INTRODUCTION:

The imbalance between the formation and impartiality of Reactive Oxygen Species (ROS) can damage nucleic acids, DNA,¹ proteins, and lipids and can trigger various chronic diseases such as atherosclerosis, cancer, diabetes, heart disease, aging, and inflammatory diseases.^2,3,4 Antioxidants are supportive to the body to release free radicals and delay or prevent oxidative stress, thus preventing various degenerative diseases such as heart disease, cancer, Alzheimer's disease, neurodegenerative diseases, and inflammatory diseases.^5,6. Such as catalase, superoxide dismutase, peroxidase glutathione⁷ system, etc., which protect against oxidative damage and repair enzymes to eliminate damaged molecules.⁴ These defenses often fail; hence the need for antioxidant foods. Nowadays, synthetic anti-oxidants are used in the food industry, which may be responsible for liver damage, and^5,6,7,8 plants play an important role in preventing disease or in controlling the effects of their vitamins and minerals, terpenoids, phenolic acids, stilbenes, tannins, flavonoids, quinone, coumarins, and alkaloids are rich in anti-oxidant activity.⁹ F. hispida (family: Moraceae) is a small but well-distributed species of the tropical fig tree. It is found all over India and in parts of Asia and southeast Australia. In India it is known as Kala umbar, Peyatti, sonatti Dumoor, and kakodumar, etc.^10,11 Almost all parts of this plant are used as a traditional remedy for various ailments.¹² In Ayurveda, the root of F. hispida is used to treat leukoderma and to check for internal bleeding. Fruits of F. hispida are used in the treatment of leukoderma. Its leaves are also used to treat coughs and asthma. In the Unani medical system, the roots of F. hispida and bark are used as a blood purifier and a powerful cleanser. Latex F. hispida is used in ringworm and is attached to ripe fruit for goiter.¹³ F. hipida leaves have neuroprotective carcinogenesis. Therefore, scientists are interested in the activity, antidiarrheal activity, and hepatoprotective activity, in finding anti-oxidants in natural sources. Antineoplastic therapeutic activity, hypolipidemic activity.^{14,15,16,17,18} Its fruits have antimicrobial activity and its roots have wound healing and antiulcer functions.^{19, 20, 21} A few studies have been conducted on its fruits and bark, in addition, a comparative study of the parts of its leaves is yet to be investigated. Therefore, the present study investigated the phytochemical and antioxidant properties of F. hispida leaves.

Figure 1

Figure 2

MATERIALS AND METHOD:

Chemicals:

1, 1-diphenyl-2-picrylhydrazyl (DPPH), 2, 2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), potassium ferricyanide, potassium acetate, phosphate buffer, catechin (CA), ferrous ammonium sulphate, butylated hydroxytoluene (BHT), gallic acid (GA), ascorbic acid (AA), AlCl3, Trichloro acetic acid (TCA), sodium phosphate, ammonium molybdate, tannic acid, quercetin (QU), EDTA, thiobarbituric acid (TBA), Methanol, ethanol, chloroform, petroleum ether, hydrochloric acids, sulfuric acid, acetylacetone, and FeCl₃ were purchased from Loba-Chemie Pvt. Ltd; vanillin was obtained from BDH; Folin-Ciocalteus phenol reagent (FCR) and sodium carbonate were obtained from HiMedia Laboratories.

Collection of plant material and authentication:

Mature F. hispida leaves were collected from the local area of Barwani Dist. Madhya Pradesh, west-central part of India (figure 1 and figure 2). The leaves of the plant were identified and authenticated by Dr. P. Jayaraman, Director, Plant Anatomy Research Centre West Tambaram, Chennai, India.

Preparation of extract:

The leaves are first washed with water to remove sticky dirt. After that, the leaves are removed and dry. After completely drying, ground into a fine powder with a fine grinding powder and then stored in an airtight container for further use. Four different solvents namely ethanol, methanol, chloroform, and petroleum ether were used for extraction. For each solvent, about 80g of the raw material was taken in clean, round glass bottles and immersed in 400ml of solvent. The contained in it was sealed with a cotton plug and aluminum foil and stored for 15days with constant movement. The resulting components were filtered through Whatman No. 1 filter paper. Then the solvents evaporated under a reduced pressure of 40°C using a rotating evaporator. Finally, the remains were stored in small empty bottles under refrigerated conditions until they could be used. A total of 4 parts were extracted from the leaves of F. hispida namely: methanol extracted from the leaves of F. hispida (ML), ethanol extracted from the leaves of F. hispida (EL), chloroform extracted by the leaves of F. hispida (CL), and petroleum ether extract of F. hispida leaves (PL).

PHYTOCHEMICAL ANALYSIS:

Phytochemical screening of the extracts:

Qualitative analysis of phytochemicals such as alkaloids, carbohydrates, flavonoids, glycosides, triterpenoids, resins, saponins, steroids, and tannins was performed by dissolving samples into specific reagents using standard methods.^22,23

Determination of total phenolics content:

The Folin-Ciocalteu method²⁴ was used to measure the total phenolic content of each extract. Briefly, 2.25ml of Folin – Ciocalteu reagent diluted (1:10) in pure water was mixed with 300μl of extract and kept for 5 minutes at room temperature. 2.25 ml of sodium carbonate solution (60g/l) was then added to the mixture and the absorption was taken at 725nm after 90 minutes of incubation at room temperature. As usual, Gallic acid (GA) was used and the total phenolic content of each extract was calculated as the equivalent of Gallic acid per gram of dry weight (mg GAE/g DW).

Determination of total flavonoids content:

Using the previously described method, ²⁴ the amount of flavonoid content is estimated. Briefly, 2.25ml of distilled water was mixed with 0.5ml of extract in a test tube followed by the mixture of 0.15ml of 5% NaNO₂ solution. The test tubes are then stored at room temperature for 6 minutes. Thereafter, 0.3ml of 10% AlCl3.6H2O solution was added and allowed to stand for 5 minutes before the addition of 1.0ml of 1M NaOH. Then the mixture was matured and the absorption was measured quickly at 510nm. As the usual Catechin (CA) was used and the results were expressed as catechism per gram of dry extract (mg CAE/g DW).

Determination of total flavonols content:

The amount of flavonol in the extract is estimated using the Kumaran and Karunakaran method.²⁵ 2.0ml of extract/standard was taken from the test tube in which 2.0ml of 2% AlCl3 (dissolved in ethanol) and 3.0ml (50 g/l) of sodium acetate solutions were added. Absorption is measured at 440nm after 2.5hours at 20°C. The amount of flavonol content expressed was proportional to quercetin, mg of QUE/g of dry extract.

Determination of total proanthocyanidins content:

The total content of proanthocyanidins was determined using a protocol reported by Sun et al.²⁶ Briefly, 0.5ml of standard extract/solution is mixed with 3ml of 4% vanillin in methanol solution, followed by the addition of 1.5ml hydrochloric acid and the mixture stored for 15 minutes at room temperature. The absorption is then measured at 500nm. Catechin was used as standard content and the total proanthocyanidins were expressed in terms of equivalent catechin, mg of CAE/g of dry form.

Evaluation of Antioxidant Activity Determination of total antioxidant capacity:

The total amount of antioxidant (TAC) extract was determined by Prieto et al.²⁷ with certain changes. Briefly, 0.5ml of extract at different concentrations was mixed with a reaction mixture (3 ml) containing 0.6M sulfuric acid, 28mM sodium phosphate, and 1% ammonium molybdate, and soaked at 95°C for 10 minutes to complete response. Then spectrophotometric readings were taken at 695nm versus blank after cooling at room temperature. The Catechin (typical) graph in various places is also constructed in the same way for comparison.

Determination of ferric reducing antioxidant capacity:

The reducing capacity was assessed following Oyaizu's method with some modifications.^28,29 250μl of samples/standard at different concentrations were mixed with 1.75ml of 0.2M phosphate buffer (pH 6.6) and 1ml of potassium ferricyanide (1%). The mixture was then placed at 50°C for 20 minutes followed by the addition of 1ml of TCA (10%). 1ml from the incubation mixture is mixed in a test tube with 1ml of distilled water and 0.2 ml of ferric chloride (0.1%). The absorption of that resulting solution is measured at 700nm after 10 minutes. Increased absorption of the reaction mixture indicates an increase in the reduction capacity.³⁰ The standard used for ascorbic acid (AA) in various concentrations is also performed similarly for comparison.

DPPH free radical scavenging assay:

DPPH free radical scavenging capacity of plant extract was determined as previously described methods ^{28, 29} with minimal modification. Immediately, 0.5 ml of the sample was mixed with 3.5 ml of methanolic solution 0.2 mM of DPPH, and the absorption was taken at 517 nm after incubation for 30 minutes at room temperature. AA and Butylated hydroxytoluene (BHT) were used as good controls. Radical disposal work is calculated by the following formula:

% Scavenging Activity =

(A control- A sample/A control) × 100

Where, A control = Absorbance of control, A sample = Absorbance of the sample.

Then the percentage of DPPH radical scavenging activity was plotted against concentration, and from the graph, IC₅₀ was calculated.

Determination of ABTS radical scavenging activity:

The antioxidant capacity of the extract plant was determined according to the ABTS radical scavenging activity following the previously described method.³⁰ ABTS radical was obtained by reaction with a 7mM ABTS stock solution containing a solution of potassium persulfate 2.45mM and the mixture was left in the dark at room temperature for 12–16 hours before use. ABTS radical solution (stable for 2 days) was diluted with water to obtain an absorption of 0.70±0.02 at 734nm. Thereafter an ABTS radical solution (3 ml) was added to 1ml of the experimental sample with various concentrations and mixed vigorously. After 6 minutes, absorbance is measured at 734nm. AA and BHT were used as positive controls. The task of extracting ABTS samples is as follows:

% Scavenging Activity =

[(A control– A sample) / A control)] ×100

Where AA control is the absorbance of the blank control (ABTS radical solution without test sample) and A sample is the absorbance of the test sample.

Determination of superoxide scavenging activity:

The activity of scavenging Superoxide was determined using the NBT method (nitro blue tetrazolium reagent) previously described by Sabu et al.³¹ (with some modifications). The experimental solution of the extracted material (20–300μg/ml) was taken from the test tube, after which 1ml (5mM) of sodium carbonate, 0.4ml (0.24 mM) of NBT, and 0.2ml of -0.1 mM EDTA solutions were added to the test tube and the absorption was taken at 560 nm immediately. Approximately 0.4 ml (1mM) of hydroxylamine hydrochloride was added to initiate the reaction and the reaction mixture was placed at 250^oC for 15 minutes, and the NBT reduction was approximately 560nm. AA and BHT were used as standards. The % of inhibitions is calculated according to the following formula:

% Scavenging Activity = [(A₀-A₁)/A₀] × 100

Where A₀ is the absorbance of the initial reading of sample/standard and A₁ is the absorbance of the final reading.

Total phenolics, flavonoids, flavonols, and proanthocyanidins contents:

The total contents of phenolic, flavonoids, flavonols, and proanthocyanidins were various extracts from leaves of F. hispida as shown in Table 1, where a relatively high number of phenolic and Flavonols are found in EL of F. hispida (75.07±1.22 mg GAE/g DW, and 49.66±2.16 mg QUE/g DW respectively), but ML contains higher concentrations of flavonoid and Proanthocyanidins (49.54±0.97mg CAE/g DW, and 10.22±0.11mg CAE/g DW) were obtained.

Table 1: Total Phenolics, Flavonoids, Flavonols, and Proanthocyanidins contents of F. hispida leaves.

Name of extracts	Phenolics (mg GAE/g DW)	Flavonoids (mg CAE/g DW)	Flavonols (mg QUE/g DW)	Proanthocyanidins (mg CAE/g DW)
EL	75.07±1.22	31.19±0.69	49.66±2.16	2.25±0.08
ML	46.86±2.09	44.88±1.20	49.89±0.89	10.22±0.11
CL	51.09±2.31	49.54±0.97	43.35±1.51	10.18±0.26
PL	13.17±0.87	40.54±1.01	34.75±2.12	8.00±0.34

Note: Results are expressed as mean ± standard deviation. DW: Dry weight of the extract.

Table 2: IC₅₀ values of F. hispida in different assays.

Name of extracts		IC₅₀ (µg/ml)
		DPPH		ABTS		Superoxide		Nitric oxide
EL		50.33±2.59		83.35±1.98		105.21±4.40		117.73±2.23
ML		79.10±3.51		103.51±3.58		104.17±3.49		108.98±4.66
CL		328.60±5.78		339.91±6.26		455.36±3.49		818.53±2.07
PL		414.61±4.46		474.40±3.01		593.29±3.21		1027.41±8.93
Standard	AA		20.07±1.36		31.41±2.97		58.10±2.23		79.07 ± 2.45
	BHT		35.24±1.86		46.73±2.37		71.59±1.96		98.47±2.73

Note: Results were expressed as mean ± standard deviation (n=3). AA: Ascorbic Acid, BHT: Butylated hydroxytoluene.

Table 3: Correlations between the antioxidative activities and total phenolic contents of the leaves extract of F. hispida.

Assay	Correlation coefficient (R²)
Assay	EL	ML	CL	PL
DPPH	0.95	0.93	0.89	0.91
ABTS	0.86	0.90	0.91	0.92
Superoxide	0.84	0.87	0.87	0.89
Nitric Oxide	0.82	0.83	0.93	0.93

DISCUSSION:

In the present study, we used four different solvents to prepare extracts from the F. hispida to detect the presence of alkaloids, flavonoids, carbohydrates, triterpenoids, glycosides, saponins, resins, and tannins and to evaluate the total value of phenolics, flavonoids, flavonols, proanthocyanidins content, and antioxidant activity. Findings from our current study vary from one extract to another. Differences in polyphenol content and biological activity depend on the type of solvent used for extraction.^32,33 Phytochemicals are chemical compounds in plants that have a prominent biological activity such as anti-inflammatory, antioxidant, anticancer, and antimicrobial properties. Phenolic and flavonoid common antioxidants are known in plants.³⁴ In addition, Proanthocyanidins play a key role in preventing various diseases, such as atherosclerosis, colon cancer, cataract, gastric ulcer, and diabetes. Flavonols also have significant health and cardiovascular health benefits.^35,36 Current studies have shown that each release of F. hispida contains significant amounts of phenolics, flavonoids, flavonols, and proanthocyanidins.

Today, polyphenols have concerned special attention because of their ability to reduce tissue damage caused by free radicals. In the present study, we used four in vitro assay models to test the antioxidant activity of F. hispida extracts. In the TAC assessment, EL showed higher antioxidant activity than other ones. DPPH is free radical scavenging due to the delocalization of a spare electron from the entire molecule. It is widely used to measure the free-radical disposal function of extracted plants and pure compounds, whereas stable DPPH free-radical is reduced by antioxidants leading to the development of a yellow pigment compound.³⁷ The ABTS radical scavenging assay is also commonly used to measure the activity of hydrogen scavenging and chain-breaking antioxidants in many released systems.³⁸ Free active radicals such as hydroxyl radical, hydrogen peroxide, and singlet oxygen can be produced by superoxide anions. Although hydrogen peroxide is less toxic to the cell, it may cause a hydroxyl radical that can interact with DNA directly and thus contribute to cancer growth, aging and cytotoxicity. Damaging Nitric Oxide is also produced in biological tissues by specific nitric oxide synthases.^{2, 39} In our recent study, EL showed high scavenging activity in the DPPH and ABTS scavenging assay, ML represents the highest antioxidant activity in Nitric Oxide scavenging assay, and Superoxide scavenging assay EL and ML produced significantly more radical scavenging compared to standard reference compounds, ascorbic acid, and butylhydroxytoluene. All other extracts also represent the average antioxidant activity compared to the standards in all these tests.

CONCLUSION:

This study concluded that F. hispida is a promising source of medicinal phytochemicals and antioxidants. It can play an important role as a therapeutic agent in preventing oxidative stress. Recent research has also recommended that methanol and ethanol are the solvents of choice to extract polyphenols from the leaves of F. hispida. Ethanol and methanolic extracted from F. hispida contain much more polyphenols compared to other extracts. Further investigation is needed to identify and identify the active compounds present in the leaves of F. hispida.

ACKNOWLEDGEMENT:

The authors are grateful to all the members of the Laboratory of Pharmacognosy for their constant support throughout this research and to the Department of Pharmacognosy, ITM (SLS) Baroda University, Gujarat, India for supplying necessary materials.

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Received on 13.06.2022 Modified on 17.12.2022

Res. J. Pharmacognosy and Phytochem. 2023; 15(3):203-208.

DOI: 10.52711/0975-4385.2023.00031