INTRODUCTION:

The use of plants for treating diseases is as old as the human species¹. Natural therapeutic agents are prepared from compounds found in nature, which contain active components in extract form obtained from plants, microbes, minerals and animals². The most dominant natural medicines source is plants due to their chemical and structural diversity and the biodiversity of their components³. Among 400000 plants species only 6% of the plants have been studied for the biological activity and few have been phytochemically investigated⁴. From ancient times, the efficacy of natural products has been associated with the chemistry, biochemistry and synthetic activities of natural products.

Thus, with scientific advancement in modern molecular and cellular biology, analytical chemistry and pharmacology, the unique properties of these natural products are being harnessed in order to exploit the chemical structural and biodiversity of these types of products in relation to their therapeutic effects⁵.

Sapindus emarginatus belongs to the family Sapindaceae. It is known locally as Soap nut tree (Ritha), is a tropical tree species distributed throughout India^6-8. Historically, it has been used in folk remedies as emetic, a mucolytic agent, paralysis of limbs and treatment of chlorosis. To our knowledge, no previous researches have been reported the extraction, study, and identification of phytochemicals present in Sapindus emarginatus seed kernel. So, the present study was carried out to identify and screen phytochemicals present in methanolic extract of Sapindus emarginatus seed kernel, and to evaluate its antimicrobial and antioxidant activity. Furthermore, to study molecular docking of some phytochemicals identified from GC-MS study of methanolic extract of plant.

MATERIAL AND METHOD:

Chemical and reagents:

All reagents and solvent were used of laboratory (LR) grade, obtained from Merk, Sigma Aldrich (Mumbai-India).

Collection of plant material:

Seeds of Sapindus emarginatus were purchased from local vender-Aurangabad. The plant was authenticated by Dr. Narayan Pandure, department of botany, Dr. Babasaheb Ambedkar Marathwada University-Aurangabad. The seeds were broken; kernel was separated from outer seed shell; finally seed kernel was ground to fine powder.

Preparation of extract:

30g of fine powder of Sapindus emarginatus seed kernel was packed in soxhlet apparatus and extracted with 95% methanol (500ml) for 6 h. Then, solvent was removed using rotary evaporator. Finally, free extract solvent was kept in amber bottle in refrigerator at 4ºC.

Phytochemical screening:

Phytochemical screening was performed to identify phytochemicals in methanol extract of sapindus emarginatus seed kernel. Methanol extract was subjected to chemical tests for the presence alkaloids, carbohydrates, proteins, glycosides, phenols and tannins, flavonoids, terpenoids, saponins and oils and fats^8-11.

Gas Chromatography- Mass Spectrometry (GC-MS):

Volatile compounds investigation of methanolic extract of plant was performed using GC-MS analysis¹²and volatile organic compounds identified based on the retention time and mass spectra with known compounds in the NIST library.

Biological activity:

Antimicrobial activity:

To evaluate antimicrobial activity of methanol extract of sapindus emarginatus seed kernel, Microbroth dilution method was used. Four strains of gram bacteria, three fungal strains were used. MIC of extract was determined as standard method^13,14.

Antioxidant activity:

The DPPHradical scavenging activity of methanol extract of sapindus emarginatus seed kernel was measured as described earlier¹⁵. EC₅₀ value of the extract was calculated. Ascorbic acid and butylated hydroxytoluene were used as standard drug.

Molecular docking study:

By comparing our compounds with other ligands and determination the pharmacophoric feature that can bind with critical amino acid of target site, target protein (PDB id: 3TYE) was selected from site protein data bank (PDB). Protein preparation; ligand preparation and docking process was done following standard protocol¹⁶.

Statistical analysis:

Free radical scavenging assay (DPPH) of methanol extract of Sapindus emarginatus seed kernel were performed in triplicate (n=3). The presented results were mean ±SE (Standard error). For Calculation of EC₅₀, the data were analysed by non Linear Regression followed by effector v/s response analysis on Graphical Prism5.0.

RESULTS AND DISCUSSION:

Phytochemical screening:

The results of preliminary phytochemical screening of different extract of Sapindus emarginatus seed kernel were presented in (Table 1). Chinnasamy S.¹⁷ studied phytochemical availability of leaf extracts of Sapindus emarginatus using soxhlet extraction method and various organic solvent. The result revealed that the present of alkaloids, carbohydrate, flavonoids, protein, and saponins in aqueous, ethanol and methanol extracts.

Table1: Phytochemical screening results of methanol extract

S. No.	Phytochemicals	Phytochemical tests	Extract
1	Alkaloids	Mayer test	+
2	Carbohydrates	a) Molish test	+++
2	Carbohydrates	b) Fehling test	+++
3	Proteins and Amino acids	a) Biuret test	-
3	Proteins and Amino acids	b) Ninhydrin test	-
4	Flavonoids	Lead acetate test	+
5	Glycosides	Killer Killan test	++
6	Phenols and Tannins	Ferric Chloride test	++
7	Terpenoids	Salkowski test	++
8	Saponins	Foam Test	+
9	Fat and oils	Spot test	++

(+) for low concentration, (++) for moderate concentration, (+++) for high concentration, (-) for absence

Gas Chromatography-Mass Spectrometry (GC-MS)

Fig. 1: GC-MS chromatogram of methanol extract of Sapindus emarginatus seed kernel

GC-MS analysis was carried out for the methanol extract of Sapindus emarginatus seed kernel (Fig 1). The different phytochemicals corresponding to different retention time can be predicted and these are enlisted in table 2. GC-MS chromatogram showed 12 peaks indicating the presence of 12 different phytoconstituents.

Table 2: Phytochemicals identified in methanol extract of Sapindus emarginatus seed kernel

Code	R.T.	Peak Area%	Molecular formula	Probable compound name
AB	12.6	4.38	C₈H₁₄O	3-Penten-2-one,3-ethyl-4-methyl
AC	14.17	0.48	C₁₁H₁₄	Benzene,[1-methylenebutyl]
AD	15.9	5.28	C₁₅H₁₀O₂	Flavone
AE	16.98	9.18	C₁₇H₃₄O₂	Hexadecanoic acid, 2-methyl-
AF	17.68	10.31	C₁₅H₂₄	Caryophyllene
AG	18.72	12.03	C₁₉H₃₆O₂	9-Octadecenoic acid, methyl ester ,[E]
AH	19.42	16.66	C₁₈H₃₄O₂	Oleic acid
AI	20.48	10.43	C₁₇H₁₂O₄	Mitoflaxone
AJ	20.72	10.13	C₁₇H₁₄O₄	4H-1-Benzopyran-4-one,5,7-dimethoxy-2-phenyl
AK	21.42	8.25	C₂₁H₂₆O	2,6-Bis[1,1-dimethyleethyl]-4-phenylmethylenecyclohexa2,5-dien-1-one
AL	22.9	6.95	C₁₉H₁₄N₂O₃	N-(3-methyl-2,7-dioxo-2,7-dihydro-3H-naphtho[1,2,3-de]quinolin-6-yl)acetamide
AM	24.87	5.91	C₁₇H₁₄O₇	4H-1-Benzopyran-4-one, 2-[3,4-dihydroxyphenyl]-5-hydroxy-3,7-dimethoxy-

9-Octadecenoic acid, methyl ester,[E] (12.03%) have been reported to have anticarinogenic activity¹⁸ and Oleic acid (16.66%) have been reported to have biological activities such as anticancer, 5-α-reductase inhibitor, antiinflammatory and antitumor^19,20. GC-MS study of petroleum ether of Sapindus emarginatus seed was done by Kadam et al.²¹, the result showed that the chromatogram possess 24 peaks of various compounds such as Oleic acid, 1-pentadecene, n-hexadecenoic acid, α-amyrin, 3- phenyl-propanol, acetate and hexadecane, 1-chloro.

Biological activity:

Antimicrobial activity:

Methanol extract of Sapindus emarginatus seed kernel screened for antibacterial and antifungal activities. The results were shown in (Table 3). MIC is defined as the lowest concentration of the antimicrobial agent that inhibits the microbial growth after 24 h²². It has reported that the mechanisms of the antimicrobial effects involves the inhibition of various cellular processes, followed by an increase in plasma membrane permeability and finally ion leakage from the cells²³.

Table 3: Minimum inhibitory concentration of plant extract and standard drugs against bacterial organisms and fungal organisms

S. No.	Microorganisms	MIC of extract (µg/mL)	MIC of standard drugs (µg/mL)
S. No.	Microorganisms	MIC of extract (µg/mL)	AM	CM	GR
1	E. coli (MTCC443)	250	100	50
2	P. aeruginosa (MTCC1688)	250	-	50
3	S. aureus (MTCC96)	250	250	50
4	S. pyogenes MTCC442	200	100	50
5	C. albicans (MMTCC227)	1000			500
6	A. niger (MTCC282)	>1000			100
7	A. clavatus (MTCC1323)	>1000			100

AM: Ampicillin, CM: Chloramphenicol, CR: Greseofulvin

The results showed that the extract have activity against gram-positive and gram-negative bacteria. The extract contains mixture of compounds, it exhibited very good inhibition (MIC=200µg/mL) against S. pyogenes compared to ampicillin (MIC=100µg/mL), chloramphenicol (MIC=50µg/mL). For antifungal activity, the extract exhibited good inhibition (MIC= 1000 µg/mL) against C. albicans compared to inhibition of antifungal standard drug Greseofulvin (MIC=500 µg/mL). It had long been documented that tannins, saponins and alkaloids are plants metabolites known for antimicrobial activity. The result of phytochemical analysis indicates that the methanolic extract of this plant contained saponins, tannins, carbohydrate, alkaloids, flavonoids and terpenoids which are responsible for its antimicrobial activity.

Antioxidant activity:

Many biochemical reactions in human body generate Reactive Oxygen Species (ROS) and other Species, which have the ability to damage crucial biomolecules^24,
25.

Table 4: Percentage of inhibition of DPPH and EC₅₀ for methanol extract of Sapindus emarginatus seed kernel

S. No.	Concentration (µg/mL)	% Inhibition of Standards		% Inhibition of extract
S. No.	Concentration (µg/mL)	BHT	Ascorbic acid	% Inhibition of extract
1	10	40.57 ± 1.53	66.44 ± 0.76	14.91 ± 0.79
2	20	78.51 ± 0.58	86.82 ± 0.39	18.64 ± 0.58
3	30	92.54 ± 0.22	100	27.19 ± 0.58
4	40	100	100	36.41 ± 0.58
5	50	100	100	46.93 ± 0.58
EC₅₀(µg/mL)		12.7 ± 0.4	11.5 ± 0.6	55.6 ± 1.4

Data represents mean ± SE (n = 3), SE: Standard error

It is known that the presence of three aromatic rings in DPPH makes its molecule very stable. This radical has absorbance maximum at 517nm. Every substance that be able to scavenge DPPH, decrease the absorbance in this wavelength, so, the DPPH assay could be considered an appropriate method for evaluation of the samples to scavenge free radical^26,27. Ascorbic acid is frequently used as a standard antioxidant and it has a strong DPPH scavenging property. The percentages of inhibition of methanol extract at different concentrations of Sapindus emarginatus seed kernel was shown in table 4. EC₅₀valueofextract was found to be 55.6±1.4µg/mL compared to 12.7±0.4µg/mL of BHT and 11.5±0.6 µg/mL of ascorbic acid. Previous studies recorded that phenolic compounds including flavonoids are associated with strong antioxidant activity (Table 4). So, the good antioxidant activity of methanol extract of Sapindus emarginatus seed kernel may be attributed to the presence of the phenolic compounds.

Molecular Docking study:

The aim of the molecular docking is the accurate prediction of the structure of a ligand inside the constraints of a receptor-binding site and to properly estimate the strength of binding²⁸. Five compounds were selected from GC-MS analysis of methanol extract of Sapindus emarginatus seed kernel for molecular docking against dihydropteroate synthase. Furthermore, ampicillin (F) was selected as a reference for docking study. Docking results between receptor and selected ligands were tabulated in (Table 5) and were shown in (Fig. 2). Among the five compounds selected from GC-MS analysis of methanol extract, the best docking score of of – 6.53kcal/mol was observed for the 4H-1-Benzopyran-4-one, 2-[3,4-dihydroxyphenyl]-5-hydroxy-3,7—dimethoxy-. The interaction is favored by the formation of hydrogen bond between O atom of ligand and residue GLU65 at distance 2.72 Å. Additionally, two ionic bonds were formed between O of ligand and active site residues LYS220 and ARG 254 at distance of 2.69 Å and 2.88 Å respectively (Fig.2).

Table 5: Molecular docking results of compounds obtained from GC-MS of extract and standard (ampicillin)

Code	Ligand	Receptor	Type of interaction	Docking score (kcal/mol)	RMSD (A°)
AK	C	OD₂-ASP101	H-donor	-5.33	0.80
AK	6-ring	CZ-PHE189	Pi-H	-5.33	0.80
AJ	O	OE1-GLU65	H-donor	-6.53	0.99
	O	NZ-LYS220	ionic
	O	NH1-ARG254	ionic
AI	O	CA-GLY216	H-acceptor	-4.11	1.77
	O	NZ-LYS220	H-acceptor
	O	NZ-LYS220	H-acceptor
	O	CA-GLY216	H-acceptor
	O	CD-ARG254	H-acceptor
	O	NZ-LYS220	ionic
	O	NH1-ARG254	ionic
	O	NE-ARG254	ionic
	O	NH1-ARG254	ionic
AL	6-ring	NE2-HIS256	Pi-H	-5.44	1.37
AH	O	NZ-LYS220	ionic	-5.86	0.855
	O	NZ-LYS220	ionic
	O	NH1-ARG254	ionic
F	S	CA-GLY216	H-acceptor	-4.48	1
	O	ND2-ASN120	H-acceptor
	O	ND2-ASN120	H-acceptor
	O	NE-ARG254	ionic
	O	NH1- ARG254	ionic
	O	NH2-ARG254	ionic

CONCLUSION:

The present study results indicate that the presence of variety of phytochemicals. Twelve of volatile compounds were separated and identified from methanolic extract of Sapindus emarginatus seed kernel using GC-MS analysis. These compounds maybe are responsible for good antibacterial and antioxidant activities of Sapindus emarginatus. Among 12 compounds identified from extract, only five compounds were docked against antibacterial target dihydro-pteroate synthase. 4H-1-Benzopyran-4-one, 2-[3,4-dihydroxyphenyl]-5-hydroxy-3,7-dimethoxy exhibited more efficiency against dihydro-pteroate synthase and docking score was – 6.53 kcal/mol.

AUTHOR’S CONTRIBUTION:

Ali alrabie, Arwa Al-dhreai, Inas al-qadsy and Vidya Pradhan carried out the experiments and wrote the manuscript. Supervisor Dr. Mazahar Farooqui conceived the original idea and corrected scientific content of the manuscript.

CONFLICT OF INTERESTS:

Authors have no conflicts of interest.

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Received on 06.01.2021 Modified on 21.07.2021

Research J. Pharm. and Tech. 2022; 15(5):2117-2121.

DOI: 10.52711/0974-360X.2022.00351