Pharmaceutical use of kava pyrones C

Abstract

Pharmaceutical use of piperine C. The effective component compound of piper nigrum Linn, especially piperine C and flavopipidol B, has good bacteriostatic effect on standard helicobacter pylori, drug-resistant and sensitive helicobacter pylori strains, and the minimum inhibitory concentration of piperine C to helicobacter pylori is 4-8 μg / mL, the inhibitory effect of piperine C on helicobacter pylori is not easy to produce drug resistance, the effect on non-helicobacter pylori is weak, the specificity is strong, the toxicity is low, and the advantages of safety, effectiveness, stability, controllability and the like are possessed. The piperine C of piper nigrum Linn can be used for treating diseases related to helicobacter pylori infection.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of medicine and biology, and particularly relates to the pharmaceutical use of piper methysticum forst. f. in resisting helicobacter pylori. BACKGROUND

[0002] Helicobacter pylori (Hp) is a gram-negative bacterium, spiral, microaerophilic, and a class I carcinogen of gastric cancer. It can also cause multiple diseases such as erosive gastritis, atrophic gastritis, gastric ulcer, and gastric lymphoma. According to the global epidemiological statistics in 2017, the infection rate of helicobacter pylori is 18.9% to 87.7%. Eradicating helicobacter pylori is the common goal of people all over the world. At present, the first-line treatment for helicobacter pylori infection is mainly triple or quadruple therapy containing two kinds of antibiotics (clamycin, amoxicillin, tetracycline, metronidazole, etc.).

[0003] Piper methysticum Forst. f. is commonly known as kava or kawa, and is different from the familiar pepper. It is a perennial evergreen shrub of the primitive flower class of the pepper family in the South Pacific Islands. Kava does not produce seeds, so the local people use its rhizome to make medicine wine and drinks. Pharmacological studies have shown that kava has antibacterial and anticancer effects. Currently, more than 10 active ingredients have been isolated from kava, including piper methysticum C, yellow kava B, dihydrokava, dihydrokava, and narcotic pepper bitter. We found that piper methysticum C has the best activity in screening the active ingredients of kava against helicobacter pylori, so we chose piper methysticum C for further study.

[0004] Although CN200910079551.0 patent of Wenkei and Sun Lijuan discloses that kava extract effervescent tablets have a calming effect on the human brain, can cause deep dreamless sleep without sleep discomfort, and have anti-inflammatory and antibacterial insecticidal effects, but it does not specify the specific active ingredients and does not clearly state which microorganisms have antibacterial effects. This study screens the antibacterial activity of piper methysticum C and other components of kava, and finds that piper methysticum C has the most obvious antibacterial activity against helicobacter pylori, and is a good lead drug against helicobacter pylori. Therefore, the related content of this study has not been reported. SUMMARY

[0005] The technical problems solved by the application: The application provides a pharmaceutical application of caravay pepperine C, i.e., an anti-Helicobacter pylori effect, and the caravay pepperine C has a good inhibiting effect on standard Helicobacter pylori strains, clinically resistant and sensitive Helicobacter pylori strains.

[0006] Technical scheme: The caravay pepperine C is applied to preparation of a Helicobacter pylori inhibiting medicine.

[0007] The caravay pepperine C is applied to preparation of a resistant Helicobacter pylori inhibiting medicine.

[0008] Preferably, the minimum inhibitory concentration of the caravay pepperine C ranges from 4 to 8 μg / mL.

[0009] Preferably, the resistant Helicobacter pylori is metronidazole resistant bacteria, clarithromycin resistant bacteria, levofloxacin resistant bacteria, levofloxacin and metronidazole resistant bacteria, clarithromycin and metronidazole resistant bacteria, levofloxacin and clarithromycin and metronidazole multiple resistant bacteria.

[0010] The resistant Helicobacter pylori inhibiting medicine mainly contains the caravay pepperine C as an antibacterial effective component.

[0011] Benefits: 1. The application finds new applications of various caravay pepper effective components, and the caravay pepper effective components can effectively inhibit growth of Helicobacter pylori; 2. The application finds that the caravay pepper effective components can be used for treating resistant or sensitive Helicobacter pylori infection, the caravay pepperine C has no significant inhibiting effect on various non-Helicobacter pylori, has high specificity for anti-Helicobacter pylori, high safety, low side effects and low drug resistance. DETAILED DESCRIPTION

[0012] Figure 1 The experiment is for Helicobacter pylori resistance to caravay pepperine C;

[0013] Figure 2 The experiment is for in-vivo inhibiting effects of the omeprazole+caravay pepperine C group and the triple therapy group on Helicobacter pylori;

[0014] Figure 3 The experiment is for H&E staining and immunohistochemical detection of gastric mucosa tissue of the omeprazole+caravay pepperine C group and the triple therapy group;

[0015] Figure 4 Toxicity of caravay pepperine C on GES-1 cells;

[0016] Figure 5 Toxicity of caravay pepperine C on MGC-803 cells;

[0017] Figure 6 In-vivo toxicity of caravay pepperine C (H&E staining). DETAILED DESCRIPTION

[0018] Example 1

[0019] 1. Materials

[0020] 1.1 Sample

[0021] 1.2 Strains

[0022] (1) Helicobacter pylori strains: standard strain 26695, strain NSH57, MSD132, G27; clinical metronidazole-resistant strains, clarithromycin-resistant strains, levofloxacin-resistant strains, levofloxacin and metronidazole-resistant strains, clarithromycin and metronidazole-resistant strains, levofloxacin and clarithromycin and metronidazole multi-drug resistant strains were provided by the Drug-Resistant Microbial Infection Prevention and Treatment Research Center of Youjiang Medical College for Nationalities.

[0023] (2) Non-Helicobacter pylori: Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Candida albicans, Enterobacter cloacae, Campylobacter jejuni, Bacillus subtilis, Proteus mirabilis, Lactobacillus curvatus, Stenotrophomonas maltophilia, Morganella morganii, Cryptococcus neoformans, Candida tropicalis, Staphylococcus haemolyticus, Acetobacter, Saccharomyces cerevisiae, Bacteroides fragilis, Bifidobacterium longum, Enterobacter cloacae, were provided by the Drug-Resistant Microbial Infection Prevention and Treatment Research Center of Youjiang Medical College for Nationalities.

[0024] 1.3 Main culture medium and reagents: Columbia medium, brain heart infusion medium, nutrient agar medium, nutrient broth medium, MH medium, Shigella medium, standard calf serum.

[0025] 1.4 Main instruments: three-gas incubator, centrifuge, microplate reader, electronic balance.

[0026] 1.5 Consumables: EP tube, Tip head, centrifuge tube.

[0027] 2. Methods and Results

[0028] 2.1 Microdilution method for detecting the minimum inhibitory concentration (MIC, 100 μL system) of Piper methysticum effective components on Helicobacter pylori

[0029] (1) Prepare Piper methysticum C, Piper methysticum B, dihydro Piper methysticum, dihydro Piper methysticum, and Piper methysticum bitter 4 mg / mL.

[0030] (2) Prepare the MIC plate. Add 173.6 μL of culture medium to the first hole, then add 6.4 μL of antibacterial drug, and dilute by 7 times to the 7th hole; the 8th hole does not add drug, and retains 90 μL of culture medium as the control of adding bacteria without adding drug.

[0031] (3) Bacterial suspension was prepared with BHI medium from Helicobacter pylori in logarithmic phase on solid plate. The concentration was adjusted to OD6 600 = 0.3 (1 x 10 8 CFU / mL) and diluted 10 times to 1 x 10 7 CFU / mL.

[0032] (4) 10 μL of the bacterial suspension was added to the first to eighth wells (the concentration of the bacterial suspension was about 1.0 x 10 6 CFU / mL in each well). The results were determined after 72 h of culture. The drug concentrations in the first to sixth wells were 128, 64, 32, 16, 8, 4 and 2 μg / mL, respectively.

[0033] (5) The lowest drug concentration that completely inhibited the growth of bacteria in the wells was taken as the MIC. The test was meaningful only when the bacteria grew obviously in the seventh well (i.e. without antibiotics) and did not grow in the eighth well (sterile). When a single hop occurred in the microdilution method, the highest drug concentration that inhibited the growth of bacteria was recorded. If multiple hops occurred, the test should be repeated. Each drug was tested in triplicate.

[0034] (6) Results: The minimum inhibitory concentration of kavacon C against Helicobacter pylori was 4-8 μg / mL, which was obviously better than that of other active components of kava and the antibacterial effect was 2-8 times that of other active components of kava. The results are shown in Table 1.

[0035] Table 1 Minimum inhibitory concentration of active components of kava against Helicobacter pylori (μg / mL)

[0036]

[0037] 2.2 Detection of minimum inhibitory concentration (MIC) of kavacon C against non-Helicobacter pylori by microdilution method (100 BL system)

[0038] (1) Kavacon C was prepared at a concentration of 4 mg / mL.

[0039] (2) The first well of the MIC plate was added with 173.6 μL of culture medium, followed by 6.4 μL of antibacterial drug, which was diluted by 7 times to the seventh well. The eighth well was not added with drug and was reserved with 90 μL of culture medium as a control without addition of drug and bacteria.

[0040] (3) Bacterial suspension was prepared with corresponding medium from bacteria in logarithmic phase on solid plate. The concentration of bacteria was adjusted to OD6 00 = 0.3 (1 x 10 8 CFU / mL) and diluted 100 times to 1 x 10 6 CFU / mL. The concentration of fungi was adjusted to OD6 6000.5 (5 x 10 6 CFU / mL), diluted 1000 times, 5 x 10 3 CFU / mL, standby.

[0041] (4) 10 μL of the inoculum was added to the first to eighth wells (the concentration of bacteria was about 1.0 x 10 5 CFU / mL, and the concentration of fungi was 5.0 x 10 2 CFU / mL). The results were determined after 24 h of culture. The drug concentrations in the first to sixth wells were 128, 64, 32, 16, 8, 4, and 2 μg / mL, respectively.

[0042] (5) The lowest drug concentration that completely inhibited the growth of bacteria in the wells was taken as the MIC. The test was meaningful only when the bacteria obviously grew in the seventh well (i.e. without antibiotics) and did not grow in the eighth well (sterile). When a single hop occurred in the microdilution method, the highest drug concentration that inhibited the growth of bacteria was recorded. If multiple hops occurred, the results should not be reported and the test should be repeated. Each drug was tested in triplicate.

[0043] (6) Results: The inhibitory effect of piperlongumine C on non-H. pylori was poor, indicating that the antibacterial spectrum was relatively narrow and specific, and piperlongumine C could specifically act on H. pylori. The results are shown in Table 2.

[0044] Table 2. Antibacterial spectrum of piperlongumine C on non-H. pylori (MIC)

[0045]

[0046] 2.3 Detection of H. pylori resistance to piperlongumine C

[0047] (1) The H. pylori G27 strain was used to detect the resistance of piperlongumine C. The MICs of metronidazole and piperlongumine C were 2 μg / mL and 8 μg / mL, respectively. The 1 / 4 MIC concentration was used for induction, and the test was performed every 3 days for a total of 24 days. The induction concentration was adjusted according to the change in MIC, for example, if the MIC of metronidazole changed to 16 μg / mL, the induction concentration was adjusted to 4 μg / mL.

[0048] (2) Results: After 24 days of induction, the H. pylori was obviously resistant to metronidazole, and the MIC increased by 128 times; the MIC of piperlongumine C did not change, indicating that H. pylori was not prone to develop resistance to piperlongumine C. See Figure 1 .

[0049] 2.4 Detection of the inhibitory effect of piperlongumine C on H. pylori in the stomach of mice.

[0050] 2.4.1 Construction of a mouse model infected with H. pylori

[0051] Thirty-two SPF 6-8 weeks old C57BL / 6 female mice were randomly divided into 6 for negative control group, and the remaining 26 for infection group. The average weight of each group of mice was calculated by weighing. Gavage: 26 mice in the infection group were fasted for 12 hours before gavage, and then gavaged with H. pylori suspension (HPBS001) prepared with BHI, the concentration of the bacterial solution was 1 x 10 9 CFU / mL, 0.5 mL per mouse, and fasted and watered for 4 hours after gavage. Gavage once a day for 5 consecutive days.

[0052] Model check: 14 days after the last gavage, the average weight of the infection group and the control group of mice was calculated by weighing. Two mice in the infection group were randomly selected, fasted for 12 hours, and then sacrificed by cervical dislocation. The stomach of the mouse was dissected, and a part of the stomach tissue was preserved in formalin for pathological examination. Another part of the stomach tissue was weighed, homogenized, and diluted by 10, 100, and 1000 times. 100 μL of the homogenate was uniformly coated on a Columbia medium plate containing 10% serum, and placed in a three-gas incubator (85% N2, 10% CO2, 5% O2) for 72-96 hours. The characteristics of bacterial colonies on the culture medium were observed, and the number of transparent bacterial colonies with needlepoint size was calculated.

[0053] Strain identification: the culture was needlepoint size and semi-transparent, and the single colony was taken for Gram staining and microscopic examination.

[0054] Model construction judgment: 10% of the mouse stomach tissue was taken to detect H. pylori colonization, and the colonization range was 1 x 10 5 ~ 1 x 10 6 CFU / g was considered to be successful colonization, and the detection results showed that H. pylori was colonized.

[0055] 2.4.2 In vivo treatment effect detection of piperlongumine C

[0056] (1) Grouping: the experiment group was evenly divided into 4 groups, namely omeprazole plus piperlongumine C group (28 mg / kg), omeprazole plus piperlongumine C group (7 mg / kg), omeprazole plus amoxicillin and clarithromycin group, PBS group, 6 mice in each group; 6 mice without H. pylori infection were taken as the negative control group.

[0057] (2) Drug administration: gavage was adopted, and omeprazole was administered 30 minutes before other drugs. The mice were fasted and watered for 4 hours after administration. The dosage was calculated according to the average weight of 20 g per mouse, and the dosage was omeprazole 138.2 mg / kg, amoxicillin 28.5 mg / kg, clarithromycin 14.3 mg / kg, piperlongumine C high concentration group 28 mg / kg, piperlongumine C low concentration group 7 mg / kg, once a day for 3 consecutive days; the negative control group was given PBS, and the dosage and frequency were the same as above.

[0058] (3) On the 3rd day after drug withdrawal, the mice in the infection group were weighed and the average weight was calculated, and then they were executed by cervical dislocation, and the gastric tissues were taken for Helicobacter pylori isolation, culture and identification, pathological sections were made and H&E staining and immunohistochemistry were performed, and the expression of IL-6, TNF-α and IL-1 inflammatory factors in the tissue samples was detected.

[0059] (4) As shown in Figure 2 , the inhibitory effect of omeprazole + kavacurin C (28 mg / kg) group and omeprazole + kavacurin C (7 mg / kg) group on Helicobacter pylori was better than that of triple therapy (omeprazole + amoxicillin + clarithromycin) group (P < 0.001). As shown in Figure 3 , according to the H&E staining and immunohistochemistry images of omeprazole + kavacurin C (7 mg / kg) group, the apoptosis cells of gastric mucosa were significantly reduced, and the inflammatory factors were significantly decreased. The expression of several inflammatory factors in the tissue samples was also detected in this study, and the results showed that the expression levels of IL-6, TNF-α and IL-1 in omeprazole + kavacurin C group were the lowest. Therefore, omeprazole + kavacurin C has good antibacterial effect in vivo.

[0060] 2.5 Toxicity detection of kavacurin C

[0061] 2.5.1 Cell toxicity detection

[0062] (1) Prepare Ges-1 and MGC-803 cell suspensions with a concentration of 1 × 10 5 .

[0063] (2) Inoculate into 96-well plates: 100 μL per well, and 3 repeats for the same sample.

[0064] (3) Incubate in a 37℃ incubator for 24 hours.

[0065] (4) Add kavacurin C 10 μL, working concentration is 32 μg / mL, 16 μg / mL, 8 μg / mL, 0 μg / mL, and set up a group without adding cells.

[0066] (5) Incubate in a 37℃ incubator for 24 hours.

[0067] (6) Add 10 μL CCK8, mix gently and incubate for 4 hours.

[0068] (7) Measure the absorbance at 450nm, and calculate the survival rate according to the formula: cell survival rate = [(As-Ab)] / [(Ac-Ab)]x100%, As is the cell culture medium, drug, CCK-8 hole, Ac is the cell culture medium containing, CCK-8, no drug hole, Ab is not only the culture medium and CCK-8 hole without cells and drugs. According to the survival rate to establish survival curve.

[0069] (8) Results: the MIC of piperlongumine C is 4-8 times, which has little damage to Ges-1 and MGC-803 cells, low toxicity and high safety, as shown in Figure 3 、 4 .

[0070] 2.5.2 Animal toxicity test

[0071] SPF level 6-8 weeks old C57BL / 6 mice were raised in SPF environment, and randomly divided into drug administration group and negative control group, 10 in each group. 10 times of therapeutic dose was given (piperlongumine C 280mg / kg), once a day for 3 consecutive days; the negative control group was given PBS, and the frequency and dose were the same as the drug administration group. The body weight of mice was measured from 1 day before administration, and the measurement was continued for 7 days. On the 3rd day after stopping administration, the body weight of mice in the infection group was measured and the average body weight was calculated, the eyeball blood was taken, and the dislocation was performed to break the neck, and the stomach, kidney, liver and spleen tissues were taken, pathological sections were made and H&E staining was performed.

[0072] The results are shown in Figure 5 , and no obvious pathological damage was found in the stomach, liver, spleen and kidney of mice in the piperlongumine C group after 10 times of high therapeutic dose (280mg / kg) gavage administration.

Claims

1. Use of piperlongumine C in the preparation of a drug for inhibiting Helicobacter pylori.

2. Use of piperlongumine C in the preparation of a drug for inhibiting drug-resistant Helicobacter pylori, wherein the drug-resistant Helicobacter pylori is metronidazole-resistant bacteria, clarithromycin-resistant bacteria, levofloxacin-resistant bacteria, levofloxacin- and metronidazole-resistant bacteria, clarithromycin- and metronidazole-resistant bacteria, levofloxacin- and clarithromycin- and metronidazole-resistant bacteria.

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