GAS CHROMATOGRAPHY-MASS SPECTROMETRY ANALYSIS OF KYLLINGA TRICEPS

Received on 12 January, 2014; received in revised form, 02 March, 2014; accepted, 26 April, 2014; published 01 July, 2014

GAS CHROMATOGRAPHY-MASS SPECTROMETRY ANALYSIS OF KYLLINGA TRICEPS

Siddabathuni Aneela ⃰ ¹, Akalanka Dey ² and Somnath De ¹

Dr. Samuel George Institute of Pharmaceutical Sciences¹, Markapur- 523316, Andhra Pradesh, India

Annamalai University, Department of Pharmacy ², Annamalai Nagar-600 802, Tamil Nadu, India

Keywords:

GC-MS analysis,Cyperaceae, Methanol extract, Soxhlet extractor

INTRODUCTION: In developing countries like India, the majority of people living in rural areas were almost exclusively using traditional medicines in treating all sorts of disease ¹. Plants make a significant contribution to health care due to the recognition of the value of traditional medicinal systems ^2-3.

Now a day’s more light is being shed on the importance of medicinal plants, many of which have been used as more traditional or folk remedies, on the other hand, they are being studied and analyzed for potential biological activities that will thus explain why the local have been used them for treating various disease and illness less ⁴. Herbal medicine is safer than synthetic medicines because the phytochemicals in the plant extract target the biochemical pathway ⁵. Plant – based natural constituents can be derived from any part of the plant like bark, leaves, flowers, roots, fruits and seeds etc ⁶. The medicinal actions of plants unique to particular plant species or groups are consistent with the concept that the combination of secondary products in a particular plant is taxonomically distinct ⁷. Screening of active compounds from plants has led to invention of new medicinal drugs which have efficient protection and treatment roles against various diseases, including cancer ⁸, heart disease ⁹, febrifuge and antidermatosis, also used to treat diabetes. The determination of phytoconstituents is largely performed by the relatively expensive and often laborious techniques such as gas (GC) and liquid chromatography (LC) combined with specific detection schemes ¹⁰.

In the last few years, GC-MS has become firmly established as a key technological metabolic profiling in both plant and non-plant species ^11-13. Kyllinga triceps is an herb belonging to the family Cyperaceae. It grows in moist places. Fresh juice of the plant is used externally to wash the wounds. It is used in the treatment of indigestion ¹⁴. Decoction of roots is used in diabetes and to relieve thirst in fevers ¹⁵. The roots yield oil which is used to promote the action of the liver and relieve pruritus ¹⁶. The literature search reveals that still no work have been done on this plant. Nobody has isolated this crude extracts from methanolic solvent and analyse the crude extract by GC-MS.  With this knowledge the present study was intended to determine the phytochemical profile of the extract of Kyllinga triceps using GC-MS.

MATERIALS AND METHODS:

Collection of the plant material: The plant Kyllinga triceps was collected from the Tirunelveli Dist, Tamil Nadu, India in the month December 2012. The plant materials was identified and authenticated by Dr. V. Chelladurai, Retired Research officer-botany, Central Council For Research In Ayurveda and Sidha (C.C.R.A.S). Govt. of India, Tiruneveli. The collected plant material was free form disease and also free from contamination of other plants.

Preparation of plant extract: 100 gms of Kyllinga triceps air dried and coarsely powdered entire plant material was extracted with 500 ml methanol by using Soxhlet extractor. The sample was kept in dark for 72 hrs with intermittent shaking. Then the solvent was evaporated under reduced pressure using rotary evaporator and to obtained viscous semi solid masses (g).

Phytochemical Screening: The methanolic extract was tested for steroids, alkaloids, sugar, phenolic compounds, flavonoids, saponins, tannins, anthraquinone and amino acids. Phytochemical screening of the extract was carried out according to the standard method ¹⁷.

GC-MS analysis: The GC-MS analysis of methanolic crude extract of Kyllinga tricepswas performed using a GC–MS equipment Thermo GC-TRACE ultra ver: 5.0, Thermo MS DSQ II. Experimental conditions of GC-MS system were as follows: TR 5-MS capillary standard non-polar column, dimension: 30Mts, ID: 0.25 mm, Film: 0.25µm was used and flow rate of mobile phase (carrier gas: He) was set at 1.0 ml/min. In the gas chromatography part, temperature programme (oven temperature) was 40^oC raised to 250^oC at 5^oC / min and injection volume was 1 micro litre. Samples which dissolved in chloroform were run fully at a range of 50-650 m/z and the results were compared by using Wiley spectral library search programme.

RESULT & DISCUSSION: Preliminary phytochemical screening of nine different metabolites (steroids, alkaloids, sugars, phenolics, flavonoids, saponins, tannins, anthraquinone and amino acids) were tested with methanolic extract of Kyllinga triceps. Hence the results showed the presence of steroids, alkaloids, sugar, phenolics, flavonoids, saponins, tannins and amino acids but not anthraquinone. The result of phytochemical screening of different secondary metabolites of Kyllinga triceps were showed in Table 1.

TABLE 1: PRELIMINARY PHYTOCHEMICAL SCREENING OF METHANOLIC EXTRACT OF KYLLINGA TRICEPS

The results are relevant to GC-MS analysis leads to the identification of number of compounds from GC fractions of the methanolic extracts of Kyllinga triceps. They were identified through mass spectrometry attached with GC.  GC-MS analysis of methanolic extract of Kyllinga triceps was putted into a Table 2. The various components present in the methanolic extract of Kyllinga triceps that were detected by the GC-MS are shown in Table 2.

TABLE 2: COMPOUNDS PRESENT IN THE METHANOLIC EXTRACT OF KYLLINGA TRICEPS USING GC-MS ANALYSIS

The results revealed  that the presence of 22 different phytocompounds viz., (E)-2-ethylidene-3-oxo-GA9 16á, 17-epoxide methyl ester (2.94 %), 3-(4'-Bromophenyl)-5,6-diphenylimidazo[2,1-b]thiazole (0.68 %), [22,23,24-trideuterio]-3á,14à,25-trihydroxy-5á-cholest-7-en-6-one (0.80 %), (Z)-4-chloro-2-methyl-1-phenylbut-3-en-1-one (2.05 %), cyclohexasiloxane, dodecamethyl-(0.72 %), 2-Propenoic acid, 1,7,7-trimethylbicyclo[2.2.1]hept-2-yl ester, exo-(4.08 %), Phenol, 2,4-bis(1,1-dimethylethyl)- (CAS)- (0.43 %), Tricyclo[4.2.1.1(2,5)]dec-7-en-9-ol (6.62 %),  Methanone, (1-hydroxycyclohexyl)phenyl-(14.88 %), 4-{2,6-Dibromo-4-[(tert-butyl)azo]phenyl}morpholine (0.47 %), Hexadecanoic acid, methyl ester (CAS) –(0.44%), n-deutero-3-ethyl-3-phenyl-2,6-dioxopiperidine-(5.19%) , 9,12-Octadecadienoyl chloride, (Z,Z)-(0.35 %), 1-(2-Allyl-phenoxy)-3-[4-(2-hydroxy-ethyl)-piperazin-1-yl]-propan-2-ol (1.29 %), 6-(2-Trimethylsilylethynyl)-2-(4-carbomethoxypyrazin-1-yl)pyridine (0.32 %), 3-Hydroxy-2-(p-tolyl)-3-phenylylisoindolin-1-one (0.44 %), 3-methyl-4-(selenophenylmethyl)cinnoline (0.34 %), Cholesta-5,7,9(11)-trien-3-ol, 4,4-dimethyl-, (3á)-( 0.50 %), 3-(3",4",5"-Trimethoxyphenyl)diosmetin(0.71%), 6-(t-Butylimino)-8-(3'-trifluoromethylphenyl)-3,4-dihyd ro-2H, 6H-pyrimido[2,1-b][1,3]thiazine-7-carbonitrile (0.61 %), diisooctylphthalate (1.89 %). The GC-MS spectrum confirmed the presence of 22 major components with the retention time 0.39, 7.46,  9.35, 12.62, 13.25, 14.33, 17.13, 17.70, 20.58, 22.43, 25.71, 27.46, 29.01, 29.32, 32.72, 33.42, 36.01, 36.55, 36.86, 37.15, 37.62, and  37.97 respectively (Fig. 1).

FIGURE 1: GC-MS CHROMATOGRAM OF METHANOLIC EXTRACT OF KYLLINGA TRICEPS

The name, molecular weight, molecular formula and structure of the component of the test materials were ascertained. The relative percentage amount of each component was calculated by comparing its average peak area to the total areas.

CONCLUSION: The present study results confirmed the presence of steroids, alkaloids, sugar, phenolics, flavonoids, saponins, tannins and amino acids. It helps to predict the formula and structure of biomolecules which can be used as drugs. It enhances the traditional usage of Kyllinga triceps which possesses several known and unknown bioactive compounds. Further investigation may lead to the development of drug formulation. Evaluation of pharmacological activity in the methanolic extract is in progress.

ACKNOWLEDGEMENT: The authors are thankful to the Management of Dr. S.G.I.P.S, Markapur for providing necessary facilities to carry out the research work and also thankful to Radiant Research Services Pvt. Ltd., Bangalore for carrying out GC-MS analysis of the sample.

REFERENCES:

1. Syder JD, Merson – MH: The magnitude of the global problems of acute diarrheal disease a review of active surveillance data, bulletin of the world health organization 1982; 60:605 – 13.
2. Mohamed Saleem TK, Azeem AK, Dilip C, Sankar C, Prasanth NV, Duraisami R: Anti-inflammatory activity of leaf extract of Genderussa vulgaris Nees, Asian pacific journal of tropical biomedicine 2011; 1(2): 147-149.
3. Pour BM, Sasidharan S: In vitro toxicity study of Lantana camara, Asian Pacific Journal of Tropical Biomedicine 2011; 1(3): 189-191.
4. Tiwari S: Plants: A rich source of herbal medicine. Journal of natural products 2008; 1: 27-35.
5. Zaidan MRS, Rain NA, Badrul AR, In vitro screening of five local medicinal plants for anti-bacterial activity using dise diffusion method. Trop biomed 2005; 22:165-170.
6. Gordon DM. Geographical structure and host specificity bacteria and the implication for tracing the source of coli form contamination. Microbiology 2001; 147: 1079-1085.
7. Wink DA, Vodovotz Y, Grisham MB, Degraff W, Cook JC, Pacelli R et al: Anti-oxidant effect of nitric oxide. Methods enzymol 1999; 301: 413-424.
8. Sheeja K, Kuttam G: Activation of cytotoxic T lymphocyte responses and attenuation of tumor growth in vivo by Andrographis paniculata extract and andrographilide, Immunopharmacol Immunotoxicol 2007; 29: 81-93.
9. Mukherjee PK, Kumar V, Houghton PJ: Screening of Indian medicinal plants for acetyl cholinesterase inhibitory activity phytother Res 2007; 21: 1142-1145.
10. Eisenhauer N, Klier M, Partsch S, Sabais ACW, Sclerber C, Weisser W, Scheu S: No interactive effects of pesticides & plant diversity on soil microbial biomass & respiration. Applied soil ecology 2009; 42: 31-36.
11. Kell BD, Brown M, Dunn WB, Spasic I, Oliver SG:Metabolic footprinting & System Biology: The medium is the message. Nature reviews microbiology 2005; 3: 557-565.
12. Janakiraman N, Johonson M, Sahaya Sathish S: GC-MS analysis of bioactive constituents of Peritrophe bicalyculata (Retz.) Nees. (Acanthaceae). Asian Pacific Journal of Tropical Biomedicine 2012; S46- S49.
13. Sahaya Sathish S, Janakiraman N, Johnson M: Phytochemical analysis of Vitex altissina L. using UV-VIS, FTIR and GC-MS. International Journal of Pharmaceutical Sciences and Drug research 2012; 4(1): 56-62.
14. Tirkey A, Khan F, Khan SS & Saify T: Medicinal plants used in treatment of indigestion in Raigarh District of Chhattisgarh. In: Biodiversity and sustainable utilization of biological resources, Scientific Publishers, Jodhpur, India. 2004; 99-104.
15. Jitendra B. J, Sheetal C K, Bhattacharya S: Medicinal flora of Madhya Pradesh and Chattisgarh – A review – Indian Journal of traditional knowledge 2006; 5(2):237- 242.
16. Kirtikar K.R, Basu B.D: Indian Medicinal Plants, International book distributors, Rajpur road, Dehradun, India 2nd Ed, 1987; 2633- 2634.
17. Brindha P, Sasikala B, Purushothaman KK: Pharmacological studies on Merugan kizhangu. Bulletin of medico-Ethno-Botanical-Research 1981; 3: 84-96.