In Vitro Free Radical Scavenging and Reducing Potentials as well as Inhibitory Potential on α-Amylase and α-Glucosidase Activities of Fruit of Morinda citrifolia (Rubiaceae).

 

Mahadeva Rao US.

Professor, School of Basic Medical Sciences, Faculty of Medicine, Universiti Sultan Zainal Abidin (UniSZA), 20400 Kuala Terengganu, Malaysia.

 

ABSTRACT:

Background: Reactive oxygen and nitrogen species produced via cellular metabolism and from exposure to environmental pro-oxidants appear to contribute to the pathogenesis of chronic disease via free radical damage to lipids, nucleic acids, and proteins. Literature review: Morinda citrifolia (MC) fruit is rich in phytochemicals necessary for treatment of diabetes. Objective: The purpose of this study was to investigate the effect of MC fruit extracts’ free radical scavenging, and reducing potentials as well as inhibitory effect on α-amylase and α-glucosidase. Methods: The of percentage yield, phytochemical screening (both qualitative and quantitative), in vitro antioxidant and antidiabetic assays, and kinetic studies were performed with different solvent extracts of MC fruit pulp. Results: MC inhibits α-glucosidase and α- amylase enzymes in the in vitro studies. The antioxidant and reducing potentials of the fruit extract gives strong biochemical rationale of their therapeutic potential. Conclusion: Therefore the fruit extract of MC may play an important role in the development of nutraceuticals and also in the management of oxidative stress induced diabetes.

 

 

 

INTRODUCTION:

Medicinal plant is an important part of traditional health care system and a veritable health care source for the vast majority of the world population. It was estimated that 70-80% of people worldwide use herb for management of mild to moderate illnesses [1-5].

 

Diabetes mellitus (DM) is an endocrine disorder resulting in obstinate elevation of blood glucose under both fasting and postprandial conditions resulting in micro and macro vascular complications [6]. The prevalence of diabetes is increasing globally and is prophesied to increase by twofold from 150 million in the year 2000 to 300 million by the year 2030 [7]. The uncharacteristic regulation of glucose metabolism that results from a malfunctioning / scarce insulin secretion is the key pathogenic event in DM.

 

Currently available drugs for normoglycemia exhibit adverse side effects on prolonged use. Hence the exploration for novel therapeutic drugs continues. Recent focus has been made towards "functional food", a natural source food purported to have a beneficial health effect for the successful treatment of various ailments especially life style diseases like diabetes.

 

Morinda citrifolia (MC) is the scientific name of the commercially known plant Noni. The name MC is also referring to the botanical name which is originally derived from the two Latin words ‘‘morus’’ imputing to mulberry, and ‘‘indicus’’ imputing to Indian, it belongs to the Rubiaceae family [8]. In Hawaii MC called Noni, whereas in Malaysia it is called Mengkudu and generally in Southeast Asia it is called nhaut, while in the Caribbean, it is called the painkiller bush or

cheese fruit [9].

 

Currently, there are two recognized varieties of MC (M. citrifolia var. citrifolia and M. citrifolia var. bracteata) and one cultivar (M. citrifolia cultivar Potteri). The most commonly found variety is M. citrifolia var. citrifolia, with the greatest health and economic importance. Traditional healers can recognize these varieties by the leaf size and shape, in addition to the fruit odor; however, most research has not distinguished between the different MC varieties yet [10]. In the early 1990s, the first commercialized products derived from MC fruit in USA were lunched [11]. Later, in 1996, MC juice was introduced as a wellness drink, due to numerous reports stating its therapeutic effects [12].

 

This present study seeks to validate the folkloric use of Noni fruit extract (NFE) in the management of diabetes mellitus (DM) and several oxidative stress induced diseases. The study also confined the kinetics of α-amylase and α-glucosidase inhibitory potentials of NFE.

 

MATERIALS AND METHODS:

Plant collection, Preparation and Extraction.

The fruits of Mengkudu were collected in Kuala Terengganu, Malaysia. They were later verified and authenticated at UniSZA Herbarium, Kuala Nerus, Malaysia [Voucher no. 00217]. The skin was peeled off and the edible part was deseeded and chopped into thin pieces, dried at 50-60⁰C, and ground into powder. Known amount of dried amount was exhaustedly extracted by the process of maceration in an aspirator using various solvents as menstruum. NFE with different extracting solvents (ethanol, hydro-ethanol, decoction and aqueous) were concentrated under reduced pressure by rotary evaporator to obtain respective thick syrup mass, and stored at 4⁰C. Working concentration of the extract was made in non-pyrogenic distilled water before use in the experiments.

 

Chemicals and reagents

Porcine pancreatic α-amylase, rat intestinal α- glucosidase, 1, 1-diphenyl-2-picrylhydrazyl, gallic acid and paranitrophenyl-glucopyranoside were products of Sigma-Adrich, Malaysia. Other chemicals and reagents were of analytical grade and the water used was glass distilled.

 

Measurement of percentage yield

The percentage yield of the NFE was calculated as ((c-b)/a) x100. Where a = weight of sample; b = weight of beaker and c= weight of beaker + sample.

 

Phytochemical Screening

1.    Qualitative Phytochemical screening

Using described procedure [13], the NFE was subjected to qualitative phytochemical screening with different extracting solvents.

 

 

 

2.    Quantitative Phytochemical Analysis

a.    Assessment of Total Phenolic Content (TPC)

The quantification of TPC with different solvents of NFE was carried out using the prescribed procedure reported by Wolfe K et al., using Folin Ciocalteu reagent [14].  Gallic acid was used as standard. TPC was expressed as mg/g gallic acid equivalent using the equation obtained from a calibration curve of gallic acid.

 

b.    Determination of Total Flavonoid Contents (TFC)

The TFC with different solvents’ extracts were determined using the method employed by Swanny [15]. TFC were calculated as quercetin (mg/g) equivalent using the equation obtained from a calibration curve of quercetin.

 

In vitro Antioxidant Assays

All experiments were conducted in triplicates and all the negative control (blank) was prepared using the same procedure replacing the NFE with distilled water. The antioxidant activity of the NFE were evaluated with various solvents based on its scavenging activities on the stable 1, 1-diphenyl-2-picrylhydrazyl (DPPH) free radical according to the method described by Braca A et al [16]. The ability of NFE to scavenge 2,2-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) cation chromophore obtained from the oxidation of ABTS solution and potassium persulphate with various solvents was determined according to the method of Re et al [17]. To these above said antioxidant assays, the percentage inhibitory / scavenging activity of the NFE / standard was calculated using [(A₀–A₁)/A₀] × 100, where A₀ is the absorbance of the control, and A₁ is the absorbance of the NFE / standard. The half maximal inhibitory concentration (IC₅₀) value were calculated from the linear regression equation using y = m x + c, where y is the percentage activity and equals 50, m is the slope, c is the intercept and x is the IC₅₀ value.

 

In vitro Antidiabetic Assays

The α-amylase and α-glucosidase inhibitory assays were carried out using the procedure of Apostolidis E et al [18]. The 50% inhibition of enzyme activity (IC₅₀) of these enzymes was expressed as % inhibition using the expression:

 

%Inhibition= [(A_control-A_NFE)/A_control]×100,

 

where A_control and A_NFE are the  absorbances of the control and NFE respectively. Concentrations of NFE / standard resulting in 50% inhibition of enzyme activity (IC₅₀) were determined graphically using the linear regression equation y = m x + c, where y is the percentage activity and equals 50, m is the slope, c is the intercept and x is the IC₅₀ value.

 

Kinetic Studies

The kinetics on inhibition of α-amylase and α-glucosidase activity by NFE with various solvents was conducted using modified methods of Ali et al, [19] and Nagmoti and Juvekar [20] respectively. The amount of reducing sugars released was determined spectrophotometrically using maltose standard curve for α-amylase and p-nitrophenol standard curve for α-glucosidase. A double reciprocal (Lineweaver–Burk) plot (1/v versus 1/[S]) where v is reaction velocity and [S] is substrate concentration was plotted to determine the mode of inhibition. Thus, reaction rates (v) were calculated and double reciprocal plots of enzyme kinetics K_m and V_max values were also calculated from Lineweaver-Burk plot (1/v versus 1/[S]) [21].

 

Statistical Analysis

Statistical analysis was performed using a Graph Pad Prism 5 statistical package (Graph Pad Software, San Diego, MA, USA). Data were expressed as means of replicate determinations ± SD, for all assays and was subjected to one-way analysis of variance (and nonparametric) followed by Bonferroni: compare all pair of column. Statistical significance was considered at P < 0.05.

 

 

RESULTS:

The percentage yield of NFE with different extracting solvents is charted out in table 1.

 

Phytochemicals (PC)

The qualitative analyses of the NFE with different extracting solvents are presented in table 2. Saponins, phenols, flavonoids, anthraquinones, alkaloid, tannins, triterpenes and phytosterols were detected at varying degree in all the tested extracts while anthraquinone and phytosterol were found in trace amount in the ethanol and hydro-ethanol extracts. The results of the quantitative phytochemical screening (TFC and TPC) of NFE with different extracting solvents are portrayed in table 3.

 

Antioxidant activity

The in vitro antioxidant potentials of the NFE with different extracting solvents are shown in figures 1- 3. NFE scavenged the generated radicals in all assays were evaluated. Ethanolic extracts showed better capability to scavenge DPPH (0.125 mg/mL) (figure 1). Its corresponding IC₅₀ value is 0.52 µg/mL which is lower and significantly different (p<0.05) from the standard (silymarin) IC₅₀: 1.09 µg/mL as seen in table 4.

 

However, the reducing power (figure 2) and ABTS cation scavenging capability (figure 3) of the NFE competed well with silymarin in a dose dependent manner (0.125 - 1 µg/mL) with the highest dose of 1 µg/mL showing the best activity (table 4).

 

 

F-1: DPPH scavenging effect of NFE with different extracting solvents.

F-2: Reducing potentials of NFE with different extracting solvents.

F-3: ABTS scavenging effect of NFE with different extracting solvents.

F-4: The inhibitory potentials of NFE with different extracting solvents on α-amylase activity.

 

F-5: The inhibitory potentials of NFE with different extracting solvents on α-glucosidase activity.

F-6: Lineweaver-Burk plot of ethanolic extract of Mengkudu fruit eliciting competitive inhibition on α- amylase activity.

F-7: Lineweaver-Burk plot of ethanolic extract of Mengkudu fruit eliciting uncompetitive inhibition on α-glucosidase activity.

Values are mean and standard deviation (SD) of triplicate determination. n=3; (p<0.05).

 

In vitro antidiabetic assays

The inhibitory potentials of NFE on both α- amylase and α- glucosidase enzymes is dose dependent (0.125-1 µg/mL), and the percentage inhibition is presented in figures 4 and 5 respectively. Ethanolic extract has the lowest IC₅₀ (0.15 µg/mL) which is significantly different (p<0.05) from all other extracts and acarbose (Table 5). Ethanol and decoction extracts show milder inhibition of α-amylase with their respective IC₅₀ value of 0.57 and 0.62 µg/mL which is higher and significantly different (p< 0.05) from acarbose and hydro-ethanol (IC₅₀:0.47 and 0.42 µg/mL) respectively. Lineweaver-Burk plot of ethanolic extract of MC fruit eliciting competitive and uncompetitive inhibition on α- amylase (figure 6) and α-glucosidase activity (figure 7)  respectively.

 

 

Table 1: The percentage yield from different extracting solvents used in Noni.

 

Table 2. Phytochemical constituents of the NFE with different extracting solvents.

Key: +: detected; +++: degree of intensity; -: not detected or in trace amount.

 

Table 3. The result of the quantitative phytochemical screening of NFE with different extracting solvents.

 

 

Table 4. The IC₅₀ values of the free radical scavenging capabilities of different extracts of Mengkudu fruit.

The values are expressed as mean ± standard deviation (SD) of triplicate determination. (p<0.05). Silymarin is the standard antioxidant agent for all the antioxidant assays except metal chelating that has citrate as the standard.

 

Table 5. The IC₅₀ values for different extracts of Mengkudu fruit on specific activities of α-amylase and α-glucosidase enzymes.

The values are expressed as mean ± standard deviation (SD) of triplicate determination. Means down vertical column not sharing a common superscript are significantly different (p<0.05) from each other.

 

DISCUSSION:

The use of plants in treating diseases is as old as civilization [22] and herbal medicine is still a major part of habitual treatment of different diseases [23]. The process in the preparation of herbs like pulverization, extraction and solvents deployed in the extraction of raw material for drugs affects the percentage yield of the biologically active compound present in the extracts. In this experiment, local solvents (ethanol, hydro-ethanol, decoction and distil water) were used in Mengkudu fruit extract preparation.

 

The percentage yield indicated that hydro-ethanol has the highest yield of 31.01% from the 30g dry weight of the fruit sample extracted while decoction extract yield 5.15% of the 30g dry weight of the sample. It is worthy of note that the traditional healer use decoction (boil the dry fruit pulp) as their method of extracting the biologically active component of the plant. It may be suggested that this method of extraction accounted for low yield of extract which may be lesser efficacious.

Result of the quantitative phytochemical assays indicated the concentration of the different quantity of the PC found in NFE though, its bioavailability is unpredictable in in vivo study, because a lot of factors like absorption barrier of the PC in the gastro intestinal tract (GIT), the effects of different enzymes such as the glucosidase, esterase, oxidase and hydrolases originating from the host and the mycobiota which may inhibit PC activity in the GIT [24]. PC are known to possess varying antioxidant activities [25-29]. Antioxidant activity of a medicinal plant cannot be concluded based on a single antioxidant test model [25] as such several in vitro antioxidant tests were conducted on the extracts using silymarin as positive control for all assays. The free radical scavenging capability of fruit of Mengkudu on the molecules of DPPH radicals, ABTS cations radical and the reducing powers were determined [30].

 

The result of the assay showed that ethanolic NFE has better performance in antioxidant activity compared to the standard and other extracts tested for DPPH while hydro ethanol showed superior activity compared to the standard and other extracts tested in ABTS and reducing power. All these divinations is based on the standard curve of percentage inhibition / scavenging effect and IC₅₀ value of the tested extract which revealed a decrease in concentration of the ROS which may be due to the scavenging ability of NFE. Similar findings have been documented for the antioxidant and anti-inflammatory properties of Noni fruit [31]. It is noteworthy that the tested extract demonstrated the ability to neutralize the ROS at different degree which may be because of the presence of PC like polyphenols which has capability to directly scavenge superoxide and other ROS like hydroxyl and peroxyl radicals [32-34]. Saponins, triterpenes and phytosterol have been demonstrated to scavenge superoxide anion [35-37]. Flavonoid are currently receiving attention as a potential protector against variety of human disease, major flavonoid has been shown to have  neutralizing effect on free radical and ROS like hydroxyl radical, superoxide radical, hydrogen peroxides [25, 36,38-40].

 

The decrease in postprandial hyperglycemia is achieved by hindered absorption of glucose by inhibition of the carbohydrate hydrolyzing enzymes in the digestive organs. The enzymes that are affected are α-amylase, that catalyses the breakdown of starch to maltose and finally to glucose, as well as α-glucosidase, present in the small intestine and catalyzing the breakdown and absorption of complex sugars. Examples of such inhibitors in clinical use are acarbose, miglitol and voglibose but they do have side effects [41].

 

 

Marked postprandial hyperglycaemia is important in the pathogenesis of T2DM, it induces mitochondrial superoxide overproduction which potently inhibit the glycolytic enzyme glyceraldehyde-3-phosphate thus, diverting upstream metabolites from glycolytic pathway into pathway of glucose overutilization resulting in formation of diacylglycerol from dihydroxylacetone phosphate (DHAP) a potent activator of protein kinase C (PKC) which ultimately causes β-cells destruction and insulin resistance [42-44]. The unregulated hydrolysis of starch by α- amylase and α-glucosidase which catalyze the rate limiting step in the conversion of oligosaccharides and disaccharides into monosaccharide’s is responsible for the elevated blood glucose seen in T2DM. Therefore, controlling hyperglycaemia via inhibition of carbohydrate hydrolysing enzymes is an important strategy in the management of T2DM [45-47]. In vitro evaluation of the inhibitory effects of the NFE on α- glucosidase and pancreatic α- amylase enzymes was carried out using acarbose as the standard to determine its percentage inhibition and their respective IC₅₀ value. Mild inhibition of α- amylase and strong inhibition of α- glucosidase enzymes is targeted as a way of reducing postprandial hyperglycaemia, and elimination of the unwanted effect like gastrointestinal discomfort flatulence, diarrhoea associated with the use of acarbose [47, 48]. In this study, ethanol and decoction extracts mildly inhibit α- amylase with their respective IC₅₀ values of 0.56 and 0.61 µg/mL which is higher and significantly different (p<0.05) from acarbose with lower IC₅₀ (0.47 µg/mL). The result of the inhibitory potentials of the extracts on α- glucosidase showed ethanol and decoction extracts has potent inhibition of the enzyme activity. Thus, it may be employed in the management of postprandial hyperglycemia. This finding is consistent with findings of many authors [49, 50] who described moderate inhibition of α- amylase and strong inhibition of α-glucosidase as a better therapeutic approach to be deployed in the delay and regulation of carbohydrate hydrolysis in the intestine which is responsible for glucose toxicity observed in T2DM.

 

The ethanolic extract which possess the highest IC₅₀ for α- amylase enzyme and lowest IC₅₀ for α- glucosidase compared to acarbose and other tested extracts of Mengkudu fruit was used to determine the mode of inhibition of α- amylase and α- glucosidase enzymes in other to investigate its enzyme inhibition kinetics. Similar findings were observed by our previous study on MC and its secondary metabolite scopoletin [50]. Nevertheless, our past research on Mengkudu’s antihyperglycemic, antidiabetic dyslipidemic and antioxidant potentials in in vivo models well lined up with the present findings [51-53]. 

Result for the mode of inhibition of α- amylase enzyme showed that the ethanolic NFE is competitively inhibiting the breakdown of disaccharides and oligosaccharides which are substrate for α- amylase. The V_max values obtained with inhibitor and without inhibitor in the reaction pathway is the same, the K_m values decreased from 4.85x10^-2 µM^-1 for reaction pathway without inhibition to 1.44x10^-2µM^-1 with inhibitor. Decreased K_m value signifies increase affinity. This result proposed competitive mode of inhibition.

 

However, the mode of inhibition of α- glucosidase by ethanolic NFE is by uncompetitive inhibition. The propose model is the binding of the NFE (inhibitor) to a site other than the active site and only when the substrate is binding to ES complex thereby inhibiting the formation of product. The kinetic further shows that there is a decrease in K_m from 7.10x10^-2 µM^-1 to 4.69 x10^-2 µM^-1without inhibitor and with inhibitor respectively and also a decrease in V_max from 19.76 µM/min without inhibition to 14.66 µM/min with inhibition which suggests a 39.74% decrease in overall activity of α- glucosidase enzyme in the presence of ethanolic extract of fruit of Mengkudu.

 

From this work, it has been conjectured that fruit of Mengkudu has great and promising potential as pharmaceutical agent, particularly to be developed as antidiabetics through the inhibition of α-glucosidase and α- amylase enzymes. This natural approach is thought to be safer and cost effective compared to its synthetic version (e.g., acarbose). Added to this, demonstrated the in vitro tests of the antioxidant activity of the fruit extract, which gives evidence and strong biochemical rationale of their therapeutic potential. Therefore, the promising results shall be carried forward to clinical trial to further corroborate the activity. Besides, data generated from these studies further promote the traditional use of plants in medicine. Therefore the fruit extract of Mengkudu may play an important role in the development of nutraceuticals and also in the management of oxidative stress induced DM.

 

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Accepted on 25.09.2018          © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(9): 4135-4142.