INTRODUCTION:

Antioxidants are compounds that bind the free radicals in human body forbid the development of many diseases and reducing the possibility of degenerative diseases like carcinogenesis and diabetes. Antioxidants are found in vegetables, fruits, nuts, grains spices etc (1,2). Growing knowledge about antioxidants, and their presence in everyday foods promote health, combined with the assumption that a high number of commonly used synthetic preservatives may have hazardous effects, has led to multiple investigating in the field of natural antioxidants (3). These natural antioxidants usually come from a diet rich in fruits and vegetables or they are carried in creams and topically applied. A majority of antioxidants naturally present in foods occur in phenolic content and especially in flavonoid (4).

The natural phenolic compounds often act as reducing agents, terminate the free radical chain reaction by removing the same, absorb light in the ultraviolet (UV) region (100-400 nm), and chelate metals, thus suppress oxidation reactions by itself being oxidized and also inhibit the production of off-odours and tastes (5). Although It is well-known that flavonoids can neutralize different types of oxidizing species including superoxide anion, hydroxyl radical or peroxy radicals (6,7). They may also act as quenchers of singlet oxygen (8). Total flavonoids, obtained significant antioxidant activity and inhibited the lipid peroxidation caused by O₂, H₂O₂ and UV irradiation (9,10). In addition of that, oxidants are added to nutrients to prevent smell, deterioration in their taste and colour. butylated hydroxytoluene (BHT), Butylated hydroxyanisole (BHA) and propyl gallate (PG) can be included in this category, which are known as synthetic antioxidants (11). The high price of natural antioxidants has led to the use of synthetic antioxidants. All of that, studies conducted subsequently have verified that synthetic antioxidants have toxic effects and consequently, restrictions have been enforced on their use. Therefore, researchers have focused their studies on plant-derived natural antioxidants (12). Plant extracts with antioxidant properties raise great interest in the phytocosmetic and neutraceutical field as they present molecules that could deactivate ROS restoring skin home-ostasis thus preventing erythema and premature aging of the skin (13,14).

Ehretia acuminata, which is commonly known as Koda, is one of the oldest spices of genus Ehretia. Nowadays, it is used in Chinese herbal medicine (15). Traditionally, it has been used in fever, sores on tongue, dysentery and many more health ailments (16,17). Some species of the Ehretia genus are used for medical purposes However, many species include phytochemicals alkaloids (18,19), flavonoids (20-22), fatty acids (23) which are responsible for anti-oxidant (24,25), anti-inflammatory (26,27), anti-allergic (28,29), anti-bacterial (30,31) activities in different species like E. laevis, E. tinifolia, E. microphylla, E. longiflora, E. obtusifolia.

The aim of the present research is comparative evaluation of antioxidant activity by ABTS, DPPH, FRAP, reducing capacity of Nitric oxide, total phenolic content and flavonoid content of Ehretia acuminata fruit extracts by different solvent systems. Moreover, Ascorbic acid and gallic acid which are synthetic antioxidant, was evaluated as a positive control and compared with all extracts of the plant.

MATERIAL AND METHODS:

Plant materials:

E. acuminata was collected from pantnagar (daytime air temperature, 26–27.9 °C) in Udham singh nagar district of Uttarakhand in Kumaun region of India in the month of April 2017 and authenticated from Botanical survey of India (BSI) Dehradun (Voucher specimen number 117138 05/2017). Fruit were dried for 15–20 days under shade until fruit seem brittle or ready for grinding and stored at room temperature, were subjected to grinding in a laboratory grinder and stored at 4 °C.

Chemicals and instruments:

Folin ciocalteu reagent (Merck), the stable free radical 2, 2'-Diphenyl-1-picryl hydrazyl (DPPH) (Sigma), 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS), ascorbic acid acid (AA) (Merck) and gallic acid (GA) (Loba chemie), All other chemicals used were of analytical grade. UV-VIS Spectrophotometer 118 (Systronic).

Preparation of crude plant extract:

Plants material was collected from kumaun region Uttarakhand. Plant material consisting of mature fruit were also collected and dried. The dried plant materials were powdered using a grinder. The extraction was done at normal temperature. About 100g of dried, ground fruit materials were soaked in different solvents (700 ml) according to increasing order of polarity (petroleum ether, chloroform, ethyl acetate, ethanol, water) for 5-8 days separately. The soaked material was stirred every 16 hours using a sterilized glass rod. The final extracts were passed through Whatman filter paper No.1. The filtrates obtained were concentrated by using vacuum on a rotary evaporator at 42 ^oC and stored at 4^oC for further use. The stock solution of different extracts (5 mg/ml) was prepared by dissolving a known amount of dry extract in 98% methanol. The working solutions of different concentrations (25, 50, 75, 100, 250, 500 and 1000 μg/ml) of different extracts were prepared from the stock solution using suitable dilution.

Antioxidant properties:

2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) assay:

The total antioxidant activity was determined by ABTS radical cation scavenging assay by the method of (Re et al., 1999) (32). ABTS radical cation was produced by ABTS (stable radical) aqueous solution with 2.4 mM potassium persulfate in the dark for 12–16 h. Prior to assay, ABTS solution was diluted in ethanol (1:89 v/v) to give an absorbance of 0.700± 0.02 at 734 nm. Triplicates of 10 µL samples (1 mg/ mL of respective organic solvents) and Ascorbic acid (concentration 0–15 µM) were added to 1 mL of diluted ABTS solution. The reaction mixture was incubated at 30ºC exactly for 30 min and the absorbance was measured at 734 nm against the ethanol(blank).

1,1-Diphenyl-2-picryl-hydrazyl (DPPH) assay:

Radical scavenging activity of the E. acuminata fruit extracts was determined by DPPH essentially as described by (Blois 1958, Jiby 2010) with some modification (33,34). The extracts of different concentrations (25, 50, 100, 150) and Ascorbic acid (25, 50, 100, 150 and 200 µg) were taken in different test tubes. The volume was adjusted to 100µL by adding MeOH. Three milliliter of 0.1 mM methanol solution of DPPH was added to these tubes and shaken vigorously. The tubes were allowed to stand at 27ºC for 30 min. The control was prepared as the same without using any extract. The changes in the absorbance of the samples were measured at 512 nm. Free radical scavenging activity was estimated as the inhibition percentage and was calculated using the following formula,

DPPH scavenging effect = Control OD − Sample OD / Control OD × 100

Ferric reducing power assay:

The ferric reducing ability of different extracts was estimated by the method of (Pulido et al., 2000) (35). The FRAP reagent was prepared by mixing 2.5 mL of 10 mM TPTZ in 40 mM HCl, 2.5 mL of 20 mM FeCl3.6H2O and 25 mL of 0.3M acetate buffer (pH 3.6). 900µL of FRAP reagent was mixed with 10µL of aliquots of plant extracts (1 mg/mL of respective organic solvents) and incubated at 37ºC. After incubation, ferric reducing ability of plant extracts was measured at 595 nm. The standard curve was prepared by ascorbic acid between 10 - 100 μM. The results were expressed as µmol Fe(II) equiv./g extract.

Nitric oxide radical scavenging assay:

Nitric oxide was generated from sodium nitroprusside and measured by Griess reaction (Marcocci L, 1994, Balakrishnan 2009) (36,37). Sodium nitroprusside (5 mM) in PBS (phosphate buffer saline) was incubated with different concentrations (25-200 µg/ml) of different the extracts, dissolved in phosphate buffer (0.25 M, pH 7.4) and the tubes were incubated at 25ºC for 5 h. Controls without using test compound, but with equivalent amounts of buffer were conducted in identical manner. After 5h 0.5ml of Griess reagent (1% sulfanilamide, 2% O-phosphoric acid and 0.1% naphthylethylene diamine dihydrochloride) was added. The absorbance was taken at 546 nm.

Total Phenolic and flavonoid contents:

The measure of total phenols in the crude extracts was determined by using Folin–Ciocalteu reagent and external calibration with gallic acid. In brief, 0.2 mL of extract solution and 0.2 mL of Folin–Ciocalteu reagent were added and mixed completely (Singelton et al., 1999) (38). After 5min, 1mL of 15% Na2CO3 was added then the mixture was allowed to stand for 2h at 27-32ºC temperature. The absorbance was measured at 765nm by using spectrophotometer (Systronic 118). The concentration of the total phenolics was measured as mg of gallic acid equivalent by using an equation obtained from gallic acid calibration curve.

The total flavonoid content (TFC) of each extract was investigated using the aluminum chloride colorimetry method described by (Chang et al., 2002) with little modifications (39). Briefly, the extract sample was diluted with methanol until 100mg/ mL. The calibration curve was prepared by diluting quercetin(QR) in methanol. The diluted extract or quercetin (2.0 mL) was mixed with 0.1 mL of 10% (w/v) aluminum chloride solution and 0.1 mL of 0.1 mM potassium acetate solution. The mixture was kept at room temperature for 30 minutes and the maximum absorbance of the mixture was measured at 415nm using a UV-VIS spectrophotometer.

Statistical analyses:

All the experiments were done in triplicates and the results were expressed as Mean± SD. The data were statistically analyzed using one way ANOVA followed by Duncan’s test. Mean values were considered statistically significant when p> 0.05.

RESULT AND DISCUSSION:

After the complete extraction of plant raw material by different solvents found different yield, nature and colour of plant extract. Highest yield found in EtOH (13gm) extract and least in chloroform (0.707gm).

Graph-1. showing Yield of extract by different solvents

ABTS^·+scavenging assay is a widely accepted method to determine the total antioxidant activity. The results were expressed as % inhibition in µg/ml. Among various solvent extracts of E. acuminata, the successive Soxhlet ethyl acetate extract possessed the highest ABTS± scavenging activity, while the petroleum ether extract showed the lowest ABTS radical cation scavenging activity. Ascorbic acid used as a standard antioxidant.

Graph 2 ABTS scavenging activity of Ehretia acuminata fruit.

DPPH radical is a stable organic free radical with an absorption band at 512 nm. It loses this absorption when accepting an electron or a free radical species, which results in a visually noticeable discoloration from purple to yellow. It can accommodate many samples in a short period and is sensitive enough to detect active ingredients at low concentrations Hseu et al., 2008 (40). Graph 3 shows the DPPH scavenging activities of the extracts in a concentration-dependent manner. The extract obtained by 100% methanol showed the significant DPPH radical scavenging activity at concentrations ranging from 25 mg/mL to 200 mg/mL. However, at concentrations ranging from 200 mg/mL to 350 mg/mL, its DPPH radical scavenging activity is not significantly different from those of the other extracts. From all extracts obtained by using a pure organic solvent, ethyl acetate gave stronger radical scavenging capacity than that of the other extracts.

Graph 3 DPPH radical scavenging activity of Ehretia acuminata fruit.

The FRAP assay is determined by the ferric reducing ability of fruit crude extracts. The successive Soxhlet ethyl acetate extract showed higher ferric reducing ability as compared to ascorbic acid. Petroleum ether and ethanol extract showed least ferric reducing ability.

Table -1 Ferric reducing antioxidant potential of Ehretia acuminata fruit

FRAP antioxidant radical scavenging activity.
S.no	Extract	FRAP value	Ferric reducing antioxidant power (100μM/ml)
1	P.E	.120	36.66
2.	CH	.667	234.31
3.	E.A	1.227	550.62
4.	EOL	.310	81.25
5.	WTR	.444	139.09
6.	A.A	2.0	788.12

Sodium nitroprusside forms nitric oxide radical in the presence of phosphate buffer solution at 25ºC. Nitric oxide reacted with Griess reagent and diazotization of nitrite with sulfanilamide and subsequently coupling with naphthylethylene diamine form colour complex. Decrease in colour intensity level is directly proportional to nitric oxide radical scavenging. Ascorbic acid used as standard showed least colour (light pink) and P.E extract showed immediate dark colour.

Graph 4 Nitric oxide scavenging activity of Ehretia acuminata fruit.

Phenolic compounds are a group of antioxidant agents, which act as free radical terminators (41-43). Total phenols were measured by Foline Ciocalteu reagent in terms of Gallic acid equivalent. The total phenolic in P.E, CH, E.A, EOL and WTR of E. acuminata was found to be 34.214, 90.521, 310.431, 56.982 and 90.225 respectively. The compounds such as flavonoids and polyphenols, which contain hydroxyls, are responsible for the radical scavenging effect of plants (44). According to this study, the high contents of this Phytochemical in ethyl acetate extract of E. acuminata fruit can explain its high radical scavenging activity.

Table -2 Total Phenolic and Flavonoid content of Ehretia acuminata fruit Phenolic and flavonoid content of E. acuminata fruit extract.

S.no	Extract	Total Phenolic Content (mg GAE/100 gm dw)*	Total Flavonoid Content (mg QRE/100 gm dw)*
1.	P.E	34.214	.071
2.	CH	90.521	.090
3.	E.A	310.431	.129
4.	EOL	56.982	.105
5.	WTR	90.225	.055

*Results are expressed as mean of 3 values ± standard deviation.

Correlation of IC₅₀in ABTS, DPPH, Nitric oxide method:

The IC₅₀ of plant extract is inversely related to its antioxidant capacity, as it expresses the amount of antioxidant required to decrease the ABTS, DPPH, Nitric oxide concentration by 50%, which is measured by interpolation from a linear regression analysis (45). A lower most IC₅₀indicates a higher antioxidant activity of a compound. Table 3 shows the IC₅₀ values in the ABTS, DPPH, Nitric oxide radical scavenging activity assay of different extracts. It was found that the ethyl acetate extract possesses the strongest radical activity (IC₅₀ 40 mg/mL) by all method. Phenolics were the crucial antioxidant components, and their total amount were directly proportional to the antioxidant activity. This research shows a correlation between total phenolic content, ABTS, DPPH and Nitric oxide antioxidant activity of the freeze-dried E. acuminata fruit extracts.

Table no- 3 Correlation of IC₅₀ by different methods of Ehretia acuminata fruit IC50 in ABTS, DPPH, Nitric oxide radical scavenging activity.

S. No	Extract	IC_50µg/ml(ABTS)	IC_50µg/ml(DPPH)	IC_50µg/ml(Nitric oxide)
1.	P.E	1520	1570	1985
2.	CH	370	400	590
3.	E.A	40	50	380
4.	EOL	500	510	750
5.	WTR	180	210	460
6.	AA	23	27	230

In this study, it was also observed that the TPC, TFC, and antioxidant activity of ethyl acetate extracts is higher than those of pure ethanol, water and the other non polar solvents.

CONCLUSION:

The ABTS, DPPH, FRAP, and nitric oxide assays gave comparable results for the antioxidant activity measured in extract of fruit of E. acuminata. The ABTS technique showed high reproducibility, was simple, rapidly performed and had highest correlation with both ascorbic acid and total phenolics. Therefore, it would be an appropriate technique for determining antioxidant in Ehretia acuminata fruit. Antioxidant activity in ethyl acetate extract is highest and in petroleum ether extract is lowest compared to all other extracts. Ascorbic acid, flovonoids and phenolics are the major contributors to antioxidant activity Ehretia acuminata.

ACKNOWLEDGEMENT:

The authors are grateful to Department of Chemistry, Kanya Gurukula campus, Gurukula Kangri University, Haridwar for providing all the necessary facilities.

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Received on 16.11.2018 Modified on 25.12.2018

Research J. Pharm. and Tech. 2019; 12(4):1811-1816.

DOI: 10.5958/0974-360X.2019.00302.0