Participants
A total of 110 ambulatory, community-dwelling Tehranian men and women volunteers between the ages of 18 and 50 were considered for initial screening. They were invited through the flyers distributed throughout the Shariati Hospital wards particular places where visible to public from January to December 2009. All volunteers were interviewed by attending physician regarding to life style, present and past medical history and using any medication or supplements.
If volunteers met initial eligibility criteria through the first interview, underwent physical exams and primary laboratory test to participate in this study. Metabolic syndrome were defined based on the AHA/NHLBI update of Adult Treatment Panel (ATPIII) criteria [22] consisting of
-
1)
Elevated waist circumference >88 cm (female), >102 cm (male)
-
2)
Reduced HDL cholesterol < 50 mg/dl (female), <40 mg/dl (male)
-
3)
Elevated triglyceride ≥ 150 mg/dl
-
4)
Elevated blood pressure ≥130/85 mmHg
-
5)
Elevated fasting blood glucose ≥ 100 mg/dl
Exclusion criteria included any history of clinical cardiovascular disease (myocardial infarction, angina, stroke, heart failure), diabetes mellitus, systemic, neurologic, psychiatric and endocrine diseases, liver function test abnormalities (aspartate aminotransferase or alanine aminotransferase levels > 3 times upper limit of normal), renal insufficiency (creatinine levels > 2.5 mg/dl) women who were pregnant, breastfed or have a pregnancy plan for the next 6 months and use of any medications, dietary supplements and herbals except of analgesics within 30 days prior to screening. Participants with a diagnosis of hypothyroidism could be included only if their level of TSH was within the normal range while they were receiving at least 3 months of a stable dose of thyroid replacement. Finally a total of 24 patients who had ≥3 of the metabolic syndrome diagnostic criteria and other eligibility criteria entered the study. Eighty six of participants were excluded because of the following reasons; in first lab results we noticed that many participants didn’t fulfill the metabolic syndrome criteria. Some of them were on diet and less inclined to participate in wash out period and a few were newly diagnosed of type 2 diabetes.
Study protocol
A double-blind, randomized, placebo-controlled trial using a parallel design with a 3-month follow up period, was established. A permuted-balanced-block randomization was employed to generate the random assignment of 24 subjects by order of entry into two different treatment groups. Each group received either Anethum graveolens extract (AGE) or placebo, one pill (600 mg) per day for 3 months. Subjects were visited during four time’s period: basal, first month, second month and third month. Twelve- hour fasting blood samples were obtained before each visiting session. During each visit anthropometric measurements and vital signs were assessed and they were asked questions about unexpected adverse events.
Body weight was assessed with a standard scale while they were lightly dressed and barefoot. Height was measured using a wall-mounted stadiometer. The body mass index (BMI) was measured by dividing the weight (kg) to height (m2). Waist circumference (WC cm) defined as the minimal abdominal circumference between the xiphoid process and iliac crest. Left hand blood pressure was measured by using standard mercury sphygmomanometer in the sitting position. The blood pressure values were confirmed by a second measurement after 5 minutes.
All study personnel were blinded to treatment assignment. All participants were told to maintain their usual dietary habits (diet stability was assessed and verified by analyzing all dietary recalls taken during each study visit by a registered dietitian). Adherence to study medication was assessed by pill count at each visit. Protocol of the study has been approved (Code: E-0046) by both the institutional review board and ethics committee of endocrinology and metabolic research institute (EMRI) of Tehran University of Medical Sciences. After giving required information about the details of the study program, all participants assigned written consent form.
Preparation of dill extract
Dried aerial parts of Anethum graveolens including leaves and steams were purchased locally from herbal markets. The plant was identified by herbaria’s expert of pharmacology department of Tehran University of Medical Sciences (TUMS). The hydro alcoholic extract of dried dill was prepared in the food and drug research laboratory of EMRI using following procedure. For the preparation of the hydro alcoholic extract, 100 gram of the dried grounded plant was suspended in 400 mL double distilled water- ethanol (2:1; v/v). The extract was filtered and the filtrate was evaporated to dryness with a rotatory vacuum evaporator. Then the dried powder (600 mg) was put into empty gelatin capsules by pharmaceutical company. More over the other gelatin capsule containing rice starch powder in the same color employed as a placebo. The toxicity assessment of hydroalcoholic extract of oral Anethum graveolens in animal was evaluated by Arbabi et al [23]. According to this study doses less than 50 mg/kg could be considered as no observable adverse effect level (NOAEL) and could be used in the future clinical investigations.
Assessment of adverse events
After initiation of the study, drug-related adverse effects were assessed by using questionnaire, physical examination and laboratory tests. The physician filled out a copy of the healthy questionnaire based on interview which was about changes in their general health since their last visit and physical examination of the patient. Besides vital sign monitoring (temperature, blood pressure, pulse rate) cardio respiratory and abdominal examination were also performed per visiting session (in each visit). Potentially adverse reaction on kidney and liver function were evaluated by measuring serum creatinine, electrolytes, liver enzyme, bilirubin (total and direct) and albumin at baseline and each visit. Drug related hematologic reaction was assessed by measuring the serum levels of all blood cells. Shifts from normal range and clinically and statistically significant deviation from baseline values of laboratory parameters were recorded as adverse events at each visit.
Biochemical analysis
White and red blood cell counts and hemoglobin levels were measured using a Multi hematologic Analysis System (Sismex, Japan). The following biochemical parameters were measured in the EMRC blood analysis laboratory using an automatic analysis system (Autoanalyzer; Hitachi Ltd, Tokyo, Japan) with enzymatic kits (Pars Azmoon, Iran): serum triglyceride (TG mg/dl), total cholesterol (TC mg/dl), high-density lipoprotein cholesterol(HDL-C mg/dl), low-density lipoprotein cholesterol (LDL-C mg/dl), fasting blood glucose (FBS mg/dl), alkaline phosphatase (ALP IU/L), alanine aminotranferease ( ALT IU/L) and aspartate aminotransferase ( AST IU/L), creatinine (mg/dl), blood urea nitrogen (mg/dl) and albumin (g/dl). All inter-assay calculated coefficients of variation were within the normal range of enzymatic kits data sheets.
Statistical analysis
Results are expressed as means ± SD. Significance of differences was evaluated using the SPSS statistical program package (SPSS; 16, Inc, USA) and defined at 0.05 level of confidence. Normality assumption of variables was assessed by Kolmogorov- Smirnov test and then the comparison between groups mean differences at baseline and each month was performed by student's t test. Within groups mean changes from baseline to final values at 4, 8 and 12 weeks in component of metabolic syndrome and other variable were analyzed by using paired t test.