SCIENTIFIC STUDIES
A. PRE-CLINICAL ANIMAL STUDIES
1. GLUCOSE TOLERANCE TEST (OGTT)
Conducted By: Amala Cancer Research Centre,Kerala
Results: Study confirmed that COGENT DB+ possess hypoglycemic property.
Wistar rats were divided into 3 Groups (Groups 1,2 and 3), each group consisted of 5 males and 5 females. Initial blood sugar was determined in all animals. All Groups were given glucose (2g/kg.b.w.) orally. Thirty minutes following glucose loading, Group 2 received COGENT DB+ orally at 500 mg/kg/b.w., whereas Group 3 received COGENT DB+ orally at 2500 mg/kg b.w. group 1, which was treated as control. Received only distilled water.
Glucose was determined in the blood of all animals of Groups 2 and 3 collected at 30 and 90 min following the administration of COGENT DB+. Glucose was determined in the blood of control animals (Group 1) also at these time points. Administration of COGENT DB+ at 2500 mg/kg b.w. decreased blood sugar at 30 and 90 m in. At 90 min. The blood sugar level reached the level of the initial sugar level, confirming that COGENT DB+ is a hypoglycemic agent.
2. EFFECT ON ALLOXAN INDUCED DIABETIC RATS
Conducted by: Amala Cancer Research Centre, Kerala.
Results: Study confirmed that COGENT DB+ is a hypoglycemic agent and also has the ability to decrease the levels of glycated haemoglobin associated with hyperglycemia.
Wistar rats were divided into 5 Groups (Groups 1,2,3,4 and 5) each group consisted of 6 animals. Groups 3,4 and 5 were treated intraperitoneally with alloxan at 120 mg/kg b.w. Five days prior to alloxan treatment Groups 4 and 5 received COGENT DB+ orally at 500 and 1000 mg/kg b.w., respectively. These animals continued to receive COGENT DB+, daily for 10 consecutive days. Similarly, Group 2 also received COGENT DB+ at 1000 mg/kg b.w. for 10 days. Group 1, which was treated as control, received only distilled water. Glucose was determined in the blood of all animals of all Groups on days 0 (prior to alloxan treatment) 3,6, and 10. Animals treated with COGENT DB+ alone at 1000 mg/kg b.w. did not show any change in blood sugar level compared to the control. COGENT DB+ at both the dose levels reverted hyperglycemic condition and increased level of glycated haemoglobin induced by alloxan administration on day 10, to normal, confirming that COGENT DB+ is a hypoglycemic agent and also has the ability to decrease the levels of glycated haemoglobin associated with hyperglycemia.
3. EFFECT ON ALLOXAN INDUCED DIABETIC RATS
Publication: Comp. Biochem. Physiol, C131,19-25(2002)
Results: OGTT performed in experimental diabetic animals showed that there was a significant improvement in glucose tolerance in rats treated with COGENT DB+.
Oral administration of 150, 300 and 450 mg/kg b.w. of the aqueous solution of COGENT DB+ for 40 days exhibited a significant reduction in blood glucose, glycated haemoglobin and increased plasma insulin, total haemoglobin along with anti-hyperlipidemic effects in diabetic rats. It also prevented body weight loss. OGTT performed in experimental diabetic animals showed that there was a significant improvement in glucose tolerance in rats treated with COGENT DB+. A comparison made with glibenclamide (600 micrograms/kg b.w.) revealed that antidiabetic effect of COGENT DB+ was better than that of glibenclamide.
4. EFFECT ON SERUM AND LIPID METABOLISM IN ALLOXAN INDUCED DIABETIC RATS
Publication: Diabetes, Obesity Metabol, 5, 1-7 (2003)
Results: COGENT db+ exhibits a strong antihyperlipidemic effect, which could exert a beneficial action against macrovascular complications associated with diabetes mellitus.
Alloxan diabetic male Wistar rats were divided into 3 groups (6 animals/group)diabetic control, diabetic rats given COGENT DB+ at 450 ng/kg b.w. diabetic rats given glibenclamide (600 microgram/kg b.w.). In addition to the above groups, a control group( 6 animals) was maintained. Oral administration of COGENT DB+ for 40 days resulted in significant reduction in blood glucose, serum and tissue (liver and kidney) lipids, whereas the activity of hepatic lipogenic enzymes were significantly increased in alloxan diabetic rats. The effect of COGENT DB+ was similar to that of glibenclamide.
From the above study, the authors concluded that COGENT DB+ exhibits a strong antihyperlipidemic effect, which could exert a beneficial action against macrovascular complications associated with diabetic mellitus.
5. EFFECT ON SERUM AND PLASMA INSULIN AND HEPATIC ENZYMES OF GLUCOSE METABOLISM IN ALLOXAN INDUCED DIABETIC RATS.
Publication: Diabetes, Obesity Metabol, 4, 1-5(2002)
Results: COGENT DB+ controls the blood glucose by increasing glycolysis and decreasing gluconeogenesis with a lower demand of pancreatic insulin.
Alloxan diabetic male Wistar rats were divided into 3 groups (6 animals/ group)-diabetic control, diabetic rats given COGENT DB+ at 450 mg/kg b.w., diabetic rats given glibenclamide (600 microgram/kg b.w.). In addition to the above groups, a control group (6 animals) was maintained.
Oral administration of COGENT DB+ for 40 days resulted in significant reduction in blood glucose and the activities of glucose-6- phosphatase and fructose-1,6-phosphatase in the liver, whereas the level of plasma insulin and hepatic hexokinase activity were significantly increased in alloxan diabetic rats. The effect of COGENT DB+ was similar to that of glibenclamide.
From the above study, the authors concluded that COGENT DB+ controls the blood glucose by increasing glycolysis and decreasing gluconeogenesis with a lower demand of pancreatic insulin.
6. EFFECT ON MUTANT OBESE RATS (WNIN/GR-OB) WITH IMPAIRED GLUCOSE TOLERANCE
Conducted By: National Institute of Nutrition, Hyderabad
Results: Study confirmed that COGENT db+ is a hypoglycemic and hypocholesterolemic agent.
WNIN/GR-Ob strains of rats are mutant, obese and glucose intolerant. They show obesity, hyperglycemia and hyperlipidemia by 35-50 days of age. Groups of the rats (3 males and 3 females per group) received distilled water (control). COGENT DB+ at 100 or 500 mg/kg b.w. daily by oral gavage for 30 consecutive days. Then the animals were maintained without any treatment for 30 days.
Compared to control animals blood glucose and plasma cholesterol levels decreased in animals treated with COGENT DB+ at 100 and 500 mg/kg b.w. at the end of 30 days. This decrease in blood glucose and plasma cholesterol levels was maintained even after the withdrawal of the treatment with COGENT DB+. It was also observed that there was an overall improvement of activity in the animals treated with COGENT DB+.
7. DOES COGENT DB+ AUGMENT HYPOGLYCEMIC CONDITION INDUCED BY INSULIN?
Conducted By: International Institute of Biotechnology and Toxicology, Chennai
Results: Study confirmed that COGENT DB+ does not cause hypoglycemia.
Male Himalayan albino rabbits were divided into 4 Groups. Group 1 received insulin at 2 units/kg. Subcutaneously. Group 2 received insulin at 2 units/kg. Subcutaneously. Followed by COGENT DB+ at 1000 mg/ kg. b.w. orally. Group 3 received COGENT DB+ along oat 1000 mg/kg ob.w. Orally. Group 4 was treated as the control. Blood was collected from individual animals prior to the treatments (0 hour) and at the end of 1,2 and 5 hours following the treatments for determination of glucose.
Group 1, which received insulin alone showed a decrease of 68% glucose at 1 hour and 46% at 2 hour, compared to 0 hour value. At 5 hours, the glucose level reached the level of 0 hour value. Group 2, which received COGENT DB+ after the insulin injection showed a decrease of 67% glucose at 1 hour and 41% at 2 hour, compared to 0 hour value. At 5 hours the glucose level reached the level of 0 hour value. These findings indicate that COGENT DB+ does not augment decrease in blood glucose, when administered with insulin. Animals treated with COGENT DB+ alone did not show any change in blood glucose levels at 0, 1,2 and 5 hours compared to 0 hour value, proving that COGENT DB+ does not cause hypoglycemia.
8. ACUTE ORAL TOXICITY IN WISTAR RATS
Conducted By: International Institute of Biotechnology and Toxicology, Chennai
Results: Study confirmed that COGENT DB+ was safe to Wistar rats even at 6000 mg/kg b.w. by oral route.
The tablets were powdered, weighed, mixed with distilled water and then given to overnight fasted groups of rats (5/sex/group) by single oral administration at the doses of 0 (control group, which received distilled water alone), 1500, 3000 and 6000 mg/kg bw. The rats were observed daily for signs and mortality for 14 days. Weekly body weight of individual animals was measured. At the end of the 14 days observation period all animals were necropsied.
Animals of none of the groups exhibited any sign. No mortality was observed in any group of animals. All the groups of animals had similar body weight gain. Gross pathology did not reveal any treatment related lesions.
It is concluded from the study that COGENT DB+ was safe to Wistar rats even at 6000 mg/kg.
9. 90-DAY REPEATED DOSE ADMINISTRATION STUDY TO EVALUATE TOXICITY
Conducted By: Toxicology Unit, Tamilnadu Veterinary and Animal Sciences University, Chennai
Wister rats were randomly distributed into 6 groups (10 rats/sex/group), viz., Group 1 (Control), Group 2 (Low dose group), Group3 (Intermediate dose group), Group 4 (High dose group), Group 5 (Control reversal group) and Group 6 (High dose reversal group). To groups 2, 3 and 4, COGENT DB+ was given at the doses of 250, 500 and 1000 mg/kg b.w., respectively, by oral intubation for 90 consecutive days. Rats belonging to group 1 were treated with distilled water alone. Rats belonging to Group 5 and Group 6 received distilled water and COGENT DB+ at 1000 mg/kg b.w., respectively.
Inlife measurements included weekly body weight change and feed consumption. Daily observations were made on clinical signs of individual rats. Blood samples were collected from individual rats of all groups on day 91 for haematological and biochemical investigations. On day 119, blood samples were collected from the reversal groups. Following blood collection rats are collected from the reversal groups. Following blood collection rats longing to groups 1-4 were sacrificed on day 91 and those belonging to groups 5-6 were sacrificed on day 119. All animals were subjected to gross ethological examination. Weight of the brain, heart, liver, kidneys, spleen, and gonads of individual rats was measured. Tissues of organs were processed for histopathology
Animals of none of the groups exhibited any signs throughout the observation period. No mortality was observed in any group of animals. All the groups +animals had similar body weight gain and feed consumption. There was significant difference in the haematological (RBC, WBC, haemoglobin, hematocrit (HCT), differential leucocyte count, thrombocyte count and coagulation time) and biochemical parameters (protein, albumin, globulin, alanine aminotransferase (ALT), alkaline phosphatase (ALP) and blood urea nitrogen (BUN) of the COGENT DB+ treated groups, when compared with the control group and the values were within their normal limits. Gross and histopathology did not reveal any treatment related lesions. Similarly, the high dose reversal group and control reversal group did not exhibit any sign during the post-treatment observation period (day 91-day 118). During this period both these groups showed similar body weight gain and feed consumption. Hematological and biochemical parameters estimated at the end of the post-treatment observation period were similar in both the groups. Reversal groups did not show any abnormal gross.
10. MUTAGENICITY EVALUATION BY DOMINANT LETHAL TEST IN MOUSE
Conducted By: International Institute of Biotechnology and Toxicology, Chennai
Results: COGENT DB+ was considered non-mutagenic by the dominant lethal assay
The ability of COGENT DB+ to induce any cytotoxic or mutagenic effect on male germinal cells was evaluated by assessing the dominant lethality in the progeny of male mice (Swiss albino strain) orally administered with the test substance in distilled water at 3000, 2000 and 1000 mg/kg b.w. Animals in the negative control group received distilled water alone. A separate positive control group was also maintained in which the animals receive a single peritoneal injection of triethylenemelamine at 0.2 mg/kg b.w. For 5 consecutive days, the mice from all the groups were mated with 3 untreated virgin females every week in the ratio of (male:female) for a period of 8 weeks to cover the entire spermatogenesis cycle. Pregnant mice were sacrificed on day 14 of gestation and the uterine contents were examined for live/dead implants and induced dominant lethality was calculated.
Females mated with males treated with COGENT DB+ at 3000, 2000 or 1000 mg/kg. b.w. did not differ significantly from the females mated by control males in any of the parameters evaluated (frequency of pregnancy, implantation egg loss, survival rate of implants and induced pre and post plantation dominant lethality).
The progeny of male mice treated with triethylenemelamine demonstrated greater induction of dominant lethality expressed by the loss of implantation zygotes as well as by the rate of the death of postimplantation embryos, thus validating the experimental procedure.
No evidence of dominant lethal effects was observed in the progeny of male mice treated with COGENT DB+ at any of the doses tested. Hence, COGENT db+ as considered non- mutagenic by the dominant lethal assay.
11. MUTAGENICITY EVALUATION BY BONE MARROW CYTOGENETIC ASSAY
Conducted By: International Institute of Biotechnology and Toxicology, Chennai
Results: COGENT DB+ was considered non-mutagenic by the dominant lethal assay
The ability of COGENT DB+ to induce numerical and structural cytogenetic anomalies was evaluated by enumerating the frequency of chromosomal aberrations in the bone marrow cells of mice (Swiss albino strain). Mice were orally administered with COGENT DB+ in distilled water at 3000, 2000 and 1000 mg/kg b.w. The animals were treated with the test substance once and sacrificed either at 18 hours or at 42 hours. Following dosing, 2 hours prior to sacrificing, mice were given colchicine (2 mg/kg b.w. i.p.). Bone marrow metaphase preparations were made following hypotonic and fixative treatment. Metaphase preparations were stained with Giemsa and aberrations are classified and scored.
Animals in the negative control group received distilled water alone and dose in the positive control group received once only a known mutagen, triethylenemelamine (TEM).
Mice treated with COGENT DB+ showed the values of numerical and structural aberrations as statistically comparable to that observed in negative controls. Mice in the positive control group showed increased frequencies of hypogenetic anomalies, thus validating the experimental procedure.
No evidence of induction of numerical and structural aberrations was observed at any of the test dose levels and at any time point of harvests in the bone marrow smears of mice treated with COGENT DB+.
It was concluded that COGENT DB+ is non-mutagenic by the mouse bone marrow cytogenetic assay.
12. MUTAGENICITY EVALUATION BY MOUSE MICRONUCLEUS ASSAY
Conducted By: International Institute of Biotechnology and Toxicology, Chennai
In order to evaluate the genotoxic potential for COGENT DB+, the mouse micronucleus test was employed. Male and female Swiss albino mice were given COGENT DB+ in distilled water orally by 1000, 2000 and 300 mg/kg b.w. The test substance was administered to mice either once (single treatment) or twice at 24 hours apart (2 days treatment). Concurrent vehicle control and positive control groups of mice were also maintained. Mice in the positive control group received a single dose of triethylenemelamine (TEM) at 1 mg/kg b.w. (ip).
Mice were sacrificed at 24 and 48 hours after the administration of COGENT DB+ in the case of single treatment, mice in the vehicle control and high dose groups were sacrificed at 24 hours after the last administration. Bone marrow smears were made for each animal. The smears were fixed in methanol, stained in the May-Gruenwald solution followed by Giesma.
A minimum of 2000 polychromatic erythrocytes per mouse were scored at each time point of sacrifice for the incidence of micronuclei. The presence of micronuclei in mature erythrocytes was also recorded.
No significant increase was observed in the frequencies of micronucleated polychromatic erythrocytes in the bone marrow smears of mice given the test substance (either given once or for 2 days) when compared with that of vehicle control mice. Similar results were recorded in mature erythrocytes.
Statistically, significant increase over control was seen in positive control group animals given TEM, thus validating the experimental procedure.
The ratio between mature and immature erythrocytes was uniform at all sacrifice points indicating absence of any inhibitory effect of the test substance on bone marrow cell division.
It was concluded that COGENT DB+ is non-mutagenic as per the mouse micronucleus assay.
13. MUTAGENICITY EVALUATION BY AMES SALMONELLA TYPHIMURIUM REVERSE MUTATION ASSAY.
Conducted By: International Institute of Biotechnology and Toxicology, Chennai
Results: It was concluded that COGENT DB+ is non-mutagenic by the Ames Salmonella typhimurium reverse mutation assay.
Five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA1538) were used in the study. COGENT DB+ dissolved in distilled water was tested at the doses of 0.5, 5, 50, 5000 and 5000 mg/ plate. Simultaneously, cultures of negative control received DMSO and cultures of positive control received mutagens like, benzo (a) pyrene, 2-nitrofluorene, sodium azide or 9-aminoacridine. In order to study the role of metabolic activation, cultures were incubated with or without S-9 mixture. The induction of histidine positive colonies was recorded.
The study indicated lack of induction of revertant colonies in any of the tester strains either with or without S-9 addition in the culture. It was concluded that COGENT DB+ is non- mutagenic by the Ames Salmonella typhimurium reverse mutation assay.
14. ONE-GENERATION REPRODUCTION STUDY IN WISTAR RATS.
Conducted By: International Institute of Biotechnology and Toxicology, Chennai
Results: It was concluded that COGENT DB+ did not affect the general reproductive process of Wistar Rats.
COGENT DB+ was tested for its effect on fertility and general reproductive performance in male and female Wistar rats. Forty males and 80 females were used in the study. They were randomly distributed into four groups, viz. G1(control), G2, G3 and G4 (experimental). Each group contained 10 males and 20 females. COGENT DB+ was powdered and mixed with distilled water and administered by oral intubation at the doses of 250, 500 and 1000 mg/kg b.w. to the rats belonging to G2, G3 and G4, respectively during the reproductive cycle. Rats in the control group were treated similarly, but with distilled water alone. The test substance was administered daily for 70 days in the case of males and for 14 days in the case of females prior to mating. The males and females were mated 1:3 ratio and the pregnancy was confirmed by vaginal smear examination.
The dosing was continued in both sexes during mating. In females, this was continued during gestation and lactation periods. After the dating, all the male rats were sacrificed and their testes, seminal vesicles, state and pituitary glands were subjected to histopathological gestations. In the case of females, after the weaning period, ovaries, uterus and vagina were subjected to histopathology. The dams were allowed to litter normally. The size, sex ratio and weight glitters were recorded. Survival and growth indices of litters on 0 th, 4th 14th and 21st day were recorded up to weaning.
One of the groups of rats showed mortality and abnormal behaviour during the entire experimental period. No changes in body weight were observed in experimental groups of rats when compared to control group of rats for the experimental period. No major variation was observed in the group mean of the size and sex ratio between the experimental groups and control group rats. The growth index of litters in control and experimental groups was to be similar. Gross pathological examination did not reveal any in the experimental groups of dams when compared with the control. Histopathological examination of the animals did not reveal any treatment related lesions.
It was concluded that COGENT DB+ did not affect the general reproductive process of Wistar rats.
B. ABSTRACT OF THE CLINICAL TRIALS
1. OZE’s AYURVEDIC MEDICAL CENTRE, KUALA LUMPUR, MALAYSIA
Several systematic clinical studies to assess the efficacy and undue effects, if any, were carried out before putting COGENT DB+ for human use.
A non-randomized clinical trial to evaluate the efficacy of COGENT DB+ was carried out in 30 Type II diabetic patients (17 males and 13 females; age 33-77 years) (treatment group). Thirty age matched Type II diabetic patients (21 males and 9 males; age 33-68 years) were considered as control. The fasting blood sugar of treatment and control groups was >126 mg/dl and random blood sugar was >200 mg/dl. All subjects in the treatment group were given COGENT DB+ (2 tablets daily after each meal) in addition to the regular allopathic drugs twice. (Daonil (Aventis Farma, SA Petaling Jaya, Selangor State, Malaysia) and Diamicron (Sevier, Bangkok, Thailand), with or without Metformin (Upha Corporation, Bangi, Selangor State, India) that they took in common with the control group for 3 months.
Thirty-two variables, viz. Liver and kidney function tests, haematological and biochemical parameters, blood sugar, insulin, C-peptide assays were carried out in treatment as well as control groups. At the end of 3 months, it was observed that there was a significant decrease in the levels of fasting and postprandial sugar, cholesterol, triglycerides, HBA1c, and increased insulin in the treatment group, when compared with the controls. Liver and kidney function tests indicated that COGENT DB+ does not exert any toxicity to these organs. The authors concluded that COGENT db+ is reliable, safe, tolerable and efficacious in the control of diabetes.
Conclusion: COGENT db+ is reliable, safe, tolerable and efficacious in the control of diabetes
2. UNIVERSITY OF KHARTOUM, SUDAN
Thirty patients (aged 30-65 years) suffering from diabetes (NIDDM) for more than 2 years were selected for the study. Prior to the study, the patients were observed for dietary management for 30 days. Five patients were on hypoglycemic drugs. Major complaints of the patients were polyurea, polydipsia and numbness. Fasting blood sugar of the patients was 130-300 mg/dl and the post prandial blood sugar was 180-400 mg./dl. After treating the patients with COGENT DB+ (2 tablets three times daily after each meal) for 3 months, fasting blood sugar dropped to 108-201 mg/dl. Liver and kidney function tests did not indicate any toxicity of COGENT DB+ to these organs.
3. MEDICAL COLLEGE HOSPITAL, KERALA
The study (double blind) was conducted in patients (100 patients) visiting the Diabetic Research Clinic of the Medical College Hospital. These patients were screened as per WHO guidelines, and only those patients not having concurrent diseases like neuropathy, hypertension, ischemic heart disease and liver disease were selected for the study. During the first month the patients were put on diet and exercise therapy.
The drug (COGENT DB+) and placebo were coded. The patients were given COGENT DB+ for placebo for 12 weeks and were followed up every 4 weeks with blood sugar, biochemical parameters for liver and kidney functions, lipid profile, haematological parameters, and glycated haemoglobin in addition to clinical evaluation. At the end of the study, after decoding the drug or placebo, it was found that 47 received placebo and 53 COGENT DB+ Demographic and clinical characteristics of patients recruited for the study are given below:
None of the patients in the Treatment group and Placebo group exhibited any side effects. All the patients of both the groups did not show changes in blood cell counts, renal and liver function tests and lipid profile.
It was concluded from the study that COGENT DB+ has a significant effect in controlling diabetes without causing any side effects.
4. DEPARTMENT OF NEPHROLOGY, UNIVERSITY HOSPITAL, BANARAS HINDU UNIVERSITY, VARANASI
One hundred and twenty four diabetic patients (mean (SD) age was 52 (13) years and mean (SD) duration of diabetes was 87*8 months were selected for the study. Ninety five percent of them were suffering from NIDDM. The baseline symptoms, blood pressure, weight, blood sugar values were recorded. Patients presented with the symptoms of fatigue (52%), polyuria (47%), polydipsia (38%) and polyphagia (23%). Patients were prescribed lesser doses of allopathic drugs and were kept on the same dose of COGENT DB+. Patients were receiving sulphonylureas, biguanides and insulin.
The patients were followed up every month for 4 months. There was a significant decrease in fasting blood sugar (about 31%) and postprandial blood sugar (about 45%) after 16 weeks of the treatment. There was no significant relationship of COGENT DB+ in decreasing the fasting blood sugar and postprandial blood sugar with regard to the type of the diabetes, duration of diabetes, age and sex of the patients.
It was concluded from the study that COGENT DB+ has a positive effect in decreasing blood sugar, irrespective of type of the diabetes, duration of diabetes, age and sex of the patients.
5. CITY HOSPITAL, GUANGZHOU, CHINA
Twenty diabetic patients (mean(SD) age was 54(5) years and mean (SD) duration of diabetes was 3(1.5) years) were selected for the study. All the patients were suffering from NIDDM (fasting blood sugar) ≥ 7.0 mmol. It was concluded from the study that there was a positive effect in reduction in FBS, PPBS and glycated haemoglobin in the patients.
6. SRI SAINATH HOSPITAL, CHENNAI
The study was conducted in 100 diabetic patients. COGENT db+ was given to the patients for 3 months. Patients showed significant reduction in glycated haemoglobin, fasting blood sugar and postprandial blood sugar and improvement in lipid profile (reduction in cholesterol, triglycerides, LDL and increase in HDL).
It was concluded from the study that COGENT db+ has a significant effect in controlling diabetes and improving lipid profile.
