Antioxidant, Antibacterial Activities and Identification of Bioactive Compounds from Terminalia chebula Bark Extracts

Background: Free radical induces numerous diseases by damaging bio-molecules such as lipids, proteins, RNA and DNA. Several scientists reported that numerous plants and plant extracts have potent antioxidant activities to scavenge free radicals. The present study was aimed to screen phytoconstituents, in vitro antioxidant and antibacterial potential in various solvent extracts of Terminalia chebula bark. Methods: Phytochemical analysis, estimation of various metabolites, in vitro antioxidant and antibacterial activity were done by adopting standard protocols. Selected bioactive (acetone) extract of T. chebula was analyzed for their phytochemical profile by GC-MS analysis. Results: The results of preliminary phytochemical screening analysis revealed that presence of various phytochemicals like, alkaloids, flavonoids, glycosides, terpenoids, phenolics, saponins and carbohydrates in most of the tested extracts. Acetone extract possess significant high quantity of both primary and secondary metabolites when compared with other extracts. Remarkable free radicals scavenging potential was observed in acetone extract with lowest IC 50 values on all tested radicals namely, DPPH . (IC 50 =144.77 µg/ml), NO . (IC 50 =149.46 µg/ml), . OH (IC 50 =121.18 µg/ml), O 2 . - (IC 50 =159.41 µg/ml), Reducing power (IC 50 =35.85 µg/ml), Fe 2+ ion chelating (IC 50 =137.56 µg/ml) and TBARS (IC 50 =201.96 µg/ml). Acetone extract expressed significant high antibacterial activity against S . typhi (15 mm). The result of GC-MS analysis of acetone extract shows the presence of 32 major bioactive compounds, including various phenolic, sesquiterpene, flavonoid, triazine and gibberellin compounds. Conclusion: The present study suggested that T. chebula bark extract serves as a good source of phytochemicals


Plant material
T. chebula bark sample were collected from Vellimalai, Tumbal, Salem District, Tamil Nadu, India.The nomenclature of collecting plant sample was identified and authenticated by Botanical Survey of India (Reference number: BSI/SRC/5/23/2011-12/Tech./32), Coimbatore, Tamil Nadu, India.The collected plant material was washed with tap water, prior to distilled water, shade dried and powdered by using electrical grinder.

Preparation of extracts
Powdered plant material (2 kg) was extracted with various solvents (hexane, chloroform, ethyl acetate, acetone, methanol and water) in increasing polarity manner in a Soxhlet apparatus until the efflux solvent became colorless.All solvent extracts were passed through Whatman (No.1) filter paper and concentrated under vacuum at 40ºC to yield plant crude extracts which stored at 4ºC until use.

Phytochemical analysis Qualitative phytochemical analysis
Preliminary qualitative phytochemical analysis of all extracts were carried out according to method of Onwukeame et al.,. 8

Quantitative phytochemical analysis Determination of total phenol content
The total phenol content of T. chebula bark extracts were estimated by spectrophotometric assay as per the method of Barreira et al.,. 9 Gallic acid was used for constructing the standard curve (20-100 µg/ml, Y=0.0129x + 0.0697, R 2 =0.9985) and the results were expressed as µg of gallic acid equivalents/ mg of extract (GAEs).

Determination of total flavonoid content
Flavonoid content of extracts was determined by using the method of Kathirvel and Sujatha. 10Different concentrations (20-160 µg/ml) of

Antioxidant, Antibacterial Activities and Identification of Bioactive Compounds from Terminalia chebula Bark Extracts
Free Radicals and Antioxidants, Vol 7, Issue 1, Jan-Jun, 2017 (±)-catechin was used as a reference compound to plot the standard curve (Y=0.0042x+ 0.0164, R 2 =0.9991) and the results were expressed as µg of (±)-catechin equivalents (CEs) per mg of extract.

Estimation of total flavonol content
Total flavonol content was determined by adopting the protocol of Grubesic et al.,. 11 Rutin was used to calculate the standard curve (20-120 µg/ml, Y=0.0043x+ 0.03682, R 2 =0.9652) and the results were expressed as µg of rutin equivalents per mg of extract.

Estimation of tannin content
Tannin content of all extracts was measured by Folin-Denis method. 12annic acid was used to plot the standard curve (20-120 µg/ml, Y=0.0003x + 0.0091, R 2 =0.9985) and the results were expressed as µg of tannic acid equivalents per mg of extract.

Estimation of protein content
Total protein content was estimated by Lowry's method. 13Bovine serum albumin was used as reference to construct standard curve (20-160 μg/ml, Y=0.0025x+0.0074,R 2 =0.9846) and the results were expressed as μg of bovine serum albumin (BSA) equivalents per mg of extract.

Estimation of ascorbic acid content
Total ascorbic acid content was estimated by the method of Omaye. 14scorbic acid was server as reference to plot standard curve (20-120 μg/ml, Y=0.0043x+0.0368,R 2 =0.9652) and the results were expressed as μg of ascorbic acid equivalents per mg of extract.

Estimation of carbohydrate content
Total carbohydrate content was estimated by Anthrone method. 15Various concentrations of standard glucose was used to construct the standard curve (20-120 μg/ml, Y=0.0114x+0.0324,R 2 =0.9914) and the results were expressed as μg of glucose equivalents per mg of extract.

In vitro antioxidant studies
Different concentrations (20-100 μg/ml) of T. chebula bark extracts were tested for various types of radicals scavenging potential.Ascorbic acid, TBHQ and BHA served as standard reference compounds for all in vitro antioxidant assays.

) scavenging assay
The DPPH radical scavenging activity was carried out according to the method of Arunika et al., 16 with some modifications.

Hydroxyl radical ( . OH) scavenging assay
The 2-deoxyribose (Fenton reaction) assay 17 was used to determine the hydroxyl radical scavenging efficacy of all extracts.

) scavenging assay
The nitric oxide radical scavenging potential of various extracts of T. chebula was determined by the method of Sfahlan et al.,. 18peroxide anion radical (O 2

. -) scavenging assay
The superoxide radical scavenging activity of different extracts of T. chebula was tested as per the modified method of Liu et al.,. 19

Ferrous ion chelating activity
Ferrous ion chelating activity was estimated according to the method of Glucin. 20

Inhibition of lipid peroxidation using thiobarbituric acid reactive substances (TBARS)
Thiobarbituric acids reactive substances analysis was measured by using the method of Barreira et al.,. 9

Reducing power
The reducing power activities of T. chebula bark extracts were determined by the method of Oyaizu. 12

GC-MS analysis
The selected bioactive (acetone) extract was subjected to GC-MS analysis using CP3800 Saturn 2200 Gas Chromatography-Mass Spectrometer (GC-MS) system.The temperature was programmed to 80°-350°C at the rate of 3°C/min and held at 350°C at 55 min.The ion source temperature was 200°C with 20-500 amu scan range.The spectrums of unknown component were compared with Wiley and NIST libraries.

Antibacterial activity
Three grams-positive bacterial strains namely, Enterococcus faecalis, Bacillus subtilis, Staphylococcus aureus and three gram-negative bacterial strains viz., Proteus vulgaris, Salmonella typhi, Shigella sonnei were used for antibacterial study.All selected bacterial strains were obtained from clinical laboratories in and around Salem District, Tamil Nadu.The antibacterial ability of various solvents bark extracts of T. chebula were evaluated using agar well diffusion method, as per the modified protocol of Srinivasan et al.,. 21

Statistical analysis
Statistical analyses were conducted using the SPSS software (16.0 versions).Analysis of Variance (ANOVA) in a completely randomized design and Tukey's multiple range tests was used to compare any significant differences between samples.The values were expressed as means ± standard deviations.All determinations were done at least in triplicate and all were averaged.The confidence limits used in this study were based on 95% (p<0.05).

Qualitative phytochemical analysis
The results of phytochemical analysis of T. chebula bark extracts revealed that presences of various classes of phytoconstituents namely, alkaloids, flavonoids, terpenoids, carbohydrates, tannins and phenolics in most of the tested extracts (Table 1).Tannins, flavonoids, phenolics, alkaloids, steroids, saponins and terpenoids were found in ethyl acetate, acetone and methanol extracts.Methanol and water extracts showed the presence of amino acids, proteins and carbohydrates.Similar findings were reported in various extracts of different parts (leaves 22 and fruits 23 ) of T. chebula.Kumar, (2006)  24 reported that T. chebula contains a number of phytoconstituents like, tannins, flavonoids, sterols, amino acids, fructose, resin and fixed oils.Fruit ethanolic extract of T. chebula shows presence of carbohydrates, proteins, anthraquinone, glycosides, saponins, triterpenoids, tannins, polyphenols, amino acids and flavonoids 25,26 which strengthens our outcome.Variety of phytochemicals present in the bark extracts may be responsible for its medicinal properties.

Quantitative phytochemical analysis
Different classes of phytochemicals (proteins, carbohydrates, vitamin C, tannins, flavonoids and phenolic compounds) were found in various concentrations in all tested extracts of T. chebula (Table 2).However, highest quantity of both primary and secondary metabolites was noticed in acetone extract such as, phenol [   High amount of total phenols, flavonols, flavonoids, tannins, ascorbic acid, carbohydrate and protein has been reported in leaf acetone extract of T. chebula 22 which supports the present findings.Similar results were noticed in various extracts of different parts of T. chebula like, seed, leaves, stem and fruit. 27,28Previous study reported that successive solvent extraction systems may leads to the differences in extraction of phytoconstituents in plant extracts. 29Moreover, reports on medicinal plant extracts state that concentration of phytochemicals harbor a positive correlation with antioxidant activity of extracts. 3Present results clearly indicates the presences of high quantity phytochemicals namely, phenols, flavonols, flavonoids, tannins, ascorbic acid, carbohydrate and protein are play important role in antioxidant potential of these extracts.

In vitro antioxidant activity
All tested extracts of T. chebula show different degrees of antioxidant ability in a dose dependent manner in all tested methods (Figure 1 and Table 3).Significant high antioxidant potential was found in acetone extract on all tested radicals scavenging assays followed by methanol extract.Acetone extract shows excellent ferrous reducing potential with lowest IC 50 value (35.85 µg/ml) followed by hydroxyl radicals scavenging activity (121.18µg/ml).Ethyl acetate and water extracts expressed sustainable antiradical potential on all tested radicals with moderate IC 50 value.Minimal free radical scavenging ability was observed in chloroform and hexane extracts with high IC 50 value.
Our results suggested that various concentrations have different activities and maximum activity was observed at 1000 µg/ml concentration.DPPH is a stable free radicals and it gives more accurate and concurred results for determination of antioxidant potential. 30The radical scavenging activity of T. chebula plant extract possessed significant activity in acetone followed by other extracts, which were capable of reducing DPPH radical into hydrogen ion, thus the concentration of the color change in the reaction depends upon the number of electrons transferred during the reaction. 31Hence, it could be assumed that the higher antioxidant potential of acetone extract was due to the electron sharing by the phenolic compounds present in them.
Reactive oxygen species formed during their reaction or with superoxide such as NO 2 , N 2 O 4 , and NO 2 -are very reactive.The reactivates of NO • and O 2•-were found to be relatively lower, but their metabolite ONOO - (peroxynitrite) is enormously reactive and directly induces toxic reactions, as well as SH-group oxidation, lipid peroxidation, protein tyrosine nitration and DNA modifications. 32In our studies, it was found that the acetone higher scavenging activity (149.46 µg) followed by the other extract (Figure 1).Similar kind of observations was observed in previous studies. 22The current results were correlated with the earlier findings 33 has shown noticeable activity against nitric oxide radicals.Hydroxyl radical scavenging activity exhibited lowest IC 50 of acetone extract was 121.18 µg/ml.The possible scavenging ability of phenolic substances might be due to the active hydrogen's donor's ability of hydroxy replacement.This activity was further compared to other related medicinal plants, i.e.Ananascosmosus 34 , Allium sativum 35 are reported potential antioxidant activity.Hence forth, the test T. chebula possesses higher amount of antioxidant potential in acetone extract.
The superoxide scavenging activity of T. chebula bark was increased markedly with the increase of concentrations.The inhibition concentration (IC 50 ) of the bark acetone extract was 159.41 µg/ml.It's very low compared with other extract.It plays a vital role in the formation of hydroxyl radical or singlet oxygen in living organisms. 36The results of our present study were comparable with the results of other reports on superoxide radical scavenging activity on Clitoria ternatea, 37 Pothos scandens are similar findings.
Reducing capacity of the bark in various extracts of the T. chebula was measured by its ability to transform Fe 3+ to Fe 2+ at various concentrations (200, 400, 600, 800 and 1000 μg/ml).The results revealed that the reducing activity considerably increased as the concentration of the extract was increased with a maximum increase at 1000 μg/ml (Figure 2).The acetone extract showed significantly higher activities than the control exhibited greater reducing power, indicating that T. chebula bark acetone extract consists of hydrophilic polyphenolic compounds that cause the greater reducing power. 38In vitro antioxidant previously reported fruit methanolic extract of T. chebula as a reducing agent and effectiveness as scavengers of free radicals. 39he main approach to avoid ROS generation that is related with redox active metal catalysis involves chelating of the metal ions.T. chebula bark acetone extract the most active extract interfered with the formation of ferrous and ferrozine complex, suggesting that it has chelating activity and captures ferrous ion before ferrozine.IC 50 values of the acetone extract for chelating activity were 137.56 μg/ml is lower than the other extract.The IC 50 of chelating effect of other extracts on Fe 2+ and ferrozine complex formation is shown in Table 3.
The results of the investigations revealed that T. chebula bark had potent lipid peroxidation inhibition activity because lower IC 50 value 201.96 μg/ml.So, it conclude that the acetone extract of T. chebula bark exhibited significant in lipid peroxide inhibition activity on compared with the standard.The activity may be related to the presence of phenols and flavonoids in the plant extract.T. chebula possess anti-lipid peroxidation, anti-superoxide radical formation and free radical scavenging activity. 40Bark and fruit acetone extract shows potential anti carcinogenic activity 41 .

Antibacterial activity
All extracts of T. chebula show a broad spectrum of antibacterial activity (5-15 mm) against most of the tested pathogens (Table 4).The results of antibacterial activity revealed that acetone extract harbor significant Medicinal plants are important source for discover a new antimicrobial agents with significant activity against infective microbes. 42Several investigators have been reported the antimicrobial potential of various parts of T. chebula. 43Leaf acetone extract of T. chebula exhibited high antibacterial activity, especially against negative strain. 22Similar findings were noticed in the present study.Acetone was reported as effective solvent for extracting alkaloids, phenolic, flavonoids, and tannin compounds  antibacterial activity against all tested pathogen and maximum growth inhibition was observed against S. typhi (15 mm) followed by E. faecalis Free Radicals and Antioxidants, Vol 7, Issue 1, Jan-Jun, 2017 which exhibited antibacterial activity 21 which are quite comparable with our results.The present results were good agreement with previous findings on antibacterial activities of T. chebula fruit extracts (Figure 3). 23,44

GC-MS analysis
The result of acetone extract GC-MS analysis indicates the presence of 32 compounds (Figure 3).Previous study has been reported that 35 and 64 different compounds were identified in T. chebula ethanolic and ethyl acetate fraction of fruit extract respectively 45,46 which strengthen the present outcome.Ellagic acid and Gallic acid are polyphenolic antioxidant compounds that occur in many plants 47 and are found to have antioxidant, antimutagenic, antiinflammatory and cardioprotective activity. 48Ferulic acid also serves as antioxidant agent. 49α-santalol which has shown outstanding chemopreventive effects against skin cancer under both in vivo and in vitro conditions. 50The presence of high quantity of these components in acetone extract of T. chebula may be the responsible for its bioactivity.

CONCLUSION
The findings of present investigation clearly indicates that acetone extract of Terminalia chebula bark possess significant antioxidant and antibacterial capacity and a good source of various phytoconstituents which recommends further research needed for isolation of bioactive principles.

Figure 1 :
Figure 1: Radical scavenging activity of different solvent crude extracts of T. chebula bark.

Figure 2 :
Figure 2: Reducing power of different solvent crude extracts of T. chebula bark.

(
14 mm).The moderate antibacterial potential was noticed in methanol and ethyl acetate extracts against most of the tested microbes.Hexane, chloroform and water extracts show only minimal/nil activity against most of the tested pathogens.

Table 2 : Quantitative phytochemical analsysis of various solvent crude extracts of T. chebula bark
*-The values are mean of triplicates with (±) standard deviation (mean ± S.D, n=3).The superscript letters (a-f) present in rows represents the effectiveness of extracts (f>e>d>c>b>a) which significantly differs when subject to Tukey's multiple comparison test (at p<0.05).