INTRODUCTION:

Free radicals are atoms or groups with unpaired electrons, formed during oxidative respiration involved in several biosynthetic processes these are highly reactive¹, maintaining the cellular homeostasis by activation of signal transduction and aiding the innate immunity².

The majority free radicals are oxygen-reactive species, such as singlet oxygen (O₂), hydrogen peroxide hydroxyl radical (OH^•) and superoxide radical (O^2-), which have the greatest biological significance^3,4. The imbalance between reactive oxygen species (ROS) and an antioxidant system causes oxidative stress⁵. During oxidative stress high levels of ROS have been concerned in more than 100 diseases, such as cardiovascular, diabetes, cancer, atherosclerosis, Alzheimer’s disease, Parkinson’s disease and arthritis^6,7. Antioxidants are used to counterbalance the effects of free radicals; therefore, they protect humans against infection and degenerative diseases. Long term use of several synthetic antioxidant agents such as butylated hydroxyanisole (BHA), butylated hydroxyl toluene (BHT), and tertiary butylated hydroquinone induce mutations and carcinogenesis in humans. There is a strong epidemiological correlation between high natural antioxidant intake and the prevention of degenerative diseases that can be attributed to these compounds' free radical scavenging proprieties⁸. Therefore, there is a strong trend to replace the synthetic with naturally occurring antioxidants that can prevent free radical-related diseases. Most of the dietary antioxidants are derived from medicinal plants, have attracted the researchers’ attention due to the risks, associated with several available synthetic antioxidants.

Balanites aegyptiaca Del medicinal plant belongs to family Zygophyllaceae is commonly known as the desert date and distributed in Africa and South Asia. The bark of this plant has been used in the treatment of skin diseases and remedy for stomach ache and jaundice⁹, the root was also reported to be used in the treatment of inflammatory-related diseases¹⁰ and antidote for snakebite¹¹ The bark of the plant is useful in curing psychiatric disorders, epilepsy, yellow fever, jaundice, and syphilis. The boiled root of the plant can be used as a soup against stomach pain, anthrax, and the infusion of the root also acts as an antidote to snakebite^12,13. Phytochemical analysis of stem bark B. aegyptiaca raveled the presence of secondary metabolites such as saponin, flavonoids, tannins, and alkaloids. The traditional importance the selected medicinal plant B. aegyptiaca was known in the study area Kafta Humera woreda, Western zone of Tigray regional state, Ethiopia, but its phytochemical analysis and in vitro antioxidant activities of ethanolic bark B. aegyptiaca are not systematically carried out. Therefore the present study aimed to explore the phytochemical screening and evaluate the radical scavenging activity of ethanolic bark B. aegyptiaca.

MATERIALS AND METHODS:

Plant material collection and Authentication:

The healthy bark of B aegyptiaca was collected in November 2018 from Humera, kafta district, Tigray regional state, Ethiopia. The stem bark of B. aegyptiaca was dried at 25°C for 10 days in the absence of sunlight. Mortar and pestle were used to make powdered and stored in an airtight container for further use¹⁴.

Preparations of the ethanolic bark extract:

About 50grams of the powdered plant sample material was taken and subject to ethanolic solvent extraction by the Soxhlet extraction method. For qualitative determination, the extracts were placed in pre-weighed flasks before drying. The remaining plant parts residues were extracted with other solvents sequentially¹⁵.

Phytochemical Screening:

The ethanolic crude stem bark extract of B. aegyptiaca was preliminarily screened to identify the secondary metabolites such as alkaloids, flavonoids, tannins, glycosides, saponins, terpenoids using standard phytochemical methods¹⁶.

In vitro antioxidant activity:

Total antioxidant capacity (TAC):

The whole antioxidant capacity of the ethanolic crude stem bark extract of B. aegyptiaca was evaluated by the Phospho-molybdenum assay method¹⁷ which is based on the reduction of Mo VI to Mo V by tested plant extract and formation of green phosphate-Mo V complex in acidic condition. Briefly, an aliquot of 0.1ml of ethanolic crude stem bark extract of B. aegyptiaca (1 mg/ml) with different concentrations ranging from 50, 100,150, 200 and 250µg/ml and ascorbic acid equivalent to 500µg was combined with 1ml of reagent solution (0.6ml sulphuric acid, 28mM sodium phosphate and 4 mM ammonium molybdate). The reaction mixture was incubated at 95⁰C for 90 min then the absorbance was measured at 695nm using a UV visible spectrophotometer against blank (0.3 ml of methanol is used as blank in the place of extract), after cooling to room temperature. The antioxidant activity was expressed as the number of gram equivalents of ascorbic acid.

Absorbance of control – Absorbance of test

% Total = ----------------------------------------------- x 100

antioxidant Absorbance of control

activity

DPPH (1-diphenyl-2-picrylhydrazyl) radical scavenging assay:

This assay is based on the reduction of DPPH in methanol in the presence of hydrogen donating antioxidant constituents of the tested plant extracts, due to the formation of the non-radical form of DPPH^18,19. The change in color is monitored at 517nm. Briefly, an aliquot of 1ml, 0.3mM DPPH ethanolic solution was added to 2.5ml of various concentrations ethanolic crude stem bark extract of B. aegyptiaca and standard ascorbic acid ranging from the 50, 100, 150, 200, and 250µg/ml) allowed to incubate at room temperature in dark condition, after 30 minutes, the absorbance was measured at 517nm. Ethanol was used as a blank. DPPH solution (1ml, 0.3mM) plus ethanol (2.5ml) serves as a negative control. All tests were performed in triplicates (n = 3) and average values are calculated. Half maximal inhibitory concentration (IC₅₀) values were also calculated. Lower absorbance of the reaction mixtures indicates higher free radical scavenging activity.

Absorbance of control – Absorbance of test

% Inhition = ------------------------------------------ x 100

Absorbance of control

Ferric reducing Antioxidant power assay:

The Fe³⁺ reducing power of the tested plant extract was determined by the method of Oyaizu et al.,²⁰, with a slight modification. Different concentrations (50, 100, 150, 200 and 250µg/ml) of ethanolic crude stem bark extract of B. aegyptiaca (0.5 ml) and standard ascorbic acid were mixed with 0.5 ml phosphate buffer (pH 6.6) and 0.5ml, 0.1% potassium hex-cynoferrate, followed by incubation at 50⁰C in a water bath for 20min. After incubation, 0.5ml, 10% TCA was added to terminate the reaction. The upper portion of the solution (1ml) was mixed with 1ml of distilled water and 0.1ml, 0.01% FeCl₃ solution was added. The reaction mixture was left for 10min at room temperature and the absorbance was measured at 700nm against appropriate blank solution. All tests were performed in triplicates (n = 3). A higher absorbance of the reaction mixture indicated greater reducing power. Ascorbic acid was used as a positive control.

Absorbance of control – Absorbance of test

% of = ----------------------------------------------- x 100

reducing Absorbance of control

power

Where,

Abs control = Absorbance of potassium ferricyanide solution (potassium ferricyanide + sodium phosphate buffer)

Abs test = Absorbance of potassium ferricyanide solution + plant extracts or ascorbic acid solutions.

Statistical Analysis:

The experimental results were expressed as mean ± SEM of three replicates, where applicable, the data were subjected to one-way analysis of variance (ANOVA) and two-way analysis of variance (ANOVA). All this analysis was done by Graph Pad prism software program (Version 6.0) and MS –Office 2010 version, P values < 0.05 were regarded as significant.

RESULTS AND DISCUSSION:

Phytochemical analysis:

As showed in table -1, the phytochemical analysis of ethanolic crude stem bark extract of B. aegyptiaca showed the presence of important secondary metabolites such as alkaloids, flavonoids, tannins and phenolic compounds, glycosides, and saponins supporting for its wide range of biological activities. Our results were agreement with previous reports of Léon et.al.,²¹ they reported that hydroethanolic stem bark extract of B. aegyptiaca showed the presence of flavonoids and Phenolic group.

Table 1: Phytochemical analysis

S. No	Phytochemicals	Result
1	Alkaloid	++
2	Flavonoids	+++
3	Tannins & Phenolic group	++
4	Glycosides	+
5	Saponins	+

Total antioxidant capacities:

The total antioxidant capacity of the ethanolic stem bark extract of B. aegyptiaca was expressed as a number of equivalents of ascorbic acid. From the standard calibration curve, as shown in figure-1, the total antioxidant activity of the ethanolic stem bark extract of B. aegyptiac was determined and reported that 82.41µg/ 250µg (table-2).

Table 2: Total antioxidant capacities of ethanolic extracts of stem bark extract of B. aegyptiaca

Description	Concentration (µg/ml)	Absorbance	Ascorbic acid equivalent μg /gm of extract
B. aegyptiaca	50	0.176	7.38
	100	0.279	41.57
	150	0.546	70.14
	200	0.817	80.04
	250	0.927	82.41

Figure 1: Calibration curve of ascorbic acid

DPPH radical scavenging activity:

The results of the DPPH radical scavenging activity of the ethanolic stem bark extract of B. aegyptiaca and the standard ascorbic acid were presented in figure-2. It was found that the tested plant extract exhibited dose-dependent DPPH radical scavenging activity with increasing concentrations. The percentage inhibition of the DPPH radical by the ethanolic bark extract of B. aegyptiac and ascorbic acid at 250μg/ml was 67.39% and 71.92% with the IC50 values of 55.41μg/ml and 21.59μg/ml respectively. Léon et.al.,²¹ reported that the hydroethanolic stem bark extract of B. aegyptiaca showed 46.83±3.01μg AAE/g DPPH scavenging activity.

Figure 2: DPPH radical Scavenging activity of ethanolic stem bark extract of B. aegyptiaca by DPPH Assay. Each value is expressed as Mean ± Standard deviation (n=3).

Reducing power:

The reducing power of tested plant extract was analyzed based on Hydrogen Atom and Electron Transfer based reactions²². The reducing power of the tested plant extract ethanolic stem bark extract of B. aegyptiaca and standard ascorbic acid at various concentrations (50,100, 150, 200 and 250μg/ml) showed a significant increase in absorbance at 700nm and during reducing the activity of tested plant extracts and standard ascorbic acid the yellow color of the reaction mixture was changed blue color while reduction Ferric (III) to Ferrous (II). As shown in figure-3, the ethanolic bark extract of B. aegyptiaca at 50, 100, 150, 200 and 250μg/ml showed significant dose depended reducing activity was found to be 0.183, 0.212, 0.324, 0.352 and 0.412 respectively. The increasing reducing activity of tested plant extract was due to the presence of electron-donating bioactive antioxidants as showed in the table-1. Krishna Chaithanya et.al.,²³ and G. Hadera brhane, et. al.,²⁴ reported that the ethanolic leaf extract of A. schimperi and A. adigratana Reynolds showed promising reducing activities respectively due to the electron-donating capacity of secondary metabolites.

Figure 3: Reducing power activity of ethanolic stem bark extract of B. aegyptiaca Each value is expressed as Mean±Standard deviation (n=3).

In current scenario existence community using plant derived diet for the management of oxidative stress related diseases. A number of plants derived substances collectively termed as phytochemicals gained prominence for their antioxidant activity. Polyphenolic compounds such as flavonoids and tannins serve protection against a variety of free radicals induced damages of biomolecules²⁵. The vital functions of antioxidants are their interface with oxidative free radicals. The phytocompounds such as terpenoids and flavonoids are the principal antioxidants that exert a scavenging effect on free radicals and reactive oxygen species by chelating of metal ions, such as iron and copper and the inhibition of enzymes responsible for free radical generation²⁶. In the present study, the ethanolic stem bark extract of B. aegyptiaca can decolorizes reaction mixture of the tested DPPH free radicals, reduces ferric (III) to ferrous (II) and Mo VI to Mo V. These results of the present study emphasize that the ethanolic ethanolic stem bark extract of B. aegyptiaca contained an substantial amount of bioactive secondary metabolites such as polyphenols, flavonoid and terpenoid contributed the free radical scavenging activity, reducing capacity and antioxidant activity.

CONCLUSION:

The results obtained in the present study have shown that stem bark extract of B. aegyptiaca have significant amount of bioactive secondary metabolites such as alkaloids, flavonoids, , poly phenolic & tannins and cardio glycosides and shown potential antioxidant activity. The selected ethanolic bark extract of B. aegyptiaca exhibited significant scavenging activity against DPPH radicals and showed promising reducing power by reducing ferric to ferrous. This study suggests that the bark extract of B. aegyptiaca phytochemical constituent will be useful for treatment free radical induced oxidative stress related diseases.

ACKNOWLEDGMENT:

The authors thank to Aksum University, Axum, Ethiopia for financial support by approved the medium-scale research project (Ref: AKUR00042/2012 E.C.)

CONFLICT OF INTEREST:

The authors confirm that this article content has no conflict of interest.

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Received on 31.05.2023 Modified on 22.07.2023

Research J. Pharm. and Tech 2023; 16(9):4302-4306.

DOI: 10.52711/0974-360X.2023.00704