Dalam kajian percubaan rawak double-blind klinikal, 50 pesakit dengan penyakit Kencing Manis Jenis 2 – Type 2 Diabetes (berusia 40-65 tahun) secara rawak telah dipecahkan kepada dua kumpulan. Peserta dalam setiap kumpulan diberi 250 mL / hari jus delima atau minuman kawalan selama 12 minggu. Penanda biokimia termasuk glukosa plasma puasa (FPG), insulin dan penanda radang telah dicerakin pada garis dasar dan sampel darah.
Kami mendapati bahawa penggunaan Jus Delima mempunyai kesan yang baik pada beberapa penanda radang pada pesakit penyakit Kencing Manis Jenis 2 – Type 2 Diabetes melalui penurunan ketara dalam IL-6 dan hs-CRP dalam plasma. Pelarasan untuk faktor membaurkan termasuk tenaga dan pengambilan karbohidrat menyediakan keputusan yang sama.
Walaupun terdapat beberapa ujian klinikal yang telah diterangkan kesan pengambilan Jus Delima ke atas tekanan darah oksidatif, [6,15,16,17] sahaja bilangan yang terhad kajian dalam vitro telah dinilai pada keradangan menggunakan delima ekstrak. [9,10] Untuk sepanjang pengetahuan kami, ini adalah kajian pertama yang mengkaji kesan pengambilan Jus Delima pada penanda keradangan pada individu dengan T2D.
Pengambilan 1 tahun PJ oleh pesakit hemodialisis menunjukkan pengurangan ketara dalam tahap semua tekanan oksidatif dan keradangan biomarkers berbanding dengan plasebo, dengan pengurangan yang ketara dalam IL6 dan TNF-α.  tahap beredar menurun vaskular lekatan sel molekul 1 (VCAM-1) dilaporkan dengan penggunaan PJ untuk tempoh 2 minggu dalam subjek hipertensi, tetapi tiada kesan ketara diperhatikan dari PJ pada tahap serum intercellular melekat molekul 1 (ICAM -1), hs-CRP, IL-6 dan profil lipid. 
Effects of pomegranate juice consumption on inflammatory markers in patients with type 2 diabetes: A randomized, placebo-controlled trial
Diabetes causes the increased concentration of circulatory cytokines as a result of inflammation. Considering that pomegranate juice (PJ) is known to have antioxidant and anti-inflammatory properties, the purpose of this study was to determine the effects of PJ consumption on markers of inflammation in patients with type 2 diabetes (T2D).
Materials and Methods:
In a randomized, double-blind clinical trial study, 50 patients with T2D (40-65 years old) were randomly assigned to one of two groups. Participants in each group received either 250 mL/day PJ or a control beverage for 12 weeks. Biochemical markers including fasting plasma glucose (FPG), insulin and inflammatory markers were assayed on the baseline and follow-up blood samples.
In all, 44 patients in two groups were included in the analysis: PJ (n = 22) and placebo (n= 22). After 12 weeks of intervention, in the PJ group, there were 32% and 30% significant decreases in plasma C-reactive protein (hs-CRP) and Interlukin-6, respectively (P < 0.05). The mean ± SD plasma interlukin-6 (7.1 ± 5.6 vs. 11.9 ± 14.4 mg/L) and hs-CRP (1791 ± 1657 and 1953 ± 1561 ng/mL) concentrations in the PJ group were significantly lower than the placebo group after intervention (P < 0.05).
PJ consumption by patients with T2D does not affect FPG or the insulin resistance index (HOMA-IR), whereas it does reduce Interlukin-6 and hs-CRP concentrations in plasma. Therefore, PJ consumption may have an anti-inflammatory effect in patients with T2D.
Type 2 diabetes (T2D) is one of the main noncommunicable chronic diseases, and its complications have become a major cause of morbidity and mortality worldwide. The increase in adipose tissue mass observed in obesity can lead to chronic activation of the innate immune system, which can lead to insulin resistance and T2D. All of the following, heart disease, metabolic syndrome and T2D, have in common the increased concentration of circulatory cytokines as a result of inflammation. Low-grade systemic inflammation is characterized by a two- to threefold increase in systemic plasma concentrations of cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-6 and C-reactive protein (CRP). The cytokines produced by adipose tissue have a key role in promoting atherosclerosis and, therefore, cardiovascular disease (CVD).
Pomegranate juice (PJ) contains diverse groups of polyphenols, including ellagitanins, gallotannins, and ellegic acid, as well as flavonoids, such as anthocyanins. However, its antioxidant activity is mainly due to hydrolysable tannin, including punicalagins, anthocyanins and ellagic acid. PJ antioxidant activity was found to be three times higher than that of red wines and green teas, and two- to six-folds stronger than the other natural beverages.
Evidence for the clinical benefits of PJ has been reported in some studies,[6,7,8] but few studies that have assessed its impact on inflammation have been conducted in vitro or used pomegranate extract.[9,10] Although patients with T2D are exposed to severe systemic oxidative stress and inflammation, data on the effect of PJ intake among these patients is limited. In this study, the effects of PJ consumption on markers of inflammation in patients with T2D were investigated.
MATERIALS AND METHODS
This study was a randomized, double-blind, placebo-controlled trial.
Subjects and ethical aspects
T2D patients, aged 40-65 years, with a diagnosis of at least 1 year, were recruited from the Charity Foundation for Special Diseases and Health Center of District 2 of Tehran, Iran. All the patients controlled their diabetes with oral hypoglycemic agents. Patients were excluded from the trial if they were smoking, suffering from any other chronic diseases and taking estrogen or progesterone (if female) or antioxidant supplements or used insulin as diabetes medication.
At baseline, patients were stratified by sex and randomly assigned to one of two groups: Group A (PJ, n = 25) and group B (placebo, n = 25). Random allocation of patients to treatment groups was performed by sequentially numbered containers. Randomization was performed by an assistant and the group allocation was blinded for the investigator and participants.
Written informed consent was obtained from all patients. Ethic approval for the trial was obtained from the ethical committee of the National Nutrition and Food Technology Research Institute (Tehran, Iran). This clinical trial has been registered in the Iranian Registry of Clinical Trials at htpp://www.irct.ir with the following identification: IRCT201206144010N8, and was conducted between November 2012 and March 2013.
At baseline, patients were stratified by sex and randomly assigned to consume 250 mL/day PJ or a control beverage of similar color and energy content for 12 weeks. The study product was packaged in single-serving bottles labeled such that neither subjects nor staff members were aware of the treatment assignment.
Subjects were advised not to change their dietary habits, physical activities or drug medication. The dietary intakes of subjects were assessed using a 3-day dietary recall (two weekdays and one weekend day) at baseline and at the end of 12 weeks. Patients’ diets were analyzed by the Nutritionist IV software (N Squared Computing, San Bruno, CA, USA).
At baseline and after 12 weeks of intervention, 10 mL blood was collected from each patient after a 12-14-h overnight fasting. Blood samples to which an anti-coagulating agent was added were centrifuged at 4000 rpm for 10 min. The plasma samples were separated into aliquots and were frozen at -70°C until they were assayed. Fasting plasma glucose (FPG) concentration was assessed using the enzymatic colorimetric method using glucose oxidase by commercial kits (Pars Azemoon, Tehran, Iran) and a Selectra 2 auto-analyzer (Vital Scientific, Spankeren, the Netherlands). The coefficient of variation (CV) for FPG was 1.3%. Plasma insulin and inflammatory markers were assessed using the enzyme-linked immunosorbent assay method (ELISA). Insulin was measured using a commercial kit (Mercodia, Uppsala, Sweden), with a CV of 5.0%. The plasma concentration of TNF-α and high-sensitive CRP (hs-CRP) were assayed by related kits (Komobiotech Inc., Gangseo-gu Seoul, Korea) and Diagnostics (Biochem, Canada Inc., Ontario, Canada), respectively. Interlukin-6 was measured using commercial kits (Biolegend, CA, USA). All immunoassays were performed by an ELISA reader Sunrise, Tecan Co. Salzburg, Austria. CVs for TNF-α, hs-CRP and IL-6 were 6.7, 6.3 and 7.1, respectively.
Weight and height were measured using a balance Seca scale with a stadiometer at the baseline and at the end of the intervention. Weight was measured while the subjects were minimally clothed and not wearing shoes, and was recorded to the nearest 100 g. Height was measured while subjects were standing without shoes, with their shoulders in a normal position, and was recorded to the nearest 0.5 cm. Body mass index (BMI) was calculated as weight (kg) divided by square of height (m).
In addition, HOMA-IR was calculated as: [Glucose (mmol/L) × Insulin (μU/mL)]/22.5.
To ascertain patient compliance, we provided each patient with a fixed number of PJ bottles and instructions to return the unused bottles at the end of the study. Based on the number of returned bottles by each patient, their compliance was determined, which was 90% for our participants. No adverse events were reported.
Pomegranate and placebo juice
In order to choose the commercial PJ with the highest polyphenol levels, several hand-squeezed and various commercially available juices were analyzed using the colorimetric assay. The phenols were determined by the Folin-Ciocalteau reagent, using Gallic acid as a standard. Total flavonoid content was measured by the aluminum chloride colorimetric assay using Catechin as a standard. PJ was diluted 1:10 (v:v) to measure its total antioxidant capacity (TAC), which was based on the inhibition percent of ABTS and comparison with bovine serum albumin (BSA) standard curve. PJ composition is shown in Table 1.
PJ and placebo was prepared by Alifard Inc., Tehran, Iran. We used special essence and formula of Wonderful Variety, Pom, supplied by Roll International Corporation, Los Angeles, CA, USA, for preparing the placebo. The sugar content of the placebo was 50% of glucose and 50% of fructose. Analyzing the placebo juice using the colorimetric assay verified that it had no polyphenols.
The juice and the placebo were kept at room temperature (<25°C) until opened, as recommended by the manufacturer.
Sample size estimation and statistical analysis
The results are expressed as mean ± SD for quantitative variables and n (%) for qualitative variables, and differences were considered significant at P ≤ 0.05.
Statistical analysis of data was performed using the Statistical Package for the Social Sciences (SPSS Inc., Chicago, IL, USA) for Windows version 16.0. A χ2 test was used to compare the qualitative variables between the two groups.
Because all quantitative parameters according to the Kolmogorov–Smirnov test had normal distributions, a paired t-test was used to compare the pre- and postintervention variables within groups, and the mean changes within groups were tested using the Student’s t-test. Adjustment for differences in baselines covariates and changes in variables during the study were performed by analysis of covariance using general linear models.
The sample size was designed to detect a 2 ng/L difference of plasma IL-6 between the groups with 95% confidence interval and 90% power. In all, 44 patients [PJ (n = 22), placebo (n = 22)] were included in the analysis as presented in the flow chart [Figure 1].
The baseline characteristics of patients did not differ significantly between the PJ-treated and the placebo groups [Table 2]. Anthropometric factors had no significant differences between the two groups at the baseline or at the end of Week 12; in addition, these factors did not change significantly within the groups during the study [Table 3].
Dietary energy and carbohydrate intake showed significant differences between the two groups at the baseline and at the end of the study (P < 0.05); however, none of the dietary factors changed significantly within each group during the study [Table 3].
No serious adverse events or side-effects were reported. All analyses were adjusted for energy and carbohydrate intake by analysis of covariance.
Plasma interlukin-6 and hs-CRP concentration reduced significantly in the PJ group at the end of the 12th week compared with baseline (P < 0.05), whereas no significant change was observed in the placebo group. Decreases in plasma interlukin-6 and hs-CRP concentrations in the PJ group were significant in comparison with the placebo group (P < 0.05; Table 4), plasma TNF-α concentration did not change significantly within each group or between groups during the study [Table 4].
Mean differences of inflammatory variables compared with baseline values are presented in Table 4. There were 30% and 32% significant decreases in plasma IL-6 and hs-CRP in the PJ and placebo groups (P < 0.05), respectively.
PJ consumption by the patients did not significantly affect the levels of parameters such as FPG, insulin and HOMA-IR [Table 4].
We found that consumption of PJ had beneficial effects on some inflammatory markers in patients with T2D through a significant decrease in IL-6 and hs-CRP in plasma. Adjustment for confounding factors including energy and carbohydrate intake provide the same results.
Although there are some clinical trials that have described the effects of PJ consumption on the oxidative state and blood pressure,[6,15,16,17] only a limited number of in vitrostudies have assessed the impact of pomegranate on inflammation using the pomegranate extract.[9,10] To the best of our knowledge, this is the first study that examines the effects of PJ consumption on markers of inflammation in individuals with T2D.
A 1-year intake of PJ by hemodialysis patients showed a significant reduction in the level of all oxidative stress and inflammatory biomarkers compared with placebo, with a significant reduction in IL6 and TNF-α. Decreased circulating levels of vascular cell adhesion molecule 1 (VCAM-1) were reported with the consumption of PJ for a period of 2 weeks in hypertensive subjects, but no significant effect was observed from PJ on the serum levels of intercellular adhesion molecule 1 (ICAM-1), hs-CRP, IL-6 and lipid profile.
In vitro studies have shown the positive inhibitory effect of pomegranate punicic acid on the production of TNF-α, induced by priming of reactive oxygen species, on the modulation of inflammatory cell signaling in colon cancer cells via pomegranate ellagitannins and reducing pro-inflammatory cytokine production via inhibiting the gene expression (pomegranate fruit extract). However, findings from in vitro studies often conflict with findings obtained from in vivo ones.
Most studies investigating the antioxidant and anti-inflammatory effects of polyphenols and flavonoids have been conducted in vitro. It has been indicated that flavonoid metabolites have different biological and antioxidant properties than their parent compounds, which suggested that data from in vitro studies using nonmetabolites of flavonoids are of limited relevance to in vivo studies.
Low-grade systemic inflammation such as diabetes is characterized by an increase in systemic plasma concentrations of cytokines such as TNF-α, IL-6 and CRP. In our study, IL-6 and hs-CRP decreased significantly but TNF-α did not show any significant reduction.
Zhao et al. have suggested that plasma TNF-α level may not be detectable in a single blood sampling as this may not reflect the host production of this inflammatory mediator. Human blood mononuclear cells are known to synthesize and secrete TNF-α. It has been suggested that activated monocytes and macrophages are important cellular sources for circulating pro-inflammatory cytokines.
The main antioxidant compounds in PJ are hydrolysable tannins, anthocyanins and ellagic acid derivatives, which contribute to the total antioxidant capacity of the juice. Some mechanisms have been also proposed for the anti-inflammatory properties of PJ, including inhibition of enzymes related to inflammation, such as peroxisome proliferators active receptors (PPARs), nuclear transcription factor kappa B (NF-kB), and NSAID activated gene-1 (NAG-1), scavenging-free radicals or production of urate, which is stimulated by fructose and other compositions of flavonoid-rich foods. In addition, pomegranate enhances endothelial nitric oxide production and bioavailability and protects nitric oxide against oxidative destruction.
Strengths of the current study are a randomized, double-blind, placebo-controlled trial design, acceptable response rate and detailed data collection through face-to-face meetings. Moreover, PJ and placebo were provided for all patients. This study had limitations because we could not measure plasma polyphenol derivatives’ levels nor did we evaluate the level of TNF-α in the blood mononuclear cells. Hence, it is suggested these two be measured in future studies.
In conclusion, our findings suggest that PJ, which is a source of natural sugars, does not affect FPG and insulin resistance index (HOMA-IR) in patients with T2D, and it also acts as an anti-inflammatory agent, lowering some inflammatory factors including IL-6 and hs-CRP. Further studies with a longer intervention period as well as a bigger sample size or using PJ with different polyphenol contents are needed in order to evaluate its protective role against diabetes and its complications and the anti-inflammatory effects of PJ.
This study was supported by the National Nutrition and Food Technology Research Institute of Iran and The Research Institute for Endocrine Sciences with research project number of 435. The authors would like to express their gratitude to the subjects for their participation and cooperation in this research and Alifard Inc., Tehran, Iran for preparing the pomegranate and placebo juices. Special thanks are due to Wonderful Variety, Pom; supplied by Roll International Corporation, Los Angeles, CA, USA for their placebo formulation. The authors wish to acknowledge Ms. Niloofar Shiva for her critical editing of English grammar and syntax of the manuscript.
Source of Support: This study was supported by the National Nutrition and Food Technology Research Institute of Iran and the Research Institute for Endocrine Sciences
Conflict of Interest: None declared.