Balasuramaniam Jaya Prasad* ,Pazhaniyandi Suramania Sharavanan,Rengaraj Sivaraj

a Department of Botany,Annamalai University,Annamalai Nagar 608002,India

b Department of Botany,Govt Arts College,Mannargudi 614001,India

c Department of Pharmacology,Aarupadai Veedu Medical College&Hospital,Puducherry 607403,India

Keywords:

Oryza punctata

α-Amylase

α-Glucosidase

High performance liquid chromatography(HPLC)

Pigmented rice

Catechin

ABSTRACT

Red rice(Oryza punctata)is a type of unpolished rice which has higher nutritional value compared to white rice or even polished rice.Owing to higher nutritive content and metabolites,dieticians strongly advise red rice for peoples with metabolic disorders including diabetics.However,the mechanism of action and contents of secondary metabolites in Indian red rice variety not reported scientifcially.Therefore,the present study aimed to evaluate its mechanism of action through inhibitory effect of α-amylase and α-glucosidase.Initially,the whole grain of red rice was macerated with methanol at room temperature for 2 weeks.Then,the dried and powdered,samples at different concentration(2.5,10,40,and 80 μg/mL)were employed to find out in vitro inhibitory effects on α-amylase and α-glucosidase.In addition,an enzyme kinetics of effective extract was calculated by Line-weaver Burk(LWB)plot analysis.Moreover,the valuable metabolites in the efficient methanolic extract were quantifeid using high performance liquid chromatography(HPLC).The results demonstrated that red rice methanolic extract(RRMEt)possess strong inhibitory activity on α-amylase and α-glucosidase compared with acarbose(P ＜0.01).The IC50 values of RRMEt was found to be 29.7±7.43 μg/mL for α-amylase and 20.4±0.25 μg/mL for α-glucosidase.LWB indicated that RRMEt is an uncompetitive inhibitor.Further,HPLC analysis revealed protocatechuic acid,catechin,and chlorogenic acids were more abundant in RRMEt among the fourteen metabolites.We conclude,the efficiency of enzyme inhibition through the influence of phenolic compounds in RRMEt.

1.Introduction

Rice(Oryza sativa)is one of the foremost necessary staple crops and provides over a fifth of the total calories consumed across the planet[1].It is a type of unpolished rice which has higher nutritional value compared to white rice or even polished rice.Owing to higher nutritive content and metabolites,dieticians strongly advise red rice for peoples with metabolic disorders including diabetics.Polished rice plays a crucial role in the budding prevalence of polygenic disorders includes diabetes mellitus(DM)in developing countries[2].Previously,unpolished rice has been reported to have lower glycemic levels[3].Red rice grains contain additional nutritional elements such as dietary fibers,vitamins E and B,and gamma amino-butyric acid(GABA)when compared to the polished rice grains[4].DM is a metabolic disorder,which causes high mortalities in developing countries Western,European and Asian origin[5].DM could be a cluster of metabolic diseases that are characterized by hyperglycemia resulting from the defects in insulin secretion,insulin action,or both,and elevated levels of blood sugar.The chronic hyperglycemia of DM is related to severe damage and failure of various organs,particularly eyes,kidneys,nerves,heart and blood vessels.The metabolism of carbohydrate,fat and proteins characterized by exaggerated fast and post alimentation of blood glucose levels[6].

The conventional treatments for diabetes comprise prompting of insulin like secretion,development action at intentional tissue and stopping degeneration of starch[7].An additional way to handle diabetes is to slow down the starch to glucose conversion through absorption besides targeting membrane bound enzymes such as α-amylase and α-glucosidase.The pancreatic α-amylase breakdown the carbohydrates,which yields maltotriose and maltose[8].α-glucosidase splits the byproducts of starch into glucose.By inhibiting both α-amylase and α-glucosidase,quite easy to delay digestion of carbohydrates,which results in suppression of postprandial symptoms[9].Currently,available antidiabetic agent,frequently produces diarrhea and other adverse effects that limit its usage among the patients with diabetes [10].Some inhibitors currently in clinical use such as acarbose and miglitol inhibit α-glucosidase and α-amylase[11].Likewise,numerous artificial antidiabetic drugs encompass their boundaries and create adverse effects and unsuccessful to combat diabetic complications.

Based on the information and limitation of available antidiabetic drugs,numerous researches are going on across the globe,for delaying the action of pancreatic enzymes.If the food itself controls glucosidase enzymes then it will be more successful than the synthetic drugs.Owing to higher nutritive content and metabolites,dieticians strongly advise red rice for peoples with metabolic disorders including diabetics.However,the mechanism of action and contents of secondary metabolites in Indian red rice variety not reported scientifically.Therefore,the present study aimed to evaluate its mechanism of action through inhibitory effect of α-amylase and αglucosidase.In addition,potential compounds in the extract quantified using high performance liquid chromatography(HPLC).

2.Materials and methods

2.1.Plant collection and extraction

Oryza punctata(Voucher specimen No.AUBOT#298) was collected from the farms at Thanjavur,Tamil Nadu,India.Briefly,rice(2 kg)was dried at 50°C for 6 days in a hot air oven and macerated with 15 L of methanol at room temperature for 2 weeks with frequent agitation until the soluble matter has dissolved.After that,the extract was filtered using Whatmann filter paper (No.1) and dried at room temperature.Then,dried and powdered red rice methanolic extract(RRMEt)(25 g)was stored at 4°C until further analysis.

2.2.α-Glucosidase inhibition assay

Inhibitory activity of RRMEt against α-glucosidase was evaluated using the reaction of p-nitrophenyl β-D-glucoside(PNPG).PNPG would be broken down by α-glucosidase into yellow coloured p-nitrophenol,which could be measured at 405 nm[12].Totally,four various concentrations of RRMEt(12.5,50,200,and 400 μg/mL) were employed in this assay.20 μL of RRMEt and 50 μL of phosphate buffer(50 mmol/L,pH 6.8)were mixed to obtain the final concentrations of 2.5,10,40,and 80 μg/mL,respectively,followed by the addition of 10 μL of α-glucosidase (1 U/mL)and pre-incubation for 5 min(37°C).Then,20 μL of PNPG(1 mmol/L) was added and incubated for 30 min(37°C)and measured at 405 nm using a microplate reader(Micro Scan MS5608A,ECIL,India).The experiments were carried out in triplicates.The results were compared with the negative control (enzymatic solution without inhibition),while acarbose was employed as the positive control.Results were calculated by using the following equation:

The IC50value was calculated to determine 50%inhibitory capacity of the reaction at a certain concentration.Therefore,the lower value of IC50indicates stronger activity.

2.3.α-Amylase inhibition assay

500 μL of sample or negative control or positive control (10-2000 μg/mL acarbose) was mixed to 500 μL of 13 U/mL α-amylase solution and then,incubated for 10 min(25°C).500 μL of soluble starch solution(potato starch dissolved in sodium phosphate buffer of pH 6.9)was added and incubated for 10 min(25°C).Finally,1 mL of dinitrosalicylic acid reagent was added and placed at 100°C in a water bath(5 min).Furthermore,it was diluted with 10 mL of distilled water and the absorbance was measured at 540 nm[13]using a Shimadzu UV-1800 spectrophotometer.Results were calculated by using the following equation:

The IC50value was calculated to determine the 50%inhibitory capacity of reaction at a certain concentration.Therefore,the lower value of IC50indicates stronger activity.The enzyme kinetics was performed through linear regression by fitting to a sigmoid dose-response equation with variables lope of Line-weaver Burk plot[14].

2.4.Extraction of phenolic compounds

The soluble and insoluble phenolic compounds extraction in the RRMEt was followed using standard hydrolysis methods[15,16].Final fraction was made up with 2 mL of 15%methanol and used to HPLC analysis.All analyses were performed in triplicate.

2.5.HPLC analysis

HPLC(Shimadzu,Japan)analysis was performed to quantify phenolic compounds in the alkaline fraction.The column conditions as follows:C18 column (250 mm × 4.6 mm × 5 μm).The sample was injected along with respective HPLC grade standards.10 μL of the sample was subjected into the column.The chromatographic separation was carried out at room temperature with a flow rate 0.8 mL/min of gradient.The column was eluted by gradient of two solvents: A (0.1% trifluoroacetic acid in water)and B(100%methanol).The samples were identified by comparing their relative retention times and UV spectra(280 and 325 nm)with authentic compounds and were detected using an external standard method[17].

3.Results

Inhibitory actions of RRMEt on α-amylase and α-glucosidase are illustrated in Fig.1 and 2,and Line weaver-Burk plot based kinetic analysis of α-amylase and α-glucosidase against RRMEt as plotted in Fig.3 and 4.RRMEt showed statistically significant(P ＜0.01)and potent inhibition against α-amylase and β-glycosidase compared to acarbose(Fig.1 and 2).IC50of RRMEt at the concentration of 40 mg/mL was 29.7 ± 7.43 and 20.4±0.25 μg/mL α-amylase and α-glucosidase,respectively.Acarbose served as a positive control that showed inhibitory potentials with IC50of 14 μg/mL.

Enzyme kinetics of RRMEt revealed it to be a non-competitive inhibitor with respect to PNPG for α-glucosidase.Inhibitor concentrations(40 and 80 μg/mL)were plotted on the X-axis and 1/V(mol/L)min-1values obtained from LWB were plotted on the Y-axis.Enzyme kinetics of RRMEt noted it to be a non-competitive inhibitor with respect to starch for α-amylase.Inhibitor concentrations(40 and 80 μg/mL)were plotted on the X-axis and 1/V(mol/L) min-1values obtained from LWB were plotted on the Y-axis.RRMEt have uncompetitive inhibitor of α-amylase and α-glucosidase and reduction of Vmaxfrom 0.93 to 0.09,0.81 to 0.12 mol/L min-1and Km50.25 to 6.51,57.75 to 6.87 mmol/L,respectively.Acarbose is a competitive inhibitor of the enzyme and it is required at a higher concentration to reduce the postprandial glucose level.The uncompetitive inhibitors bind to the enzyme-substrate complex,lowering the Kmand the maximum enzyme activity(Vmax).

Fig.1.Inhibitory action of RRMEt on α-amylase.

Fig.2.Inhibitory action of RRMEt on α-glucosidase.

Fig.5 illustrated that 15 different soluble and insoluble phenolic compounds present in the RRMEt,which includes protocatechuic acid(26.5±3.6),catechin (73.7±8.5),chlorogenic acids (20.5±5.6),vanillic acid(5.66±0.64),hydroxybenzoic acid(2.71±0.38),coumaric acids(0.55 ± 0.09),ferulic acid (5.56 ± 0.26),quercetin (4.26 ± 0.34),myricetin (5.16 ± 0.48),luteolin (7.55 ± 0.45),apigenin (11.63 ±0.69),cinnamic acid(6.03±0.38),and syringic acid(6.23±0.37)mg/100 g of RRMEt.

4.Discussion

Carbohydrates (CHOs) in general stand for the mainstream of the human diet.They form the major quantity and energy resources in humans as rice,wheat,potato,etc.[18].Before absorption,the poly-and oligosaccharides go through enzymatic breakdown into simple monosaccharide forms.This breakdown occurs in two steps-starch: first sliced by αamylases,then,followed by the breakdown of polysaccharides using membrane-bound enzymes in the epithelium of the small intestine,and final products of this breakdown are monosaccharide,i.e.,glucose,galactose and fructose[19].Enzymes are secreted by the epithelium of the maturing enterocytes in jejunum.Sucrose,isomaltase (usually complex together),maltase,glucoamylase,trehalase and lactase are collectively referred to as alpha-glucosidases [20].Many reports have demonstrated that α-amylase and α-glucosidase inhibitory actions on different extracts[21,22].Pigmented rice is a high quality source of valuable components,which offer vital advantages besides common diet[23].Earlier,many researchers have carried their research on both humans and animals,and proved that consumption of brown rice could reduce the risk of cardiovascular disease(CVD),and cancer.These protective health effects have been linked to the bioactive compounds present in the bran layer of rice grains such as antioxidants,polyphenols,minerals,vitamins and dietary fibers[24-26].In the present study,RRMEt showed the presence of protocatechuic acid,catechin,chlorogenic acids,vanillic acid,hydroxybenzoic acid,coumaric acids,ferulic acid,quercetin,myricetin,luteolin,apigenin,cinnamic acid,and syringic acid.Momilactone,naturally occurring diterpenes found in rice varieties possessed strong α-amylase and α-glucosidase inhibitory activities[27].Rice flour contains low protein and easily digestible components,preferably,the gluten free food products[28].Similarly,red rice methanolic extract has shown strong α-amylase and α-glucosidase inhibitions.Chung et al.[29]added that pigmented rice could be used as a functional food for the management of diabetes as it could inhibit the level of digestive enzymes due to its high content of polyphenol components.Low glycemic index and delayed digestion could be the possible mechanisms behind the antidiabetic activity of the pigmented rice[30].

Fig.3.Line weaver-Burk plot based kinetic analysis of α-amylase against RRMEt.

Fig.4.Line weaver-Burk plot based kinetic analysis of α-glucosidase against RRMEt.

Fig.5.Phenolic compounds present in the RRMEt.

5.Conclusion

We concluded that red rice extract possesses strong in vitro antidiabetic effects through inhibition of α-amylase and α-glucosidase activities and catechin may be responsible for its antidiabetic activity.

Declaration of Competing Interest

Authors declare that they do not have any conflict of interest.

Acknowledgement

Author Balasubramaniam Jaya Prasad would like to thank Department of Science and Technology,Government of India,for providing INSPIRE Fellowship and authorities of Annamalai University for providing necessary support.