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Archives of Medical Research
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ORIGINAL ARTICLE
Anti-Inflammatory Effects of Resveratrol in Patients with Ulcerative Colitis: A Randomized, Double-Blind, Placebo-controlled Pilot Study Maryam Samsamikor,a Naser Ebrahimi Daryani,b Parisa Rezanejad Asl,c and Azita Hekmatdoosta a
Department of Clinical Nutrition and Dietetics, Faculty of Nutrition and Food Technology, National Nutrition and Food Technology, Research Institute Shahid Beheshti University of Medical Sciences, Tehran, Iran b Digestive Disease Research Institute, cDepartment of Biostatistics, Tehran University of Medical Sciences, Tehran, Iran Received for publication January 10, 2015; accepted May 12, 2015 (ARCMED-D-15-00019).
Background and Aims. Ulcerative colitis (UC) is a chronic idiopathic inflammatory disease in which reducing pro-inflammatory and/or increasing anti-inflammatory molecules is the mainstay of treatment. The aim of this study was to evaluate the effects of supplementation with resveratrol as an antiinflammatory and antioxidant agent on inflammation and quality of life in patients with active UC. Methods and Results. In this randomized, double-blind, placebo-controlled study, 50 eligible patients with active mild to moderate UC were supplemented with either a 500-mg resveratrol or placebo capsule for 6 weeks. Serum inflammatory markers, activity of NF-kB in peripheral blood mononuclear cells (PBMC) and quality of life were assessed at baseline and at the end of the study. Resveratrol supplementation led to a significant reduction in plasma levels of TNF-a (19.70 12.80 to 17.20 10.09 pg/mL) and hs-CRP (4764.25 2260.48 to 2584.50 1792.80 ng/mL) and activity of NF-kB in PBMCs (0.19 0.05 to 0.10 0.04 OD) ( p ! 0.001), whereas there were no significant changes of these factors in placebo group. Also, the score of inflammatory bowel disease questionnaire -9 (IBDQ-9) increased, whereas the clinical colitis activity index score decreased significantly in the resveratrol group (32.72 7.52 to 47.64 8.59) ( p ! 0.001) and when compared with the placebo group (35.54 9.50 to 41.08 6.59) ( p ! 0.001). Conclusion. Our results indicate that 6 weeks supplementation with 500 mg resveratrol can improve quality of life and disease clinical colitis activity at least partially through inflammation reduction in patients with UC. Whether these effects will be continued in longer duration of treatment remains to be determined. Ó 2015 IMSS. Published by Elsevier Inc. Key Words: Ulcerative colitis, Resveratrol, Inflammation, Anti-oxidant.
Introduction Inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn’s disease are chronic intestinal inflammatory disorders in which reducing proinflammatory and/or increasing anti-inflammatory molecules is the cornerstone of treatment (1). The etiology of Address reprint requests to: Dr. Azita Hekmatdoost, Department of Clinical Nutrition and Dietetics, Faculty of Nutrition and Food Technology, National Nutrition and Food Technology Research Institute, Shahid Beheshti University of Medical Sciences, P.O. Box: 19395-4741, Tehran, Iran; Phone: þ98 21 22360658; FAX: þ98 2122360657; E-mail: [email protected]
this disease has not yet been well understood. It seems that UC develops by a defect in the regulation of the immune response to intestinal flora in genetically susceptible individuals and influenced by environmental factors (1). Currently, patients undergo some types of drug therapies including amino salicylates, glucocorticosteroids, immune modulators, antibiotics and anti-TNF drugs, which cause severe adverse effects such as increasing anti-antibody reactions, risk of allergy, infection and mutagenesis. Thus, there has always been a big challenge to find a way to decrease the toxicity of these drugs (2,3). Resveratrol (trans-5,3,4’-trihydroxystilbene) is a polyphenolic phytoalexin natural compound found in plants
0188-4409/$ - see front matter. Copyright Ó 2015 IMSS. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.arcmed.2015.05.005 ARCMED2015_proof ■ 17-5-2015 13-31-10
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such as grapes, berries and peanuts. A vast range of biological functions are attributed to this molecule, which focuses mainly on modulating oxidation and inflammation (4). It is believed that resveratrol could be one of the possible compounds in preventing and treating chronic inflammation as well as autoimmune abnormalities (5). In experimental models of UC, resveratrol improved the disease activity index and decreased oxidative stress and inflammatory biomarkers, whereas it could increase the activity of tissue antioxidant enzymes (3,5e9). However, there is no study evaluating the effects of resveratrol in patients with UC. Hence, we designed this study to investigate the effects of resveratrol supplementation on inflammatory biomarkers and quality of life in patients with UC.
Materials and Methods Study Design This study was conducted as a double-blind placebocontrolled randomized clinical trial. Patients who met the inclusion criteria were invited to take part in the study. At the first visit, the benefits of participation were explained and they were informed of the research goals, method of intervention and duration of the study. Among 165 patients with UC who were interviewed, 50 eligible patients were recruited in the study (Figure 1). All signed an informed consent form, which was approved by the ethics committee of the National Nutrition and Food Technology Research Institute (NNFTRI), Shahid Beheshti University of Medical Sciences (SBMU), Tehran, Iran. All subjects were informed that they would either be in the supplement group or in the placebo group. At the beginning and at the end of the study, a general information questionnaire, 3-day food record (2 ordinary days and one weekend day), the Simple Clinical
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Colitis Activity Index Questionnaire (SCCAIQ) (10), and inflammatory bowel disease questionnaire -9 (IBDQ-9) were completed (11,12). It has been shown that the SCCAIQ has a good correlation with existing more complex scoring systems and therefore could be useful in the initial assessment of patients with ulcerative colitis (10,12). IBDQ-9 assesses the patients’ quality of life from the four aspects of gastrointestinal, systemic, emotional and social disturbances using nine questions. Responses for each question are scored on a 7-point Likert scale in which 7 corresponds to the highest level of functioning. It has been shown that this questionnaire score is statistically significantly correlated with clinical and colonoscopy scores (11). This questionnaire is linguistically validated in the Iranian population (13). Patients were asked not to change their diet and their physical activities during the study period. They were also asked to inform the researcher of any changes made in the type or dosage of the drugs or of any disease relapse causing hospitalization and prevent taking supplements afterwards. Ethical Considerations The study protocol was approved by the medical ethics committee of NNFTRI, SBMU with certificate No 46477. The research has also been registered in the Iranian Center for Clinical Trials (No. IRCT201209154010N10) and is accessible at the following website: www.irct.ir. Patients Patients were selected from those who were referred to the Gastroenterology Clinic of Imam Khomeini Hospital, Tehran, Iran, considering underlying inclusion criteria: their tendency to take part in the study, mild to moderate active ulcerative colitis, males and females $18 years of age and
Figure 1. Patient enrollment flow chart.
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Anti-inflammatory Effects of Resveratrol on Ulcerative Colitis
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BMI (body mass index) 18.5-30 kg/m2, whereas exclusion criteria included drug type and dosage change in the past month, evident affliction with other intestinal diseases or disorders, cancer, inflammatory diseases and infectious diseases, pregnancy or lactation, history of taking supplements such as multivitamin-mineral, omega-3, polyphenolic and antioxidants, taking anti-coagulation drugs such as heparin and warfarin or NSAIDs (nonsteroidal anti-inflammatory drugs), antihistamines and calcium channel antagonists such as nifedipine within the past month, relapse of the disease that required hospitalization and changing the type and dosage of the drugs during the intervention. Intervention Patients were randomly assigned to take either a capsule of resveratrol or placebo once daily for 6 weeks. Resveratrol capsules contained 500 mg pure trans-resveratrol (Sumabe Company, Australia). Trans-form was used in this study because of the better efficiency and functionality it has compared to the cis-form (14). Placebo capsules contained MCT (medium-chain triglycerides) similar in shape and color to the resveratrol capsules. Before beginning, the bottles containing capsules for the both groups were marked by an individual other than the researchers with A and B codes in order to keep the researchers and patients uninformed about the type of each group (considering the double-blind characteristic of the study). Compliance was evaluated by counting the leftover capsules. If patients had taken O90% of their capsules they were considered compliant.
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was carried out through enzyme-linked immunosorbent assay (ELISA) method using a kit produced by Cell Signaling (US). Plasma concentration of TNF-a was measured using the kit produced by KOMA BIOTECH Inc. (Korea) and the plasma concentration of hs-CRP was measured using the kit produced by Diagnostic Biochem (Canada) through the ELISA method. Statistical Analysis Data from a 3-day food record were analyzed using Nutritionist IV software (v.4.1; First Data Bank Division, The Hearst Corporation). The normality of data distribution was assessed using Shapiro-Wilks test. To compare the qualitative confounding variables of the two groups, chi square test was used. To compare the mean of quantitative variables of each group (when the distribution of data was normal), paired t-test was applied for those data that measured for two times and for comparing the means of the two groups, independent t-test was used. When the distribution of data was not normal to compare them at each group, Wilcoxon test was applied and to compare them between groups the ManneWhitney test was used. To remove the impact of those confounding factors, which had been revealed to be significantly different between groups at the beginning of the study, analysis of covariance test was used; p value was considered to be statistically significant when !0.05. STATA (Statistics & Data) software, 12th ed., was applied for the statistical analysis of data without being aware of the group that received treatment.
Clinical Measurements Anthropometric characteristics including height (without shoes with the precision of 0.5 cm and only at the beginning of the study), weight (with light clothes and with the precision of 100 g using Seca scale), waist circumference (WC) (distance between the lowest rib to the iliac spine) and hip circumference (HC) (the greatest circumference size of the hip) were measured. Patients’ BMI calculated by dividing the weight (kg) by the height squared (m2) and waist-tohip ratio (WHR) was computed by dividing WC/HC.
Results During the 6-week follow-up, only one patient in the placebo group withdrew the study due to personal reason. Forty nine patients remained until the end of the study. Variable distribution method proved that all variables considered in this study, except the patients’ drug dosage, had normal distribution. No significant differences were found between sex, age and height at the beginning of the study Table 1. Personal characteristics and anthropometric measurements of the resveratrol and placebo group at baselinea
Biochemical Measurements Blood samples were collected from patients after 12-14 h fasting at the baseline and after the treatment. To separate plasma from PBMCs (peripheral blood mononuclear cells), the blood samples were first centrifuged, which resulted in separation of plasma. Next, PBMCs were separated based on the Ficoll concentration slope following separation of buffy coat and adding PBS (phosphate-buffered saline), which served as buffer and ultimately addition of Ficoll. Plasma samples and PBMCs were frozen at 80 C for the next procedures. Separated PBMCs were lysed to measure the NF-kB activity in the nucleus. This measurement
Variables Age (years) Height (cm) Weight (kg) Waist circumference (cm) Hip circumference (cm) BMI (kg/m2) Waist/hip ratio
Resveratrol supplement (n [ 25) 37.72 167.27 69.26 82.56 99.6 24.74 0.82
16.23 8.86 12.42 10.83 6.58 3.73 0.09
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p
0.317 0.396 0.656 0.552 0.907 0.992 0.492
39 164.85 67.56 84.58 99.83 24.73 0.84
Values are reported as mean standard deviation.
a
Placebo (n [ 24) 11.81 10.88 14.07 12.80 7.36 3.58 0.08
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Table 2. Confounding dietary factors in the two groups before and after interventiona Placebo
Resveratrol supplement Variable Dietary Dietary Dietary Dietary Dietary Dietary
intake intake intake intake intake intake
Before intervention of of of of of of
vitamin E (mg/day) vitamin C (mg/day) folate (mg/day) carotenoids (mg/day) zinc (mg/day) selenium (mg/day)
21.07 75.30 308.59 695.76 7.73 0.03
(5.58) (30.26) (106.43) (590.21) (1.84) (0.01)
After intervention 22.33 58.73 259.11 582.25 6.97 0.03
(6.64) (31.40) (87.67) (576.97) (1.51) (0.01)
P1b 0.874 0.276 0.89 0.138 0.666 0.358
Before intervention 21.33 72.31 263.72 478.15 7.52 0.03
(5.96) (32.66) (69.61) (394.81) (1.44) (0.01)
After intervention 20.31 78.04 282.71 654.76 7.88 0.03
(5.74) (55.58) (88.67) (517.04) (1.48) (0.01)
P1b
P2c
0.263 0.139 0.353 0.646 0.038 0.764
0.255 0.028 0.053 0.204 0.065 0.324
Values are reported as mean standard deviation. P1 value, difference compared with the value at the beginning of study within groups (paired t-test). c P2 value, mean difference of changes between the two groups (independent t-test). a
b
and between weight, WC, HC, BMI and WHR of the groups at the baseline and at the end of the study (Table 1). Mean and standard deviation of dietary intakes of vitamin C and E, folate, carotenoids, zinc and selenium are displayed in Table 2. Only dietary intake of zinc increased significantly in the placebo group; however, this difference was not significant between the placebo and supplement group. The difference between the two groups was significant just in dietary intake of vitamin C. Because the medication dosage distribution was nonnormal, Wilcoxon nonparametric test was used for intragroup comparisons and Mann-Whitney nonparametric test was used for comparisons between groups. In the resveratrol group, a significant difference in the dosage of all three drugs consumed from 1 month prior to the trial and during the intervention was observed, whereas the difference in the placebo group was significant for the dosages of azathioprine and mesalazine. These differences between groups were not significant (Table 3). As shown in Table 4, the plasma level of hs-CRP in the resveratrol group revealed a significant reduction at the end of the 6th week compared to the beginning of the study ( p # 0.01), whereas no significant decrease was observed in placebo group. These changes were significant between groups ( p # 0.001). The plasma level of TNF-a showed a significant decrease in both groups at the end of the study; however, reduction in the resveratrol group was more than that of the control group ( p # 0.001). At the end of intervention, the total mean concentration of NF-kB p65 revealed a significant decrease between groups after
6 weeks ( p # 0.001). All observed changes remained significant after data adjustment for vitamin C intake. The IBDQ-9 score increased, whereas the SCCAI score decreased significantly in the resveratrol group compared with the placebo group ( p ! 0.001 for each one) and no significant changes were seen after adjustment for vitamin C intake. No complications were observed due to the consumption of resveratrol supplement or placebo during the intervention. Discussion According to our knowledge, this is the first double-blind randomized clinical trial that investigated the effects of resveratrol supplementation on inflammatory factors, clinical disease activity index, and quality of life in patients with UC. Our results have shown that 500 mg resveratrol could decrease the clinical activity index score, serum level of inflammatory factors and the activity of NF-kB in PBMCs, whereas it increased the IBDQ-9 scores. Previous studies have shown the beneficial roles of resveratrol in experimental models of UC. Resveratrol could improve the disease activity index, decrease MDA (malondialdehyde) and MPO (myeloperoxidase) activity and increase the activity of tissue SOD (superoxide dismutase) and GSH-PX (glutathione peroxidase), and decrease the concentration of TFN-a, IFN-g (interferon-g) and proinflammatory interleukins (3,5e9). Martın et al. showed that 10 mg/kg/day resveratrol significantly reduced serum
Table 3. Medicinal confounding factors in the two groups 1 month prior to and during the intervention Placebo
Resveratrol supplement Variable Dose of azathioprine Dose of corticosteroid Dose of mesalazin a
Before intervention
After intervention
P1a
Before intervention
After intervention
P1a
P2b
31 (49.62) 0.16 (0.374) 3040 (1008.54)
53 (52.70) 0.52 (0.510) 3540 (828.15)
0.007 0.003 0.001
52.08 (50.50) 0.42 (0.504) 2916.67 (789.42)
68.75 (42.51) 0.58 (0.504) 3312.5 (777.57)
0.003 0.102 0.011
0.936 0.182 0.552
P1 value: difference compared with the value at the beginning of study within groups (Wilcoxon test). P2 value: mean difference of changes between the two groups (Mann-Whitney Test).
b
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þOD, optical density. Note: Values are reported as mean standard deviation and range. Difference compared with the beginning of the research with Wilcoxon test and NS*; **p value !0.005; ***p value !0.01. a p value for efficiency of the supplement: with Mann-Whitney Test. b p value for efficiency of supplement after adjustment with ANCOVA test adjusted for vitamin C intake.
hs-CRP 4764.25 2260.48 (2503.77e7024.73) 2584.50 1792.80 (791.70e4377.30)*** 3158.67 2419.553 (739.11e5578.22) 3538.42 2348.93 (1189.49e5887.35)* #0.001 !0.0001 (ng/mL) TNF-a 19.70 12.80 (6.90e32.50) 17.20 10.09 (7.11e27.29)*** 20.53 13.34 (7.19e33.87) 23.59 14.82 (8.77e38.40)*** #0.001 !0.0001 (pg/mL) NF-kB p 65 0.19 0.05 (0.140e0.240) 0.10 0.04 (0.060e0.140)*** 0.10 0.05 (0.050e0.150) 0.12 0.03 (0.090e0.0150)** #0.001 !0.0001 (þOD)
b
p a
After intervention Before intervention After intervention Before intervention Variable
Placebo(mean ± SD [range]) Resveratrol supplement(mean ± SD [range])
Table 4. Inflammatory factors in the two groups, before and after intervention
455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509
p
Anti-inflammatory Effects of Resveratrol on Ulcerative Colitis
5
TNF-a and cyclooxygenase-2 (COX-2) and NF-kB gene expression and histological damage of colon, whereas it increased colonocyte apoptosis (4). Singh et al. reported that resveratrol supplementation could decrease COX-2 and increase SIRT-1 (silent mating type information regulation1) gene expression in the colonic immune cells (15). Resveratrol has been applied in many cardiovascularrelated clinical trials; however, there are limited studies evaluating its effects in inflammatory disorders (16,17). Bo et al. reported that supplementation with 500 mg/day resveratrol significantly reduced serum hs-CRP, whereas it increased total antioxidant capacity in smokers (18). Timmers et al. showed that 1 month supplementation with 150 mg/day resveratrol in obese subjects decreased some inflammatory factors such as IL-1b, hs-CRP, TNF-a and IL-6; however, this reduction was significant only in TNF-a (19). We have shown that 500 mg/day resveratrol significantly attenuated inflammatory markers in patients with fatty liver (16). Ren et al. demonstrated that in a dose-related response, resveratrol can suppress NF-kB activation. According to their findings, resveratrol can inhibit IkappaB kinase (IKK), which results in inhibition of NF-kB activation. Thus, this compound can be assumed as a new approach in treatment of inflammatory diseases (20). Busch et al. reported that resveratrol can inhibit NF-kB and phosphatidylinositol 3 kinase (PI3K) induced by IL-1b, through inhibiting IKK activation (21). The current study has several strengths including high participation rate (O90%), the moderately low drop-out rate, successful blinding, its double-blind, placebocontrolled design, and assessment of NF-kB activity in PBMCs to find a mechanism of action for resveratrol effects on inflammatory cytokines in the body. This study also has some limitations. We could not evaluate patient disease activity by colonoscopy because they did not agree to undergo the procedure; however, we used the SCCAIQ, which has been shown that it has a strong correlation with colonoscopy and other clinical assessments in patients with UC. Furthermore, we could not assess TNF-a gene expression in colon tissue because it also needed an invasive procedure to obtain the tissue samples (22). Moreover, we did not measure the serum concentration of resveratrol; however, patient compliance was evaluated by counting the remaining capsules and, if patients had taken !90% of their capsules, it was recommended that they should be excluded from the study. In conclusion, this randomized, double-blind, placebocontrolled trial found some evidence that 6 weeks, 500 mg/day resveratrol supplementation can improve quality of life in patients with active mild to moderate UC, at least partially through attenuation of NF-kB activity resulting in reduction of inflammatory markers. Further studies with longer duration and different dosage of supplementation are needed to confirm and increase the clinical application of the present results.
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Table 5. IBDQ-9 and SCCAI scores in the two groups before and after the intervention Resveratrol supplement Variable IBDQ-9 Score SCCAIQ Score
Placebo
Before intervention
After intervention
Before intervention
After intervention
32.72 7.52 12.34 2.51
47.64 8.59*** 8.14 2.1
35.54 9.50 10.76 2.55
41.08 8.59*** 9.34 2.65
a
p
!0.001 !0.001
p
b
!0.001 !0.001
IBDQ-9, Inflammatory Bowel Disease Questionnaire -9; SCCAI, Simple Colitis Clinical Activity Index. Note: Values are reported as mean standard deviation. Difference compared with the beginning of the research with paired t-test and NS*; **p value !0.005; ***p value !0.01. a p value for efficiency of the supplement with Student t test. b p value for efficiency of supplement after adjustment with ANCOVA test adjusted for vitamin C intake.
Uncited Table Table 5.
Acknowledgments Q1 This study was supported by a grant from the National Nutrition and Food Technology Research Institute of the Shahid Beheshti University and the Digestive Disease Research Institute of the Shariati Hospital. Competing interests: The authors declare that they have no competing interests.
References
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1. Danese S, Fiocchi C. Ulcerative colitis. N Engl J Med 2011;365: 1713e1725. 2. Rutgeerts P. A critical assessment of new therapies in inflammatory bowel disease. J Gastroenterol Hepatol 2002;17:S176eS185. 3. Yao J, Wang JY, Liu L, et al. Anti-oxidant effects of resveratrol on mice with DSS-induced ulcerative colitis. Arch Med Res 2010;41:288e294. 4. Martın AR, Villegas I, Sanchez-Hidalgo M, et al. The effects of resveratrol, a phytoalexin derived from red wines, on chronic inflammation induced in an experimentally induced colitis model. Br J Pharmacol 2006;147:873e885. 5. Cui X, Jin Y, Hofseth AB, et al. Resveratrol suppresses colitis and colon cancer associated with colitis. Cancer Prev Res (Phila) 2010;3: 549e559. 6. Abdallah DM, Ismael NR. Resveratrol abrogates adhesion molecules and protects against TNBS-induced ulcerative colitis in rats. Can J Physiol Pharmacol 2011;89:811e818. 7. Sanchez-Fidalgo S, Cardeno A, Villegas I, et al. Dietary supplementation of resveratrol attenuates chronic colonic inflammation in mice. Eur J Pharmacol 2010;633:78e84. 8. Singh UP, Singh NP, Busbee B, et al. Alternative medicines as emerging therapies for inflammatory bowel diseases. Int Rev Immunol 2012;31:66e84. 9. Singh UP, Singh NP, Singh B, et al. Role of resveratrol-induced CD11bþ Gr-1þ myeloid derived suppressor cells (MDSCs) in the reduction of CXCR3þ T cells and amelioration of chronic colitis in IL-10/ mice. Brain Behav Immun 2012;26:72.
10. Walmsley RS, Ayres RC, Pounder RE, et al. A simple clinical colitis activity index. Gut 1998;43:29e32. 11. Verissimo R. Quality of life in inflammatory bowel disease: psychometric evaluation of an IBDQ cross-culturally adapted version. J Gastrointestin Liver Dis 2008;17:439e444. 12. Casellas F, Alcala MJ, Prieto L, et al. Assessment of the influence of disease activity on the quality of life of patients with inflammatory bowel disease using a short questionnaire. Am J Gastroenterol 2004; 99:457e461. 13. Gholamrezaei A, Haghdani S, Shemshaki H, et al. Linguistic Validation of the Inflammatory Bowel Disease Questionnaire-Short Form (IBDQ-9) in Iranian Population. J Isfahan Med School 2011;28. 14. Malekyian EF, Shavandi N, Saremi A. The effect of one session Resvin (resveratrol) supplementation on total antioxidant capacity’ superoxide dismutase and creatine kinase in elite women volleyball players. ZUMS J 2013;21:64e75. 15. Singh UP, Singh NP, Singh B, et al. Resveratrol (trans-3,5,4’-trihydroxystilbene) induces silent mating type information regulation-1 and down-regulates nuclear transcription factor-kappaB activation to abrogate dextran sulfate sodium-induced colitis. J Pharmacol Exp Ther 2010;332:829e839. 16. Faghihzadeh F, Adibi P, Rafiei R, et al. Resveratrol supplementation improves inflammatory biomarkers in patients with nonalcoholic fatty liver disease. Nutr Res 2014;34:837e843. 17. Udenigwe CC, Ramprasath VR, Aluko RE, et al. Potential of resveratrol in anticancer and anti-inflammatory therapy. Nutr Rev 2008;66: 445e554. 18. Bo S, Ciccone G, Castiglione A, et al. Anti-inflammatory and antioxidant effects of resveratrol in healthy smokers a randomized, doubleblind, placebo-controlled, cross-over trial. Curr Med Chem 2013;20: 1323e1331. 19. Timmers S, Konings E, Bilet L, et al. Calorie restriction-like effects of 30 days of resveratrol supplementation on energy metabolism and metabolic profile in obese humans. Cell Metab 2011;14:612e622. 20. Ren Z, Wang L, Cui J, et al. Resveratrol inhibits NF-kB signaling through suppression of p65 and IkappaB kinase activities. Pharmazie 2013;68:689e694. 21. Busch F, Mobasheri A, Shayan P, et al. Resveratrol modulates interleukin-1beta-induced phosphatidylinositol 3-kinase and nuclear factor kappaB signaling pathways in human tenocytes. J Biol Chem 2012;287:38050e38063. 22. Tibble J, Bjarnason I. Non-invasive investigation of inflammatory bowel disease. World J Gastroenterol 2001;7:460e465.
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ORIGINAL ARTICLE
Anti-Inflammatory Effects of Resveratrol in Patients with Ulcerative Colitis: A Randomized, Double-Blind, Placebo-controlled Pilot Study Maryam Samsamikor,a Naser Ebrahimi Daryani,b Parisa Rezanejad Asl,c and Azita Hekmatdoosta a
Department of Clinical Nutrition and Dietetics, Faculty of Nutrition and Food Technology, National Nutrition and Food Technology, Research Institute Shahid Beheshti University of Medical Sciences, Tehran, Iran b Digestive Disease Research Institute, cDepartment of Biostatistics, Tehran University of Medical Sciences, Tehran, Iran Received for publication January 10, 2015; accepted May 12, 2015 (ARCMED-D-15-00019).
Background and Aims. Ulcerative colitis (UC) is a chronic idiopathic inflammatory disease in which reducing pro-inflammatory and/or increasing anti-inflammatory molecules is the mainstay of treatment. The aim of this study was to evaluate the effects of supplementation with resveratrol as an antiinflammatory and antioxidant agent on inflammation and quality of life in patients with active UC. Methods and Results. In this randomized, double-blind, placebo-controlled study, 50 eligible patients with active mild to moderate UC were supplemented with either a 500-mg resveratrol or placebo capsule for 6 weeks. Serum inflammatory markers, activity of NF-kB in peripheral blood mononuclear cells (PBMC) and quality of life were assessed at baseline and at the end of the study. Resveratrol supplementation led to a significant reduction in plasma levels of TNF-a (19.70 12.80 to 17.20 10.09 pg/mL) and hs-CRP (4764.25 2260.48 to 2584.50 1792.80 ng/mL) and activity of NF-kB in PBMCs (0.19 0.05 to 0.10 0.04 OD) ( p ! 0.001), whereas there were no significant changes of these factors in placebo group. Also, the score of inflammatory bowel disease questionnaire -9 (IBDQ-9) increased, whereas the clinical colitis activity index score decreased significantly in the resveratrol group (32.72 7.52 to 47.64 8.59) ( p ! 0.001) and when compared with the placebo group (35.54 9.50 to 41.08 6.59) ( p ! 0.001). Conclusion. Our results indicate that 6 weeks supplementation with 500 mg resveratrol can improve quality of life and disease clinical colitis activity at least partially through inflammation reduction in patients with UC. Whether these effects will be continued in longer duration of treatment remains to be determined. Ó 2015 IMSS. Published by Elsevier Inc. Key Words: Ulcerative colitis, Resveratrol, Inflammation, Anti-oxidant.
Introduction Inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn’s disease are chronic intestinal inflammatory disorders in which reducing proinflammatory and/or increasing anti-inflammatory molecules is the cornerstone of treatment (1). The etiology of Address reprint requests to: Dr. Azita Hekmatdoost, Department of Clinical Nutrition and Dietetics, Faculty of Nutrition and Food Technology, National Nutrition and Food Technology Research Institute, Shahid Beheshti University of Medical Sciences, P.O. Box: 19395-4741, Tehran, Iran; Phone: þ98 21 22360658; FAX: þ98 2122360657; E-mail: [email protected]
this disease has not yet been well understood. It seems that UC develops by a defect in the regulation of the immune response to intestinal flora in genetically susceptible individuals and influenced by environmental factors (1). Currently, patients undergo some types of drug therapies including amino salicylates, glucocorticosteroids, immune modulators, antibiotics and anti-TNF drugs, which cause severe adverse effects such as increasing anti-antibody reactions, risk of allergy, infection and mutagenesis. Thus, there has always been a big challenge to find a way to decrease the toxicity of these drugs (2,3). Resveratrol (trans-5,3,4’-trihydroxystilbene) is a polyphenolic phytoalexin natural compound found in plants
0188-4409/$ - see front matter. Copyright Ó 2015 IMSS. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.arcmed.2015.05.005 ARCMED2015_proof ■ 17-5-2015 13-31-10
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such as grapes, berries and peanuts. A vast range of biological functions are attributed to this molecule, which focuses mainly on modulating oxidation and inflammation (4). It is believed that resveratrol could be one of the possible compounds in preventing and treating chronic inflammation as well as autoimmune abnormalities (5). In experimental models of UC, resveratrol improved the disease activity index and decreased oxidative stress and inflammatory biomarkers, whereas it could increase the activity of tissue antioxidant enzymes (3,5e9). However, there is no study evaluating the effects of resveratrol in patients with UC. Hence, we designed this study to investigate the effects of resveratrol supplementation on inflammatory biomarkers and quality of life in patients with UC.
Materials and Methods Study Design This study was conducted as a double-blind placebocontrolled randomized clinical trial. Patients who met the inclusion criteria were invited to take part in the study. At the first visit, the benefits of participation were explained and they were informed of the research goals, method of intervention and duration of the study. Among 165 patients with UC who were interviewed, 50 eligible patients were recruited in the study (Figure 1). All signed an informed consent form, which was approved by the ethics committee of the National Nutrition and Food Technology Research Institute (NNFTRI), Shahid Beheshti University of Medical Sciences (SBMU), Tehran, Iran. All subjects were informed that they would either be in the supplement group or in the placebo group. At the beginning and at the end of the study, a general information questionnaire, 3-day food record (2 ordinary days and one weekend day), the Simple Clinical
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Colitis Activity Index Questionnaire (SCCAIQ) (10), and inflammatory bowel disease questionnaire -9 (IBDQ-9) were completed (11,12). It has been shown that the SCCAIQ has a good correlation with existing more complex scoring systems and therefore could be useful in the initial assessment of patients with ulcerative colitis (10,12). IBDQ-9 assesses the patients’ quality of life from the four aspects of gastrointestinal, systemic, emotional and social disturbances using nine questions. Responses for each question are scored on a 7-point Likert scale in which 7 corresponds to the highest level of functioning. It has been shown that this questionnaire score is statistically significantly correlated with clinical and colonoscopy scores (11). This questionnaire is linguistically validated in the Iranian population (13). Patients were asked not to change their diet and their physical activities during the study period. They were also asked to inform the researcher of any changes made in the type or dosage of the drugs or of any disease relapse causing hospitalization and prevent taking supplements afterwards. Ethical Considerations The study protocol was approved by the medical ethics committee of NNFTRI, SBMU with certificate No 46477. The research has also been registered in the Iranian Center for Clinical Trials (No. IRCT201209154010N10) and is accessible at the following website: www.irct.ir. Patients Patients were selected from those who were referred to the Gastroenterology Clinic of Imam Khomeini Hospital, Tehran, Iran, considering underlying inclusion criteria: their tendency to take part in the study, mild to moderate active ulcerative colitis, males and females $18 years of age and
Figure 1. Patient enrollment flow chart.
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Anti-inflammatory Effects of Resveratrol on Ulcerative Colitis
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BMI (body mass index) 18.5-30 kg/m2, whereas exclusion criteria included drug type and dosage change in the past month, evident affliction with other intestinal diseases or disorders, cancer, inflammatory diseases and infectious diseases, pregnancy or lactation, history of taking supplements such as multivitamin-mineral, omega-3, polyphenolic and antioxidants, taking anti-coagulation drugs such as heparin and warfarin or NSAIDs (nonsteroidal anti-inflammatory drugs), antihistamines and calcium channel antagonists such as nifedipine within the past month, relapse of the disease that required hospitalization and changing the type and dosage of the drugs during the intervention. Intervention Patients were randomly assigned to take either a capsule of resveratrol or placebo once daily for 6 weeks. Resveratrol capsules contained 500 mg pure trans-resveratrol (Sumabe Company, Australia). Trans-form was used in this study because of the better efficiency and functionality it has compared to the cis-form (14). Placebo capsules contained MCT (medium-chain triglycerides) similar in shape and color to the resveratrol capsules. Before beginning, the bottles containing capsules for the both groups were marked by an individual other than the researchers with A and B codes in order to keep the researchers and patients uninformed about the type of each group (considering the double-blind characteristic of the study). Compliance was evaluated by counting the leftover capsules. If patients had taken O90% of their capsules they were considered compliant.
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was carried out through enzyme-linked immunosorbent assay (ELISA) method using a kit produced by Cell Signaling (US). Plasma concentration of TNF-a was measured using the kit produced by KOMA BIOTECH Inc. (Korea) and the plasma concentration of hs-CRP was measured using the kit produced by Diagnostic Biochem (Canada) through the ELISA method. Statistical Analysis Data from a 3-day food record were analyzed using Nutritionist IV software (v.4.1; First Data Bank Division, The Hearst Corporation). The normality of data distribution was assessed using Shapiro-Wilks test. To compare the qualitative confounding variables of the two groups, chi square test was used. To compare the mean of quantitative variables of each group (when the distribution of data was normal), paired t-test was applied for those data that measured for two times and for comparing the means of the two groups, independent t-test was used. When the distribution of data was not normal to compare them at each group, Wilcoxon test was applied and to compare them between groups the ManneWhitney test was used. To remove the impact of those confounding factors, which had been revealed to be significantly different between groups at the beginning of the study, analysis of covariance test was used; p value was considered to be statistically significant when !0.05. STATA (Statistics & Data) software, 12th ed., was applied for the statistical analysis of data without being aware of the group that received treatment.
Clinical Measurements Anthropometric characteristics including height (without shoes with the precision of 0.5 cm and only at the beginning of the study), weight (with light clothes and with the precision of 100 g using Seca scale), waist circumference (WC) (distance between the lowest rib to the iliac spine) and hip circumference (HC) (the greatest circumference size of the hip) were measured. Patients’ BMI calculated by dividing the weight (kg) by the height squared (m2) and waist-tohip ratio (WHR) was computed by dividing WC/HC.
Results During the 6-week follow-up, only one patient in the placebo group withdrew the study due to personal reason. Forty nine patients remained until the end of the study. Variable distribution method proved that all variables considered in this study, except the patients’ drug dosage, had normal distribution. No significant differences were found between sex, age and height at the beginning of the study Table 1. Personal characteristics and anthropometric measurements of the resveratrol and placebo group at baselinea
Biochemical Measurements Blood samples were collected from patients after 12-14 h fasting at the baseline and after the treatment. To separate plasma from PBMCs (peripheral blood mononuclear cells), the blood samples were first centrifuged, which resulted in separation of plasma. Next, PBMCs were separated based on the Ficoll concentration slope following separation of buffy coat and adding PBS (phosphate-buffered saline), which served as buffer and ultimately addition of Ficoll. Plasma samples and PBMCs were frozen at 80 C for the next procedures. Separated PBMCs were lysed to measure the NF-kB activity in the nucleus. This measurement
Variables Age (years) Height (cm) Weight (kg) Waist circumference (cm) Hip circumference (cm) BMI (kg/m2) Waist/hip ratio
Resveratrol supplement (n [ 25) 37.72 167.27 69.26 82.56 99.6 24.74 0.82
16.23 8.86 12.42 10.83 6.58 3.73 0.09
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p
0.317 0.396 0.656 0.552 0.907 0.992 0.492
39 164.85 67.56 84.58 99.83 24.73 0.84
Values are reported as mean standard deviation.
a
Placebo (n [ 24) 11.81 10.88 14.07 12.80 7.36 3.58 0.08
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Table 2. Confounding dietary factors in the two groups before and after interventiona Placebo
Resveratrol supplement Variable Dietary Dietary Dietary Dietary Dietary Dietary
intake intake intake intake intake intake
Before intervention of of of of of of
vitamin E (mg/day) vitamin C (mg/day) folate (mg/day) carotenoids (mg/day) zinc (mg/day) selenium (mg/day)
21.07 75.30 308.59 695.76 7.73 0.03
(5.58) (30.26) (106.43) (590.21) (1.84) (0.01)
After intervention 22.33 58.73 259.11 582.25 6.97 0.03
(6.64) (31.40) (87.67) (576.97) (1.51) (0.01)
P1b 0.874 0.276 0.89 0.138 0.666 0.358
Before intervention 21.33 72.31 263.72 478.15 7.52 0.03
(5.96) (32.66) (69.61) (394.81) (1.44) (0.01)
After intervention 20.31 78.04 282.71 654.76 7.88 0.03
(5.74) (55.58) (88.67) (517.04) (1.48) (0.01)
P1b
P2c
0.263 0.139 0.353 0.646 0.038 0.764
0.255 0.028 0.053 0.204 0.065 0.324
Values are reported as mean standard deviation. P1 value, difference compared with the value at the beginning of study within groups (paired t-test). c P2 value, mean difference of changes between the two groups (independent t-test). a
b
and between weight, WC, HC, BMI and WHR of the groups at the baseline and at the end of the study (Table 1). Mean and standard deviation of dietary intakes of vitamin C and E, folate, carotenoids, zinc and selenium are displayed in Table 2. Only dietary intake of zinc increased significantly in the placebo group; however, this difference was not significant between the placebo and supplement group. The difference between the two groups was significant just in dietary intake of vitamin C. Because the medication dosage distribution was nonnormal, Wilcoxon nonparametric test was used for intragroup comparisons and Mann-Whitney nonparametric test was used for comparisons between groups. In the resveratrol group, a significant difference in the dosage of all three drugs consumed from 1 month prior to the trial and during the intervention was observed, whereas the difference in the placebo group was significant for the dosages of azathioprine and mesalazine. These differences between groups were not significant (Table 3). As shown in Table 4, the plasma level of hs-CRP in the resveratrol group revealed a significant reduction at the end of the 6th week compared to the beginning of the study ( p # 0.01), whereas no significant decrease was observed in placebo group. These changes were significant between groups ( p # 0.001). The plasma level of TNF-a showed a significant decrease in both groups at the end of the study; however, reduction in the resveratrol group was more than that of the control group ( p # 0.001). At the end of intervention, the total mean concentration of NF-kB p65 revealed a significant decrease between groups after
6 weeks ( p # 0.001). All observed changes remained significant after data adjustment for vitamin C intake. The IBDQ-9 score increased, whereas the SCCAI score decreased significantly in the resveratrol group compared with the placebo group ( p ! 0.001 for each one) and no significant changes were seen after adjustment for vitamin C intake. No complications were observed due to the consumption of resveratrol supplement or placebo during the intervention. Discussion According to our knowledge, this is the first double-blind randomized clinical trial that investigated the effects of resveratrol supplementation on inflammatory factors, clinical disease activity index, and quality of life in patients with UC. Our results have shown that 500 mg resveratrol could decrease the clinical activity index score, serum level of inflammatory factors and the activity of NF-kB in PBMCs, whereas it increased the IBDQ-9 scores. Previous studies have shown the beneficial roles of resveratrol in experimental models of UC. Resveratrol could improve the disease activity index, decrease MDA (malondialdehyde) and MPO (myeloperoxidase) activity and increase the activity of tissue SOD (superoxide dismutase) and GSH-PX (glutathione peroxidase), and decrease the concentration of TFN-a, IFN-g (interferon-g) and proinflammatory interleukins (3,5e9). Martın et al. showed that 10 mg/kg/day resveratrol significantly reduced serum
Table 3. Medicinal confounding factors in the two groups 1 month prior to and during the intervention Placebo
Resveratrol supplement Variable Dose of azathioprine Dose of corticosteroid Dose of mesalazin a
Before intervention
After intervention
P1a
Before intervention
After intervention
P1a
P2b
31 (49.62) 0.16 (0.374) 3040 (1008.54)
53 (52.70) 0.52 (0.510) 3540 (828.15)
0.007 0.003 0.001
52.08 (50.50) 0.42 (0.504) 2916.67 (789.42)
68.75 (42.51) 0.58 (0.504) 3312.5 (777.57)
0.003 0.102 0.011
0.936 0.182 0.552
P1 value: difference compared with the value at the beginning of study within groups (Wilcoxon test). P2 value: mean difference of changes between the two groups (Mann-Whitney Test).
b
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þOD, optical density. Note: Values are reported as mean standard deviation and range. Difference compared with the beginning of the research with Wilcoxon test and NS*; **p value !0.005; ***p value !0.01. a p value for efficiency of the supplement: with Mann-Whitney Test. b p value for efficiency of supplement after adjustment with ANCOVA test adjusted for vitamin C intake.
hs-CRP 4764.25 2260.48 (2503.77e7024.73) 2584.50 1792.80 (791.70e4377.30)*** 3158.67 2419.553 (739.11e5578.22) 3538.42 2348.93 (1189.49e5887.35)* #0.001 !0.0001 (ng/mL) TNF-a 19.70 12.80 (6.90e32.50) 17.20 10.09 (7.11e27.29)*** 20.53 13.34 (7.19e33.87) 23.59 14.82 (8.77e38.40)*** #0.001 !0.0001 (pg/mL) NF-kB p 65 0.19 0.05 (0.140e0.240) 0.10 0.04 (0.060e0.140)*** 0.10 0.05 (0.050e0.150) 0.12 0.03 (0.090e0.0150)** #0.001 !0.0001 (þOD)
b
p a
After intervention Before intervention After intervention Before intervention Variable
Placebo(mean ± SD [range]) Resveratrol supplement(mean ± SD [range])
Table 4. Inflammatory factors in the two groups, before and after intervention
455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509
p
Anti-inflammatory Effects of Resveratrol on Ulcerative Colitis
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TNF-a and cyclooxygenase-2 (COX-2) and NF-kB gene expression and histological damage of colon, whereas it increased colonocyte apoptosis (4). Singh et al. reported that resveratrol supplementation could decrease COX-2 and increase SIRT-1 (silent mating type information regulation1) gene expression in the colonic immune cells (15). Resveratrol has been applied in many cardiovascularrelated clinical trials; however, there are limited studies evaluating its effects in inflammatory disorders (16,17). Bo et al. reported that supplementation with 500 mg/day resveratrol significantly reduced serum hs-CRP, whereas it increased total antioxidant capacity in smokers (18). Timmers et al. showed that 1 month supplementation with 150 mg/day resveratrol in obese subjects decreased some inflammatory factors such as IL-1b, hs-CRP, TNF-a and IL-6; however, this reduction was significant only in TNF-a (19). We have shown that 500 mg/day resveratrol significantly attenuated inflammatory markers in patients with fatty liver (16). Ren et al. demonstrated that in a dose-related response, resveratrol can suppress NF-kB activation. According to their findings, resveratrol can inhibit IkappaB kinase (IKK), which results in inhibition of NF-kB activation. Thus, this compound can be assumed as a new approach in treatment of inflammatory diseases (20). Busch et al. reported that resveratrol can inhibit NF-kB and phosphatidylinositol 3 kinase (PI3K) induced by IL-1b, through inhibiting IKK activation (21). The current study has several strengths including high participation rate (O90%), the moderately low drop-out rate, successful blinding, its double-blind, placebocontrolled design, and assessment of NF-kB activity in PBMCs to find a mechanism of action for resveratrol effects on inflammatory cytokines in the body. This study also has some limitations. We could not evaluate patient disease activity by colonoscopy because they did not agree to undergo the procedure; however, we used the SCCAIQ, which has been shown that it has a strong correlation with colonoscopy and other clinical assessments in patients with UC. Furthermore, we could not assess TNF-a gene expression in colon tissue because it also needed an invasive procedure to obtain the tissue samples (22). Moreover, we did not measure the serum concentration of resveratrol; however, patient compliance was evaluated by counting the remaining capsules and, if patients had taken !90% of their capsules, it was recommended that they should be excluded from the study. In conclusion, this randomized, double-blind, placebocontrolled trial found some evidence that 6 weeks, 500 mg/day resveratrol supplementation can improve quality of life in patients with active mild to moderate UC, at least partially through attenuation of NF-kB activity resulting in reduction of inflammatory markers. Further studies with longer duration and different dosage of supplementation are needed to confirm and increase the clinical application of the present results.
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Table 5. IBDQ-9 and SCCAI scores in the two groups before and after the intervention Resveratrol supplement Variable IBDQ-9 Score SCCAIQ Score
Placebo
Before intervention
After intervention
Before intervention
After intervention
32.72 7.52 12.34 2.51
47.64 8.59*** 8.14 2.1
35.54 9.50 10.76 2.55
41.08 8.59*** 9.34 2.65
a
p
!0.001 !0.001
p
b
!0.001 !0.001
IBDQ-9, Inflammatory Bowel Disease Questionnaire -9; SCCAI, Simple Colitis Clinical Activity Index. Note: Values are reported as mean standard deviation. Difference compared with the beginning of the research with paired t-test and NS*; **p value !0.005; ***p value !0.01. a p value for efficiency of the supplement with Student t test. b p value for efficiency of supplement after adjustment with ANCOVA test adjusted for vitamin C intake.
Uncited Table Table 5.
Acknowledgments Q1 This study was supported by a grant from the National Nutrition and Food Technology Research Institute of the Shahid Beheshti University and the Digestive Disease Research Institute of the Shariati Hospital. Competing interests: The authors declare that they have no competing interests.
References
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10. Walmsley RS, Ayres RC, Pounder RE, et al. A simple clinical colitis activity index. Gut 1998;43:29e32. 11. Verissimo R. Quality of life in inflammatory bowel disease: psychometric evaluation of an IBDQ cross-culturally adapted version. J Gastrointestin Liver Dis 2008;17:439e444. 12. Casellas F, Alcala MJ, Prieto L, et al. Assessment of the influence of disease activity on the quality of life of patients with inflammatory bowel disease using a short questionnaire. Am J Gastroenterol 2004; 99:457e461. 13. Gholamrezaei A, Haghdani S, Shemshaki H, et al. Linguistic Validation of the Inflammatory Bowel Disease Questionnaire-Short Form (IBDQ-9) in Iranian Population. J Isfahan Med School 2011;28. 14. Malekyian EF, Shavandi N, Saremi A. The effect of one session Resvin (resveratrol) supplementation on total antioxidant capacity’ superoxide dismutase and creatine kinase in elite women volleyball players. ZUMS J 2013;21:64e75. 15. Singh UP, Singh NP, Singh B, et al. Resveratrol (trans-3,5,4’-trihydroxystilbene) induces silent mating type information regulation-1 and down-regulates nuclear transcription factor-kappaB activation to abrogate dextran sulfate sodium-induced colitis. J Pharmacol Exp Ther 2010;332:829e839. 16. Faghihzadeh F, Adibi P, Rafiei R, et al. Resveratrol supplementation improves inflammatory biomarkers in patients with nonalcoholic fatty liver disease. Nutr Res 2014;34:837e843. 17. Udenigwe CC, Ramprasath VR, Aluko RE, et al. Potential of resveratrol in anticancer and anti-inflammatory therapy. Nutr Rev 2008;66: 445e554. 18. Bo S, Ciccone G, Castiglione A, et al. Anti-inflammatory and antioxidant effects of resveratrol in healthy smokers a randomized, doubleblind, placebo-controlled, cross-over trial. Curr Med Chem 2013;20: 1323e1331. 19. Timmers S, Konings E, Bilet L, et al. Calorie restriction-like effects of 30 days of resveratrol supplementation on energy metabolism and metabolic profile in obese humans. Cell Metab 2011;14:612e622. 20. Ren Z, Wang L, Cui J, et al. Resveratrol inhibits NF-kB signaling through suppression of p65 and IkappaB kinase activities. Pharmazie 2013;68:689e694. 21. Busch F, Mobasheri A, Shayan P, et al. Resveratrol modulates interleukin-1beta-induced phosphatidylinositol 3-kinase and nuclear factor kappaB signaling pathways in human tenocytes. J Biol Chem 2012;287:38050e38063. 22. Tibble J, Bjarnason I. Non-invasive investigation of inflammatory bowel disease. World J Gastroenterol 2001;7:460e465.
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