Phytochemical Profiling of Erythrina variegata Leaves by Gas Chromatography-mass Spectroscopy

Biologically active compounds derived from plants have become an important source of drugs due to the increasing recognition of herbal medicine as an alternative form of health care (Afolayan et al., 2008). Numerous plants contain a range of phytopharmaceuticals, which have been shown to have significant uses in agriculture, human and veterinary medicine and the prevention of disease (Newman et al., 2003). Plant components have been used as a key source of medicine from ancient times and have been the primary source of drugs in Indian systems of medicine and other ancient systems across the world (Devi et al., 2011). Phytochemicals are the naturally occurring bioactive substances found in plants and the most substantial bioactive constituents of plants are alkaloids, tannins, flavonoids, steroids, terpenoids, carbohydrates and phenolic compounds (Sowmya et al., 2015).

“Erythrina variegata” L. (Fabaceae) also known as tiger’s claw, indian coral tree, farad and parijata is a species of Erythrina that is indigenous to the tropical and subtropical areas of the Indian subcontinent. In India, its leaves are generally utilized for curing (Kumar et al., 2011). According to a previous study on the same species of plant contains hepatoprotective and anti-leprosy properties. Its extract has been shown to have sedative, antidiuretic, antioxidant and antihyperlipidemic activities (Hemmalakshmi et al., 2017). Erythrina variegata leaves, bark and root are used in India for the treatment of various diseases viz., as antioxidant activity, antiosteoporotic, anthelmintic, antiulcer, diuretic, analgesic, cytotoxic, cardiovascular effect and respiratory effect (Baranitharan et al., 2019). However, there haven’t been many thorough, scientific investigations on the phytochemical screening and isolation of secondary metabolites in Erythrina variegata.

Antioxidant activity has been proposed to play roles in various pharmacological activities such as anti-aging, anti-inflammatory, anti-atherosclerosis and anti-cancer activities. Inhibition of free radical-induced damage by supplementation of antioxidants has become an attractive therapeutic strategy for reducing the risk of diseases (Sakat and Juvekar, 2010). Although there are many synthetic antioxidants, they are quite unsafe and their toxicity is of concern (Sakat and Juvekar, 2010). It is possible to employ natural products in place of synthetic ones if they have antioxidant, antifungal, antibacterial, antimicrobial and insecticidal properties. Due to their usefulness, low toxicity to vertebrates and great biodegradability, plant-borne compounds have received significant attention in recent years as potential replacements for synthetic ones (Baranitharan et al., 2019). The bioactive chemicals found in plants can be exploited to create ecologically friendly vector and pest management products. 

In order to explore the antioxidant, antifungal, antibacterial, antimicrobial and insecticidal activity using various solvents including aqueous, ethanol and hexane extracts obtained from Erythrina variegata leaves, the current work was done. GC-MS analysis was used to identify the phyto-components present in the extract in order to assess the medicinal characteristics of the plant.

The laboratory experiment was carried out at the Department of Seed Science and Technology, Tamil Nadu Agricultural University, Coimbatore during 2019-2020. Fresh matured leaves of Erythrina variegata were obtained from Agricultural Engineering College and Research Institute, Kumulur,  Tiruchirappalli, Tamil Nadu, India were used as the base leaf material for this study.
 
Preparation of extraction
 
The collected Erythrina variegata leaves were washed well with distilled water and shade dried at room temperature. The dried leaves were ground into fine powder in an electric blender. From the powdered leaf material, the extracts were prepared using solvents such as water, ethanol and hexane. The water, ethanol and hexane extracts were prepared by mixing twenty-five grams of powdered leaves in 250 mL of water, ethanol and hexane were taken separately in 1:10 w/v. Then it was shaken for 48 hours at a speed of 190 to 220 rpm. The extracts were filtered through Whatman No.1 filter paper and collected in brown bottles separately. Following that, the different extract solvents were evaporated by Rota-vapour sepearately, where the water bath’s temperature was maintained at 40°C and then lyophilized by lyophilizer which gave rise to a solid mass of the extract. The solid mass was refrigerated under -20°C until it was used for the GC-MS analysis (Preeti and Chandrawati, 2017); (Suriyavathana et al., 2016); (Hemmalakshmi et al., 2016).

GC-MS Analysis of leaf extracts 
 
GC-MS analysis of leaf extracts was carried out by following the method of Hema et al. (2010). The Perkin-Elmer GC Clarus 500 system, Agilent Co., USA and a Gas Chromato graph Interfaced to a Mass Spectrometer (GC-MS) equipped with an Elite-I, fused silica capillary column (30 mm X 0.25 mm 1D X 1 μMdf, formed of 100% Dimethyl polysiloxane) were used to conduct the GC-MS analysis of the extract. An electron ionization device with an ionising energy of 70 eV was employed for GC-MS detection. The carrier gas, helium (99.999%), was utilized with an injection volume of 2 μl and a constant flow rate of 1 ml/min (split ratio: 10:1); 250°C for the injector and 280°C for the ion source. The oven temperature was configured to begin at 110°C (isothermal for 2 minutes), rise at a rate of 10°C/min to 200°C, then 5°C/min to 280°C and ending with a 9 minute isothermal at 280°C. At 70 eV, 0.5 seconds of scanning time and fragments ranging in size from 45 to 450 Da, mass spectra were recorded. The GC ran for 36 minutes in total. The software used to handle mass spectra and chromatograms was called Turbomass and it was utilized to compute the relative percent amount of each component by comparing its average peak area to the total areas.
 
Identification of components
 
The National Institute of Standard and Technology (NIST) database, which contains more than 62,000 patterns, was used for the GC-MS experiment. For the interpretation of the mass spectrum, comparison was made between the spectra of the unknown component and the spectrum of the known components in the NIST collection.

The extracted Erythrina variegata leaves samples were analyzed with GC-MS to detect phyto-components. Based on their specific retention time, probability and peak area %, chemical compounds were identified and quantified using the GC-MS technique. A wide range of compounds were identified by GC-MS, as shown in Table 1 (aqueous leaf extract), Table 2 (ethanol leaf extract) and Table 3 (hexane leaf extract). The compound prediction is based on National Institute Standard and Technology Database (Table 1, 2 and 3).







The results revealed that, in aqueous leaf extract presence of 17-octadecynoic acid (1.52%), hexadecanoic acid, methyl ester (1.10%), methyl 9-cis,11-trans-octadecadienoate (1.03%), 11-octadecenoic acid, methyl ester (1.22%), dasycarpidan-1-methanol, acetate (ester) (1.08%), 9-octadecenamide, (Z)- (1.29%), oleic Acid (1.23%), 9,12-octadecadienoic acid (Z,Z)-, 2-hydroxy-1-(hydroxymethyl) ethyl ester (11.98%), 9-octadecenoic acid (Z)-, 2,3-dihydroxy propyl ester (3.30%), 9-octadecenoic acid (Z)-, 2-hydroxy-1- hydroxymethyl) ethyl ester (8.75%). The spectrum profile of GC-MS confirmed the presence of ten major components with the retention time 15.5, 21.1, 24.2, 24.4, 25.5, 25.7, 25.9, 29.1, 29.3 and 29.5 respectively (Table 1 and Fig 1).



The ethanolic leaf extract showed the presence of eight major different organic compounds. The major phytochemical compounds among them were 1,2,3-butanetriol (1.90%), diethyl phthalate (35.04%), 6-hydroxy-4,4,7a-trimethyl-5,6,7,7a-tetrahydro benzofuran-2(4H)-one (2.16%), n-hexadecanoic acid (1.85%), eicosanoic acid (1.73%), 9, 12, 15-octadecatrienoic acid, (Z,Z,Z)- (3.45%), 9,12-octadecadienoic acid (Z,Z)-, 2-hydroxy-1- (hydroxymethyl) ethyl ester (1.55%), 9-octadecenoic acid (Z)-, 2,3-dihydroxy propyl ester (2.12%) with the retention time of 4.7, 14.2, 17.9, 21.8, 23.6, 25.0, 29.1 and 29.3 respectively (Table 2 and Fig 2).



Whereas, the most prevailing nine major compounds present in the hexane leaf extract were 2-pyrrolidinone, 1-methyl- (1.56%), hexadecanoic acid, 2-hydroxy-1- (hydroxymethyl) ethyl ester (1.83%), n-hexadecanoic acid (2.78%), 9,12-octadecadienoic acid (Z,Z)- (3.20%), 9,12,15-octadecatrienoic acid, (Z,Z,Z)- (5.44%), 9,12-octadecadienoic acid, ethyl ester (3.44%), 9,12,15-octadecatrienoic acid, ethyl ester, (Z,Z,Z)- (4.43%), 9,12-octadecadienoic acid (Z,Z), 2-hydroxy-1-(hydroxymethyl) ethyl ester (6.84%), 9-octadecenoic acid (Z)-, 2,3-dihydroxypropyl ester (13.41%), tetratetracontane (1.07%) confirmed with the retention time of 5.0, 18.0, 21.7, 24.9, 25.0, 25.5, 25.6, 29.1, 29.3 and 29.7 respectively (Table 3 and Fig 3).



The gas chromatogram shows that the relative concentrations of various compounds are getting eluted as a function of retention time. Peak heights indicated the relative amounts of the chemicals found in the plant. To determine the kind and structure of the compounds, the mass spectrometer examines the compounds eluted at different times.

In general, the effectiveness of medicinal plants is assessed by correlating the phytochemical compounds to the biological activities of the plants (Belkacem et al., 2013). In the current investigation, the GC-MS analysis of the leaf extracts of Erythrina variegata in water, ethanol and hexane revealed the presence of 24, 26 and 22 chemicals, respectively. In this account, the aqueous, ethanol and hexane leaf extract contained ten, eight and nine major components having antioxidant, nematicide, pesticide insecticidal, larvicidal, insect repellent, antimicrobial, antibacterial and antifungal activities, 5-alpha reductase inhibitor, hemolytic, alpha-glucosidase inhibitors activity, free radical scavenger, peroxidase substrate, reductant and lipid peroxidase inhibitor, plasticizer, surfactant and emulsifying properties, anticancer activity, hypocholesterolemic, hepatoprotective and cytoprotective activities (Table 4, 5 and 6), had very important medicinal values as shown.







This plant has the potential to serve a wide range of medicinal roles, thus according to GC-MS analysis, which revealed the presence of these biomolecules. The presence of some of the important components resolved by GC-MS analysis and their biological activities were reported in this study. Consequently, this type of GC-MS analysis is a step toward comprehending the nature of the active principles in this medicinal plant and this kind of study will be useful for more in-depth investigation.

The present phytochemical study of Erythrina variegata leaf extract was studied using three different solvent extracts revealed the presence of various bioactive components in leaf extracts of Erythrina variegata. By considering the above data, the ethanol leaf extract showed the potent more bioactive compounds with highest peak area percentage compared with aqueous and hexane extract. In which the GC-MS analysis of leaf extract exhibited a profiles and this may helpful for the development of several herbal formulations with antioxidant, insecticidal, antimicrobial and antifungal properties and it is used for various ailments by traditional practitioners. Therefore, more research on the bioactivity, toxicity profile, isolation and identification of specific constituents are very much needed. It is also time to explore its pharmacological values at the molecular level with the help of various biotechnological techniques in the future

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