Characterization and Evaluation of Vernonia amygdalina Extracts for its Antidiabetic Potentials

Background: There is increase in the demand for the use of medicinal plants for the treatment of various ailments such as cancer, hypertension and diabetes locally as a result of the inability of the synthesized drugs to completely eradicate the ailments and also with the attendant side effects attached to the use of drugs. Objective: The aim of this research work was to investigate the Aldose reductase, α-glucosidase, β -glucosidase and maltase glucoamylase inhibitory potential of the crude extracts of Vernonia amygdalina, and characterizing the extracts. Materials and method: The leaves of the plant was obtained, air dried and turned to powdered form using commercial grinding machine with dimension 34×38×75. The extracted crudes were subjected into in-vitro studies of the inhibitory potentials using recommended protocols. The IC50 values were calculated using Graph pad prism 5.0 software and the identified compounds were screened for drug properties using ONLINE OSIRIS server explorer. Results: The (IC50 0.6 ± 0.03 μg/mL) of the chloroform extract was better than the methanolic extract of IC50 (1.532±0.63μg/mL) and the results were better than the acarbose standard (IC50 234.6 + 2.01μM). The maltase glucoamylase inhibitory potentials of the chloroform and methanolic extracts in the range of IC50 1.112 ± 0.90 μg/mL and 1.315 ± 0.7 μg/mL. The β-glucosidase screening of the extracts results indicated that they do not have good selective inhibitor properties. IC50 of Aldose reductase (ALR2) of chloroform extract IC50 (1.339+0.264μg/mL) was better than that of methanolic extract of IC50 (1.437+0.6μg/mL) and these values were better when compared with the standard, sorbinil of IC50 (3.10 +0.20μM).The aldehyde reductase (ALR1) of methanolic extract IC50 (0.325+0.02μg/mL) was better than that of chloroform extract of IC50 (0.964+0.16μg/mL) and when compared with standard 10mM vaproic acid IC50 (57.4 +10μM).The GC-MS of the chloroform extract revealed compounds which were screened computationally to for various drug properties such as drug likeness, cLogS, cLogP, H-bond acceptor and H-bond Donor. Conclusion: The promising inhibitory potential of the plant extracts and characterization of the extract is an indication of the usefulness of the plant for the treatment of diabetes. Keyword: Diabetes mellitus, drug properties, aldose reductase, α-glucosidase, maltase glucoamylase, β-glucosidase Introduction There are widely research studies on the use of medicinal plants for the treatment of diabetes and many studies have revealed the efficacies of crude plant extracts as well as their bioactive compounds in reducing blood glucose levels as reported by Kaushik [1]. It has been reported that decreasing the post prandial glucose level by slowing down the absorption of glucose by inhibiting the carbohydrate hydrolyzing enzymes ( αand β-glucosidases) in digestive tract is one of the therapeutic approaches to treat diabetes [2].There are many synthetic antidiabetic drugs being used for the management of the ailment however, it has not been reported that novel drugs have been found to completely eradicate the disease [3] and this has justified the continuously searching for the plants with good potent inhibitors of α-glucosidase, β-glucosidase and maltase glucoamylase with no side effect as being characterized with synthetic drugs available. αglucosidase inhibitors are defined as oral antidiabetic drugs that are widely used for type 2 diabetes Characterization and Evaluation of Vernonia amygdalina Extracts for its Antidiabetic Potentials http://www.ijSciences.com Volume 7 – January 2018 (01) 32 mellitus and function primarily by disallowing the digestion of carbohydrates and thus reduces the impact on the blood sugar. There are widely use of manufactured α-glucosidase inhibitors this includes acarbose, miglitol and voglibose. It is has been proofed that α-glucosidase inhibitors are used to establish higher glycemic control over hyperglycemia in DM type 2 patients[4]. Moreover, it is has been reported that Maltase glucoamylase, is an αglucosidase digestive enzyme and comprises two subunits with different substrate precision[4]. Research has revealed that the recombinant enzyme studies had shown that its N-terminal catalytic domain has highest activity against maltose, while the C-terminal domain has a broader substrate precision and activity against glucose oligomers [5]. It has been earlier reported that Aldose reductase having two isoforms (ALR1 and ALR2) has been implicated in the cause of diabetic problems that may be connected to a much significance flux of glucose through the polyol pathway, caused in tissues such as lens, kidney, retina and nerves at high blood glucose levels. Therefore, the inhibitory property of aldose reductase is gaining recognition as a major therapeutic tool for the treatment of hyperglycemiainduced cardiovascular pathologies [6].Long term drawbacks of diabetes include among others; cataractogenesis and microangiopathy (including nephropathy, retinopathy and neuropathy) thought to be linked to excess free glucose in corresponding tissues[7]. Widely research work has provided evidence of aldose reductase (AR) implication in diabetes severalty [8, 9]. Scientific investigation to provide inhibitors of AR has been a major task for the chemists in order to effectively treat various degrees of complications associated with diabetes. V. Amygdalina is one of the widely consumed leaf vegetables in Nigeria and well known as a medicinal plant with many pharmacological uses such as diabetes, fever reduction and recently a non pharmaceutical solution to consistent headache and joint pains connected to AIDS ((an infusion of the plant is taken as needed) [10, 11]. It has been scientifically documented that the roots of the plant has been used as toothache and gingivitis as a result of its proven antimicrobial activity [12]. V. amygdalina plant has been researched upon and the experimental evidence for its use in diabetes management as reported by Udoamaka [13] included; 80 mg/kg aqueous extract of the leaves produced a dose dependent decrease in blood glucose levels in normal and alloxan-induced diabetic rabbits [14]; chronic intake of 400mg/kg ethanolic extract of the fresh leaves significantly decreased fasting blood glucose levels, increased serum and pancreatic insulin levels, increased the activity of liver antioxidant enzymes as well as increased the expression and distribution of Glut 4 receptors in STZ-induced diabetic rats [15]; 150ml of a (1:1:1) decoction mix of the leaves of Vernonia amygdalina, Ocimum gratissimum and Gongronema latifolium decreased baseline blood glucose levels in normal subjects when preadministered to normal subjects 45 minutes before an OGTT [16], however the selected plant has not been evaluated simultaneously for its potent inhibition of αand βglucosidases, maltase glucoamylase, aldose reductase (ALR1 and ALR2) and identifying the bioactive compounds of the chloroform crude extract.


Introduction
There are widely research studies on the use of medicinal plants for the treatment of diabetes and many studies have revealed the efficacies of crude plant extracts as well as their bioactive compounds in reducing blood glucose levels as reported by Kaushik [1]. It has been reported that decreasing the post prandial glucose level by slowing down the absorption of glucose by inhibiting the carbohydrate hydrolyzing enzymes ( αand β-glucosidases) in digestive tract is one of the therapeutic approaches to treat diabetes [2].There are many synthetic antidiabetic drugs being used for the management of the ailment however, it has not been reported that novel drugs have been found to completely eradicate the disease [3] and this has justified the continuously searching for the plants with good potent inhibitors of α-glucosidase, β-glucosidase and maltase glucoamylase with no side effect as being characterized with synthetic drugs available. αglucosidase inhibitors are defined as oral antidiabetic drugs that are widely used for type 2 diabetes mellitus and function primarily by disallowing the digestion of carbohydrates and thus reduces the impact on the blood sugar. There are widely use of manufactured α-glucosidase inhibitors this includes acarbose, miglitol and voglibose. It is has been proofed that α-glucosidase inhibitors are used to establish higher glycemic control over hyperglycemia in DM type 2 patients [4]. Moreover, it is has been reported that Maltase glucoamylase, is an αglucosidase digestive enzyme and comprises two subunits with different substrate precision [4]. Research has revealed that the recombinant enzyme studies had shown that its N-terminal catalytic domain has highest activity against maltose, while the C-terminal domain has a broader substrate precision and activity against glucose oligomers [5]. It has been earlier reported that Aldose reductase having two isoforms (ALR1 and ALR2) has been implicated in the cause of diabetic problems that may be connected to a much significance flux of glucose through the polyol pathway, caused in tissues such as lens, kidney, retina and nerves at high blood glucose levels. Therefore, the inhibitory property of aldose reductase is gaining recognition as a major therapeutic tool for the treatment of hyperglycemiainduced cardiovascular pathologies [6].Long term drawbacks of diabetes include among others; cataractogenesis and microangiopathy (including nephropathy, retinopathy and neuropathy) thought to be linked to excess free glucose in corresponding tissues [7]. Widely research work has provided evidence of aldose reductase (AR) implication in diabetes severalty [8,9]. Scientific investigation to provide inhibitors of AR has been a major task for the chemists in order to effectively treat various degrees of complications associated with diabetes.
V. Amygdalina is one of the widely consumed leaf vegetables in Nigeria and well known as a medicinal plant with many pharmacological uses such as diabetes, fever reduction and recently a non pharmaceutical solution to consistent headache and joint pains connected to AIDS ((an infusion of the plant is taken as needed) [10,11]. It has been scientifically documented that the roots of the plant has been used as toothache and gingivitis as a result of its proven antimicrobial activity [12]. V. amygdalina plant has been researched upon and the experimental evidence for its use in diabetes management as reported by Udoamaka [13] included; 80 mg/kg aqueous extract of the leaves produced a dose dependent decrease in blood glucose levels in normal and alloxan-induced diabetic rabbits [14]; chronic intake of 400mg/kg ethanolic extract of the fresh leaves significantly decreased fasting blood glucose levels, increased serum and pancreatic insulin levels, increased the activity of liver antioxidant enzymes as well as increased the expression and distribution of Glut 4 receptors in STZ-induced diabetic rats [15]; 150ml of a (1:1:1) decoction mix of the leaves of Vernonia amygdalina, Ocimum gratissimum and Gongronema latifolium decreased baseline blood glucose levels in normal subjects when preadministered to normal subjects 45 minutes before an OGTT [16], however the selected plant has not been evaluated simultaneously for its potent inhibition of αand βglucosidases, maltase glucoamylase, aldose reductase (ALR1 and ALR2) and identifying the bioactive compounds of the chloroform crude extract.

Research Laboratory
The research work was carried out at the centre for the Advanced Drug Research (CADR), Department of Pharmacy, COMSATS Institute of Information Technology, Abbottabad, Pakistan in the month August, 2017.

Materials and Instruments
All the reagents, solvents used are of analytical grade, α-glucosidase (from Saccharomyces cerevisiae), substrate p-nitrophenylα-D-glucopyranoside (pNPG), β-glucosidase (from sweet almonds) and 96 well plates were purchased from Sigma Aldrich.

Plants source
Vernonia amygdalina leaves were collected from farm and were authenticated at the Department of Agricultural Science, Afe Babalola University Ado Ekiti, Nigeria.

Crude extracts preparation
The extraction was done by soaking two hundred grams of powdered samples in 1000ml of chloroform and methanol for a period of five days and filtered through whatman filter paper. The extracts were concentrated using a rotary evaporator at 35°C and the dried extract was stored at room temperature for further use. The stock sample was prepared by dissolving 10mg of dried crude extracts in 1mL of 100% dimethyl sulfoxide (DMSO) and labelled as stock (10mg/ml), working solution was made as 1 mg/ml.

Extraction of Aldose Reductase Enzymes
Material and Instruments used for the extraction procedure All the chemicals needed for the enzyme extraction were of analytical grade. Substrates (D,Lglyceraldehyde and sodium-D-glucoronate) and nicotinamide adenine dinucleotide phosphate (NADPH) as co-factor were purchased from Sigma Aldrich. Eliza microplate reader was used with a UV range of 340nm for the enzymatic reaction.

Isolation and purification of Aldose reductase enzyme (ALR2)
This was done according to Hayman [17] with minor modification; the modification to the method was done by adding different mass of ammonium sulphate to the supernatant depending on the volume of supernatant collected as percentage of saturation increases.

Isolation and purification of ALR1 (Aldehyde reductase)
Kidneys were removed from the calf soon after slaughtering and dissolved in 3 volumes of 10mM sodium phosphate buffer, pH 7.2, containing 0.25M sucrose, 2.0mM EDTA dipotassium salt and 2.5 mM β-mercaptoethanol. The homogenate was Centrifuged the at 12,000×g at 0-4°C for 30 minutes. The precipitate was discarded as it contained insoluble lipids. Collected supernatant layer was subjected to 40% ammonium sulphate saturation to isolate ALR1.This 40% saturated liquid was centrifuged at 12,000×g at 0-4°C for 30 minutes. Again, precipitate was discarded and supernatant was subjected to 50% saturation with ammonium sulphate salt. The same procedure was repeated with this and in the last step; ammonium sulphate was added to increase saturation up to 75%. Centrifuge the supernatant at 12,000×g at 0-4°C for 30 minutes, causing the precipitation of ALR1.Now precipitated material was collected and supernatant discarded. The precipitated material containing enzyme was re-dissolved in 10mM sodium phosphate buffer, pH 7.2 containing 2.0mMEDTA dipotassium salt and 2.0mM βmercaptoethanol and dialyzed over night using the same buffer. The dialyzed material containing ALR1 enzyme was stored at -80°C before use [18].

Total Protein Evaluation in the Extracted Enzymes
Test Procedure using total protein kit (Laboratory kit made by: (Chemelex, S.A. Pol.ind.Can,Castells. Industrial 113 NAu J.08420, Canovelles, Barcelona Spain) The instrument was adjusted to zero with distilled water (iii) Pipette into a cuvette Determination of aldose reductase (ALR2 and ALR1) inhibitory activities UV spectrophotometer was used at 340 nm in order to determine the activity of aldose reductase by measuring the NADPH consumption. Each well of the 96-well plate contains exactly 100 μL of assay mixture containing phosphate buffer 100 mM at pH 6.2 (10μL), with 10μL of 1mg/ml of crude extracts followed by addition of 35 μL of enzyme and 20 μL of substrate (D,L-glyceraldehyde for ALR2 and sodium-D-glucoronate for ALR1). The mixture was incubated at 37°C for 5 min and for the enzymatic reaction to run properly 0.5mM NADPH (20 μL) as a cofactor was added and reading was taken at 340 nm. The mixture was incubated again at 37 °C for 10 min and reading was taken at the respective UV range in ELIZA plate reader. For ALR2 (10 mM Sorbini) and ALR1 (vaproic acid) of 10 μL each was used as positive control and 20μL buffer solution as negative control respectively. The enzymatic reaction was run in triplicate with a final volume of 100μL in each well. Absorbance was noted and results were analysed.

α-glucosidase inhibition study
The inhibitory effect of the 1mg/ml of the working solution was performed according to a previously published method by Gao [19].

β-glucosidase inhibition study
The evaluation of inhibitory activity against βglucosidase was performed following the previously published method by Pérez [20].

Extraction of intestinal maltase-glucoamylase enzyme
Intestinal maltase-glucoamylase was extracted by following the literature reported procedure [21,22]. The enzyme was extracted from white male rats (1-2 months) weighing 150-250 g and starved for 12 hours before death. Rats were killed by cervical dislocation. Whole intestines were gently removed and washed with ice cold 0.9% NaCl. For extraction, intestines were cleaned and cut longitudinally, and mucosal scrapings of 5-6 rats were combined and homogenized in 50 volumes of 5 mM Ethylenediaminetetraacetic Acid (EDTA); pH 7.0. All the steps were carried out at 4 °C. Homogenized intestines were centrifuged at 15,000 rpm for 45 min at 4 °C and the supernatant was discarded. Pellet was re-suspended in 90 mL of ice-cold water followed by 5 mL of 0.1M-EDTA/ 0.2 M potassium phosphate, pH 7, and 5 mL of 0.1 M cysteine was added. Mixture was incubated at 37 °C for 30 min. Pellet was collected by centrifugation at 15,000 rpm for 45 min and the supernatant was discarded. Intestinal suspension was re-dissolved in 10 mM potassium phosphate buffer, pH 6.8, to which 4 mg papain and 0.4 mg cysteine were added. The activated papain solution was immediately used. Mixture was incubated at 37 °C for 40 min with continuous shaking. It was then centrifuged at 15,000 rpm for 90 min. Supernatant was collected and precipitated with ammonium sulphate to 80% saturation. It was dialyzed overnight against distilled water with three changes of water (40 vol every time). The residue was re-dissolved in 4 mL of 10 mM-potassium phosphate (pH 7) and protein concentration was determined. The extracted enzyme was stored at -80 °C until further use.

Maltase-glucoamylase inhibition assay
Maltase-glucoamylase inhibition assay was carried out using the substrate p-nitrophenyl α-d-glucoside using reported procedure by Tanaka [23]. The activity of the test compounds against maltase glucoamylase was determined by measuring pnitrophenol at a wavelength of 405 nm. Acarbose was used as a positive control.

Statistical Analysis
The total percentage inhibitions were calculated by the formula: %Inhibition= control absorbance testwell absorbance [19] IC 50 ≥ 50% values of potent inhibitors were determined by testing the serial dilutions of inhibitors and were calculated by using the program PRISM 5.0 (GraphPad, San Diego, California, USA).

GC/MS analysis
GC-MS analysis of methanol and chloroform extracts of were performed using TurboMass GC System, fitted with an Elite-5 capillary column (30 m, 0.25 mm inner diameter, 0.25 μm film thickness; maximum temperature, 350°C coupled to a Perkin Elmer Clarus 600C MS. Helium was used as gas carrier at a constant flow rate of 1.0 ml/min. The injection, transfer line and ion source temperatures were 280°C. The ionizing energy was 70 eV. The oven temperature was programmed from 70 °C (hold for 2 min) to 280 0 C (hold for 10 min) at a rate of 5°C/min. The crude extract was solubilised with chloroform and filtered with syringe filter (Corning, 0.45 μm). Volumes of 1 μl of the crude extracts were injected with a split ratio 1:20. The data were obtained by collecting the mass spectra within the scan range 50-550 m/z. The identification of chemical compounds in the extracts were based on GC retention time; the mass spectra matched those of standards available at NIST library.     [26]. In addition, the methanolic extract of B.micrantha (IC 50 1.06+0.1μg/mL) showed good potent and selective inhibitor than A.calamus (IC 50 1.26mg/mL) and N.sativa (IC 50 1.53mg/mL) as reported by Balaji [27] against α-glucosidase. The IC 50 (1.112+0.90 μg/mL) of methanolic extract of Vernonia amygdalina was better than the chloroform extract of IC 50 (1.315+0.700 μg/mL) when tested against maltase glucoamylase and the results were better than the standard acarbose (IC 50 234.6 ±2.01μM). However, the IC 50 both for the extracts of V.amygdalina against maltase glucoamylase showed good inhibitory potentials than the methanolic extracts of Artocarpus altilis (IC 50 118.88+11.14μg/mL), A.heterophyllus (IC 50 70.58+9.66 μg/mL), Cinnamomus zeylanicum (IC 50 130.55+10.5 μg/mL) and piper betel (IC 50 84.63+13.09 μg/mL) at concentrations ranged from 20 to 100 μg/mL as reported by Sindhu [26]. The βglucosidase screening of the Vernonia amygdalina extracts showed that they do not potent and selective inhibition, the methanolic extract had 12.89+8.21% and chloroform extract had 15.58+4.00% inhibitory potential against β-glucosidase and these values were less than the Castonospermine standard (59.98%) as reported by Verma [28]. IC 50 of Aldose reductase (ALR2) of chloroform extract IC 50 (1.339+0.264μg/mL) was better than that of methanolic extract of IC 50 (1.437+0.6μg/mL) and these values were better when compared with the standard, sorbinil of IC 50 (3.10 +0.20μM).The aldehyde reductase (ALR1) of methanolic extract IC 50 (0.325+0.02μg/mL) was better than that of chloroform extract of IC 50 (0.964+0.16μg/mL) and when compared with standard 10mM vaproic acid IC 50 (57.4 +10μM).

Osiris Drug Properties
Some of the identified compounds were found to have various drug characteristics when screened using OSIRIS Online server explorer [29] and this include; drug likeness, cLogS, cLogP, mutagenic, tumorigenic, irritability, H-bond acceptor and H-bond Donor. The promising inhibitory effect of the plant extracts against aldose reductase, aldehyde reductase, α-glucosidase, glucoamylase and the identified bioactive compounds could be taken as a tool for further insight into the usefulness of the plant for the treatment of diabetes mellitus.

Conclusion
This study discovered the promising and excellent antidiabetic potential of methanolic and chloroform extracts of V. amygdalina that can be beneficial for the treatment of diabetes mellitus. This study will help the researcher to uncover the critical areas of using the various drug properties as revealed as a tool for gaining more insight onto the isolation of compounds in the plant with therapeutic properties that many researchers were not able to explore. Thus a new theory on antidiabetic agent may be arrived at.