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"Indigo"
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Efficacy and safety of short-term therapy with indigo naturalis for ulcerative colitis: An investigator-initiated multicenter double-blind clinical trial
Indigo naturalis (IN) is a blue pigment extracted from Assam indigo and other plants and has been confirmed to be highly effective for ulcerative colitis (UC) treatment in several clinical studies.
We conducted a multicenter double-blind study to confirm the efficacy and safety of short-term IN administration.
A multicenter, randomized controlled trial was conducted between December 2015 and October 2018 in our facilities. Forty-six patients with mild to moderate active UC (Lichtiger index: 5-10) were randomly assigned to the IN group or the placebo group and received 5 capsules (500 mg) twice a day for 2 weeks. We investigated the efficacy according to blood tests and the Lichtiger index before and after administration, and we also examined adverse events.
The analysis included 42 patients (20 males, 22 females) with an average age of 45 years. Nineteen patients were assigned to the placebo group, and 23 were assigned to the IN group. After treatment administration, in the placebo group, no change in the Lichtiger index was observed (7.47 to 6.95, p = 0.359), and hemoglobin was significantly reduced (12.7 to 12.4, p = 0.031), while in the IN group, the Lichtiger index (9.04 to 4.48, p = 0.001) and albumin (4.0 to 4.12, p = 0.022) improved significantly. Mild headaches were observed in 5 patients and 1 patient in the IN and placebo groups, respectively.
Short-term administration of IN is highly effective without serious adverse events such as pulmonary hypertension or intussusception and may prevent the occurrence of serious adverse events.
Journal Article
Indigo Naturalis ameliorates murine dextran sodium sulfate-induced colitis via aryl hydrocarbon receptor activation
Background
Indigo Naturalis (IN) is used as a traditional herbal medicine for ulcerative colitis (UC). However, the mechanisms of action of IN have not been clarified. We aimed to evaluate the efficacy of IN for ameliorating colonic inflammation. We further investigated the mechanisms of action of IN.
Methods
Colitis severity was assessed in dextran sodium sulfate-induced colitis and trinitrobenzene sulfonic acid-induced colitis models with or without the oral administration of IN or indigo, which is a known major component of IN. Colonic lamina propria (LP) mononuclear cells isolated from IN-treated mice were analyzed with quantitative reverse transcription polymerase chain reaction (qRT-PCR) and flow cytometry. LP and splenic mononuclear cells cultured in vitro with IN or indigo were also analyzed. The role of the candidate receptor for indigo, the aryl hydrocarbon receptor (AhR), was analyzed using
Ahr
-deficient mice.
Results
Colitis severity was significantly ameliorated in the IN and indigo treatment groups compared with the control group. The mRNA expression levels of interleukin (
Il
)-
10
and
Il-22
in the LP lymphocytes were increased by IN treatment. The treatment of splenocytes with IN or indigo increased the expression of anti-inflammatory cytokines and resulted in the expansion of IL-10-producing CD4
+
T cells and IL-22-producing CD3
−
RORγt
+
cells, but not CD4
+
Foxp3
+
regulatory T cells. The amelioration of colitis by IN or indigo was abrogated in
Ahr
-deficient mice, in association with diminished regulatory cytokine production.
Conclusions
IN and indigo ameliorated murine colitis through AhR signaling activation, suggesting that AhR could be a promising therapeutic target for UC.
Journal Article
Ferroptosis in the colon epithelial cells as a therapeutic target for ulcerative colitis
BackgroundFerroptosis, a type of programmed cell death triggered by oxidative stress, was suspected to play a role in ulcerative colitis. Indigo naturalis is highly effective against ulcerative colitis, but its mechanism is unclear. This study found that indigo naturalis treatment suppressed ferroptosis.MethodsWe analyzed 770 mRNA expressions of patients with ulcerative colitis. Suppression of ferroptosis by indigo naturalis treatment was shown using a cell death assay. Malondialdehyde levels and reactive oxygen species were analyzed in CaCo-2 cells treated with indigo naturalis. Glutathione metabolism was shown by metabolomic analysis. Extraction of the ingredients indigo naturalis from the rectal mucosa was performed using liquid chromatograph—mass spectrometry.ResultsGene expression profiling showed that indigo naturalis treatment increased antioxidant genes in the mucosa of patients with ulcerative colitis. In vitro analysis showed that nuclear factor erythroid-2-related factor 2-related antioxidant gene expression was upregulated by indigo naturalis. Indigo naturalis treatment rendered cells resistant to ferroptosis. Metabolomic analysis suggested that an increase in reduced glutathione by indigo naturalis. The protein expression of CYP1A1 and GPX4 was increased in the rectum by treatment with indigo naturalis. The main ingredients of indigo naturalis, indirubin and indigo inhibited ferroptosis. Indirubin was detected in the rectal mucosa of patients with ulcerative colitis who were treated with indigo naturalis.ConclusionsSuppression of ferroptosis by indigo naturalis in the intestinal epithelium could be therapeutic target for ulcerative colitis. The main active ingredient of indigo naturalis may be indirubin.
Journal Article
Removal of naphthol green B and indigo carmine from wastewater by wheat bran and urea-modified rice husk
Dye-related water contamination is a profound environmental issue, primarily because of the toxic nature of dyes and their harmful effects on living organisms. These pollutants can have severe consequences for ecosystems and human health. In response to this challenge, natural adsorbents have emerged as a highly promising solution. The novelty of my work lies in the use of wheat bran and urea-modified rice husk as biosorbents for the removal of naphthol green B and indigo carmine dyes from wastewater. While agricultural waste materials have been explored for wastewater treatment and rice husk modification with urea to enhance adsorption capacity is a unique approach. This innovative method offers a cost-effective and environmentally friendly solution for treating dye-contaminated wastewater, contributing to sustainable wastewater management practices. Their cost-effectiveness, ease of application, and high removal efficiency make them attractive options for mitigating dye pollution. The results are notable, with wheat bran and urea-modified rice husk achieving removal rates of 96% and 98% for naphthol green b, respectively. Similarly, indigo carmine removal rates reached 92% and 91% with wheat bran and urea-modified rice husk, respectively. Using Fourier transform infrared spectroscopy and scanning electron microscopy, various mechanisms behind the adsorption process of both dyes onto the adsorbent’s surfaces have been uncovered. These mechanisms encompass electrostatic interactions and the active roles of functional groups. The study results underscore that wheat bran and urea-modified rice husk are not just cost-effective but also highly efficient adsorbents for removing acidic dyes from wastewater.
Journal Article
Clinical outcomes of patients with remitting ulcerative colitis after discontinuation of indigo naturalis
Indigo naturalis is an effective treatment for ulcerative colitis. However, long-term use of indigo naturalis causes adverse events, such as pulmonary hypertension. The natural history of patients with ulcerative colitis who discontinued indigo naturalis after induction therapy is unknown. Moreover, the clinical features of patients who relapsed within 52 weeks after the discontinuation of indigo naturalis are unclear. This study aimed to assess the clinical outcomes of patients with ulcerative colitis after discontinuation of indigo naturalis and to identify potential markers responsible for relapse. This single-center retrospective study investigated the follow-up of 72 patients who achieved a clinical response 8 weeks after indigo naturalis treatment. We observed relapse in patients with ulcerative colitis after the discontinuation of indigo naturalis. We analyzed the factors predicting long-term outcomes after discontinuation of indigo naturalis. Relapse was observed in 24%, 57%, and 71% of patients at 8, 26, and 52 weeks, respectively. There were no predictive markers in patients who relapsed within 52 weeks after the discontinuation of indigo naturalis. The ulcerative colitis relapse rate after indigo naturalis discontinuation was high. Follow-up treatment is required after the discontinuation of indigo naturalis in patients with ulcerative colitis.
Journal Article
The activity of indigo carmine against bacteriophages: an edible antiphage agent
Bacteriophage infections in bacterial cultures pose a significant challenge to industrial bioprocesses, necessitating the development of innovative antiphage solutions. This study explores the antiphage potential of indigo carmine (IC), a common FDA-approved food additive. IC demonstrated selective inactivation of DNA phages (P001, T4, T1, T7, λ) with the EC
50
values ranging from 0.105 to 0.006 mg/mL while showing no activity against the RNA phage MS2. Fluorescence correlation spectroscopy (FCS) revealed that IC selectively binds to dsDNA, demonstrated by a significant reduction in the diffusion coefficient, whereas no binding was observed with ssDNA or RNA. Mechanistically, IC permeates the phage capsid, leading to genome ejection and capsid deformation, as confirmed by TEM imaging. Under optimal conditions (50 °C, 220 rpm), IC achieved up to a 7-log reduction in phage titer, with kinetic theory supporting the enhanced collision frequency induced by agitation. Additionally, IC protected
E. coli
cultures from phage-induced lysis without affecting bacterial growth or protein production, as demonstrated by GFP expression assays. IC’s effectiveness and environmental safety, combined with its FDA approval and cost-effectiveness, make it a promising antiphage agent for industrial applications.
Key points
•
Indigo
carmine effectively inactivates a broad spectrum of bacteriophages, offering protection to bacteria in industrial cultures.
•
A novel application of indigo carmine as a food-grade, environmentally safe, and FDA-approved antiphage agent protecting bacterial cultures.
•
Antiphage activity arises from indigo carmine’s interaction with DNA within the phage capsid without harming bacterial cells or compromising protein production in bacterial cultures.
Graphical abstract
Journal Article
Indigo Naturalis Ameliorates Dextran Sulfate Sodium-Induced Colitis in Mice by Modulating the Intestinal Microbiota Community
Indigo naturalis (IN) is a traditional Chinese medicine, named Qing-Dai, which is extracted from indigo plants and has been used to treat patients with inflammatory bowel disease (IBD) in China and Japan. Though there are notable effects of IN on colitis, the mechanisms remain elusive. Regarding the significance of alterations of intestinal flora related to IBD and the poor water solubility of the blue IN powder, we predicted that the protective action of IN on colitis may occur through modifying gut microbiota. To investigate the relationships of IN, colitis, and gut microbiomes, a dextran sulfate sodium (DSS)-induced mice colitis model was tested to explore the protective effects of IN on macroscopic colitis symptoms, the histopathological structure, inflammation cytokines, and gut microbiota, and their potential functions. Sulfasalazine (SASP) was used as the positive control. Firstly, because it was a mixture, the main chemical compositions of indigo and indirubin in IN were detected by ultra-performance liquid chromatography (UPLC). The clinical activity score (CAS), hematoxylin and eosin (H&E) staining results, and enzyme-linked immunosorbent assay (ELISA) results in this study showed that IN greatly improved the health conditions of the tested colitis mice, ameliorated the histopathological structure of the colon tissue, down-regulated pro-inflammatory cytokines, and up-regulated anti-inflammatory cytokines. The results of 16S rDNA sequences analysis with the Illumina MiSeq platform showed that IN could modulate the balance of gut microbiota, especially by down-regulating the relative quantity of Turicibacter and up-regulating the relative quantity of Peptococcus. The therapeutic effect of IN may be closely related to the anaerobic gram-positive bacteria of Turicibacter and Peptococcus. The inferred metagenomes from 16S data using PICRUSt demonstrated that decreased metabolic genes, such as through biosynthesis of siderophore group nonribosomal peptides, non-homologous end-joining, and glycosphingolipid biosynthesis of lacto and neolacto series, may maintain microbiota homeostasis during inflammation from IN treatment in DSS-induced colitis.
Journal Article
Antioxidant effects of bis-indole alkaloid indigo and related signaling pathways in the experimental model of Duchenne muscular dystrophy
Indigo is a bis-indolic alkaloid that has antioxidant and anti-inflammatory effects reported in literature and is a promissory compound for treating chronic inflammatory diseases. This fact prompted to investigate the effects of this alkaloid in the experimental model of Duchenne muscular dystrophy. The main aim of this study was to evaluate the potential role of the indigo on oxidative stress and related signaling pathways in primary skeletal muscle cell cultures and in the diaphragm muscle from mdx mice. The MTT and Neutral Red assays showed no indigo dose-dependent toxicities in mdx muscle cells at concentrations analyzed (3.12, 6.25, 12.50, and 25.00 µg/mL). Antioxidant effect of indigo, in mdx muscle cells and diaphragm muscle, was demonstrated by reduction in 4-HNE content, H₂O₂ levels, DHE reaction, and lipofuscin granules. A significant decrease in the inflammatory process was identified by a reduction on TNF and NF-?B levels, on inflammatory area, and on macrophage infiltration in the dystrophic sample, after indigo treatment. Upregulation of PGC-1a and SIRT1 in dystrophic muscle cells treated with indigo was also observed. These results suggest the potential of indigo as a therapeutic agent for muscular dystrophy, through their action anti-inflammatory, antioxidant, and modulator of SIRT1/PGC-1α pathway.
Journal Article