International Journal of Pharmacological Research ISSN: 2277-3312 www.ssjournals.com Journal DOI:10.7439/ijpr Bioactive Compound Evaluation of Ethanol Extract from Geodorum densiflorum (Lam.) Schltr. by GC-MS analysis Keerthiga M* and Anand S. P Department of Biotechnology, National College (Autonomous & CPE), Tiruchirappalli – 01, Tamil Nadu, India Corresponding author* Keerthiga Manohar, Research Scholar, Department of Biotechnology, National College (Autonomous & CPE), Tiruchirappalli – 01, Tamil Nadu, India E-mail: keerthigamanohar@gmail.com Abstract The phytochemical constituents are responsible for medicinal value of the plant species. The present investigation was carried out to analyze the bioactive components from the whole plant of Geodorum densiflorum (Lam.) Schltr. using GC-MS technique. The chemical compositions of the ethanolic extract of G. densiflorum were investigated using Perkin - Elmer Gas Chromatography – Mass Spectrometry and about twenty one bioactive phytochemical compounds were identified. The prevailing compounds where Hexadecanoic acid, Ethyl ester (38.884 %), Ionone (7.125 %), 3-Deoxy-d-mannoic lactone (7.4441 %), 2,3-Butanediol (4.725 %) and 2-Piperidinone, N-[4-bromo-n-butyl]- (4.004 %), (E)-9-Octadeconoic acid ethyl ester (3.891 %), 1H-Pyrrole-2-Carbonitrile (3.778 %), Pyridinium, 1-amino-, chloride (3.305 %), 4H- Pyran-4-one, 3,5-dihydroxy-2-methyl- (3.274) and having various biological activities. This was the first report on the identification of bioactive compounds from ethanol extract of G. densiflorum. Keywords: Phytochemical Compounds, GC-MS analysis and Geodorum densiflorum. 1. Introduction From ancient times higher plants are best sources of bioactive compounds, play principal role in the maintenance of human health. Literature studies represent the herbal plants are reservoir of effective chemotherapeutants, these are more systemic, easily biodegradable and non-phytotoxic.[1]-[3] In general, secondary metabolites from plants were having interesting biological activities. These secondary metabolites are act as lead compound for new drugs because of its variety of structural arrangements and properties.[4] Knowledge on the phytoconstituents of plants is desirable for the discovery of therapeutic agents, new sources of economic phytocompounds for the synthesis of complex chemical substances and for disclosing the actual significance of folkloric remedies.[5] The standardization of the natural drugs has emerged as a new branch of science as the phytochemicals have complementary and overlapping mechanism of action; hence a thorough validation of the herbal drugs was emphasized and prioritized. Mass Spectrometry coupled with chromatographic separations, Gas Chromatography (GC/MS) is normally used for direct analysis of chemical components existing in herbal medicines. For the analysis of medicinal plants, GC-MS technique have been proved to be highly commended analysis for non-polar components and volatile essential oil, fatty acids, lipids[6] and alkaloids[7]. Recently there has been tremendous progress in medicinal plants research; however orchids have not been exploited fully for their medicinal application. Recent researches reported the presence of Anthocyanins, Stikbnoids, Triterpenoids, Orchinol, Hircinol, Cyperpedinl, Jibantine, Nidemin, Lorogcossin, Gymopusin in Bulbophyllum gumpous; alkyl perulate and β-sistosero D-glucoside in Vanda tessellate; Alkanes and Alkanol Sistosterol, Resin, Saponin, Tannins, Fatty Acids, Colouring Agents etc.[8] Geodorum densiflorum (Lam.) Schltr. (Family: Orchidaceae) is an endangered terrestrial orchid widely distributed in humid tropical forests of south India. Literature studies indicate that no reports on G. densiflorum has so far been undertaken to provide enough scientific data in favour of reported traditional uses. Traditional use varies among the local practitioners for menstrual cycle regulation, joint pain and arthritis, diabetic, applied externally to cure Carbuncles, etc.[9] As part of the endeavor for search of therapeutic properties of G.densiflorum we herein reported the GC-MS analysis of ethanol extract from the whole plant. 2. Materials and Methods The plant Geodorum densiflorum (Lam.) Schltr. were collected from Periyakombai hill area of Kolli hills in Namakkal District of Tamil Nadu, India. The botanical identity of this plant was confirmed by Dr. G. V. S. Murthy, Scientist – F & Head of Office, Botanical Survey of India (Southern Circle), Coimbatore, Tamil Nadu. The voucher IJPR Volume 5 Issue 6 (2015) 139 Keerthiga M and Anand S. P Research Article specimen was submitted at the Department of Botany, National College (Autonomous), Tiruchirappalli-620 001, Tamil Nadu, India. 2.1 Plant Sample Extraction The whole plant material was shaded dried and made into fine powder. 20 g powdered plant material is soaked in 50 ml of absolute ethanol overnight and then filtered through whatmann filter paper No.41 along with 2gm Sodium Sulfate to remove the sediments and traces of water in the filtrate. Before filtering, the filter paper along with Sodium Sulfate is wetted with ethanol the filtrate is then concentrated and reduces the volume to 1ml the extract contains both polar and nonpolar Phytocomponents. 2.2 GC-MS Analysis GC MS analysis was carried out on a GC clarus 500 Perkin Elmer system comprising a AOC-20i auto sampler and Gas Chromatography interfaced to a Mass Spectrometer (GC-MS) instrument employing the following conditions: column RTX 5Ms (Column diameter is 0..32mm, column length is 30 m and column thickness 0.50 μm), operating in electron impact mode at 70eV; Helium gas (99.999%) was used as carrier gas at a constant flow of 1.73 ml /min and an injection volume of 0.5 μl was employed (split ratio of 10:1) injector temperature 270 ºC; ion-source temperature 200 ºC. The oven temperature was programmed from 40 ºC (isothermal for 2 min), with an increase of 8ºC/min, to 150 ºC, then 8 ºC/min to 250 ºC, ending with a 20 minutes isothermal at 280 ºC. Mass spectra were taken at 70eV; a scan interval of 0.5 seconds and fragments from 40 to 450 Da. Total GC running time is 26 minutes. The relative percentage amount of each component was calculated by comparing its average peak area to the total areas. Software adopted to handle mass spectra and chromatograms was a Turbo Mass Ver 5.2.0 [10]. 2.3 Identification of components Interpretation on GCMS was conducted using the database of National Institute Standard and Technology (NIST) having more than 62,000 patterns. The spectrum of the unknown component was compared with the spectrum of the known components stored in the NIST library. The name, molecular weight and structure of the components of the test materials were ascertained (Dr. Duke’s. Phytochemical and Ethnobotanical Databases). 3. Results Twenty one compounds were identified in ethanol extract of G. densiflorum by GC-MS analysis. The bioactive compounds with their Retention Time (RT), Molecular structure, Molecular Formula, Molecular Weight, Concentration (peak area %), nature of compounds and biological activity are presented in (Table 1 & 2 and Fig 1). The prevailing compounds where Hexadecanoic acid, ethyl ester (38.884 %), Ionone (7.125 %), 3-Deoxy-d-mannoic lactone (7.4441 %), 2,3-Butanediol (4.725 %) and 2-Piperidinone, N-[4-bromo-n-butyl]- (4.004 %), (E)-9-Octadeconoic acid ethyl ester (3.891 %), 1H-Pyrrole-2-Carbonitrile (3.778 %), Pyridinium, 1-amino-, chloride (3.305 %), 4H- Pyran-4-one, 3,5-dihydroxy-2methyl- (3.274), Undecanal, 2-methyl- (2.128 %), 5-Hydroxymethylfurfural (2.005 %), Propanoic acid, 2-hydroxyl-, 2methylpropyl ester (1.628 %) and p-Ethoxybenzyl alcohol (1.457 %). The presence of various bioactive compounds justifies the use of the whole plant for various ailments by traditional practitioners. However isolation of individual phytochemical constituents and subjecting to biological activity will definitely give fruitful results. Fig-1: Chromatogram obtained from GC-MS with the ethanolic extract of G.densiflorum (Lam.) Schltr. IJPR Volume 5 Issue 6 (2015) 140 Keerthiga M and Anand S. P Research Article Table 1: Components detected in ethanolic extract of G.densiflorum (Lam.) Schltr. Molecular Formula Molecular Weight Peak Area % 5-Dodecanol C12H26O 186.334 0.132 4.061 1-Pentanol C5H12O 88.15 0.437 3 7.154 Guanidine CH5N3 59.07 0.261 4 8.532 D-Mannitol, 1-o-heptyl C13H28O6 280.357 0.275 5 9.440 2,5-Dimethyl-5-ethyl-2oxazoline C7H13NO 127.184 0.346 6 10.705 2-Piperidinone, N-[4bromo-n-butyl]- C9H16BrNO 234.136 4.004 7 12.416 1,2-Cyclopentanediol, 3methyl- C6H12O2 116.158 0.305 8 12.726 Ionone C13H20O 192.30 7.125 9 14.126 Propanoic acid, 2hydroxy-, 2-methylpropyl ester C7H14O3 146.184 1.628 10 14.791 2,3-Butanediol C4H10O2 90.121 4.725 11 15.239 Pyrimidine-4,6(3H, 5H)dione, 2-buttylthio- C8H12N2O2S 200.258 0.401 12 16.236 Undecanal, 2-methyl- C12H24O 184.318 2.128 13 17.450 1,3-Dioxolane, 2pentadecyl- C18H36O2 284.477 0.553 14 19.030 1H-pyrrole-2-Carbonitrile C5H4N2 92.1 3.778 15 20.189 Pyridinium, 1-amino-, chloride C5H7N2.Cl 138 3.305 16 20.656 Hexadecanoic acid, ethyl ester C18H36O2 284.477 38.884 S. No RT Compound Name 1 4.020 2 IJPR Volume 5 Issue 6 (2015) Molecular Structure 141 Keerthiga M and Anand S. P Research Article 17 21.494 4H-Pyran-4-one, 3,5dihydroxy-2-methyl- C6H6O4 142.109 3.274 18 21.853 3-Deoxy-d-mannoic lactone C6H10O5 162.140 7.444 19 22.821 (E)-9-Octadeconoic acid ethyl ester C20H38O2 310.514 3.891 20 23.933 5-Hydroxymethylfurfural C6H6O3 126.11 2.005 21 25.371 P- Ethoxybenzyl alcohol C9H12O2 152.190 1.457 Table 2: GC-MS analysis showed phytochemical compounds, their nature and their biological activities of ethanol extract of G. densiflorum. S. No Compound Name Nature of Compound Biological Activity 1 2 5-Dodecanol 1-Pentanol Fatty alcohol Alcohol 3 Guanidine Nitrogenous Compound 4 d-mannitol, 1-o-heptyl 2,5-Dimethyl-5-ethyl-2oxazoline 2-Piperidinone, N-[4-bromon-butyl]1,2-Cyclopentanediol, 3methyl- Sugar alcohol Heterocyclic Compound Alcoholic Compound Antimicrobial [16] Ionone Monoterpenes Antibacterial, Sedative, Antitumor, Cytotoxic, Antiinflammatory, Insecticidal and Molluscidal activity [17] Alkene Compound Anticancer activity [18] Volatile Compound [19] 5 6 7 8 9 10 Propanoic acid, 2-hydroxy-, 2-methylpropyl ester 2,3-Butanediol Alkaloid Flavour Antimicrobial Anticancer, Antidiabetic, Antiviral, Antiinflammatory,Antibiotic,Antileishmenial, Antiprotozoal, Antihistaminic and Antihypertensive Osmotic diuretic agent, Renal vasodilator Antibacterial, Anti-inflammatory, Anti-tumour and Antioxidant Antimicrobial, Antioxidant, Anti-inflammatory References [11] [11] [12] [13] [14] [15] 11 Pyrimidine-4,6(3H, 5H)dione, 2-buttylthio- Heterocyclic Compound 12 13 Undecanal, 2-methyl1,3-Dioxolane, 2-pentadecyl- 14 1H-pyrrole-2-Carbonitrile Aldehydes Aromatic Compound Heterocyclic aromatic Compound Pesticides Hypoglycemic, Antimalarial, Analegesic, Antiinflammatory, PDE4 inhibitor and Spasmolytic activity Pesticides, Flavors and Fragrances Antibacterial and Antifungal activity Hypolipidemic, Antimicrobial, Anti-inflammatory and Antitumor activity 15 Pyridinium, 1-amino-, chloride Nitrogen Compound Antimicrobial and Anti-inflammatory activity [24] 16 Hexadecanoic acid, ethyl ester Palmitic acid ester Antioxidant, Hypocholesterolemic, Nematicide, Pesticide, Lubricant, Anliandrogenic, Flavor, Hemolytic, 5 Alpha reductase inhibitor [25] Flavonoid Fraction Antimicrobial, Anti-inflammatory and Antioxidant Cyclic ester Antimicrobial activity Anti-inflammatory, Anticancer, hypocholesterolemic, 5-Alpha reductase inhibitor and antiandrogenic activity Antioxidant, Antiproliferative activity Antimalarial 17 18 4H-Pyran-4-one, 3,5dihydroxy-2-methyl3-Deoxy-d-mannoic lactone 19 (E)-9-Octadeconoic acid ethyl ester Oleic acid ester 20 21 5-Hydroxymethylfurfural P- Ethoxybenzyl alcohol Sugar Compound Alcoholic Compound IJPR Volume 5 Issue 6 (2015) [20] [21] [22] [23] [16] [26] [27] [28] [29] 142 Keerthiga M and Anand S. P Research Article 3. Discussion The identified compounds with more percentage like Hexadecanoic acid, ethyl ester (Palmitic acid ester) (38.884 %), Ionone (Monoterpenes) (7.125 %), 3-Deoxy-d-mannoic lactone (Cyclic ester) (7.4441 %), 2,3-Butanediol (Volatile Compound) (4.725 %) and 2-Piperidinone, N-[4-bromo-n-butyl]- (Alkaloid) (4.004 %),showed a wide range of potent bioactivity. Among the twenty one identified compounds 10 showed Anti-microbial activity, 9 showed Anti-inflammatory, 6 showed Anti-cancer and 4 showed anti-oxidant and also showed activities such as hypocholesterolemic, antiandrogenic, Anliandrogenic, antiproliferative, Hemolytic, 5 Alpha reductase inhibitor, Hypolipidemic, Antimalarial, Nematicide, Pesticide, Insecticidal and Molluscidal activity and also having compounds for Lubricant and Flavor. Unsaturated fatty acids are important to every cell for normal growth, to support the lubricating quality of skin [30] and to lower cholesterol levels of the blood.[31] Pure isolated plant alkaloids are used as basic medicinal agent for analgesic, anti-spasmodic, antibacterial properties, etc. For example Quinine (alkaloid) extracted from Cinchona is used to treat malaria.[32] The previous study on G. densiflorum showed antibacterial, antioxidant, cytotoxic, thrombolytic, sedative, analgesic and anxiolytic activities.[33] The presences of these phytocompound are responsible for the properties of plant. 4. Conclusion The GC-MS studies carried out on ethanol extract of Geodorum densiflorum (Lam.) Schltr. showed the presence of bioactive compounds responsible for potent activity of medicinal orchid. Further work regarding specific activity of the identified compound will provide more insight about the role of plant. References [1] Vyas GD. Soil Fertility Deterioration in Crop Land Due to Pesticide. Journal of Indian Botanical Society, 1999; 78: 177-78. [2] Kaushik JC, Arya Sanjay, Tripathi NN, Arya S. Antifungal properties of some plant extracts against the damping off fungi of forest nurseries. Indian Journal of Forestry, 2002; 25: 359-361. [3] Chaman Lal and Verma LR. Use of certain bio-products for insect-pest control. Indian Journal of Traditional Knowledge, 2006; 5(1): 79- 82. [4] de-Fatima A, Modolo LV, Conegero LS, Pilli RA, Ferreira CV, Kohn LK, de-Carvalho JE. Lactones and their derivatives: biological activities, mechanisms of action and potential leads for drug design. Curr. Med. Chem. 2006; 13: 3371-84. [5] Milne A. Inhalational and local anesthetics reduce tactile and thermal responses in Mimosa pudica Linn. Masui, 1993; 1190-3. [6] Jie MSF and Choi CYC. J. Int. Fed. Clin. Chem 1991; 3: 122. [7] Betz JM, Gay ML, Mossoba MM, Adams S and Portz BS. Chiral gas chromatographic determination of ephedrinetype alkaloids in dietary supplements containing Má Huáng. J AOAC Int 1997; 80: 303. [8] Kumar S, Subramoniam A and Pushpangadan P. Aphrodisiac activity of Vanda tesellata extract in mice. Indian Journal of Pharmacol. 2000; 32: 300-04. [9] Keerthiga M and Anand SP. Physicochemical, Preliminary Phytochemical Analysis and Antibacterial Activity against Clinical Pathogens of Medicinally Important Orchid Geodorum densiflorum (Lam) Schltr. International Journal of Pharmacy and Pharmaceutical Sciences 2014; 6(8): 558-61. [10] Srinivasan R, Natarajan D and Shivakumar M S. Antimicrobial and GC-MS Analysis of Memecylon edule Leaf Extracts. International Journal of Current Pharmaceutical Review and Research, 2014; 5(1): 1-13 [11] Dr. Duke's Phytochemical and Ethnobotanical Databases. http://www.ars-grin.gov/duke/chem-activities.html [12] Muhammad Said, Amin Badshah, Naseer Ali Shah, Hizbullah Khan, Ghulam Murtaza, Boris Vabre, Davit Zargarian and Muhammad Rashid Khan. Antitumor, Antioxidant and Antimicrobial Studies of Substituted Pyridylguanidines. Molecules, 2013; 18: 10378-96. [13] Ramalakshmi S and Muthuchelian K. Analysis of Bioactive Constituents from the Leaves of Mallotus tetracoccus (Roxb.) Kurz, by Gas Chromatography - Mass Spectrometry. International Journal of Pharmaceutical Science and Research. 2011; 2(6): 1449-54. [14] Shivani Bansal and Halve AK. Oxazolines: Their Synthesis and Biological Activity. IJPSR, 2014; 5(11): 4601-16. [15] Meenakshi VK, Gomathy S, Senthamarai S, Paripooranaselvi M and Chamundeswari KP. GC-MS Determination of the Bioactive Components of Microcosmus exasperatus Heller, 1878. J. Curr. Chem. Pharm. Sc., 2012; 2(4): 271-76. [16] Jananie RK, Priya V, Vijayalakshmi K. Determination of Bioactive Components of Cynodon dactylon by GC-MS Analysis. New York Science Journal, 2011; 4 (4): 16-20 IJPR Volume 5 Issue 6 (2015) 143 Keerthiga M and Anand S. P Research Article [17] Zhang H, Qiu M, Chen J, Sun Y, Wang C, Fong HHS. Plant terpenes, in Phytochemistry and Pharmacognosy section, Chemical Sciences, Engineering and Technology Resources, Encyclopedia of Life Support Systems (EOLSS), Developed under the Auspices of the UNESCO, EOLSS Publishers, Oxford, UK, 2011. (http://www.eolss.net). [18] Paul John Peter M, Yesu Raj J, Prabhu Sicis VP, Joy V, Saravanan J and Sakthivel S. GC-MS Analysis of bioactive components on the Leaves extract of Stylosanthes fruticosa- A potential folklore medicinal plant. Asian Journal of Plant Science and Research, 2012; 2 (3): 243-53. ord.web.ym.edu.tw/ezcatfiles/b024/img/img/31/916643624.xls [19] Choong-Min Ryu, Mohamed A. Farag, Chia-Hui Hu, Munagala S. Reddy, Han-Xun Wei, Paul W. Paré, and Joseph W. Kloepper. Bacterial volatiles promote growth in Arabidopsis. Proc. Natl. Acad. Sci. USA, 2003; 100: 4927– 32. [20] Yuriy M. Kononevich, Ludmila S. Bobkova, Alexander S. Smolski, Anatoly M. Demchenko. Synthesis and Antioxidant Activity of 7-Thio Derivatives of 6,7-Dihydro-1H-cyclopenta[d]pyrimidine-2,4(3H,5H)-dione. Sci Pharm. 2015; 83: 41–48. [21] Sujatha, Karthika, Sivakamasundari, Mariajancyrani and Chandramohan. GC-MS Analysis of Phytocomponents and Total Antioxidant Activity of Hexane Extract of Sinapis alba. IJPCBS, 2014; 4(1): 112-17. [22] Hatice Baspınar Küçük, Ayse Yusufoglu, Emel Matarac and Sibel Dösler. Synthesis and Biological Activity of New 1,3-Dioxolanes as Potential Antibacterial and Antifungal Compounds. Molecules, 2011. 16: 6806-15 [23] Dharmesh R. Dhameliya and Mukesh C. Patel. Synthesis, Characterization and Biological Activity of Novel Pyrrole Compounds. Rasayan J Chem. 2011; 4(3): 580-84. [24] Vildan Alptüzün, Sülünay Parlar, Hüseyin Taşl and Ercin Erciyas. Synthesis and Antimicrobial Activity of Some Pyridinium Salts. Molecules, 2009; 14: 5203-15. [25] Farina Mujeeb, Preeti Bajpai and Neelam Pathak. Phytochemical Evaluation, Antimicrobial Activity, and Determination of Bioactive Components from Leaves of Aegle marmelos. BioMed Research International. 2014; 1-11 [26] Shobana S, Vidhya VG, Ramya M. Antibacterial activity of garlic varieties (Ophioscordon and Sativum) on enteric pathogens. Curr Res J Biol Sci. 2009; 1: 123–6. [27] Manonmani R and Catharin Sara S. GC-MS Analysis of Bioactive Components of an Important Medicinal Fern Actiniopteris radiata (Swartz) Link. World Journal of Pharmaceutical Research, 2015; 4(4): 1860-69. [28] Zhao.L, Chen J., Su J., Li L., Hu S., Li B., Zhang X., Xu Z and Chen T. In vitro antioxidant and antiproliferative activities of 5-hydroxymethylfurfural. J. Agric. Food Chem. 2013; 61(44): 10604-11. [29] Wright AD, Kong GM, Angerhofer CK, Greenidqe P, Linden A and Desqueyroux-Faundez R. Antimalarial activity: the search for marine-derived natural products with selective antimalarial activity. J. Nat. Prod. 1996; 59(7): 710-16. [30] Okwu, D. E., & Morah, F. N. I. The potentials of Garcinia kola seed as source for nutraceuticals. J. Med. Arom. Plant Sci, 2006; 28, 605-11. [31] Igwe, O. U., & Okwu, D. E. GC-MS evaluation of bioactive compounds and antibacterial activity of the oil fraction from the seeds of Brachystegia eurycoma (HARMS). Asian Journal of Plant Science and Research, 2013; 3(2), 47-54. [32] Esimone, C. O., Nworu, C. S., Ekong, U. S., & Okereke, B. Evaluation of the antiseptic properties of Cassia alatabased herbal soap. Internet J Altern Med, 2008; 6(1): 1-8. [33] Keerthiga M and Anand SP. A review on Ethnomedicinal, Phytochemical and Pharmacological studies of Geodorum densiflorum (Lam.) Schltr. - an endangered orchid. Journal of Research in Biology, 2014; 4(8):1543-48. IJPR Volume 5 Issue 6 (2015) 144