Preventative and Therapeutic Potential of Flavonoids in Peptic Ulcers

Peptic ulcer disease is a common gastrointestinal tract disorder that affects up to 20% of the population of the world. Treatment of peptic ulcer remains challenging due to the limited effectiveness and severe side effects of the currently available drugs. Hence, natural compounds, owing to their medicinal, ecological, and other safe properties, are becoming popular potential candidates in preventing and treating peptic ulcers. Flavonoids, the most abundant polyphenols in plants, exhibit gastroprotective effects against peptic ulcer both in vivo and in vitro. In this review, we summarized the anti-ulcer functions and mechanisms, and also the bioavailability, efficacy, and safety, of flavonoid monomers in the gastrointestinal tract. Flavonoids exerted cytoprotective and rehabilitative effects by not only strengthening defense factors, such as mucus and prostaglandins, but also protecting against potentially harmful factors via their antioxidative, anti-inflammatory, and antibacterial activities. Although controlled clinical studies are limited at present, flavonoids have shown a promising preventable and therapeutic potential in peptic ulcers.


Introduction
A peptic ulcer is characterized as a mucosal break induced by acid or pepsin secretion in the gastrointestinal tract, especially the stomach and proximal duodenum [1]. Furthermore, peptic ulceration infiltrates through the mucosa layer to induce mucosal lesions, resulting in inflammation of the digestive tract [1,2]. Until the second half of the 20th century, Helicobacter pylori (H. pylori) infection and use of non-steroidal anti-inflammatory drugs (NSAIDs) were found to be the main risk factors of peptic ulcer [3]. Subsequently, various conditions, such as ischemia, inflammatory bowel disease (IBD), and renal diseases, and poor lifestyle, including stress, smoking, and excessive consumption of caffeine or alcohol, were also found to be risk factors of peptic ulcer [4]. Recently, a high incidence rate of 20% has been reported on peptic ulcers, which are mainly observed in 30-60-year-old people [4]. While the mortality rate of peptic ulcer is low, it is becoming prevalent and causes pain and severe complications. Peptic ulcer patients usually suffer from epigastric pain, such as burning or gnawing, and typical dyspeptic symptoms, such as bloating, nausea, fullness, and heartburn. Alternatively, some patients may experience complications, such as bleeding, perforation, and gastric

Anti-Ulcer Mechanisms of Flavonoids
Peptic ulcer is caused by an imbalance in gastrointestinal defense factors, such as prostaglandins, mucus, and bicarbonate, and potentially harmful factors, such as pepsin, acid, and H. pylori infection ( Figure 2). Anti-ulcer effects of flavonoids include functions such as anti-acid secretion, inhibition of pepsin level and activity, and increasing gastric mucus and bicarbonate secretion. Additionally, flavonoids boost mucosal cytoprotective, antioxidative, anti-inflammatory, and antibacterial defenses against peptic ulcer. Usually, one type of flavonoid can exhibit anti-ulcer roles through multiple mechanisms.

Anti-Ulcer Mechanisms of Flavonoids
Peptic ulcer is caused by an imbalance in gastrointestinal defense factors, such as prostaglandins, mucus, and bicarbonate, and potentially harmful factors, such as pepsin, acid, and H. pylori infection ( Figure 2). Anti-ulcer effects of flavonoids include functions such as anti-acid secretion, inhibition of pepsin level and activity, and increasing gastric mucus and bicarbonate secretion. Additionally, flavonoids boost mucosal cytoprotective, antioxidative, anti-inflammatory, and antibacterial defenses against peptic ulcer.
Usually, one type of flavonoid can exhibit anti-ulcer roles through multiple mechanisms.

Anti-Ulcer Mechanisms of Flavonoids
Peptic ulcer is caused by an imbalance in gastrointestinal defense factors, such as prostaglandins, mucus, and bicarbonate, and potentially harmful factors, such as pepsin, acid, and H. pylori infection ( Figure 2). Anti-ulcer effects of flavonoids include functions such as anti-acid secretion, inhibition of pepsin level and activity, and increasing gastric mucus and bicarbonate secretion. Additionally, flavonoids boost mucosal cytoprotective, antioxidative, anti-inflammatory, and antibacterial defenses against peptic ulcer. Usually, one type of flavonoid can exhibit anti-ulcer roles through multiple mechanisms.

Flavonoids Exert Anti-Ulcer Effects by Regulating Gastric Secretion Pathways
Normally, the stomach secretes a number of molecules, including gastric acid, pepsin, and gastric mucus. Stomach acid and pepsin promote digestion of ingested foods and gastric mucus protects the epithelial cells from damage due to gastric acid and pepsin [37] (Figure 3). However, a high concentration of gastric acid aggravates mucosal damage in peptic ulcer [38]. Therefore, inhibition of gastric acid excessive secretion is essential in peptic ulcer treatment. Gastric acid secretion is regulated by gastrointestinal hormones. Acetylcholine, gastrin, histamine are the main hormones that stimulate parietal cells to secrete acid. Additionally, somatostatin inhibits acid secretion and exerts a tonic restraint on parietal, enterochromaffin-like, and gastrin cells via acting on sst2 receptors [38,39]. More importantly, in the final step of gastric acid secretion, H + K + -ATPase, a proton pump in the membrane of parietal cells, catalyzes H + transport at the expense of ATP hydrolysis. Figure 3. Flavonoids exert anti-ulcer effects through regulating gastric secretion pathways and prostaglandin levels. Flavonoids (1) decrease acetylcholine, gastrin, histamine, and somatostatin levels and inhibit H+K+-ATPase activities, therefore inhibiting gastric acid secretion; (2) promote mucus and bicarbonate secretion; (3) inhibit pepsin activity; (4) exhibit cytoprotective activity by regulating prostaglandin levels.
Flavonoids could exert anti-ulcer effects by inhibiting gastric acid secretion, similar to how histamine (H2)-receptor antagonists and PPIs work. Catechins, the most abundant polyphenol in tea, showed gastroprotective effects by regulating gastric secretion pathways. Wistar rats treated with 0.1% and 1% crude catechin for 2 weeks had a reduced gastric lesion index (from 23 ± 9 to 16 ± 5 and 9 ± 7, respectively) in a water immersion restraint stress model and inhibited the release of gastrin (from 108 ± 21 to 56 ± 12 and 46 ± 9 pg/dL, respectively), somatostatin (from 169 ± 23 to 74 ± 11 and 70 ± 25 pg/dL, respectively), and histamine (from 139 ± 21 to 92 ± 18 and 79 ± 19 nmol/L, respectively) in an isolated rat stomach infusion model [40]. It assumed that catechin might confer a protective effect by regulation of gastrointestinal hormones. However, caution is required for the decrease in . Flavonoids exert anti-ulcer effects through regulating gastric secretion pathways and prostaglandin levels. Flavonoids (1) decrease acetylcholine, gastrin, histamine, and somatostatin levels and inhibit H+K+-ATPase activities, therefore inhibiting gastric acid secretion; (2) promote mucus and bicarbonate secretion; (3) inhibit pepsin activity; (4) exhibit cytoprotective activity by regulating prostaglandin levels.
Flavonoids could exert anti-ulcer effects by inhibiting gastric acid secretion, similar to how histamine (H 2 )-receptor antagonists and PPIs work. Catechins, the most abundant polyphenol in tea, showed gastroprotective effects by regulating gastric secretion pathways. Wistar rats treated with 0.1% and 1% crude catechin for 2 weeks had a reduced gastric lesion index (from 23 ± 9 to 16 ± 5 and 9 ± 7, respectively) in a water immersion restraint stress model and inhibited the release of gastrin (from 108 ± 21 to 56 ± 12 and 46 ± 9 pg/dL, respectively), somatostatin (from 169 ± 23 to 74 ± 11 and 70 ± 25 pg/dL, respectively), and histamine (from 139 ± 21 to 92 ± 18 and 79 ± 19 nmol/L, respectively) in an isolated rat stomach infusion model [40]. It assumed that catechin might confer a protective effect by regulation of gastrointestinal hormones. However, caution is required for the decrease in somatostatin as it may lead to an increased acid amount to some extent. In ischemia reperfusion-induced gastric ulcer rats, administration of 50 mg/kg catechins for 3 days reduced the level of H + K + -ATPase from 1.15 ± 0.05 to 0.51 ± 0.03 mmol Pi liberated min −1 (mg protein) −1 and increased the plasma histamine level when compared to the model group [41]. Quercetin is a common flavonol

Flavonoids Show Gastric Cytoprotective Activity by Regulating Prostaglandins Levels
Prostaglandins (PGs), such as prostaglandin E2 (PGE2), are the main arachidonic acid metabolites. PGs regulate production of gastric mucus and bicarbonate and reduction in acid output, restore the gastric mucosa by dilating vessels, improve mucosal blood flow, and accelerate mucosal healing [59,60]. Two isoforms of cyclooxygenase (COX), cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), are key enzymes in the biosynthesis of PGs. The COX-1 isoform is expressed in most tissues, including the gastrointestinal tract, and produces PGs. In contrast, COX-2 has no or little expression in most tissues but is rapidly induced in the inflammatory setting. Usually, traditional NSAIDs, such as indomethacin, non-selectively inhibit both COX-1 and COX-2 and cause peptic damage with a marked decrease in gastric PGE2 content [61].
Flavonoids show gastric cytoprotective activities by regulating PGs' biosynthesis pathways ( Figure 3). From the studies, Alcaraz et al. [49] suggested that the gastroprotective effects of the flavone derivative hypolaetin-8-glucoside against the NSAID indomethacin could be involved in the cytoprotective effects of endogenous PGs. This flavonoid also increased the COX activity in in vitro experiments. A 48 mg/kg indomethacin treatment significantly decreased PGE2 level by 85.5% in Sprague-Dawley rats. By pretreatment with 50 and 100 mg/kg chrysin, the gastroprotective PGE2 level increased by 87% and 90%, respectively [57]. Isoliquiritigenin, mentioned above, not only decreased the gastric lesion area and increased the gastric mucus secretion but also increased gastric COX-2 expression in indomethacin-induced ulcer in mice. However, the gastric PGE2 content was not evaluated in the study [56]. Genistein, a soy-derived isoflavone, also increased the gastric PGE2 level to 210.3 ± 5.4 ng/g tissue when compared to the indomethacin treatment model group (113.3 ± 4.6 ng/g tissue) [62]. By administration of 100 mg/kg diosmin (a flavonoid abundant in citrus fruits), the PGE2 level was significantly increased when compared with the ethanol treatment group, despite ethanol also evoking depletion of PGE2 [63]. Nobiletin, a polymethylated flavonoid from citrus fruits, exhibited a gastric cytoprotective effect through regulating PGE2. Pretreatments with 10 and 20 mg/kg nobiletin both increased the serum PGE2 level significantly in an ethanol-induced gastric model in mice [64]. Furthermore, treatment with 0.02, 0.07, and 0.21 g/d licoflavone from Glycyrrhiza upregulated the levels of arachidonic acid and PGE2 to protect the gastric mucosa and accelerate mucosal healing in an acetic acid-induced gastric ulcer model [65].
These species are normal byproducts of cellular metabolism, such as mitochondrial oxidative phosphorylation. When ROS concentration exceeds an organism's antioxidative capacity, cells enter an oxidative stress state, in which ROS cause oxidative damage to cellular components. They lead to lipid peroxidation and damage cell membranes, resulting in the release of intracellular components and tissue damage. ROS also cause degradation of gastric epithelial base membrane components and change intracellular metabolism and DNA damage [67][68][69]. As potent antioxidants, flavonoids scavenge free radicals and decrease their formation, thus providing positive effects against peptic ulcer. Early studies showed that ternatin, a tetramethoxy flavone isolated from Egletes viscosa Less, could act against gastric mucosal damage induced by ethanol but not indomethacin or hypothermic restraint stress after pretreatment at concentrations of 25 and 50 mg/kg in rats, indicating that ternatin affords a gastroprotection effect through a PGs-independent mechanism, probably involving free radical scavenging and anti-inflammatory actions [70]. In one in vitro experiment, catechins (10 −5 -10 −1 g/100 mL) developed O 2 − scavenging activity in a concentration-dependent manner. In vivo, oral administration of catechins in rats at doses of 50, 100, and 200 mg/kg reduced ethanol-induced gastric mucosal injury by 49%, 70%, and 100%, respectively. Higher doses of 300 and 400 mg/kg catechins reduced stress-induced gastric injury by 80% and 93%, respectively [55]. In a H 2 O 2 /NaOH/DMSO-generated ROS system, compared to DL-α-tocopherol, a common natural antioxidant, garcinol, a flavonoid isolated from Garcinia indica, had a stronger effect in scavenging hydroxyl radicals, a weaker activity in scavenging methyl radicals, and a comparable ability in scavenging superoxide anions. Furthermore, oral administration of 200 mg/kg garcinol prevented acute gastric ulceration by radical formation induced by both indomethacin-and water-immersion stress [71]. Pretreatments for 24 h with 25 and 50 µM of quercetin, also known as 3,5,7,3 ,4 -pentahydroxy flavone, both attenuated the increase in H 2 O 2 -induced oxidative stress in GES-1 cells. Quercetin also reduced gastric ROS accumulation in ethanol-induced gastric ulcer in Balb/c mice. Images of mice injected with ROS-sensitive L-012 were used to show the oxidative stress state of different groups. Obvious chemiluminescence signals were observed in the gastric region of mice treated with ethanol, but only weak chemiluminescence signals were detected after pretreatment with quercetin, suggesting that quercetin alleviated gastric ROS accumulation in ethanol-induced gastric injury [72].
Flavonoids not only scavenge ROS directly but also protect and activate antioxidant enzymes which, in turn, protect against oxidative damage in peptic ulcer. Antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), combat free radicals and alleviate oxidative damage [73]. SOD catalyzes the highly reactive superoxide free radical (O 2 − ) into less reactive hydrogen peroxide (H 2 O 2 ) and molecular oxygen (O 2 ), which is the first line of defense against ROS [74]. CAT breaks down H 2 O 2 into water and molecular oxygen, consequently completing the detoxification process initiated by SOD [75]. GPx catalyzes the breakdown of H 2 O 2 and inhibits lipid peroxidation [76]. Glutathione (GSH) is essential in maintaining gastric mucosal integrity, and depletion of GSH may induce mucosal ulceration [77]. It was found that in animal models of peptic ulcer induced by alcohols, stress, and NSAIDs, the activities of the antioxidant enzymes were decreased.
In an ischemia reperfusion-induced gastric ulcer model, pretreatment with 50 mg/kg (+)-catechins increased CAT and SOD activities (from 15.5 ± 1.3 to 32.4 ± 1.8 U, from 87.6 ± 12.4 to 145.0 ± 7.5 U, respectively) and reduced the level of malondialdehyde (MDA), an end-product of lipid peroxidation in peptic ulcer, from 0.48 ± 0.02 to 0.30 ± 0.01 nmol [41]. Catechins also showed gastroprotective effects in 95% ethanol-induced acute gastric ulcer by preventing the depletion of SOD activity and GSH level and by reducing lipid peroxidation at the doses of 25 and 50 mg/kg [78]. Quercetin accounted for the anti-ulcer ability of methanolic extract from Madhuca indica J. F. Gmel. (Sapotaceae) leaves. Treatment with 5 and 10 mg/kg quercetin for 14 days showed significant and dose-dependent healing effects on the ulcerated area that was caused by acetic acid in comparison to the control group in rats. At the same time, gastric SOD and GSH levels were also elevated significantly while the level of MDA decreased [79]. Treatment with low doses of rutin (20,40, and 80 mg/kg), a flavonol of Ruta graveolens, reduced the ulcer index in all ethanol-, acetic acid-, and stress-induced ulceration models by increasing vitamin C and GPx activity and decreasing MDA levels [80]. In the 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH) free radical scavenging assay, anthocyanins of Rubus coreanus exhibited free radical scavenging activities of 8.46%, 20.47%, 37.31%, and 69.17% at the doses of 10, 25, 50, and 100 µg/mL, respectively. In the naproxen-induced gastric ulcer model in rats, pretreatment with 20, 50, and 80 mg/kg anthocyanins twice daily for 3 days reduced gastric MDA levels and increased CAT and SOD activity [81]. Isoorientin, derived from Eremurus spectabilis, significantly decreased the MDA level (p < 0.05) and increased SOD activity and the GSH level in gastric tissues at doses of 25, 50, and 100 mg/kg [82]. Flavonoids, including hesperidin [47,48,54,83], diosmin [63], nobiletin [64], and genistein [62], aromadendrin-4 -O-methyl-ether and kaempferide [84] from Brazilian green propolis, and biochanin A [85] from soy and red clover also showed positive effects in treating peptic ulcer by increasing activities of antioxidant enzymes in vivo.
Flavonoids can also regulate nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway to improve oxidative stress. Nrf2 is a key transcription factor that regulates phase II detoxification and upregulates antioxidant genes HO-1. Upregulation of HO-1 expression results in increased accumulation of iron, bilirubin, and carbon monoxide, which in turn reduces the sensitivity of gastrointestinal cells to oxidative damage [86,87]. Treatment of Int-407 cells with 100 µM catechin decreased the ROS and lipid peroxidation, increased the activity of antioxidant enzymes, and upregulated nuclear/cytosol Nrf2 ratio and HO-1 protein expression in a time-dependent manner when compared with the ketoprofen-exposed model group. Sprague-Dawley rats pretreated with 35 mg/kg catechin for 21 days before the administration of ketoprofen also exhibited a reduced gastric ulcer area [88]. The flavonoid hesperidin was found to increase gastric expression of HO-1 and Nrf-2 in stress-induced gastric ulcer in diabetic rats [48]. Silymarin, a flavonoid mixture from the Silybum marianum (milk thistle) plant, prevented oxidative stress by enhancing GSH and SOD activities, upregulating the Nrf2 gene and inhibiting lipid peroxide production in indomethacin-induced gastric ulcer in albino rats [89].
This section showed that flavonoids have beneficial effects on treating peptic ulcer via antioxidative activity ( Figure 4). Flavonoids increase the activities of antioxidant enzymes SOD, CAT, GPx, GSH, and the nuclear Nrf2 protein level to scavenge ROS, then prevent the lipid peroxidation and protect the integrity of cell membranes and gastric tissue. Besides, the increased Nrf2 protein upregulates HO-1 to increase the iron, bilirubin, and carbon monoxide to minimize oxidative damage of gastric tissue.  [81]. Isoorientin, derived from Eremurus spectabilis, significantly decreased the MDA level (p < 0.05) and increased SOD activity and the GSH level in gastric tissues at doses of 25, 50, and 100 mg/kg [82]. Flavonoids, including hesperidin [47,48,54,83], diosmin [63], nobiletin [64], and genistein [62], aromadendrin-4′-O-methyl-ether and kaempferide [84] from Brazilian green propolis, and biochanin A [85] from soy and red clover also showed positive effects in treating peptic ulcer by increasing activities of antioxidant enzymes in vivo. Flavonoids can also regulate nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway to improve oxidative stress. Nrf2 is a key transcription factor that regulates phase II detoxification and upregulates antioxidant genes HO-1. Upregulation of HO-1 expression results in increased accumulation of iron, bilirubin, and carbon monoxide, which in turn reduces the sensitivity of gastrointestinal cells to oxidative damage [86,87]. Treatment of Int-407 cells with 100 μM catechin decreased the ROS and lipid peroxidation, increased the activity of antioxidant enzymes, and upregulated nuclear/cytosol Nrf2 ratio and HO-1 protein expression in a time-dependent manner when compared with the ketoprofen-exposed model group. Sprague-Dawley rats pretreated with 35 mg/kg catechin for 21 days before the administration of ketoprofen also exhibited a reduced gastric ulcer area [88]. The flavonoid hesperidin was found to increase gastric expression of HO-1 and Nrf-2 in stress-induced gastric ulcer in diabetic rats [48]. Silymarin, a flavonoid mixture from the Silybum marianum (milk thistle) plant, prevented oxidative stress by enhancing GSH and SOD activities, upregulating the Nrf2 gene and inhibiting lipid peroxide production in indomethacin-induced gastric ulcer in albino rats [89].
This section showed that flavonoids have beneficial effects on treating peptic ulcer via antioxidative activity ( Figure 4). Flavonoids increase the activities of antioxidant enzymes SOD, CAT, GPx, GSH, and the nuclear Nrf2 protein level to scavenge ROS, then prevent the lipid peroxidation and protect the integrity of cell membranes and gastric tissue. Besides, the increased Nrf2 protein upregulates HO-1 to increase the iron, bilirubin, and carbon monoxide to minimize oxidative damage of gastric tissue. Flavonoids increase the activities of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione (GSH). and the nuclear Nrf2 level to exert gastroprotective effects through scavenging reactive oxygen species (ROS) and up-regulating the phase II detoxification and antioxidant genes HO-1.2.4 Flavonoids ameliorate peptic ulcer by regulating inflammatory pathways.
Nitric oxide (NO) plays a multifaceted role in gastric mucosal stability. Low concentration of NO produced by constitutive nitric oxide synthase (cNOS) helps to retain gastric mucosal integrity, mediate gastric blood flow, and inhibit gastric acid secretion [96]. However, inflammation triggers upregulation of inducible nitric oxide synthase (iNOS) in macrophages and neutrophils, which increases NO levels and results in cytotoxic effects and gastric oxidative damage [96][97][98]. Pretreatment with rutin at doses of 50, 100, and 200 mg/kg exhibited gastroprotective effects against gastric ulcer induced by ischemia-reperfusion by preventing elevation of iNOS activity and by inhibiting cNOS and MPO activity in the gastric mucosa [94]. Indomethacin administration decreased the total nitrite/nitrate level in gastric mucosa, which may be related to a decreased production of cNOS in gastric tissue and upregulation of iNOS in neutrophils and macrophages. The catechin monomer epigallocatechin gallate (EGCG) showed ulcer-healing action against indomethacin-induced gastric ulcer by regulating NO levels. Treatment with 2 mg/kg EGCG reduced serum nitrite levels, suppressed the serum nitric oxide synthase activity, reversed increased iNOS expression, and reduced endothelial NOS expression in gastric tissues damaged by indomethacin [99].
Neutrophil infiltration leads to the production of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β), which is associated with the nuclear factor kappa B (NFκB) pathway and mitogen-activated protein kinases (MAPK) signaling cascades [100]. Stimulations such as ROS and NO or external stimuli activate NFκB by degrading iκB-α and phosphorylating NFκB p65/p50 subunits. Activated NFκB p65/p50 then translocate from the cytoplasm to the nucleus and promote gene expression of pro-inflammatory cytokines [101]. The MAPK cascade (ERK, extracellular-signal-regulated kinases; JNK, c-Jun N-terminal kinases; p38, p38 mitogen-activated protein kinases) is closely related to inflammation response. As mentioned above, COX-2 is induced primarily during inflammation and regulates the inflammatory response in gastric injury [102]. Pro-inflammatory cytokines, such as TNF-α and IL-1β, upregulate the expression of COX-2 via the NFκB pathway. Complicatedly, COX-2 not only plays roles in the production of PGs but also increases leukocyte adhesion and neutrophil activation, which aggravates peptic ulceration [103].
Diosmin was found to suppress gastric inflammation by reducing MPO activity and TNF-α and NF-κB levels. Levels of anti-inflammatory interleukin-10 (IL-10) were augmented by diosmin in ethanol-induced gastric ulcer in rats [63]. In the ethanol-induced gastric ulcer model, kaempferol, a flavanol from Kaempferia galanga L, decreased the plasma level of TNF-α by 33%, 43%, and 48%, and that of IL-1β by 46%, 43%, and 37% at doses of 40, 80, and 160 mg/kg, respectively [93]. Gastric injury induced by HCl/ethanol and upregulation of neutrophil infiltration triggered by aspirin were both ameliorated by oral administration of kaempferol (3 and 30 mg/kg) which decreased the levels of iκB, JNK, and p38 [104]. Hesperidin exhibited anti-inflammatory activities by reducing the gastric TNF-α and COX-2 levels in ethanol-induced peptic ulcer in rats compared to the model group after administration at 50 mg/kg for 15 days [83]. Anthocyanins from Korean blackberries (Rubus coreanus) have shown anti-gastric ulcer effects in association with the antioxidative and anti-inflammatory activity. In a naproxen-induced gastric ulceration rat model, oral administration of anthocyanins (20, 50, and 80 mg/kg b.w.) twice daily for 3 days attenuated the expression of pro-inflammatory cytokines TNF-α and IL-1β and activated the expression of metalloproteinase-2 (MMP-2), a zinc-dependent endoproteinase associated with inflammation [81]. Nobiletin, a major polymethoxyflavone in citrus fruits, reduced the MPO activity and pro-inflammatory cytokines via the MAPK pathway in ethanol-induced acute gastric ulcer in mice [64].

Figure 5.
Flavonoids play key roles in inhibiting the occurrence and development of inflammation through the MAPK/P65 pathway by reducing inflammatory cytokine levels. In total, some factors activate MAPK upstream kinase and IKK complex (a, β, γ), MAPKKK activates downstream cascade MEK and Erk, JNK and P38 step by step. On the other hand, the IKK complex phosphorylates IκB proteins and frees NF-κB/Rel complexes to translocate to the cell nucleus. The activated MAPK downstream kinases and NF-κB p65/p50/52 would increase the expression of pro-inflammatory TNFα, IL-1β, and IL-6 and augment anti-inflammatory IL-10. Flavonoids would regulate kinases involved in these two signaling pathways and inhibit pro-inflammatory cytokines to ameliorate inflammatory symptoms in peptic ulcer.

Flavonoids Possess Anti-H Pylori Activities for Peptic Ulcer Healing
Bacteria H. pylori infection is the strongest known risk factor for peptic ulcer and even gastric cancer. This bacterium produces urease to maintain an alkaline environment for survival in acidic stomach. Urease catalyzes urea to ammonia which is toxic to intercellular junctions and causes tissue injury [105]. H. pylori expresses adhesins, such as blood group antigen adhesin (BabA) and outer inflammatory protein adhesin (OipA), which are needed for the attachment of these bacteria to the gastric epithelium [1,106,107]. H. pylori induces secretion of chemokines and pro-inflammatory cytokines, such as monocyte chemotactic protein-1 (MCP-1), IL-6, and TNF-α, which induces an inflammatory response in the early stage of infection [108]. H. pylori also induces oxidative burst in neutrophils recruited to the gastric injury sites and causes mucosal damage [109,110].
Both in vitro and in vivo studies suggested that many kinds of flavonoids possess anti-H. pylori activities, thus providing a benefit in peptic ulcer healing. Six tea catechins, including EGCG, epicatechin gallate, epigallocatechin, epicatechin, crude catechin (Polyphenon70SR), and crude theaflavins, possessed anti-H. pylori activities, with EGCG being the most active one. In vivo studies further proved that a Mongolian gerbil feeding diet containing catechins showed positive effects against H. pylori and protected the gastric mucosal [111]. After oral administration of 200 mg/kg quercetin for 15 days, a significant decrease of H. pylori infection was found in both the antrum and . Flavonoids play key roles in inhibiting the occurrence and development of inflammation through the MAPK/P65 pathway by reducing inflammatory cytokine levels. In total, some factors activate MAPK upstream kinase and IKK complex (a, β, γ), MAPKKK activates downstream cascade MEK and Erk, JNK and P38 step by step. On the other hand, the IKK complex phosphorylates IκB proteins and frees NF-κB/Rel complexes to translocate to the cell nucleus. The activated MAPK downstream kinases and NF-κB p65/p50/52 would increase the expression of pro-inflammatory TNF-α, IL-1β, and IL-6 and augment anti-inflammatory IL-10. Flavonoids would regulate kinases involved in these two signaling pathways and inhibit pro-inflammatory cytokines to ameliorate inflammatory symptoms in peptic ulcer.

Flavonoids Possess Anti-H Pylori Activities for Peptic Ulcer Healing
Bacteria H. pylori infection is the strongest known risk factor for peptic ulcer and even gastric cancer. This bacterium produces urease to maintain an alkaline environment for survival in acidic stomach. Urease catalyzes urea to ammonia which is toxic to intercellular junctions and causes tissue injury [105]. H. pylori expresses adhesins, such as blood group antigen adhesin (BabA) and outer inflammatory protein adhesin (OipA), which are needed for the attachment of these bacteria to the gastric epithelium [1,106,107]. H. pylori induces secretion of chemokines and pro-inflammatory cytokines, such as monocyte chemotactic protein-1 (MCP-1), IL-6, and TNF-α, which induces an inflammatory response in the early stage of infection [108]. H. pylori also induces oxidative burst in neutrophils recruited to the gastric injury sites and causes mucosal damage [109,110].
Both in vitro and in vivo studies suggested that many kinds of flavonoids possess anti-H. pylori activities, thus providing a benefit in peptic ulcer healing. Six tea catechins, including EGCG, epicatechin gallate, epigallocatechin, epicatechin, crude catechin (Polyphenon70SR), and crude theaflavins, possessed anti-H. pylori activities, with EGCG being the most active one. In vivo studies further proved that a Mongolian gerbil feeding diet containing catechins showed positive effects against H. pylori and protected the gastric mucosal [111]. After oral administration of 200 mg/kg quercetin for 15 days, a significant decrease of H. pylori infection was found in both the antrum and corpus mucosa in H. pylori-induced gastric ulcer in guinea pigs. Meanwhile, quercetin ameliorated inflammation and lipid peroxidation [112]. Moon et al. [113] tested anti-H. pylori activities of natural flavonoids by a paper disc diffusion test. The results showed that quercetin, kaempferol, naringenin, luteolin (flavonoids from Resedaceae plants), and hesperetin (flavonoid from the peels of Citrus maxima) inhibited the growth of H. pylori, and therein, 7-O-Butylnaringenin, a novel flavonoid modified from naringenin, was the most effective one. Moreover, 7-O-Butylnaringenin and hesperetin also inhibited the urease activities of H. pylori. In contrast, up to 20 mM of hesperidin, apigenin (a flavonoid rich in Apium graveolensvar. dulce, Selaginella tamariscina, and Sabinachinenesis), and genkwanin (also known as 4 5-dihydroxy-7-methoxyflavone from Daphne genkwa Sieb. et Zucc.) showed no effects on the growth of H. pylori. Fukai et al. [114] isolated 15 known flavonoids and 3 new isoflavonoids from licorice, a medicinal plant used for the treatment of peptic ulcer. Among these flavonoids, vestitol, licoricone, 1-methoxyphaseollidin, gancaonol C, glycyrin, formononetin, isolicoflavonol, glyasperin D, 6,8-diprenylorobol, gancaonin I, dihydrolicoisoflavone A, and gancaonol B exhibited anti-H. pylori activities in vitro. Ustün et al. [115] isolated three flavonoids, namely isorhamnetin (quercetin 3-methyl ether), quercetin 3,7-dimethyl ether, and kaempferol 3,7-dimethyl ether, from the chloroform extract of Cistus laurifolius. All of these three flavonoids showed in vitro anti-H. pylori activities. Isoflavone aglycones, irisolidone, tectorigenin, and genistein from flowers and rhizomes of Leguminosae (Pueraria thunbergiana) were also found to inhibit H. pylori growth in an in vitro study [116].

Other Mechanisms
Flavonoids also prevent or treat peptic ulcer by regulating amino acid metabolism, gastrointestinal motor activity, or other factors. One study showed that treatment with licoflavone caused a change in the content of amino acids, histidine, tryptophan, lysine, and glycine in plasma in acetic acid-induced gastric ulcer rats [65]. Intragastric administration of marmin and nobiletin from Aurantii fructus immaturus at a dose of 25 mg/kg significantly inhibited gastric motor activity, which could be helpful for gastric emptying during gastric ulcer [117]. Besides anti-acid secretory, cytoprotective, antioxidative, and anti-inflammatory activities, chrysin also showed gastroprotective effects by promoting angiogenesis by upregulating the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and adhesion molecule CD31 (platelet endothelial cell adhesion molecule-1) [57].

Combination Therapy of Flavonoids and Approved Drugs
In recent years, the combination therapy of flavonoids and approved drugs was an alternative strategy for the treatment of peptic ulcer. It is beneficial to overcome some disadvantages, especially drug resistance, when using antibiotics in treating H. pylori eradication [1,13,14]. Isomoto et al. studied the combination treatment of the flavonoid sofalcone and standard triple therapy in a clinical trial of 165 patients with peptic ulcer with H. pylori infection. Combination treatment of the flavonoid sofalcone (100 mg twice daily) and standard triple therapy with rabeprazole (10 mg twice daily), clarithromycin (200 mg twice daily), and amoxicillin (750 mg twice daily) for 7 days significantly improved the cure rate (94%) compared to the typical triple therapy without sofalcone (84.9%) and compared to the combination treatment of polaprezinc (anti-ulcer drug) and typical triple therapy (84.9%) using per protocol analysis [118]. The evidence also showed that pretreatment with a combination of famotidine and quercetin improved the gastroprotective effects in indomethacin-induced gastric ulcer in rats when compared to famotidine treatment alone. Famotidine is one of the most potent antagonists for peptic ulcer and it has rare side effects. However, its low oral bioavailability (40-50%) and short biological half-life (2-4 h) limit its efficacy [119]. Treatment with a combination of 12 mg/kg famotidine beads and 50 mg/kg quercetin significantly reduced the ulcer index and MPO level and prevented GSH, SOD, and CAT level decrease (p < 0.05) compared with the model group and the famotidine group alone [77].
Studies of combined drug-flavonoid therapies of peptic ulcer are still limited. However, a combination of drugs and flavonoids showed improved effectiveness in the peptic ulcer treatment, suggesting a novel treatment strategy for peptic ulcers.

Bioavailability Improvement of Flavonoids on Peptic Ulcer
Poor bioavailability is one of the major limitation of flavonoids in both in vivo study and clinical application. For example, Choi et al. [56] studied the in vivo gastroprotective effects and pharmacokinetics, tissue distribution, and metabolism of isoliquiritigenin in mice. Due to the metabolism, the absolute bioavailability of isoliquiritigenin was low, but the absorbed fraction of isoliquiritigenin was high. One thing we want to emphasize is that isoliquiritigenin was highly distributed in the stomach in the tissue profiles. Considering the above problems and characteristics of flavonoids, some studies have explored new technologies to improve its efficiency.
Novel formulation strategies, such as nanoencapsulation technology, including liposomes, microspheres, and nanocapsules, have a great potential to improve bioavailability of flavonoids [4,120,121]. Polymeric nanocapsuled-quercetin had about a 20-fold higher efficacy than the free one in inhibiting the upregulation of the matrix metalloproteinase and infiltration of inflammatory cells and oxidative stress in ethanol-induced gastric ulcer in rats. It also reduced the gastric ulcer by 90%, which was better than the effects of famotidine (80%) [122]. Similarly, in the case of diosmin, the coated chitosan-poly (d,l-lactide-co-glycolide) (PLGA) nanoparticle version showed greater anti-ulcer activity by reducing the ulcer area and suppressing inflammation in ethanol-induced gastric ulcer than the free diosmin due to a prolonged residence time and better bioavailability [123]. Hence, nanoencapsulation technology would attract much more attention to enhance cell uptake of flavonoids with minimal systemic side effects in peptic ulcer treatment.

Safety Assessment of Flavonoids on Peptic Ulcer
The long history of flavonoid-daily intake (about 4 million years) of human beings reflects the safety of flavonoids through evolution [124]. The balanced diet including a daily intake of vegetables, fruits, or beverages containing a considerable amount of flavonoids, which keep us healthy, also proved the long-term safety of flavonoids [25,30]. Recent scientific research also suggested that natural flavonoids have a wide safety margin and hardly cause acute toxic effects [30,125,126], although we have no qualms about saying that it should be concerned that in some extreme circumstances, such as intravenous injection of a large amount of flavonoids could be dangerous [30,127].
From the following data, it is not difficult to find that the effective concentration of flavonoids is far lower than the concentration of its toxicity or side effects. Specifically, several studies assessed the gastroprotective effects and toxicity of flavonoid monomers in peptic ulcer. According to the guidelines of the Organization for Economic Co-operation and Development, the toxic class of hesperidin in Wistar rats was assessed and there was no toxicity sign during the 14-day observation, and the lethal dose of hesperidin was higher than 2000 mg/kg, which is far beyond the effective concentration of 300 and 400 mg/kg [47]. The toxicity and effectiveness of biochanin A were assessed by orally feeding rats using six dosages (from 250 to 5000 mg/kg). The behavioral, neurological, and autonomic behaviors of animals were under observation for 2 weeks without any sighs of diarrhea, weakness, tremors, seizures, or loss of controlled movement. There were no statistically significant differences of kidney and liver parameters (such as total protein, albumin, globulins, chloride, anion gap, potassium, sodium, urea, and creatinine) between the rats given vehicle 10% Tween 20 and rats given 250 and 5000 mg/kg biochanin A (p < 0.05) [85]. Following oral administration of 30, 300, and 3000 mg/kg/d quercetin for 28 days in Swiss mice, no histopathological changes were found in the organs and the biochemical variables were normal at any doses of quercetin [128]. Intraperitoneal administration of myricetin at an extreme dose of 1000 mg/kg did not cause any death of mice [125]. In acute toxicity studies conducted in Wistar rats, up to 500 mg/kg/d genistein was considered safe without any adverse effects [129]. In histamine-induced gastric acid secretion in mice, oral administration of 50 mg/kg myricetin attenuated gastric ulcer and caused no irregular behavioral symptoms [44].
In addition to flavonoid monomers, there are many toxicological studies on flavonoid-rich compounds, which also fully demonstrated the safety of flavonoids within a certain range. One acute toxicological study on grape seed extract (with proanthocyanins as the main component) showed that the oral dose of 4 g/kg body weight of grape seed extract had no physiological effect on rats [130]. Oral administration of 1000 mg/kg of rutin-rich (76 ± 3%) Dimorphandra mollis dry extract was considered safe in rodents by acute and chronic (180 days) toxicity evaluation [131]. When studying the gastroprotective effects of (+) -catechin hydrate on gastric ulcer induced by ethanol in rats, the safety of oral administration of 125 mg/kg and 250 mg/kg (+)-catechin hydrate was also assessed. Rats given the two dosages of (+) -catechin hydrate did not show any indications of toxicity during the 14-day observation. No marks of toxicity were shown in the liver and kidney either [78].
Though more clinical data are needed to prove the safety of flavonoids, the effectiveness of flavonoids at low concentrations showed that they have a high prospect in drug development.

Conclusions
Here, we listed a total of 60 kinds of flavonoids which exerted gastroprotective effects in different peptic ulcer models. Of note, except catechins, theaflavins, and anthocyanins with definite chemical composition and structure, there are 29 and 37 flavonoids monomers were displayed in prevention (Table 1) and treatment ( Table 2) strategies, respectively. From the tables, we clearly found that tea, fruits, soy, licorice, and honey were the main sources of flavonoids. It is no surprise that catechins and their monomers from tea, one of the most popular drinks worldwide, have drawn most attention both in preventative and therapeutic treatment. Quercetin and its derivative also played important roles in the improvement of gastric ulcer. Moreover, the preventive effect of flavonoids on gastric ulcer received much more attention than the therapeutic research, and it has almost been verified through in vivo experiments. In recent years, more reports that flavonoids also have remarkable therapeutic effects on peptic ulcer have gradually emerged, although they were more often proved in vitro, which showed that flavonoids have a prospect in the treatment after injury. Per os (p.o.) and intragastric administration (i.g.) are the most frequently used methods at the concentration of 3~100 mg/kg range of flavonoids, which are far below the LD50 at the 2000 mg/kg in acute toxicity test discussed above. However, patients with peptic ulcer are underrepresented in clinic trials. At present, most data came from laboratory model tests. The tables clearly revealed that the human intestinal epithelial cell line (int-407 cells) and the human gastric mucosal epithelial cell line (GES-1 cells) were usually used as in vitro models to assess the protective effects of flavonoids. Moreover, the in vivo models of peptic ulcer include ulcers caused by oxidative damage, ethanol, NSAIDs, stress, and H. pylori or acid-ethanol (ethanol or ethanol/HCl)-induced acute gastric ulcer models. These models reflected the causes and phenotypes of human peptic ulcer disease, although the protective effects of the flavonoids in these models are often determined by the route of administration, animal species, duration, and the dose of administration.           Flavonoids are abundant secondary metabolites in nature with potentially beneficial effects on human health. These compounds have been shown to protect the gastrointestinal mucosa against ulcer in animal studies and in limited clinical trials. A combination of flavonoids and existing drugs or the nanoencapsulation of flavonoids were found to exhibit better therapeutic effects on peptic ulcer when compared to the single or standard treatment. Flavonoids exhibit several anti-ulcer protective mechanisms, such as anti-acid secretory activity, cytoprotective effects, antioxidative activity, anti-inflammatory, and antibacterial activity ( Figure 6). Although future controlled clinical studies and bioavailability improvements are needed to assess the efficacy of flavonoids in preventing and/or treating peptic ulcer disease, it is still undeniable that flavonoids, especially the monomers, are suitable candidates in preventing as well as treating peptic ulcers.  The funders did not have any role in the study design, data collection, or data analysis.

Conflicts of Interest:
The authors declare that they have no conflicts of interest concerning this article.