Use of the composition in preparing a drug for preventing / treating hand, foot and mouth disease

Abstract

The present invention proposes the use of a composition in preparing a medicine for preventing or treating hand, foot and mouth disease, and the medicine comprises: ephedra, bitter almond, liquorice, platycodon, houttuynia cordata, scutellaria baicalensis, perilla seed, bombyx batryticatus, asarum and tea. The composition of the present invention can effectively prevent or treat hand, foot and mouth disease, lays a foundation for the scientific research and clinical application of hand, foot and mouth disease, and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to use of a composition in preparing a drug for preventing / treating hand, foot and mouth disease. Background Art

[0002] Hand, foot and mouth disease is an infectious disease caused by enterovirus. There are more than 20 types of enterovirus that can cause hand, foot and mouth disease, among which Coxsackievirus A16 (Cox A16) and enterovirus 71 (EV71) are the most common. It often occurs in children under 5 years old, with symptoms such as mouth pain, anorexia, low fever, and small herpes or ulcers on the hands, feet, and mouth. Most children recover on their own in about a week, while a few children may develop complications such as myocarditis, pulmonary edema, and aseptic meningoencephalitis. Some severely ill children develop rapidly and die.

[0003] Currently, drugs for the treatment of hand, foot and mouth disease are still under research. Summary of the invention

[0004] The present invention aims to solve the technical problems existing in the prior art to at least a certain extent. To this end, the present invention proposes the use of a composition in the preparation of a medicine, and the composition containing ephedra, bitter almond, liquorice, platycodon, houttuynia, scutellaria, perilla seed, bombyx batryticatus, asarum and tea can effectively prevent or treat hand, foot and mouth disease, laying a foundation for the scientific research and clinical application of hand, foot and mouth disease, and has a good application prospect.

[0005] The present invention proposes the use of a composition in preparing a medicine. According to an embodiment of the present invention, the medicine is used for preventing or treating hand, foot and mouth disease, and the composition comprises: ephedra, bitter almond, liquorice, platycodon, houttuynia cordata, scutellaria baicalensis, perilla seed, bombyx batryticatus, asarum and tea.

[0006] Ephedra is pungent, warm and slightly bitter, and can open and promote lung qi, relieve cough and asthma, and is the main drug. Scutellaria baicalensis and Houttuynia cordata are bitter and cold, and can clear the lungs and purge fire, relieve restlessness and thirst, and remove phlegm and heat, and are the assistant drugs. Bitter almonds, perilla seeds, platycodon, bombyx batryticatus, tea, and asarum can lower qi and resolve phlegm, relieve cough and asthma, and stop wind and stop spasm, and are adjuvant drugs. Licorice can both invigorate qi and harmonize the middle, and can also reconcile the various drugs, and is the guiding drug. The combination of various drugs can play the role of clearing heat and promoting lung qi, resolving phlegm and relieving cough, and lowering adverse qi and relieving asthma, which can effectively inhibit the survival of hand, foot and mouth disease virus, thereby preventing or treating hand, foot and mouth disease, laying a foundation for the scientific research and clinical application of hand, foot and mouth disease, and has a good application prospect.

[0007] According to an embodiment of the present invention, the mass ratio of ephedra, bitter almond, scutellaria, perilla seed, bombyx batryticatus, houttuynia cordata, asarum, tea, licorice and platycodon is (80-100): (140-170): (140-160): (170-190): (170-190): (340-360): (5-25): (5-25): (90-110): (140-160).

[0008] According to an embodiment of the present invention, the use of the above composition in preparing a medicine may also have the following additional technical features:

[0009] According to an embodiment of the present invention, the composition comprises: ephedra, fritillaria cirrhosa, bitter almond, scutellaria baicalensis, jasmine, perilla seed, bombyx batryticatus, hawthorn, radish seed, gypsum, houttuynia cordata, asarum, tea, liquorice and platycodon.

[0010] Ephedra is pungent, warm and slightly bitter, it can open and clear the lung qi, relieve cough and asthma; Gypsum is pungent, sweet and very cold, and it is combined with ephedra to clear the lung without adding heat, clear the lung without retaining evil, and is a monarch drug. Scutellaria baicalensis and Houttuynia cordata are bitter and cold, clear the lung and purge fire, relieve restlessness and quench thirst, remove phlegm and heat, and strengthen the lung clearing and heat relieving effect of gypsum, and are a minister drug. Bitter almond, Fritillaria cirrhosa, Radix dahliae, Perilla seed, Radish seed, Platycodon grandiflorum, Bombyx batryticatus, tea, Asarum can descend qi and resolve phlegm, relieve cough and asthma, stop wind and stop spasm; Hawthorn can eliminate food accumulation, wake up the spleen, and resolve phlegm turbidity, and are adjuvant drugs. Licorice can both invigorate qi and harmonize the middle, and can also reconcile various drugs, and is a guiding drug. The inventor found that the use of the above components can better inhibit the survival of hand, foot and mouth virus, thereby preventing or treating hand, foot and mouth disease.

[0011] According to an embodiment of the present invention, the mass ratio of ephedra, fritillaria, bitter almond, scutellaria, dahlia, perilla seed, bombyx batryticatus, hawthorn, radish seed, gypsum, houttuynia, asarum, tea, licorice and platycodon is (80-100): (80-100): (140-170): (140-160): (140-160): (170-190): (170-190): (170-190): (170-190): (290-330): (340-360): (5-25): (5-25): (90-110): (140-160). Thus, the survival of hand, foot and mouth disease can be better inhibited, thereby preventing or treating hand, foot and mouth disease.

[0012] According to an embodiment of the present invention, the drug is used to inhibit EV71 virus.

[0013] According to an embodiment of the present invention, the medicine is used to prevent or treat diseases caused by infection with EV71 virus.

[0014] According to an embodiment of the present invention, the disease is selected from hand, foot and mouth disease, viral pharyngitis, myocarditis, pulmonary edema or encephalitis.

[0015] According to an embodiment of the present invention, the drug is used for reducing swelling, relieving pain, lowering temperature, and reducing vascular permeability.

[0016] According to an embodiment of the present invention, the dosage form of the drug is pills, tablets or capsules.

[0017] According to an embodiment of the present invention, the drug is in a single-dose form, and the drug contains a daily dose of 13.6 to 81.6 g of the composition. Thus, at this dose, the survival of the hand, foot and mouth virus can be effectively inhibited.

[0018] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0020] Figure 1 The toxicity determination of EV71 according to one embodiment of the present invention is shown, A: the effect of EV71 on RD cells; B: the effect of different concentrations on cell survival; C: nonlinear analysis of cell TCID 50 ;

[0021] Figure 2 The toxicity test of the extracts according to one embodiment of the present invention on RD cells is shown, A: TD50B (half toxic dose) of the three extracts; B: the effects of the three extracts on cell survival at dilution ratios of 1 / 64, 1 / 256 and 1 / 1024, extract 1: XRKC; extract 2: B1; extract 3: B2;

[0022] Figure 3 The direct killing effect of the extract according to one embodiment of the present invention on EV71 is shown, specifically the effect of 1 / 64, 1 / 128, 1 / 265, and 1 / 512 times dilution of the drug on cell survival rate;

[0023] Figure 4 The effect of the Chinese medicine granules according to one embodiment of the present invention on the latency and frequency of writhing in mice is shown, wherein A: the Chinese medicine granules prolong the latency of writhing in mice; B: the Chinese medicine granules inhibit the frequency of writhing in mice; Sham: control group; XRKC: Chinese medicine granules;

[0024] Figure 5 It shows that the Chinese medicine granules according to one embodiment of the present invention prolong the pain range of the plantar of mice, wherein Sham: control group; XRKC: Chinese medicine granules;

[0025] Figure 6The figure shows that the Chinese medicine granules according to one embodiment of the present invention reduce the swelling of rat toes caused by egg white, wherein A: a comparison between a low-dose Chinese medicine granule and a control group; B: a comparison between a medium-dose Chinese medicine granule and a control group; C: a comparison between a high-dose Chinese medicine granule and a control group; sham: control group; XRKC: Chinese medicine granule;

[0026] Figure 7 It shows that the Chinese herbal granules according to one embodiment of the present invention improve the increased capillary permeability in mice, wherein Sham: control group; XRKC: Chinese herbal granules;

[0027] Figure 8 The results show that the Chinese medicine granules according to one embodiment of the present invention reduce blood sugar and increase the body temperature of rats caused by exogenous 2,4-dinitrophenol, wherein A: a comparison between a low-dose Chinese medicine granule and a control group; B: a comparison between a medium-dose Chinese medicine granule and a control group; C: a comparison between a high-dose Chinese medicine granule and a control group; sham: control group; XRKC: Chinese medicine granule;

[0028] Fig. 9 The survival curve of young mice according to one embodiment of the present invention is shown, wherein 10 -0 , 10 -1 , 10 -2 , 10 -3 Respectively represent dilution of 10 -0 , 10 -1 , 10 -2 , 10 -3 times of EV71-MA-V virus stock solution; Control is the normal control group;

[0029] Fig.10 The effect of the Chinese medicine granules according to one embodiment of the present invention on the survival rate of young mice infected with EV71 is shown, wherein Sham: control group; XRKC: Chinese medicine granules; EV71: EV71 virus;

[0030] Fig.11 The effect of the Chinese medicine granules according to one embodiment of the present invention on the clinical scores of young mice infected with EV71 is shown; A: clinical score of the sham group, B: clinical score of the EV71 group, C: clinical score of the high-dose group, D: clinical score of the medium-dose group, and E: clinical score of the low-dose group;

[0031] Fig.12 The effect of the Chinese medicine granules according to an embodiment of the present invention on the inflammatory factors in the brain tissue of young mice infected with EV71 is shown; wherein, A: IL-1β level; B: TNF-α level; Sham: control group; XRKC: Chinese medicine granules; EV71: EV71 virus. DETAILED DESCRIPTION

[0032] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0033] Example 1

[0034] Recipe: Ephedra 90g, Fritillaria 90g, bitter almond (fried) 150g, Scutellaria 150g, Gardenia 150g, Perilla seed (fried) 180g, Bombyx batryticatus (fried) 180g, Hawthorn (fried) 180g, Radish seed (fried) 180g, Gypsum 300g, Houttuynia cordata 360g, Asarum 15g, Tea leaves 15g, Licorice 90g, Platycodon 150g.

[0035] Preparation method: Among the 15 medicinal materials, volatile oils are extracted from Asarum and Houttuynia cordata by distillation, and the distilled aqueous solution is collected in another container; the medicinal residues and the remaining 13 medicinal materials such as Ephedra sinica and Fritillaria cirrhosa (crushed) are decocted in water twice, each time for 1.5 hours, the decoctions are combined, filtered, the filtrate is combined with the above aqueous solution, and concentrated to obtain extract 1.

[0036] Comparative Example 1

[0037] The Houttuynia cordata and Scutellaria baicalensis in Example 1 were replaced with Forsythia suspensa, Honeysuckle and Isatis indigotica.

[0038] Recipe: Ephedra 90g, Fritillaria 90g, bitter almond (fried) 150g, Forsythia suspensa 180g, Honeysuckle 180g, Isatis root 150g, Gardenia 150g, Perilla seed (fried) 180g, Bombyx batryticatus (fried) 180g, Hawthorn (fried) 180g, Radish seed (fried) 180g, Gypsum 300g, Asarum 15g, Tea leaves 15g, Licorice 90g, Platycodon 150g.

[0039] Preparation method: Among the sixteen medicinal materials, volatile oils are extracted from Asarum and Houttuynia cordata by distillation, and the distilled aqueous solution is collected in another container; the medicinal residues and the remaining fourteen medicinal materials such as Ephedra sinica and Fritillaria cirrhosa (crushed) are decocted in water twice, each time for 1.5 hours, the decoctions are combined, filtered, the filtrate is combined with the above aqueous solution, and concentrated to obtain extract 2.

[0040] Example 2

[0041] Recipe: Ephedra 90g, bitter almond (fried) 150g, Scutellaria 150g, Perilla seed (fried) 180g, Bombyx batryticatus (fried) 180g, Houttuynia cordata 360g, Asarum 15g, tea leaves 15g, licorice 90g, Platycodon 150g.

[0042] Preparation method: Among the ten medicinal materials, volatile oils from Asarum and Houttuynia cordata are distilled and the distilled aqueous solution is collected in another container; the medicinal residues and the other eight medicinal materials including Ephedra are decocted twice with water, each time for 1.5 hours, the decoctions are combined, filtered, the filtrate is combined with the above aqueous solution, concentrated, and extract 3 is obtained.

[0043] Example 3

[0044] Recipe: Ephedra 80g, Fritillaria 60g, Bitter Almond (fried) 140g, Scutellaria baicalensis 140g, Gardenia jasminoides 140g, Perilla seed (fried) 170g, Bombyx batryticatus (fried) 170g, Hawthorn (fried) 170g, Radish seed (fried) 170g, Gypsum 290g, Houttuynia cordata 340g, Asarum 5g, Tea leaves 5g, Licorice 90g, Platycodon 140g.

[0045] Preparation method: Among the 15 medicinal materials, distill and extract volatile oil from Asarum and Houttuynia cordata, and collect the distilled aqueous solution in another container; add water to the residue and the other 13 medicinal materials such as Ephedra sinica and Fritillaria cirrhosa (crushed) and boil them twice, each time for 1.5 hours, combine the decoctions, filter, combine the filtrate with the above aqueous solution, and concentrate.

[0046] Example 4

[0047] Recipe: Ephedra 100g, Fritillaria 100g, bitter almond (fried) 170g, Scutellaria 160g, Gardenia 160g, Perilla seed (fried) 190g, Bombyx batryticatus (fried) 190g, Hawthorn (fried) 190g, Radish seed (fried) 190g, Gypsum 330g, Houttuynia cordata 360g, Asarum 25g, Tea leaves 25g, Licorice 110g, Platycodon 160g.

[0048] Preparation method: Among the 15 medicinal materials, distill and extract volatile oil from Asarum and Houttuynia cordata, and collect the distilled aqueous solution in another container; add water to the residue and the other 13 medicinal materials such as Ephedra sinica and Fritillaria cirrhosa (crushed) and boil them twice, each time for 1.5 hours, combine the decoctions, filter, combine the filtrate with the above aqueous solution, and concentrate.

[0049] Example 5

[0050] Recipe: Ephedra 85g, Fritillaria 60g, Bitter Almond (fried) 145g, Scutellaria baicalensis 145g, Gardenia jasminoides 145g, Perilla seed (fried) 175g, Bombyx batryticatus (fried) 175g, Hawthorn (fried) 175g, Radish seed (fried) 175g, Gypsum 295g, Houttuynia cordata 345g, Asarum 10g, Tea leaves 10g, Licorice 100g, Platycodon 145g.

[0051] Preparation method: Among the 15 medicinal materials, distill and extract volatile oil from Asarum and Houttuynia cordata, and collect the distilled aqueous solution in another container; add water to the residue and the other 13 medicinal materials such as Ephedra sinica and Fritillaria cirrhosa (crushed) and boil them twice, each time for 1.5 hours, combine the decoctions, filter, combine the filtrate with the above aqueous solution, and concentrate.

[0052] Example 6

[0053] Recipe: Ephedra 95g, Fritillaria 95g, Bitter Almond (fried) 160g, Scutellaria 160g, Gardenia 160g, Perilla Seed (fried) 185g, Bombyx Batryticatus (fried) 185g, Hawthorn (fried) 185g, Radish Seed (fried) 185g, Gypsum 320g, Houttuynia cordata 355g, Asarum 20g, Tea 20g, Licorice 105g, Platycodon 155g.

[0054] Preparation method: Among the 15 medicinal materials, distill and extract volatile oil from Asarum and Houttuynia cordata, and collect the distilled aqueous solution in another container; add water to the residue and the other 13 medicinal materials such as Ephedra sinica and Fritillaria cirrhosa (crushed) and boil them twice, each time for 1.5 hours, combine the decoctions, filter, combine the filtrate with the above aqueous solution, and concentrate.

[0055] Example 7

[0056] 1. Determination of EV71 toxicity

[0057] MDCK (canine kidney cells, AIPR8134 host cells) and RD (human rhabdomyosarcoma cells, EV71 and Ech07 host cells) were digested with 0.25% trypsin in the logarithmic growth phase and the cell concentration was adjusted to 1×10 4 100 μL per well was inoculated into a 96-well plate. 2 When the cells grow to a monolayer in the incubator, discard the culture medium. Add 100 μL of the corresponding virus solution diluted 10 times in series, repeat 6 wells for each concentration, and set up a cell control. Incubate at 37°C, 5% CO 2 The cells were cultured in a constant temperature incubator for 24 h under the same conditions. 10 μL of 5 mg / mL CCK8 solution was added to each well. After culturing for 1-2 h, the OD value at 450 nm was measured by a microplate reader. The TCID50 infection concentration of the virus was calculated according to the Reed-Muench formula. 50 .

[0058] Cell survival rate = OD value of each group × 100% / OD value of normal cells

[0059] Cell ratio distance = (lesion rate above 50% - 50%) × 100% / (lesion rate above 50% - lesion rate below 50%)

[0060] TCID 50=Antilg (Ig higher than 50% CPE percentage virus dilution ± ratio × dilution factor logarithm)

[0061] According to the experimental plan, RD cells were selected as EV71 host cells for virulence determination. The results are as follows Figure 1 As shown in Figure 2, EV71 has good virulence, and its half infectious concentration (TCID 50 ) is 10 -3.27±0.08 / 0.1ml. For subsequent experiments, 100 times TCID 50 The concentration of EV71 is about 10 -1.5 / 0.1ml concentration.

[0062] 2 Toxic effect of extract on RD cells

[0063] Extract 1, Extract 2 and Extract 3 were diluted 64, 256 and 1024 times with culture medium (DMEM), respectively, and inoculated into 96-well plates where RD cells had grown into a monolayer, and each concentration was repeated for 6 wells. A normal cell control group was set up at the same time. The cells were cultured in a constant temperature incubator at 37°C and 5% CO2 for 24 h. The degree of cell pathology was observed under an inverted microscope. 10 μL of CCK8 solution was used. After culturing for 1-2 h, the OD value at 450 nm was measured by a microplate reader. The cell half-toxic concentration TC was calculated according to the Reed-Muench formula 50 , find the maximum non-toxic concentration TC 0 .

[0064] The results are as follows Figure 2 As shown, the toxicity relationship is extract 1 = extract 3 > extract 2; extract 1, extract 2 and extract 3 all have a protective effect on RD cells, and the survival rate is improved, specifically extract 1 > extract 2 > extract 3.

[0065] 3. Direct killing effect of extract on EV71 virus

[0066] Extract 1, Extract 2, and Extract 3 were diluted 64, 128, 256, and 512 times with culture medium (DMEM) and mixed with an equal volume of 100 times TCID 50 The virus solution was allowed to interact for 2 h, and 100 μL per well was inoculated in a 96-well plate grown into a monolayer of cells. Four replicate wells, a cell control well, and a blank control well were set up at 37°C and 5% CO. 2 The cells were cultured in a virus incubator and the culture was terminated after 24 h. The cell survival rate was calculated by MTT method and the half effective concentration (EC) of the drug was calculated by Reed-Muench formula. 50 ) and therapeutic index (TI).

[0067] The results are as follows Figure 3As shown, at the same dilution concentration, the maximum effect of extract 1 is at 256 times dilution concentration and the virus direct killing effect of the other concentrations of extracts is relatively stable. The maximum effect of extract 2 is at 64 times dilution concentration, and the effect decreases rapidly with the decrease of concentration. The maximum effect of extract 3 is at 512 times dilution concentration, and the effect gradually increases with the decrease of concentration. It can be seen that the anti-EV71 virus effect of extract 1 may last the longest.

[0068] In summary, Extract 1 has the strongest protective effect on RD cells and its anti-EV71 virus effect may last the longest. Therefore, Extract 1 has the best overall anti-EV71 virus effect and can be effectively used in the treatment of hand, foot and mouth disease.

[0069] Example 8

[0070] The extract of Example 1 was granulated to obtain Chinese medicine granules. The effects of the Chinese medicine granules on abdominal pain and foot pain in mice, swelling of rat toes, permeability of mouse abdominal capillaries, and body temperature of rats were investigated to evaluate the antipyretic, analgesic, and anti-inflammatory effects of the Chinese medicine granules.

[0071] 1. Experimental Methods

[0072] 1Analgesic effect of Chinese herbal granules

[0073] 1.1 Effect on pain caused by glacial acetic acid: 40 mice were taken, 10 mice in each group, half male and half female, and randomly divided into a control group and three dose groups of Chinese medicine granules (19g / kg, 9.5g / kg, 4.8g / kg) according to body weight. The drug-administered group was gavaged once a day for 5 consecutive days, and a 6% acetic acid saline solution was injected intraperitoneally after the last administration, causing a large area of ​​deep abdominal pain and causing the mice to have a "writhing reaction", which was manifested as a depression in the abdomen, extension of the trunk and hind limbs, and elevation of the buttocks. The number of animals with writhing reactions in each group and the number of writhing times in 30 minutes were observed and recorded, and the differences between the groups were compared with the average number of writhing times and the percentage of drug analgesia in each group.

[0074]

[0075] 1.2 Effect on hot plate pain: Place the aluminum lunch box in a 55±5℃ water bath and preheat for 10 minutes. Take 40 female mice with a pain threshold of 5-30 seconds, measure the pain threshold twice and take the average value as the pre-drug pain threshold of the mouse. According to the pain threshold, they were randomly divided into 4 groups, with 10 mice in each group. Control group, three dosage groups of Chinese medicine granules (19g / kg, 9.5g / kg, 4.8g / kg). The drug-administered group was gavaged once a day for 5 consecutive days, and the pain threshold of the mice was measured after the last administration. If there is no pain reaction within 60 seconds, take it out immediately and calculate according to 60 seconds. Compare the difference in pain domain before and after administration.

[0076] 2 Anti-inflammatory effects of Chinese herbal granules

[0077] 2.1 Effect on egg white-induced swelling of rat toes: 40 male rats were randomly divided into groups of 10 rats per group according to body weight. There were three dosage groups (10g / kg, 5g / kg, and 2.5g / kg) of the control group and Chinese medicine granules. The drug-administered group was gavaged once a day for 5 consecutive days. A mark was made on the left hind foot of the rat on the fourth day after gavage to measure the normal toe volume. On the fifth day after administration, 1 mouse was subcutaneously injected with 0.05ml of 10% fresh egg white in the left hind toe to induce inflammation. The volume of the left hind toe of the rat was measured at 0.5h, 1h, 2h, 4h, and 6h after injection, and the percentage of toe swelling was calculated, and the intergroup comparison was made with the model group.

[0078] Toe swelling percentage = (volume after egg white injection - volume before egg white injection) × 100% / volume before egg white injection

[0079] 2.2 Effect on the permeability of peritoneal capillaries in mice: 40 male mice were randomly divided into groups of 10 mice each according to body weight. There were three dose groups (19g / kg, 9.5g / kg, and 4.8g / kg) of the control group and the Chinese medicine granules. The drug-administered group was gavaged once a day for 5 consecutive days. 30 minutes after the last administration, 0.6% glacial acetic acid was injected intraperitoneally, 0.01mL / 10g. 30 minutes later, 0.5% Evans blue saline solution was injected into the tail vein of the mice, 0.1ml / 10g. After 20 minutes, the mice were killed by supporting the cervical vertebra, the abdominal cavity was opened, and the abdominal cavity was washed several times with 6ml of saline. The washing solution was precipitated with a pipette. After collection, saline was added to 10ml, centrifuged at 3000rpm for 15min, the supernatant was aspirated and colorimetrically determined at 590nm, and the absorbance value of the washing solution was read for inter-group t-test.

[0080] 3 Antipyretic effect of Chinese herbal granules

[0081] 40 male rats were randomly divided into 4 groups of 10 rats each according to body weight. There were three dose groups (10g / kg, 5g / kg, and 2.5g / kg) of control group and Chinese medicine granules. The drug-administered group was gavaged once a day for 5 consecutive days. The anus peripheral temperature of the rats was measured with an infrared thermometer one day before administration, once in the morning and afternoon, and once before modeling. The average of the above three temperatures was taken as the normal body temperature. 30 minutes after the last administration, 2,4-dinitrophenol 24mg / kg was subcutaneously injected into the back of the rats, and the anus temperature was measured once every 30 minutes, and each rat was measured for 3 hours, for a total of 6 times. The temperature difference before and after modeling was used as an indicator for inter-group comparison.

[0082] 2. Data Statistics

[0083] The experimental data were expressed as mean ± SD. SPSS 18.0 statistical software was used for processing, and the comparison of means between groups was performed using t-test. P < 0.05 was considered statistically significant.

[0084] 3. Experimental Results

[0085] 1. Chinese herbal granules inhibit glacial acetic acid-induced abdominal pain in mice

[0086] The mice were tested according to the experimental plan, and the weight of each group of mice, the latency of the mice to writhing after administration of glacial acetic acid, the number of animals with writhing reactions, and the number of writhing times in 30 minutes were recorded. The results showed that under the condition that there was no difference in the weight of each group of mice, the high-dose group of Chinese medicine granules (19g / kg) could significantly prolong the latency of mice to writhing compared with the control group ( Figure 4 A).

[0087] The number of body twisting of each group of mice in 30 minutes was counted, and the results showed that the high dose (19g / kg), medium dose (9.5g / kg), and low dose (4.8g / kg) of Chinese medicine granules could reduce the average number of body twisting of mice in 30 minutes ( Figure 4 B).

[0088] (Data see Table 1)

[0089] Table 1 Chinese herbal granules inhibit glacial acetic acid-induced abdominal pain in mice

[0090]

[0091] 2. Chinese herbal granules increase the pain threshold of mice

[0092] The plantar pain threshold of mice was measured using a 55±5℃ aluminum iron plate. The plantar pain threshold of mice in each group was statistically analyzed before and 5 days after administration of the drug. The results are as follows: Figure 5 As shown in Table 2, after 5 days of administration, the mice in the three dose groups of Chinese medicine granules were significantly improved compared with the control group.

[0093] Table 2 Chinese herbal granules extend the pain range of the plantar surface of mice

[0094]

[0095]

[0096] 3. Chinese herbal granules reduce egg white-induced toe swelling in rats

[0097] 40 male SD rats were divided into a control group and three Chinese medicine granule dosage groups (10g / kg, 5g / kg, 2.5g / kg). Each group of drugs was intragastrically administered for 5 consecutive days, and a mark was made on the left hind paw of the rat on the fourth day to measure the normal toe volume. One hour after administration on the fifth day, 0.05ml of 10% fresh egg white was subcutaneously injected into the left hind toe to induce inflammation. The volume of the left hind toe of the rat was measured at 0.5h, 1h, 2h, 4h, and 6h after injection. The experimental results are shown in Table 3 and Figure 6Compared with the control group, the medium and large doses of Chinese herbal granules could significantly improve the degree of toe swelling in rats (P<0.05, P<0.01, P<0.001), and the inhibitory effect of the medium and large doses of Chinese herbal granules was more obvious in the early stage of toe swelling, indicating that Chinese herbal granules can effectively fight against the induction of inflammation.

[0098] Table 3 Effect of Chinese herbal granules on reducing egg white-induced toe swelling in rats

[0099]

[0100] 4 Chinese herbal granules improve increased capillary permeability in mice

[0101] The OD value of the peritoneal lavage fluid of each group of mice was measured by an enzyme marker. Figure 7 The results showed that compared with the control group, the high dose of Chinese medicine granules could significantly inhibit the increase of capillary permeability of mice induced by glacial acetic acid, and the OD value of peritoneal lavage fluid was significantly reduced (P<0.001). The effects of the three dose groups of Chinese medicine granules on vascular permeability were positively correlated with the increase of dose.

[0102] (Data see Table 4)

[0103] Table 4 Chinese herbal granules improve the increase of capillary permeability in mice

[0104] Grouping Number of cases (only) OD value Control group 10 0.232±0.021 Chinese medicine granules (4.8g / kg) 10 0.205±0.020 Chinese medicine granules (9.5g / kg) 10 0.168±0.023 Chinese medicine granules (19g / kg) 10 0.076±0.019

[0105] 5 Chinese herbal granules for hypoglycemic effect Exogenous 2,4-dinitrophenol induces increased body temperature in rats

[0106] 2,4-Dinitrophenol was used to induce the increase of body temperature in rats, and the antipyretic effect of three dose groups (10g / kg, 5g / kg, and 2.5g / kg) of Chinese medicine granules was observed. Figure 8 The results showed that 2,4-dinitrophenol can continuously induce an increase in body temperature in rats, and the peak temperature appears 1.5 hours after administration, and then slowly returns to normal. A large dose of Chinese medicine granules can significantly inhibit the increase in body temperature in rats, reducing the body temperature of rats 1h, 1.5h, 2h, 2.5h and 3h after administration, respectively, while the trend of reducing the body temperature of rats by small and medium doses of Chinese medicine granules is less than that of a large dose. The results show that Chinese medicine granules have a certain antipyretic effect. (Data in Table 5)

[0107] Table 5 Chinese herbal granules for hypoglycemic effect Exogenous 2,4-dinitrophenol induces increase in body temperature in rats

[0108]

[0109] 4. Experimental Conclusion

[0110] Hand, foot and mouth disease in children is often characterized by herpes on the skin of the hands and feet and the oropharynx, accompanied by pathological manifestations such as limb pain, increased inflammation, and elevated body temperature. The above experimental results show that Chinese medicine granules can play an antiviral role by directly killing the hand, foot and mouth disease EV71 virus, inhibiting the invasion of the hand, foot and mouth disease EV71 virus into cells, and reducing the activity of the hand, foot and mouth disease EV71 after invading cells. At the same time, it can play a dose-dependent antipyretic, analgesic, and anti-inflammatory effect.

[0111] Example 9

[0112] The extract of Example 1 was granulated to obtain Chinese medicine granules. The in vivo antiviral effect of the Chinese medicine granules on hand, foot and mouth disease virus (EV71) was evaluated, and the therapeutic effect on cough and asthma in children was evaluated.

[0113] 1. Experimental Methods

[0114] 1. Virus inoculation and passaging

[0115] 100 μl of EV71 virus stock solution (approximately 1×10 3.27 TCID50) was injected intraperitoneally into 1-day-old suckling mice, and the behavior of the suckling mice was observed. The hind limb muscles were taken after hind limb paralysis or 9 days later, and the tissue grinding fluid was obtained after grinding to infect Vero cells. When more than 90% of the cells showed cytopathic effects, the virus was collected, and the EV71-MA-I virus was obtained after repeated freezing and thawing at -80℃ for 3 times and centrifuged. 100μl of EV71-MA-I virus stock solution was injected intraperitoneally to infect 6-day-old mice, and the behavior of the suckling mice was observed. The hind limb muscles were taken after hind limb paralysis or 7 days later, and the tissue grinding fluid was obtained after grinding to infect Vero cells, and the EV71-MA-II virus was collected. Then 100μl of EV71-MA-II virus stock solution was used to infect 12-day-old mice. The hind limb muscles were taken after hind limb paralysis or 7 days later, and the tissue grinding fluid was obtained after grinding to infect Vero cells, and the EV71-MA-III virus was collected. 12-day-old mice were intraperitoneally infected with 100 μl of EV71-MA-III virus stock solution, and the hind limb muscles were taken after paralysis of the hind limbs or 7 days later, and the tissue grinding fluid was obtained after grinding, and then infected with Vero cells to collect EV71-MA-IV virus. 2-week-old mice were infected with 100 μl of EV71-MA-IV virus stock solution, and the hind limb muscles were taken after paralysis of the hind limbs or 7 days later, and the tissue grinding fluid was obtained after grinding, and then infected with Vero cells to collect EV71-MA-V virus, which is the highly efficient fifth-generation adapted strain, and stored at -80°C for use.

[0116] 2. Determination of LD50 of the fifth-generation adapted strain

[0117] The EV71-MA-V virus was diluted tenfold to obtain four dilutions of the virus, which were injected intraperitoneally into two-week-old mice (100 μl / mouse). The mortality of the mice was observed, the number of deaths was recorded, and the LD50 infection dose of EV71 for two-week-old mice was calculated, which served as the basis for the infection dose in subsequent experiments.

[0118] 3. Criteria for judging the virulence of in vivo viruses

[0119] The clinical manifestations are divided into five levels, and the toxicity is evaluated by a points system. The levels are as follows:

[0120] Table 6 Grading of clinical manifestations of hand, foot and mouth disease in rats

[0121]

[0122]

[0123] 4. Effect of Chinese herbal granules against EV71 in young mice

[0124] Six litters of suckling mice were selected, with no less than 6 suckling mice in each litter, and no limit on females. The mice were randomly divided into three dose groups of Chinese medicine granules (10g / kg, 5g / kg, 2.5g / kg), a virus control group and a normal control group. Each group began to be gavaged one day before infection for five consecutive days. The virus control group and the normal control group were gavaged with equal volumes of saline. On the second day of administration, the fifth-generation virus adaptation strain was intraperitoneally injected with the toxin-enhanced virus. The activity and eating of the young mice were observed daily, and the young mice were killed 7 days after infection with the virus. Brain tissue was collected to determine the inflammatory factors IL-1β and TNF-α.

[0125] 2. Data Statistics

[0126] The experimental data were expressed as mean ± SD. SPSS 18.0 statistical software was used for processing, and the comparison of means between groups was performed using t-test. P < 0.05 was considered statistically significant.

[0127] 3. Experimental Results

[0128] 1.EV71 LD50 determination

[0129] According to the experimental plan, the EV71-MA-V virus was diluted to 10 -0 , 10 -1 , 10 -2 , 10 -3 Times the original solution, and intraperitoneally inject into 2-week-old mice (100 μl / mouse). Observe the mortality of the mice, record the number of deaths and calculate the LD50 infection dose of EV71 for 2-week-old mice.

[0130] The results show Fig. 9As shown in the figure, the mice in each group died at different time periods after being infected with the virus. The mice in group 10-0 died first 3 days after infection, and the number of deaths increased immediately, until all died on the 7th day. -1 The first group of mice to die 4 days after infection, and the number of deaths increased immediately, until all died on the 9th day. -2 The first group of mice to die 7 days after infection, and the number of deaths increased immediately. -3 After one death occurred in the young mice of the group on the 9th and 10th days of infection, no death occurred afterwards. Fig.11 The survival curve of Zhongtian young mice and the principle of 5-day administration and 7-day observation in subsequent experiments were used. The LD50 value after 7 days of virus infection was selected as 10 -1 Conduct follow-up experiments.

[0131] 2. Effects of Chinese herbal granules on the survival rate and physical signs of young mice infected with EV71

[0132] Six litters of suckling mice were selected, with no less than 6 suckling mice in each litter, regardless of females. They were randomly divided into three dose experimental groups (10g / kg, 5g / kg, 2.5g / kg), a virus control group and a normal control group. Each group began to be gavaged with the drug one day before infection for five consecutive days. The virus control group and the normal control group were gavaged with equal volumes of saline. On the second day of drug administration, the fifth-generation virus adaptation strain was intraperitoneally injected after the virus was increased, and the activity and eating of the young mice were observed day by day.

[0133] The results are as follows Fig.10 and 11 As shown, the high dose can significantly reduce the mortality rate of young mice and significantly improve the activity of young mice. The mortality rate of young mice in the medium and small dose groups did not improve significantly, but the death time was significantly prolonged by 1-2 days.

[0134] 3. Effects of Chinese herbal granules on inflammatory factors in young mice infected with EV71

[0135] The brain tissues of rats in each group were collected and stored in liquid nitrogen. The brain tissues were homogenized before the experiment and the supernatant was removed for determination.

[0136] The results are as follows Fig.12 As shown, compared with the control group, IL-1β and TNF-α in the brain tissue of rats in the EV71 group increased significantly. Chinese herbal granules can reduce inflammation in brain tissue and effectively inhibit EV71-induced inflammatory response in the brain tissue of young mice.

[0137] 4. Experimental Conclusion

[0138] Hand, foot and mouth disease in children is often characterized by herpes on the skin of the hands and feet and the oropharynx, accompanied by pathological manifestations such as limb pain, increased inflammation, and increased body temperature. The above experimental results show that Chinese medicine granules can effectively reduce the death of young mice induced by hand, foot and mouth disease EV71 virus and effectively inhibit the occurrence of inflammation in the brain tissue of young mice.

[0139] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0140] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. Use of the composition in the preparation of medicines, It is characterized in that The medicine is used to prevent or treat hand, foot and mouth disease. The composition is made of the following components in the following mass ratio: ephedra, fritillaria cirrhosa, bitter almond, scutellaria baicalensis, jasmine, perilla seed, bombyx batryticatus, hawthorn, radish seed, gypsum, houttuynia cordata, asarum, tea, liquorice and platycodon, and the mass ratio is (80-100): (80-100): (140-170): (140-160): (140-160): (170-190): (170-190): (170-190): (170-190): (170-190): (290-330): (340-360): (5-25): (5-25): (90-110): (140-160).

2. The use according to claim 1, It is characterized in that The dosage form of the drug is pill, tablet or capsule.

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