CN114224989B - Traditional Chinese medicine composition for resisting helicobacter pylori - Google Patents
Traditional Chinese medicine composition for resisting helicobacter pylori Download PDFInfo
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Abstract
The invention discloses a traditional Chinese medicine composition with an anti-helicobacter pylori effect. The feed is prepared from the following raw materials in parts by mass: 6-10 parts of radix bupleuri, 8-12 parts of scutellaria baicalensis, 3-6 parts of coptis chinensis, 15-30 parts of semen plantaginis, 10-15 parts of gardenia, 15-30 parts of dandelion, 6-9 parts of cardamon, 8-12 parts of mangnolia officinalis, 10-20 parts of poria cocos, 15-30 parts of semen coicis, 6-9 parts of pinellia ternate and 15-30 parts of calcined concha arcae. In vitro experiments show that the Chinese medicinal composition has strong activity of resisting helicobacter pylori and helicobacter pylori adhesion. The decoction of the Chinese medicinal composition has remarkable antibacterial effect on the standard strain of helicobacter pylori (NCTC 11637), and the minimum inhibitory concentration (MIC value) is 64mg/ml of the decoction. Under lower dosage, the traditional Chinese medicine composition can obviously inhibit the helicobacter pylori from adhering to the gastric mucosal epithelium of the mouse, and the main link of the action is the direct contact stage of the helicobacter pylori and the gastric mucosal epithelium.
Description
Technical Field
The invention relates to a traditional Chinese medicine composition for resisting helicobacter pylori, belonging to the technical field of medicines.
Background
Helicobacter pylori is the most common pathogenic bacterium of upper digestive tract diseases such as acute and chronic gastritis, peptic ulcer and the like, is also a class I carcinogenic factor, and is one of the most important risk factors for gastric cancer. Helicobacter pylori can be spread through oral-oral/fecal-oral routes, people are generally susceptible, and infection cannot be eliminated by self if active intervention measures are not taken. At present, the successful eradication of helicobacter pylori is considered to reduce the risk of the occurrence of intestinal type gastric cancer, and screening and eradication treatment of helicobacter pylori should be carried out in areas with high risk of gastric cancer. As the high helicobacter pylori infection rate countries in China, the incidence rate and the death rate of the gastric cancer are in the forefront of the whole world, so that the eradication treatment of patients with treatment indications has great significance for the prevention and treatment of the gastric cancer.
In recent years, the situation of helicobacter pylori resistance to antibiotics is getting more severe in China, and new therapeutic drugs or schemes are urgently needed to be developed to meet the challenges in treatment. The traditional Chinese medicine is considered as one of new paths for treating helicobacter pylori, and can play a role in multiple links of helicobacter pylori invasion, adhesion, colonization and pathogenesis, so that the multi-target anti-helicobacter pylori effect is realized.
Disclosure of Invention
The invention aims to provide a traditional Chinese medicine composition.
The traditional Chinese medicine composition provided by the invention is prepared from the following raw materials in parts by mass:
6-10 parts of radix bupleuri, 8-12 parts of radix scutellariae, 3-6 parts of coptis chinensis, 15-30 parts of semen plantaginis, 10-15 parts of fructus gardeniae, 15-30 parts of dandelion, 6-9 parts of round cardamon seed, 8-12 parts of mangnolia officinalis, 10-20 parts of poria cocos, 15-30 parts of semen coicis, 6-9 parts of pinellia ternate and 15-30 parts of calcined concha arcae.
Specifically, the traditional Chinese medicine composition is prepared from the following raw materials in parts by mass: 6 parts of radix bupleuri, 10 parts of radix scutellariae, 6 parts of coptis chinensis, 15 parts of semen plantaginis, 10 parts of fructus gardeniae, 15 parts of dandelion, 6 parts of fructus cardamomi, 12 parts of cortex magnoliae officinalis, 10 parts of poria cocos, 30 parts of semen coicis, 6 parts of pinellia ternate and 30 parts of calcined concha arcae.
The traditional Chinese medicine composition can be in various dosage forms, and specifically can be decoction or granules.
The decoction of the traditional Chinese medicine composition can be prepared according to the following steps: decocting the above Chinese medicinal materials in water twice, mixing decoctions, filtering, concentrating the filtrate into fluid extract with concentration of 7.375g/mL (measured at 20 deg.C),
the amount of water used for decocting each time can be 2000ml/200g of Chinese medicinal materials;
the time for the first decoction may be 1.5 hours, and the time for the second decoction may be 1 hour.
The application of the traditional Chinese medicine composition in preparing the following products also belongs to the protection scope of the invention:
1) Agents that inhibit the growth of helicobacter pylori;
2) Agents that inhibit the adhesion of helicobacter pylori;
3) Drugs for preventing diseases caused by helicobacter pylori;
4) A medicament for treating a disease caused by helicobacter pylori;
in the 2), the adhesion can be the adhesion of helicobacter pylori to the gastric mucosa;
in the above 3) and 4), the disease caused by helicobacter pylori may specifically be acute or chronic gastritis, gastric or duodenal ulcer, gastric cancer (mainly gastric adenocarcinoma), gastric mucosa-associated lymphoid tissue lymphoma.
The invention also provides a medicament for resisting helicobacter pylori, which takes the traditional Chinese medicine composition as an active ingredient.
In vitro experiments show that the Chinese medicinal composition has strong activity of resisting helicobacter pylori and helicobacter pylori adhesion. The decoction of the Chinese medicinal composition has remarkable antibacterial effect on the standard strain of helicobacter pylori (NCTC 11637), and the minimum inhibitory concentration (MIC value) is 64mg/ml of the decoction. At a lower dosage, the traditional Chinese medicine composition can obviously inhibit the helicobacter pylori from adhering to the gastric mucosal epithelium of the mouse, and the main action link is the direct contact stage of the helicobacter pylori and the gastric mucosal epithelium.
The research result of the invention shows that the traditional Chinese medicine composition has the activity of resisting the helicobacter pylori and the activity of resisting the adhesion of the helicobacter pylori under low concentration, which shows that the traditional Chinese medicine composition is a medicine with great development potential for diseases such as acute and chronic gastritis, gastric ulcer, duodenal ulcer and the like closely related to the helicobacter pylori, and can be directly used for treating corresponding diseases and preparing related medicines thereof.
The inventor summarizes the characteristic that helicobacter pylori has damp-heat evil qi by combining the previous traditional Chinese medicine syndrome research results and published documents, and explores a traditional Chinese medicine composition for treating helicobacter pylori emergent infection in clinical practice. Through further basic experiments, the traditional Chinese medicine composition is found to be capable of inhibiting the growth of helicobacter pylori under in vitro conditions and inhibiting the adhesion of the helicobacter pylori to gastric mucosal epithelium, thereby achieving the treatment purposes of reducing the colonization of the helicobacter pylori and eliminating the helicobacter pylori infection. The present invention has been completed by combining the obtained experimental results.
The application of the traditional Chinese medicine composition in preparing the anti-helicobacter pylori medicine is disclosed for the first time, the traditional Chinese medicine composition has the inhibition activity on the helicobacter pylori, has the inhibition effect on the adhesion process of the helicobacter pylori to the gastric mucosa, and can be used for preventing and treating the helicobacter pylori infection.
Drawings
FIG. 1 is a graph showing the results of determining the MIC of the Chinese medicinal composition for helicobacter pylori using the agar dilution method in example 1.
Fig. 2 shows the results of the toxicity test in example 2, which indicates significant difference from the blank control group, P <0.05.
FIG. 3 is a fluorescence image (x 100 times) of different drug concentrations and different intervention methods [ A ] for pre-treating gastric mucosa with 0,1,2,5mg/mL of a Chinese medicinal composition, respectively; [B] pretreating helicobacter pylori with 0,1,2,5mg/mL of the Chinese medicinal composition respectively; [C] respectively intervening in the adhesion process by using 0,1,2,5mg/mL of the traditional Chinese medicine composition.
FIG. 4 shows the pretreatment of gastric mucosa with 0 and 5mg/mL of the Chinese medicinal composition, and the positive control of 0.1mg/mL of rebamipide.
FIG. 5 shows the results of fluorescence quantitative analysis, wherein the composition is used for pre-treating gastric mucosa, the composition is used for pre-treating helicobacter pylori, the composition is used for intervening adhesion process, and the composition is used for pre-treating gastric mucosa and positive control. * Representing significant difference from blank control group, P <0.05; * Indicates significant difference from blank control group, P <0.001.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 inhibitory Effect of Chinese medicinal composition on helicobacter pylori
1. Materials and methods
1.1 materials
1.1.1 test strains
Helicobacter pylori NCTC11637 standard strain.
1.1.2 Experimental drugs and Primary reagents
The traditional Chinese medicine composition is composed of radix bupleuri, radix scutellariae, rhizoma coptidis, semen plantaginis, fructus gardeniae, dandelion, cardamom seed, magnolia officinalis, poria cocos, semen coicis, pinellia ternate and calcined concha arcae (conventional medicinal parts), wherein the mass ratio of the Chinese medicine composition is (6):
decocting the above Chinese medicinal materials with water twice, wherein the amount of water used for each decoction is 2000ml/200g of the Chinese medicinal materials, the first decoction is carried out for 1.5 hours, the second decoction is carried out for 1 hour, the decoction liquids are merged and filtered, the filtrate is concentrated, and the final concentration of clear paste per ml is equivalent to 7.375g (measured at 20 ℃) decoction pieces.
Columbia agar (lot # CM 0331) and Naoxing liquid (lot # CM 0225) were purchased from OXOID, UK, and Brookfield broth (lot # 3182288) was purchased from Bidi medical devices (Shanghai) Co., ltd, and prepared according to the instructions; defibrinated sheep blood was purchased from Beijing Baote medical instruments, inc.; fetal bovine serum (batch No. 20140623) was purchased from Beijing Feimer Biotech limited; a microaerophilic gas generating bag (batch No.: CN 0025A) was purchased from Beijing Xin Longfu medicine sales Co., ltd.
1.1.3 Experimental facility
Clean bench (Shanghai Smart laboratory facilities, ltd.), constant temperature incubator (Shanghai-constant technology, ltd.), microaerophilic culture tank (Beijing glass works), constant temperature shaking table (Shanghai-constant instruments, ltd.), liquid culture tank purchased from OXOID, england, mixed gas (85% N) 2 、10％CO 2 、5％O 2 )。
1.2 methods
1.2.1 helicobacter pylori culture method and strain identification method
Uniformly inoculating helicobacter pylori NCTC11637 into Columbia blood agar culture medium containing 8% defibrinated sheep blood, culturing for 3 days at 37 deg.C in microaerophilic environment by air-bleed ventilation method, and subculturing positive ones; the strain was identified by observing colony morphology, gram stain, and rapid urease, hydrogen peroxide, and oxidase assays.
1.2.2 drug bacteriostasis test
Agar dilution method: collecting fresh helicobacter pylori cultured for 72 hr, diluting to 3 × 10 8 cfu/ml, bacteria were inoculated with 1. Mu.l inoculating loop onto drug-containing agar ( drug gradient 4, 8, 16, 32, 64, 128 mg/ml) plates and simultaneously inoculated into drug-free medium as control. After inoculation, the culture medium is placed in a microaerophilic environment at 37 ℃ and observed after 72 hours of culture, and the minimum effective concentration on an agar plate without colonies is the Minimum Inhibitory Concentration (MIC).
2. Results
As shown in figure 1, when the concentration of the medicine in the agar is 64mg/ml, the agar plate is aseptically grown, so that the minimum inhibitory concentration of the traditional Chinese medicine composition on helicobacter pylori is 64mg/ml.
3. Conclusion
The MIC of the traditional Chinese medicine composition on helicobacter pylori is determined by adopting an agar dilution method, and whether the traditional Chinese medicine composition has an antibacterial effect on helicobacter pylori in vitro is studied. The result shows that the traditional Chinese medicine composition has a remarkable bacteriostatic effect on the standard strain NCTC 11637.
Example 2 inhibitory Effect of Chinese medicinal composition on adhesion of helicobacter pylori to gastric mucosal epithelium
1. Materials and methods
1.1 materials
1.1.1 test strains
Helicobacter pylori NCTC11637 standard strain.
1.1.2 Experimental drugs and Primary reagents
The preparation method of the traditional Chinese medicine composition is the same as that of the embodiment 1.
Columbia blood agar (OXOID; no: 2152465), defibered sheep blood (Beijing Baote Hao science and technology Co., ltd.; no: 2018/092701), brookfield powder (OXOID; no: 7128995), fetal bovine serum (Beijing Feimer biotechnology Co., ltd.; no:313B 051), fluorescein isothiocyanate FITC (Beijing Lanbo biotechnology Co., ltd., no: 3326-32-7), rebamipide (Zhejiang Otsuka Pharmaceutical Co., ltd., 180511R), paraffin slicer (Taiva, R135), centrifuge (Dynamica, VELOCITY-18R), constant temperature incubator (Shanghai Henan apparatus Co., ltd., THZ-98B), fluorescence microscope (Nikon, ti-U). Healthy KM mice gastric mucosa paraffin-embedded tissues were provided by the Experimental animals center of the first Hospital, beijing university.
1.2 methods
1.2.1 toxicity test
9.5mL of Brookfield broth (containing 10% fetal bovine serum) and 0.5mL of the drug solution were added to the plate to give a drug concentration of 100, 50, 20, 10,5,2,1mg/mL in the plate, and an equal volume of Brookfield broth was used as a blank. Freshly cultured H.pylori were collected with a sterile inoculating loop, adjusted to a concentration of 3X 10 8 CFU/mL. 100 mu L of the bacterial liquid is respectively added into the culture solution of the plate and cultured for 2 hours at 37 ℃ under the condition of micro-aerobic shaking. Collecting the liquid in the plate, centrifuging at 4000r/min for 10min, and washing off the liquid medicine. Each 100. Mu.L of each was plated on Columbia blood agar medium in triplicate, and the total number of Colony Forming Units (CFU) was counted after 48 hours of culture. The total number of CFUs in the blank control group was set to 100%.
1.2.2 in situ adhesion experiments
1.2.2.1 preparation of sections: the paraffin-embedded tissue of the gastric mucosa of the mouse was cut into sections with a thickness of 3mm, deparaffinized in the order of xylene (30 min × 2 times) -100% alcohol (10 min × 2 times) -95% alcohol (3 min × 2 times) -80% alcohol (3 min × 2 times), and placed in PBS for use.
1.2.2.2fitc-labeled helicobacter pylori: freshly cultured H.pylori was collected, suspended in Brookfield broth and adjusted to a concentration of 3X 10 8 CFU/mL, 100. Mu.L of FITC (10 mg/mL) in DMSO was added to the suspension and incubated at 37 ℃ in a microaerophilic environment with shaking in a dark room for 1 hour. Centrifuging at 4000r/min for 5min, washing off unlabeled FITC, and repeating for 3 times.
1.2.2.3 Co-incubation: 1mL FITC-labeled H.pylori was diluted 15-fold with Brookfield broth and incubated with gastric mucosal tissue sections in a microaerophilic environment at 37 ℃ for 2 hours in a dark room. Washed 4 times with PBS before observation.
1.2.2.4 drug intervention: the experiment sets 3 medicine intervention methods: the gastric mucosa is intervened in advance (gastric mucosa slices are pretreated by the traditional Chinese medicine composition liquid for 2 hours), the helicobacter pylori is intervened in advance (the helicobacter pylori marked by FITC is pretreated by the traditional Chinese medicine composition liquid for 2 hours and is carried out in a microaerophilic environment at 37 ℃ in a dark room), and the adhesion process is intervened (the gastric mucosa slices and the helicobacter pylori are not pretreated in advance, and the traditional Chinese medicine composition liquid is added during co-incubation). An equal volume of buchner broth was used as a blank and the experiment was repeated 3 times.
1.2.3 interpretation of results
Observing the adhesion quantity of the helicobacter pylori under a fluorescence microscope, reading the helicobacter pylori film by a multi-person blind method, and performing visual simulation scoring on the adhesion quantity according to the report of LENGSFELDC. The most significant number of adhesions was scored as (+++++), and then progressively decreased as (+++++, ++++, ++). Meanwhile, the fluorescence intensity was quantitatively analyzed by using Image J software, and the fluorescence intensity of the blank control group was set to 100%.
2. Results
2.1 toxicity test
As shown in FIG. 2, the 5mg/mL and lower concentration liquid of the Chinese medicinal composition has no significant difference compared with the blank control group, and does not affect the growth of helicobacter pylori. The drug concentration was set to 3 groups accordingly (high dose group 5mg/mL, medium dose group 2mg/mL, low dose group 1 mg/mL).
2.2 in situ adhesion test
As shown in FIG. 3,4, white highlight is FITC-labeled H.pylori, and since the adhesion sites of H.pylori are mainly distributed in gastric mucosal epithelium and stomach fovea, the intercepting visual field is mainly a gastric mucosal epithelium part. As shown in Table 1, the amount of adhesion of H.pylori can be significantly reduced by the pretreatment of gastric mucosa with drugs and the intervention of the adhesion process through visual observation, and the amount of adhesion of H.pylori with drug pretreatment is not significantly changed. The effects of the drug pretreatment of gastric mucosa and the positive control drug rebamipide were comparable.
TABLE 1 visual simulation scoring results
Through statistical analysis of fluorescence quantification, pretreatment of gastric mucosa with 5mg/mL of the traditional Chinese medicine composition can significantly inhibit adhesion of helicobacter pylori (P = 0.034) (fig. 5A); the traditional Chinese medicine compositions of 2mg/mL (P = 0.016) and 5mg/mL (P = 0.001) can also remarkably inhibit the adhesion of the helicobacter pylori during the adhesion process (figure 5C); pretreatment of h.pylori showed no inhibition (P = 0.422) (fig. 5B). The 5mg/mL (P = 0.022) drug-pretreated gastric mucosa significantly inhibited h.pylori adhesion compared to the known positive control drug rebamipide (P = 0.02), and there was no significant difference (P = 0.998) (fig. 5D).
3. Conclusion
The Chinese medicinal composition can inhibit helicobacter pylori from adhering to gastric mucosa epithelium, has anti-adhesion effect not directly against helicobacter pylori, and has main action when gastric mucosa and gastric mucosa contact helicobacter pylori.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific examples, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is made possible within the scope of the claims attached below.
Claims (4)
1. The application of the traditional Chinese medicine composition in preparing the following products,
1) Agents that inhibit the growth of helicobacter pylori;
2) Agents that inhibit the adhesion of helicobacter pylori;
3) Drugs for preventing diseases caused by helicobacter pylori;
4) A medicament for treating a disease caused by helicobacter pylori;
the traditional Chinese medicine composition is prepared from the following raw materials in parts by mass: the traditional Chinese medicine composition is prepared from radix bupleuri, coptis chinensis, plantain seeds, gardenia, dandelion, cardamom seeds, mangnolia officinalis, poria cocos, semen coicis, pinellia ternate and calcined concha arcae, wherein the mass ratio is (6);
in said 2), the adhesion is the adhesion of helicobacter pylori to the gastric mucosa;
in the above 3) and 4), the disease site caused by helicobacter pylori is gastric mucosa.
2. Use according to claim 1, characterized in that: the traditional Chinese medicine composition is a decoction or a granule.
3. Use according to claim 2, characterized in that: the decoction of the traditional Chinese medicine composition is prepared according to the following steps: decocting the above Chinese medicinal materials in water twice, mixing decoctions, filtering, and concentrating the filtrate into fluid extract.
4. Use according to claim 3, characterized in that: the dosage of the decoction water is 2000ml/200g of the Chinese medicinal raw materials each time;
the time for the first decoction is 1.5 hours, and the time for the second decoction is 1 hour;
the concentration of the clear paste after concentration is 7.375 g/mL.
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| 复方清热化湿制剂对幽门螺杆菌的体外抗菌作用研究;于靖等;《中国中西医结合消化杂志》;20160430;第24卷(第04期);245-248 * |
| 清热化湿方对幽门螺杆菌感染小鼠的抗菌和抗炎作用;于靖等;《中成药》;20170131;第39卷(第01期);7-14 * |
| 清热化湿方联合三联疗法治疗脾胃湿热型幽门螺杆菌感染的随机对照临床研究;杨闪闪等;《世界中西医结合杂志》;20200928;第15卷(第09期);1734-1738 * |
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