Assam University Journal of Science & Technology: Biological Sciences Vol. 4 Number I
29-34,2009
Isolation, Characterization and Bio-activity Screening ofCompound from Clerodendrum viscosum Vent. M. Dutta Choudhury!, Satya B. PauF, Sudip ChoudhurylShuvasish Choudhury!, Pinak Pani Nath Choudhury4.· Department of Life Science, Assam University, Silchar, Assam - 788011, India 2 D~partment of Chemistry, Assam University, Silchar, Assam - 788011, India 3 Department of Chemistry, G. C. College, Silchar, Assam - 788004, India 4 Department of Zoology, S S College, Hailakandi, Assam - 788151, India * Author for correspondence; email:
[email protected] 1
Abstract Studies on the extracts of different species of the genus Clerodendron have been carried out by a number of researchers round the globe. The present paper reports the isolation, structure elucidation and antimicrobial activity of a novel compound (Viscosene) obtainedfrom the plant Clerodendron viscosum Vent. The structure of the isolated compound is elucidated by spectral analysis.
Keywords: Clerodendron viscosum, Natural product, Viscosene
Introduction Plants, microorganisms, vertebrates and invertebrates are fine biochemical factories for the biosynthesis of both primary and secondary metabolites. Their ingenuity in creating diverse structures with generous sprinkling of functionalities and delicacies is thrilling. This is the outcome of evolution through millions of years during which they have been withstanding many tough tests of survival (Valiathan, 1998). Studies on the extracts of different species of the genus Clerodendron have been carried out by a number of researchers round the globe (Shrivastava and Patel T., 2007). Roots and leaf extracts of C. indicum, C. phlomidis, C. serratum, C. trichotomum, C. chinense and C. petasites have been used for the treatment of rheumatism, asthma and other inflammatory diseases (Hazekamp et. aI, 2001; Kang et. aI, 2003). Plant species such as C. indicum and C. inerme were used to treat coughs, serofulous infection, buboes problem, venereal infections, skin diseases and as a vermifuge, febrifuge and also to treat Beriberi disease (Panthong et.al, 2003).
The plant Clerodendron viscosum Vent. is an indigenous medicinal plant widely distributed in various parts of India, Ceylon, Malaya and Bangladesh (AI-Amin et.al, 2007). In Bangladesh, among the shrubs, the highest density (53.57 plants/1 00 m2 ) and frequency (35.71 %) were found in Clerodendrum viscosum (Moore and Chapman, 1986).Repellent response of Clerodendron viscosum to the larvae and adults of flour Beetle, Tribolium confusum was studied by Husain and Hasan. They observed that both the larvae and adults were repelled by contact with different food media when mixed with leaf dusts of the plant (Husain and Hasan, 2008). The commonly reported useful parts of the plant for therapeutic uses are Root and Leaves (Ayurvedic Medicinal Plants website). The antifungal activity ofthe ethanolic extract of leaf of C. viscosum, against Botryodiplodia was reported in (Anwar et.al, 2007). The free radical scavenging potential of the roots of C. viscosum was studied by Narayanasamy and his co-workers (Narayanasamy et.al, 2007) using different
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antioxidant models of screening. The ethanolic extract of the root (1000 iglml concentration) showed maximum scavenging of the radical cation, 2,2-azinobis-(3-ethylbenzothiazoline-6sulphonate)(ABTS) observed upto 98.92% followed by scavenging of nitric oxide radical (96.75%),f~rric ion radical (94.43%), 1,1diphenyl, 2-picryl hydrazyl (DPPH) (92.25%) and anti lipid peroxidation potential (81.13%). The aqueous extract showed only moderate activity. They commented that this finding justifies the . therapeutic application of the plant in the indigenous system of medicine, augmenting its therapeutic value. Description of the Plant
Clerodendrum viscosum, Vent. (local name Bhant) is a perennial shrub with bluntly 4-angled stern. Leaves in whorls, sessile, narrow lanceolate, subentire, glabrous, rather hard. Flowers bluish-purple often white in pyramid shaped terminal panicles. The plant is slightly woody shrub with bluntly quadrangular sterns and branches, leaves usually three at a node, sometimes opposite oblong or elliptic, serrate; flowers blue, many in long cylindrical thyrsus; fruits 4 lobed purple durpe, somewhat succulent with one pyrene in each lobe (Assian Medicinal Plant website; genebank website). For the present work, green leaves of the plant were collected from several growing areas in Barak Valley during March-April, 2008. The plant material was air dried at ambient temperature (-25°C) and than powdered. The powdered material was used for further experimentations. Experimental Section Extraction The desiccated and grinded aerial parts of Clerodendron viscosum was extracted exhaustively with petroleum ether and ethyl acetate by soxhlet apparatus. In each batch of exhaustive extraction, 250 g material was taken in soxhlet. The material was first defatted by extraction with petroleum ether (300ml). The defatted plant material was then extracted with ethyl acetate (300m!). Both the ethyl acetate extracts was dried in vacuo using rotary evaporator to get the crude extract. The ethyl acetate extract,
was suspended in hexane. After filtration and concentration, the soluble phase was fractionized by column chromatography over activated silica gel (100-200 mesh) using a gradient of petroleum ether : ethyl acetate as eluent (petroleum ether : EtOAc = 100:0 to 98:2). The first fraction after drying in vacuo, was further purified by Preparative Thin Layer Chromatography (PTLC) using Silica gel G coated on glass substrate and Hexane: Ethyl acetate (99: 1) as the mobile phase. The compound obtained from PTLC was subjected to hyphenated technique GC-MS. Gas Chromatography-Mass Spectroscopy resolved the material to be a mixture of two compounds and provided mass spectra for both sepa~ately (Compound 1 at 42.2 minutes and major amount of Compound 2 at 48.4 minutes). The material was then further purified carefully by another PTLC keeping the polarity slightly lesser. Finally the Compound 2 was isolated in pure form (checked by GC-MS). From the spectral analysis the compound seemed to be a new compound and is named Viscosene. The Compound 1 was identified by MS Library search to be a known compound Squalene. Structure Elucidation of Viscosene Viscosene exhibited clean molecular ion peak at rnIz 410 in the mass spectra obtained from GCMS. The peak was associated with peaks at mlz 411 (33% ofthe molecular ion peak) and 412 (5% of the molecular ion peak) indicating that they were the peaks corresponding to [M+ 1] and [M+2] respectively. The compound exhibited peak at mlz 395 corresponding to [M-15] peak due to dissociation of CH3 moiety from the compound. As the compound does not seem to contain nitrogen as even number of nitrogen atoms (according to Nitrogen Rule) could not be justified in the MS fragmentation or from the !H NMR spectrum of the compound. Thus the molecular formula of the compound seems to be C30H so and hence the compound is having 6(six) double bond equivalents (DBE). The FT-IR spectrum of the compound exhibited band at about 2950 cm-! due to v(C-H) asymmetric stretching of methyl group. The peak at 2926 cm-! is due to v(C-H) asymmetric stretching of methylene moiety (>CH2). The peak
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at 2850 cm-! is characteristic to v(C-H) symmetric stretching of methylene moiety (>CH2). The band at 1630 cm-! is due to v(C=C). The band with m6derate intensity centered at about 759 cm-! corresponds to C-H bending in 1,3- disubstituted benzene ring. The !H NMR spectrum of the compound exhibited peaks at 07.0 to 0 7.5 indicative ofthe presence
of 1,3- disubstituted benzene ring. The peak at 0 4.7 is due to vinylic proton. The ·peak at 03.4 seems to be due to the benzal proton. The peak centered at 0 0.9 corresponds to terminal methyl protons. The !H NMR spectrum assignment of the compound is presented below:
1.18
1.56
1.563
7.06
The ElMS fragmentations of the compound are presented below:
--@-=---tl~~ [C30H50 ] • +
[C29H47 ]®
-2~
+
mlz = 395
Molecular ion mlz = 410
Chemical Fonnula: C sHlI+ mlz: 71
327
~ 83
69
I
~ -14
Chemical Fonnula: C 2s H39+ mlz: 339
Chemical Fonnula: C24 H 3' mlz: 325
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The high 'percentage abundance' of the peak at mlz 243 indicates the high stability of the corresponding fragment. This cationic fragment seems to get its high stability due to resonance.
The proposed tentative structure of Viscosene is presented below:
II
Ij
I-methyl-3-(2,6, 14, 18-tetramethylnonadeca-7, 17~dien-9-yl)benzene
Viscosene paper disc of 6mm diameter containing 200 1111 disc of sample was placed over the inoculated medium. The plates were allowed to remain in the room temperature for 2 hours. After two hours the plates were incubated at 37°C for 24 hours. The zone of inhibition was measured using a Zone Reader.
Antimicrobial analysis of the crude extract and the isolated compound Viscosene The activity of the crude extracts against different microorganisms was monitored. The microorganisms undertaken were Bacillus subtilis, Klebsilla pneumonae, E. coli, Proteus vulgaris. Disc diffusion method was used to determine the zone of inhibition. The sterile molten Nutrient agar cooled to 45°C was inoculated with different organism. The inoculums used were the young cultures. Under aseptic technique the inoculums was uniformly inoculated over the molten agar by using sterile cotton swab. Wattman No 2 filter
The standard used were, Sparfloxacin Chephaloxin, Oxacillin, Amphicilin Disc containing the solvent (Petroleum ether, ethyl acetate, hexane) was also used to monitor its influence on the antimicrobial activity of the materials, if any. The results are tabulated in Tablel
Table 1 : Antimicrobial activity of crude extracts of C. viscosum SI No
Microbe
Standard (mm)
Ethyl acetate extract
Sparfloxacin Chephaloxin Oxacillin Amphicilin
1 Bacillus subtilis 2 Klebsilla pneumonae 3 E coli 4 Proteus vulgaris
Crude extract
Visco sene
Solvent (Hexane)
39 35 49
N.M
N.M
17
N.M
N.M
N.M
7 9
N.M
N.M
N.M
N.M
7 7 7
N.M
7
9 7
N.M
9 9 7
N.M N.M
N.M.- Not Measurable. It has been observed that, the ethyl acetate extract is exhibiting antimicrobial activity almost similar to that of the isolated compound viscosene. Both· failed against Bacillus subtilis. One major point of importance in this study is that, the antimicrobial activity of the EtOAc extract retained on fractionation. Even the fractions from
this extract were active against Proteus vulgaris, though the crude extract was inactive. The isolated compound viscosene showed good inhibitory effect against Klebsilla pneumonae, E. coli, Proteus vulgaris but found to be inactive against Bacillus subtilis. Its activity was similar or better than that shown by the standard used (except
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Sparfloxacin). It was also found to be active against Proteus vulgaris, whereas the crude extract was inactive against it. These observations indicate that viscosene isolated from the EtOAc extract
probably one of the active principle( s) of the plant, as the antimicrobial activity was retained on separation of compound. Photographs of culture plates are presented in Figure 1.
E. coli
Klebsilla pneumoni
Proteus vulgaris
Bacillus sp
Figure 1 : Antimicrobial activity of crude extracts of C. viscosum Conclusion Studies on the isolation, structure elucidation and antimicrobial activity of compounds from the medicinally important plant Clerodendron viscosum Ventis reported here. One of the isolated compounds was a known compound squalene. The other compound (viscosene) is probably a novel one. The tentative structure of the isolated compound viscosene is elucidated by spectral analysis. The ethyl acetate extract as well as the isolated compound viscosene exhibited good antimicrobial activity.
2. A.nwar, M. N.; Begum, J.; Chowdhury, M.Y.J.U.; Khan, S.(2007). Antifungal Activity of Forty Higher Plants against Phytopathogenic Fungi, Bangladesh, J Microbiol, 24 (1): 76-78
Acknowledgements The authors are grateful to the Director and scientific officers ofSAlF, Shillong for recording the spectra of the compounds.
5. Husain, M. M.; Hasan, M. R. (2008). Repellency of Indigenous Plant, Bhant (Clerodendron Viscosum Vent.) Leaf on Tribolium Confusum Duval, Bangladesh 1. Sci. Ind. Res. 43(2): 2671 272
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