The effects of Bunium Persicum (Black Caraway) supplementation on glycemic indices, lipid proles and serum levels of nesfatin-1 in overweight and obese patients with type 2 diabetes: a double-blind randomized placebo-controlled clinical trial

Background: Diabetes mellitus is the most common metabolic disorder worldwide. Our aim was to determine the effects of bunium persicum (BP) on serum glucose indices, lipid prole, and nesfatin-1 levels among overweight or obese T2DM patients. Methods: The place of participant recruitment was the diabetic clinic of Bu-Ali hospital in Zahedan. Based on the eligibility criteria, 60 participants were randomly divided into two groups as BP (n=30) or placebo (n=30). The supplementation was one 1000 mg capsules 2 times/day BP with launch and dinner for 8 weeks. Bodyweight, Waist circumference, serum nesfatin-1, fasting blood sugar (FBS), insulin (FBI), total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured. Quantitative insulin sensitivity checks index (QUICKI), homeostasis model assessment-insulin resistance (HOMA-IR) and Body Mass Index (BMI) were also calculated. Results: In comparison with placebo, PB signicantly increased QUICKI and decreased FBS, HOMA-IR, BMI and WC (P<0.05). At the end of the study after adjustment for confounders, the changes were similar (P<0.05) with an exception for QUICKI which had a trend (P=0.054) and WC (P > 0.05). The differences in the FBI, TG, TC, LDL, HDL and Nesfatin-1 were not signicant (P>0.05). Conclusion: PB supplement improved serum glucose indices and decreased BMI among overweight or obese T2DM patients; though, further trials are suggested to conrm results.

belonging to the Apiaceae family, is a widely distributed annual herbaceous plant (12) . Its seed is generally known in Iran as 'Zireh Siah' and has been used extensively in local foods and Iranian traditional medicine to cure several disorders (13) .
Furthermore, this plant is called with different names throughout the world as ''Black zire''," Black caraway", ''Carum carvi'', ''Persian Cumin'', "Zire kuhi", " Shah zira "," Kala Zeera", "Black Persian cumin", "Wild caraway" and "wild cumin" (14,15) . This herb has been used since ancient times, particularly in the treatment of digestive disorders and is known worldwide as antibacterial, antiulcerogenic, and is traditionally used to treat atulence, colic pain, bronchitis, diabetes, cardiovascular diseases, hypertension, and some gastrointestinal illnesses, including diarrhea and in ammatory bowel disease (16)(17)(18) . Furthermore, it may serve as a dietary source of natural antioxidants for health improvement and may be used as a natural antioxidative food additive to increase food quality and stability (19) . The known main constituents of BP have been demonstrated as gamma-terpinene, p-cymene, cuminal and cumin aldehyde (20,21) .
Nesfatin-1 is a newly identi ed 82 amino acid peptide derived from its greater precursor protein, NEFA/nucleobindin 2 (NUCB2) (22) . Nesfatin-1 dramatically decreases food intake when administered into the third cerebral ventricle of rats at picomole doses. The result of a recent report by Shimizu showed the potential physiological relevance of endogenous nesfatin-1 in the prevention of food intake was suggested by the stimulation of food intake induced by I.C.V injection of a nesfatin-1 antibody (22,23) .
fasting nesfatin-1 was signi cantly decreased in T2DM patients compared to healthy subjects and maybe one of the appetite-related hormones involved in diabetic Polyphagia (24) . Accordingly, this study is based on the hypothesis that bioactive compounds found in Bunium Persicum have antidiabetic and Appetite-Suppressing properties. The hypoglycemic, antihyperlipidemic and appetite reducing effects of caraway in humans and rats have been shown before (13,25) . Because bunium persicum is used widely in Iranian traditional medicine and as a common spice in Iranian foods, and to the best of our knowledge, there had been no clinical study on the effect of Bunium persicum on metabolic status of overweight and obese patients with type 2 diabetes and also, previous related studies have reported some inconsistent results; the present study was conducted to evaluate the effect of Bunium Persicum powder on glycemic indices, lipid pro les and serum levels of nesfatin-1 in overweight and obese patients with type 2 diabetes. disability, uncontrolled hypertension (>140/90 mmHg), Treatment with statins, antihypertensive, probiotics, multivitamin-mineral and antioxidant supplements during the three past months, pregnancy or lactation, expert athlete, intake of statins, caraway interacting drugs, losing weight during the past 3months and not taking more than 10% of the intervention supplement.

Methods
This trial adhered to CONSORT guidelines and included a completed CONSORT checklist as an additional le.
-Randomization and Intervention: According to the block randomization method, participants were divided into two equal groups by an assistant (BP [n=30] or placebo [n=30] groups). The strati ed randomization method was utilized for matching age (30-45 and 46-65 yrs) and gender. The ratio of the two groups was 1:1. Three patients from the PB group and 2 patients from the placebo group declined to participate after randomization and before the beginning of the study (Figure 1). The T2DM patients were identi ed, the eligibility criteria checked, the study details were clari ed, and informed consent was obtained by the main investigator (BSC student). The questionnaires including the general questionnaire, 24-hour food recall (at the beginning and end), and short-form IPAQ (SF-IPAQ) questionnaire (at the beginning and end) were administered through interviewing.
The dietary status was determined using gram per day of values from 24-hour food recall (valid in Iran (27) ) by the Nutritionist 4 software (27,28) .
The IPAQ questionnaire provides information on physical activity that people do as part of their everyday lives. The questions are about the time when the person has been active during the last 7 days. This questionnaire addresses the activities in the workplace or as part of the homework and the garden, place to place movement, exercises, and leisure activities. It also considers all the intense activities over the past 7 days. Intense activities require a great deal of physical power and more intense breathing. The IPAQ addresses only continuous activities for at least 10 minutes. Its short form has 7 classi ed questions determining the three activity levels (1-3 or low-to-high levels). This questionnaire had been validated in Iran (29,30) .
Anthropometric measurements: Weight (at the beginning and end), height (at the beginning) and waist circumference (at the beginning and end) were determined by using a digital scale and stadiometer (Seca ® Germany, Model: 755 1021994). They were measured as such: Weight without shoes, with minimal clothing, and with 100-gram accuracy; height without shoes, standing, heels sticking to the wall, at and forward head, and with 0.5centimeter accuracy; and waist circumference with minimal clothing, at the middle of the last rib and the iliac crest. Body mass index (BMI) was calculated via dividing weight in kilograms by squared height in meters.

Blood biomarkers measurement:
At the beginning and the end of the study, 10 ml of blood (at the beginning and end) was taken from the peripheral vein after 12-hour fasting during the night and centrifuged for 20 min (3000 g). Serum glucose was determined on the same day of blood withdrawal. The remaining serums were frozen and stored at -80°C up to the analysis.
Serum nesfatin was determined using the sandwich ELISA and kit as Shanghai Crystal Day Biotech Co.
Ltd ® ; Intra-assay CV<8 %, Inter-assay CV<10 % by an automatic device (Elisys Uno Human ® ). Similarly, the ELISA kit for FBI was DiaMetra ® Co of Italy, DCM076-8; Intra-assay CV≤5 %, Inter-assay CV≤10 %. Serum glucose was measured according to the glucose oxidase method using the Hitachi analyzer device (q17 ® ) and the speci c kit as Bionik ® , Liquid Stable, Glucose oxidase GOD-POD, Mono-reagent; Intraassay CV≤2.10 %, Inter-assay CV≤3.09 %. Also, the serum levels of TC, TG, LDL-C, and HDL-C were measured using Hitachi analyzer device (q17 ® ) and the speci c kits as 1-Bionik ® , Liquid Stable, as regards, no article was found similar to our study, the sample size determined by considering the sample size of the similar studies in obese and overweight patients (31,32) and using the extract of antiobesity factors (25) , The sample size determined from 8 to 44 in each group. The sample size for this study according to study limitations (control of various factors such as powder consumption, diet control, physical activity, nutritional counseling, cost) in each group 27 patients are considered but accounting to 10% missing during the study, the number of patients is 30 in each group.
-Data Analysis and Accessibility: Data management including entry, security, coding, and storage was performed at this stage. The missing data of the follow-up stage and baseline of one patient were estimated by modi ed-intention to treat (m-ITT) analysis and regression imputation method. The Kolmogorov-Smirnov, Chi-square, Fisher Exact, and t or Mann-Whitney tests assessed normality of continuous variables as well as categorical and continuous baseline characteristics, respectively. Two-way repeated measures analysis of variance (TWRM-ANOVA) was used to determine time effects and time by treatment interaction effects on all dependent variables. Moreover, TWRM-ANOVA was adjusted for dietary intake of vitamin B12. Also, 95% con dence interval (CI) and a P-value<0.05 were considered for reporting the measurements. Data analysis was conducted using SPSS 16 (statistical package for the social sciences) and STATA 11SE (general-purpose statistical software package by Stata Corp) software. The main investigator had access to the nal dataset and the results were presented by the publication.

Results -Participants' characteristics
According to Figure 1, overall, 680 people were screened based on medical history. Speci cally, 82 subjects had the eligibility criteria, of whom 12 declined and 4 could not participate. Also, 66 subjects were randomized, with 3 subjects in the BP group and 3 subjects in the placebo group refusing to participate and as such did not receive the intervention. Thus, the rst visit was completed for 60 subjects (bunuim persicum n = 30; placebo n = 30). In addition, 6 subjects could not continue the follow-up stage (for personal reasons and travel; bunuim persicum n = 3; placebo n = 3). Further, the baseline serum sample of two subjects in the placebo group was not available. Eventually, data analysis was performed for 52 subjects according to the modi ed-ITT analysis.
The general characteristics and physical activity level of the patients are presented in Table 1. Most of the participants had similar education, high economic and low physical activity level. Both groups used more than 95% of the prescribed supplements.
-Changes in dietary intake and blood biomarkers The dietary intake of carbohydrates, protein percent and iron in the baseline were higher in the BP group, while the other baseline features were similar between the two groups (Tables 3).
The dietary intake of vitamin B12 during the study was higher in the BP group (P<0.05,  (Table 4). -Safety Any side effects related to the treatment were reported, and only vomiting was observed for one patient in the BP group in one of his follow-ups.

Discussion
This trial is the rst to assess the bene cial effects of Bunium persicum on blood glucose indices, lipid pro le and Serum levels of nesfatin-1 in overweight or obese patients with type 2 diabetes mellitus (T2DM). The different clinical usages and the lack of awareness concerning the advantages and disadvantages of Bunium Persicum in patients with T2DM made this study very pertinent. According to both unadjusted and adjusted analysis models, consumption of 2,000 mg of BP for 8 weeks caused a signi cant reduction in FBG, HOMA-IR, BMI and augmented QUICKI in the intervention group compared with placebo. Furthermore, the decrease in WC was signi cant in the unadjusted model but not signi cant in the adjusted model. Limited human studies have investigated the effect of Bunium persicum on glycemic indices and lipid pro les and according to our knowledge, no study that examined the effects of Bunium persicum on serum Nesfatin-1 level in these patients.
However, some studies have been conducted on the effect of caraway (Bunuim Persicum and Carum Carvi) on diabetic rats. In one of these studies, no signi cant differences were seen in terms of dietary intake between the groups before and after the intervention except vitamin b12. In another study by Kazemipoor et al., no signi cant differences were found in dietary intake at baseline, but at the end of the intervention, the results showed a signi cant reduction in carbohydrate intake in the caraway group (25) .
The means of FBS levels decreased from 175.4±69.9 mg/dl to 142.6±53.8 mg/dL in BP group (p < 0.05). The decrease in FBS was signi cantly observed at the intervention group compared with the placebo (p < 0.05). Likewise, caraway consumption leads to a reduction of fasting blood insulin levels in the intervention group from 9.4±6.8 to 5.6±2.5, (p <0.05) and HOMA-IR from 4.2±3.7 to 2.3±1.3 (p <0.05).
Even though, the mean differences of the FBI were not signi cant in the intervention group as compared to the control group at the end of the study (p>0.05). The blood glucose lowering effect of Bunium persicum is maybe for the prohibition of hepatic glucose production or stimulation of glucose usage by muscle and adipose tissue (33) or inhibition of glucose reabsorption in the kidney (34) . Another hypothesis for the possible mechanism of hypoglycemic activity of this plant may be through its main bioactive compounds. The main components of bunuim persicum (KF1141) oils were γ-terpinene, cumin aldehyde, ρ-cymene and limonene (35) . The hypoglycemic effect of limonene has been previously reported in diabetic rats by decreasing the activities of gluconeogenic enzymes, increasing the glycolytic enzymes and stimulate insulin secretion in pancreatic β-cells (36,37) . Soundharrajan et al (2018) indicated that Limonene like insulin, could stimulate glucose absorption in differentiated cells through 2-Deoxy-Dglucose uptake by activation of complex Protein kinase B signaling pathways (38) . D-limonene can signi cantly decrease the activities of glucose 6-phosphatase and fructose 1, 6-bisphosphatase which can lead to decreased gluconeogenesis and thereby reducing the endogenous production of glucose (39). The presence of terpenoids is more signi cant in bunium persicum since they are reported to possess hypoglycemic activity in diabetic and normal mammals. The results revealed that the methanolic extract of Bunium persicum showed 36% inhibition in α-amylase at a concentration of 100 mg/mL. High differences were found in the activity of ethyl acetate extracts of Bunium persicum which showed 40% of inhibition at 250 mg/mL, respectively. Although, the mechanism of action is not clear (15,40). Also, cumin aldehyde in the bunium persicum oilseeds has a signi cant inhibitory activity against the α-glucosidase enzyme that catalyzes the nal step in the digestive process of carbohydrates. Its inhibitory effect of cuminaldehyde can postpone glucose uptake and reduce hyperglycemia (41) . The hypoglycemic effect of caraway seed has been previously shown by investigators. Haidari et al reported that oral consumption of caraway caused a signi cant decrease in blood glucose levels in streptozotocin-induced diabetic rats (13) . Kazemipoor et al. (2015) studied the consumption of Caraway extract in overweight and obese women patients. After 12 weeks, no signi cant effect was found for FBS levels (42) . In another research by Seyed Sadjadi et al. (2014), Short-Term Extract Administration of Caraway signi cantly decreased FBS levels in STZ-induced diabetic rats compared with control diabetic rats (43) . The anti-hyperglycaemic effect of caraway has been shown by M. Eddouks et al in diabetic rats previously. Oral administration of the aqueous caraway extracts (20 mg/kg) signi cantly decreased blood glucose levels in STZ diabetic rats but no changes were observed in fasting plasma insulin concentrations after treatment in either normal or STZ diabetic rats. these results indicated that the underlying mechanism of this pharmacological activity seems to be independent of insulin secretion (44) . Nevertheless, controversial results of some studies showed that consumption of Ethanolic Extract of caraway and Bunium Persicum Seeds signi cantly increased fasting blood insulin levels in streptozotocin-induced diabetic rats (45,46) .
In this study, the effect of caraway powder consumption on TG, cholesterol, HDL and LDL levels was not signi cant. Similarly, Kazemipoor et al. (2015), reported that consumption of caraway extract in overweight and obese women was not effective on lipid pro les (42) . Ghorbani et al. (2017) investigated the effect of high intensity interval training along with consumption of caraway seeds (Carum carvi L.) on liver enzymes, lipid pro le, and blood glucose in obese and overweight women and found signi cant differences in triglyceride(p=0.043) between groups and Paired t-test results showed that there is a signi cant difference between pre and post levels of triglyceride in overweight and obese groups, but no signi cant difference was observed in the other variables between the groups. However, HDL was signi cantly increased in the overweight group compared to the control and obese groups (31) . Our results are not consistent with the various studies on animals that have shown that caraway (bunium persicum and C. carvi) has signi cant hypolipidemic effects (43,(47)(48)(49) . In the study of Lemhadri et al., It was found that the extract of caraway produced a signi cant decrease in triglycerides levels at normal rats and a decrease in cholesterol levels in STZ diabetic rats. On the other hand,15 days after repeated oral administration of caraway extract exhibited a signi cant triglyceride and cholesterol-lowering activities In both groups of normal and STZ diabetic rats (48) . In another animal study, there was a signi cant inverse relationship between oral administration of caraway and total cholesterol (p <0.05) and low-density lipoprotein levels (p=0.001) in the treated animals compared with the diabetic control rats. but no signi cant changes were reported in the levels of high-density lipoprotein and triglycerides (13) .
With regard to the ndings of the present study, caraway signi cantly reduced body weight, Body Mass Index and Waist Circumference compared with the placebo group. Results in our study are almost consistent with the results of the Kazemipoor et al. (2015) study, During the 3-month intervention, the waist circumference (WC), the mean body weight and BMI had signi cantly decreased in the caraway extract group compared with the placebo group (42) . In another human study in obese women, Mohammadkhani et al. (2019) found that 8 weeks of aerobic exercise and taking a caraway supplement can improve weight and WHR in the supplement-exercise group compared with placebo-exercise and control groups (50) . Recently, Khaksari et al reported no signi cant changes in body weight after 6 weeks of Bunium persicum aqueous extract administration in hypercholesterolemic male mice (47) .
Weight, BMI and WC lowering effect of BP in our study may be related to Nesfatin-1 hormone levels that improved within the BP group in this study (p <0.05). Nesfatin-1 may contribut to the energy homeostasis, decrease appetite and reduce body fat mass (51-53) . Thus, there is a possibility that nesfatin-1 plays a role in the decrease in body weight in the BP group of our study (51) . In line with our ndings, A.Stengel et al reported that injection of nesfatin-1 can reduce body weight in rats (54) . Also intracerebroventricular (ICV) and peripheral nesfatin-1 injection in rats can inhibit food intake (55,56) .
The novelty would make this study very relevant. As the side-effects of the PB (up to 2 gram/day) had not been reported previously, it may be practically feasible for patients to continue taking it in the long run. Nevertheless, the effects and durability of this intervention in the long run should be investigated. The use of the PB in some diseases especially T2DM needs to be further studied. In addition, the emergence of obesity and, consequently, T2DM should also be considered.
The important strengths of this study were: the earliest assessment of PB effects in overweight or obese T2DM patients especially by assessing nesfatin-1 levels, the double-blinded strati ed blocked randomization design and assessing dietary intakes and physical activity status and adjusting for them.
However, some limitations were the self-reporting of diet and physical activity, measuring hemoglobin A1c (HbA1c), body composition, and bioavailability and serum levels PB or its components, and determining the durability of the intervention in the long run, and 24-hrs food recall which is not appropriate for determining the usual food intake. The personal information of patients was kept secret before, during, and after the study.

Conclusion
-Consent to publish: Not applicable.
-Availability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on a reasonable request.
-Competing Interests: There is no potential con ict of interests with respect to research, authorship, and publication.
-Funding: Funding was supported by the Zahedan University of Medical Sciences. The funding body did not have a role in the design of the study and collection, analysis, interpretation of data and writing the manuscript.
-Authors Contributions: SJM, MS, MDM, and ZM conceived and developed the idea for the paper and revised the manuscript. ASG wrote numerous drafts. ADP contributed to statistical interpretations. All authors read and approved the nal manuscript.      Figure 1