1. INTRODUCTION:

Natural medicine from plants source are established to be safe and effective. Plants species have been used in folkloric medicine to treat various ailments. Even today compounds from plants continue to play a major role in primary health care as therapeutic remedies in many developing countries¹. Among them few plants are identified as poisonous plants. However, they could be exploited for significant pharmacological purposes. For example C. collinus is a toxic plant belongs to the family Euphorbiaceae. All parts of this plant are reported to be highly toxic. C. collinus is a rich source of lignans of which cleistanthin A and B were reported as having the property of cytotoxicity, anticancer and larvicidal activity. C. collinus poisoning usually occurs by intentional ingestion of the leaves with mortality as high as 30%, usually occurs 3-7 days after ingestion^2-3. Cleistanthin A and B glycosides are very important toxins of it. Extract of the various plant parts yielded a multitude of compounds of which the glycosides, arylnaphthalenelignan lactones are toxic⁴.

Aqueous extract of C. collinus prepared in the form of decoction (100-200ml) is used for suicidal purposes; it has been reported as slow poison. This specific property of the plant might be attributed to the secondary metabolites. Perusal of literature revealed that, there is no study on spectroscopic analysis of aqueous, ethyl acetate, ethanol and methanol extract and the total number of compounds present in the extract is not yet reported⁵. For this reason the present study was designed to identify the phytocompounds from aqueous, ethyl acetate, ethanol and methanol dried leaf extracts of C. collinus to understand the chemical composition.

2. MATERIALS AND METHODS:

Collection of sample and extraction:

Leafs of C. collinus was harvested in August, 2011 from plains of Virallimalai, Trichy (Dt), Tamilnadu, India. The aqueous, methanol, ethanol and ethyl acetate hot crude leaf extracts of sample was prepared from 100gm of leaf powder by using Soxhlet apparatus. Excess solvents were removed by concentrating the extracts and stored in desiccators until use.

GC- MS analysis:

GC-MS analysis was carried out by Agilent-7890A GC instrument coupled with MS-5975 inert MSD and triple axis mass selective ion detector. The DB-5MS column with dimensions of 30m x 0.2mm capillary column was used for the analysis. The initial temperature was kept at 150° C and the maximum of 300° C. One µL of sample was injected with split mode (10:1). Helium gas used as a carrier gas at flow rate of 0.8 ml/min and the total run time was 22 mins. GC-MS for identification of phytocomponents were conducted using the database of National Institute Standard and Technology MS library (NIST- MS library).

3. RESULTS:

The phytocompounds present in all the crude extracts of C. collinus were identiﬁed by GC–MS analysis. The GC–MS chromatograms showed the presence of most important phytocompounds with highest concentrations (Figure. I). Phytocompounds with their retention time (RT), molecular formula and molecular weight (MW) in the leaf extracts of C. collinus are given in Table I, II, III and IV. The diversity of phytocompounds extracted in various solvents depends on the polarity of the solvent. Four major chemical compounds were found in aqueous extract (Table I), 4-O-Methylmannose (70.12%) and 1,2,3-Benzenetriol (21.76%), were the major constituents in aqueous extract. Fifteen compounds were found in ethyl acetate extract among them Silane, trimethyl[5-methyl-2-(1-methyl ethyl) phenoxy]- Anthracene (7.06%) was found as major compounds (Table II). 4-C-methyl- (Methylthio)-acetonitrile 4-O-Methylmannose (58.53%) and 5-Methyl-2-trimethylsilyloxy-aceto phenone (7.35 %) were found at higher level in the ethanol extract followed by other five compounds (Table III). In methanolic extract ten major phytocompounds were screened. Among that Thiophene, tetrahydro-2-methyl- Myo-Inositol (48.54%), 1,2,3-Benzenetriol (7.51%) and Vitamin E(dl-.alpha.-Tocopherol) (6.52%) were identified as a major constituents (Table IV and V).

Figure 1: GC-MS Chromatograph of plant leaf extracts

Table I. Chemical composition of C. collinus aqueous leaf extract.

Pk#	RT	Area%	Library/ID(C:\Database\NIST08.L)	Mol. Formula	Mol. Wt.
1	2.864	21.76	1,2,3-Benzenetriol	C₆H₆O₃	194
2	4.279	70.12	4-O-Methylmannose	C₇H₁₄O₆	194
3	14.177	2.89	4-Hydroxy-6,7-dimethoxy-9-[3,4 (methylenedioxy)phenyl]naphtho[2,3-c]furan-1(3H)-one	C₂₁H₁₆O₇	380
4	18.143	5.23	Benzeneacetic acid, .alpha.-[3-methoxy-4-(4-methoxyphenyl)-5-oxo-2(5 H)-furanylidene]-, methyl ester	C₂₁H₁₈O₆	366

Table II. Chemical composition of C. collinus ethyl acetate leaf extract.

Pk#	RT	Area%	Library/ID(C:\Database\NIST08.L)	Mol. Formula	Mol. Wt.
1	2.895	0.76	Heneicosane,11(1ethylpropyl)Tridecane	C₂₆H₅₄	366
2	3.110	0.85	Ethanol, 2,2'-dithiobis-N-[2,3-Difluoro-2,3-di(difluoroamino)aziridino] difluoroaminofluoro methylene imine	C₄H₁₀O₂S₂	154
3	3.510	1.61	Phenol, 3,5-bis(1,1-dimethylethyl)	C₁₄H₂₂O	206
4	4.770	2.96	Decanoic acid, silver(1+) salt	C₁₄H₂₂O	206
5	5.016	2.78	Hepta-2,4-dienoic acid, methyl ester	C₈H₁₂O₂	140
6	5.447	4.06	2,4,6-Trimethylmandelic acid	C₁₁H₁₄O₃	194
7	6.000	5.78	Didodecyl phthalate	C₃₂H₅₄O₄	502
8	7.260	1.57	Butanoic acid	C₁₅H₂₈O₂	240
9	8.674	2.16	Quinazoline	C₉H₈N₂O	160
10	9.105	1.51	Eicosane(3-ethyl-5-(2-ethylbutyl))	C₂₀H₄₂	282
11	9.627	2.45	Bumetrizole	C₁₇H₁₈ClN₃O	315
12	14.485	0.80	Silicic acid, diethyl bis(trimethy lsilyl) ester Cyclotrisiloxane	C₁₀H₂₈O₄Si₃	296
13	16.759	0.86	Cyclotrisiloxane	C₆H₁₈O₃Si₃	222
14	17.159	5.31	1,1,1,3,5,5,5-Heptamethyltrisiloxane	C₇H₂₂O₂Si₃	222
15	18.143	7.06	Silane	C₁₃H₂₂OSi	222

Table III. Chemical composition of C. collinus ethanol leaf extract.

Pk#	RT	Area%	Library/ID(C:\Database\NIST08.L)	Mol. Formula	Mol. Wt.
1	2.895	5.05	1,2,3-Benzenetriol	C₆H₆O₃	126
2	3.049	2.14	Pyrazole-5-carboxylic acid, 3-meth yl	C₅H₆N₂O₂	126
3	4.186	58.53	4-C-methyl-(Methylthio)-acetonitrile 4-O-ethylmannose	C₇H₁₄O₆	194
4	5.815	9.23	Benzoic acid, 3,4,5-trihydroxy-1,3,6-Cycloheptatriene-1-carboxyli c acid, 3,4,6-trihydroxy-5-oxo-Acetic acid	C₇H₆O₅	170
5	6.768	1.79	1,3-Cyclooctadiene	C₈H₁₂	108
6	17.159	3.24	Methyltris(trimethylsiloxy)silane Acetamide, N-[4-(trimethylsilyl)phenyl]-Cyclotrisiloxane,	C₁₀H₃₀O₃Si₄	310
7	18.143	7.37	5-Methyl-2-trimethylsilyloxy-aceto phenone	C₁₂H₁₈O₂Si	222

Table IV. Chemical composition of C. collinus methanol leaf extract.

Pk#	RT	Area%	Library/ID(C:\Database\NIST08.L)	Mol. Formula	Mol. Wt.
1	2.833	7.51	1,2,3-Benzenetriol	C₆H₆O₃	126
2	3.510	1.51	Phenol, 2,4-bis(1,1-dimethylethyl)	C₁₄H₂₂O	206
3	4.709	48.54	Thiophene, tetrahydro-2-methyl- Myo-Inositol	C₅H₁₀S	102
4	5.446	2.41	Benzeneacetonitrile	C₈H₅F₂N	153
5	5.662	1.54	Hexadecanoic acid	C₁₇H₃₄O₂	270
6	5.815	4.56	n-Hexadecanoic acid	C₁₆H₃₂O₂	256
7	6.645	6.88	Phytol	C₂₀H₄₀O	296
8	6.799	7.62	9,12,15-Octadecatrienoic acid	C₁₈H₃₀O₂	278
9	15.284	6.52	Vitamin E(dl-.alpha.-Tocopherol)	C₂₉H₅₀O₂	430
10	18.17	5.15	Cholesta-5,17(20)-dien-3-ol, acetate	C₂₉H₄₆O₂	426

Table V. Major phytocompound profile of C. collinus leaf extracts.

Name of the Phytocompound		Biological properties
4-O-Methylmannose		Antibacterial activity¹⁹
1,2,3-Benzenetriol		Antioxidant,Antiseptic, Antibacterial, Antidermatitic, Fungicide, Pesticide, Antimutaginic Dye and Candidicide²⁰
Silane		Coupling Agent²¹
Didodecyl phthalate		Antimicrobial and Antifouling²²
4-C-methyl (Methylthio)acetonitrile 4-O Methylmannose		Secondary messenger²³
Benzoic acid		Food preservative²⁴
Thiophene		Antimicrobial activity²⁵
9,12,15-Octadecatrienoic acid		Anti-inflammatory, Cancer preventive, Hepatoprotective, Antiarthritic and Antieczemic²⁶

4. DISCUSSION:

The GC-MS analysis of all extracts were given in the figure I and table I, II, III and IV. This is in concerned with the finding of several authors reported early as some important phytocompounds in genus of Cleistanthus^6-7. 1, 2, 3-Benzenetriol (C₆H₆O₃) a major compound was present at peak area (5.05-21.76%) in aqueous, ethanol and methanol extracts except ethyl acetate extract. Similarly, this compound is present in Mussaenda frondosa and Excoecaria agallocha extracts ^8-9. The 4-O-Methylmannose (C₇H₁₄O₆) a major compound was found in aqueous extract with a high peak area (70.12 %); also it is present in the methanol stem extract of Sauropus androgynus¹⁰. The phyto compounds of Silane (C₁₃H₂₂OSi), 1,1,1,3,5,5,5 Heptamethyltrisiloxane (C₇H₂₂O₂Si₃), Didodecyl phthalate 1, 2-Benzenedicarboxylic acid (C₃₂H₅₄O₄) and 2, 4, 6-Trimethylmandelic acid (C₁₁H₁₄O₃) were found at the peak area (7.06-4.06) in ethyl acetate extract. Such groups of chemical compounds have been reported as first time in C. collinus ethyl acetate extract. The 4-C-methyl- (Methylthio)-acetonitrile 4-O-Methylmannose (C₇H₁₄O₆) and Benzoic acid (C₇H₆O₅) was revealed at high peak area (59.53 and 9.23 %) in ethanol extract of C. collinus, which is coincided with the report registered in Arabidopsis thaliana ¹¹. In the methanolic leaf extract Thiophene, tetrahydro-2-methyl- Myo-Inositol (C₅H₁₀S), 9,12,15-Octadecatrienoic acid (C₁₈H₃₀O₂), Phytol (C₂₀H₄₀O) and dl-.alpha.-Tocopherol (C₂₉H₅₀O₂) were found at peak area between 48.54 – 6.52% , similarly reported in plant extracts of Tagetes patula, Chlorella vulgaris and Cimicifuga racemosa^12-15. Exclusive preliminary phytochemical screening followed by analysis of the potential biological property^16-18 may not be sufficient enough to justify the activity is attributed to the presence of the active principles. Further chromatographic fractionation of the crude extract using various organic solvents combinations and concentrations is needed to isolate and characterize pure compounds. In this present work, major phytochemical constituents were identified from aqueous, ethyl acetate, ethanol, and methanol leaf extracts of C. collinus by Gas Chromatography Mass Spectrometry (GC-MS) method. Similar kind of phytocompounds were isolated and identified from various other plant resources. Pharmacological studies revealed that they have highly significant biological properties^19-26. It is obvious that the leaf extracts contains various phytocompounds which may have the appropriate biological property to use in pharmaceutical industry. However, further studies like bio-prospecting are essential to support its biological properties.

5. ACKNOWLEDGEMENT:

The authors are grateful to the University Grants Commission, Govt. of India, New Delhi for financial support under Rajiv Gandhi National Fellowship. The authors are also grateful to PRIST University for continuous encouragement during the work and provided laboratory facilities.

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Received on 31.07.2013 Modified on 15.09.2013

Research J. Pharm. and Tech. 6(11): November 2013; Page 1173-1177