HERBAL FORMULATION FOR THE PREVENTION AND MANAGEMENT OF TYPE-2 DIABETES MELLITUS AND ASSOCIATED MICROVASCULAR COMPLICATIONS

Abstract

Disclosed is an herbal formulation for the prevention and management of Type 2 diabetes and associated risk factors containing an effective amount of a hydro-alcoholic extract of Salacia roxburghii and an hydro-alcoholic extract of Salacia oblonga. Also disclosed are methods of preparing the formulation and using the formulation for the prevention and management of Type 2 diabetes and associated risk factors.

Claims

1) An herbal formulation for the prevention and management of Type 2 diabetes and associated risk factors comprising: an effective amount of a hydro-alcoholic extract of Salacia roxburghii and a hydro-alcoholic extract of Salacia oblonga.

2) The herbal formulation of claim 1, further comprising one or more of minerals, vitamins, salts, filler and binders.

3) The herbal formulation of claim 1, comprising TABLE-US-00027 Salacia roxburghii 450-900 mg/day Salacia oblonga 400-900 mg/day.

4) The herbal formulation of claim 1, wherein the extracts are hydro-alcoholic extracts of Salacia roxburghii root and Salacia oblonga root.

5) A method of managing Type 2 diabetes and associated CHD risk factors in a subject suffering from diabetes, comprising administering the herbal formulation of claim 1 to the subject.

6) The method of claim 5, wherein the formulation provides alpha glucosidase, amylase inhibitory properties and lipid lipase lowering effects in the subject.

7) The method of claim 5, wherein the extract of Salacia roxburghii acts as an agonist for peroxisome proliferaters activators (PPAR) activated receptor 7 and a, and regulates insulin responsiveness gene transcription involved in glucose production transport and utilization, thus reducing blood glucose level in the subject.

8) The method of claim 5, wherein the hydro-alcoholic extract of Salacia roxburghii has potent anti-diabetic role in the subject.

9) The method of claim 5, wherein the hydro-alcoholic extract of Salacia roxburghii and Salacia oblonga reduces elevated triglycerides along with blood glucose level in the subject.

10) The method of claim 5, wherein the formulation reduces Apolipo B and increases good cholesterol HDL-c in the subject.

11) The method of claim 5, wherein the formulation has anti-atherosclerotic property by reducing oxidized LDL-c in diabetes and improving endothelial dysfunction in the subject.

12) The method of claim 5, wherein the formulation reduces vascular inflammation by reducing IL-6, TNF-α and CRP in the subject.

13) The method of claim 5, wherein the the formulation has anti-atherosclerotic and anti-obesity effects in the subject.

14) The method of claim 5, wherein the formulation provides antioxidant potentials through homocysteine regulation in the subject.

15) A process for the preparation of an herbal formulation for the prevention and management of Type 2 diabetes and associated risk factors, comprising preparing a hydro-alcoholic extract of Salacia roxburghii and a hydro-alcoholic extract Salacia oblonga by extracting the Salacia roxburghii and Salacia oblonga with aqueous ethanol in 30:70 ratio of water to ethanol at 70-80° C. while maintaining pH of solution between 7-10, subjecting the hydro-alcoholic extracts to separation by chromatography, and characterizing molecules in the extracts by using IR and NMR.

16) The method of claim 15, wherein the extracts are root extracts.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0028] FIG. 1 is a flow chart showing extraction steps of the invention,

[0029] FIG. 2 shows the beneficial role of the herbal formulation on fasting blood glucose level among Type 2 Diabetes Mellitus cases,

[0030] FIG. 3 shows the decrease in post prandial blood glucose level with the herbal formulation in the case of Type 2 Diabetes Mellitus cases,

[0031] FIG. 4 shows the decrease in Interleukin 6 with the herbal formulation in Type 2 Diabetes Mellitus cases,

[0032] FIG. 5 depicts the effect of herbal composition on CRP level in of Type 2 Diabetes Mellitus cases,

[0033] FIG. 6 depicts the decrease in inflammatory biomarker following herbal formulation in treatment of Type 2 Diabetes Mellitus cases,

[0034] FIG. 7 shows the beneficial role of the herbal formulation on endothelin level among Type 2 Diabetes Mellitus cases,

[0035] FIG. 8 shows decrease in Apolipoprotein B following herbal formulation treatment in Type 2 Diabetes Mellitus cases,

[0036] FIG. 9 depicts the beneficial role of the herbal formulation in decreasing the Homocysteine level among Type 2 Diabetes Mellitus cases,

[0037] FIG. 10 shows the decrease in the Leptin level after the treatment of herbal drug formulation in Type 2 Diabetes Mellitus cases,

[0038] FIG. 11 shows the modulation of adiponectin after the administration of the herbal formulation in Type 2 Diabetes Mellitus cases.

DETAILED DESCRIPTION OF THE INVENTION

[0039] The hydro-alcoholic extract of two Ayurvedic plants Salacia roxburghii and Salacia oblonga by using 30:70 ratio of water and ethanol 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.

[0040] 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, homocysteine etc.

[0041] The pre-clinical toxicological studies were carried out to determine the safety profile of individual plant candidate as well as all two 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.

[0042] The beneficial role of test formulation on fasting and postprandial blood glucose level, α-glucosidase inhibitory activity, 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.

Extraction Procedure:

[0043] The shed dried root of Salacia roxburghii and Salacia oblonga were utilized for obtaining extracted material of the plants. The water and ethanol 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.

[0044] The extraction was done at the temperature of 70-80C. The pH of the solution was maintained between 7-10. FIG. 1 shows the steps followed and carried out to isolate the active compound and preparation of test drug.

[0045] According to this invention there is provided an Ayurvedic formulation for the prevention and management of Type 2 diabetes and associated cardiac risk factors with the object to prevent the diabetic patients from cardiac death and also from various type of morbidity caused due to impaired glucose tolerance. The present test formulation comprising of the following two ingredients—

TABLE-US-00001 1. Salacia roxburghii - Root 2. Salacia oblonga - Root

[0046] Preferably the aforesaid plants are present in the test drug in following doses—

TABLE-US-00002 Name of the plants Dose 1. Salacia roxburghii 450-900 mg/day 2. Salacia oblonga 400-900 mg/day

[0047] The formulation may also comprise known additives such as minerals, vitamins, salts, filler (for capsulation or tablet) and binders, if required to present in trace amount.

[0048] 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 tablet.

[0049] Preferably but without implying any limitation the preparation comprises—

TABLE-US-00003 Name of the plants Dose 1. Salacia roxburghii 450 mg/day 2. Salacia oblonga 450 mg/day

[0050] The present herbal formulation is based on the combined effect of two plant extract namely Salacia roxburghii and Salacia oblonga. This novel formulation has shown α-glucosidase inhibitory activity, PPAR agonist activity, modified abnormal lipids including triglyceride, reduced inflammatory cytokines, regulates adipokines, improves cardiac functions. Those effect are mediated through alpha-glucosidase and amylase inhibition including reduction in lipid lipase concentration, PPAR agonist 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, decreases leptin level and enhances adiponectin. All these beneficial effects ultimately slowed down/improved the atherosclerotic process among diabetes patients and has potential role in prevention of adverse cardiac event.

[0051] Salacia roots modulate multiple targets like peroxisome proliferator-activated receptor-alpha-mediated lipogenic gene transcription, angiotensin II/angiotensin II type 1 receptor, alpha-glucosidase, aldose reductase and pancreatic lipase. These multi-target actions may mainly contribute to Salacia root-induced improvement of Type 2 diabetes and obesity-associated hyperglycemia, dyslipidemia and related cardiovascular complications seen in humans and rodents. The results of bioassay-guided identification indicate that mangiferin, salacinol, kotalanol and kotalagenin 16-acetate are at least in part responsible for these multi-target regulatory activities of Salacia roots. The evidence suggests that this unique traditional medicine fulfils a multiple-target strategy in the prevention and treatment of diabetes and obesity. Although toxicological studies have suggested minimal adverse effects of the herbal medicine in rodents, a clinical trial is crucial to further confirm the safety of Salacia roots. In addition, further mechanistic studies are necessary in order to allow a better understanding of how use of Salacia root may interact with other therapeutic interventions. Salacia oblonga containing various active components have been found to meet multiple targets in diabetes and obesity through modulating PPAR-α mediated lipogenic gene transcription and AT1 signalling and inhibiting α-glucosidase

[0052] As diabetes increases the risk of myocardial infarction and risk of stroke, therefore the CHD risk factors particularly lipid abnormalities must be treated aggressively to reduce the risk of atherosclerosis. Further, it is reported that α-glucosidase inhibitors do not increase insulin level and their main effect is to lower postprandial blood glucose, prevent from weight gain. It is hypothesized that present test formulation improves the glycemic load by improving insulin sensitivity, as a PPAR-α agonist, stimulates beta-cells, increases glucose uptake in tissues, may have activity on cathepsin and increases cyclic AMP count on islets. As synergism test drug has antioxidant effects.

[0053] In the present study, treatment with test formulation significantly reduced the level of homocysteine indicating increased endothelium dependent flow-mediated dilation in patient with Type 2 diabetes mellitus. Atherosclerosis occurs due to a number of factors in diabetic individuals. Both insulin resistance and elevated lipid levels are the triggering factors for atherogenic 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, HDL-c and triglyceride is the major risk factor for CHD. Therefore, the prevention and management of risk factors of cardiovascular is the basic and essential principle among diabetes patients to prevent them from cardiac arrest.

About the Plants:

[0054] 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 is a member of the Hippocrastaceae family commonly known as “saptarangi” due to its golden colored root bark. It is widely distributed in south region of India and Sri Lanka. Salacia oblonga is a perennial wild, woody, climbing plant found in foot hills of the mountain of Eastern and Western Ghats of Tamilnadu and Kerla. The seed of Salacia oblonga is light brown and orange in colour and its fruits are globose and tuberculata. In Ayurvedic system of Medicine it has been used as a treatment of diabetes. This is due to salacia's “mangiferin” component, which inhibits sugar absorption and also increases the body's sensitivity to insulin. The roots and stems of Salacia oblonga have been used as a treatment for diabetes in the ayurvedic system of medicine. A highly potent α-glucosidase inhibitor, kotalanol 1, has now been isolated from this plant. One of the conventional therapeutic approaches to diabetes management is through the use of alpha-glucosidase inhibitors that lower glucose levels by blocking the enzymes that digest starches in the intestines.

EXAMPLES

Example-I

[0055] When the hydroalcoholic extract of Salacia roxburghii (100 mg/kg) was given to Sprague Dawley rats 46 percent α-glucosidase inhibitory activity was recorded. Further, amylase and lipid lipase lowering effects were also noticed.

Example-II

[0056] When the hydro-alcoholic extract of Salacia roxburghii (100 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-III

[0057] When the hydro-alcoholic extract of Salacia roxburghii in the dose of 100 mg/kg was given to streptozotocin induced diabetic rats, the drugs exerted agonist for PPAR activated receptors resulting in insulin regulated gene transcription.

Example-IV

[0058] When the organic extract of Salacia roxburghii in the dose of 900 mg/day (in two divided doses) was given 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

[0059] When the hydro-alcoholic extract of Salacia roxburghii (475 mg/day) and Salacia oblonga (450 mg/day) was mixed and given to Type 2 diabetes patients a marked decrease in triglycerides content was estimated along with blood glucose lowering property of the test formulation.

Example-VI

[0060] When the hydro-alcoholic extract of Salacia oblonga (525 mg/day) and Salacia roxburghii (425 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

[0061] When the hydro-alcoholic extract of Salacia oblonga (550 mg/day) and Salacia roxburghii (350 mg/day) mixed and given to diagnosed cases of Type 2 diabetes subjects the oxidized LDL-c level decreased indicating improvement in atherosclerotic process among Type 2 diabetes patients.

Example-VIII

[0062] When the hydro-alcoholic extract of Salacia roxburghii (575 mg/day) and Salacia oblonga (350 mg/day) was mixed and given to diabetes patients decrease in proinflammatory markers like IL-6, TNF-α including hs.CRP was estimated indicating improvement in vascular inflammation in diabetes cases.

Example-IX

[0063] When the hydro-alcoholic extract of Salacia roxburghii (450 mg/day) and Salacia oblonga (450 mg/day) was mixed and administered to selected diabetes patients increase in adiponectin and decrease in leptin level indicated the anti-atherosclerotic and antiobesity role of test formulation.

Example-X

[0064] When the hydro-alcoholic extract of Salacia roxburghii (625 mg/day) and Salacia oblonga (325 mg/day) mixed and given to diabetes patients the elevated homocysteine level markedly decreased following treatment. This study indicated the anti-oxidant activity of this combined drug.

Example-XI

[0065] When the organic extract of Salacia roxburghii (625 mg/day) and Salacia oblonga (325 mg/day) mixed and orally administered to selected Type 2 diabetes patients a better effectivity of test formulation was recorded. The blood glucose level is 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 and elevated Hcy decreased. This novel combined formulation has shown potential role in the prevention and management of cardiovascular risk factors among diabetic patients through regulation of PPAR-α and PPAR-7 and also due to its aglucosidase inhibitory activity resulting in control of glycemic index and enhancing insulin sensitivity. Thus it is proposed as a better safer remedial measure for the management of Type 2 diabetes mellitus patients.

[0066] 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.

TABLE-US-00004 Alpha-glucosidase inhibitory activity of single as well as combined formulation Percent inhibition Concentration Salacia Salacia Combined (μg/ml) roxburghii oblonga formulation  50 mg/kg 25.88 22.36 29.75 100 mg/kg 41.82 42.29 58.55 150 mg/kg 44.6 32.21 63.94

Experimental Study-I

[0067]

TABLE-US-00005 TABLE I-2 Reduction in blood glucose level following test drug treatment in Streptozotocin (STZ) treated diabetic rats Blood glucose level (mg/d ) Treated group 7.sup.th Days 14.sup.th Days 28.sup.th Days Normal control (N = 6) * 58.36 ± 8.24 57.25 ± 9.31  59.24 ± 11.27 Td. With STZ (65 mg/kg) 328.68 ± 26.97 285.60 ± 25.65 255.45 ± 34.69 N = 6 ** Td. With STZ + Test drug 247.49 ± 21.37 205.32 ± 24.43 165.80 ± 19.27 (N = 6)*** Td. With STZ + 228.33 ± 19.44 192.74 ± 15.18 157.51 ± 10.24 Acarbose**** 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-US-00006 TABLE I-2 Reduction in hs C-reactive protein following test drug treatment in STZ treated diabetic rats hs CRP (mg/L) Treated group 7.sup.th Days 14.sup.th Days 28.sup.th Days Normal control (N = 6) * 1.45 ± 0.35 1.36 ± 0.28 1.25 ± 0.39 Td. With STZ (65 mg/kg) 8.26 ± 2.36 7.65 ± 2.08 5.36 ± 1.67 N = 6 ** Td. With STZ + Test drug 6.27 ± 1.57 5.27 ± 0.82 3.97 ± 0.72 (N = 6)*** Td. With STZ + 6.78 ± 1.52 5.43 ± 0.75 4.97 ± 0.87 Acarbose**** 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-US-00007 TABLE I-3 Reduction in Interleukin-6 following test drug treatment in STZ treated diabetic rats IL-6 (pg/ml) Treated group 7.sup.th Days 14.sup.th Days 28.sup.th Days Normal control (N = 6) * 0.79 ± 0.12 0.89 ± 0.12 0.82 ± 0.18 Td. With STZ (65 mg/kg) 4.05 ± 1.10 3.84 ± 0.97 3.27 ± 0.79 N = 6 ** Td. With STZ + Test 2.58 ± 0.27 2.12 ± 0.32 1.60 ± 0.24 drug (N = 6)*** Td. With STZ + 2.87 ± 0.25 2.49 ± 0.32 2.23 ± 0.26 Acarbose**** 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-US-00008 TABLE I-4 Effect of Herbal formulation on adiponectin level in STZ treated diabetic rats Treated group Adiponectin (μg/ml) 7.sup.th Days 14.sup.th Days 28.sup.th Days Normal control (N = 6)* 13.28 ± 1.90 14.36 ± 1.98 14.40 ± 2.08 Td. With STZ  6.79 ± 1.12  5.75 ± 0.98  5.11 ± 0.85 (65 mg/kg) N = 6 ** Td. With STZ + Test  9.27 ± 1.98 10.22 ± 1.69 12.63 ± 2.08 drug (N = 6)*** Td. With  8.87 ± 2.10  9.65 ± 1.63 10.23 ± 2.14 STZ + Acarbose ****

TABLE-US-00009 TABLE II-3 Role of Herbal formulation on body weight following cafeteria diet in experimental rats Comp. Initial Body Weight (grams) vs After 30 Treated group Initial After 15 Days After 30 Days days Normal control 104.48 ± 3.75 109.68 ± 6.22 116.25 ± 4.93 P < 0.001 (N = 6) Cafeteria diet only  98.28 ± 4.35 127.58 ± 6.14  156.83 ± 11.20 P < 0.001 (N = 6) Cafeteria diet + test 112.63 ± 9.43 118.26 ± 8.11 125.67 ± 7.52 P < 0.001 formulation (N = 6)

Experimental Study-II

[0068]

TABLE-US-00010 TABLE-II-2 Effect of herbal formulation on total cholesterol and triglycerides following cafeteria dies in experimental animals TC (mg/dl) Comp. TG (mg/dl) Comp. After Initial vs After Initial vs 30 After 30 30 After 30 Treated group Initial Days days Initial Days days Normal control 82.68 ± 86.38 ± P > 0.05 81.75 ± 82.23 ± P > 0.05 (N = 6)  4.36  5.27  7.98  9.28 Cafeteria diet 86.21 ± 90.46 ± P < 0.05 78.58 ± 94.25 ± P < 0.05 only (N = 6)  6.43  8.95  5.73  4.65 Cafeteria diet + 84.74 ± 80.37 ± P < 0.05 82.15 ± 73.48 ± P < 0.05 test formulation  7.98  5.93  8.73  7.29 (N = 6)

TABLE-US-00011 TABLE-II-3 Effect of Herbal formulation on blood glucose and adiponectin level following cafeteria dies in experimental rats Blood glucose Comp. Adiponectin Comp. level (mg/dl) Initial vs (pg/ml) Initial vs Treated After 30 After 30 After After 30 group Initial Days days Initial 30 Days days Normal 59.85 ± 57.70 ± P > 0.05 11.78 ± 12.264 ± P > 0.05 control  6.29  6.38  1.92  2.16 (N = 6) Cafeteria 55.67 ± 72.28 ± P < 0.001  6.54 ±  7.85 ± P < 0.001 diet only  6.08  5.97  0.97  1.10 (N = 6) Cafeteria 58.28 ± 65.36 ± P < 0.01  7.82 ±  10.17 ± P < 0.01 diet + test  6.27  4.85  1.03  1.84 formulation (N = 6)

TABLE-US-00012 TABLE II-4 Role of Herbal 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 (N = 10)* 65.42 ± 7.79 64.87 ± 6.48 65.65 ± 8.37 High cholesterol diet 654.31 ± 50.24 887.29 ± 48.37 482.49 ± 38.58 (N = 10)** High cholesterol diet + 567.97 ± 78.29 658.36 ± 72.48 405.28 ± 46.15 (N = 10)*** test formulation High cholesterol diet + 691.52 ± 78.85 691.52 ± 78.85 280.50 ± 16.80 statin (2.5mg/kg/day) (N = 10)**** Comp. *vs** P > 0.05 P < 0.001 P < 0.001 **vs*** P < 0.001 P < 0.001 P < 0.001 ***vs**** P < 0.001 P < 0.001 P < 0.001

TABLE-US-00013 TABLE II-5 Effect of Herbal 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 (N=10)* 22.25 ± 4.30 23.12 ± 2.68 22.12 ± 3.49 High cholesterol diet — 16.39 ± 4.85 12.97 ± 1.68 (N = 10)** High cholesterol diet + — 17.58 ± 2.46 19.46 ± 2.72 test formulation (N = 10)*** High cholesterol diet + — 20.24 ± 4.76 21.43 ± 3.25 statin (2.5 mg/kg/day) (N = 10)**** 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-US-00014 TABLE II-6 Effect of Herbal 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 23.74 ± 4.59 22.27 ± 5.12 23.75 ± 6.19 (N = 10)* High cholesterol diet 298.65 ± 62.34 328.17 ± 58.27 312.33 ± 46.27 (N = 10)** High cholesterol diet + 197.32 ± 34.18 265.18 ± 39.11 137.98 ± 29.10 test formulation (N = 10)*** High cholesterol diet + 168.34 ± 24.36 253.68 ± 36.45 107.78 ± 16.34 statin (2.5 mg/kg/day) (N = 10)**** Comp. *vs** P > 0.05 P < 0.001 P < 0.001 **vs*** P < 0.05 P < 0.05 P < 0.001 ***vs**** P < 0.05 P > 0.05 P < 0.05

TABLE-US-00015 TABLE II-7 Effect of Herbal formulation on Triglycerides level among high cholesterol diet treated rats Tr glycerides level (mg/dl) Treated group Initial After 15 Days After 1 months Normal control (N = 10)* 27.82 ± 8.65 30.64 ± 7.73 28.22 ± 5.47 High cholesterol diet 301.68 + 59.12 338.67 ± 63.75 297.12 ± 38.48 (N = 10)** High cholesterol diet + 201.65 ± 49.37 242.17 ± 59.35 187.90 ± 26.87 test formulation (N = 10)*** High cholesterol diet + 195.34 ± 19.67 227.65 ± 30.12 113.63 ± 18.84 statin (2.5 mg/kg/day) (N = 10)**** Comp. *vs** P > 0.05 P < 0.001 P < 0.001 **vs*** P < 0.05 P < 0.05 P < 0.01 ***vs**** P < 0.05 P > 0.05 P < 0.05

Clinical Study

[0069]

TABLE-US-00016 TABLE-CS-1 Effect of Herbal formulation on Body Mass Index among Type 2 diabetes mellitus cases Comp. Body Mass Index Initial vs No. of 6 Months 1 Year After 1 year Treatment sex Cases Initial Treatment Treatment treatment Conventional M 61 31.62 ± 30.71 ± 29.62 ± P < 0.001 Drug Treated  3.01  3.04  3.01 F 38 29.26 ± 28.97 ± 28.04 ± P < 0.001  2.94  2.76  2.16 Conventional M 72 29.75 ± 28.63 ± 27.73 ± P < 0.001 Drug +  2.89  2.95  2.90 Herbal F 43 30.26 ± 29.63 ± 27.68 ± P < 0.001 Formulation  2.83  2.82  2.15

TABLE-US-00017 TABLE CS-2 Beneficial Role of Herbal Formulation on Fasting Blood Glucose Level among Type 2 Diabetes Mellitus Cases Fasting Blood Glucose Level Comp. No. (mg/dL) Initial vs of 6 Months 1 Year After 1 year Treatment sex Cases Initial Treatment Treatment treatment Conventional M 61 148.45 ± 121.91 ± 116.75 ± P < 0.001 Drug Treated  29.82  23.73  21.41 F 38 143.75 ± 124.87 ± 121.92 ± P < 0.001  20.84  18.66  20.43 Conventional M 72 183.75 ± 117.34 ± 110.45 ± P < 0.001 Drug +  24.91  19.28  14.77 Herbal F 43 154.73 ± 122.92 ± 108.73 ± P < 0.001 Formulation  23.47  21.03  16.85 Normal range: 70-120 mg/dl

TABLE-US-00018 TABLE CS-3 Decrease in Post Prandial Blood Glucose Level Following Herbal Formulation Treatment in Type 2 Diabetes Mellitus Cases Post Prandial Blood Glucose Comp. No. Level (mg/dL) Initial vs of 6 Months 1 Year After 1 year Treatment Cases Initial Treatment Treatment treatment Conventional M 61 314.91 ± 188.72 ± 169.42 ± P < 0.001 Drug Treated  41.68  30.91  33.71 F 38 331.83 ± 204.87 ± 178.91 ± P < 0.001  45.62  40.11  38.92 Conventional M 72 329.75 ± 178.45 ± 158.74 ± P < 0.001 Drug +  44.90  33.77  40.82 Herbal F 43 334.12 ± 182.96 ± 164.91 ± P < 0.001 Formulation  32.64  30.11  29.42 Normal range: <140 mg/dl

TABLE-US-00019 TABLE CS-4 Decrease in Interleukin 6 Following Herbal Formulation Treatment in Type 2 Diabetes Mellitus Cases Comp. No. Interleukin 6 (pg/ml) Initial vs of 6 Months 1 Year After 1 year Treatment Cases Initial Treatment Treatment treatment Conventional M 61 1.34 ± 1.17 ± 1.14 ± P < 0.001 Drug Treated 0.20 0.18 0.21 F 38 1.08 ± 1.29 ± 1.22 ± P < 0.05 0.24 0.18 0.31 Conventional M 72 1.42 ± 1.13 ± 0.87 ± P < 0.001 Drug + 0.23 0.34 0.22 Herbal F 43 1.34 ± 0.96 ± 0.81 ± P < 0.001 Formulation 0.41 0.37 0.22 Normal range: <1 pg/ml

TABLE-US-00020 TABLE CS-5 Effect of Herbal Formulation on CRP Level in Type 2 Diabetes Mellitus Cases Comp. No. CRP Level (mg/L) Initial vs of 6 Months 1 Year After 1 year Treatment Cases Initial Treatment Treatment treatment Conventional M 61 2.84 ± 2.91 ± 2.58 ± P < 0.05 Drug Treated 0.70 0.65 0.66 F 38 3.11 ± 2.97 ± 2.93 ± P > 0.05 0.45 0.34 0.39 Conventional M 72 3.21 ± 2.58 ± 2.13 ± P < 0.001 Drug + 0.42 0.39 0.29 Herbal F 43 2.93 ± 2.18 ± 1.73 ± P < 0.001 Formulation 0.42 0.41 0.32 Normal range: 1-3 mg/L

TABLE-US-00021 TABLE CS-6 Decrease in Inflammatory Biomarker TNF□ Following Herbal Formulation Treatment in Type 2 Diabetes Mellitus Cases Comp. No. TNF-□ (pg/mL) Initial vs of 6 Months 1 Year After 1 year Treatment Cases Initial Treatment Treatment treatment Conventional M 61 162.41 ± 157.39 ± 159.75 ± P > 0.05 Drug Treated  30.13  38.44  33.91 F 38 171.20 ± 168.96 ± 173.75 ± P > 0.05  25.73  31.04  35.08 Conventional M 72 168.97 ± 134.73 ± 121.91 ± P < 0.001 Drug +  31.64  25.13  18.99 Herbal F 43 182.73 ± 148.96 ± 136.94 ± P < 0.001 Formulation  38.11  29.75  23.84 Normal range: 25-800 pg/ml

TABLE-US-00022 TABLE CS-7 Beneficial Role of the Herbal Formulation on Endotheline Level among Type 2 Diabetes Mellitus Cases Comp. No. Endotheline (pg/mL) Initial vs of 6 Months 1 Year After 1 year Treatment Cases Initial Treatment Treatment treatment Conventional M 61 613.91 ± 590.84 ± 578.94 ± P < 0.01 Drug Treated  63.54  71.90  73.84 F 38 438.75 ± 416.22 ± 387.81 ± P < 0.001  41.97  53.17  58.27 Conventional M 72 578.89 ± 409.64 ± 373.91 ± P < 0.001 Drug +  64.34  52.14  35.87 Herbal F 43 641.93 ± 538.87 ± 413.92 ± P < 0.001 Formulation  51.34  48.90  58.25 Normal range: 0.32-1000 pg/ml

TABLE-US-00023 TABLE CS-8 Decrease in Apolipoprotein B Following Herbal Formulation Treatment in Type 2 Diabetes Mellitus Cases Comp. No. Apolipoprotein B (mg/dL) Initial vs of 6 Months 1 Year After 1 year Treatment Cases Initial Treatment Treatment treatment Conventional M 61 145.97 ± 151.39 ± 148.92 ± P > 0.05 Drug Treated  30.21  25.97  26.82 F 38 161.31 ± 173.14 ± 164.45 ± P > 0.05  25.75  28.72  21.87 Conventional M 72 168.99 ± 145.90 ± 132.78 ± P < 0.001 Drug +  32.42  25.82  30.01 Herbal F 43 158.72 ± 124.84 ± 116.72 ± P < 0.001 Formulation  27.08  24.88  17.91 Normal range: 55-159 mg/dl

TABLE-US-00024 TABLE CS-9 Beneficial Role of the Herbal Formulation in Decreasing the Homocysteine Level among Type 2 Diabetes Mellitus Cases Comp. No. Homocysteine (mmol/L) Initial vs of 6 Months 1 Year After 1 year Treatment Cases Initial Treatment Treatment treatment Conventional M 61 38.79 ± 37.24 ± 37.91 ± P > 0.05 Drug Treated  4.11  3.89  4.24 F 38 35.82 ± 36.14 ± 36.82 ± P > 0.05  6.31  5.91  5.32 Conventional M 72 41.02 ± 33.71 ± 27.94 ± P < 0.001 Drug +  4.92  4.06  3.75 Herbal F 43 37.85 ± 31.04 ± 26.42 ± P < 0.001 Formulation  5.17  3.21  3.81 Normal range: 5-15 mmol/L

TABLE-US-00025 TABLE CS-10 Decrease in Leptin level following test drug treatment in Type 2 Diabetes Mellitus Cases Comp. No. Leptin(μgm/L) Initial vs of 6 Months 1 Year After 1 year Treatment Cases Initial Treatment Treatment treatment Conventional M 61 23.74 ± 21.92 ± 20.85 ± P < 0.001 Drug Treated  3.11  2.87  2.93 F 38 26.11 ± 24.93 ± 23.40 ± P < 0.001  4.01  4.11  3.05 Conventional M 72 25.83 ± 20.75 ± 14.82 ± P < 0.001 Drug +  3.14  3.28  2.64 Herbal F 43 28.93 ± 23.91 ± 19.87 ± P < 0.001 Formulation  3.21  2.94  3.01 Normal range: 5-12 μgm/L

TABLE-US-00026 TABLE CS-11 Mmodulation of adiponectin following Herbal formulation treatment in Type 2 Diabetes Mellitus Cases Comp. No. Adiponectin(μg/ml) Initial vs of 6 Months 1 Year After 1 year Treatment Cases Initial Treatment Treatment treatment Conventional M 61 16.88 ± 17.82 ± 17.20 ± P > 0.05 Drug Treated  3.03  2.61  2.72 F 38 13.84 ± 14.64 ± 14.82 ± P > 0.05  2.71  3.11  2.84 Conventional M 72 15.35 ± 18.11 ± 23.91 ± P < 0.001 Drug +  2.62  4.01  3.73 Herbal F 43 12.96 ± 16.35 ± 19.11 ± P < 0.001 Formulation  3.09  2.87  2.93 Normal range: 5-30 μg/ml