The Effects of Coriander Seed Supplementation on Serum Glycemic Indices, Lipid Prole and Parameters of Oxidative Stress in Patients with Type 2 Diabetes Mellitus: A Randomized Double-Blind Placebo-Controlled Clinical Trial

Background: This research aimed to study the effect of coriander seed supplementation on serum glycemic indices, lipid prole and oxidative stress parameters in patients with type 2 diabetes mellitus (T2DM). Methods: In this randomized double-blinded, placebo-controlled trial, eligible 40 T2DM patients aged 30-60 years were recruited from Sina Hospital (Tabriz, Iran) and randomly assigned into two groups to receive either coriander seed powder (1000 mg/day, n=20) or placebo (1000 mg/day, n=20) for 6 weeks. Anthropometric measurements, dietary intake, and biochemical parameters including fasting blood sugar (FBS), serum insulin, Homeostatic Model Assessment for Insulin Resistance (HOMA-IR), total cholesterol (TC), triglyceride (TG), high- and low-density lipoprotein cholesterols (HDL-C and LDL-C), malondialdehyde (MDA), and total antioxidant capacity (TAC) were assessed before and after supplementation. Results: Anthropometric measurements were not signicantly different between intervention and placebo groups. Coriander seed supplementation led to signicant within-group reductions in FBS (156.15±23.19 to 130.30±21.15), serum insulin (17.72±0.47 to 17.12±0.76), HOMA-IR (6.82±0.95 to 5.52±0.99), TC (183.85±55.68 to 145.20±31.36), TG (152.50±37.59 to 130.40 ±27.96), LDL-C (127.35±23.45 to 111.40±25.71), and MDA (1.65±0.15 to 1.49±0.15), whereas there were signicant increases observed in serum TAC (1.93±0.12 to 1.97±0.09) (P<0.05). Post-dose comparisons showed signicant between-group differences for FBS, serum insulin, HOMA-IR, TC, TG, level were observed in coriander seed group (p < 0.001). In the placebo group, except for serum TC level which had a statistically signicant increase (p = 0.042), all other variables did not change signicantly (p > 0.05) compared with baseline. At the end of the study, results of ANCOVA test showed statistically signicant differences between the two studied groups in FBS (p < 0.001), serum insulin (p = 0.001), HOMA-IR (p < 0.001), TC (p < 0.001), TG (p < 0.001), LDL-C (p < 0.001), TAC (p < 0.001) and MDA (p < 0.001) levels, after adjusting for baseline values. Age, sex, drug use and physical activity were controlled for as confounding factors.

Hyperglycemia, lipid pro le abnormalities [6][7][8], impaired oxidant/antioxidant balance [9,10], increased production of free radicals [11,12], and impaired antioxidant defense system [13,14] are among the main contributing factors in the etiology of diabetes and could potentially associate with diabetes-related complications. Oxidative stress is a prominent risk factor condition associated with the pathogenesis of T2DM [15]. Oxidative damages occur by the over-production of free radicals beyond the antioxidant capacities to impose cells with oxidative stress partly affects formation of lipid peroxidation products such as malondialdehyde (MDA) [17]. Several studies showed that MDA can potentially lead to the risk of insulin resistance and subsequently development of cardiovascular diseases [16]. Moreover, oxidative stress and insulin resistance are associated with increased concentrations of in ammatory biomarkers [17]. Therefore, controlling the oxidative stress might in uence hyperlipidemia to better manage T2DMrelated complications [17,18].
The oral hypoglycemic agents could associate with various side effects, hence, adherence to these treatments is low; therefore, recently there is much attention paid to natural herbal remedies [20]. Herbal remedies can improve glycemic responses [19]. Coriander is one of the important herbs that has healing properties [20]. The main medicinal part of the plant is its fruit called coriander seed [22]. Coriander seeds are rich in bioactive compounds with strong antioxidant properties including avonoids, polyphenols, carotenoids, steroids, tannins, limonene, linalool, α-pinen, ß-caryophyllene, and γ-terpene [21]. The antioxidant properties of coriander seeds are mainly related to polyphenols and linalool (coriandol) [24].
Polyphenols can inhibit the oxidative stress by suppressing free radicals and coriandol by increasing the antioxidant defense system e ciency [22]. Recently, studies on animal models indicated bene cial effects of coriander seed on glycemic indices [23], lipid pro le [22][23][24][25], and oxidative stress [25][26][27]. Only one clinical trial has been conducted on the effect of coriander seeds on type 2 diabetes [28]. Therefore, there is a limited number of studies regarding the effects of coriander seed on diabetes mellitus in human studies particularly no data exist to show its ameliorating effects on oxidative stress in T2DM patients, this clinical trial was planned to study the effects of coriander seed powder supplementation on serum glycemic indices, lipid pro le and oxidative stress parameters in T2DM patients.

Subjects
This randomized double-blind placebo-controlled trial was conducted at Diabetes Clinic of Sina Hospital administered by Tabriz University of Medical Sciences, Tabriz, Iran.
Inclusion criteria were: age 30-60 years, having T2DM for more than 3 years and satisfaction with the project. The exclusion criteria were: smokers, pregnant women, breast-feeding, any thyroid, liver, digestive, kidney and immune system dysfunction, cardiovascular diseases, uncontrolled T2DM and/or insulindependent diabetic patients, using non-steroidal anti-in ammatory drugs, hormone therapy, and taking antioxidant supplements during the last three months before the study.
The study protocol was approved by the Ethics Committee of Tabriz University of Medical Sciences (no.IR.TBZMED.REC.1398.677) and registered on the Iranian Registry of Clinical Trials website (IRCT20190224042821N2). All subjects were fully explained before recruitment and a written informed consent was obtained from each subject prior to the study enrolment.
Sample size was calculated based on FBS results reported by Parsaian et al. [30]. Considering a con dence level of 95% and power of 80%, 18 subjects were calculated for each group taking into account the likelihood of a dropout (20%), 22 subjects in each group were recruited (total sample size: 44 T2DM patients).

Study design and measurements
Diabetic patients referred to the Sina Diabetes Clinic who had a medical record in this center and were eligible for inclusion were explained about the plan. Individuals who were willing to participate in the project were divided into case and placebo groups based on the numbers inserted on their case (couple and individual). Of the 44 patients who met the inclusion criteria, there was lost to follow-up (n=4) due to dissatisfaction ( Figure 1). Finally, 20 controls and 20 cases in each group were completed the interventions to conduct statistical analysis. First coriander seeds were washed, dried at room temperature, and then powdered with an electric mill. Then coriander seed powder was packed in 500 mg gelatin capsules. Corn starch was used to prepare the placebo (500 mg). The capsules were prepared using capsule ller under the aseptic conditions and 70% ethanol sterilized tool to prevent secondary contamination. The quality of the capsules was checked and cleaned with sterile cotton. The intervention or placebo group allocation was hidden from the researchers, and the coriander seed and placebo capsules were similar in appearance. Therefore, neither the participants nor the researchers were aware of the therapeutic assignments in this study. Patients were asked not to change the number and dosage of their medication (metformin and glibenclamide) and their physical activity during the study period. They were asked to take the capsules twice daily (30 min before lunch and dinner) during six weeks. The capsule intake guidelines were assessed by telephone interview once a week. At the end of study (the 6 th week), patients were undergone re-examination.

Anthropometric assessments
At the beginning of trial, demographic characteristics were obtained and International Physical Activity Questionnaire (IPAQ) was completed to assess patients' physical activity level [29]. Furthermore, anthropometric measurements were performed at the beginning and at the end of the trial. Body weight was measured to the nearest 0.5 kg using a Seca scale (Hamburg, Germany), with the patients being barefoot and wearing light clothing. Height was also measured using a mounted tape, with the participants' arms hanging freely by their sides and recorded to the nearest 0.5 cm. Body mass index (BMI) was calculated by dividing weight (in kilograms) by the square of height (in meters). Waist circumference (WC) was obtained using an inelastic tape measure to the nearest 1 mm. The mid-point between the last rib and the iliac crest was recorded as WC. Hip circumference (HC) was measured at the widest point of the hip. The waist to hip ratio (WHR) was calculated.

Nutritional assessments
Information regarding dietary intake was gathered using a 24-hour recall method for 3 days (including 2 working days and 1 weekend) a week before and at the end of supplementation. Total energy, macronutrients, and antioxidant vitamins intake were determined with the Nutritionist IV software program (First Databank Inc, Hearst Corp, San Bruno, CA, USA).

Laboratory assessments
At the beginning and at the end of the trial period, 5 mL of blood samples were taken from each patient after 12-hour overnight fasting. All serum samples were stored at -70 ° C until assay. Fasting blood sugar (FBS), serum total cholesterol (TC), triglyceride (TG), and high-density lipoprotein cholesterol (HDL-C) were measured using the standard enzymatic colorimetric method by Auto-analyzer Bio-systems (Authoanalyzer, BS-200, MINDRAY chemistry analyzer, Germany, 2009) and Pars-Azmoon Diagnostic Kits (Tehran, Iran). Friedewald's formula was used to calculate low density lipoprotein cholesterol (LDL-C) [30].
Serum insulin concentration was determined by ELISA kit (Diameter, Italy and Bioassay Technology Laboratory, China). To measure insulin resistance, we used homeostatic model assessment of insulin resistance (HOMA-IR) based on the following formula: HOMA-IR = fasting insulin (µU/mL) × fasting glucose (mg/dL) / 405 [31]. Serum concentration of malondialdehyde (MDA) was determined via thiobarbituric acid reactive substances (TBARS) method described by Bilici et al. [32]. Total antioxidant capacity (TAC) was measured using spectrophotometry method with a Randox kit (Randox Laboratories, Ltd., UK).

Statistical analysis
Data were analyzed by SPSS software version 21.0 (SPSS, Inc, Chicago, IL, USA). Kolmogorov-Smirnov test was used to check the normality of the data. Quantitative and qualitative data were reported as mean ± standard deviation and frequency (percentage), respectively. Differences between variables before and after study were compared by paired t-test. Comparisons between groups were made by chi-squared test, independent sample t-test or Mann-Whitney U test, as appropriate. Analysis of covariance (ANCOVA) was used to nd any differences between the two groups at the end of the study, adjusting for baseline values. P-value less than 0.05 was considered statistically signi cant.

Results
Forty patients with T2DM completed this clinical trial (20 patients in each coriander seed and placebo groups as described in Fig. 1). No adverse events were reported in patients supplemented with coriander seed and/or placebo. General characteristics, medication intake and physical activity level of study patients are shown in Table 1. The variables been normalized before the analyses. At baseline, there was no signi cant difference between the two groups in terms of general characteristics, medication intake and physical activity level (Table 1). Anthropometric measurements and dietary intake analysis of patients throughout the study are shown in Table 2 and Table 3, respectively. At baseline, there was no signi cant difference between the two groups in anthropometric measurements and dietary intake (p > 0.05). Furthermore, no signi cant changes were Page 8/20 seen within and/or between coriander seed and placebo groups in anthropometric measurements and dietary intake after 6 weeks (p > 0.05).  As illustrated in Table 4, there was no signi cant difference in serum glycemic indices, lipid pro le and oxidative stress parameters between the two study groups at baseline (p > 0.05). Compared to baseline, FBS, serum insulin and HOMA-IR levels decreased signi cantly in the coriander seed group (p < 0.001, p = 0.003 and p < 0.001, respectively). Moreover, signi cant decrease in serum TC, TG, and LDL-C was observed in the coriander seed group (p < 0.001), whilst changes in serum HDL-C were not signi cant (p = 0.428). In addition, signi cant increase in serum TAC level and signi cant decrease in serum MDA level were observed in coriander seed group (p < 0.001). In the placebo group, except for serum TC level which had a statistically signi cant increase (p = 0.042), all other variables did not change signi cantly (p > 0.05) compared with baseline. At the end of the study, results of ANCOVA test showed statistically signi cant differences between the two studied groups in FBS (p < 0.001), serum insulin (p = 0.001), HOMA-IR (p < 0.001), TC (p < 0.001), TG (p < 0.001), LDL-C (p < 0.001), TAC (p < 0.001) and MDA (p < 0.001) levels, after adjusting for baseline values. Age, sex, drug use and physical activity were controlled for as confounding factors. Data were expressed as mean ± SD. P < 0.05 was considered signi cant.
* P values indicate comparison between groups (and ANCOVA test, adjusted for baseline values, after 6 weeks). Data were expressed as mean ± SD. P < 0.05 was considered signi cant.
* P values indicate comparison between groups (and ANCOVA test, adjusted for baseline values, after 6 weeks). ** P values indicate comparison between groups (independent sample t-test at baseline) *** P values indicate comparison within groups (paired t-test).

Discussion
Findings showed acceptable e cacy of supplementation by coriander seeds among the study population with T2DM. Serum glucose levels decreased signi cantly in the coriander seed group compared to baseline measures and those in the placebo group. Accordingly, Aissaoui et al. [33] study who indicated signi cant post-intervention changes in coriander seed supplemented obese hyperlipidemic and hyperglycemic rats. Similar results were also discussed by Parsaian et al. in T2DM patients [30], showed the hypoglycemic effect of coriander seed including stimulating glucose utilization by peripheral tissues, especially muscle and adipose tissue, increasing hepatic glucose uptake, decreasing hepatic glucose production and gluconeogenesis, and increasing the activities of glucose 6-phosphatase and glycolysis by polyphenols and avonoids extracted from coriander [33]. Furthermore, inhibiting glucosidase and amylase activities as well as decreasing glucose release and absorption in the gastrointestinal tract via SGLT1 transporter suppression have also been suggested to play a role in reducing glycemia [28]. By contrast, Al-Suhaimi et al. [34] showed no changes in serum glucose level after coriander seed administration in healthy adult male rabbits. Contrast to present ndings, Nyakudya et al. [35], showed increased serum levels of glucose after ve weeks of coriander intervention but this increase was not statistically signi cant. Present results showed that coriander seed supplementation led to signi cant decrease in serum levels of insulin compared to baseline and placebo group. In a study by Aissaoui et al. [33], reduction in serum insulin was also observed following coriander seed supplementation but was not statistically signi cant. In addition, insulin resistance improved signi cantly in the coriander seed group compared to baseline and placebo group. This nding was also in accordance with a previous investigation [33]. It seems that coriander seed increases insulin signaling, thereby reducing insulin resistance by enhancing insulin sensitivity [33].
Dyslipidemia is an important risk factor for the development of atherosclerosis in patients with T2DM [24].
Signi cant decreases in serum levels of TC, TG, and LDL-C were found in the intervention group supplemented coriander seed compared to baseline and placebo group. Our study was consistent with results of previous studies in animal models [22][23][24][25] that indicated a signi cant decrease in serum cholesterol after coriander seed administration. Moreover, Parsaian et al. [28] reported the potential effects of coriander seed supplementation in lowering serum cholesterol in T2DM patients. Cholesterol lowering mechanisms of coriander seed are presumably attributable to the decreased cholesterol biosynthesis particularly by inhibition of β-Hydroxy β-methylglutaryl-CoA (HMG-CoA) reductase, a key enzyme in cholesterol biosynthesis, increasing cholesterol degradation to fecal bile acids and increasing lecithin-cholesterol acyl transferase (LCAT) [25,28]. In addition, avonoids and polyphenols that present in coriander seed may be responsible for its hypolipidemic and hypoglycemic effects [28]. Decrease in serum LDL-cholesterol can also be due to decreased LDL synthesis or an increased LDL metabolism [25].
Moreover, decrease in serum TG may be due to the stimulation of TG degradation through increased lipoprotein lipase expression and activity as well as decreased TG synthesis and its secretion [25]. Inconsistent with our results, Al-Suhaimi et al. [34] study showed no decrease in serum levels of cholesterol after the intervention in healthy male rabbits. This lack of cholesterol reduction might be attributed to the low dose of coriander seed used in that study. According to our study, changes in serum levels of HDL-C were not signi cant compared to baseline and placebo group. Our study was in line with Parsaian et al. [28] study who reported no signi cant changes in serum HDL-C after coriander seed supplementation. Our study was not similar with Dhanapakiam et al. [24] who proved that HDL-C levels increased after coriander seed consumption probably due to the removal of cholesterol from the tissues and directing it to the liver by HDL-C. In addition, Aissaoui et al. [33] indicated that coriander seed decreased serum HDL-C signi cantly which was probably due to the large changes in blood cholesterol levels.
Diabetes mellitus is associated with increased reactive oxygen species, lipid peroxidation, and impaired . To the best of our knowledge, present study was the rst research assessing the effects of coriander seed supplementation in T2DM. According to our results, coriander seed increased serum levels of TAC and decreased serum levels of MDA signi cantly compared to baseline and placebo group. Previous animal studies showed the role of coriander seed in scavenging superoxide anion and hydroxyl radicals [25][26][27]. Coriander seed contains phenolic compounds which might exert antioxidant activity, thereby it could reduce lipid peroxidation and related markers in tissues [28]. This indicates that coriander seed extract might interact with the accumulation of peroxyl radicals and act as chain breaking antioxidant against lipid peroxidation [26]. The active components of coriander seed can take part as electron donors, which can react with free radicals to generate stable forms of radicals and thereby terminate the radical chain reaction [26].
The limitations of the current study were small number of participants and short duration of supplementation. The strengths of our study were conducting a double-blind, randomized, placebocontrolled design. We evaluated dietary energy and nutrients intake at baseline and at the end of trial to nd any confounders. Also, coriander seed powder seemed to be well tolerated by participants.

Conclusion
Present study demonstrated that coriander seed powder supplementation decreased serum glucose, insulin, insulin resistance, TC, TG, LDL-C, and MDA levels and increased serum TAC level signi cantly in T2DM patients. These ndings suggest that coriander seed can be useful in management of diabetic patients.

Declarations
Ethics approval and consent to participate All procedures followed were in accordance with the ethical standards of the Ethical Committee of Tabriz University of Medical Sciences.

Consent for publication
Not applicable Availability of data and materials