CN113559226B - Medicinal and edible dual-purpose composition for resisting helicobacter pylori and application thereof - Google Patents
Medicinal and edible dual-purpose composition for resisting helicobacter pylori and application thereof Download PDFInfo
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- CN113559226B CN113559226B CN202110772248.XA CN202110772248A CN113559226B CN 113559226 B CN113559226 B CN 113559226B CN 202110772248 A CN202110772248 A CN 202110772248A CN 113559226 B CN113559226 B CN 113559226B
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Classifications
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- A—HUMAN NECESSITIES
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- 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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- A61K36/9068—Zingiber, e.g. garden ginger
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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Abstract
An anti-helicobacter pylori medicine and food dual-purpose composition and an application thereof are disclosed, wherein the composition comprises the following components in parts by weight: 3-10 parts of ginger extract, 0-3 parts of sophora flower bud extract and 0-5 parts of mulberry leaf extract. The combined use can resist helicobacter pylori synergistically, has good anti-urease activity, and has good effects on treating sensitive and drug-resistant helicobacter pylori infected related stomach diseases and the like. The composition has the advantages of definite curative effect, simple formula, low price and less toxic and side effects on preventing and treating helicobacter pylori related gastropathy.
Description
Technical Field
The invention belongs to the field of food therapy and health care products, and particularly relates to a helicobacter pylori resistant medicine-food dual-purpose composition and application thereof.
Background
About half of the world's population is currently infected with H.pylori (Hp). Hp infection causes diseases of the digestive tract such as gastritis and gastric and duodenal ulcers, and is one of the major risk factors for the development of gastric cancer. Many studies have shown that eradication of Hp can significantly reduce the incidence of gastric cancer. Currently, the world health organization recommends a treatment regimen for Hp infection by triple or quadruple therapy, i.e. the simultaneous administration of a proton pump inhibitor (omeprazole, etc.) plus two antibiotics (two selected from clarithromycin, amoxicillin, tetracycline, metronidazole, etc.) or a bismuth agent (bismuth potassium citrate, etc.) plus a proton pump inhibitor plus two antibiotics. However, with the long-term use of antibiotics, Hp is likely to generate different degrees of drug resistance to antibiotics, which results in failure of radical treatment of triple or quadruple therapy, and also often causes side effects such as dysbacteriosis, and the like, thus increasing the burden of patients. For refractory gastritis, gastric ulcer and the like caused by drug-resistant helicobacter pylori infection, the treatment by using antibiotics generating drug resistance is not suitable, the antibiotics are required to be adjusted, but few candidate antibiotics effective to the helicobacter pylori exist, and a new scheme is urgently required to be explored.
The production of a large amount of highly active urease is one of the biological properties of Hp. Urease hydrolyzes urea to generate ammonia, and an 'ammonia cloud' protective layer is formed around the thalli to resist the killing effect of gastric acid. Therefore, urease plays a role in colonization and toxicity in H.pylori infection and is also an important target for the treatment of H.pylori. Thus, the screening of the inhibitor for resisting Hp urease has great application prospect in resisting helicobacter pylori.
The theory of homology of medicine and food (also called as homology of medicine and food) exists in traditional Chinese medicine since ancient times, and the theory of homology of medicine and food means that many foods, namely medicines, can prevent and treat diseases like the foods and the medicines, and have wide application in daily health preservation and clinical treatment. Based on the antibacterial activity, the medicine and food dual-purpose composition is screened and prepared from the antibacterial activity, the growth of the helicobacter pylori and the activity of urease are inhibited, the aim of daily prevention or treatment of stomach diseases caused by helicobacter pylori infection is fulfilled, a theoretical basis is provided for application of traditional Chinese medicinal materials in resisting the helicobacter pylori, and a reference is provided for better clinical application of the traditional Chinese medicine.
Disclosure of Invention
The technical problem to be solved is as follows: the invention aims to overcome the defects of the prior art, and provides a helicobacter pylori resistant composition used as both medicine and food and an application thereof, namely a ginger extract, a sophora flower bud extract and a mulberry leaf extract, by adopting a natural plant extract used as both medicine and food as a raw material according to the principle of 'homology of medicine and food' of Chinese medical science food therapy.
The technical scheme is as follows: a medicine and food dual purpose composition for resisting helicobacter pylori is composed of the following raw materials by weight: 3-10 parts of ginger extract, not more than 3 parts of sophora flower bud extract and not more than 5 parts of mulberry leaf extract.
Preferably, the composition comprises 3-7 parts of ginger extract, 1-3 parts of sophora flower bud extract and 1-5 parts of mulberry leaf extract in parts by weight.
The composition can be used for preparing food and health product for preventing or treating digestive tract diseases caused by helicobacter pylori infection.
The composition can be made into tablet, capsule, pill, granule or powder.
Has the advantages that: the composition can be used for preparing medicines for preventing or treating stomach diseases such as acute and chronic gastritis, gastric ulcer, duodenal ulcer and the like caused by drug resistance or sensitive helicobacter pylori infection, and has small toxic and side effects and low medical cost. The composition has reasonable compatibility, has better inhibiting effect on helicobacter pylori antibiotic sensitive strains and drug-resistant strains, effectively inhibits the activity of helicobacter pylori urease, has the action characteristics of multiple components, multiple ways and multiple targets, and is suitable for Hp infected persons. In conclusion, the application of the composition can reduce the use of antibiotics so as to effectively relieve the problem of helicobacter pylori resistance.
Detailed Description
EXAMPLE 1 screening of plant extracts for anti-helicobacter pylori Activity
In order to eradicate the helicobacter pylori, 76 kinds of plant extracts with dual purposes of medicine and food are selected firstly, in-vitro activity screening of the helicobacter pylori resistance is carried out, and the plant extract with the Hp resistance activity is obtained by measuring the Minimum Inhibitory Concentration (MIC) of the helicobacter pylori.
1.1 materials
1) Chemicals and plant extracts: amoxicillin, metronidazole, clarithromycin, levofloxacin and the like are available from MCE. 76 plant extracts including clove, cassia seed, medlar, angelica dahurica, ginkgo, phaseolus calcaratus, lily, smoked plum, linseed, finger citron, tuckahoe, liquorice, galangal, kudzu root, lotus leaf, fructus momordicae, yellow mustard seed, sealwort, black sesame, black pepper, sophora flower, wrinkled gianthyssop, honeysuckle, ginger, exocarpium citri rubrum, orange peel, mint, platycodon grandiflorum, chrysanthemum, chicory, kelp, radish seed, lotus seed, lophatherum gracile, purslane, pawpaw, fructus cannabis, scaphium scaphigerum, dandelion, elsholtzia, gorgon fruit, Chinese olive, mulberry, nutmeg, cinnamon, yam, sea-buckthorn, fructus amomi, mulberry leaf, common fennel, field thistle, citron, houttuynia, fresh lalang grass rhizome, allium macrostemon, polygonatum, emblica, bunge, houttuynia, bitter caraway, coix seed, semen hovenia dulcis, bamboo leaf flavone, coriander, cordyceps militaris, loquat leaf, moringa leaf, short stalk, maca powder, maca powder, margarita, Chinese magnoliavine fruit powder, Chinese magnoliavine fruit, red sage root, Chinese magnoliavine fruit, red sage root, Chinese magnoliavine fruit, Chinese magnol, The broccoli powder, the assai fruit, the acanthopanax brachypus, the coix seed, the perilla seed and the like are purchased from Xian Xiaocao plant science and technology Limited liability company.
2) The strain is as follows: helicobacter pylori standard strains 26695, G27 and ATCC 43504; other clinical strains Hp129, Hp159 and JRES00015 were isolated and characterized from clinical patient gastric mucosa samples at the first Hospital and the Yifu Hospital affiliated to the Nanjing medical university.
3) Media and main reagents: brain heart infusion Broth (BHI), columbia medium, calf serum (FCS), and 100% dimethyl sulfoxide (DMSO).
4) The main apparatus is as follows: BINDER CB160 three-gas incubator, ultraviolet spectrophotometer, constant temperature shaking table (Thermo), centrifuge, electronic balance, etc.
1.2 method: detecting minimum inhibitory concentration (MIC, 100 μ L system) of plant extract on helicobacter pylori by broth dilution method
1) Preparing 100mg/mL plant extract stock solution respectively, and the solvent is sterile water or 100% DMSO.
2) Preparing bacterial liquid: a suspension of the logarithmic phase-grown H.pylori on a solid plate was prepared from BHI (containing 10% FCS) and adjusted to a concentration OD600 of 0.2 (approximately 1X 10 cell concentration)8CFU/mL), diluted 10 times, the bacterial load was about 1X 107CFU/mL, spare.
3) Preparation of 96-well plate: the first well was diluted to well 8 by a double dilution method with 176. mu.L of BHI broth (containing 10% FCS) and 4. mu.L of each stock solution of plant extract.
4) Inoculating a bacterial liquid: adding 10 μ L of the above-mentioned bacterial suspension to each well (the bacterial concentration per well is about 1.0 × 10)6CFU/mL), the final concentration of the plant extract was 2000. mu.g/mL, 1000. mu.g/mL, 500. mu.g/mL, 250. mu.g/mL, 125. mu.g/mL, 62.5. mu.g/mL, 31.3. mu.g/mL, and 16. mu.g/mL, in that order. In another row, 100. mu.L of BHI medium (10% FCS) was added to each well as a sterile control group; as a positive control, 90. mu.L of BHI culture medium (containing 10% FCS) and 10. mu.L of the above-mentioned stock solutions were added to each well of the other row. Placing the mixture in a three-gas incubator for culture.
5) And (5) judging a result: the results were interpreted after 48 or 72h incubation, with the lowest concentration that completely inhibited bacterial growth in the wells being the MIC. The experiment was only significant when bacteria were significantly growing in the positive control wells (i.e., no drug) and the sterile control group was growing aseptically. The experiment was repeated 3 times.
1.3 results
The results are shown in Table 1. The results show that the three extracts including ginger, sophora flower bud and mulberry leaf have the activity of resisting helicobacter pylori, and the MIC range is 250-1000 mu g/mL; while the MICs of the other 73 plant extracts to Hp were all greater than 1000. mu.g/mL.
TABLE 1 minimum inhibitory concentration (MIC, μ g/mL) of plant extract
Example 2 detection of Combined anti-Hp Effect
Three plant extracts (ginger, sophora flower bud and mulberry leaf extract) with anti-helicobacter pylori activity are selected for carrying out combined antibacterial effect detection aiming at helicobacter pylori. By means of standard checkerboard titration [ Tanaka M, Isogai E, Isogai H, et al, synthetic effect of genomic reagents and proton pump inhibitors on Helicobacter pylori. J antisense Chemother 2002,49(6): 1039-]The effect of the combination was evaluated by using a fractional inhibition concentration index (FIC). FIC is calculated as follows: sigma FIC ═ FICA+FICBOf which FICA＝MICA(in the presence of B)/MICA(alone), FICB＝MICB(in the presence of A)/MICB(alone). FIC index interpretation criteria are as follows: less than or equal to 0.5, synergistic effect; 0.5-1, additive effect; 1-4.0, independent effect;>4.0, antagonism. The experiment was repeated 3 times.
The results are shown in Table 2. The results show that the ginger extract, the sophora flower bud extract and the mulberry leaf extract can resist helicobacter pylori synergistically.
TABLE 2 Combined anti-Hp Effect of ginger, Sophora flower bud and Mulberry leaf
EXAMPLE 3 medicinal and edible composition formulation and determination of anti-helicobacter pylori Activity thereof
A medicine and food dual-purpose composition (composition 1) for resisting helicobacter pylori comprises the following components in parts by weight: 3 parts of ginger extract, 2 parts of sophora flower bud extract and 5 parts of mulberry leaf extract.
A medicine and food dual purpose composition (composition 2) for resisting helicobacter pylori comprises the following components in parts by weight: 4 parts of ginger extract, 3 parts of sophora flower bud extract and 3 parts of mulberry leaf extract.
A medicine and food dual-purpose composition (composition 3) for resisting helicobacter pylori comprises the following components in parts by weight: 5 parts of ginger extract, 1 part of sophora flower bud extract and 4 parts of mulberry leaf extract.
A dual-purpose composition (composition 4) for medicine and food for resisting helicobacter pylori comprises the following components in parts by weight: 6 parts of ginger extract, 3 parts of sophora flower bud extract and 1 part of mulberry leaf extract.
A medicine and food dual purpose composition (composition 5) for resisting helicobacter pylori comprises the following components in parts by weight: 7 parts of ginger extract, 1 part of sophora flower bud extract and 2 parts of mulberry leaf extract.
The MIC of the above 5 compositions for H.pylori were determined by the method of example 1, i.e., broth dilution method, and the results are shown in Table 3. The results show that 5 compositions all had anti-helicobacter pylori activity, with compositions 4 and 5 being the most potent, stronger than each individual plant extract.
TABLE 3 minimum inhibitory concentration (MIC, μ g/mL) for each composition
EXAMPLE 4 determination of anti-helicobacter pylori urease Activity
4.1 materials
1) Chemical products: urea, Jack Bean Urease (JBU) standards, Acetohydroxamic Acid (AHA), ethylenediaminetetraacetic Acid (EDTA), Sodium Nitrosoferricyanide (SNP), Sodium Salicylate (SSA), and sodium hypochlorite (NaOCl) were purchased from Sigma.
2) The strain is as follows: helicobacter pylori standard strain G27.
3) The main apparatus is as follows: an enzyme-labeling analyzer, an ultrasonic crusher and an electronic balance.
4.2, method: the anti-urease activity was measured by Bertholt color development.
1) Extracting Hp urease: the G27 strain was inoculated into 200 ml of BHI medium (containing 10% FCS), cultured in a three-gas incubator at 37 ℃ for 48 hours, centrifuged to collect cells, washed twice with phosphate buffered saline PBS (pH7.4), and the Hp cells were stored at-80 ℃ for 24 hours. Then taken out to return to room temperature, added with 12 ml of 50mM phosphate buffer (containing 300mM sodium chloride, pH7.0) for heavy suspension, and added with protease inhibitor, ultrasonication for 10 minutes, 4 ℃ 14000rpm centrifugation for 30 minutes, supernatant through Sephadex G-25 column desalination, to the crude enzyme added with equal volume of glycerol, and at 4 ℃ storage, for activity determination.
2) Drawing a jack bean urease activity standard curve: the jack bean urease standard was diluted with PBS-EDTA solution to 0.128, 0.064, 0.032, 0.016 and 0.008U/ml. 100 μ l of jack bean urease standard and 100 μ l of 100mM urea solution were added to a 1.5ml centrifuge tube, and reacted at room temperature for 20 minutes, and then Bertholt color developing solution was added, that is, 100 μ l A reagent (9.73mM SNP and 700mM SSA) and 100 μ l B reagent (125mM NaOH and 11.3mM NaOCl) were added in this order, mixed well, and developed in the dark at room temperature for 10 minutes, and PBS-EDTA was used as a negative control. After the color development is finished, the solution is absorbed onto a 96-well plate, an enzyme-labeling instrument is used for detecting OD635, and the OD under each concentration is calculatedRelative value＝ODAbsolute value-ODNegative control。
3) The Bertholot color method is used for determining the urease activity: the extracted Hp urease activity was detected and calculated using the Berthelot color method and standard curve described above. The urease concentration was then measured by the Bradford method and the extracted urease activity was finally calculated in U/mg.
4) Urease half-inhibitory concentration (IC50) determination: mu.L of urease solution (10U/mL), 25. mu.L of the plant extract or composition to be tested (the concentration is set according to the experiment) and 50. mu.L of 100mM phosphate buffer solution containing urea are added into a 96-well plate, mixed uniformly, protected from light, reacted at room temperature for 20 minutes, then 100. mu.L of the reagent A and the reagent B are added, mixed uniformly, protected from light, reacted at room temperature for 20 minutes, and the absorbance value is measured at 635 nm. The inhibition rate and median inhibitory concentration IC50 were calculated from a blank group, a normal control group, and a positive control group (AHA).
4.3 results
The results are shown in Table 4. The results show that each composition has a certain anti-helicobacter pylori urease activity.
TABLE 4 half inhibitory concentration of each sample on urease (IC50)
Claims (3)
1. The medicine and food dual-purpose composition for resisting helicobacter pylori is characterized by comprising the following raw materials in parts by weight: 3-7 parts of ginger extract, 1-3 parts of sophora flower bud extract and 1-5 parts of mulberry leaf extract.
2. The use of the composition of claim 1 in the preparation of anti-helicobacter pylori health products.
3. The composition of claim 1, wherein the composition is formulated as a tablet, capsule, pill, granule, or powder.
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