Antioxidant and Antidiabetes Capacity of Hexane, Ethylacetate and Ethanol Extracts of Durio zibethinus Murr. Root

Evary and Nur: Antioxidant and Antidiabetes Capacity of Hexane, Ethylacetate and Ethanol Extracts of Durio zibethinus Murr. Root

Authors

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INTRODUCTION

The normal metabolic process resulted the formation of Reactive Oxygen Species (ROS) and free radicals in human body.1 The high formation of ROS could lead to human disease including diabetes and its complications.2 The high incidence of diabetes make it be an urgent health problem to cure. The most frequently diabetes incidence is type II diabetes which showed with the high postprandial glucose concentration. One of possible mechanisms of medicinal plants to cure diabetes is reducing postprandial glucose concentration by inhibiting the α-glucosidase enzyme in the intestinal surface.3

Bioactive compounds from plants have searched for their antioxidant and antidiabetic properties. The large quantity of Polyphenols plants exhibited high antioxidant and α-glucosidase activity.4 Some antioxidant mechanism has been found to have a good relationship with the α glucosidase inhibitory activity of medicinal plants, especially for DPPH scavenging activity and inhibiting lipid peroxidation. A research has been proof that the high DPPH and hydroxyl radical scavenging activity of Murraya koenigii leaf extracts resulted the high of their α-glucosidase inhibitory properties.5

Some medicinal properties of Durio zibethinus Murr. have been found early. The chloroform extract of Durio zibethinus fruit pulp and wood bark had been found to show a great antibacterial activity against Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis.6,7 The other medicinal properties of Durio zibethinus Murr. including antioxidant and anti-inflammatory effect of the fruit pulp,8 antidiabetic and antihipercholesterolemia activity of its fruit peel extract.9

In this study, we determine the DPPH scavenging activity and inhibition of β-carotene bleaching activity of difference Durio zibethinus Murr. root extracts. In addition, we examine the α-glucosidase inhibitory activity of the extracts, in vitro. We also perform phytochemical identification of the extracts.

MATERIALS AND METHODS

Chemicals

1, 1 diphenyl-2picril-hydrazyl (DPPH), α-glycosidase from Saccharomyces cerevisiae and para-nitrophenyl- glucopyranoside were products of Sigma-Aldrich. Other chemicals and reagents used were of analytical grade while the water used was glass-distilled.

Plant material

Durio zibethinus Murr. root was obtained from South Sulawesi, Indonesia.

Preparation of extract

The fresh Durio zibethinus Murr. root was washed, dried (using herbs drier) and ground using wood machine. The dried sample was extracted using terraced extraction method with hexane, ethylacetate and ethanol sequentially, by maceration on a sonicator at room temperature and solvent was replenished every 24 h for 3 days to ensure that all possible compounds were extracted. The resulted extracts were filtered and concentrated using a rotary evaporator (Buchi) under reduced pressure at approximately 50oC. The concentrated extracts were air dried using electric fan until they dried well.

Phytochemical constituens determination
Total Phenolic Content

Total phenolic content of the extracs were examined by Folin-Ciocalteu method with slight modification.10 The extract were prepared in methanol and 100 µl of each concentration in 5 ml volumetric flask was added with 2.5 ml Folin Ciocallteu’s reagent (7,5%). The mixtures were vortexted and incubated at room temperature for 10 min. After incubation, 2 ml of NaOH 1% was added and the mixture was vortexted. The volume was adjusted to 5 ml and incubated for 1 h in the dark room. The absorbance was measured at 750 nm against blank with a UV/Vis spectrofotometer (UV 1800 Shimadzu, Japan). Gallic acid was used as standard. The results were expressed as mg gallic acid equivalents (GAE)/g extract. The determination of total penolic compound in the extracts were done in triplicate.

Total Flavonoid Content

Total flavonoid content was determined using aluminium-chloride colorimetric assay.11 Briefly, 1 ml of extract solution in suitable solvent was mixed with 1.5 ml ethanol, 100 l of AlCl3 (10%) and 100 µl of sodium acetate 1M, respectively. The volume was adjusted to 5 ml with ethanol in a volumetric flask and incubated for 30 min. Quercetin was used as standard. The absorbance was measured at 431 nm with a 1800-UV/Vis spectrofotometer (Shimadzu). All measurements were done in triplicate. Total flavonoid content was expressed as mg of quercetine equivalent (mgQE) per gram extract.

DPPH Radical Scavenging Assay12

Evaluation of antioxidant activity was done using DPPH radical scavenging method. In brief, 10mg/ml solution of DPPH in methanol was prepared. This solution (100 µl) was added to 20 µl of extracts with concentration of 10, 50, 100, 150, 200 ppm. Solutions were made up to 200 µl with methanol in a 96 well plate. The mixture was shaken vigorously and allowed to stand at room temperature for 30 min, in a dark room. The absorbance was measured at 515 nm by using Biotek micro plate reader. Reference standard compound being used was ascorbic acid and experiment was done in triplicate. The IC50 value of the sample, which is the concentration of sample required to inhibit 50% of the DPPH free radical, was calculated using Log dose inhibition curve. Lower absorbance of the reaction mixture indicated higher free radical activity. The percent DPPH scavenging effect was calculated by using following equation:

DPPH scavenging effect (%) or Percent inhibition=A0A1A0×100https://s3-us-west-2.amazonaws.com/jourdata/pj/PharmacognJ-10-5-937_g000.jpg

Where A0 was the absorbance of control reaction and A1 was the Absorbance in presence of test or standard sample.

β-Carotene-Linoleic acid Assay

β-Carotene-Linoleic acid Assay was done according to the method developed by Mammadov13 with slight modification. Three mililiter of β-carotene solution (0.2 mg/ml in chloroform) was pippeted into a flask (100 ml) containing 0.12 ml linoleic acid and 1.2 ml of 100% Tween 20. The mixture was evaporated 40oC with agitation for 10 min to remove chloroform. After evaporation, the mixture was immediately diluted with 50 ml distilled water. The distilled water was added slowly to the mixture with vigorous agitation to form emulsion. The assay was done in 96 microplate. Into 180 µl of β-carotene emulsion was added 20 µl of extract solution in 5 different concentration. The mixture was gently shaked using thermomixer at 50oC for 120 min. Absorbance was measured at 450 nm using Biotek Microplate Reader. Blank solution was prepared containing β-carotene emulsion without sample. The total antioxidant activity was calculated using the following equation :

AA = ( 1 – (Ab0 - Abt)/ (As0 – Ast)) × 100%

Where AA is antioxidant activity, Ab0 and As0 are the absorbance of blank and sample at the initial time of incubation respectively. WhileAbt and Ast are absorbance of blank and sample at 120 min of incubation.

α-Glycosidase inhibitory assay14

The α-glucosidase inhibitory activity was determined by measuring the release of 4-nitrophenol from P-nitrophenyl α-D-glucopyranoside. The assay mixtures for these experiments contained 20 µl of sample (extracts) in different concentration added with 14 µl of 5 mM p-nitrophenyl α-glucopyranoside and 52 µl of phosphate buffer pH 7. The mixtures were pre-incubated for 5 min at 37°C. After that 14µl of enzyme solution 0.5 U/ml was added and incubated for 15 min at 37oC. The reaction was terminated by the addition of 100 µl of 100 m Msodium carbonate. The liberated p-nitrophenol was determined at 405 nm using Biotek Microplate Reader. The % inhibition rates were calculated using the formula,

Inhibition(%)=Abs405(control)Abs405(extract)×100Abs405(control)https://s3-us-west-2.amazonaws.com/jourdata/pj/PharmacognJ-10-5-937_g001.jpg

The IC50 values were determined from plots of percent in hibition versus log inhibitor concentration. All tests were performed in triplicate.

Statistical Analysis

All data were analyzed by analysis of variance (ANOVA)

RESULTS AND DISCUSSION

The bioactive compound is summarized in Table 1, antioxidant activity assay in Table 2 and the in vitro α-glucosidase inhibitory activity in Table 3. The quantitative chemical compound analysis of Durio zibethinus showed that the root is rich in total phenolics and total flavonoids content according to the data shown in Table 1. Total Phenolics content of different extracts was significant and varying between 19.55 ± 0.058 to 102.92 ± 0.331 mg GAE/g extract. The flavonoids contents of the extracts in the terms of standard quersetin equivalents (QE) were between 0 to 1.88 ± 0.003 mg QE/g extract. Among the tested extracts, EED has the highest amount of total phenolics and flavonoids compound.The phenolics compound contained of hydroxyl groups which made it soluble in higher polarity solvent such as ethanol.15 The hexane extract showed the lowest content of polyphenol (19.55 GAE mg/g dw) and flavonoid (failed to extract flavonoid content 0 mg QE/g dw) compounds due to its low polarity.

Table 1

Total Polyphenol and Flavonoid of the extracts.

ExtractTotal polyphenol content (mg/g GAE)Total flavonoid content (mg/g QE)
EHD19.55 ± 0.0580
EEAD90.62 ±0,1011.004 ± 0,016
EED102.92 ± 0.3311.88 ± 0.003
Table 2

Antioxidant activity of hexane, ethyl acetate, and ethanol extract of Durio zibethinus Murr

Extractβ-Caroten-Linoleic Acid AssayDPPH Scavanging Acivity
Concentration (µg/ml)Percent Inhibition (%)IC50 (µg/ml)Concentration (µg/ml)Percent Inhibition (%)IC50 (µg/ml)
EED10038.521166.83218.70211.213
20058.495525.926
30064.7211044.236
40075.3561569.330
50080.8042083.354
EEAD10040.337139.5332513.73383.951
20061.3495029.713
30069.6497545.277
40080.28510061.506
50084.43612574.365
EHD10025.628273.5850036.288541.279
20040.73660051.144
30052.74970058.718
40068.95380067.000
50080.01290072.742
Table 3

α-Glucosidase Inhibitory Activity of Durio zibethinus Murr. Extracts.

Extractα-Glucosidase Inhibitory Activity
Concentration (µg/ml)Percent Inhibition (%)IC50 µg/ml
EED567.0123.346
463.629
359.093
224.567
18.997
EEAD25075.98823.693
20074.241
15073.283
10072.701
5055.514
EHD25086.874119.84
20076.232
15060.124
10031.014
5022.029

Polyphenols is a major antioxidant compound in plants.Based on the high phenolics and flavonoids content of the extracts, they could be a potential antioxidant resource. The EED extract has the best antioxidant activity against DPPH with IC50 of 11.21µg/ml compared to standard ascorbic acid which exhibited 50% inhibition at the concentration of 6.56 µg/ml because of the high phenolics and flavonoids content in EED allow it to donate electron such as hydroxyl group to the reactive species to form a non radical DPPH-H form in the homogenous system.13,16

The EEAD extracts also revealed a quite potential antioxidant with IC50 of 83.951 ppm followed with hexane extract 541.28 ppm. The hexane extract showed the lowest antioxiant activity because it contained of low poliphenolic and flavonoid compound. The same pattern also shown from the determination of antioxidant activity of the Durio zibethinus Murr extacts using β-carotene-linoleic acid assay. The three extracts exhibited an antioxidant capacity by protecting oxidation of linoleic acid in β-carotene emulsion to form hidroperoxides which would caused discoloration of β-carotene. The high absorbace of β-carotene emulsion after 120 min of incubation means the high antioxidant capacity of the extract13. Poliphenol compounds exhibited a high benefit for protection of lipid peroxidation as have been studied by Dey17 showed that poliphenol from grapes decreased the liver triglicerol of a rat fed high-fat-high-sucrose diet.

The chemical component in Durio zibethinus Murr are promised as the source of α glcosidase inhibitor because it contained of high of polyphenols and revealed a high antioxidants capacity. In this study, we investigate the activity of different extracts from Durio zibethinus Murr. It was found that the extracts showed different inhibitory activity against α glucosidase enzyme which ranged between 3.346 to 119.84 µg/ml. The EED exhibit the highest activity to inhibit α glucosidase enzyme with IC50 3.346 µg/ml followed with EEAD with IC50 23.693 µg/ml and EHD 119.84 µg/ml.

In this study, the high polyphenol content of the extracts showed potential activity to be usefull for diabetes treatment by two possible mechanisms which were inhibited the α-glucosidase enzyme to decrease and control post prandial hyperglycemia and protect hyperglycemia mediated hepatic injury by decrease generation of superoxide and prevent lipid peroxidation as their capacity to prevent lipid peroxidation in vitro.17

CONCLUSION

In conclusion, this study clearly resulted that Durio Zibethinus Murr. has high potency as antioxidant activity against free radicals in vitro. The antioxidant activity of this potent medicinal plant may be attributed to its compounds potency to scavange the free radicals as well as to inhibit lipid peroxidation. This medicinal plants also has a high potency as antidiabetic agents based on its high power to inhibit the α-glucosidase enzyme in vitro.

ACKNOWLEDGEMENT

The authors Yayu Mulsiani Evary and Muh Nur Amir acknowledges the financial support from Hasanuddin University, Indonesia. We are thankful to Laboratory of Biofarmaka for supporting this research for all instruments needed.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

ABBREVIATIONS

EHD

Hexane extract of Durio zibethinus Murr. root

EEAD

Ethylacetate extract of Durio zibethinus Murr. root

EED

Ethanol extract of Durio zibethinus Murr. Root

DPPH

1,1-Diphenyl 2-picrylhydrazine

GAE

Gallic acid equivalent

QE

Quercetin equivalent

UV-Vis

Ultraviolet-visible.

REFERENCES

1. 

Moskovitz J, Yim MB, Chock PB , authors. Free Radicals and Disease. Archives of Biochemistry and Biophysics. 2002;397(2):354–9

2. 

Giacco F, Brownlee M , authors. Oxidative stress and diabetic complications. Circ Res. 2010;107(9):1058–70

3. 

Hisseyni ES, Kashani HH, Asadi MH , authors. Mode of action of medicinal plants on diabetic disorder. Life Science Journal. 2012;(94):2276–3

4. 

Gao J, Xu P, Wang Y, Wang Y, Hochstetter D , authors. Combined effects of green tea extracts, green tea polyphenols or epigallocatechin gallat with acarbose on inhibition against α-amilase and α-glucosidase in vitro. Molecules. 2013;18(9):11614–23

5. 

Gul MZ, Attuluri V, Qureshi IA, Ghazi IA , authors. Antioxidant and α-glucosidase inhibitory activities of Murraya koenigii leaf extracts. Phcog J. 2012;4(32):65–72

6. 

Sah BP, Pathak T, Sankar S, Suresh B , authors. Phytochemical investigations on the Fruits of Durio zibenthinus Linn. for antimicrobial activity. International Journal of Pharma Sciences and Research (IJPSR). 2014;5(12):878–82

7. 

Sivananthan M, Elamaran M , authors. in vitro evaluation of antibacterial activity of chloroform extract Andrographis paniculata leaves and roots, Durio zibethinus wood bark and Psidium guajava leaves against selected bacterial strains. International Journal of Biomolecules and Biomedicine (IJBB). 2013;3(1):12–19

8. 

Chingsuwanrote P, Muangnoi C, Parengam K, Tuntipopipat S , authors. Antioxidant and anti-inflammatory activities of durian and rambutan pulp extract. International Food Research Journal. 2016;23(3):939–47

9. 

Muhtadi M, Haryoto H, Sujono TA, Suhendi A , authors. Antidiabetic and antihypercholesterolemia activities of rambutan (Nephelium lappaceum L.) and durian (Durio zibethinus Murr.) Fruit Peel Extracts. Journal of Applied Pharmaceutical Science. 2016;6(4):190–4

10. 

Wang ST, Gao W, Fan Y, Liu K, Du Y, Wang LL , authors. et al. Phenol profiles and antioxidant capacities of Bistort Rhizoma (Polygonum bistorta L.) extracts. Royal Society of Chemistry. 2016;6(33):27320–8

11. 

Agbo MO, Uzor PF, Akazie-nNeji UN, Eze-Odorukwe CU, Ogbatue UB, Mbaoji EC , authors. Antioxidat, Total Phenolic and Flavonoid content of Selected Nigerian Medicinal Plants. J Pharm Sci. 2015;14(1):35–41

12. 

Shekhar TC, Anju G , authors. Antioxidant activity by DPPH radical scavenging method of Ageratum conyzoides Linn. leaves. American Journal of Ethnomedicine. 2014;1(4):244–9

13. 

Ramazan M, Pinar I, Vaizogullar E , authors. Havser. Antioxidant Activity and Total Phenolic content of Gagea fibrosa and Romulea ramiflora. Iran. J Chem Chem Eng. 2011;30(3):57–62

14. 

Alagesan K, Thennarasu P, Kumar V, Sankarnarayanan S, Balsamy , authors. Identification of α-glucosidase in hibitors from Psidium guajava Leaves and Syzygiumcumini Linn. Seeds. International Journal of Pharma Sciences and Research (IJPSR). 2012;3(2):316–22

15. 

Castellano G, Tena J, Torrens F , authors. Classification of Phenolic Compounds by Chemical Structural Indicators and Its Relation to Antioxidant Properties of Posidonia Oceanica (L.) Delile. MATCH Communications in Mathematical and in Computer Chemistry. 2012;67:231–50

16. 

Rice-Evans CA, Miller NJ, Paganga G , authors. Structure-antioxidant activity reletionship of flavonoids and phenolic acids. Free Radical Bio. Med. 1996;20(7):933–56

17. 

Dey A, Lakshmanan J , authors. The role of antioxidants and other agents in alleviating hyperglycemia mediated oxidative stress and injury in liver. Food and Function. 2013;4(8):1148–84

GRAPHICAL ABSTRACT

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SUMMARY

  • Active natural products from medicinal plants are good sources for the antioxidant and antidiabetic agent. Natural antioxidants protect human health against oxidative stress and free radicals which cause degenerative disease such as diabetes. One approach to treat diabetes is to reduce gastrointestinal glucose absorption through the inhibition of the α-glucosidase enzyme. In this study, we used Durio zibethinus Murr roots which taken from Wotu, South Sulawesi, Indonesia. We make three different root extracts from Durio zibethinus Murr root by gradual extraction system. The extracts were examined for their DPPH radical scavenging activity, antioxidant activity using β carotene-linoleic acid assay, and their α-glucosidase inhibitory activity by standar method. The measurements were done using Biotek Microplate Reader. Additionally, we were also determined their Total polyphenols and total favonoids quantitatively using spectofotometer UV-Vis. The root extracts of Durio Zibethinus Murr were effectively scavenged DPPH radicals in varied rate. The hexane (EHD), ethyl acetate (EEAD), and ethanol (EED) extracts revealed DPPH radical scavenging activity with IC50 of 541.28, 83.95 and 11.24 µg/ml respectively and their β-carotene-linoleic acid assay showed activity with IC50 of 0, 139.53, and 166.83 µg/ml, respectively. In vitro assay of the α-glucosidase inhibitory activity of the EHD, EEAD, and EED extracts showed an IC50 of 119.84, 23.69, and 3.35 µg/ml, respectively. In this present study, we found that ethanol extract revealed the most active antioxidant and the highest inhibitory activity against α-glucosidase enzyme. The total phenolics contents of the EHD, EEAD, and EED extracts were 19.55 ± 0.058; 90.62 ±0,101; 102.92 ± 0.331 mg GAE/g extracts. The total flavonoid contents of the EHD, EEAD, and EED extracts were 0; 1.004 ± 0,016; 1.88 ± 0.003 mg QE/g extracts. The ethanol extract have the highest polyphenols and flavonoids content than that of other extracts and in accordance with its antioxydant and antidiabetes activity. The high polyphenol content of the extracts showed potential activity to be usefull for diabetes treatment by two possible mechanisms which were inhibited the α-glucosidase enzyme to decrease and control post prandial hyperglycemia and protect hyperglycemia mediated hepatic injury by decrease generation of superoxide and prevent lipid peroxidation as their capacity to prevent lipid peroxidation in vitro. This study proofs the medicinal potencies of Durio zibethinus Murr. root extracts.