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Analysis of phytocomponents in the methanolic extract of Eupatorium triplinerve by GC-MS method

G. Selvamangai1 Anusha Bhaskar2*
  1. Department of Biotechnology, Alpha Arts and Science College, Chennai
  2. Department of Biotechnology, PRIST University, Vallam, Thanjavur 614 403.
Corresponding Author: G. Selvamangai E-mail: [email protected]
Received:12 February2013 Accepted: 28 February 2013
Citation: G. Selvamangai, Anusha Bhaskar* “Analysis of phytocomponents in the methanolic extract of Eupatorium triplinerve by GC-MS method” Int. J. Drug Dev. & Res., January-March 2013, 5(1): 384- 391.
Copyright: © 2013 IJDDR, G. Selvamangai et al. This is an open access paper distributed under the copyright agreement with Serials Publication, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Abstract

Objective: To characterize the phytochemical constituents of Eupatorium triplinerve using GC – MS and to study the ability of the metabolites to serve as an antagonist to caspase 3 receptors to ascertain its anticancer properties. Methods: Ten grams of the powdered sample was subjected to column chromatography over silica gel (100-200 mesh) and eluted with n-hexane, chloroform, ethanol and methanol respectively. n- Hexane and Chloroform did not elute much of the compounds. The methanol fraction of the Eupatorium triplinerve was taken for GCMS analysis. The analysis was carried out on a GC Clarus 500 GC system with a column packed with Elite – 1 (10% dimethyl poly siloxane, 30 x 0.25 mm ID x 1 EM df), the compounds are separated using with Helium as carrier gas at a constant flow 1ml/min. sample extract (2 μL) injected into the instrument was detected by Turbo gold mass detector (Perkin Elmer) with the aid of the Turbo mass 5.1 software. The important compounds obtained from GC-MS were further studied in silico to study its anticancer activity by docking with caspase 3 receptor of four important metabolites neophytadiene, nitrocyclohexane, octadecane and tetradecanoic acid. Results: The GC MS analysis provided peaks of eleven different phytochemical compounds namely hexadecanoic acid (14.65%), 2,6,10-trimethyl,14-ethylene-14-pentadecne (9.84%), Bicyclo[4.1.0]heptane, 7-butyl- (2.38%), Decanoic acid, 8-methyl-, methyl ester (3.86%), 1-undecanol (7.82%), 1-hexyl-1- nitrocyclohexane (2.09%), 1,14-tetradecanediol (6.78%), Octadecanoic acid, 2-hydroxy-1,3-propanediyl ester (19.18%) and 2- hydroxy-3-[(9E) -9-octadecenoyloxy] propyl(9E)-9-octadecenoate (8.79%). From the docking assay it was found that nitrocyclohexane and neophytadiene compounds exhibited good docking score. Conclusion: The bioactive compounds in the methanolic extract of Eupatorium triplinerve have been screened using this analysis. Isolation of individual components would however, help to find new drugs.



 

Key words

GC-MS,Caspase,Eupatorium triplinerve

INTRODUCTION

In recent years the use of plants in the management and treatment of diseases has gained considerable importance. Plants and fruits are considered as one of the main sources of biologically active compounds. An estimate of the World Health Organization (WHO) states that around 85 – 90% of the world’s population consumes traditional herbal medicines (1). Plants are capable of synthesizing an overwhelming variety of low-molecular weight organic compounds called secondary metabolites, usually with unique and complex structures. Many metabolites have been found to possess interesting biological activities and find applications, such as pharmaceuticals, insecticides, dyes, flavors and fragrances.
Eupatorium triplinerve Vahl is familiarly known as Ayappana belongs to Asteraceae family. It is a slender herb with narrow lanceolate leaves and large number of pedicelled flower-heads at the top of the branch. The methanolic extract of E triplinerve is reported to have hepatoprotective effect and antioxidant effect against carbon tetracholoride induced hepatotoxicity in rats (2), while the ethanolic extract had analgesic effect in inflammatory model of pain (3), antibacterial and antifungal activity (4), antiseptic and in the treatment of various ulcers and haemorrhages (5). Although the plant is used in Ayurvedic medicine for the treatment of ailments there are no reports on the constituents that are responsible for the therapeutic effect. With this background the present study was aimed to identify the phytoconstituents present in E triplinerve using GC-MS analysis.
The anticancer activity of the metabolites obtained as a result of GC-MS analysis were analyzed by docking with caspase3 receptor which is responsible for apoptosis. The CASP3 protein is a member of the cysteine-aspartic acid protease (caspase) family. Sequential activation of caspases plays a central role in the execution-phase of cell apoptosis. Caspases exist as inactive proenzymes that undergo proteolytic processing at conserved aspartic residues to produce two subunits, large and small, that dimerize to form the active enzyme. This protein cleaves and activates caspases 6 and 7; and the protein itself is processed and activated by caspases 8, 9, and 10 (6).

MATERIALS AND METHODS

Collection and preparation of plant material

Fresh plants of E. triplinerve were collected from the natural habitats of Tiruchirappalli, Tamil Nadu, India. The samples were washed thoroughly in running tap water to remove soil particles and other adhered debris and finally washed with sterile distilled water. The whole plants were shade dried and ground into fine powder. The powdered materials were stored in air tight polythene bags until use.

Plant sample extraction

Plant sample extraction and Column chromatography

Ten grams of powdered sample was extracted with 50 mL methanol overnight and filtered through ash less filter paper with sodium sulphate (2 g). The crude extract was subjected to column chromatography over silica gel (100-200 mesh) and eluted with nhexane, chloroform, ethanol and methanol respectively. n-Hexane and Chloroform did not elute much of the compounds. The methanol fraction of the Eupatorium triplinerve was taken for GC-MS analysis.

Gas Chromatography- Mass Spectrum Analysis (GC-MS)

GC-MS analysis was carried out on a GC Clarus 500 Perlin Elmer system comprising a AOC-20i autosampler and gas chromatograph interfaced to a mass spectrophotometer (GC – MS) instrument employing the following conditions: column Elite – 1 fused silica capillary column (30 x 0.25 mm ID x 1 EM df, composed of 100% Dimethyl polysiloxane), operating in electron impact mode at 70 eV; helium (99.999%) was used as carrier gas at a constant flow of 1 ml/min and an injection volume of 0.5 EI was employed (split ratio of 10:1 injector temperature 250 ºC; ion-source temperature 280 ºC. The oven temperature was programmed from 110 ºC (isothermal for 2 min). with an increase of 10 C/min, to 200 ºC then 5 ºC/min to 280 ºC, ending with a 9 min isothermal at 280 ºC. Mass spectra were taken at 70 eV; a scan interval of 0.5s and fragments from 40 to 550 Da.

Identification of components

Interpretation on mass spectrum GC-MS was conducted using the database of National Institute Standard and Technology (NIST) having more than 62,000 patterns. The spectrum of the unknown component was compared with the spectrum of the known components stored in the NIST library. The name, molecular weight and structure of the components of the test materials were ascertained.

Molecular docking

Preparation of ligands

The secondary metabolites obtained from GC-MS analysis were used for docking. The two-dimensional structures of the ligands were generated using the ACD/ChemSketch tool. The data are converted and saved in mol format and is used for docking analysis.

Protein Data Bank (PDB)

Source: www.rcsb.org

The PDB is the single, global archive for information about the 3D structure of biomacromolecules and their complexes, as determined by X-ray crystallography, NMR spectroscopy and cryo-electron microscopy. Caspase 3 receptor was downloaded from Protein data bank with the specific resolution and the PDB id is 2X70.

Docking

Auto-dock (modeling and simulation application was used for the study which focuses on optimizing the drug discovery process. The mechanism for ligand placement is based on fitting points. Fitting points are added to hydrogen bonding groups on the protein and ligand. A molecular mechanics like scoring function which includes terms of hydrogen bonds is employed by DS to rank the docked posses. The docking algorithm was also accessed in order to know the binding sites and the number of rotatable bonds of the ligand.

RESULTS

The identified compounds of the leaves of E triplinerve, their retention indices, percentage composition, chemical structure and activities are given in Table 1. The compound prediction is based on Dr. Duke’s Phytochemical and Ethnobotanical Databases. The results showed the presence of hexadecanoic acid (14.65%), 2,6,10-trimethyl, 14- ethylene-14-pentadecne (9.84%), bicycle[4.1.0]heptanes (2.38%), decanoic acid (3.86%), 1-undecanol (7.82%), 1-hexyl-1- nitrocyclohexane (2.09%), 1,14-tetradecanediol (6.78%), octadecanoic acid (19.18%) and 2-hydroxy- 3-[(9E)-9-octadecenoyloxy]propyl(9E)-9- octadecenoate (8.79%). The spectrum profile of GCMS confirmed the presence of 10 major components with retention time15.084, 15.75, 16.2,16.40,16.96,17.15,18.38,19.986,20.148 and 21.619 respectively (Figure 1). The individual fragmentation of the components is illustrated in (Figures 2A-2J).
The 3D structure of compounds namely Neophytadine, Nitrocyclohexane, Octadecane and Tetadecanoic acid which were present in notable amounts in the extract were docked with Caspase 3 receptor. The results of interaction with these compounds are shown in Fig 3. All amino acid residues are displayed as ball and stick and the receptor as a ribbon. The interaction between them is given in table 2.

DISCUSSION

In the present study, the GC-MS analysis of the methanolic extract of E triplinerve showed the presence of ten compounds. In terms of percentage amounts hexadecanoic acid, tetradecanoic acid and octadecanoic acid were predominant in the extract. These three major compounds have all shown to have hypocholesterolemic activity, antioxidant and lubricating activity. Anticancer and antiproliferative are shown by tetradecanoic acid and 2,6,10,- trimethyl,14-ethylen-14-pentadecne, while 1-hexyl-1- nitrocyclohexane and 1,14-tetradecanediol other compounds show antimicrobial and antiinflammatory activities.
There is growing awareness in correlating the phytochemical components and their biological activities [7,8,9]. E triplinerve is a plant used in Ayurvedic medicine however there are no reports on the thorough phytochemical analysis of the plant. We report the presence of some of the important components resolved by GC-MS analysis and their biological activities.
Docking is the process of fitting together of two molecules in 3-dimensional space. Docking allows the scientist to virtually screen a database of compounds and predicts the strongest binders based on various scoring function. It explores ways in which two molecules such as drug and receptor together and dock to each other well. The molecules binding to a receptor, inhibits function, and acts as a drug [10]. Verlinde and Hol, [11], suggested that when a drug binds to a target in molecular modeling and molecular design software, the lower the energy value the higher is the affinity of the drug. Keeping this in this view, it is clear from the results (table-4) that nitrocyclohexane and neophytadiene have a higher affinity towards the receptor, since they produce a lower energy value while interacting with the receptor.
Thus this type of GC-MS analysis is the first step towards understanding the nature of active principles in this medicinal plant and this type of study will be helpful for further detailed study.

Tables at a glance

Table icon Table icon
Table 1 Table 2
 

Figures at a glance

Figure 1 Figure 2 Figure 2a Figure 4
Figure 1 Figure 2 Figure 2a Figure 3
 

References

1) WHO Report, World Health Organization, Geneva, WHO/EDM/TRM/ 2002, 21, 19.

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3) CheriyanBV,Venkatadri N, Viswanathan S and Kamalakannan P. Screening of alcoholic extract of Eupatorium triplinerveVahl and its fractions for its antinociceptive activity. Indian Drugs. 2009;46 (10): 797-802.

4) Garg SC, Nakhare S. Studies on the essential oil from the flowers of Eupatorium triplinerve. Indian Perfumer 1993; 37(4): 318 – 323.

5) Ghani, A. 1998. Medicinal plants of Bangladesh: Chemical constituents and uses. 1st edn.Asiatic Society of Bangladesh.pp. 174.

6) Alnemri ES, Livingston DJ, Nicholson DW, Salvesen G, Thornberry NA, Wong WW, Yuan J. Human ICE/CED-3 protease nomenclature. Cell 1996; 87(2): 171.

7) Fernie AR, Trethewey RN, Krotzky AJ, Willmitzer L. Innovation – Metabolite profiling: from diagnostics to system biology. Nat Rev Mol Cell Biol. 2004; 5: 763 – 769.

8) Sumner LW, Mendes P, Dixon RA. Plant metabolomics: largescalephytochemistry in the functional genomics era. Phytochem 2003; 62(6): 817 – 836.

9) Robertson DG. Metabonomics in toxicology: A review. ToxicolSci 2005;85: 809 – 822.

10) Sivakumar R, Pradeepchandran RV, Jayaveera KN. Computer aided discovery of benzimidazole derivatives on peptide deformylase. as antimicrobial agent using hex. Eur J Chem 2011; 2 (4): 558 – 560.

11) Verlinde CL, Hol WG. Structure-based drug design: progress, results and challenges. Structure 1994; 2: 577−587.
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