PRELIMINARY PHYTOCHEMICAL SCREENING AND GC-MS ANALYSIS OF METHANOLIC LEAF EXTRACT OF TEPHROSIA FALCIFORMIS RAMASWAMI FROM INDIAN THAR DESERT

PRELIMINARY PHYTOCHEMICAL SCREENING AND GC-MS ANALYSIS OF METHA-NOLIC LEAF EXTRACT OF TEPHROSIA FALCIFORMIS RAMASWAMI FROM INDIAN THAR DESERT

Vandana ¹ and G. S. Deora ^{* 2}

Taxonomy and Plant Diversity Laboratory ¹, Center of Advanced Study, Department of Botany, Jai Narain Vyas University, Jodhpur - 342005, Rajasthan, India.

Department of Botany ², Mohanlal Sukhadia University, Udaipur - 313001, Rajasthan, India.

ABSTRACT: Present study was conducted to identify and characterize the phytoconstituents of methanolic extract of leaves of Tephrosia falciformis (Pers.) Ramaswami of family Fabaceae. The methanolic extract of leaves was subjected to qualitative screening for primary and secondary metabolites as per standard methods; further GC-MS analysis was carried out for the identification of secondary metabolites. The preliminary analysis of leaf extract confirmed the presence of primary as well as secondary phytoconstituents such as phenolic compounds, alkaloids, flavonoids, terpenoids, phytosterols, etc. in methanolic extract. GC-MS analysis of extract revealed the presence of 53 phytochemicals. The presence of various compounds with different chemical groups like quinolene, piperdinone (alkaloids), Neophytadiene (sesqueterpenens), phytol (diterpene alcohol), sitosterol, stigmasterol (sterols) and many fatty acids including their derivatives in considerable amount gives the good prospect for the use of this plant in pharmaceutical preparations to cure health-related problems.

Tephrosia falciformis, Methanolic extract, Phytochemicals, GC-MS

INTRODUCTION: Every plant is rich in phytochemical compounds; consequently, they are a potential source of drugs, but a biological screening is essential to know more about the activities of these compounds. The plant contains phytochemicals in form of primary and secondary metabolites. Primary metabolites are involved in vital metabolic pathways, whereas the secondary metabolites accomplished non-vital functions in plant. Secondary constituents are involved in chemical defense against pathogens and predators and assists in pollination and dispersal. They also act as photoprotectants and allelopathic agents.

Activities of these constituents are also beneficial to humans in prevention of diseases in the form of medicine. The important bioactive phytochemicals include alkaloids, phenols, flavonoids, terpenes, steroids, and glycosides. Family Fabaceae is the third-largest family of flowering plants. It consists of 650 genera and 18000 species ¹. Genus Tephrosia of this family has 400 species throughout the world ². About twenty-seven species of Tephrosia are reported in India ³. This genus is broadly distributed in tropical, subtropical and arid regions of world ⁴. Plants of this genus are herb to undershrub and grow like a weed.

Genus Tephrosia is well known for its richness in bioactive compounds, especially in flavonoids ^{5 6}. This genus has been used for the treatment of syphilis, stomach ache, dropsy, inflammation, rheumatic pain and respiratory disorders and as an abortifacient, diuretic laxative also ^{7, 8}.

Tephrosia falciformis (Pers.) Ramaswami, commonly known as rati biyani ⁹ is 3-4 feet high under shrub grown in sandy plains. It is a rare and threatened plant species of Rajasthan, especially in the Indian Thar desert region Fig. 1. The leaves are imparipinnate with 7-9 pairs of leaflets. Both surfaces of leaflets are densely covered with silky grey hair.

This plant is medicinally underexplored, earlier some phytochemicals reported from the seeds of its pods namely triacontanol, Pongamol, Sitosterol, Lanceolatin-B and Lanceolatin-A along with two flavanoid namely Falciformin (7methoxy-8-(3-hydroxy-3-methyl-but-1-enyl) and 7-hydroxy-8-(γ,γ-dimethylallyl) ^{10, 11}. Any phytoconstituents is not reported from leaves previously, but the present study first time revealed the presence of primary and secondary metabolites in leaf extract of T. falciformis.

FIG. 1: T. FALCIFORMIS: GROWING IN NATURAL HABITAT

MATERIALS AND METHODS:

Plant Collection: The fully matured fresh leaves of T. falciformis (Pers.) Ramaswami was collected from Jaisalmer district of Rajasthan, India in August. The identification of plant was done by taxonomist of BSI, Jodhpur with authentication no.BSI/AZRC/I.2012/Tech/2019-20 (PI.Id)/526. Collected leaves were washed with distilled water and dried in shade at room temperature for 15 days. Dried leaves were coarsely powdered with the help of a grinder. Leaf powder was stored in airtight containers for phytochemical analysis.

Preparation of Crude Extract: About 5 gm dried leaf powder was weighed using an electric balance and extracted into 100 ml methanol of HPLC grade for 72 h with frequent shaking. The extract was filtered through a muslin cloth and Whatman no. 1 filter paper, which was followed by centrifugation for 20 min on 2000 rpm. The extract was again filtered by Whatman no. 1 filter paper and left to evaporate on room temperature till crude extract obtained. The crude extract was transferred into sterile, airtight containers, which were stored in the refrigerator for further use ¹².

Preliminary Phytochemical Screening of Leaf Powder: Preliminary phytochemical screening of methanolic extract was done as per the standard methods ^{13, 14} to prove the presence of phyto-chemicals.

GC-MS Analysis of Extract: The extract was also subjected to GCMS analysis to find out the bioactive compound of the leaf. The sample was prepared by reconstituting crude extract in methanol at the concentration of 1 mg/ml. The gas chromatography-mass spectroscopy (GC-MS) analysis of leaf extract was done on Shimadzu QP-2010 plus system with a thermal desorption system.

GC system was equipped by a fused silica capillary column having dimensions of 30m × 0.25mm × 0.25µm. Helium gas (99.99%) was used as carrier gas at a constant flow rate of 1.21 ml/min. in the split ratio 10:0.An injection volume of 1 µl of the sample was injected into the column. The pressure was kept at 69.0 k Pa. Ionization energy was set on 70Ev. The column oven temperature was initially set on 50 ºC to withhold time of 3 min; the oven temperature was increased to 280 ºC at the rate of 10º withhold time of 24 min. For GC program, Ion source temperature and interface temperature were 220 ºC and 270 ºC respectively. The total running time for GC-MS was 60 min.

The sample was injected in splitless mode and analyzed in MS full scan mode with start m/z 50 & end m/z 650 with a scan speed of 1250.

Interpretation on unknown mass spectrum GC-MS was done by comparing the fragmentation patterns of the mass spectra with the known, and standard compound provided in the database of NIST 16 (National Institute of Standard and Technology) and Wiley 8 library. The compound was identified by their GC retention time. The relative percentage of the amount of each compound was obtained by comparing the average peak area with the total peak area. The name, molecular formula and molecular weight of each detected compound were determined.

RESULTS: Preliminary phytochemical screening reveals the presence of primary and secondary metabolites. Results of preliminary phytochemicals screening are shown in Table 1. 

TABLE 1: QUALITATIVE PHYTOCHEMICAL SCREENING OF METHANOLIC LEAF EXTRACT OF T. FALCIFORMIS

Secondary phytoconstituent reported in GC-MS analysis are shown in Table 2 with peak area percentage, molecular weight and molecular formula. Bioactivity of some significant compounds with molecular structures listed in Table 3 and chromatogram in Fig. 2.

The GC-MS chromatogram of leaf extract of T. falciformis revealed the presence of 53 phyto chemicals, which includes alkaloids, terpens, sterols, phenols, flavonoids, and essential oils etc. Among these phytochemicals 6-(2-ethoxyphenyl)-5-nitro-2-piperidinone (23.86%) exibits the highest peak percentage followed by 9,12,15-Octadeca-trienoic acid, (Z,Z,Z)- (14.34%), n-Hexadecanoic acid (13.89%),2-Phenyl-4-anilino-6[1H]-pyrimidi-none (10.04%), gamma.-Sitosterol  (3.18%), naph-thalene(2.30%), Phytyl tetradecanoate (2.21%), Stigmasterol (1.68%), 1h, 3h-furo[3,4-c]furan, 1,4-bis (3,4-dimethoxyp (1.48%), 2-(3-methoxy-5-methyl-benzyliden)-5,7-dimeth (1.31%),3,6-hepta-nooxepin-4,5-dicarbonsaure-dimeth (1.29), [1] ben-zothieno [2, 3-c]naphtho[1,2-g]quinoline (1.14%), Phytol (1.01%). Lowest peak with 0.18% was reported for two compounds2-Hydroxy-1-(1'-pyr-rolidiyl)-1-buten-3-one and 1,d2,4-Cyclopentane-trione, 3-(2- pentenyl).

TABLE 2: PHYTOCHEMICALS REPORTED IN GC-MS ANALYSIS OF METHANOLIC LEAF EXTRACT OF T. FALCIFORMIS

FIG. 2: CHROMATOGRAM OF METHANOLIC EXTRACT OF T. FALCIFORMIS

TABLE 3: BIOACTIVITY OF SOME COMPOUND REPORTED IN METHANOLIC EXTRACT OF LEAF OF T. FALCIFORMIS

S. no.	Compound name	Chemical group	Molecular structure	Biological activity
1	Benzoic acid, methyl ester	Methyl Ester		Antimicrobial properties 34
2	Benzofuran, 2,3-dihydro-	Essential oils		Antifungal activity32, Antioxidant activity35
3	Naphthalene	Aromatic hydrocarbon		Antimicrobial 36
4	2-Methoxy-4-vinylphenol	Phenols		Anticancer 37
5	2-Hydroxy-1-(1'-pyrrolidiyl)-1-buten-3-one	Alcohol		Antiseptic, Anesthetic, allergic dermatitis 38
6	Neophytadiene	Sesquiterpene		Antimicrobial 39, Anti-inflammatory 31
7	n-hexadecanoic acid	Fatty acid		Anti-inflammatory 40
8	Palmitic acid, TMS derivative	Fatty acid derivative		Antitumor 41
9	Phytol	Diterpene alcohol		anticancer, antioxidant, antitumor, antimicrobial 23
10	Hexadecanoic acid, methyl ester	Fatty acid derivative		Anticancer 42
11	Octadecanoic acid	Fatty acids		Antimicrobial 28
12	[1]benzothieno[2,3-c]naphtho[1,2-g]quinoline	Alkaloid		Antinociceptive 16, antifungal activity 17, 43.  Antimalarial, antibacterial, anthelmintic, cardiotonic, anticonvulsant, anti-inflammatory and analgesic activity 43
13	gamma.- Sitosterol	Sterols		Anticancer 44  Antidiabetic 45
14	Stigmasterol	Sterols		Anti-inflammatory 25,46

DISCUSSION: Bioactive compounds are responsible for the medicinal characteristics of the plants ¹⁵. The presence of many significant secondary metabolites shows the potential of this plant for various therapeutical and pharmaceutical applications. The present analysis reveals that leaf contains terpene compounds in abundance along with alkaloids. Alkaloids are generally toxic to man and many of them have shown physiological activities; hence they are widely used in medicine ¹³. Quinolene alkaloid also reported in leaf extract of T. falciformis, previous studies on Quinolene reveals that it has antinociceptive ¹⁶ and antifungal activity ¹⁷. Likewise, terpenoids are isoprene molecules that are significant not only in plant growth and ecology but also provide a shield against insects.
These ranges from volatile (mono and sesquiterpenes), less volatile (diterpenes), and involatile compounds (triterpenoids steroids and carotenoid). Plant-based terpenoids are used in food, chemical industries, and pharmaceuticals and also used in the development of biofuel product ¹⁸. Several studies reported the antimicrobial ^{19, 20}, antioxidant, anti-inflammatory, antiparasitic, anticarcinogenic ²¹, antimalarial activity ²² of sesquiterpenes. Diterpenes Phytol reported in leaf extract, which shows anticancer, antioxidant, anti-inflammatory, diuretic, antitumor, chemopreventive, antimicrobial properties and also use in vaccine formation ²³. Other reported bioactive compound phytosterols are biogenetic precursors of many hormones and oviposition stimulants of some insects ¹³. Additionally, phytosterols show lipid-lowering, anticancer, anti-inflammatory, and anti-allergy effects ²⁴. Stigmasterol is useful in the treatment of asthma ²⁵.

Many studies have reported the advantages of phenolic compounds like antiaging, antioxidant, anti-inflammatory, anti-proliferative agents ²⁶. The fatty acids are active metabolites, and responsible for various medicinal properties, like hexadecanoic acid shows antibacterial and antifungal activity ²⁷ and octadecanoic acid is antimicrobial in nature ²⁸  palmitic acid and linolenic acid are known for their antibacterial and antifungal activity ^{29, 30}. The essential oils are found to be rich in sesquiterpenes and responsible for anti-inflammatory ³¹, antifungal ^32, and anticancer activity ³³.

CONCLUSION: The GC-MS analysis of leaf extract of T. falciformis, which is an underexplored plant from the medicinal point of view, revealed the presence of 53 bioactive compounds. The medicinal utility of some of the compounds are already discussed which can justify the use of this plant for pharmaceutical preparations; however, the isolation of individual compound and study of its biological potential against various microorganisms is imminent for innovation of effective and safe medicines for treatment.

ACKNOWLEDGEMENT: Authors are thankful to the University grant commission, New Delhi, for providing financial assistance in the form of CAS program in the Department of Botany, Jai Narain Vyas University, Jodhpur, Rajasthan.

CONFLICTS OF INTEREST: The authors declare no conflicts of interest.

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    How to cite this article:

    Vandana and Deora GS: Preliminary phytochemical screening and GC-MS analysis of methanolic leaf extract of Tephrosia falciformis Ramaswami from Indian Thar Desert. Int J Pharm Sci & Res 2020; 11(6): 3040-46. doi: 10.13040/IJPSR.0975-8232.11(6).3040-46.

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