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ORIGINAL ARTICLE
Year : 2017  |  Volume : 8  |  Issue : 1  |  Page : 20-24  

Low inhibition of alpha-glucosidase and xanthine oxidase activities of ethanol extract of Momordica charantia fruit


Department of Pharmaceutical Chemistry, Kulliyah of Pharmacy, International Islamic University , Kuantan, Pahang DM, Malaysia

Date of Web Publication21-Apr-2017

Correspondence Address:
A Khatib
Department of Pharmaceutical Chemistry, Kulliyah of Pharmacy, International Islamic University Malaysia, Kuantan, Pahang DM, Malaysia.
Malaysia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0976-9234.204906

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   Abstract 

Objective: To evaluate the α-glucosidase and xanthine oxidase inhibitory activities of different ethanolic and aqueous extracts of Momordica charantia fruit. Materials and Methods: The M. charantia fruits were extracted using different concentrations of ethanol in water (0, 20, 40, 60, 80 and 100 %, v/v). The samples from different ethanolic and aqueous extracts of M. charantia fruit (0, 20, 40, 60 and 100 % ethanol in water [v/v] extracts) were tested for α-glucosidase and xanthine oxidase inhibitory activities. Quercetin and allopurinol were used as positive controls for α-glucosidase and xanthine oxidase inhibitory assay, respectively. The samples were also tested for phytochemicals screening such as alkaloid, phenol, steroid, tannin, coumarins and flavonoid. The significant difference was determined by one-way ANOVA test with a Tukey comparison at confidence interval of 95%. Results: Phytochemical screening showed the presence of coumarin, alkaloid, steroid and phenol in the extracts. The six different M. charantia extracts showed low inhibition on α-glucosidase and xanthine oxidase activities. Conclusions: The antidiabetic activity of the ethanolic and aqueous extracts of M. charantia fruit was not through the inhibition of α-glucosidase and xanthine oxidase.

Keywords: α-glucosidase, antidiabetes, antioxidant, Momordica charantia, xanthine oxidase


How to cite this article:
Khatib A, Perumal V, Ahmed Q U, Uzir B F, Murugesu S. Low inhibition of alpha-glucosidase and xanthine oxidase activities of ethanol extract of Momordica charantia fruit. J Pharm Negative Results 2017;8:20-4

How to cite this URL:
Khatib A, Perumal V, Ahmed Q U, Uzir B F, Murugesu S. Low inhibition of alpha-glucosidase and xanthine oxidase activities of ethanol extract of Momordica charantia fruit. J Pharm Negative Results [serial online] 2017 [cited 2017 Sep 25];8:20-4. Available from: http://www.pnrjournal.com/text.asp?2017/8/1/20/204906


   Introduction Top


M. charantia belongs to the Cucurbitaceae family.[1],[2] Native people from all over the country use fruit juice and leaf tea of this plant as a laxative and stimulant, as well as for the treatment of diabetes, colic, wounds, infections and hepatitis. It has been reported scientifically to be used as anti-diabetic and anti-inflammatory agents.[3],[4],[5],[6] However, the mechanism of action of this plant as an anti-diabetic agent is still unclear. The objective of this study was to evaluate the mechanism of action of M. charantia fruit for its anti-diabetic activity through the α-glucosidase and xanthine oxidase inhibitory activities.


   Materials and Methods Top


Chemicals

Ethanol and acetone were purchased from R and M Marketing (Essex, UK), while quercetin, p-nitrophenyl-α-D-glucopyronase (PNP-glucoside), and xanthine were obtained from Sigma-Aldrich Chemie (Missouri, USA). Potassium dihydrogen phosphate, α-glucosidase from Saccharomyces cerevisiae and dimethyl sulfoxide (DMSO) were purchased from HmbG Chemicals Inc (Hamburg, Germany), Megazyme (Wicklow, Ireland) and Fisher Scientific (Loughborough, UK), respectively. Glycine and allopurinol were obtained from Nacalai-Tesque, Inc (Kyoto, Japan). Xanthine oxidase from buttermilk was obtained from Merck (New Jersey, U.S.A).

Plant collection and extraction

The fruits of M. charantia were collected from the farm located in Perak, Malaysia. It was deposited in the Herbarium Kulliyyah of Pharmacy, International Islamic University for species verification (PIIUM 0215). The twelve weeks old fruit samples were randomly collected from the farm. The fruit obtained were cut to remove the seeds. Then, the fruits were washed and frozen using liquid nitrogen prior freeze drying. It was followed by grinding into a fine powder and kept at -80°C in deep freezer before further analysis. A total of 36 samples for the extraction were arranged in which six duplicates for each of six different ethanolic and aqueous extracts were prepared. The samples were placed inside the conical flask with mass of five g each. The samples were mixed with 150 mL of different percentage of ethanol in water (0, 20, 40, 60, 80 and 100 %, v/v) and sonicated for 30 min. After sonication, the filtration procedure was done using filter paper followed by evaporation of solvent using rotatory evaporator (Buchi, Flawil, Switzerland) at 40oC ± 1oC before freeze drying. The samples were then prepared and stored at -80 C until further analysis. The yield of extraction was calculated by the following equation:



Phytochemical analysis

The M. charantia ethanolic and aqueous extracts were subjected to phytochemical screening for the presence of various constituents such as phenol, tannin, alkaloid, steroid, flavonoid and coumarin.[7],[8],[9]

Assay for α -glucosidase inhibitory activity

Briefly, as much as 5, 50, 100, 150 and 200 mg of the M. charantia's fruits of six different ethanolic and aqueous extracts were dissolved in one mL of DMSO. While for the positive control, one mg of quercetin was dissolved in one mL of DMSO. A solution of 30 mM phosphate buffer (pH 6.5) was used to prepare stock solutions of the plant extracts to form a final concentration of 160 µg/mL. Then ten µL/well of sample was mixed with 15 µL/well of α-glucosidase type 1 from Saccharomyces cerevisiae enzyme (Megazyme, Ireland) which was prepared from 50 mM buffer (pH 6.5). Then 115 µL/well of 30 mM buffer (pH 6.5) was added and incubated for five min at room temperature. Then 75 µL/well of PNP-glucoside was added to initiate reaction and incubated at room temperature for 15 min. Lastly, about 50 µL glycine at pH ten was added to stop the reaction. The final volume of 250 µL of plant extract, positive control, enzyme, 30 mM buffer and substrate were located in 96-well microplates. The absorbance (A) was measured using microplate reader (Tecan, Männedorf, Switzerland) at 405 nm to represent the amount of p-nitrophenol released from PNP-glucoside (A).10 The IC50, the sample concentration needed to inhibit 50% α-glucosidase was determined using linear regression analysis. The equation given below was used to calculate the inhibitory activity (%):

Inhibitory activity (%) = [(Acontrol – Asample) / (Acontrol) x 100 %]

Assay for xanthine oxidase inhibitory activity

Xanthine oxidase (XO) inhibitory activity was measured using a spectrophotometer (Tecan Nanoquant Infinite M200, Switzerland) at 295 nm under aerobic condition. The reaction mixture contained 50 mM phosphate buffer (pH 7.5), 0.15 mM xanthine and 0.3 U/ml enzymes (xanthine oxidase from buttermilk). The absorbance (A) increment at 295 nm indicates the formation of one mmol of uric acid/ min at 25 °C. Different concentrations (0.69, 1.39, 2.78, 5.56, and 6.94 mg/ml) of M. charantia crude extracts were dissolved in five % dimethyl sulfoxide (DMSO) in buffer, which gives sample concentration of 100 µg/ml, 200 µg/ml, 400 µg/ml, 800 µg/ml and 1000 µg/ml, respectively in the assay. Allopurinol (100 µg/ml), a known XO inhibitor, was used as the positive control.11,12 All determinations were in triplicates and half maximal concentration (IC50) values were calculated from the percentage of inhibition. XO inhibition activity was expressed as the percentage inhibition of XO as follows:

Inhibitory activity (%) = [(Acontrol – Asample) / (Acontrol) x 100 %]


   Results Top


Phytochemical Analysis

[Table 1] shows the result of phytochemical screening of M. charantia fruit extracted using different percentage of ethanol in water (0, 20, 40, 60, 80 and 100%, v/v). It was observed that 100% of ethanolic extract of M. charantia fruit had phenols, alkaloids, steroids and coumarins in appreciable amounts. However, 80% of ethanolic extract of M. charantia fruit showed the presence of coumarins in abundance and the least number of alkaloids and steroids. The other ethanolic extracts of M. charantia fruits i.e. 0, 20, 40 and 60% showed only the presence of alkaloids and coumarins in little amounts.
Table 1: The result of phytochemicals screening of Momordica charantia fruit extracted using different percentage (v/v) of ethanol in water

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Assay for α -glucosidase inhibitory activity

The IC50 values for α-glucosidase inhibitory activity and the yield of each extract are shown in [Table 2]. The results showed that all extracts have a low α-glucosidase inhibitory activity with the IC50 values higher than 1.00 mg/mL. The 80% of ethanol extract was the extract with the most potent inhibitory activity (the IC50 value = 3.97 ± 12.29 mg/mL). The α-glucosidase inhibitory activity was reduced in solvent mixtures up to aqueous extract, with IC50 value of 6.98 ± 33.80 mg/mL, followed by 20% ethanol extract, which had a value of 6.53 ± 29.52 mg/mL. The 40, 60 and 100% ethanol extracts had values of 5.59 ± 21.19 mg/mL, 5.26 ± 21.28 mg/mL and 4.45 ± 17.17 mg/mL, respectively. The differences in IC50 values and extraction yields are due to the different type of metabolites extracted in different polarity of the solvents. The highest yields were obtained with 80% ethanolic extract (62.03%) while the 40% ethanolic extract gave the lowest yields (23.36%).
Table 2: Comparison of yields of extraction and α-glucosidase inhibitory activity of ethanolic extracts of Momordica charantia fruit

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Xanthine oxidase inhibitory activity

XO inhibitory activity was evaluated using microplate reader measurement through the formation of uric acid from xanthine. It was observed that all the M. charantia fruit ethanolic extracts have no inhibition on XO at the highest assay sample concentration of the extract (1.0 mg/ml) in [Table 3]. While the positive control, allopurinol showed a potent XO inhibitory activity (the IC50 = 0.16 μg/mL). Among various concentrations of M. charantia fruit extracts tested, aqueous extract showed the highest XO inhibitory activity (percentage of inhibition= 25.66%) compared to others at 1000 µg/mL of assay sample concentration. These were followed by 40, 60, 20, 100 and 80% ethanolic extracts of M. charantia fruit with the percentage of inhibition 23.66, 23.28, 18.67, 14.11 and 9.6% respectively.
Table 3: Percentage of xanthine oxidase inhibitory assay of different concentrations of ethanol extracts of Momordica charantia fruit

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   Discussion Top


The goals of treatment for patients with chronic hyperglycemia such as diabetes are to avoid from any threatening mortality, to allay the symptoms, to make the sugar level within or near the normal range.[13] M. charantia is found to be one of the most popular medicinal plants traditionally used to treat diabetes due to its hypoglycemic effect that helps reduce blood sugar level to a normal range and prevents or delays the progression of the diabetic complications.[14] Our recent finding using diabetic induced rat showed that the ethanolic extract of M. charantia fruit was able to normalize the glucose level.[15] This finding is in line with other researches, both through animal model and human clinical trial.[16],[17],[18],[19]

The mechanism of action on how this fruit treats diabetes has been extensively studied.[20],[21],[22] Some of possible mechanisms were through the inhibition of carbohydrate-hydrolyzing enzyme α-glucosidase and suppression of oxidative stress enzyme xanthine oxidase.[23],[24] Inhibition of α-glucosidase enzyme delays the digestion of carbohydrates which will consequently require more time to digest carbohydrates to produce glucose, thus, decreasing the rate of sugar absorption and reversing the increase in postprandial plasma glucose.[25] While the inhibition of XO will suppress the production of uric acid and superoxide through the oxidation of hypoxanthine to xanthine.[26] XO is one of the main potential sources of vascular superoxide in oxidative stress causing several vascular diseases including diabetes.[27] Oxidative stress in diabetes mellitus has been shown to coexist with a reduction in the antioxidant status and glycation of proteins, inactivation of enzymes, and alteration in structural functions of collagen basement membrane of pancreatic β-cell.[28]

In the present study, it was noteworthy that different ethanolic and aqueous extracts of M. charantia fruits showed a low inhibition of α-glucosidase. It indicates that these extracts did not promote the anti-diabetic effect through α-glucosidase inhibitory activity. However, the present finding is contradictory to the previous reported studies which might be influenced by different origin, maturity, post-harvest and processing conditions. Different ethanolic extracts of cultivated M. charantia fruit from Hamyang Korea prepared by extraction using 50 and 70% of ethanol under temperature 50 and 70oC were reported to have α-glucosidase inhibitory activity with IC50 less than 1.0 mg/ml.[29] While the aqueous and methanol extracts of M. charantia fruit obtained from a local market in Mauritius was also reported to have α-glucosidase inhibitory activity. The aqueous extract was prepared using soxhlet apparatus for 5 hours at 40oC, while the methanol extract was prepared by triple soaking in 80% methanol at room temperature for 3 days. The reported IC50 values of both extracts ranged between 10 to 20 μg/mL. Both extracts found to inhibit α-glucosidase activity similar to the standard drug i.e. 1-Deoxynojirimycin (IC50= ± 20 μg/mL).[30] Nevertheless, the maturity and the postharvest condition of these fruits were not mentioned. This α-glucosidase inhibitory activity was attributed to the presence of trehalose and polypeptide k in M. charantia fruits.[31] Meanwhile, catechin, α-linolenic acid, α-D-glucopyranoside, vitamin E, D-fructose and squalene were reported as α-glucosidase inhibitors from different plant which are also present in the M. charantia fruit.[32],[33]

In the present study, M. charantia fruit showed low inhibition on XO which is contrary to the previous study.[34] The water and ethanol extracts of M. charantia were reported to have potent XO inhibitory activity with IC50 7.90 and 7.60 μg/mL, respectively. While the IC50 value of a standard reference ascorbic acid was 0.70 μg/mL. The aqueous extract was prepared by extracting with boiling water for one hour, while the ethanol extract was prepared by maceration in ethanol for six days. In addition, prior evidence by another research, a new cucurbitane-type triterpene glycosides 23, 24, 25, 26, 27-pentanorcucurbitane Taiwacin B and a known steroid glycoside [35] isolated from M. charantia significantly inhibited XO. The contradictory result on both α-glucosidase and XO inhibition between the present study and the aforementioned studies were caused by many factors which have been mentioned earlier


   Conclusion Top


The bioactivity of ethanolic and aqueous extracts of M. charantia fruits on α-glucosidase and XO inhibitions was very low. From this study, we can conclude that although the ethanolic extract of M. charantia fruit showed anti-diabetic activity through the rat model in our previous research, [15] the anti-diabetic mechanism should not be through the inhibition of both enzymes.

Acknowledgement

This research was funded by RACE 14-011-0017 and Malaysian My Ph.D Scholarship.

Financial support and sponsorship

Race-14-011-0017 and Malaysian My Ph.D Scholarship

Conflicts of interest

There are no conflicts of interest



 
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    Tables

  [Table 1], [Table 2], [Table 3]



 

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