Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the gastrointestinal tract with complex etiology and limited therapeutic options. It is reported that IBD can be caused by defects in genes related to the innate immune function of the epithelium, which plays a crucial role in maintaining intestinal immune homeostasis [1, 2]. Apart from congenital factors, if the intestinal epithelium, acting as a selective permeability barrier, is physically damaged or experiences issues with selective permeability due to environmental stimulation, it can trigger an excessive mucosal immune response, leading to IBD [2]. Furthermore, changes in the composition and function of the intestinal microbiota have been observed in IBD patients [3]. There is accumulating evidence suggesting that this imbalance is a potential cause of IBD, and the control of IBD is associated with the proliferation of beneficial bacteria [4].

Ulcerative colitis (UC), like Crohn’s disease, is one of the IBDs and is a chronic idiopathic condition with a low remission rate [5, 6]. UC has a higher incidence than Crohn’s disease and is characterized by inflammation limited to the mucosal surface, which begins in the rectum and extends continuously proximally [5]. The main clinical symptoms include bloody diarrhea, stool urgency, fecal incontinence, and abdominal pain [1, 7]. While there is still an epidemiological gradient in UC between Western and Asian countries, the incidence and prevalence of IBD have significantly increased in Asia in the 21st century. The substantial rise in patients over just a few decades is primarily attributed to UC and environmental factors, suggesting a close relationship [8, 9]. Therefore, UC can be understood as a multifactorial disease resulting from the interaction of genetic defects regulating intestinal epithelial immunity, damage to the barrier and mucosa caused by external stimuli (environmental factors, etc.), and an imbalance in the intestinal microflora as the primary causes.

In recent years, traditional herbal medicine has gained traction as a potential source of innovative and effective treatments for inflammatory conditions. Among these, IN has emerged as a compelling candidate for investigation in IBD therapeutics. IN, known as “청대” in Korea and “Qingdai” in Chinese, is a blue powder made from the leaves or stems of Strobilanthes cusia (Nees) Kuntze, Persicaria tinctoria (Aiton) Spach, and Isatis tinctoria L [10]. As of now, 63 compounds have been isolated from IN. Active components include indirubin [11-15], indigo [11, 15, 16], tryptanthrin [17], isatin [18], etc. Previous studies have revealed that indigo, indirubin, and tryptanthrin are the main components of IN through HPLC or NMR analysis [19, 20]. These main components have been reported to possess pharmacokinetic and pharmacological effects, including anti-tumor, anti-angiogenic, anti-inflammatory, and anti-microbial effects [10]. According to the growing interest in IN, related research continuously accumulates, reporting various effects and pharmacological mechanisms. This study aims to explore IN’s effects and pharmacological mechanisms on UC comprehensively.

In this study, 11 papers were selected, encompassing research on both IBD and UC. Compounds of IN used in IBD research include indirubin, isatin, indigo, and tryptanthrin. The most frequently employed extract was a mixed extract of IN, and among the individual ingredients, indirubin was predominantly utilized. For the experimental model of IBD, mice were used in which the epithelial barrier was chemically damaged with DSS, leading to the spread of pro-inflammatory substances to the periphery and inducing colitis [21]. In DSS-induced colitis, IN improved the DAI score, increased colon length, alleviated mucosal damage, and reduced macrophage infiltration, ultimately ameliorating overall colitis lesions (Table 1).

Table 1

Preclinical evidence and mechanisms of action.

No.	Compound	Study	Model	Inducer	Results	Ref.
1	Indigo	In vivo In vitro	Male C57BL/6JJmsSlc mice, Caco-2 cell	ND	1. Induce Cyp1a1 and Cyp1a1 mRNA 2. Induce CYP1A1 and CYP1A1 mRNA, AhR is induced to transfer nucleus 3. Strengthening wound closure (inducing cell proliferation and actin polymerization)	[20]
2	Indirubin	In vivo	Male BALB/c mice	DSS 2%	1. Decrease NLRP3, IL-1β 2. Decrease of IL-17A mRNA expression and IL-17A secretion 3. Inhibition of NF-κB pathway (down regulation of NF-κB p65, Igkv) 4. Up regulation of Cyp1a1 and Gsta5 5. Supression of lipid peroxidation (decrease HBA and 4-HNE)	[21]
3	IN, indigo, or indirubin	In vivo	Male BALB/c mice	DSS 3%	1. Decrease IL-1β, IL-6 levels in serum and tissues 2. Reduce IgG concentration in serum and tissues 3. Reduce IgM concentration in tissues 4. Reduction of TLR4, TLR2, MyD88, p65, p-p65, IKBα, p-IKBα proteins in tissues 5. The dominant bacteria in IN and indigo are Butyricimonas, Eubacterium, Coprococcus, Ruminococcus and other probiotics, and in Indirubin, proteobacteria 6. Decrease MPO level in serum and tissue (IN and indirubin) 7. Decrease IgA level in serum (IN and indigo)	[24]
4	Indirubin with isatin	In vivo	Male BALB/c mice	DSS 3%	1. Reduction of TNF-α, IL-6 and IFN-γ/increase of IL-10 (singleness decreases, combination largely increases) 2. Reduction of COX-2, iNOS, PGE2, NO 3. Suppression of CD4+ T cell infiltration 4. Increase of Foxp3 expression 5. Decrease of MPO activity 6. Attenuate oxidative stress (decrease MDA/increase SOD, GSH) 7. Suppression cell death (decrease Tunel positive cell ratio, Bax level and cleaved-caspase-3 and increase Bcl-2 level) 8. Inhibition of NF-κB and MAPK signal pathway (decrease p-IκB, p-p38, p-ERK, p-JNK level, inhibition of IκB degradation and NF-κB p65 transfer to nucleus)	[25]
5	Tryptanthrin	In vivo	Male SD mice	DSS 5%	1. Suppression of IL-6, TNF-α 2. Decrease of p-STAT3, NF-κBp65 expression level 3. Increase of IκBα	[27]
6	Indirubin	In vivo In vitro	Male C57BL mice, bone marrow dendritic cell (BMDC)	DSS 2%, LPS	1. Inhibition of MPO activity 2. Suppression of CD80, CD86, CD40 and MHC-II expression 3. Decrease of TNF-α expression and increase of TGF-β expression 4. Increase of Treg proliferation (FOXP3 expression), decrease Th17 ratio, reduction of RORγt 5. Decrease IL-17 and increase IL-10 6. When αVβ8 siRNA treat, reverse 1-3 situation	[30]
7	IN	In vivo In vitro	Male C57BL/6 mice, naive CD4 T cell	DSS 2%, IL-6, TGF- β, anti-IFN-γ, anti-IL-4	1. Decrease MPO activity 2. Increase T-SOD, CAT, GSH-Px activity 3. Activation of p-AMPK and Nrf-2 4. Inhibition of transcription factor of Th1 and Th17 (decrease IFN-γ, IL-17A, T-bet, RORγt mRNA expression) 5. Decrease pro-inflammatory cytokine (decrease IFN-γ, IL-17A/F, TNF-α and IL-1β) 6. Decrease Th1 and Th17 cell frequency 7. Inhibition of p-STAT1 and p-STAT3 phosphorylation 8. Suppression of Th1 and Th17 cell differentiation (in vitro)	[33]
8	IN	In vivo	C57/BL6 mice	DSS 2.5%	1. Suppression of GSK3-β mRNA expression 2. Decrease pro-inflammatory cytokine (TNF-α, IL-1β and IL-17a)	[38]
9	Indigo or indirubin	In vivo	Male BALB/c mice	DSS 3%	1. Reduction of pro-inflammatory cytokine level included TNF- α , IFN- γ , IL-12, IL-23, IL-17A 2. Attenuation of ROS/RNS production 3. Protection of physical barrier of colitis epithelium (increase ZO-1, occludin and E-cadherin expression) 4. Regulation of colitis permeability (increase MUC2 expression) 5. Regulation of mucosal immune homeostasis 5-1. Decrease neutrophils (CD11b+ Gr-1+), reduction of regulation neutrophils 5-2. Decrease dendritic cells (CD11b+ CD11c+) and macrophages (CD11b+ F4/80+) 5-3. Increase relative amount of NK cells (CD335+ CD11b+) 5-4. Decrease ILC2, ILC3 6. Regulation of intestinal microbiome (increase Firmicutes, Norank_f_Muribaculaceae, Lactobacillus and Alloprevotella)	[45]
10	IN	In vivo	Male Sprague-Dawley rats	DSS 4.5%	1. Decrease MPO activity and pro-inflammatory cytokine TGF-β level in serum 2. Recovery of changed microbiome by DSS in colitis(abundantly relative increase of Firmicutes, Actinobacteria, Bifidobacteriaceae, Ruminococcaceae, Uminococcus_1, Ruminococcaceae_UCG-005, Norank_f__Erysipelotrichaceae, Butyricicoccus and Bifidobacterium)(relatively decrease of Bacteroidetes, Escherichia-Shigella as normal level) 3. Butyric acid (one of the SCFAs): significantly increase in treatment group compare to control group⇒ Elevated level of GPR41 and GPR43 in treatment group compare to control 4. When removed microbiome by treating antibiotics cocktail, no anti-inflammation effect of IN⇒ The anti-colitis effect of IN appears to be dependent on the presence of the intestinal microbiome	[46]
11	IN	In vivo	Male Kunming (KM) mice	DSS 3%	1. Decrease IL-6, IL-8, TNF-α and increase IL-10 (upregulation of anti-inflammation and down-regulation of pro-inflammation) 2. Recovery of divers microbiome damaged by DSS 3. Recovery of microbiome species changed by colitis: after IN treat, decrease Bacteroidetes, Proteobacteria and increase Firmicutes 4. Increase Peptococcus and decrease Turicibacter	[47]

1. Anti-inflammatory mechanisms

Studies have demonstrated that compounds of IN may exert anti-inflammatory effects by modulating key pathways involved in the inflammatory cascade. This includes the inhibition of pro-inflammatory cytokines and signaling molecules associated with the pathogenesis of IBD.

Indirubin and indigo play a crucial role in regulating multiple inflammatory pathways by activating the AhR pathway, which involves the expression of genes such as CYP1A1. The insolubility of indigo in the intestines hinders its absorption, preventing the induction of CYP1A1 in the liver. Instead, indigo activates AhR by inducing it in the intestines [22]. Liu et al. [23] provided additional validation for the activation of the AhR pathway by demonstrating upregulation of CYP1A1 and Gsta5 following indirubin administration. Moreover, indirubin exhibited an inhibitory effect on NF-κB/NLRP3/IL-1β as evidenced by the downregulation of NLRP3, NF-κB p65, IL-1β, and Igkv.

NF-κB, a transcription factor crucial in inflammation and innate immune responses [24], is induced through the MYD88-dependent pathway of TLR4. Upon recognizing LPS stimulation, TLR4 recruits interleukin-1 receptor-associated kinase 4 (IRAK-4) through interaction with TIR domains like MYD88, initiating downstream signaling involving tumor necrosis factor receptor-associated factor 6 (TRAF6), transforming growth factor-β-activated kinase 1 (TAK1), IKKs, NF-κB, and mitogen-activated protein kinase (MAPK) [25]. Particularly noteworthy is the observation that indirubin administration within the compounds of IN alleviated colitis, coinciding with a reduction in the expression of proteins related to the TLR4/MYD88/NF-κB pathway [26]. Administration of indirubin and isatin, either alone or in combination, also led to the inhibition of the MAPK pathway alongside NF-κB [27]. The interplay between signal transducer and activator of transcription 3 (STAT3) and NF-κB involves mutual activation, with IL-6 expression induced in this process [28]. Additionally, tryptanthrin reduced the proteins (IL-6, TNF-α, p-STAT3, NF-κBp65) central to these mechanisms [29].

2. Immunomodulation

The immunomodulatory properties of IN are crucial in IBD management. Evidence suggests it can regulate immune responses, potentially suppressing the exaggerated immune reactions observed in IBD patients.

Integrin αvβ8, a cell adhesion molecule expressed by dendritic cells, is crucial in activating transforming growth factor-β (TGF-β) and mediating T-cell differentiation [30]. This deficiency in integrin αvβ8 has been associated with the induction of colitis [31]. In line with this, Zhang et al. [32] reported that indirubin induces dendritic cell activation and apoptosis by upregulating αvβ8 expression. This induction not only facilitates antigen uptake but also inhibits antigen presentation and T-cell activation.

Th17 and Treg cells are differentiated from naive CD4 T cells through the TGF-β signaling pathway. Th17 cells promote inflammatory responses by producing pro-inflammatory cytokines like IL-17, while Treg cells suppress immune responses by generating anti-inflammatory cytokines such as IL-10 and TGF-β [33]. Cytokine expression in Treg cells is regulated through the transcription factor Foxp3 and in Th17 cells through retinoid orphan receptor gamma t (RORγt) [34]. Therefore, maintaining the balance between Th17 and Treg cells is crucial in immune-mediated inflammatory diseases. An increase in the Th17/Treg ratio was reported in DSS-induced IBD mice, and IN regulated this imbalance. In the study by Zhang et al. [32], the administration of indirubin to DSS-induced mice increased Foxp3 and IL-10 expression, accompanied by a decrease in RORγt and IL-17 expression. Gao et al. [27] further supported these findings by demonstrating elevated levels of Foxp3 and IL-10 following the single or combined administration of indirubin and isatin. These results strongly suggest that IN improves UC by modulating the Th17 and Treg cell action balance. Additionally, Xiao et al. [35] expanded on this understanding, revealing that the administration of IN to mice with DSS-induced colitis not only led to a decrease in the frequency of Th1 and Th17 cells but also resulted in the inhibition of the phosphorylation of STAT1 and STAT3, as well as a reduced expression of IFN-γ, IL-17A, T-bet, and RORγt. The observed reduction in expression indicates an immunosuppressive effect by inhibiting Th1 and Th17 differentiation by IN.

GSK3-β, a regulator of various immune system signaling pathways, has been implicated in the control of chronic intestinal inflammation [36, 37]. After GSK3 inhibition, the number of Th17 cells reduced in the intestines of healthy mice [38], and forkhead box P3 (FoxP3) expression was prolonged in Treg cells treated with a GSK3-β inhibitor [39]. Yue et al. [40] demonstrated reduced pro-inflammatory factors (TNF-α, IL-1β, and IL-17a) and decreased GSK3-β expression after administering IN to mice with DSS-induced colitis. Notably, the transcription level of Foxp3 was maintained.

3. Modulation of intestinal microbiota

In recent studies, an imbalance in the intestinal microbiome has emerged as a significant factor contributing to IBD, notably observed in individuals with IBD, where the biodiversity of the intestinal microbiota diminishes. This imbalance includes a decrease in Firmicutes and an increase in Gamma-proteobacteria, resulting in heightened oxidative metabolism, reduced production of short-chain fatty acids (SCFAs), and increased mucin degradation [41]. SCFAs, crucial metabolites produced by intestinal microorganisms through dietary fiber, play a pivotal role in maintaining intestinal homeostasis, with lower SCFA levels associated with an elevated risk of developing IBD [42-44]. Moreover, the gut microbiota’s influence extends to intestinal innate and adaptive T cell differentiation and activation, where an imbalance between Th17 and Tregs has been reported in correlation with disruptions in the intestinal microbiota [45, 46]. Consequently, an imbalance in the gut microbiota can lead to immune dysregulation, contributing to IBD, such as UC.

This study specifically aimed to confirm the impact of IN on regulating the intestinal microbiome. Xie et al. [47] noted a decrease in the diversity of the intestinal microbiome and an increase in pathogenic bacteria (Proteobacteria, Verrucomicrobiota) during DSS treatment. Following IN administration, pathogenic bacteria decreased, while beneficial bacteria, including Firmicutes, Norank_f_Muribaculaceae, Lactobacillus, and Alloprevotella, increased. In studies conducted by Sun et al. [48] and Liang et al. [49], the diversity and ratio of the intestinal microbiota composition, disrupted by DSS, were effectively restored through IN administration. Furthermore, there was a down-regulation of pro-inflammatory cytokines and an up-regulation of anti-inflammatory cytokines. Sun et al. [48] additionally demonstrated a significant increase in butyrate acid, accompanied by elevated levels of GPR41 and GPR43, which are butyrate acid-related factors and part of the SCFAs. Interestingly, no relief of colitis symptoms was observed when IN was administered to mice with DSS-induced colitis after removing the intestinal microbiome through an antibiotic cocktail treatment. This suggests that the anti-colitis effect of IN appears to depend on the presence of the intestinal microbiome. In the research conducted by Yang et al. [26], the composition of the intestinal microbiota in the DSS group and the indirubin group closely resembled that of other groups. However, there was no overlap between the IN and indigo groups in non-metric multidimensional scaling (NMDS) analysis samples and the control and DSS groups. Notably, compared to indigo, indirubin exhibited a weaker ability to regulate the composition of the intestinal microbiome.

4. RCT (randomized controlled trials)

A systematic analysis of RCT involving IN in patients with UC is presented. The review assesses study designs, patient populations, and outcomes, offering a comprehensive overview of the current state of clinical research on IN in the context of UC (Table 2).

Table 2

Randomized controlled trials.

Disease	Patients	Treatment intervention	Period	Outcome	Results	Ref.
Ulcerative colitis	41	CurQD (curcumin 500 mg, Qindai 500 mg) 3 capsule (curcumin 1.5 g, qingdai 1.5 g)/ oral	8 weeks	[Co-primary outcome] - Clinical response (SCCAI, a decrease ≥ 3 point) - Mayo score (a decrease ≥ 3 point & a decrease ≥ 30% from baseline) [Secondary outcomes] 1. Clinical remission (SCCAI, ≤ 2) 2. Clinical response 3. Mayo subscore (a decrease ≥ 1 point from baseline) 4. Fecal calprotectin level (a decrease ≥ 50% from baseline) 5. The median calprotectin levels 6. The onset of the reduction in SCCAI of ≥ 3 points 7. The onset of no rectal bleeding	[Co-primary outcome] 43 (12/28) : 8 (1/13) p = 0.033 [Secondary outcomes] (CurQD : placebo) 1. 50 (14/28) : 8 (1/13) p = 0.01 2. 85.7 (24/28) : 30.7 (4/13) p < 0.001 3. 70 : 25 p = 0.036 4. 46.8 (13/28) : 15.4 (2/13) p = 0.08 5. Significantly reduce (p < 0.001) : no significant (p = 0.8) 6. Day 16 (p < 0.01) : no significant 7. Day 12 (p < 0.05) : no significant	[48]
Ulcerative colitis	42	The IN powder 5 capsule (500 mg)/ oral	2 weeks	1. Lichtiger index 2. Lichtiger index decreased by more than 30% compared to baseline 3. Lichtiger index decreased by more than 50% compared to baseline	1. Significantly reduce (p = 0.001) : no significant (p = 0.359) 2. 82.6% and 26.5%, respectively (p = 0.0003) 3. 60.9% and 5.3%, respectively (p = 0.0002) 5 people in the IN group and 1 person in the placebo group experienced mild headaches	[49]
Ulcerative colitis	86	62.5 mg, 125 mg, or 250 mg IN 4 capsules twice daily for 8 weeks (total of 0.5 g, 1 g, or 2 g of IN)/oral	8 weeks	[Primary outcome] 1. Clinical response [Secondary outcomes] 1. Clinical remission 2. Mucosal healing 3. Mayo score 4. MTWSI score 5. Fecal calprotectin level 6. Fecal immunochemical blood	[Primary outcome] (0.5 g IN : 1.0 g IN : 2.0 g IN : placebo) 1. 69.6% (p = 0.0002) : 75.0% (p = 0.0001) : 81.0% (p = 0.0001) : 13.6% [Secondary outcomes] (0.5 g IN : 1.0 g IN : 2.0 g IN : placebo) 1. 26.1% (p = 0.0959) : 55.0% (p = 0.0004) : 38.1 (p = 0.0093) : 4.5% 2. 56.5% (p = 0.0045) : 60.0% (p = 0.0032) : 47.6% (p = 0.0217) : 13.6% 3. Mayo scores compared to placebo; –2.83 (p = 0.0024) : –3.64 (p = 0.0002) : –3.42 (p = 0.0004) 4. MTWSI scores compared to placebo; –4.67 (p < 0.0001) : –5.46 (p < 0.0001) : –6.4 (p < 0.0001) 5. 60.0% (p = 0.0022) : 57.6% (p = 0.0051) : 36.8% (p = 0.1245) : 10.5% 6. 38.1% (p = 0.0670) : 40.0% (p = 0.0648) : 44.4% (p = 0.0265) : 10.0% 10-20% of patients experienced mild liver dysfunction (reversible) except for one patient (1 g IN group) due to nausea No serious side effects requiring hospitalization	[50]

In a research study by Ben-Horin et al. [50], an eight-week randomized, double-blind trial was conducted with active UC patients in Israel and Greece. The trial used a combination of Curcumin-QingDai, with 41 patients randomly assigned to either the treatment group (CurQD) or the control group (placebo) in a 2:1 ratio. The co-primary outcome, focusing on the percentage of patients meeting both clinical response and Mayo score criteria, revealed a significant disparity: CurQD; 43%: placebo; 8% (p = 0.033). Secondary outcomes, assessed through various instruments, demonstrated achievement rates of clinical remission and clinical response as CurQD; 50%: placebo; 8% (p = 0.01) and CurQD; 85.7%: placebo; 30.7% (p < 0.001), respectively. Additionally, the proportion of patients with a Mayo subscore decrease of more than one point was CurQD; 70%: placebo; 25% (p = 0.036). Furthermore, the proportion of patients experiencing a more than 50% decrease in fecal calprotectin levels from the baseline value was CurQD; 46.8%: placebo; 15.4% (p = 0.08). While the median calprotectin level significantly decreased in the CurQD group (p < 0.001), no noteworthy treatment effect was observed in the placebo group (p = 0.8). In the CurQD group, the time for the SCCAI score to decrease by more than three points or for rectal bleeding to cease was 16 days (p < 0.01) and 12 (p < 0.05), respectively, with no significant results obtained in the control group.

Uchiyama et al. [51] investigated 42 patients with mild to moderately active UC, randomly assigning them to the IN group (23 patients) or the placebo group (19 patients). After treatment administration, the Lichtiger index of the IN group significantly improved (p = 0.001), while no change was observed in the placebo group (p = 0.359). The proportion of patients whose Lichtiger index decreased by more than 30% compared to baseline was 82.6% and 26.5%, respectively (p = 0.0003), and the proportion of patients whose Lichtiger index decreased by more than 50% compared to baseline was 60.9% and 5.3%, respectively (p = 0.0002). The IN group recorded a significantly higher rate than the placebo group. During the two-week experiment, five people (21.7%; 5/23) in the IN group and one person (5.26%; 1/19) in the placebo group experienced mild headaches, and no severe adverse reactions such as pulmonary hypertension and intussusception occurred.

Furthermore, Naganuma et al. [52] conducted a multicenter randomized controlled trial examining the efficacy of IN in 86 patients with active UC in Japan. Patients were randomly assigned to four groups and administered 62.5 mg, 125 mg, or 250 mg of IN or placebo, four capsules twice daily, for eight weeks (total of 0.5 g, 1 g, or 2 g of IN). After an eight-week clinical trial, the primary endpoint, clinical response, was 0.5 g IN; 69.6% (p = 0.0002), 1.0 g IN; 75.0% (p = 0.0001), 2.0 g IN; 81.0% (p < 0.0001), placebo; 13.6%, confirming a dose-dependent linear trend for IN. Examining the secondary endpoint, clinical remission, a higher treatment effect was found in the patient group administered 1 g IN daily than in the patient group administered 2 g IN daily (0.5 g IN; 26.1% [p = 0.0959], 1.0 g IN; 55.0% [p = 0.0004], 2.0 g IN; 38.1% [p = 0.0093], placebo; 4.5%). Mucosal healing also showed similar results (0.5 g IN; 56.5% [p = 0.0045], 1.0 g IN; 60.0% [p = 0.0032], 2.0 g IN; 47.6% [p = 0.0217], placebo; 13.6%). Patients receiving IN daily had greater improvements in Mayo scores compared to patients receiving placebo (0.5 g IN; –2.83 [p = 0.0024], 1.0 g IN; –3.64 [p = 0.0002], and 2.0 g IN; –3.42 [p = 0.0004]). The MTWSI score was also significantly improved in the IN group compared to the placebo group (0.5 g IN; –4.67 [p < 0.0001], 1.0 g IN; –5.46 [p < 0.0001], 2.0 g IN; –6.4 [p < 0.0001]). At week eight, the proportion of patients with fecal calprotectin levels less than 150 μg/g and the proportion of patients with fecal immunochemical blood levels less than 100 ng/mL were respectively 60.0% (p = 0.0022); 57.6% (p = 0.0051); 36.8% (p = 0.1245); 10.5%, and 38.1% (p = 0.0670); 40.0% (p = 0.0648); 44.4% (p = 0.0265); 10.0%. During eight weeks of treatment, no serious side effects requiring hospitalization occurred. 10-20% of patients receiving 0.5 to 2.0 g of IN daily for eight weeks experienced mild liver dysfunction, which was reversible. The patients recovered, and, except for one patient (1 g IN group) who terminated early due to nausea, there were no serious side effects requiring hospitalization.

Despite the emergence of various biological agents and small molecule drugs for inflammatory diseases, a significant number of patients do not respond to these new drugs, experience side effects, or have difficulty receiving treatment due to issues such as cost [50]. IN, which is proposed as a new drug candidate for these diseases, is a multi-ingredient herbal medicine containing both organic and inorganic compounds and is effective in suppressing inflammation, tumors, bacteria, and psoriasis [10]. In this study on DSS-induced colitis, IN improved the DAI score, increased colon length, alleviated mucosal damage, and reduced macrophage infiltration, thereby improving overall colitis lesions. In addition, the anti-inflammatory efficacy of IN has been proven in various RCT studies [50-54].

In this study, the anti-inflammatory mechanism of IN for UC was summarized. The three main compounds of IN, indirubin, indigo, and tryptanthrin, were mainly used as a single ingredient. Indirubin regulates various inflammatory pathways by activating the AhR pathway by expressing genes such as CYP1A1. In addition, inhibition of the MAPK pathway was also observed along with NF-κB inhibition by reducing the expression of proteins related to the TLR4/MYD88/NF-kB pathway. Upregulating the cell adhesion molecule integrin αvβ8, an important factor related to T cell-mediated immunity, induced DC activation and apoptosis and antigen absorption to suppress antigen presentation and T cell activation. In addition, after indirubin administration, the expression of Foxp3 and IL-10 increased, and the expression of RORγt and IL-17 decreased, suggesting that indirubin suppresses the immune response by regulating the Treg/Th17 ratio. Furthermore, the intestinal microbiota analysis revealed an overlap in the composition of the indirubin group and the DSS group in terms of intestinal microbiota, suggesting that the anti-inflammatory mechanism of indirubin in colitis is primarily associated with suppressing cellular immunity rather than regulating intestinal microbiota. Like indirubin, indigo also showed AhR pathway activation and a clearer recovery of the intestinal microbiota composition damaged by DSS than indirubin. Tryptanthrin showed a decrease in IL-6/STAT3/NF-κB related proteins (IL-6, TNF-α, p-STAT3, NF-κBp65). In addition, IN extract reduced the expression of Th1-related IFN-γ and T-bet, which are involved in inflammatory responses other than Th17, and decreased the expression of GSK3-β and decreased proinflammatory factors (TNF-α, IL-1β, and IL-17a). In addition, it has been reported that the imbalance of the intestinal microbiome, which has recently been attracting attention as a cause of IBD, can be improved through IN. After IN administration, intestinal microbiota diversity was restored, pathogenic bacteria decreased, and beneficial bacteria such as Firmicutes, Norank_f_Muribaculaceae, Lactobacillus, and Alloprevotella increased. Considering that the effect was suppressed when IN was administered after removing the intestinal microbiome through antibiotic cocktail treatment, the anti-colitis mechanism of IN appears to be executed dependent on the intestinal microbiome.

IN may offer potential benefits in treating IBD compared to well-established medications such as mesalazine, balsalazide, and olsalazine. These conventional medications alleviate inflammatory symptoms and slow the progression of IBD but do not effectively control beneficial bacteria or restore intestinal function [55-57]. However, IN’s clinical efficacy and safety profiles lack extensive validation compared to the well-established medications.

The side effects reported in the studies of IN included headache and nausea, which were minimal in two of the three studies. In the remaining study, early termination due to worsening colitis occurred in four out of 28 patients in the treatment group and appeared in six out of 13 control subjects. In the same study, one case was reported where liver levels increased 10 times the upper limit of normal. Recently, many cases of pulmonary hypertension have been reported in patients taking green tea for UC [58-60]. These cases have a common rapid improvement after stopping taking IN. Although the exact mechanism between IN and the development of pulmonary arterial hypertension (PAH) has not yet been elucidated, expected hypotheses include that indigo and indirubin are AhR ligands [22, 23]. Indigo contains serotonin, which can act as a pulmonary vasoconstrictor. There are claims that it is involved in the occurrence of PAH due to its similar structure [60, 61]Although IN is a promising drug candidate for inflammatory diseases, its side effects may hinder its development. Therefore, to develop new drugs for IN, follow-up research is necessary to uncover the mechanism of developing severe side effects, such as pulmonary hypertension, related to IN and to find ways to improve them.

Furthermore, research on IN has predominantly focused on these three active ingredients, neglecting other compounds in IN. IN contains indirubin, tryptanthrin, indigo, and other organic and inorganic ingredients. Therefore, utilizing IN as a therapeutic agent necessitates genetic, developmental, and innovative research to understand the genetics, development, efficacy, and mechanisms of action of its various ingredients [62].

This study highlights the potential of IN as a promising therapeutic effect for IBD. Preclinical evidence and clinical trials demonstrate that IN has anti-inflammatory and immunomodulatory effects, improving disease activity and promoting mucosal healing. However, concerns about potential side effects warrant further investigation. Overall, IN emerges as a multifaceted candidate for IBD treatment, emphasizing the need for continued research to optimize its efficacy and safety profile.

The authors declare no conflicts of interest in this work.