Comparative study on DPPH free radical scavenging and alpha-glucosidase inhibitory activities of ethanolic extracts from different parts of durian plant ( Durio zibethinus

F U L L P A P E R Comparative study on DPPH free radical scavenging and alpha-glucosidase inhibitory activities of ethanolic extracts from different parts of durian plant (Durio zibethinus Murr.) Evary, Y.M., Nugroho, A.E. and Pramono, S. Master of Pharmaceutical Science, Faculty of Pharmacy, Universitas Gadjah Mada, 55821, Indonesia Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Hasanuddin University, 90245, Makassar, Indonesia Department of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy Universitas Gadjah Mada, 55821, Yogyakarta, Indonesia Department of Biology Pharmacy, Faculty of Pharmacy, Universitas Gadjah Mada, 55821, Yogyakarta, Indonesia


Introduction
Oxidation process as an essential part in aerobic metabolism resulting in radicals which actually needed in the primer immunity response.However, the imbalance of radicals and the existing antioxidant in the body could harm the cells and cause various diseases such as immunodeficiency, heart disease, stroke, diabetes and cancer (Gülçin, 2012).Type II diabetes mellitus which was resulted from unhealthy lifestyle, high carbohydrate consumption, high stress, and reactive oxygen species exposure has been a high prevalence disease.Treatment of this disease was directed to the reduction of postprandial blood glucose levels.An alphaglucosidase inhibitor which inhibits the metabolism of carbohydrate in the intestine has the benefit in reducing blood glucose and lowering diabetes complication risk (Dewi and Maryani, 2015).
Natural antioxidants such as flavonoid and phenolic compounds have been proved for their benefit in treating those diseases due to their antioxidant activity (Hay et al., 2004;Kang et al., 2012;Nugroho et al., 2013).Phenolic compounds have the ability to inhibit carbohydrate digestion and absorption.Phenolic compounds also can stimulate insulin secretion from βpancreas cells by increasing hepatocellular AMPdependent protein kinase (AMPK) and inhibit the accumulation of lipids in HepG2 cells which can increase glucose levels through gluconeogenesis process (Dey and Lakshmanan, 2013).Quercetin has been proven its ability to repair the proliferation of pancreas cells by inhibiting the cyclin-dependent kinase 1a (cdk1a) expression (Dey and Lakshmanan, 2013).Some research has been conducted to durian (Durio zibethinus Murr.).An examination of the ethanolic extract of durian peels in vivo showed that the extract has the ability to reduce the blood glucose of alloxan-induced diabetic mouse with a dose of 500 mg/ Kg BW.It was stated by Muhtadi et al. (2015) that a high dose of durian peels extract is required to achieve hypoglycemia effect.In vitro examination of the methanol extract of Durio kutejensis and Durio dulcis wood bark showed inhibition of alpha-glucosidase enzyme by 63.82% (in an amount of 5 µg/mL extract) and by 61.90% (in an amount of 15 µg/mL extract) respectively (Yusro et al., 2016).

Durio zibethinus
Murr. is a widely spread species of durian in Indonesia and the root have been traditionally used by Wotu society, South Sulawesi as a traditional medicine to treat diabetes.Besides that, the DFRS activity of ethanolic fruit peels extracts of Durio zibethinus Murr.also has been done and it was revealed that the IC 50 value was 372.08±73.02µg/mL, indicating a low antiradical activity.Therefore, a comparative study was conducted on the DFRS and AGI activities of the different parts of durian extract (Durio zibethinus Murr.) to obtain a proper natural source of antioxidant and antidiabetic agent.

Plant materials
The roots, twigs, leaves and inner fruit barks of durian (Durio zibethinus Murr.) were obtained from Wotu, South Sulawesi, Indonesia.The samples were taken on June 2018.

Preparation of extract
Root, twig, leaves and inner fruit bark of durian (Durio zibethinus Murr.) were sorted, cleaned, and dried using oven.Dried samples were ground and macerated with 70% ethanol for three times.The macerate was collected and concentrated with the rotary evaporator 60°C and air-dried until thick extract obtained.

Determination of total phenolic content
The quantitative phenolic content of the extracts was measured using Folin-Ciocalteu assay.Briefly, 2.5 mL Folin-Ciocalteu 7.5% and 400 µl of samples were mixed and incubated for 8 mins.After the addition of 2 mL of 5% sodium hydroxide, the mixture was left for 1 hr at room temperature.The absorbance of each solution was read using spectrophotometer UV-Vis at 653.5 nm.The standard gallic acid (4-20 µg/mL) was plotted to conduct calibration curve.The total phenolic content was expressed in the term of Gallic Acid Equivalents (GAE) in mg/g dry extract (Ministry of Health RI, 2012).

Determination of total flavonoid content
The total flavonoid level of the extracts was evaluated using aluminium trichloride colorimetric method.Briefly, 200 µL of sample, 1.5 mL of 95% of ethanol, 100 µL of 10% aluminium trichloride and 100 µL of 1M sodium acetate were homogenized with ethanol until the volume reaches 5 mL in a volumetric flask.The solution was incubated for half an hour.The absorbance of each solution was read spectrophotometrically at 443 nm.The standard calibration curve (2-10 µg/mL) was plotted using quercetin.The total flavonoid content was expressed in the term of Quercetin Equivalents (QE) in mg/g dry extract (Ministry of Health RI, 2012).

DPPH radical scavenging assay
Free radical scavenging activity of Durio zibethinus Murr.root, twig, inner fruit bark and leaves extracts were carried out using the DPPH scavenging method.In brief, 16 mg DPPH was dissolved in 250 mL methanol p.a. and vortexed until completely dissolved.To 100 µL of methanolic DPPH solution was added 100 µL of sample.The mixture was left at room temperature for half an hour and the absorbance was observed at 515 nm.FULL PAPER Vitamin C was the positive control of the measurement.DPPH solution at the same concentration without sample addition was used as the negative control.The measurement was done in triplicate.The DFRS activity of each solution was calculated according to the following equation: Where A control was the absorbance of negative control and A sample was the absorbance in presence of test or standard sample (Anthony and Saleh, 2013).

Alpha-glucosidase inhibitory assay
The AGI activity was evaluated by measuring the release of 4-nitrophenol from P-nitrophenyl α-Dglucopyranoside (pNPG) as the substrate of alphaglucosidase enzyme.The measurements contained 100 µl of phosphate buffer pH 6.8, 10 µL of the αglucosidase enzyme (0.5 U/mL), and 20 µL of sample (extracts or acarbose) in different concentration.The mixture was preincubated for 15 min at 37°C.After that 20 µL of 5 mM substrate was added and leave for 20 mins at 37°C.Addition 50 µL of 100 mM sodium carbonate was used to end the reaction.The release of pnitrophenol was determined at 404 nm using microplate reader.The % inhibition level were calculated using the formula: IC 50 values were calculated using SPSS 20 version software.The probit score of percent inhibition was plotted with log concentration of inhibitor.All tests were performed in triplicate (Telagari and Hullatti, 2015).

Statistical analysis
All data were shown as mean ± SEM for at least three replications for each prepared sample.The SPSS ver.20.for windows was used to determine the significant differences of comparisons of each extract evaluated.One-way analysis of variance (ANOVA) followed by Tukey's and Bonferroni test was done.Results with p-value less than 0.05 were considered as statistically significant.

Determination of total phenolic content
Phenolics are ubiquitously secondary metabolites present in plants.They have been found to have high antioxidant and antidiabetic activity (Dewi and Maryani, 2015).This assay is a simple method and useful for describing and standardizing natural materials.The total phenolic content of durian root, twig, inner fruit bark and leaves extracts is demonstrated in Table 1.The DR extract revealed the highest total phenolic content at 94.01±0.018mg GAE/g dry extract, approximately three -fold more than the other three extracts.Khan et al. (2013) previously compare several parts of methanolic white mulberry extracts (flowers, root bark, leaves and stem bark) and found the root bark extract also shown to have a high phenolic content (165 mg GAE/g dry extract).The DT, DIFB, and DL exhibited phenolics compound of 31.65,12.96, and 27.38 mg GAE/g dry extract, respectively.Statistical analysis of mean of phenolic content of the extracts evaluated revealed significant differences with p< 0.05.It indicated that the extracts consist of significantly different phenolic content, respectively.

Determination of total flavonoid content
Flavonoids are the widely spread group of plant phenolic compounds, indicated with a benzopyrone structure.The total flavonoid content of the extracts was expressed as quercetine equivalent in milligrams per gram dry extract and showed in Table 1.The result showed that the DL extract had the highest flavonoid content at a concentration of 2.97 mg QE/g dry extract, followed with DR, DT, DIFB extracts at a concentration of 0.44, 0.37, and 0.25 mg QE/g dry extract, respectively.Statistically, the durian root and twig extract showed a closely the same flavonoid content.Nevertheless, the overall extracts contained relatively low flavonoid compounds.

DPPH free radical scavenging activity
The DFRS assay is a fast and simple method to determine the intrinsic ability of an extract or a substance to donate proton or electron to the DPPH radical in a homogenous system (Siriwardhana and Shahidi, 2002).The DFRS activity of the durian part extracts was demonstrated in   FULL PAPER showed a strong radical scavenging activity while the other two (inner fruit bark and leave) extracts showed a low capacity as presented in Figure 1.This phenomenon might be attributed to their phenolic content as have been found by Rabeta et al.The high scavenging potential of Vitex negundo might be due to its high phenolic content in the methanolic extract (Rabeta et al., 2013).In this assay, vitamin C was used as positive control with IC 50 of 5.05±0.02µg/mL.
The relationship between DFRS activity (Y) with total phenolic content revealed a coefficient of determination (R 2 ) of 0.7194 (Figure 2.).The result suggested that phenolic compounds have a significant correlation to the DPPH free radical scavenging activity of the extracts.Previous studies have explained the role of phenolic compounds with free hydroxyl group in scavenging DPPH free radicals.The higher phenolics content of the extracts, the stronger their scavenging activity (Siriwardhana and Shahidi, 2002;Khan et al., 2013;Dewi and Maryani, 2015).

Alpha-glucosidase inhibitory (AGI) activity assay
One approach in diabetes management is to maintain the normal blood glucose through inhibition of its absorption and metabolism.The AGI activity of the extracts were 0.58 µg/mL for DR extract, 1.55 µg/mL DT extract, and 64.43 µg/mL DIFB extract (presented in Table 2).The leaves extract did not show any inhibition against the enzyme.The AGI of durian part extracts varied but they were more potent (except leaves extract) than acarbose which was used as a positive control with IC 50 of 781.63 µg/mL (0.782 mg/mL).In the previous study performed by Telagari and Hullatti (2015) using the similar method, the acarbose activity has been examined and showed inhibitory activity with IC 50 of 0.93 mg/mL (Telagari and Hullatti, 2015).The relationship between the concentration and AGI activity is as shown in Figure 3.In this study, the most potent alpha-glucosidase inhibitor extract was the DR extract, followed with DT and DIFB extracts.The DR and DT extracts had a potential activity based on their high content of phenolic compounds.Jang et al. (2015) and Choi et al. (2015) also reported that the root bark and twig extracts of mulberry revealed a high antioxidant and AGI activities due to its potential phenolic compounds.

Conclusion
The result of this study clearly demonstrated that phenolic compounds from DR and DT extracts contributed to their DFRS and AGI activities.The findings indicated that durian extracts especially DR and DT extracts could be considered as potential natural sources of antidiabetes and antioxidant agents.

Figure 1 .
Figure 1.Relationship between concentration of root (A), twig (B), inner fruit bark, and leave extracts of durian plant with their DPPH free radical scavenging activity

Table 2 .
The root and twig extract

Table 1 .
Total phenolic and flavonoid content of ethanolic extracts from different parts of Durio zibethinus Murr.
*Expressed as mg GAE /g extract, **Expressed as mg QE /g extract.Means with different superscript in each column are significantly different at p<0.05 according to Tukey's test (n=3) eISSN: 2550-2166 © 2019 The Authors.Published by Rynnye Lyan Resources

Table 2 .
). DPPH free radical scavenging and alpha-glucosidase inhibitory activities of ethanolic extracts from different parts of Durio zibethinus Murr.Means with different superscript in each column are significantly different at p<0.05 according to Tukey's test (n=3)