Study design and procedures

The protocol of the present trial has been previously published in the BMJ Open Journal [23]. Informed consent was obtained from all patients. This study was a randomized, placebo-controlled, triple-blind with two parallel groups on forty-six T2DM patients (30 to 50 years old, 29 females and 17 males) with fasting plasma glucose (FPG) levels ≥126 mg/dL that were recruited among those referring to the Iranian Diabetes Society and the Afzalipour Hospital and other clinics in Kerman, Iran, from May 2022 to October 2022 and randomly assigned to a treatment or placebo group. Public platforms such as WhatsApp, posters, telephone calls, and introductions from the medical staff were used to recruit participants. An overview of the research is shown in Fig. 1. All eligible patients were included in the study based on inclusion and exclusion criteria. Inclusion criteria were a T2DM history for over six months, body mass index (BMI) of 25 to 35 kg/m2 during the previous three months, no weight changes during the last three months, using metformin or glibenclamide or both to control blood sugar, not taking insulin, consent to consume DSP, having stable physical activity level (PAL), and adhering to a regular dietary regimen. Exclusion criteria encompassed any changes in diet, lifestyle, class, or dose of hypoglycemic drugs, receiving insulin therapy, taking glucocorticoids, laxatives, anti-obesity, lipid-lowering non-steroidal anti-inflammatory (NSAIDS) drugs, multivitamins, and antioxidants during the last three months prior to the onset of the research, currently consuming prebiotics, probiotics, or antibiotics, adherence to a weight-loss or a special diet within the last six months, a history of intestinal diseases such as bowel inflammation, intestinal cancer, and digestive problems, suffering from thyroid problems, a history of pulmonary, hepatic, renal, cardiac, or infectious diseases, being under radiotherapy or chemotherapy for cancers, being pregnant, breastfeeding, smoking, alcohol consumption, having regular vigorous physical activity, being a professional athlete, developing gastrointestinal issues during the research, and reluctance to consume DSP. The patients were required to follow their recommended diets as well as their PAL, during the course of the study.

Fig. 1Randomization and blinding

Forty-six participants, after a 2-week run-in course, were equally and randomly allocated to the placebo and intervention groups (n = 23 per group) using a random sequence of numbers prepared by an independent researcher. The patients were allocated in a 1:1 ratio to receive either DSP or placebo, which were identical in terms of shape, color, and taste. The independent researcher packaged DSP and placebo sachet in boxes and consecutively numbered them according to a computer-generated randomization list. The randomization scheme consisted of a sequence of four blocks such that each block contained two treatment arms (A and B)., and the participants were matched for BMI, sex, and age. The randomization sequence was prepared by the random allocation software (RAS). Each block’s arm sequence was assigned in random order using the OnCore randomization algorithm. For concealment allocation, opaque sealed sequentially numbered envelopes placed in order. In order to maintain a random sequence, numbering was done on the outer surface of the envelopes in the same order. Finally, the lid of the letter envelopes was glued and placed in a box in order. At the time of starting registration of participants, based on the order of entry of eligible participants into the study, one of the envelopes opened and the assigned group of that participant was revealed.

The boxes were delivered to the study’s supervisor in sealed envelopes. Each participant was assigned according to the allocation list to receive either the supplement or placebo, delivered in pre-packed boxes every week. The allocation was masked from the participants, study supervisor, and those involved in outcome assessment and data analysis. The patients were requested to decrease the intake of any substance or supplements that might affect blood sugar and lipid profile during the run-in phase and to maintain their diet and PAL as recommended.

Intervention

The intervention group received 5 gr of the DSP daily (date seed, Flavinea Co, Iran). The same amount of Maltodextrin was given to each participant in the placebo group (maltodextrin, Jiujiang Hurirong Trade Co, China). Both sachets could not be distinguished by color, odor, or taste. The powders were divided into sachet of 2.5 gr. Participants added each sachet to semi-solid foods like yogurt to reach the dose of 5 g for eight weeks. It was explained how to incorporate the supplement into their regular diet. Both powders were odorless and flavorless that were provided in identical opaque packages to participants every four weeks. Participants were contacted twice weekly to ensure compliance, keep up with physical activity, and resolve supplement administration issues. Any change in diet, lifestyle, class, or dose of hypoglycemic drugs the patient made during the intervention were asked to be reported. Also, a checklist was provided for participants to mark after each consumption of the prescribed powder to evaluate for cases of non-compliance. Adherence to the regular consumption of powders (DSP or placebo) was also determined by counting packages, also the acceptance rate of individuals was calculated by determining the ratio of the number of consumed sachets to the total number of sachets. Consequently, patients with an acceptance rate below 90% were excluded from the study. Also, patients were excluded if they altered their diet or lifestyle, or changed the type or dosage of their hypoglycemic medications.

Chemical analysis of DSP (Phoenix dactylifera)

The composition and antioxidants per 100 gr of DSP was analyzed using Folin-Ciocalteu colorimetric analysis and the aluminum chloride colorimetry method and is shown in Table 1.

Table 1 Chemical composition, total phenolic acid, and flavonoid content of date seeds and placebo (100 g) and each supplement package (5 g).Sample size

The sample size was calculated based on changes in mean (SD) of hemoglobin A1c (HbA1c) level as the primary outcome in the study by Hashem zadeh et al. (−0.3%) by PASS 22.0 software according to the parameters: α = 0.05, β = 0.1, CI = 0.95 which results in 19 participants per arm. Considering a 20% drop-out rate throughout the study, 46 total patients enrolled and were randomly assigned to two equal groups based on the block randomization method.

Body weight, physical activity, and dietary intake measurements

With the least amount of clothes and without shoes, weight was measured using a reliable digital scale to the nearest 0.1 kg (Seca, Hamburg, Germany). The International Physical Activity Questionnaire short form was used to evaluate the participant’s PAL before and after the intervention [24]. A 3-day questionnaire record was used to evaluate dietary intake before and after taking supplements (mean intake of one holiday and two weekdays) [25]. Also, patients were instructed to daily report the food consumed to expert on WhatsApp daily. Data on dietary intake were analyzed by a dietician using Nutritionist IV software.

Measurements of biochemical parameters

All patient communication was handled by a research nurse and a research dietician in the diabetes clinic. Before and after the trial, 10 mL of venous blood was collected from participants after 10–12 h of fasting. Serum samples used to determine the parameters including fasting plasma glucose (FPG), hemoglobin A1c (HbA1c), insulin, uric acid, lipopolysaccharide (LPS), pentosidine, carboxymethyl-lysine (CML), soluble receptor for advanced glycation end products (s-RAGE), high-sensitivity C-reactive protein (hs-CRP), total antioxidant capacity (TAC), malondialdehyde (MDA), glutathione peroxidase (GPX) and superoxide dismutase (SOD). FPG, HbA1c, and uric acid were quantified immediately using commercial diagnostic kits (Pars Azmoon, Tehran, Iran). The remaining serum was stored at −70 °C until the end of the study. HbA1c was determined using the chromatographic method with the HPLC D-10 system (Bio-Rad Laboratories, Hercules, CA, USA). The level of hs-CRP in serum was measured using an immunoturbidimetric technique (Pars Azmoon, Tehran, Iran). Serum levels of pentosidine, CML, s-RAGE (American Life Technol-ogies Inc., Palo Alto, CA), and LPS (LAL kit endpoint-QCL1000; Cambrex BioScience, Walkersville, Maryland, USA) were determined using an ELISA (an enzyme-linked immunosorbent assay) kit. An automatic analyzer used the colorimetric approach to assess the GPx and SOD activities (RANSEL kits and RANSOD kits, respectively). To determine TAC, as a marker of total antioxidant status, Randox kit [RANDOX Laboratories Ltd, UK] was used. The technique is based on the redox reaction of the blue green color (2,2’-azinobis-3-ethylbenzo-thiazoline-6-sulfonic acid (ABTS)) radical cation with antioxidants [26]. The MDA concentration was measured by spectrofluorometer via reaction with thiobarbituric acid (as a TBARS) [27]. The homeostasis model assessment of β-cell function (HOMA-β), Homeostatic model assessment of insulin resistance (HOMA-IR) and quantitative insulin sensitivity check index (QUICKI) were used to assess insulin resistance through the following formula [28]:

$$}-}=[}(}/})\times }(}/})]/22.5$$

$$}-}=[}(}/})\times }(}/})]/405$$

$$}-}=(360* }(}/}))/(}(}/})-63)$$

$$}=1/(\log (},}/})+\log (},}/}))$$

Primary/secondary outcomes

The primary outcomes were to evaluate the impact of DSP versus placebo on the glycemic control (FPG and HbA1c) insulin, insulin resistance (HOMA-IR, HOMA-B, QUICKI, C-peptide), TAC, MDA, CML, pentosidine, s-RAGE, GPX, and SOD in patients with T2DM. The secondary outcomes included LPS, uric acid, and hs-CRP.

Ethical consideration

This study was performed according to the principles of the Declaration of Helsinki. This study was approved by the ethics committee of Tabriz University of Medical Science (IR.TBZMED.REC.1400.752) and registered with the Iranian registry of clinical trials (www.irct.ir/IRCT20150205020965N10).

Statistical analysis

The data were analyzed using SPSS version 20.0. The results for quantitative and qualitative variables were expressed as mean, standard deviation (SD), and percentage, respectively. The Kolmogorov–Smirnov test used to check if variables are normally distributed. A Chi-square test and an unpaired sample student T-test were used to assess between-group qualitative and quantitative data differences at baseline. To compare quantitative variables between-group post-intervention an analysis of covariance (ANCOVA) was performed. Also, Within-group comparisons performed via paired sample student T-test. A significance level of less than 0.05 was considered for all tests.