# Patent

## Title
Herbal formulation for the prevention and management of type-2 diabetes mellitus and vascular complications associated with diabetes

## Abstract

This invention relates to a novel herbal formulation for the prevention and management of type-2 diabetes mellitus and vascular complications associated with diabetes comprising, preparing of hydromethanolic extract of at least one plant selected form Salacia roxburghii, Salacia oblonga, Garcinia Indica and Lagerstroemia parviflora at 70°-80° C., maintaining ph of the solution between 7-10, separating the active compounds by using TLC, HPLC, HPTLC, subjecting the active compounds to the step of molecular characterization.

## Background
FIELD OF INVENTION
This invention relates to a novel herbal formulation for the prevention and management of type-2 diabetes mellitus and vascular complications associated with diabetes.
BACKGROUND OF INVENTION
Type-II diabetes mellitus (DM) is a metabolic disorder that is primarily characterized by insulin resistance, relative insulin deficiency and hyperglycemia. It is rapidly increasing in the developed countries and there is also evidence that this pattern will be followed in much of the rest of the parts of the world in coming years. About 90-95% of all North Americans are suffering from type-II diabetes and 20% of the population are over the age of 65 years. Diabetes affects over 150 million people worldwide and this number is expected to double by 2025. Now it has been proven that the Islet Amyloid Polypeptide genes are responsible for the early on set of type-II diabetes.
Type II diabetes is the predominant form of diabetes accounting for 90% of case globally. In type II diabetes, two impairments are found to increase blood glucose levels; impaired insulin action and impaired pancreatic insulin secretion. In addition, liver also plays key role in adjusting blood glucose levels via. gluconeogenesis in fasting and glucogenolysis in postprandial conditions. In type II diabetes glucogenolysis increases disproportionally which further contribute in elevated blood glucose level.
About 55% patients of type-II diabetes mellitus are obese, with the result obesity leads to increased insulin resistance that can develop into diabetes. Further, type-II diabetes mellitus often associated with obesity, hypertension and dyslipidemia. Additional factors founds to be associated with type-II diabetes include ageing, high fat diets and less active life style.
Diabetes is associated with factors which directly contribute to cardiovascular disorders including insulin resistance, dyslipidemia, atherosclerosis, hypertension, excessive oxidations, endothelial dysfunction, vascular inflammation and growth factors may affect platelet aggregation. Obesity is another risk factor in the development of diabetes and CHD.
Both diabetes and impaired glucose tolerance are associated with increased risk of reno-Cardio vascular diseases (CVD). Approximately 80% of death in diabetes patients are due to CVD which is significantly associated with dyslipidemia.
Inflammation plays a vital role in pathogenesis of CHD among diabetes patients. Interleukin-6 and C-reactive protein are scientific bio-markers associated with hyperglycemia, insulin resistance and type-2 diabetes mellitus. Both the factors are responsible for development of cardiovascular disorders. IL-6 has been shown to induce gluconeogenesis subsequently hyperglycemia and hyperinsulinemia. Levels of CRP are increased in type-2 diabetes and can cause CHD death independently. Hyperhomocysteinemia (Hyper Hcy) is also recognized as independent risk factor for CVD in type-2 diabetes. Hyper Hcy exerts toxic effects via elevated oxidative stress which induces endothelial dysfunction. In addition adipokine leptin has been found to be positively associated with insulin resistance, diabetes risk, triglyceride level, CRP, blood pressure, obesity etc. Adiponectin, a protein secreted by adipose cells regulates insulin sensitivity with energy metabolism. A decreased level of adiponectin concentration is recorded in above conditions. Recently peroxisome proliferator activator receptor (PPAR-α)-α and PPAR-γ are found to be associated with diabetes that regulated insulin responsive gene transcription involved in glucose production, transport and utilization.
The available oral anti-hyperglycemic drugs are in practice for the management of diabetes but application of these agents is limited because of adverse reaction of these synthetic chemicals.
Similarly treatment with statin for dyslipidemia causes risk of hepatic or muscle enzyme abnormalities.
Keeping the above facts in view it was thought to propose a plant based formulation showing beneficial role in the management of type-2 diabetes mellitus and associated disorder. In Ayurvedic system of medicine, several plants have been advocated for their hypoglycemic effects and are still in practice. Taking the lead from ancient literature four plants Salacia roxburghii (Saptarangi), Salacia oblonga, Garcinia indica (Kokum) and Lagerstroemia parviflora (Jarul) were selected out of various screened plants carried out for this purpose and the novel formulation was prepared following standard norms.
In experimental studies the safety and efficacy profile of single plant candidate and also combined formulation has been established. The test drug was found to have an agonist for PPAR activated receptor and regulates insulin responsive gene transcription involved in glucose production, transport and utilization and thus reduced blood glucose and reduces hyper-insulinemia. Our claims are established on the basis of experimental and clinical trials.
OBJECTS OF INVENTION
The main object of this invention is to propose plant based herbal formulation in the prevention and management of type-2 diabetes mellitus and associated vascular complications with the purpose of preventing CHD death among diabetic patients.
Another object of present invention is to propose a plant based Ayurvedic formulation showing role as an agonist for PPAR activated receptor and can regulate insulin responsive gene transcription.
Still, another object of present invention is to propose an Ayurvedic formulation showing beneficial role on abnormal lipid metabolism (dyslipidemia) particularly oxidized LDL and triglycerides among type-2 diabetes cases.
Yet another object of present invention is to propose a novel formulation which can retard the pro-inflammatory markers TNF-α, IL-6 and also CRP in type-2 diabetes cases.
Another object of present invention is to propose a plant based formulation showing beneficial role in increasing adiponectin among type-2 diabetes cases to prove the anti-obesity and anti-atherosclerotic property of present novel formulation.
Further, object is to propose a plant based formulation effective in the prevention and management of hyperglycemia, ultimately that can prevent major organ system i.e. brain, heart, kidney in diabetic patients.
STATEMENT OF INVENTION
According to this invention there is provided a novel herbal formulation for the prevention and management of type-2 diabetes mellitus and vascular complications associated with diabetes comprising hydromethanolic extract of at least one plant out of four plants i.e Salacia roxburghii, Salacia oblonga, Garcinia indica and Lagerstroemia parviflora in effective doses and a process for the preparation of thereof comprising; preparation of hydro-methanolic extract of Salacia roxburghii and Lagerstroemia parviflora by mixing aqueous and methanol in 30:70 ratio at 70-80° C., maintaining pH of the solution between 7-10, separating chromatographically active compounds/molecules by using TLC, HPLC, HPTLC, molecular characterization by using IR and NMR separating the active chemical constituents present in plants.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a flow diagram of the process.



DETAILED DESCRIPTION OF INVENTION
The present invention relates to the novel Ayurvedic formulation and to the process thereof for the prevention and management of dyslipidemia, atherosclerosis, obesity, and endothelial dysfunction in type-2 diabetes mellitus cases. The preparation of present invention is advantageous if used for the prevention and management of hyperglycemia, abnormal lipids, altered adipokines, elevated inflammatory cytokines, obesity, hyperhomocystinemia, hypertension and atherosclerosis involved with diabetic patients. The beneficial role of novel Ayurvedic formulation can be assessed on blood glucose level, as an agonist PPAR-α activated receptor activity, inflammatory pro-cytokines including CRP, adipokine, adiponectin, homocysteine, oxidized LDL-c, triglycerides and endotheline level.
The hydro-methanolic extract of four Ayurvedic plants Salacia roxburghii, Salacia oblonga, Garcinia indica and Lagerstroemia parviflora by using 30:70 ratio of water and methanol respectively is used for the experimental and clinical studies. The water utilized for extraction was decontaminated for any type of bacterial or abnormal growth by using reverse osmosis plant. After extraction the active molecules was identified and separated by HPLC, HPTLC and NMR procedures.
The biological activity was studied on the basis of mode of action of the test drug and effect of the drug on various parameters undertaken for this clinical condition. The molecular characterization was done by using. NMR and bio-molecular reaction following the interaction between the chemical and biological markers like insulin resistance (blood glucose levels), inflammatory cytokines, adipokines, various fractions of lipids including triglycerides, the PPAR-α and PPAR-γ.
The pre-clinical toxicological studies were carried out to determine the safety profile of individual plant candidate as well as all four plant candidates combined to prepare novel formulation before using the drug for human beings. The mode of action of single plant and combined formulation was carried out in animal models.
The beneficial role of test formulation on fasting and postprandial blood glucose level, PPAR-α agonist activity, insulin resistance, abnormal lipids, atherosclerosis, altered adipokines and inflammatory cytokines etc. were established in animal models before using the drug for human consumption.
Mechanism of Action of Present Formulation:
The present formulation containing the hydro-methanolic extract of Salacia roxburghii, Salacia oblonga, Garcinia indica and Lagerstroemia parviflora has shown anti-diabetic property in various experimental and clinical trials. The present formulation contains phyto-molecules that target various receptors responsible for diabetes. It is agonist for the PPAR-α & γ, with the result circulating level of triglycerides, LDL-c and total cholesterol decreases. It has been observed that extract of Salacia roxburghii enhances the level of PPAR-α mediated lipogenic gene expression resulting in the decreased level of triglycerides, oxidized LDL and total cholesterol.
The mode of action of present formulation is similar to the action of statin. The test formulation significantly influences the starch metabolizing enzyme as it reduces the hexokinase and lactate dehydrogenase. These liver enzymes play a major role in the metabolism of glucose.
Mangiferine and corosolic acid both have shown significant effect in reducing glycemic index both in animals and human subjects. Salcinol an important phyto-constituents found in Salacia roxburghii and Salacia oblonga have shown inhibition of cardiac angiotensin-II type-I receptor preventing the atherosclerosis and endothelial dysfunction thus prevents vascular changes in diabetes patients.
Extraction Procedure:
The shed dried root and fruits of Salacia roxburghii, Salacia oblonga, fruits and seeds of Garcinia indica and bark of Lagerstroemia parviflora were utilized for obtaining extracted material of the plants. The water and methanol extract (30:70 ratio) were utilized for the extraction of active compound found in the plants. After extraction the extracted materials were taken for chromatographic separation by using TLC, HPLC, and HPTCL. After identification and separation of the active compound the molecular characterization was carried out by using IR and NMR.
The extraction was done at the temperature of 70-80° c. The pH of the solution was maintained between 7-10. The following steps were followed and carried out to isolate the active compound, preparation of test drug as well as to develop a new drug entity. FIG. 1 is a flow diagram showing the steps involved in the extraction.
According to this invention there is provided an Ayurvedic formulation for the prevention and management of type-2 diabetes mellitus and associated vascular complications with the object to prevent the diabetic patients from CHD death and also from various type of morbidity caused due to hyperglycemia. The present test formulation comprising of the following four ingredients—
















Name of the plants
Part used









1. 
Salacia roxburghii (Saptarangi) 
Root and fruits



2. 
Salacia oblonga (Saptachakra)
Root



3.
Garcini indica (Kokum)
Fruits & Seeds



4. 
Lagerstroemia parviflora (Jarul)
Leaves










Preferably the aforesaid plants are present in the test drug in following doses—
















Name of the plants
Dose









1. 
Salacia roxburghii
225-400 mg/day



2. 
Salacia oblonga
200-425 mg/day



3. 
Garcinia indica
175-300 mg/day



4. 
Lagerstroemia parviflora
175-325 mg/day










The formulation may also comprise known additives such as minerals, vitamins, salts, filler (for capsulation or to prepare syrup) and binders, if required to present in trace amount.
Thus any known additive or supplement is added to prepare the final formulation as required and present in trace amount. Reference is made here in capsule form. However, it would be apparent that the preparation may also be in the form of syrup/tablet.
Preferably but without implying any limitation the preparation comprises—
















Name of the plants
Dose









1. 
Salacia roxburghii
275 mg/day



2. 
Salacia oblonga
325 mg/day



3. 
Garcinia indica
225 mg/day



4. 
Lagerstroemia parviflora
225 mg/day










Hypothesis:

The present Ayurvedic formulation is based on the combined effect of four plant extract namely Salacia roxburghii, Salacia oblonga, Garcinia indica and Lagerstroemia parviflora. This novel formulation is an agonist for PPAR receptor, modified abnormal lipids including triglyceride, reduced inflammatory cytokines, regulates adipokines, and improves renal cardiac functions. Those effect are mediated through PPAR-α & γ against activity that results in insulin responsive gene transcription involved in glucose production, transport and utilization ultimately reduces blood glucose level, reduces inflammatory process by reducing CRP, IL-6, TNF-α, it regulates abnormal lipids including triglycerides concentration, enhances adiponectin and also reduces homocysteine. All these beneficial effects ultimately slowed down/improved atherosclerotic process among diabetes patients and has potential role in prevention of adverse cardiac event.
As documented diabetes increases the risk of myocardial infarction and risk of stroke, it is hypothesized that present test formulation improves the glycemic load by improving insulin sensitivity, as an agonist for PPAR-α activated receptors, stimulates beta-cells, increases glucose uptake in tissues, may have activity on cathepsin-b and increases cyclic AMP count on islets. As synergism test drug has anti-oxidant effects.
Atherosclerosis occurs due to a number of factors in diabetic individuals. Both insulin resistance and elevated lipid levels are the triggering factors for athrogenic injury, endothelial dysfunction in diabetics otherwise is more prone to atherogenic injury due to decreased production of endothelial nitric oxide and increased production of plasminogen activator inhibitor. Lipid disorders like elevated total cholesterol, low HDL-c and high triglyceride level is the major risk factor for CHD. Therefore, the prevention and management of vascular complications is the basic and essential principle among diabetes patients to prevent them from CHD death.
As the present test formulation contains of extracts of four plants Salacia roxburghii, Salacia oblonga, Garcinia indica and Lagerstroemia parviflora therefore, to describe the action of individual plant is helpful in understanding the specific action on specific target involved in diabetes. Garcinia indica extract contained 0.67 percent of garcinol and 0.17 percent of hydroxycitric acid. ((−)-Hydroxycitric acid (−)-HCA) has a potential effect in stimulating beta cells of islet langerhans. Similarly gracinol have shown potential effect in reducing pro-inflammatory cytokines mainly responsible for endothelial dysfunction among diabetic patients. The corosilic acid present in extract of Lagerstroemia parviflora in the dose of 1.02 percent showed potential effect in minimizing the inflammatory markers in type-2 diabetes mellitus cases. The proportion of mangiferin and salacinol varies in Salacia roxburghii and Salacia oblonga. The Salacia roxburghii exerted more potential effect in the prevention and management of diabetic complications particularly the vascular complications. The Salacia oblonga has specific action in regulation of postprandial glycemic index, PPAR-α & γ as well as it prevents the cardiac hypertrophy by regulating angiotensin-II receptors. Mangiferin modulates the liver enzyme like hexokinase, lactate dehydroxygenase, pyruvate kinase and also the metabolizing enzyme glucose-6-phosphatase, fructose-6bisphosphatase, glucose-6phosphatase dehydrogenase. Therefore, it is proven that combined formulation acted on different targets involved in type-2 diabetes mellitus.
C-peptide has no relation with blood glucose level but it has strong relationship with status of glomerular damage in diabetes. It has been demonstrated that C-peptide infusion increases the utilization of glucose among the diabetic patients. In healthy individuals C-peptide level indicates the better renal function suggesting measurement of C-peptide level might be helpful in the prevention of renal complications among diabetes.
The present novel Ayurvedic formulation has been developed on the basis of hypothesis that certain plants having bio-molecules responsible for enhancing the general body resistance by arresting the process of inflammation, catabolism and preventing oxidative damage due to reactive oxygen species. It is postulated that some of the plant based products have potentiality to prevent the comments complications like neuropathy, retinopathy and nephropathy associated with diabetes. It has been observed that adiponectin induces TNF-α, IL-6 and promotes tolerance to itself and other pro-inflammatory stimuli. It has been considered as an important bio-marker for insulin resistance, obesity and generalized atherosclerotic changes in body. Therefore our main target is to find out the
specific target which can arrest inflammation and prevents various complications associated with type-2 diabetes mellitus.
The investigators have established that combined formulation of above mentioned plants in specific dose not only enhances the level of adiponectin but also reduces the glycemic index and prevents from damage of vital organs by reducing inflammatory cytokines responsible for the endothelial dysfunction in type-2 diabetes mellitus. Adiponectin the important biomarker has potentiality to regulate multiple targets involved in type-2 diabetes mellitus.
About the Plants:
1. Salacia roxburghii: Mainly found in Eastern part of India. Salacia species belongs to family Hippocrastaceae. Fruits and root are used for medicinal purpose. Salaretin is the main active compound isolated from this plant is responsible for breakdown of starch in diet. Mangiferin is also an important component of Salacia roxburghii. This drug has shown anti-diabetic, anti-inflammatory, anti-oxidant, anti-obesity and homocysteine lowering activity in diabetes patients. Further, the drug enhanced PPAR-α—mediated lipogenic gene expression. Mangiferin lowers blood lipids in diabetes.

2. Salacia oblonga: Mainly found in Eastern part of India & Sri Lanka. Salacia species belongs to family Hippocrastaceae. The roots and stems of Salacia oblonga have been used extensively in Ayurveda and traditional Indian medicine for the treatment of Diabetes. Historically, Salacia oblonga is in use from the centuries by traditional practitioners. Recently, Salacia oblonga is being used to treat diabetes as it seems to lower blood sugar and insulin levels in a manner similar to prescription drugs. After recent pharmacological investigations it has been proved that Salacia roots modulate multiple targets mainly; peroxisome proliferator-activated receptor-alpha-mediated lipogenic gene transcription, angiotensin II/angiotensin II type 1 receptor, alpha-glucosidase, aldose reductase and pancreatic lipase. The multi-targeted actions of Salacia root ultimately induced the improvements in type 2 diabetes mellitus and obesity-associated hyperglycemia, dyslipidemia and other related cardiovascular complications.

3. Garcinia indica (Kokum):

Belongs to the family Clusiaceae. Seeds and fruits of Garcinia indica is used for medicines and cosmetics. Hydroxycitric acid (HCA) is extracted from the rind of the fruit which is used as hypocholesterolaemic agent. It is also a potential anti-obesity agent. The anti-oxidant property of aqueous extract of the plant is observed. Garcinia indica extract is having anti-allergic effects. It contains oxalicacid, malic acid, polyphenols, carbohydrates, anthocyanin and ascorbic acid. Anti-oxidant property is due to Garcinol. Isogarcinol also shows anti-inflammatory, lipase inhibitor, anti-obesity and neuroprotective effect.
4. Lagerstroemia parviflora is commonly known as Jarul belongs to family Lytheraceae. It is very common tree found in the moist and dry forest mainly of Andhra Pradesh. The active compounds found in Lagerstroemia parviflora are—Flosin-B, Stachyurin, Casuarinin, casuarin, epipunicacortein-A, ellargic acid, corosolic acid, tannin etc. Leaves possess anti-diabetic, antibacterial, anti-obesity, anti-oxidant effects. It maintains the normal weight, promotes healthy insulin level. According to recent studies it increases glucose uptake and has positive role in glycemic control. The chemical constituents mainly ellagic acid has shown an inhibitory effect on glucose transport assay. The other active compounds corosolic acid has exhibited potent anti-diabetic activity as it reduced both fasting and postprandial blood glucose level.
EXAMPLE-I
In non-clinical efficacy profile evaluation when the hydro-methanolic extract of Salacia roxburghii in the dose of 50 mg/kg and Lagerstroemia parviflora 50 mg/kg and Garcinia indica 40 mg/kg was given to streptozotocin induced diabetic rats, the drug exerted as an agonist for PPAR activated receptors resulting in insulin regulated gene transcription.
EXAMPLE-II
When the hydro-methanolic extract of Salacia roxburghii 50 mg/kg and Salacia oblonga 50 mg/kg was given to Sprague Dawley rats, 63 percent α-glucosidase inhibitory activity was recorded. Further, amylase and lipid lipase lowering effects were also noticed.
EXAMPLE-III
When the hydro-methanolic extract of Salacia roxburghii 60 mg/kg and Garcinia indica 60 mg/kg was given to streptozotocin induced diabetic rats a significant reduction in blood glucose level was measured indicating anti-diabetic role of the drug.
EXAMPLE-IV
In clinical study when the hydro-methanolic extract of Salacia roxburghii in the dose of 525 mg/day and Lagerstroemia parviflora 350 mg/day combined and given in two divided doses to diagnosed cases of type-2 diabetes a significant decrease in postprandial blood glucose level was noticed. An average 40 percent depletion of blood glucose level indicated the anti-diabetic activity of test drug.
EXAMPLE-V
When the hydro-methanolic extract of Salacia oblonga (475 mg/day) and Lagerstroemia parviflora (450 mg/day) was mixed and given to type-2 diabetes patients a marked decrease in triglycerides content was estimated along with glycemic control property of the test formulation as glycosylated hemoglobin improved following treatment in diabetes patients.
EXAMPLE-VI
When the hydro-methanolic extract of Garcinia indica 250 mg/day, Lagerstroemia parviflora (325 mg/day) and Salacia oblonga (325 mg/day) mixed and administered to type-2 diabetes patients a significant decrease in Apolipo-B and moderate increase in HDL-c level was noticed.
EXAMPLE-VII
When the hydro-methanolic extract of Garcinia indica 250 mg/day, Lagerstroemia parviflora (350 mg/day) and Salacia roxburghii (350 mg/day) mixed and given to diagnosed cases of type-2 diabetes patients the oxidized LDL-c level decreased indicating improvement in atherosclerotic process among type-2 diabetes patients.
EXAMPLE-VIII
When the hydro-methanolic extract of Salacia roxburghii (325 mg/day) and Lagerstroemia parviflora (275 mg/day) and Salacia oblonga (350 mg/day) was mixed and given to diabetes patients decrease in pro-inflammatory markers like IL-6, TNF-α including hs. CRP was estimated indicating improvement in vascular inflammation in diabetes cases. It also reduces inflammatory marker resistin in diabetic patients.
EXAMPLE-IX
When the hydro-methanolic extract of Salacia oblonga (450 mg/day) and Lagerstroemia parviflora (450 mg/day) was mixed and administered to selected diabetes patients increase in adiponectin level indicated the anti-atherosclerotic and anti-obesity role of test formulation.
EXAMPLE-X
When the hydro-methanolic extract of Salacia roxburghii (275 mg/day), Lagerstroemia parviflora (225 mg/day), Salacia oblonga (325 mg/day) and Garcinia indica (225 mg/day) mixed and orally administered to selected type-2 diabetes patients a better effect of test formulation was recorded as the blood glucose level decreased, abnormal lipids modified including apolipo-B, the inflammatory cytokines IL-6 and TNF-α decreased, CRP and leptin also reduced, where as adiponectin level increased. On the whole this novel combined formulation has shown potential role in the prevention and management of type-2 diabetes mellitus and associated vascular complications through regulation of PPAR-α and PPAR-γ resulting in glycemic control and enhancing insulin sensitivity. Thus it is proposed as a better safer remedial measure for the prevention and management of type-2 diabetes mellitus and associated vascular complications in diabetes patients thus CHD death can be prevented among diabetes patients.










Alpha-glucosidase inhibitory activity of single as well as 


combined formulation








Concen-
Percent inhibition












tration
Salacia
Lagerstroemia
Salacia
Garcinia
Combined


(μg/ml)
roxburghii
parviflora
oblonga
indica
formulation















 50 mg/kg
23.79
28.72
28.40
25.03
31.61


100 mg/kg
37.82
43.09
39.61
23.75
61.80


150 mg/kg
41.6
48.22
46.22
25.85
72.32









Experimental Study-I







TABLE 1







Reduction in blood glucose level following test drug treatment 


in Streptozotocin (STZ) treated diabetic rats









Blood glucose level (mg/dl)










Treated group
7th Days
14th Days
30th Days





Normal control (N = 6) *
63.91 ±
61.94 ±
64.82 ±



8.73
10.11
12.04


Td. With STZ
334.74 ±
293.71 ±
268.45 ±


(65 mg/kg) N = 6 **
28.99
31.22
37.02


Td. With STZ + Test
258.97 ±
209.85 ±
172.75 ±


drug (N = 6)***
22.84
26.90
21.65


Td. With STZ +
237.90 ±
194.93 ±
164.35 ±


Acarbose ****
21.64
16.87
12.86





Comp.





* vs **
P < 0.001
P < 0.001
P < 0.001


** vs***
P < 0.001
P < 0.001
P < 0.001


***vs ****
P > 0.05
P < 0.05
P < 0.05













TABLE 2







Reduction in hs C-reactive protein following test drug treatment 


in STZ treated diabetic rats









hs CRP (mg/L)










Treated group
7th Days
14th Days
30th Days





Normal control (N = 6) *
1.51 ±
1.43 ±
1.38 ±



0.24
0.30
0.45


Td. With STZ (65 mg/kg)
9.11 ±
8.83 ±
6.89 ±


N = 6 **
2.04
2.12
1.72


Td. With STZ + Test drug
7.34 ±
5.73 ±
4.08 ±


(N = 6)***
1.54
0.84
0.74


Td. With STZ + Acarbose
7.58 ±
5.85 ±
5.11 ±


****
1.61
0.68
0.93





Comp.





* vs **
P < 0.001
P < 0.001
P < 0.001


** vs***
P < 0.001
P < 0.001
P < 0.001


***vs ****
P > 0.05
P > 0.05
P > 0.05













TABLE 3







Reduction in Interleukin-6 following test drug treatment in 


STZ treated diabetic rats









IL-6 (pg/ml)










Treated group
7th Days
14th Days
30th Days





Normal control (N = 6) *
0.83 ±
0.91 ±
0.87 ±



0.11
0.13
0.20


Td. With STZ
4.13 ±
3.94 ±
3.54 ±


(65 mg/kg) N = 6 **
1.02
0.92
0.88


Td. With STZ + Test
2.65 ±
2.16 ±
1.73 ±


drug (N = 6)***
0.24
0.31
0.27


Td. With STZ + Acarbose
2.94 ±
2.58 ±
2.21 ±


****
0.26
0.30
0.29





Comp.





* vs **
P < 0.001
P < 0.001
P < 0.001


** vs***
P < 0.001
P < 0.01
P < 0.001


***vs ****
P > 0.05
P > 0.05
P > 0.05













TABLE 4







Effect of test formulation on adiponectin level in STZ 


treated diabetic rats









Adiponectin (μg/ml)










Treated group
7th Days
14th Days
30th Days





Normal control (N = 6)*
13.45 ±
14.13 ±
14.42 ±



1.87
2.06
2.11


Td. With STZ (65 mg/kg)
 6.86 ±
 5.83 ±
 5.09 ±


N = 6 **
1.13
0.97
0.86


Td. With STZ + Test drug
 9.11 ±
10.12 ±
12.93 ±


(N = 6)***
2.04
1.75
2.14


Td. With STZ + Acarbose
 8.83 ±
 9.82 ±
10.66 ±


****
2.02
1.65
2.01





Comp.





*vs **
P < 0.001
P < 0.001
P < 0.001


** vs***
P < 0.05
P < 0.001
P < 0.001


***vs ****
P > 0.05
P > 0.05
P > 0.05






Experimental Study-II







TABLE 1







Effect of Test formulation on body weight following cafeteria 


diet in experimental rats










Body Weight (grams)
Comp. Initial













After 15 
After 30 
vs After 30


Treated group
Initial
Days
Days
days





Normal control
104.93 ±
110.82 ±
117.36 ±
P < 0.001


(N = 6)
3.88
6.03
4.91



Cafeteria diet only
 99.22 ±
128.92 ±
158.90 ±
P < 0.001


(N = 6)
4.37
6.11
12.13



Cafeteria diet + test
114.97 ±
119.93 ±
128.72 ±
P < 0.001


formulation (N = 6)
9.82
8.41
7.04
















TABLE 2







Effect of Test formulation on total cholesterol and triglycerides following cafeteria


dies in experimental animals
















Comp.


Comp.












TC (mg/dl)
Initial vs
TG (mg/dl)
Initial vs















After 30
After 30

After 30
After 30


Treated group
Initial
Days
days
Initial
Days
days





Normal control
84.78 ± 4.69
88.36 ± 5.11
P > 0.05
81.89 ± 8.63
83.01 ± 9.34
P > 0.05


(N = 6)








Cafeteria diet
87.11 ± 6.94
91.35 ± 9.12
P < 0.05
79.74 ± 5.80
98.34 ± 4.93
P < 0.05


only (N = 6)








Cafeteria diet +
86.90 ± 8.30
81.45 ± 6.03
P < 0.05
83.22 ± 8.91
74.90 ± 7.85
P < 0.05


test formulation








(N = 6)
















TABLE 3







Effect of Test formulation on body weight following cafeteria dies in experimental


rats












Blood glucose level
Comp.

Comp.



(mg/dl)
Initial vs
Adiponectin (μg/ml)
Initial vs















After 30
After 30

After
After 30


Treated group
Initial
Days
days
Initial
30 Days
days





Normal control
58.90 ± 7.02
55.70 ± 6.88
P > 0.05 
12.87 ± 1.91
13.16 ± 2.08 
P > 0.05 


(N = 6)








Cafeteria diet
54.93 ± 6.12
71.11 ± 5.90
P < 0.001
—
7.82 ± 1.03
P < 0.001


only (N = 6)








Cafeteria diet +
56.35 ± 6.31
64.01 ± 4.87
P < 0.01 
—
11.02 ± 1.94 
P < 0.01 


test formulation








(N = 6)
















TABLE 4







Role of test formulation on Total Cholesterol among high 


cholesterol diet treated rats









Total cholesterol level (mg/dl)










Treated group
Initial
After 15 Days
After 1 months





Normal control
64.32 ±
 63.80 ±
 64.70 ±


(N = 10)*
7.89
6.52
8.42


High cholesterol diet
—
895.42 ±
480.82 ±


(N = 10)**

49.75
40.72


High cholesterol diet +
—
688.59 ±
402.71 ±


test formulation

101.22
45.90


(N = 10)***





High cholesterol diet +
—
691.52 ±
280.50 ±


statin (2.5 mg/kg/day)

78.85
16.80


(N = 10)****





Comp.





*vs**
P > 0.05
P < 0.001
P < 0.001


**vs***

P < 0.001
P < 0.001


***vs****

P < 0.001
P < 0.001













TABLE 5







Effect of test formulation on HDL-c level among high


cholesterol diet treated rats









HDL-c level (mg/dl)










Treated group
Initial
After 15 Days
After 1 months





Normal control
22.50 ±
23.32 ±
22.37 ±


(N = 10)*
4.33
2.85
3.85


High cholesterol diet
—
17.82 ±
13.85 ±


(N = 10)**

5.32
1.85


High cholesterol diet +
—
18.45 ±
20.65 ±


test formulation (N = 10)***

2.91
2.85


High cholesterol diet + statin
—
20.32 ±
21.85 ±


(2.5 mg/kg/day) (N = 10)****

4.85
3.85





Comp.





*vs**
P > 0.05
P < 0.05
P < 0.001


**vs***

P > 0.05
P < 0.001


***vs****

P > 0.05
P < 0.05













TABLE 6







Effect of test formulation on LDL-c level among high


cholesterol diet treated rats









LDL-c level (mg/dl)










Treated group
Initial
After 15 Days
After 1 months





Normal control
23.85 ±
 22.75 ±
 24.22 ±


(N = 10)*
4.78
5.72
6.85


High cholesterol diet
—
341.50 ±
314.40 ±


(N = 10)**

62.32
48.34


High cholesterol diet +
—
270.15 ±
139.22 ±


test formulation (N = 10)***

40.23
30.12


High cholesterol diet + statin
—
255.80 ±
108.85 ±


(2.5 mg/kg/day) (N = 10)****

37.38
16.85





Comp.





*vs**
P > 0.05
P < 0.001
P < 0.001


**vs***

P < 0.05
P < 0.001


***vs****

P > 0.05
P < 0.05













TABLE 7







Effect of test formulation on Triglycerides level among 


high cholesterol diet treated rats









Triglycerides level (mg/dl)










Treated group
Initial
After 15 Days
After 1 months





Normal control
26.85 ±
30.32 ±
28.40 ±


(N = 10)*
8.70
7.85
5.52


High cholesterol diet
—
340.70 ±
298.50 ±


(N = 10)**

64.80
39.32


High cholesterol diet +
—
241.93 ±
188.93 ±


test formulation (N = 10)***

59.75
28.45


High cholesterol diet + statin
—
228.50 ±
112.85 ±


(2.5 mg/kg/day) (N = 10)****

31.80
19.30













Comp.





*vs**
P > 0.05
P < 0.001
P < 0.001


**vs***

P < 0.05
P < 0.01


***vs****

P > 0.05
P < 0.05






Clinical Study







TABLE 1







Effect of test formulation on Body Mass Index among type-II 


diabetes mellitus cases













Comp. 




Body Mass Index
Initial














After 6 
vs After 



No. of

months
6 months


Groups
cases
Initial
therapy
therapy





Conventional treatment
34
31.62 ±
30.71 ±
P > 0.05




3.01
3.04



Conventional treatment +
45
29.75 ±
27.73 ±
P < 0.05


test formulation

2.89
2.90



Test formulation treated
43
31.25 ±
27.94 ±
P < 0.01




2.97
2.41
















TABLE 2







Change in Fasting Blood Glucose level following test formulation 


treatment in type-2 diabetes mellitus cases













Comp. 




Fasting blood glucose (mg/dl)
Initial















After 3
After 6
vs After 



No. of

months
months
6 months


Groups
cases
Initial
therapy
therapy
therapy





Conventional
34
172.37 ±
133.23 ±
116.44 ±
P < 0.001


treatment

24.90
23.60
26.24



Conventional
45
173.90 ±
122.87 ±
110.45 ±
P < 0.001


treatment + test

25.82
26.90
12.82



formulation







Test formulation
43
161.04 ±
130.45 ±
123.97 ±
P < 0.001


treated

16.42
10.82
8.41
















TABLE 3







Changes in postprandial blood glucose level following test 


formulation treatment in type-2 diabetes mellitus cases













Comp. 




Postprandial blood glucose (mg/dl)
Initial













No.

After 3 
After 6 
vs After 



of

months
months
6 months


Groups
cases
Initial
therapy
therapy
therapy





Conventional
34
280.90 ±
167.03 ±
142.94 ±
P < 0.001


treatment

29.90
32.05
22.36



Conventional
45



P < 0.001


treatment + test

287.90 ±
174.82 ±
148.34 ±



formulation

29.82
41.02
31.84



Test formulation
43
261.39 ±
219.47 ±
169.42 ±
P < 0.001


treated

17.84
13.80
10.87
















TABLE 4







Changes in Total cholesterol following test formulation 


treatment among type-2 diabetes mellitus cases













Comp. 




Total cholesterol (mg/dl)
Initial













No. 

After 3 
After 6
vs After 



of

months
months
6 months


Groups
cases
Initial
therapy
therapy
therapy





Conventional
34
209.59 ±
202.63 ±
202.66 ±
P > 0.05


treatment

42.97
40.30
39.19



Conventional
45
223.54 ±
188.01 ±
183.68 ±
P < 0.05


treatment + test

32.41
28.32
25.56



formulation







Test formulation
43
227.10 ±
189.45 ±
173.08 ±
P < 0.01


treated

19.80
20.73
13.97
















TABLE 5







Role of test formulation on the HDL-c levels among type-2


diabetes mellitus cases













Comp. 




HDL-c (mg/dl)
Initial















After 3 
After 6 
vs After 



No. of

months
months 
6 months


Groups
cases
Initial
therapy
therapy
therapy





Conventional
34
46.03 ±
46.24 ±
45.90 ±
P > 0.05


treatment

6.60
5.70
6.00



Conventional
45
48.90 ±
50.91 ±
53.90 ±
P < 0.02


treatment + test

3.84
3.56
3.22



formulation







Test formulation
43 
41.99 ±
46.39 ±
49.02 ±
P < 0.05


treated

4.11
3.75
2.88
















TABLE 6







Effect of test formulation on LDL-c levels among type-2


diabetes mellitus cases












LDL-c (mg/dl)
Comp. Initial 















After 3 
After 6 
vs After 



No. of

months 
months
6 months


Groups
cases
Initial
therapy
therapy
therapy





Conventional
34
131.52 ±
130.63 ±
129.60 ±
P > 0.05


treatment

6.90
5.68
6.02



Conventional
45
136.04 ±
122.45 ±
109.35 ±
P < 0.01


treatment + test

8.11
6.93
6.79



formulation







Test formulation
43
138.45 ±
116.93 ±
99.85 ±
P < 0.01


treated

6.90
7.84
6.35
















TABLE 7







Changes in Triglyceride content following test formulation 


treatment in type-2 diabetes mellitus cases












Triglyceride (mg/dl)
Comp. Initial 















After 3 
After 6 
vs After 



No. of

months
months
6 months


Groups
cases
Initial
therapy
therapy
therapy





Conventional
34
220.46 ±
216.20 ±
217.63 ±
P > 0.05


treatment

45.34
39.27
41.46



Conventional
45
241.87 ±
165.82 ±
132.42 ±
P < 0.01


treatment + test

41.90
46.08
20.52



formulation







Test formulation
43
262.75 ±
223.78 ±
174.90 ±
 P < 0.001


treated

38.41
29.45
31.65
















TABLE 8







Changes in Apolipo (B) under influence of test formulation 


treatment in type-2 diabetes mellitus cases












Apolipo (B) (mg/dl)
Comp. Initial 















After 3 
After 6 
vs After 



No. of

months
months
6 months


Groups
cases
Initial
therapy
therapy
therapy





Conventional
34
122.69 ±
128.76 ±
133.83 ±
P < 0.05 


treatment

20.53
22.44
17.80



Conventional
45
131.90 ±
115.90 ±
84.90 ±
P < 0.001


treatment + test

22.87
21.35
16.82



formulation







Test formulation
43
128.65 ±
113.82 ±
93.82 ±
P < 0.001


treated

17.94
10.77
8.36
















TABLE 9







Changes in TNF-α following test formulation treatment in 


type-2 diabetes mellitus cases












TNF-α (pg/ml)
Comp. Initial















After 3 
After 6 
vs After 



No. of

months
months
6 months 


Groups
cases
Initial
therapy
therapy
therapy





Conventional
34
623.60 ±
535.67 ±
480.33 ±
P < 0.01 


treatment

97.35
106.64
106.62



Conventional
45
735.92 ±
428.90 ±
364.75 ±
P < 0.001


treatment + test

114.87
94.34
71.38



formulation







Test formulation
43
687.42 ±
554.82 ±
461.30 ±
P < 0.001


treated

85.90
72.85
58.97
















TABLE 10







Effect of test formulation on pro-inflammatory bio-markers 


Interleukin-6 among type-2 diabetes mellitus cases












Interleukin-6 (pg/ml)
Comp. Initial 















After 3 
After 6 
vs After 



No. of

months
months
6 months


Groups
cases
Initial
therapy
therapy
therapy





Conventional
34
1.81 ±
1.79 ±
1.71 ±
P > 0.05 


treatment

0.31
0.29
0.31



Conventional
45
2.05 ±
1.64 ±
1.38 ±
P < 0.001


treatment + test

0.23
0.31
0.28



formulation







Test formulation
43
1.92 ±
1.52 ±
1.26 ±
P < 0.01 


treated

0.41
0.31
0.28
















TABLE 11







Effect of test formulation on inflammatory marker C-reactive 


protein among type-2 diabetes mellitus cases












CRP (mg/L)
Comp. Initial 















After 3 
After 6 
vs After 



No. of

months
months
6 months


Groups
cases
Initial
therapy
therapy
therapy





Conventional
34
2.36 ±
2.26 ±
2.06 ±
P > 0.05


treatment

0.56
0.53
0.51



Conventional
45
3.84 ±
2.35 ±
2.09 ±
P < 0.01


treatment + test

1.02
0.82
0.51



formulation







Test formulation
43
3.28 ±
2.38 ±
1.93 ±
P < 0.01


treated

0.92
0.71
0.24
















TABLE 12







Homocysteine lowering effect of test formulation in type-2 diabetes 


mellitus cases














Homocysteine (μmol/L)
Comp. Initial















No. of

After 6
After 12
vs After 12


Treatment group
Sex
cases
Initial
months
months
months

















Conventional
M
58
41.09 ±
38.42 ±
37.95 ±
t = 
3.72


treatment


3.16
2.95
3.10
P < 
0.001



F
29
35.75 ±
31.62 ±
30.88 ±
t = 
4.403





5.01
3.17
3.23
P < 
0.001


Conventional +
M
95
43.71 ±
37.71 ±
31.22 ±
t = 
19.394


Test formulation


4.88
5.06
3.97
P < 
0.001



F
42
38.89 ±
30.42 ±
27.46 ±
t = 
15.508





3.82
2.97
2.88
P < 
0.001


Treated with test
M
118
31.54 ±
29.08 ±
26.11 ±
t = 
15.083


formulation


3.17
2.92
2.32
P < 
0.001



F
56
28.77 ±
28.90 ±
26.08 ±
t = 
5.689





2.85
2.98
2.11
P < 
0.001









It is to be noted that the present invention is susceptible to modifications, adaptations and changes by those skilled in the art. Such variant embodiments employing the concepts and features of this invention are intended to be within the scope of the present invention, which is further set forth under the following claims—

## Claims

1. A herbal formulation for the treatment of vascular complications associated with type-2 diabetes mellitus consisting essentially of a therapeutically effective amount of a hydromethanolic extract of Salacia roxburghii, Garcinia indica and Largerstoremia parviflora.

2. The herbal formulation as claimed in claim 1, wherein said hydro-methanolic extract is water: methanol in a ratio of 30:70.

3. The herbal formulation as claimed in claim 1, wherein parts of the plants used for preparing the extracts are Salacia roxburghii Root and Fruits, Garcinia indica Fruits and Seeds and Largerstroemia paryiflora Leaves.

4. The herbal formulation as claimed in claim 1, wherein the plant extracts are present in the herbal formulation in the following amounts:
















Salacia roxburghii
225-400 mg,



Garcini indica
175-300 mg, and



Largerstroemia parviflora
175-325 mg.

5. The herbal formulation as claimed in claim 1, wherein the plant extracts are present in the herbal formulation in the following amounts:
















Salacia roxburghii
275 mg,



Garcinia Indica
225 mg, and



Largerstroemia parviflora
225 mg.

6. A herbal formulation for the treatment of vascular complications associated with type-2 diabetes mellitus consisting essentially of a therapeutically effective amount of a hydromethanolic extract of Salacia roxburghii and Garcinia indica.

7. The herbal formulation as claimed in claim 6, wherein said hydromethanolic extract is water: methanol in a ratio of 30:70.

8. The herbal formulation as claimed in claim 6, wherein parts of the plants used for preparing the extract are Salacia roxburghii Root and Fruits, and Garcinia indica Fruits and Seeds.

9. The herbal formulation as claimed in claim 6, wherein the plant extracts are present in the herbal formulation in the following amounts:
















Salacia roxburghii
225-400 mg, and



Garcinia indica
175-300 mg.

10. The herbal formulation as claimed in claim 6, wherein the plant extract are present in the herbal formulation in the following amounts:















Salacia roxburghii
275 mg, and



Garcinia indica
225 mg.

11. A herbal formulation for the treatment of vascular complications associated with type-2 diabetes mellitus consisting essentially of a therapeutically effective amount of a hydromethanolic extract of Salacia roxburghii, Salacia oblonga and Garcinia indica.

12. The herbal formulation as claimed in claim 11, wherein said hydromethanolic extract is water: methanol in a ratio of 30:70.

13. The herbal formulation as claimed in claim 11, wherein parts of the plants used for preparing the extract are Salacia roxburghii Root and Fruits, Salacia oblonga Root and Garcinia indica Fruits and Seeds.

14. The herbal formulation as claimed in claim 11, wherein the plant extracts are present in the herbal formulation in the following amounts:















Salacia roxburghii
225-400 mg



Salacia oblonga
200-425 mg, and



Garcinia indica
175-300 mg.

15. The herbal formulation as claimed in claim 11, wherein the plant extracts are present in the herbal formulation in the following amounts:















Salacia roxburghii
275 mg,



Salacia oblonga
325 mg, and



Garcinia indica
225 mg.

16. A herbal formulation for the treatment of vascular complications associated with type-2 diabetes mellitus consisting essentially of a therapeutically effective amount of a hydromethanolic extract of Salacia roxburghii, Salacia oblonga, Garcinia indica and Lagerstroemia parviflora.

17. The herbal formulation as claimed in claim 16, wherein said hydromethanolic extract is water: methanol in a ratio of 30:70.

18. The herbal formulation as claimed in claim 16, wherein parts of the plants used for preparing the extract are Salacia roxburghii Root and Fruits, Salacia oblonga Root, Garcinia indica, Fruits and Seeds, and Largerstroemia paryiflora Leaves.

19. The herbal formulation as claimed in claim 16, wherein the plant extracts are present in the herbal formulation in the following amounts:















Salacia roxburghii
225-400 mg,



Salacia oblonga
200-425 mg,



Garcinia indica
175-300 mg, and



Largerstroemia parviflora
175-325 mg.

20. The herbal formulation as claimed in claim 16, wherein the plant extracts are present in the herbal formulation in the following amounts:















Salacia roxburghii
275 mg,



Salacia oblonga
325 mg,



Garcinia indica
225 mg, and



Largerstroemia parviflora
225 mg.