Antioxidant and antidiabetic activities of Terminalia bellirica fruit in alloxan induced diabetic rats

South African Journal of Botany 130 (2020) 308
315

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Antioxidant and antidiabetic activities of Terminalia bellirica fruit in alloxan induced diabetic rats Ashutosh Gupta, Ramesh Kumar, Abhay K. Pandey* Department of Biochemistry, University of Allahabad, Allahabad 211002, India

A R T I C L E

I N F O

Article History: Received 13 August 2019 Revised 26 November 2019 Accepted 11 December 2019 Available online xxx Edited by V Kumar Keywords: Terminalia bellirica DPPH Reducing power Hydroxyl radical scavenging assay Total antioxidant capacity a-amylase inhibitory activity Antidiabetic activity

A B S T R A C T

Excessive production of free radicals in the living system leads to oxidative stress which is associated with degenerative disorders including diabetes. Diabetes is increasing at a rapid pace despite the availability of synthetic anti-hyperglycemic drugs in the market. Hence antioxidant and anti-diabetic compounds of natural origin have attracted attention for drug development. The aim of the present study was to investigate the phytochemical composition, antioxidant potential, a-amylase inhibitory and antidiabetic activity of aqueous (AQ) and ethyl acetate (EA) extracts of Terminalia bellirica fruit. Chemical methods were employed for phytochemical analysis. Antioxidant activities of extracts were measured using in vitro assays viz., DPPH free radical scavenging, reducing power, hydroxyl radical scavenging and phosphomolybdate assays. The antidiabetic potential was measured by in vitro a-amylase inhibitory activity and in vivo serum biochemical assays in alloxan-induced diabetic rats. Chemical analysis showed the presence of phenols, flavonoids, alkaloids, terpenoids, saponins and glycosides as a major phytochemical in AQ and EA extracts of T. bellirica fruit. Quantitatively EA extract showed the presence of higher content of phenolics and flavonoids as compared to AQ extract. Further EA extract exhibited considerable free radical scavenging abilities in DPPH and HRSA assays (up to 94%), reducing power assay and appreciable total antioxidant power in phosphomolybdate assay (78 mgPGE/g). The EA extract exhibited comparatively better a-amylase inhibitory activity (IC50 43.5 mg/ml) as compared to AQ extract (IC50 74.8 mg/ml). The activity was comparable to standard drug acarbose. Antidiabetic activity of extracts was studied in alloxan-induced diabetic rats by monitoring the body weight, blood glucose, lipid profile and other biochemical parameters for 28 days. In diabetic rats both the extracts showed a restorative effect on body weight and blood biomarkers such as glucose, creatinine, total protein, total cholesterol, LDL, HDL, triglyceride, urea and uric acid. The ethyl acetate extract also exhibited superiority over the aqueous extracts during in vivo antidiabetic assays. The results revealed that T. bellirica fruit extracts possess antioxidant, a-amylase inhibitory and antidiabetic activities and hence it could be useful for the management of hyperglycemia and oxidative stress. © 2019 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction In a living system, various biochemical processes lead to the generation of reactive oxygen species (ROS) during metabolism. However, excessive production of ROS causes an imbalance between oxidant and antioxidant equilibrium that is accountable for damaging vital biomolecules like lipid, protein, DNA etc., and the condition is referred to as oxidative stress (Sharma et al., 2017; Gupta and Pandey, 2019). Free radicals such as hydroxyl, superoxide and peroxyl radical induced oxidative damage have been supposed to be the primary cause of numerous diseases such as diabetes, stroke, cancer, arteriosclerosis, and cardiovascular diseases. ROS targets the unsaturated fatty acids in the bio-membranes that result in lipid * Corresponding author. E-mail addresses: [email protected], [email protected] (A.K. Pandey). https://doi.org/10.1016/j.sajb.2019.12.010 0254-6299/© 2019 SAAB. Published by Elsevier B.V. All rights reserved.

peroxidation, decrease in fluidity, loss of enzymes and receptor activity, and damage membrane proteins ultimately leading to cell degradation (Kumar and Pandey, 2014; Kumar et al., 2019a). Diabetes is one of the top five most significant diseases in developed countries. By definition, diabetes is described as a metabolic disorder characterized by chronic hyperglycemia resulting from defects in insulin secretion, insulin action, or both (Kumar et al., 2019b). Insulin deficiency at the metabolic level causes derangement of carbohydrate, protein and lipid metabolism which eventually leads to a number of secondary complications such as hyperlipidemia, coronary artery disease, renal failure, stroke, neuropathy, retinopathy and blindness (Chait and Bornfeldt, 2009; Joshi et al., 2019). It is estimated that nearly 75% of the world’s population extensively depends on plant based medicines (Kasabri et al., 2010). In past decades, plant derived antioxidants and hypoglycemic agents have attracted a great deal of attention particularly in the management of degenerative

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diseases associated with oxidative damage. Antioxidants prevent oxidative damage mainly by four mechanisms. These include reducing the concentration of ROS, breaking chain reactions, scavenging free radicals and chelating transition metals that catalyze free radicals formation (Sharma et al., 2019). a-Amylase is accountable for the conversion of starch to simple sugar (dextrin, maltotriose, maltose and glucose). Inhibitors of these enzymes lower the carbohydrate absorption and delay the overall time of carbohydrate digestion which leads to a marked reduction in the rate of glucose absorption thereby moderating the postprandial plasma glucose rise (Rhabasa and Chiasson, 2004; Singh et al., 2019). Acarbose, miglitol and voglibose are some commonly used inhibitors in clinical practice for diabetes management (Bailey, 2003). Although, these drugs are identified to be associated with numerous gastrointestinal side effects including diarrhea, abdominal pain and flatulence in the patients (Fujisawa et al., 2005; Singh et al., 2007). Hence, it is the need of time to identify and explore the amylase inhibitors from natural sources having fewer side effects. Terminalia bellirica Roxb. (Combretaceae) commonly known as belleric myrobalan, is found throughout the different region of Asia (Kapoor, 1990). Its fruit is rich in bioactive compounds and has been used for the treatment of various ailments in the indigenous systems of medicine (Jadon et al., 2007). It contains termilignan, thannilignan, 7‑hydroxy‑30 ,40 -(methylenedioxy) flavone, anolignan B, gallic acid, ellagic acid, corilagin, ß-sitosterol, arjungenin, belleric acid, bellericoside and cannogenol 3-O-ß-Dgalactopyranosyl-(1!4)-O-a-L rhamnopyranoside (Lobo et al., 2010). T. bellirica crude extracts as well as Triphala, an ayurvedic preparation containing dried powder of Emblica officinalis, T. chebula and T. bellirica fruits have shown appreciable antioxidant and hypoglycemic activities in alloxan-induced diabetic rats (Sabu and Kuttan, 2002). In addition, the aqueous extract of T. bellirica was found to stimulate insulin secretion in the clonal pancreatic cell line (Kasabri et al., 2010). Hence, it is necessary to validate the phytomedicine ingredients used in traditional system of medicine to cure diabetic complications. The present study aims to evaluate the antioxidant and antidiabetic potential of aqueous and ethyl acetate extracts of T. bellirica fruit. To the best of our knowledge it is first reported on the antidiabetic efficacy of the ethyl acetate extracts of T. bellirica fruit against alloxan-induced diabetes. 2. Materials and methods 2.1. Chemicals Glibenclamide was obtained from Sigma Chemical CO., USA. Total cholesterol, low-density lipoproteins (LDL), high-density lipoproteins (HDL), triglycerides, total protein, creatinine, urea and uric acid were assayed using commercially available kits supplied by Erba Diagnostics, Mannheim GmbH, Germany. All other unlabelled laboratory chemicals such as allaxon, a-amylase, 3,5-dinitrosalicylic acid (DNSA), 1,1-Diphenyl-2-picrylhydrazyl(DPPH), aluminum chloride, sodium hydroxide, ferric chloride, mayer`s reagent and draggendorff`s reagent, potassium hexacyanoferrate, butylated hydroxyanisole, ferrous ammonium sulfate, starch, nash reagent, ammonium molybdate, propyl gallate etc. were purchased from Sisco Research Laboratory Pvt. Ltd. New Delhi, India. 2.2. Plant material and preparation of extract T. bellirica (Tb) fruits were purchased from local markets of Allahabad and identification was confirmed by Prof. D. K. Chauhan from the Department of Botany, University of Allahabad. The fruits were shade dried at room temperature for 10
15 days and ground into fine powder in a mixer grinder. The powdered sample was extracted sequentially with ethyl acetate and water in Soxhlet apparatus (Mishra et al., 2009). The extract was centrifuged at 4000 rpm for 10 min at 4 °C

309

and filtered using Whatman filter paper. The excess solvent in extracts was removed under reduced pressure. The percentage yield of ethyl acetate (EA) extract was 4.27% while aqueous (AQ) extract was 28.43%. The dried extracts were constituted in distilled water for the assessment of phytochemicals, antioxidant and antidiabetic activities. 2.3. Phytochemical screening The chemical screening was carried out on AQ and EA fractions of Tb fruit using standard procedures to identify the active constituents like phenolics and tannins, flavonoids, alkaloids, saponins, cardiac glycosides, carbohydrates, terpenoids and steroids (Trease and Evans, 1989; Mishra et al., 2009). 2.4. Total phenolic and flavonoid content of extracts 2.4.1. Determination of total phenolics Total phenolic content in both the extracts was determined according to the standard protocol (Singh et al., 2002). 0.2 ml of the sample was diluted to 3 ml with water. A small amount (0.5 ml) of two-fold-diluted Folin
Ciocalteau reagent was added and the contents were mixed. After 3 min, 2 ml of 20% sodium carbonate solution was added and the tubes were placed in a boiling water bath for 1 min followed by cooling at room temperature. The absorbance was measured at 650 nm against a reagent blank using a spectrophotometer (Evolution 201, Thermo Scientific, Waltham, MA, USA). The concentration of phenolics in the test sample was expressed as milligram catechol equivalents/g (mg CE/g). 2.4.2. Determination total flavonoid content The aluminum chloride colorimetric method (Chang et al., 2002) as previously modified by us (Mishra et al., 2011) was used for the determination of flavonoids in Tb fruit extracts. A small amount (0.2 ml) of extract in pure DMSO was separately mixed with 1.8 ml of methanol, 0.1 ml of 10% aluminum chloride, 0.1 ml of 1 M potassium acetate and 2.8 ml of distilled water. Tubes were incubated at room temperature for 30 min and then the absorbance of the reaction mixture was measured at 415 nm using a spectrophotometer (Evolution 201, Thermo Scientific, Waltham, MA, USA). The calibration curve was prepared with a quercetin solution. The flavonoid content in the test sample was expressed as milligram quercetin equivalent/g sample (mg QE/g). 2.5. In vitro antioxidant activity of plant extracts 2.5.1. DPPH radical scavenging activity The free radical scavenging activity of the EA and AQ extracts was measured in vitro by 1, 1-diphenyl-2-picrlhydrazyl (DPPH) assay (Singh et al., 2002; Mishra et al., 2011). Water was used as a solvent for dissolving extracts instead of methanol. 3 ml of 0.1 mM DPPH solution prepared in methanol was added to 0.1 ml of the test extracts. The final concentration of extracts in the reaction mixture was 6.66
33.33 mg/ml. The content was mixed and allowed to stand at room temperature for 30 min in dark. The reduction of DPPH free radical was measured by recording the absorbance at 517 nm. The percentage scavenging activities (% inhibition) at different concentrations of the extract fractions were calculated using the following formula. % Radical scavenging activity ¼

ðAc
AsÞ  100 Ac

Whereas, AC and AS represent absorbance of the control and the sample, respectively.

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2.5.2. Reducing power assay The reducing power of test extracts was determined by the method of Oyaizu (1986) with slight modifications (Mishra et al., 2013a). 1 ml aliquot of extracts (1.31
13.15 mg/ml) prepared in DMSO was taken in test tubes. To each test tube, 2.5 ml of phosphate buffer (0.2 M, pH 6.6) and 2.5 ml of 1% potassium hexacyanoferrate [K3Fe(CN)6] were added and contents were mixed. Tubes were incubated at 50 °C in a water bath for 20 min. The reaction was terminated by adding 2.5 ml of 10% trichloroacetic acid and then centrifuged at 4000 g for 10 min. 1 ml of the supernatant was mixed with 1 ml of distilled water and 0.5 ml of FeCl3 solution (0.1%, w/v) and kept at 25 °C for 2 min. The absorbance was measured at 700 nm. The butylated hydroxyanisole (BHA) was used as a positive control. 2.5.3. Hydroxyl radical scavenging activity (HRSA) HRSA of extracts was determined by the method of Klein et al. (1981). Aliquots of 100 ml extracts (2.66
13.33 mg) were taken in different test tubes. One milliliter of Fe
EDTA solution (0.13% ferrous ammonium sulfate and 0.26% EDTA), 0.5 ml of 0.018% EDTA and 1 ml of 0.85% (v/v) DMSO (in 0.1 M phosphate buffer, pH 7.4) were added to the test tubes, followed by 0.5 ml of 0.22% (w/v) ascorbic acid. The tubes were capped tightly and incubated on a water bath at 85 °C for 15 min. After incubation, 1 ml of ice-cold TCA (17.5% w/v) was added immediately. Three milliliters of Nash reagent (7.5 g of ammonium acetate, 3 ml glacial acetic acid and 2 ml acetylacetone were mixed and made up to 100 ml with distilled water) was added to all the tubes and incubated further at room temperature for 15 min. Absorbance was measured at 412 nm. BHT was used as a standard compound for comparison. Percentage (%) HRSA was calculated by the following formula: ðA0
A1Þ %HRSA ¼  100 A0 Where A0 is the absorbance of the control and A1is absorbance of the sample. 2.5.4. Total antioxidant capacity by phosphomolybdate assay The total antioxidant activity of the extracts was evaluated by phosphomolybdate assay (Mishra et al., 2011). An aliquot of 10 ml of the extract solution was mixed with 1 mL of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate) in a microcentrifuge tube. The tubes were incubated in a water bath at 95 °C for 90 min. After cooling, the absorbance of the mixture was measured at 695 nm against a blank. The propyl gallate was used as a reference to draw the standard curve. The reducing capacities of the extracts were expressed as mg of propyl gallate equivalents/g of extract (mg PGE/g). 2.5.5. In vitro a-amylase inhibitory assay A starch solution (1% w/v) was prepared by stirring 1 g starch in 100 ml of 20 mM of phosphate buffer (pH 6.9) containing 6.7 mM of sodium chloride. The enzyme solution was prepared by mixing 27.5 mg of porcine pancreatic a-amylase in 100 ml of 20 mM of phosphate buffer (PBS, pH 6.9) containing 6.7 mM of sodium chloride. To 100 ml of (20
100 mg/ml) plant extracts, 200 ml porcine pancreatic amylase was added and the mixture was incubated at 37 °C for 20 min. To the reaction mixture 100 ml (1%) starch solution was added and incubated at 37 °C for 10 min. The reaction was stopped by adding 200 ml DNSA (1 g of 3,5 dinitrosalicylic acid, 30 g of sodium-potassium tartrate and 20 ml of 2 N sodium hydroxide was added and made up to a final volume of 100 ml with distilled water) and kept it in a boiling water bath for 5 min. The reaction mixture was diluted with 2.2 ml of water and absorbance was read at 540 nm. Blank was prepared by replacing the enzyme solution with 200 ml in distilled water. Control, representing 100% enzyme activity was prepared in a similar manner, without extract (Ali et al., 2006).

2.6. Experimental animals Healthy albino Wistar rats of either sex, approximately of the same age (weight 230
260 g) were procured from Indian Institute of Toxicological Research, Lucknow. They were kept in the departmental animal house in a well cross (23 § 2 °C) with light and dark cycles of 12 h of 1 week before and during experiments. Animals were provided with standard pellet diet and water was given ad libitum. The in vivo study was performed in accordance with the Guide for the Care and Use of Laboratory Animals, as adopted and promulgated by the Institutional Animal Care Committee, Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) India.

2.7. Induction of diabetes The overnight fasted rats were injected intraperitoneally (i.p.) with allaxon monohydrate (Sisco Research Laboratories Private Limited, Mumbai, India) dissolved in sterile normal saline (0.9% NaCl) at a single dose of 80 mg/kg to induce diabetes. After a week, diabetes was confirmed by the determination of fasting blood glucose level with the help of one-touch electronic glucometer (Accu-Check, Roche Diabetes Care GmbH SandhoferStrasse Mannheim, Germany) using commercially available glucose strips. Rats with a fasting plasma glucose range of 250
300 mg/dl were selected for the study.

2.8. Experimental design and treatment schedule The rats were divided into five groups (n = 5). The group details included Group-1 normal rats; Group-2 alloxan-induced diabetic rats; Group-3 diabetic rats administered with the standard drug (glibenclamide 5 mg/kg); Group-4 diabetic rats administered with AQ extract (250 mg/kg) and Group-5 diabetic rats administered with EA extract (250 mg/kg) of Tb fruit. Single dose of glibenclamide and extracts were administered every day orally for 28 days.

2.9. Biochemical analysis Blood was drawn from the veins of the rat tail at set time intervals till the end of the study. Blood glucose and body weight measurements were performed on days 1, 7, 14, 21 and 28 of the study. Blood glucose estimation was performed by glucometer. Estimation of total protein, creatinine, urea, uric acid, total cholesterol, LDL, HDL and triglyceride were done by using commercially available standard kits supplied by Erba Diagnostics on the fully automated analyzer (ERBAEM 200).

2.10. Statistical analysis All assays were carried out in triplicate. Statistical significance was analyzed using the student t-test. Results were expressed as mean§ standard deviation (SD). The graphs were prepared using GraphPad Prism software 5.01 version (GraphPad Software, San Diego, CA, USA).

3. Results 3.1. Phytochemical screening Tb fruit AQ and EA extracts revealed the presence of flavonoids, terpenoids, carbohydrate, alkaloids, cardiac glycosides, phenolic and tannin and saponins while steroids were absent in both extracts.

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*

Inhibition (%)

80

*

*

* 60

dependent response. At the lowest test concentration (1.31 mg/ml) BHA exhibited considerable reducing power (not shown in the figure). At higher concentrations, reducing power of EA extract was comparable to that of BHA.

ethyl acetate extract

aqueous extract

100

311

*

40

3.5. Hydroxy radical scavenging assay

20 0 6.66

13.33

20

26.66

33.33

concentration of extracts (µg/ml) Fig. 1. Free radical scavenging activity of T. bellirica fruit aqueous and ethyl acetate extracts by DPPH assay. The results are expressed as mean § SD of three replicates (*p < 0.05).

3.2. Total phenolic and flavonoid contents Total phenolic content in AQ and EA extracts was 220 § 0.72 mg CE/g and 360 § 0.34 mg CE/g sample, respectively. Whereas flavonoid content was 127 § 0.63 mg QE/g and 190 § 0.97 mg QE/g sample, respectively. 3.3. DPPH radical scavenging activity Free radical scavenging activity of Tb fruit extracts at different concentrations in the reaction mixture (6.66
33.33 mg/ml) was evaluated by the DPPH method (Fig. 1). The degree of discoloration indicates the scavenging potentials of the extracts. EA extracts (EC50 = 5.79 mg/ml) showed superior free radical scavenging potential (57
94%) as compared to AQ extract (EC50 = 14.67 mg/ml) (27
77%). In general, with increasing concentration of extracts, there was a gradual increase in free radical scavenging activity. Standard antioxidant namely BHA (EC50 = 0.89 mg/ml) has already been reported to produce 95% scavenging activities (Kumar et al., 2013). 3.4. Reducing power assay Considerable reducing power was observed in ethyl acetate fraction in comparison to aqueous extract (Fig. 2). Higher absorbance values indicated higher reducing power. The reducing power of the extract increased with increasing concentration of extracts in the reaction mixture (1.3
13.15 mg/ml) in both AQ extract (absorbance 0.04
0.375) and EA extract (absorbance 0.04
0.50) exhibiting dose

Absorbance at 700 nm

0.6

aqueous extract

Radical scavenging potential of AQ and EA extracts was determined in the concentration range 2.66
13.33 mg/ml (Fig. 3). An increasing trend in radical scavenging activity was observed with increasing concentration of all the extracts suggesting concentration dependent response. EA extracts (EC50 = 8.79 mg/ml) at all test concentrations demonstrated greater activity than AQ extract (EC50 = 24.23 mg/ml). Scavenging activity of EA and AQ extracts was comparable to the activity shown by standard antioxidant BHT (EC50 = 4.06 mg/ml). The hydroxyl radical scavenging potential of BHT ranged between 44 and 80% at test concentrations.

3.6. Total antioxidant capacity determination by phosphomolybdate assay The total antioxidant capacity of Tb fruit extracts was measured at different concentrations of test extracts (6.66, 13.33, 20.00, 26.66 and 33.33 mg/ml). At all test concentrations, EA extract showed comparatively superior antioxidant potential as compared with AQ extract (Fig. 4). The total antioxidant capacity for AQ extract at different concentrations was in the range 7.5
37 mg PGE/g while for EA extract it was in the range 23.5
78 mg PGE/g. The activity of EA extract at each concentration was almost double of the AQ extract value.

3.7. In vitro a-amylase inhibitory assay A dose-dependent increase in the inhibitory activity of Tb fruit extracts against a-amylase enzyme was observed (Fig. 5). EA extracts in the concentration range (20
100 mg/ml) displayed comparatively better a-amylase inhibitory potential (34.72 § 1.42 to 85.89 § 2.64%) with IC50 value of 43.5 mg/ml as compared to the activity of AQ extracts (19.91 § 2.31 to 67.13 § 2.44%) having IC50 74.8 mg/ml. Acarbose, an a-amylase inhibitor was used as a standard drug for comparison which showed 80.39§2.17% inhibition of enzyme at 33.33 mg/ml.

ethyl acetate extract

0.5 0.4 0.3 0.2 0.1 0.0 1.31

2.63

5.26

7.89

10.52

13.15

concentration of extracts (µg/ml) Fig. 2. The reducing power of T. bellirica fruit aqueous and ethyl acetate extracts. The results are expressed as mean § SD of three replicates.

312

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Aqueous extract

*

60

% HRSA

300

Ethylacetate extract

*

* *

40

*

Rat bodyweight (g)

80

20

280

Group 1 Group 2

260

Group 3 Group 4

240

Group 5

220 200 0

0 2.66

5.33

8

10.66

concentration of extracts (µg/ml) Fig. 3. Hydroxyl radical scavenging activity (HRSA) at different concentration of T. bellirica fruit aqueous and ethyl acetate extracts. The results are expressed as mean § SD of three replicates (*p  0.05).

µg PGE/g of sample

100

Aqueous extract

Ethyl acetate extract

*

80

*

*

60 40

* *

20 0

6.66

13.33

20

26.66

7

13.33

33.33

Concentration of extracts (µg/ml)

Fig. 4. Total antioxidant capacity determination of T. bellirica fruit aqueous and ethyl acetate extracts by phosphomolybdate method. The AO capacity was expressed as mg PGE/g of sample (propyl gallate equivalent/g). Each value is represented as mean § SD (*p  0.05).

14 21 Number of days

28

Fig 6. Effect of administration of T. bellirica fruit extracts on the body weights of normal and alloxan-induced diabetic rats. The results are expressed as mean § SD (n = 5). Group1: normal control; Group2: diabetic control; Group3: diabetic rats treated with glibenclamide (5 mg/Kg); Group 4: diabetic rats fed with aqueous extract (250 mg/kg); Group5: diabetic rats fed with ethyl acetate extract (250 mg/kg).

extract treated diabetic rats, the daily administration of EA extract exhibited relatively greater weight gain than AQ extract after 28 days. An increase in blood glucose level of Group 2 diabetic rats was observed from day 1 (280 mg/dl) to day 28 (370 mg/dl) while in the normal control group it was virtually constant between 96
98 mg/dl during the experimental period (Fig. 7). A significant difference was observed in the blood glucose level between normal and diabetic rats. Up to 2 weeks minor decline in sugar level was observed in glibenclamide-treated diabetic rats (Group 3) as compared to diabetic rats. However, in the next two weeks, a rapid reduction in blood glucose level (145 mg/dl) was seen in group 3 rats signifying noteworthy hypoglycemic action of glibenclamide. Tb AQ and EA extracts (250 mg/kg) treatment in hyperglycemic rats for 4 weeks led to a marginal reduction in blood glucose from 280 mg/dl to 237 mg/dl.

3.9. Biochemical analysis 3.8. Effects of extracts on body weight and blood glucose level in diabetic rats In normal control rats (Group 1) gradual increase in body weight of rats from 250 g to 288 g was observed over a period of 28 days while in diabetic rats (Group 2) body weight regularly decreased from 248 g to 222 g (Fig. 6). Group 3 rats (glibenclamide treated diabetic rats) showed an increase in body weight. Similarly, Group 4 and Group 5 diabetic rats fed with AQ and EA extracts (250 mg/kg), respectively revealed weight gain. However, in extract-treated rats weight gain was a little lower than that of Group 3 rats. Among

100

aqueous extract

ethyl acetate extract

400

*

80

*

*

60 40

Blood glucose mg/dl

% Inhibition

There was a significant upsurge in total cholesterol, LDL and triglycerides levels and a decline in HDL level in alloxan treated rats (Group 2) in comparison to the control group. Diabetic rats treated with EA extract showed a maximum reduction in total cholesterol, LDL and triglycerides coupled with significant increment in HDL levels as compared to AQ extract (Table 1). Serum total protein was significantly (p < 0.05) reduced while serum urea, creatinine and uric acid levels were elevated significantly in alloxan-induced diabetic rats in comparison to the normal rats. In comparison to AQ extract, EA extract accounted for significant restoration (p < 0.05) of total protein serum creatinine, urea and uric acid levels in diabetic rats (Table 2).

*

*

20

300

Group 1 Group 2 Group 3

200

Group 4 Group 5

100

0 0

0

20

40

60

80

100

concentration of extracts (µg/ml) Fig. 5. a-amylase inhibitory effect of aqueous and ethyl acetate extracts of T. bellirica fruit. The results are expressed as mean § SD of three replicates (*p  0.05).

7

14 21 Number of days

28

Fig. 7. Effect of administration of T. bellirica fruit extracts on blood glucose of normal and alloxan-induced diabetic rats. The results are expressed as mean § SD (n = 5). Group1: normal control; Group2: diabetic control; Group3: diabetic rats treated with glibenclamide (5 mg/Kg); Group 4: diabetic rats fed with T. bellirica aqueous extract (250 mg/kg); Group5: diabetic rats fed with T. bellirica ethyl acetate extract (250 mg/kg).

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Table 1 Effect of administration of T. bellirica fruit extracts on the serum lipid profile of normal and alloxan-induced diabetic rats. Group

Total cholesterol (mg/dl)

LDL (mg/dl)

HDL (mg/dl)

Triglyceride (mg/dl)

Group 1 Group 2 Group 3 Group 4 Group 5

76.21 § 0.45 140.65 § 0.68a 87.38 § 0.25b 114.54 § 0.42b 95.26 § 0.31b

39.53 § 0.58 90.39 § 0.35a 51.18 § 0.48b 65.81 § 0.48b 57.17 § 0.56b

40.28 § 0.41 25.76 § 0.57a 35.62 § 0.51b 28.47 § 0.49b 30.51 § 0.42b

99.37 § 0.58 140.23 § 0.41a 103.38 § 0.51b 120.87 § 0.57b 108.29 § 0.39b

Abbreviations: LDL
low-density lipoprotein; HDL
high-density lipoprotein. Each value represents mean § SD where n = 5. a Comparison between group 1 and group 2 (p < 0.05). b Comparison between group 2 and group 3, 4 and 5 (p < 0.05). Group 1: normal control; Group 2: diabetic control; Group 3: diabetic rats treated with glibenclamide (5 mg/Kg); Group 4: diabetic rats fed with T. bellirica aqueous extract (250 mg/kg); Group 5: diabetic rats fed with T. bellirica ethyl acetate extract (250 mg/kg).

4. Discussion Medicinal plants contain therapeutically active compounds that can act as free radical scavengers, thus mitigating oxidative stressrelated pathologies (Firuzi et al., 2011). Chemical analysis of Tb fruit extracts revealed the presence of secondary metabolites like phenolics, flavonoids, terpenoids, carbohydrate, alkaloids, cardiac glycosides, and saponins (Lobo et al., 2010). In the current study, it was observed that total phenolic content in EA extracts was about 1.6 times higher than the AQ extract. Similarly, EA extract was also rich in flavonoids content (190 § 0.97 mg QE/g sample) in comparison to AQ extract. Our results regarding the number of phenolics and flavonoids are in agreement with the reports on aerial parts of Tb with minor deviations (Rashed et al., 2014). Plant secondary metabolites including phenolics and flavonoids are bioactive compounds that have been reported to possess antimicrobial, antioxidant, antiinflammatory and other pharmacological activities (Mishra et al., 2013b). Substantial free radical scavenging, reducing power, hydroxyl radical scavenging and total antioxidant capacity observed during the current study validated the antioxidant efficacy possessed by Tb fruit (Figs. 1
4). Both the extracts displayed the concentration dependent discoloration of the reaction mixture in DPPH assay demonstrating appreciable free radical scavenging capacity which suggests their proton donating ability (Kumar and Pandey 2014). Comparatively superior DPPH radical scavenging activity (EC50 5.79 mg/ml) and HRSA (EC50 8.79 mg/ml) of EA extract could be attributed to the presence of higher content of phenolic and flavonoids (Fig. 1) (Kumar et al., 2012; Oyaizu 1986). The extracts also exhibited considerable reducing power during in vitro assays. Comparatively better efficacy was observed for EA extract (Fig. 3). The study showed that test extract had an appreciable ability to convert Fe3+ into Fe2+, that is, the capability to donate hydrogen atom that breaks the free radical chain, a property of antioxidants (Pandey et al., 2012). Reports on other natural products have emphasized a direct correlation between antioxidant activity and reducing the power of plant extracts (Kumar and Pandey, 2015). The phosphomolybdenum method is used for the assessment of total antioxidant power based on the reduction of molybdenum (VI) by the antioxidants and the formation of a green molybdenum (V) complex, which shows maximum absorbance at 695 nm (Prieto et al., 1999). The activity shown by EA extract was nearly double of the value shown by AQ extract in the phosphomolybdenum assay (Fig. 4). The difference in the antioxidant capacity of both the extracts may be attributed to differences in the amount of bioactive compounds viz., phenolics and flavonoids. Results of in vitro antioxidant assays suggested over all superior antioxidant capability in EA extracts of Tb fruit. The extracts were also evaluated for a-amylase inhibitory activity. EA extract exhibited a noteworthy reduction in starch breakdown from 34% to 85% at lowest to highest concentrations (20
100 mg/ml) while AQ extract showed 19
67% inhibition at the same

concentration range (Fig. 5). Lower IC50 values of EA (43.5 mg/ml) and AQ (74.8 mg/ml) suggested promising a-amylase inhibitory activity in Tb extracts. This action of extracts could be helpful in managing type 2 diabetes. Besides conventional treatment of diabetes, there is another group of drugs introduced for the control of type 2 diabetes is denoted by the inhibitors of a-amylase. Acarbose and miglitol are a-amylase inhibitors currently used in clinical practice. These drugs diminish the digestion of starch to more simple sugars (Banerjee et al., 2017). Hence inhibition of a-amylase activity by extracts may lead to a decrease in blood glucose level because of lesser availability of the monosaccharide for absorption in the mucosal border of the small intestine. The delay in the glucose absorption rate is accountable for maintaining the blood glucose level in hyperglycemic individuals. Several natural compounds including castonospermine, an alkaloid isolated from Castano sperumaustrale seed have well-known a-glucosidase inhibitory action (Day, 1990). The therapeutic agent miglitol, an a-amylase inhibitor is derived from moranoline (1-deoxynojirimycin) which was isolated from mulberry root bark (Yoshikunni, 1998; Kurihara et al., 2003). These a- amylase inhibitors are also referred to as starch blockers as they hinder or slow down the absorption of starch into the body mainly by obstructing the hydrolysis of 1,4-glycosidic linkages of starch and other oligosaccharides into maltriose, maltose and other simple sugars. It has been widely recognized that polyphenols have a-amylase and a-glucosidase inhibitory activity (Kwon et al., 2008; Kang et al., 2014). Flavonoids are a major group of polyphenolic compounds that have been reported to possess inhibitory activity against a-amylase and a-glucosidase (Tadera et al., 2006; Williams, 2013). In relation to their structure, number and position of their hydroxyl groups in the molecule which is determining factors for enzyme inhibition. The inhibitory activity increased considerably with an increase in the number of the hydroxyl group on the B ring (Tadera et al., 2006). The present study revealed the presence of a larger amount of polyphenolics in both the extracts of Tb fruit. Hence these phenolics and flavonoids might be associated with a-amylase inhibitory action of Tb extracts. Since both the extracts displayed significant in vitro a-amylase inhibitory activity, their anti-diabetic efficacy was further evaluated in alloxan-induced diabetic rats. The alloxan causes free radical generation in the pancreas that damages b-cells which in turn leads to insulin deficiency and consequently results in hyperglycemia (Sharma et al., 2010). Alloxan also causes oxidative stress in liver tissue and decreases the level of antioxidant enzymes which are responsible for a significant increase in aldehyde products of lipid peroxidation that finally leads to hepatotoxicity in rats (El-Missiry and El-Gindy, 2004). Diabetic rats showed a significant elevation in glucose, total cholesterol, LDL triglycerides, creatinine, urea and uric acid with a simultaneous decrease in HDL and total protein. The daily administration of the glibenclamide and Tb fruit extracts (EA and AQ) in groups 3
5 for 4 weeks caused a decline in blood glucose level and further led to

314

A. Gupta et al. / South African Journal of Botany 130 (2020) 308
315 Table 2 Effect of administration of T. bellirica fruit extracts on the serum markers in normal and alloxan-induced diabetic rats. Group

Total protein (mg/dl)

Uric acid (mg/dl)

Creatinine (mg/dl)

Urea (mg/dl)

Group 1 Group 2 Group 3 Group 4 Group 5

6.54 § 0.42 4.18 § 0.29a 6.35 § 0.48b 5.43 § 0.32b 5.95 § 0.42b

1.78 § 0.31 2.75 § 0.37a 1.89 § 0.24b 2.21 § 0.37b 2.04 § 0.29b

0.94 § 0.17 2.19 § 0.21a 1.39 § 0.32b 1.18 § 0.29b 1.08 § 0.15b

24.35 § 3.29 52.20 § 4.04a 30.48 § 3.64b 39.43 § 3.69b 34.05 § 3.32b

Each value represents mean § SD where n = 5. a Comparison between group 1 and group 2 (p < 0.05). b Comparison between group 2 and group 3, 4 and 5 (p < 0.05). Group 1: normal control; Group 2: diabetic control; Group 3: diabetic rats treated with glibenclamide (5 mg/Kg); Group 4: diabetic rats fed with T. bellirica aqueous extract (250 mg/kg); Group 5: diabetic rats fed with T. bellirica ethyl acetate extract (250 mg/kg).

the maintenance of body weight in the diabetic rats (Figs. 6 and 7). Glibenclamide in hyperglycemic condition exhibits blood glucose lowering effect via stimulating insulin secretion from the remaining b-cells of the pancreas. The hypoglycemic activities of Tb fruit extracts may be attributed due to their a-amylase inhibitory and antioxidant activities. The antioxidant activity could be responsible for quenching of the alloxan mediated free radical generation in the pancreas and thereby protecting further damages to b-cells. Several studies in the past have verified the effectiveness of antioxidant food supplements as an approach for controlling diabetic complications (Sabu and Kuttan, 2002; Latha and Daisy, 2010). The study revealed that oral administration of EA extract of Tb fruit significantly decreased blood glucose and lipid levels i.e., total cholesterol, LDL and triglycerides, and increased HDL cholesterol level as compared to AQ extract in diabetic rats (Table 1). Abnormalities in lipid profiles are one of the most common complications in diabetes mellitus. High levels of total cholesterol and more importantly LDL-cholesterol in the blood are major coronary risk factors (Tchobroutsky, 1978). Insulin deficiency causes an increase in free fatty acid mobilization from adipose tissue which results in increased production of cholesterol rich LDL particles and dyslipidemia. The decrease in total serum protein in diabetic rats could be correlated with deficiency of insulin resulting in increased protein catabolism and decreased protein synthesis (Latha and Daisy, 2010). Hyperglycemia leads to renal dysfunction which is represented by elevated serum creatinine, urea and uric acid. Present work demonstrated that Tb extracts were effective in restoring these renal function markers to near normal value suggesting its therapeutic efficacy in hyperglycemia (Table 2). In general EA extracts showed better antioxidant and anti-diabetic activity. The antidiabetic effect of Tb fruit extract may be due to the synergistic action of more than one compound present in the extract. Preliminary phytochemical analysis of fruit extracts has revealed the presence of phenolic compounds and tannins as major constituents. Fruit contains 23.60
37.36% tannins such as chebulinic acid, chebulagic acid, 1,3,6-trigalloylglucose and 1,2,3,4,6-pentagalloyl glucose, glucogallin, ellagic acid, gallic acid etc. (Singh et al., 2016; Iwai, 2008). The hypoglycemic activity of Tb fruit extracts might be due to the presence of polyphenolic compounds that suppress the elevated plasma glucose level (Iwai, 2008). Tannins have been shown to exert insulin-like glucose transport stimulatory activity. Moreover, gallotannins such as pentagalloyl glucose are more potent and effective in insulin receptor (IR) binding, IR activation and glucose transport induction (Klein et al., 2007).

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