The formulation of lozenge using black mulberries (Morus nigra L.) leaf extract as an α-glucosidase inhibitor.

BACKGROUND: Diabetes mellitus is a chronic metabolic disease, which possibly leads to kidney, brain, heart failure, and other organ complications, subsequently harming human health. These symptoms have been prevented using the leaf of black mulberry (BM), as a traditional medicine, because the phenolic compounds contained are able to decrease blood glucose concentration. Meanwhile, previous reports have shown that BM contains 1-deoxynojirimycin, with strong activity as an α-glucosidase inhibitor. The aim of this study, therefore, was to formulate and evaluate BM leaf extract in lozenge dosage form as an α-glucosidase inhibitor.
MATERIALS AND METHODS: The leaves of BM were extracted using the maceration method, where ethanol (70%) served as a solvent, and the inhibitory activity of the sourced α-glucosidase enzyme was determined through in vitro study. Subsequently, the extract was formulated into lozenge dosage form and evaluated for physical stability and also the effect of α-glucosidase enzyme.
RESULTS: The result showed an inhibitory activity of BM leaf extract against the enzyme α-glucosidase, with a half maximal inhibitory concentration (IC50) value of 357.6 μg/mL, whereas the lozenge formulation containing 43% of extract as well as 5% polyvinylpyrrolidone showed the best physical stability as compared to other formulas. However, the lozenge inhibits α-glucosidase enzyme with an IC50 value of 549.7 μg/mL.
CONCLUSION: It was established that the lozenge of BM leaf extract possesses activity as an α-glucosidase inhibitor.

INTRODUCTION

Diabetes mellitus is a chronic metabolic disease with a tendency to cause kidney, brain, and heart failure, as well as other organ complications, which is harmful to health. Furthermore, the inception is often affiliated with insulin resistance or a limitation in secretion and also low sugar use.[1] This form of resistance further impairs the body cell responsiveness,[2] categorized as a prediabetic stage associated with obesity, subsequently leading to type 2 diabetes mellitus.[3]

It is expected that the worldwide estimation of sufferers in 2030 increases by over twofold from the statistics obtained in 2005.[4] This is a potentially serious medical concern, and the drug choices during treatment have continuously been improved with the aim of attaining healing. However, resistance remains a huge challenge in the quest to achieve success, leading to interests in specific targeting, especially for type 2 diabetes mellitus.[5]

The control of postprandial glucose levels is a strategy to prevent diabetes mellitus, which is achieved by inhibiting the carbohydrate hydrolysis enzymes, including α-glucosidase in digestive organs. Therefore, it is possible for specific inhibitors, consisting of voglibose and acarbose, to restrain the liberation of glucose from oligosaccharides, and subsequently decreasing postprandial glucose levels and insulin responses.[6] This class of drugs are, therefore, applied in the control of patient blood glucose, combined with dietary modifications and other antidiabetic agents.[5] Furthermore, the use of combination drug therapy tends to cause side effect and is possibly detriment to human physiology;[1] hence, it is important to identify and develop new α-glucosidase inhibitors.

Morus nigra L. (black mulberry [BM]) is well-known natural plant used as antidiabetic agents. Previous reports have shown the possibility for the leaf powder to reduce very low density lipoprotein cholesterol, low-density lipoprotein cholesterol triglycerides, fatty acids, and blood glucose in patients with type 2 diabetes mellitus. Also, the water extract caused substantial decline in the glucose levels of induced rats, subsequently leading to the regulation of oxidative stress levels, improved hexokinase activity, synthesis of glycogen, and reduction in glucose-6-phosphate formation in the liver of animals.[27]

The mechanisms of BM Leaf tends to be multidirectional, as 1-deoxynojirimycin (DNJ) has been identified as the best-known main component, possessing the tendency to inhibit α-glucosidase enzyme present in the small intestine.[8] The aim of this study, therefore, was to formulate and evaluate BM leaf extract in the lozenge dosage form and also to estimate the activity of α-glucosidase inhibitor, using in vitro study. Conversely, a lozenge is a solid dosage form intended to disintegrate or dissolve slowly in the mouth, which has the following advantage: easing of consumption for pediatric and geriatric patients, and also ensuring an extended contact period between the active drug and the oral cavity.

MATERIALS AND METHODS

Materials

Plant material

The leaf of BM was collected from Maribaya Timur, Cibodas, West Java and authenticated by the Department of Biology, Faculty of Science, Universitas Padjadjaran, Bandung, Indonesia.

Chemicals

The α-glucosidase enzyme was purchased from Sigma-Aldrich (Saint Louis, MI. USA), and all other chemicals used were of technical grade.

Extraction

The leaves of BM were dried in an oven at 35oC–40oC, and subsequently extracted using 70% ethanol with a maceration method for 24 hours (×3) at ambient temperature. The ethanol was removed by a rotary evaporator (IKA RV 10, IKA Company, Staufen, Germany) at 40°C to obtain the crude extract.[910]

Phytochemical screening extract

Phytochemical screening was conducted to evaluate the presence of secondary metabolites, including flavonoids, alkaloids, polyphenols, tannins, saponins, quinones, steroids/triterpenoids, monoterpenes, and sesquiterpenes.[11]

Formulation of lozenge

The lozenge formulation consisting of BM leaf extract and all other ingredients was prepared through wet granulation, and the granules were subsequently dried in the oven at 40oC for 6h. These were then directly compressed into tablets with a press, filling an average weight of 650mg, with the composition shown in Table 1.

Table 1

Formulation of black mulberry leaf extract lozenges

Ingredients	Composition of lozenges (%)
	FI	FII	FIII	FIV	FV	FVI
BM leaf extract	43	43	43	43	43	43
Aerosil	2	2	2	2	2	2
Polyvinylpyrrolidone	3	3	3	5	5	5
Stevia	4	5	6	4	5	6
Maltodextrin	1	1	1	1	1	1
Avicel	2	2	2	2	2	2
Magnesium stearate	4	4	4	4	4	4
Mannitol	41	41	41	41	41	41

BM = black mulberry

Evaluation of black mulberry leaf extract granules (pre-compression parameters)

The granules consisting of BM leaf extract and excipient were evaluated using flow properties, determined with angle of repose and compressibility parameters by Carr’s index, as well as tapped and bulk density.[1213]

Evaluation of black mulberry leaf extract lozenge (post-compression parameters)

The thickness and weight variation

A total of 20 lozenges per formula were measured for thickness testing, using a Vernier Caliper. Also, 20 tablets were individually weighed using an electronic balance, and the values obtained were compared to the average tablet weight, and the results presented as mean ± standard deviation (SD).[13]

Hardness

The hardness of 10 lozenges for each formula was determined using the tablet hardness tester instrument (VK 200 Tablet Hardness Tester, Varian, North Carolina, USA), and the results presented as mean ± SD.[14]

Friability

Twenty lozenges from each formula were accurately weighed and placed in tablet friability tester (Varian Friabilator 25–4000, North Carolina, USA), which rotated at 25rpm for 4min. These were subsequently brushed and reweighed, and then the percent of weight loss was calculated using the formula: % friability = ([initial weight – final weight]/initial weight) × 100.[13]

Disintegration time

The disintegration time analysis of lozenges was conducted according to USP 30, using a disintegration tester instrument (Erweka ZT6-1-D, Langen, Germany) containing the phosphate buffer medium maintained at pH 6.2 and 37°C ± 0.5°C.[15]

Stability test

Stability studies were conducted at 40oC with relative humidity (RH) 75%, and also at room temperature (25oC) with 75% RH for 30 days. Also, other physical parameters of lozenges were evaluated, including hardness, friability, and dissolution time.[16]

Determination of α-glucosidase inhibitory activity

Ten μL of BM leaf extract and the formulated lozenges were briefly dissolved, respectively, in dimethyl sulfoxide at varying concentrations. This was then mixed with 40 μL of phosphate buffer (pH 7.0) and 25 μL of p-nitrophenyl-α-D-glucopyranoside, followed by incubation at 37°C for 5min, and the addition of 25-μL α-glucosidase solution. The mixture was, therefore, incubated at 37°C for 15min, prior to the incorporation of 100 μL of Na2CO3 solution (0.2 M) to terminate the reaction, which was then monitored at 405nm using a microplate reader. Furthermore, the half maximal inhibitory concentration (IC50) value was calculated from the concentration–effect linear regression curve, where acarbose was adopted as a positive control.[1718]

Statistical analysis

The experimental data, including in vitro study and physical stability presented as mean of samples ± SD, were statistically analyzed using one-way analysis of variance (ANOVA) method. However, Kruskal–Wallis analysis method was used on instances where the data were not normally distributed.[19]

RESULT AND DISCUSSION

The determination results obtained at the Department of Biology, Faculty of Science, Universitas Padjadjaran showed Morus nigra L. as the species of BM leaves used in this study. Also, maceration method was adopted in the extraction process, in an attempt to protect compounds, especially those responsible for the inhibition of α-glucosidase enzyme, contained in the BM leaves from thermal decomposition.[11] Furthermore, 70% ethanol was used as a solvent, based on the universal dissolution characteristics for both polar and nonpolar constituents,[20] producing a rendement value of 21.83%.

The phytochemical screening conducted to determine the presence of secondary metabolites detected flavonoids, polyphenols, tannins, steroids and triterpenoids, and also saponins. This result supports the assumption that α-glucosidase inhibitory compounds exist in BM leaf, encompassing flavonoids, phenolic acid, flavonol derivative, and polyphenols.[18]

α-Glucosidase is a determinant enzyme affiliated with the inception of postprandial hyperglycemia, attained by the hydrolysis of oligosaccharides (type 2 diabetes mellitus).[21] Furthermore, acarbose and voglibose are well-known commercial brands used in treatment, which show numerous side effect, including liver disorders, flatulence, and hepatic injury.[2223] Also, the leaf of BM has been reported to possess effective α-glucosidase inhibitory activity.[24]

This potential was determined through tests and a comparison was made using acarbose as a positive control. Furthermore, the inhibition profile was ascertained with the IC50 values using the dose–response curves obtained through the serial dilution of BM leaf extract and acarbose, at concentrations of 4000–62.5 μg/mL.[6] The IC50 calculated for BM leaf extract was 228.5 μg/ mL ± 11.4, whereas the value for kojic acid was 357.6 μg/ mL ± 10.5. This, therefore, indicates the potential for the material studied to serve as a α-glucosidase inhibitor, although statistical analysis showed no significant difference (P < 0.05) in activity, compared to IC50 of acarbose.

The 1-deoxynojirimycin (DNJ) content of BM leaf extract has been affiliated with the enzyme inhibitory properties, due to the ability to effectively reduce postprandial blood glucose levels.[25] Also, some previous studies showed the strong inhibitory effect of disaccharidase present in human digestive tract, implicated in the reduced conversion of disaccharides to glucose in the body. Conversely, this does not affect the absorption of sugars,[26] whereas α-glucosidase inhibitors diminish carbohydrate absorption in the intestine, subsequently decreasing blood glucose levels.[2]

The result of granules pre-compression evaluation is shown in Table 2.

Table 2

Evaluation of pre-compression

Parameters	F1 (1:1)	F2 (1:2)	F3	F4	F5	F6
Angle of repose (o)	24.7 ± 0.72	23.77 ± 1.47	22.30 ± 1.44	21.56 ± 0.63	20.16 ± 1.43	20.16 ± 0.51
Flow rate (g/s)	12.96 ± 0.80	12.32 ± 0.73	11.96 ± 0.94	16.01 ± 1.13	15.97 ± 0.60	15.97 ± 0.60
Carr’s index (%)	20.74 ± 4.36	21.27 ± 4.88	22.27 ± 3.70	17.05 ± 1.34	15.07 ±3.61	16.17 ± 2.48

All the values were calculated as mean ± standard deviation

The angle of repose for all formulations ranged from approximately 20o to 25o, whereas the flow rate was over 10g/s, with Carr’s compressibility index of approximately 16%–21%. These are indicative of good granule flow property for the BM leaf extract and all excipients.[13]

The results of post-compression, encompassing weight variation, diameter, thickness, hardness, friability, and disintegration time are reported in Table 3.

Table 3

Evaluation of post-compression

Formula	Weight variation (mg)	Diameter (mm)	Thickness (mm)	Hardness (N)	Friability (%)	Disintegration time (min)
F1	650.8 ± 0.71	12.99 ± 0.03	5.27 ± 0.05	35.05 ± 2.26	0.79 ± 0.19	5.65 ± 0.02
F2	651.3 ± 1.13	13.00 ± 0.02	5.28 ± 0.04	36.00 ± 2.45	0.87 ± 0.13	5.62 ± 0.02
F3	651.2 ± 1.00	13.00 ± 0.02	5.28 ± 0.04	35.65 ± 2.08	0.87 ± 0.29	5.68 ± 0.02
F4	651.2 ± 0.96	13.00 ± 0.02	5.26 ± 0.05	55.75 ± 2.57	0.41 ± 0.08	6.10 ± 0.16
F5	651.6 ± 1.08	12.99 ± 0.02	5.29 ± 0.04	55.95 ± 2.52	0.44 ± 0.14	6.16 ± 0.13
F6	651.1 ± 0.91	13.00 ± 0.02	5.29 ± 0.04	56.10 ± 2.55	0.38 ± 0.11	6.18 ± 0.16

All the values were calculated as mean ± standard deviation

The mechanical properties of tablet/lozenges serve as an important test in pharmaceuticals, with reference to pharmacopoeial requirements; hence, all formulations result in very low weight products, which lie within the limits. Furthermore, the friability recorded was <1%, whereas the diameter of all formula was not more than three times and not <1 1/3 the thickness. Conversely, the disintegration time for all formulas was <15min, indicating that official requirements for tablets were met.[27]

The stability test for lozenge, including friability, hardness, and disintegration time is shown in Figures 1–3.

Figure 1

Friability measurement result of black mulberry leaf extract lozenges (all sample values were determined as mean ± standard deviation; n = 3)

Figure 3

Disintegration time result for black mulberry leaf extract lozenges (all sample values were determined as mean ± standard deviation; n = 3)

Hardness measurement result of black mulberry leaf extract lozenges (all sample values were determined as mean ± standard deviation; n = 3)

On the basis of friability measurements, the lozenges remained within the acceptable official requirements, which is <1%. Furthermore, statistical analysis using ANOVA showed a significance value of 0.010 (P < 0.05), which indicates the absence of any storage effects on the product friability.

On the basis of hardness measurements, some formulated lozenges remained within the official range recommended for tablets (3–40 Kp). However, the results of statistical analysis by ANOVA showed a significance value of 0.015 (P < 0.05), which indicates that there was no effect of storage on lozenge hardness.

On the basis of the disintegration time measurement, the formulated lozenges were maintained within the official range recommended for tablet (<15min). Furthermore, statistical analysis obtained using ANOVA showed a significance value of 0.010 (P < 0.05), which indicates the absence of any storage effects on lozenge disintegration time.

The formulation of lozenge containing 43% BM extract and 5% polyvinylpyrrolidone (PVP) (FVI) was selected for the determination of α-glucosidase inhibitory activity, due to the high convenience of this formula, as compared to others. Therefore, the result of α-glucosidase inhibitory activity determination is shown in Table 4.

Table 4

Result of α-glucosidase inhibitory activity determination for formulated lozenges

Sample	IC50 (μg/mL)
Acarbose	228.60 ± 6.23
BM leaf extract	357.60 ± 8.44
Lozenges of BM leaf extract	549.72 ± 10.45
Lozenges without BM leaf extract	6773.92 ± 11.62

BM = black mulberry, IC50 = half maximal inhibitory concentration

All sample values were determined as mean ± standard deviation; n = 3

The result showed lower activity in the BM leaf extract lozenges, as compared with acarbose and BM leaf extract, although it specifically manifested potential tyrosinase inhibitory activity. This result indicates the presence of DNJ, which possesses remarkable α-glucosidase inhibition potentials. Furthermore, statistical analysis showed significant difference (P < 0.05) between the IC50 of the formulated lozenges and the blank preparation.

CONCLUSION

On the basis of the results and discussion, it is concluded that BM leaf extract possesses an α-glucosidase inhibitory activity, due to the IC50 value of 357.6 μg/mL. Furthermore, the formulation containing 43% BM extract and 5% PVP showed the best physical stability, whereas BM leaf extract lozenges showed high α-glucosidase inhibitory activity, with an IC50 value of 549.7 μg/mL.

Acknowledgement

The authors are grateful to Universitas Padjadjaran for the provision of financial support.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.