Abstract

   Anthocyans, containing anthocyanins and anthocyanidins, play a crucial
   role in preventing and treating inflammatory bowel disease (IBD). Most
   anthocyanins and their basic elements, namely anthocyanidins have been
   recognized for the effective treatment of IBD, but the key biomarkers
   of anthocyan-treated IBD remain unclear. In this study, a
   bioinformatics analysis based on network pharmacology was performed to
   demonstrate the core-targets, biological functions, and signaling
   pathways of most common anthocyanidins that existed in anthocyans to
   reveal their potential or major mechanisms. The network pharmacology of
   the multi-target drug molecular design with specific signal nodes was
   selected, which was used to analyse core targets and complete the
   bioinformatics analysis of core targets. The network assays indicated
   44 common targeted genes, 5 of which were core targets of both six most
   common anthocyanidins and IBD. These 44 common targets related to major
   signaling mechanisms of the six most common anthocyanidins in IBD may
   involve following processes: promotion of intracellular metabolism and
   proliferation, inhibition of cell necrosis, anti-inflammation and
   regulation of intestinal epithelial survival mainly via pathways such
   as, the EGFR tyrosine kinase inhibitor resistance pathway, platelet
   activation, microRNAs in cancer, arachidonic acid metabolism and the
   cGMP-PKG signaling pathway. Thus, our findings may provide other
   molecular details about anthocyans in the treatment of IBD and
   contribute towards the use of anthocyanidins, which will be meaningful
   shedding light on the action mechanisms of anthocyanidins in treating
   IBD.
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   Anthocyans, containing anthocyanins and anthocyanidins, play a crucial
   role in preventing and treating inflammatory bowel disease (IBD).[32]
   graphic file with name d0ra09117k-ga.jpg

1. Introduction

   Inflammatory bowel disease (IBD) is chronic and intestinal inflammation
   with unknown etiology and pathogenesis, which is mainly defined as
   Crohn's disease (CD) or ulcerative colitis (UC).^[33]1 UC is an
   idiopathic and chronic inflammatory disorder of the colonic mucosa,
   which starts in the rectum and extends to the proximity in an
   uninterrupted pattern through the entire colon or part of it, and
   bloody diarrhea, abdominal pain, and urgency are the typical presenting
   symptoms of UC.^[34]2 Mainly affected by the change of environmental
   factors and gut microbiota, CD often causes disorders of innate and
   adaptive immune responses, ranging from mildly active diseases such as
   occasional diarrhea and rectal bleeding to severely active diseases
   that may cause 10–20 translation of bloody bowel per day. Besides,
   people with CD have an increased risk of cancer, osteoporosis, anemia,
   nutritional deficiency, depression, infection, and thrombosis.^[35]3
   However, the location of inflammation is different between CD and UC.
   In CD, inflammation is usually transmural, and CD can be associated
   with intestinal granuloma, strictures, and fistulas, while in UC,
   inflammation is usually limited to the mucosa, and the symptoms in CD
   are not typical in UC.^[36]4 In addition, IBD, including two idiopathic
   types of CD and UC, and atypical types of collagen colitis and
   refractory colitis, is characterized as chronic gastrointestinal
   inflammation, alternating clinical remission recurrence, and patients
   with severe IBD need to be treated by the colorectal resection.^[37]5
   IBD has affected over 1 million individuals in the United States, while
   more than 2.5 million in Europe. The prevalence of IBD is highest in
   the Western world, affecting up to 0.5% of the total population, with a
   treatment cost of over 6 billion in 2004.^[38]6 There are mainly
   5-aminosalicylic acid, 6-mercaptopurine, sulfasalazine,
   glucocorticoids, azathioprine, cyclosporine, thioguanine, methotrexate,
   and tacrolimus. However, long-term usage is limited due to different
   side effects and other adverse effects of these drugs, including,
   diarrhea, abdominal pain, nausea, and vomiting.^[39]7 Consequently, it
   is essential to highlight the exploit of new treatment and drugs for
   IBD to relieve the current risk and reduce costs.

   It is noteworthy that many patients with IBD begin to turn to
   alternative medicine and turn to traditional plant-based drugs;
   consequently, anthocyan-rich foods are highly emphasized. Anthocyans,
   including anthocyanins and anthocyanidins, are a group of water-soluble
   plant pigments found in different parts of higher plants, mainly
   flowers and fruits, including leaves, stems, and roots. Depending on
   the pH value and the presence of chelating metal ions, they are
   intensely coloured in purple, red, blue, or violet.^[40]8 Notably,
   anthocyanins and anthocyanidins have antioxidative, antimicrobial,
   anti-inflammatory, and anti-mutagenic properties, which have been
   recognized as bioactive substances with health benefits. Anthocyanins
   and anthocyanidins play a crucial role in preventing and treating many
   chronic diseases such as metabolic disorders, cancer, eye diseases, and
   cardiovascular diseases, particularly IBD.^[41]9,10 Anthocyans play an
   anti-inflammatory role by protecting intestinal mucosal integrity,
   epithelial barrier function, and recovery of immune regulation, and by
   regulating gut microbiota, which is beneficial to IBD
   treatment.^[42]11,12 Moreover, anthocyanin degradation products and
   colonic metabolites, such as anthocyanidins, can suppress
   pro-inflammatory cytokine production.^[43]13 As the bioavailability of
   consumed anthocyanins and anthocyanidins to the plasma seems to be less
   than 2%, it is recognized that the anthocyans are either absorbed
   through the gastrointestinal tract or metabolized in the intestines or
   liver.^[44]8 Numerous studies have demonstrated the anti-inflammatory
   effects of anthocyan-rich fruits and plants (ESI Table 1[45]†).
   Increasing evidence suggests anthocyans as dietary supplements, whose
   role in managing IBD and its associated oncogenesis is deemed to be
   crucial.^[46]14 Due to the existence of flavonoid ions in the
   anthocyanin structure and its unique electronic distribution, multiple
   anthocyanins are derived from natural anthocyanidins and will be
   hydrolyzed into anthocyanidins. There are only six anthocyanidins that
   have been commonly found in fruits and vegetables, namely, cyanidin,
   peonidin, delphinidin, pelargonidin, malvidin, and petunidin.^[47]15,16
   The six most common anthocyanidins are highlighted as the basic
   elements of anthocyanins, and they are produced upon anthocyanin
   hydrolysis. Otherwise, early research has pointed out that the
   anthocyanidins could very well be the immediate metabolites after
   ingestion of anthocyanins because the h-glucosidase enzyme found in
   intestinal bacteria can easily hydrolyze respective anthocyanins to
   anthocyanidins.^[48]17,18 In addition, another research found that the
   anthocyanins assayed did not inhibit cell proliferation of cell lines
   tested at a specific concentration (200 μg ml^−1), but anthocyanidins
   showed cell proliferation inhibitory activity.^[49]19 Therefore,
   herein, we summarize the anti-inflammatory activity or potential of the
   six most common anthocyanidins (ESI Table 2[50]†), and their actions by
   inhibiting the downstream genes are shown in [51]Fig. 1, highlighting
   that the downstream genes are closely connected with the
   anti-inflammation of the mentioned six anthocyanidins, as described in
   the following research.

Fig. 1. Anti-inflammatory activity of the six most common anthocyanidins. ↓
represents inhibition. Red circles represent the most common anthocyanidins
and yellow circles the downstream genes.

   [52]Fig. 1
   [53]Open in a new tab

   Cyanidin and delphinidin acted by mitigating TNFα-triggered activation
   of transcription factor nuclear factor kappa B (NF-κB), and downstream
   phosphorylation of myosin light chain (MLC), which support a particular
   capacity in the protection of the intestinal barrier against
   inflammation-induced permeabilization, in part through the inhibition
   of the NF-κB pathway.^[54]20 Another research pointed out that cyanidin
   and delphinidin supplementary in mice fed with a high-fat diet can
   reduce lipid deposition and inflammation in the liver, potentially
   related to the activation of the redox-sensitive signals IKK/NF-κB and
   JNK1/2, and increased expression of the PTP1B phosphatase regulated by
   NF-κB.^[55]21,22 In the research of anti-inflammatory activities of
   peonidin, treatment of JB6 P(+) cells with peonidin inhibited
   12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cytochrome c oxidase
   assembly protein-2 (COX-2) expression, and also decreased TPA-induced
   neoplastic transformation and blocked TPA-induced phosphorylation of
   extracellular signal-regulated kinases (ERKs) in the cells to meet the
   anti-inflammatory effect.^[56]23 However, malvidin, peonidin, and
   petunidin exhibited an inhibitory effect of secretory phospholipase A
   (2) (sPLA2).^[57]24 It is known that sPLA2 contributes to the mediation
   of the inflammation, which is the main factor leading to the excessive
   production of arachidonic acid under inflammatory conditions.^[58]25,26
   Thus, these anthocyanidins play a modulatory role, realizing their
   anti-inflammatory activities. Otherwise, pelargonidin accomplishes its
   anti-inflammatory effects by inhibiting the inducible nitric oxide
   synthase (iNOS) protein and mRNA expression and nitric oxide (NO)
   production.^[59]27 Studies have shown that compounds that inhibit the
   expression or activity of iNOS have potential anti-inflammatory
   effects.^[60]28,29

   After collecting and analysing the effects of anthocyan-rich foods on
   IBD, the relevant anti-inflammatory activities of the six most common
   anthocyanidins were studied. However, the mentioned mechanism about the
   anthocyanidins is still unclear, and the treatment of IBD still
   urgently needs to be studied as the early research has pointed out the
   anti-inflammatory effects of anthocyanidins^[61]20–26 and also other
   effects on various types of cancers,^[62]17,19 making it essential to
   completely explore the usefulness of this natural ingredient. Thus, we
   aimed to find out the potential mechanisms of IBD treatment by
   analysing the most existing anthocyanidins. We consider that the common
   targets and potential signaling mechanisms of these 6 anthocyanidins
   for treating IBD will contribute to relevant information to IBD
   treatment. To explore the common targets and major signaling mechanisms
   of the anthocyanidins in IBD treatment, we tried to integrate
   bioinformatics and network pharmacology tools to predict the target
   genes and analyse the significant interactions between the six most
   common anthocyanidins and IBD. The investigative flowchart is shown in
   [63]Fig. 2.

Fig. 2. Investigate flowcharts for prioritizing biotargets and signaling
pathways of anthocyans in treatment of IBD.

   [64]Fig. 2
   [65]Open in a new tab

2. Materials and methods

2.1. Screening potential targets of the six most common anthocyanidins and
IBD

   The potential targets of the most common anthocyanidins (cyanidin,
   delphinidin, malvidin, petunidin, pelargonidin, and peonidin) were
   acquired through analysis of the Traditional Chinese Medical Systems
   Pharmacology (TCMSP), SuperPred, and Swiss Target Prediction. TCMSP, a
   unique system pharmacology platform and a form of Chinese herbal
   medicines is used to capture the relationships between drugs, targets,
   and diseases. TCMSP selects the connected targets of anthocyanidins by
   using the SysDT formulas of Herbal Ingredients' Targets Database (HIT),
   and the information of TCMSP mainly comes from the Therapeutic Target
   Database (TTD) and PharmGKB databases.^[66]30 SuperPred is a prediction
   webserver for anatomical therapeutic chemical code and target
   prediction of compounds, which allows prognoses about the medical
   indication area of novel compounds and finds new leads for known
   targets.^[67]31 Swiss Target Prediction is an online tool since 2014.
   It aims to predict the most probable small molecular protein targets
   according to the principle of similarity, and possible targets can also
   be found through material structures' input on it.^[68]32 SuperPred and
   Swiss Target Prediction accomplish the work based on the methods,
   namely, 2D fragment and 3D similarity searching. Otherwise, there are
   authoritative and comprehensive disease analysis databases. GeneCards
   has been regarded as a searchable and integrative database, considered
   the most comprehensive genetic data database because of the
   comprehensive, easy-to-use information about all annotated and
   predicted human genes.^[69]33 The other one, Online Mendelian
   Inheritance in Man (OMIM), is a major repository of comprehensive and
   selected information on genes and genetic phenotypes and their
   relationships. Through this database, diseases are linked to genes
   related to the human genome and provide the relevant references for