Human interferon gynecological antibacterial compound preparation and application thereof

Abstract

The invention discloses a human interferon gynecological antibacterial compound preparation and application thereof, and belongs to the technical field related to traditional Chinese medicine, the compound preparation comprises a component A containing interferon alpha1b and a component B containing a sophora flavescens extract, the volume ratio of the component A to the component B is 1: (8-10), the ph of the compound preparation is 6.5-7.0, and the pH value of the compound preparation is 6.5-7.0. The compound preparation disclosed by the invention is combined with the antiviral / immunoregulation effect of interferon and the antibacterial / anti-inflammatory effect of radix sophorae flavescentis, and can be used for simultaneously aiming at viral infection, bacterial infection and inflammatory reaction of cervicitis and reducing the drug resistance risk.

Description

Technical Field

[0001] The present invention belongs to the technical field related to traditional Chinese medicine, and particularly relates to a human interferon gynecological antibacterial composite preparation and its application. Background Art

[0002] Cervicitis is an inflammatory disease that occurs in the cervix (the part of the lower end of the uterus leading to the vagina). Its essence is a local inflammatory reaction caused by pathogen infection or mechanical damage to the vaginal part of the cervix or the mucosa of the cervical canal. It is a common gynecological disease, mostly seen in women of childbearing age.

[0003] Interferon α1b can be used for the treatment of viral chronic cervicitis, such as combined with HPV infection, herpes virus, etc. 25. Its antiviral mechanism can inhibit pathogen replication and relieve the inflammatory reaction. However, in the case of mixed bacterial infection, directly using interferon may reduce the antiviral effect due to the continuous reproduction of pathogens. Summary of the Invention

[0004] In view of the above situation, to overcome the defects of the prior art, the present invention provides a human interferon gynecological antibacterial composite preparation and its application. The composite preparation involved combines the antiviral / immunomodulatory effect of interferon with the antibacterial / anti-inflammatory effect of Sophora flavescens, and can simultaneously target viral infections, bacterial infections and inflammatory reactions of cervicitis and reduce the risk of drug resistance.

[0005] Specifically, the present invention is achieved through the following solutions:

[0006] In the first aspect, the present invention provides a human interferon gynecological antibacterial composite preparation, which includes component A containing interferon α1b and component B containing Sophora flavescens extract. The volume ratio of component A to component B is 1:(8 - 10), and the pH of the composite preparation is 6.5 - 7.0.

[0007] Further, the preparation method of component A is: adjusting the concentration of the purified interferon α1b solution to 1×10 5 -1×10 6 IU / mL, and adding 0.1 - 1.0% human albumin (w / v) as a stabilizer, and adjusting the pH to 6.5 - 7.0.

[0008] Further, the preparation method of component B is: preparing Sophora flavescens extract and dissolving the extract in pH 6.5 sterile phosphate buffer, and the concentration of Sophora flavescens extract is 1% - 3%.

[0009] In the second aspect, the present invention provides a preparation method of a human interferon gynecological antibacterial composite preparation, including the following steps:

[0010] S1, mixing component A and component B at a volume ratio of 1:(9 - 10), and the final concentration of interferon α1b is 1×105 -1×10 6 IU / mL;

[0011] Adjust the pH of the mixed solution to 6.5 - 7.0 with 0.1M sodium bicarbonate;

[0012] The composite preparation is prepared by filtering and sterilizing the mixed solution prepared from S2 and S1 through a 0.22μm filter membrane.

[0013] In the third aspect, the present invention provides the application of the above composite preparation in the preparation of drugs for treating cervicitis. Animal experiments show that the composite preparation of human interferon α1b and sophora flavescens extract can significantly improve the pathological state of cervical inflammation by synergistically inhibiting the release of inflammatory factors, promoting mucosal repair and enhancing local immune response.

[0014] Compared with the prior art, the present invention has the following beneficial effects: in terms of antiviral and immunomodulatory effects, the composite preparation can inhibit virus infection, perform immunomodulation and enhance local immune response; in terms of anti-inflammatory and tissue repair, the composite preparation can relieve the inflammatory reaction and promote mucosal repair. The composite preparation combines the antiviral / immunomodulatory effects of interferon and the antibacterial / anti-inflammatory effects of sophora flavescens, and can simultaneously target viral infections, bacterial infections and inflammatory reactions of cervicitis. The multi-component characteristics of sophora flavescens extract can reduce the drug resistance of single components and prolong the effectiveness of the drug. Detailed implementation mode

[0015] The present invention discloses a human interferon gynecological antibacterial composite preparation and its application. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate modifications and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0016] Example 1:

[0017] The present invention provides a human interferon gynecological antibacterial composite preparation, which includes component A containing interferon α1b and component B containing sophora flavescens extract. The volume ratio of component A to component B is 1:10, and the pH of the composite preparation is 6.5 - 7.0.

[0018] The preparation method of component A is: adjust the concentration of the purified interferon α1b solution to 1×10 5 -1×10 6 IU / mL, and add 0.1 - 1.0% human serum albumin (w / v) as a stabilizer, and adjust the pH to 6.5 - 7.0.

[0019] Among them, the preparation method of interferon α1b is as follows: the human interferon α1b gene is introduced into Escherichia coli (such as BL21 strain), and an inducer (such as IPTG) is used to initiate protein expression. The fermentation temperature is 25 - 30 °C, and the fermentation period is 18 - 24 hours; the periplasmic space expression technology is adopted to avoid the contamination of host proteins after cell disruption and improve the purity of the target protein; interferon α1b in the periplasmic space is released by the osmotic pressure method, and purified by ion exchange chromatography and gel filtration chromatography in sequence, with a purity ≥ 95%.

[0020] The preparation method of component B is as follows: prepare the Sophora flavescens extract, and dissolve the extract in a sterile phosphate buffer solution with a pH of 6.5. The concentration of the Sophora flavescens extract is 1%.

[0021] Among them, the preparation method of the Sophora flavescens extract is as follows:

[0022] After drying and pulverizing the Sophora flavescens root, it is refluxed and extracted twice with 70% ethanol (volume ratio 1:10) at 60 °C for 1 hour each time. The extraction solutions are combined and concentrated under reduced pressure until the alcohol smell disappears; the concentrated solution is subjected to column chromatography on macroporous adsorption resin (such as D101), eluted with 30% ethanol, and the enriched section of total alkaloids of Sophora flavescens is collected, and the content of total alkaloids is controlled to be ≥ 10%.

[0023] The preparation method of the compound preparation:

[0024] S1, mix component A and component B at a volume ratio of 1:10, and the final concentration of interferon α1b is 1×10 5 IU / mL;

[0025] Adjust the pH of the mixed solution to 6.5 - 7.0 with 0.1M sodium bicarbonate;

[0026] S2, the mixed solution prepared in S1 is filtered and sterilized through a 0.22 μm filter membrane to obtain the compound preparation;

[0027] S3, dispense it into sterile brown glass bottles, seal it, and store it in the dark at 2 - 8 °C.

[0028] Example 2

[0029] The present invention provides a human interferon gynecological antibacterial compound preparation, which includes component A containing interferon α1b and component B containing Sophora flavescens extract. The volume ratio of component A to component B is 1:9, and the pH of the compound preparation is 6.5 - 7.0.

[0030] The preparation method of component A is as follows: adjust the concentration of the purified interferon α1b solution to 1×10 6 IU / mL, and add 0.1 - 1.0% human albumin (w / v) as a stabilizer, and adjust the pH to 6.5 - 7.0.

[0031] Among them, the preparation method of interferon α1b is the same as that in Example 1;

[0032] The preparation method of Component B is as follows: Prepare Sophora flavescens extract, and dissolve the extract in sterile phosphate buffer at pH 6.5, with the concentration of Sophora flavescens extract being 3%.

[0033] Among them, the preparation method of Sophora flavescens extract is the same as that in Example 1.

[0034] Preparation method of the compound preparation:

[0035] S1, Mix Component A and Component B according to a volume ratio of 1:9, and the final concentration of interferon α1b is 1×10 6 IU / mL;

[0036] Adjust the pH of the mixed solution to 6.5 - 7.0 with 0.1M sodium bicarbonate;

[0037] S2, The mixed solution prepared in S1 is filtered and sterilized through a 0.22μm filter membrane to obtain the compound preparation;

[0038] S3, Aliquot into sterile brown glass bottles, seal and store in the dark at 2 - 8°C.

[0039] Comparative Example 1: Without adding Component B

[0040] The present invention provides a human interferon gynecological antibacterial compound preparation, including Component A containing interferon α1b;

[0041] The preparation method of Component A is as follows: Adjust the concentration of the purified interferon α1b solution to 1×10 5 IU / mL, and add 0.1 - 1.0% human albumin (w / v) as a stabilizer, and adjust the pH to 6.5 - 7.0.

[0042] Among them, the preparation method of interferon α1b is the same as that in Example 1.

[0043] Comparative Example 2: Without adding Component A

[0044] The present invention provides a human interferon gynecological antibacterial compound preparation, including Component B containing Sophora flavescens extract. The preparation method of Component B is as follows: Prepare Sophora flavescens extract, and dissolve the extract in sterile phosphate buffer at pH 6.5, with the concentration of Sophora flavescens extract being 1%.

[0045] Among them, the preparation method of Sophora flavescens extract is the same as that in Example 1.

[0046] Experimental Example 1 Physical and chemical, activity detection and safety detection of the compound preparation of the present invention.

[0047] Potentiometric method for detecting the pH value range: The pH range of the compound preparations prepared in Example 1 and Example 2 is 6.5 - 7.0;

[0048] Cytopathic effect inhibition method (CPE) to verify interferon activity: Example 1, interferon activity 1×10 5 IU / mL; Example 1, interferon activity 1×10 6 IU / mL;

[0049] Determination of total alkaloids in Sophora flavescens by HPLC method ≥ 10%;

[0050] Safety detection: endotoxin < 0.25 EU / mL (limulus reagent method);

[0051] Microbial limit: the total number of aerobic bacteria, molds and yeasts all meet the aseptic preparation standards of the Chinese Pharmacopoeia.

[0052] Experimental Example 2, the effect of the compound preparation of the present invention on the rat model of cervicitis.

[0053] 2.1 Construction of a rat model of cervicitis: Female SD rats (6 - 8 weeks old) were selected to establish a cervicitis model. The cervical inflammatory and infectious environment was simulated by local injection of lipopolysaccharide (LPS) or human papillomavirus (HPV).

[0054] 2.2 Experimental grouping:

[0055] Compound preparation group: Vaginal local administration was given daily, and the compound preparation prepared in Example 1 (interferon α1b 1×10 5 IU / mL + total alkaloids in Sophora flavescens 1% concentration) 0.1 mL / time, for 7 consecutive days; n = 10;

[0056] Control group 1: The preparation prepared in Comparative Example 1 (interferon α1b with the same concentration as the compound preparation group) was given, and the dose was the same as that of the compound preparation group; n = 10;

[0057] Control group 2: The preparation prepared in Comparative Example 2 (Sophora flavescens extract with the same concentration as the compound preparation group) was given, and the dose was the same as that of the compound preparation group; n = 10;

[0058] Blank control group: Healthy animals (without induced cervicitis); n = 10;

[0059] Model group: Animals with cervicitis model, without drug treatment; n = 10;

[0060] 2.3 Experimental methods and test indicators

[0061] Histopathological analysis: After the administration was completed, cervical tissues were taken, and HE staining was used to evaluate the infiltration of inflammatory cells, epithelial integrity and degree of vascular dilation.

[0062] Detection of inflammatory factors: ELISA method was used to detect the levels of IL-6, TNF-α, and IL-1β in the cervical tissue homogenate.

[0063] Immunohistochemical analysis: Detect CD68 in cervical mucosa + Degree of macrophage infiltration and activation of STAT signaling pathway.

[0064] 2.4 Analysis of experimental results

[0065] Referring to the data in Table 1, the inflammation scores, IL-6 and TNF-α levels in the model group were significantly higher than those in the blank control group (P<0.01), and the integrity of cervical epithelium decreased to 30%, indicating the successful establishment of the disease model.

[0066] The levels of inflammatory factors (IL-6, TNF-α) in the compound preparation group decreased by 78%-82% compared with the model group, and were significantly better than those in the single drug group (P<0.05); the cervical epithelial repair rate (90%) was close to that of the blank control group. Animal experiments showed that the compound preparation of human interferon α1b and Sophora flavescens extract significantly improved the pathological state of cervical inflammation by synergistically inhibiting the release of inflammatory factors, promoting mucosal repair and enhancing local immune response. Its efficacy and safety data provided a theoretical basis for subsequent clinical trials.

[0067] It can be seen from the experimental data that the compound preparation can significantly reverse the pathological state of the cervicitis model, and the effect is better than that of single drug treatment; its mechanism of action is closely related to inhibiting the release of inflammatory factors, reducing immune cell infiltration and promoting epithelial repair.

[0068] Table 1: Key index data of each group.

[0069]

[0070] Note: Data are expressed as mean ± standard deviation, *P<0.05 (vs model group), **P<0.01 (vs model group), #P<0.05 (vs single drug group).

[0071] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A human interferon gynecological antibacterial compound preparation, characterized in that: The invention comprises component A containing interferon alpha 1b and component B containing sophora flavescens extract, wherein the volume ratio of component A to component B is 1:(8-10), and the pH value of the composite preparation is 6.5-7.

0.

2. A human interferon gynecological antibacterial compound preparation according to claim 1, characterized in that: The preparation method of component A is as follows: the concentration of the purified interferon α1b solution is adjusted to 1×10 5 -1×10 6 IU / mL, and 0.1-1.0% human albumin (w / v) was added as a stabilizer, and the pH was adjusted to 6.5-7.

0.

3. A human interferon gynecological antibacterial compound preparation according to claim 1, characterized in that: The preparation method of the B component is as follows: prepare the Sophora flavescens extract, and dissolve the extract in a sterile phosphate buffer solution with a pH of 6.5, wherein the concentration of the Sophora flavescens extract is 1%-3%.

4. The method for preparing a human interferon gynecological antibacterial compound preparation according to claim 1, characterized in that: The following steps are involved: S1, mix component A and component B in a volume ratio of 1:(9-10), and the final concentration of interferon α1b is 1×10 5 -1×10 6 IU / mL; Adjust the pH of the mixture to 6.5-7.0 with 0.1 M sodium bicarbonate; The mixed solution prepared in S2 and S1 was sterilized by filtration through a 0.22 μm filter membrane to obtain the composite preparation.

5. Use of the composite preparation according to any one of claims 1 to 3 in the preparation of drugs for treating cervicitis.

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