GC-MS Analysis of Methanolic Extracts of Leaf and Stem of Marsilea minuta (Linn.)

Aims: To analyze the chemicals composition of methanolic extracts of leaf and stem of M. minuta collected from Uppur Village, Tamilnadu, India by GC-MS. Study Design: Experimental study. Methodology: The Methanolic extracts were prepared and concentrated at 40°C using hot air oven. The concentrated methanolic extracts were subjected to GC-MS analysis using the instrument Perkin Elmer Clarus 500. Results: The methanolic extract of leaf of M. minuta showed the presence of 36 phyto compounds including n-Hexadecanoic acid (44.41%) (C16H32O2); (Z)6,(Z)9-Pentadecadien-1-ol (35.49%) (C15H28O); Phytol (5.10%) (C20H40O); 2-Cyclohexane-1-one,4-hydroxy-3,5,6-trimethyl-4 (3-oxo-1butenyl) (2.25%) (C13H18O3); 9,12,15-Octadecatrienoic acid (Z,Z,Z) (2.0%) (C18H30O2); 3,7,11,15Tetramethyl-2-hexadecan-1-ol (1.99%) (C20H40O) and Benzofuran, 2,3-dihydro(1.1%) (C8H8O). The methanolic extract of stem of M. minuta showed the presence of 27 bioactive compounds Original Research Article Sabithira and Udayakumar; JOCAMR, 3(1): 1-13, 2017; Article no.JOCAMR.30871 2 including 1,19-Eicosadiene (40.94%) (C20H38); n-Hexadecanoic acid (20.13%) (C16H32O2); 2(R)3(S)-1,2,3,4-Butanetetrol (16.31%) (C4H10O4); Glycerin (6.70%) (C3H8O3); Octadecanoic acid (4.24%) (C18H36O2); Benzofuran, 2,3-dihydro(1.73%) (C8H8O); 3,7,11,15 – Tetramethyl-2 hexane decen-1-ol (1.55%) (C20H40O); n-Hexadecanoic acid methyl ester (1.15%) (C17H34O2) and 10Octadecenoic acid methyl ester (1.42%) (C19H36O2). Conclusion: The results of the present study concluded that the presence of various phytocompounds in leaf and stem of M. minuta. Therefore, plants are the rich sources of chemicals but largely unknown and unexplored. So, it is our hope that this study will encourage further research on isolation and purification of therapeutically important phytocompounds from M. minuta


INTRODUCTION
Plant is a source of medicinal agents for a long time. Medicinal plants have been used for years in daily life to treat various diseases [1]. India has the richest and most diverse cultural traditions especially with the use of traditional systems of medicine. Nutraceuticals are the alternative forms of conventional medicine with a view of accomplishing desirable therapeutic outcomes and reduction in side effects compared with synthetic therapeutic agents [2]. People have been using medicinal plants based on their acclaimed therapeutic values and till date over 85,000 medicinal plants with various therapeutic benefits have been identified and documented globally [3]. The traditional medicine in India consists of different components such as Ayurveda, Siddha, Unani, Homeopathy and Naturopathy [4]. Medicinal plants are the source of drugs in traditional systems of medicine, modern medicines, food supplements, folk medicines, pharmaceutical intermediates and chemical entities for drug synthesis [5]. Medicinal plants have been used in the folkloric medicine for the treatment of number of diseases [6]. Medicinal plants and their purified compounds have shown pharmacological properties [7]. The analysis of plant extracts has been a great interest for researchers to identify new pharmacologically important molecules of new drugs that will be useful to treat diseases [8].
The identification of bioactive chemical compounds from medicinal plants is an important task in the pharmaceutical industry for drug development and preparation of medicines [9]. Some microorganisms are the primary source for the synthesis of antibiotics which are used in the treatment of diseases. However, the screening of medicinal plants for bioactive compounds against diseases is an important field and it may be serve as an alternatives means for the synthesis of antibiotics [10,11].
Marsilea minuta is a common aquatic or subaquatic fern. It is a member of Marsileaceae family [12,13]. M. minuta is an important medicinal plant and it is distributed worldwide. It comprises 53 well defined living and 10 fossil species, among these 9 species are found in India [14,15]. M. minuta leaves and shoots are commonly used as vegetables. It is used in the treatment of cough and respiratory disorders. Juice of fresh shoots and decoction of leaves of M. minuta are used to treat cough [16,17]. Traditionally, M. minuta is used to treat indigestion, nose bleeding, kidney infection, toxicity and hepatitis [18]. M. minuta is also used to treat psychopathic conditions, diarrhoeal, respiratory and skin diseases [19,20]. The whole plant of M. minuta is used as astringent, expectorant, digestive and diuretic drug [21].
The medicinal plant M. minuta is used to treat insomnia and mental problems. The regular usage of the plant M. minuta as green vegetables is believed to exert favorable effects on hypertension, sleeping disorder, bronchitis, fever and headache [12]. It is also recommended for the treatment of spastic condition of leg muscle, epilepsy and migraine [12,19,22]. Leaves of M. minuta are prescribed by folk medical practitioners to treat diabetes and gastrointestinal disorders [23,24]. In some mental clinics, the decoction of M. minuta could be prescribed to patients with psychological disorders along with their meal [25]. M. minuta is reported to possess CNS active principles, so it is recommended for the treatment of various neurological disorders [26]. It has also been reported to possess hepatoprotective and antiaggressive properties [27,28].
In vitro antibacterial activity of leaf extract of M. minuta against human pathogens has been reported [29]. Marsiline is an important active compound and its chemical name is ester of 1triacontanol and hexacosanoic acid isolated from M. minuta . The researchers reported that the  marsiline possess sedative, anticonvulsant,  antidepressant,  hypocholesterolemic  and  antifertility properties [30,31,32]. The phytochemical constituents of leaf of M. crenata using GC-MS have been reported [33].
The anti-stress activity of M. minuta may be potentially valuable for the treatment of stress and stress related disorders [34]. M. minuta has also been reported to possess antihepatotoxic properties [35].
The antimicrobial activity of rhizome of M. minuta against some human bacterial and fungal pathogens has been reported [36] and the extract of M. minuta has been shown to possess antioxidant [37] and antidiabetic [38] properties. Several studies have been conducted using M. minuta, but no study has characterized the active phytocompounds present in the leaf and stem of M. minuta. So, the present study was designed to characterize the phytocompounds present in the leaf and stem of M. minuta using GC-MS.

Collection of Plant Material
The fresh plants of M. minuta were collected from natural habitats of Uppur Village, Thiruvarur District, Tamilnadu, India. The collected plant was identified by Rev. Dr. S. John Britto, Director, Rabinet Herbarium and Centre for Molecular Systematics, St. Joseph's College, Tiruchirappalli, Tamilnadu, India and deposited in the herbarium (Voucher specimen number: KG 002). The collected plants were brought into the laboratory and washed thoroughly in running tap water to remove the soil particles and adhered debris and then finally washed with sterile distilled water. The leaf and stem of M. minuta were separated and dried under shade for 10 days at room temperature. Then the plant materials were pulverized into powder. The powdered materials were stored in air tight containers until the time of use.

Preparation of Plant Extracts
The leaf and stem of M. minuta extracts were prepared according to previously reported procedure [39]. For this, 50 g of leaf and stem powder of M. minuta was soaked in 500 ml of methanol and kept in orbital shaker for 48h. After 48h, it was filtered through Whatman no. 1 filter paper (125 mm) and then the supernatant was concentrated at 40°C till the solvent evaporated completely using hot air oven. The concentrated methanolic extracts of leaf and stem of M. minuta were subjected to GC-MS analysis.

GC-MS Analysis
The GC-MS analysis was performed to identify the chemical compounds present in the leaf and stem of M. minuta by using an instrument Perkin Elmer Clarus 500 [39]. The data were obtained on a Capillary Column Elite-5MS [5% phenyl 95% dimethyl poly siloxane]. Helium (99.999%) was used as the carrier gas with a flow rate of 1ml/min in the split mode (10:1). An aliquot of 1µl of methanol solution of the sample was injected into the column with the injector temperature maintained at 270°C. GC oven temperature started at 110°C and holding for 2min and it was raised to 200°C at the rate of 10°C/min without holding. Holding was allowed at 280°C for 9min with the program rate of 5°C/min (60°C@8°C/min to 230°C (5min)@6°C/min to 280°C (10min)). GC interface and ion source temperature was maintained at 200°C. The mass spectrum of compounds in the sample was obtained by electron ionization at 70eV and the detector was operated in scan mode from 40-450 amu (atomic mass units). A scan interval of 0.5 second and fragments from 40 to 450Da were maintained. The total running time was 36 minutes.

Identification of Chemical Compounds
Interpretation of mass spectra of the extracts of leaf and stem of M. minuta was conducted using the database of National Institute of Standard and Technology [NIST] library. The library has more than 62,000 spectral patterns. The spectrum of the compound was compared with the spectrum of NIST library database. The identity of the spectra above 95% was needed for the identification of compounds. The name, molecular weight and structure of the compounds identified and characterized from the extracts of leaf and stem of M. minuta were ascertained. The relative percentage amount of each component was calculated by comparing its average peak area with the total area. The spectrum of the unknown component was compared with the spectrum of the component stored in the NIST library using the Turbomass version 5.2.0.

RESULTS
The

DISCUSSION
Plants are an important part of our everyday diet, and the plant constituents and their nutritional value have been intensively studied for decades. Secondary metabolites are characterized by enormous chemical diversity and every plant has its own characteristic set of secondary metabolites. So, plants synthesize an extensive array of secondary metabolites often highly complex structures. The chemical investigations of medicinal plants have largely been driven to find new drugs to treat human disease. The secondary metabolites have been of interest to humans as flavors, fragrances, dyes, pesticides and pharmaceuticals.
Currently, a number of modern drugs have been isolated from natural sources. Plant derived pharmacological compounds have recently become a great interest owing to their versatile applications. Ethnobotanical research has increased considerably in the last few years and is presently considered a subject of great interest. Medicinal plants have been good source for the synthesis of many drugs. There is a growing awareness in correlating the active principles from the medicinal plants with their biological activities [5]. Phytocompounds are possessing biological properties and demonstrated to exert beneficial effects. Nature is and will still serve as the man's primary source for the cure of his aliments. However the potential of higher plants are as sources for new drugs. There are many thousand plant species in the world, but only a small proportion has been investigated both phytochemically and pharmacologically. In this study, the selected medicinal plant M. minuta is an important and used in the treatment of many diseases by tribal people. So, there is need to identify and characterize the active phytochemicals from M. minuta.
The bioactive compounds of leaf and stem of M. minuta were determined by GC-MS analysis and the identified bioactive compounds were documented in Tables 1 and 2. The gas chromatogram of the leaf and stem of M. minuta showed that the relative concentration of various compounds getting eluted at different retention time. The height of the peak indicates that the relative concentration of the compound present in the extract of leaf and stem of M. minuta. The mass spectrometry analysis was carried out to identify the compounds in the leaf and stem of M. minuta eluted at different retention time. The mass spectra are the fingerprints of phytocompounds of leaf and stem of M. minuta, which were identified by NIST library. Similarly, the bioactive compounds of stem and leaf of Tiliacora acuminata [41], leaf of Cassia italica [42] leaf, stem and seed of Cajanus cajan [43], leaf of Allamanda cathartica [44] and stem bark of Dolichandrone atrovirens [45] were determined by GC-MS analysis.
The biological activities of phytocompounds of leaf and stem of M. minuta were predicted by Duke's Ethnobotanical Databases [40]. As per Duke's Ethnobotanical Database, the identified phytochemicals of leaf and stem of M. minuta possess antibacterial, antifungal, antimalarial, antioxidant, antitumor, anti-inflammatory, hypocholesterolemic, anticancer, diuretic, antihyperglycaemic and analgesic activities. So, the presence of phytochemicals in leaf and stem of M. minuta may be responsible for controlling diseases. Similarly, the presence of many phytocompounds in leaf and stem of M. quadrifolia with their biological activities were reported [39]. Due to their large pharmacological activities, phytochemicals have been used for centuries in traditional medicine.
Bioactive compounds from plants belong to various chemical groups such as phenolic compounds, flavonoids, alkaloids, tannins, saponins, glycosides, lignans, terpenoids, etc. The methanol is generally used as a first solvent for extraction of bioactive compounds in medicinal plants, because lots of polar compounds and certain group of non-polar compounds are dissolving in it. So in this study, the methanol was used for extraction of bioactive compounds. The results of GC-MS profile of leaf and stem of M. minuta were also showed that the presence of phenolic compounds and flavonoids. No other studies are available on phytochemicals of leaf and stem of M. minuta using GC-MS. So, up to date of our knowledge the present study may be the first report on phytochemicals of leaf and stem of M. minuta using GC-MS. In this study, the identified bioactive compounds of leaf and stem of M. minuta were reported as new compounds.
The uses of medicinal plants and phytomedicines have led to need for the analysis of plant compounds. In this study, we used the GC-MS technique for the analysis of secondary metabolites in leaf and stem of M. minuta. Similarly in our previous studies, we used the GC-MS technique for the analysis of the phytocompounds in leaf, flower and stem of Aerva lanata [46] and leaf, fruit and latex of Croton bonplandianum [47]. Continuing our research on the chemical composition of the different parts of medicinal plants, we now reported the results of phytocompounds from leaf and stem of M. minuta. We are particularly hopeful that this study will help guide research on further analysis of chemical compounds of leaf and stem of M. minuta.

CONCLUSION
The results of this study confirmed that the methanolic extracts of leaf and stem of M. minuta possess many bioactive constituents and which may be responsible for the pharmacological activities. Further studies are needed to isolate and purify the phytocompounds possess pharmacological properties. So, the present study may be useful in the identification of novel drugs from leaf and stem of M. minuta.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.