WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Sivakumar et al. SJIF Impact Factor 5.210 Volume 4, Issue 11, 2031-2039 Research Article ISSN 2278 4357 GC-MS ANALYSIS OF BIOACTIVE COMPONENTS FROM ETHANOL EXTRACT OF ANDROGRAPHIS PANICULATA. V. Sivakumar* and G. Gayathri PG & Research Department of Biochemistry, Adhiparasakthi College of Arts & Science, Kalavai, TN, India. Article Received on 21 Sep 2015, Revised on 10 Oct 2015, Accepted on 31 Oct 2015 *Correspondence for Author Dr. V. Sivakumar PG & Research Department of Biochemistry, Adhiparasakthi College of Arts & Science, Kalavai, TN, India. ABSTRACT Medicinal plants are sources of important therapeutic aids for alleviating human ailments. Andrographis paniculata is one of the medicinally important plants belonging to the family Acanthaceae, commonly known as Nilavembu in Tamil. The present investigation was carried out to determine the chemical components present in the Andrographis paniculata medicinal plant by GC-MS technique. The major phytocomponents found in 3,7,11,15-tetramethyl-2-hexadecen- 1-ol (14.82), phytol (9.67), 2,6,10,14,18,22-tetracosahexaene, 2,6,10,15,19,23-hexamethyl-, (all-e) (2.80), heptacosane (3.99), 2,4,4- trimethyl-3-hydroxymethyl-5a-(3-methyl-but-2-enyl)-cyclohexene (10.54), 10,12-tricosadiynoic acid, trimethylsilyl ester (4.12), tetratetracontane (18.73), 3beta.-hydroxyguaia-4(15),10(14),11(13)- trien-6,12-olide 8-(.alpha.,.beta.-dihydroxybutyrate) (11.66), dl-.alpha.-tocopherol (4.42), pentatriacontane (4.45), beta.-sitosterol (7.73). The presence of some of these constituents in the plant extract provides the scientific evidences for the antimicrobial, antitumor, antiviral, antispasmodic, antiperistatic, stomachic and antioxidant properties of the plant. KEYWORDS: Andrographis paniculata, Phytocompounds, Medicinal plant, GC-MS technique. INTRODUCTION Medicinal plants and natural products have been used by human beings since early ages in folk medicine due to their therapeutic potential and the search on medicinal plants had to the discovery of noval drugs used against many diseases. Crude phytoextracts are better medicinal alternatives, because plants owe curative value to hoard of chemical substance www.wjpps.com Vol 4, Issue 11, 2015. 2031
present in various plant tissues. [1] At the same time certain plants also synthesised toxic chemicals apparently as primary defence against pathogens and predators. There is considerable evidence indicating that many of plant products are toxic to human. [2] It is thus obvious that effects on risks and curing efficiency revaluation of therapeutic activity of traditional medicinal plants should address both aspects, an assessment of phytoextracts as used traditionally; and the effects of active compounds purified from them. In recent years Gas Chromatography - Mass Spectrum (GC-MS) studies have been increasingly applied for the analysis of medicinal plants as this technique has proved to be a valuable method for the analysis of various chemical compounds. Andrographis paniculata (A. paniculata) is an herbaceous plant of family Acanthaceae, genus Andrographis native to India and Srilanka. It is widely cultivated in southern Asia. Andrographis paniculata or kalmegh is one of the most widely used plants in Ayurvedic formulations. [3] A. paniculata was recommended in Charaka Samhita dating to 175 BC for treatment of jaundice along with other plants in multi plant preparations. [4] It is used as antispasmodic, antiperistatic, stomachic and also an anthelmintic. The juice of fresh leaves is a domestic remedy in the treatment of colic pain, loss of appetite, irregular stools and diarrhea. Long known in traditional Asian medicine as an immune system booster, Andrographis has demonstrated significant activity in fighting common cold, flu and upper respiratory infections. [5] The pharmacological studies suggest anti-inflammatory, [6] antipyretic, [7] anti-viral, [8] immunostimulatory, [9] potential cancer therapeutic agent, [10] antihyperglycemic [11] and antioxidant [12] properties. The herb, A. paniculata is the main source of the bitter principle. The extremely bitter and characteristic taste of A. paniculata of the Acanthaceae family, gives it the term kings of bitters. Several recent studies have validated some of the medicinal properties of this plant and its use in traditional medicine; such properties include its antimicrobial activity, [13] hepatoprotective capacity, [14] antimalarial activity [15] and antdiarrhoeal potential. Hence the aim of the present study is to identify the phytocomponents of this plant and subjecting the methanol extract of the plant leaves to Gas chromatography Mass Spectrum analysis. www.wjpps.com Vol 4, Issue 11, 2015. 2032
MATERIALS AND METHODS Plant Material The medicinal plant Andrographis paniculata were collected from Adhiparasakthi Agricultural College, medicinal garden, kalavai, Vellore district, Tamil nadu, India. The plants were authentified by the herbarium of Botany Directorate in National Institute of Herbal Science, Plant Anatomy Research Center, Chennai. A voucher specimen no: PARC/2015/ 3001. Preparation of plant extract Preparation of plant extract was done by following the method of Anwar. [16] The plant was dried and pulverized to powder in a mechanical grinder. Required quantity of plant sample was weighed, transferred to flask, treated with the ethanol until the powder was fully immersed, incubated over night and filtered through a Whatman No.41 filter paper. Filtrate is then concentrated till dry residue was remained. After weighing the residue, respective amount of methanol was added to make the final solution. This solution was further used for GC-MS analysis. GC - MS Analysis of bioactive compounds from Andrographis paniculata The methanol extract obtained from sample was subjected to Gas Chromatography and Mass Spectroscopy for the determination of bioactive volatile compounds. Some of the important features are summarized below. GC-MS analysis of the sample was carried out using Perkin Elmer Make clarus 680 with Elite-5MS (30.0 m, 0.25mmID, 250μm df) Column. Helium was used as the carrier gas and the temperature programming was set with Initial temp 60 C for 2 min, ramp 10 C/min to 300 C, hold 6 min., 1 μl samples were injected with split less mode. Mass spectra was recorded over 35-650 amu range with electron impact ionization energy 70 ev. The total running time for a sample was 32.00 min. Quantitative determinations were made by relating respective peak areas to TIC areas from the GC-MS. Identification of phytoconstituents Interpretation on mass spectrum of GC-MS was done using the database of National Institute Standard and Technology (NIST) having more than 71,000 patterns. The mass spectrum of the unknown component was compared with spectrum of known component stored in NIST library. Quantitative determinations were made by relating respective peak areas to TIC areas from the GCMS. The name, molecular weight, retention time and peak area percentage of the test materials was ascertained. www.wjpps.com Vol 4, Issue 11, 2015. 2033
RESULT AND DISCUSSION GC-MS is one of the best techniques to identify the constituents of volatile matter, long chain, branched chain hydrocarbons, alcohols acids, esters etc. The GC-MS analysis of Andrographis paniculata revealed the presence of eleven compounds (Phytochemical constituents) that could contribute the medicinal quality of the plant. The identification of the phytochemical compounds was confirmed based on the peak area, retention time and molecular formula. The active principles with their Retention time (RT), Molecular formula, Molecular weight (MW) and peak area in percentage are presented in Table 1 and Fig 1. Fig 1. GC-MS Chromatogram of ethanol extract of Andrographis paniculata. Eleven compounds were identified in the ethanol extract of Andrographis paniculata. The first compound identified with retention time (16.24min) was 3, 7, 11, 15-tetramethyl -2- hexadecen-1-ol, whereas beta-sitosterol was the last compound which took longest retention time (28.40min) to identify. The prevailing compounds were 3,7,11,15-tetramethyl-2- hexadecen-1-ol (14.82), phytol (9.67), 2,6,10,14,18,22-tetracosahexaene, 2,6,10,15,19,23- hexamethyl-, (all-e) (2.80), heptacosane (3.99), 2,4,4-trimethyl-3-hydroxymethyl-5a-(3- methyl-but-2-enyl)-cyclohexene (10.54), 10,12-tricosadiynoic acid, trimethylsilyl ester (4.12), tetratetracontane (18.73), 3beta.-hydroxyguaia-4(15),10(14),11(13)-trien-6,12-olide 8- (.alpha.,.beta.-dihydroxybutyrate) (11.66), dl-.alpha.-tocopherol (4.42), pentatriacontane (4.45), beta.-sitosterol (7.73). www.wjpps.com Vol 4, Issue 11, 2015. 2034
Table 1: Phytocomponents in the ethanol extract of Andrographis paniculata. S.No. Retention Time Name of the Compound Molecular Formula MW Peak Area (%) 1. 16.24 3,7,11,15-TETRAMETHYL-2- HEXADECEN-1-OL C 20 H 40 O 296 14.820 2. 18.95 PHYTOL C 20 H 40 O 296 9.671 3. 24.40 2,6,10,14,18,22- TETRACOSAHEXAENE, 2,6,10,15,19,23-HEXAMETHYL- C 30 H 50 410 2.807, (ALL-E)- 4. 24.97 HEPTACOSANE C 27 H 56 380 3.990 5. 25.09 2,4,4-TRIMETHYL-3- HYDROXYMETHYL-5A-(3- METHYL-BUT-2-ENYL)- C 15 H 26 O 222 10.549 CYCLOHEXENE 6. 26.18 10,12-TRICOSADIYNOIC ACID, TRIMETHYLSILYL ESTER C 26 H 46 O 2 Si 418 4.125 7. 26.28 TETRATETRACONTANE C 44 H 90 618 18.732 8. 26.41 3BETA.-HYDROXYGUAIA- 4(15),10(14),11(13)-TRIEN-6,12- OLIDE 8-(.ALPHA.,.BETA.- C 19 H 24 O 7 364 11.667 DIHYDROXYBUTYRATE) 9. 26.67 DL-.ALPHA.-TOCOPHEROL C 29 H 50 O 2 430 4.429 10. 27.82 PENTATRIACONTANE C 35 H 72 492 4.457 11. 28.40 BETA.-SITOSTEROL C 29 H 50 O 414 7.731 Figures 2, 3,4 and 5 show the mass spectrum of 3,7,11,15-tetramethyl-2-hexadecen-1-ol (RT -16.24), 2,4,4-trimethyl-3-hydroxymethyl-5a-(3-methyl-but-2-enyl)-cyclohexene (RT- 25.09), tetratetracontane (RT - 26.28), 3beta.-hydroxyguaia-4(15),10(14),11(13)-trien-6,12- olide 8-(.alpha.,.beta.-dihydroxybutyrate) (RT - 26.41) respectively. Figures 2. The mass spectrum of 3,7,11,15-tetramethyl-2-hexadecen-1-ol (RT -16.24). www.wjpps.com Vol 4, Issue 11, 2015. 2035
Figures 3. The mass spectrum of 2,4,4-trimethyl-3-hydroxymethyl-5a-(3-methyl-but-2- enyl)-cyclohexene (RT - 25.09). Figures 4. The mass spectrum of tetratetracontane (RT - 26.28). www.wjpps.com Vol 4, Issue 11, 2015. 2036
Figures 5. The mass spectrum of 3beta.-hydroxyguaia-4(15),10(14),11(13)-trien-6,12- olide 8-(.alpha.,.beta.-dihydroxybutyrate) (RT - 26.41). The phytochemicals identified through GC-MS analysis showed many biological activities relevant to this study are listed in Table 2. The biological activities listed are based on Dr. Duke s Phytochemical and Ethnobotanical Databases created by Dr. Jim Duke of the Agricultural Research Service/USDA. Table 2: GC-MS analysis showed phytochemical compounds, their nature and their biological activities of methanol extract of Andrographis paniculata. S.No. R/T Name of the Compound Activity 1. 16.24 3,7,11,15-TETRAMETHYL-2-HEXADECEN-1-OL 2. 18.95 PHYTOL Anticancer, Cancer preventive, Diuretic 3. 24.40 2,6,10,14,18,22-TETRACOSAHEXAENE, 2,6,10,15,19,23- HEXAMETHYL-, (ALL-E)- 4. 24.97 HEPTACOSANE Antibacterial 5. 25.09 6. 26.18 2,4,4-TRIMETHYL-3-HYDROXYMETHYL-5A-(3- METHYL-BUT-2-ENYL)-CYCLOHEXENE 10,12-TRICOSADIYNOIC ACID, TRIMETHYLSILYL ESTER 7. 26.28 TETRATETRACONTANE Hypoglycaemic, Antioxidant 8. 26.41 3.BETA.-HYDROXYGUAIA-4(15),10(14),11(13)-TRIEN- www.wjpps.com Vol 4, Issue 11, 2015. 2037
6,12-OLIDE 8-(.ALPHA.,.BETA.-DIHYDROXYBUTYRATE) 9. 26.67 DL-.ALPHA.-TOCOPHEROL Antioxidant activity. 10. 27.82 PENTATRIACONTANE Antioxidant activity, 11. 28.40 BETA.-SITOSTEROL Antihyper cholesterolemia, hypoglycaemic activity, Antioxidant, Anticancer, In the present study, 11 phytocomponents have been identified from ethanol extracts of Andrographis paniculata by Gas chromatography and Mass spectrometry analysis. So it is recommended as a plant of phytopharmaceutical importance. However, further studies will need to be undertaken to ascertain fully its pharmacological activity. ACKNOWLEDGEMENT The authors thank to VIT, University, Vellore, Tamilnadu, for providing facilities and support used to carry out the work. REFERENCE 1. Milne A, Inhalational and local anesthetics reduce tactile and thermal responses in Mimosa pudica linn, Masui, 1993; 1190-1193. 2. Yamin Bibi., Sobia Nisa, Abdul Waheed, Muhammad Zia, Sadia Sarwar, Sabbir Ahmed and Fayyaz Chaudhary, M. Evaluation of Viburnum foetens for anticancer and antibacterial potential and phytochemical analysis. African Journal of Biotechnology, 2010; 9(34): 5611-5615. 3. Coon JT and Ernst ET. Andrographi paniculata in the treatment of upper respiratorytract infections: assystemic review of safety and efficacy. Planta Medica, 2004; 70(4): 293-298. 4. Sharma, P. V. Charka Samhita, Ed. Chankhambhia Orientalia, 1983; II: Varanasi. 5. Melchior, J., Spasov, A. A., and Ostrovskij, O. V. Double blind placebo controlled pilot and phase III study of activity of standardized Andrographis paniculta Herba Nees extract fixed combination (Kan jang) in the treatment of uncomplicated upper espiratory tract infection. Phytomedicine, 2000; 7: 341-350. 6. Shen, Y. C., Chen, C. F., and Chiou, W. F. Andrographolide prevents oxygen radical production by human neutrophils: possible mechanism (s) involved in its anti - inflammatory effect. British Journal of Pharmacology, 2002; 135: 399-406. www.wjpps.com Vol 4, Issue 11, 2015. 2038
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