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Formulation of Mucuna pruriens seed extract as tablets and the in vivo antidiabetic properties of the tablets

Stephen O. Majekodunmi1, Ademola A. Oyagbemi2, Oluwatoyin A. Odeku 3*
1Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy, University of Uyo, Uyo, Nigeria,
2Department of Veterinary Physiology, Biochemistry & Pharmacology, University of Ibadan, Nigeria
3Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, University of Ibadan, Ibadan, Nigeria
				


*Corresponding author
Email: HYPERLINK "mailto:pejuodeku@yahoo.com" \t "_blank"pejuodeku@yahoo.com; o.odeku@ui.edu.ng
Tel: +234�8057320466


					
ABSTRACT
Diabetes mellitus and its complications continue to be one of the highest causes of morbidity and mortality in recent time. Mucuna pruriens L. extract has been formulated into tablets using direct compression (DC) and wet granulation (WG) methods  and the in vivo antidiabetic properties of the tablet have been evaluated in alloxan induced diabetic rabbits. Mechanical properties of the tablets were assessed using crushing strength, friability and the crushing strength-friability ratio while the drug release properties were evaluated using the disintegration and dissolution times. The in vivo antidiabetic properties of the tablets was evaluated in alloxan induced diabetic rabbits and compared with the effects glibenclamide. The results showed that M. pruriens tablets prepared by direct compression exhibited higher mechanical and faster drug release properties than tablets prepared by wet granulation. The properties of the tablets depended on the method of preparation, compression pressure, type of excipient/binder and the concentration of the binding agent used in the formulation. The results of the in vivo studies indicates that M. pruriens tablets exhibited hypoglycaemic properties in alloxan-induced diabetic rabbits with the highest reduction in blood glucose level of 51.5 and 47.4% observed for tablets prepared by DC and WG respectively, 50.8% reduction for glibenclamide d 72h after drug administration. Statistical analysis indicated that there were no significant (p>0.05) difference in the reduction in the blood glucose levels achieved for M. pruriens tablets and glibenclamide although faster reduction in blood glucose level was achieved by glibenclamide. This indicates that M. pruriens tablets could be successfully formulated and  used for the treatment of diabetes. Furthermore, the method of preparation and the type and concentration of excipients/ binders employed in the formulation need to be carefully selected to enable the production of tablets with the desired drug release properties.  


Keywords: Diabetes, Mucuna pruriens, Tablet, Drug release properties, Mechanical properties.



Introduction
Diabetes mellitus, a metabolic disorder of the pancreas in which the blood glucose levels are abnormally high (hyperglycaemia), is a growing threat to public health in modern society due to urbanization, population growth, aging and increasing prevalence of obesity and physical inactivity [1]. While some Non Insulin Dependent Diabetic Mellitus (NIDDM) patients can be managed by diet alone, other forms require hypoglycaemic therapy and/or insulin. The drawbacks of insulin such as oral ineffectiveness, short shelf life, requirement of constant refrigeration, parenteral therapy with its attendant abscesses and fatal hypoglycaemia in the event of excess dosage has limited its usage and pharmacotherapy with sulphonylureas, biguanides and thiazolidones is also associated with side effects [2]. 
The use of medicinal plants in the treatment of diabetes has been reported in several studies in African region and up to 80% usage has been reported in Morocco among the type II diabetic patients [3-6]. Ethnobotanical studies of plants used in the treatment of diabetes in parts of African have shown that about 49 plant species are used in Lagos, South West Nigeria[7], 34 plant species in North West Nigerian plants [8], 14 species in Enugu, South East Nigeria [9], 28 plant species in Cote d�ivoire [10, 32 species in South Africa [11] and 54 plant species in North Central region of Morocco[12]. 
One of the plants used in the treatment of diabetic is Mucuna pruriens L (Fam. Leguminoseae) also known as velvet beans or Cowitch in English, Werepe in Yoruba; Karara in Hausa and Agbara in Ibo. It is an annual climbing plant indigenous to tropical regions, especially Africa, India and the West Indies where it is found in bushes, bush paths and hedges, and is eaten as food during famine and specialty food in North Eastern India. Studies have demonstrated the hypoglycaemic properties of the alcohol extract of the seed of M. pruriens in normal rats and streptozotocin [13, 14, 15],  and in alloxan-induced diabetic rats [16]. A recent study has shown that acute and chronic administration of the extract resulted in a dose dependent reduction in the blood glucose level in alloxan-induced diabetic rats with 55% reduction in blood glucose level achieved 8h after administration of 100mg/kg body weight of the extract while the administration of glibenclamide (5mg/kg body weight) resulted in 60% reduction [17]. Acute toxicity studies revealed that the extract was relatively safe at low doses, although some adverse reactions were observed at higher doses (8-32 g/kg body weight), no death was recorded. Furthermore, oral administration of M. pruriens seed extract significantly (p<0.001) reduced the weight loss associated with diabetes.
Herbal medicinal products have been widely criticised in spite of their efficacy due to lack of standardization and good presentation. In traditional medicine, the powdered seed of M. pruriens is usually soaked in hot water or alcohol and unspecified quantities of the decoction are ingested without recourse to toxicological and other side effects [6, 16]. However, to improve patient compliance and acceptance, there is the need to formulate the extract of the seed of M. pruriens into tablet dosage form. Thus in the present study, the ethanolic extract of the seed of M. pruriens has been formulated into tablet dosage form by direct compression (DC) and wet granulation (WG) methods to determine the most suitable method for the preparation of the tablets and the in vivo antidiabetic properties of the tablets has been investigated in alloxan induced diabetic rabbits and compared with those of glibenclamide.

Materials and methods
The materials used are microcrystalline cellulose co-processed with �-lactose monohydrate, (Cellactose�, Meggle Group, Wasserburg, Germany), corn starch B.P (BDH Chemicals, Poole, London, UK), microcrystaline cellulose (Avicel� RH 102, FMC International Co. Cork, Ireland), lactose (DMV, Veghel, Netherlands), magnesium stearate (Hopkins and Williams, Chadwell, Health, Essex, UK), alloxan monohydrate (Sigma Chemical Co., St. Louis, M.O., USA), the seeds of Mucuna pruriens were collected at Tosa village near Moniya at Akinyele Local Government of Oyo State, Ibadan, Nigeria in the month of January and authenticated (Voucher No. UIH 22305) by the Herbarium Unit of the Department of Botany and Microbiology, University of Ibadan, Ibadan, Nigeria. A description of the extraction and characterisation of the extract has been give elsewhere [17]. All other chemicals and reagents used are of analytical grade.

Preparation of powder mixtures for direct compression
Batches (100g) consisting of M. pruriens extract (20%) and Cellactose, or Avicel (80%) as diluents were thoroughly mixed for ten (10) minutes using a cubic mixer. The degree of mixing was determined and found to be ( 95% [18].		

Preparation of granules
Batches (100 g) consisting of M. pruriens extract (20% w/w), lactose (70%w/w) and corn starch (10%w/w) were dry-mixed for 5 min in a Kenwood planetary mixer and then moistened with 1%w/w or 4%w/w binder solution (PVP or corn starch mucilage). Massing was continued for 5 min and the wet masses were granulated by passing them manually through a No.12 mesh sieve (1,400 (m), dried in hot air oven for 18 hour at 50oC, and then re-sieved through a No. 16 mesh size (1,000 (m). The moisture content of the granules was determined with an Ohaus moisture balance (Ohaus Scale Corporation, USA). The degree of mixing was determined and found to be ( 95%. The granules were stored in airtight containers.

Preparation of tablets
Tablets (500 mg) were prepared from the powder mixtures and granules (500-1000�m size fractions) by compressing the materials for thirty seconds with predetermined loads of 5 and 10kN using a Carver Hydraulic Hand Press (Model C, Carver Inc, Menomonee Falls, WI, USA). Before each compression, the die (10.5 mm diameter) and the flat-faced punches were lubricated with a 2%w/w dispersion of magnesium stearate in ethanol: ether (1:1) solution. After ejection, the tablets were stored over silica gel for 24 hours to allow for hardening and elastic recovery.

Crushing strength and friability tests
The load required to diametrically break the tablet (crushing strength, CS) was determined at room temperature using a PTB 301 crushing strength tester (Pharmatest, Switzerland). 
The friability of the tablets was determined using a friabilator (Model TF 2D, Scientific Equipment Ltd., Bombay, India) operated at 25 revolution per minute (rpm) for 4 min. 

Disintegration test
	The disintegration times (DT) of the tablets were determined in distilled water at 37 ( 0.50C using an Erweka disintegration apparatus (Model: Copley ZT2, Erweka Apparatebau GMBH, Heusenstamm, Germany).


Determination of UV absorption for Mucuna pruriens seed extract
Ethanol extract (1g) was weighed and placed in 100 mL flask and 0.1M HCl was added to make up to volume and the flask was agitated in a shaker for about 1 hour, then filtered through a Whatman filter paper No1 and the filtrate was scanned using a UV spectrophotometer (Model Cintra 6, Type GBC UV �Visible, GBC, Scientific Equipment Limited, Victoria, Australia) and the wavelength for maximum absorption was determined. 
Different concentrations of the ethanolic extract of Mucuna pruriens were prepared to contain between 0.02 to 0.1mg/mL of extract in 0.1M HCL. The absorbance of each concentration was taken at the wavelength of 281nm and plotted against the various concentrations to obtain the calibration curve for the extract. 

Dissolution test
The dissolution times of the tablets were determined at 37(0.5oC in 900 mL of 0.1M HCl using a dissolution test apparatus (Model DA-6D, Veego Scientific Devices, Mumbai, India) with the rotating basket positioned 25 mm above the bottom of the round-bottomed flask and operated at 50 rpm. Samples (5mL) were withdrawn and replaced with fresh medium at different time intervals and the samples were appropriately diluted .The amount of drug released was determined spectrophotometrically at wavelength of 281nm (Model Cintra 6, Type GBC UV �Visible, GBC, Scientific Equipment Limited, Victoria, Australia). The results are the mean of four determinations.
	
In vivo antidiabetic studies
Animals
 Rabbits (1.00 - 1.65 kg) of both sexes bred in the animal house of the Department of Veterinary Pharmacology, Faculty of Veterinary Medicine, University of Ibadan (Ibadan, Nigeria) were used for the in vivo studies. The animals were grouped randomly into 5 groups (A - E) consisting of four rabbits each and kept at room temperature (25(2oC) with good ventilation, and allowed free access to water and food. The animals were acclimatized for one week before the commencement of the experiment.


Induction of diabetes
The animals were fasted for 16 hours but given free access to water ad libitum. Diabetes was induced by a single intraperitoneal injection of 100 mg/kg of alloxan monohydrate (dissolved just before use in 0.9% normal saline) to the rabbits in groups B, C, D and E while group A served as normal control. Hyperglycemia was confirmed 72 hour after alloxan injection (blood glucose level range of 450-485 mg/dL). 

In vivo antidiabetic properties
For the in vivo studies, Group A consisted of untreated rabbits (normal control), Group B consisted of diabetic control rabbits which did not receive any medication, Groups C and D were given one tablet each (containing 100 mg M. pruriens seed extract) produced by direct compression and wet granulation respectively, while Group E were given glibenclamide tablet as standard reference drug (5 mg/kg body weight). Tablets were inserted directly into the guts of the rabbit using pointed forceps. Blood samples were collected from marginal ear vein of all the animals onto glucometer strips and the blood glucose level was determined at 0 min (pre dose) and at various time intervals for 192 hours. 
	The rate of elimination of glucose was fitted into the First-order equation:
	
		 ln Qt =  ln Qo -  kt                                     (1)

where k1 is the first-order elimination rate constant, Qt is the concentration of glucose at time t, and Qo is the initial blood glucose concentration. The blood glucose elimination rate was obtained from a plot of Log blood glucose level at time t (Qt) versus time. The slopes of the regression analysis is the elimination rate constant.

Statistical analysis                                
Statistical analysis was done using ANOVA followed by Dunnett�s multiple comparison tests. Data were expressed as mean � S.D. p value < 0.05 was considered significant.



Results and Discussion
Formulation studies
The results of the mechanical properties of M. pruriens tablets prepared using direct compression (DC) and wet granulation (WG) are shown in Table 1. The results indicate that crushing strength (CS) which provides a measure of tablet strength, increased while friability (F) which provides a measure of tablet weakness [20], decreased with increase in the compression force for tablets prepared by both methods. Mucuna pruriens extract was directly compressible on its own and formed intact tablet without addition of any diluents. However, tablets containing the directly compressible excipient exhibited higher mechanical properties than  those made with the extract alone while tablets containing Cellactose exhibited higher mechanical strength than those containing Avicel. On the other hand, there was an increase in the CS and decrease in friability of tablets with increase in the concentration of binding agent used in the formulation. Formulations containing polyvinylpyrrolidone exhibited higher CS and lower F than those containing cornstarch. The results showed that all M. pruriens tablets prepared by both methods passed the BP requirements for friability by showing friability values less than 1%w/w [19, 21]. Statistical analysis revealed that there were no significant difference (p>0.05) in the mechanical properties of tablet prepared using the two binders. Thus, M. pruriens tablets with good mechanical strength that will be able to withstand the rigours of transport and handling could be prepared by both direct compression and wet granulation. 
The crushing strength-friability ratio, CSFR, also increased with increasing compression force and concentration of binder employed in the formulation. The ranking of CSFR values for tablets prepared by direct compression was Avicel > Cellactose > extract alone while M. pruriens tablets containing PVP as binders had higher CSFR values than tablets prepared by corn starch. Generally, tablets prepared by direct compression exhibited higher mechanical strength than tablets prepared by wet granulation.
The results drug release properties of pharmaceutical tablets which have been characterized using the disintegration and dissolution times for M. pruriens tablets are presented in Table 1. The disintegration time of M. pruriens tablets prepared containing the extract alone was significantly (p<0.05) higher than those of the tablets containing the directly compressible excipients. However, there was no significant (p>0.05) difference in the disintegration time containing Avicel and Cellactose. On the other hand, the disintegration time of tablets prepared by wet granulation containing corn starch as binding agent was significantly (p<0.05) higher than those containing PVP at the same concentration. Furthermore, tablets prepared by wet granulation exhibited higher disintegration times than those prepared by direct compression. The results showed that all formulations of M. pruriens tablets complied with the official requirement on disintegration by disintegrating within 15 minutes [21].
The result of the spectrophotometric analysis showed that the ethanolic extract of M. pruriens exhibited a principal absorption maximum at 281nm. The calibration curve of absorbance versus concentration at wavelength of 281nm was linear regression with equation of y = 9.150x � 0.015 and coefficient of determination, r2 = 0.998. The amount of drug released was plotted against time and representative plots for M. pruriens tablets containing the extract alone prepared by direct compression, Cellactose as directly compressible excipient and corn starch (5%) as binder prepared at compression force of 5kN are presented in Fig. 1. The dissolution times (t50 and t80- time required for 50% and 80% of M. pruriens to be released respectively) were obtained from the dissolution profiles of the tablets. The ranking of the dissolution times for tablets prepared by direct compression was generally extract alone>Cellactose>Avicel while for wet granulation, the dissolution times increased with increase in the concentration of the binding agent with formulations containing PVP generally showing higher values. However, only M. pruriens containing corn starch as binding agent complied with the official requirement on dissolution times for uncoated tablets by releasing 80%w/w of the drug within 45min (21). There were generally significant (p < 0.05) differences in the dissolution times of tablets prepared by both methods. 
It is noteworthy that M. pruriens tablets prepared by both methods produced tablets of acceptable mechanical properties, however only tablets produced by wet granulation containing corn starch as binding agent at the concentration of 1%w/w, fulfilled the pharmacopoeial requirements mechanical and drug release properties. Wet granulation appears to produce tablets of acceptable mechanical and drug release properties than those prepared by direct compression. Thus, the methods of preparation of the M. pruriens tablets need to be carefully selected to ensure the production of tablets with adequate bond strength to withstand the rigors of handling and at the same time release the active compound (s) for biological action. Furthermore, the type and concentration of excipient and binder employed in the formulation of M. pruriens the tablets need to be carefully chosen to enable the production of suitable tablets.
In vivo antidiabetic properties of M. pruriens tablets
The in vivo antidiabetic properties of M. pruriens tablets prepared by direct compression and wet granulation methods was compared with that of glibenclamide after single administration to alloxan-induced diabetic rabbits. Plots of Log blood glucose level versus time in rabbits administered with M. pruriens tablets prepared by direct compression, wet granulation and glibenclamide tablets are presented in Figure 2 while the parameters obtained from the First order elimination rate of blood glucose are presented in Table 2. The results showed that there was a gradual decrease in the blood glucose level of the diabetic rats treated with M. pruriens tablets and glibenclamide. Three phases of linearity was observed at 0-10h, 10-72h and 72-192h, with correlation coefficients generally (0.95. The elimination of glucose from the rabbits exhibited an initial fast rate of reaction in the first 10 h after which the rate becomes slower. The ranking of the of the elimination rate constant k1 was glibenclamide> DC > WG. Thus, glibenclamide facilitated faster elimination of glucose form the blood while M. pruriens tablets prepared by wet granulation facilitated the slowest elimination rate. The rate of elimination of glucose in the second phase, k2, became slower after 10 h (t1) and the lowest blood glucose level was achieved at 72 h for M. pruriens tablets and 54 h for glibenclamide tablet. Statistical analysis indicates that k1 was significantly higher (p < 0.05) than k2. After 72 h there was a rebound as the blood glucose level increased as a result of the decrease in the concentration of the hypoglycaemic agent in the blood. The blood glucose level increased to the diabetic concentration after 192 hour when the experiment was terminated. This is expected since only a single dose of the tablets was administered. In antidiabetic therapy, repeated administration of the drug is done at specified time intervals ensuring that the blood glucose level is maintained at the desired level over the period of treatment.
The chart of the blood glucose level in rabbits 72h after administration of M. pruriens tablets prepared by direct compression, wet granulation and glibenclamide to the diabetic rabbits, the diabetic control rabbits which did not receive any medication and normal control rabbits where diabetes was not induced are presented in Figure 3. The results showed that the blood glucose level was consistently high in the diabetic rabbits whereas, there was a significant (p<0.01) decrease in the blood glucose level of the treated animal. The results also showed that for the treated groups, the reduction in blood glucose level of 44.0 and 39.8% was achieved for M. pruriens tablets prepared by direct compression and wet granulation respectively while 52.5% reduction was observed for glibenclamide. However, after 72h, the highest reduction in blood glucose level of 51.5 and 47.4% was observed for M. pruriens tablets prepared by DC and WG respectively while glibenclamide exhibited 50.8% reduction. Statistical analysis indicated that there were no significant (p>0.05) difference in the reduction in the blood glucose levels achieved for M. pruriens tablets and glibenclamide although faster reduction in blood glucose level was achieved by glibenclamide. This indicates that M. pruriens tablets could be successfully formulated and  used for the treatment of diabetes.

Conclusion
Mucuna pruriens tablets with good mechanical and drug release properties have been successfully formulated by direct compression and wet granulation. The properties of the tablets depended on the method of preparation, compression pressure, type of excipient/binder and the concentration of the binding agent used in the formulation. The results of the in vivo studies indicates that M. pruriens tablets exhibited hypoglycaemic properties in alloxan-induced diabetic rabbits for over 72 h. The result showed that the methods of preparation of M. pruriens tablets and the type and concentration of excipients/ binders employed in the formulation of M. pruriens need to be carefully chosen to enable the production of tablets with the desired drug release properties.  

References
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Table 1: Mechanical and drug release properties of M. pruriens tablets prepared by direct compression and wet granulation methods (mean (SD, n=4)

Direct compression
CP
(kN)DiluentCrushing strength (kN)Friability (%)   CSFRDT
(min)t50
(min)t80
(min)5Extract alone33.4 � 0.00.6 � 0.052.2 � 0.06.73� 0.0241.56� 0.7183.41� 0.141036.5 � 0.00.5 � 0.071.4 � 0.013.17� 0.0154.73� 0.6187.48� 0.215Avicel33.7 � 0.10.5 � 0.167.4 � 0.03.44� 0.0258.21� 0.5264.12� 0.371036.2 � 0.00.4 � 0.090.5 � 0.14.56� 0.0362.11� 0.4768.17� 0.475Cellactose35.1 � 0.00.7 � 0.047.5 � 0.03.18� 0.0147.23� 0.4570.23� 0.481039.1 � 0.00.5 � 0.078.2 � 0.04.10� 0.0461.15� 0.5786.18� 0.31

Wet granulation
CP
(kN)BinderBinder conc. (%w/w)Crushing strength (N)Friability (%)CSFRDT
(min)t50
(min)t80
(min)5PVP131.0 � 0.10.7 � 0.143.7 � 0.03.78� 0.0136.50� 0.1755.12� 0.211032.6 � 0.00.7 � 0.049.4 � 0.14.13� 0.0343.13� 0.1258.32� 0.655433.5 � 0.10.5 � 0.064.4 � 0.05.00� 0.0242.10� 0.1956.50� 0.421034.5 � 0.00.4 � 0.086.3 � 0.17.36� 0.0143.00� 0.1661.17� 0.725Corn starch130.5 � 0.10.9 � 0.033.9 � 0.09.27� 0.1124.20� 0.2540.14� 0.611032.0 � 0.00.7 � 0.145.7 � 0.09.80� 0.1227.13� 0.3244.13� 0.565432.9 � 0.00.6 � 0.154.8 � 0.010.48� 0.0142.14� 0.4152.18� 0.411033.6 � 0.00.5 � 0.067.2 � 0.111.17� 0.0160.32� 0.3476.15� 0.26


Table 2: Glucose kinetics: Different rate constants (k) and corresponding points of change from one component to the other (t) and half-lives (t1/2) for tablets prepared by direct compression, wet granulation and glibenclamide tablet

DrugMethod of preparationk1
(hr-1)t1
(h)k2
(hr-1)t2
(hr)k3
(hr-1)M. pruriensDC0.02810.00.00571.00.007WG0.02110.50.00571.00.005Glibenclamide
(Daonil�)-0.03510.00.00552.00.005




 

Figure 1: The release profiles of M. pruriens tablets containing the extract alone, (, Cellactose as diect compression excipient,(, and corn starch 1% as binder,(, prepared at compression force of 5kN. (mean � SD, n = 4)


 


Figure 2: Plots of Log blood glucose level versus time in rabbits administered with M. pruriens tablets prepared by direct compression using Avicel as excipient, (; wet granulation using 4% PVP as binder ,(, and glibenclamide tablets, (. (Mean( SD, n=4)
 
 

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