Qualitative and GC-MS Analysis of Phytoconstituents of Parthenium hysterophorus Linn.

 

Chanchal Kumari¹, J. Deepalakshmi²

¹University of Madras, Chennai, Tamilnadu, India

²Assistant Professor (Biochemestry), Department of Biochemistry, Mohamed Sathak College of Arts and Science, University of Madras, Sholinganallur, Chennai, Tamilnadu, India

 

ABSTRACT:

Parthenium hysterophorus L. distributed throughout plain land includes roadsides in India and the tropics of the world. It infests pastures and farmland causing often disastrous loss of yield, is known as in common names such as famine weed, congress grass. Methanol extract of Parthenium hysterophoruslinn. was screened for their phytochemical constituents. Results on the qualitative analysis on the plant P. hysterophorus showed that the presence of steroids, sugars, alkaloids, tannins, quinines and phenols in all their three organs. The GC-MS analysis revealed that presence of 45 compounds in the leaves. The more predominated peak in phytol (30.27 %) in leaves, and this activity might be attributed to its higher content of diterpenes. The leaf proteins are reported to be better than cereal and legume proteins. It is used as spices in many parts of the world. Parthenin free dried fibres of plants are used as cattle feed. In Maharashtra and Gujarat (India) it is used in the treatment of diabetes mellitus,antibacterial, antitumor activity and also used as folk remedy for the treatment of infectious and degenerative diseases and find application in traditional, ethnomedicine for treatment of wounds, ulcerated sores, fever, anemia, fecundity and behavioral response and also against inflammatory, skin, neural diseases and female reproductive problems.

 

 

 

INTRODUCTION:

The life of each organism present in the world is based on the green vegetation which it lives around. Every organism in this universe has a specified role to play. Many play a conservative role, among those plants are being the prime base as they are sustaining our environment.

 

Even several centuries before the invention of modernized equipments and drugs, plants provided cures for many severe medical illnesses. Even today, plant materials continue to play a major role in primary health care as therapeutic remedies in many developing countries. [Akash PA, et.al. 2003]. In the recent years, we were hugely depended on the commercial and synthetic drugs which have resulted in the adverse side effects, resistance among several pathogenic organisms and much more. This scenario pushed us to go back to our mother of all producers, the plants to look for effective medicine of lesser or no side effect. Parthenium hysterophorus L. from the family of Asteraceae, popularly known as Congress weed, Carrot weed, Star weed, Fever few, White top, ChatakChandani, Bitter weed. The ability of its seeds to germinate in any season of the year makes it a constantly flourishing component of the vegetation. [Bhatt J.R.Singh, 2012] Parthenium is also reported as a promising remedy against hepatic amoebiasis. South American Indians use the decoction of roots to cure ambioticdysentry, whereas parthenin, a toxin of Parthenium, is found pharmacologically active against neuralgia and certain types of rheumatism (Bhowmik PC, 2003). It is applied externally on skin disorders and decoction of the plant is often taken internally as a remedy for a wide variety of ailments [Cunningham, S. D, 1996; Das B, 1999]. In this study, we had evaluated the phytochemical screening and antimicrobial activity of methanol extract of Parthenium hysterophorus L. leaves against various clinical isolates of pathogenic bacteria and fungi.

 

MATERIALS AND METHODS:

Collection and Identification of Plant:

Fresh whole plant of Parthenium hysterophoruslinn were collected from the Kelambakkam, Kanchipuram District, Tamilnadu. The collected plant was identified and authenticated by Dr.D.Aravind Assistant Professor, Department of Medicinal Botany , National Institute of Siddha,An Autonomous body under the Ministry of AYUSH, Govt. of India, Tambaram, Sanatorium, Chennai-600047, Tamil Nadu, India vide authentication certificate number NISMB2182O16 dated on 01.03.2016.

 

Preparation of Extract:

Parthenium hysterophorus L. leaves were collected from well grown plant. Collected leaves were washed thoroughly with running tap water then with distilled water and dried at room shade for a week. Then it was homogenized to a fine powder and stored in an airtight glass container, protected from sunlight, stored at 4°C until the further process. The methanol extraction of the plant leaves were carried out using a Soxhlet extractor in the ratio 1:10(w/v). The extract liquid was subjected to rotary evaporation in order to remove the solvent. The semisolid extract obtained was stored in an airtight container at 4º C in freezer for further use. The dried extract were exposed to UV light (200-400 nm) for 24 hrs and checked frequently for sterility by streaking on nutrient agar plates [Datta, S, 2001]. For antimicrobial activity, a volume of 20mg of the extract was dissolved in 1ml of 5 percent dimethyl sulphoxide (DMSO). It was sterilized by filtration using 0.22 μmillipore filter.

 

Phytochemical Analysis:

The preliminary phytochemical analysis was carried out on the methanol extract using standard procedures [Dembitsky V (2003),; Kong, C.H,2007,; Krishnaveni M,2014] to identify the phytochemical constituents. They are

Detection of Alkaloids:

0.5g of each sample were dissolved individually with 5ml of 2N HCL and filtered.

 

Dragendroff's reagent:

Filtrate was treated with Dragendroff's reagent. Formation of red precipitate indicates the presence of alkaloids.

 

Test for Flavonoids:

Extracts were treated with few drops of sodium hydroxide solution. Formation of intense yellow colour, which becomes colourless on addition of dilute acid, indicates the presence of flavonoids.

 

Test for Phenols:

Xtracts were treated with 3-4 drops of ferric chloride solution. Formation of bluish black colour indicates the presence of phenols.

 

Test for Carbohydrates: Benedict's test:

To 1 ml of the filtrate, 5 ml of Benedict's reagent were added. The mixture was heated; appearance of red precipitate indicated the presence of reducing sugars.

 

Test forGlycosides:

0.5g of each extract was stirred with 10ml of boiling distilled water. This was filtered and 2ml of the filtrate hydrolized with a few drops of concentrated HCL and the solution rendered alkaline with a few drops of ammonia solution. 5 drops of this solution was added to 2ml of Benedict`s qualitative reagent and boiled. Appearance of reddish brown precipitate showed the presence of glycosides.

 

Test for Terpenoids:

Five ml of extract was mixed in 2ml of chloroform, and concentrated sulphuric acid (3 ml) was carefully added to form a layer. A reddish brown colouration of the inter face was formed to show positive results for the presence of terpenoids.

 

Test for Saponins:

The extract was mixed with 5 ml of distilled water in a test tube and it was shaken vigorously. The formation of stable foam was taken as an indication for the presence of saponins.

 

Test for Steroids:

The extract was mixed with 2ml of chloroform and concentrated sulphuric acid was added sidewise. A red colour produced in the lower chloroform layer indicated the presence of steroids.

 

Test for Proteins: Millon’s test:

Small portion of the extract when mixed with 2ml of Millon's reagent, White precipitate appeared which turned red upon gentle heating that confirmed the presence of Protein. Test for Tannins: The extract was mixed with 2 ml of Ferric chloride solution. A blue-green coloration indicated the presence of Tannin.

 

GC-MS Analysis:

GC-MS Gas chromatography – Mass spectrometry (GC- MS) is an analytical method that combines the features of gas chromatography and mass spectrometry to identify different substances with in a test sample. Applications of GC-MS include drug detection, fire investigation, environmental analysis, explosives investigation and identification of unknown samples. Additionally, it can identify trace elements in materials that were previously thought to have disintegrated beyond identification. Volatiles can be identified by GC- MS. Volatiles play a vital role in healthcare systems of medicinal plants.  GC-MS analysis of the extract was performed using a Perkin Elmer GC Claurus 500 system and Gas Chromatograph interfaced to a Mass Spectrometer equipped with an Elite 5MS fused silica capillary column (30 × 0.25 mm ID. ×1 Mm df, composed of 5% Diphenyl/ 95% Dimethyl poly siloxane). For GC-MS detection, an electron ionization system with ionization energy of 70 eV was used. Helium gas (99.999%) was used as the carrier gas at a constant flow rate of 1 ml/min and an injection volume of 3 Ml was employed (split ratio of 10:1). Injector temperature 250ºC; Ion-source temperature 280ºC. The oven temperature was programmed from 110°C (isothermal for 2 min) with an increase of 10ºC/min to 200°C, then 5ºC/min to 280 ºC ending with a 9 min isothermal at 280°C. Mass spectra were taken at 70 eV, a scan interval of 0.5seconds and fragments from 45 to 450 Da. The relative percentage amount of each component was calculated by comparing its average peak area to the total areas. Software adopted to handle mass spectra and chromatograms was a Turbo Mass V. 5.2.0.(Praveen Kumar P.,et al., 2010)

 

RESULT:

Phytochemical Screening of Methanol Extract of Parthenium Hysterophorus Linn.

The dried powder material was extracted successively with methanol. The percent yield of methanol was found to be 25%. Secondary metabolites were found in good proportion in methanol extract of Parthenium hysterophoruslinn. The results of preliminary phytochemical constituents of the Parthenium hysterophoruslinn are presented in Table-1 carbohydrates, fatty acids, proteins, aminoacids, Saponins, Tannins, Carotenoids, Flavonoids, Alkaloids, Glycosides, polyphenols, Anthraquinone and steroids as been observed as the active phytoconstituents of Parthenium hysterophoruslinn with the methanolic extract of Parthenium hysterophoruslinn.. The presence of these constituents in most plant has been reported to have proven medicinal implication.

 

Table-1: Preliminary Phytochemical Tests of Parthenium Hysterophorus Linn.

 

GC-MS STUDY:

The GC-MS study of Parthenium hysterophorouslinn has shown many phytochemicals which contributes to the medicinal activity of plants.GC-MS chromatogram shows the retention time. The peak area percent was calculated using height and width of the peak obtained (Fig -1).

 

Figure 1: GC-MS Chromatogram of Parthenium Hysterophorus Linn:

In the present investigation 45 phytocompounds were obtained. All the 45 compounds have been identified in the methanolic extract of Parthenium hysterophorouslinn.

 

Table-2: Phytoconstituents Identified From The Methanolic Leaf Extract of Parthenium Hysterophorus Linn. Through Gc-Ms Analysis.

 

Table-3:Pharmacological Properties ofImportant Phytocomponents Identified InThe Methanolic Leaf Extract Of PartheniumHysrerophorusLinn ByGC-MSAnalysis.

S.No	RT	Name of the compound	Compound Nature	Biological activity
1.	10.134	Hydantoin	Hetero cyclic organic compound	Anticancer, Antioxidants
2.	13.269	Acetamide	Acetic acid	Narcolepsy treatment
3.	14.301	Caryophyllene oxide	Sesquiterpene	Anti-inflammatory,Antibiotic,Anti -oxidant, Anti-carcinogenic
4.	15.586	Benzaldehyde,3-phenoxy	Benzal chloride	Anti-bacterial, Anti-fungal infecticidal
5.	16.210	Isoaromadendrene epoxide	Alkaloids	Anti-fungal,Anti-microbial
6.	16.389	3-Carene,4,acetyl	Organic compound	Anti-aging, Anti-cholinergic activity
7.	16.441	Ethanone,1-(2,6,6-trimethyl-1- cyclohexen-1-yl)	Ketones	Anti-cancer research

 

Table 3 Countineu

S.No	RT	Name of the compound	Compound Nature	Biological activity
8.	16.953	Benzoic acid,2-(4-aminophenyl)	Phytol	Anti-bacterial, Anti-tubercular
9.	17.050	2-Pentadecanone,6,10,14- trimethyl	Flavonoid	Anti-microbial
10.	17.080	1,3,6-Heptatriene,2,5,5- trimethyl	Artemisia triene	Antimicrobial activity
11.	17.451	Culmorin	Sesquiterpenoids	Antibiotic activity
12.	17.540	2-Ethylphenethyl phenyl sulfide	Alcohol	Anti-hypertensive
13.	17.852	Alloaromadendrene oxide-(1)	Alkane	Anti-microbial activity
14.	17.897	Hexadecanoic acid	Palmitic acid	Anti-inflammatory, Ulcerogenic
15.	18.224	Dibutyl phthalate	Phthalic acid	Anti-microbial
16.	4.993	Glycerin	Polyol, colorless, odorless	Food preserver, Lubricating agent
17	18.298	n-Hexadecanoic acid	Palmitic acid	Anti-inflammatory, Analgesic
18	18.788	6-Heptenoic acid,4-isopropenyl- 6-methyl-2-oxo-,ethyl ester	Fatty acid	Anti-microbial
19	19.234	Valeric acid,tridec-2-ynyl ester	Ester	Anti-bacterial activity
20	19.457	Phytol	Diterpene	Anti-microbial, Anti-cancer, Anti-inflammatory,Analgesic,Hepatoprotective,Anti- androgenic.
21	19.531	8,11-Octadecadienoic acid, methyl ester	Monoterpene	Anti-cancer activity
22	19.590	9,12,15-Octadecatrienoic acid, methyl ester,(Z,Z,Z)	Linolenic acid	Anti-viral activity
23	19.635	11,12,-Dibromo-tetradecan-1-ol acetate	Fatty acid	Anti-microbial activity
24	19.954	Fumaric acid	Fumaric acid esters	Anti-cancer activity
25	20.341	Octadecanamide	Amide compounds	Antioxidant
26	20.512	1-Methylene-2b-hydroxymethyl- 3,3-dimethyl-4b-(3-methylbut-2 -enyl)-cyclohexane	Organic compounds	Anti-microbial activity
27	21.314	9-Octadecenoic acid,12-hydroxy -,methyl ester	Esters	Antiviral activity
28	21.403	Trifluroacetyl-alpha.-fenchol	Crystalline compound	Antibacterial activity
29	21.633	4-Allyloxyimino-2-carene	Carotenoids	Antiinflammatory, Antimicrobial
30	21.916	9-Octadecanamide	Stearic acid amide	Antiinflammatory, Antibacterial activity, Anticancer activity.
31	22.599	Spiro[4.5]decane-2,6-dione	Steroids	Anticancer activity
32	22.837	Cyclotetradecane,1,7,11- trimethyl-4-(1-methylethyl)	Alkaloid	Antimicrobial
33	25.377	Squalene	Triterpene	Anti inflammatory, ulcerogenic
34.	23.684	Azuleno (4,5-b) furan 2,9-dione, decahydro-6a-hydroxy-6,9a-dimethyl-3-methylene, [3aS,-(3a alpha – 6 beta -6a alpha- 9a beta, 9b beta)]	Monoterpenes	Anti inflammatory, anti cancer
35.	21.730	Bicyclol (3.1.1.) heptan 3-one (6,6-dimethyl-2-2-methyl-propy)l	Carotenoids	Antioxidants
36.	21.128	Benzothiophene-3-Carbo-nitrite 4,5,6,7, tetrahydro 2-(4-tert-butyl benzyl idenamino)	Phenol	Pesticides
37.	20.185	2-oxo-3(3-Methoxybenzoyl) propanoic acid, ether ester	Phenol	Antimicrobial
38.	20.051	Methyl 8,11,14-Heptadecatrienoate	Carotenoids	Anti inflammatory,
39.	17.258	4-Bromobutanoic acid tridec-2-ynyl ester	Alkyl alcohol	Anticancer agent
40.	16.552	1-Methyl 3,5-di-isopropoxybenzene	Alkaloids	Anti microbial effects
41.	15.765	Chlortoluron	Carotenoids	Herbicides
42.	15.401	Spiro-10-(2,11-dioxabicyclo 4,4-1(undeca 3,5-diene)-2’-(oxirane) 1,3,7,7, tetra methyl	Saponins	Anti-toxicity
43.	14.168	2-phenyl-2-4, Octadienol	Carotenoids	Anti-oxidants.
44.	16.432	Pentyl -undecyl ester	Fatty acid esters	Antioxidants

 

DISCUSSION :

In earlier study Gupta et al.(1977) reported that same plant shows the presence of carbohydrates, aminoacids, and saponins,inmethanolic extract of Parthenium Hysterophorus Linn. Leaf. In the present study along with the above mentioned compounds, it has been found  that methanolic extract of Parthenium Hysterophorus Linn. Leaf had additional phytoconstituents, when tested qualitatively through GC-MS analysis. These are terpenes, glycosides,tannins,flavonoids, carbohydrates, protein, steroids, alkaloids, aminoacids, saponins, terpenes and phenolics. Most of these compounds will play major role in various biological activities right from inhibiting the microbial growth to destroying cancer cells (Lakshmanan Krishnavignesh, et,al;2013).

 

CONCLUSION:

The present research established that, the Parthenium Hysterophorus Linn. Leaf contain most of the phytocomponents when tested qualitatively through GC-MS analysis and showed considerable amount of terpenes, carbohydrates, protein, steroids, alkaloids,aminoacids, saponins, tannins, terpenes and phenolics. Further study can be carried out to genetically modified Parthenium hysterophoruslinn. For better understanding about the metabolic pathway of secondary metabolites production and these molecular studies greatly help in understanding the mechanism of drug molecule action and enhancing the efficiency of treatment in medical.

 

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Received on 05.04.2017          Modified on 28.04.2017

Accepted on 09.05.2017      ©AandV Publications All right reserved