Application of burdock extract in preparation of anti-helicobacter pylori product
By preparing anti-Hercicycline products from burdock extract, combined with commonly used clinical antibiotics, the problems of antibiotic resistance and low bioavailability of burdock pylori in anti-Hercicycline treatment were solved, and the Helicobacter pylori was achieved. Effective removal of bacteria and improved therapeutic effects.
Patent Information
- Application Number
- CN202411439268.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-15
- Publication Date
- 2024-12-27
AI Technical Summary
The existing anti-Herrelic pylori treatment methods rely on antibiotics, resulting in an increase in bacterial resistance. It is of great significance to seek new anti-Herrelic pylori drugs, and the bioavailability of burdock is low, affecting its therapeutic effect.
The burdock extract is prepared by preparing anti-Herrelic pylori products from burdock extract, including drying, crushing, sieving, water bath extraction and under-pressure concentration, and burdock extract is prepared, combined with commonly used clinical antibiotics to enhance inhibition and killing pylori. The effect of hematobacterium.
Burdock extract has a direct killing effect on Helicobacter pylori. In animal experiments, Helicobacter pylori was significantly removed from the stomach of mice, and there was no organic solvent residue. The effect was better than the active monomer Burdock pylori, which broadened the application field of Burdock , providing a new direction for Helicobacter pylori treatment.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of medical technology, and particularly relates to application of burdock fruit extract in preparing anti-Helicobacter pylori products. Background Art
[0002] Helicobacter pylori is a Gram-negative bacillus, one of the few bacteria that can survive and colonize in the low pH environment of the human stomach. There is a cluster of flagella on one side of the bacteria, which can support its rapid movement in the stomach. The urease contained in Helicobacter pylori can decompose urea, increase the pH of the stomach and reduce the effect of gastric acid on the bacteria. Helicobacter pylori can also induce inflammatory reactions in gastric epithelial cells and damage the gastric mucosa through the chemotaxis of multiple chemical receptors and multiple signal transduction pathways. Therefore, Helicobacter pylori often causes chronic active gastritis, and there is a certain probability of causing diseases such as peptic ulcer, gastric mucosa-associated lymphoid tissue lymphoma and gastric cancer. At present, the eradication treatment of Helicobacter pylori is mainly based on triple and quadruple therapy with antibiotics, that is, two antibiotics, a proton pump inhibitor, and a bismuth agent is added to the quadruple therapy for treatment. However, too frequent use of antibiotics for treatment will lead to an increase in bacterial resistance. The resistance rate of Helicobacter pylori to some antibiotics, such as clarithromycin, metronidazole, levofloxacin, etc., is increasing year by year, and the primary resistance rate is basically higher than 20%, and even double, triple or even multiple resistance occurs. Therefore, it is of great significance to discover new anti-Helicobacter pylori drugs.
[0003] Burdock is a plant of the genus Arctium in the Asteraceae family. It is distributed worldwide and is mainly distributed in Northeast China, North China and Southwest China in China. Burdock seeds are the dried mature fruits of burdock, which can also be called powerful seeds and evil fruits. In autumn, when the fruits are ripe, the fruit sequences are harvested, dried, the fruits are beaten, impurities are removed, and then dried again. As a traditional Chinese medicine, burdock seeds have a history of application of nearly 2,000 years in my country and have been included in the "Famous Doctors' Records" as early as. It is pungent and bitter in taste, cold in nature, and belongs to the lung and stomach meridians. It can evacuate wind-heat, promote lung function and clear rash, detoxify and relieve sore throat. It is often used for wind-heat colds, coughs with sputum, measles, rubella, sore throat, mumps, erysipelas, carbuncle, sore and poisonous. In modern pharmacological studies, burdock seeds have been proven to have anti-inflammatory, antiviral, hypoglycemic and hypolipidemic effects.
[0004] Burdock seeds contain a variety of ingredients, mainly lignans, phenolic acids, fatty oils, terpenes, etc. Arctigenin is one of the main active ingredients of burdock seeds and has a variety of pharmacological activities, including in vitro inhibition of Helicobacter pylori. However, after oral administration, arctigenin can be metabolized in large quantities in the intestine and has a low bioavailability. Summary of the invention
[0005] In order to solve the above technical problems, the present invention proposes the use of burdock fruit extract in the preparation of anti-Helicobacter pylori products to expand the application value of burdock fruit in anti-Helicobacter pylori.
[0006] In order to achieve the above object, the present invention is achieved through the following technical solutions:
[0007] The preparation method of burdock fruit extract comprises the following steps:
[0008] S1: drying and crushing mature burdock seeds in sequence, and sieving to obtain burdock seed powder;
[0009] S2: adding water to the burdock seed powder at a solid-liquid ratio of 1:(10-30) and soaking for 20-40 minutes, then extracting in a water bath at 80-100°C for 1-3 hours, filtering while hot, adding water to the residue for secondary extraction, combining the filtrates, concentrating under reduced pressure and drying to obtain the burdock seed extract.
[0010] Preferably, in S1, the crushed burdock seeds are sieved using a 40-mesh sieve to obtain burdock seed powder.
[0011] Preferably, the solid-liquid ratio of burdock seed powder to water in S2 is 1:20, and the mixture is soaked in water for 30 minutes.
[0012] Preferably, the water bath extraction temperature in S2 is 90° C. and the time is 1 h.
[0013] The present invention also provides application of the burdock fruit extract in preparing an anti-Helicobacter pylori product.
[0014] The beneficial effects of the present invention are:
[0015] The invention extracts the burdock seed extract from the mature dry fruit of the burdock plant of the Asteraceae family. The in vitro antibacterial experiment finds that the burdock seed extracts from various sources have a direct killing effect on Helicobacter pylori, and the combination with four commonly used clinical antibiotics shows an additive effect. In animal experiments, the burdock seed extract can eliminate most of the Helicobacter pylori colonized in the stomach of mice. The burdock seed extract prepared by the preparation method of the invention has no organic solvent residue, is simple and easy to obtain, and has a significantly higher effect on the elimination of Helicobacter pylori colonized in the stomach of mice than its active monomer arctigenin. The invention broadens the application field of burdock seeds and provides a new direction for the treatment of Helicobacter pylori. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.
[0017] Figure 1 This is the time-inhibitory kinetic curve of the low-concentration burdock fruit extract in Example 2.
[0018] Figure 2 This is the time-bactericidal kinetic curve of the high concentration burdock fruit extract in Example 2.
[0019] Figure 3 This is a diagram showing the staining results of the Warthin-Starry silver staining method for gastric mucosal tissues of mice in different groups in Example 3. DETAILED DESCRIPTION
[0020] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.
[0021] Example 1
[0022] Preparation of burdock extract:
[0023] Select dry, mature, complete and full burdock fruit, crush it into powder, and pass it through a 40-mesh sieve. Add 20 times the amount of water (solid-liquid ratio: g / mL) to the burdock powder, soak it for 30 minutes, then place it in a water bath at 90°C for reflux extraction for 1 hour, collect the filtrate by centrifugation, add an equal amount of water to the filter residue and repeat the extraction once, combine the filtrates obtained from the two extractions, concentrate them by rotary evaporation, and then dry them to solid powder to obtain the burdock extract.
[0024] Example 2
[0025] In vitro inhibitory activity of burdock seed extract against Helicobacter pylori:
[0026] (1) The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of burdock extracts from different sources against standard strains (ATCC43504, ATCC700392, SS1) and clinical strains (CS01, QYZ-001, QYZ-003, QYZ-004, ICDC111001) of Helicobacter pylori were detected by broth microdilution method.
[0027] Experimental method: The burdock extract solution was prepared with brain heart infusion soup (BHI), ultrasonicated for 10 minutes to fully dissolve it, filtered through a 0.22μm filter membrane for sterilization, and gradiently diluted to obtain 640, 320, 160, 80, 40, 20, 10, and 5μg / mL concentrations of the drug solution. At the same time, a positive control drug was set: clarithromycin was dissolved in DMSO to obtain a 2560μg / mL clarithromycin stock solution, which was diluted with BHI to different concentrations (0.128, 0.064, 0.032, 0.016, 0.008, 0.004μg / mL) of the drug solution when used (DMSO final concentration <1%).
[0028] Pipette 50 μL of the prepared drug solution into a 96-well plate, set up 3 replicate wells for each drug concentration, and set up negative control wells (containing only drug, no inoculated strain), growth control wells (containing no drug, inoculated strain), blank control wells [containing no drug, only BHI and BHI containing 20% fetal bovine serum (FBS)]
[0029] Take the Helicobacter pylori strain cultured on the Columbia blood agar plate for 48-72h, scrape the bacterial moss into BHI broth, and adjust the turbidity of the bacterial suspension to 1Mc F with BHI containing 20% FBS. After diluting 10 times, inoculate 50μL per well in the above-mentioned 96-well plate containing the drug solution, place the 96-well plate in a three-gas incubator with the gas conditions set to 85% nitrogen, 10% carbon dioxide and 5% oxygen, and culture at 37°C and 150rpm for 72h. Then take out and check the results. The drug concentration when the bacterial turbidity is significantly reduced by naked eye observation is the MIC concentration.
[0030] Take out the 96-well plate after 72h of culture, aspirate 100 μL of the liquid in the wells with MIC, 2MIC, 4MIC, and 8MIC drug concentrations and evenly spread it on a fresh blood plate. After 72h of culture, take out and observe the results. The drug concentration at which the bacterial colony is reduced by 99.9% compared with the growth control group is the MBC concentration.
[0031] Experimental results: As shown in Table 1 below, the minimum inhibitory concentration of raw burdock extracts from 5 different sources against Helicobacter pylori ATCC700392 is 40μg / mL, and the minimum bactericidal concentration is 80μg / mL; the minimum inhibitory concentration of stir-fried burdock extracts from 3 different sources against Helicobacter pylori ATCC700392 is 80-160μg / mL, and the minimum bactericidal concentration is 160-320μg / mL. This shows that both raw burdock extract and stir-fried burdock extract have a strong inhibitory effect on Helicobacter pylori, and the source has little effect on the effect of burdock extract against Helicobacter pylori, while the effect of raw burdock extract against Helicobacter pylori is better than that of stir-fried burdock extract after processing.
[0032] Table 1 Minimum inhibitory concentration and minimum bactericidal concentration of burdock extracts from different sources and processing methods against Helicobacter pylori standard strain ATCC700392
[0033]
[0034] The burdock extract (Nbz) with the origin code Jilin 1 was used as an example to test the antibacterial and bactericidal effects of burdock extract on different strains. As shown in Table 2 below, the minimum inhibitory concentration of burdock extract for standard strains of Helicobacter pylori (ATCC43504, ATCC700392, SS1) is 40 μg / mL, and the minimum inhibitory concentration for clinical strains of Helicobacter pylori (CS01, ICDC111001, QYZ-001, QYZ-003, QYZ-004) is between 10-40 μg / mL. The minimum bactericidal concentration of burdock extract against standard strains of Helicobacter pylori (ATCC43504, ATCC700392, SS1) is 80-160 μg / mL, which is 2-4 times the MIC; the minimum bactericidal concentration for clinical strains of Helicobacter pylori (CS01, ICDC111001, QYZ-001, QYZ-003, QYZ-004) is 10-80 μg / mL, which is 1-2 times the MIC, indicating that burdock extract exhibits strong antibacterial and bactericidal effects on clinical strains.
[0035] Table 2 Minimum inhibitory concentration and minimum bactericidal concentration of burdock extract against standard and clinical strains of Helicobacter pylori
[0036]
[0037] *: R indicates resistance, S indicates sensitivity; MTZ: metronidazole, CLR: clarithromycin, LEF: levofloxacin, AMO: amoxicillin.
[0038] (2) Monitoring the antibacterial effect of burdock extract on Helicobacter pylori ATCC700392 based on the time-inhibition curve
[0039] Experimental method: Helicobacter pylori ATCC700392 was used as the test strain, and the drug-containing BHI of burdock extract with concentrations of 1 / 4MIC, 1 / 2MIC, and MIC were prepared by the two-fold dilution method, and the drug-free BHI was used as the growth control group. The ATCC700392 strain plate was taken out from the incubator, and the bacterial lawn was scraped into the BHI broth. The turbidity of the bacterial suspension was adjusted to 1McF with BHI containing 20% FBS and then diluted 10 times. According to the dosage of bacterial suspension: drug-containing BHI / drug-free BHI = 1:1, 2mL was inoculated into each well in a 6-well plate, and then placed in a three-gas incubator, and shaken and cultured at 37℃, microaerobic environment and 150rpm. 100μL was taken at 0h, 8h, 12h, 24h, 28h, 32h, 36h, 48h, 56h, and 72h to measure the absorbance value at OD600nm. The larger the absorbance value, the more bacteria there are.
[0040] Experimental results: Figure 1 It can be seen that the inhibitory activity of burdock seed extract on Helicobacter pylori ATCC700392 is dose-dependent. At 10 μg / mL, it slightly inhibits bacterial growth, at a concentration of 20 μg / mL, it inhibits most of the bacterial growth, and at 40 μg / mL, it completely inhibits bacterial growth.
[0041] (3) Monitoring the bactericidal effect of burdock extract on Helicobacter pylori ATCC700392 based on the time-bactericidal curve
[0042] Experimental method: Helicobacter pylori ATCC700392 was used as the test strain, and the drug-containing BHI of burdock extract with concentrations of 2MIC, 4MIC, and 8MIC was prepared by the two-fold dilution method, and the drug-free BHI was used as the growth control group. The ATCC700392 strain plate was taken out from the incubator, and the bacterial lawn was scraped into the BHI broth. The turbidity of the bacterial suspension was adjusted to 1Mf with BHI containing 20% FBS, and then diluted 10 times. According to the dosage of bacterial suspension: drug-containing BHI / drug-free BHI = 1:1, 2mL was inoculated into each well in 6 plates, and then placed in a three-gas incubator, and shaken and cultured at 37°C, microaerobic environment and 150rpm. 50 μL of culture medium was taken at 0h, 12h, 24h, 36h, 48h, 60h and 72h, respectively, and inoculated into blood agar plates after 10-fold gradient dilution (1:10-1:100000), and cultured for 4-5 days. After the bacteria grew into complete single colonies, the bacterial colonies were counted, and the bactericidal curves under different drug concentrations were obtained by plotting.
[0043] Experimental results: Figure 2As shown, the bactericidal effect of Arctium lappa extract on Helicobacter pylori shows a dose-dependence. At a drug concentration of 320 μg / mL, all bacteria can be killed in 36 h. At a drug concentration of 160 μg / mL, all bacteria can be killed in 48 h. At a drug concentration of 160 μg / mL, all bacteria can be killed in 60 h.
[0044] (4) The combined effects of Arctium lappa extract and antibiotics commonly used in clinical treatment of Helicobacter pylori infection were determined by the checkerboard dilution method.
[0045] Experimental method: Prepare Arctium lappa extract solutions with concentrations of 5 - 80 μg / mL according to the two-fold dilution method, and prepare antibiotics with different concentrations [amoxicillin (AMO) 0.125 - 8 μg / mL, clarithromycin (CLR) 0.004 - 0.256 μg / mL, metronidazole (MTZ) 0.05 - 3.2 μg / mL, levofloxacin (LEF) 0.025 - 1.6 μg / mL] in the same way.
[0046] Using ATCC700392 as the test strain, scrape the bacterial lawn on the plate, adjust the turbidity of the bacterial suspension to 1 McF with brain heart infusion containing 20% FBS, dilute it 10 times, and then according to the microbroth checkerboard dilution method, inoculate 120 μL per well into a 96-well plate according to the ratio of bacterial suspension: extract solution: antibiotic of 2:1:1. Place it in a triple-gas incubator and incubate it with shaking at 37 °C, microaerophilic environment, and 150 rpm for 72 h. The well with obvious clarity observed by the naked eye is the minimum combined inhibitory concentration.
[0047] Among them, combined inhibition means FICI = MIC Nbz(联用) / MIC Nbz(单用) +MIC 抗生素(联用) / MIC 抗生素(单用) . When FICI ≤ 0.5, it indicates a synergistic effect; when 0.5 < FICI ≤ 1, it is an additive effect; when 1 < FICI ≤ 2, it is an indifferent effect; when FICI > 2, it is an antagonistic effect.
[0048] Experimental results: As can be seen from Table 3 below, when Arctium lappa extract is combined with the four antibiotics, an additive effect is shown, indicating that Arctium lappa extract can be combined with commonly used clinical antibiotics.
[0049] Table 3 Inhibitory effects of the combination of Arctium lappa extract (Nbz) and antibiotics on Helicobacter pylori ATCC700392
[0050]
[0051]
[0052] Example 3
[0053] Inhibitory activity of burdock seed extract against Helicobacter pylori in mice:
[0054] Experimental methods: Forty-four SPF C57BL / 6 male mice aged 3-5 weeks were randomly divided into 7 groups, including 7 blank groups (no oral administration of Helicobacter pylori suspension + no medication), 7 infection model groups (oral administration of Helicobacter pylori suspension), 6 triple positive control groups (oral administration of Helicobacter pylori suspension + omeprazole, amoxicillin, and clarithromycin), 6 low-dose burdock extract groups (oral administration of Helicobacter pylori suspension + 58.5 mg / kg of burdock extract), 6 medium-dose burdock extract groups (oral administration of Helicobacter pylori suspension + 117 mg / kg of burdock extract), 6 high-dose burdock extract groups (oral administration of Helicobacter pylori suspension + 234 mg / kg of burdock extract), and 6 arctiin groups (oral administration of Helicobacter pylori suspension + 19.93 mg / kg of arctiin). The dose settings of the low, medium and high dose groups of burdock extract correspond to the daily clinical doses of 3, 6 and 12 g calculated by the surface area method, respectively. The dose setting of the arctiin group corresponds to the total proportion of arctiin and arctiin contained in the dose group of burdock extract. After all mice adapted to growth for 1 week, except for the blank group, all other mice were gavaged with 0.1-0.2 ml of Helicobacter pylori suspension of a certain density every 2 days for 7 times, and colonized for 2 weeks. On the 14th day, one mouse was randomly selected from the blank group and the infection model group for spinal dislocation and sacrificed. After dissection, the pathological tissue of the stomach was taken for physiological and biochemical detection to confirm that Helicobacter pylori had successfully colonized in the mouse stomach. Then the mice in the blank group and the infection model group were gavaged with normal saline, and the mice in other groups were gavaged with corresponding drugs once a day for 14 days.
[0055] All mice were killed on the second day after the last oral gavage, and gastric tissues were collected. Half of them were ground into homogenate and then blood plate colony counts were used to test Helicobacter pylori infection and clearance, and the clearance rate of Helicobacter pylori in each group of mice was calculated; the other half were fixed with tissue fixative and sliced, and the Helicobacter pylori in gastric mucosal tissue was stained and observed using Warthin-Starry silver staining. Wherein, clearance rate (%) = (bacterial load in infection model group - bacterial load in drug administration group) / bacterial load in infection model group * 100%
[0056] Experimental results: Table 4 below shows the clearance of Helicobacter pylori in the stomach of mice under the administration of different dose groups. The average clearance rate of the positive control group was the highest, which could basically clear all bacteria in the stomach of mice; the average clearance rate of the low-dose group of burdock extract was 72.15%, the medium-dose group was 90.78%, and the high-dose group was 55.53%, indicating that the burdock extract at different doses had a certain clearance effect on Helicobacter pylori in the stomach of mice; the average clearance rate of the arctiin group was 16.91%, and the clearance effect was relatively low.
[0057] Table 4 Clearance rate of Burdock extract on Helicobacter pylori in mouse stomach
[0058]
[0059] *:n represents different mouse individuals
[0060] like Figure 3 As shown, Helicobacter pylori colonization in the gastric mucosal tissue of mice in different groups is shown, indicated by blue arrows. The mucosal tissue morphology of mice in the blank group was normal, and there was no Helicobacter pylori colonization in the gastric pits; Helicobacter pylori was seen hiding in the mucosa in the infection model group. There was no Helicobacter pylori in the field of vision in the triple positive control group and the medium-dose group of burdock extract, and the tissue morphology was close to that of the blank group; only a few bacteria were occasionally seen in the low-dose and high-dose groups of burdock extract; Helicobacter pylori colonization was seen in the arctiin group, and the removal effect was poor.
[0061] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all details in detail, nor do they limit the invention to only the specific embodiments described. Obviously, many modifications and changes can be made according to the content of this specification.
Claims
1. A method for preparing a burdock fruit extract, characterized in that: The following steps are involved: S1: drying and crushing mature burdock seeds in sequence, and sieving to obtain burdock seed powder; S2: adding water to the burdock seed powder at a solid-liquid ratio of 1:(10-30) and soaking for 20-40 minutes, then extracting in a water bath at 80-100°C for 1-3 hours, filtering while hot, adding water to the residue for secondary extraction, combining the filtrates, concentrating under reduced pressure and drying to obtain the burdock seed extract.
2. The method for preparing the burdock fruit extract according to claim 1, characterized in that: In the step S1, the crushed burdock seeds are sieved using a 40-mesh sieve to obtain burdock seed powder.
3. The method for preparing the burdock fruit extract according to claim 1, characterized in that: The solid-liquid ratio of burdock seed powder to water in S2 is 1:20, and the mixture is soaked in water for 30 minutes.
4. The method for preparing the burdock fruit extract according to claim 1, characterized in that: The water bath extraction temperature in S2 is 90° C. and the time is 1 h.
5. Use of the burdock fruit extract according to any one of claims 1 to 4 in the preparation of an anti-Helicobacter pylori product.