Clinical outcomes of flavonoids for immunomodulation in inflammatory bowel disease: a narrative review

IBD

IBD is defined as a chronic, relapsing, debilitating and incurable disease that leads to the disruption of various gastrointestinal processes. IBD is characterized by a set of diseases that were recognized, but not fully understood in the antiquities of westernized countries during the advent of the industrial revolution [1-3]. While both conditions, UC and CD, are classified under IBD, they have distinguishable features notwithstanding mucosal inflammation. The first identified subtype of IBD was UC, which is a continuous mucosal inflammatory disease restricted to the colon, extending from the rectum to, frequently, the periappendiceal region (Fig. 1) [1,2]. It is often associated with bloody diarrhea and tenesmus [1,2]. CD is a transmural inflammation involving all layers of the wall of the gastrointestinal tract, with segmental inflammatory lesions that can extend from the oral cavity to the anus, and is often a cause of terminal ileitis, strictures, and fistulas (Fig. 1) [1].

Figure 1 Demonstration of mucosal involvement in inflammatory bowel disease. Crohn’s disease (A) is shown to demonstrate inflammation that is transmural, extending from the mouth to the anus in discontinuity (e.g., skip lesions), whereas ulcerative colitis is a continuous inflammation that can extend from the ascending colon to the rectum (B)

The exact pathophysiology of the disease remains to be fully understood. One risk factor for the predisposition to the disease is familial [2,3]. It often occurs in genetically susceptible individuals, with an incidence that peaks in early adulthood, although presentation can occur at all ages. Genome-wide association studies have identified variants of nucleotide-binding oligomerization domain protein 2 (NOD2) DNA among hundreds of genes that increase individual susceptibility to the pathogenesis of CD, although the concordance rate in monozygotic twins is 10-15% in UC and 30-35% in CD, suggesting a greater likelihood of non-inheritable contributors to the development of IBD [2,3].

T-cell migration, and its interdependent association with increased amounts of intestinal tumor necrosis factor (TNF)-α, have been shown to play a role in the chronicity of intestinal inflammation, owing to its involvement in weakening epithelial barrier function [4,5]. This process is mediated by environmental factors such as diet, smoking and antibiotic use, as they can affect the function of the epithelial barrier via dysregulation of the immune response and overreaction to autophagy [6]. Consequently, this leads to an inappropriate immune-mediated inflammatory response to commensal gut microbes, causing a greater abundance of entero-invasive strains and other potentially pathogenic species of preexisting gut microbes; however, specific organisms have not been identified as causal agents of IBD (Fig. 2) [6].

Figure 2 Pictographic representation of the Th-mediated inflammatory pathway in inflammatory bowel disease. On the left-hand side is the Th1 mediated inflammatory process observed in Crohn’s disease, whereas the right-hand side depicts the role of Th2 cells in ulcerative colitis. A diet high in fat and refined sugars is seen increasing the production of the proinflammatory cytokines TNF-α and IFN-γ. The mucous layer is reduced on both the flanked epithelial cells, with a consequent increase in the abundance of entero-invasive gut bacteria. This process can be seen as being induced by a low soluble fiber diet. The epithelial cells in the center depict the expected cellular environment of the normal gastrointestinal tract

IFN, interferon; IL, interleukin; Th, T helper; TNF, tumor necrosis factor

The role of environmental factors has been substantiated by an observed increase in incidence among first-generation immigrants from countries with low IBD prevalence to ones with higher proportions of affected individuals [7,8]. There has also been a positive correlational shift in the rates of affected individuals in countries that are progressively becoming more industrialized [3]. The role of diet has been supported by reports that it in fact plays an integral part in the modification of IBD progression, via either perpetuation or improvement of the disease [9]. However, nutritional assessments, guidelines for nutritional therapy and dietary recommendations are scarce in the clinical setting, as focus is often placed on the elimination of certain foods, with limited regard to the implementation of diet-based therapies for the amelioration of IBD [10].

The role of nutrition in IBD

Nutrition is regarded as playing an important role during IBD, as it can modulate its pathogenesis via alterations of the intestinal microbiota, immune system, and barrier function of the colonic epithelium [11]. Westernized diets, which tend to be high in fat and refined carbohydrates, are believed to induce epigenetic changes, such as altered expression of microRNAs (miRNAs), DNA methylation and histone modification in the development of IBD [8]. Due to inflammation, accelerated rates of DNA methylation secondary to increased cellular recycling have been observed in colonic epithelial cells in comparison to unafflicted cohorts, whereas variable activity of miRNAs has been identified in the dysregulation of autophagy and intracellular bacterial processing [8,12]. Thus, dietary intake is purported to have a strong association with the disruption of the host’s cellular machinery and defense system.

Diet low in soluble fiber has resulted in a similar dysregulation, as it can lead to an increase in mucolytic bacteria known to degrade the colonic mucus layer, which can render the host epithelium vulnerable to enteric pathogens (Fig. 2) [11]. Similarly, diets high in fat and refined sugars lead both to decreased levels of butyrate (a favorable short-chain fatty acid) and increased expression of TNF-α and interferon-γ (Fig. 2). These alterations consequently decrease the overall abundance of anti-inflammatory cytokines and protective T-regulatory cells in the lamina propria of colonocytes [11]. In view of such disruptions of the gut mucosal homeostasis, it is prudent to suggest avoidance of obesogenic or western-industrialized foods, while encouraging greater consumption of foods rich in specific micronutrients. However, it should be noted that the available data have only demonstrated an association between dietary consumption and disease onset or burden. Considering the heterogeneity of outcomes and unaccounted confounders, possibly owing to biases or ineffective study methods, causal relationships cannot be determined. Nonetheless, given the concerns of limited drug response and the nutritional adequacy of enteral and parenteral nutrition in the management of IBD, there is a greater demand for the employment of nutritional assessments and focus on diet-based therapies [10].

Flavonoids

Flavonoids are benzo-γ-pyrone-derived bioactive polyphenolic compounds, synthesized from phenylalanine during plant stress. They are responsible for the admirably rich colors that typify many plants, and have been purported to exert antioxidant, anti-inflammatory, and anti-allergic effects [13-15]. There are more than 5000 types of flavonoids: they have been subdivided into varying classes, based on structural differences in their heterocyclic oxygen ring, and include: 1) anthocyanidins; 2) flavanols; 3) flavanones; 4) flavonols; 5) flavones; 6) isoflavones; 7) neoflavonoids; and 8) proanthocyanidins [16-18]. Their phytochemical properties, which cannot be synthesized by humans, are involved in enzymatic inhibition of pathogenic microbes and regulation of gene expression. They are often found at variable concentrations in food plants, such as flowers, stems, fruits, legumes, cereals, vegetables, seeds, herbs, nuts, tea, wine, and cocoa [16,18].

The phytochemical property of flavonoids was a sought-after component in traditional medicinal preparations, such as propolis honey and royal jelly [19,20]. In recent years, flavonoids have received increased attention, due in part to the compound’s antiangiogenic and antiproliferative/anticancer potential [21]. Higher intakes of foods containing such compounds have been linked to a reduction in all-cause mortality in age-adjusted models (odds ratio [OR] 0.81, 95% confidence interval [CI] 0.71-0.93), with the strongest associations observed in wine and tea consumers [22]. Populations with a higher dietary intake of foods and beverages containing flavonoids have shown an inverse correlation in the incidence of cardiovascular disease [23]. Moreover, there is a growing body of data demonstrating the compounds’ ability to regulate the intestinal immune system through suppression of immune cells and effector molecules (e.g., chemokines, cytokines, and TNF). These effectors have been shown to all play a role in the inflammatory process of IBD. Specific flavonoids, such as apigenin, epigallocatechin gallate, quercetin and rutin, have been identified as compounds with the ability to exert such properties (Table 1) [15,16,24].

Table 1 Flavonoids that have been shown to exert anti-inflammatory properties, their heterocyclic ring structure, and overall immunomodulatory effects

The role of flavonoids in IBD

Fruits and vegetables account for 300 mg/kg of fresh weight content in flavonoids, with the average daily adult consumption approximating 20-1000 mg [25,26]. However, a great deal of the flavonoid concentration is lost in food preparation and processing. Additionally, the bioavailability of the compounds is often partial for specific types of flavonoids, as a large proportion remain unabsorbed and are found in greater concentrations in the gastrointestinal lumen [27]. The rate of absorption is seemingly determined by the presence or absence of glycosylation of the compounds, although the validity of this notion has been questioned in light of the variability in absorption among the classes of flavonoids [28]. For example, the predominantly glycosylated form of quercetin is more readily absorbed, while catechin, a non-glycosylated flavonoid, is often absorbed with the same relative efficiency [29,30]. Nonetheless, the reduction in the absorptive capacity of specific flavonoids, leading to greater concentrations in the colon, suggests these compounds have proximal roles in subverting the pathogenesis of IBD.

Immunomodulatory effects of flavonoids

Inflammation is a normal biological process that is activated in response to infection, tissue injury and chemical irritation [31]. It is normally transient with resolution of the inciting event; however, dysregulation and derangement of this process can cause continued inflammation, leading to the onset of many diseases. In the case of IBD, an alteration in the colonic epithelial barrier leads to the recognition of luminal antigens by T helper (Th) cells, which propagates a dramatic cascade of proinflammatory cytokines (e.g., interleukin [IL]-1, IL-4, IL-5, IL-6, IL-10, IL-17A, TNF-α, and interferon-γ) thereby disrupting intestinal homeostasis (Fig. 2) [31,32]. CD is believed to be a Th1-mediated response, with ensuing activation of IL-1, IL-6, TNF-α, and interferon-γ, whereas UC is mediated by Th2 cells and the release of IL-4, IL-5, IL-10, IL-13, and TNF-α (Fig. 2) [31,33-36]. The immunomodulatory role of flavonoids has been exhibited in in vitro studies, in which the levels of IL-1β and TNF-α were reduced with administration of quercetin or baicalin; a similar reduction in TNF-α and interferon-γ was also observed with quercetin use [32]. However, given the paucity of clinical trials, it is difficult to confirm whether similar results at the molecular level have been appreciated in human models, though human trials have seen a reduction in inflammatory markers, disease severity scores and indices, and endoscopic changes.

Clinical trials

Epigallocathechin-3-gallate (EGCG)

In a 9-week open pilot trial that enrolled a total of 13 patients with mild-to-moderate UC to receive anthocyanin-rich bilberries, 90.9% of patients exhibited a positive response and 63.4% achieved remission, as determined by a decrease in endoscopic Mayo score (P<0.001), histologic Riley index, and fecal calprotectin (P=0.049) [37,38]. An increase in fecal calprotectin with cessation of bilberry consumption was observed during follow up [37]. A similar finding was observed with the administration of poorly absorbed EGCG in a double-blind, placebo-controlled pilot study, where 20 patients with mild to moderately active UC were randomized to receive 56-days of daily oral polyphenon E (400 mg, or 800 mg in split doses) or a green tea extract containing 50% EGCG, vs. placebo (Table 2). The results yielded a favorable response rate in the treatment arm, as 66.7% in the polyphenon E group experienced a decrease in the UC disease activity index (P=0.03), with 53.3% of participants achieving statistically non-significant active remission (P=0.10) (Table 2) [39]. Despite the poor systemic bioavailability of the compound with oral administration, there was an appreciable reduction in the inflammatory burden of the disease, probably due to the increased colonic mucosal exposure of EGCG [39]. In both in vivo and in vitro studies, EGCG combined with apigenin was shown to inhibit cytokines and immune cells [40]. However, the results did not demonstrate statistically significant findings, possibly because the studies were of limited power.

Table 2 Clinical outcomes of flavonoid therapy

On the other hand, bioavailability was seen to increase by >3.5-fold when polyphenon E capsules were consumed on an empty stomach preceding an overnight fast, in a study that assigned 30 healthy volunteers with an average age of 42 years to 400, 800 or 1200 mg daily doses [41]. Consumption of polyphenon E capsules containing 800 mg of EGCG was overall well tolerated on an empty stomach [41]. However, as this clinical trial was used to determine tolerance and side-effects associated with flavanol dosage in healthy controls, there were no data on clinical improvement in IBD symptoms or in inflammatory markers. Side-effects of EGCG-containing foods include nausea in patients taking 1200 mg and an increased risk of hepatotoxicity, characterized by elevated liver function tests, with doses of 800 mg daily [41,42]. Other published case reports have also noted a dose-independent risk for hepatotoxicity with intake concentrations between 140 mg and 1000 mg. However, the risk was seemingly increased by consumption on an empty stomach, while it is believed that the varying responses to the compound were dependent on inter-individual variability and genetic factors [43].

Curcumin

Curcumin, an antioxidant and polyphenolic compound, a food additive that produces the major pigment in turmeric, was used as a nutraceutical in a pilot study that recruited 10 patients with IBD [44]. The patient cohorts, who were aged 28 to 54 years, included 5 patients with ulcerative proctitis or proctosigmoiditis and 5 others with CD. The group with UC was treated with 550 mg of curcumin b.i.d. for 1 month, followed by 550 mg t.i.d. for an additional month, whereas the CD cohort was administered 360 mg of curcumin t.i.d. for 1 month and 360 mg q.i.d. for 2 months (Table 2). The study found that all 5 subjects with UC had an overall improvement in symptoms, determined by a global score from an administered questionnaire (P<0.02), along with stool frequency and quality. Additionally, while no quantified values were provided, patients with UC had a normalized erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) (Table 2). The CD cohort had a mean reduction of 10 mm/h in ESR, 0.1 mg/dL in CRP, and 55 points in their CD activity index (CDAI) [44]. Limitations of this study include a lack of both controls and elimination of confounding variables, and a lack of assessment of the compounded effect of flavonoids with concomitant pharmacotherapy.

A multicenter randomized, placebo-controlled, double-blind study assessing curcumin’s ability to induce remission in 50 mesalamine-treated participants with active mild-to-moderate UC found that 14 of 26 patients receiving 3 g/day of curcumin for 1 month achieved clinical remission after an intention-to-treat analysis was conducted (OR 42, 95%CI 2.3-760; P=0.01) [45]. The primary outcome was defined by a reduction in Simple Clinical Colitis Activity Index (SCCAI) of ≥3 points, which was achieved in 17 patients in the curcumin group and 3 in the placebo group (OR 13.2, 95%CI 3.1-56.6; P<0.001) (Table 2). Endoscopic remission, as determined by a partial Mayo score ≤1, was observed in 8 of 22 patients in the curcumin group and 16 in the placebo group (OR 20.7, 95%CI 1.1-393; P=0.43). Nine of the participants (curcumin n=4; placebo n=5) in the study, however, underwent concomitant treatment with mesalamine, but no difference in outcomes was observed by the authors, who noted that the small size of this subgroup precluded statistical analysis [45].

A pilot study assessed the use of 140 mg/20 mL water curcumin enema (NCB-02) in 23 of 45 patients with mild-to-moderately active ulcerative proctitis and proctosigmoiditis, with concurrent usage of 800 mg of oral 5-aminosalicylic (5-ASA) acid b.i.d. [46]. The results found that, following 8 weeks of treatment, 92.9% of the treatment group achieved improved outcomes, compared to 50% in the placebo group (P=0.01), while 71.4% vs. 31.3% (P=0.03), and 85.7% vs. 50% (P=0.04), respectively, achieved clinical remission and improvement in their endoscopic disease activity (Table 2). These findings, however, were obtained using a per protocol analysis, as the initial intention-to-treat analysis demonstrated a non-inferior outcome between the treatment and placebo group who also received 5-ASA [46].

Curcumin was also explored as maintenance therapy in a randomized, multi-center, double-blind, placebo-controlled trial that enrolled 89 patients with quiescent UC [47]. Forty-five of the patients received a total of 2 g/day, 1 g of curcumin after breakfast and 1 g after an evening meal, plus sulfasalazine, while 44 received placebo along with sulfasalazine or mesalamine for 6 months (Table 2). The recurrence rates using an intention-to-treat analysis were 2 of 43 (4.65%) in the treatment group and 8 of 39 (20.51%) participants in the placebo group (P=0.040) during the designated 6 months of therapy. The treatment arm also saw an improvement in both the clinical activity index (P=0.038) and the endoscopic index (P=0.0001). However, during a 6-month follow up, 8 additional patients in the treatment arm were noted to have had a disease flare in comparison to 6 in the placebo group [47]. Nevertheless, the treatment arm was observed to have a greater likelihood of maintaining disease remission.

A recent retrospective multicenter cohort study, evaluating the effectiveness of curcumin and QingDai (QD) in 87 patients who had active UC with an SCCAI ≥3 points, observed both clinical and biomarker remission [48]. The primary outcome of the study was to achieve clinical remission within 8-12 weeks of treatment with 500 mg of herbal extract dry powder daily containing varying doses of curcumin, ranging from 2-3 g, admixed with 0.5-2 g of QD. Sixty patients received 2 g curcumin and 1 g QD. Fifteen patients with moderate-severe UC (SCCAI scores >10) received 3 g curcumin and 2 g QD, whereas 13 patients with milder UC (SCCAI 3-4) received 2 g curcumin and 0.5 g QD (Table 2). Forty-one (46.5%) achieved clinical remission, whereas 53 (60.2%) had a clinical response. Twenty-six patients were on corticosteroids at baseline, of whom 7 experienced corticosteroid-free remission. In addition, of the participants who were on biologics, 39.5% achieved clinical remission, with 58.1% achieving a clinical response [48]. Median SCCAI decreased from 7 (interquartile range [IQR] 5-9) to 2 (IQR 1-3) (P<0.001), while the median fecal calprotectin decreased from 1000 μg/g (IQR 392-2772) to 75 μg/g (IQR 12-136). Of the 25 patients who had baseline endoscopic data (85% had moderate-severe UC) 9 maintained stable therapy. Eight of the 9 patients had endoscopic improvement (score decrease of ≥1 in Mayo endoscopic subscore), while 4 patients had endoscopic remission (Mayo endoscopic subscore of 0) (Table 2). Major adverse outcomes included 4 patients who experienced liver transaminase increases of >3 times the upper limit of normal, whereas 4 patients were hospitalized in the setting of worsening SCCAI scores. One patient was omitted from the efficacy analysis after starting ustekinumab [48]. Limitations of this study include its lack of clarity regarding the induction and maintenance doses, as well as the lack of a placebo control.

Plantago major seed

In a randomized controlled trial in which 3600 mg/day (two 600 mg capsules before each meal, t.i.d.) of Plantago major (large plantain) seed was administered to a group of 31 of 51 UC subjects with mild and moderate disease, the treatment group showed an improvement in abdominal tenderness (P=0.011), distension (P=0.001), anal pain (P=0.051), and visible blood (P=0.001) after 8 weeks of treatment (Table 2) [49]. However, there were no overall statistically significant differences between the treatment and placebo group randomized to receive wheat flour, for any of the respectively aforementioned primary outcomes (P=0.224, P=0.283, and P=0.455). Disease activity was assessed using the Lichtiger Colitis Activity Index at weeks 4 and 8 (Table 2). Initially 61 participants were recruited, with 51 completing the trial, (n=28 in the treatment group, n=23 in the placebo group). No side-effects were reported in either of the 2 groups, and seemingly discontinuation from the study was due to loss to follow up. Nonetheless, limitations of the study were the frequency of recommended intake, the low sample size, and the concomitant use of routine medications [49]. Additionally, the use of the per-protocol analysis reduced the relevance for real-world applications regarding patient adherence patterns.

Quercetin

The edible part of the mango, called Mangifera indica L., which contains quercetin, gallic acid, and kaempferol and galloyl glycosides, was used in an 8-week open label trial involving 10 participants with mild-to-moderate CD or UC at concentrations of 200-400 g of mango pulp daily (Table 2) [50]. There was an observed reduction in SCCAI from 4.4 to 2.8 (P=0.0447) without statistically significant findings in terms of overall improvement in symptoms and quality of life, based on participant responses from the Short IBD Questionnaire [53]. The following inflammatory markers were found to have been lowered with the mango pulp regimen: IL-8 by 16.2% (P=0.0475), growth-regulated oncogene (GRO) by 25.0% (P=0.0375), and granulocyte macrophage colony-stimulating factor (GM-CSF) by 28.6% (P=0.0485) (Table 2). Shortcomings of this study were its open-label nature and the fact that participants were advised to weigh and record the exact amount of mango pulp they consumed daily, which may have been a factor in the statistically non-significant quality-of-life scores noted in the study [50].

Rose oil

In a pilot double-blind study that randomized 1000 mg rose oil soft capsules t.i.d. for a total of 2 months to 20 of 40 UC participants (mean age 41±10 years) vs. placebo (liquid paraffin soft capsules; n=20), the treatment group experienced significant improvement in partial Mayo score (P<0.05) and the IBD questionnaire (IBDQ-9) (P<0.05) (Table 2) [51]. However, although mean fecal calprotectin levels were decreased by the completion of the study, the difference was not statistically significant (P=0.229). Additionally, the results for primary outcomes, demographic factors and disease course were not statistically significant when an intergroup analysis was conducted (P>0.05). Overall satisfaction was greatly improved among the treatment group (7.46±2.33 vs. 6.79±2.01), although this also showed no statistical significance. Only 28 patients completed the study, with ultimately 14 participants remaining in both groups. The most common side-effects of rose oil were increased frequency of defecation and abdominal cramping (10% in the treatment and 15% in the placebo groups), which were reportedly the leading causes of participant withdrawal from the study (P=0.56) [51].

Silymarin

Silymarin, a polyphenolic flavonoid derived from milk thistle, was studied in a randomized double-blind, placebo-controlled clinical trial for the management of UC in 80 patients considered to be in remission [52]. Forty-two of the participants were assigned to 140 mg of silymarin daily, while 38 participants received placebo (composed of lactose monohydrate, corn starch magnesium steerage) daily for 6 months (Table 2). Ten patients were lost from the study because of nausea in the silymarin group (n=4) and disease flare-up, while 6 in the placebo group left because of abdominal pain. The study observed an overall improvement in the disease activity index in the silymarin group from 11.3±3.5 to 10.7±2.8 (P<0.05). Thirty-five of 38 patients in the silymarin group achieved complete remission after 6 months, in contrast to 21 of 32 from the placebo group (P=0.50). Improvements were also seen in hemoglobin level (11.8±1.6 g/dL vs. 13.4±1.2 g/dL; P<0.05) and ESR (23.7±11.5 mm/h vs. 10.8±3.2 mm/h; P<0.05) in the silymarin group (Table 2) [52].