TWI406668B - A pharmaceutical composition for inhibition of helicobacter pylori growth and helicobacter pylori-induced inflammation in human gastric epithelial cells - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for inhibiting Helicobacter pylori and H. pylori-induced inflammatory responses in gastric epithelial cells, which contains ovatodiolide compound and a pharmaceutically acceptable carrier.

Description

Medicinal composition for inhibiting growth of Helicobacter pylori and inhibiting inflammatory reaction of Helicobacter pylori-induced gastric epithelial cells

The present invention relates to a pharmaceutical composition for inhibiting the growth of Helicobacter pylori and inhibiting the inflammatory response of Helicobacter pylori-induced gastric epithelial cells, comprising an ovatodiolide compound or a pharmaceutically acceptable carrier.

Anisomeles indica is a perennial erect herb that grows in the wild and is widely found in tropical Asia, such as China, India and Taiwan. Yucai has been widely used in Chinese herbal medicines to relieve gastrointestinal diseases, skin inflammation, liver protection and immune regulation. At present, it has been pointed out that the methanol extract of S. chinensis can inhibit macrophage inflammatory reaction and inhibit tumor cell proliferation such as colorectal cancer, prostate cancer and breast cancer (Hsieh, SC, Fang, SH, Rao, YK and Tzeng, YM, 2008). Inhibition of pro-inflammatory mediators and tumor cell proliferation by Anisomeles indica extracts. J Ethnopharmacol 118, 65-70). The chemical composition of the needle grass has been analyzed one after another, not only has an anti-inflammatory response and can inhibit the cell cycle of cancer cells. The most important chemical component, ovatodiolide, has also been found to resist the secretion of nitric oxide by macrophages and inhibit the activation of T lymphocytes (Rao, YK, Fang, SH, Hsieh, SC, Yeh). , TH and Tzeng, YM, 2009. The constituents of Anisomeles indica and their anti-inflammatory activities. J Ethnopharmacol 121, 292-296). However, the research on the inhibition of Helicobacter pylori growth or the regulation of bacterial-induced gastric epithelial inflammatory response from the purified fish needles (ovatodiolide).

The present invention relates to a pharmaceutical composition for inhibiting the growth of Helicobacter pylori and inhibiting the inflammatory response of Helicobacter pylori-inducing gastric epithelial cells, comprising an ovatodiolide compound, wherein the fish needle lactone compound is a fish needle The extract of the grass ( Anisomeles indica ) is isolated or chemically synthesized, wherein the extract of the needle grass is a hot water extract of the needle grass. The pharmaceutical composition of the present invention, wherein the Helicobacter pylori strain comprises a standard Helicobacter pylori strain and a clinical multi-antibiotic anti-drug Helicobacter pylori strain. The pharmaceutical composition of the present invention inhibits Helicobacter pylori-induced inflammation of gastric epithelial cells by inhibiting bacterial adhesion, invasion, translocation of cytotoxin associated gene A (CagA), phosphorylation of cytotoxin-related proteins, or inhibition of Helicobacter pylori The mechanism of induction of nuclear factor-kappa B (NF-κB) activation and secretion of interleukin-8 (IL-8) was achieved.

The present invention demonstrates the degree of growth inhibition of Helicobacter pylori by the extracted crude extract of the needles and the hot water extract of the needle grass. It was found from the results that the concentration of the inhibitory circle of the needles and the hot water extract was 19 to 20 mm at a concentration of 1 mg/ml (Table 1). In this invention, it was confirmed that the effective concentration of the hot water extract of the needles and the needles of the needles is similar to the standard drug clarithromycin (CLR) for treating H. pylori infection - at a concentration of 0.05 mg/ml. The inhibition zone is 22 mm. Therefore, it can be confirmed that the crude hot water extract of the needles and the needle grass has the effect of inhibiting the growth of H. pylori.

The present invention also confirms that the hot water extract of the needle grass and its purified oxytodiolide act on the gastric epithelial cells (AGS cells), and are not toxic to the AGS cell. This material is shown to be quite safe.

The present invention uses the needle needle lactone to inhibit the minimum concentration of Helicobacter pylori (Hp26695) (minic bactericidal concentration (MBC)), and acts on a multi-drug resistant strain (v633, v1254, v1354), and can be found regardless of the Helicobacter pylori standard. The effective concentration of MBC of the strain or the multi-drug resistant strain, the needle of the needle, falls between 50 and 100 micromoles (μM). Its effective concentration is similar to clarithromycin and much less than the effective concentration of clarithromycin in multi-drug resistant strains. The above results show that the needles can not only inhibit the standard strain of Helicobacter pylori, but also inhibit the growth of multi-drug resistant strains.

The present invention further demonstrates that the erigerin can inhibit the ability of Helicobacter pylori to adhere to gastric epithelial cells and the ability of Helicobacter pylori to invade gastric epithelial cells. It is shown that the fish needle lactone has an effect of affecting the adhesion of H. pylori and invading the gastric epithelial cells.

The present invention also demonstrates that the erigerin has an inflammatory response caused by inhibition of H. pylori infection. The nuclear factor kappa B-cold light enzyme structure kit was transferred into gastric epithelial cells, and after co-cultivation with fish needle lactone and Helicobacter pylori, the activity of cold light enzyme was inhibited. It was also found that the needles inhibited the secretion of inflammatory cytokines, interleukin-8, by Helicobacter pylori infection.

In addition, the present invention also demonstrates that the needles can effectively inhibit the translocation, phosphorylation and cytopathic effects of cytotoxin-related proteins of Helicobacter pylori in gastric epithelial cells such as hummingbird phenotype. effect. These results show that the needles play a very important role in inhibiting the pathogenesis of Helicobacter pylori infection of gastric epithelial cells.

The pharmaceutical composition of the present invention can be produced by a conventional pharmaceutical preparation method, and a compound of the active ingredient is mixed with one or more carriers to prepare a desired dosage form, and the pharmaceutical composition dosage form of the present invention includes but is not limited to Capsules, lozenges, powders, granules or other liquid preparations.

The pharmaceutical composition of the present invention can be used as a health care medicine or food. The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier comprising an excipient, a filler, a binder, a diluent, a disintegrant, an absorption enhancer or a flavoring agent.

The embodiments of the present invention will be further described in conjunction with the accompanying drawings. The embodiments of the present invention are not intended to limit the scope of the present invention, and those skilled in the art, without departing from the spirit of the invention. In the scope of the invention, the scope of the invention is defined by the scope of the appended claims.

The invention may be embodied in different forms and is not limited to the examples mentioned below. The following examples are merely representative of the different aspects and features of the present invention. The examples are not intended to limit the scope of the invention as described in the claims.

Example 1: Separation of the fish needle lactone compound from the needle grass

The source of Anisomeles indica is Yuli, Hualien County, Taiwan, and was deposited as a voucher sample at the Biochemical Science and Technology Research Institute of Chaoyang University of Science and Technology. The dried fish needle grass (2.8 kg) was added to methanol (4 liters × 4) to extract the extract. After thorough extraction, about 293 grams (9.6% based on the dry weight of the whole plant) will be obtained, and the unprocessed extract will be dissolved in sterile water, degreased in n-hexane, and then chloroform and n-butyl The alcohol was layered, and a chloroform layer (65 g) was taken into a cerium oxide gel column containing n-hexane/ethyl acetate to perform chromatography, and five kinds of extracts (fractional 1-fraction 5) were produced. Take F2 and add n-hexane/ethyl acetate (98:2) and separate to perform 7-methoxy-3',4',5,6-tetrahydroxyflavone (paraffin, pedalitin, compound 1 , 25). Mg) and apigenin (apigenin, compound 2 , 16 mg). Fractionation 3 (2.9 g) was separated from the erbium-containing gel column by the addition of n-hexane/ethyl acetate (8:2) to isolate the chlorfenapyr compound ( Compound 3 , 120 mg) (Shahidul Alama et al ., 2000). . Fractionation 4 (8 g) was separated from n-hexane/ethyl acetate by a ruthenium oxide gel column to separate methylgallate ( compound 4 , 75 mg) and 4-dihydroxybenzoic acid (4-dihydroxybenzoic acid, Compound 5 , 55 mg). Soluble n-butanol (124 g) was separated from the six extracts (F1-F6) by addition of chloroform/methanol (100:10 to 30:70) through a cerium oxide gel column. 2 and fractionated by flash CC 3 fractionating containing chloroform / methanol (8: 2) isolating the reference element -one 7- O -β-D- glucuronide methyl ester (scutellarein 7- O -β-D -glucuronide methyl ester, compound 6 , 3.2 mg) and quercetin 7- O -glucuronide (apigenin 7- O- glucuronide, compound 7 , 32 mg). Fractionation 4 Purification of the purified desrhamnosylverbascoside (calceolarioside, compound 8 , 78.9 mg) by HPLC (CH3CN-0.1% TFA in H2O, 17:83, UV detection at 220 nm) (Damtoft and Jensen, 1994) , Cistanoside F ( compound 9 , 17 mg), phenylpropanol A ( compound 10 , 13 mg), (Miyae et al., 1996), and campaneoside II (campneoside II) , compound 11 , 200 mg). Fractionation 5 Separation of ergot (acteoside, compound 12 , 245 mg) by HPLC (methanol in water - 0.1% trifluoroacetic acid, 13:87) (Wu et al ., 2004), isosomeoside (isoacteoside) , compound 13 , 10 mg) (Wu et al., 2004) and quercetin 7- O- β-d-(6" -O - p -coumarin glucoside) (terniflorin, compound 14 , 15 Mg).

Example 2, other test materials

1. Cell line: human gastric epithelial cells (AGS cell, ATCC CRL1739).

2. Medium: F12 medium.

3. Strains: Helicobacter pylori strain 26695 (ATCC 700392) and multi-drug resistant strains v633, v1254, v1354 (Lai, CH, Kuo, CH, Chen, PY, Poon, SK, Chang, CS and Wang, WC, 2006. Association of antibiotic resistance and higher internalization activity in resistant Helicobacter pylori isolates. J Antimicrob Chemother 57, 466-471).

4. Pharmacy:

Gentamicin, vancomycin, amphotericin B, 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) (Sigma-Aldrich, St. Louis, MO, USA), Lipofectamine 2000, Invitrogen , Carlsbad, CA), decomposition buffer, luciferase substrate, beta-galactosidase expression vector (Promega, Madison, MA, USA) and dimethyl hydrazine (DMSO).

Example 3: Anti-Helicobacter pylori growth activity test

The activity of the fish needle grass and its purified substance against H. pylori was tested by agar diffusion method. Three antibiotics were used: amoxicillin (0.05 mg/ml), clarithromycin (0.05 mg/ml) and nitromethazole ethanol (0.8 mg/ml) as positive control. Agar diffusion analysis confirmed the degree of inhibition of the growth of Helicobacter pylori by 14 extracted compounds and crude extracts of Stipa. From the results, it was found that at the concentration of 1 mg/ml, the most inhibitory factor for Helicobacter pylori was compound 3, which was nalinchol. The present invention also confirmed that the effective concentration of the hot water extract of the compound 3 and the needle grass was similar to that of the standard drug clarithromycin for treating H. pylori infection (22 mm at 0.05 mg/ml).

Example 4: Minimum bactericidal concentration (MBC)

Microanalysis was used to test the minimum bactericidal concentration of the needlefish lactone against Helicobacter pylori strains and multi-drug resistant strains, including three standard antibacterial drugs (amine ampicillin, clarithromycin and nitromethazole ethanol). The minimum bactericidal concentration is minimized to achieve a concentration that inhibits bacterial growth. The present invention utilizes the action of the needle needle lactone on the multi-drug resistant strains, v633, v1254 and v1354, respectively. In Table 2, the present invention demonstrates that the effective bactericidal concentration of the minimum bactericidal concentration of the needles of ricutine lactone falls between 50 and 100 micromoles regardless of Hp 26695 or the multi-drug resistant strain. The effective concentration is similar to that of clarithromycin and much less than the effective concentration of nitromethoxazole in multi-drug resistant strains (Table 2). From the above results, it has been shown that the needles can not only affect the antibiotic-susceptible H. pylori strain, but also affect the growth of multi-drug resistant strains.

Example 5: Injectance of erythromycin to inhibit the adhesion of Helicobacter pylori and invasion into gastric epithelial cells

The present study confirmed that luterolactone affects the growth of H. pylori and further tests whether or not Lactone can affect the ability of H. pylori to adhere to gastric epithelial cells. H. pylori was infected with gastric epithelial cells at a bacterial infection dose (MOI) of 50 for 6 hours under different concentrations of luteolin. The standard antibiotics used were used as positive control groups: ampicillin, clarithromycin and nitromethazole ethanol at a concentration of 15 micromoles. A total of six experiments were performed and the numerical analysis was expressed as mean ± standard deviation. Statistics were analyzed by Student's t- test. A sample of dimethyl hydrazine was used for comparison. * P <0.05 is statistically significant. In Fig. 2A, the effect concentration of the needles of 15 to 60 micromolar inhibits the adhesion of H. pylori to 21-78% as compared with the dimethyl sulfoxide as the control group. Next, the present invention tests whether the fishlactone can affect the invasion ability of the Helicobacter pylori-infected gastric epithelial cells. In Figure 2B, the concentration of the needles of 15 to 60 micromolar inhibits the invasion of H. pylori. Capacity, and as a control group, dimethyl sulfoxide does not affect adhesion and invasion. The results show that the needles have an effect on the adhesion of H. pylori and invasion into gastric epithelial cells.

The present invention further confirms whether the needles have an inflammatory response caused by inhibition of H. pylori infection. The nuclear factor kappa B-luciferase construct was used to test the expression of luciferase after the addition of the fish needle lactone and Helicobacter pylori culture. When the nuclear factor kappa B-luciferase construct was transferred into gastric epithelial cells and co-cultured with fish needle lactone and Helicobacter pylori, luciferase activity was inhibited (Fig. 3A). Compared with the control group dimethyl sulfoxide, the needles lactone can achieve 18, 42 and 64% inhibition of luciferase activity at 15, 30 and 60 micromolar. Following the present invention, after testing for infection of gastric epithelial cells by H. pylori, the needles lactone inhibits the expression of interleukin-8. Gastric epithelial cells infected with H. pylori in co-culture with 15 to 60 micromolar of luterolactone, and then tested for the amount of interleukin-8, the results are the same as the nuclear factor kappa B activity test, will also be the needle grass Lactone inhibited the production of interleukin-8 by H. pylori infection (Fig. 3B). These results show that the activity of NF-κB caused by H. pylori can be inhibited by the action of hemeroside and then by H. pylori. The standard antibiotics used were positively regulated with ampicillin, clarithromycin and nitromethazole ethanol at a concentration of 15 micromoles to test for luciferase activity and interleukin-8 production. A total of six experiments were performed and the numerical analysis was expressed as mean ± standard deviation. Statistics were analyzed by Student's t- test. A sample of dimethyl hydrazine was used for comparison. * P <0.05 is statistically significant.

Example 6: Ovatodiolide inhibits the pathogenic effect of Helicobacter pylori cells

The present invention further demonstrates that the needles can inhibit the effect of Helicobacter pylori cytotoxin associated protein (CagA) on translocation and phosphorylation of gastric epithelial cells. In Figure 4A, cytotoxin-related protein translocation and tyrosine-phosphorylated cytotoxicity were inhibited by different concentrations of chlorfenapyr (0 micromoles - 60 micromoles). And the phenomenon of this inhibition has a concentration-dependent situation. Compared with cells that are completely free of richitolide, the translocation and phosphorylation of nearly 80% of cytotoxin-related proteins is inhibited by 60 micromolar (Fig. 4B and C). ). Figure 4A, gastric epithelial cells were infected with Helicobacter pylori for 6 hours under different concentrations of erigeron (0, 15, 30, 60 micromoles), then the whole cell lysate was collected and used for immunization. Transfer analysis detects the performance of cytotoxin-associated proteins. The detection of cytotoxin-associated protein and phosphorylated cytotoxin-related protein was performed by using mouse anti-cytotoxin-related protein antibody and mouse anti-phosphotyrosine antibody. Detection of actin expression was performed using a goat anti-actin antibody as an internal control group. Figure 4B shows the translocation of cytotoxin associated proteins and Figure 4C shows the extent of cytotoxin associated protein phosphorylation. A total of three experiments were performed, and numerical analysis was expressed as mean ± standard deviation. Statistics were analyzed by Student's t- test. It is based on a sample of dimethyl hydrazine. * P <0.05 is statistically significant. Moreover, the cytotoxin-related protein of Helicobacter pylori induced hummingbird phenotype reaction was also significantly inhibited. In the results, nearly 52% of hummingbird cells were inhibited by the action of the needles (Fig. 5). Figure 5A, mock infection; Figure 5B, Helicobacter pylori infection of gastric epithelial cells; Figure 5C, infection of gastric epithelial cells with H. pylori for 6 hours under the action of 60 micromolar needles; Figure 5D, hummingbird cells proportion. A total of three experiments were performed, and numerical analysis was expressed as mean ± standard deviation. Statistics were analyzed by Student's t- test. The arrow in Fig. 5B refers to gastric epithelial cells of hummingbird cells. The scale bar is 20 microns. These findings confirm that the separation of the needles of Semena sinensis plays a very important role in inhibiting the pathogenic effect of Helicobacter pylori cytotoxin-related proteins on gastric epithelial cells.

Figure 1. Chemical structure of the fish needle lactone compound obtained by purifying the needle grass.

Figure 2. The needlefish lactone and standard antibiotics inhibit the adhesion of Helicobacter pylori to gastric epithelial cells (A) and invasion (B).

Figure 3. The needlefish lactone affects the inflammatory response of Helicobacter pylori-induced gastric epithelial cells. The effect of activation of nuclear factor kappa B (A) and the production of interleukin-8 (B) under the action of fish needle lactone.

Figure 4. The needlefish lactone inhibits the translocation and phosphorylation of Helicobacter pylori cytotoxin-associated proteins.

Figure 5. The needlefish lactone reduces the inflammatory response of gastric epithelial cells induced by Helicobacter pylori cytotoxin associated proteins. The cytotoxin-associated protein of Helicobacter pylori induces the degree of humectal cells in gastric epithelial cells under the action of acupuncture.

Claims (7)

A pharmaceutical composition for preparing a medicament for inhibiting invasion of Helicobacter pylori into gastric epithelial cells and inhibiting Helicobacter pylori-induced inflammation of gastric epithelial cells, wherein the pharmaceutical composition comprises an ovatodiolide compound; Spirulina induces inflammation in gastric epithelial cells by inhibiting bacterial adhesion, invasion, cytotoxin associated gene A (CagA) translocation, cytotoxin associated gene A (CagA) phosphorylation or inhibition of Helicobacter pylori The mechanism of induction of nuclear factor-kappa B (NF-κB) activation and secretion of interleukin-8 (IL-8) was achieved. The use of the first aspect of the patent application, wherein the fish needle lactone compound is isolated or chemically synthesized from an extract of Anisomeles indica . The use of the first aspect of the patent application, wherein the Helicobacter pylori comprises a standard Helicobacter pylori strain and a clinical multi-antibiotic anti-drug Helicobacter pylori strain. The use of the first aspect of the patent application, wherein the pharmaceutical composition can be used as a health care medicine or food. For the use of the first item of the patent application, wherein the pharmaceutical composition can be further The step includes a pharmaceutically acceptable carrier. The use according to claim 5, wherein the pharmaceutically acceptable carrier comprises an excipient, a filler, a binder, a diluent, a disintegrant, an absorption enhancer or a flavoring agent. The use of the pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises a capsule, a lozenge, a powder, a granule or other liquid preparation.