Gas Chromatography-Mass Spectrometry (GC-MS) Analysis of Ethanolic Extracts of Aerva lanata (L.)

Aim: To determine the phytochemical constituents present in the different parts of Aerva lanata using Gas Chromatography – Mass Spectrometry (GC-MS). Study Design: GC-MS analysis of bioactive compounds in different Methodology: 15 g of powdered plant material of leaf, flower and root were soaked with 60 mL of 95% ethanol for 24 hrs. After 24 hrs, the extract was filtered and the filtrate was concentrated to 1 mL by bubbling nitrogen gas into the solution. 2 µL of ethanolic extracts of leaf, flower and root of A. lanata was used for GC-MS analysis. Results: The GC-MS analyses showed that the presence of four different phytocompounds in Original Research Article ethanolic extract of leaf of A. lanata . The highest peak area of 74.73% for isophytol was identified in leaf of A. lanata . The ethanolic flower extract of A. lanata showed that the presence of twelve different phytocompounds. Flower extract contains the highest amount of phytocompound was 6, 9,12 –octadecatrienoic acid, phenyl methyl ester (z,z,z)- with the peak area of 25%. The root extract of A. lanata showed that the presence of eight different bioactive compounds. The root of A. lanata showed more quantity of lanost-9 (11)-en-12-one with the highest peak area of 45.11%. Conculsion: The present study confirmed that the presence of active compounds in different parts of A. lanata. In future, the isolation of above mentioned bioactive compounds from the different part of A. lanata would be useful to find out the novel drugs.


INTRODUCTION
In the recent past, there has been growing interest in exploiting the biological activities of different ayurvedic medicinal herbs, owing to their natural origin, cost effectiveness and lesser side effects [1]. Medicinal plants are expensive gift from nature to human. The approval of traditional medicine is an alternative form of health care and the development of microbial resistance to the existing antibiotics has induced the researchers to scrutinize the antimicrobial and other biological activities of compounds from plants [2]. Herbal medicines are safer than synthetic medicines because the phytocompounds of the plant extract has no side effects. Medicinal plants have been used all over the world for the treatment and prevention of various ailments, particularly in developing countries [3]. Plant-based natural constituents can be derived from any part of the plant like bark, leaves, flowers, roots, fruits, seeds, etc [4]. The medicinal properties of plants unique to particular plant species or groups are consistent with the concept that the combination of secondary products in a particular plant is taxonomically distinct [5]. There is a growing awareness in correlating the phytochemical constituents of a medicinal plant with its pharmacological activity [6]. Screening of active compounds from plants has led to the invention of new medicinal drugs which has efficient protection and used for the treatment of various diseases.
Today natural products derived from plants are being tested for presence of new drugs with new modes of pharmacological action. A special feature of higher plants is the capacity to produce a large number of secondary metabolites [7]. Recent studies are involved in the identification and isolation of new therapeutic compounds of medicinal importance from higher plants for specific diseases [8,9]. Knowledge of the chemical constituents of plant is helpful in the discovery of therapeutic agent as well as new sources of economic materials like oil and gums. The most important bioactive constituents of the plants are alkaloids, tannins, flavonoids and phenolic compounds. In India large number of plant species had been screened for their pharmacological properties but still a vast wealth of endangered species are unexplored. Medicinal plants are interested in the field of biotechnology, because most of the drug industries are depending on the plants for the production of pharmaceutical compounds [10].
Plants are rich source of secondary metabolites with interesting biological activities. In general these secondary metabolites are an important source with a variety of structural arrangements and properties [11]. Natural products from microbial sources have been the primary source of antibiotics, but with the increasing recognition of herbal medicine is an alternative form of health care. The screening of medicinal plants for active compounds has become very significant because they may serve as talented source of bulk antibiotic prototypes [12,13].
Aerva lanata (L.) belongs to Amaranthaceae family, known as Polpula is a prostrate to decumbent, sometimes erect herb, found throughout tropical parts of India as a common weed in the fields and wasteland. Traditionally A. lanata leaves are used as sap for eye complaints, an infusion is given to cure diarrhea and kidney stone, and root is used in the treatment of snake bite. A leaf decoction is used as gargle for treating sore throat and also used in various complex treatments against guinea worm [14]. A variety of pharmacological functions of this plant like anti-inflammatory, diuretic, expectorant, hepatoprotective and nephroprotective activities were reported [15]. Alcoholic extract of shoots of A. lanata has shown significant antidiabetic and antihyperglycaemic activities [16]. Antimicrobial, cytotoxic, urolithiatic, antihyperlipidaemic, antiparasitic, antihelmentic activities of A. lanata were also reported [17,18]. The preliminary phytochemical studies were conducted and revealed that the presence of various bioactive compounds. GC-MS analysis of acetone extract of leaves [19] and methanolic extracts of root, flower, stem and leaves of A. lanata [20] were reported. But, there is no phytochemical study on ethanol extract of different parts of A. lanata. So, the present study was aimed to analyze the phytocompounds of ethanol extract of different parts like leaf, flower and root of A. lanata using gas chromatography-mass spectrometry (GC-MS).

Collection and Preparation of Plant Material
The medicinal plant Aerva lanata was collected from in and around Mayiladuthurai at Nagapattinum District, Tamilnadu, India. The plant was identified and authenticated (RV/001/A.L Juss/2012) by Dr. S. John Britto, Director, Rapinat Herbarium and Centre for Molecular Systematics, Department of Botany, St. Joseph's College, Tiruchirappalli, Tamilnadu, India. The leaf, flower and root were separated and washed thoroughly in running tap water to remove soil particles and adhered debris and then finally washed with sterile distilled water. The parts leaf, flower and root of A. lanata were shade dried separately and ground well into powder. The powdered materials were stored in air tight containers at 4°C.

Plant Sample Extraction
15 g of the powdered plant material of leaf, flower and root were soaked in 60 mL of 95% ethanol for 24 hrs. After 24 hrs, the extracts were filtered through Whatmann filter paper No. 1 along with 2 gm of sodium sulfate to remove the sediments and traces of water in the filtrate. Before filtering, the filter paper along with sodium sulphate was wetted with 95% ethanol. The filtrate was then concentrated to 1 ml by bubbling nitrogen gas into the solution. From this, 2 μL of ethanolic extract of different parts of A. lanata was subjected to GC-MS analysis [21].

GC-MS Analysis
GC-MS analysis of the ethanol extracts of different parts of A. lanata (leaf, flower and root) were performed using a Perkin Elmer GC Clarus 500 system comprising AOC-20i auto-sampler and a Gas Chromatograph interfaced to a Mass Spectrometer (GC-MS) equipped with an Elite-1MS (100% Dimethyl poly siloxane) fused capillary column (30 m x 0.25 mm ID x 1EMdf). For GC-MS detection, an electron ionization system was operated in electron impact mode with ionization energy of 70eV. Helium gas (99.999%) was used as carrier gas at a constant flow rate of 1ml/min, and an injection volume of 0.5 EI was employed (split ratio of 10:1). The injector temperature was maintained at 250°C, the ion-source temperature was 280°C, the oven temperature was programmed from 110°C (isothermal for 2 min), with an increase of 10°C/min to 200°C, then 5°C/min to 280°C, ending with a 9min isothermal at 280°C. Mass spectra were taken at 70eV; a scan interval of 0.5 seconds and fragments from 45-450Da [22]. The solvent delay was 0 to 2 min and the total GC-MS running time was 36 min. The relative percentage amount of each component was calculated by comparing its average peak area to the total areas. The Mass detector used in this analysis was Turbo-Mass Gold-Perkin Elmer and the software adopted to handle mass spectra and chromatogram was a Turbo-Mass ver-5.2.

Identification of Components
The relative percentage amount of each component was calculated by comparing its average peak area to the total areas. The detection employed by using the NIST (National Institute of Standards and Technology) library ver.2.0 (2005). The prediction of biological activity of compounds was based on Dr. Duke's Phytochemical and Ethnobotanical Databases created by Dr. Jim Duke of the Agricultural Research Service/USDA. Interpretation of GC-MS was conducted using the database of NIST library having more than 62,000 patterns. The spectrum of the unknown component was compared with the spectrum of the known the components stored in the NIST library ver. 2.0. The name, molecular weight and molecular formula of components of the test materials were ascertained.

RESULTS
The identification of the phytocompounds was carried out based on the retention time and molecular formula. The name of identified compounds in the different parts of A. lanata with their retention time (RT), molecular formula (MF), molecular weight (MW) and peak area percentage were represented in Tables 1, 2 and 3.
Similarly, in the present study many phytocompounds were identified in the ethanolic extract of root, flower and leaves of A. lanata. The identified compounds possess biological and pharmacological properties were predicted from Dr Duke's Phytochemical and Ethnobotanical Databases [21]. In this study, the identified phytocompounds from the ethanolic extract of leaf possess antimicrobial and anti-inflammatory activities.
Six phytocompounds has pharmacological activities out of eight compounds were observed in the root of A. lanata. Dihydrotachysterol (DHT) is an analog of Vitamin D and it was reported as systemic effectors of calcium metabolism and promotes calcification of bones. Vitamin D and DHT are administered in the case of hyperparathyroidism to activate calcification. However, high dose of DHT induces, the pathologic calcification leading to excessive accumulation of calcium [42]. The compounds such as lanost-9(11)-en-12-one, cholest-22-ene-21-ol, 3, 5-dehydro-6-methoxyl-pivalate, urs-12en-24oic acid, and 3-oxo-methyl ester (+)-were present in the root of A. lanata are steroid in nature and possess antimicrobial, antiinflammatory, antiarthritic, diuretic and antiasthma activities. Many steroids are used as medicine for the treatment of cancer, arthritis, allergy and in birth control [43,44]. Yamunadevi et al. [45] reported that the presence of different types of steroids in the methanolic extracts of root, stem, flower, leaves and seeds of A. lanata. Similarly, the steroidal compounds urs-12-en-24oic acid and 3-oxo-, methyl ester, (+)-were reported in the ethanolic extract of Canscora perfoliata [46] and also in ethanol extract of leaf of Barleria montana [47].

CONCLUSION
The results of the present investigation revealed that the presence of phytocompounds in the ethanol extracts of different parts of A. lanata by GC MS analysis. The phytoconstituents present in the different parts of A. lanata may be attributed to the medicinal characteristics. In future, the isolation and purification of above mentioned phytocompounds would be useful in the preparation of novel drugs for treating diseases.