Antiadhesion and Anti-inflammation Effects of Noni (Morinda citrifolia

Article pubs.acs.org/JAFC

Antiadhesion and Anti-inflammation Effects of Noni (Morinda citrifolia) Fruit Extracts on AGS Cells during Helicobacter pylori Infection Hsin-Lun Huang, Chien-Hui Ko, Yeong-Yu Yan, and Chin-Kun Wang* School of Nutrition, Chung Shan Medical University, 110, Sec. 1, Jianguo North Road, 40203 Taichung, Taiwan ABSTRACT: Helicobacter pylori is a human gastric pathogen that adheres to host cells and injects cytotoxin-associated gene A (CagA) to induce interleukin-8 (IL-8), inducible nitric oxide (iNOS), and cyclooxygenase 2 (COX-2). Noni (Morinda citrifolia) is found to possess antibacteria, anti-inflammation, and antioxidation activities, but its effect on H. pylori infection is still unknown. Ethanol and ethyl acetate extracts of noni fruit were used in this study. The inhibitory effect on CagA and H. pyloriinduced IL-8, iNOS, and COX-2 were determined. The coculture medium was collected for measuring neutrophil chemotaxis. Both extracts of noni fruit showed weak inhibition on H. pylori. Both ethanol and ethyl acetate extracts provided antiadhesion of H. pylori to AGS cells and down-regulation on the CagA, IL-8, COX-2, and iNOS expressions. Results also indicated both extracts relieved neutrophil chemotaxis. Noni fruit extracts down-regulated inflammatory responses during H. pylori infection, and the phenolic compounds play key role in antiadhesion. KEYWORDS: Helicobacter pylori, noni, phenolics, antiadhesion, CagA, IL-8

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negative strains on host cells.10 In addition, the lipopolysaccharide (LPS) of H. pylori also increases the secretion of IL-8 through the activation of transcription factors, TNF-α, or nitric oxide (NO), which aggravates local inflammation.11 H. pylori cannot be eradicated from the human immune system, and uninterrupted secretion of IL-8 is the main reason for gastric disease formation. Numerous studies have shown up-regulated expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthesis (iNOS) in host cells or tissue during H. pylori infection.12,13 Normally, moderate inflammation enhances the defense against bacterial infection and contributes to wound healing. However, H. pylori causes severe inflammation by endotoxin-like molecules, including CagA, vacuolating cytotoxin A (vacA), LPS, and urease. These molecules increase the risk of gastric disease and impair gastric epithelial structure and tissue healing. Therefore, the suppression of the growth or adhesion of H. pylori is important to relieve the inflammation of gastric epithelial cells. Clarithromycin, amoxicillin, tetracycline, and metronidazole with antacid agents are common treatments for gastric ulcer or gastritis patients (with H. pylori infection).14 However, antibiotic resistance of H. pylori has been found. The resistance of H. pylori to amoxicillin is 29% in Brazil and 100% in Nigeria.15 Antibiotics also possess side effects, such as allergic reaction and diarrhea, because the balance of intestinal bacteria is destroyed. Prevent H. pylori-induced inflammation is the first priority to restrain gastric disease formation.

INTRODUCTION Helicobacter pylori belongs to Gram-negative bacteria, which spread by mouth or food from contact with the carrier. H. pylori penetrates mucosa to colonize in human gastric epithelial cells and induces pro-inflammatory cytokines. Persistent infection with H. pylori could cause chronic inflammation and lead to gastritis, gastric ulcers, or gastric cancer.1 Cytotoxin-associated gene A (CagA) positive strains have higher pathogenicity than negative strains.2 This means that CagA protein changes the growth and morphology of gastric epithelial cells and regulates the host immune response.3,4 CagA protein is injected to the host cells via a type IV secretion system (T4SS) after H. pylori has attached to the host cells. CagA can be activated by intracellular tyrosine phosphorylation or nonphosphorylation pathways when it enters host cells.5 The activated-CagA protein causes structural irregularity and cell proliferation to regulate apoptosis of host cells, which is conducive to the growth and adhesion of H. pylori.6 CagA also activates nuclear factor kappa-B (NF-κB) and the nuclear factor of activated T cells (NFAT) to increase the expression of tumor necrosis factor-alpha (TNF-α), interleukin1 beta (IL-1β), and interleukin-8 (IL-8) proteins, which cause severe inflammation in gastric tissue.6 IL-8 is synthesized from macrophages and endothelial and epithelial cells, which play an important role in the progress of gastric diseases.7 IL-8 is a chemotactic factor, which attracts neutrophils from the bloodstream to infiltrate the target site and further eliminate foreign antigens through phagocytosis.8 IL-8 is a potent angiogenic factor to increase local inflammation that is associated with chronic diseases and tumor formation.8 Several studies have shown that IL-8 protein is significantly increased in gastric tissue from gastric ulcer, gastric cancer, or gastric atrophy patients with H. pylori.9,10 In particular, CagA positive strains of H. pylori can induce higher levels of IL-8 protein secretion and mRNA expression than © 2014 American Chemical Society

Received: Revised: Accepted: Published: 2374

November 18, 2013 January 6, 2014 February 14, 2014 February 14, 2014 dx.doi.org/10.1021/jf405199w | J. Agric. Food Chem. 2014, 62, 2374−2383

Journal of Agricultural and Food Chemistry

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with various concentrations of noni extracts (0.05, 0.096, 0.189, 0.375, 0.75, 1.25, 2.5, 5, or 10 mg/mL) cultured in an anaerobic pack, at 37 °C for 24 h. One hundred microliters of suspension was detected using ELISA at 600 nm for the minimum inhibitory concentration (MIC) test. One hundred microliters of suspension was mixed with 100 μL of urease reagent (3 mM PBS, pH 5.8; and 2% urea and 7 μg/mL phenol red, pH 5.0) at 37 °C for 4 h, and the minimum bactericidal concentration (MBC) was measured at 560 nm. Antiadhesion Assay. AGS cells were seeded in a 24-well plate with 5 × 105/mL cells, and 400 μL of medium was added to each well overnight. The H. pylori suspension (15 μL) and samples (200, 400, or 600, or 800 μg/mL) were added to each well. After 6 h, nonadherent bacteria were washed off, using PBS. The amount of adherent bacteria was estimated using urease reagent and H. pylori antibody−FITC (no. GTX36341, GenTex, USA). The suspension was collected to determine the amount of nonadherent bacteria at 600 nm and to measure the bacterial viability in the urease reagent at 560 nm. Determination of IL-8 Protein. The level of IL-8 protein from the cell-cultured medium was measured using a commercial enzymelinked immunosorbent assay kit (no. K0031216, Koma Biotech Inc., Korea). CagA, iNOS, and COX-2 Protein Expression Assay. AGS cells were seeded in a 10 cm dish with 1 × 105/mL. H. pylori suspension was added and cocultured with the extracts for 6 h. All cells were collected and disintegrated by using cell lysis buffer (no. 9803, Cell Signaling, USA). Cell proteins were collected after centrifuged (6708g) for 10 min at 4 °C. CagA, iNOS, and COX-2 protein expression were detected by Western blot.20 The protein was resolved in 10% SDS−polyacrylamide gels and electroblotted on a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked in 5% fatfree dried milk, dissolved in TBS buffer (25 mM Tris, pH 7.4, 150 mM NaCl, 0.3% Tween-20). The membrane was soaked with anti-COX-2 polyclonal antibody (ab52237, Abcam, UK), anti-iNOS polyclonal antibody (ab3523, Abcam, UK), or anti-CagA monoclonal antibody (ab90490, Abcam, UK) overnight at 4 °C. The removed antibody and membrane were soaked with an appropriate HRP-conjugated secondary antibody (ab6721, Abcam, UK) for 1 h at room temperature. The reacted bands of the target proteins were revealed by enhanced chemiluminescence, using an enhanced chemiluminescence (ECL) commercial kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Neutrophil Isolation. Whole blood was collected from a human, mixed with 4.5% dextran, and left to subside for 40 min at 37 °C (the ratio of whole blood to 4.5% dextran was about 4:1). An upper layer was obtained, which contained neutrophils and residual red blood cells (RBCs), and this was then mixed with Ficoll hypaque and centrifuged at 400g for 10 min at 25 °C. The leukocytes pallet was mixed with Ficoll hypaque (the ratio of pallet to Ficoll hypaque was about 2:1) at 400g for 10 min at 25 °C, and the upper layer was removed. RBC lysis buffer was added for 5 min to remove erythrocyte (the ratio of pallet to RBC lysis buffer was about 1:4). After centrifuging at 250g for 10 min at 25 °C, the cell residue was mixed with RPMI medium (containing 15% FBS) and cultured at 37 °C for 24 h. Cell viability and morphology were determined by MTT and Giemsa stain (no. 32884, Sigma, Germany).21 Neutrophil Chemotaxis Assay. Neutrophil chemotaxis was determined by two-chamber techniques. Thirty microliters of supernatant of H. pylori cocultured with AGS cells or various concentrations of extracts were poured into the lower layer. The IL8 protein (1000 pg/mL) was used as a positive control. Fifty microliters of neutrophils (2 × 105/mL) was added to the upper layer. A PVP-free membrane was placed between the lower and upper layers and cultured for 1 h at 37 °C. The degree of migration of neutrophil was calculated by Giemsa stain.22 Statistical Analysis. All results are presented as the mean ± SD (n = 6). The difference in the results for this study was assessed using a one-way ANOVA of SPSS (version 12.0; SPSS Inc.). A significant difference is identified for p < 0.05.

Noni (Morinda citrifolia) is native to lands from Southeast Asia to Australia and is also cultivated in Polynesia, India, the Caribbean, South America, and southern Taiwan.16 Noni can inhibit various pathogens, such as Staphylococcus aureus, Pseudomonas aeruginosa, Proteus morgaii, Bacillus subtilis, Escherichia coli, and different strains of Salmonella and Shigella.17,18 Noni juice also provides an anti-inflammatory effect to relieve locally acute inflammation and selectively inhibits COX-1 and COX-2 expression.16,19 A previous study has shown that noni fruit contains abundant polyphenols and alkaloids.16 Noni exhibits various biological activities, but its effect on H. pylori are still unknown. This study determines the effect of noni fruit extracts on the adhesion of H. pylori and H. pyloriinduced inflammation.

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MATERIALS AND METHODS

Preparation of Ethanol and Ethyl Acetate Extracts. One hundred grams of noni fruit powder was extracted with 1 L of 95% ethanol or 99% ethyl acetate for 24 h at room temperature. The residues were removed by filtration, and the extracts were evaporated to dryness, under vacuum, at 37 °C. The dried extracts were transferred into airtight amber bottles and stored at −80 °C. The phenolic compounds of both extracts were measured using highperformance liquid chromatography (HPLC). HPLC Analysis of Phenolic Compounds Present in Noni Fruit Extracts. The phenolic compounds were separated using HPLC with a binary gradient intelligent pump (L6200A, Hitachi) and a UV−vis detection system (L4250, Hitachi). A Lichrospher 100 RP-18e (Merck) column (5 μm, 25 cm × 4 mm i.d.) and security guard RP-18e (5 μm, 4 × 4 mm i.d.) were used. The mobile phase consisted of 2.5% (v/v) acetic acid water solution (solvent A) and acetonitrile (solvent B). The gradient program consisted of 3% B, initially, changing to 21% B after 4 min, was maintained at 21% B until 10 min and raised to 22% B after 11 min, was maintained at 22% B until 15 min and raised to 35% B after 16 min, was maintained at 35% B until 35 min and raised to 100% B after 36 min, and was maintained at 100% B until 40 min and then reduced to 3% B after 45 min. The injection volume for all samples was 20 μL. Simultaneous monitoring was performed at 266 nm, and the flow rate was 0.8 mL/min. Isolation and Culture of H. pylori. H. pylori was isolated from gastric ulcer patients. The gastric mucus of gastric ulcer patients was collected after executed gastroscopy. The H. pylori-positive tissues were detected by a urease rapid test. The gastric mucus (H. pyloricontained) was seeded in H. pylori selection medium (10 mg/mL vancomycin, 2.5 U/mL polymyxin B, 5 mg/mL trimethoprim, and 2 mg/mL amphotericin B in 22 g/L brucella broth) in an anaerobic pack at 37 °C for 72 h. The activity and purity of the H. pylori was determined using morphology, urease, and oxidase test (genotype belong to CagA and urease positive strains). H. pylori was stored in 30% glycerin-containing medium at −80 °C. Cell Line and Culture. Human gastric cancer cell lines (AGS cells, BCRC 60102) were cultured using F12 nutrition mixture medium containing 10% FBS fetal bovine serum and 1% penicillin− streptomycin at 37 °C in a humidified incubator containing 5% CO2. Cell Viability Assay. The effect of extracts on cell viability was studied using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). AGS cells were seeded in a 96-well plate with 200 μL (5 × 105/mL) of cells in each well and left overnight. The cells were treated with various concentrations of noni extracts (200, 400, 600, or 800 μg/mL) or H. pylori and 0.05 mg/mL amoxicillin at 37 °C for 24 h. The medium was replaced by fresh medium, containing 0.5 mg/mL MTT, and cultured at 37 °C for 4 h. One hundred microliters of isopropanol was added to each well to dissolve the crystals. The optical density of each sample was read at 630 nm against the blank prepared from the cell-free well. Bacterial Drug Sensitivity Test. The H. pylori suspension (optical density = 0.3 at 600 nm for 1 × 108 CFUs/mL) was treated 2375

dx.doi.org/10.1021/jf405199w | J. Agric. Food Chem. 2014, 62, 2374−2383


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Figure 1. HPLC profiles of phenolic compounds present in ethanol (A) and ethyl acetate (B) extracts of noni fruit.

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RESULTS AND DISCUSSION Phenolic Profile of Both Extracts. Phenolics have manifold bioactive effects, such as antibacterial, antiadhesion, and anti-inflammation, in animal or cell models.23,24 Noni fruit has been found to contain several phenolics and flavonoids.25 Ethanol extract contained 48.81 ± 7.06 mg/g total phenolics and 40.91 ± 1.17 mg/g total flavonoids. Ethyl acetate extract contained 68.82 ± 4.62 mg/g total phenolics and 39.38 ± 0.44 mg/g total flavonoids. Ethanol extract contained 2.8 ± 0.02 mg/g and ethyl acetate contained 6.9 ± 0.06 mg/g condensed tannins. The ethyl acetate extract was more abundant in phenolics, flavonoids ,and condensed tannins than the ethanol extract. The phenolic profiles of ethanol and ethyl acetate extracts were measured by HPLC. Large amounts of coumaric acid and quercetin were found in the ethanol extract (23.8 and 4.4 mg/g extract), whereas the ethyl acetate extract contained abundant quercetin (28 mg/g extract) (Figure 1). Effect of Noni Fruit Extracts on H. pylori. Ethanol and ethyl acetate extracts of noni fruit showed poor inhibition on the density of H. pylori at lower doses (0.04−2.5 mg/mL) (Figure 2A). Similar results were also found for bacterial viability (Figure 2B). The ethanol extract had a better MIC (5 mg/mL) than the ethyl acetate extract (10 mg/mL). Both extracts had the same MBC (10 mg/mL), but it was much poorer than that for amoxicillin (0.04 mg/mL). Amoxicillin is widely used to eradicate H. pylori; it was deemed a positive control.26 Noni fruit extracts showed bactericidal activity at

high concentrations (Figure 2B). The effects of noni fruit extracts on the growth of H. pylori were weak, even though both extracts contained abundant coumaric acid and quercetin (Figure 1). Actually, H. pylori could resist fractional antibiotics, such as macrolides, metronidazole, tetracycline, and quinolones.27 It is difficult to eradicate H. pylori from humans using antibiotics. In this study, a moderate way to prevent the colonization of H. pylori was demonstrated. Antiadhesion of H. pylori to Gastric Epithelial Cells. H. pylori adhesion clearly influences local inflammation and disease formation. Thus, the adhesion of H. pylori on AGS cells was measured after treatment with both extracts. First, the cell viability of noni fruit extracts and appropriate ratio of H. pylori to AGS cells were determined. Both extracts had no effect on cell viability (200−800 μg/mL) after 24 h of culture. The appropriate ratio of H. pylori to cells was 300:1 (data not shown). Panels A and B of Figure 3 show the antiadhesion activity of both extracts on H. pylori, which appeared to be dependent on concentration. Nonadhering H. pylori was detected in the cocultured suspension (Figure 3C,D). The viability of H. pylori in the suspension remained unchanged after treatment with both extracts except for amoxicillin (Figure 3E,F). Noni fruit extracts showed weak inhibition of bactericidal activity on H. pylori, but greatly reduced its adhesion to epithelial cells. Adhesion is an important step for the growth, colonization, and transportation of bacteria pathogen-associated molecules to the host cell. The CagA protein is transported to 2376



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Figure 2. Density (A) and viability (B) of H. pylori after noni fruit extract treatment. H. pylori was cultured with extracts for 24 h. The MIC and MBC of treatments were measured. (∗) Significantly different as compared with 0 mg/mL. E, ethanol extract; EA, ethyl acetate extract; Am, amoxicillin.

the host cell via a T4SS after H. pylori adheres to the gastric epithelial cell.28 Therefore, the inhibition of the adhesion of H. pylori may relieve the inflammation and decrease the risk of gastric diseases. Inhibition on IL-8 and CagA. H. pylori induces inflammation and releases cytokines from host cells or immune cells. IL-8 is an important factor in the development of gastric diseases during H. pylori infection.2 IL-8 protein allows neutrophil infiltration, which causes severe inflammation and tissue disruption and further leads to gastritis, gastric ulcer, and gastric cancer.8 CagA, iNOS, COX-2, and IL-8 proteins were induced during H. pylori infection. Time-dependent increases of CagA, COX-2, iNOS, and IL-8 protein production were found, and these molecules reached the maximum at 6 h (data not shown). Indeed, the production of IL-8, iNOS, and COX-2 on AGS cells was not changed during both extract treatments separately (data not shown). Furthermore, the effects of noni fruit extracts on H. pyloriinduced inflammatory response were determined. Ethanol extract significantly inhibited intercellular CagA protein, but there was a difference among 200, 400, 600, and 800 μg/mL

(Figure 4A). Ethyl acetate extract inhibited CagA protein at concentrations >400 μg/mL (Figure 4B). Similar results also appeared on the IL-8 production; both extracts significantly inhibited IL-8 secretion (Figure 4C,D). CagA protein is produced by H. pylori, which is transported into host cell via a T4SS. CagA can induce IL-8 protein secretion, and it also regulates transcription factors via CagA phosphorylation or phosphorylation-independent pathways.5,6 The intracellular CagA protein was determined to ascertain the severity of H. pylori infection. These results showed the change of CagA and IL-8 resulted in antiadhesion for H. pylori, which prevented CagA transportation and modified IL-8 production. Inhibition of COX-2 and iNOS. H. pylori induces the expressions of iNOS and COX-2 proteins, which causes tissue damage. Furthermore, iNOS and COX-2 increase the risk of gastritis, gastric ulcer, and gastric cancer. Therefore, the effect of noni fruit extracts on inflammation of H. pylori-induced on AGS cells was evaluated. The expression of COX-2 protein was significantly lower for both extract treatments than with H. pylori infection only (Figure 5A,B). The ethanol extract did not affect the expression 2377



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Figure 3. Adhesion of H. pylori to AGS cells after ethanol extract (A) and ethyl acetate extract (B) treatment, density of H. pylori from suspension after ethanol (C) and ethyl acetate extract (D) treatment, and viability of H. pylori from suspension after ethanol (E) and ethyl acetate extract (F) treatment. Different letters above bars indicate significant difference. Hp only, H. pylori cocultured only; Am, amoxicillin used as a positive control.

tissue inflammation.31 Interestingly, the ethanol extract demonstrated antiadhesion, but did not restrain the expression of iNOS at 200−600 μg/mL (Figures 3A and 5C). This result showed that iNOS expression may not be relevant to the adhesion of H. pylori. The inhibition of COX-2 and iNOS expression by noni extracts probably proceeded via antiadhesion and regulates the inflammatory signal in the host cell during H. pylori infection.

of iNOS protein. Ethyl acetate extract showed significant inhibition at concentrations >400 μg/mL (Figure 5C,D). The expression of COX-2 and iNOS protein in gastritis, gastric ulcer, or gastric cancer patients with H. pylori infection is higher than in H. pylori-negative patients.29 The overexpression of COX-2 protein causes host cell invasion, proliferation, and apoptosis, which increase the risk of tissue lesions.30 The iNOS protein induces the secretion of IL-8 protein and increases 2378



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Figure 4. Transportation of CagA on AGS cells induced by H. pylori after ethanol (A) and ethyl acetate extract (B) treatment and expression of IL-8 induced by H. pylori after ethanol (C) and ethyl acetate extract (D) treatment. Extracts and H. pylori were cocultured with AGS cells for 6 h. Different letters above bars indicate significant difference. C, control, non-H. pylori; Hp, H. pylori cocultured only; Am, amoxicillin.

layer, and the neutrophil isolated from human peripheral blood was seeded in the upper layer. Undeniably, IL-8 protein caused numerous migrations of neutrophils, and H. pylori groups also induced neutrophil chemotaxis (Figure 6). This result indicated that both extracts inhibited neutrophil migration (significantly different as compared with the H. pylori group) from a coculture medium. The ethanol extract showed powerful inhibition of neutrophil migration (significantly different to the antibiotic group) (Figure 6B). Neutrophil is attracted during H. pylori infection, but the H. pylori cannot be eradicated. Long-term neutrophil infiltration causes tissue damage and local structural failure. The abundant infiltration of neutrophil is thought to increase the development of gastritis, gastric ulcer, or gastric cancer and to cause recurrence of gastric disease.34 Therefore, the prevention of IL8 secretion and neutrophil chemotaxis may decrease the risk of gastric disease.

Moreover, noni root extracts are found to reduce the expression of COX-2, iNOS, and TNF-α in LPS-induced cells and animal models, and a similar composition is found in both root and fruit.32 The ethyl acetate extract of noni fruit suppresses the expression of COX-2 and iNOS on LPS-induced macrophages.19 The high polar extract of noni fruit also inhibits iNOS and COX-2 expression of gastric ulcer rat.33 These results indicated that noni fruit extracts could regulate the H. pyloriinduced inflammation. Inhibition of Neutrophil Chemotaxis on H. pylori Infection. IL-8 plays a crucial role in neutrophil chemotaxis.8 Noni fruit extracts showed inhibition on IL-8 during H. pylori infection (Figure 4C,D). To further demonstrate that the chemotaxis of neutrophil was relieved by noni fruit extracts, the cultured medium was collected after both extracts (800 μg/ mL) and H. pylori (the ratio of H. pylori to AGS cells was the same as that for the antiadhesion assay) were cocultured with AGS cells for 6 h. The cultured medium was added to the lower 2379



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Figure 5. Expression of COX-2 on AGS cells induced by H. pylori after ethanol (A) and ethyl acetate extract (B) treatment and expression of iNOS induced by H. pylori after ethanol (C) and ethyl acetate extract (D) treatment. Noni fruit extracts and H. pylori were cocultured with AGS cells for 6 h. Different letters above bars indicate significant difference. C, control, non-H. pylori; Hp, H. pylori cocultured only; Am, amoxicillin.

Contribution of Phenolic Compounds to Antiadhesion. Many studies demonstrate antiadhesion on various bacteria for plants or natural products, such as cranberry, green tea, and isolates of red wine.35−37 The active ingredients are caseinoglycopeptides, fucosylated saccharides, glycoprotein, oligosaccharides, sialylgalactosides, proanthocyanidins, and other phenolic compounds.37,38 Previous studies indicate that condensed tannins (such as proantocyanidin) show antiadhesion of H. pylori.35,36,39,40 The contents of condensed tannin were scarce in both extracts of noni fruit, and their role in antiadhesion of H. pylori needs to be determined. To confirm the importance of the phenolics on antiadhesion of H. pylori from noni fruit extracts, polyvinylpyrrolidone (PVP) was mixed with both extracts separately to precipitate phenolics. Both extracts lost the antiadhesion of H. pylori when

the phenolics were removed (E-PVP and EA-PVP groups) (Figure 7). Accordingly, the phenolics of noni fruit extracts may play key role on antiadhesion of H. pylori. However, the effects of quercetin and coumaric acid on antiadhesion of H. pylori remain unknown. To replicate the extracts, pure coumaric acid and quercetin were conversed to levels of each extract and were used to evaluate the effect on the antiadhesion of H. pylori infection. The group of equivalent contents of coumaric acid (19.04 μg/ mL) in 800 μg/mL ethanol extract (E-C) provided antiadhesion on H. pylori (significant difference as compared with the Hp extract group), but the effect was weaker than the ethanol extract group (Figure 7). The group of equivalent contents of coumaric acid (19.04 μg/mL) and quercetin (3.52 μg/mL) in 800 μg/mL ethanol extract (E-CQ) demonstrated 2380



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Figure 6. Effect of noni fruit extracts on neutrophil chemotaxis (A) and quantification (B). Different letters above bars indicate significant difference. (∗) Significantly different as compared with the control. C, control, non-H. pylori; IL-8, IL-8 protein as positive control, 1000 pg/mL; Hp, H. pylori cocultured only; Hp+E, H. pylori and ethanol extract cocultured; Hp+EA, H. pylori and ethyl acetate extract cocultured; Hp+Am, H. pylori and amoxicillin cocultured.

coumaric acid-rich ethanol extract also inhibited the expression of IL-8 and COX-2 proteins induced by H. pylori (Figures 4C and 5A). Coumaric acid probably interacted with other compounds to regulate inflammation during H. pylori infection. These results indicated that coumaric acid showed the potential to prevent formation of gastric diseases. Undoubtedly, quercetin has a key role in the ethyl acetate extract against adhesion and inflammation during H. pylori infection. Quercetin is confirmed to selectively activate proinflammation signals, such as PGE2 and NO production, to relieve inflammatory responses, and also to promote wound healing in animal models.43,44 Quercetin also reduces inflammation and lipid peroxidation in H. pylori infection of gastric mucosa.45 The effect of quercetin on the adhesion of bacteria to AGS cells is still unknown. The mechanisms for the antiadhesion on bacteria are multifarious, such as target coating, adhesin inhibition, receptor inhibition, and receptor competitive inhibition.46 The particular flavonoids, such as catechin, proanthocyanins, and anthocyanins, have been shown to change the surface modification and biofilm detachment to cause bacterial adhesion failure.47,48 Thus, quercetin probably suppresses the adhesion of H. pylori via the similar pathway of catechin or proanthocyanin. The chemical structure of

great inhibition on adhesion (significant difference as compared with the Hp extract group) but was lower than that of the ethanol extract group (Figure 7). On other hand, the group of equivalent contents of quercetin (22.4 μg/mL) in 800 μg/mL ethyl acetate extract (EA-Q) provided inhibition on adhesion (significant difference as compared with Hp extract group) (Figure 7). These results confirmed that quercetin and coumaric acid played key roles in the antiadhesion of H. pylori infection. However, the equivalent contents of coumaric acid showed weaker than ethanol or ethyl acetate extracts, respectively (Figure 7). Interestingly, the ethanol extract showed excellent inhibition on CagA transportation and antiadhesion at low dose (200 μg/mL) (Figure 4A). Thus, there are unknown components in ethanol extract of noni fruit that contribute to the antiadhesion and blocking CagA signal of H. pylori. Coumaric acid can inhibit intestinal inflammation in dextran sulfate sodium (DSS)-induced rats by obstructing the COX-2 signal, but its effect on the expression of COX-2 and iNOS is weak.41 Coumaric acid showed weak capability in prevention for H. pylori owing to no effect on growth of H. pylori.42 In this study, the coumaric acid group (E-C) showed significant inhibition on the adhesion of H. pylori even though that was weaker than the ethanol extract group (Figure 7). The 2381



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probably played key roles. Further works to identify the unknown components are needed. Noni fruit is an agent or supplement that prevents H. pylori adhesion or related diseases. Further work is required to confirm the actual mechanism for antiadhesion.

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AUTHOR INFORMATION

Corresponding Author

*(C.-K.W.) E-mail: wckjff@gmail.com. Phone: +886424730022, ext. 11751. Notes

The authors declare no competing financial interest.

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Figure 7. Contribution of phenolic compounds on antiadhesion. Different letters above bars indicate significant difference. C, control, non-H. pylori; Hp, H. pylori cocultured only; E, ethanol extract; EA, ethyl acetate extract; E-PVP, ethanol extract reacted with PVP before treated; EA-PVP, ethyl acetate extract reacted with PVP before treated; E-C, equivalent contents of ethanol extract; E-CQ, equivalent contents of coumaric acid and quercetin of ethanol extract; EA-Q, equivalent contents of quercetin of ethyl acetate extract; AM, amoxicillin.

coumaric acid is different from that of quercetin, and this could dominate the capacity of antiadhesion of bacteria.42,43 In this study, quercetin not only contributed to antiinflammation but also inhibited the adhesion of H. pylori. It probably represented a new way to prevent or inhibit inflammation or pathogenesis that was caused by microorganisms. Indeed, the ethyl acetate fraction of noni fruit contains other phenolic compounds, such as scopoletin and rutin.19 In this study, quercetin was found to be the major phenolic compound in ethyl acetate extract (Figure 1B). This was probably due to the variety of noni or sample extraction. On the other hand, abundant coumaric acid and quercetin were found in the ethanol extract of noni fruit (Figure 1A). The group of equivalent contents of coumaric acid and quercetin in 800 μg/mL of ethanol extract showed weaker antiadhesion of H. pylori than that of ethanol extract treatment (Figure 7). This indicated that some other substances in the ethanol extract could contribute to the antiadhesion. Previous studies verify the various components in noni fruit, such as asperulosidic acid and anthraquinones, that may have the potential to affect the adhesion and inflammation for bacterial infection.49,50 Moreover, the condensed tannin was found in ethanol extract. The antiadhesion of H. pylori disappeared in the E-PVP group (Figure 7). Accordingly, the high-polar condensed tannin probably played a critical role in the antiadhesion of H. pylori. Further studies are required. Phenolics provide antioxidant, anti-inflammatory, or bactericidal activity and are rarely studied for their antiadhesion for bacteria. In this study, the phenolics of noni fruit were shown to regulate inflammation by impeding the attachment of H. pylori to the host cells. These results showed that coumaric acid and quercetin inhibited inflammation by reducing the adhesion of H. pylori. Conclusion. Ethanol and ethyl acetate extracts of noni fruit showed excellent antiadhesion and anti-inflammation for AGS cells from H. pylori infection and decreased the risk of gastric diseases. Coumaric acid, quercetin, and unknown components 2382



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Antiadhesion and Anti-inflammation Effects of Noni (Morinda citrifolia

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