Components by Gas Chromatography/ Mass Spectroscopy. Allured Publishing Corporation, Carol Stream, IL, USA. Afzal, A., Oriqat, G., Akram Khan, M., Jose, J., Afzal, M., 2013. Chemistry and biochemistry of terpenoids from Curcuma and related species. J. Biol. Act. Prod. Nat. 3, 1–55. Angel, G.R., Menon, N., Vimala, B., Nambisan, B., 2014. Essential oil composition of eight starchy Curcuma species. Ind. Crops Prod. 60, 233–238.

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