Phytochemical, Antioxidant and Antimicrobial Potential of Azadirachta Indica leaves

Pharmacology is a branch of pharmaceutical sciences that deals with the study of natural substances, particularly those derived from plants, which possess therapeutic properties.  This discipline integrates aspects of botany, chemistry, biology and medicine to explore the medicinal potential of bioactive compounds exist in plants.  The importance of Pharmaceutical has grown as the world turns to nature for treatments, especially with the increasing concern over synthetic drugs & their side effects along with the sustainability of chemical-based therapies.  Through the biochemical assessment of medicinal plants, scientists evaluate the chemical composition, therapeutic effects, and safety profiles of plant-based compounds.  This comprehensive approach helps identify new drugs, understand the mechanisms of their action and ensure their efficacy and safety for human use. Too much medicinal plants have been currently used in the Bhutanese Traditional Medicine (BTM) as a chief ingredient of polyherbal formulations and these plants have been individually indicated for treating various types of infections including malaria, tumour and microbial (Phurpa Wangchuk et al., (2011).  Extensive phytochemical analysis has led to the characterization and identification of major components of essential oils, which are of wide interest, especially to cosmetic and pharmaceutical industries (Raut et al., 2014).  Performing pharmacological and toxicological studies, many features inherent in herbal medicinal products need to be considered in order to guarantee valid results: concerning in vitro studies, difficulties are often related to lacking knowledge of ADME (Absorption Distribution Metabolism and Excretion) characteristics of the bioactive constituents, nuisance compounds producing false positive and false negative results, and solubility problems (Eva-Maria Pferschy-Wenzig & amp; Rudolf Bauer 2015).

However, the leaf extracts of Azadirachta indica were screened for antifungal accomplishment along with various dermatophytes, Trichophyton, and Epidermophyton floccosum, and they indicated high capability in hindering these organisms (Natrajan et al., 2002). Thus, in the study the presence of flavonoids and phenolic compounds in the A indica leaves extract could be considered responsible for conferring antioxidant ability (Ramesh kumar et al., 2018). The crude methanolic extracts of A. indica and M. azedarach have significant phytochemical properties assessed to other extracts and hence the phytochemicals of M. azedarach and A. indica can be exploited for plant based anticancer and antimicrobial agents soon (T.R.J.  Jeba Malaret al., 2019). It is suggested that A indica plays pivotal role in the prevention and treatment of cancer via its hepatic antioxidant activity. Indeed, application of neem extract can decrease tumor growth via restore of the antioxidant disturbances close to the control ones in the liver. (Maryam Imran et al.,2022). This study helps in identifying plant species with therapeutic efficacy, determining the active ingredients responsible for their medicinal properties, and assessing their safety and effectiveness.  By investigating the biochemical composition of medicinal plants, the study provides valuable insights into their mechanisms of action, potential side effects, and their role in traditional and modern healthcare.

MATERIALS AND METHODS

Sample collection

The fresh leaves of A. indica were collected from Gwalior district, MP India in the month of July 2025.  The healthy leaves of A. indica were then taken, washed with distilled water and shade dried for 4 weeks.

Preparation of extract

Dried leaves of plant were ground in a sterile blender to fine powder.  30-gram powder was soaked in 300 ml of methanol and water in conical flask and stored at room temperature for 72h separately.  The extract was filtered through Whatman No.1 filter paper, and the fine power product was obtained finally after evaporation to dryness using a rotary evaporator.

Preliminary phytochemical screening

Test of alkaloids was done to determine its presence or absence, according to the method described by Anjanal et al., 2012.  Test of glycosides was done to determine its presence or absence according to the method described by (Sharma V, Singh M 2012).  Test of phenol and tannins was determined by the method described by Slinkdard et al., 1977.  Test of steroids was done to determine its presence or absence as described by (Sharma V, Singh M 2012).  Test was done to determine the presence or absence of flavonoids according to the method described by Piyanete et al., 2009.  Presence or absence of volatile oil was determined as described by.  Presence or absence of carotenoids was determined according to the method described by Kikuzaki et al., 1993. Test of saponins was done to determine its presence or absence according to the method described by Kikuzaki and Nakatani (Pardeshi et al., 2019).

Quantitative determinations of leaves extract of A. Indica

Estimation of Tannins

Estimation of Tannins was carried out using Folin-Denis reagent according to the method of (AOAC, 1980) with some modifications.  To 0.20 g of the fine power product was added in 20 ml of 50% methanol.  This was shaken thoroughly and placed in a water bath at 80°C for 1 h to ensure uniform mixing.  The extract was filtered into a 100-ml volumetric flask, followed by the addition of 20 ml of distilled water, 2.5 ml of Folin-Denis reagent and 10 ml of 17% aq. Na₂CO₃ and was thoroughly mixed.  The mixture was made up to 100 ml with distilled water; it was then mixed and allowed to stand for 20 min.  The bluish-green colour developed at the end of the reaction mixture of different concentrations ranges from 0 to 10 ppm.  The absorbance of the tannic acid standard solutions as well as sample was measured after colour development at 760 nm using the Lab Tronics model LT 2203 UV-VIS Spectrophotometer.  Results were expressed as mg/g of tannic acid equivalent using calibration.

Alkaloid estimation

Alkaloids are basic nitrogenous compounds with certain biological and pharmacological activity.  Alkaloid solution produces white yellowish precipitate when a few drops of Mayer’s reagents are added (Siddiqui et al.,1997).  Most alkaloids are precipitated from neutral or slightly acidic solution by Mayer’s reagent (Evans, W.C, Trease,2002).  The alcoholic extract was dispersed to dryness, and the residue was heated on a boiling water bath with 2% hydrochloric acid.  After cooling, the mixture was filtered and treated with a few drops of mayer’s reagents.  The sample were then observed for the presence of turbidity or yellow precipitation.

Determination of flavonoids according to the Christ-Müllers method

Total flavonoid content was measured according to the Christ-Müller's method (Christ and Müller, 1960; Polish Pharmacopoeia, 2002) using a Lab Tronics model LT 2203 UV-VIS Spectrophotometer.  Content of flavonoids in all sources was calculated as quercetin and hyperoxide and samples for the analysis were prepared according to the Polish Pharmacopoeia VI.

Saponins determination

A 40-gram fine power product was kept with 100 mL of 20% aqueous ethanol contained in a conical flask.  The mixture was slept in a water bath and heat up at a temperature of 55 °C for 4 h with incessant stirring.  The mixture was filtered and re-extracted with 300 mL of 20% ethanol.  The filtrates were combined and the volume was reduced to 60 mL using a water bath at a temperature of 95 °C.  The concentrated filtrate was transferred into a 500 mL separating funnel and 25 mL of diethyl ether was added to it.  This was shaken actively and allowed to separate into two layers.  This extraction was carried out three times, and the aqueous layer was recovered.  The aqueous layer was extracted three times with 60 mL of n-butanol.  The n-butanol extract was washed three times with 10 mL of 5% NaCl.  The washed n-butanol extract was heated in a water bath to evaporate the n-butanol.  The n-butanol extract was dried in the oven at a temperature of 50 °C to a constant weight to give the saponins (Theresa Ibibia Edewor et al., 2016).

Determination of total phenols by spectrophotometer

The total phenolic content was decided by the spectrophotometric method (Kim et al., 2003).  In brief, a 1 ml of the fine power product (1 mg/ml) was mixed with 1 ml of Folin-Ciocalteu’s phenol reagent.  After 5 min, 10 ml of a 7% Na2CO3 solution was added to the mixture followed by the addition of 13 ml of deionized distilled water and mixed methodically.  The mixture was kept in the dark for 90 min at 23°C, after which the absorbance was read at 750 nm.  The Total Phenol Contain was determined from extrapolation of calibration curve which was made by preparing gallic acid solution.  The estimation of the phenolic compounds was carried out in triplicate.  The Total Phenolic Contants was stated as milligrams of gallic acid equivalents per g of dried sample.

Determination of invitro antioxidant activity

1,1-Diphenyl-2-picryl hydrazyl (DPPH) radical scavenging activity

DPPH radical scavenging activity was measured according to the method of (Shimada et al.  1992).  The antioxidant activity of the plant leaves methanol and water extract was prepared in 95% ethanol.  A stock solution of methanolic and water extracts and ascorbic acid were prepared in the concentration of 10mg/1ml (100µg/ml) from stock solution.   The plant leaves extract in methanol and water at several concentrations (20, 30, 40, 50 and 60μl) were mixed with an aliquot of 2 ml of 600 μM DPPH solution in ethanol and incubated at 25°C for 30 min and absorbance of the test mixture was read at 517 nm using a spectrophotometer against a DPPH control containing only 1 ml of ethanol in place of the extracts.  All experimentations were determined thrice, and the results were.  Ascorbic acid was used as a standard.  Percent inhibition was computed using the following expression:

 Inhibition (%) =[(A control-A sample)(A control)

Where, A control and A sample stand for absorbance of the control and absorbance of tested extract solution, respectively.

ABTS  2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonate) Assay

Free radical scavenging activity of plant fine power product was determined by ABTS radical cation decolorization assay (Re et al., 1999). ABTS + cation radical was produced by the reaction between 7mM. ABTS in water 2.45 mM potassium persulphate (1:1) stored in the dark at room temperature for 12-16h before use. ABTS + solution was then dilutated with methanol to obtain an absorbance of 700 at 733nm. After the addition of 5 µl of plant extract to 3.995 ml of diluted ABTS solution the absorbance was measured at 30 ml after the initial mixing. An appropriate solvent blank was run in each assay. All the measurements were carried out at least three times. Percent inhibition of absorbance at 733 nm was calculated using the formula,

ABTS·+ scavenging effect (%)=(AB-AA)(AB) ×100

where AB is absorbance of ABTS radical + methanol; AA is absorbance of ABTS radical + sample extract/standard. Trolox was used as standard substance.

Thio barbituric acid-reactive species

The Thiobarbituric acid-reactive species (TBARS) assay as described by previous methods Kikuzaki and Nakatani 1993 was used to measure the lipid peroxide formed, using mixture containing 4mg of the methanolic and water leaves extract in 4 ml of 99.5% ethanol (final conc 0.02%) 4.1 ml of 2.52 linoleic acid in 99% ethanol 8ml of 0.05M phosphate buffer (PH-7.0) and 3.9 ml water was placed in a test tube and then placed in incubator at 40C in the dark separately.  To 2ml of this mixture. 2ml of 20% trichloroacetic acid and 2ml of 67% TBA solutions were added.  This mixture was kept in water bath (100?C) for 15 minutes and after cooling at room temperature it was centrifuged at 3000rpm for 30 minutes.  Antioxidant activity was based and the absorbance of the supernatant at 534nm.  The percentage of antioxidant was calculated by following formular for TBA by (Demirtas et al., 2013 and Toth et al., 2018).

Percentage of Activity=A of Control-A of TestA of Control ×100

Antimicrobial screening of the plant extract

The antimicrobial activity of plant extract was evaluated by well diffusion methods given by Boyle et al., (1973).  The microbial strains of six bacterial samples which were used for the test were taken, the six different strains employed were Gram positive Staphylococcus aureus and Gram-negative Escherichia coli, Salmonella typhi, Klebsiella pneumoniae, Pseudomonas aeruginosa and Proteus vulgaris.  They were taken and cultured on broth containing nutrient agar.  A McFarland 0.5 standard was prepared, and the bacterial suspension was compared with the 0.5 McFarland standards to adjust the turbidity of the inoculums for the susceptibility test.  The inoculums of bacteria were transferred into Muller-Hinton agar plates using sanitary cotton swab.  Wells were prepared by striking the agar plate already inoculated with a pure culture of the test organism with the help of sterile glass pipe.  Then 20, 30, 40, 50 μl of samples were added in wells from the stock solution of 10 mg/ml.  In the two wells, 30 μl of methanol (as negative control) and 30 μl of reference antibiotic solution (as positive control) were added.  Gentamycin (1%) and vancomycin (1%) were used as reference antibiotic for Gram-negative bacteria and Gram-positive bacteria, correspondingly.  Dissemination of extracts, antibiotics and methanol and water were allowed at room temperature for 1.5h.  All the plates were then covered with tops and incubated at 35- 37°C for 18-24h and zone of inhibitions were measured.

Results

Primary Phytochemical Screening

The preliminary phytochemical screening of methanolic and water extracts of A. indica leaves showed the presence of various secondary metabolites.  Alkaloids, phenol, tannin, flavonoids, saponins were the most prominent.  Flavonoids, alkaloids and phenolic compounds are a major group of compounds that act as primary antioxidants or free radical scavenger.  Successively, it may be used for the preparation of drug in a competent way which may command to the remedy of many diseases in the future.  So, due to the existence of such secondary metabolites, A. indica may have higher therapeutic value.  Primary phytochemical exhibiting of the water and methanolic leaves extract is presented in Table 1.  The following results exhibited the appropriate amount of alkaloid, flavonoids, tannins, saponins and phenols are present in both extracts. 

Table 1. Quantitative estimation of phytoconstituents in leaves extracts of A Indica.

Values are expressed Mean ± SEM (n = 3)

Antioxidant activity

The antioxidant activity of both water and methanolic leaves extract of A. Indica were determined by spectrophotometric analysis of DPPH (2,2’ –diphenyl-1- picrylhydrazyl) Scavenging activity, ABTS  2,2′-azinobis-(3-ethylbenzothiazoline-6 sulphonate) assay and TBARS (Thiobarbituric reactive substance) assay.

DPPH Radical Scavenging Activity

In this method, when antioxidant reacts with the free radical DPPH (1,1-diphenyl-2-picrylhydrazyl deep violet colour) convert it to faint colour 1, 1-diphenyl-2-picrylhydrazine.  The degree of fain colour indicates the free radical scavenging potentials of the sample.  The DPPH radical Scavenging activity was recorded in terms of % inhibition as shown in Figure1.  IC50 values were interpolated from linear regression equation (D.  Yang et al., 2007). The higher radical scavenging activity of the extract showed by lower IC50 value.  The results showed that water extract, methanolic extract and ascorbic acid (standard drug) has an IC50 of (62.00, 78.00 and 12.00 mg/ml).  The results showed that water and methanolic leaves extract of A. Indica possessed antioxidants.  The values are presented in Mean ± SEM, (n=3).

ABTS Assay

The methanolic and water leave extract of A indica examined for radicals scavenging and antioxidant activities using three different assay methods.  The percentage inhibitions of the methanolic and water leave extract of A indica were concentration dependent.  The percentage inhibitions for DPPH assay are given in Figure 1.  At all concentrations (20 – 50 mg/mL), the methanol and water extracts showed stronger DPPH radicals scavenging activity than the Standard.  Interestingly, the methanol extract at 20 mg/mL and 40 mg/mL were far above average and demonstrated comparable scavenging activity to Ascorbic acid.  Unlike in DPPH assay, the ascorbic acid was less active in scavenging ABTS radicals, while ascorbic acid activities were superior to all the concentrations of methanol and water extract.  However, the methanol and water extracts scavenging activity at 40 mg/mL was exceeding average (Figure 2).

Thiobarbituric Acid Reactive Substances (TBARS)

In TBARS, the total antioxidant activity exhibited by the extracts and standard were shown in Figure 3.  The results showed that both extracts possessed antioxidant activity compared to ascorbic acid.  The values are presented in Mean ± SEM, (n=3).

Antibacterial activity

The result for antibacterial activity of methanolic and water leave extract of A indica is shown in Table 2,3.  The antibacterial potential may be due to various phytochemicals like phenols and flavanols.  A. indica methanol extract was the most effective against S. aureus and S. typhi, whereas it was less effective against E. coli and K. pneumoniae and was moderately effective against Proteus vulgaris and Pseudomonas aeruginosa.  The zone of inhibition (ZOI) diameter was measured, and it was found that A. indica leaves methanol showed maximum zone of inhibition against Gram positive S. aureus and minimum zone of inhibition against Gram negative E. coli. The zone of inhibition was further increased on increasing concentration with maximum inhibition of 20±3 mm on S. aureus when 300μg extract was used. Result of the antibacterial activity of A. indica leaves methanol extract.

Table 2. Result of the antibacterial activity of A. indica leaves water extract.

Table 3. Result of the antibacterial activity of A. indica leaves methanol extract.