Available online at www.sciencedirect.com

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Journal of Taibah University for Science 10 (2016) 805–812

Antioxidant activity of polyphenolic extract of

Retzius fruits

∗

Sarmistha Saha , Ramtej J. Verma

Department of Zoology, University School of Sciences, Gujarat University, Ahmedabad 380009, India

Available online 25 November 2014

Abstract

The objective of this study was to investigate the in vitro antioxidant activities of polyphenolic extract of Terminalia chebula

Retzius (Combretaceae) fruits. The polyphenolic extract of T. chebula fruits was evaluated for antioxidant activity by determining

the reducing power, total antioxidant capacity, DPPH radical concentration (IC50 14 ␮g/mL), nitric oxide radical concentration

(IC50 30.51 ␮g/mL) and hydrogen peroxide scavenging activity (IC50 265.53 ␮g/mL) under in vitro conditions. Moreover, the

characterisation of the extract was also measured by determining the total phenolic, flavonoid, and ascorbic

acid contents. Characterisation of the extract was also performed by HPLC profiling with the standard . The present study

demonstrated that the extract has significant reducing capacity and nitric oxide scavenging activity. It also scavenges hydrogen

peroxide-induced radicals. The activity of the extract may be due to the total polyphenolic content. The antioxidant activity of the

extract is significantly higher than the standard ascorbic acid, and its activity is concentration-dependent. It is concluded that a

polyphenolic-rich fraction of T. chebula fruits is a potential source of natural antioxidants.

© 2014 The Authors. Production and hosting by Elsevier B.V. on behalf of Taibah University. This is an open access article under

the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Keywords: Terminalia chebula; Ascorbic acid; Total antioxidative capacity; DPPH radical scavenging activity; Nitric oxide radical scavenging

assay; Hydrogen peroxide scavenging assay

1. Introduction ROS generation or decreased antioxidant availability can

result in a net increase in intracellular ROS. Normally,

It is currently hypothesised that many diseases are intracellular molecules including mitochondrial antiox-

due to oxidative stress that results from an imbalance idants prevent cellular damage produced by endogenous

between the formation and detoxification of pro- ROS. In living systems, free radicals and ROS are con-

oxidants. Oxidative stress is initiated by reactive oxygen stantly generated and cause extensive damage to tissues

species (ROS), which are produced as a by-product of and in various disease conditions, particularly degen-

electron transport in mitochondria [1]. The increase in erative diseases, and also lead to extensive lysis [2].

Therefore, the mechanism of action of many synthetic

∗ drugs involves free radical scavenging, which protects

Corresponding author. Tel.: +91 8460619412.

against oxidative damage but has adverse side effects [3].

E-mail address: sarmistha [email protected] (S. Saha).

An alternative is the consumption of natural antioxidants

Peer review under responsibility of Taibah University.

from various food supplements and traditional medicines

[4], and there is increased interest among phytother-

apy researchers to use medicinal plants with antioxidant

ELSEVIER Production and hosting by Elsevier

activity for protection against oxidative stress.

http://dx.doi.org/10.1016/j.jtusci.2014.09.003

1658-3655 © 2014 The Authors. Production and hosting by Elsevier B.V. on behalf of Taibah University. This is an open access article under the

CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

806 S. Saha, R.J. Verma / Journal of Taibah University for Science 10 (2016) 805–812

Terminalia chebula Retzius (Combretaceae), com- Ascorbic acid was obtained from Merck Specialties Pvt.

monly known as haritaki, exhibits a number of medicinal Ltd., Mumbai, India. HPLC-grade methanol and acetoni-

activities due to the presence of a large number of dif- trile, as well as acetic acid were obtained from Merck

ferent phyto-constituents. The plant has a round crown Specialties Pvt. Ltd., Mumbai, India. All of the chemi-

and spreading branches. The bark is dark brown with cals used in this study were of analytical reagent grade,

some longitudinal cracks. The leaves are ovate and ellip- unless specified.

tical, with two large glands at the top of the petiole.

The fruit or drupe is approximately 1–2 inches in size. 2.2. Plant material and extract preparation

It has five lines or five ribs on the outer skin. The

The T. chebula fruits were collected from a local

fruits of T. chebula are reported to have hepatoprotective

market of Ahmedabad, India, during January 2012 and

activity against CCl4 and tert-butyl hydroperoxide [5].

were taxonomically identified and authenticated as T.

The fruits also display cytoprotective [6], antidiabetic

chebula fruits by Dr. Yogesh T. Jasrai, Department of

[7,8], antioxidant [9], antibacterial [10,11], anti-arthritic

Botany, Gujarat University, India. A voucher specimen

[12], hypo-cholesterolaemic [13] and anti-inflammatory

was deposited in the herbarium for future reference (Ref. activities [14].

No. GU/2013/53).

Antioxidant activities increase proportionally with

The T. chebula fruits were thoroughly cleaned,

the content, primarily because of their redox

dried under the shade and coarsely powdered. The

properties [15]. Among the diverse roles of polyphe-

polyphenolic-rich extract was prepared according to a

nols, they protect cell constituents against destructive

previously reported method [20]. The powdered plant

oxidative damage, thus limiting the risk of various

material was mixed with 70% methanol and stored at

degenerative diseases associated with oxidative stress by

room temperature for 5 days. After 5 days, it was filtered

acting as potent free radical scavengers. The polyphenol

and the solvent was evaporated. The residue was dis-

antioxidant activity is due to the chemical structure and

solved in water, and the aqueous layer was washed with

ability to donate/accept electrons, thereby delocalising

petroleum ether several times until a clear upper layer of

the unpaired electron within the aromatic structure [16].

petroleum ether was obtained. The lower layer was then

Recent studies showed the antioxidant properties of dif-

treated with ethyl acetate containing glacial acetic acid

ferent extracts of T. chebula fruits. In an earlier report, a

(10 mL/L). Extraction of the was performed

70% methanol extract of T. chebula fruits was found to

for 36 h at room temperature, and the combined ethyl

have good efficacy in radical scavenging abilities [17].

acetate layer was then concentrated. The residue was

In another report, chloroform, ethanolic, n-butanolic and

lyophilised. The extract obtained was dried and stored in

organic aqueous extracts were investigated for anti-lipid ◦

an airtight container at 4 C. The yield of the dry poly-

peroxidation, anti-superoxide radical formation and free

phenolic extract was 30.5% (w/w). The dried extract was

radical scavenging activities [18]. The results showed

dissolved in Milli-Q water and used for further study.

that all tested extracts exhibited antioxidant activity at

different magnitudes of potency [18]. , chebu-

2.3. Phytochemical studies

lanin, and 1,6-di-O-galloyl-b-d-,

isolated from T. chebula, were also tested in this study

Qualitative analysis for determining the presence of

and showed significant antioxidant activity [18]. Chang

, , glycosides, flavonoids, steroids and

and Lin [19] reported antioxidant activities of water,

alkaloids in the polyphenolic extract was performed

methanol and 95% ethanol extracts of the air-dried fruit

using standard methods [21].

of T. chebula. However, the antioxidant activity of the

polyphenolic-rich extract of T. chebula fruit has not been 2.3.1. Total phenolic content (TPC)

previously evaluated. Therefore, the objective of this The total phenolic content of the polyphenolic extract

study was to assess the free radical scavenging potential of T. chebula was estimated by the reported method [22].

of the polyphenolic extract of T. chebula fruits. Various concentrations of gallic acid were used to plot the

standard curve. The total phenolic content of the extract

2. Materials and methods is expressed as mg gallic acid equivalents/g dry weight

of extract.

2.1. Chemicals

2.3.2. content

Gallic acid standard (Batch No. 90618) was procured The total flavonoid content in the T. chebula polyphe-

from Himedia Laboratories Pvt. Ltd., Mumbai, India. nolic extract was estimated by the standard method [23].

S. Saha, R.J. Verma / Journal of Taibah University for Science 10 (2016) 805–812 807

The flavonoid content of the extract is expressed as mg spectrophotometer method at concentrations of

quercetin equivalents/g dry weight of extract. 50–500 ␮g/mL [27]. Ascorbic acid equivalents were

calculated using a standard curve of ascorbic acid. The

2.3.3. Tannin content experiment was conducted in triplicate, and the values

␮

The tannin content of the T. chebula polyphenolic are expressed as g equivalents of ascorbic acid per

␮

extract was estimated by the method described by Hager- g/mL of extract.

man and Butler [24]. The tannin content of the extract is

expressed as mg rutin equivalents/g dry weight of extract. 2.7. DPPH radical scavenging assay

2.3.4. Ascorbic acid content The ability of T. chebula polyphenolic

Ascorbic acid, also called vitamin C, is one of the extract/ascorbic acid to scavenge 1,1-diphenyl-2-

most abundant antioxidants in T. chebula extract and was picrylhydrazyl (DPPH) radical was measured by the

quantified according to the previously reported method reported method Shimada et al. [27]. The addition of

[25]. The ascorbic content of the extract is expressed as 0.1 mM DPPH solution in various concentrations (10,

␮ ␮

g/g dry weight of extract. 25, 50, 75, 100 and 150 g/mL) of plant extract/ascorbic

acid in the presence of Tris–HCl buffer (50 mM, pH 7.4)

2.4. Standardisation of extract resulted in decreased absorbance, which was measured

at 517 nm. A mixture of methanol and extract served

The polyphenolic extract of T. chebula fruits was

as the blank. The per cent inhibition was calculated

standardised using gallic acid as a standard. For

by measuring the absorbance of extract/ascorbic acid

reverse-phase separation of gallic acid from the other

treated samples against the blank. The IC50 values for

constituents, a Spectra-Physics HPLC-UV system (San

the polyphenolic extract were calculated and compared

Jose, CA, USA) with a Spectra system P1000 pump,

with the standard reference compound ascorbic acid.

a Spectra system UV1000 detector and a Rheodyne

7125 injector with a 20-␮L loop was used. Chro-

2.8. Nitric oxide radical scavenging assay

matographic separation was achieved on a COSMOSIL

C18 column (250 mm × 4.6 mm, length × inner diame-

◦ The ability of the polyphenolic extract of T. chebula

ter with 5-␮m particle size), maintained at 35 C in a

at graded concentrations of 50–500 ␮g/mL to scavenge

column oven. The mobile phase consisted of deionised

nitric oxide radical was determined by the modified

water:acetonitrile:acetic acid (88:10:2, v/v/v). The flow

method [28]. Various concentrations of polyphenolic

rate of the mobile phase was maintained at 1.0 mL/min,

extract were added to 10 mM sodium nitroprusside and

and the wavelength of the detector was set at 280 nm.

incubated for 2.5 h. After incubation, Griess reagent

was added to the tubes and the absorbance of the chro-

2.5. Reducing power assay

mophore formed was read at 590 nm. The blank solution

contained a mixture of 0.5 mL PBS and 0.5 mL extract.

The reducing power was determined based on the

The IC50 values and per cent inhibition by various con-

ability of the antioxidant to form a coloured complex

centrations of extract were calculated by comparing the

with potassium ferricyanide, TCA and FeCl3. It was

absorbance values of the control and test compounds.

measured by a modified method [26] in which the extract

of T. chebula fruits (1 mL) at various concentrations

2.9. Hydrogen peroxide scavenging assay

(25, 50, 100, 150, 200 and 250 ␮g/mL) was mixed with

potassium ferricyanide and phosphate buffer (pH 6.6)

◦ The hydrogen peroxide scavenging activity of T.

and incubated at 50 C for 20 min. Then, TCA (10%)

chebula at concentrations of 50–500 ␮g/mL was esti-

was added and centrifuged at 3000 rpm for 10 min. The

mated using a hydrogen peroxide solution [29]. A

supernatant was removed and mixed with FeCl3 (0.1%).

solution of hydrogen peroxide (2 mmol/L) was pre-

The absorbance was then measured at 700 nm. A higher

pared in phosphate buffer (pH 7.4). The polyphenolic

absorbance of the reaction mixture indicates a higher

extract/ascorbic acid (25, 50, 75, 100, 125 and

reducing power.

150 ␮g/mL) was added to the hydrogen peroxide solu-

tion (0.6 mL). A mixture of phosphate buffer (3.3 mL)

2.6. Total antioxidant capacity

and extract (0.5 mL) served as the blank. The absorbance

The total antioxidant capacity of the polyphe- of hydrogen peroxide at 230 nm was determined after

nolic extract of T. chebula was measured using a 10 min against a blank solution containing phosphate

808 S. Saha, R.J. Verma / Journal of Taibah University for Science 10 (2016) 805–812

buffer without hydrogen peroxide and compared to

ascorbic acid, which was the reference compound.

2.10. Statistical analysis

The results are expressed as the mean ± standard error

of the mean (SEM). The statistical analysis and lin-

ear regression analysis were performed using GraphPad

Instat, software, version 5.0. The IC50 values were cal-

culated and compared by paired t tests. p < 0.05 was

considered significant.

3. Results and discussion

3.1. Phytochemical screening

The qualitative phytochemical screening of the poly-

phenolic extract revealed the presence of tannins,

saponins, flavonoids and alkaloids. The total phenolic

content of the extract of T. chebula was 134.47 mg gallic

acid equivalent/g dry weight. The flavonoid content of

the extract was 7.934 mg of quercetin equivalent/g dry

weight. The tannin content of the polyphenolic extract of

T. chebula was 31.47 mg rutin equivalent/g dry weight.

The ascorbic acid content of the extract was 8.74 ␮g/g

dry weight of extract. Phytochemical analysis of T.

chebula showed the presence of gallic acid, ,

tannic acid, ethyl gallate, , ,

, mannitol, ascorbic acid (vitamin C), and other

compounds [30]. Another report found T. chebula with

32% tannin content [31]. Polyphenols are widely dis-

tributed in plants, and phenolic antioxidants act as free

radical scavengers and metal chelators [32]. Recently,

bioflavonoids and polyphenols of plant origin have been

used extensively for free radical scavenging and to inhibit

Fig. 1. (a) Chromatogram of the standard gallic acid and (b) chro-

membrane lipid peroxidation [33].

matogram of the polyphenolic extract of T. chebula fruits.

3.2. Standardisation of extract

separation of polyphenolic compounds, such as gallic

acid and ellagic acid, from T. chebula fruits by sub-

The polyphenolic extract of T. chebula fruits showed

critical water extraction. The decoction of T. chebula

the presence of gallic acid with a retention factor of 2.50

fruit contained 3,4,6-tri-O-galloyl-d-glucose, chebulic

(Fig. 1b), which is comparable to the retention factor of

acid, ␤-, corilagin, ␣-punicalagin, chebu-

2.40 of the pure standard gallic acid (Fig. 1a). More-

lagic acid, gallic acid, 1,3,4,6-tri-O-galloyl-␤-d-glucose,

over, the polyphenolic extract of T. chebula fruits also

chebulinic acid, 1,2,3,4,6-penta-O-galloyl-d-glucose,

showed the presence of other polyphenolic compounds,

ellagic acid, and 1,6-di-O-galloyl-d-glucose [38].

with retention factors of 1.40 and 1.80. Recently, many

Flavonoids also have significant antioxidant activity

natural antioxidants have been isolated from different

under both in vivo and in vitro conditions [39].

plant materials [34,35], and some types of polyphe-

nols, such as chebulanin, corilagin, neochebulinic acid,

3.3. Reducing power assay

ellagic acid, gallic acid, chebulagic acid, and chebulinic

acid, are present in the fruits of T. chebula [36]. In an The reducing capacity of the polyphenolic extract

earlier study, Rangsriwong et al. [37] investigated the of the T. chebula fruits was measured by its ability to

S. Saha, R.J. Verma / Journal of Taibah University for Science 10 (2016) 805–812 809

Fig. 2. The reducing power activity of the polyphenolic extract of T.

Fig. 3. Total antioxidant capacities of the polyphenolic extract of

chebula fruits. Data are presented as the means ± SEM of triplicates.

T. chebula fruits and ascorbic acid. Data are presented as the

***p < 0.001 from the control.

means ± SEM of triplicates. *p < 0.05; ***p < 0.001 from the control.

3+ 2+

transform Fe to Fe at various concentrations (25, 50,

100, 150, 200 and 250 ␮g/mL). The results revealed that

the reducing activity significantly increased as the con-

2

centration of the extract was increased (r = 0.989), with

␮

a maximum increase at 250 g/mL (Fig. 2). The reduc-

tive capacity of a compound depends on the presence

of reductones, which exhibit antioxidative potential by

breaking the free radical chain and donating a hydrogen

atom. Therefore, reducing activity leads to the termina-

tion of the radical chain reactions that may otherwise be

very damaging [40]. The presence of antioxidant reduc-

tants in the polyphenolic extract of T. chebula causes

3+

the reduction of the Fe /ferricyanide complex to the

ferrous form, indicating that the polyphenolic extract of

T. chebula has significant reducing power. Li et al. [41] Fig. 4. DPPH radical scavenging activity of the T. chebula extract and

ascorbic acid. Data are presented as the means ± SEM of triplicates.

reported the existence of a similar linear co-relationship

***p < 0.001 from the control.

between the reducing power and total phenolic content

(TPC).

activity of the extract was the highest at the max-

imum dose of 150 ␮g/mL, with an IC50 value of

3.4. Total antioxidant capacity 2

14 ± 0.05 ␮g/mL (r = 0.976). Ascorbic acid showed

2

an IC50 value of 16 ± 0.06 ␮g/mL (r = 0.987). The

The total antioxidant capacity of the polyphenolic

effect was concentration-dependent. DPPH is a stable,

extract of T. chebula fruits ranged from 0.140 to 0.387 at

2 nitrogen-centred free radical, which, upon accepting

graded concentrations of 50–500 ␮g/mL (r = 0.963;

hydrogen from the antioxidants present in the poly-

Fig. 3). Gallic acid, a polyphenyl present in T. chebula,

phenolic extract, is converted into a stable diamagnetic

was previously evaluated for free radical scavenging

molecule, diphenyl-picryl hydrazine [43]. The observed

activity and has significant reducing power and antioxi-

reduction of DPPH by the extract was either due to the

dant activity [42].

transfer of a hydrogen atom or the transfer of an electron.

Phenolic compounds are also effective hydrogen donors,

3.5. DPPH radical scavenging assay

which makes them good antioxidants [44].

The DPPH radical scavenging activity of the poly-

3.6. Nitric oxide radical scavenging assay

phenolic extract of T. chebula at different concentrations

is shown in Fig. 4 and is compared to the refer- In the present study, the nitric oxide radical quench-

ence compound ascorbic acid. The radical scavenging ing activity of the polyphenolic extract was detected and

810 S. Saha, R.J. Verma / Journal of Taibah University for Science 10 (2016) 805–812

Fig. 6. Hydrogen peroxide radical scavenging activity of the polyphe-

Fig. 5. Nitric oxide radical scavenging activity of the extract of

nolic extract of T. chebula fruits and ascorbic acid. Data are presented

T. chebula fruits and ascorbic acid. Data are presented as the

as the means ± SEM of triplicates. **p < 0.01; ***p < 0.001 from the

means ± SEM of triplicates. ***p < 0.001 from the control. control.

compared with the standard ascorbic acid. The extract can cross cell membranes rapidly, and once inside the

2+ 2+

exhibited the maximum per cent inhibition of 267% cell, H2O2 likely reacts with Fe , and possibly Cu

at a concentration of 500 ␮g/mL, with an IC50 value ions, to form hydroxyl radicals, which then become

of 30.51 ␮g/mL, in a concentration-dependent manner powerful oxidising agents. This may cause many toxic

2

(r = 0.977; Fig. 5). However, ascorbic acid exhibited effects. The phenolic compounds may act as free radical

maximum per cent inhibition of 189%, with an IC50 scavengers because of their hydrogen-donating ability

2

value of 42 ␮g/mL (r = 0.955; Fig. 5). The scavenging and scavenging ability [46].

activity of the extract against nitric oxide was detected

by its ability to inhibit the formation of nitrite through 3.8. Correlation between the total phenolic content

direct competition with oxygen and oxides of nitro- and antioxidant activity

gen in the reaction mixture [40]. The decrease in the

concentration of the nitric oxide radical was more signif- The total phenolic content of the polyphenolic extract

icant than ascorbic acid, which is due to the antioxidant of T. chebula is significantly correlated with its total

activity of the polyphenolic extract. Nitric oxide is a antioxidant capacity (R = 0.992, p < 0.05), DPPH rad-

potent pleiotropic mediator of physiological processes, ical scavenging activity (R = 0.971, p < 0.05), nitric

such as smooth muscle relaxation, neuronal signalling, oxide radical quenching activity (R = 0.995, p < 0.05)

inhibition of platelet aggregation and regulation of cell- and hydrogen peroxide scavenging activity (R = 0.990,

mediated toxicity. It is a diffusible free radical that plays p < 0.05). This result indicates that the phenolic contents

many roles as an effector molecule in diverse biological of T. chebula are responsible for its antioxidant activity. It

systems, including neuronal communication, vasodilata- has been proposed that the antioxidant activity of plants

tion and antimicrobial and antitumor activities [45]. may be due to their phenolic compounds [47]. Many

Moreover, in pathological conditions, nitric oxide reacts plants exhibit efficient antioxidant properties because of

with superoxide anion and forms potentially cytotoxic their phenolic constituents [48].

molecules, such as peroxynitrite.

4. Conclusion

3.7. Hydrogen peroxide scavenging activity

Polyphenols are valuable plant constituents for the

The polyphenolic-rich extract of T. chebula scavenging of free radicals because of their phenolic

showed significant scavenging activity of H2O2 in a hydroxyl groups [49]. This, together with the obtained

2

concentration-dependent manner (r = 0.990, Fig. 6), results, suggests that as the amount of polyphenolic com-

␮

with an IC50 value of 265.53 g/mL, whereas the IC50 pounds increases, the antioxidant activity also increases.

2

␮

value for ascorbic acid was 278 g/mL (r = 0.993, In conclusion, the present study demonstrates that the

Fig. 6). Hydrogen peroxide is a weak oxidising agent polyphenolic extract of T. chebula fruits can protect the

and can directly inactivate a few enzymes, usually by body from oxidative stress from ROS, which may be

oxidation of essential thiol groups. Hydrogen peroxide due to the phyto-chemicals in the form of polyphenols

S. Saha, R.J. Verma / Journal of Taibah University for Science 10 (2016) 805–812 811

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