WO2018223922A1 - 药物组合物在制备用于抗幽门螺杆菌的药物中的用途 - Google Patents

药物组合物在制备用于抗幽门螺杆菌的药物中的用途 Download PDF

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WO2018223922A1
WO2018223922A1 PCT/CN2018/089762 CN2018089762W WO2018223922A1 WO 2018223922 A1 WO2018223922 A1 WO 2018223922A1 CN 2018089762 W CN2018089762 W CN 2018089762W WO 2018223922 A1 WO2018223922 A1 WO 2018223922A1
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pharmaceutical composition
use according
extract
beeswax
culture
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PCT/CN2018/089762
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English (en)
French (fr)
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李俐
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李俐
北京荣祥再生医学研究所有限公司
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Priority to KR1020197038417A priority Critical patent/KR20200011976A/ko
Priority to US16/617,458 priority patent/US20210121502A1/en
Publication of WO2018223922A1 publication Critical patent/WO2018223922A1/zh

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    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/201—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having one or two double bonds, e.g. oleic, linoleic acids
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Definitions

  • the present invention relates to the use of a pharmaceutical composition for the preparation of a medicament for use against Helicobacter pylori.
  • the invention further relates to the use of a pharmaceutical composition for the preparation of a medicament for the treatment/prevention of a disease caused by Helicobacter pylori.
  • Cigar Patent ZL 02105541.6 discloses a pharmaceutical composition suitable for oral administration, which comprises a homogeneous mixture of edible oil with beeswax and ⁇ -sitosterol, wherein the beeswax forms microcrystals in the composition, according to the combination
  • the total wax weight is 0.5-50% for beeswax and at least 0.1% for beta-sitosterol.
  • the composition may also contain other pharmaceutical ingredients and be used to deliver other active ingredients to the gastrointestinal tract for the treatment of various diseases.
  • this pharmaceutical composition is mainly used to protect mucosal tissues from damage caused by irritants, and to promote the repair and regeneration of damaged or incomplete gastrointestinal mucosal tissues, especially for the treatment of gastrointestinal dysfunction, such as gastritis. , peptic ulcer, reflux esophagitis, dyspepsia and gastric cancer, as well as the physiological structure and function used to reconstruct mucosal tissue.
  • pharmaceutical composition means a pharmaceutical composition comprising edible oil and beeswax, ⁇ -Valley A homogeneous mixture of sterols, wherein the beeswax in the composition forms microcrystals having a beeswax content of from 0.5 to 50% and a beta-sitosterol of from 0.1% to 20%, based on the total weight of the composition.
  • HHP Helicobacter Pylori
  • MALT gastric mucosa-associated lymphoid tissue
  • Helicobacter pylori is parasitic in gastric mucosa, and 67% to 80% of gastric ulcers and 95% of duodenal ulcers are caused by Helicobacter pylori.
  • the technical problem to be solved by the present invention is to inhibit or kill Helicobacter pylori using the above-mentioned known pharmaceutical composition, thereby treating diseases caused by Helicobacter pylori.
  • one aspect of the invention relates to the use of a pharmaceutical composition for the preparation of a medicament for use against Helicobacter pylori, wherein the pharmaceutical composition is a pharmaceutical composition suitable for oral administration comprising edible oil and beeswax, beta a homogeneous mixture of sitosterol, wherein the beeswax in the composition forms microcrystals having a beeswax content of from 0.5 to 50% and a beta-sitosterol of from 0.1% to 20%, based on the total weight of the composition.
  • anti-Helicobacter pylori refers to the inability of H. pylori to grow and multiply, slow reproduction of Helicobacter pylori, Helicobacter pylori mutation, Helicobacter pylori death, and/or decreased Helicobacter pylori pathogenicity.
  • the inability of H. pylori to grow and multiply means that the pharmaceutical composition of the present invention is capable of directly killing Helicobacter pylori, and Helicobacter pylori is completely unable to grow and reproduce.
  • the slow reproduction of Helicobacter pylori means that the pharmaceutical composition of the present invention can cause Helicobacter pylori to exhibit a certain degree of reproduction, but the breeding time is short, followed by morphological variation, the mutation is a transitional stage before death, and the bacteria eventually die.
  • the reduced pathogenicity of H. pylori means that the pharmaceutical composition of the present invention is capable of inhibiting the killing effect of H. pylori on cells, i.e., reducing its toxicity.
  • the effect of normal cultured Helicobacter pylori on cells is significant, and the effect of Helicobacter pylori cultured on the cells after addition of the pharmaceutical composition can be divided into different cases: the higher the concentration of the pharmaceutical composition, the stronger the inhibition of the bacteria The smaller the effect on cell growth, the lower the concentration of the pharmaceutical composition, the smaller the inhibition of the bacteria, the greater the effect on cell growth, and the greater the killing effect on the cells.
  • the present invention relates to the use of a pharmaceutical composition for the preparation of a medicament for the treatment/prevention of a disease caused by Helicobacter pylori, wherein the pharmaceutical composition is a pharmaceutical composition suitable for oral administration, comprising a homogeneous mixture of edible oil with beeswax and ⁇ -sitosterol, wherein the beeswax in the composition forms microcrystals, the beeswax content is 0.5-50%, and the ⁇ -sitosterol is 0.1% by weight of the total composition. -20%.
  • diseases caused by Helicobacter pylori include: gastritis caused by Helicobacter pylori infection, gastric ulcer, duodenal ulcer, gastric cancer, gastric non-Hodgkin's lymphoma, and gastric mucosa-associated lymphoid tissue lymphoma.
  • the disease caused by Helicobacter pylori is a disease caused in a mammal, which is preferably a human.
  • the beta-sitosterol content of the pharmaceutical composition is between 0.5 and 20% by weight.
  • the beta-sitosterol content of the pharmaceutical composition is between 1-10% by weight.
  • the beeswax content of the pharmaceutical composition is between 3 and 30% by weight.
  • the amount of beeswax in the pharmaceutical composition is between 5 and 20% by weight.
  • the amount of beeswax in the pharmaceutical composition is between 6 and 10% by weight.
  • the edible oils in the pharmaceutical composition are corn oil, malt oil, soybean oil, rice bran oil, rapeseed oil, sesame oil, and fish oil.
  • the pharmaceutical composition further comprises propolis in an amount of from 0.1 to 30% by weight.
  • the pharmaceutical composition contains water in an amount of 1% or less by weight.
  • the oral administration is selected from the group consisting of a tablet, a pill, a capsule, an emulsion, a gel, a syrup, or a suspension.
  • the pharmaceutical composition further comprises a xanthine or xanthine extract having a content of 2-5% of xanthine or xanthine of 0.1 to 0.5%, based on the total weight of the composition.
  • the Astragalus membranaceus extract is an extract of Astragalus water, an organic solvent such as an oil and ethanol, or an extract of a combination of water and an organic solvent. More preferably, the extract is an extract of 1 to 50% by weight of xanthine in an edible oil, preferably sesame oil. It is best to use Astragalus root, which is one or more of the genus Astragalus, Astragalus membranaceus, Astragalus membranaceus, Astragalus membranaceus, Lijiang jaundice, and Astragalus membranaceus.
  • the pharmaceutical composition further comprises a cork or cork extract, 2-5% cork or a cork extract containing 0.1-1% paisolide, based on the total weight of the composition.
  • the cork extract is an extract of cork water, an organic solvent such as an oil and ethanol, or an extract of a combination of water and an organic solvent. More preferably, the extract is an extract of 1 to 50% by weight of Cork in edible oil, preferably sesame oil. It is advisable to use cork bark, and one or more of yellow saplings, yellow-skinned, yellow-skinned, yellow-skinned, and yellow-skinned trees.
  • the pharmaceutical composition further comprises, based on the total weight of the composition, 2-5% of Coptidis Rhizoma, or Coptidis Rhizoma extract containing 0.1-1% berberine.
  • the extract of Coptidis Rhizoma is an extract of Coptidis water, an organic solvent such as an oil and ethanol, or an extract of a combination of water and an organic solvent.
  • the composition is an extract of 1 to 50% by weight of berberine in an edible oil, preferably sesame oil. It is best to use the roots of Coptis chinensis, and the plants are selected from one or more of the genus Trichosanthes, the Emei, or the Ranunculaceae.
  • the pharmaceutical composition further comprises 2-5% xanthine or xanthine in an amount of 0.1-0.5% of xanthine extract, 2-5% cork or 0.1 in terms of total weight of the composition.
  • 2-5% Phellodendron extract of Cork Lactone 2-5% of Coptidis Rhizoma or Coptidis Rhizoma extract containing 0.1-1% berberine, 2-10% poppy shell or poppy shell extract containing 0.1-1% papaverine And 2-10% earthworm or earthworm extract containing amino acids.
  • the poppy shell extract is water of a poppy shell, an extract of an organic solvent such as oil and ethanol, or an extract of a combination of water and an organic solvent.
  • the extract is an extract of 1 to 50% by weight of the poppy shell in an edible oil, preferably sesame oil.
  • the extract of the earthworm is an extract of earthworm, an organic solvent such as an oil and ethanol, or an extract of a combination of water and an organic solvent. More preferably, the composition is an extract of 1-50% by weight of Dijon in edible oil.
  • the extraction method of the extracts of Astragalus, Phellodendron, Rhizoma Coptidis, Poppy, and Dijon can be obtained by the method described in Chinese Patent ZL 93100276.1 or Chinese Patent ZL 02105541.6.
  • the pharmaceutical composition comprises, based on the total weight of the composition, 7% beeswax, 1% sterol, 0.5% phellodone, 0.3% xanthine, and 0.5% by weight berberine.
  • the beeswax has microcrystals having a length of from 0.1 to 100 microns.
  • At least two microcrystals of beeswax in the pharmaceutical composition polymerize into a microcrystalline composite.
  • the microcrystals of the beeswax are sufficiently uniformly dispersed in the edible oil.
  • the clinical application value of the pharmaceutical composition of the present invention is that the pharmaceutical composition of the present invention strongly inhibits the strong antibacterial effect of the growth of Helicobacter pylori on Helicobacter pylori, and indicates the direction for future research and development.
  • the results of the present invention demonstrate that the pharmaceutical combination of the present invention is an excellent "antibiotic" against Helicobacter pylori, and can be used for treating gastritis, gastric ulcer, duodenal ulcer, gastric cancer, and gastric mucosa-associated lymphoid tissue lymphoma. disease.
  • Figure 1A In Example 2, HP cultured in Columbia medium was grown normally and the morphology was normal (DIC, x 1000).
  • Fig. 1B In Example 2, HP was cultured in a Columbia medium containing 20% of the pharmaceutical composition, and cultured for 72 hours, and it was revealed that there was no bacterial growth (DIC, ⁇ 1000) in the medium.
  • Fig. 1C In Example 2, HP was cultured in a Columbia medium containing 5% of the pharmaceutical composition, and cultured for 72 hours, and it was revealed that there was no bacterial growth (DIC, ⁇ 1000) in the medium.
  • Fig. 2A In Example 2, on day 3 after the culture, HP cultured in a Columbia medium containing 1.25% of the pharmaceutical composition was normal in morphology and did not mutate (DIC, ⁇ 1000).
  • Fig. 2B In Example 2, HP cultured in a Columbia medium containing 1.25% of the pharmaceutical composition on the 5th day after the culture had mutated, mainly showing the growth of the cells (DIC, ⁇ 1000).
  • Fig. 2C In Example 2, on day 7 after the culture, HP cultured in a Columbia medium containing 1.25% of the pharmaceutical composition showed significant variation, mainly as the cells became longer. The death of mutant bacteria increased. The background is dead HP (DIC, ⁇ 1000).
  • Fig. 2D In Example 2, on day 9 after the culture, HP cultured in a Columbia medium containing 1.25% of the pharmaceutical composition showed less and less mutant bacteria and markedly died. The background is dead HP (DIC, ⁇ 1000).
  • Figure 3A In Example 3, 3 ml of normal HP suspension on day 4 after co-culture, no OMEC (DIC, x 600) was observed under the microscope.
  • Figure 3B In Example 3, 3 ml of the mutated HP suspension on day 4 after co-culture, OMEC (DIC, x 600) was observed under the microscope.
  • Fig. 4A Co-culture results of HP and OMEC cultured in a Columbia medium to which no pharmaceutical composition was added on day 17 after co-culture in Example 4 (DIC, ⁇ 600).
  • Figure 4B Co-culture results of HP and OMEC cultured in Columbia medium containing 0.3125% of the pharmaceutical composition on day 17 after co-cultivation (DIC, x 600).
  • Fig. 4C Co-culture results of HP and OMEC cultured in Columbia medium containing 1.25% of the pharmaceutical composition in Example 4 on the 17th day after co-culture (DIC, ⁇ 600).
  • Figure 4D In Example 4, the normal control group (DIC, x 600) on the 17th day after co-culture.
  • Fig. 5A In Example 5, on the 46th day after co-cultivation of OMEC and the mutant HP suspension, OMEC was not completely dead, and a typical form of OMEC (DIC, x 600) was still observed.
  • Fig. 5B In Example 5, on day 46 of the normal control, OMEC was still grown normally, and the morphology was typical (DIC, x 600).
  • Figure 6 Photograph of the medium recorded by a stereomicroscope in Example 6, no bacterial growth in Columbia medium (spectroscopic mirror, x 8).
  • Fig. 7A In Example 8, HP cultured in Columbia medium was normal in growth and normal morphological staining (DIC, x 1000).
  • Fig. 7B In Example 8, HP was cultured in a Columbia medium containing 2.5% of the pharmaceutical composition, and cultured for 72 hours, and it was revealed that there was little bacterial growth (DIC, ⁇ 1000) in the medium.
  • Fig. 8A In Example 8, HP cultured in Columbia medium was normal in growth and normal morphological staining (DIC, x 1000).
  • Fig. 8B In Example 8, HP cultured in a Columbia medium containing 1.25% of the pharmaceutical composition was in the early stage of variation, mainly showing the growth of the cells (DIC, ⁇ 1000).
  • Fig. 8C In Example 8, HP cultured in a Columbia medium containing 1.25% of the pharmaceutical composition was in the late stage of variation, mainly in that the cells became thinner and longer, and the dead HP was shown (DIC, ⁇ 1000). .
  • Example 1 Preparation of a pharmaceutical composition
  • a pharmaceutical composition was obtained according to the method disclosed in Example 1 of Chinese Patent ZL 02105541.6.
  • step 1 Put the refined sesame oil and astragalus (100kg: 5kg) into the reaction tank, heat the reaction tank, when the temperature rises to 120 ° C, stop heating, keep warm for 50 minutes, while fully stirring, filtering, removing the dregs , the extraction oil is obtained, that is, the medicated oil I is prepared.
  • Step 2 Press the medicated oil I into another reaction tank, heat it, add the cleaned beeswax when the temperature rises to 85 °C, weigh the medicinal oil I 93kg: 7kg of beeswax, stir well, wait until the temperature rises to 120 °C, stop heating, continuous stirring, heat preservation for 20 minutes, that is, medicated oil II.
  • Step 3 Grinding the medicated oil II with a colloid press having a pitch of 0.6-0.8 mm and an output speed of 15 kg/15 min. Alternatively, it can be homogenized for 15-20 minutes at a speed of 6000–10000 rpm using a homogenizer at 40 ⁇ 2 °C. The homogenate was stirred at 100 rpm, evacuated to 0.09 MP or less, and cooled to 40 ⁇ 2 ° C for 50 minutes. When the temperature is lowered to 20 ° C, when the degree of vacuum reaches 0.6 to 0.8 MP, it is continuously maintained for 20 minutes, and the pharmaceutical composition is completed.
  • Example 2 The pharmaceutical composition of the present invention inhibits Helicobacter pylori and mutates Helicobacter pylori
  • Ultrapure water system (Milli-Q type, Millipore, USA); two-stage reverse osmosis purified water system (Beijing Yingnuo Green Technology Co., Ltd.); electronic balance (AUW220D type, Japan Shimadzu); electronic balance (SCOUT SL SPN402F type, Ohaus Authorized, Mettler-Toledo Changzhou Weighing Equipment System Co., Ltd.); electronic balance (AB135-S type, Mettler-Toledo, Switzerland); electronic balance (ES-1000HA type, Changsha Xiangping Technology Development Co., Ltd.); floor-type high-speed refrigerated centrifuge Machine (J20-XP type, American Beckman-Coulter); desktop high speed refrigerated centrifuge (1-15K type, Sigma, Germany); desktop high speed centrifuge (type 1-14, Sigma, Germany); ultra low temperature refrigerator (Forma925 type, American Thermo) ; three gas incubator (CB150 type, Germany Binder); hybrid box (Maxi14 type, American The
  • Colony The colony on the plate is needle-like, the glass is translucent and moist, with a diameter of 1 to 2 mm. When the inoculum is large, the colonies are fused on the surface of the plate to form a translucent lawn.
  • Morphology Take a clean glass slide, drop 1 drop of normal saline in the center of the slide, use a sterile ring to scrape the appropriate amount of bacteria, place it in physiological saline and apply it to a thin membrane, dry naturally or alcohol light. Drying, Gram staining, steps: crystal violet solution for 1 min, water washing, iodine solution for 1 min, water washing, 95% ethanol for 30 s, water washing, blush solution for 1 min, washing with water, drying, observation under a microscope, under the microscope for gram Negative staining, showing a red-colored, spiral, curved or s-shaped, rod-shaped bacteria of varying lengths.
  • Oxidase reaction reagent preparation: prepare 1% TMPD solution, weigh 0.02606g, dissolve in 2.61ml of sterile ultra-pure water, dissolve and store at 4°C in the dark, and set aside. At the time of identification, prepare a slide, take a filter paper and fix it on the slide. Scrape a ring of suspected bacteria onto the filter paper, and quickly add 1 drop of the 1% TMPD solution prepared above. The positive person will soon appear dark blue in the bacteria. Black reaction.
  • Catalytic reaction Prepare a clean concave slide, scrape a ring of suspected bacteria to the center of the concave surface, and quickly add 1 drop of 3% H 2 O 2 . The positive person sees rapid and continuous generation of oxygen bubbles, one after another.
  • Urease reaction scrape 1 ring of suspected bacteria onto the HP test strip and apply a little. The positive coated area immediately turns into a bright red.
  • Preparation of cryopreservation solution Prepare a clean 150ml conical flask, accurately weigh the brain heart extract (BHI) 1.85g into a conical flask, add 50ml of ultrapure water, and pass the induction cooker to dissolve BHI in boiling water.
  • BHI brain heart extract
  • the cotton thread is tightly fastened, autoclaved at 121 ° C for 15 minutes, cooled to room temperature, added with 5.6 ml of FBS, mixed, and dispensed in a 15 ml centrifuge tube, 5 ml per tube, and stored at -20 ° C for later use.
  • Cryopreserved bacteria Add 0.5ml of frozen solution to the cryotube, scrape a large amount of bacteria in the logarithmic growth phase with the bacteria ring, and grind the bacteria against the tube wall in the frozen solution, so that the bacteria are not allowed.
  • bacteria blocks which are tightly capped, marked, placed in a foam box that has been equilibrated at room temperature, and placed in a -70 ° C ultra-low temperature freezer.
  • Revitalizing bacteria Remove the bacteria from the -70 ° C ultra-low temperature freezer, quickly melt in a 37 ° C water bath, clean the surface of the disinfection tube, mix the bacterial solution, take 30 ⁇ l of bacterial solution and add to the center of the Columbia medium plate, with a triangular glass coating rod The film was coated and cultured in a three-gas incubator at 37 ° C, 10% CO 2 , 5% O 2 , 85% N 2 , 98% relative humidity.
  • HP was cultured in a special medium for culturing HP, and the bacteria grew normally, and the morphology, staining and biochemical reactions were normal.
  • Columbia medium containing different concentrations of the drug composition
  • the bacteria could not grow at all in the medium.
  • a concentration of the pharmaceutical composition of at least 5% (w/v, i.e., 5 g of the pharmaceutical composition added to 100 ml of Columbia medium), HP still failed to grow.
  • HP cultured in Columbia medium was normal in growth and normal morphology staining (see Fig. 1A).
  • HP was cultured in a Columbia medium containing 20% (w/v) of the pharmaceutical composition, and cultured for 72 hours, and it was revealed that there was no bacterial growth in the medium (see Fig. 1B).
  • HP was cultured in a Columbia medium containing 10% (w/v) of the pharmaceutical composition, and cultured for 72 hours, and it was revealed that there was no bacterial growth in the medium.
  • HP was cultured in a Columbia medium containing 5% (w/v) of the pharmaceutical composition, and cultured for 72 hours, and it was revealed that there was no bacterial growth in the medium (see Fig. 1C).
  • HP cultured in a Columbia medium containing 1.25% (w/v) of the pharmaceutical composition was normal in morphology and did not mutate (see Fig. 2A).
  • HP cultured in a Columbia medium containing 1.25% (w/v) of the pharmaceutical composition had mutated, mainly as the cells became elongated (see Fig. 2B).
  • HP cultured in a Columbia medium containing 1.25% (w/v) of the pharmaceutical composition had undergone significant mutation, and the cells became long and filamentous, and the background was dead HP.
  • Example 3 Effect of normal and variant H. pylori on oral mucosal epithelial cells (OMEC)
  • each total amount is equally divided into 10 equal portions of the dispensing liquid, and the dispensing liquid is set to 4 ml, which is easy to store, easy to handle, and easy to handle.
  • the specific steps are as follows: Take four sterile 1.5ml Eppendorf tubes, wrapped in aluminum foil, mark well, and weigh vancomycin 10mg, soluble amphotericin B 10mg, polymyxin B 0.42mg, and an electronic balance. Oxybenzidine 5mg.
  • the other three water-soluble antibiotics weighed were placed in an Eppendorf tube, and the trimethoprim was treated as follows: placed in a sterile large glass test tube, and 10 ml of sterile ultrapure water was added to completely rinse the drug. Down to the bottom of the tube, add 20 ⁇ l of DL-lactic acid, clamp with a test tube holder, cover the tampon, and heat the alcohol lamp to boiling for 10 minutes, then cool to room temperature. Add 1 ml of sterile ultrapure water to each of the other three antibiotic tubes, cover, shake, and dissolve. Prepare a 50ml centrifuge tube, transfer the other three antibiotics separately, and rinse the Eppendorf tube with sterile ultrapure water for 1 or 2 times.
  • the cooled trimethoprim solution is also transferred into it, and the ultrapure water is washed and recovered.
  • Residue finally add sterile ultrapure water to 40ml, filter this 40ml mixed antibiotic solution into another 50ml sterile centrifuge tube with a needle filter, and dispense into 10 sterile 15ml centrifuge tubes, 4ml per tube, Sealed, marked, and stored at 20 °C.
  • 100ml of medium is prepared, only 96ml of ultrapure water should be added when dissolving the medium, and 1 tube (ie 4ml) should be added before the plate is mixed with antibiotic solution.
  • CBAB Columbia Blood Agar Foundation
  • the above cell suspension was equally divided into each well of a 6-well plate, and an appropriate amount of 10% FBS DMEM cell culture solution was added to each well, and the total amount of each well was 5 ml.
  • HP was cultured 3 days and 5 days in advance to obtain normal HP and variant HP, respectively.
  • Normal HP was cultured in a Columbia medium without a pharmaceutical composition, and on the 3rd day after the culture, obvious lawn was formed, and half of the total area of the lawn was scraped with cells, and transferred to 4 ml of 10% FBS DMEM.
  • the B1 well does not aspirate the culture supernatant, and 1 ml of normal HP suspension is added;
  • A1 well was used to discard 1 ml of the culture supernatant, and 3 ml of the variant HP suspension was added;
  • the B2 well does not aspirate the culture supernatant, and 1 ml of the variant HP suspension is added;
  • the amount of culture fluid was filled in each well, and the A3 and B3 wells were normal blank control wells;
  • the plates were incubated continuously at 37 ° C in a 5% CO 2 incubator, and cell growth, cell morphology and structure were recorded once or twice a day;
  • the cells were observed with a Nikon TE2000U inverted microscope, and images were recorded with a Nikon DMX1200.
  • the image resolution was adjusted as needed, the observation time was shortened as much as possible, and the experimental results were properly preserved.
  • HP cultured normally in the Columbia medium without the addition of the pharmaceutical composition was typical on the 3rd day after the culture, and the Gram stain showed that the bacterial morphology was normal and not mutated, and the obvious lawn was formed at this time.
  • HP cultured in a Columbia medium supplemented with a 0.3125% (w/v) pharmaceutical composition showed that the bacterial morphology had undergone significant variation.
  • the effect of normal and mutant HP cultures on the growth of OMEC was observed by OMEC and HP co-culture, specifically:
  • Example 4 Effect of the pharmaceutical composition of the present invention on the virulence of Helicobacter pylori under in vitro culture conditions
  • the above cell suspension was equally divided into each well of a 6-well plate, and an appropriate amount of 10% FBS DMEM cell culture solution was added to each well, and the total amount of each well was 5 ml.
  • HP was cultured 3 days in advance, the medium was, respectively, the Columbia medium without the added pharmaceutical composition, the second was the Columbia medium containing 0.3125% (w/v) of the pharmaceutical composition, and the third It is a Colombian medium containing 1.25% (w/v) of the pharmaceutical composition.
  • the medium was, respectively, the Columbia medium without the added pharmaceutical composition
  • the second was the Columbia medium containing 0.3125% (w/v) of the pharmaceutical composition
  • the third It is a Colombian medium containing 1.25% (w/v) of the pharmaceutical composition.
  • On the third day after the culture obvious lawns have been formed, and the total area of all the lawns or the total area of the lawn is scraped with cells.
  • Half transferred to 4 ml of 10% FBS DMEM and gently ground to a uniform HP suspension with a dripper. Thus three different HP suspensions were obtained, ready for use.
  • HP culture suspension was added to different culture wells of the culture plate.
  • the specific arrangement was:
  • the plates were incubated continuously at 37 ° C in a 5% CO 2 incubator, and cell growth, cell morphology and structure were recorded once or twice a day;
  • the cells were observed with a Nikon TE2000U inverted microscope, and images were recorded with a Nikon DMX1200.
  • the image resolution was adjusted as needed, the observation time was shortened as much as possible, and the experimental results were properly preserved.
  • HP was cultured in three different pharmaceutical composition contents in Columbia medium, and on the third day after the culture, it was found that the first HP cultured in the Columbia medium without the added pharmaceutical composition was negative for Gram stain, indicating that the bacteria were many.
  • OMEC morphology is normal; the second HP cultured in Columbia medium containing 0.3125% (w/v) pharmaceutical composition, Gram stain negative, many bacteria, OMEC morphology is normal; the third contains 1.25% (w /v) Pharmaceutical composition of Columbia medium cultured HP, Gram stain negative, OMEC morphology is normal, but few bacteria. The first and second colonies are typical at this time, and obvious lawns have formed.
  • HP cultures cultured in medium with different concentrations of the drug composition on the growth of OMEC was observed.
  • the first HP culture was found to have a significant effect on OMEC, indicating that HP has a strong virulence; the second HP culture also has a significant effect on OMEC, and OMEC death is obvious, indicating that the proliferation of HP is due to the low concentration of the pharmaceutical composition.
  • the effect of virulence was not obvious; the third HP culture had no obvious effect on OMEC, indicating that the pharmaceutical composition played a role and significantly inhibited the reproduction and virulence of HP.
  • the above cell suspension was equally divided into each well of a 6-well plate, and an appropriate amount of 10% FBS DMEM cell culture solution was added to each well, and the total amount of each well was 5 ml.
  • HP was cultured 6 days in advance with Columbia medium containing 0.3125% (w/v) of the pharmaceutical composition to obtain mutant HP. At this time, obvious lawn was formed, and half of the total area of the lawn was scraped with cells, and 4 ml of 10% was transferred. In FBS DMEM medium, gently grind into a uniform 4 ml variant HP suspension with a dripper and set aside.
  • the plates were incubated continuously at 37 ° C in a 5% CO 2 incubator, and cell growth, cell morphology and structure were recorded once or twice a day;
  • the cells were observed with a Nikon TE2000U inverted microscope, and images were recorded with a Nikon DMX1200.
  • the image resolution was adjusted as needed, the observation time was shortened as much as possible, and the experimental results were properly preserved.
  • HP cultured in Columbia medium supplemented with 0.3125% (w/v) of the pharmaceutical composition on the 6th day after the culture, Gram stain showed that the morphology had undergone significant variation, and was added to the culture well of the cultured OMEC to observe the OMEC. Growth and the effect of mutant HP cultures on OMEC growth.
  • OMEC On the first day after co-cultivation of OMEC and the mutant HP suspension, OMEC was not completely dead, and a typical form of OMEC was observed. On the 20th day after the co-culture of OMEC and the mutant HP suspension, OMEC was not completely dead, and the morphologically intact OMEC was observed. On the 24th day after co-culture of OMEC and mutant HP suspension, OMEC was not completely dead, and more typical OMECs were observed. On day 46 after co-cultivation of OMEC with the mutant HP suspension, OMEC was not completely dead, and a typical morphology of OMEC was still visible (see Figure 5A). Normal control On day 46, OMEC still grew normally with a typical morphology (see Figure 5B).
  • Example 6 Effect of DMEM medium on the growth of Helicobacter pylori
  • Embodiment 3 Same as Embodiment 3. Also included are microplate readers (Multiskan Ascent, Finnish Labsystems), stereo microscopes (SMZ1000 type, Nikon, Japan), and ELISA plates (Costar, USA).
  • HP was cultured 3 days earlier with Columbia medium. On the day of the experiment, HP was harvested by cell scraping, and all the lawns of the total area were scraped with cells, transferred to 4 ml of 10% FBS DMEM, and gently ground to a uniform HP suspension with a dripper. After vortexing, add 4 ml of 10% FCS DMEM. Take a detachable microplate, add three kinds of analytes, mix and add 200ul each, respectively:
  • the OD 620 value was measured using a Labsystems Multiskan Ascent microplate reader at 620 nm wavelength.
  • HP suspensions were mixed and divided equally into the A1 and A2 wells of a 6-well plate. Each well was cultured in a cell culture incubator of about 3 ml, 5% O 2 , 37 ° C.
  • HP should be proliferated in co-culture with cells, but in the experiment, HP was directly suspended in cell culture medium DMEM, cultured in a 5% O 2 , 37 ° C cell culture incubator, and the OD 620 of HP suspension was found. The value did not increase with the prolongation of the culture time, but decreased significantly. A total of 3 times were detected, one at a time lower than the other one.
  • Example 7 Preparation of a pharmaceutical composition containing xanthine and Phellodendron and its action against Helicobacter pylori
  • a pharmaceutical composition was prepared according to the method of Example 1, in which the refined sesame oil and xanthine and cork (100 kg: 5 kg: 4 kg) were placed in a reaction tank in the same manner as in Example 1.
  • the pharmaceutical composition obtained in the present example was used in the detection method in Example 2, and the following results were obtained:
  • HP was cultured in a culture medium of culture medium for culturing HP, and the bacteria grew normally, and the morphology, staining and biochemical reaction were normal; while HP was cultured in a Columbia medium containing different concentrations of the pharmaceutical composition of the present example, and the bacteria were in the medium. It does not grow at all, and HP still cannot grow when the concentration of the pharmaceutical composition is at least 5%.
  • HP cultured normally in the Columbia medium without the addition of the pharmaceutical composition was typical on the 3rd day after the culture, and the Gram stain showed that the bacterial morphology was normal and not mutated, and the obvious lawn was formed at this time.
  • HP stained with Columbia medium supplemented with 0.3125% of the pharmaceutical composition of the present example showed that the bacterial morphology had undergone significant variation.
  • the effect of normal and mutant HP cultures on OMEC growth was observed by co-cultivation of OMEC and HP. On day 4, day 15, and day 41 after co-culture, 3 ml and 1 ml of normal HP suspension, no OMEC was seen under the microscope, indicating that OMEC died, and HP culture had killed OMEC.
  • Example 8 Preparation of a pharmaceutical composition containing astragalus and berberine and its anti-Helicobacter pylori effect
  • a pharmaceutical composition was prepared according to the method of Example 1, in which the refined sesame oil and xanthine, and berberine (100 kg: 5 kg: 4 kg) were placed in a reaction tank in the same manner as in Example 1.
  • Example 7 The pharmaceutical composition obtained in the present Example was used in the detection methods in Example 2 and Example 3, and a test result similar to that obtained in Example 7 was obtained, which was capable of inhibiting H. pylori.
  • Example 9 Preparation of a pharmaceutical composition containing astragalus, cork and berberine and its anti-Helicobacter pylori effect
  • a pharmaceutical composition was prepared according to the method of Example 1, in which the refined sesame oil and xanthine, and berberine (100 kg: 5 kg: 5 kg: 5 kg) were placed in a reaction tank in the same manner as in Example 1.
  • the pharmaceutical composition obtained in the present Example was used in the detection methods in Example 2 and Example 3, and the detection results similar to those obtained in Example 7 were obtained, and the present examples containing different concentrations were used.
  • the pharmaceutical composition of the Columbia medium is cultured with HP, the bacteria are completely unable to grow in the medium, and HP is still unable to grow at a concentration of the pharmaceutical composition of at least 5%.
  • Example 10 Preparation and antibiotics of pharmaceutical compositions containing astragalus, berberine, cork, poppy shell and earthworm The role of Helicobacter pylori
  • a pharmaceutical composition was prepared according to the method of Example 1, wherein in step 1, refined sesame oil and xanthine, berberine, cork, poppy shell and earthworm (100 kg: 5 kg: 4 kg: 4 kg: 5 kg: 5 kg) were placed in a reaction tank. The rest are the same as in the first embodiment.
  • HP was cultured in a special medium for culturing HP, and the bacteria grew normally, and the morphology, staining and biochemical reaction were normal.
  • HP was cultured in a Columbia medium containing different concentrations of the pharmaceutical composition of the present example, and two high and medium were found. The concentration is completely unable to grow, and only a very small number of bacteria grow at low concentrations.
  • the pharmaceutical composition of the present example is shown to have a strong inhibitory effect on HP.
  • HP cultured in Columbia medium was normal in growth and normal morphological staining (see Fig. 7A).
  • HP was cultured in a Columbia medium containing 10% of the pharmaceutical composition of the present example, and cultured for 72 hours, and it was revealed that there was no bacterial growth in the medium.
  • the HP was cultured in a Columbia medium containing 5% of the pharmaceutical composition of the present example, and cultured for 72 hours, and it was revealed that there was no bacterial growth in the medium.
  • the HP was cultured in a Columbia medium containing 2.5% of the pharmaceutical composition of the present example, and cultured for 72 hours, and the results showed that there was little bacterial growth in the medium (see Fig. 7B).
  • HP was cultured in a culture medium of culture medium for culturing HP, and the bacteria grew normally, and the morphology, staining and biochemical reaction were normal; while HP was cultured in a Columbia medium containing 1.25% of the pharmaceutical composition of the present example, and the morphology of the bacteria in the medium was observed. Significant variation occurred. Variations range from general variation to significant variation, from atypical to typical, never obvious to obvious.
  • HP cultured in Columbia medium was normal in growth and normal morphological staining (see Fig. 8A).
  • HP cultured in a Columbia medium containing 1.25% of the pharmaceutical composition of the present example was in the early stage of variation, mainly in that the cells became elongated (see Fig. 8B).
  • HP cultured in a Columbine medium containing 1.25% of the pharmaceutical composition of the present example was in the late stage of variation, mainly in that the cells became thinner and longer, and the dead HP was shown (see Fig. 8C).
  • the pharmaceutical composition obtained in the present Example was used in the detection method in Example 3, and the following results were obtained: HP cultured in a Columbia medium supplemented with a pharmaceutical composition of the present example as low as 0.3125%, co-culture On days 4, 15, and 41, 3 ml and 1 ml of normal HP suspension, no OMEC was seen under the microscope, indicating that OMEC died and the HP culture had killed OMEC.
  • Example 11 Effect of pharmaceutical composition against Helicobacter pylori in humans
  • Twenty-two patients with upper gastrointestinal ulcer and inflammation were diagnosed by gastroscopy and 12 males. 10 females, aged 29 to 71 years old. Of the 22 cases, 15 were single lesions, and the other 7 were double or multiple lesions. That is, gastric ulcer and duodenal ulcer in 5 cases (where), chronic gastritis in 16 cases (where), gastric mucosal erosion in 4 cases (where), esophageal ulcer inflammation in 1 case (at).
  • Example 1 According to the principle of voluntary patient, the pharmaceutical composition prepared in Example 1 (Group A) was separately administered, a total of 15 cases; the pharmaceutical composition prepared in Example 1 was added to the conventional medical treatment group (B). Group), a total of 7 cases, observed the clinical symptoms and signs changes in the two groups of patients, and the results of gastroscopy after one month, compare the treatment effect.
  • Group A esophagitis, gastric ulcer, inflammation: The pharmaceutical composition prepared in Example 1, 2.5 g each time, 4 times/day, half an hour before 3 meals, and before going to sleep, patients with esophagitis It should be chewed and swallowed; duodenal ulcer: The pharmaceutical composition prepared in Example 1 was taken 4.0g, 4 times/day, 3 meals before and before going to sleep, and the total course of treatment was one month.
  • Group B The pharmaceutical composition prepared in Example 1 was administered in the same manner as in the experimental group A.
  • General medical treatment includes: metoclopramide 20mg, 3 times / day, ranitidine 150mg, 2 times / day, orally.
  • the therapeutic drugs of the group A are the pharmaceutical compositions prepared in the first embodiment alone, and the therapeutic drugs of the group B are in addition to the pharmaceutical compositions prepared in the first embodiment, and the western medicine is accepted. , ranitidine, amoxicillin, etc.) treatment.
  • the pharmaceutical composition of the present invention can be substituted for the therapeutic effects of the above-mentioned western medicines. These effects include alleviating clinical symptoms, protecting the gastrointestinal mucosa, promoting upper gastrointestinal ulcers, healing inflammatory diseases, and controlling Helicobacter pylori infection. According to the results of this study.
  • the pharmaceutical composition of the present invention can quickly relieve symptoms such as ulcers and pain of an inflammatory patient by preventing the colonization of HP or changing the pathogenicity of HP, from re-injury of the injury factor and stimulation of the nerve endings.
  • the local inflammation is obviously improved, and at the same time, the physiological environment and living substance for regenerative repair are provided for the lesion. It promotes the physiological regeneration of ulcers and improves the quality of repair healing.

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Abstract

一种药物组合物在制备用于抗幽门螺杆菌的药物中的用途，其中所述的药物组合物是一种适用于口服的药物组合物，包括食用油与蜂蜡、β-谷甾醇的均质混合物，其中，该组合物中蜂蜡形成微晶体，按该组合物总重量计算，蜂蜡的含量为0.5-50%，β-谷甾醇为0.1-20%。该药物组合物可用于抑制或杀灭幽门螺杆菌，以及用于治疗或预防由幽门螺杆菌引起的疾病。

Description

药物组合物在制备用于抗幽门螺杆菌的药物中的用途 技术领域
本发明涉及药物组合物在制备用于抗幽门螺杆菌的药物中的用途。本发明还涉及药物组合物在制备用于治疗/预防由幽门螺杆菌引起的疾病的药物中的用途。
背景技术
中国专利ZL 02105541.6披露了一种适用于口服的药物组合物，该药物组合物包括食用油与蜂蜡、β-谷甾醇的均质混合物，其中，该组合物中蜂蜡形成微晶体，按该组合物总重量计算，蜂蜡的含量为0.5-50％，β-谷甾醇至少为0.1％。此外，该组合物还可以含有其它药物成分，并用于将其它有效成分传输到胃肠道，用以治疗各种疾病。
此外，这一药物组合物主要用于保护粘膜组织免受刺激物造成损伤，以及促进受损的或功能不全的胃肠道粘膜组织的修复和再生，尤其用于治疗胃肠功能失调，如胃炎、消化性溃疡、回流性食管炎、消化不良和胃癌，以及用于重建粘膜组织的生理性结构和功能。
在本申请中，“药物组合物”、“本发明所述的药物组合物”或者“本发明的药物组合物”指一种药物组合物，该药物组合物包括食用油与蜂蜡、β-谷甾醇的均质混合物，其中，该组合物中蜂蜡形成微晶体，按该组合物总重量计算，蜂蜡的含量为0.5-50％，β-谷甾醇为0.1％-20％。
幽门螺杆菌(Helicobacter Pylori，HP)是一种螺旋状或S状、微需氧的革兰氏阴性细菌，专一定居于人胃，构成人类急慢性胃炎、消化性溃疡(胃溃疡和十二指肠溃疡)、胃癌、胃非何杰金氏淋巴瘤和胃粘膜相关淋巴样组织(MALT)淋巴瘤的主要病因，HP在人群中的感染率非常高，达40～90％，通常在儿童期感染，而且一经感染，将终生携带，携带者是HP的传染源。
人们通常是在幼年时就受到感染，5岁以下达到50％。这种细菌感染首先引起慢性胃炎，并导致胃溃疡和胃萎缩，严重者则发展为胃癌。据统计，初次感染幽门螺杆菌年龄较早的人群萎缩性胃炎及胃癌发生率高，幽门螺杆菌感染与胃癌死亡率的高低呈现平行关系。幽门螺杆菌寄生在胃粘膜组织中，67％～80％的胃溃疡和95％的十二指肠溃疡是由幽门螺杆菌引起的。慢性胃炎和消化道溃疡患者的普遍症状为：食后上腹部饱胀、不适或疼痛，常伴有其他不良症状，如暖气、腹胀、反酸和食欲减退等。有些患者还可出现反复发作性剧烈腹痛、上消化道少量出血等。
由于HP在消化系统有很强的致病性，不断发现有效的药物已成为当务之急。
现有技术中，幽门螺杆菌的药物治疗方案通常使用抗菌药物，例如奥美拉唑 阿莫西林、甲硝唑片等。目前在世界范围内，临床上针对幽门螺杆菌使用的抗生素价格昂贵、容易产生耐药性、毒副作用大，整体效果不理想。
发明内容
本发明要解决的技术问题是采用上述的公知药物组合物抑制或杀灭幽门螺杆菌，进而治疗由幽门螺杆菌引起的疾病。
因此，本发明一方面涉及药物组合物在制备用于抗幽门螺杆菌的药物中的用途，其中所述的药物组合物是一种适用于口服的药物组合物，其包括食用油与蜂蜡、β-谷甾醇的均质混合物，其中，该组合物中蜂蜡形成微晶体，按该组合物总重量计算，蜂蜡的含量为0.5-50％，β-谷甾醇为0.1％-20％。
具体地，抗幽门螺杆菌是指幽门螺杆菌不能生长繁殖、幽门螺杆菌繁殖缓慢、幽门螺杆菌变异、幽门螺杆菌死亡和/或幽门螺杆菌致病性降低。
在具体的实施方案中，幽门螺杆菌不能生长繁殖是指，本发明的药物组合物能够直接杀灭幽门螺杆菌，幽门螺杆菌完全不能生长繁殖。幽门螺杆菌繁殖缓慢是指，本发明的药物组合物能够使幽门螺杆菌表现一定程度的繁殖，但繁殖时间短，随后形态变异，变异是死亡前的过渡阶段，细菌最终死亡。
在具体的实施方案中，幽门螺杆菌致病性降低是指，本发明的药物组合物能够抑制幽门螺杆菌对细胞的杀伤作用，即降低其毒性。
正常培养的幽门螺杆菌对细胞的作用是显著杀伤，而加入药物组合物之后再进行培养的幽门螺杆菌对细胞的作用可分为不同情况：药物组合物浓度越高，细菌受到的抑制越强，对细胞生长的影响越小，药物组合物浓度越低，细菌受到的抑制越小，对细胞生长的影响越大，细胞受到的杀伤作用就越大。
另一方面，本发明涉及药物组合物在制备用于治疗/预防由幽门螺杆菌引起的疾病的药物中的用途，其中所述的药物组合物是一种适用于口服的药物组合物，其包括食用油与蜂蜡、β-谷甾醇的均质混合物，其中，该组合物中蜂蜡形成微晶体，按该组合物总重量计算，蜂蜡的含量为0.5-50％，β-谷甾醇为0.1％-20％。
具体地，其中由幽门螺杆菌引起的疾病包括：幽门螺杆菌感染引起的胃炎、胃溃疡、十二指肠溃疡、胃癌、胃非何杰金氏淋巴瘤和胃粘膜相关淋巴样组织淋瘤。
具体地，由幽门螺杆菌引起的疾病是在哺乳动物中引起的疾病，所述哺乳动物优选是人。
在具体的实施方案中，所述药物组合物中β-谷甾醇的含量按重量计算，介于0.5-20％之间。
在具体的实施方案中，所述药物组合物中β-谷甾醇的含量按重量计算，介于1-10％之间。
在具体的实施方案中，所述药物组合物中蜂蜡的含量按重量计算，介于3-30％ 之间。
在具体的实施方案中，所述药物组合物中蜂蜡的含量按重量计算，介于5-20％之间。
在具体的实施方案中，所述药物组合物中蜂蜡的含量按重量计算，介于6-10％之间。
在具体的实施方案中，所述药物组合物中食用油是玉米油、麦芽油、豆油、米糠油、油菜籽油、芝麻油和鱼油。
在具体的实施方案中，所述药物组合物中还含有蜂胶，含量按重量计算，介于0.1-30％。
在具体的实施方案中，所述药物组合物中含有水，其含量按重量计算，为小于等于1％。
在具体的实施方案中，所述的口服选自下列剂型，包括：片剂、丸剂、胶囊、乳状液、凝胶体、糖浆、或悬浮液。
在具体的实施方案中，所述药物组合物进一步包括黄芩或黄芩提取物，按组合物总重量计算，2-5％的黄芩或黄芩甙的含量为0.1-0.5％的黄芩提取物。
所述黄芩提取物是黄芩的水、有机溶剂如油和乙醇的提取物，或水和有机溶剂两者组合的提取物。更优选的是，该提取物是1-50重量％的黄芩在食用油，优选是芝麻油中的提取物。最好是用黄芩根，这种植物选用唇形科的粘毛黄芩、滇黄芩、甘肃黄芩、薄叶黄芩、丽江黄芩、川黄芩中的一种或多种均可。
在具体的实施方案中，所述药物组合物进一步包括黄柏或黄柏提取物，按组合物总重量计算，2-5％黄柏的或含0.1-1％黄柏内酯的黄柏提取物。
所述黄柏提取物是黄柏的水、有机溶剂如油和乙醇的提取物，或水和有机溶剂两者组合的提取物。更优选的是，该提取物是1-50重量％的黄柏在食用油中，优选是芝麻油中的提取物。采用黄柏树皮为宜，黄柏选用黄皮树、秃叶黄皮树、峨嵋黄皮树、云南黄皮树、镰刀叶黄皮树中的一种或多种。
在具体的实施方案中，所述药物组合物进一步包括，按组合物总重量计算，2-5％的黄连，或者含0.1-1％小檗碱的黄连提取物。
所述黄连提取物是黄连的水、有机溶剂如油和乙醇的提取物，或水和有机溶剂两者组合的提取物。优选的是，该组合物是1-50重量％的黄连在食用油，优选为芝麻油中的提取物。最好选用黄连根，植物选用毛茛科三角叶黄连、峨眉黄连或者毛茛科植物云连中的一种或多种。
在具体的实施方案中，按组合物总重量计算，所述药物组合物进一步包括2-5％的黄芩或黄芩甙的含量为0.1-0.5％的黄芩提取物、2-5％黄柏或含0.1-1％黄柏内酯的黄柏提取物、2-5％的黄连或含0.1-1％小檗碱的黄连提取物、2-10％罂粟壳或含0.1-1％罂粟壳碱的罂粟壳提取物和2-10％地龙或含氨基酸的地龙提取物。
所述罂粟壳提取物是罂粟壳的水、有机溶剂如油和乙醇的提取物，或水和有 机溶剂两者组合的提取物。优选的是，所述的提取物是1-50重量％的罂粟壳在食用油，优选是芝麻油中的提取物。
所述地龙提取物是地龙的水、有机溶剂如油和乙醇的提取物，或水和有机溶剂两者组合的提取物。更优选的是，该组合物是1-50重量％的地龙在食用油中的提取物。
黄芩、黄柏、黄连、罂粟壳及地龙的提取物的提取方法可参见中国专利ZL 93100276.1或中国专利ZL 02105541.6中所描述的方法进行提取获得。
在具体的实施方案中，所述药物组合物按组合物总重量计算，包括7％蜂蜡、1％甾醇、0.5％黄柏内酯、0.3％黄芩甙和0.5重量％小檗碱。
在具体的实施方案中，所述的蜂蜡具有微晶体，其长度是0.1-100微米。
在具体的实施方案中，所述药物组合物中蜂蜡的至少两个微晶体聚合成微晶复合物。
在具体的实施方案中，所述的蜂蜡的微晶体充分地均匀分散在食用油中。
本发明的药物组合物的临床应用价值在于：本发明的药物组合物强烈抑制幽门螺杆菌的生长对幽门螺杆菌的强力抗菌作用，为未来研发指明了方向。本发明的结果说明本发明的药物组合即是极好的针对幽门螺杆菌的“抗生素”，可以用于治疗胃炎、胃溃疡、十二指肠溃疡、胃癌以及胃粘膜相关淋巴样组织淋巴瘤等疾病。
附图说明
图1A：实施例2中，用哥伦比亚培养基培养的HP，生长正常，形态染色正常(DIC，×1000)。
图1B：实施例2中，用含20％药物组合物的哥伦比亚培养基培养HP，培养72h，结果显示培养基中没有细菌生长(DIC，×1000)。
图1C：实施例2中，用含5％药物组合物的哥伦比亚培养基培养HP，培养72h，结果显示培养基中没有细菌生长(DIC，×1000)。
图2A：实施例2中，培养后第3天，用含1.25％药物组合物的哥伦比亚培养基培养的HP，形态正常，未发生变异(DIC，×1000)。
图2B：实施例2中，培养后第5天，用含1.25％药物组合物的哥伦比亚培养基培养的HP，已经发生变异，主要表现为菌体变长(DIC，×1000)。
图2C：实施例2中，培养后第7天，用含1.25％药物组合物的哥伦比亚培养基培养的HP，变异明显，主要表现为菌体变长。变异菌死亡增加。背景为死亡的HP(DIC，×1000)。
图2D：实施例2中，培养后第9天，用含1.25％药物组合物的哥伦比亚培养基培养的HP，变异细菌越来越少，死亡明显。背景为死亡的HP(DIC，×1000)。
图3A：实施例3中，共培养后第4天，3ml正常HP混悬液，镜下未见OMEC (DIC，×600)。
图3B：实施例3中，共培养后第4天，3ml变异HP混悬液，镜下可见OMEC(DIC，×600)。
图4A：实施例4中，共培养后第17天，用未添加药物组合物的哥伦比亚培养基培养的HP与OMEC的共培养结果(DIC，×600)。
图4B：实施例4中，共培养后第17天，用含0.3125％药物组合物的哥伦比亚培养基培养的HP与OMEC的共培养结果(DIC，×600)。
图4C：实施例4中，共培养后第17天，用含1.25％药物组合物的哥伦比亚培养基培养的HP与OMEC的共培养结果(DIC，×600)。
图4D：实施例4中，共培养后第17天，正常对照组(DIC，×600)。
图5A：实施例5中，OMEC与变异HP混悬液共培养后第46天，OMEC未完全死亡，仍可见形态典型的OMEC(DIC，×600)。
图5B：实施例5中，正常对照第46天，OMEC仍正常生长，形态典型(DIC，×600)。
图6：实施例6中，体视显微镜记录的培养基的照片，哥伦比亚培养基中无细菌生长(体视镜，×8)。
图7A：实施例8中，用哥伦比亚培养基培养的HP，生长正常，形态染色正常(DIC，×1000)。
图7B：实施例8中，用含2.5％的药物组合物的哥伦比亚培养基培养HP，培养72h，结果显示培养基中只有极少的细菌生长(DIC，×1000)。
图8A：实施例8中，用哥伦比亚培养基培养的HP，生长正常，形态染色正常(DIC，×1000)。
图8B：实施例8中，用含1.25％的药物组合物的哥伦比亚培养基培养的HP，处于变异早期，主要表现为菌体变长(DIC，×1000)。
图8C：实施例8中，用含1.25％的药物组合物的哥伦比亚培养基培养的HP，处于变异晚期，主要表现为菌体变细变长，图中显示死亡的HP(DIC，×1000)。
具体实施方式
以下结合附图，通过实施例进一步说明本发明，但不作为对本发明的限制。以下提供了本发明实施方案中所使用的具体材料及其来源。但是，应当理解的是，这些仅仅是示例性的，并不意图限制本发明，与如下试剂和仪器的类型、型号、品质、性质或功能相同或相似的材料均可以用于实施本发明。下述实施例中所使用的实验方法如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1：药物组合物的制备
按照中国专利ZL 02105541.6实施例1中所披露的方法，获得药物组合物。
简要地，步骤1：将精制的芝麻油和黄芩(100kg:5kg)放入反应罐，加热反应罐，当温度升到120℃时，停止加热，保温50分钟，同时充分搅拌，过滤，去除药渣，留取提取油液，即制成药油I。
步骤2：将药油I压入另一反应罐，加热，当温度升至85℃时加入清制后的蜂蜡，按比例称取药油I 93kg：蜂蜡7kg，充分搅拌，待温度升到120℃，停止加热，连续搅拌，保温20分钟，即制成药油II。
步骤3：用胶体压榨机将药油II进行研磨，压榨机的齿距为0.6-0.8mm，输出速度为15Kg/15min。或者，也可用均质机在40±2℃条件下，以每分钟6000–10000转速的速度均质15-20分钟。在100rpm条件下搅拌均质物，抽真空至0.09MP以下，降温至40±2℃时，保温50分钟。等温度降到20℃时，真空度达到0.6～0.8MP时，连续保持20分钟，药物组合物即制作完成。
参见中国专利ZL 02105541.6实施例2，上述方法制备的药物组合物的功效成份参见表1：
表1
成份 每100g含量
天然维生素E 15mg～50mg
总黄酮 20mg～60mg
β-谷甾醇 0.20g～1.0g
亚油酸 35g～55g
油酸 25g～45g
实施例2：本发明的药物组合物抑制幽门螺杆菌并使幽门螺杆菌发生变异
1.材料与方法
1.1 仪器、设备、材料及试剂
超纯水系统(Milli-Q型，美国Millipore)；双级反渗透纯化水系统(北京英诺格林科技有限公司)；电子天平(AUW220D型，日本Shimadzu)；电子天平(SCOUT SL SPN402F型，Ohaus授权，Mettler-Toledo常州称重设备系统有限公司)；电子天平(AB135-S型，瑞士Mettler-Toledo)；电子天平(ES-1000HA型，长沙湘平科技发展有限公司)；落地式高速冷冻离心机(J20-XP型，美国Beckman-Coulter)；台式高速冷冻离心机(1-15K型，德国Sigma)；台式高速离心机(1-14型，德国Sigma)；超低温冰箱(Forma925型，美国Thermo)；三气培养箱(CB150型，德国Binder)；杂交箱(Maxi14型，美国Thermo)；颗粒制冰机(SIM-F124型，日本Sanyo)；电子恒温水浴锅(CS501-3C型)、烘干箱(重庆四达实验仪器有限公司)；倒置显微镜(TE2000U型，日本Nikon)；正置显微镜(E800型，日本Nikon)；显微成像系统(DXM 1200型，日本Nikon)；普 通倒置显微镜(XDS-1B型)、普通光学显微镜(BK1201型)(重庆光学仪器厂)；生物洁净工作台(BCN-1360B型)、生物安全柜(BSC-IIA2型)、生化培养箱(HPS-200B型)(北京东联哈尔仪器制造有限公司)；幽门螺杆菌(ATCC43504，上海北思生物科技有限公司)；哥伦比亚血琼脂基础(CBAB，CM0331，英国OXOID公司)；脑心浸出物(BHI，CM1135，英国OXOID公司)；营养琼脂培养基(NAM，北京三药科技开发公司)；载玻片和盖玻片(国药集团北京化学试剂公司)；营养琼脂(NA)、革兰氏染色液(结晶紫、碘液、95％乙醇、番红)、大张滤纸、无菌脱纤维绵羊血、二甲苯、3％过氧化氢、N,N,N,N-四甲基对苯二胺二盐酸盐(TMPD)、甲氧苄氨嘧啶TMP、硫酸多粘菌素B、可溶性两性霉素B、盐酸万古霉素、DL-乳酸、三角形玻璃涂布棒(北京索莱宝科技有限公司)；一分钟快速幽门螺杆菌试纸(化学反应法)(珠海市克迪科技开发有限公司)；一次性培养皿(α plus，青岛金典生化器材有限公司)；各种型号注射器；6孔培养板(美国Costar)；50ml离心管；Eppendorf离心管；微量加样器(1000μl、200μl、20μl、10μl，均为法国Gilson)；大小滴头；培养皿(φ5cm、φ6cm)；0.22μm微孔滤膜；针头滤器；有盖三角烧瓶；有盖小试管；针头；DMEM培养基(GIBCO，美国Invitrogen Corporation)；胎牛血清(FBS，ExCell公司)；注射用青霉素钠；注射用硫酸链霉素；大玻璃试管。
1.2方法
1.2.1 含药物组合物的哥伦比亚培养基的制备
备一洁净250ml锥形瓶，精确称取3.9g哥伦比亚血琼脂基础(CBAB)置于锥形瓶中，加入100ml超纯水，通过电磁炉，使CBAB在沸水中溶解，盖好棉塞线绳扎紧，121℃15min高压灭菌，当高压灭菌器压力归零时立即取出培养基，立即无菌操作加入一定量药物组合物，无菌盖好棉塞，覆上无菌牛皮纸，频繁旋转摇动培养基，使药物组合物融化溶解均匀，进一步冷却至约50℃，无菌加入8ml无菌脱纤维绵羊血，混匀，趁热快速浇板。盖好，冷却，标记，倒放，4℃保存备用。
1.2.2 HP的鉴定
菌落：平板上的菌落呈针尖样，毛玻璃样半透明，湿润，直径1～2mm，接种菌量大时，菌落在平板表面融合成一层半透明的菌苔。
形态：取一张洁净玻片，将1滴生理盐水滴在玻片中央，用取菌环无菌刮取适量细菌，点在生理盐水中并涂成薄薄的菌膜，自然干燥或酒精灯火烘干，进行革兰氏染色，步骤：结晶紫液1min，水洗，碘液1min，水洗，95％乙醇30s，水洗，番红液1min，水洗，干燥，在显微镜下观察，镜下为革兰氏染色阴性，显示为紫红色、螺旋状、弯曲状或S状、长短不一的杆状菌。
生化反应
氧化酶反应：试剂配制：配制1％TMPD溶液，称取0.02606g，溶于2.61ml 无菌超纯水中，溶解后4℃避光保存，备用。鉴定时，备一玻片，取一条滤纸固定在玻片上，刮取1环可疑细菌沾至滤纸上，快速加1滴上述配制的1％TMPD溶液，阳性者在有菌部位很快出现深蓝/黑色反应。
触媒反应：备一洁净凹玻片，刮取1环可疑细菌点至凹面中央，快速加1滴3％H 2O 2,阳性者见到快速的连续不断的氧气泡生成，此起彼伏。
尿素酶反应：刮取1环可疑细菌至HP检测试纸上，稍加涂布，阳性者涂布区立刻变为鲜艳的红色。
1.2.3 HP的保存和复苏
冻存液的配制：备一洁净150ml锥形瓶，精确称取脑心浸出物(BHI)1.85g置于锥形瓶中，加入50ml超纯水，通过电磁炉，使BHI在沸水中溶解，盖好棉塞线绳扎紧，121℃15min高压灭菌，冷却至室温，加入5.6ml FBS，混匀，分装于15ml离心管中，每管5ml，－20℃保存备用。
冻存细菌：将0.5ml冻存液加入冻存管中，用取菌环刮取较多量的处于对数生长期的细菌，在冻存液中将细菌贴着管壁研磨下来，不使细菌有菌块存在，拧紧盖好，标记好，置于已经在室温下平衡的泡沫盒中，放入－70℃超低温冰箱中。
复苏细菌：将细菌从－70℃超低温冰箱中取出，快速在37℃水浴中融化，清洁消毒管表面，混匀菌液，取30μl菌液加至哥伦比亚培养基平板中央，用三角形玻璃涂布棒涂成菌膜，37℃，10％CO 2，5％O 2，85％N 2，98％相对湿度三气培养箱中培养。
2.结果
2.1 用培养HP的专用培养基哥伦比亚培养基培养HP，细菌正常生长，形态、染色及生化反应正常；而用含不同浓度药物组合物的哥伦比亚培养基培养HP，细菌在培养基中完全不能生长，在药物组合物浓度最低为5％(w/v，即5g药物组合物加入100ml哥伦比亚培养基)时，HP仍然不能生长。
具体地，用哥伦比亚培养基培养的HP，生长正常，形态染色正常(参见图1A)。用含20％(w/v)药物组合物的哥伦比亚培养基培养HP，培养72h，结果显示培养基中没有细菌生长(参见图1B)。用含10％(w/v)药物组合物的哥伦比亚培养基培养HP，培养72h，结果显示培养基中没有细菌生长。用含5％(w/v)药物组合物的哥伦比亚培养基培养HP，培养72h，结果显示培养基中没有细菌生长(参见图1C)。
2.2 用培养HP的专用培养基哥伦比亚培养基培养HP，细菌正常生长，形态、染色及生化反应正常；而用含低浓度药物组合物的哥伦比亚培养基培养HP，细菌在培养基中形态发生明显变异。变异有一个明显过程，最后变异的细菌均归于消亡。
具体地，培养后第3天，用含1.25％(w/v)药物组合物的哥伦比亚培养基培养的HP，形态正常，未发生变异(参见图2A)。培养后第5天，用含1.25％(w/v) 药物组合物的哥伦比亚培养基培养的HP，已经发生变异，主要表现为菌体变长(参见图2B)。培养后第6天，用含1.25％(w/v)药物组合物的哥伦比亚培养基培养的HP，已发生明显变异，菌体变长明显，呈丝状，背景为死亡的HP。培养后第7天，用含1.25％(w/v)药物组合物的哥伦比亚培养基培养的HP，变异明显，主要表现为菌体变长。变异菌死亡增加。背景为死亡的HP(参见图2C)。培养后第8天，用含1.25％(w/v)药物组合物的哥伦比亚培养基培养的HP，变异明显，主要表现为菌体变长，呈丝状，变异菌减少。背景为死亡的HP。培养后第9天，用含1.25％(w/v)药物组合物的哥伦比亚培养基培养的HP，变异细菌越来越少，死亡明显。背景为死亡的HP(参见图2D)。培养后第10天，用含1.25％药物组合物的哥伦比亚培养基培养的HP，变异的HP所剩无几，背景为大量死亡的HP。
实施例3：正常和变异幽门螺杆菌对口腔粘膜上皮细胞(OMEC)的作用
1.材料与方法
1.1 仪器、设备、材料及试剂
同实施例2。
1.2方法
1.2.1 混合抗生素的配制
按照盐酸万古霉素10mg/L培养基、可溶性两性霉素B 10mg/L培养基、硫酸多粘菌素B 2500U/L培养基、甲氧苄氨嘧啶5mg/L培养基的浓度，计算出制备总量1L哥伦比亚培养基需要的量，即：万古霉素10mg，可溶性两性霉素B 10mg，硫酸多粘菌素B先换算成mg数，由于1mg＝6000U，所以需要0.42mg，甲氧苄氨嘧啶5mg。现在由于每次只制备100ml哥伦比亚培养基，所以把各个总量均分至10等份的分装液中，分装液设为4ml，易存、易取、易操作。具体步骤如下：取四个无菌1.5ml的Eppendorf管，铝箔包住，标记好，分别用电子天平称取万古霉素10mg、可溶性两性霉素B 10mg、硫酸多粘菌素B 0.42mg、甲氧苄氨嘧啶5mg。将称取的其它三种水溶性抗生素置于Eppendorf管中，而对甲氧苄氨嘧啶做如下处理：将其置于无菌大玻璃试管中，加入10ml无菌超纯水，将药品全部冲洗下至试管底部，加20μl DL-乳酸，用试管夹夹住，盖好棉塞，酒精灯火加热至沸腾计时开始共10min，后冷却至室温。向另三种抗生素管中各加1ml无菌超纯水，盖好，震荡，溶解。备一50ml离心管，将另三种抗生素分别移入，并用无菌超纯水涮洗Eppendorf管1～2次，最后将已冷却的甲氧苄氨嘧啶溶液也移入其中，超纯水涮洗回收残留，最后补加无菌超纯水至40ml，将此40ml混合抗生素液用针头滤器过滤至另一50ml无菌离心管中，并分装至10只无菌15ml离心管中，每管4ml，封好，标记好，20℃保存备用。每次制备100ml培养基，溶解培养基时只需加入96ml超纯水，临浇板前加入1管(即4ml)混合抗生素液即可。
1.2.2 哥伦比亚培养基的制备
备一洁净250ml锥形瓶，精确称取3.9g哥伦比亚血琼脂基础(CBAB)置于锥形瓶中，加入100ml超纯水，通过电磁炉，使CBAB在沸水中溶解，盖好棉塞线绳扎紧，121℃15min高压灭菌，冷却至约50℃，无菌加入8ml无菌脱纤维绵羊血，混匀，趁热快速浇板。盖好，冷却，标记，倒放，4℃保存备用。
1.2.3 含混合抗生素哥伦比亚培养基的制备
备一洁净250ml锥形瓶，精确称取3.9g哥伦比亚血琼脂基础(CBAB)置于锥形瓶中，加入96ml超纯水，通过电磁炉，使CBAB在沸水中溶解，盖好棉塞线绳扎紧，121℃15min高压灭菌，冷却至约50℃，无菌加入4ml混合抗生素液和8ml无菌脱纤维绵羊血，混匀，趁热快速浇板。盖好，冷却，标记，倒放，4℃保存备用。
1.2.4 含药物组合物的哥伦比亚培养基的制备
备一洁净250ml锥形瓶，精确称取3.9g哥伦比亚血琼脂基础(CBAB)置于锥形瓶中，加入100ml超纯水，通过电磁炉，使CBAB在沸水中溶解，盖好棉塞线绳扎紧，121℃15min高压灭菌，当高压灭菌器压力归零时立即取出培养基，立即无菌操作加入一定量药物组合物，无菌盖好棉塞，覆上无菌牛皮纸，频繁旋转摇动培养基，使药物组合物融化溶解均匀，进一步冷却至约50℃，无菌加入8ml无菌脱纤维绵羊血，混匀，趁热快速浇板。盖好，冷却，标记，倒放，4℃保存备用。
1.2.5 HP的鉴定
同实施例2中的1.2.2部分。
1.2.6 HP的保存和复苏
同实施例2中的1.2.3部分。
1.2.7 OMEC的培养以及与正常和变异HP的共培养
用无菌一次性植绒拭子，刮取颊部OMEC，并释放细胞于冰浴中含双抗的PBS中；
细胞计数。4℃，2000rpm，离心5min，弃上清；
加入3ml预冷的10％FBS DMEM细胞培养液，漩涡震荡，充分混匀细胞，后补加10ml同样DMEM培养液；
4℃，2000rpm，离心5min，弃上清；
先加入3ml预冷的10％FBS DMEM细胞培养液，漩涡震荡，充分混匀细胞，后补加10ml同样DMEM培养液；
将上述细胞混悬液均分至6孔板的每个孔中，每孔补加适量10％FBS DMEM细胞培养液，每孔总量5ml。
37℃、5％CO 2细胞培养箱中培养；
提前3天和5天培养HP，分别得到正常HP和变异HP。用未添加药物组合物的哥伦比亚培养基培养正常的HP，在培养后第3天，此时已经形成明显菌苔，用 细胞刮刮取总面积一半的菌苔，移入4ml 10％FBS DMEM中，用滴头轻轻研磨成均匀的正常HP混悬液，备用；而用添加0.3125％(w/v)药物组合物的哥伦比亚培养基培养的HP，在培养后第5天，也用同样方法得到4ml变异HP混悬液，备用。
OMEC培养后第1天，在培养板不同的培养孔中加入不同的HP混悬液，具体安排是：
A1孔吸弃1ml培养上清，加入3ml正常HP混悬液；
B1孔不吸弃培养上清，加入1ml正常HP混悬液；
A2孔吸弃1ml培养上清，加入3ml变异HP混悬液；
B2孔不吸弃培养上清，加入1ml变异HP混悬液；
各孔补齐培养液量，A3、B3孔为正常空白对照孔；
将培养板置于37℃、5％CO 2温箱中连续孵育，每天一次或两次观察记录细胞生长情况、细胞形态和结构；
细胞照相时选取不同培养孔的相同位置；
用Nikon TE2000U倒置显微镜观察细胞，用Nikon DMX1200记录图像，图像分辨率可根据需要而适当调整，观察时间尽量缩短，妥善保存实验结果。
2.结果
用未添加药物组合物的哥伦比亚培养基正常培养的HP，在培养后第3天，菌落典型，革兰氏染色显示细菌形态正常，未变异，此时已经形成明显菌苔。而用添加0.3125％(w/v)药物组合物的哥伦比亚培养基培养的HP，在培养后第5天，革兰氏染色显示细菌形态已经发生明显变异。通过OMEC和HP共培养观察正常和变异HP培养物对OMEC生长的影响，具体地：
共培养后第4天，3ml(图3A)及1ml正常HP混悬液，镜下未见OMEC，说明OMEC死亡，HP培养物已经将OMEC杀死。而共培养后第4天，3ml(图3B)及1ml变异HP混悬液镜下可见OMEC，说明OMEC未死亡，HP培养物未将OMEC杀死。共培养后第4天，正常空白对照孔镜下可见正常OMEC，生命状态良好。
共培养后第15天，3ml及1ml正常HP混悬液，镜下未见OMEC，说明OMEC死亡，HP培养物已经将OMEC杀死。共培养后第15天，3ml及1ml变异HP混悬液，镜下可见OMEC，说明OMEC未死亡，HP培养物未将OMEC杀死。共培养后第15天，正常空白对照孔，镜下可见正常OMEC，生命状态良好。
共培养后第41天，3ml及1ml正常HP混悬液，镜下未见OMEC，说明OMEC死亡，HP培养物已经将OMEC杀死。共培养后第41天，3ml及1ml变异HP混悬液，镜下可见OMEC，说明OMEC未死亡，HP培养物未将OMEC杀死。共培养后第41天，正常空白对照孔，镜下可见正常OMEC，生命状态良好。
实施例4：本发明的药物组合物在体外培养条件下对幽门螺杆菌毒力的影响
1.材料与方法
1.1 仪器、设备、材料及试剂
同实施例3
1.2方法
1.2.1-1.2.6同实施例3。
1.2.7 OMEC的培养以及与HP培养物的共培养
用无菌一次性植绒拭子，刮取颊部OMEC，并释放细胞于冰浴中含双抗的PBS中；
细胞计数。4℃，2000rpm，离心5min，弃上清；
加入3ml预冷的10％FBS DMEM细胞培养液，漩涡震荡，充分混匀细胞，后补加10ml同样DMEM培养液；
4℃，2000rpm，离心5min，弃上清；
先加入3ml预冷的10％FBS DMEM细胞培养液，漩涡震荡，充分混匀细胞，后补加10ml同样DMEM培养液；
将上述细胞混悬液均分至6孔板的每个孔中，每孔补加适量10％FBS DMEM细胞培养液，每孔总量5ml。
37℃、5％CO 2培养箱中培养；
提前3天培养HP，培养基分别是，第一种是用未添加药物组合物的哥伦比亚培养基，第二种是用含0.3125％(w/v)药物组合物的哥伦比亚培养基，第三种是用含1.25％(w/v)药物组合物的哥伦比亚培养基，在培养后第3天，此时已经形成明显菌苔，用细胞刮刮取总面积的全部菌苔或总面积菌苔的一半，移入4ml 10％FBS DMEM中，用滴头轻轻研磨成均匀的HP混悬液。因此得到三种不同的HP混悬液，备用。
OMEC培养后第3天，在培养板不同的培养孔中加入HP培养物悬液，具体安排是：
A1孔吸弃2ml培养上清，加入第一种HP混悬液4ml。
A2孔吸弃2ml培养上清，加入第二种HP混悬液4ml。
A3孔吸弃2ml培养上清，加入第三种HP混悬液4ml。
B1、B2、B3孔各补加2ml 10％FBS DMEM，作为正常空白对照孔。
将培养板置于37℃、5％CO 2温箱中连续孵育，每天一次或两次观察记录细胞生长情况、细胞形态和结构；
细胞照相时选取不同培养孔的相同位置；
用Nikon TE2000U倒置显微镜观察细胞，用Nikon DMX1200记录图像，图像分辨率可根据需要而适当调整，观察时间尽量缩短，妥善保存实验结果。
2.结果
用三种不同药物组合物含量的哥伦比亚培养基培养HP，在培养后第3天，发现，第一种用未添加药物组合物的哥伦比亚培养基培养的HP，革兰氏染色阴性，显示细菌很多，OMEC形态正常；第二种用含0.3125％(w/v)药物组合物的哥伦比亚培养基培养的HP，革兰氏染色阴性，细菌很多，OMEC形态正常；第三种用含1.25％(w/v)药物组合物的哥伦比亚培养基培养的HP，革兰氏染色阴性，OMEC形态正常，但细菌很少。第一种和第二种此时菌落典型，且已形成明显菌苔。共培养后观察含不同浓度药物组合物培养基培养的HP培养物对OMEC生长的影响。发现第一种HP培养物对OMEC作用明显，说明HP的毒力很强；第二种HP培养物对OMEC作用也很明显，OMEC死亡明显，说明由于药物组合物浓度很低，对HP的繁殖和毒力影响不明显；第三种HP培养物对OMEC作用不明显，说明药物组合物发挥了作用，明显抑制了HP的繁殖和毒力。
1)共培养后第2天，用未添加药物组合物的哥伦比亚培养基培养的HP与OMEC的共培养，镜下未见OMEC，说明OMEC死亡，HP培养物已经将OMEC杀死。共培养后第2天，用含0.3125％(w/v)药物组合物的哥伦比亚培养基培养的HP与OMEC的共培养，镜下未见OMEC，说明OMEC死亡，HP培养物已经将OMEC杀死。共培养后第2天，用含1.25％(w/v)药物组合物的哥伦比亚培养基培养的HP与OMEC的共培养，镜下OMEC生长正常，说明HP培养物未影响OMEC生长。共培养后第2天，正常对照组的OMEC生长正常。
2)共培养后第17天，用未添加药物组合物的哥伦比亚培养基培养的HP与OMEC的共培养，镜下未见OMEC，说明HP培养物显著抑制OMEC生长(图4A)。共培养后第17天，用含0.3125％(w/v)药物组合物的哥伦比亚培养基培养的HP与OMEC的共培养，镜下未见OMEC，说明HP培养物显著抑制OMEC生长(图4B)。共培养后第17天，用含1.25％(w/v)药物组合物的哥伦比亚培养基培养的HP与OMEC的共培养，镜下OMEC生长正常，说明HP培养物未影响OMEC生长(图4C)。共培养后第17天，正常对照组，镜下OMEC生长正常(图4D)。
3)共培养后第51天，用未添加药物组合物的哥伦比亚培养基培养的HP与OMEC的共培养，镜下未见OMEC生长，说明HP培养物显著抑制OMEC生长。共培养后第51天，用含0.3125％(w/v)药物组合物的哥伦比亚培养基培养的HP与OMEC的共培养，镜下未见OMEC生长，说明HP培养物显著抑制OMEC生长。共培养后第51天，用含1.25％(w/v)药物组合物的哥伦比亚培养基培养的HP与OMEC的共培养，镜下可见OMEC生长，说明HP培养物未影响OMEC生长。共培养后第51天，正常对照组，镜下可见OMEC正常生长。
实施例5：变异幽门螺杆菌毒力减弱
1.材料与方法
1.1 仪器、设备、材料及试剂
同实施例3。
1.2方法
1.2.1-1.2.6同实施例3。
1.2.7 OMEC的培养以及和HP的共培养
用无菌一次性植绒拭子，刮取颊部OMEC，并释放细胞于冰浴中的含双抗PBS中；
细胞计数。4℃，2000rpm，离心5min，弃上清；
加入3ml预冷的10％FBS DMEM细胞培养液，漩涡震荡，充分混匀细胞，后补加10ml同样DMEM培养液；
4℃，2000rpm，离心5min，弃上清；
先加入3ml预冷的10％FBS DMEM细胞培养液，漩涡震荡，充分混匀细胞，后补加10ml同样DMEM培养液；
将上述细胞混悬液均分至6孔板的每个孔中，每孔补加适量10％FBS DMEM细胞培养液，每孔总量5ml。
37℃、5％CO 2培养箱中培养；
用含0.3125％(w/v)药物组合物的哥伦比亚培养基提前6天培养HP，得到变异HP，此时已经形成明显菌苔，用细胞刮刮取总面积一半的菌苔，移入4ml 10％FBS DMEM培养液中，用滴头轻轻研磨成均匀的4ml变异HP混悬液，备用。
OMEC培养后第4天，在培养板的培养孔中吸弃2ml培养上清，加入上述变异HP培养物悬液，对照孔用10％FBS DMEM培养液补齐。
将培养板置于37℃、5％CO 2温箱中连续孵育，每天一次或两次观察记录细胞生长情况、细胞形态和结构；
细胞照相时选取不同培养孔的相同位置；
用Nikon TE2000U倒置显微镜观察细胞，用Nikon DMX1200记录图像，图像分辨率可根据需要而适当调整，观察时间尽量缩短，妥善保存实验结果。
2.结果
用添加0.3125％(w/v)药物组合物的哥伦比亚培养基培养的HP，在培养后第6天，革兰氏染色显示，形态已经发生明显变异，加入培养OMEC的培养孔中，观察OMEC的生长情况以及变异HP培养物对OMEC生长的影响。
结果发现，变异HP培养物对OMEC有一定作用，但不能完全杀死OMEC，说明变异的HP毒力已经减弱。
具体地，OMEC与变异HP混悬液共培养后第1天，OMEC未完全死亡，可见形态典型的OMEC。OMEC与变异HP混悬液共培养后第20天，OMEC未完全死亡，可见形态完整的OMEC。OMEC与变异HP混悬液共培养后第24天，OMEC未完全死亡，可见较多形态典型的OMEC。OMEC与变异HP混悬液共培养后第46天，OMEC未完全死亡，仍可见形态典型的OMEC(参见图5A)。正常对照 第46天，OMEC仍正常生长，形态典型(参见图5B)。
实施例6：DMEM培养基对幽门螺杆菌生长的影响
1.材料与方法
1.1 仪器、设备、材料及试剂
同实施例3。还包括酶标仪(Multiskan Ascent，芬兰Labsystems)、体视显微镜(SMZ1000型，日本Nikon)、酶标板(美国Costar)。
1.2方法
1.2.1-1.2.6同实施例3。
1.2.7 DMEM培养基培养HP
用哥伦比亚培养基提前3天培养HP。实验当日，用细胞刮收获HP，用细胞刮刮取总面积的全部菌苔，移入4ml 10％FBS DMEM中，用滴头轻轻研磨成均匀的HP混悬液。漩涡震荡后，再加4ml 10％FCS DMEM。取一可拆卸的酶标板，加入三种被测物，混匀，各加200ul，分别是：
1)HP混悬液(HP+DMEM)       3个平行孔
2)PBS                     3个平行孔
3)DMEM                    3个平行孔
用Labsystems Multiskan Ascent酶标仪620nm波长测OD 620值。
将其它HP混悬液混匀，均分至6孔板的A1和A2孔中。每孔约3ml，5％O 2、37℃细胞培养箱培养。
36h后，用上述培养过程中的HP混悬液进行细菌培养，均为哥伦比亚培养基，每皿加30ul上述HP混悬液，点在中央，用三角形玻璃涂布棒涂布均匀。10％CO 2、5％O 2、85％N 2、37℃三气培养箱培养，5天后观察HP生长情况。同时用同样方法第二次检测OD 620值。
72h后用同样方法第三次检测OD 620值。
2结果
实验开始之初，第一次检测OD 620值。结果如下：
1)HP+DMEM 3个孔：0.484；0.463；0.473
2)PBS 3个孔：0.036；0.037；0.037；
3)DMEM 3个孔：0.081；0.066；0.062
36h后，第二次检测OD 620值结果如下。结果如下：
1)HP+DMEM 3个孔：0.325；0.350；0.301
2)PBS 3个孔：0.035；0.035；0.036；
3)DMEM 3个孔：0.060；0.064；0.068
72h后用同样方法第三次检测OD 620值。结果如下：
1)HP+DMEM 3个孔：0.284；0.271；0.267
2)PBS 3个孔：0.035；0.035；0.036；
3)DMEM 3个孔：0.059；0.062；0.058
一般意义上认为，HP应在与细胞共培养中增殖，但在实验中，将HP直接悬浮于细胞培养液DMEM中，5％O 2、37℃细胞培养箱培养，发现HP悬液的OD 620值随着培养时间的延长没有提高，反而明显降低，共检测3次，一次比一次低，又发现将孵育36h的HP菌液直接涂布在5个哥伦比亚培养基上，10％CO 2、5％O 2、85％N 2、37℃三气培养箱培养，5天后观察，未见一个菌落，未发现HP生长(见图6)，上述结果说明，HP在与OMEC共培养过程中对细胞增殖的抑制是HP本身的致病性所致，而并非是因为HP大量增殖消耗大量营养致使细胞营养不足所致。
实施例7：含黄芩和黄柏的药物组合物的制备及其抗幽门螺杆菌的作用
按照实施例1的方法制备药物组合物，其中在步骤1中将精制的芝麻油和黄芩和黄柏(100kg：5kg：4kg)放入反应罐，其余同实施例1。
将本实施例中获得的药物组合物，用于实施例2中的检测方法中，获得以下结果：
用培养HP的专用培养基哥伦比亚培养基培养HP，细菌正常生长，形态、染色及生化反应正常；而用含不同浓度的本实施例的药物组合物的哥伦比亚培养基培养HP，细菌在培养基中完全不能生长，在药物组合物浓度最低为5％时，HP仍然不能生长。
将本实施例中获得的药物组合物，用于实施例3中的检测方法中，获得以下结果：
用未添加药物组合物的哥伦比亚培养基正常培养的HP，在培养后第3天，菌落典型，革兰氏染色显示细菌形态正常，未变异，此时已经形成明显菌苔。而用添加0.3125％的本实施例的药物组合物的哥伦比亚培养基培养的HP，在培养后第5天，革兰氏染色显示细菌形态已经发生明显变异。通过OMEC和HP共培养观察正常和变异HP培养物对OMEC生长的影响。共培养后第4天、第15天和第41天，3ml及1ml正常HP混悬液，镜下未见OMEC，说明OMEC死亡，HP培养物已经将OMEC杀死。而共培养后第4天、第15天和第41天，3ml及1ml变异HP混悬液镜下可见OMEC，说明OMEC未死亡，HP培养物未将OMEC杀死。共培养后第4天，正常空白对照孔镜下可见正常OMEC，生命状态良好。
实施例8：含黄芩和黄连的药物组合物的制备及其抗幽门螺杆菌的作用
按照实施例1的方法制备药物组合物，其中在步骤1中将精制的芝麻油和黄芩、和黄连(100kg：5kg：4kg)放入反应罐，其余同实施例1。
将本实施例中获得的药物组合物，用于实施例2和实施例3中的检测方法中，获得与实施例7中获得的组合物相似的检测结果，该组合物可以抑制幽门螺杆菌。
实施例9：含黄芩、黄柏和黄连的药物组合物的制备及其抗幽门螺杆菌的作用
按照实施例1的方法制备药物组合物，其中在步骤1中将精制的芝麻油和黄芩、和黄连(100kg：5kg：5kg：5kg)放入反应罐，其余同实施例1。
将本实施例中获得的药物组合物，用于实施例2和实施例3中的检测方法中，获得与实施例7中获得的组合物相似的检测结果，而用含不同浓度的本实施例的药物组合物的哥伦比亚培养基培养HP，细菌在培养基中完全不能生长，在药物组合物浓度最低为5％时，HP仍然不能生长。用添加0.3125％的本实施例的药物组合物的哥伦比亚培养基培养的HP，共培养后第4天、第15天和第41天，3ml及1ml正常HP混悬液，镜下未见OMEC，说明OMEC死亡，HP培养物已经将OMEC杀死。
实施例10：含黄芩、黄连、黄柏、罂粟壳和地龙的药物组合物的制备及其抗 幽门螺杆菌的作用
按照实施例1的方法制备药物组合物，其中在步骤1中将精制的芝麻油和黄芩、黄连、黄柏、罂粟壳和地龙(100kg：5kg：4kg：4kg：5kg:5kg)放入反应罐，其余同实施例1。
将本实施例中获得的药物组合物，用于实施例2的检测方法中，获得以下结果：
用培养HP的专用培养基哥伦比亚培养基培养HP，细菌正常生长，形态、染色及生化反应正常；而用含不同浓度本实施例的药物组合物的哥伦比亚培养基培养HP，发现高、中两个浓度完全不能生长，低浓度只有极少数细菌生长。说明本实施例的药物组合物对HP具有强烈抑制作用。
具体地，用哥伦比亚培养基培养的HP，生长正常，形态染色正常(参见图7A)。用含10％本实施例的药物组合物的哥伦比亚培养基培养HP，培养72h，结果显示培养基中没有细菌生长。用含5％本实施例的药物组合物的哥伦比亚培养基培养HP，培养72h，结果显示培养基中没有细菌生长。用含2.5％本实施例的药物组合物的哥伦比亚培养基培养HP，培养72h，结果显示培养基中只有极少的细菌生长(参见图7B)。
用培养HP的专用培养基哥伦比亚培养基培养HP，细菌正常生长，形态、染色及生化反应正常；而用含1.25％本实施例的药物组合物的哥伦比亚培养基培养HP，细菌在培养基中形态发生明显变异。变异从一般变异到显著变异，从不典型到典型，从不明显到明显。
具体地，用哥伦比亚培养基培养的HP，生长正常，形态染色正常(参见图8A)。用含1.25％本实施例的药物组合物的哥伦比亚培养基培养的HP，处于变异早期，主要表现为菌体变长(参见图8B)。用含1.25％本实施例的药物组合物的哥伦比 亚培养基培养的HP，处于变异晚期，主要表现为菌体变细变长，图中显示死亡的HP(参见图8C)。
将本实施例中获得的药物组合物，用于实施例3中的检测方法中，获得以下结果：用添加低至0.3125％的本实施例的药物组合物的哥伦比亚培养基培养的HP，共培养后第4天、第15天和第41天，3ml及1ml正常HP混悬液，镜下未见OMEC，说明OMEC死亡，HP培养物已经将OMEC杀死。
实施例11：药物组合物在人体内抗幽门螺杆菌的作用
1.材料与方法
1.1 临床资料
选择胃镜检查诊断为上消化道溃疡与炎症病人22例，男12例。女10例，患病年龄29岁～71岁。22例中15例为单发病变，其他7例为双发或多发病变。即胃溃疡和十二指肠溃疡各5例(处)，慢性胃炎16例(处)，胃黏膜糜烂4例(处)，食道溃疡炎症1例(处)。
1.2 分组与治疗方法
(1)分组方法：根据病人自愿原则，分为单独服用实施例1中制备的药物组合物(A组)，共15例；服用实施例1中制备的药物组合物加内科常规治疗组(B组)，共7例，观察两组病人的临床症状与体征变化，和一个月后的胃镜复查结果，比较治疗效果。
(2)服用方法：A组：食管炎、胃溃疡、炎症：实施例1中制备的药物组合物，每次2.5g，4次/日，3餐前半小时及临睡前服用，食管炎患者宜咀嚼吞服；十二指肠溃疡：实施例1中制备的药物组合物每次4.0g，4次/日，3餐前及临睡前服用，总疗程一个月。B组：实施例1中制备的药物组合物的服用方法同实验A组。内科常规治疗包括：胃复安20mg，3次/日，雷尼替丁150mg，2次/日，口服。病变重伴有出血倾向者改用洛赛克20mg，1次/日，口服。为加速清除HP感染，加服阿莫西林500mg，1次/日，连服7天，或克林霉素0.5g，2次/日，连服7天。疗程1个月。
(3)记录服药前症状、体征与治疗后的相应变化及不良反应，一个疗程结束后作电子胃镜检查。
1.3 疗效判断标准
(1)治愈：服药后10天上腹痛等症状体征明显改善及黑便消失，20天症状体征消失，一个月胃镜检查提示黏膜炎症消失，溃疡生理愈合，无明显瘢痕；HP试验转阴；
(2)好转：服药后10天上腹痛等症状体征稍有改善，黑便停止或减轻；20天上腹痛等症状体征明显改善，黑便停止，OB试验转阴或呈弱阳性；一个月胃镜下提示炎症减轻，溃疡面积缩小1/2以上，或为瘢痕性愈合，HP试验由强阳性转为阴 性或弱阳性；
(3)无效：服药后20天症状体征没有明显变化，一个月胃镜下提示炎症病变未见好转，溃疡愈合面积小于1/2。HP试验仍为阳性。
2.结果
A、B两组治疗10天，病人自觉症状均有改善.即症状改善率均为100％。治疗20天：A组症状消失率为93.3％，B组为100％。治疗一月胃镜复查结果：A组HP转阴率为53.3％(8/15)，B组为42.9％(3/7)；好转率：A组为40.0％(6/15)。B组为42.9％(3/7)；无效率：A组为6.7％(1/15)，B组为14.3％(1/7)。溃疡治愈：A组与B组均为100％，其中瘢痕愈合分别为：A组6.7％、B组14.3％，炎症性病变均有明显改善(A组与B组均为100％)。
可见，两组结果虽然相似，但A组的治疗药物为单纯实施例1中制备的药物组合物，B组的治疗药物除实施例1中制备的药物组合物，同时接受了西药(胃复安、雷尼替丁、阿莫西林等)治疗。进而说明本发明的药物组合物可替代上述西药的治疗作用.这些作用包括缓解临床症状、保护胃肠黏膜、促进上消化道溃疡、炎症性病麦愈合、控制幽门螺杆菌感染等。根据本研究结果分析。认为本发明的药物组合物能通过阻止HP的定植或改变HP的致病性，免受损伤因子的再损伤和神经末梢受刺激而能较快的缓解溃疡、炎症病人疼痛等不适症状。局部炎症明显改善，同时为病变部位提供再生修复的生理环境和生命物质。而促进溃疡生理再生修复并提高了修复愈合质量。

Claims (23)

  1. 药物组合物在制备用于抗幽门螺杆菌的药物中的用途，其中所述的药物组合物是一种适用于口服的药物组合物，其包括食用油与蜂蜡、β-谷甾醇的均质混合物，其中，该组合物中蜂蜡形成微晶体，按该组合物总重量计算，蜂蜡的含量为0.5-50％，β-谷甾醇为0.1-20％。
  2. 根据权利要求1所述的用途，其中抗幽门螺杆菌是指幽门螺杆菌不能生长繁殖、幽门螺杆菌繁殖缓慢、幽门螺杆菌变异、幽门螺杆菌死亡和/或幽门螺杆菌致病性降低。
  3. 药物组合物在制备用于治疗/预防由幽门螺杆菌引起的疾病的药物中的用途，其中所述的药物组合物是一种适用于口服的药物组合物，其包括食用油与蜂蜡、β-谷甾醇的均质混合物，其中，该组合物中蜂蜡形成微晶体，按该组合物总重量计算，蜂蜡的含量为0.5-50％，β-谷甾醇为0.1-20％。
  4. 根据权利要求3所述的用途，其中由幽门螺杆菌引起的疾病包括幽门螺杆菌感染引起的胃炎、胃溃疡、十二指肠溃疡、胃癌、胃非何杰金氏淋巴瘤和胃粘膜相关淋巴样组织淋瘤。
  5. 根据权利要求3或4所述的用途，其中由幽门螺杆菌引起的疾病是在哺乳动物中引起的疾病，所述哺乳动物优选是人。
  6. 根据权利要求1-5中任一项所述的用途，其特征在于：所述药物组合物中β-谷甾醇的含量按重量计算，介于0.5-20％之间。
  7. 根据权利要求1-5中任一项所述的用途，其特征在于：所述药物组合物中β-谷甾醇的含量按重量计算，介于1-10％之间。
  8. 根据权利要求1-5中任一项所述的用途，其特征在于：所述药物组合物中蜂蜡的含量按重量计算，介于3-30％之间。
  9. 根据权利要求1-5中任一项所述的用途，其特征在于：所述药物组合物中蜂蜡的含量按重量计算，介于5-20％之间。
  10. 根据权利要求1-5中任一项所述的用途，其特征在于：所述药物组合物中蜂蜡的含量按重量计算，介于6-10％之间。
  11. 根据权利要求1-5中任一项所述的用途，其特征在于：所述药物组合物中食用油是玉米油、麦芽油、豆油、米糠油、油菜籽油、芝麻油和鱼油。
  12. 根据权利要求1-5中任一项所述的用途，其特征在于：所述药物组合物中还含有蜂胶，含量按重量计算，介于0.1-30％。
  13. 根据权利要求1-5中任一项所述的用途，其特征在于：所述药物组合物中含有水，其含量按重量计算，为小于等于1％。
  14. 根据权利要求1-5中任一项所述的用途，其特征在于：所述的口服选自下列剂型，包括：片剂、丸剂、胶囊、乳状液、凝胶体、糖浆、或悬浮液。
  15. 根据权利要求1-5中任一项所述的用途，其特征在于：所述药物组合物进一步包括黄芩或黄芩提取物，按组合物总重量计算，2-5％的黄芩或黄芩甙的含量为0.1-0.5％的黄芩提取物，所述黄芩选自唇形科的粘毛黄芩、滇黄芩、甘肃黄芩、薄叶黄芩、丽江黄芩、川黄芩。
  16. 根据权利要求1-5中任一项所述的用途，其特征在于：所述药物组合物进一步包括黄柏或黄柏提取物，按组合物总重量计算，2-5％黄柏的或含0.1-1％黄柏内酯的黄柏提取物，所述的黄柏选自黄皮树、秃叶黄皮树，峨嵋黄皮树、云南黄皮树、镰刀叶黄皮树。
  17. 根据权利要求1-5中任一项所述的用途，其特征在于：所述药物组合物进一步包括，按组合物总重量计算，2-5％的黄连，或者含0.1-1％小檗碱的黄连提取物。
  18. 根据权利要求15-17中任一项所述的用途，其特征在于：所述的黄芩提取物是黄芩的芝麻油提取物，所述的黄柏提取物是黄柏的芝麻油提取物，所述的黄连提取物是黄连的芝麻油提取物。
  19. 根据权利要求1-5中任一项所述的用途，其特征在于：按组合物总重量计算，所述药物组合物进一步包括2-5％的黄芩或黄芩甙的含量为0.1-0.5％的黄芩提取物、2-5％的黄柏或含0.1-1％黄柏内酯的黄柏提取物、2-5％的黄连或含0.1-1％小檗碱的黄连提取物、2-10％的罂粟壳或含0.1-1％罂粟壳碱的罂粟壳提取物和2-10％的地龙或地龙提取物。
  20. 根据权利要求1-5中任一项所述的用途，其特征在于：所述药物组合物按组合物总重量计算，包括7％蜂蜡、1％甾醇、0.5％黄柏内酯、0.3％黄芩甙和0.5重量％小檗碱。
  21. 根据权利要求1-5中任一项所述的用途，其特征在于：所述的蜂蜡具有微晶体，其长度是0.1-100微米。
  22. 根据权利要求21中任一项所述的用途，其特征在于：所述药物组合物中蜂蜡的至少两个微晶体聚合成微晶复合物。
  23. 根据权利要求22所述的用途，其特征在于：所述的蜂蜡的微晶体充分地均匀分散在食用油中。
PCT/CN2018/089762 2017-06-06 2018-06-04 药物组合物在制备用于抗幽门螺杆菌的药物中的用途 WO2018223922A1 (zh)

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