INTRODUCTION:

Plants are vital source of various types of chemical compounds which are naturally formed as product of secondary metabolism. These compounds are primarily not necessary for growth and reproduction but used by plants in defence activities against several pathogens or to overcome various environmental stresses. These phytochemicals are of low molecular weight and known as secondary metabolites.

They show diverse chemical structure and bio activities. Since ancient times, phytochemical compounds have been continuously explored by human for their medicinal properties and applied for the treatment of various types of diseases. Although allopathic treatment is an effective and speedy medical treatment but due to mild to severe toxic side effects of many conventional or modern medicinal drugs and emerging drug resistance in microorganisms, an alternative and plant-based medicines are preferred for treatment in many cases. Hence, plant-based medicinal treatment has very little or no harmful side effects, it is a popular method of treatment in many countries. Besides this, plant-based medicines are always economical and accessible to majority of the people mainly in developing countries in comparison to conventional medicines. Phytochemical compounds become an efficient source of infinite raw materials for the detection and formulation of novel drugs¹.

However phytochemicals possess various bioactivity but they can be toxic in nature sometime, hence phytochemical screening and toxicity studies are necessary for any plant²before these are considered for any drug preparation.Perliminary phytochemical screening of some Fabaceae plants like, Tephrosia purpurea³, Cajans cajan⁴, Sesbania grandiflora⁵ and Glycyrrhiza glabra⁶reveals the presence of various phytochemicals like carbohydrates, amino acids alkaloids, phenolic compounds, flavonoids, terpenoids, phytosterols and glycosides etc.

Bioactive phytochemical compounds are secondary metabolites which are effective against many pathogens and this therapeutic potential is used for novel drug development to cure different type of diseases or health disorders. For the identification of phytoconstituent various sophisticated technique are available and GC-MS is such a technique that applied for identification of chemical compounds in many plants of different families like - Tephrosia villosa⁷, Diospyros virginiana⁸, Lantana camara⁹, Andrographis peniculata¹⁰, Piper longum¹¹.

Members of the family Fabaceae are well known for possessing medicinal properties like anti-inflammatory, antibacterial, antioxidant, anti-fungal, laxative, anti-osteoporotic, anti-cancer, anti-nephritic, anti-diabetic, sedative, chemo-preventive, neuroprotective and insecticidal activities¹².

Tephrosia is a large pantropical genus comprising more than 350 species which is widely distributed in tropical, sub-tropical and arid areas of the world¹³. This genus is significantly rich in bioactive compounds like rotenone, isoflavones, flavanols, flavanones, prenylated flavonoids, chalcones and glucosides^{14,15,16,17,18,19,20,21,22}. Most of the Tephrosia species are considered poisonous due to the higher concentration of Rotenone²³.

Tephrosia wallichii Graham is a perennial, erect herb with imparipinnate compound leaves, terminal raceme, densely hairy calyx, bright pink-purple corolla, stamen 9+1, staminal tube short, ovary densely pubescent with medium length hairs, glabrous style and penicillate stigma. Pods of T. wallichii are linear, dark brown, sparsely hairy with small hairs, 4-7 seeds per pod, seeds reniform, pale brown- reddish-brown, smooth²⁴ Fig (1).

Figure -1 (a) Plant in field (b) Flower

MATERIALS AND METHODS:

Plant collection and identification:

Fresh and mature leaves were collected from healthy plants of Tephrosia wallichii in the rainy season. Flowering twigs were also collected from the same plant and field for the identification and authentication of plant species. Authentication of the collected specimen was done by comparing the flowering twig with the earlier identified herbarium sheet of Tephrosia wallichii with accession number 729, deposited in the herbarium of the Botany Department of Jai Narain Vyas University, Jodhpur.

Preparation of leaf extract:

Collected leaf samples were thoroughly washed under running tap water to remove dirt or dust particles for the preparation of leaf powder. After washing, leaves were left for air drying in shadow for 15 days to reduce the moisture content of leaves. Completely air-dried leaves were homogenized with the help of an electrical grinder. The leaf powder was again sieved to remove any remaining unnecessary parts like veins or rachis fragments to obtain a fine leaf powder. This fine powder of leaves was stored in air-tight bottles for phytochemical analysis.

Extraction of phytochemicals:

The preparation of methanolic leaf extract was done by following the maceration process²⁵. For leaf extract 5 gm of leaf powder was dissolved in 100ml of 70% of HPLC grade methanol covered with the aluminium foil and finally left for 72hours with occasional stirring after. After 72hours extract was filtered by a muslin cloth to remove leaf fragments. The filtered extract was re-filtered by a Whatman paper 1 to remove any tiny leaf fragments. Further, this filtrate was centrifuged at the rate of 2500rpm for 20 minutes. The supernatant obtained after centrifugation was left to evaporate in a porcelain bowl at 40ºC in a water bath. After evaporation, a brownish-black crude extract remained in a bowl, which was further used to prepare a stock solution for GC-MS analysis. For preliminary phytochemical tests the crude extract was re-dissolved in distilled water; while for GC-MS investigation, the stock solution of the injecting sample was prepared by re-dissolving the crude extract into mother solvent methanol. Only 1 µl of stock solution was used for GC-MS analysis. Preliminary phytochemical tests were performed as per the standard methods of ^26,27,28.

Gas chromatography-mass spectroscopy of leaf extract was performed at AIRF, JNU, New Delhi. GC-MS analysis was carried out by using the Shimadzu-QP 2010 system equipped with a capillary column. 1µl volume was used for GC-MS analysis. Helium gas was used as carrier with a flow rate of 1.21ml/min. The initial column temperature was kept at 50ºC and a 3-minute hold time. The final temperature was fixed at 280ºC with 24 minutes a hold time. The injection temperature was kept at 260º. The ionization energy was 70eV and pressure was kept at 69 kPa. The ion source temperature at 220ºC and interface temperature at 270ºC were fixed for the gas chromatography analysis. The total running time of GC program was 60 minutes.

A mass spectrum was generated for each separated component where the quantity of each component was represented by a peak in the mass spectrum and identified by their retention time. The unknown mass spectrum was evaluated by comparing the fragmentation patterns of mass spectra with the identified standard compound present in the Wiley and NIST database²⁹.

RESULTS:

Investigation of methanolic leaf extract of T. wallichii by preliminary phytochemical tests confirmed the presence of amino acids, proteins, carbohydrates, alkaloids, phenols, flavonoids, phytosterols, saponin, terpenes, and glycosides. The results of preliminary phytochemical tests are presented in Table 1.

GC-MS of methanolic leaf extract of T. wallichii disclosed a total of 53 compounds, all phytochemical compounds except one were identified. The chromatogram of phytochemicals of methanolic leaf extract is presented in Fig.2. Molecular formula and molecular weight of phytoconstituents with retention time and per cent peak area is given in Table 2.

The principle phytoconstituent with the highest peak area i.e. 15.03% was identified as 1H-Indole-2,3-dione, 7-(3-methyl butyl)- followed by 9,12,15-Octadecatrienoic acid, (Z,Z,Z)-(14.54%) and Naphthalene (14.46%). Lowest peak area was reported for 1-Heneicosanol (0.06%) and 2,6-Dimethoxy-4-methyl-nicotinonitrile (0.06 %) followed by (1R,3E,7E,11R)-1,5,5,8-Tetramethyl-12-oxabicyclo [9.1.0] dodeca-3,7-diene (0.09%) and 4-Methyl-4-phenyl-2,3:5,6-diepoxycyclo hexanone (0.09%).

Table 1: Preliminary phytochemical screening of methanolic leaf extract of T. wallichii

Phytochemical tests	Phytochemical compounds	Results
Fehling’s test Molisch’s test	Carbohydrates	+
Ninhydrin test Xanthoproteic test	Amino acids	+
Ferric chloride test Lead Acetate test	Phenols	+
Shinoda test and Alkaline reagent test	Flavonoids	+
Dragendorff’s test Wagner’s test	Alkaloids	+
Salkowaski test Liebermann burchard’s test	Terpenoids and Phytosterols	+
Olive oil test Foam test	Saponin	+
Ruthenium Red test Alcohol test	Gum and Mucilage	+
Glycosides test Keller Killani test	Glycosides	+

Table 2: Phytochemicals reported in GC-MS analysis of methanolic leaf extract of T.wallichii

Peak#	R. Time	Area	Area%	Name	Molecular Weight	Molecular Formula
1	10.468	1634047	0.85	Un-identified
2	11.067	27785777	14.46	Naphthalene	128	C₁₀H₈
3	12.883	524871	0.27	2-Methoxy-4-vinylphenol	150	C₉H₁₀O₂
4	14.008	210206	0.11	4H-Inden-4-one, 1,2,3,5,6,7-hexahydro-1,1,2,3,3-pentamethyl-	206	C₁₄H₂₂O
5	15.566	712928	0.37	1,3-Cyclopentanedione, 4-methyl-5-pentyl-	182	C₁₁H₁₈O₂
6	15.842	462947	0.24	2(4H)-Benzofuranone, 5,6,7,7a-tetrahydro-4,4,7a-trimethyl-	180	C₁₁H₁₆O₂
7	16.108	271981	0.14	3',5'-Dimethoxyacetophenone	180	C₁₀H₁₂O₃
8	16.426	362190	0.19	Ketone, 7-methoxy-2-benzofuranyl methyl	190	C₁₁H₁₀O₃
9	16.810	182452	0.09	(1R,3E,7E,11R)-1,5,5,8-Tetramethyl-12-oxabicyclo[9.1.0]dodeca-3,7-diene	220	C₁₅H₂₄O
10	17.196	950191	0.49	Pyrrolidine, 1-(1-cyclohexen-1-yl)-	151	C₁₀H₁₇N
11	17.543	410562	0.21	Cyclopentane-1-carboxylic acid, 2-hydroxy-1,2,3-trimethyl-, ethyl ester	200	C₁₁H₂₀O₃
12	18.354	941629	0.49	Tetradecanoic acid	228	C₁₄H₂₈O₂
13	18.646	921016	0.48	2(4H)-Benzofuranone, 5,6,7,7a-tetrahydro-6-hydroxy-4,4,7a-trimethy	196	C₁₁H₁₆O₃
14	19.130	1889009	0.98	Neophytadiene	278	C₂₀H₃₈
15	19.411	800956	0.42	Pentadecanoic acid	242	C₁₅H₃₀O₂
16	19.578	570533	0.30	3,7,11,15-Tetramethyl-2-hexadecen-1-ol	296	C₂₀H₄₀O
17	20.040	3099585	1.61	Hexadecanoic acid, methyl ester	270	C₁₇H₃₄O₂
18	20.515	23759019	12.36	n-Hexadecanoic acid	256	C₁₆H₃₂O₂
19	21.177	668971	0.35	Maltose	342	C₁₂H₂₂O₁₁
20	21.393	537018	0.28	Heptadecanoic acid	270	C₁₇H₃₄O₂
21	21.482	664546	0.35	Arabino-Hex-1-enitol, 1,5-anhydro-2-deoxy-	146	C₆H₁₀O₄
22	21.677	570385	0.30	9,12-Octadecadienoic acid (z,z)-, methyl ester	294	C₁₉H₃₄O₂
23	21.736	2011192	1.05	9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)-	292	C₁₉H₃₂O₂
24	21.842	3182942	1.66	Phytol	296	C₂₀H₄₀O
25	21.970	1199796	0.62	Methyl stearate	298	C₁₉H₃₈O₂
26	22.206	27954400	14.54	9,12,15-Octadecatrienoic acid, (Z,Z,Z)-	278	C₁₈H₃₀O₂
27	22.376	5577902	2.90	Octadecanoic acid	284	C₁₈H₃₆O₂
28	22.539	227172	0.12	7,10-Hexadecadienoic acid, methyl ester	266	C₁₇H₃₀O₂
29	23.973	256830	0.13	4,8,12,16-Tetramethylheptadecan-4-olide	324	C₂₁H₄₀O₂
30	24.456	447818	0.23	1H-Indole-3-carboxaldehyde, 5-methoxy-	175	C₁₀H₉NO₂
31	25.108	108156	0.06	1-Heneicosanol	312	C₂₁H₄₄O
32	25.326	13793696	7.18	Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester	330	C₁₉H₃₈O₄
33	25.569	28883301	15.03	1H-Indole-2,3-dione, 7-(3-methylbutyl)-	217	C₁₃H₁₅NO₂
34	26.033	285053	0.15	4-Methoxy-2(1H)-quinolone	175	C₁₀H₉NO₂
35	26.166	12563962	6.54	Pyridazin-3(2H)-one, 4,5-di(ethylthio)-2-phenyl-	292	C₁₄H₁₆N₂OS₂
36	26.241	562240	0.29	2-Phenyl-furo[b]benzopyran-4(4h)-one	262	C₁₇H₁₀O₃
37	26.700	123021	0.06	2,6-Dimethoxy-4-methyl-nicotinonitrile	178	C₉H₁₀N₂O₂
38	26.960	7876974	4.10	Octadecanoic acid, 2,3-dihydroxypropyl ester	358	C₂₁H₄₂O₄
39	27.347	230214	0.12	2,8,8-Trimethyl-3-phenyl-8,9-dihydro-7h-pyrazolo[1,5-a]quinazolin-6-	305	C₁₉H₁₉N₃O
40	27.465	178400	0.09	4-Methyl-4-phenyl-2,3:5,6-diepoxycyclohexanone	216	C₁₃H₁₂O₃
41	27.793	406973	0.21	Squalene	410	C₃₀H₅₀
42	28.134	3215752	1.67	.alpha.-Tocospiro B	462	C₂₉H₅₀O₄
43	28.371	1298696	0.68	.alpha.-Tocospiro B	290	C₂₉H₅₀O₄
44	29.028	2005093	1.04	1.beta.-Acetoxyeudesma-4(15),7(11)-dien-8.alpha.,12-olide	290	C₁₇H₂₂O₄
45	29.217	1500981	0.78	pyrrolidine, 1,1'-(5-methyl-1,3-phenylene)bis-	230	C₁₅H₂₂N₂
46	32.111	761212	0.40	Vitamin E	430	C₂₉H₅₀O₂
47	34.009	651931	0.34	Reynosin	248	C₁₅H₂₀O₃
48	34.702	1967834	1.02	Stigmasta-5,22-dien-3-ol	412	C₂₉H₄₈O
49	36.165	1450925	0.75	.gamma.-Sitosterol	414	C₂₉H₅₀O
50	37.194	1250500	0.65	24-Noroleana-3,12-diene	394	C₂₉H₄₆
51	37.696	909218	0.47	Lup-20(29)-en-3-one	424	C₃₀H₄₈O
52	38.544	3088217	1.61	Lupeol	426	C₃₀H₅₀O
53	39.894	319394	0.17	Vitamin E	430	C₂₉H₅₀O₂
		192220614	100.00

Figure 2- Chromatogram showing peaks of phytochemicals present in methanolic leaf extract of T. wallichii

DISCUSSION:

Plant-derived phytochemical compounds which possess pharmacological activities are known as phytopharmaceuticals and they work in various ways such as they can target specific receptors, interrupting disease pathways and disrupting pathogenic life cycles³⁰.

The present study reported fifty-two phytochemical compounds present in the methanolic leaf extract of T. wallichii by Gas chromatography and mass spectroscopy technique. These phytochemicals possess various types of biological activities that can be utilised for novel drug development. Leaf extract contains phyto chemical compounds of different chemical groups like alkaloids, phenols, flavonoids, terpens etc. GC-MS revealed the presence of some alkaloids in leaf extract of T. wallichii such as - 1H-Indole-3-carboxaldehyde, 5-methoxy-; 1H-Indole-2,3-dione,7-(3-methyl buty; 4-Methoxy-2(1H)-quinolone; Pyridazin-3(2H)-one,4,5-di(ethylthio)-2-phenyl-; 2,6-Dimethoxy-4-methyl-nicotinonitrile; 2,8,8-Trimethyl-3-phenyl-8,9-dihydro-7H pyrazolo [1,5-A] quinazolin-6 – and these alkaloids may have high medicinal potential, therefore, must be explored for their bioactivity and tested against disease causing microorganisms.

Quinolones containing alkaloids possess antimicrobial activity³¹. Indole-containing alkaloids display various activities against cancer cells and microbes³². Pyrrolidine alkaloids express a broad spectrum of biological activities such as analgesic, antioxidant, anti-inflammatory, cytotoxic, antimicrobial and antihistaminic activities³³. Quinazoline alkaloids exhibit insecticidal, anti-inflammatory, antiplatelet and antifungal activities³⁴. Few alkaloids contain alkaloids and flavonoids both components known as flavoalkaloid like Pyrrolidine, 1-(1-cyclohexen-1-yl)- and pyrrolidine, 1,1'-(5-methyl-1,3-phenylene) bis-, found in leaf extract of T. wallichii. A flavone namely 2-Phenyl-furo [B] benzopyran-4(4H)-one present in the leaf extract of T. wallichii displays antibacterial activity³⁵. Methanolic leaf extract of T. wallichii is rich in terpene compounds and methanolic leaf extract. Terpenoids are natural pesticides and display antimicrobial, anti-inflammatory, and diuretic activities, additionally, they are useful in gastrointestinal contractions, pains and itching³⁶. Many terpenes and terpene alcohols are found in methanolic leaf extracts of T.wallichii such as Neophytadiene, Reynosin, Squalene, Phytol, Lupeol and Lup-20(29)-en-3-one. Neophytadien is a potent antimicrobial, anti-inflammatory, analgesic, antipyretic and anti-oxidant activity-containing compound³⁷. Squalene is a triterpene compound that contains antioxidant properties³⁸and is also the main component of skin surface lipids³⁹. Reynosin is a sesquiterpene which exhibits an antimycobacterial effect⁴⁰. Phytol is an acyclic diterpene alcohol which shows antioxidant, anticancer and antimicrobial activity⁴¹. Lupeol is a pentacyclic triterpenoid which acts as a cholesterol-lowering agent and also exhibits anti-inflammatory, anti-microbial, anti-invasive, anti-angiogenic, and anti-protozoal properties⁴². A long-chain fatty alcohol namely 1-Heneicosanol exhibits antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa and antifungal activity against Candida albicans and Candida krusei⁴³. alpha.-Tocospiro B and Vitamin E are Tocopherols found in the leaf extract of T. wallichii. Tocopherols show high antioxidative activity therefore they are highly significant in cardiovascular disease and cancer prevention^44,45,46. Phytosteroids belong to the triterpene family and are precursors of steroid hormones and part of plant defence mechanisms^47,48. Phytosterols contain anti-inflammatory, antidiabetic, anticancer and hypocholesterolemic activity⁴⁹. Stigmasta-5, 22-dien-3-ol and gamma.-Sitosterol are phytosterols present in the leaf extract of T. wallichii.

CONCLUSION:

The present study revealed the presence of several phytochemicals in the leaves of T. wallichii. This plant is unexplored for its medicinal properties; hence the complete biochemical characterization of this plant can provide new drug formulations against many disease-causing microorganisms and also for many health disorders. These bioactive compounds show a wide array of therapeutic activities. Sometimes, a few modifications in the natural structure of phytochemicals can increase their stability and biological activities therefore further exploration of the biological activities of these compounds is strongly recommended which opens doors for effective drug development to cure diseases.

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Received on 23.10.2023 Revised on 13.03.2024

Accepted on 10.06.2024 Published on 20.01.2025

Available online from January 27, 2025

Research J. Pharmacy and Technology. 2025;18(1):159-164.

DOI: 10.52711/0974-360X.2025.00024

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