The institutional ethics committee reviewed and approved the research protocol and the informed consent vide review letter No. IRB/NIMS/080/2010. It is also confirmed that the ethics committee of Nizam’s Institute of Medical Sciences is constituted and functions as per Good Clinical Practice guidelines issued by Central Drug Standard Control Organisation and Ethical guidelines for Biomedical research on Human subjects, issued by Indian Council of Medical Research.
Selection of the subjects
The proposed study was carried out in the Diabetes Day Care Center of the University Department of Endocrinology and Metabolism at Nizam's Institute of Medical Sciences, Hyderabad. The research protocol and the informed consent was reviewed and approved by the Institutional Ethics Committee of Nizam’s Institute of Medical Sciences University, Hyderabad, India.
Men and women aged between 30–70 years with body mass index (BMI) ≥ 19 kg/m2, fasting plasma glucose (FPG) 100–125 mg/dl (IFG) (or) post 75 g oral glucose load, plasma glucose (oral glucose tolerance test, OGTT) 140–199 mg/dl (IGT) and those who were willing to give informed consent form were included in the study. Type 1 diabetes subjects, those who were taking drugs that could alter glucose tolerance, whose fasting triglycerides (TG) were >400 mg/dl, those who had a history of cancer or any major illness of the liver, kidney and central nervous system and women who were pregnant, breast feeding or planning a pregnancy during the course of the study were excluded from the study.
A single blinded (subjects were blinded to allocation), 3-year follow-up of randomized controlled trial of Fenugreek in 66 control and 74 study (Fenugreek) subjects was initiated in nondiabetic people with prediabetes (Fig. 1 and Tables 1 and 2). The proposed study design included investigation of long-term intake of Fenugreek intervention in persons with prediabetes. Study subjects and matched-controls were selected and monitored once in every 3 months, for identifying study-specific changes during and after study period.
All subjects in control as well as study groups were given similar instructions of physical activity and diet (adaptation of standard life style measures advised were, on dietary modification appropriate for weight and activity, weight reduction as necessary and exercise/physical activity for at least 30 minutes every day for a minimum of 5 days every week). Control and study subjects were counseled once in 3 months with advice on appropriate lifestyle and dietary practices.
After steps of consenting, screening, and diet and lifestyle training, all subjects were randomly assigned to either the Fenugreek group or control group using a fixed randomization scheme with assignment based on computer-generated random numbers performed by an independent researcher. The allocation scheme was sealed in opaque and consecutively numbered envelopes. Envelopes were opened sequentially by the independent person.
Fenugreek intervention program
The use of Fenugreek has been limited by its bitter taste and pungent odor. Isolation of biologically active components or production of a debitterized extract, which would allow greater use of the plant, has been investigated . Debitterized, defatted and deodorized Fenugreek fiber with vitamins, minerals and amino acids was supplied (single batch) by SMS Pharmaceuticals limited (Jeedimetla, Hyderabad, India). The compound was in the form of a fine powder. Fenugreek powder (debitterized and processed) 5 g twice a day, was given to the subjects along with 200 ml of water half an hour before meals and they were asked to follow the same dosage regime up to the end of study. Number of Fenugreek packs supplied to and returned by the subjects at the follow-up visit was reported to calculate the compliance of study medication. Efficacy parameters were assessed at each visit.
Data collection and measurable methods
Measurements were made at baseline (before treatment) and once in every 3 months during the study period. Demographic data was recorded at the baseline; a questionnaire on medical history and medication was administered, and body weight, height, and vital signs were measured. Height, weight, BMI, waist-to-hip ratio (WHR), FPG, post prandial plasma glucose (PPPG), lipid profile - serum cholesterol, serum triglycerides (TG), high density lipoprotein cholesterol (HDLc), low density lipoprotein cholesterol (LDLc) and serum insulin were recorded at baseline and during follow-up visits.
By measuring with tape horizontally, the WHR was calculated, waist as the minimal abdominal circumference located midway between the lower rib margin and the iliac crest and hip as the widest circumference over the great trochanters. The diagnosis of CAD (coronary artery disease), based on the presence of angina symptoms and abnormalities in resting electrocardiogram, was also assessed at baseline and after each year during the follow-up. Hypertension was determined by history of high blood pressure (≥130/85 mmHg).
Dyslipidemia was defined by any of the following: total cholesterol ≥200 mg/dl, triglycerides ≥150 mg/dl, HDL cholesterol ≤35 mg/dl, and/or LDL cholesterol ≥100 mg/dl or taking lipid-lowering drugs. OGTT at 2 h was performed in all subjects by taking 75 g oral glucose solution after overnight fasting; and then 2 h later, blood glucose level was measured. Blood was collected at 8:00 AM from the antecubital vein while the subjects were in the recumbent position after an overnight fasting. FPG, HbA1c, total cholesterol, triglyceride, HDLc, LDLc and serum insulin levels were measured according to the standard procedures and the sample analyses were done at Vimta Labs Ltd which is approved by the College of American Pathologists. HOMA-IR was calculated to assess change of IR.
It was assumed that during a 3-year follow-up period, 36 % subjects with prediabetes (IFG or IGT) develop clinical diabetes, with annual incidence rates of conversion ranging between 10 and 12 % [14–16]. In the proposed 3-year study it is expected that administration of Fenugreek reduces the risk of diabetes development in 14 % study subjects. Assuming the risk errors of α = 0.05 and β = 0.20, sample size was calculated based on the normal approximation to the binominal and was found to be 73 subjects per group including 20 % drop outs during the study.
For analysis of outcome variables, values of mean (SD) at baseline and at the end of 3 years were presented for both the groups. Statistical analysis was performed by two-tailed paired t-test for numerical variables, chi-square test for ordinal variables as appropriate, multivariate analysis; significance was set at p < 0.05. Cumulative incidence rate of diabetes during the study period was calculated as the proportion of individuals who developed diabetes for every six months (E/N, number of events (E)/number of persons (N)). Relative risk reduction rate (RRR, control event rate―study event rate/control event rate) for developing diabetes was calculated at the end of 3 years. Insulin secretion and insulin sensitivity were calculated using a pre-validated formula, homeostasis model assessment (HOMA). Statistical analysis was performed using the Statistical Package for Social Sciences 13.0 software ((SPSS Inc., Chicago, IL).)