American Journal of Ethnomedicine, 2015, Vol. 2, No. 6 ISSN: 2348-9502 Available online at http://www.ajethno.com © American Journal of Ethnomedicine

Phytochemical Screening and GCMS Studies of the Medicinal Linn.

S. Suresh1, G.Pradheesh2 and Dr. V. Alex Ramani*3

1Assistant Professor, Department of Chemistry, Vetri Vinayaha College of Engineering and Technology, Tholurpatti, Thottiam, Trichirappalli – 621215, Tamilnadu, India. 2Assistant Professor, Department of Chemistry, SNS College of Technology, Coimbatore – 641035, Tamilnadu, India. 3Associate Professor, Department of Chemistry, St. Joseph’s College, Trichirappalli – 620002, Tamilnadu, India

*Corresponding author e-mail: [email protected] ABSTRACT

Objective: The plant Pavetta indica Linn.is variable shrub (or) small tree belonging to the of , reported to have medicinal properties. The leaves and roots of this plant are used in poultices for boils and itches, to cure hemorrhoidal pain, constipation, jaundice etc. The present work is aimed at the phytochemical screening and GCMS Studies for the presence of secondary metabolites like alkaloids, flavonoids, terpenoids, steroids, tannins, etc. Methods: The Phytochemical screening of the leaf extracts were carried out applying the standard methods and tests. It shows the presence of metabolites like alkaloids, carbohydrate, tannins, steroidal glycosides, steroids, flavonoids, etc. The ethanolic extract was subjected to GCMS studies. Results: The phytochemical screening reveals that the both ethanolic and methanolic extracts of Pavetta indica Linn. contains the phytoconstituents - alkaloids, carbohydrate, tannins, steroidal glycosides, steroids, flavonoids, etc. The GCMS analysis of ethanolic extracts indicates the presence of 36 phytoconstituents belonging to the types of acids, alkanes, amines, esters and phenolic compounds. Conclusion: The phytochemical screening and GCMS analysis of the extracts are in good agreement with the presence of alkaloids; four alkaloids are reported to be present by the GCMS studies. The medicinal properties of Pavetta indica Linn. may are attributed to the presence of alkaloids.

Keywords- Medicinal values, GCMS studies, Pavetta indica Linn., Phytochemical screening.

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INTRODUCTION Phytochemical Screening10,11

1,2 The phytochemical screening of the Pavetta indica Linn (Tamil: Kattu leaf extracts were carried out applying the thirani, Panna pavadai, Sirukonnai, Pavattai) standard methods and tests as prescribed by is a shrub or small tree belongs to the family J B Harbone12. Hence, the presence or of Rubiaceace. The leaves very variable absence of various phytoconstituents were elliptic – oblong to elliptic – lanceolate and determined. The experimental procedures obovate – oblong, glossy – green flowers are and the results are given in the Table No -1. white. The roots are said to possess purgative, aperient, diuretic and tonic Gas Chromatography and Mass properties and are prescribed in visceral spectrometry13 obstructions, jaundice, headache, urinary The ethanolic extract was subjected diseases and dropsical affections. The 3 to GC-MS analysis of the instrument GCMS phytochemical investigation , chemical (Schimadz U Japan) with Elite – DB – 5M composition of essential oil4 and physio – 5 Column and the GCMS solution version phytochemical screening had been reported 2.53SV3 software. Initially oven to this plant. The plant was studied and temperature was maintained at 30°C for 2 found to possess anti – inflammatory minutes and the temperature was increased potential6, analgestic7, antimicrobial8 , 9 gradually up to 200ºC at 10.0/35.0 min and antipyretic activities . The aim of the present 4.0 µL of sample was injected for analysis. study was to identify the phytocomponents Helium gas 99.9% of purity was used as of the ethanolic and methanolic extracts of carrier gas as well a elution. The flow rate of the plant leaves applying the GCMS and the helium gas set to 1.5ml /min. The phytochemical screening techniques. temperature was maintained at 230ºC. The sample injector with split ratio was 20 MATERIALS AND METHODS throughout the experiment periods. The mass spectroscopic analysis was done at 70 The leaves of Pavetta indica Linn. eV. The spectra were recorded for mass were collected from Elamanur region (Near range 40 – 1000 m/z for about 35 minutes. Trichy) from the month of July at The separated compounds were identified by 5.00pm.They were identified and comparing their mass spectra with the mass authenticated by the Rapinet Herbarium, St. spectral data of the compounds present in Joseph college (Autonomous)Trichy -02, the data bank. The GCMS chromatogram is Tamilnadu, India. attached in Figure No. 1.

Sample preparation The leaves of Pavetta indica Linn. RESULTS AND DISCUSSION were shade dried and pulverized well. About Phytochemical screening 20g of the plant leaves were soaked in The results of the phytochemical 100ml of ethanol and methanol. It was left screening of the plant Pavetta indica Linn. for 24 hours in order to extract the and its GCMS profiling are presented here. phytoconstituents- alkaloids, carbohydrate, The plant Pavetta indica Linn .was analysed tannins, steroidal glycosides, steroids, qualitatively for the phytochemically active flavanoids, acids and others. The above compounds and the results are given in the extracts were filtered using Whatmann Table No: 2. The ethanolic and methanolic No.1filter paper the residue was removed. extracts of the leaves of Pavetta indica Linn. showed the presence of phytochemically

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American Journal of Ethnomedicine ______ISSN: 2348-9502 active compounds such as alkaloids, peak area ranging from 1 to 5%. The data of carbohydrate, tannins, steroidal glycosides, GCMS studies are given in the Table No: 3. steroids, flavonoids. The following metabolites were analysed to be absent in CONCLUSION the ethanolic and methanolic extracts saponins, sapanin glycosides, cumarin, The results of the phytochemical anthocyanin and flavones. The details are screening revealed that both ethanolic and given in the Table No: 2. methanolic extracts of Pavetta indica Linn. contained the phytoconstituents - alkaloids, GCMS Study carbohydrate, tannins, steroidal glycosides, GCMS analysis was carried out on steroids, flavonoids, etc. the ethanolic extracts of Pavatta indica The GCMS analysis of ethanolic Linn. showed as many as 36 compounds to extracts indicated the presence of 36 present. The lists of compounds are given in phytoconstituents belonging to the types of Table No – 3. The GCMS analysis was done acids, alkanes, amines, esters and phenolic using the instrument GCMS (Schimadz U compounds. Hence, the medicinal plant QP2010 with GCMS solution version 2.53 Pavetta indica Linn had been found to software. The sample volumes was 4.0µL. possess significant phytoconstituents that The sample of ethanolic extract was run for might be attributed to the medicinal 35 minutes. The chromatogram (Figure characteristics. No:1) showed prominent peaks in the retention time ranging 4.0 – 38.0minutes. ACKNOWLEDGEMENT Based on the percentage peak area We acknowledge Dr. M. the compounds 1,2- benzene dicarboxylic Karthikeyan, Principal and Mr. D. Johnson, acid, diethylester(CAS) Ethyl phthalate, 2,4- Head of General Engineering, Vetri Imidazolidinedione, 1-[[(5-nitro-2-furanyl) Vinayaha College of Engineering and methane]amino]-(CAS)upiol, phalic acid, Technology for their constant allyl ethyl ester(CAS) Ethylallylphthalate, 1, encouragement in doing this part of my 3-dioxoline, tartronic acid, (P- research work. Ethoxyphenyl) diethyl ester were found to We place on record once sincere be significantly in higher quantities with the acknowledgement Rev. Fr. Andrew Francis, peak areas ranging from 59.63 to 60%. Principal, St. Joseph’s College The compounds methane, sulfinyl (Autonomous) for permitting us to do bis – (CAS) dimethyl sulfoxide, propane, 2- laboratory facilities in the Department of chloro-(CAS) 2-chloropropane, n-butyric Chemistry. D7acid were observed to be in moderate We gratefully acknowledge the quantities with the peak area ranging from timely help offered by L. Rakesh Sharma 20 to21%. The following compounds 1, 2- and S. Saraswathi doing the GCMS studies benzenedicarboxylic acid, phthalic acid, at the Ampigen Laboratories, Tanjavur- butyl ester, di isobutyl benzene – 1, 2 – 613501, Tamilnadu, India. dicarboxylate, hydrazine, hexadecanoic acid, palmitic acid, octadecanoic acid, strearic REFERENCES acid, 3, 4 – hexanediol, tetradecanoic acid, myristic acid, decanoic acid, capric acid, 1- 1. The Wealth of India, “A dictionary of Indian propanamine, n-propylamine, formamide, raw materials and industrials products, Raw nonane, 3-bromodecane, 4-heptane were materials” 1991, Vol – 7, N – Pe, pp.282. quatified to be in lower amounts with the

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2. Bur Kill, H.M,”The Useful of western 10. Evans W C, Trease and Evans, tropical, Africa”, 1985, Vol – 4. Pharmacology, 15th Edition, Reed Elsevier 3. Ramamoorthy J, Venkatramanan S, Meena India Pvt Ltd., New Delhi, 2006. R, Satkar Prasad and Devi A.P, 11. Harbone J B, A Guide to Modern “Phytochemical investigation of pavetta Techniques of Plant Analysis, 3rd Edition, indica”, Indian Journal of Chemical Kluwer Academic Publisher U.S.A.,1998. Science, 2011,9(1), pp.397 – 402. 12. Harbone J B, Phytochemical Methods, 2nd 4. K Prasad, K.Moulekhi and G Bisht Edition1928. “Chemical composition of the essential oil 13. Ganesh S and Jannet Vennila J., of Pavetta indica Linn.L leaves”, Research Phytochemical Analysis of Acanthus Journal of Phytochemistry, 2011,5,pp.66 – ilicifolius and Avicennia officinalis by GC- 69. MS, Research Journal of Phytochemistry, 5. Ramamoorthy J “Physio – Phytochemical 2011,5(1) pp60 – 65. screening and Diuretic activity of leaves of 14. F.W Mclafferty, Interprewtation of Mass Pavetta indica Linn.Linn” Journal of Spectra, 2nd Edition, W. A. Benjamin. Inc. Pharmaceutical Science and Research, 2010 Publishers, New York,1984. Aug – 1. 15. Prasad K, Moulekhi K and Bisht G., 6. Subhash C , Mandal et al “Evaluation of anti “Chemical composition of the Essential oil – inflammatory potential of Pavetta indica of Pavetta indica Linn. Leaves”, Research Linn.Linn. leaf extract (family: Rubiaceae) Journal of Phytochemistry, 2011, 5(1), in rats”, Phytotherapy Research, Vol 17 pp.66-69. issue 7, pp817 – 820 / D O I 10.1002/ 16. Prasad K and Bisht G., “Evaluation of Ptr.1095. Nutritive, Antioxidant and Mineral 7. Golwala D K et al, “Analgestic activity of Composition of Pavetta indica Linn. Ethanolic leaf Extract of Pavetta indica”, Leaves”, Research Journal of International Journal of Pharmaceutical Phytochemistry, 2011, 5(1), pp.54-59. Science and Drug Research 2009; I(2) 17. N.Bidyanada Sing and N. Saravanan, the pp.119 – 120. effect of Pavetta indica Linn.in CCl4 8. Vinod Kumar Gupta, Charanjeet Kaur, induced Hepatotoxicity in Rats / Pharmacie Aritra Simlai and Amit Roy , “Antimicrobial Globale (IJCP), 2012, 6(04). activity of Pavetta indica Linn.leaves”, 18. Natarajan. P, Thanga thirupathi A, Journal of Applied Pharmaceutical Science, Ramarajan S and Jaya S.,”Preliminary Study 2013 April, Vol – 3 (04), pp.078 – 082 / of Antidiabetic activity of Methanolic DOI : 10.7324/JAPS.2013.3414 extract of Pavetta indica Linn. in diabetic 9. Lakshmi S , Mohana et al,” Antipyretic rats”. Asian Journal of Pharmacetical and Activity of Pavetta indica Linn.Linn clinical Research,2013, 6(1) (Rubiaceae) Leaf Extract”, Selected issues 19. Sujatha S and Gowri Prakash, “Bioactive in ethnopharmacology / Division of Screening and antimicrobial activity of Pharmacognosy and Phytochemistry, flowers from the medicinal plant Pavetta Department of Pharmaceutical Technology , indica Linn.”,International Journal of Jadavapur University, Kolkata – 700032, Current Microbiology and Applied India / Subhash mandal @ yahoo.com . Sciences,2013,5,pp.211-221, http://www. [email protected]. IJCMAS.com

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Table 1. Details of Phytochemical Screening of the extracts of Pavetta indica Linn.

S. No Name of the Test Experimental Procedure Phytoconstituent

0.5 ml of extract was treated with Mayer's reagent 1 a) Mayer's test (potassiomercuric iodide solution) to gave cream colored Alkaloids precipitate. 0.5 ml of extract was treated with Dragendroff’s reagent b) Dragondraff (potassium bismith iodide). Formation of orange or orange red Alkaloids test precipitate was obseved. 0.5 ml of extract was treated with Wager’s reagent (solution of c) Wagner test Alkaloids iodine with KI) and it gave an brown or reddish brown precipitate. 0.5ml of extract was treated with 1ml of alpha – napthol and con. 2 a) Molisch test Carbohydrates H2SO4, which gave purple coloation. 0.5 ml of extract to which equal quantity of Fehling solution – A (copper sulphate) & B (potassium ammonium tartate) were b) Fehling test Carbohydrates added. The content was heating to give brick red precipitate was obtained. Dilute 1ml of alcohol in 0.5 ml of extract. The mixture was diluted 3 Foam test to 20 ml of distilled water. It was shaken well for 15min. The Saponins formation of foam was observed. 0.5 ml of alcoholic or aqueous extracts was treated with lead 4 Lead acetate test Tannins acetate solution. White precipitate was observed. Ferric chloride 0.5 ml of alcoholic extracts was treated with 2 drops of neutral Pseudo Tannin 5 test ferric chloride. brownish green coloration was observed (Condensed tannin) 0.5 ml of extract was treated with aqueous ammonia solution. It 6 Ammonia test Chlorogenic acid was exposed to air which gradually develops a green color. 0.5 ml of extract was treated with aqueous sodium hydroxide 7 NaOH test Anthocyanin solution formation of blue violet coloration 0.5ml of extract was dissolved in 2ml chloroform. The mixture Libermann's 8 was treated with acetic acid, acetic anhydride and conc. H SO Steroidal glycosides Burchard test 2 4 gave bluish green coloration. 0.5 ml of extract was treated with 80% H SO , gave deep yellow 9 H SO test 2 4 Saponins glycosides 2 4 color. 0.5 ml of extract was mixed 2ml of ammonia. The mixture was 10 Ammonia test Flavonoids observed under UV and visible lights - formation of fluorescence. 0.5 ml of alcoholic extract was treated with magnesium foil and 11 Shinoda's test Flavones conc. HCl. It gave intense cherry red coloration 0.5 ml of alcoholic extract was treated with 10% Sodium 12 NaOH test Coumarin hydroxide solution, yellow coloration was observed. 0.5 ml of extract was dissolved in 1ml of chloroform. The mixture 13 Salkowaski test Steroids was treated with conc. H2SO4. It gave red coloration.

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Table 2. Details of Phytochemical Screening of the extracts of Pavetta indica Linn.

Phytochemical Methanol S. No Name of the test Ethanol Extract constituents Extract Mayer’s test + +

Alkaloids Dragondraff test + + 1 Wagner test + + Molish test + +

Fehling test - + 2 Carbohydrates Benedicts test - - 3 Saponins Foam test - - 4 Tannins Lead Acetate test + + Condensed Condensed 5 Pseudo tannins Ferric chloride. Tannin Tannin 6 Chlorogenic acid Ammonia test + + 7 Anthocyanin NaOH test - - Liebermann’s 8 Steroidal Glycosides + + Burchard test

9 Saponins glycosides H2SO4 test - -

10 Flavonoids Ammonia test + +

11 Flavones Shinoda’s test - -

12 Coumarin Sodium chloride test - - Anthracene - 13 Ammonia test - glycoside 14 Steroids Salkowaski test + + Note: + = Present - = Absent

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Table 3. Phytoconstituents of Pavetta indica Linn. by GCMS Study.

Molecular Molecular % Peak Compound S. No RT Name of the compound Formula Weight area Nature Methane, sulfinylbis- (CAS) Dimethyl Organo 1 6.418 C H O S 78 20.52 sulfoxide 2 6 sulphur Propane, 2-chloro- (CAS) 2- 2 6.418 C H Cl 78 20.52 Haloalkane Chloropropane 3 7

3 6.418 n-Butyric-D7 acid C7 H8 92 20.52 Fatty acid 1,2-Benzenedicarboxylic acid, Aromatic di 4 24.191 C H O 194 1.84 dimethyl ester (CAS) Methyl phthalate 10 10 4 carboxylic acid Methyl o- 5 24.191 C H Br Cl O 296 1.84 Ester (Bromochloroacetyl)benzoate 10 8 3 Alkane 6 25.210 Docosane (CAS) n-Docosane - C H 310 1.18 22 46 Alkane 7 25.210 Nonane, 5-methyl-5-propyl C H 184 1.18 13 28

8 25.210 3-Bromodecane C10 H21 Br 220 1.18 Haloalkanes 4-Heptanone, 3-methyl- (CAS) 3- 9 25.210 C H O 128 1.18 Ketone Methyl-4-heptanone 8 16 Alkane 10 25.210 Hexadecane (CAS) n-Hexadecane C H 226 1.18 16 34 hydrocarbon Phthalate 1,2-Benzenedicarboxylic acid, diethyl 11 28.250 C H O 222 59.63 ester ester (CAS) Ethyl phthalate 12 14 4

2,4-Imidazolidinedione, 1-[[(5-nitro-2- Hetero cyclic 12 28.250 furanyl)methylene]amino]- (CAS) C H N O 238 59.63 8 6 4 5 compound Upiol Phthalic acid, allyl ethyl ester (CAS) Phthalate 13 28.250 C H O 234 59.63 Ethylallylphthalate 13 14 4 ester 1,3-dioxolane, 2-phenyL-2- 14 28.250 C H O 240 59.63 Dioxy ether (phenylmethyl)- 16 16 2 tartronic acid, (p-ethoxyphenyl)-, 15 28.250 C H O 296 59.63 Ester diethyl ester 15 20 6 Phthalic acid , butyl ester with ester butyl glycolate (CAS) 1,2-

16 34.38 Benzenedicarboxylicacid , 2- butoxy – C18H24 O6 336 3.52 Ester 2- oxoethyl butyl ester (CAS) butyl (butoxycarbonyl)methyl phthalate Hydrazine, (phenylmethyl ) - (CAS) Amino 17 35.41 C H O 122 3.52 Benzylhydrazine - 95% 8 10 Compound

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18 35.41 Headecanoic acid (CAS) Palmitic acid C16 H32 O2 256 1.19 Fatty acids Octadecanoic acid (CAS) Stearic acid , 19 35.41 C H O 284 1.19 Fatty acids n-OCtadecanoic acid 18 36 2

20 35.410 10-bromo-7-hydroxy-11-iodolaurene C15 H18 Br I O 420 1.19 Alcohol 3,6,9-trimethyl-7-nitro-2,3- Hetro cyclic 21 35.410 C H N O 257 1.19 dihydronaphtho[1,8-bc]pyran 15 15 3 compound 3,4-Hexanediol, 2,5-dimethyl- (cas) Alcohol 22 35.410 C H O 146 1.19 2,5-dimethyl-3,4-hexandiol 8 18 2 -

23 35.410 Tetradecanoic acid (CAS) Myristic acid C14 H28 O2 228 1.19 Fatty acids Saturated fatty 24 35.410 Decanoic acid (CAS) Capric acid C H O 172 1.19 10 20 2 acids

25 35.562 butyl-2-ethylhexyl phthalate C20H30O4 334.44 1.52 Ester 2-methyl-6-beta-d- Hetero cyclic 26 35.562 ribofuranosylimidazo[1,2-c]pyrimidin- C H N O 281 1.52 12 15 3 5 compound 5(6H)-one 3-methylhomoadamantane

27 35.562 Tricyclo[4.3.1.13,8]undecane, 3- C12 H20 164 1.52 Alkane methyl- (CAS) (3R*,4S*)-3-(2-Nitro-4- Hrtro cyclic 28 35.562 methoxyphenyl)-4-(4- C H N O 329 1.52 19 23 4 compound hydroxyphenyl)hexane Butanoic acid, 2-hydroxy-, methyl 29 37.97 C H O 118 2.67 Ester ester (CAS) methyl 2-hydroxybutyrate 5 10 3 4-p-chorophenyl-2-dimethylamino-5- 247 2.67 Hetero cyclic 30 37.973 C H N O S nitrosothiazole 12 13 3 compound

31 37.973 1-Propanamine (CAS) n-Propylamine C3 H9 N 59 2.67 Amine Formamide, N-methyl- (CAS) N- 32 37.973 C H N O 59 2.67 Amide methylformamide Methylformamide 2 5 1-germa-2-silabutane ( ethylsilyl) Alkane 33 37.973 C H GE SI 136 2.67 germane 2 10 N-[1,2,2,2-tetrafluoro-1- C H F N O 2.67 34 37.973 (trifluoromethyl)ethyl]sulfimide- 6 9 7 2 2 306 Sulfamide S trimethylamine adduct Ethyl 2-(1'-hydroxy-1'-methylethyl)- 35 37.973 C H O 254 2.67 Alkane 5,6,6-trimethyl-3,4-heptadienoate 15 26 3 3-Fluoro-2-methoxy-3- 36 37.973 C H F O 258 2.67 Haloketone (trifluoromethyl)nonan-4-one 11 18 4 2

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Fig. 1: GCMS Chromatogram of Pavetta indica Linn.

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