RUHUNA JOURNAL OF SCIENCE Vol 8: 103-111, December 2017 eISSN: 2536-8400  Faculty of Science DOI: http://doi.org/10.4038/rjs.v8i2.29 University of Ruhuna  Faculty of Science, University of Ruhuna Sri Lanka 103 Short Paper Comparative GC-MS analysis of all Curcuma species grown in Sri Lanka by multivariate test H.M.I.C. Herath, T.D.C.M.K. Wijayasiriwardene*, G.A.S. Premakumara Industrial Technology Institute, Bauddaloka Mawatha Colombo, Sri Lanka *Correspondence: drchandima@iti.lk Received: 26 th October 2017, Revised: 28 th December 2017, Accepted: 30 th December 2017 Abstract Curcuma is clinically valuable genus in Traditional Medicine. People use various plants under the same vernacular name may lead to adulteration or substitution. Whole plants of Curcuma species were collected in 2016 in the flowering season. Voucher specimens of the plants were authenticated from the National Herbarium, Peradeniya. Essential oils were extracted from Clevenger’s apparatus and analyzed separately by GC-MS. The analyses were carried out with RTX WAX capillary column. Sampling and experiments were done according to WHO guidelines. One hundred sixty four phytochemicals were analyzed by simple correspondence and by cluster variable method. By cluster varibale as per phytochemicals present, mainly two groups were identified. C. albiflora and C. oligantha were identified as one group and the rest of the three plants were kept in the other group. A total of 64 constituents of essential oil obtained from whole plant of C. albiflora were identified by GC-MS, where α-pinene (10.87 %), caryophyllene oxide (8.85 %), alcanfor (5.12 %), aromadendrene oxide-(1) (4.81 %), n-hexadecanoic acid (4.74 %), α-famesene (3.93 %), camphene (3.52 %), and isoborneol (3.4 %) were detected as major compounds. The essential oil of C. aromatica possesses 7–methanoazulene (13.75 %) and curcumene (25.71%). Caryophyllene (15.07%), phytol (13.38%), humulene (8.24%), elemene (6.11%), caryophyllene oxide (5.82%) were found in C. oligantha. This preliminary study has identified chemical markers present in all Curcuma species grown in Sri Lanka. Keywords: Curcuma species, essential oils, GC-MS, multivariate. 1 Introduction Curcuma is an important genus in Traditional Medicine of Sri Lanka. Commonly known as turmeric, five species are reported in Sri Lanka (e.g. C. albiflora Thw., C. aromatica Salisb., C. Longa L., C. oligantha Trimen., C. H.M.I.C. Herath et al. GC-MS analysis of five Curcuma species in Sri Lanka Ruhuna Journal of Science Vol 8: 103-111, December 2017 104 zedoaria Roscoe). These plant materials are known to be used for centuries to manage various health conditions. People use turmeric in culinary as a flavor or color. In addition, Curcuma species show antioxidant, anti-inflammatory, hypocholestraemic, choleratic, antimicrobial, antirheumatic, antifibrotic, antivenomous, antidiabetic, antihepatotoxic, anticancerous, larvicidal, pheromone, insecticidal, anti-plasmodium, hyperprotective, platelet aggregation inhibitory, antiarthritic, COX-1 inhibitory, antiviral and antiproliferative activity etc (Afzal et al. 2013; Tholkappiyavathi et al. 2013; Abbasi and Shah 2015; Krup et al. 2013; Sikha, et al. 2015;). However, many plants are reported under the same Sinhalese vernacular name. For example, the people in Mahiyanganaya area use both C. aromatica, and C. zedoaria as ‘Harankaha’, while the people in Erathna area (Kegalle district) name C. aromatica as ‘Beheth-harankaha’. In literature, Zingiber zerumbet, C. albiflora, and C. zedoaria are reported as ‘Harankaha’ (Dassanayake 1983). Regionally people have used various plants under the same vernacular name, even those plants have different chemical compositions and medicinal uses. Furthermore, C. albiflora and C. oligantha are unexplored plants. This is the first report of the chemical constituents of these different species of Curcuma, and the study was conducted to understand the similarities in terms of phytochemical constituents by analyzing data using multivariate test on five species available in Sri Lanka, C. albiflora, C. aromatica, C. longa, C. oligantha, and C. zedoaria. Therefore, the present study will provide important information on similarities and dissimilarities of Curcuma plants grown in Sri Lanka by chemical analysis. 2 Material and Methods 2.1 Samples and chemical analysis Whole plants of Curcuma species were collected in the year 2016 from wet and dry zones of Sri Lanka in the flowering season; C. albiflora (Kitulgala, Kegalle and Erathna, Ratnapura district), C. aromatica (Erathna, Rathnapura), C. longa (Kahathuduwa, Colombo district), C. oligantha (Hebarawa, Badulla district), C. zedoaria (Gonapola, Colombo district). Voucher specimens of the plants were authenticated and deposited in the National Herbarium, Peradeniya, Sri Lanka, and Herbal Technology Section, Industrial Technology Institute for future reference. Phytochemicals in essential oil extracted from Clevenger’s apparatus of five Curcuma species were analyzed by GC-MS. GC-MS analysis was carried out on a THERMOSCIENTIFIC TRACE 1300 detector and with RTX WAX capillary column. Mode of H.M.I.C. Herath et al. GC-MS analysis of five Curcuma species in Sri Lanka Ruhuna Journal of Science Vol 8: 103-111, December 2017 105 operating conditions was split (1:50), and the oven temperature program was 60 o C (after 10.00 min) to 240 o C at 5 o C/min with helium as carrier gas. Identification of constituents was done by matching 1700 eV mass spectra, 250 o C quad temperature, 250 o C source temperature, 50 - 450 (amu) scan parameters, and matching with NIST library. The fragmentation pattern and the retention time were compared with the standards to confirm the presence of a particular phytochemical. Sampling and analysis were done according to WHO guidelines (WHO 2012). 2.2 Statistical Analysis Statistical tests were performed using Minitab 17. Multivariate test was used to determine the complex relationship among variables (Amel 2015). 164 phytochemicals were analyzed by simple correspondence analysis and by cluster variable method. But trace elements were discarded. Statistical analysis examined the relationships between the 5 species and the associations between variables in two dimensions, and similar phytochemical contents were identified from their positions as described in Greenacre (1983). 3 Results and Discussion Phytochemicals detected in the extracts prepared from Curcuma albiflora, C. aromatica, C. longa, C. oligantha, and C. zedoaria are summarized in Table 1. Table 1: Chemical profiles of five Curcuma species grown in Sri Lanka (AF: Curcuma albiflora, AR: C. aromatica, CL: C. longa, CO: C. oligantha, CZ: C. zedoaria). Compound Area% AF AR CL CO CZ alpha ylangene 0.47 0 0 0 0 Guaia-1(10),11-diene 1.07 0 0 0 0 myrtenal 0 0 0.1 0 0 o-Cymene 0.2 0 0 0 0 p-cymene 0 0 13.3 0 0 thymol 0 0 0.2 0 0 (-)-Alcanfor 5.12 0 0 0 0 (-)-Myrtenol 0.81 0 0 0 0 (-)-Spathulenol 0 0 0 1.97 0 H.M.I.C. Herath et al. GC-MS analysis of five Curcuma species in Sri Lanka Ruhuna Journal of Science Vol 8: 103-111, December 2017 106 Table 1. Continued. Compound AF AR CL CO CZ (1R)-(+)-Nopinon 0.2 0 0 0 0 (E)-bocimene 0 0 0.3 0 0 (E)-nerolidol 0 0 0.5 0 0 (E)-p-famesene 0 0 0.2 0 0 (Z)-p-ocimene 0 0 0.1 0 0 1- bisabolone 0 1.5 0 0 0 1( 10),4-furanodien-6-one 0 0 0 0 0 2(10)-Pinen-3-ol 0.75 0 0 0 0 2-heptanol 0 1 0 0 0 2-octanol 0 0 0 0 0 2-Tridecanone 0.15 0 0 0 0 3-carene 0 0 0.9 0 0 6 - Camphenol 0.12 0 0 0 0 8-tunnerone 0 0 0 0 0 á-copaene 0.85 0 0 0 0 a-curcumene 0 0 0.2 0 0 a-humulene 0 0 0.2 0 0 á-Linalool 0.69 0 0 0 0 alpha- Bourbonene 0 0 0 0.06 0 alpha Copaene 0 0 0 1.89 0 alpha Terpineol 0 0 0.14 0 0 alpha-Bisabolene epoxide 0 0 0 0 0 alpha-Famesene 4.01 0 0 0 0 á-Myrcene 0 0 0 0 0 Andrographolide 0 0 0 0.07 0 a-phellandrene 0 0 18.2 0 0 a-pinene 14.5 0.3 2.6 0 0.4 ar-curcurnene 0 3.1 0 0 0 Aromadendrene oxide-(1) 4.81 0 0 0.38 0 ar-turmerol 0 0.4 0.2 0 0 ar-turnerone 0 6.3 0.1 0 0 Asarone 0 0 0 0.98 0 a-terpinene 0 0 0.4 0 0 a-terpineol 0.64 0.6 0.9 0 0 a-thujene 0 0 0.1 0 0 a-turmerone 0 6.7 0.3 0 0 Azulene 0 0 0 0.37 0 bisabolene 0 0 0.2 0 0 B-elernene 0 1.4 0 0 0 B-sesquiphellandrene 0 1.7 0 0 0 carnphene 0 0.7 0 0 0 Caryophyllene 2.57 0 0 15.07 0 Caryophyllene oxide 9.35 0 0.4 5.82 0 H.M.I.C. Herath et al. GC-MS analysis of five Curcuma species in Sri Lanka Ruhuna Journal of Science Vol 8: 103-111, December 2017 107 Table 1. Continued. Compound AF AR CL CO CZ cawacrol 0 0 0.1 0 0 ç-Elemene 0 0 0 6.11 0 cineole 0 0 14.6 0 0 Cinnamal 0 0 0.4 0 0 cis-&elernenone 0 0.2 0 0 0 cis-sabinol 0 0 1 0 0 cis-Verbenol 0.28 0 0 0 0 cubedol 0 0 0 0 0 curcuphenol 0 0.2 0 0 0 curdione 2.83 0 0.5 0 0 curzerene 0 0.2 0 0 0 curzerenone 0 11 0 0 0 de hydro-p-cymene 0 0 0.1 0 0 Denderalasin 0.6 0 0 0 0 d-Mannose 0 0 0 0.08 0 Doconexent 0.5 0 0 0 0 Elemene 0 0 0 1.86 0 elemicin 0 0 0.2 0 0 endo-Borneol 1.44 0 0 0 0 epicurzerenone 0 0 0 0 0 Eremophila-1(10),11-diene 0.91 0 0 0 0 Eucalyptol 2.21 0 0 0 0 Falcarinol 0.13 0 0 0.13 0 Gamolenic Acid 0 0 0 0.1 0 gerrnacrone 0 0.5 0 0 0 Humulene 0 0 0 8.24 0 Ionone 0 0 0 0.19 0 Isoaromadendrene epoxide 0.69 0 0 0 0 Ledene oxide-(II) 2.11 0 0 0.52 0 limonene 0 1 3.3 0 0 linalool 0 0.2 1.2 0 0 myrcene 0 0 1.8 0 0 Myrtanal 0.11 0 0 0 0 myrtenol 0 0 0 0 0 Naphthalene 0 0 0 0.78 0 Neocurdione 0.87 0 0 0 0 n-Hexadecanoic acid 4.74 0 0 0 0 Patchoulane 0.16 0 0 0 0 p-bisabolene 0 1.8 0 0 0 p-caryophyllene 0 0.3 0.5 0 0 p-cymen-8-01 0 0 2.4 0 0 Phytol 0.99 0 0 13.38 0 Pinocarvone 0.46 0 0 0 0 H.M.I.C. Herath et al. GC-MS analysis of five Curcuma species in Sri Lanka Ruhuna Journal of Science Vol 8: 103-111, December 2017 108 Table 1. Continued. Compound AF AR CL CO CZ ppinene 0 0 7.2 0 0 p-pinene 0 0.4 0 0 0 p-sesquiphellandrene 0 0 0.2 0 0 p-turrnerone( curlone) 0 0.9 0 0 0 myrcene 0 0.2 0 0 0 terpinen-4-01 0.25 0.5 0.8 1.82 0 terpinolene 0 0 11.6 0 0 trans-á-Ionone 0 0 0 0.2 0 trans-Carvone oxide 0.11 0 0 0 0 trans-Geranylacetone 0.15 0 0 0 0 T-rnuurolol 0 0.1 0 0 0 y-terpinene 0 0 1 0 0 zingiberene 0 0.8 0.5 0 0 2-Undecanone 0 0 0 0 0.02 Verbenone 0 0 0 0 0.03 1-Carvon 0 0 0 0 0.04 Carvon 0 0 0 0 0.05 Pichtosine 0 0 0 0 0.05 1 Phelendrine 0 0 0 0 0.07 Cyclene 0 0 0 0 0.08 beta Myrcene 0 0 0 0 0.15 Carbinol 0 0 0 0 0.15 2-Nonanone 0 0 0 0 0.27 2-Nonanol 0 0.5 0 0 0.29 Phenyldihydropyran 0 0 0 0 0.31 Linderazulene 0 0 0 0 0.32 Terpineol 0 0 0 0 0.33 beta Pinene 0 0 0 0 0.41 1-Limonene 0 0 0 0 0.54 dl-Limonene 0 0 0 0 0.61 Sabinene 0 0 0.4 0 0.61 1 ,8-cineole 0 5.5 0 0 1.01 delta Elemene 0 0 0 0 1.05 Borneol 0 1.1 0.3 0 1.69 Camphene 3.64 0 0 0 1.87 beta Elemene 0 0 0 0 1.98 Germacrene B 0 0.3 0 0 3.98 Isoborneol 3.46 3.4 0 0 4.04 beta Eudesmene 0 0 0 0 4.97 Germacrone 0 0 0.2 0 5.17 Benzofuran 0 0 0 0 8.84 Aromadendrene 0 0 0 0.19 11.75 Camphor 0 32.3 0 0 11.82 Debromofiliforminol 0 0 0 0 31.46 H.M.I.C. Herath et al. GC-MS analysis of five Curcuma species in Sri Lanka Ruhuna Journal of Science Vol 8: 103-111, December 2017 109 Among chemicals in C. albiflora, alpha-pinene (14.5%), caryophyllene oxide (9.35%), and alconfor (5.12%) have been found as major compounds. Caryophyllene (15.07%), humulene (8.24%), and phytol (13.38) were found as major compounds in C. oligantha. Camphor (32.3%), debromofiliforminol (31.46%), and alpha-phellendrene (18.2%) were found as major compounds in C. longa, C. zedoaria, and C. aromatica essential oil. By cluster varibale analysis of phytochemicals present in five speces, mainly two subgroups were clustered; C. albiflora and C. oligantha into one and C. aromatica and C. zedoaria into another. These two subgroups are separated from C. longa (Figure 1). Fig. 1: Cluster variable analysis of dendrogram obtained from phytochemicals present in five Curcuma species in Sri Lanka (AF: C. albiflora, AR: C. aromatica, CL: C. longa, CO: C. oligantha, CZ: C. zedoaria). This is in agreement with the seperation of Curcuma species according to morphological characters explained by Dassanayake (1983). Symmetric row and column plot and column plot from Simple Correspondence Analysis showed comparable results (Figure 2). Variables S im il a r it y CZARCLCOAF 48.47 65.65 82.82 100.00 Dendrogram with Single Linkage and Correlation Coefficient Distance H.M.I.C. Herath et al. GC-MS analysis of five Curcuma species in Sri Lanka Ruhuna Journal of Science Vol 8: 103-111, December 2017 110 Fig. 2: Column and symmetric plot obtained from phytochemicals present in five Curcuma species in Sri Lanka (AF: C. albiflora, AR: C. aromatica, CL: C. longa, CO: C. oligantha, CZ: C. zedoaria). The present study has identified chemical markers present in all Curcuma species grown in Sri Lanka. However, collection of more samples of same species from different locations is needed to be analyzed for the establishment of chemo-taxonomical identity and distribution pattern by multivariate test. 4 Conclusion Cluster variable method and simple correspondence analysis enabled the clustering of Curcuma species grown in Sri Lanka into two major groups based on their phytochemical composition. The chemo-taxonomy of Curcuma plants grown in Sri Lanka could be established with the extension of number samples. References Abbasi K, Shah AA. 2015. Biological evaluation of Turmeric (Curcuma longa). International Journal of Current Microbiology and Applied Science 4(11): 236-249. H.M.I.C. Herath et al. GC-MS analysis of five Curcuma species in Sri Lanka Ruhuna Journal of Science Vol 8: 103-111, December 2017 111 Afzal A, Oriqat G, Khan MA, Jose J, Afzal M. 2013. 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