GC-MS IDENTIFICATION OF ENDEMIC HERB CURCUMA ALBIFLORA THW. (SRI LANKA)

GC-MS IDENTIFICATION OF ENDEMIC HERB CURCUMA ALBIFLORA THW. (SRI LANKA)

H. M. I. C. Herath, T. D. C. M. K Wijayasiriwardene ^* and G. A. S. Premakumara

Industrial Technology Institute, Colombo, Sri Lanka.

ABSTRACT: Curcuma albiflora Thw. is an important anti-inflammatory medicinal plant, which is used in Sri Lankan Traditional Medicine. The plant is endemic to Sri Lanka and unexplored. Therefore it is necessary to conduct a proper systematic study to establish a chemical profile of C. albiflora. Therefore, the current study was conducted GC-MS study on various extracts of C. albiflora. The whole plant parts of the plant C. albiflora were collected from June to October in the flowering season at Sabaragamuwa province, Sri Lanka. Samples were authenticated by National Herbarium, Peradeniya. Hydro-distillation by Clevenger's apparatus and continuous extraction by Soxhlet apparatus were carried out on various solvents (petroleum ether, acetone, and methanol). GC-MS analyses were performed on various extracts (water, petroleum ether, acetone, and methanol) of C. albiflora. Retention index was calculated of each compound based on standard alkane chromatogram. The whole plant of C. albiflora essential oil consisted mainly of α-pinene (14.51%), caryophyllene oxide (9.35%), alcanfor (5.12%), aromadendrene oxide-I (4.81%), α-famesene (4.01%) and camphene (3.64%). While petroleum ether extract of C. albiflora consisted mainly neocurdione (5.15%), trans-z-α-bisabolene epoxide (4.69%) and caryophyllene oxide (3.9%),  acetone extract consisted mainly  α-pinene (9.76%), 2,4-octadiyne (6.1%), and (3α,5α) 2-methylene cholestanol (3.73%) and methanol extract consisted mainlycaryophyllene oxide(16.18%), 5α-androstanone, cyclic ethylene-mercaptole (11.04%), acetonyl-di-methyl carbinol (10.29%), aromadendrene oxide-(1) (9.15%), α-pinene (6.52%), neocurdione (5.85%), (e)-α-famesene (4.82%). The current study was the first chemical analysis of plant C. albiflora, and it is suggested to study TLC profile of various extracts, antioxidant bio assays and pharmacological properties in future.

Curcuma albiflora, Retension index, Neocurdione, Mercaptole

INTRODUCTION: Plants of genus Curcuma are known to have anti-inflammatory, and antioxidant properties and which is given from acute to chronic situations ^{1, 2, 3}. Among more than 90 species are accounted for Curcuma, 5 species (C. albiflora, C. zedoaria, C. longa, C. aromatica and C. oligantha) are reported in Sri Lanka ^{4, 5}.

C. albiflora is endemic to Sri Lanka, and its chemical profile has not been established. Therefore, current GC-MS study was conducted to establish a chemical profile of the whole plant of C. albiflora on its various extracts.

MATERIALS AND METHODS: Matured whole plants (height 35-50 cm) of C. albiflora were collected in the flowering season from 2016 to early 2017 in Sabaragamuwa from June to October in Sabaragamuwa province (Erathna: N 6^o 50' 07'', E 80^o 24' 41'', Kitulgala: N 6° 59' 41'', E 80° 24' 20'' and Bopathella: N 6^o 48' 07'', E 80^o 22' 12''). Since, C. albiflora is an endemic threatened species, approval was taken from Forest department, Sampathpaya, Battaramulla (My ref. R&E/RES/ NFSRC/12). Voucher specimens of the plants (herbariums) were authenticated and deposited at the National Herbarium, Peradeniya, Sri Lanka (My ref 6/01/H/03). Collected samples were cleaned by tap water and cut into small pieces (2 cm long). All procedures were carried according to WHO guidelines and other published data ^{6, 7}. Cut samples were dried under shade to obtain 8-10% moisture content.

Then they were ground and sieved from 40 mesh size sieve. The powder was kept in proper plastic containers with silica gel sachets to remove atmospheric moisture. Powdered samples (40#) were extracted by hydrodistillation with Clevenger's apparatus (6 h), and with Soxhlet apparatus (6 h) by various solvents (200ml petroleum ether, 200 ml acetone and 200ml methanol). Extracts were concentrated by rot evaporator, and 2 ml was sent to analyze by GC-MS. The GC-MS analysis was carried out on a Thermo Scientific Trace 1300 detector and with RTX WAX capillary column. Mode of operating conditions was split (1:50) and the oven temperature program was 60 °C (after 10.00 min) to 240 °C at 5 °C /min with helium as the carrier gas. Identification of constituents was done by matching 1700 eV mass spectra, 250 °C quad temperature, 250 °C source temperature, 50 - 450 (amu) scan parameters, and direct matching and reverse matching with NIST library.

Determination of compounds in GC-MS requires matching the retention time of the same compounds with standard on two chromatographic columns.  Kovat (1958) introduced a retention index scheme. The retention index (I) of a particular compound was calculated according to the formulae;

I = 100 [n + (N-n) log t_u – log t_s /log t_L – log t_s ]

Where; n: number of carbon atoms in the smaller alkane, N: number of carbon atoms in larger alkane, t_u: retention time of unknown compound, ts: retention time of smaller carbon atom, and t_L: retention time of larger carbon atom.

Standard alkane series was used a number of carbons from 10 to 27 (e.g., 10, 11, 12 …etc.). To calculate the retention index of unknown compounds for the current study, above formulae, modified as follows;

I = 100 [n + t_u - t_s  /  t_L - t_s  ]

Where, n is the number of carbon atoms in the smaller alkane, t_u: retention time of unknown compound, ts: retention time of smaller carbon atom, and t_L: retention time of larger carbon atom.

RESULTS AND DISCUSSION: Volatile compounds of preliminary phytochemical analysis are identified with GC-MS analysis.  The C. albiflora essential oil was extracted by hydro-distillation, and solvent extraction was performed by continuous extraction (Soxhlet apparatus) using petroleum ether, dichloromethane, acetone and methanol as solvents. Extracted fractions were subjected to GC-MS identification. A total of 64 constituents of essential oil were identified by GC-MS. 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%) as major compounds, which were more than 3%. Identified chemical constituents in the essential oil of C. albiflora were listed in Table 1.

TABLE 1: PERCENTAGE COMPOSITION OF ESSENTIAL OIL OF C. ALBIFLORA

MW- molecular weight, RI: retention index

A total of 26 constituents of petroleum ether extract were identified by GC-MS; Hexadecanoic acid (28.85%), 2-methylhexadecan-1-ol (5.52%), neocurdione (5.15%), trans-Z-α-bisabolene epoxide (4.69%), m-ethyl toluene, (4.52 %), and caryophyllene oxide (3.9 %) as major compounds. Identified chemical compounds in petroleum-ether extract of C. albiflora were listed in Table 2. A total of 11 compounds were found in DCM extract; 1, 8 cineol (34.29 %), α-pinene (14.94 %), camphene (12.66%), caryophyllene oxide (10.19 %), and caryophyllene (4.88%) were found as major compounds Table 3.

TABLE 2: PERCENTAGE COMPOSITION OF PETROLEUM ETHER EXTRACT OF C. ALBIFLORA

MW- molecular weight, R_t- retention time

TABLE 3: PERCENTAGE COMPOSITION OF DCM EXTRACT OF C. ALBIFLORA THW

MW- molecular weight, R_t- retention time

A total of 12 compounds were found in acetone extract of C. albiflora; 4-hydroxy-4-methyl pentane-2-one (15.12%), d-mannose (11.67%), α-pinene (8.73%), 2, 4-octadiyne (4.76%), 2-methylene-(3α, 5α)-cholestan-3-ol (3.73%), as major compounds (>3%). In the preliminary phytochemical analysis, sugar was reported in acetone extract of C. albiflora; this may correlate with d-mannose found. Identified chemical compounds in acetone extract of C. albiflora were listed in Table 4.

TABLE 4: PERCENTAGE COMPOSITION OF ACETONE EXTRACT OF C. ALBIFLORA, SRI LANKA

MW- molecular weight, R_t - retention time

TABLE 5: PERCENTAGE COMPOSITION OF METHANOL EXTRACT OF C. ALBIFLORA

MW- molecular weight, R_t- retention time

A total of 29 compounds were found in methanol extract of C. albiflora; Caryophyllene oxide (16.18%), cyclic ethylene mercaptole (11.04%), acetonyldimethylcarbinol (10.29%), aromadend-rene oxide-1 (9.15%), α-pinene (6.52%), neocurdione (5.85%), (e)-α-famesene (4.82%), methyl octadecadiynoate (2.34%), ethyl benzene (2.31%), (-)-alcanfor (3%) as major compounds. Identified chemical compounds found in methanol extract of C. albiflora were listed in Table 5.

Monoterpenes such as α-myrcene (0.02 %), 1,8 cineol (2.21 %), camphene (3.64%), (-)-carvone (0.05%), pinocarvone (0.46%) in the essential oil and d-1,8 cineol (0.32%), camphene (1.45%), and limonene (1.44%) were found in acetone extract of C. albiflora. These plant constituents are reported to have various pharmacological properties such as toxicant, antibacterial, antioxidant, antimicrobial, antimitogenic, anti-cancer, antiprotozoal, and hepatoprotective properties etc. (Makabe et al., 2006); Nerolidol (antinociceptive and anti-inflammatory), Caryohylene oxide (analgesic and anti-inflammatory), euclyptol (anti-inflammatory), myrtinol (antinflammatory), bisabalool (anti-nosiceptive and anti-inflammatory), caryophyllene (anti-inflammatory), α-terpineol (anti-inflammatory), D-limonene (anti-inflammatory and antioxidant), α-pinene (anti-inflammatory).

Secondary metabolites of plants are important chemical compounds in medicine. Among them, some compounds have a potent therapeutic value. Curcuma species possess phytochemicals which have reported to have medicinal properties; 1, 8 cineole (toxicant, antioxidant, antimicrobial), turmerone (insecticidal, antiplasmodium), bisabolol oxide (antimicrobial mutagenic, genotoxic), borneol (antimicrobial, antioxidant, hyper-protective, larvicidal), camphene (antimicrobial, antioxidant), camphor (larvicidal, antioxidant), caryophyllene (anticancer, antioxidant, anti-inflammatory), curdione (hyper-protective, platelet aggregation inhibitor, antimicrobial), curzerene (antimicrobial, antioxidant, antioxidant) etc.¹ Falcarinol, mercap-tole and cubedol were reported first time in C. albiflora essential oil. The rhizomes of C. aromatica contain phytosterols, alkaloids and saponins ⁸. C. longa is commercially very popular species which is used in cookery, cosmetology and medicinally. C. longa consists of curcuminoids, glycosides, terpenoids, and flavonoids. These phytochemicals are responsible for its therapeutic effect. Curcumin present in C. longa, and C. aromatica, an effective anti-inflammatory agent inhibited lysosomal enzymes and is effective in inhibiting lipid peroxide formation. It has cytotoxic and antioxidant activities. It reduces cholesterol level and helps control blood sugar ⁹.

TABLE 6: GC-MS PROFILE OF C. OLIGANTHA (FROM MAHIYANGANAYA)

The essential oil of C. aromatica rhizome possesses 7-methanoazulene (13.75%) and curcumene (25.71%). Various extracts of C. aromatic possesses 7-methanoazulene and curcumene; hexane extract {7–methanoazulene (21.81%) and curcumene (30.02%)} and ethyl ecetate extract {7–methanoazulene (35.08%) and curcumene (13.65%)} ¹⁰. They can be easily differentiated with TLC or gas chromatography (GC) due to the presence of camphene and camphor and high boiling alcohol in the volatile oil of C. aromatics which are absent in C. longa. The rhizome of C. oligantha possesses aliphatic constituents, β-sitosterol, curcumin, etc ¹¹. C. oligantha is a poorly explored plant and Sri Lankan study of the plant has not been found. Therefore, the GC-MS profile of C. oligantha was obtained and reported in Table 6. Major chemical constituents found in the rhizome of C. oligantha are caryophyllene (15.07%), phytol (13.38%) and humulene (8.24%). Phytol and humulene inhibit inflammatory responses by reducing cytokine production ^{12, 13}. Dry rhizome of C. zedoaria under steam distillation shows 1.0-1.5% greenish red colour volatile oil which contains monoterpenes (cineol, borneol, camphene etc.), sesquiterpenes (d-camphor). Rhizome of C. zedoaria contains 1.5% d-α pinene, 3.5% d-camphor, 9.6% cineol, 4.2% d-camphor, 1.5% d-borneol, 10.0% sesquiterpene, 48.0% sesquiterpene-alcohols, 21.0% residue. Classified chemical compounds in five Curcuma species were reported in the Table 7 ^{14, 15, 16, 17, 18}.

TABLE 7: CLASSIFIED CHEMICAL COMPOUNDS OF FIVE CURCUMA SPECIES

CONCLUSION: Curcuma species grown in Sri Lanka have significant therapeutic value. But C. albiflora and C. oligantha have not been studied to that extent. To validate the medicinal value of C. albiflora, detailed pharmacological research has to be conducted.

ACKNOWLEDGEMENT: National Science Foundation (Sri Lanka) for funding facility (Grant Number: NSF/SCH/2018/03.

CONFLICT OF INTEREST: Nil

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    How to cite this article:

    Herath HMIC, Wijayasiriwardene TDCMK and Premakumara GAS: GC-MS identification of endemic herb Curcuma albiflora Thw. (Sri Lanka). Int J Pharmacognosy 2018; 5(7): 419-25. doi link: http://dx.doi.org/10.13040/IJPSR.0975-8232.IJP.5(7).419-25.

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