In a randomized, placebo-controlled, double-blind clinical trial, DMT2 patients aged 30-60 years old were recruited for the current study from Firoozgar hospital, Tehran, Iran. This study was designed with 90% power, with 2- sided a=0.05 (type I error), to detect a 5% difference in serum glucose between the 2 group. On the basis of SDs observed in the current study, the number of subjects needed to treat to detect this difference was 16/group. Given an anticipated dropout rate of 25 percent, we set the enrollment target at 20 subjects. The inclusion criteria were as follows, fasting blood glucose≥126 mg/dl, age of participants rages 30-60 years old and unique medication plan as Metformin+Glibenclamide. The diagnosis was performed by an endocrinologist via fasting blood glucose more than 126 mg/dl. Exclusion criteria were as follows, insulin treatment, sensitivity to the extract of Rheum ribes, pregnancy during the study, variation in the dosage of the medicines during the study, consumption of any dietary supplements, smoking, alcoholism, green tea consumption, and use of other herbal medicine. A history of diseases including liver, kidney and cardiovascular diseases, thyroid disorders, gastrointestinal problems, lipid-lowering or anti-hypertension treatment, using corticosteroids, cyclosporine, non-steroidal anti-inflammatory or immunosuppressive drugs, warfarin and anti-epileptic medications, pregnancy or breastfeeding, allergy to plants of ragweed species was taken for each subject.
All participants provided written informed consent after receiving an explanation of the purposes of the study, inclusion criteria, exclusion criteria, disadvantages and advantages of the study which were approved by the ethics committee of the Iran University of Medical Sciences. Meanwhile, all of participants were requested to don’t alter their usual dietary intake and physical activity during the study. The study was recorded in the clinical trial record center of Iran under the registration number of IRCT201410142709N31
Plant material and extract preparation
Rheum ribes were obtained from the Shahroud region (Semnan, Iran). The plants were washed, air-dried for 12 hours, and turned into small pieces. Identification of the plant was confirmed at the Department of Medicinal Plants, Faculty of Pharmaceutical Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Then, the pieces were equally assigned to each of the ethanolic or aqueous groups and placebo. The extraction was carried out using a maceration method according to previous studies (18). For this purpose, 100 g of dried rhubarb flowers were poured into a dish after grinding, and 1000 ml of ethyl alcohol and 1000 ml of water were added to it. After 4 hours, the contents of the container were stacked with flat paper in a clean lab and then the container was placed on a bain-marie. After evaporation of the solvent, an extract was obtained, as for every 100 grams of rhubarb, 8 grams of extract was obtained. The alcoholic group was immersed in ethanol alcohol in dark and closed containers, and, were stored for 4 days. After that, it was filtered off, the pulp was removed, and the purified extract was evaporated by using a rotary evaporator. Moreover, to prepare an aqueous extract, the immersed plant in water was boiled in the metal tank. Then, it was cool down at the laboratory temperature, passed through filter paper, concentrated on bain-marie. After these steps, extracts were turned in to dry extract, converted into granular and powder by corn starch. Ultimately, the capsulated step was done.
Regarding chemical composition, Rhubarb plant was analyzed in Faculty of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran, by High Performance Liquid Chromatography or HPLC analysis method and its constituents were obtained: α-Pinene (13.5%), α-Terpinene (1.3%), p-Cymene (10.6%), Limonene (8.6%), trans-β-Ocimene (1.4%), Terpinolene (12.4%), Isoterpinolene (1.4%), cis-Isopulegone (2.1%), Sabinyl acetate (4.3%), 4-Vinyl-2-methoxy-phenol (2.1%), β-Elemene (1.5%), Germacrene-d (22.3%), Bicyclogermacrene (9.6%), and γ-Dodecadienolactone (3.7%). The others were in trace amounts
After selecting the samples, a general information questionnaire was completed. Body weight was measured using a scale (Seca, Hamburg, Germany) for all subjects, without shoes and wearing light clothing. Height was measured with a mounted tape and without shoes. BMI was assessed as weight in kilograms divided by height in meters squared.
Information about daily energy and macronutrients intake was obtained by 3-day dietary recall, including 2 regular days and 1 weekend day. Three days average dietary data were analyzed by Nutritionist 4 software for all subjects at the beginning of the study and the end of 6th weeks of study (First Databank Inc., HearstCorp., San Bruno, CA).
At the study baseline and after stratification for gender, pre-intervention weight and age, subjects were randomly assigned to three groups of 20 subjects each as follows: The aqueous extract group (AG, n= 20) and the ethanolic extract group (EG, n= 20) were given 3 Rheum ribes capsules daily for 6 weeks. Each capsule contained 450 mg of Rheum extract (3×450 mg daily). The placebo group (PG, n= 20) was given 3 placebo capsules (contain starch) daily for the same period which was similar to capsules of AG and EG in appearance, shape, and color. The capsules of Rheum ribes was prepared by Department of Medicinal Plant, Faculty of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Compliance of the volunteers with the study protocol was evaluated via phone interviews once per week and by counting returned capsules every 2 weeks.
Fasting blood samples (10ml) were drawn from the brachial artery of all participants after 12 hours fasting for biochemistry tests at the beginning and 6th weeks of intervention. Serum samples were immediately centrifuged (Sigma, UK) at 10000 rpm for 20 min to separate serum. Then the serum samples were stored at -70c ͦ before analysis at the Iran University of Medical Sciences reference laboratory. Serum glucose was measured by standard enzymatic method of glucose oxidase. Serum levels of ApoB and ApoA1 were measured by the method of immunoturbidimetry. The serum levels of insulin were measured by the ELISA method. Commercial kits were used to measure serum levels of insulin, glucose, ApoA1 and ApoB (Pars Azmoon, Tehran, Iran). Insulin resistance was assessed by Homeostasis Model Assessment (HOMA-IR) and beta cells function assessed by Homeostasis Model Assessment-beta (HOMA-B) with the formula: [glucose (nmol/L) x insulin (microU/L) /22.5] and [20 x Insulin (μIU/ml) /glucose (mmol/ml) -3.5]%, respectively. Interventions were followed by phone contact.
Data were analyzed by SPSS software, version 22. In this study, less than 0.05 was considered as a meaningful level. All quantitative variables were reported as mean± SD and qualitative variables were reported as a number (percent). The Kolmogorov–Smirnov test was used for determining the normality of the data. The paired T-test test was used for comparing the mean of pre-intervention and post-intervention data with a normal distribution. The ANOVA method was used for comparing the mean of the three groups. Besides, ANCOVA were used for adjustment of the confounding factors such as age and pre-intervention variants. The method of analysis of variance with repetitive measurements and follow-up tests were used for evaluating the meaningfulness of diet determined by 2 measurements during the intervention period. The confidence level for all variables was considered 95%. The statistically meaningful level was P<0.05.