Research Journal of Chemical Sciences ______________________________________________ ISSN 2231-606X Vol. 4(3), 21-26, March (2014) Res. J. Chem. Sci. International Science Congress Association 21 13-methyl-27-norolean-14-en-3-ol, a Triterpeneisolated from the stem of Euphorbia Hirta (linn) Possess an Anti-Asthmatic Properties Saxena Prachi1 and Tiwari Pradeep2 Department of Chemistry, Dr. Harisingh Gour University Sagar, MP, INDIA Department of Chemistry, Vindhya Institute of Technology and Science, Jabalpur, MP, INDIAAvailable online at: www.isca.in, www.isca.me Received 14th January 2014, revised 24th January 2014, accepted 13th February 2014Abstract The present study deals with the phytochemical screening of the alcoholic extract of stem of Euphorbia hirta revealed the presence of triterenoid which were subjected to physical, chemical and spectral identification using IR, 1HNMR, 13CNMR and FABMS. On the basis of the spectral data analysis and chemical reactions, the compounds have been established as 13-methyl-27-norolean-14-en-3-ol namelytaraxerol. The present study has been further designed to evaluate the anti-asthmatic activity of the compound taraxerol. The anti-asthmatic activity was carried out onhistamine induced bronchospasm in guinea pigs which showed that, the alcoholic extract of the stem significantlyinhibited (p 0.05) the contractile effect of histamine. Thus the alcoholic extracts were found to possess potentantiasthmatic property against guineapig with p values less than 0.05, suggesting their potential for further investigation as anti-asthmatic agent. Keywords: Euphorbia hirta, euphorbiaceae, triterpenoid, taraxerol, spectroscopic analysis, antiasthmatic activity. Introduction A number of herbal drugs have been used in the treatment of asthma and proven therapy in the treatment of this disease have been tried to give combined beneficial effects. The pharmacological actions of active principles isolated from various medicinal plants have been studied in our world. Presently various synthetic drugs and physical medicine have been discovered and are being used on a large scale but the significant of the natural drugs have not been relegated.Euphorbia hirta Linnbelongs to natural order Euphorbiaceae. It is an annual herbaceous plant with a slender hairy stem. Its flowers are small and yellow in colour. The plant produces milky latex which is irritating to the mucus membrane2,3. The leaves of this plant have relaxing action upon the smooth muscles of lungs. It possesses great therapy in bronchial asthma. The whole plant is used for the treatment of diseases in children especially in warms,bowels complain and cough. Its juice is used in bronchial asthma affections and chronic coughs. The latex of this plant is used as an application for warts. The roots of this plant possess on anti-emetic properties. Its juice is used in dysentery, colic and also in warm infection4,5. The decoction of its leaves is used in bronchial asthma and chronic coughs. This plant has been found to possess anti-asthmatic, anti-histaminic and spasmolytic properties. In view of its medicinal importance it was worthwhile to carry out systematic phytochemical investigation on the stem of this plant. Material and Methods The stems of the Euphorbia hirtaLinn.were collected locally and identified by taxonomist of Botany Department of Dr. H.S. Gour University Sagar (M.P.). The plant material was collected in the month of September and October-2002 and herbarium specimen has been XXXV deposited in Chemistry Department room no. 36 of this University. Extraction and Isolation: The air-dried powdered stem (4.0 kg) of the plant Euhorbiahirta was extracted with 95% ethanol. The total ethanolic extract was concentrated under reduced pressure to get a light brown viscous mass, which was successively and sequentially fractionated with various solvent from nonpolar to polar as petroleum ether (60–80°C), benzene, chloroform, ethyl acetate, acetone and methanol. The ethyl acetate extract on concentration under reduced pressure yielded a brown viscous mass, which on addition of excess of solvent ether gave a precipitate. The precipitate was then subjected to TLC21 examination, using n-butanol: acetic acid: water (4 : 1 : 5) as solvent system and I2 vapour as visualising agent when depicted two spots. Therefore, it was subjected to column chromatography over silica gel G for purification and eluted with benzene, benzene: chloroform (1 : 1), benzene : chloroform: ethyl acetate (1 : 1 : 1) and studied separately. Compound: Colourless needles which analysed for the mf 3050O, mp 280-282°C [M] 426, IR KBrmaxcm–1 3422.8 (—OH group), 3018.6 (= C-H str), 2932.4 (–C–H str of CH), 2856.6 (–C–H str),1608.6 (–CH–CH group) ,1400.0, 13195 (triterpenoidal nature), 813 (� C = CH). FABMS m/z 426 [M] 408, 393, 284, 269, 204, (base peak)133. 13C-NMR (400 MHz, Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 4(3), 21-26, March (2014) Res. J. Chem. Sci. International Science Congress Association 22 CDCl3) : 39.5 (C–1), 33.8 (C–2), 216.8 (C–3), 47.3 (C–4), 54.6 (C–5), 38.6 (C–6), 36.6 (C–7), 36.5 (C–8), 52.7 (C–9), 38.9 (C–10), 51.6 (C–11), 58.1 (C–12), 37.6 (C–13), 156.5 (C–14), 119.2 (C–15), 38.3 (C–16), 35.5 (C–17), 48.2 (C–18), 20.1 (C–19), 28.7 (C–20), 33.2 (C–21), 35.2 (C–22), 21.5 (C–23), 26.2 (C–24), 16.2 (C–25), 26.7 (C–26), 19.5 (C–27), 30.2 (C–28), 33.7 (C–29), 29.9 (C–30).H-NMR (400 MHz, CDCl3) 2.03 (1H, m, H–1a), 2.10 (1H, m, H–1b), 2.38 (1H, ddd, J= 15.2, 6.2, 2.4, H–2a), 2.63 (1H, ddd, J= 16.4, 11.0, 6.6, H–2b), 3.62(1H, t, H-3) , 1.29 (1H, m, H–5), 1.71 (1H, m, H–6), 1.17 (1H, m, H–7a), 1.46 (1H, m, H–7b), 1.02 (1H, d, J= 4.8, H–9), 3.14 (1H, t, J= 4.8, H–11), 2.80 (1H, d, J= 4.8, H–12), 5.53 (1H, dd, J= 8.0, 3.5, H–15), 1.69 (1H, dd, J= 14.6, 8.2, H–16a), 1.89 (1H, dd, J= 14.6, 3.4 H–16b), 1.17 (1H, m, H–18), 1.55 (1H, m, H–19), 1.42 (1H, m, H–19), 0.98 (1H, m, H–22a), 1.07 (1H, m, H–22b), 1.09 (1H, s, H–23), 0.93 (1H, s, H–24), 1.22 (1H, s, H–25), 1.10 (1H, s, H–26), 0.78 (1H, s, H–27), 0.85 (1H, s, H–28), 0.98 (1H, s, H–29), 0.95 (1H, s, H–30). Results and Discussion The air-dried powdered stem of the plant 'Euphorbia hirta' were extracted with ethanol (95%) and the total ethanolic extract was concentrated to light brown viscous mass. The ethylacetate soluble fraction of the ethanolic extract was chromatographed over Si-gel G column. On elution with benzene: CHCl: ethyl acetate (1:1:1), it gave a compound which analysed for mf 3050O. [M+] 426 (FABMS) mp 282-283°C The significant peaks which appeared at 1400.0 cm–1 and 1319.5 cm –1 in the IR spectrum of the compound showed, it to be of triterpenoidal nature16. It responded to all the characteristic colour reactions of triterpenoid6,7The IR spectrum of the compound shows a peak at 3422.8 cm-1 which implied the presence of –OH group(s) in it. The compound was found to form acetyl derivative C3252, [M+] 468 mp.304-306°C. Estimation of acetyl group was found to be 91.8% as described by Wiesenberger method8 as stated by Belcher and Go, and so impressed the presence of one —OH group in the compound. The compound on oxidation with CrO3/pyridine afforded a ketone of mf C3049O, [M+] 425,mp 242-244°C. The compound gave positive Zimmerman test10 for the C-3 keto group which confirmed the presence of one —OH group at C-3 and further indicated that this —OH group was secondary in nature. On the basis of above deliberations it has concluded that the C-3 —OH group of compound was secondary in nature and present at C-3 position. An IR peak which appeared at 813cm–1 in the IR spectrum of compound indicated the presence of double bond in the compound. On catalytic hydrogenation with Pd/C, it afforded a dihydro derivative having mf C3052O, mp.186-187°C. This indicated the presence of one double bond in it. In HNMR spectrum of the compound olefin proton signal appeared at 553 as (dd) at low field which indicated the presence of vinyl proton at C–15 owing to the double bond between C–14 and C–15. This also suggested that this compound was a taraxerene type triterpene11-17. Further the double bond was confirmed by the reaction of compound with tetranitromethane (RUZICKA'S REACTION)26 gave yellow colour which showed the presence of double bond in the nucleus. An IR peak appeared at 2932.4 cm–1 for angular methyl groups which were estimated by Zeisel's method18 (21.73%) and confirmed the presence of eight methyl groups in it. Various singlets are obtained in the H-NMR spectrum at 109,107, 122, 110, 0.78, 0.85 0.98 and 0.95 indicated the presence of methyl groups. Chemical shift in 13C-NMR spectrum at 21.5, 26.2, 16.2 , 26.7, 19.5, 30.2, 33.7, 29.9 showed methyl groups in it .The compound was further identified by mass spectrum .The base peak at m/z 204 which originated from the fragmentation of ring C and D of an 14 taraxerene while another peak observed at m/z 284 originated from retro-Diels-Alder (RDA) fragmentation of ring D, which suggested the presence of unsaturation 19,20. The above IR, 1HNMR, 13CNMR and FABMS spectral data and their comparison with those described in the literatures showed the structure of isolated compounds are to be the Taraxerol (13methyl-27-norolean-14-en-3-ol). Figure-1Taraxerol Test Animals: Guinea pigs of either sex weighing 250-500 gm of pir bright white strain were used for the biological study.The animals were housed under standard conditions of temperature and humidity for 24 hrs and fasted with fresh leafy vegetables and carrots and with water adlibitum. Induction of Histamine23 Animals were divided into four groups The guinea pigs of either sex were exposed to histamine aerosol (0.5% histamine diphosphate in saline) in an airtight histamine chamber by using a glass sprayer to induce the bronchospasm. The guinea pigs after exposure to the histamine aerosol indicated the sign of progressive immobilization and bouts of coughing leading to convulsions when fell on its back. Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 4(3), 21-26, March (2014) Res. J. Chem. Sci. International Science Congress Association 23 The time taken by the guinea pigs to fall on its back after exposure to aerosol was called as exposition time which was noted for each animals Exposition time also known as PCD (pre-convulsive dyspnoea). After recording the exposition time the animals were immediately removed from the chamber and placed in a fresh air so that the animals came back to its normal state. Anti-Asthmatic Screening of Isolated Compounds: Anti-asthmatic screening of the isolated compounds were carried out on histamine induced bronchonstricted guinea pigs22. A detail method is elaborated below Drug Treatment Schedule24,25 When the histamine was induced later after 1 hour the first three group of animals were administered separately with increasing doses of compounds and the fourth group animal was received salbutamol (050 mg/kg) by the intraperitoneal route. After 1 hour of drug administration the animal was again exposed to histamine and exposition times were noted. The fourth group animal was treated as a standard group of animal. The animals in a first three groups were treated with isolated compounds. The animal of first group was given 50 mg/kg of compound second group was treated with 100 mg/kg and third group was administered with 200 mg/kg of compound Drug was administered intraperitoneally. Later after 1 hour each of the animals were exposed to aerosol and their exposition time were recorded. Each time the animals were fed with fresh green leafy vegetables and tap water and adlibitum and made them refresh for one week. After then the animals were ejected intraperitoneally with an increasing doses. After 30 min of drugs administration they were again exposed to histamine and the exposition were noted for each animal. The difference in the exposition time before and after extract administration was taken as a measure of the protective effect of the drugs. Table- l H-NMR and 13C-NMR Spectrum of the Triterpenoid in CDCLS No dd-Value Pattern Assignments S No dd Value Carbon No Carbon type 1 2.03 m H-1a 1 39.5 C-1 CH 2 2 2.10 m H-1b 2 33.8 C-2 CH 2 3 2.38 ddd H-2a 3 21.68 C-3 CH 4 2.63 ddd H-2b 4 47.3 C-4 C 5 3.62 t H-3 5 54.6 C-5 CH 6 1.29 m H-5 6 38.6 C-6 CH 2 7 1.71 m H-6 7 36.6 C-7 CH 2 8 1.17 m H-7a 8 36.5 C-8 C 9 1.46 m H-7b 9 52.7 C-9 CH 10 1.02 d H-9 10 38.9 C-10 C 11 3.14 t H-11 11 51.6 C-11 CH 2 12 2.80 d H-12 12 58.1 C-12 CH 2 13 5.53 dd H-15 13 37.6 C-13 C 14 1.69 dd H-16a 14 15.65 C-14 C 15 1.89 dd H-16b 15 11.92 C-15 CH 16 1.16 m H-18 16 38.3 C-16 CH 2 17 1.55 m H-19a 17 35.5 C-17 C 18 1.42 m H-19b 18 48.2 C-18 CH 19 0.98 m H-22a 19 20.1 C-19 CH 2 20 1.07 m H-22b 20 28.7 C-20 CH 2 21 1.09 s H-23 21 33.2 C-21 CH 2 22 0.93 s H-24 22 35.2 C-22 CH 2 23 1.22 s H-25 23 21.5 C-23 CH 3 24 1.10 s H-26 24 26.2 C-24 CH 3 25 0.78 s H-27 25 16.2 C-25 CH 3 26 0.85 s H-28 26 26.7 C-26 CH 3 27 0.99 s H-29 27 19.5 C-27 CH 3 28 0.95 s H-30 28 30.2 C-28 CH 3 30 29.9 C-30 CH 3 Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 4(3), 21-26, March (2014) Res. J. Chem. Sci. International Science Congress Association 24 Figure-2 Mass Spectrum of Triterpene Figure– 3 13C-NMR Spectrum of the Triterpenoid in CDCL3 Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 4(3), 21-26, March (2014) Res. J. Chem. Sci. International Science Congress Association 25 Table-2 Effect of Compound Extract on Histamine Aerosol induced Bronchospasm in Guinea Pig Treatment Dose mg/kg ip Pre-treatment exposition time in seconds ±± SE Post-treatment exposition time in seconds ±± SE % protection Compound 50 115.26 ± 10.06 130.21 ± 10.95 11.48% Compound 100 116.23 ± 25.12 145.65 ± 12.57 20.86% Compound 200 122.23 ± 10.99 230.35 ± 46.93 30.71 % Salbutamol 0.65 145.22 ± 5.16 386.42 ± 14.26 62.24 % Calculation: The percentage protection offered by drugs in PCD was calculated by the formula Percentage protection = 100 Etb EtbEta - (% Protection offered by the extract) Where: Eta is the mean exposition time after administration of drug, Etb is the mean exposition time before administration of drug Statistics: The results were expressed as mean SD The significance between post- treatment and pre-treatment exposition times was analysed by student test The values are considered to be significantly different when the p value was less than 0.05. The compound offered the dose- dependent protection to 20.86 % and 30.71 % at doses 100 and 200 mg/kg respectively. Thus it can conclude that the plants drugs isolated from the stem of Euphorbia hirta Linn showed a significant protection against histamine challenge. Thus they can have a very potent anti-asthmatic effect. They produce bronchoprotective effect due to its H1-blocking effect. Conclusion13-methyl-27-norolean-14-en-3-ol, a taraxerol isolated from the extract of stem of Euphorbia hirta possess an antiasthmatic property. Acknowledgement We extend gratitude to the director of Regional Sophisticated Instrumentation Centre (CDRI Lucknow) for recording various spectra. 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