INTRODUCTION:

The ancient Indian medicinal framework extensively utilized India's diverse array of flora, which included plants, shrubs, trees, and seeds. Medicinal plants include several naturally occurring bioactive chemicals that contribute to their therapeutic properties. Numerous organisms depend on the process of oxidation to provide the necessary energy for their metabolic activities. Free radicals, which consist of an oxygen core and various reactive oxygen species, have the potential to induce tissue damage and persistent cell death when generated within a living organism¹. Antioxidant components have the potential to reduce free radical-mediated cellular and tissue deterioration, making them the most essential food ingredients in a living thing^2,3. Although almost all organism have developed antioxidant defence and repaired mechanisms to secure them fromthe mechanisms of oxidative damage are insufficient to dramatically reduce the damage.

Numerous antioxidants that are present in plants naturally and act as active oxygen or free radical scavengers^4,5. Recently, there has been a significant development in interest in identifying the use of naturally occurring antioxidants in place of manufactured antioxidants for incorporation into meals or pharmaceuticals, whose use is restricted due to their undesirable side effects like carcinogenicity^6,7. Natural antioxidants can defend against free radicals, slow the progression of various chronic disorders, and prevent food lipid oxidative rancidity^8,9. Therefore, the study of natural antioxidants has become increasingly significant. Reactive oxygen species are regulated by a network of substances found in plant tissues containing phenolic substances, vitamins C and E, glutathione, and enzymes. The tissues of natural plants contain a variety of phenolic substances, such as lignin, hydroxycinnamate esters, tannins, and flavonoids¹⁰. According to a study, the amount of phenolic compounds in plant materials had a well correlation with the antioxidant activity of those materials¹¹. The potential of phenolics to scavenge free radical, chelate metals and inhibit lipoxygenase may be connected to their bioactivity¹². Polyphenols appear to be significant metabolic modulators due to their ability to affect a number of the biological pathways and compounds identified and suggested as prospective objectives for the investigation of polyphenolic compounds. Flavonoids, a specific type of polyphenol, form a cluster of phytochemicals widely recognized for their significant antioxidant properties, rendering them highly abundant and influential in our dietary intake. Researchers have demonstrated numerous pharmacological and biochemical effects of flavonoids, such as antibacterial, antithrombotic, antimutagenic, and anticarcinogenic activity^13,14,15. Flavonoids have the ability to function as free radical scavengers, exerting a preventive effect on the chain reactions of radicals that result from triglyceride oxidation. The compound 2,2-diphenyl-1-picrylhydrazyl, which exhibits free radical behaviour, serves as a commonly used method to assess the capacity of flavonoids to provide radical species with hydrogen atoms¹⁶. Most polyphenols also possess antibacterial properties. Bacterial species and polyphenol structure both have an impact on how well bacteria tolerate them^17,18.

Hardwickia binata, sometimes known as "Anjan," is a delightful medium to large deciduous ornamental tree with beautiful drooping branchlets. Harongana madagascariensis Chois and Hardwickia trapeziformis (R. Grah.) are synonyms for this monotypic genus of flowering plants. The Detarioideae subfamily of legumes exists. It is an important plant for both economic and medicinal reasons.¹⁹^,²⁰. It has traditionally been used in folklore remedies for a variety of illnesses, such as fungus, gram-positive and gram-negative bacteria, cancer, diarrhoea, leprosy, worm infections, dyspepsia, leucorrhoea, chronic cystitis, and gonorrhoea.^21-25. Researchers have reported that Hardwickia binata has antimicrobial activity.^26,27. The development of innovative antimicrobial chemotherapeutic medicines has become a significant area of medical research.²⁸ The study's goal was to find out how well ethyl acetate, acetone, chloroform, and ethanolic leaf extract of Hardwickia binata fight free radicals and bacteria. To orient future research towards the discovery of novel, powerful compounds, we present the results of such studies in this paper and secure antibacterial and antioxidant compounds.

MATERIAL AND METHODS:

Chemicals and reagents:

We procured all analytical quality solvents and chemicals for this investigation from reputable companies like Sigma-Aldrich (USA), Merck (Germany), E-Merck, and other well-known manufacturers.

Plant Material:

Hardwickia binata leaves were collected from Amravati Road, Nagpur. The head of the R.T.M. Nagpur University Botany Department recognized and validated the plant specimen. With collection number 1072, the Department of Botany's Herbarium has properly kept a voucher specimen. The leaves were shade-dried and coarsely pulverized.

Preparation of extracts:

Defatted the 250g powdered crude material using petroleum ether. Then extracted it sequentially using ethyl acetate, chloroform, acetone, and ethanol in a Soxhlet extractor and made concentrated extracts using a rotating vacuum evaporator. Storage in desiccator for analysis.

Bacterial culture used:

Pure culture of Klebsiella pneumonia, Bacillus subtilis, Escherichia coli, and Staphylococcus aureus were obtain from Rajiv Gandhi biotechnology center, LIT, Nagpur.

Physicochemical Screening:

The purity and strength of leaf powder were measured using physicochemical parameters. This research investigated loss on drying, yield to alcohol and water (95%), total ash, acid-insoluble ash, water soluble ash, and sulphated ash. Water and alcohal-soluble extractive levels are also measured. The mean and standard deviation are presented from triple trials.

Preliminary phytochemical screening:

The pilot phytochemical study used a standardized method to identify terpenoids, flavonoids, saponins, phenols and tannins, steroids, alkaloids, glycosides, resins, carbohydrates, and proteins²⁹.

Quantification of phenolic and flavonoid content:

Procedures were given to measure the involvement of the extraction of flavonoid and phenolic compounds using various solvent extracts.

Estimation of Total Phenolic content:³⁰

Hagerman et al. (1998) utilized gallic acid as a phenolic standard. Dissolve 100milligrams of the drug in 100mL of distilled water (TDW) to make the stock solution. The original solution was diluted by five, yielding a 1mL solution with 200μg/ml extracts in a test tube. In this experiment, mixed 0.5mL of 2N Folin-Cilcalteau eagent (FCR) and 8mL of TDW in a flask. After 5 minutes, added 1.5mL of 20% sodium carbonate. We left the mixture for 2 hours, stirring it occasionally. A UV spectrophotometer at 765nm measured absorbance on a blank sample. We measured the total phenolic content in triplicate using the gallic acid equivalent method.

Determination of total flavonoid content³¹

Flavonoids were measured using aluminium chloride colorimetry. The experiment requires 1mL of extract to 4mL of distilled water in a 10mL volumetric flask. After 5 minutes of agitation with 5% sodium nitrite, the flask received 0.3mL of 10% aluminium chloride. After addition of2 mL of1M sodium hydroxide, the flask was diluted with distilled water to 10mL. Reference standard solution had quercetin concentrationof 20,40, 60, 80, and 100µg/mL. The test and standard solutions absorbance was measured at 510nm relative to the reagent blank using a UV/visible spectrophotometer. Flavonoids were quantified in mg quercetin equivalents/g extract. Estimated triplicates.

Antioxidant assay methods:

DPPH Free radical scavenging method:

The 50μM DPPH solution was chosen because of its linearity range. For the experiment, 4.9 mL of buffered 50 M DPPH was used. Next, 0.1mL of different concentrations (50–1000µg/mL) were added to the solution.The purpose of these modifications was to test samples or standard solutions. Absorbance was measured at 517nm and compared to test and standard blanks. After a 30minute light-free incubation at 37°C, this evaluation was done. Testing was done three times. A linear regression curve was used to calculate the IC₅₀(concentration resulting in 50% inhibition) and compare it to ascorbic acid, a common antioxidant. Free radicals DPPH inhibition (I%) was calculated using the equation below:

I%=100X (A_blank- A_sample) / A_blank

The absorbance of the test compound is denoted as A_sample, while the absorbance of the control reaction is denoted as A_blank. Interpolating from a linear regression analysis yielded the IC₅₀ value (gml^-1), which scavenged 50% of DPPH radical³².

Nitric oxide radical inhibition method:

The experimental procedure involved the use of sodium nitroprusside as a reagent for the generation of nitric oxide, then identified by the implementation of the Greiss-Ilosovog Reaction³³^,3⁴. Sodium nitroprusside (5 mM, 4mL), phosphate buffer saline (1mL), and extract solution (1mL) were added to a 6-ml reaction mixture. We incubated this mixture at 250°C for 150 minutes. After incubation, 0.2mL of the nitrile-containing reaction mixture was with drawn. Combine 2 mL of 0.33% sulphanilic acid with 20% glacial acetic acid. The mixture was diazotized for 5 minutes. After mixing, two mL of naphthyl ethylene diamine dihydrochloride was added and incubated for 30 minutes. Different diffused light conditions produce a pink chromophore. Unlike the test and blank solutions, the UV spectrophotometer assessed absorbance at 550nm. Graphs and linear regression determined 50% inhibition.

Reducing power assay method:³⁵

Hardwickia binata extract (100-1000µg) was mixed with 1mL of distilled water, phosphate buffer (0.2M, pH 6.6), and 1% potassium ferricyanide [K₃Fe(CN)₆]. The solution was incubated at 50°C for 30 minutes. After adding 2.5 millilitres of 10% trichloroacetic acid, the mixture was centrifuged at 3000 RPM for 10 minutes. The top layer of solution (2.5mL) was combined with distilled water (2.5mL) and FeCl₃(0.5mL, 0.1%), and spectrophotometer absorbance was measured at 700nm. Increased reaction mixture absorbance reduces power.

Antimicrobial activity:

Agar well diffusion assay as described in Indian Pharmacopoeia³⁶ is adopted for antibacterial activity against the selected pure culture of Klebsiella pneumonia, Bacillus subtilis, Escherichia coli, and Staphylococcus aureus Mullier-Hinton plates (Hi-Media, Mumbai) were utilized. Each plate included 5 mm wells with 0.1mL of each plant extract at 100mg/mL in Dimethylformamide (DMF). The extract diffused into the media for one hour at room temperature. Bacteria were cultured at 37c for 24hours. Results were measured in mm by measuring growth inhibition zones around the disc. Gentamycin sulphate was used as standard. Clear inhibition zones surrounding discs suggest antibacterial action. After 24hours of incubation, the inhibitory zone (mm) was measured and reported as a mean±standard error of three measurements.

RESULTS:

Extractive values:

Table 1 shows Hardwickia binata leaf extractive values.

Table 1: Extractive values of Hardwickia binata leaf extracts

Sr. No	Extract	Sample Code	Solvent	% yield
1	Hardwickia binata leaf Ethyl acetate extract	HBEA	Ethyl acetate	3.2
2	Hardwickia binata leaf chloroform extract	HBC	Chloroform	3.4
3	Hardwickia binata leaf acetone extract	HBA	Acetone	4.2
4	Hardwickia binata leaf ethanol extract	HBE	Ethanol	6.5

Physicochemical evaluation:

The outcomes of the following tests: water-soluble, alcohol-soluble, acid-soluble, and insoluble ash, extractive value, moisture content, sulphated ash of leaves of Hardwickia binataare given in Tables 2.

Table 2: Physicochemical Analysis of Hardwickia binata leaves

Sr. No	Physicochemical parameter	Weight of sample taken (g)	% value
1	Total Ash content	2.0	8.05±0.408
2	Acid insoluble ash content	2.0	0.24±0.624
3	Water soluble ash content	2.0	4.6±0.321
4	Water soluble extractive value	5.0	23.96±0.678
5	Alcohol soluble extractive value	5.0	5.6±0.456
6	Moisture content	5.0	7.8±0.308
7	Sulphated ash content	1.0	11.04±0.236

Table 3: Preliminary screening of phytochemicals of the investigated extracts of Hardwickia binata leaves.

Sr. No	Phytochemicals	Method	HBEA	HBC	HBA	HBE
1	Carbohydrate	Molisch's test Fehling test Benedict’s test	- - -	- - -	- - -	+ + -
2	Protein	Biuret test, Xanthoproteic test, Millon's test	+ + +	- - -	+ + +	+ + +
3	Amino acid	Ninhydrin test	-	-	+	+
4	Saponin	Foam test	-	-	-	+
5	Tannin and phenolic compound	Ferric chloride test	+	+	+	+
6	Steroid	Salkowski test	-	-	-	+
7	Alkaloid	Dragendorff”s test Mayer's test. Wagner’s test Mayer’s test	- - - -	- - - -	- - - -	- - - -
8	Glycoside	Keller Killiani test.	+	+	+	+
9	Flavonoid	Alkaline test	+	+	+	+

+ = Present, - = Absent

Table 4: Total phenolic and total flavonoid content of Hardwickia binata leaf extract in various solvents.

Sr. No.

Phytoconstituents

Ethyl acetate extract

Chloroform extract

Acetone extract

Ethanol Extract

Total Phenolic content

(mg/g Gallic acid equivalent)

14.51±1.85

17.42±0.308

38.54±0.247

51.24±0.528

Total Flavonoid content

(mg/g Rutin equivalent)

28.25±0.165

45.67±0.276

87.76±0.285

117.43±0.236

Values are mean ± SEM of 3 parallel measurements

Qualitative phytochemical screening:

The results showed that there were several different types of phytochemicals present, as shown in Table 3.

Quantification studies:

The results presented in Table 4 demonstrate the quantitation of Total flavonoids and phenols.

Total phenolic content:

Figure 1 and Table 4 indicate plant total phenolic content (TPC). The quantification of phenolic compounds was carried out using gallic acid equivalents. The phenolic content of Hardwickia binata ethyl acetate extract was 14.51±1.85mg/g GAE; chloroform extract yielded 17.42±0.308mg/g GAE; acetone extract 38.54±0.247mg/g; and Hardwickia binata ethanolic extract 51.24±0.528mg/g GAE.

Total Flavonoid content:

Table 4 displays the Total flavonoid content (TFC) of the extracts. The amount of flavonoid content was calculated as rutin equivalents. According to the study, the ethanolic extract had the most flavonoid (117.43±0.236mg GAE/g dry material). It was followed by the acetone, chloroform, and ethyl acetate extracts, which all had polyphenolic chemicals.

Antioxidant activity:

Scavenging of DPPH radicals:

Ascorbic acid, the positive control, had strong scavenging action with an IC₅₀ of (9±0.01µg/mL). Ethanol extract had the highest IC50 (211±0.41µg/mL), followed by acetone (315±0.38µg/mL), chloroform (416±0.43µg/mL), and ethyl acetate (421±0.43µg/mL)

Figure 1: DPPH assay method for Hardwickia binatain ethyl acetate, chloroform, acetone, ethanol extract and Ascorbic acid

Nitric oxide radical scavenging:

Table 5 shows the NO• scavenging activity, with an IC₅₀ of (1049±0.09)μg/mL of ethanol extract, Ascorbic acid, acetone, chloroform, and ethyl acetate extracts had IC₅₀ values of (322±0.08), (1122±0.06), (1149±0.10), and (1350±0.48)μg/mL.

Figure 2: Nitric oxide scavenging assay method for Hardwickia binata in ethyl acetate, chloroform, acetone, ethanol extract and Ascorbic acid

Reducing power assay method:

The reducing power sequence was ascorbic acid > ethanol > acetone > chloroform > ethyl acetate. All extracts prevented ferrous and ferrozine complex formation. Ascorbic acid had an IC₅₀value of 142.0± 1.23%µg/mL, while the IC₅₀ values for ethanol, acetone, chloroform, and ethyl acetate extract were 198 ± 1.01, 201±1.07, 330±1.82, and 350±1.52µg/mL. Figure 3 and Table 5 revealed the results.

Table 5: Antioxidant activity of Hardwickia binata by using different methods.

Sr. No	Content	DPPH Method IC ₅₀ (μg/ml)	Nitric oxide scavenging method IC ₅₀ (μg/ml)	Reducing power assay method IC ₅₀ (μg/ml)
1	Ascorbic acid	9	322	142
2	Ethyl acetate	421	1350	350
3	Chloroform	416	1149	330
4	Acetone	325	1122	201
5	Ethanol	211	1049	198

Antimicrobial activity:

Ethyl acetate, chloroform, acetone, and ethanol extracts from Hardwickia binata Roxb. Leaves at 100mg/mL were tested against gram-positive and gram-negative bacteria like Staphylococcus aureus, Bacillus subtilis, Eschericha coli, and Klebsiella pneumonia. Table 6 shows antibacterial study results.

Figure 3: Reducing power assay method for Hardwickia binata in ethyl acetate, chloroform, acetone, ethanol extract and Ascorbic acid

DISCUSSION:

Recovery from many ailments requires traditional medicine. Herbal medicines are becoming more popular because they work well and have few adverse effects. Researchers initially studied herbal plants for their numerous medicinal uses, particularly in ancient times. The difference in extract yields may be due to the solvent polarity used, which promotes phytochemical component solubility³⁷. Hardwickia binata ethanol extract (HBE) has the highest phytochemical solubility in alcohol. Physicochemical characterization may help determine plant quality and purity. Ash content (total, acid-insoluble, and water-soluble) and moisture determination are two ways to measure plant minerals. However, medicinal plant humidity is below the recommended 17%³⁸. While plant components need moisture, crude medications should have as little as feasible. Ash values reflect the medicine's quality, care, and purity, whereas extractive values reveal its element content³⁹.

Using statistical methods, phytochemical identification can predict plant pharmacology; however, early phytochemical research still employs qualitative evaluations. Phytochemical research is required to generate biomarker molecules for plant material identification and quality evaluation⁴⁰. Flavonoids include antioxidants that protect the brain from oxidative damage⁴¹. Antimicrobials have prevented microbial illnesses for decades. Reduced economic incentives, difficult regulatory constraints, and other reasons hinder antimicrobial resistance drug development⁴². Antimicrobials have prevented microbial illnesses for decades. Reduced economic incentives, difficult regulatory constraints, and other reasons hinder antimicrobial resistance drug development^43,44,45.

CONCLUSION:

This study proved antioxidant and antibacterial properties. This study found that Hardwickia binata leaf extracts are antioxidant and rich in phenolics and flavonoids. The high polyphenolic content of the ethanol extract may explain its outstanding free radicals scavenging and antioxidant activity. Additionally, the plant may contain natural antioxidants that might prevent oxidative stress-related diseases. The antibacterial properties of Hardwickia binata effectively combat disease-causing this plant's bioactive compounds may possess antibacterial properties, enabling the creation of antimicrobial agents for bacterial infections. To make new chemotherapeutic agents, more research should be done to separate, identify, and clean up these phytoconstituents, as well as to find out how well they work as medicines and how safe they are.

ABBREVIATIONS:

HBEA: Hardwickia binata ethyl acetate extract, HBC: Hardwickia binata chloroform extract, HBA: Hardwickia binata acetone extract, HBE: Hardwickia binata ethanol extract, DPPH: 2,2-diphenyl-2-picrylhydracyl hydrate, TDW: Triple distilled water, FCR: Folin-Ciocalteau reagent, GAE: Gallic acid equivalents, QE: Quercetin equivalent, TPC: Total phenolic content; TFC: Total flavonoid content; IC₅₀: half-maximal inhibitory concentration.

CONFLICT OF INTEREST:

The authors declare that there is no conflict of interest.

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Received on 25.09.2023 Modified on 16.04.2024

Research J. Pharm. and Tech 2024; 17(11):5267-5273.

DOI: 10.52711/0974-360X.2024.00806

Sr. No.	Microorganism used	Zone of inhibition in mm
Sr. No.	Microorganism used	Ethyl acetate	Chloroform	Acetone	Ethanol	Gentamycin
1	Staphylococcus aureus,	21±0.89	23±0.24	23±0.74	24±0.26	25±0.24
2	Bacillus subtilis	19±0.56	18±0.41	18±0.66	19±0.22	24±0.19
3	Escherichia coli	18±033	16±0.24	22±0.27	23±0.59	25±0.28
4	Klebsiella pneumonia	15±0.45	14±0.67	17±0.45	16±0.23	26±0.46