Sequential Fractionation by Organic Solvents Enhances the Antioxidant and Antibacterial Activity of Ethanolic Extracts of Fruits and Leaves of Terminalia bellerica from North Western Himalayas , India

Background: Terminalia bellerica belonging to family combretaceae is one of the major components of “Triphala”, an Ayurvedic formulation. Objective: To evaluate antioxidant potential in the ethanolic extract and its active fraction (chloroform, ethyl acetate, nbutanol and aqueous fraction) from fruits and leaves of Terminalia bellerica. Materials and Methods: Folin-Ciocalteau method and aluminium chloride method was used to quantify total phenolic and flavonoid content respectively in ethanolic extract and their fractions from fruits and leaves of T. bellerica. The antioxidant activity was evaluated using total antioxidant activity, DPPH, FRAP and total antioxidant activity methods. Results: Total phenolics (254.72±3.03 mg/g GAE and 227.52±1.38 mg/g GAE) and flavonoids (64.77±1.24 mg/gm RE and 75.57±1.38 mg/gm RE) content was higher in ethyl acetate fraction of both fruits and leaves sample of T. bellerica, respectively. The order of free radical scavenging activities was ethyl acetate fraction > crude ethanolic extract > n-butanol fraction > chloroform fraction > aqueous fraction. Similarly, ethyl acetate fraction of both fruits and leaves exhibited more antimicrobial activity as compared to that of ethanolic extract as revealed from agar well diffusion method with diameter of zone of inhibi¬tion of 14.0±1.41 mm, 21.0±1.41 mm, 14.0±1.41 mm, 14.5±0.71 mm in fruits and 18.0±1.41 mm, 22.5±2.12 mm, 15.5±2.12 mm, 14.5±3.53 mm in leaves against B. subtilis, S. aureus, E. coli, K. pneumoniae, respectively. MIC values for fruits were 3.125 mg/ml, 0.375 mg/ml, 3.125 mg/ml, 3.125 mg/ml and for leaves were 1.5625 mg/ml, 0.19 mg/ml, 0.78 mg/ ml, 0.78 mg/ml against B. subtilis, S. aureus, E. coli, K. pneumoniae, respectively. Conclusion: The present study provides the evidence for comparative antioxidant and antibacterial potential of ethanolic extracts of fruits and leaves of T. bellerica. . Moreover, leaves can be promoted to be used for therapeutics and natural antioxidants.


INTRODUCTION
The Indian Himalayan Region (IHR) comprises of a wide spectrum of biodiversity in diverse groups of flora, fauna and microorganisms.2] This biodiversity is being utilized by the inhabitants of the IHR for various purposes such as medicine, food, fuel, fodder, timber, making agricultural tools, fiber, religious and various other purposes. 2Plants with medicinal properties enjoyed the highest reputation in the indigenous systems of medicine all over the world and still constitute one of the major sources of drugs in modern as well as tradi-tional systems of medicine in spite of tremendous development in the field of synthetic drugs and antibiotics. 3In the remote areas of IHR where primary health centers are not available, inhabitants mostly rely on plants for the treatments of various ailments/ diseases, that is, cancer, kidney disorders, asthma, skin diseases, piles, etc. 4 Phenolic compounds are secondary plant metabolites, present in plants act as powerful antioxidants which can protect the cellular mechanism from free radicals by acting as hydrogen donors and radical scavenger. 5Secondary metabolites are play important role for the normal growth, development and defense mechanisms of plants. 68] Therefore, it is necessary to find out medicinal plants to validate their use and to reveal the active principle by isolation and characterization of their active constituents.Bioactive compound of natural products play major role in drug discovery.Nowadays, natural antioxidant has been promoted over synthetic antioxidant due to safety concerns.Natural antioxidant such as flavonoids, polyphenols and phenolics possess antioxidant properties due to their chelating and reducing capability.Antioxidants play important role to protect the body from damage caused by free radicals.Therefore, there is an increasing interest among researchers to identifying natural source of antioxidant.Conventionally practiced, natural antioxidants are already exploited commercially, but still there is demand to find more medicinal plants concerning the antioxidant potential.T. bellerica is one of the major components of "Triphala", an Ayurvedic formulation which play important role to encourage health, immunity and long life. 9This antioxidant rich preparation is used in Ayurvedic medicine for the treatment of numerous diseases like anaemia, asthma, constipation, fever, jaundice and chronic ulcers. 10The fruits of T. bellerica have been phytochemically reported to contain beta-sitosterol, ellagic acid, galloyl glucose, mannitol, glucose, gallic acid, ethyl gallate, chebulagic acid, galactose, fructose, Rhamnose, 11 Arjungenin, belleric acid, bellericoside, [12][13] cannogenol and three lignans and one flavan. 14Therefore, the current study was focused on the comparative antioxidant and antibacterial activity of ethanolic extracts and active fractions of fruits and leaves of Terminalia bellerica and to promote the utilization of leaves in therapeutics.

Processing of fruit and leaves of T. bellerica
The fruit and leaves of T. bellerica were collected from the Kangra region of Himachal Pradesh (30°22′40″-33°12′40″ N to 75°45′55″-79°04′20″ E), India during the month of October, 2017.The collected fruits and leaves were thoroughly washed with running tap and then dried in hot air oven at 35-40ºC and ground to fine powder.

Cold maceration for extract preparation
The dried powder of fruits and leaves (10 g) of T. bellerica were defatted and then ethanolic extract was prepared using a cold maceration method on a rotary shaker at 120 rpm for 5 days to ensure complete extraction. 15

Screening of phytocompounds in fruits and leaves extracts and its solvent fractions
Quantitative analysis of total phenolic content (TPC) and total flavonoid content (TFC) Total phenolic content (TPC) and total flavonoid content (TFC) of the ethanolic extract and fractions of fruit and leaves of T. bellerica were quantified by using Folin-Ciocalteu method 19 and aluminum chloride method. 20Total phenolic content was calculated from calibration curve of gallic acid (25-100 μg) and expressed in terms of gallic acid equivalents (GAE) per gram of dry extract, whereas, TFC was quantified from the standard curve of rutin (25-100 µg/ml) and expressed as rutin equivalents (RE) per gram of dry extract.
In-vitro antioxidant activity of different solvent fractions of fruits and leaves of T. bellerica Ethanolic extract and its active fractions such as chloroform, ethyl acetate, n-butanol and aqueous fraction were dissolved at a concentration of 1 mg/ml in ethanol.Then different dilutions were prepared (2.5-10 µg/ml) using ethanol.Ascorbic acid was used as a standard antioxidant compound.

DPPH radical scavenging activity
The ethanolic crude extract and active fraction were determined DPPH radical scavenging activity described by Barros et al. 21The percentage of inhibitory activity was calculated using the following equation-% Inhibition= (A c -A s /A c ) × 100 where A c is the absorbance of the control.A s is the absorbance of the extract/standard.

Ferric reducing antioxidant power (FRAP) assay
The reducing power of crude extract and its active fraction was measured using the method described by Benzie and Strain. 22The antioxidant capacity based on the ability to reduce ferric ions of extracts and its fractions were calculated from the linear calibration curve of FeSO 4 (2.5-20 µM) and expressed as µM Fe (II) equivalents per gram of extract.

Total antioxidant activity assay
The total antioxidant activity of the extracts was measured by the phosphomolybdenum method described by Prieto et al. 23 Ascorbic acid was used as reference standard.The antioxidant activity is expressed as the number of equivalents of ascorbic acid (AAE).

Antibacterial activity of ethanolic extracts of fruits and leaves and its solvent fractions
Antibacterial activity was analysed against both Gram's positive (Staphylococcus aureus and Bacillus subtilis) and Gram's negative (Escherichia coli and Klebsiella pneumoniae), whereas, antifungal potential of extract was done against Saccharomyces cerevisiae (H1086), Candida albicans (ATCC90028) and Candida albicans (MTCC277).All the bacterial and fungal strains were revived from Yeast Biology Lab, Shoolini University, Solan, Himachal Pradesh, India.Antibacterial activity of ethanolic extract of fruits and leaves and its solvent fractions was done using agar well diffusion method. 24In this method, nutrient agar (NA) plates were prepared and uniformly spreaded with the bacterial culture of 0.5 McFarland Standard using sterile cotton swabs.The wells were punched with the cork borer (6 mm) in the agar and 50 μl of ethanolic extract and their fractions extract of T. bellerica (25 mg/ml) were loaded in the wells.After the incubation of 18 h at 37ºC, the zone of inhibition was measured using HiAntibiotic Zone scale-C (Himedia Biosciences, Mumbai (India).Amoxyclav (10 µg) was used as a positive control and DMSO (solvent) was used as negative control in the antibacterial assay.The tests were performed in triplicate and results were recorded as mean± S.D. The minimum inhibitory concentration (MIC) of the extract was evaluated by broth dilution method described under CLSI. 25guidelines using 2, 3, 5-tripheny tetrazolium chloride.The methanolic extract were dissolved in DMSO and geometric dilutions ranging from 12.5-0.025mg/ml of extract were prepared in a 96-welled micro titer plate, including one growth control (nutrient broth containing DMSO) and a positive control (NB broth inoculated with bacterial culture and containing Amoxyclav).Plates were incubated under normal atmospheric conditions at 37°C for 24 h for bacteria.After incubation, dye was added to each well and incubated for 2h.The color change was then observed visually.The growth was indicated by changes in color from purple to pink or colorless.The Pharmacognosy Journal, Vol 11, Issue 1, Jan-Feb, 2019 lowest concentration at which color change appeared was taken as the MIC value.

Statistical analysis
Total phenolic content, flavonoid content and half of minimum inhibitory concentration (IC 50 ) was determined by linear regression analysis method.Each sample was analyzed individually in triplicates and the results are expressed as the mean value (n = 3) ± standard deviation.The values of zones of inhibition were expressed as mean± S.D. (n=2).

Sequential fractionation of selected plant extracts
The yield of extracts obtained from 2 g of ethanolic extract of fruits and leaves ranged from 3.3 % to 12.8 %.Among fruit extracts of T. bellerica, maximum extractive yield was observed in n-butanol fraction (19.5 %) and in case of leaves extracts, maximum extractive yield was obtained in aqueous fraction (12.8 %) (Table 1).

Qualitative analysis of phytoconstituents of fruits and leaves extracts
Phytochemical analysis revealed that flavonoids and phenolics were present in crude extract and its active fractions, except aqueous fractions.Similarly, alkaloids and steroids were absent in the chloroform and aqueous fraction in both ethanolic extracts.However, proteins were present in crude extract and active fraction (ethyl acetate and n-butanol fraction), but amino acid were absent in both ethanolic extract and their fractions (Table 2).

Quantitative analysis of phytoconstituents of fruits and leaves extracts
Total phenolic content was higher in ethyl acetate fraction (254.72±3.03mg/g GAE and 227.52±1.38 mg/g GAE), followed by ethanolic crude extracts (227.89±2.038mg/gm GAE and 206.07±1.89mg/gm GAE) of both fruits and leaves sample of T. bellerica, respectively.In case of flavonoids, higher amount was found in ethyl acetate fraction of fruits (64.77±1.24mg/gm RE) and leaves (75.57±1.38 mg/gm RE) as compared to other fractions (Figure 1).

In vitro antioxidant activity of ethanolic extract of fruits and leaves of T. bellerica and its fractions
The antioxidant potential was determined by various methods such as total antioxidant activity, DPPH radical scavenging assay and FRAP assay.

Total antioxidant activity
Total antioxidant activity of ethanolic extract and its derived fraction was calculated from standard curve of ascorbic acid (y = 0.0111x -0.0092; R 2 =0.9995) (Figure 4).Order of total antioxidant activity for fruit extract       3).

Antimicrobial activity of T. bellerica and its active fractions
The ethanolic extracts and its derived fractions exhibited the good antibacterial activity against tested bacteria using disc diffusion method.The antimicrobial activity followed the pattern of variation of total phenolic and flavonoid content.The results from the agar well diffusion method, followed by measurement of minimum inhibitory concentration (MIC), indicated that ethyl acetate fraction of both fruits and leaves exhibited more antimicrobial activity as compared to that of ethanolic extract with diameter of zone of inhibition in mm were 14±1.41,21±1.41,14±1.41,14.5±0.71 in fruits, whereas zone of inhibition diameters in mm were 18±1.41,22.5±2.12,15.5±2.12,14.5±3.53 in leaves and MIC values for fruits were 3.125 mg/ml, 0.375 mg/ml, 3.125 mg/ml, 3.125 mg/ml and for leaves were 1.5625, 0.19 mg/ml, 0.78 mg/ml, 0.78 mg/ml against Pharmacognosy Journal, Vol 11, Issue 1, Jan-Feb, 2019 B. subtilis, S. aureus, E. coli, K. pneumoniae, respectively.The leaves of T. bellerica showed more antibacterial potential in comparison to that of fruits extracts, clearly showing the importance of leaves in therapeutics (Table 4).

DISCUSSION
Medicinal plants have been used from ancient times for treatments for several diseases.The market use of medicinal plants is very high due to presence of many bioactive components, which cannot be synthesized artificially. 26Therefore recognition of antioxidant potential along with phytochemical variations for sustainable utilization of T. bellerica is the prime motive of the present study.Secondary metabolites such as phenolics and flavonoids are powerful antioxidant and have been used in many therapeutics such as diabetes, cardiovascular disorders, etc. 27 Phenolic and flavonoids are the major secondary metabolites in the medicinal plants which are responsible for antioxidant activity. 28In present analysis, ethanolic crude extract and active fractions of fruits and leaves of T. bellerica revealed that flavonoids and phenolics were present in crude extract and its active fractions except aqueous fractions.However, proteins were present in crude extract and active fraction (ethyl acetate and n-butanol fraction), but amino acid were absent in both ethanolic extract and their fractions.][34][35] Total phenolic (254.72±3.03mg/gm GAE and 227.52±1.38 mg/gm GAE) and flavonoids (64.77±1.24mg/gm RE and 75.57±1.38mg/gm RE) content was higher in ethyl acetate fraction of both fruits and leaves sample of T. bellerica, respectively.The present study showed that fruits extracts possessed maximum amount of TPC as compared to that of leaves.The results of study from Arya et al.33 also showed that methanolic fruits (278.50 ± 19.28 mg/gm GAE) extracts showed higher amount of phenolic compound as compared leaves extract (259.28 ± 6.42 mg/gm GAE).In the present study, higher amount of flavonoid content was found to be in the extracts of leaves, which was in accordance with the concept that higher phenolic content was not always accompanied with higher amount of flavonoids. 36The order of free radical scavenging activities was ethyl acetate fraction>crude ethanolic extract>n-butanol fraction>chloroform fraction > aqueous fraction with DPPH and FRAP assay for both fruits and leaves.However, the order was ethyl acetate fraction> n-butanol fraction>crude ethanolic extract>chloroform fraction > aqueous fraction in case of fruits and the order was ethyl acetate fraction>crude ethanolic extract>n-butanol fraction>chloroform fraction>aqueous fraction in leaves of T. bellerica with total antioxidant assay.][40] Evaluation of free radical scavenging activity and antioxidant potential of acetone extract of T. bellerica fruit was done by using in-vitro assays. 41imilarly, ethyl acetate fraction was found to be more effective as compared to crude acetone extracts in all antioxidant assays, i.e., DPPH, β-carotene bleaching inhibition and reducing power whereas for chelating ability on Fe +2 ion, crude acetone extract showed higher activity.2] Rastogi, investigated fruits, leaves and bark of T. bellerica for their antioxidant potential.It was observed that the leaves of T. bellerica, which are rich in phenolics and flavonoids, exhibit the highest antioxidant potential as evidenced by the better DPPH radical scavenging ability as well as the total antioxidant capacity. 43imilar to present study, ethanolic extract of Ficus pseudopalma into chloroform, ethyl acetate and aqueous fraction and found that ethyl acetate fraction rich in phenolic and flavonoids also showed more antibacterial activity. 44Similarly, studied the effect of T. bellerica and its fraction against S. typhi and S. typhimurium and it was found that alcoholic and aqueous extracts showed significant anti-microbial activity. 45Also, showed good antibacterial activity and antioxidant activity of crude methanolic extract of the fruits of T. bellerica Roxb along with its various organic fractions with moderate toxicity. 46Chanda et al. 47 also investigated the antibacterial activity of T. bellerica stem and leaf acetone extracts and its fractions and found that both the extracts showed better activity towards Gram negative bacteria than Gram positive bacteria.49]38 This difference in activity is because of the difference in the cell wall structure of Gram positive and negative bacteria.Baliah and Astalakshmi 50 studied the effect of solvents on the antibacterial activity of crude extract of T. chebula Retz.Using disc diffusion method against gram-negative bacteria such as E. coli, P. aeruginosa and K. pneumoniae and gram-positive bacteria such as B. subtilis, S. aureus.Of the eleven solvent used methanol, ethanol and acetone extracts showed more antibacterial activity than all other solvent extracts. 50Similar to present study, evaluation of antimicrobial activity of ethyl acetate, ethanol and aqueous extracts of the leaves from T. bellerica against selected Gram positive and Gram negative bacteria (E.coli, P. aeroginosa, S. aureus, B. subtilis).They found that T. bellerica extract shows significant inhibitory effect against Gram positive and Gram negative bacteria at high concentration.The study shows that ethanol and ethyl acetate leaf extracts of T. bellerica can be used as a potential source of antimicrobial agents. 49nhancement of antibacterial activity in fruits and leaves extract may be attributed due to enrichment of phenolic and flavonoid content in ethyl acetate fraction as compared to all other fractions.similar to present study, study on ethanolic extract and its fraction of T. arjuna showed that the fraction enriched with phenolic and flavonoid content showed more antimicrobial and antioxidant activity as compared to crude ethanolic extract. 51The results suggested that antibacterial and antioxidant activity of plant extracts also depend on the type of solvent also.

Figure 1 :
Figure 1: Quantification of total phenolics and flavonoids content in crude ethanolic extract of fruits and leaves and their fractions.Total Phenolic content was expressed as mg/gm gallic acid equivalents (GAE), whereas flavonoid content was expressed in terms of mg/gm rutin (RE).

Figure 2 :
Figure 2: DPPH radical scavenging activity of ethanolic extract and its different fractions.% DPPH activity was determined for fruits extract and leaves extracts and its fractions.The values represent mean ± S.D. of three independent experiments.

Figure 3 :
Figure 3: FRAP assay of ethanolic extract and its different fractions.FRAP assay was performed for fruits and leaves extracts of T. bellerica along with standard, ascorbic acid.The values represent mean ± S.D. of three independent experiments.Figure 4: Total antioxidant assay of ethanolic extract and its different fractions.Total antioxidant assay was performed for fruits and leaves extracts of T. bellerica and expressed in terms of Ascorbic acid equivalents (AAE).The values represent mean ± S.D. of three independent experiments.

Figure 4 :
Figure 3: FRAP assay of ethanolic extract and its different fractions.FRAP assay was performed for fruits and leaves extracts of T. bellerica along with standard, ascorbic acid.The values represent mean ± S.D. of three independent experiments.Figure 4: Total antioxidant assay of ethanolic extract and its different fractions.Total antioxidant assay was performed for fruits and leaves extracts of T. bellerica and expressed in terms of Ascorbic acid equivalents (AAE).The values represent mean ± S.D. of three independent experiments.