World Journal of Zoology 9 (2): 129-133, 2014 ISSN 1817-3098 IDOSI Publications, 2014 DOI: 10.5829/idosi.wjz.2014.9.2.84306 Screening of Phytochemical Using Gas Chromatograph and Mass Spectrometer GC-MS Analysis and Nematicidal Activities of Leaf Extract of Halmeti, Ervatamia heyneana C. Azhagumurugan and M.K. Rajan Department of Zoology, Ayya Nadar Janaki Ammal College (Autonomous), Sivakasi-626 124 Abstract: The present study was to investigate the phytochemical constituents screened by Gas chromatograph and Mass spectrometer (GC-MS) method. In the GC-MS analysis, 9 bioactive phytochemical compounds were identified in the methanolic leaf extract of Ervatamia heyneana revealed the presence of alkaloids, sterols, triterpenoid, flavonoids and different concentrations (5, 10, 15, 20 and 25 ppm) of leaf extract were prepared and nematicidal activity was evaluated by determination of nematode egg hatchability and nematode larval mortality of the methanolic leaf extract of E.heyneana were analysed. Key words: Alkaloids Sterols Triterpenoid Flavonoids Ervatamia heyneana INTRODUCTION persistent in environment and are safe for mammals and other non target organisms. Botanical pesticides are Ervatamia heyneana is a shrub or small tree readily available in many places, often cheaper than their belonging to the family apocynaceae. It is found synthetic counter parts and their crude extracts are easy distributed in open forests of Konkan, North Kanara, to prepare even by farmers. In the present study, Western Ghats, in Malabar and Travancore up to an methanolic leaf extract of E. heyneana have shown altitude of 3000 ft. It is being used traditionally by local nematicidal activity against root knot nematode population as remedy for various ailments. Alkaloids, Meloidohyne incognita. sterols, triterpenoids have been reported from latex, root bark, roots, seeds and stem bark of the plant E. heyneana. MATERIAL AND METHODS Anti-cancer activities of the extract of the root, stem and leaf have been reported from E. heyneana [1] Medicinal Plant Material: The leaves of E. heyneana were collected plants generally contain several compounds which may from Ayya Nadar Janaki Ammal College Campus, Sivakasi, serve as an alternative, effective, cheap and safe Tamil Nadu and India. The leaves were washed nematicide for the treatment of root knot nematode thoroughly with sterile distilled water, leaf material was infections. then air dried under shade conditions and powder with Root-knot nematodes (RKN) are the most damaging the help of mixer grinder. nematode pests of field and vegetable crops because of their ability to feed on a range of plants including field Preparation of Extract: The crude powdered sample of and vegetable crops, ornamentals, fruit trees and weeds E. heyneana leaves (20 g) were weighed and subjected [2,3]. Nematode control is far more complex than any other to solvent extraction for 8-10hrs repeatedly. The powder kind of pathogens because nematodes mainly attack was extracted by Vacuum rotatory evaporator with underground parts of plants [4]. The control of plant 200 ml of methanol as a solvent. The condensed parasitic nematodes has mainly depended on synthetic extracts were used for preliminary screening of nematicides [5]. Bioactive products of plants being less phytochemicals. Corresponding Author: Azhagu Murugan, Department of Zoology, Ayya Nadar Janaki Ammal College (Autonomous), Sivakasi-626 124, India. 129
Collection of Egg Masses and Nematode Larva: The egg to handle mass spectra and chromatograms was a masses and nematode larvae, pure culture of M.incognita Turbomass. Interpretation on mass spectrum GC-MS was maintained on tomato plants in sterilized soil. Effect on conducted using the database of national Institute egg hatchability was evaluated on five mature uniform Standard and technology (NIST) having more than 62,000 size egg masses of M. incognita were suspended in the patterns. The spectrum of the unknown component was extracts and water (control), replicated three times in compared with the spectrum of the known components cavity blocks. The blocks were kept at room temperature. stored in the NIST library. The Name, Molecular weight The total number of larvae hatched was recorded at 24, 48 and structure of the components of the test materials were and 72 hours intervals. Hatched larvae were counted ascertained. under the inverted compound microscope. For effect of nematode mortality on 30 freshly hatched J2 of RESULTS AND DISCUSSION M. incognita were placed in each dilutions and control, replicated three times in cavity blocks. The blocks were Screening of Phytochemicals: The results pertaining to kept at room temperature. the GC-MS analysis are given in Figures 1, 2, 3, 4, 5 & 6 and Table 1. Seven compounds were detected in methanol GC-MS Analysis: GC-MS analysis of these extracts were extract of E. heyneana leaves. Among the identified performed using a Perkin-Elmer GC Clarus 500 system and phytochemicals, Digitoxin, Dodecanoic acid, 10 methyl, Gas chromatograph interfaced to a Mass spectrometer methyl ester, 3-Dimethylamino -2 -(4 - chlorphenyl) - (GC-MS) equipped with a Elite-I, fused silica capillary thioacrylic acid, thiomorpholide, 10-Octadecenoic acid, column (30mmX0.25mm 1D X 1 µmdf, composed of 100% methyl ester, Oleic acid, 9-Octadecenoic acid (Z), Dimethyl poly siloxane). For GC-MS detection, an electron tetradecyl ester, Dasycarpidan - 1- methanol, acetate ionization system with ionizing energy of 70 ev was (ester), 5H-Cyclopropal (3,4)benz(1,2-e)azulene-5-one,9- used. Helium gas (99.999%) was used as the carrier gas (acetyloxy)-3- [( acetyloxy)methyl]1,1a,1b,4a,7a,7b,8,9,9aa t constant flow rate 1ml/min and an injection volume decahydro-4a,7b,9a-trihydroxy-1,1,6,8-etramethyl, of 2µl was employed (split ratio of 10:1); Injector [1ar(1aa,1ba,4aa,7aa,7ba,8a,9a,9aa)], 2,6,10,14,18,22- temperature 250 C; Ion-source temperature 280 C. The Tetracosahexaene,2,6,10,15,19,23-hexamethyl-,(all-E) were oven temperature was programmed from 110 C antihelminthic Anti-Inflammatory and Anti microbial (isothermal for 2 min.), with an increase of 10 C/min, to activities and anti cancerus activity of the leaf 200 C, then 5 C/min to 280 C, ending with a 9min extract. isothermal at 280 C. Mass spectra were taken at 70 ev; a Grover and Patni [6] reported that twenty one scan interval of 0.5seconds and fragments from 45 to 450 biologically active phytochemicals were screened in the Da. methanolic extract of Woodfordia fruticosa leaves using Total GC running time was 36 minutes. The relative % GC-MS analysis similarly Praveen Kumarm et al. [7] amount of each component was calculated by comparing reported the activities of 9.12.15 octadecatrienoic acid, its average peak area to the total areas, software adopted ethyl ester (z,z,z), ethyl palmitate and N-hexadecanoic Table 1: Phytocomponents identified in the leaf methanol extracts of Halmeti, Ervatamia heyneana S.No RetentionTime Name of the Compound Molecular Formula Molecularweight Peak Area % 1 12.32 Digitoxin C41H64O13 764.93 11.5 2 15.3 Dodecanoic acid, 10 methyl, methyl ester C14H28O2 228.37 100 3 15.73 3-Dimethylamino -2 -(4 - chlorphenyl) - thioacrylic acid, thiomorpholide - - 13.5 4 17.02 10-Octadecenoic acid, methyl ester C19H36O2 296.48 100 5 17.18 Oleic acid CH 3(CH 2) 7CH=CH (CH 2) 7COOH 282.46 42.4 6 18.53 9-Octadecenoic acid (Z), tetradecyl ester C32H62O2 478.83 17.7 7 19.85 Dasycarpidan - 1- methanol, acetate (ester) C20H26N2O2 326.43 15.6 8 21.48 5H-Cyclopropal (3,4)benz(1,2-e)azulene-5-one,9-(acetyloxy)-3- [(acetyloxy)methyl]1,1a,1b,4a,7a,7b,8,9,9a-decahydro-4a,7b 9a-trihydroxy-1,1,6,8-tetramethyl,[1ar(1aa,1ba,4aa,7aa,7ba,8a,9a,9aa)] C24H 32 O9 0 9.6 9 26.25 2,6,10,14,18,22-Tetracosahexaene,2,6,10,15,19,23-hexamethyl-,(all-E) C30H50 410.71 100 130
Egg Hatchability: The nematicidal effects of leaf extract of E. heyneana are shown in Table 2 (Fig 1). The results show that larval hatch commence within 24 hours of exposure to water extracts of the Leaf and distilled water (Control). The results show a decrease in larval hatch as the concentration of the extract increases and also increasing exposure period also increase larvae hatch. The higher larvae hatch was recorded at the control than concentrations of plants extracts. However, more larvae were observed with decrease in the concentration of the Fig 1: Egg hatchability extracts especially from twenty four hours of exposure. Flavonoid compounds like naringenin and rutin have also been indicated to be effective in suppressing egg hatch [9]. Jesse [10] reported the suppression of egg-hatch on root knot nematode with African locust bean (P. biglobosa) and Lannea (L. acida). The mixture of African locust bean husks and Lannea leaves extracts were most suppressive (9.63%) follow by lannea (10.50%) and the African locust bean (11.56%). The effect of the extract as a result of the phytochemical content of the fruit which tend to inhibit egg hatching. It has been reported Fig 2: Larval mortality that extracts of plants that contained saponins tannins tend to inhibit nematodes egg hatch because of their acid as Anti-inflammatory, hypocholesterolemic cancer ovicidal property [11, 12]. preventive, hepatoprotective, Antioxidant and hypocholesterolemic. It is therefore not unlikely that these Larval Mortality: E. heyneana exhibited more mortality phytochemicals found in F. longispina are responsible of (75-85%) after 48 hrs exposure (Table 3 & Fig 2). Larval the traditional applications of this medicinal plant. mortality decreased with increasing concentration of the Senthilkumar et al. [8] analyzed some compound from the leaf extract. Nematode larval mortality increased with leaves of Trichilia connaroides through GC and MS. increased time of exposure. Caudra et al. [13], Thus, this type of GC-MS analysis is the first step evaluated the nematicidal properties of the by-products towards understanding the nature of active principles in from pyroligneous acid, neem (Azadirachta indica), this medicinal plants and this type of study will be helpful Chinaberry (Melia azaderach) and spirobolus for further detailed study. marginatus and of the colloid compounds of Table 2: Effect of different concentrations of leaf extract on Egg hatchability and larval mortality in the root knot nematode Meloidogyne incognita. Egg hatchability at different concentrations (ppm) of Halmeti, Ervatamia heyneana ----------------------------------------------------------------------------------------------------------------------------------------- Exposure Time (Hours) Control 5 ppm 10 ppm 15 ppm 20 ppm 25 ppm Total 24 22 17 13 11 6 1 70 48 25 21 16 8 4 1 75 72 29 23 12 7 3 0 74 Larval mortality (%) at different concentrations (ppm) of Halmeti, Ervatamia heyneana ------------------------------------------------------------------------------------------------------------------------------------------------------------ Exposure Time (Hours) Control 5 ppm 10 ppm 15 ppm 20 ppm 25 ppm 24 0 6.0 10.6 15.0 19.0 23.33 48 0 12.0 12.33 18.0 21.0 26.0 72 0 15.0 15.0 17.33 22.6 27.0 131
Table 3: Toxic effect of leaf extracts Ervatamia heyneana against the root knot nematode Meloidogyne incognita Plant Hours LC50 LC 90 Slope ± SE 2 Chi square (X ) Spontaneous response rate Ervatamia 24 16.481 149.95 2.10 ± 0.45 1.75 0.00 heyneana 48 12.88 345.95 1.61 ± 0.43 4.89 0.00 72 09.69 165.98 1.66 ± 0.44 3.18 0.00 Fig. 1: GC-MS chromatogram of methanolic extracts of Leaf of Ervatamia heyneana\ inorganic salts. The greatest percentage of mortality Ayya Nadar Janaki Ammal College, Sivakasi for was obtained with neem ceil, secretions of S. marginatus and the inorganic compound S-1. Nematicidal property of neem seed formulations against Radopholus similis was also reported by Kosma et al. [14]. Earlier studies by Gamal et al. [15] showed that leaf extract of providing necessary facilities to carry out project. REFERENCES this Faidherbia albida fruit was found effective on second 1. Dighe, V., G. Parekh and D. Mestry, 2012. stage juveniles of M. incognita. The mechanisms of Quantitation of camptothecin from ervatamia the plant extracts action may include denaturing and heyneana (wall.) T. Cooke stem powder using high degrading of proteins and other compounds and performance thin layer chromatography, Int Journal results in the death of nematodes. The control being Pharm Bio Sci, 3: 230-237. distilled water showed no effect on mortality of nematodes. 2. Anwar, S.A., A. Zia, M. Hussain and M. Kamran, 2007. Host suitability of selected CONCLUSION plants to Meloidogyne incognita in the Punjab, Pakistan. International Journal of Nematology, From this study it can be concluded that the methanolic leaf extract of E. heyneana possess nematicidal activity against root-knot nematode Meloidogyne incognita also been proved. ACKNOWLEDGEMENT We profoundly thank UGC for providing fund to carry out this project under Major Research Project Scheme. We also thank the management, 17: 144-150. 3. Anwar, S.A. and M.V. McKenry, 2010. Incidence and reproduction of Meloidogyne incognita on vegetable crop genotypes. Pakistan Journal of Zoology, 42: 135-141. 4. Sikora, R.A and E. Fernandez, 2005. Nematode parasites of vegetables. In: Luc, M., Sikora, R.A. & Bridge, J. (Eds). Plant-parasitic nematodes in subtropical and tropical agriculture. CABI Publishing, Wallingford, UK, 319-392. 132
5. Akhtar, M. and A. Malik, 2000. Roles of organic soil 11. Adegbete, A.A and S.O. Adesayan, 2005. Root amendments and soil organisms in the biological extract of plant to control root nematode on edible control of plant-parasitic nematodes: A review. soybeans. World Journal of Agricultural Sciences, Bioresource Technology,74: 35-47. 1: 18-23. 6. Grover, N. and V. Patni, 2013. Phytochemical 12. Umar, I., 2013. Nematicidal potential of Syzgnum characterization using various solvent extracts and aromaticum extract on M. javanica on tomao. Taraba GC-MS analysis of methanolic extract of Woodfordia Journal of Agricultural Sciences, 1: 97-99. fruticosa (L.) kurz. leaves, Int. J. Pharm. Pharm. Sci., 13. Cuadra, R., X. Cruz., E. Perera, E. Martin and A. Diaz, 5: 291-295. 2000. Some Natural Compound with Nematicidal 7. Praveen Kumarm, P., S. Kumaravel and C. Lalitha, effect. Revista-de-Proteccion-Vegetal, 15: 31-37. 2010. Screening of antioxidant activity, total 14. Kosma, P., Z. Ambang, B.A.D. Begoude, G.M. phenolics and GC-MS study of Vitex negundo. Hoopen, J. Ten Kuate and A. Akoa, 2011. African Journal of Biochemistry, 4: 191-195. Assessment of nematicidal properties and 8. Senthilkumar, N., S. Murugesan and K.B. phytochemical screening of neem seed formulations Vijayalakshmi, 2012. GC-MS-MS analysis of Trichilia using Radopholus similis, parasitic nematode of connaroides (Wight & Arn.) Bentv (Meliaceae): A plantain in Cameroon, Crop Protection, 30: 733-738. tree of ethnobotanical records. Asian J. Plant Science 15. Gamal, A.E., W.L. Dong, C.P. Juang, B.Y. Hwuang and Research, 2: 193-197. and Y.C. Ho, 2008. Evaluation of various extracts for 9. Bajaj, S.O., 1999. Nematicidal activity of some their nematicidal efficacies against juveniles of phenolic compounds against root-knot nematodes Meloidogyne incognita, Journal of Asia-Pacific Meloidogyne incognita. Pyhtochemistry, 79: 209-214. Entomology, 11: 99-102. 10. Jesse, Y. A., 2005. Suppression of egg-hatch on rootknot nematodes (M. incognita) with plants extracts. Journal of Research in Agriculture, 2: 63-65. 133