Research Article - (2013) Volume 3, Issue 1
1Department of Botany, Kanchi Mamunivar Centre for Post Graduate Studies, Lawspet, Puducherry - 605008. (India)
2Department of Plant Science, Tagore Arts College, Lawspet, Puducherry - 605 008. (India)
Hugonia mystax L. is woody evergreen liana distributed throught India, in dry forest area. It is locally knows as Modirakanni (Tamil). Ethnobotanically, the stem bark is used for stomach pain, vomiting and indigestion. Powdered materials were subjected to successive extraction with petroleum ether, chloroform and ethanol by soxhlet method to determine the successive extractive values and GC-MS analysis to investigate the chemical components present in it. Totally 62 chemical compounds were identified of which 18 compounds were identified from petroleum ether extract, 29 compounds from chloroform extracts and 21 compounds from ethanol extract of which di-n-octyl phthalate (24.32%), 2-methyl-7-nonadecene (20.83%), α-D-Glucopyranoside, methyl (21.10%) were major constituent with the biological activities like antimicrobial, antifungal and antioxidant activity present in the stem extracts.
GC-MS analysis, Hugonia mystax L., petroleum ether extract, ethanol stem extracts.
The genus Hugonia L., of family Linaceae comprise about 40 species in the world; of which two species namely Hugonia mystax L., and H. ferruginea Wight & Arn., were reported from India [1,2]. The plant Hugonia mystax is a woody evergreen liana distributed throughout India in dry topical forest it is also known as Modirakanni. Ethnobotanically the bark is made in to a decoction with Curcuma aromatica and is given with honey for inflammations in the stomach, vomiting, stomach pain, indigestion [3]. The aerial parts used as herbal remedies for diabetes [4,5]. Review of literature revealed less work on this plant. Hence in the present study, the successive extractive value and Gas Chromatography-Mass Spectrometry (GC-MS) analysis of petroleum ether, chloroform and ethanol extract of stem of Hugonia mystax were done.
Collection of Plant material
The plant of Hugonia mystax L., was collected from the Marakanam forest vicinity, of Villupuram district, Tamil Nadu. The collected plant materials was botanically identified and confirmed by using flora such as Flora of Tamil Nadu Vols. 1-3 [6] and ‘An Excursion Flora of Central Tamil Nadu Carnatic‘[7]. The species conformation was engaged at French Institute Herbarium (HIFP), Puducherry. The herbarium specimen was prepared and deposited at the Department of Botany, Kanchi Mamunivar Centre for Post Graduate Studies, Lawspet, Puducherry, for future reference.
Preparation of the Extracts
The collected materials (stem) were chopped into small pieces separately, shade-dried, and coarsely powdered using a pulverizor. The coarse powders were subjected to successive extraction with organic solvents of increasing polarity such as petroleum ether, chloroform and ethanol by Soxhlet method. The extracts were collected and distilled off on a water bath at atmospheric pressure and the last trace of the solvents was removed in vacuo and stored at 4ºC. The resulted extracts were subjected to GC-MS analysis.
Determination of Extractive values
All the successive extracts of stem were subjected to determine the successive extractive values by following standard methods [8].
Gas Chromatography-Mass Spectrometry (GC-MS) analysis
GC-MS analysis was performed with GC Clarus 500 Perkin Elmer equipment. Compounds were separated on Elite- 1 capillary column (100% Dimethylpolysiloxane). Oven temperature was programmed as follows: isothermal temperature at 50ºC for 2min, then increased to 200ºC at the rate of 10ºC/min, then increased up to 280ºC at the rate of 5ºC/min held for 9 min. Ionization of the sample components was performed in the El mode (70 eV). The carrier gas was helium (1ml/min) and the sample injected was 2μl. The detector was Mass detector turbo mass gold-Perkin Elmer. The total running time for GC was 36 min and software used was Turbomass 5.2.
Using computer searches on a NIST Ver.2.1 MS data library and comparing the spectrum obtained through GC – MS compounds present in the plants sample were identified.
Identification of Compounds
The individual compounds were identified from ethanol extracts based on direct comparison of the retention times and their mass spectra with the spectra of known compounds stored in the spectral database, NIST (version year 2005).
Successive Extractive values
In stems, extractive values recorded were 4 % in petroleum ether, 5 % in chloroform and 7 % in ethanol.
The results of GC-MS analysis on petroleum ether, chloroform and ethanol extracts of stem were given in the Table 1-3.
Table 1. GC-MS analysis of petroleum ether extract of the stem.
Table 2. GC-MS analysis of chloroform extract of the stem.
Table 3. GC-MS analysis of ethanol extract of the stem.
(i) Petroleum ether extract
In petroleum ether extract, 18 compounds were identified, of which 6 compounds were belonged to the class dicarboxylic acid, 5 compounds were belonged to the class fatty acids and their esters, 3 compounds were belonged to fatty alcohols and one compound each belonged to the class cardiac glycoside, secosteroid and aromatic acid and hydrocarbon respectively. Among this classes, di-n-octyl phthalate was found to be present as major constituent with the peak area 24.32% and retention time 21.28 minutes, followed by digitoxin with the peak area 12.16% and retention time 33.30 minutes and followed by 9,12-octadecadienoic acid, methyl ester, (E,E)-with the peak area 11.26% and retention time 15.07 minutes. Compounds such as 1,2-benzenedicarboxylic acid, diheptyl ester; 1,2- benzenedicarboxylic acid, dipentyl ester and benzoic acid, 2-(1-oxopropyl)- was found to be present as least quantity with the peak area 0.90 % and retention time 14.03, 14.79 and 13.95 respectively (Table 1; Figure 1).
Figure 1: GC-MS Chromatogram of petroleum ether extract of stem of Hugonia mystax
(ii) Chloroform extract
In chloroform extract 29 compounds were identified, of which 5 compounds were belonged to the class hydrocarbons, 4 compounds were belonged to the class fatty alcohols, 3 compounds were belonged to the class fatty acids and their esters, 2 compounds each belonged to the class steroids, unsaturated hydrocarbons, aliphatic aldehydes and triterpene alcohols, one compound each belonged to the class aromatic ester, phenolic ester, dicarboxylic ester, carboxylic acid, alkaloid, ketones, aromatic hydrocarbon and pesticide respectively. Among this classes, 2-methyl-7-nonadecene was found to be presents as major constituent with the peak area 20.83% and retention time 23.72 minutes, followed by dehydrodiconiferyl alcohol with the peak area 13.54% and retention time 31.79 minutes and followed by 1-eicosanol with the peak area 8.39% and retention time 20.80 minutes. In this class, 1H-cycloprop[e]azulene,1a,2,3,4,4a,5,6,7b-octahydro-1,1,4,7-tetramethyl-,[1aR(1aα,4α,4aα,7bα)]- was found to be least quantity with the peak area 0.34% and retention time7.07 minutes respectively (Table 2; Figure 2).
Figure 2: GC-MS Chromatogram of chloroform extract of stem of Hugonia mystax
(iii) Ethanol extract
In ethanol extract 21 compounds were identified, of which 4 compounds were belonged to the class fatty acids and their esters, 3 compounds each belonged to the class sugars and aromatic alcohols, 2 compounds were belonged to the class phenols and phenolic esters, one compound each belonged to the class triol, alkene hydrocarbon, hydrocarbon, fatty alcohol, terpene, sesquiterpene, aromatic esters, lactames and dicarboxylic ester respectively. Among this classes, α-D-glucopyranoside, methyl was found to be present as major constituent with the peak area 21.10% and retention time 9.90 minutes, followed by glycerin with the peak area 18.91% and retention time 2.36 minutes, and followed by 2,7-dioxa-tricyclo[4.4.0.0(3,8)]deca-4,9-diene with the peak area 9.63% and retention time 7.59 minutes. In this class, 3, 5-Dimethoxy-4-hydroxybenzyl alcohol was found to be as least quantity with the peak area 0.35% and retention time 10.60 minutes respectively (Table 3; Figure 3).
Figure 3: GC-MS Chromatogram of ethanol extract of stem of Hugonia mystax
(iv) Comparative GC-MS analysis of compounds in various extracts of stem.
Comparatively in total 62 compounds were identified with the petroleum ether, chloroform and ethanol extract of stem. Among these, the compound such as di-n-octyl phthalate were found to be present in petroleum ether and chloroform extract where as it is absent in ethanol extract, compounds 4-((1E)-3-hydroxy-1-propenyl)-2- methoxyphenol and dehydrodiconiferyl alcohol were found to be present in chloroform and ethanol extract where as it is absent in petroleum ether extract, 1,2-benzenedicarboxylic acid, butyl 2-ethylhexyl ester present repeatedly in same petroleum ether extract where as it is absent in chloroform and ethanol extract, 1-tridecene also present repeatedly in chloroform extract whereas it is absent in petroleum ether and ethanol extract. Compounds dibutyl phthalate; 1,2-benzenedicarboxylic acid, dipentyl ester; 1,2-benzenedicarboxylic acid, butyl 2-ethylhexyl ester; 1,2- benzenedicarboxylic acid, butyl 2-ethylhexyl ester; 1,2-benzenedicarboxylic acid, diheptyl ester; digitoxin; vitamin D3; benzoic acid, 2-(1-oxopropyl)-; 13-tetradecynoic acid, methyl ester; cyclopentaneundecanoic acid, methyl ester; hexadecanoic acid, ethyl ester; 9,12-octadecadienoic acid, methyl ester, (E,E)-; 9,12,15-octadecatrienoic acid, 2- [(trimethylsilyl)oxy]-1-[[(trimethylsilyl)oxy]methyl]ethyl ester, (Z,Z,Z)-; 2,6,10-dodecatrien-1-ol, 3,7,11-trimethyl-, (E,E)-; Z,Z-2,5-pentadecadien-1-ol; 9,12-octadecadien-1-ol, (Z,Z)-; heptadecane, 9-hexyl- were present only in petroleum ether extract. The compounds 2-butenoic acid, 3-(phenylthio)-, ethyl ester; phenol, 2,5-bis(1,1- dimethylethyl)-; 2-acetylbenzoic acid; 2-piperidinone, N-[4-bromo-n-butyl]-; 9,9-dimethoxybicyclo[3.3.1]nona-2,4- dione; 1H-cycloprop[e]azulene, 1a,2,3,4,4a,5,6,7b-octahydro-1,1,4,7-tetramethyl-, [1aR-(1aα,4α,4aα,7bα)]-; cinerin II; 2-methyl-7-nonadecene; 10-heneicosene (c,t); 1-docosene; tetracontane, 3,5,24-trimethyl-; 2-dodecanol; cyclododecanemethanol; hexadecen-1-ol, trans-9-; 1-eicosanol; octadecanoic acid, ethyl ester; 11,14-eicosadienoic acid, methyl ester; docosanoic acid, ethyl ester; 1-cyclohexylnonene; stigmasterol; pentadecanal-, cis-11- hexadecenal and squalene were present only in chloroform extract. The compounds 3,5-dimethoxy-4-hydroxybenzyl alcohol; 2-methoxy-4-vinylphenol; phenol, 4-ethenyl-, acetate; benzenepropanoic acid, 2,5-dimethoxy-; 2- pyrrolidinone, 1-methyl-; 1,2-benzenedicarboxylic acid, diisooctyl ester; 1,2,3-propanetriol, monoacetate; acetic acid, trifluoro-, 3,7-dimethyloctyl ester; decanoic acid, decyl ester; hexanedioic acid, bis(2-ethylhexyl) ester; Lsorbose; α-D-glucopyranoside, methyl; D-glycero-d-tallo-heptose; glycerin; 2,7-dioxa-tricyclo[4.4.0.0(3,8)]deca- 4,9-diene; 1-undecene, 7-methyl-; 2-methyl-1-undecanol; 3-decyn-2-ol and widdrol were present only in ethanol extract (Table 4).
Table 4. Comparative GC-MS analysis of compounds in various extracts of stem.
In the present study, comparatively 62 components identified of which all the compounds are medicinally valuable. The compound like α-D-Glucopyranoside, methyl used as a preservative [9]; Phenolics usually possess antimicrobial and antifungal activities and consequently defensive functions [10]; cardiac glycosides used as inhibition of malignant cells [11-13]; anticancer effect in humans[14] and also digitoxin is a lower risk for leukemia/lymphoma as well as lower incidence of cancer of the kidney/urinary tract [15]; Squalene, an isoprenoid from the group of polyphenyl compounds, is an intermediate metabolite in cholesterol synthesis possessing antioxidant, immunostimulating, hypolipidemic, cholesterol reducing, anticarcinogenic and antiinflammatory activity [16]; antimicrobial activity particularly against to tuberculosis mycobacteria [17]. In addition, squalene the main component of skin surface polyunsaturated lipids, shows some advantages for the skin as an emollient and antioxidant, and for hydration and its antitumor activities [18]. Many naturally occurring polyhydroxylated sterols exhibit potent biological activities showed potent cytotoxicity to cancer cells [19-22]; stigmasterol isolated from plants were reported to be involved in the synthesis of many hormones like progesterone, androgens, estrogens and corticoids [23] with several pharmacological prospects such as antiosteoarthritic, antihypercholestrolemic, antitumor, hypoglycaemic, antimutagenic, antioxidant, anti-inflammatory and CNS effects [24-28]. Moreover, the presence of various bioactive compounds confirms the application of H. mystax for various ailments by traditional practitioners. Similarly, the same studies were previously reported by the plants like Alseodaphne semecarpifolia, Stylosanthes fruticosa, Cassia auriculata, Wrightia tinctoria, Vernonia cinerea [29-33]. However, isolation of individual phytochemical constituents may proceed to find a novel drug. In addition to this, the results of the GCMS profile can be used as phytochemical tool for the identification of the bioactive components.
From the present study, it was concluded that the plant Hugonia mystax L. are highly valuable in medicinal usage for the treatment of various human ailments along with the chemical constituents present in it. The compounds needs further research on toxicological aspects to develop safe drug.
Authors thanks Mr. S. Kumaravel, Manager, Quality Control, Food Testing Laboratory, Indian Institute of Crop Processing Technology (IICPT), Thanjavur for providing facilities to carry out the work. The first author (A. Vimalavady) specially thanks Pondicherry Adi Dravidar Development Corporation (PADCO), Puducherry, for the financial assistance to carry out this research.
