Anthocyanins in Colorectal Cancer Prevention Review

Colorectal cancer (CRC) is still a big health burden worldwide. Nutrition and dietary factors are known to affect colorectal cancer development and prognosis. The protective roles of diets rich in fruits and vegetables have been previously reported to contain high levels of cancer-fighting phytochemicals. Anthocyanins are the most abundant flavonoid compounds that are responsible for the bright colors of most blue, purple, and red fruits and vegetables, and have been shown to contribute to the protective effects of fruits and vegetables against cancer and other chronic diseases. Berries and grapes are the most common anthocyanin-rich fruits with antitumor effects. The antitumor effects of anthocyanins are determined by their structures and bioavailability as well as how they are metabolized. In this review, we aimed to discuss the preventive as well as therapeutic potentials of anthocyanins in CRC. We summarized the antitumor effects of anthocyanins and the mechanisms of action. We also discussed the potential pharmaceutical application of anthocyanins in practice.


Introduction
Colorectal cancer (CRC) is the third most common tumor and the second leading lethal tumor worldwide [1]. The risk factors for CRC include heritable genetic changes, environmental and food-borne mutagens, excess weight and obesity, inflammatory bowel disease, heavy alcohol use, smoking, physical inactivity, compromised gut microbiota, and poor diet. Nutrition and dietary factors are known to affect CRC. Epidemiological investigations have revealed that diets high in fruits, vegetables, and whole grains are associated with reduced risk of CRC, while diets high in red and processed meat are associated with increased risk [2]. The American Cancer Society recommends limiting the intake of red and processed meats and eating more vegetables and fruits to help lower the risk of CRC. The protective roles of diets rich in fruits and vegetables have been previously reported. Fruit and vegetable-rich diets are associated with reduced oxidation, DNA damage, cell growth inhibition, cell cycle arrest, and low-grade inflammation as well as the promotion of beneficial gut microbiota [3]. Phytochemicals, naturally occurring plant chemicals that provide fruits and vegetables with color, odor, and flavor, show the potential to reduce oxidative stress, prevent DNA damage, impact DNA repair, slow cancer cell growth, promote cancer cell death, reduce inflammation, and boost immune function [4]. The most popular types of phytochemicals reported exhibiting anticancer efficacy contain carotenoids, flavonoids, isoflavones, polyphenols, and isothiocyanates. Anthocyanins are the most abundant flavonoid compounds that are responsible for the colors of most fruits and vegetables, and they contribute to the protective effects of fruits and vegetables against chronic diseases and cancer [5].
Anthocyanins are derived from flavonol with a basic structure of a flavylium ion lacking a ketone oxygen at the 4-position [6,7]. Depending on their skeleton, there are six The chemical structure of anthocyanins is significantly associated with their stability and bioavailability, both of which determine their health benefits. Most anthocyanins are not stable and are sensitive to light, temperature, pH, enzymes, oxygen, antioxidants, and other factors, necessitating high standards for the anthocyanin regimen and supplement product.
The bioavailability and metabolism of anthocyanins are of great research interest in biomedicine, clinic, and pharmacology. The rate and extent of anthocyanin absorption are affected by the anthocyanidin, sugar moiety, and acylated groups in the chemical structure. Anthocyanins can be absorbed via the mouth, stomach, and small intestine. Anthocyanins can be taken up, hydrolyzed, and conjugated by oral epithelial cells, and oral bacteria [9]. Anthocyanins can permeate the gastric mucosa through a bilitranslocase-mediated mechanism [10]. Anthocyanin glycosides can be rapidly and efficiently absorbed in the small intestine, involving transportation mechanisms such as glucose transporter SGLT1, glucoseassociated transporters (GLUTs), and intestinal bilitranslocase [10][11][12][13]. In the colon, the absorption of anthocyanins is zero, but the metabolism of anthocyanins will occur due to spontaneous degradation under physiological conditions or microbial metabolism [11,14]. However, only a small amount of anthocyanins can be found in the circulatory system and in the urine. The gap between the poor bioavailability and the positive health effects of anthocyanins is still a big challenge for the pharmacology research and development of anthocyanin-derived drugs. Extensive first-pass metabolism of anthocyanins is one of the reasons for the poor bioavailability, which means that most anthocyanins enter the circulatory system as metabolites [8,11,12,15]. The total bioavailability of anthocyanins should be higher than the estimated approximate 1% in previous reports when accounting for unmetabolized parent compounds, phase I and phase II metabolites, conjugated products, and microbe-generated metabolites [8,11,12,15]. However, the complex mixtures of unmetabolized parent anthocyanins, phenolic degradation products, glucuronide conjugates, sulfides conjugates, and post-colonic bacterial metabolites may coexist in the biofluids and tissues. Which anthocyanins are subject to the specific metabolism is not well known, and the technology to detect all the metabolites is also limited. Scientific understanding of the bioavailability and metabolism of anthocyanins is constantly evolving.
In our former studies, we demonstrated the cancer preventive effects of strawberries and black raspberries in colon cancer and esophageal cancer and found that anthocyanins are the main components in both fruits [16,17]. The roles of strawberry and black raspberry (BRB) extracts and anthocyanin compounds in colon cancer cell lines or animal models are summarized in our latest review on strawberries and BRB in inflammation-related CRC prevention [5]. Anthocyanins exist abundantly in fruits, vegetables, legumes, nuts, tea, and wine. Aside from strawberries and BRB, other fruits and vegetables rich in anthocyanins such as cherry, grape, pomegranate, purple sweet potato, eggplant, and some dark leafy greens also showed anticancer effects against CRC in both in vitro and in vivo models [16,17]. In this review, we summarize the anticancer effects of anthocyanins in CRC, and then discuss the pharmaceutical application of anthocyanins in CRC prevention and therapy.

Epidemiology Study
Some epidemiology studies have found an inverse association between the high consumption of anthocyanins and the decreased risk of CRC. A large multicentric case-control study conducted in Italy found a significant inverse relationship between anthocyanidins and CRC, with a hazard ratio of 0.78 (95% CI, 0.61-0.99) adjusting age, sex, fruit and vegetable intake, BMI, physical activity, alcohol intake, and education level [18]. In another case-control study in China, no significant association was found between the total flavonoid intake and CRC risk, but an inverse association between anthocyanidins and CRC risk was verified with an adjusted OR of 0.80 (95% CI 0.64, 1.00) for the highest quartile for anthocyanidin consumption compared with the lowest quartile [19]. However, other studies have shown no association between anthocyanin intake and CRC risk [20][21][22][23]. A more recent meta-analysis indicates that anthocyanin consumption is inversely associated with the risk of developing CRC with a pooled RR at 0.78 (95% CI, 0.64-0.95) [24]. Regarding the critical roles of systematic review and meta-analysis in evidence-based medicine, this finding significantly supports the active role of anthocyanins in CRC prevention.

Anthocyanin-Rich Whole Fruits and Vegetables in Colon Cancer Prevention
In our previous study, we detected the role of lyophilized strawberries in azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced and inflammation-associated colon carcinogenesis in the murine model. We found that strawberries decreased tumor incidence, reduced a bundle of proinflammatory mediators, and the nitrosative stress, at least, through inhibiting phosphatidylinositol 3-kinase (PI3K)/AKT and nuclear factor kappa B (NFkB) signaling cascades, as indicated in Table 1 [16]. Other groups also found that lyophilized blackberries and strawberries reduce the tumor multiplicity and pro-inflammatory gut bacteria in the AOM/DSS murine model [25]. In our recently published review, we summarized the roles of strawberries, BRB, and anthocyanin components in the chemoprevention of inflammatory bowel disease (IBD)-related CRC [5]. In the rat model of colitis-associated carcinogenesis induced by 1,2-dimethylhydrazine (DMH) and 2,4,6-trinitrobenzene acid (TNBS), lyophilized acai pulp can reduce aberrant crypt foci (ACF) multiplicity, tumor cell proliferation, and incidence of tumors with high-grade dysplasia [26]. IBD patients are at an increased risk of developing CRC. CRC that develops in IBD patients tends to have a worse prognosis and survival rate compared to patients with sporadic CRC in the advanced stage. Compared to the modest efficiency of some chemical inhibitors, the anthocyanin-rich whole fruits showed better efficiency in preventing CRC in IBD patients due to additive and synergistic activities of multiple bioactive phytochemicals [5].
For sporadic CRC, BRB showed preventive effects in the non-inflammation-promoted CRC model, the AOM rat model. BRB decreased the ACF burden, ACF, and adenocarcinoma multiplicities, and reduced urinary 8-hydroxy-2 -deoxyguanosine (8-OHdG) levels [27]. Pomegranate and baked purple potato were also shown to prevent CRC development in the AOM-induced CRC in animals. Pomegranate juice suppressed the number of ACF and dysplastic ACF, lowered proliferation of mucosa cells and decreased expression levels of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), NF-κB (p65), and vascular cell adhesion molecule 1 (VCAM-1) [28]. Baked purple potato suppressed the tumor incidence, promoted apoptosis, and increased cancer stem cell markers [29]. Apc Min mouse is a popular animal model for studies of human colon cancer. With a mutation in the APC gene, the primary phenotype of Apc Min mice is the development of multiple intestinal adenomas [30]. Tart cherry significantly decreased the number and volume of adenomas in Apc Min mice [31]. As in IBD-related CRC, anthocyanin-rich whole fruits and vegetables are efficient strategies to prevent sporadic CRC. Table 1. Anthocyanin-rich whole fruits and vegetables in colon cancer prevention.

Fruit
Model Findings Ref.

Anthocyanin Extract in Colon Cancer Prevention
The pros for whole foods in animal studies are the additive and synergistic activities of multiple bioactive phytochemical profiles. These pros are also cons for the whole food study, as it is unclear as to which components are responsible for chemoprevention. We considered anthocyanins to be largely responsible for the chemoprevention effects of anthocyanin-rich fruits and vegetables. The anthocyanin extract from these foods showed significant efficiency in preventing colon cancer in cell lines, animal models, and clinical trials ( Table 2).

Black Raspberry
In CRC cell lines, the anthocyanin-enriched extract of BRB can suppress cell proliferation, induce apoptosis, and decrease the activity of DNMT1 and DNMT3B, and demethylate CDKN2A, SFRP2, SFRP5, and WIF1 in the Wnt pathway [32,33]. In the AOM/DSS colon cancer mouse model, the BRB anthocyanin extraction in the diet decreased tumor multiplicity, modulated the composition of gut commensal microbiota, and changed the inflammation index and the methylation status of the SFRP2 gene [34].

Bilberry
The bilberry anthocyanin extract showed antioxidative activity in Caco-2 cells, with an IC 50 at 0.53 ± 0.04 mM [35]. Bilberry showed an antiproliferation effect in HT-19 cells and the mechanisms associated with increasing the expression of p21 [38][39][40]. Bilberry anthocyanin extract induced mitochondrial damage, activated apoptosis, inhibited cell proliferation in the mouse colon cancer cell line MC38 [41]. Bilberry extract has the potential to reduce the side effects of some chemotherapy drugs by suppressing the activity of topoisomerase I and the DNA strand breaks [42]. In the AOM/DSS-induced and IBDrelated CRC mouse model, the bilberry anthocyanin extract decreased the tumor number and inflammation [43]. In the AOM rat model, the bilberry extract was shown to reduce total ACF, colonic cellular proliferation, and COX-2 mRNA expression [44].

Cranberry
The cranberry anthocyanin extract inhibited the cell proliferation of HT-29, SW620, and SW480 cells derived from the primary tumor and the metastatic site of the same patient with CRC [33,45]. Interestingly, the cranberry anthocyanin extract exhibited more potent inhibitory effects in the progressive and metastatic SW620 cells, but not SW480 cells [45].

Other Berries
Other berries like red raspberry and strawberry extract, lingonberry, elderberry, and black currant showed antitumor effects in CRC cell lines by inhibiting cell growth and promoting cell apoptosis [33,52]. The anthocyanin extract from flowering plant berries such as Prunus spinosa drupes (blackthorn) could inhibit the growth and colony formation, promote apoptosis in HCT-116 cells, and reduce tumor growth in xenograft mice [53].  Curly kale Caco-2, HT-29, and HCT 116 cells Inhibited cell proliferation [70] Pistacia atlantica sub kurdica HT 29 cell Inhibited cell growth; arrested S phase [72] Chinese eggplant HT 29 cell Antioxidant, protected cell from DNA damage [71] Apple Rat Inhibited ACF, regulated apoptosis-related genes Aurka, p53 and COX-2, and cell migration-related genes MMP-2 and 9 [73] 2.3.8. Grapes Besides berries, grapes are of great interest for research due to their rich anthocyanin profiles and noted health benefits for humans. In HT-29 and CaCo-2 cells, the anthocyaninrich extracts from grapes could inhibit cell growth and promote apoptosis [38,54,55]. The grape extract has the potential to reduce the side effects of some chemotherapy drugs by suppressing the activity of topoisomerase I and the DNA strand breaks [42]. Compared to phenolic acids, tannins, and flavonol components, the grape anthocyanin extract showed the greatest antiproliferation effects. In another study, however, the authors found that the non-anthocyanin fraction showed a better antitumor effect [56]. In the AOM rat model, the grape extract reduced the total burden of ACF and COX-2 mRNA expression [44]. In APC min mice, the grape pomace extract decreased adenoma burden, adenoma number, and the expression of AKT and Ki-67 [57].

Red Wine
Red wine contains anthocyanins as well as flavonoids and their derivatives and shows various promising biological effects. The methanol extracts of red wine could suppress HCT-15 cell growth and block the S, G2, and M phases [58]. The anthocyanin fraction showed higher efficiency than the non-anthocyanin fractions. In HCT-116 cells, the lyophilized red wine extract reduced cell proliferation, increased the expression of p53, p21 cell cycle gatekeepers, induced antioxidant response by activating transcriptional factor, nuclear factor erythroid 2-related factor 2 (Nrf2), and promoted differentiation through E-cadherin and the epithelial-mesenchymal transition (EMT) pathway [59].

Tart Cherry
Similar to whole tart cherry, anthocyanin-rich extract from tart cherry could decrease bodyweight loss and tumor burden when combined with sulindac in APC min mice [60].

Plum
Polyphenol-rich extract from Illawarra plum (Podocarpus elatus Endl.) suppressed cell growth, arrested S cell phase, increased apoptosis, and decreased telomerase activity and telomere length in HT-29 cells [61]. The anthocyanin extract from Java plum (Eugenia jambolana) suppressed cell proliferation, promoted apoptosis in HCT-116 cells, and elevated the proportion of colon cancer stem cells [62].

Vitis coignetiae Pulliat
Grape family fruit Vitis coignetiae Pulliat (Meoru in Korean) has been used in Korean folk medicine to treat inflammatory disorders and cancer for a long time. The intense dark red hue of Vitis coignetiae Pulliat indicates an abundance of anthocyanin pigments [63]. Anthocyanins isolated from Meoru inhibited cell viability, proliferation, motility, and invasiveness, and induced cell apoptosis in HCT-116 and HT-29 cells [63][64][65][66]. In the HT-29 xenograft mouse model, the anthocyanins isolated from Meoru significantly suppressed tumor growth [65].

Purple Fleshed Sweet Potato
Purple fleshed sweet potato is a specific sweet potato rich in anthocyanins [67]. The anthocyanin extract from purple fleshed sweet potato decreased cell number, inhibited cell growth, and promoted cell cycle arrest in WiDr and SW480 cells [67,68]. In colon cancer stem cells, anthocyanin extraction suppressed proliferation, promoted apoptosis, and suppressed the WNT signaling pathway [29]. In the mouse model of AOM-induced CRC, anthocyanin extraction decreased the ACF number, cell proliferation, and promoted apoptosis [67].

Other Fruits and Vegetables
The anthocyanin extract from purple-shoot tea [69], curly kale [70], Chinese eggplant [71], and Pistacia atlantica sub kurdica [72], and apple [73] showed antitumor effects in cell lines or carcinogenesis animal models. The antitumor effects are associated with cell proliferation inhibition, apoptosis induction, cell migration inhibition, cell cycle arrest, DNA damage protection, and antioxidation.

Cyanidin
Cyanidin is one of the most widespread anthocyanidins in fruits and vegetables. Aglycone cyanidin can inhibit CRC cell growth and proliferation [74][75][76]. In Cvorovic's study, they found that the cell toxicity was higher in drug-resistant cancer cells than that in their parental cells [76]. In Briviba's study, the author found that aglycone cyanidin showed better effects than its glycosides, cyanidin chloride-3-5-diglucoside and cyanidin chloride-3galactoside [75]. In the study by Renis, they reported that cyanidin-3-O-β-glucopyranoside can inhibit cancer cell growth and proliferation with different molecular effects than the aglycone cyanidin [74].
The purified cyanidin-3-glucoside from strawberries showed antioxidant effects as well as the ability to inhibit the cell growth of HT-29 and HT-116 cells. Moreover, cyanidin-3-glucoside had the best antioxidant effects among the anthocyanins and other phenolics extracted from strawberries [77]. The cyanidin-3-glucoside single component promoted colon cancer cell attachment to fibronectin and inhibited 3D spheroid growth [78]. Cyanidin-3-Oβ-glucoside extracted from purple corn can decrease the development of colonic mucosal lesions and ACF induction in DMH-PhIP-treated animals [79].

Delphinidin
Compared to cyanidin, delphinidin differs in the number and position of hydroxyl groups on the b ring in the molecular structure. Delphinidin inhibited cell growth, decreased cell viability, arrested the G1 and G2/M cell phases, and promoted apoptosis in the Caco-1 and HCT-116 cells [80][81][82]. The cell toxicity of delphinidin is higher in drug-resistant cancer cells than that in their mother cells [76].

Antioxidant
The most well-known mechanism associated with the health-protective effects of anthocyanins is antioxidant activity. Oxidative stress is a complex condition involving reactive oxygen species (ROS) (ONOO − , O 2− , H 2 O 2 , and LPO) and antioxidants (GPx and SOD 2 ). Oxidative stress occurs as a response to increased ROS or decreased antioxidants, leading to a failure to prevent ROS-induced oxidative damage [86]. Anthocyanins are reported to have superior antioxidant properties compared with other related polyphenols and well-recognized antioxidants such as ascorbic acid and α-tocopherol [77,87]. Anthocyanins were reported to suppress oxidative damage, DNA strand-breaking effects, transepithelial electrical resistance in colon cancer cells, and decreased oxidative stress marker urinary 8-OHdG levels in animal models of AOM-induced CRC [27,35,42,47,51,63,71,76,81,86,88].
Anthocyanins are antioxidative by scavenging ROS, promoting glutathione reductase expression involved in metal chelation, inhibiting lipoprotein oxidation, inhibiting peroxyl radical-induced apoptosis, and protecting the formation of oxidative DNA damage [47,51,71,76,86,88]. The antioxidant capacity of anthocyanins is affected by their structures. Hydroxyl groups in position 3 of ring C and the 3 , 4 and 5 positions in ring B are the major components responsible for the antioxidant activity. Generally, cyanidin with groups 3 , 4 di-OH substituted has higher antioxidant activity when compared to pelargonidin, malvidin, and peonidin with a single OH group in ring B [89]. However, delphinidin still has low antioxidant activity even with groups 3 and 4 di-OH substituted. Types of glycosylation of anthocyanidins are also important for their antioxidant activities. Different glycosylation patterns either enhanced or reduced the antioxidant capacity [71]. In addition, the antioxidant capacity of anthocyanins is impacted by the food intake of anthocyanins.

Antiproliferation
Anthocyanin-rich whole foods, anthocyanin extracts, anthocyanin single compounds, and anthocyanin metabolites have all exhibited antiproliferation activities in multiple colon cancer cell types and animal models. In parallel with the antiproliferation effects, anthocyanins arrested the cell cycle at G1/G0 and G2/M, and modulated cell cycle-related genes (cyclin A, cyclin B, cyclin E, cyclinD1, p21, p25, p53, and cyclin-dependent kinase inhibitors) [46,58,61,80,81]. The MAPK pathway (ERK, c-Jun-NH2 kinase, and p38) represents a superfamily of proteins that regulate cell differentiation, proliferation, and survival during carcinogenesis [16]. Blocking activation of the MAPK pathway was reported as one of the molecular mechanisms for the anticarcinogenic action of anthocyanidins in colon cancer [16,63,64,91]. Inhibition of the receptor tyrosine kinases (RTKs) such as epidermal growth factor receptor (EGFR), ErbB2, ErbB3, vascular endothelial growth factor receptor-2 (VEGFR-2), VEGFR-3, and insulin-like growth factor 1 receptor (IGF1R) might be another molecular event associated with the antiproliferation effects of anthocyanins in cancers [92,93]. Cyanidin-3-Oglucoside and delphinidin-3-O-glucoside significantly inhibited EGFR in HCT-116 and HT-29 cells [55]. AKT/ERK/NFκB signaling cascades play an important role in cell proliferation and survival [5]. Lyophilized acai pulp increased the negative regulators of cell proliferation such as Dlc1 and AKT3 [26]. Pomegranate juice inhibited the phosphorylation of PI3K/AKT and mTOR expression and suppressed NFκB in the AOM rat model [28]. Lyophilized strawberries decreased PI3K/AKT signaling and NFkB in the AOM/DSS mouse model [16]. Grape anthocyanin extract from red grape (Vitis coignetiae Pulliat) has inhibitory effects on PI3K/AKT and mTOR [57,63,65]. A decrease in both hTERT expression and telomere length was reported as one of the mechanisms of the antitumor effects of Illawarra plum in colon cancer [61].
The antiproliferation effects of anthocyanins are also affected by their structures. The ortho-dihydroxyphenyl structure on the B-ring was considered as the functional group contributing to the inhibitory action [94]. The nonacylated monoglycosylated anthocyanins have higher effects than anthocyanins with pelargonidin, triglycoside, and/or acylation with cinnamic acid exerted [40,71]. For each anthocyanin, the exact mechanisms for these potential anticancer activities differ from one another.

Induction of Apoptosis
Apoptosis is another key target for antitumor activities. Anthocyanin-rich whole foods, anthocyanin extract, anthocyanin single compounds and anthocyanin metabolites have all exhibited apoptosis-inducing activities in multiple colon cancer cell types and animal models. The pro-apoptotic effect of anthocyanins can be attributed to the ROS-mediated mitochondrial caspase-independent pathway [62,82,88]. Anthocyanin extract from purpleshoot tea promoted apoptosis through cleavage of poly adenosine diphosphate-ribose polymerase (PARP), activation of caspase 3, and an increased expression of the Bax/Bcl-2 ratio [69]. Anthocyanin extracts and single compounds promoted apoptosis by decreasing the expression of anti-apoptotic proteins (survivin, cIAP-2, and XIAP) [55]. Anthocyanins could promote apoptosis in a p53-independent manner in colon cancer stem cells by elevating Bax and cytochrome c, proteins-mediating mitochondrial apoptosis [29,82].

Anti-Invasive Activity
Cancer cells can spread beyond the layer of tissue where the initial tumor developed and grow into nearby healthy tissues. Anthocyanin extract from Vitis coignetiae Pulliat inhibited HT-29 cell invasion by suppressing the PI3K/AKT pathway and NFkB-MMP2/MMP-9 axis [64,66]. The EMT broadly regulates cancer cell invasion and metastasis. Anthocyanin extract from grapes can induce morphological change, increase the epithelial marker Ecadherin, and reduce the cell migration activity in HCT-116 cells [59]. Cancer stem cells (CSCs) are a subpopulation of cancer cells with unique properties of self-renewal, infinite division, and multi-directional differentiation potential [95]. CSCs play critical roles in colon cancer invasion and metastasis. Anthocyanin extract from grapes can significantly decrease the expression of colon CSCs markers CD44 and CD133 [59]. Anthocyanins extracted from baked purple-fleshed potato and Eugenia jambolana can inhibit proliferation and promote apoptosis in both colon cancer cells and colon CSCs [29,62]. The suppression of β-catenin, the WNT pathway effector and the key regulator of CSCs proliferation, contributed to the inhibition of CSCs by anthocyanins. β-catenin and E-cadherin regulation were also found in CRC patients consuming BRB powder [96].

Anti-Inflammation
Chronic inflammation induces oncogenic mutations, genomic instability, immune microenvironment changes, early tumor promotion, angiogenesis, and increased risk of CRC [5]. Reduction in inflammation by anti-inflammatory pharmaceuticals such as NSAIDs and 5-aminosalicylate showed chemopreventive effects for CRC. Anthocyanin-rich whole foods showed anti-inflammatory effects in multiple animal models.

Microbiota
Alterations in the composition and function of bacterial microbiota are considered significant factors for developing inflammation-promoted CRC [5]. The colon is the major place for anthocyanin metabolism due to microbial metabolism [11,14]. Anthocyanins showed both bacteriostatic and bactericidal activities in vitro [99]. Therefore, investigations on anthocyanins and microbiota play critical roles in elucidating the health benefits of anthocyanins as they relate to colon cancer. A cross-sectional study showed that the higher intake of anthocyanin-rich food was associated with higher microbial diversity [100]. Diets containing the anthocyanin extract from blackberries and strawberries significantly suppressed the populations of the pro-inflammatory Bilophila wadsworthia in AOM/DSS-treated animals [25]. Diets containing BRB modulate the imbalance in gut microbiota during carcinogenesis by decreasing the pathogenic bacteria Desulfovibrio sp. and Enterococcus spp., and increasing probiotics Eubacterium rectale, Faecalibacterium prausnitzii, and Lactobacillus in AOM/DSS-induced inflammation-associated CRC [34]. In the colitis mouse model, anthocyanins can reverse the DSS-induced reduction in Porphyromonadaceae, Rikenellaceae Prevotellaceae, Firmicutes/Bacteroidetes ratio, and decrease Ruminococcus gnavus [101,102]. The beneficial microbiota activities of anthocyanins are frequently reported in multiple disease animal models and human clinical trials. In C57BL/6 mice, blueberry anthocyanin extract increased Bifidobacterium, Lactobacillus, Roseburia, Faecalibaculum, and Parabacteroides [103]. In the mouse model, bileberry anthocyanins reduced the ratio of Firmicutes/Bacteroidetes (F/B), and increased Akkermansia and Parabacteroides [104]. In human clinical trials, anthocyanin-rich apple products increased Sutterella, Butyricicoccus, and Lactobacillus and reduced Streptococcus, Ruminococcus, Blautia, and Roseburia [105]. More research is needed to better understand how anthocyanins modulate the gut microbiota and thus regulate their bioavailabilities and antitumor activities.

Encapsulation
The low bioavailability of anthocyanins is a pitfall for clinical applications. Encapsulation of anthocyanins or their bioactive metabolites has the potential to increase the localized concentration and boost their health benefits such as chemoprevention for CRC. Anthocyanin nanocomplexes with chitosan hydrochloride (CHC), carboxymethyl chitosan (CMC), and beta-Lactoglobulin (beta-Lg) can significantly improve the stability and bioavailability of anthocyanins in a simulated gastrointestinal tract [106]. Oidtmann's study also showed that encapsulation can inhibit the early degradation of anthocyanins in the intestinal system [107]. The biological properties, especially the antioxidant effects, of bilberry extract-loaded pectin amide core-shell capsules, whey protein matrix capsules, and coated apple pectin matrix capsules were comparable to those of the non-encapsulated bilberry extract [108]. In colon cancer cell line HT-29, anthocyanins encapsulated within the FA-g-MD wall showed higher antioxidant activities than anthocyanins alone [109]. Endogenous nanoparticle exosomes can be used to encapsulate anthocyanins in order to increase their availability and efficiency. In Aqil's study, milk exosome-encapsulated anthocyanins had higher antiproliferation efficacy in ovarian cancer cell lines [110]. Moreover, encapsulation may have the possibility of regulating the gut microbial composition, and the biosynthesis of short-chain fatty acids by the gut microbiota [111].

Combination
The antitumor activity of anthocyanins can be affected by their structures. Methylation by catechol-O-methyltransferase (COMT) during metabolism will decrease the efficiency of anthocyanins [112]. The antiproliferative activity of anthocyanins extracted from grape anthocyanins, cyanidin-3-glucoside and delphinidin-3-glucoside, was improved when used in combination with the drug Entacapone, a COMT inhibitor [113]. The improved efficacy may be due to the higher bioavailability by inhibition of COMT and an increase in ROS.
To counteract their low bioavailability, anthocyanins can be combined with different anthocyanins, other polyphenols, and chemotherapy drugs [114,115]. Luteolin is a common plant flavonoid with the potential for cancer prevention and treatment. The combination of cyanidin-3-O-glucoside chloride and luteolin had higher efficiency of antiproliferation and apoptosis induction than either compound on its own [116]. The binding of pectin to anthocyanins can improve the stability of anthocyanins to enhance their colonic accessibility and gut microbial fermentation [117]. When combined with chemotherapy drugs, anthocyanins had the potential to decrease their side effects and improve efficacy. In the APC min mouse model, anthocyanin-rich tart cherry extract combined with sulindac had less significant body weight loss, fewer tumors, and a smaller total tumor burden [60]. Delphinidin showed the activity of autophagy induction in hepatocellular carcinoma cells. When delphinidin was combined with an inhibitor of autophagy, 3-methyladenine, the macroautophagy was significantly magnified [118]. Petunidin-3-O-glucoside combined with a PI3K inhibitor significantly increased cell death in glioblastoma cells [119]. Delphinidin selectively sensitized leukemia cell NB4 to As(III) by decreasing intracellular arsenic accumulation and strengthening intrinsic/extrinsic pathway-mediated apoptosis induction [120,121].

Gut Microbiota Fermentation
Low bioavailability and complex metabolite profiles suggest that metabolites play an important role in the chemopreventive effects of anthocyanins. Gut microbiota is crucial in anthocyanin metabolism, as gut microbiota fermentation can help to formulate active metabolites of anthocyanins with antitumor activities. The metabolites produced by fermentation varies by the composition of microbiota and the chemical structure of anthocyanins [122]. New phenolic compounds such as syringic acid, rhamnetin, hippuric acid, cinammic acid, protocatechuic acid, caffeic acid, kaempferolrhamnoside, chlorogenic acid, cryptochlorogenic acid, ferulic acid, etc., are commonly found as the metabolites of anthoycanins after gut microbiota fermentation. Interestingly, some disparities in the metabolite profile of anthocyanins by different gut microbial strains were also noticed [123]. The alternation in gut microbiota and the bioavailability of anthoycanins by microbiota are two key elements in investigating the health benefits of anthocyanins. However, because collective knowledge of the gut microbiome still necessitates expansion, further research is needed to determine the association between the health benefits of anthocyanins and microbiota fermentation.

Conclusions
Epidemiology evidence, cell line investigations, and animal preclinical studies all showed the antitumor effects of anthocyanins in colon cancer. Anthocyanin-rich whole foods, anthocyanin extracts, anthocyanin single compounds, and the metabolites of anthocyanins all exhibited antitumor effects against CRC. The antitumor effects of anthocyanins are associated with multiple biological processes including proliferation, apoptosis, cell invasion, methylation, inflammation, and gut microbiota modification involving multiple genes and signaling pathways. The antitumor effects of anthocyanins are affected by their structures. Low bioavailability is a large pitfall in the research on anthocyanins. Phase I and phase II metabolites, conjugated products, and microbe-generated metabolites should be considered in the total bioavailability. The antitumor effects of anthocyanins may be attributed to the involvement of multiple genes, integrated roles in cell proliferation, apoptosis, invasion and metastasis, epigenetics, inflammation and gut microbiota modification. Using encapsulation strategies can increase the local bioavailability and target tissue specificity. Combining different anthocyanins, and anthocyanins with other polyphenols or drugs, and harnessing the gut microbiota fermentation, may enhance the health benefits of anthocyanins.
Author Contributions: N.S. conceptualized the idea, drafted, and reviewed the manuscript. X.C. reviewed the manuscript and revised the manuscript. T.C. conceptualized the idea, supervised the project, reviewed, and revised the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: This work is supported by the internal funds of The Ohio State University Comprehensive Cancer Center.