CN114129621A - Application of tartary buckwheat flavone in preparation of antibacterial or gastritis preventing and treating medicine or health-care product - Google Patents
Application of tartary buckwheat flavone in preparation of antibacterial or gastritis preventing and treating medicine or health-care product Download PDFInfo
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- CN114129621A CN114129621A CN202111330975.7A CN202111330975A CN114129621A CN 114129621 A CN114129621 A CN 114129621A CN 202111330975 A CN202111330975 A CN 202111330975A CN 114129621 A CN114129621 A CN 114129621A
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- Prior art keywords
- tartary buckwheat
- buckwheat flavone
- helicobacter pylori
- flavone
- health
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- 150000002212 flavone derivatives Chemical class 0.000 title claims abstract description 124
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
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Abstract
The invention belongs to the technical field of medicines and foods, and particularly relates to application of tartary buckwheat flavone in preparation of medicines or health-care products for inhibiting bacteria or preventing and treating gastritis. The invention aims to solve the technical problem of providing the application of the tartary buckwheat flavone in preparing the medicines or health-care products for inhibiting bacteria or preventing and treating gastritis. Test results show that the tartary buckwheat flavone can inhibit the growth of helicobacter pylori and the expression of virulence genes of the helicobacter pylori; in addition, the tartary buckwheat flavone can also treat and prevent the expression of inflammatory factors caused by helicobacter pylori infection of gastric mucosal epithelial cells; furthermore, rutin, fireworks glycoside, quercetin and kaempferol which are main components of the tartary buckwheat flavone can also effectively inhibit helicobacter pylori and inflammation caused by infection of the helicobacter pylori. The application of the tartary buckwheat flavone in preparing the medicament or the health-care product for inhibiting bacteria or preventing and treating gastritis provides a new choice for health care and clinical medication.
Description
Technical Field
The invention belongs to the technical field of medicines and foods, and particularly relates to application of tartary buckwheat flavone in preparation of medicines or health-care products for inhibiting bacteria or preventing and treating gastritis.
Background
Tartary buckwheat (L.) Gaertn is a crop widely planted on Yunyuan plateau and loess plateau in China, and is a small coarse cereal which is edible and medicinal in dicotyledonous plants of Fagopyrum (Fagopyrum Mill) in Polygonaceae (Polygonaceae). The tartary buckwheat contains various nutritional ingredients and pharmacological active ingredients, and besides being used as a grain crop, the tartary buckwheat flavone has proven to have the effects of resisting oxidation, clearing free radicals, reducing blood sugar, blood fat, blood pressure and cholesterol, preventing atherosclerosis and the like. The flavonoid compound is the main active ingredient in the tartary buckwheat, and the research on the flavonoid compound also shows that the flavonoid compound has the effects of preventing and treating various human diseases.
Helicobacter pylori (h. pylori) is a gram-negative bacterium that colonizes in various regions of the stomach and duodenum, plays an important role in the occurrence and development of digestive tract diseases, and is a causative factor of chronic gastritis and gastric ulcer. Pyrori promotes the progression of chronic inflammation by producing cytotoxic pathogenic factors such as cytotoxic associated proteins, vacuolar toxin A, urease, etc., activating host inflammation and producing immune factors (such as IL-6, TNF-alpha, etc.). Currently, effective and widely used regimens for eradication therapy of h.pyri include standard triple therapy, bismuth quadruple PBMT therapy (PPI, bismuth, metronidazole and tetracycline), and combined PPI, clarithromycin and metronidazole therapy. Because of the wide use of antibiotics, the drug resistance of H.pyri is increasing, and the action of antibiotics causes the imbalance and functional disorder of gastrointestinal flora, and various adverse reactions are often accompanied in the treatment process, so that the curative effect often fails to achieve the expected effect.
Many researches find that compared with pure western medicine treatment, the traditional Chinese medicine and western medicine combined treatment has better curative effect on the inflammation caused by H.pyrori and higher clinical application value; partial research even finds that a single Chinese medicinal prescription can also eradicate H.pyriri, in addition, research related to the tartary buckwheat flavone is mostly the research and development of the application and the health care wine preparation process on reducing blood fat and blood sugar, but the tartary buckwheat flavone is not found in the traditional Chinese medicinal composition for treating H.pyriri at present, and the report of inhibiting H.pyriri and inflammation caused by H.pyriri infection by combining rutin, fireworks glycoside, quercetin, kaempferol and the like which are extracts of the tartary buckwheat flavone is not reported.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. The invention aims to provide the application of the tartary buckwheat flavone in preparing antibacterial drugs or health-care products, and the invention also aims to provide the application of the tartary buckwheat flavone in preparing drugs or health-care products for preventing and treating gastritis.
The technical scheme for solving the technical problems comprises the following steps: provides an application of tartary buckwheat flavone in preparing antibacterial drugs or health products.
Further, the medicine or health product is used for inhibiting the growth of helicobacter pylori.
Furthermore, the medicine or health care product inhibits the growth of the helicobacter pylori by inhibiting the expression of the helicobacter pylori virulence gene.
Furthermore, the drug or health product inhibits the growth of helicobacter pylori by inhibiting the expression of urease.
The invention also provides the application of the tartary buckwheat flavone in preparing the medicine or the health-care product for preventing and treating the gastritis.
Further, the medicine or health care product prevents and/or treats the inflammation caused by helicobacter pylori infection by reducing inflammatory factors generated by helicobacter pylori infection gastric mucosal epithelial cells.
Further, the medicine or health product can inhibit the mRNA expression of inflammatory factors IL-6, IL-8 and CXCL-1.
Wherein, the medicine or the health care product is prepared by taking the buckwheat flavonoids as active ingredients and adding pharmaceutically or dietetically acceptable auxiliary materials or auxiliary ingredients.
Furthermore, the invention also provides application of the tartary buckwheat flavone in preparation of antibacterial or anti-inflammatory daily necessities or cosmetics.
Further, in the application of the tartary buckwheat flavone in preparing a medicament or a health-care product for inhibiting bacteria or preventing and treating gastritis or preparing an antibacterial or anti-inflammatory daily product or a cosmetic, the tartary buckwheat flavone is a tartary buckwheat flavone extract.
Preferably, the tartary buckwheat flavone extract is a crude extract or a refined extract.
Further, the tartary buckwheat flavone extract comprises rutin, fireworks glycoside, quercetin and kaempferol.
Has the advantages that: the invention takes the buckwheat flavone as a research object to discuss the influence of the buckwheat flavone on H.pyriri infection and inflammation caused by the H.pyriri infection. Firstly, determining the bacteriostatic activity of the tartary buckwheat flavone extract on different H.pyri strains by a drug sensitive tablet method and a two-fold dilution method, and finding that the tartary buckwheat flavone extract has certain bacteriostatic activity on H.pyri standard strains and drug-resistant strains, wherein the bacteriostatic effect on clinical drug-resistant strains is generally better than that on the standard strains, which shows that the tartary buckwheat flavone extract has a inhibitory effect on H.pyri; meanwhile, four flavone monomers (rutin, fireworks glycoside, quercetin and kaempferol) respectively have certain bacteriostatic activity on H. Then, the tartary buckwheat flavone extract is added into the H.pyriri flat plate, the change of the expression of the virulence factor mRNA of the thallus is detected, and the result shows that the expression level of mRNA of urease genes UreA and UreA genes of two H.pyriri standard strains can be reduced by adding the tartary buckwheat flavone extract, and the inhibition of the growth of H.pyriri by the tartary buckwheat flavone extract can be regulated and controlled by inhibiting the expression of the urease. Finally, the mRNA expression changes of the cell inflammatory factors IL-6, IL-8 and CXCL-1 after treatment are measured by adding H.pylori and the tartary buckwheat flavone extract to GES-1 cells, and the increase of the inflammatory factors IL-6, IL-8 and CXCL-1 caused by H.pylori infection can be inhibited by adding the tartary buckwheat flavone extract; meanwhile, through the addition of H.pylori and four flavone monomers to GES-1 cells respectively, mRNA expression changes of treated cell inflammatory factors IL-6, IL-8 and CXCL-1 are measured, and the increase of the expression of the inflammatory factors IL-6, IL-8 and CXCL-1 caused by H.pylori infection can be inhibited by the addition of the flavone monomers, so that the tartary buckwheat flavone can prevent and/or treat inflammatory reaction caused by H.pylori infection of gastric epithelial cells, and the tartary buckwheat flavone extract can be used as an effective component for inhibiting H.pylori infection and preventing and treating inflammatory reaction caused by H.pylori infection.
Drawings
FIG. 1 is a schematic diagram of the inhibition zone plate of the buckwheat flavone extract in 3.1 of the example;
FIG. 2 is a histogram showing changes in H.pyrori urease genes UreA, UreB in example 3.3; wherein a is h.pyloi strain SS1, B is h.pyloi strain 26695;
FIG. 3 is a graph showing the effect of different concentrations of Tartary buckwheat flavone extract on the activity of GES-1 cells in example 3.4;
FIG. 4 is a bar graph of the effect of the extract of Tartary buckwheat flavones in example 3.5 on the change in H.pyriri treatment of the GES-1 cytokines IL-6, IL-8, CXCL-1; a, B shows that the influence of flavone extract of Fagopyrum tataricum on the expression of mRNA of inflammatory factor in GES-1 cells treated by strains SS1 and 26695;
FIG. 5 is a bar graph of the effect of rutin in 3.6 of the example on the change in H.pyrori treatment of the GES-1 cytokines IL-6, IL-8, CXCL-1; a, B shows that rutin influences the expression change of the inflammatory factor mRNA of GES-1 cells treated by strains SS1 and 26695 respectively;
FIG. 6 is a bar graph of the effect of Firework glycoside on the change in H.pyri treated GES-1 cytokines IL-6, IL-8, CXCL-1 in example 3.6; a, B shows that the influence of the nicotiflorin on the expression of the inflammatory factor mRNA of the GES-1 cells treated by the strains SS1 and 26695 respectively is changed;
FIG. 7 bar graph of the effect of quercetin on the change in H.pyrori treatment of GES-1 cytokines IL-6, IL-8, CXCL-1 in example 3.6; a, B wherein the effect of quercetin on the expression of mRNA for inflammatory factors in the treatment of GES-1 cells by strains SS1 and 26695, respectively;
FIG. 8 is a bar graph of the effect of kaempferol on the change in H.pyrori treatment of the GES-1 cytokines IL-6, IL-8, CXCL-1 in example 3.6; wherein A, B is kaempferol affecting changes in mRNA expression of inflammatory factors in GES-1 cells treated by strains SS1 and 26695, respectively.
Detailed Description
At present, the main effects of the buckwheat flavonoids are reported to be antioxidation, scavenging free radicals, reducing blood sugar, blood fat, blood pressure and cholesterol, preventing atherosclerosis and the like, but no research shows that the buckwheat flavonoids have inhibition capability on H.pyriri and inflammation caused by infection of H.pyriri, and no report exists that the buckwheat flavonoids extract containing rutin, fireworks glycoside, quercetin and kaempferol can inhibit the growth of H.pyriri and can prevent and/or treat the inflammation of the stomach caused by H.pyriri infection by reducing inflammatory factors generated by the H.pyriri infecting gastric mucosal epithelial cells.
In the invention, the tartary buckwheat flavone extract can be prepared by adopting a method in the prior art, such as the in vitro antioxidant activity and the in vivo hangover alleviating and liver protecting effect of tartary buckwheat bran total flavone (Tongyu, Shushu, Niumann, Wangsong billon, Yanxuejing, Sun Qin, Relabong, J, food industry science and technology, 2020, 41(17): 314-. And through detection, the tartary buckwheat flavone extract mainly contains four monomer components of rutin, fireworks glycoside, quercetin and kaempferol.
The raw materials and equipment used in the embodiment of the present invention are known products and commercially available products, unless otherwise specified.
Examples
1 Material
1.1 major drugs and reagents
Refined extract of tartary buckwheat flavone (self-made); vancomycin, amphotericin, polymyxin B, trimethoprim, nalidixic acid were purchased from Sigma; rutin, fireworks glycoside, quercetin and kaempferol are purchased from Shanghai Mecline Biotechnology, Inc., Chengdou Pusi Biotechnology, Inc., Sigma and Shanghai Mecline Biotechnology, Inc respectively; columbia blood agar medium, brain heart extract were purchased from Oxoid; aseptic defibered sheep blood was purchased from Zhengzhou Yikang bioengineering, LLC; DMSO was purchased from Solarbio; chloroform was purchased from Chongqing Chuandong chemical group; isopropanol was purchased from the Chengdong chemical group; TRIZOL was purchased from Invitrogen; DMEM high glucose medium, fetal bovine serum was purchased from Gibco;
super total RNA extraction kit was purchased from Promega; TB
Premix Ex TaqTMPrimeScript RT reagent Kit with gDNA Eraser from TaKaRa; CCK8 kit was purchased from Biosharp; SS1 and 26695 are H.pyri standard strains, SCU-HP-0916A, SCU-HP-0916C, SCU-HP-1230A, SCU-HP-1230B is a drug-resistant H.pyri strain, and the above are laboratory deposited strains; the normal human gastric mucosal epithelial cell line GES-1.
1.2 Main instruments
Anaerobic incubators (E200, GeneScience); turbidimeters (WGZ-2XJ, Shanghai Rui apparatus); continuous wavelength plate reader (Multiskan go, Thermo); a biological safety cabinet (1389, Thermo); fluorescent quantitative PCR instrument (Quant Studio 3, Thermo); low speed centrifuges (TDZ5B-WS, Shanghai Luxiang apparatus); inverted microscope (DIAPHOT, Nikon); light microscope (DP73, OLYMPUS); CO2 incubator (3111, Thermo).
2 method
2.1 culture of H.pyri
Culturing H.pyri blood plate of 3d in anaerobic incubator, inoculating H.pyri colony with moist lawn and smooth edge with sterile inoculating loop, inoculating by marking method, sealing with sealing film, transferring to 37 deg.C constant temperature anaerobic incubator with optimal humidity of 80-90%, and culturing in microaerobic environment (5% O)2，10％CO2，85％N2)3d。
2.2 method for determining antibacterial activity of Tartary buckwheat flavone extract and four flavone monomers by drug sensitive tablet method and two-fold dilution method
The drug sensitive tablet method is used for determining the bacteriostatic zone: the experiments were divided into 2 groups. Buckwheat flavone group: the concentration of buckwheat flavonoids in the drug sensitive tablet is 200mg/mL (the concentrations of four flavonoid monomers, namely rutin, fireworks glycoside, quercetin and kaempferol, are all 50 mg/mL); blank control group: soaking in DMSO to obtain blank tablet. Preparation of 1 McLee unit (3X 10) on H.pyri plates cultured for 3d8CFU/mL), coating 90 μ L of the bacterial suspension on a flat plate, attaching a corresponding drug sensitive tablet on the surface of the flat plate, culturing in an anaerobic incubator at 37 ℃ for 3d, and measuring the diameter of a bacteriostatic circle. The experiment was repeated 3 times with 3 replicates in each group.
Determination of MIC by two-fold dilution method: the experiments were divided into 2 groups. The concentration of 1X 10 prepared from H.pyri plates cultured for 3d8CFU/mL of bacterial suspension. Buckwheat flavone group: preparing blood plate in 24-well plate, wherein the concentration of flavone extract of Fagopyrum tataricum is 200, 100, 50, 25, 12.56.25mg/mL, and adding 90 mu L of bacterial suspension into each hole; blank control group: adding 90 μ L of sterile water into blood plate without tartary buckwheat flavone extract; negative control group: adding 90 μ L bacterial suspension into blood plate without tartary buckwheat flavone extract. Minimum concentration for no h.pyri growth was MIC for 3d anaerobic incubator at 37 ℃. The experiment was repeated 3 times with 3 replicates in each group.
2.3 real-time fluorescent quantitative nucleic acid amplification (qPCR) method for determining H.pyri thallus virulence metabolic factor expression after treatment of tartary buckwheat flavone extract
The experiments were divided into 2 groups. Buckwheat flavone group: preparing blood plate with tartary buckwheat flavone extract concentration of 10mg/mL, and preparing 1 McLeod unit (3X 10) by culturing H.pyri plate for 3d8CFU/mL), and coating 90 mu L of the bacterial suspension on a flat plate; blank control group: preparation of 1 McLee unit (3X 10) on H.pyri plates cultured for 3d8CFU/mL), and 90 mu L of the bacterial suspension is coated on a blood plate without the tartary buckwheat flavone extract. After 3d of culture in an anaerobic incubator at 37 ℃, colonies on the plate are scraped, RNA is extracted, and the mRNA expression levels of UreA and UreB genes are detected by a qPCR method. The experiment was repeated 3 times with 3 replicates in each group.
2.4 cell culture
For 5% CO at 37 ℃2GES-1 cells with normal growth state in the incubator are cultured in DMEM culture solution (containing 10% FBS) every 2 d; every 4d, 0.25% trypsinization, subculture and cell cryopreservation.
2.5 CCK-8 method for selecting flavone concentration of radix Et rhizoma Fagopyri Tatarici
The experiments were divided into 3 groups. Collecting 80% GES-1 cells grown in T25 culture flask, digesting, counting, inoculating into 96-well culture plate to make the number of cells per well be 4 × 103And (4) respectively. Buckwheat flavone group: the concentrations of the tartary buckwheat flavone extract in the GES-1 cell suspension are respectively 500, 250, 125, 62.5, 31.25, 15.6, 7.8, 3.9, 1.95 and 0.97 mu g/mL; blank control group: DMEM medium only; negative control group: containing only GES-1 cell suspension. Placing in 5% CO2Culturing at 37 deg.C for 24h, adding 10 μ L CCK-8 into each well, incubating for 50min, and detecting and recording OD450 value of each well by use of microplate reader. The experiment was repeated 3 times with 3 replicates per group.
2.6 determination of intracellular inflammatory factors
(1) Influence of tartary buckwheat flavone extract on H. The experiments were divided into 6 groups. Collecting 80% GES-1 cells grown in T25 culture flask, digesting, counting, inoculating into 12-well culture plate to make the number of cells per well be 3 × 105And (4) respectively. Blank control group: a suspension containing only GES-1 cells; group of helicobacter pylori: in GES-1 cell suspension, the ratio of multiplicity of infection was 100: 1 bacteria/cell number ratio h.pyri; buckwheat flavone group: the concentration of the tartary buckwheat flavone extract in the GES-1 cell suspension is 500 mug/mL; tartary buckwheat flavone for treating helicobacter pylori groups: in GES-1 cell suspension, the ratio of multiplicity of infection was 100: inoculating H.pyriri into the bacteria/cell number ratio of 1, treating for 2h, changing into fresh culture medium, and adding tartary buckwheat flavone extract to make the concentration of the tartary buckwheat flavone extract 500 μ g/mL; the tartary buckwheat flavone can prevent helicobacter pylori groups: the concentration of the tartary buckwheat flavone extract in the GES-1 cell suspension is 500 mug/mL, the tartary buckwheat flavone extract is changed into a fresh culture medium after being treated for 2 hours, and the concentration of the tartary buckwheat flavone extract in the GES-1 cell suspension is changed into the concentration of the tartary buckwheat flavone extract in the fresh culture medium according to the infection complex number of 100: 1 bacteria/cell number ratio h.pyri; the co-processing group of the tartary buckwheat flavone extract and the helicobacter pylori comprises: the concentration of the tartary buckwheat flavone extract in the GES-1 cell suspension is 500 mug/mL, and the ratio of the multiplicity of infection is 100: 1 bacteria/cell number ratio was inoculated into h. Placing in 5% CO2Culturing at 37 deg.C for 24h, extracting cell RNA, and detecting the expression level of mRNA of IL-6, IL-8, and CXCL-1 genes by qPCR method. The experiment was repeated 3 times with 3 replicates in each group.
(2) The effect of flavone monomers on the inflammatory response of h. The experiments were divided into 6 groups. Collecting 80% GES-1 cells grown in T25 culture flask, digesting, counting, inoculating into 12-well culture plate to make the number of cells per well be 3 × 105And (4) respectively. Blank control group: a suspension containing only GES-1 cells; group of helicobacter pylori: in GES-1 cell suspension, the ratio of multiplicity of infection was 100: 1 bacteria/cell number ratio h.pyri; rutin (Firework glycoside, Quercetin, Kaempferol) group: the drug concentration in the GES-1 cell suspension is 100 mug/mL; rutin (Firework glycoside, Quercetin, Kaempferol) treats the helicobacter pylori group: in GES-1 cell suspension, the ratio of multiplicity of infection was 100: inoculating H.pyri in the bacterial/cell number ratio of 1, treating for 2h, and changing to fresh cultureNourishing the base, and adding the medicine with the concentration of 100 mug/mL; rutin (Firework glycoside, Quercetin, Kaempferol) prevents helicobacter pylori groups: the drug concentration in the GES-1 cell suspension is 100 mug/mL, the medium is changed into a fresh medium after being treated for 2 hours, and the ratio of the multiplicity of infection is 100: 1 bacteria/cell number ratio h.pyri; rutin (Firework glycoside, Quercetin, Kaempferol) and helicobacter pylori co-treatment group: the drug concentration in the GES-1 cell suspension was 100 μ g/mL, while the multiplicity of infection was 100: 1 bacteria/cell number ratio was inoculated into h. Placing in 5% CO2Culturing at 37 deg.C for 24h, extracting cell RNA, and detecting the expression level of mRNA of IL-6, IL-8, and CXCL-1 genes by qPCR method. The experiment was repeated 3 times with 3 replicates in each group.
3 results
3.1 inhibitory Effect of Tartary buckwheat flavone extract on H.pyriri
The schematic diagram of the bacteriostatic circle of the tartary buckwheat flavone extract is shown in figure 1. As can be seen from table 1, the tartary buckwheat flavone extract has certain bacteriostatic activity on both h.pyri standard strains and clinical drug-resistant strains, wherein the bacteriostatic effect on the clinical drug-resistant strains is generally better than that on the standard strains. The inhibition activity of the tartary buckwheat flavone extract on the strain SS1 is the weakest, the diameter of an inhibition zone is the smallest, and the MIC is 100mg/mL and is higher than that of other strains; the tartary buckwheat flavone extract has the strongest bacteriostatic activity on the strain SCU-HP-0916C, the diameter of a bacteriostatic zone exceeds two standard strains, and the MIC value is the minimum and is 25 mg/mL.
Table 1 inhibition zones and MIC determination of different h.pyriri strains treated with tartary buckwheat flavone extract: (
n＝3)
| Group of | Antibacterial circle (mm) | MIC(mg/mL) |
| Control group | - | - |
| SS1 | 7.667±0.8819 | 100 |
| 26695 | 15.33±0.6667 | 50 |
| SCU-HP-0916A | 12.33±0.8819 | 50 |
| SCU-HP-0916C | 21.67±0.8819 | 25 |
| SCU-HP-1230A | 17.33±0.8819 | 50 |
| SCU-HP-1230B | 15.67±1.202 | 50 |
Note: the diameter (6mm) of the drug sensitive tablet is deducted; "-" indicates that the set of no detection values
3.2 inhibitory Effect of four flavone monomers on H.pyri
As can be seen from table 2, the four flavone monomers rutin, fireworks glycoside, quercetin and kaempferol have certain bacteriostatic activity on different h.pyriri strains, wherein the bacteriostatic effect on clinical drug-resistant strains is generally better than that on standard strains.
Table 2 zone of inhibition assay for flavone monomer treatment of different h
n＝3)
Note: the diameter (6mm) of the drug sensitive tablet is deducted; "-" indicates that the set of no detection values
3.3 H. Pyricularia thallus UreA, UreB Gene expression level detection
As can be seen from table 3 and fig. 2, compared with the blank control group, the expression level of UreA gene mRNA of the tartary buckwheat flavone group SS1 strain was significantly reduced by 72.3% (p < 0.05), and the expression level of UreA gene mRNA was reduced by 49.3%, but there was no significant difference (p ═ 0.146); compared with a blank control group, the expression level of UreA gene mRNA of the tartary buckwheat flavone group 26695 strain is remarkably reduced by 73.3 percent (p is less than 0.0001), and the expression level of UreB gene mRNA is reduced by 72.3 percent (p is less than 0.0001). Urease is a cytosolic enzyme required for h.pyrori to survive in the gastro-polar environment, which plays a role not only in nitrogen metabolism, but also in acid resistance and virulence. After the tartary buckwheat flavone extract is added, the urease genes UreA and UreB of the two H.pyriri standard strains are reduced in expression, and the fact that the tartary buckwheat flavone extract inhibits the growth of the H.pyriri possibly through inhibiting the expression of urease to regulate the growth of the H.pyriri.
Table 3 influence of buckwheat flavonoids extract on the UreA, UreA b gene mRNA levels of h
n＝3)
| Group of | Blank control group | Tartary buckwheat flavone group |
| UreA(SS1) | 1 | 0.28±0.132* |
| UreB(SS1) | 1.05±0.047 | 0.56±0.249 |
| UreA(26695) | 1 | 0.26±0.032**** |
| UreB(26695) | 0.89±0.023 | 0.17±0.06**** |
Note:*indicates p < 0.05 compared to the blank control group;****indicates p < 0.0001 compared to the blank control.
3.4 optimum action concentration of Tartary buckwheat flavone extract screened by CCK-8 method
The low-concentration tartary buckwheat flavone extract of 0.97 mu g/mL and 1.95 mu g/mL hardly influences the growth of GES-1 cells; the tartary buckwheat flavone extract of 3.9-500 mug/mL can promote the proliferation of human normal gastric mucosa epithelial cells GES-1 and enhance the cell activity, the cell activity is in positive correlation with the concentration of the tartary buckwheat flavone extract, the cell activity is significantly enhanced by 57.02% (p is less than 0.001) at 500 mug/mL, the tartary buckwheat flavone extract of 3.9-250 mug/mL has enhancement effect on the GES-1 cell activity but has no significant difference, and in consideration of ensuring better activity of GES-1 cells, 500 mug/mL is finally selected as the effect concentration of the tartary buckwheat flavone extract in the cell experiment (see table 4 and figure 3).
TABLE 4 Effect of Tartary buckwheat flavone extracts at various concentrations on GES-1 cell growth (S)
n＝3)
| Group of | Absorbance of the solution | Cellular activity |
| Control group | 0.81±0.07 | - |
| 500 | 1.18±0.017*** | 157.02％ |
| 250 | 1.04±0.031 | 135.22％ |
| 125 | 1.02±0.028 | 132.92％ |
| 62.5 | 0.96±0.019 | 123.84％ |
| 31.25 | 0.92±0.012 | 117.25％ |
| 15.6 | 0.95±0.04 | 121.08％ |
| 7.8 | 0.92±0.01 | 116.95％ |
| 3.9 | 0.83±0.048 | 103.11％ |
| 1.95 | 0.80±0.093 | 98.77％ |
| 0.97 | 0.77+0.059 | 94.18％ |
Note: p < 0.001, as compared to the blank control group
3.5 Effect of Tartary buckwheat flavone extract on expression of inflammatory factor of H.pyriri infected GES-1
Compared with a blank control group, the strain SS1 treatment can obviously improve the expression (p is less than 0.0001) of inflammatory factors IL-6(p is less than 0.0001), IL-8(p is less than 0.001) and CXCL-1, and the expression levels of the IL-6, IL-8 and CXCL-1 in the tartary buckwheat flavone group are respectively reduced by 60.7%, 79% and 82.3% (p is less than 0.0001); compared with a helicobacter pylori group, the expression levels of IL-6, IL-8 and CXCL-1 in a tartary buckwheat flavone treatment group are respectively and remarkably reduced by 92.9 percent, 98.8 percent and 95.1 percent (p is less than 0.0001), the expression levels of IL-6, IL-8 and CXCL-1 in a tartary buckwheat flavone prevention group are respectively and remarkably reduced by 80.5 percent, 85.1 percent and 57.1 percent (p is less than 0.0001), and the expression levels of IL-6, IL-8 and CXCL-1 in a tartary buckwheat flavone and pylorus co-treatment group are respectively and remarkably reduced by 32.0 percent, 89.6 percent and 87.7 percent (p is less than 0.0001). The above experiment results show that the tartary buckwheat flavone extract can reduce the expression level of inflammatory factors IL-6, IL-8 and CXCL-1 secreted by GES-1 cells infected by SS1, and the anti-inflammatory effect of the tartary buckwheat flavone treatment group is superior to that of the tartary buckwheat flavone prevention group and the tartary buckwheat flavone and pylorus co-treatment group (see table 5 and figure 4A).
TABLE 5 Effect of Tartary buckwheat flavone extract on the expression of inflammatory factors of SS1 infected GES-1 cells ((
n＝3)
| Group of | IL-6 | IL-8 | CXCL-1 |
| |
1 | 0.92±0.035 | 0.98±0.06 |
| Group of helicobacter pylori | 4.26±0.399**** | 5.84±0.43**** | 1.65±0.148*** |
| Tartary buckwheat flavone group | 0.39±0.049 | 0.13±0.006 | 0.16±0.012**** |
| Tartary buckwheat flavone treatment pyloric group | 0.30±0.015#### | 0.10±0.003#### | 0.08±0.012#### |
| Tartary buckwheat flavone preventive pyloric group | 0.83±0.035#### | 0.87±0.055#### | 0.71±0.058#### |
| Tartary buckwheat flavone and pylorus co-processing group | 1.07±0.04#### | 0.61±0.046#### | 0.20±0.026#### |
Note: p < 0.001, as compared to the blank control group; p < 0.0001 in comparison to the blank control group; # # indicates p < 0.0001 compared to H.pylori groups.
Similarly, the strain 26695 treatment can significantly improve the expression of inflammatory factors IL-6(p < 0.0001), IL-8(p < 0.05) and CXCL-1(p < 0.0001) compared with the blank control group, while the expression levels of IL-6, IL-8 and CXCL-1 in the tartary buckwheat flavone group are respectively reduced by 72.0% (p < 0.05), 92.40% and 89.2% (p < 0.05); compared with a helicobacter pylori group, the expression levels of IL-6, IL-8 and CXCL-1 in the tartary buckwheat flavone treatment group are respectively and remarkably reduced by 92.0% (p is less than 0.0001), 97.2% (p is less than 0.01) and 99.5% (p is less than 0.0001), and the expression levels of IL-6, IL-8 and CXCL-1 in the tartary buckwheat flavone and pylorus co-treatment group are respectively and remarkably reduced by 87.1% (p is less than 0.0001), 94.9% (p is less than 0.01) and 99.8% (p is less than 0.0001). The experimental results show that the tartary buckwheat flavone extract can reduce the expression level of inflammatory factors IL-6, IL-8 and CXCL-1 secreted by 26695 infected GES-1 cells, and the anti-inflammatory effect of the tartary buckwheat flavone treatment group is generally better than that of the tartary buckwheat flavone and pylorus co-treatment group. The above results indicate that the tartary buckwheat flavone extract can be used as an effective component for reducing inflammatory reaction caused by H.pyriri infection (see table 6 and figure 4B).
TABLE 6 influence of Tartary buckwheat flavone extract on the expression of inflammatory factors of 26695 infected GES-1 cells (C)
n＝3)
| Group of | IL-6 | IL-8 | CXCL-1 |
| |
1 | 1.31±0.049 | 0.89±0.023 |
| Group of helicobacter pylori | 3.17±0.228**** | 2.89±0.036* | 10.73±0.277**** |
| Tartary buckwheat flavone group | 0.28±0.002* | 0.1±0.002 | 0.10±0.003* |
| Tartary buckwheat flavone treatment pyloric group | 0.25±0.026#### | 0.06±0.003## | 0.06±0.002#### |
| Tartary buckwheat flavone and pylorus co-processing group | 0.41±0.002#### | 0.15±0.003## | 0.02±0.002#### |
Note:*indicates p < 0.05 compared to the blank control group;****indicates p < 0.0001 compared to the blank control;##p < 0.01, compared to the group of H.pylori;####indicates that p < 0.0001 compared with the group of H.pylori.
3.6 Effect of flavone monomers on expression of H.pyripri-infected GES-1 inflammatory factors
Compared with the blank control group, the expression levels of IL-6, IL-8 and CXCL-1 in the rutin group are respectively reduced by 70.7 percent (p is less than 0.05), 86 percent (p is less than 0.001) and 92.7 percent (p is less than 0.0001); compared with helicobacter pylori group, rutin treatment group, rutin prevention group, and rutin and pylorus co-treatment group have reduced mRNA expression levels of IL-6, IL-8, and CXCL-1. The above experiment results show that rutin, one of the main flavone components in the extract of flavone of Fagopyrum tataricum, can reduce the expression levels of inflammatory factors IL-6, IL-8 and CXCL-1 secreted by SS1 infected GES-1 cells (FIG. 5A).
Similarly, the expression levels of IL-6, IL-8 and CXCL-1 in the rutin groups are respectively reduced by 73.0% (p < 0.05), 84.1% (p < 0.0001) and 93.7% (p < 0.0001) compared with the blank control group; compared with helicobacter pylori group, rutin treatment group, rutin prevention group, and rutin and pylorus co-treatment group can significantly reduce the expression levels of IL-6, IL-8 and CXCL-1(p is less than 0.0001). The above experiment results show that rutin can reduce the expression levels of inflammatory factors IL-6, IL-8 and CXCL-1 secreted by 26695 infected GES-1 cells (FIG. 5B).
Compared with a blank control group, the expression levels of IL-6, IL-8 and CXCL-1 in the nicotianoside group are respectively reduced; compared with helicobacter pylori groups, the mRNA expression levels of the Firework glycoside treatment groups, the Firework glycoside and pylorus co-treatment groups IL-6, IL-8 and CXCL-1 are obviously reduced. The above experiment results show that as one of the main flavone components in the tartary buckwheat flavone extract, the nicotiflorin can reduce the expression levels of inflammatory factors IL-6, IL-8 and CXCL-1 secreted by GES-1 cells infected by SS1 and 26695 strains (figure 6).
Compared with a blank control group, the expression levels of IL-6, IL-8 and CXCL-1 in the quercetin group are respectively reduced; compared with helicobacter pylori group, mRNA expression levels of IL-6, IL-8 and CXCL-1 in the quercetin treatment group and the quercetin and pylorus co-treatment group are obviously reduced. The above experiment results show that quercetin, one of the main flavone components in the extract of flavone of Tartary buckwheat, can reduce the expression levels of inflammatory factors IL-6, IL-8 and CXCL-1 secreted by GES-1 cells infected by SS1 and 26695 strains (FIG. 7).
Compared with a blank control group, the expression levels of IL-6, IL-8 and CXCL-1 in the kaempferol group are respectively reduced; compared with helicobacter pylori group, the mRNA expression levels of IL-6, IL-8 and CXCL-1 in the kaempferol treatment group, the kaempferol prevention group and the kaempferol and pylorus co-treatment group are obviously reduced. The above experiment results show that kaempferol, one of the main flavone components in the extract of tartary buckwheat flavone, can reduce the expression levels of inflammatory factors IL-6, IL-8 and CXCL-1 secreted by GES-1 cells infected by SS1 and 26695 strains (figure 8).
The results suggest that rutin, fireworks glycoside, quercetin and kaempferol are main flavone components in the tartary buckwheat flavone extract, and may be the reason for reducing the inflammatory reaction caused by H.
Claims (12)
1. Application of Tartary buckwheat flavone in preparing antibacterial drugs or health products is provided.
2. Use according to claim 1, characterized in that: the medicine or health product is used for inhibiting the growth of helicobacter pylori.
3. Use according to claim 2, characterized in that: the medicine or health product inhibits the growth of helicobacter pylori by inhibiting the expression of helicobacter pylori virulence genes.
4. Use according to claim 2, characterized in that: the medicine or health product can inhibit the growth of helicobacter pylori through inhibiting the expression of urease.
5. Application of buckwheat flavonoids in preparing medicine or health product for preventing and treating gastritis is provided.
6. Use according to claim 5, characterized in that: the medicine or health care product prevents and/or treats inflammation caused by helicobacter pylori infection by reducing inflammatory factors generated by helicobacter pylori infection gastric mucosal epithelial cells.
7. Use according to claim 6, characterized in that: the medicine or health product can inhibit mRNA expression of inflammatory factors IL-6, IL-8 and CXCL-1.
8. Use according to any one of claims 1 to 7, characterized in that: the medicine or health product is prepared by taking the buckwheat flavonoids as active ingredients and adding pharmaceutically or dietetically acceptable auxiliary materials or auxiliary ingredients.
9. Application of Tartary buckwheat flavone in preparing antibacterial or anti-inflammatory daily necessities or cosmetics is provided.
10. Use according to any one of claims 1 to 9, characterized in that: the radix Et rhizoma Fagopyri Tatarici flavone is radix Et rhizoma Fagopyri Tatarici flavone extract.
11. Use according to claim 10, characterized in that: the tartary buckwheat flavone extract is a crude extract or a refined extract.
12. Use according to claim 10, characterized in that: the tartary buckwheat flavone extract comprises rutin, fireworks glycoside, quercetin and kaempferol.
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