DESC:FIELD OF THE INVENTION:
The present invention relates to an anti-diabetic composition and a process thereof. More particularly, present invention relates to an anti-diabetic composition and a process thereof comprising the extracts of Salacia species and Gymnema Species for the treatment, prevention and mitigation of hyperglycemia like conditions of Diabetes mellitus.

BACKGROUND OF THE INVENTION:
Glucose is important for health as it is the major source of energy for the cells of the body and a fuel for the brain cells. The pancreas secretes insulin which helps the sugar to be absorbed in the cells of the body.

Diabetes Mellitus is a chronic medical condition wherein the human body is not able to absorb sugar into its cells to meet the cell’s energy requirements. This is either due to the pancreas not producing enough insulin or the body not being able to use the insulin to absorb the excess sugar. An excess sugar remains in blood which is very detrimental to the functioning of the various organs in the body. This can lead to serious medical problems like heart disease, vision loss and kidney disease.

Types of Diabetes mellitus
There are two types of Chronic Diabetes: Type 1 and Type 2. In type 1, the diabetes is caused by an autoimmune reaction by the body which stops the body from making the required insulin. This is comparatively a rarer type and happens in younger adults and children. Everyday administration of insulin is needed for survival by the patient.
Type 2 diabetes is wherein the body is not able to use the insulin due to which the blood sugar is not controlled in the body to normal levels. This category constitutes 90-95 % of the total diabetes patients. This condition is preventable by making lifestyle changes like getting proper exercise, controlling body weight, eating healthy etc.

Conventional treatment:
For type 1 Diabetes the main treatment option is intake of insulin by the patient. The conventional treatment of type 2 Diabetes mellitus comprises of managing the disease by changing the lifestyle and using medications to reduce and keep the sugar levels in blood under the acceptable levels.
There are several types of drugs to control the blood sugar like alpha-glucosidase inhibitors (AGIs), biguanides, bile acid sequestrants (BASs), DPP-4 inhibitors, meglitinides, SGLT2 inhibitors, sulfonylureas, thiazolidinediones, GLP-1 agonists etc.

AGIs are a class of drugs that are used in the treatment of type 2 diabetes mellitus, alone or combined with other antidiabetic drugs. They, however, do not affect fasting insulin and serum triglyceride concentrations.

AGIs inhibit the absorption of carbohydrates from the small intestine. They competitively inhibit enzymes that convert complex non-absorbable carbohydrates into simple absorbable carbohydrates. These enzymes include glucoamylase, sucrase, maltase, and isomaltase.

Acarbose is the most commonly used drug of this class, and also the most widely studied one. Others include voglibose and miglitol.

There is evidence showing increased risk of liver transaminases (AST and ALT) associated with the use of AGIs in patients with type 2 diabetes exists (Zhang et al., 2016). Elevations greater than 3.0-fold upper limit of AST and ALT levels were 6.86 and 6.48 times higher in patients receiving AGIs compared to patients in the control arms and the findings may suggest caution in the use of AGIs for those who are at substantial risk for hepatic dysfunction (Zhang et al., 2016).

Herbal extracts
Manisha et al in the article titled as “Indian Herbs and Herbal Drugs Used for the Treatment of Diabetes,” compared the effects of conventional medications and deduced that the medicinal formulation from herbal plants has less side effects and are more cost effective.

Salacia Reticulata
Various literatures has been reported in the art which discloses the treatment of the diabetes mellitus at large for example article titled as “Antidiabetic Effects of Salacia reticulata (Kothala himbutu), Emblica officinalis (Anmaroku) and Ipomoea batatas (Suiou) on KK-Ay Mice”. The article shows that the use of a formulation consisting of extracts of Salacia Reticulata resulted in suppressed blood insulin levels, significantly decreased levels of HbA1c.

Also, a prior art titled “Oral hypoglycemic activity of some medicinal plants of Sri Lanka.” showed that the extract of S. reticulata has a significant effect on carbohydrate metabolism.

An article titled “A polyherbal formulation in the management of hyperglycemia and hyperlipidemia conditions in patients with type 2 diabetes” mentions that a polyherbal product containing S. oblonga was used to manage hyperglycemia in patients with type II diabetes at a dose of 1000 mg per day for 8 weeks to 89 patients. Additional plant components included Tinospora cordifolia, Emblica officinalis, Curcuma longa, and Gymnema sylvestre. Fasting and postprandial blood glucose levels as well as glycosylated hemoglobin (HbA1c) were significantly decreased.

Gymnema Sylvestre:
An article titled as “Antidiabetic effect of a leaf extract from Gymnema sylvestre in non-insulin-dependent diabetes mellitus patients” by Baskaran et al., 1990, investigated the effectiveness of extract from the leaves of Gymnema Silvestre, in controlling hyperglycemia in Type 2 diabetic patients on conventional oral anti-hyperglycemic agents. GS4 (400 mg/day) was administered for 18-20 months as a supplement to the conventional oral drugs. The patients showed a significant reduction in blood glucose, glycosylated haemoglobin and glycosylated plasma proteins and raised insulin levels.

Polyherbs using Salacia reticulata and Gymnema Sylvester
A patent document bearing application no. JP2002051735A discloses a combination of carbohydrate inhibitor digestive enzyme inhibitors (includes a-amylase inhibitors and a-glucosidase inhibitors) and the sugar absorption inhibitors are used in their diabetic food for controlling blood sugars. a-glucosidase inhibitor includes 1-deoxynojirimycin and salacinol wherein 1-Deoxynojirimycin is contained in mulberry leaves and the like, and salacinol is contained in Salacia reticulata and the like. The sugar absorption inhibitor of the said invention includes saponin, conduritol A, gurmarin and dietary fiber. Saponin is contained in Gymnema Silvestre, Gymnema inodrum, cod, thonburi and the like. Conduritol A and gurmarin are contained in Gymnema Silvestre and the like. The formulation proved increase in postprandial blood glucose elevation.

Another patent literature bearing application no.: IN2012DE00907A relates to regulation of insulin and normalizing blood sugar, high cholesterol. The literature mentions the use of extracts from the plant Gymnema Sylvestre, Commiphora Mukul, Azadirachta Indica, Curcuma Longa, Terminalia Chebula, Tinospora Cordifolia, Emblica Officinalis, Tribulus Terrestris, Momordica Charantia and Salacia Reticulata. This literature mentions that the above said formulation helps to bring down the fasting and post-prandial blood glucose levels in people with type 2 diabetes.

In view of above existing state of art, there arises a need for a composition which would not only control the blood sugar without any side effects but also have an improved positive effect on other parameters like lipid profile, hepatic profile, HbA1c and insulin.

Accordingly, the present invention provides an effective and efficacious anti-diabetic composition for diabetes. More particularly, the present invention provides a therapeutic herbal composition for treating, mitigating and curing Diabetes mellitus without any side effects.

OBJECT OF THE INVENTION:
The main object of the present invention is to provide an anti-diabetic composition for diabetes mellitus and a process thereof.

Yet another object of the present invention is to provide an anti-diabetic composition comprising the extracts of Salacia species and Gymnema Species.

Yet another object of the invention is to provide a process of preparing said anti-diabetic composition comprising extracts of Salacia species such as Salacia Chinensis, Salacia reticulata, Salacia oblonga either alone or in combination and Gymnema Sylvestre.

Yet another object of the present invention is to provide a composition against Diabetes mellitus which is efficacious and effective.

Yet another object of the present invention is to provide an anti-diabetic composition which shows synergistic and unexpected effect.

Yet another object of the present invention is to provide a process for preparing anti-diabetic composition which is easy to scale and cost-effective.

Yet another object of the present invention is to provide an anti-diabetic composition which shows positive effect on other parameters like lipid profile, hepatic profile, HbA1c and insulin.

SUMMARY OF THE INVENTION:
Accordingly, the present invention relates to an anti-diabetic composition and a process thereof for treating, curing, and mitigating Diabetes mellitus.

The present invention provides a composition and a process thereof comprising extracts of Salacia species such as Salacia Chinensis, Salacia reticulata, Salacia oblonga either alone or in combination and Gymnema Sylvestre to provide an efficacious and improved composition against Diabetes mellitus.

The present invention also provides a synergistic composition showing unexpected effects against Diabetes mellitus.

The present invention relates to a composition comprising extracts of Salacia species such as Salacia Chinensis, Salacia reticulata, Salacia oblonga either alone or in combination and Gymnema Sylvestre. The specific combination of these herbal extracts has shown significant decrease in the blood glucose, HbA1C, serum triglycerides, total cholesterol, LDL, VLDL, AST, ALT and ALP while significantly alleviating the diabetes induced body weight loss. The observed changes in the composition treated group was comparable to that of standard (Metformin 150 mg/kg with Vildagliptin 4 mg/kg) in blood glucose, serum insulin level, reversal of body weight loss while superior to that of standard group in reversing the HbA1C, lipid profile and hepatic profile.

The studies conducted confirm that the antidiabetic potential of said composition improves blood glucose, glycated hemoglobin, prevented muscle wasting, improved lipid and hepatic profile.

The present invention also discloses a process/method of making said composition. The process of extraction includes a simultaneous extraction from Salacia species dried roots and Gymnema species dried leaves. The ratio of the raw materials taken is preferably 5:1.
The % yield of Gymnema Sylvestre individually is in the range of 10% to 20%, preferably around 12% to 15%, more preferably at around 14% and the % yield of Salacia reticulata is in the range of 1% to 10%, preferably around 3% to 7%, more preferably at around 4%.

The composition can be formulated in various dosage forms including parenteral form, oral forms, however, a preferred form is an oral dosage form for consumption by the Diabetic patients. The oral form is preferably a capsule made from Vcap HPMC size “0” capsules clear cap/clear body.

The preferable method of making said oral composition involves mixing the extract from Salacia Reticulata with a microcrystalline cellulose. After this wet granulation is performed with purified water and the extract from Gymnema Sylvestre is added extra granularly to avoid sticking. Blending is done using a colloidal silicon dioxide. For the purpose of lubrication, a suitable additive is used preferably magnesium stearate.

This formulation as prepared in the above-mentioned manner includes preferably 200 mg of Salacia extract, 200 mg of Gymnema extract and 200 mg of excipients per capsule.

The present invention thus discloses a novel process of preparing an anti-diabetic composition comprising the extracts of Salacia species and Gymnema Sylvestre to provide an efficacious and improved composition for therapeutic use against the chronic disease of Diabetes Mellitus.

BRIEF DESCRIPTION OF DRAWINGS
The disclosure will be described and explained with additional specificity and detail with the accompanying figures in which:

Figure 1 is a graphical representation of the effect of anti-diabetic composition on Body weight (g) and Blood glucose (mg/dL) in streptozotocin-nicotinamide induced diabetes in rats in an experiment conducted related to the invention.

Figure 2 is a graphical representation of effect of anti-diabetic composition on HbA1C (%) and Serum Insulin (µU/ml) in streptozotocin-nicotinamide induced diabetes in rats in an experiment conducted related to the invention.

Figure 3 is a graphical representation of the effect of anti-diabetic composition on lipid profile and hepatic profile in streptozotocin-nicotinamide induced diabetes in rats in an experiment conducted related to the invention.

Figure 4 is a graphical representation of the comparison of glucosidase inhibition by Sava anti-diabetic formula with and without excipients.

Figure 5 is a graphical representation of inhibition of a-glucosidase activity by SAVA extract and other commercial formulations.

DETAILED DESCRIPTION OF THE INVENTION
It will be understood by those skilled in the art that the following detailed description is exemplary and explanatory of the invention and is not intended to be restrictive thereof.

The present invention relates to an anti-diabetic composition and the process of making thereof for preventing, curing and mitigating Diabetes mellitus. In particular, the present invention provides an anti-diabetic composition and a process thereof comprising the extracts of Salacia species and Gymnema Sylvestre to provide an efficacious and improved composition for therapeutic action in Diabetes mellitus.

Salacia reticulata Wight (Hypocrataceae) is a woody climbing shrub with greenish-brown bark that is indigenous to India and Sri Lanka, while other species as S. chinensis and S. oblonga are also found in Asia and other parts of the world.

According to an article in the ‘National library of medicine’ titled “Anti-diabetic and Anti-hyperlipidemic effects and safety of Salacia reticulata and Related Species,” Salacia extracts modulate multiple targets that influence carbohydrate and lipid metabolism including a-glucosidase, aldose reductase, pancreatic lipase, peroxisomal proliferator activated receptor a, glucose transporter 4 mediated glucose uptake, and angiotensin II type 1 receptor. Furthermore, Salacia extracts exhibit free radical scavenging, antioxidant and hepatoprotection activities. Constituents from Salacia species that have been identified as exhibiting anti-diabetic effects include salacinol, kotalanol, ponkorinol, salaprinol, and their corresponding desulfonated compounds.

Gymnema Sylvester is a woody climbing shrub that is native to the tropical forests of India, Africa and Australia. Its leaves have been used in the ancient Indian medicinal practice Ayurveda for thousands of years. It has been a traditional remedy for various ailments, including diabetes, malaria and snakebites.

Research is conducted to study the effects of the composition comprising both the herbal components of said invention on Diabetes induced rats. Treatment with said embodiment caused significant decrease in the blood glucose, HbA1C, serum triglycerides, total cholesterol, LDL, VLDL, AST, ALT and ALP while significantly alleviated the diabetes induced body weight loss.

The observed changes in the treated group are comparable to that of standard (Metformin 150 mg/kg with Vildagliptin 4 mg/kg) in blood glucose, serum insulin level, reversal of body weight loss while superior to that that of standard group in reversing the HbA1C, lipid profile and hepatic profile. The study confirms the antidiabetic potential of the said embodiment which improved blood glucose, glycated hemoglobin, prevented muscle wasting, improved lipid and hepatic profile. The detailed execution of the research is as reported below.

The study was done to evaluate said embodiment in Streptozotocin-Nicotinamide induced diabetes mellitus in rats. The objectives of study were to induce diabetes mellitus by intraperitoneal administration of Streptozotocin-Nicotinamide and thereafter determine the effect of treatment of the composition on blood glucose, lipid profile, hepatic profile, HbA1c and insulin. Further, the effectiveness of the present invention was compared with standard metformin with vildagliptin.

The animals on which the study was conducted were divided into three groups of eight animals each as follows:
• Group I – Diabetic Control (streptozotocin 65 mg/kg ip. (STZ)+ nicotinamide 110 mg/kg (NIC)) induced treated with water orally once daily.
• Group II – STZ-NIC Diabetic animals treated with standard (metformin 150 mg/kg and vildagliptin 4 mg/kg, orally once daily)
• Group III – STZ-NIC Diabetic animals treated with the composition (165 mg/kg) orally once daily (suspended in water to make 30 mg/ml)

The first step was to induce the rats with Diabetes mellitus. This was done by dissolving Streptozotocin in a freshly prepared 0.1 M citrate buffer (pH 4.5) and nicotinamide was dissolved in normal physiological saline. The overnight fasted (16 h fasting with free access to water) animals were induced with diabetes by intraperitoneal injection of 65 mg/kg of Streptozotocin (STZ), thereafter, intraperitoneal injection of 110 mg/kg of nicotinamide was injected after 15 minutes. The vehicle control group (Group I) were injected with buffer alone. After 72 h, blood was withdrawn by retroorbital puncture under anesthesia and the blood glucose level was estimated to confirm the induction of diabetes mellitus. After 1 week of induction, blood glucose level was estimated again, and the fasting blood glucose level of more than 200 mg/dL was considered as diabetic. The animals were given respective treatment for 3 weeks. The blood glucose and body weight were determined every week and the lipid profile, hepatic profile, HbA1C and serum insulin were determined at the end of 3 weeks treatment.

The results of the study on various aspects like on body weight, blood glucose, HbA1C, serum insulin, lipid profile and hepatic profile are illustrated here in the following paragraphs.

Effect on body weight:
After two weeks of induction of diabetes, the body weight of the diabetic control had significantly (p<0.01) decreased, which was further decreased in the third week. Treatment with said embodiment significantly (p<0.001) prevented the loss in body weight. when compared to diabetic control on day 14 and 21 of treatment. The observed effect was as comparable to that of standard treated group. Please refer to Table 1 and Figure 1.

Effect on blood glucose:
Treatment with composition of present invention significantly (p<0.001) decreased the blood glucose level by 35% from day 14 onwards, which was further decreased by 53% on day 21 when compared to diabetic control group of animals. The observed effect was compared comparable to that of standard treated group which had significantly (p<0.001) decreased the blood glucose level by 27.5 % on day 14 and 47.9 % on day 21 when compared to diabetic control. The data is further depicted in Table 1 and Figure 1. This confirms the antihyperglycemic potential of the composition of the present invention which is more effective and efficacious to that of the standard.

Table 1: Effect of said composition of the embodiment on Body weight (g) and Blood glucose (mg/dL) in streptozotocin-nicotinamide induced diabetes in rats
Parameters Days Vehicle Control Standard (Metformin 150 mg/kg + Vildagliptin 4 mg/kg) Composition
(165 mg/Kg)
Body weight (g)
Day 0 172.16 ± 1.96 172.00 ± 5.54 174.16 ± 3.18
Day 7 169.32 ± 4.86 170.00 ± 5.54 174.32 ± 2.96
Day 14 158.32 ± 2.36 175.50 ± 4.82** 177.00 ± 3.60***
Day 21 150.32 ± 2.10 177.66 ± 4.90** 182.00 ± 2.94***
Blood Glucose (mg/dL) Day 0 215.32 ± 3.10 225.00 ± 6.42 247.66 ± 11.82
Day 7 297.82 ± 20.04 282.82 ± 17.46 251.66 ± 21.96
Day 14 320.16 ± 15.20 232.00 ± 15.32*** 207.33 ± 22.06***
Day 21 368.66 ± 14.64 192.00 ± 10.22*** 172.82 ± 17.30***

Values are expressed as mean ± SEM, n = 6
One way ANOVA followed by Dunnet’s multiple comparison test
**p<0.01, ***p<0.001 when compared to diabetic control group
The composition – Test compound

Effect on HbA1C:
The Treatment was done with the present invention composition for 3 weeks. Said treatment significantly (p<0.001) decreased the HbA1C levels by 51.2 % when compared to diabetic control group of animals. The observed effect was as comparable to that of standard treated group which had significantly (p<0.001) decreased the HbA1C levels by 33.2 % when compared to diabetic control. Said comparative data is shown in Table 2 and Figure 2. There was also a significant decrease (p<0.05) in HbA1C levels of the composition treated group of animals when compared to standard group of animals. This confirms that administration of the composition resulted in round the clock antihyperglycemic effect and was superior to that of the standard.

Effect on Serum Insulin
After treatment with the composition of present invention for 3 weeks there was no change in the serum insulin when compared to diabetic control group. The serum insulin of standard group of animals were also not affected on 3 weeks of treatment (Table 2: Figure 2). The streptozotocin-nicotinamide model causes only 40 % decrease in the ß cells sparing 60 % of the ß cells. Hence any drug acting through insulinogenic effect has been found to increase the serum insulin level. However, the observed serum insulin in the present study suggests that the antihyperglycemic activity of the composition is not related to alteration in insulin secretion and the observed decrease in glucose level is without any extra load on pancreatic ß cells.

Effect on Lipid Profile:
Serum triglycerides (mg/dL)
The normal level of serum triglycerides is less than 150 mg/dL. Treatment with the composition of present invention for 3 weeks significantly (p<0.001) decreased the serum triglycerides levels by 48.6 % when compared to diabetic control group of animals. The observed effect was better than that of standard treated group which had decreased the serum triglycerides levels by 14 % when compared to diabetic control. The data is also represented in Table 2 and Figure 3.

Total cholesterol (mg/dL)
The normal level of serum total cholesterol is less than 170 mg/dL. Treatment with the composition of present invention for 3 weeks significantly (p<0.001) decreased the serum total cholesterol levels by 27.33 % when compared to diabetic control group of animals. The observed effect was better than that of standard treated group which had significantly (p<0.05) decreased the serum total cholesterol levels by 11.34 % only when compared to diabetic control. The data is shown in Table 2 and Figure 3.

HDL cholesterol (mg/dL)
The normal level of serum HDL cholesterol is more than 40 mg/dL. Treatment with the composition of present invention or standard for 3 weeks did alter the HDL cholesterol levels when compared to diabetic control as shown in Table 2 and Figure 3.

VLDL cholesterol (mg/dL)
The normal level of serum VLDL cholesterol is 5-51 mg/dL. There was no notable change in the serum VLDL cholesterol level of the diabetic control group, which was found to be 43.50 mg/dL in 3 weeks of induction of diabetes in diabetic control. Treatment with the composition for 3 weeks significantly (p<0.001) decreased the serum VLDL cholesterol by 48.59 % when compared to diabetic control group of animals. The observed effect was better than that of standard treated group which had decreased the serum VLDL cholesterol levels by 14.02 % when compared to diabetic control as shown in Table 2 and Figure 3. Though the VLDL levels were decreased upon treatment with composition and standard, the observed serum VLDL level in diabetic animals were not elevated considering clinically normal VLDL values in the diabetic control group of animals.

LDL cholesterol (mg/dL)
The normal level of serum LDL cholesterol is less than 100 mg/dL. Treatment with the composition of present invention for 3 weeks significantly (p<0.05) decreased the serum LDL cholesterol by 27.52 % when compared to diabetic control group of animals. The observed effect was better than that of standard treated group which had decreased the serum LDL cholesterol levels only by 13.24 % when compared to diabetic control. The data is also shown in Table 2 and Figure 3. Though the LDL levels were decreased upon treatment with and standard, the observed serum LDL level in diabetic animals were not elevated considering clinically normal LDL values in the diabetic control group of animals.

The most commonly observed lipid abnormalities in diabetes are hypertriglyceridemia and hypercholesterolemia. A marked increase in triglyceride levels were observed in the diabetic control animals which confirms the failure to activate lipoprotein lipase in diabetics which lead to hypertriglyceridemia. The decrease in the lipid profile upon treatment with the composition confirms the antidiabetic potential of the composition. The observed alterations in lipid profile of the composition treated groups is superior to the alterations observed in standard treated group.

Table 2: Effect of the composition on lipid profile and hepatic profile in streptozotocin-nicotinamide induced diabetes in rats
Parameters Diabetic Control Standard (Metformin 150 mg/kg + Vildagliptin 4 mg/kg) Composition
(165 mg/Kg)
HbA1C (%) 16.04 ± 0.58 10.70 ± 0.56*** 7.82 ± 0.80***,$
Serum Insulin (µU/ml) 5.30 ± 0.14 5.48 ± 0.08 5.48 ± 0.10
Triglycerides (mg/dL) 217.50 ± 21.48 187.00 ± 8.80 111.8 ± 7.12***
Total Cholesterol (mg/dL) 180.70 ± 4.14 160.20 ± 2.76* 131.30 ± 8.54***
HDL (mg/dL) 32.32 ± 3.14 31.82 ± 2.64 33.00 ± 1.80
VLDL (mg/dL) 43.50 ± 4.28 37.40 ± 1.76 22.36 ± 1.42***
LDL (mg/dL) 104.80 ± 9.58 90.92 ± 4.34 75.96 ± 7.68*
ALT (U/L) 146.80 ± 10.00 75.50 ± 4.36*** 71.16 ± 4.86***
AST (U/L) 248.70 ± 10.96 204.30 ± 5.22** 159.00 ± 2.42***
ALP (U/L) 587.80 ± 69.64 335.20 ± 16.70** 209.80 ± 22.56***

Values are expressed as mean ± SEM, n = 6
One way ANOVA followed by Dunnet’s multiple comparison test
*p<0.05, **p<0.01, ***p<0.001 when compared to diabetic control group
$p<0.05 when compared to standard group
COMPOSITION– Test compound

Effect on Hepatic Profile
Alanine Transaminase ALT (U/L)
The normal level of serum ALT is 1-34 U/L. Treatment with the composition of present invention for 3 weeks significantly (p<0.001) decreased the serum ALT levels by 51.52 % when compared to diabetic control group of animals. The observed effect was comparable to that of standard treated group which had significantly (p<0.001) decreased the serum ALT levels by 48.56 % when compared to diabetic control as shown in Table 2 and Figure 3. However, the observed decrease in both treatment groups did not reverse ALT to clinically normal levels.

Aspartate Transaminase AST (U/L)
The normal level of serum AST is 8-60 U/L. Treatment with the composition of present invention for 3 weeks significantly (p<0.001) decreased the serum AST levels by 36.06 % when compared to diabetic control group of animals. The observed effect was superior to the standard treated group which had significantly (p<0.001) decreased the serum AST levels by 17.85 % when compared to diabetic control. The data is also represented in Table 2 and Figure 3. However, the observed decrease in both treatment groups did not reverse AST to clinically normal levels.

Alkaline Phosphatase ALP (U/L)
The normal level of serum ALP is 48-406 U/L. Treatment with the composition for 3 weeks significantly (p<0.001) decreased the serum ALP levels by 64.30 % when compared to diabetic control group of animals. The observed effect was superior to the standard treated group which had significantly (p<0.001) decreased the serum ALP levels by 42.97 % when compared to diabetic control (Table 2: Figure 3). Furthermore, the observed decrease in both treatment groups reversed the ALP to clinically normal levels.

Aminotransferases, such as alanine aminotransferase and aspartate aminotransferase indicate the intracellular hepatic enzymes that have leaked into the circulation and denotes hepatocyte injury. It is hypothesized that elevation in ALT, AST and ALP are considered as predictors of diabetes. Further, the elevation in the levels of these gluconeogenic enzymes whose gene transcription is suppressed by insulin could indicate impairment in insulin signaling and elevated blood glucose levels rather than purely hepatocyte injury. The increased activities of ALT, AST, and ALP in the serum of diabetic rats may be primarily due to the leakage of these enzymes from liver cytosol into blood stream as a consequence of the hepatotoxic effect of STZ. However, diabetic rats treated with the composition for 3 weeks diminished the activity of these enzymes to, suggesting the protective nature of the composition.

Extraction and making of composition
In accordance with a preferred embodiment of the invention, the extracts of Salacia Reticulata are obtained from its dried roots and water is used as a solvent. Also, the extracts of Gymnema Sylvester are obtained from its dried leaves and are obtained as 70 % ethanolic extract.

The process of formulation was done in 4 batches wherein in three batches (1195-04, 1195-05, 1195-06) the Salacia extract and the Gymnema extract added during granulation process which gave desirable assay of salacinol as well as Gymnemic acid-IV. However due to hygroscopic nature of gymnema extract the sticking was observed after addition of water during granulation process.

The Salacia reticulata extract and gymnema extract possess minimum microbial load as shown in Table 3 below.

Table 3: The microbial load details below:
Sr. No. Name of Extract Microbial load (cfu/gm)
1 Salacia reticulata extract 300 cfu/gm
2 Gymnema sylvestre extract 3.8 x102 cfu/gm

Table 4: Formulation process for three batches: 1195-04, 1195-05, 1195-06
Sr. no Ingredients mg/cap
1 Salacia reticulata extract 200.00
2 Gymnema sylvestre extract 25% 200.00
3 Microcrystalline cellulose (Avicel PH 101) 190.00
Wet granulation
4 Purified water q. s
Blending
5 Colloidal silicon dioxide (Aerosil 200) 5.00
Lubrication
6 Magnesium stearate 5.00
Total 600.00
7 Vcap HPMC size “0” capsules clear cap/clear body 1

Table 5: Observations made for three batches: 1195-04, 1195-05, 1195-06
Sr. no Parameters Initial 6 Month (at 2-8°C)
1 Appearance Brown colored granules Brown colored granules
2 Water content (%) 7.69 7.42
3 Gymnemic acid-IV (%w/w) 0.20 0.18
4 Salacinol (%w/w) 0.252% 0.219%
5 Microbiological parameters:
Total plate count (cfu/g)
5.6 x 104
2100

As per an optimized embodiment the extraction process of the composition and the portions of the ingredients used for formulation is as tabulated below:

Table 6: Formulation process for batch 1195-09
Sr. no Ingredients Mg/cap
1 Salacia reticulata extract 200.00
2 Microcrystalline cellulose (Avicel PH 101) 190.00
Wet granulation
3 Purified water q. s
Blending
4 Gymnema sylvestre extract 25% 200.00
5 Colloidal silicon dioxide (Aerosil 200) 5.00
Lubrication
6 Magnesium stearate 5.00
Total 600.00
7 Vcap HPMC size “0” capsules clear cap/clear body 1

Gymnema extract was mixed extra granularly to avoid sticking. The blend was found to be free flowing and the assay of Gymnemic acid-IV was found to be stable till 5 months. The total stability data after 6 months will confirm the stability of optimized formulation.

Table 7: Observations made for batch 1195-09
S. no. Parameters Initial 5 Month (at 2-8°C)
1 Appearance Brown coloured granules Brown coloured granules
2 Water content (%) 7.69 7.74
3 Gymnemic acid-IV (%w/w) 0.20 0.25

The formulation batch 1195-09 using wet granulation method including 200 mg Salacia extract, 200 mg gymnema extract and 200 mg excipients per capsule was finalized for future bulk batches.

Various extraction batches were formulated using different ratios of the raw materials of Salacia Reticulata and Gymnema Sylvester.

Table 8: Extraction batches for Salacia-Gymnema polyherbal extraction:
Sr. No. Sample Batch number Raw material (g) Raw material ratio (SR:GS) Extracting Solvent Condition
1 Gymnema sylvestre RDP/PH/034 50 - 50% Ethanol Reflux 60 °C, 3 hrs, 3 cycles
2 Salacia reticulata RDP/PH/033 50 -
3 Salacia+Gymnema RDP/PH/036 25+25 1:1
4 Salacia+Gymnema RDP/PH/037 125+25 5:1
5 Salacia+Gymnema RDP/PH/038 190+25 7.5:1

It was observed that the % yield of Gymnema sylvestre was found to be around 14% and the % yield of Salacia reticulata was about 4%.

The simultaneous polyherbal extracts with 1:1, 5:1 and 7.5:1 gave 14%, 6.6% and 6.5% yields, respectively. The % content of Gymnemic acid-IV and Salacinol in the respective extract is in the range of 0.1% to 1.5%, preferably, the % content of Gymnemic acid-IV and Salacinol are given below.

Table 9: The % content of Gymnemic acid-IV and salacinol are given in table below.
Sr. no Parameters Salacia reticulata Gymnema sylvestre Polyherb
RDP/PH/036 RDP/PH/037 RDP/PH/038
1 Appearance Dark brown coloured extract Black coloured extract Dark brown coloured extract Dark brown coloured extract Dark brown coloured extract
2 Yield (%) 4.2 14.0 14.0 6.6 6.5
2 Gymnemic acid-IV (%w/w) - 0.84 0.84 0.53 0.44
3 Salacinol (%w/w) 0.54 - 0.138 0.305 0.387
4 Bulk density (g/ml) 0.784 0.588 - 0.703 -
5 Tapped density (g/ml) 0.886 0.645 - 0.816 -
6 Carr’s index (%)
Flowability: 11.5
Good 8.83
Excellent - 13.84
Good -
7 Microbiological parameters:
Total plate count (cfu/g) - - 1.32 x 105 5.0 × 105 4.5 × 106

The results suggest that the 1:1 ratio of raw material will have higher yield of Gymnema extract which is reflecting in the % assay of Gymnemic acid –IV from batch no. RDP/PH/036. The salacinol content was found to be on lower side as the % yield of Salacia was only 4% in the total extract obtained.

Thus, to achieve equal amount of both the extracts, the starting material of Salacia was kept higher. Hence two different ratios 5:1 and 7.5:1 for Salacia: Gymnema were tried.

It was found that the 5:1 ratio gave the final extract containing equal amount of two extracts which was highlighted through the % assay of Salacinol and Gymnemic acid-IV. The % assays were found to be half in the mix extract as compared to individual extracts. Hence the 5:1 ratio was concluded as optimum to yield 1:1 ratio of both extracts in the mix extract.

Also, the bulk density and tapped density were found to be in range of 0.5-0.8 g/ml and carr’s index showed good flowability of the extract.

Comparison of present invention anti-diabetic composition with Alpha-glucosidase inhibitors (AGIs):
Acarbose, voglibose and miglitol are the most commonly used drug of this class of AGIs, and also the most widely studied one.

There is evidence showing increased risk of liver transaminases (AST and ALT) associated with the use of AGIs in patients with type 2 diabetes exists. Elevations greater than 3.0-fold upper limit of AST and ALT levels were 6.86 and 6.48 times higher in patients receiving AGIs compared to patients in the control arms and the findings may suggest caution in the use of AGIs for those who are at high risk for hepatic dysfunction.

As per the preferred embodiment of the present invention said anti-diabetic composition does not show any increase in ALT and AST levels, in fact the animals treated with this extract show lesser levels of AST and ALT.

Comparison of glucosidase inhibition by said anti-diabetic composition with and without excipients
• The estimation of alpha glucosidase inhibition as plain extracts and also as a formulation has been done and there is no effect of excipients seen on alpha glucosidase inhibition (Figure 4).

There is a widespread need for providing a safe formulation for tackling the chronic conditions of Diabetes mellitus which is a major health hazard in the world.

Thus, present invention provides an efficacious and effective anti-diabetic composition for treating, preventing and mitigating heath conditions related to Diabetes mellitus and also provides a scalable and cost-effective process for the preparing the said anti-diabetic composition comprising Gymnema sylvestre and Salacia reticulata.

DPPIV Enzyme Inhibition by Salacia and Gymnemic Acid Extract, their Phyto-constituents and Capsule Contents
DPPIV Enzyme Inhibition Assay
Background: DPPIV (DPP4, CD26) is a member of the class of proteases known as propyl peptidases, which cleave proteins after proline residues. DPPIV, a serine dipeptidyl peptidase, cleaves the N-terminal X-Ala or X-Pro from target polypeptides such as GLP-1. Major DPPIV substrates in the body are so-called incretin hormones, which are key regulators of the post prandial insulin release. Thus DDPIV inhibition leads to greater availability of these proteins. Therefore prolongs the half life of insulin action. The majority of effects seen upon DPPIV treatment are ascribed to an increase in GLP-1 levels. Because of this DPPIV becomes major target for treatment in T2DM, Example of Inhibitors: Sitagliptin, Vildagliptin, Saxagliptin etc.

Principle: The kit contains chromogenic substrate (H-Gly-Pro-pNA). The enzyme cleaves p-nitroaniline (pNA) from the chromogenic substrate which causes increase in absorbance at 405 nm. In presence of inhibitor which binds to the enzyme, the substrate is not cleaved and thus in there is decreased absorbance at 405 nm as compared to control where there is only enzyme and substrate. Percentage enzyme inhibition is calculated based on the reduction in absorbance as compared to control. The kit is useful to screen inhibitors of DPPIV, a potential therapeutic target for Diabetes. Known Enzyme Inhibitor provided in the kit is P32/98 (Isoleucyl thiazolidide)

Table 10: Protocol
Sample Assay Buffer DPPIV (17.3µU/µl) Inhibitor Substrate
Blank 50 µl 0 0 50 µl
Control 35 µl 15 µl 0 50 µl
Inhibitor* 25 µl 15 µl 10 µl 50 µl
Test Sample # X µl 15 µl Y µl 50 µl
*Refers to 10 µl of the diluted P32/98 provided in the kit
# Test sample is the experimental inhibitor. Dissolve/dilute experimental inhibitor into assay buffer and add to appropriate wells at desired volume “Y”. Adjust the volume “X” to bring total volume to 100 µl (X+Y = 35 µl)
As per the protocol Blank, Control, Inhibitor and Test Sample is prepared by adding assay buffer, DPPIV Enzyme and Inhibitor as mentioned above and incubated for 10 mins at desired reaction temperature to allow inhibitor-enzyme interaction. After 10mins incubation substrate is added to all the wells and absorbance is measured at A405nm in a microplate reader after 1 min intervals for a total of 20 mins. Plot data A405nm versus time for each sample. Obtain a best fit line for the data points and determine the slope.
% activity remaining is calculated = (Slope of +inhibitor sample/control slope) x 100
% Inhibition = 100 - % activity

Table 11: % Inhibition of various Formulation against the standard
Name of the compound Concentration % Inhibition
P32/98 (Isoleucyl thiazolidide) – Known DPPIV Inhibitor 0.08 mg/ml 97.93
Salacia extract (SE) 0.2 mg/ml 40.00
Salacinol 0.5 mg/ml 33.33
Mangiferin 0.5 mg/ml 96.00
Gymnema Extract (GE) 0.1 mg/ml 54.64
Gymnemic Acid IV 0.35 mg/ml 55.26
SAVA Formulation (SE+GE) SE - 0.1 mg/ml & GE - 0.1 mg/ml 36.67

Conclusion:
Salacia extract and Gymnema Extract and their phyto-constituents are known to possess DPPIV enzyme inhibition properties as shown above. It can be seen from the above table that SAVA formulation of SE+GE at just 0.1 mg/ml shows 36.67 % inhibition, thus can act as a potent DPPIV enzyme inhibitor upon consumption of entire dosage of the formulation thereby helping to control diabetes.

Comparison of the a--glucosidase Inhibition By Commercial Anti-Diabetic Formulations And Sava Formulation a-glucosidase inhibition
Protocol: a-glucosidase Inhibition
The inhibition of a-glucosidase enzyme was determined in 100 mM sodium phosphate buffer pH 6.8 by reacting 0.1 U/mL of the enzyme with 1.25 mM pNPG in the absence or presence of Salacia/Gymnema/Capsule extracts in a final reaction volume of 200 µL at 37 °C. The reaction was initiated by adding pNPG and the released pNP was determined by measuring absorbance at 410 nm using a microplate reader after 10 min of reaction time. Background readings were eliminated by subtracting the absorbance of the mixture without enzyme. The measured absorbance was considered directly proportional to the enzymatic activity and a-glucosidase inhibition was determined as follow:

Enzyme inhibition %= (Absorbance negative control - Absorbance Reaction) X 100
Absorbance negative control

Table 12: Percent inhibition of a-glucosidase activity by SAVA extract and other commercial antidiabetic formulations
S. no. Formulation Concentration (mg/ml) % inhibition (Mean ± SE) p-value
1 Baidyanath Madhumehari 0.1 15.89 ± 3.57 < 0.001
2 D.B.T. (Sharangdhar) 51.67 ± 0.94
3 Diasalve 13.54 ± 3.02
4 SAVA formulation 99.67 ± 0.12
5 SugCare 97.77 ± 0.23
7 Baidyanath Madhumehari 0.05 14.24 ± 1.64 < 0.001
8 D.B.T. (Sharangdhar) 28.00 ± 5.46
9 Diasalve ND
10 SAVA formulation 99.33 ± 0.33
11 SugCare 92.84 ± 0.31
Values represent the mean ± standard error. ND: Not detected.
Values represent the mean ± standard error. * and ** indicate p < 0.001.

Table 13: Percent inhibition of a-glucosidase activity by Salacia, Gymnema and SAVA Formulation
S. no. Content Concentration (mg/ml) % inhibition (Mean ± SE) p-value
1 Salacia extract 0.05 82
3 SAVA Formulation (SE+GE) 0.05 81
2 Salacia extract 0.10 99.2
4 SAVA Formulation (SE+GE) 0.10 99
5 Mangiferin 0.75 59

Comparison Of Maltase Inhibition By Commercial Anti-Diabetic Formulations And Sava Formulation

Table 14: Percent inhibition of maltase activity by SAVA extract and other commercial antidiabetic formulations
S. No. Formulation (1 mg/ml) % inhibition (Mean ± SE) p-value
1 Baidyanath Madhumehari 52.39 ± 2.62 < 0.05
2 D.B.T. (Sharangdhar) 74.02 ± 1.36
3 Diasalve 85.27 ± 7.17
4 SAVA formulation 96.65 ± 2.4
5 SugCare 87.34 ± 5

Values represent the mean ±standard error. ND: Not detected.
Values represent the mean ± standard error. * and ** indicate p < 0.05.

Conclusion:
Baidyanath Madhumehari contains G. sylvestre and Trigonella foenum-graecum along with other herbal extracts. D.B.T. contains Eugenia jambolana, Gymnema sylvestre, and other extracts. Diasalve contains Salacia reticulata, Gymnema sylvestre, and other extracts. Sugcare contains extracts derived from Mangifera indica, Syzygium cumini, Gymnema sylvestre, etc. SAVA extract significantly inhibits the activities of a-glucosidase, DPPIV and maltase. When compared to other commercial formulations for a-glucosidase and maltase SAVA formulation gives better results for their inhibition. It contains the extracts of Salacia reticulata and Gymnema sylvestre. It inhibits both alpha-glucosidase and maltase by 96.65 and 99.67% at 0.1 mg/ml and 1 mg/ml, respectively.

Alpha-glucosidases hydrolyze various carbohydrates present in the diet into glucose. Maltase acts on the disaccharide, maltose to yield glucose. An important strategy to treat type-2 diabetes is inhibition of the activity of alpha-glucosidases using synthetic drugs. The main problems associated with these drugs are the gastrointestinal side effect. Hence, the use of herbal extracts that act as inhibitors of these enzymes hyperglycemia. Thus, SAVA extract with significantly higher inhibition of both theses enzymes can be an effective antidiabetic formulation.

Compositions of various formulations used:
Table 15: Composition of Baidyanath Madhumehari
Baidyanth Madhumehari g
Gudmar 10
Methi 10
Jamun 5
Karela 5
Kalmegh 5
Kala jeera 5
Giloy 5
Haldi 5
Vijaysar 5
Vat 5
Kutki 5
Bilva 5
Gular 5
Neem 5
Babool 5
Tejpatta 5
Shilajeet 5
Yashad Bhasma 3
Vang Bhasma 2
Excipients
Total 100

Table 16: Composition of D.B.T. (Sharangdhar)
D.B.T. mg
Eugenia jambolana 40
Gymnema sylvestre 40
Caesalpinia bonducella 40
Casearia esculenta 40
Curcuma longa 40
Plumbago zeylanica 20
Picrorhiza kurrao 20
Balasmodendron mukul 20
Jasad Bhasma 10
Vanga Bhasma 5
Nag Bhasma 5
Excipients
Total 280

Table 17: Composition of Diasalve
Diasalve mg
Salacia reticulata 250
Gymnema sylvestre 150
Psidium guajava 75
Berberis aristate 75
Tinospora cordifolia 50
Rubia cordifolia 25
Pterocarpus marsupium 25
Aegle marmelos 25
Moringa oleifera 25
Excipients
Total 700

Table 18: Composition of Sugcare
Sugcare (contains extracts derived from) mg
Mangifera indica 625
Syzygium cumini 625
Gymnema sylvestre 625
Asphaltum Punjabium(Shilajit) 167
Momordia charantia 625
Excipients
Total 2667
,CLAIMS:We claim:
1. An anti-diabetic composition comprising of extracts of Salacia Reticulata and Gymnema Sylvestre along with other excipients including microcrystalline cellulose, an anti-caking agent, an additive and purified water (q.s)
wherein the composition is used effectively against heath conditions related to Diabetes mellitus by reducing the blood sugar, HbA1c while keeping lipid profile and hepatic profile values under acceptable limits without affecting the liver transaminases (ALT and AST).

2. The composition as claimed in claim 1, wherein salacinol is obtained from the water extracts of said Salacia Reticulata and Gymnemic acid-IV is obtained from the ethanolic extracts of said Gymnema Sylvestre.

3. The composition as claimed in claim 1, wherein the weight in grams of salacia extract per dosage ranges from 1 to 300 milligrams.

4. The composition as claimed in claim 1, wherein the Gymnemic acid-IV containing ethanolic extract of gymnema Sylvester ranging between 1 and 10%.

5. The composition as claimed in claim 1, wherein the weight in grams of Gymnemic acid-IV per dosage ranges from 1 to 300 milligrams.

6. The composition as claimed in claim 1, wherein the said composition is as shown below:
Sr. no Ingredients Mg/cap
1 Salacia reticulata extract 200.00
2 Microcrystalline cellulose (Avicel PH 101) 190.00
3 Purified water q. s
4 Gymnema sylvestre extract 25% 200.00
5 Colloidal silicon dioxide (Aerosil 200) 5.00
6 Magnesium stearate 5.00
Total 600.00
7 Vcap HPMC size “0” capsules clear cap/clear body 1

7. A process for preparing the antidiabetic composition wherein the steps comprise of:
a. extracting salacinol as a water extract from the plant source;
b. extracting gymnemic acid-IV as an ethanolic extract from the plant source;
c. mixing salacinol, gymnemic acid-IV and the microcrystalline cellulose to obtain a mixture;
wherein the Gymnemic acid-IV is added to the mixture granularly after wet granulation to avoid sticking,
d. wet granulating the mixture as obtained in step (c ) by adding purified water;
e. blending the mixture as obtained in step (d) by adding anti-caking agent to the above mixture;
f. conducting lubrication of the mixture as obtained in step (e) by using additive for lubrication; and
g. filling the final mixture in the capsule.

8. The process as claimed in claim 7, wherein the Salacinol and Gymnemic acid-IV are extracted simultaneously in ratios of 1:1,5:1, 7.5:1 with extracting solvent of 50% ethanol.

9. The process as claimed in claim 7 wherein the extract has the following microbial load:
Name of Extract Microbial load (cfu/gm)
Salacia reticulata extract 300 cfu/gm
Gymnema sylvestre extract 3.8 x102 cfu/gm

10. The process as claimed in claim 7 wherein the % content of salacinol in Salacia extract is in the range of 0.1% to 1.5%.

11. The process as claimed in claim 7 wherein the % content of Gymnemic acid-IV in Gymnema extract is in the range of 0.1% to 1.5%.