Formulating anti-diabetic nutraceutical tablets based on edible plants from Tripura, India

: Nutraceuticals are food-based drugs that are used as dietary supplements to minimize chronic diseases. Diabetes is one of the most common chronic diseases all over the world. Recently, herbal nutraceuticals have taken a promising role in treating diabetes. We aimed to develop herbal nutraceutical tablets and evaluate its anti-diabetic activity using ob/ob mice. Five plant species were collected by field survey methods based on oral interviews with traditional healers of Tripura. The wet granulation method was applied to formulate the herbal nutraceutical tablet. Water- and fat-soluble vitamins were determined by reversed-phase high-performance liquid chromatography. Trace elements were analyzed by atomic absorption spectrophotometry. To evaluate the anti-diabetic activity of the herbal tablets, we determined serum hemoglobin, glycosylated serum protein, and oral glucose tolerance. The newly formulated herbal nutraceutical tablets provided the optimal energy level. It contained sufficient amounts of essential minerals, such as iron (74.6 ± 2.7 mg/g), sodium (4.4 ± 0.4 mg/g), potassium (5.3 ± 0.7 mg/g), calcium (163.1 ± 2.2 mg/g), magnesium (39.2 ± 1.7 mg/g), and phosphorus (14.6 ± 2.1 mg/g). We also found optimal quantities of water-soluble vitamins, such as vitamin C (27.2 ± 4.3 mg/g), vitamin B1 (0.6 ± 0 mg/g), vitamin B3 (0.6 ± 0.2 mg/g), vitamin B6 (1.1 ± 0.2 mg/g), vitamin B12 (0.6 ± 0.2 mcg/g), and folic acid (82.6 ± 7.6 mcg/g), as well as fat-soluble vitamins, such as vitamin A (287.4 ± 6.3 mcg/g), vitamin D3 (2.6 ± 0.6 mcg/g), and vitamin E (0.7 ± 0 ng/g). Finally, the herbal nutraceutical tablet (200 mg/kg) significantly improved the anti-hyperglycemic effect on ob/ob mice (type 2 diabetes), compared to the standard drug, metformin (200 mg/kg). The results suggest that the newly formulated herbal tablet may be recommended as an anti-diabetic nutraceutical drug .

the regulations of the National Institutes of Health. The Institutional Ethics Committee (No. 1667/GO/a/12/ CPCSEA) approved the study protocol [18].
Acute toxicity test. Acute toxicity tests of five different plant powder samples were measured by the method described by Ali et al. with some modification [19]. The Swiss albino mice (18-25 g) of both sexes were divided into two groups, the control group and the experimental group, ten animals in each. The control group received only distilled water, whereas the experimental group received different doses (5, 50, 300, 800, 1200, and 2000 mg/kg body weight) of the powder sample orally. The mice were then kept under observation up to 72 h for mortality or symptoms of toxicity [19].
Formulation of herbal tablet. We used the wet granulation method to prepare novel herbal antidiabetic nutraceutical tablets. Equal amounts of previously prepared plant materials (M. paradisiaca fruit, M. paradisiaca stem, C. paeoniifolius corn, A. heterophyllus seed, and C. esculenta leaf) were placed in a rapid mixture granulator, with a 10% starch solution added dropwise into the binder. The damp masses were screened through a sieve #10 and then dried. The dried granules were screened through a sieve #20 and stored in a desiccator until they were ready for tablet compression. The prepared granules were compressed in a single punch tablet press machine (Manesty Type F3, Liver Poole, England) with a punch diameter of 0.75 cm and a compression pressure of 933 Pa (N/m 2 ). The die volume matched the tablets' weight to confirm that 600 mg was obtained [20].
Evaluation of granules. Tablet granules were evaluated by Wadher et al. methods with some modification. Particularly, we determined their bulk density, tapped density, Hausner quotient, Carr's compressibility index, flow rate, and angle of repose [21].
Evaluation of herbal tablets. Weight variation, thickness, hardness, friability, and in vitro disintegration time were determined by Wadher et al. methods with some modification [21].
Total moisture, total carbohydrate, complete protein, total fat, total ash, and total caloric value of the herbal tablets were determined by Debnath et al. method with some modification [17]. Total dietary fiber was measured by Ozoliņa et al. method with some modification [22].
Concentrations of minerals were determined by an atomic absorption spectrophotometer. Debnath et al. method with some modification was applied to measure the content of minerals in the tablets [17].
Vitamins C, B 1 , B 3 , B 6 , and folic acid were determined by Antakli et al. method with some modification, using the RP-HPLC system [23].
Determination of fat-soluble vitamins. Vitamins A, D 3 , and E were determined by using the reversed-phase high-performance liquid chromatography as reported by Xue et al. with some modification [24].
Anti-diabetic activity. Experimental design. The animals were randomly divided into four groups of six animals for test purposes, namely: a) a normal group (completely healthy mice) treated with 0.5% sodium carboxymethyl cellulose; b) a vehicle control group (ob/ob mice) treated with 0.5% sodium carboxymethyl cellulose; c) a positive control group treated with 200 mg/kg of metformin via gavage; and d) an experimental group treated with 200 mg/kg herbal nutraceutical tablet via gavage. The experiment lasted four weeks. At the end of the experiment, all the animal groups fasted overnight, and blood samples were collected from the tail vein. Before blood collection, the animals were given pentobarbital as an anesthetic agent [18].
Determination of serum hemoglobin and glycosylated serum protein. Hemoglobin (HbA1c) and glycosylated serum protein were measured by respective kits (Merck Millipore, Germany) according to the manufacturer's instruction [18].
Oral glucose tolerance test. After four weeks of treatment with herbal nutraceutical tablets, the animals were made to fast overnight, and glucose solution (2 g/kg of body weight) was administered orally. After that, their blood samples were collected every 30 min (0, 30, 60, 90, and 120 min). The blood glucose was measured by a glucose meter (i-QARE DS-W®) [18].
Statistical analysis. For the analysis of granules, herbal tablets, proximate compositions, minerals, and vitamins, the data were expressed as mean ± SDs. For the evaluation of anti-diabetic activity in ob/ob mice, the data were expressed as mean ± S.E.M. One-way ANOVA was used to determine significant differences among groups, after which the modified Student's t-test with the Bonferroni correction was applied to compare individual groups. All statistical analyses were performed with SPSS 17.0 software. P < 0.05 was considered statistically significant.

RESULTS AND DISCUSSION
Ethnomedicinal study. The field survey showed that the five edible medicinal plants were applied by the healers of Tripura to cure different diseases ( Table  1). Ethnomedicinal studies play a vital role in finding medicinal plants that can be used to produce novel crude drugs. They also verify the protection of cultural heritage [11]. The data that we collected from the informants of healers clearly proved that the plants we had selected were of medicinal importance.
Acute toxicity test. Our results indicated no changes of behaviour or mortality caused by the plant samples at the highest dose of 2000 mg/kg b.wt. This meant that this dose had no lethal or toxic effect. Toxicity assessment is one of the crucial steps prior to human uses of any pharmaceuticals or food ingredients. Acute toxicity studies determine adverse effects of any active compound after oral ingestion of a single or multiple doses [19]. We found that the five plant samples under study had no toxic effect.
Evaluation of granules. All the physical parameters of the herbal granules under study were found to be satisfactory ( Table 2). The Carr's compressibility index indicates the strength of the powder/granules, while the Hausner ratio determines the powder/granules' inter-particulate friction. Both parameters are used to analyze the powder/granules' flow rate. The Carr's compressibility index of less than 10 or the Hausner ratio of less than 1.11 indicate an "excellent" flow rate, while the Carr's compressibility index higher than 38 or the Hausner ratio higher than 1.60 indicate a "very poor" flow rate. Both parameters depend on the bulk density and the tap density of the powder/granules [25]. The angle of repose is another parameter used to evaluate the powder/granules' flow rate. Its value of less than 30° indicates an "excellent" flow rate, while the value greater than 56° indicates a "very poor" flow rate [26]. Our results showed an excellent flow rate in the granules prepared with a 10% starch solution ( Table 2). Evaluation of herbal tablets. All the physical parameters of the herbal tablets under study were found to be satisfactory ( Table 3). The weight variation test is employed to ensure that each tablet/capsule in the batch contains the same amount of drug ingredients. Checking a tablet's thickness is necessity for packaging since very thick tablets are not suitable for packaging. A tablet's friability and hardness tests are closely related to each other. They determine the physical strength of a tablet. Disintegration refers to the mechanical breakup of a compressed tablet into small granules at a specific time point. The disintegration test provides critical safety data on the drug's bioavailability in the body [27,28].
In our experiments, 20 tablets were used to check the average uniformity of weight and 10 tablets to check the average uniformity of thickness and hardness. For the friability and disintegration tests, the samples were prepared in triplicate. According to the results (Table 3), the physical parameters of our newly formulated tablets were within the limits established by the United States Pharmacopoeia.
Determination of proximate composition. The proximate composition of the herbal nutraceutical tablets is represented in Table 4. The moisture content is an essential parameter because high moisture affects the physical stability of food products. Their shelf life also depends on the total moisture content [29]. As we can see in Table 4, our newly formulated herbal tablets had a low moisture content (62.44 ± 2.11 %), indicating high physical stability.
Carbohydrates, proteins, and fats hold a special place in human nutrition. The human body requires them in relatively large amounts for normal functioning. These three macronutrients provide energy (measured in calories) in the human body [30]. Our newly formulated herbal nutraceutical tablet contained optimal amounts of carbohydrates (1.88 ± 0.52 %), proteins (0.95 ± 0.65 %), and fats (0.43 ± 0.53 %), as well as provided a good amount of energy (15.19 kcal).
So, the herbal nutraceutical tablets developed may be recommended to people with unbalanced energy levels. Dietary fiber is a complex mixture of polysaccharides. Diets with a high content of fiber alleviate constipation [31]. Our herbal nutraceutical tablet contained a good amount of dietary fiber (1.66 ± 0.67%), so it may be used to reduce constipation.
Minerals determination. Iron is an essential microelement for producing blood. Anemia is the most common disease caused by iron deficiency. Every year, approximately 30% of patients suffer from     Table 6 Profiles of water soluble and fat-soluble vitamins in herbal nutraceutical tablets Vitamin Total amount C (ascorbic acid), mg/g 27.2 ± 4.3 Vitamin B 1 (thiamin), mg/g 0.6 ± 0.0 Vitamin B 3 (niacin), mg/g 0.6 ± 0.2 Vitamin B 6 (pyridoxine), mg/g 1.1 ± 0.2 Vitamin B 12 (cobalamin), mcg/g 0.6 ± 0.2 Vitamin B 9 (folic acid), mcg/g 82.6 ± 7.6 Vitamin A (retinol), mcg/g 287.4 ± 6.3 Vitamin D 3 (cholecalciferol), mcg/g 2.6 ± 0.6 Vitamin E (tocopherol), ng/g 0.7 ± 0.0 *The values represent mean ± SD for three samples anemia globally, particularly 51% in India [32]. Our herbal nutraceutical tablet had a significant amount of iron (74.6 ± 2.7 mg/g), so it may alleviate the effects of iron deficiency (Table 5). Dietary potassium and sodium are two electrolytes that play a vital role in regulating fluid and blood volume [33]. The tablets developed contained a fair amount of sodium (4.4 ± 0.4 mg/g) and potassium (5.3 ± 0.7 mg/g), so it may be used to regulate the body's fluid and blood volume. Calcium, phosphorus, and magnesium are three crucial micronutrients for healthy bone and teeth formation, as well as metabolic functions [34]. Our herbal nutraceutical tablets contained a considerable amount of these minerals, namely 163.1 ± 2.2, 14.6 ± 2.1, and 39.2 ± 1.7 mg/g, respectively. Therefore, the newly developed herbal tablets can help bone formation and improve metabolic functions in the human body. Determination of vitamins. We determined the values of water-soluble (C, B 1 , B 3 , B 6 , B 12 , and folic acid) and fat-soluble vitamins (A, D 3 , and E) in our herbal nutraceutical tablets (Table 6). We found rich amounts of vitamin C (27.2 ± 4.3 mg/g) and vitamin A (287.4 ± 6.3 mcg/g). Vitamin C is a water-soluble vitamin that works as an antioxidant and improves the immune function of the human body [35]. Vitamins B 1 , B 3 , B 6 , and B 9 are essential for maintaining the nervous system, digestion, protein metabolism, red blood cells,  [39]. The oral administration of the herbal tablet to ob/ob mice significantly reduced their blood glucose level (Fig. 2). Therefore, this tablet may be used as an anti-diabetic drug.
We also found that the herbal nutraceutical tablet (200 mg/kg) significantly enhanced oral glucose tolerance (Fig. 2). This means that this tablet can be used to alleviate type 2 diabetes mellitus through progressing insulin sensitivity.

CONCLUSION
Our study showed that the newly formulated herbal tablet contained optimal amounts of macro-and micronutrients, water, and fat-soluble vitamins. The tablet also provided significantly higher hypoglycemic activity compared to the standard drug, metformin. The results suggested that the herbal tablets developed may be recommended as an anti-diabetic herbal remedy.

CONTRIBUTION
and skin health. Vitamins A, D 3 , and E are common fat-soluble vitamins that support such body functions as vision, hair growth, bone maintenance, immune system regulation, oxidative stress prevention, etc. [36]. Our herbal nutraceutical tablets contained fair amounts of vitamins D 3 , E, and B group (Table 6). Therefore, they may be used to treat diseases caused by their deficiency.
Anti-diabetic activity. The treatment of ob/ob mice with our herbal nutraceutical tablets (200 mg/kg body weight) significantly decreased their fasting blood glucose, serum hemoglobin, and glycosylated serum protein (Fig. 1). The effectiveness of the herbal tablet was comparable to that of metformin, a standard drug (200 mg/kg body weight).
A fasting blood glucose test, which is generally called a fasting plasma glucose test, measures the amount of glucose in the blood and determines the patient's risk of prediabetes or diabetes [37]. Our newly formulated herbal tablet (200 mg/kg) reduced the fasting blood glucose level ( Fig. 1a and 1b). Serum hemoglobin and glycosylated serum protein are important indicators for the long-term glycemic control [38]. Our experiment showed that the herbal tablet (200 mg/kg) reduced their levels, compared to metformin (200 mg/kg) ( Fig. 1c and 1d).
An oral glucose tolerance test determines the body's response to glucose. This test can be used to detect