CN115969874A - Pharmaceutical composition for resisting influenza virus - Google Patents
Pharmaceutical composition for resisting influenza virus Download PDFInfo
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Abstract
The invention provides a pharmaceutical composition for resisting influenza virus, relating to the field of pharmaceutical compositions, and the raw materials of the composition comprise: chlorogenic acid, caffeic acid, hyperoside, protocatechuic acid, p-hydroxyphenylacetic acid and Senecio cannabifolius less polysaccharide. In addition, the composition has excellent effects when used in combination with isoquercitrin and p-hydroxybenzoic acid. The composition has excellent efficacy, has a certain action relation to influenza viruses, and can play a role in efficiently resisting the influenza viruses.
Description
Technical Field
The invention relates to the field of pharmaceutical compositions, and in particular relates to a pharmaceutical composition for resisting influenza viruses.
Background
The herba Senecionis Cannabifolii granule is a Chinese medicinal preparation prepared from water extract of herba Senecionis Cannabifolii (SCH) of Senecio of Compositae. Contains flavonoid, phenolic acid, and alkaloid. Has the functions of clearing heat, eliminating phlegm, relieving cough and relieving asthma, and is clinically used for intrapulmonary infection, chronic bronchitis, asthmatic bronchitis, acute respiratory infection and the like. The literature reports that the Senecio cannabifolius less has the function of resisting influenza viruses.
The existing preparations taking Senecio cannabifolius less as main raw materials comprise Feining granules, feining dropping pills, feining dispersible tablets and the like, generally have certain functions of clearing heat, eliminating phlegm and relieving cough, and are mainly used for relieving symptoms such as chronic bronchitis cough and the like. Relevant studies, such as literature: gao Xiao Ming, zhang Ying, wangdi, et al, fengning tablets influence Th1/Th2 cytokines of cough patients after cold [ J ] Liaoning university of traditional Chinese medicine, 2016, stress study the influence of Fengning tablets on Th1/Th2 cytokines of cough patients after cold. The literature: the Houbao lung Ning particle secondary development [ D ] Jilin university, 2012, adopts a High Performance Liquid Chromatography (HPLC) method to respectively carry out quantitative analysis on phenolic acid components in the Senecio cannabifolius less by using chlorogenic acid and flavonoid compounds by using quercetin. Provides scientific basis for developing new generation lung-calming granules and optimizing the preparation extraction process.
Such as the patent: CN101278958A (China; published: 2008.10.8) discloses a medicine for treating influenza, laryngopharyngitis, pneumonia and other diseases, which is prepared from Senecio cannabifolius less as raw material and medicinal carrier as matrix, and has the advantages of quick release, quick effect and low cost. For another example, CN101278959A (country: china; published: 2008.10.8) discloses a dispersible tablet for treating lung diseases similar to the above patent and its preparation method, and the related dispersible tablet also has the effect of treating influenza, pharyngolaryngitis, pneumonia, etc. In addition, the patents CN109646473A (country: china; published: 2019.4.19) and CN103417593A (country: china; published: 2013.12.4) and the like have conducted research on related drugs. However, the research of the related preparations at present focuses on the optimization development of the process, or the matching use of the preparations with other auxiliary materials or raw material medicines, so that the deep research of related functional components is rarely carried out, or further developed medicaments have various problems of poor efficacy, low safety, weak pertinence to influenza viruses and the like.
Aiming at the problems, a composition which has more excellent efficacy, strong pertinence to influenza viruses and safety is required to be found for preventing and treating the influenza virus infection.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a pharmaceutical composition for resisting influenza virus, which has more excellent efficacy, strong pertinence to the influenza virus and safety, and can effectively prevent and treat influenza virus infection.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the invention provides a composition, which comprises the following raw materials: chlorogenic acid, caffeic acid, hyperoside, protocatechuic acid, p-hydroxyphenylacetic acid and Senecio cannabifolius less polysaccharide.
Further, the composition comprises the following raw materials in parts by weight: 0.18-0.25 parts of chlorogenic acid, 0.01-0.02 parts of caffeic acid, 0.1-0.2 parts of hyperin, 0.01-0.02 parts of protocatechuic acid, 0.15-0.2 parts of p-hydroxyphenylacetic acid and 8-9.5 parts of Senecio cannabifolius less polysaccharide.
Preferably, the composition comprises the following raw materials in parts by weight: 0.2 part of chlorogenic acid, 0.015 part of caffeic acid, 0.16 part of hyperoside, 0.01 part of protocatechuic acid, 0.18 part of p-hydroxyphenylacetic acid and 9 parts of Senecio cannabifolius less polysaccharide.
Further, the weight ratio of the chlorogenic acid to the hyperin to the senecio cannabifolius less polysaccharide ranges from 0.18 to 0.25 to 8 to 9.5. Preferably 0.2.
Further, the invention also provides a preparation method of the composition, which comprises the following steps: mixing chlorogenic acid, caffeic acid, hyperoside, protocatechuic acid, p-hydroxyphenylacetic acid, and herba Senecionis Cannabifolii polysaccharide.
Further, the composition also comprises isoquercitrin and p-hydroxybenzoic acid.
Preferably, the composition further comprises, in parts by weight: 0.005-0.009 parts of isoquercitrin and 0.001-0.005 parts of p-hydroxybenzoic acid. More preferably, isoquercitrin is 0.007 parts, and parahydroxybenzoic acid is 0.003 parts.
Further, the invention also provides a preparation method of the composition, which comprises the following steps: mixing chlorogenic acid, caffeic acid, hyperoside, protocatechuic acid, p-hydroxyphenylacetic acid, herba Senecionis Cannabifolii polysaccharide, isoquercitrin, and p-hydroxybenzoic acid.
Further, the composition provided by the invention can be used for preparing a medicament for resisting influenza viruses.
Further, the influenza virus is influenza a virus H1N1.
Further, the present invention also provides a medicament for resisting influenza virus, comprising: the composition and medically usable auxiliary materials; the excipients include, but are not limited to, binders.
Further, the dosage form of the medicament comprises tablets, capsules, granules, paste, liquid, pills or suspensions.
Preferably, the dosage form is a granule.
The technical effects obtained by the invention are as follows:
1. the medicinal composition for resisting influenza virus is prepared by carrying out further intensive research on the basis of the Senecio cannabifolius less, and matching various effective extraction components in the Senecio cannabifolius less, has a certain action relationship on influenza virus, can play a role in efficiently resisting the influenza virus, and can be used for preparing related medicaments such as feining particles and the like.
2. In the research, the invention discovers that the synergistic effect exists among partial active ingredients in the Senecio cannabifolius less, such as chlorogenic acid, hyperin and Senecio cannabifolius less polysaccharide, and meanwhile, partial active ingredients are further matched with isoquercitrin and p-hydroxybenzoic acid for use together, so that the finally prepared composition also has more excellent anti-influenza virus effect.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not to be limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It should be noted that the raw materials used in the present invention are all common commercial products, and thus the sources thereof are not particularly limited.
TABLE 1 compositions and proportions of the compositions of examples 1-6 of the present invention
TABLE 2 compositions and proportions of comparative examples 1-4 of the present invention
The preparation methods of examples 1 to 3 and comparative examples 1 to 4 include the following steps: mixing chlorogenic acid, caffeic acid, hyperoside, protocatechuic acid, p-hydroxyphenylacetic acid, and Senecio cannabifolius less polysaccharide (prepared by phase synchronization after replacing raw materials in comparative example 4).
The preparation process of examples 4-6 includes the following steps: mixing chlorogenic acid, caffeic acid, hyperoside, protocatechuic acid, p-hydroxyphenylacetic acid, senecio cannabifolius less polysaccharide, isoquercitrin, and p-hydroxybenzoic acid.
Application example 1
Granules: the composition prepared in example 1 is mixed with auxiliary materials (including but not limited to binders), and then the mixture is granulated, dried, sized and classified to obtain granules.
Application example 2
Granules: the composition prepared in example 3 is mixed with auxiliary materials (including but not limited to binders), and then the mixture is granulated, dried, sized and classified to obtain granules.
1. Materials and methods
1. Experimental Material
1.1 test drugs
Feining granules: supplied by Jilin Huakang pharmaceutical products, inc. (national Standard Z22021801, batch No. 220301).
Comparative examples 1-4, examples 1-6 were prepared as described above and supplied by Jilin Huakang pharmaceutical industry, inc.
1.2 animals, cells and viral strains
(1) BALB/c mice, SPF grade, purchased from Yinss laboratory animal technology, inc., catharan city, animal production certification number: SCXK (Ji) -2020-0002.
(2) Animal feeding and experiments are carried out in the experimental animal center of the basic medical college of Jilin university, the whole process accords with the requirements of animal ethical examination, and animals are fed with common maintenance feed and freely drink water.
(3) Vero cells were cryopreserved by the basic medical institute of gilin university.
(4) Virus strains: influenza a virus H1N1, purchased from ATCC, college of basic medicine, jilin university.
1.3 reagents
The interleukin 6 (IL-6) detection kit, the tumor necrosis factor alpha (TNF-alpha) detection kit and the interleukin-10 (IL-10) detection kit are all purchased from Shanghai enzyme-linked biotechnology, inc.
2. Method for producing a composite material
2.1 grouping and administration of Experimental animals
(1) BALB/c mice were randomly divided into: the body weight of mice was recorded in the blank group, model group, pulmonic group, comparative example 1 group, comparative example 2 group, comparative example 3 group, comparative example 4 group, example 1-high dose group, example 1-medium dose group, example 1-low dose group, example 2 group, example 3 group, example 4 group, example 5 group, example 6 group, each group consisting of 6 mice. After the mice are anesthetized by 10% sodium pentobarbital, normal groups are dripped into the nose by 0.05mL of physiological saline, and the other groups of mice are infected by 0.05mL of H1N1 influenza virus solution, so that an influenza model is established.
(2) The groups were as above except for the normal group, and 100-fold TCID was performed by nasally dropping H1N1 virus solution into each group of mice 50 Infection, 0.05 mL/mouse. After 24h of infection, the administration was performed by gavage for 3 times/d/mouse for 6 consecutive days, wherein the administration was performed 180 mg/time in the example 1-high dose group, 90 mg/time in the example 1-medium dose group, 45 mg/time in the example 1-low dose group, and the other groups were identical to the example 1-low dose group, and the same amount of physiological saline was administered by gavage in the normal group. No animal died during the experiment, the final weight of the mouse was counted at 6d, the final weight change was calculated, the mouse was dissected, blood was taken and kept in a low temperature freezer at-80 ℃ for future use.
2.2 detection of mouse serum cytokine content
And (3) taking mouse serum, and detecting the content of the inflammatory factor IL-6 in the mouse serum of each group by adopting an interleukin 6 (IL-6) detection kit.
2.3 toxicity test of groups of drug Vero cells
The medicines are dissolved in 100mL cell maintenance liquid preheated at 37 ℃, and the dissolved medicine liquid is filtered by sterile filter heads with the diameters of 0.45 mu m and 0.22 mu m respectively for standby. Respectively diluting the mother liquor according to concentration gradient to 8 liquid medicines with concentration of the diluted liquid and the mother liquor, wherein the concentration of the diluted liquid is 40 mg/mL, 30 mg/mL, 20 mg/mL, 10 mg/mL, 5 mg/mL, 1 mg/mL and 0.1 mg/mL, and the liquid medicines are used for standby. Cells in logarithmic growth phase were inoculated into 96-well plates (1X 10) 5 one/mL, 100. Mu.L per well). 24 After h, each concentration of drug solution, 100. Mu.L/well, 3 multiple wells per concentration, was added, simultaneouslyBlank control wells. After 24h of drug action, the medium was discarded and 100. Mu.L of the maintenance solution and 10. Mu.L of CCK8 reagent were added to each well. The 96-well plate was incubated for an additional 2 hours and the concentration determined to have no effect on cell survival was the maximum non-toxic concentration (TC 0) for each group of drugs, as shown in table 5. We finally used 5 mg/mL, 10 mg/mL, 20 mg/mL as the low, medium, and high concentrations in example 1 for subsequent experiments, and the toxicity results of the remaining groups were examined by subsequent experiments using 10 mg/mL concentrations, as detailed in Table 6.
2.4 groups of drugs act against influenza viruses in vitro
Will be 1 × 10 5 Vero cells/mL were seeded in 96-well plates at 100. Mu.L/well. After 24h, the culture medium was aspirated, washed 3 times with PBS, and each of the example and model groups was inoculated with 10 -2.5 Dilution multiple influenza A virus H1N1 to Vero cells (TCID 50 10 each) -5 ) The blank group is only inoculated with equal amount of Vero cells, each hole is 100 mu L, the absorption is 1 h, PBS is washed for 3 times, PBS is absorbed and discarded, each example group is added with each group of medicine solution, the blank group and the model group are added with equal amount of normal saline, 100 mu L/hole, each 5 multiple holes, the subsequent experiments are carried out by adopting 5 mg/mL, 10 mg/mL and 20 mg/mL as the low, medium and high concentrations of the example 1, the rest groups are carried out by adopting 10 mg/mL concentration for subsequent experiments, the disease rate is observed, and the influence of each group of medicine on the cytopathic effect of the influenza virus is counted.
2.5 detection of the content of various inflammatory factors in Vero cells
In each example, 3 groups of samples were taken for cell supernatants, and the contents of inflammatory factors IL-6, TNF- α and IL-10 in each group were measured using an interleukin 6 (IL-6) assay kit, a tumor necrosis factor α (TNF- α) assay kit and an interleukin-10 (IL-10) assay kit.
3. Statistical analysis
All experimental data in this study were statistically analyzed using Graphpad prism V6.0 and the experimental results were expressed as x (mean + -s (standard deviation); homogeneity of variance and normal distribution of data were first examined and then analyzed using t-test. P <0.05 shows statistical differences, P <0.01 shows statistical differences.
2. Results
1. Protective effect of each group of medicines on H1N1 infected mice
TABLE 3 Final body weight changes in mice
Note: * Represents significant difference compared with the model group, namely P <0.05, and is extremely significant difference, namely P <0.01; # indicates a significant difference compared to the feining granule group, i.e., P <0.05, # # is the most significant difference, i.e., P <0.01.
TABLE 4 changes in the expression level of IL-6 inflammatory factors
Note: * The representation has significant difference compared with the model groupP<0.05 as a very significant difference i.e.P<0.01; # shows that there is a significant difference from the granule group of FEININGP<0.05, # # is a very significant difference, namelyP<0.01。
2. The anti-virus function of each group in vitro
The maximum non-toxic concentrations of each group of drugs are shown in the following table:
TABLE 5 maximum non-toxic concentration of each group of drugs
The results are shown in Table 6, "+" indicates toxic and "-" indicates non-toxic.
TABLE 6 toxicity of each group of drugs to cells
TABLE 7 Effect (CPE) of groups of drugs on cytopathic Effect of influenza Virus (n = 5)
Note: cytopathic effect is judged according to 6-grade standard: -indicates that the cells are growing normally and no lesions are present; 1 represents cytopathic less than 10% of the entire monolayer; 2 indicates that the cytopathic effect is less than about 25% of the total monolayer of cells; 3 indicates that the cytopathic effect is less than about 50% of the total monolayer of cells; 4 indicates that the cytopathic effect is less than about 75% of the total monolayer of cells; + indicates that the cytopathy accounts for more than about 75% of the total monolayer of cells.
TABLE 8 changes in expression levels of IL-6 inflammatory factor in vitro following viral infection
Note: * Represents significant difference compared with the model group, namely P <0.05, and is extremely significant difference, namely P <0.01; # indicates a significant difference compared to the granule group of feinin, i.e., P <0.05, # # is a very significant difference, i.e., P <0.01.
TABLE 9 changes in the expression levels of TNF-alpha inflammatory factor in vitro following viral infection
Note: * The representation is significantly different from the model group thatP<0.05 as a very significant difference i.e.P<0.01; # shows a significant difference from the FEINING granule group, namelyP<0.05, # # is a very significant difference, namelyP<0.01。
TABLE 10 changes in expression levels of IL-10 inflammatory factor in vitro following viral infection
Note: * The representation has significant difference compared with the model groupP<0.05 as a very significant difference i.e.P<0.01; # shows that there is a significant difference from the granule group of FEININGP<0.05, # # is the most significant difference, i.e.P<0.01。
3. Conclusion
The composition of the present invention has a better anti-influenza a virus H1N1 effect both in vivo and in vitro, and its possible mechanism is to promote the expression of the cytokine IL-10 inhibiting inflammation to slow down the virus-induced inflammatory response (reduced levels of IL-6 and TNF- α), wherein the effect of the examples is better than that of the comparative example and the feining granule group, the high, medium and low doses of example 1 are better than those of examples 2-3, and the optimal dose is the high dose of example 1; the comparative example and the FENGNING granule have the same effect.
Finally, it should be noted that the above-mentioned contents are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, and that the simple modifications or equivalent substitutions of the technical solutions of the present invention by those of ordinary skill in the art can be made without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. A composition characterized by: the raw materials comprise: chlorogenic acid, caffeic acid, hyperoside, protocatechuic acid, p-hydroxyphenylacetic acid and Senecio cannabifolius less polysaccharide.
2. The composition of claim 1, wherein: the raw materials comprise the following components in parts by weight: 0.18-0.25 parts of chlorogenic acid, 0.01-0.02 parts of caffeic acid, 0.1-0.2 parts of hyperoside, 0.01-0.02 parts of protocatechuic acid, 0.15-0.2 parts of p-hydroxyphenylacetic acid and 8-9.5 parts of Senecio cannabifolius less polysaccharide.
3. The composition of claim 1, wherein: the raw materials comprise the following components in parts by weight: 0.2 part of chlorogenic acid, 0.015 part of caffeic acid, 0.16 part of hyperoside, 0.01 part of protocatechuic acid, 0.18 part of p-hydroxyphenylacetic acid and 9 parts of Senecio cannabifolius less polysaccharide.
4. The composition of claim 1, wherein: the weight ratio of the chlorogenic acid to the hyperoside to the Senecio cannabifolius less polysaccharide is 0.18 to 0.25 and is (8 to 9.5).
5. The composition of claim 1, wherein: also comprises 0.005-0.009 parts of isoquercitrin and 0.001-0.005 parts of p-hydroxybenzoic acid.
6. A process for preparing a composition according to any one of claims 1 to 4, characterized in that: the method comprises the following steps: mixing chlorogenic acid, caffeic acid, hyperoside, protocatechuic acid, p-hydroxyphenylacetic acid, and herba Senecionis Cannabifolii polysaccharide.
7. A method of preparing the composition of claim 5, wherein: the method comprises the following steps: mixing chlorogenic acid, caffeic acid, hyperoside, protocatechuic acid, p-hydroxyphenylacetic acid, herba Senecionis Cannabifolii polysaccharide, isoquercitrin, and p-hydroxybenzoic acid.
8. Use of a composition according to any one of claims 1 to 5 in the manufacture of a medicament for use against an influenza virus.
9. A medicament for use against an influenza virus, characterized by: comprising a composition according to any one of claims 1 to 5 and a medically acceptable excipient.
10. The medicament of claim 9, wherein: the dosage form of the medicine comprises tablets, capsules, granules, paste, liquid, pills or suspensions.
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