CN116509938A - Traditional Chinese medicine compound composition with enterovirus 71-type resisting effect and preparation method and application thereof - Google Patents
Traditional Chinese medicine compound composition with enterovirus 71-type resisting effect and preparation method and application thereof Download PDFInfo
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- A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
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Abstract
The invention belongs to the technical field of medicines and foods, and particularly relates to a traditional Chinese medicine compound composition with an enterovirus 71-type resisting effect, and a preparation method and application thereof. The traditional Chinese medicine compound composition is prepared from the following raw materials in parts by weight: 6-12 parts of lucid ganoderma, 10-20 parts of grifola frondosa, 5-10 parts of chrysanthemum, 6-15 parts of honeysuckle and 15-25 parts of cordate houttuynia. Experimental results show that the traditional Chinese medicine compound composition can effectively prevent enterovirus 71 from entering vero cells in vitro and inhibit virus-related mRNA from replicating in the cells, and can be used for preparing medicines, foods or sanitary products for resisting enterovirus 71.
Description
Technical Field
The invention belongs to the technical field of medicines and foods, and particularly relates to a traditional Chinese medicine compound composition with an enterovirus 71-type resisting effect, and a preparation method and application thereof.
Background
Enterovirus 71 (EV-71) belongs to the genus enterovirus of the family picornaviridae, is an enterovirus which is discovered at the latest at present, and has strong infectivity and high pathogenicity. EV-71 is frequently generated in infants under 5 years old, hand-foot-mouth disease and central nervous system infection are two common clinical symptoms caused by EV-71 infection, and complications such as myocarditis, pulmonary edema, aseptic meningoepithymen and the like can be caused when the infection is severe, which is one of the important causes of death of infants nowadays. As specific prevention and treatment methods are lacking in the aspect of resisting EV-71 viruses at present, the method depends on vaccines to a great extent, and meanwhile, the currently used medicines have the defects of large toxic and side effects, easy drug resistance, incapacity of thoroughly eliminating viruses and the like, for example, a great deal of researches show that ribavirin has obvious teratogenic toxic effects. Therefore, the research on safe and efficient antiviral drugs in natural drugs is particularly important.
Ganoderma lucidum is a fungus of Polyporaceae, namely Ganoderma lucidum, and dried fruiting body thereof, has the effects of tonifying qi, soothing nerves, relieving cough and asthma, contains active ingredients which have wide immunoregulatory activity, antioxidation and anti-aging, and some proteoglycan and triterpene compounds in Ganoderma lucidum have broad-spectrum antiviral effects. Grifola frondosa is a fruiting body of Polyporus frondosus of Polyporaceae. Is a precious dual-purpose fungus for medicine and food, and has the effects of replenishing qi and strengthening spleen; the efficacy of reinforcing deficiency and strengthening body resistance is widely applied to the treatment of spleen deficiency. For spleen deficiency and qi weakness; the effects of tiredness and hypodynamia are obvious, and the grifola frondosa also has stronger effects on improving immunity, resisting tumors, resisting viruses and the like. The chrysanthemum is used as a food and a Chinese medicinal material, has the effects of dispelling wind and clearing heat, suppressing hyperactive liver and improving eyesight, clearing heat and detoxicating, and the like, and modern researches find that the chrysanthemum has various effects of enhancing the immunity of organisms, delaying aging, resisting oxidation, resisting viruses, resisting bacteria, and the like; the honeysuckle is used as a medicine and food dual-purpose traditional Chinese medicine, and has the effects of resisting bacteria, resisting viruses, clearing heat and detoxicating, resisting inflammation, enhancing immunity and the like; herba Houttuyniae is a medicinal and edible plant, also called as herba Latifoliae, herba Potentillae Anserinae and the like, and has the effects of clearing heat and detoxicating, removing carbuncles and expelling pus, promoting urination and treating stranguria, and is known as a broad-spectrum antibiotic in traditional Chinese medicines, and researches show that the herba Houttuyniae has various pharmacological effects of resisting viruses, bacteria, tumors, radiation and immunity.
The invention prepares the traditional Chinese medicine compound composition with the effect of resisting enterovirus 71 by lucid ganoderma, grifola frondosa, chrysanthemum, honeysuckle and cordate houttuynia, and the product is used as a plant extract with homology of medicine and food, has the advantages of good effect, quick response and no toxic or side effect on antivirus, has good popularization and use values and has wide development prospect.
Disclosure of Invention
The invention aims to provide a traditional Chinese medicine compound composition with an enterovirus 71-type resisting effect, and a preparation method and application thereof.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a traditional Chinese medicine compound composition with an enterovirus 71-type resisting effect is prepared from the following raw materials in parts by weight: 6-12 parts of lucid ganoderma, 10-20 parts of grifola frondosa, 5-10 parts of chrysanthemum, 6-15 parts of honeysuckle and 15-25 parts of cordate houttuynia.
Preferably, the traditional Chinese medicine compound composition is prepared from the following raw materials in parts by weight: 6 parts of lucid ganoderma, 15 parts of grifola frondosa, 5 parts of chrysanthemum, 6 parts of honeysuckle and 15 parts of houttuynia cordata.
The preparation method of the traditional Chinese medicine compound composition with the effect of resisting enterovirus 71 comprises the following steps:
1) Taking 6-12 parts by weight of ganoderma lucidum according to a feed liquid ratio of 1: mixing 15m/v with ultrapure water, adjusting pH to 8.5, adding alkaline protease according to the proportion of 2% of Ganoderma, oscillating at 55deg.C for 2 hr, inactivating enzyme in boiling water bath for 10min, cooling to room temperature, filtering, concentrating the filtrate, and lyophilizing to obtain Ganoderma alkaline protease zymolyte;
2) Taking 10-20 parts by weight of grifola frondosa according to a feed liquid ratio of 1: mixing 15m/v with ultrapure water, adjusting pH to 7.8, adding compound protease according to the proportion of 4% of the weight of Grifola frondosa, oscillating for 2h at 53 ℃, inactivating enzyme in boiling water bath for 10min, cooling to room temperature, filtering, concentrating the filtrate, and lyophilizing to obtain Grifola frondosa compound protease zymolyte;
3) Taking 5-10 parts by weight of chrysanthemum, 6-15 parts by weight of honeysuckle and 15-25 parts by weight of cordate houttuynia according to a feed liquid ratio of 1: mixing 20m/v with ultrapure water, soaking at room temperature for 2h, decocting for 30min in a micro-boiling state, cooling to room temperature, and filtering to obtain primary filtrate and primary filter residue; adding ultrapure water with the mass 10 times of that of the primary filter residue into the primary filter residue, keeping a micro-boiling state, decocting for 30min, cooling to room temperature, and filtering to obtain secondary filtrate and secondary filter residue; combining the primary filtrate and the secondary filtrate, centrifuging, collecting the supernatant, concentrating, and freeze-drying to obtain the chrysanthemum and honeysuckle and houttuynia cordata composite water extract;
4) Uniformly mixing the ganoderma lucidum alkaline protease zymolyte obtained in the step 1) and the grifola frondosa compound protease zymolyte obtained in the step 2) and the chrysanthemum and honeysuckle cordate houttuynia compound aqueous extract obtained in the step 3) to obtain the traditional Chinese medicine compound composition with the enterovirus 71-resisting effect.
Application of the traditional Chinese medicine compound composition in preparation of anti-enterovirus 71 type products.
The invention has the remarkable advantages that:
1) The invention adopts natural substances as raw materials.
2) The invention has scientific compatibility and high safety performance, and is suitable for industrial production.
3) The traditional Chinese medicine compound composition has enterovirus 71-resistant activity, can better achieve an antiviral effect by improving the immunity of organisms, has better health care value, has a certain commercial value and has wide development prospect.
Drawings
Fig. 1 shows the substance toxicity of the herbal compound composition to vero cells.
FIG. 2 shows the effect of a herbal composition on the morphology of EV-71 virus-infected vero cells.
FIG. 3 shows the effect of a herbal compound composition on the viability of EV-71 virus-infected vero cells.
FIG. 4 shows the effect of a herbal compound composition on EV-71 virus capsid protein VP 1.
Detailed Description
In order to make the contents of the present invention more easily understood, the technical scheme of the present invention will be further described with reference to the specific embodiments, but the present invention is not limited thereto.
In the following examples, alkaline protease was purchased from Beijing Soy Bao technology Co., ltd, with a product number of B8360, and its enzyme activity/potency was not less than 200000U/g.
In the following examples, the complex protease was purchased from Beijing Soy Bao technology Co., ltd, with a product number of C8800, and its enzyme activity/potency was not less than 100units/mg.
Example 1
A Chinese medicinal compound composition with anti-enterovirus 71 type effect is prepared from the following raw materials in parts by weight: 6g of lucid ganoderma, 15g of grifola frondosa, 5g of chrysanthemum, 6g of honeysuckle and 15g of cordate houttuynia.
The preparation method of the traditional Chinese medicine compound composition comprises the following steps:
1) Mixing 6g of ganoderma lucidum with 90mL of ultrapure water, adjusting the pH value to 8.5, adding alkaline protease according to the proportion of 2% of the weight of the ganoderma lucidum, oscillating for 2 hours in a water bath at 55 ℃, inactivating the enzyme in the boiling water bath for 10 minutes, cooling to room temperature, and filtering to obtain filtrate; concentrating the filtrate at 70deg.C under reduced pressure to 12mL to obtain concentrated solution; pre-freezing the concentrated solution at-80deg.C for 12 hr, and freeze drying at-70deg.C for 24 hr to obtain Ganoderma alkaline protease zymolyte.
2) Mixing 15g of grifola frondosa with 225mL of ultrapure water, adjusting the pH value to 7.8, adding compound protease according to the proportion of 4% of the weight of the grifola frondosa, oscillating water bath at 53 ℃ for 2h, inactivating enzyme in boiling water bath for 10min, cooling to room temperature, and filtering to obtain filtrate; concentrating the filtrate at 70deg.C under reduced pressure to 15mL to obtain concentrated solution; pre-freezing the concentrated solution at-80deg.C for 12 hr, and freeze-drying at-70deg.C for 24 hr to obtain Maitake Mushroom compound protease zymolyte.
3) Mixing 5g of chrysanthemum, 6g of honeysuckle, 15g of cordate houttuynia with 520mL of ultrapure water, soaking for 2 hours at room temperature, then decocting for 30 minutes in a micro-boiling state, cooling to room temperature, and filtering to obtain primary filtrate and primary filter residue; adding ultrapure water with the mass 10 times of that of the primary filter residue into the primary filter residue, keeping a micro-boiling state, decocting for 30min, cooling to room temperature, and filtering to obtain secondary filtrate and secondary filter residue; combining the primary filtrate and the secondary filtrate, centrifuging at 4000r/min for 10min, collecting supernatant, and concentrating under reduced pressure at 80deg.C to 15mL to obtain concentrated solution; pre-freezing the concentrated solution at-80deg.C for 12 hr, and freeze-drying at-70deg.C for 24 hr to obtain flos Chrysanthemi and flos Lonicerae herba Houttuyniae compound water extract.
4) Uniformly mixing the ganoderma lucidum alkaline protease zymolyte obtained in the step 1) and the grifola frondosa compound protease zymolyte obtained in the step 2) and the chrysanthemum and honeysuckle cordate houttuynia compound aqueous extract obtained in the step 3) to obtain the traditional Chinese medicine compound composition with the enterovirus 71-resisting effect.
Example 2
The inhibiting effect of the traditional Chinese medicine compound composition on enterovirus type 71 is shown in the following experiment:
1. the experimental method comprises the following steps:
1. experimental materials
Vero cells (laboratory preservation); the EV-71 virus strain is given away by a teacher of Fujian university Zhao Chao; DMEM medium, trypsin, diabody, fetal Bovine Serum (FBS) were purchased from fujian midwifery.
2. Grouping and dose design
(1) Positive drug control: ribavirin is added with a dosage of 25 mug/ml;
(2) Negative control: 2% DMEM
(3) The Chinese medicinal compound composition is dissolved in 2% DMEM to a concentration of 0.195, 0.391, 0.781, 1.562 and 3.125mg/ml.
3. Cytopathy
EV-71 virus can cause vero cells to find lesions, known as CPE effect, with significantly reduced cell viability. The negative control group can see obvious pathological effect after virus is added, and if the sample can effectively inhibit virus infection, few pathological effect can appear, and the cell state is still good.
4. Toxicity of the traditional Chinese medicine compound to vero cells:
after vero cells grow over 90% in a T25 flask and are digested for 2 minutes, they are blown into a centrifuge tube and collected, and a cell suspension is prepared by adding a culture medium, and cell counting is performed. Adjusting the number of cells to about 5000 cells per hole, paving the cells into 96-well plates, adding PBS (phosphate buffer solution) around the cells to prevent pollution, discarding old culture medium after the cells grow to 60-70% of fusion degree, adding 2% DMEM (dmem) culture medium containing Chinese herbal compound compositions with different concentrations to continue culturing the cells, setting 6 repeated holes for each gradient, sequentially setting the gradients to 0.195, 0.391, 0.781, 1.562 and 3.125mg/ml, placing the 96-well plates into an incubator to normally culture for 48 hours, and detecting the activity of the cells by adopting a CCK-8 kit to further judge the toxicity of the compound to the cells.
Cell viability calculation formula:
cell viability (%) = [ (dosing) - (blank) ]/(control)/(blank) ] x100%
Adding the medicine: absorbance of wells with cells, CCK-8 solution and drug solution
Blank: absorbance of wells with medium and CCK-8 solution without cells
Control: absorbance of wells with cells, CCK-8 solution without drug solution
5. The effect of the Chinese herbal compound on the activity of EV-71 infected cells:
inoculating vero cells into 96-well plate with 5000 cells per well, culturing in cell culture box for 24 hr, discarding old culture medium, and adding 100ul 100TCID 50 Cells were infected with/ml EV-71 virus, and after 2 hours, the cells were continuously cultured by changing 2% DMEM medium containing different concentrations of the Chinese herbal compound composition, 6 duplicate wells were set for each gradient, the gradients were set to 0.0975, 0.195, 0.391, 0.781, 1.562mg/ml in order, and positive drug groups, blank control group and virus control group were set, and after 96-well plates were placed in an incubator for normal culture for 24 hours, cell activity was detected with CCK-8 kit.
6. Effects of traditional Chinese medicine compound on EV-71 viral capsid protein VP 1:
plating vero cells in a 6-hole plate, placing the 6-hole plate into an incubator for culturing for 24 hours until the cells are full of 80-90%, adding EV-71 virus for infecting for 2 hours, discarding the virus liquid, changing the virus liquid into a traditional Chinese medicine compound composition of 0.097, 0.391 and 1.5mg/ml, placing the 6-hole plate into the incubator for culturing for 24 hours, adding Trizon lysate for lysing the cells, collecting the cells into a centrifuge tube, extracting RNA, and detecting the expression of VP1 by adopting fluorescence quantification qpcr.
2. Experimental results
1. The effect result of the compound Chinese medicine composition on vero cell toxicity is shown in figure 1, and the compound Chinese medicine composition has no toxicity to cells below the concentration of 3.125mg/ml, so that no error is generated in the subsequent antiviral experiment performed below the concentration.
2. The effect of the compound traditional Chinese medicine composition on the morphology of vero cells infected by EV-71 virus is shown in figure 2, and compared with the control group, the virus group has obvious CPE effect, however, when the compound traditional Chinese medicine composition is added, the CPE effect can be obviously reduced, and as the concentration is increased, the CPE effect is smaller.
3. The effect of the compound Chinese medicine composition on the activity of EV-71 infected cells is shown in figure 3, and the inhibiting rate of the compound Chinese medicine composition on EV-71 viruses gradually becomes higher and the vero cell survival rate gradually increases along with the increase of the concentration of the compound Chinese medicine composition.
4. The effect of the compound Chinese medicine composition on the capsid protein VP1 of EV-71 virus is shown in figure 4, and compared with ribavirin, the compound Chinese medicine composition can obviously inhibit VP1 expression at low concentration.
In conclusion, the traditional Chinese medicine compound composition provided by the invention can be obviously inhibited in the aspect of EV-71 virus infection. Meanwhile, the Chinese herbal compound composition can be used as both medicine and food and can be used for green health-care food.
The foregoing description is only of the preferred embodiments of the invention, and all equivalent changes and modifications made by the applicant of the present invention shall fall within the scope of the present invention.
Claims (4)
1. A Chinese medicinal compound composition with anti-enterovirus 71 type effect is characterized in that: the traditional Chinese medicine compound composition is prepared from the following raw materials in parts by weight: 6-12 parts of lucid ganoderma, 10-20 parts of grifola frondosa, 5-10 parts of chrysanthemum, 6-15 parts of honeysuckle and 15-25 parts of cordate houttuynia.
2. The traditional Chinese medicine compound composition with anti-enterovirus 71 type effect according to claim 1, which is characterized in that: the traditional Chinese medicine compound composition is prepared from the following raw materials in parts by weight: 6 parts of lucid ganoderma, 15 parts of grifola frondosa, 5 parts of chrysanthemum, 6 parts of honeysuckle and 15 parts of houttuynia cordata.
3. A method for preparing a traditional Chinese medicine compound composition with anti-enterovirus 71 type efficacy according to claim 1, which is characterized in that: the method comprises the following steps:
1) Taking 6-12 parts by weight of ganoderma lucidum according to a feed liquid ratio of 1:15 mixing m/v with ultrapure water, adjusting pH to 8.5, adding alkaline protease according to the proportion of 2% of Ganoderma, oscillating at 55deg.C for 2 hr, inactivating enzyme in boiling water bath for 10min, cooling to room temperature, filtering, concentrating the filtrate, and lyophilizing to obtain Ganoderma alkaline protease zymolyte;
2) Taking 10-20 parts by weight of grifola frondosa, and mixing according to a feed liquid ratio of 1:15 mixing m/v with ultrapure water, regulating pH to 7.8, adding compound protease according to the proportion of 4% of the weight of Grifola frondosa, oscillating for 2h at 53 ℃, inactivating enzyme in boiling water bath for 10min, cooling to room temperature, filtering, concentrating the filtrate, and lyophilizing to obtain Grifola frondosa compound protease zymolyte;
3) Taking 5-10 parts by weight of chrysanthemum, 6-15 parts by weight of honeysuckle and 15-25 parts by weight of cordate houttuynia according to a feed liquid ratio of 1:20 mixing m/v with ultrapure water, soaking at room temperature for 2h, decocting for 30min in a micro-boiling state, cooling to room temperature, and filtering to obtain primary filtrate and primary filter residue; adding ultrapure water with the mass 10 times of that of the primary filter residue into the primary filter residue, keeping a micro-boiling state, decocting for 30min, cooling to room temperature, and filtering to obtain secondary filtrate and secondary filter residue; combining the primary filtrate and the secondary filtrate, centrifuging, collecting the supernatant, concentrating, and freeze-drying to obtain the chrysanthemum and honeysuckle and houttuynia cordata composite water extract;
4) Uniformly mixing the ganoderma lucidum alkaline protease zymolyte obtained in the step 1) and the grifola frondosa compound protease zymolyte obtained in the step 2) and the chrysanthemum and honeysuckle cordate houttuynia compound aqueous extract obtained in the step 3) to obtain the traditional Chinese medicine compound composition with the enterovirus 71-resisting effect.
4. The use of the compound Chinese medicinal composition according to claim 1 in the preparation of anti-enterovirus 71 type products.
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