A Study of Antioxidant and Anti-bacterial activities of Borassus flabellifer

The present study was designed to estimate the antimicrobial and antioxidant activities of ethanolic and aqueous extract of Borassus flabellifer fruit, juice, leaves and sap. The antioxidant evaluation was carried out by various radical scavenging assays and antimicrobial activity by the disk diffusion method. For antibacterial activity, bacterial species such as Escherichia coli and Staphylococcus aureus were tested for all extracts. The extract showed moderate antioxidant activity when compared to the standard vitamin C. In both DPPH and nitric oxide oxide assay when related IC 50 value, ethanolic fruit extract showed promising results of 132 and 119 μg/mL respectively with ascorbic acid as standard which showed 25 and 35 μg/mL respectively. In Ferric Thiocyanate (FTC) Method and Thiobarbituric Acid (TBA) Method the radical scavenging activity of ethanolic fruit extract showed satisfactory results of 160 and 148 μg/mL ascorbic acid being the standard which showed 94 and 97 μg/mL respectively. The total flavonoid contents were 32.7 /100g for ethanolic sap extracts and 53.9/100g in aqueous extract of sap. The fruit juice extract showed Original Research Article Nayak et al.; JPRI, 33(25B): 53-60, 2021; Article no.JPRI.67137 54 maximum inhibition zone with 31.4 and 32.1 mm against E. coli and S.aureus correlated other extracts .Ethanolic and aqueous sap extracts zone of inhibition was 25.2 and 26.8 against E. coli and zone of inhibition was 28.3 against S.aureus in both ethanolic and aqueous sap. Ethanolic and aqueous leaves extracts zone of inhibition was 28.5 and 24.1 against E. coli. Ethanolic and aqueous leaves extracts zone of inhibition was 30.4 and 25.8 against S.aureus. Herein, the results suggest that the Borassus flabellifer plant extracts have potential antioxidant and antimicrobial properties.


INTRODUCTION
The use of herbal plants for infection prevention and treatment is a common remedy. It ranges from ancient and general medicines of each nation to consistent and titrated herbal extracts. Even though traditional medicine has become a popular treatment of health care, the main drawback is the lack of final and complete data about the composition of extracts and their pharmacological activities. For this purpose plant-based obtained medications require a thorough evaluation of their pharmacological activities.
To establish their herbal pharmacological activities, conventional testing methods in many plant extracts have been effective for specific ailments [1].
Antioxidants or oxidation inhibitors are compounds that delay or prevent oxidation, generally prolonging cells' lives. Overproduction of free radicals causes oxidative stress which results in the form of diseases or disorders. Free radicals are agents with a very short half-life, high reactivity and are responsible for harmful activity towards macromolecules such as proteins, DNA and lipids [2]. In general, the reactive oxygen species have higher reactivity with some other molecules than that of groundstate oxygen, due to the additional electron they carry, Which affects various enzyme systems and cause harm that can further lead to conditions such as cancer, ischemia, ageing, adult syndromes of respiratory distress, rheumatoid arthritis, etc [3].A diet mainly composed of medicinal herbs protects against chronic illnesses linked to oxidative stress. Variable chemical families and quantities of antioxidants include dietary plants. Animal antioxidants suggest leading to the positive health effects of nutritional plants [4].
Borassus flabellifer belongs to the Arecaceae family, generally referred to as Palmyra palm, which is native to tropical Africa but is grown in India ( Fig. 1) [5].Customarily the various parts of the plant, for example, root, leaves, organic product, and seeds are utilized for different human health issues. Blossoms of B. flabellifer were examined for antipyretic impacts and pain relieving, mitigating movement, hematological, biochemical boundaries, and immunosuppressant property [6]. The various portions of the herb are used for healing properties like antihelminthic and diuretic [7].
B. flabellifer fruit pulps are typical in traditional, and sap in diabetic patients as a sweetener. Phytochemical investigations of the plant uncovered spirostane-type steroid saponins; steroidal glycoside also contains an unpleasant compound called flabelliferrins. However, more studies are necessary for the comprehension and use of these compounds [8].
Iron(III) chloride(FeCl 3 ), Ethylene diamine tetraacetic acid (EDTA), Tetracholoro acetic acid, Thiobarbituric acid, Potassium ferricyanide, Ammonium molybdate, Folin-Ciocalteu, Sodium carbonate, Nitro blue tetrazolium (NBT) and Dimethyl sulfoxide (DMSO) were procured from Lab India, Thane Mumbai. incorporated. The ascorbic acid was used as a standard and prepared as mentioned above without the extracts. For 30 minutes, the samples were incubated at room temperature, and later absorbance of all the samples, including blank, were measured at 517 nm using shimadzu uvspectrophotometer 1300 series .Determined the percentage of inhibition activity from the following equation:

Collection of Plant Materials and Preparation of Extracts
Where A o is the control absorbance, and A 1 is the extract/ standard absorbance [9,10]. Here ascorbic acid was used as a standard. The experiments were performed in triplicate.

Nitric oxide radical inhibition assay (NO°)
The use of the Griess Illosvoy reaction is possible to estimate NO° inhibition. The assay was carried out by a modified GriessIl losvoy reagent using 0.1 % N-(1-Naphthyl) ethylene diamine dihydrochloride and ascorbic acid as standard [11,12].
The samples were measured at 540 nmusing shimadzu uv-spectrophotometer 1300 series wavelength were compared to the blank using the following formula:

Scavenging activity of Nitric oxide radical = (A o blank -A 1 sample) × 100 / A o blank
Where Ao is the blank absorbance, and A 1 is the sample absorbance Ascorbic acid was used as standard.

Ferric thiocyanate (FTC) method
The assay was developed by Kikuzaki  To a test tube, 0.1 mL of the reaction mixture was transferred from the above solution to a test tube, later added 9.7 mL of 75% hydroethanolic solution, followed by 0.1 mL of 30 % aqueous mL ammonium thiocyanate and 0.1 mL of 0.02 M ferrous chloride in 3.5 % hydrochloric acid. Once the red color is found in the samples indicating ferric thiocyanate presence, the samples were measured spectrometrically to get the absorbance at 500 nm using shimadzu uv-spectrophotometer 1300 series [13].
The ascorbic acid value was used as a positive control, while the reactant without the herbal drug was used as a negative control. The FTC assay was done using the following formula. % Inhibition= 100-(A 1 /A 0 ) ×100) A 0 is the absorbance of the control and A 1 is the absorbance of the sample extracts/standard.

Thiobarbituric Acid (TBA) Method
This experiment, added 2 mL of 20% of TCA and 2 mL of 0.67% of TBA was added to 1 ml of the FTC method for sample plant extract solution. As a standard, we used ascorbic acid. The resultant was kept in the water bath for 10 minutes then cooled and certrifuged at 3000 rpm. The supernatant's absorbance was measured at 552 ηm and using shimadzu uv-spectrophotometer 1300 series [14].

Estimation of total phenolic content
The Folin-Ciocalteu method is used to evaluate total phenolic content. In an ultrasonic water bath, extracted 200 mg of finely ground plant material for 20 minutes with 10 mL of 50 %methanol. Under cold condition, the content was centrifuged for 10 minutes and the supernatant was collected. The remaining residue again extracted for 20 minutes with 5 mLof 50% methanol and finally both supernatants were resting for 20 minutes at room temperature [15].

(A) Analysis of total phenolics (TP)
Tannic acid standard (0.02-0.10mg/mL) and extract / fraction (0.005 mL) were prepared with triple distilled water (TDW) up to 0.5 mL volume. It was vigorously mixed and kept at room temperature for 40 minutes. Measured the absorption of all tested samples at 725 nm using shimadzu uv-spectrophotometer 1300 series and the total amount of tannic acid equivalent total phenols were determined from the standard tannic acid curve.

Analysis of simple phenolics (SP) -
Weigh100 mg polyvinyl pyrrolidone into a test tube. Add to it 1.0 ml distilled water and then 1.0 ml of herbal extract. It was vigorously mixed and kept at room temperature for 40 min. The absorption of all tested samples were measured at 725 nm using shimadzu uvspectrophotometer 1300 series and the total amount of tannic acid equivalent simple phenols were determined from the standard tannic acid curve.
Total tannins were calculated as follows: Total tannin content = Total phenols -Simple phenols

Estimation of total flavonoids
The aluminum chloride colorimetric assay estimated total flavonoids. For the experiment 250 μL of each sample with 1.25mL of deionized water and 0.075mL of 5% sodium nitrite were incubated for 6 minutes. Posteriorly, we added, 0.15mL of 10% aluminum chloride waiting for 6 minutes, and then included 0.5 mL of 1M sodium hydroxide and 2.5mL of deionized water in the solution. After 6 minutes, read the samples at 510 nm and the total flavonoids were estimated using shimadzu uv-spectrophotometer 1300 series [16].

Total flavonoids = (A×M o . /A 0 × M)
A is the absorbance of the plant extract solution, A o is the absorbance of the standard ascorbic acid solution, M is the weight of plant extract, and M o is the weight of ascorbic acid solution.

Antibacterial Activity
Several extracts of plants were performed on antibacterial activity, mainly to Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923.The method of disk diffusion was able to assess the antibacterial activity of B. flabellifer. We prepared the culture plates by pouring 20mL (nutrient media containing Müller Hinton Agar, MHA) molten media into sterile Petri plates, with ciprofloxacin (5 mg) standard or control. The experiment included extracts of B. flabellifer in the holes of inoculated plates at 37 o C for 24 hours, posteriorly inhibition zones around the area of holes were measured in millimeters [17]. In both DPPH and nitric oxide oxide assay when IC 50 value were compared, ethanolic fruit extract showed results of 132 and 119 µg/ml respectively as compared to standard ascorbic acid which showed 25 and 35 µg/ml as shown in Fig 2 . As compared to the other tested extracts, the fruit extracts of Borassus flabellifer results revealed good antioxidant properties.

RESULTS AND DISCUSSION
The IC 50 results of FTC and TBA assay for all the extracts along with ascorbic acid are represented in Fig.3. Radical scavenging activity of Ethanol fruit extract showed good results of 160 and 148 µg/mL as compared to standard ascorbic acid 94 and 97 µg/mL. Fair results were observed which can be interpreted as good antioxidant agent.
The antioxidant activity of herbal drugs was reported that phenolic compounds play an important role in stabilizing lipid peroxidation. Table 2 shows the estimated values of the assay. The total phenolic contents of ethanol extracts of leaves are highest (80.23 ± 3.19) and total tannins were also highest (59.77 ± 1.45) as compared to fruit and sap extracts. The aqueous leaf extract showed good results (16.27 ± 4.91) and total tannins in sap extract of water were found to be good with 11.69 ± 5.3.
TFC activity is the process which tells the amount of flavanoid content in the samples. Phenolic compounds that contained in the plants have redox properties, and the properties allow them acting as antioxidants.
As quantified and analysed in Table 3 the ethanol and aqueous extracts of plant, the total flavonoid contents were found to be 42.8µg/100g of aqueous fruit extracts and 53.9 µg/100g of aqueous sap extracts and 21.5µg/100g of ethanol fruit extracts and 32.7 µg/100g of ethanol sap extracts respectively.   The extracts prepared were investigated to evaluate their antibacterial property against two strains of organisms, E. coli (Gram negative) S. aureus (Gram positive) by cup plate method. Evaluation of antibacterial activity of these plant extracts was recorded in Table 4 and illustrated in Fig.4 and Fig.5.
The results showed the suppression of the growth of bacteria by the plant extracts. The growth inhibition property was found to be 31.4 mm in ethanolic fruit extract which was compared with ciprofloxacin as standard which showed the zone of inhibition of 32.4 mm against case of s-aureus, ciprofloxacin showed 34.8 mm of zone of inhibition and zone of inhibition for ethanol fruit extract was found to be 32.1 mm.
Thus it was observed, that both ethanolic aqueous fruit extracts more potent results as compared to all other plant extracts

CONCLUSION
In the present study, Borassus flabellifer juice, sap and leaves were tested for antioxidant

Zone of inhibition of ethanolic fruit Fig. 5. Zone of inhibition of and aqueous fruit extracts
These properties of plant are because of the phytochemical compounds are responsible for Borassus flabellifer.
prepared were investigated to evaluate their antibacterial property against two (Gram negative) and by cup plate method. Evaluation of antibacterial activity of these plant 4

and illustrated
The results showed the suppression of the growth of bacteria by the plant extracts. The growth inhibition property was found to be 31.4 mm in ethanolic fruit extract which was compared ard which showed the zone of inhibition of 32.4 mm against e-coli. In ciprofloxacin showed 34.8 mm and zone of inhibition for ethanol fruit extract was found to be 32.1 mm. Thus it was observed, that both ethanolic and aqueous fruit extracts more potent results as compared to all other plant extracts analyzed.
Borassus flabellifer fruit, juice, sap and leaves were tested for antioxidant and antibacterial activities. Extracts we prepared by Soxhlet extractor. Ethanol and water extracts of Borassus flabellifer fruit have been shown to promote antioxidant function in conventional pharmaceutical products for the treatment of inflammatory conditions. This study also verified that extracts of Borassus flabellifer fruit are possible source of antioxidants and could be used to prevent free radical illnesses.
However, the isolation and characterization of the active responsible for the above activities require further work. Antimicrobial studies of both ethanolic and aqueous fruit extract also showe a potent zone of inhibition when compared to the standard. The fruit extract results promising antimicrobial and antioxidant properties.

CONSENT
Not applicable.

Zone of inhibition (mm) for S.aureus inhibition of and aqueous fruit
and antibacterial activities. Extracts were extractor. Ethanol and water fruit have been shown to promote antioxidant function in conventional pharmaceutical products for the treatment of inflammatory conditions. This study Borassus flabellifer are possible source of antioxidants and e used to prevent free radical-related illnesses.
However, the isolation and characterization of the active constituents responsible for the above activities require further work. Antimicrobial studies of both ethanolic and aqueous fruit extract also showed a potent zone of inhibition when compared to the dard. The fruit extract results revealed promising antimicrobial and antioxidant