WO2007006171A1 - Composition de médecine chinoise traditionnelle, préparation et procédé de contôle de la qualité de celle-ci - Google Patents

Composition de médecine chinoise traditionnelle, préparation et procédé de contôle de la qualité de celle-ci Download PDF

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Publication number
WO2007006171A1
WO2007006171A1 PCT/CN2005/000999 CN2005000999W WO2007006171A1 WO 2007006171 A1 WO2007006171 A1 WO 2007006171A1 CN 2005000999 W CN2005000999 W CN 2005000999W WO 2007006171 A1 WO2007006171 A1 WO 2007006171A1
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water
solution
mobile phase
epimedium
test
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PCT/CN2005/000999
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Chinese (zh)
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WO2007006171A8 (fr
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Sik Kwan To
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Guangzhou Perfect Set Medicine Limited
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Publication of WO2007006171A8 publication Critical patent/WO2007006171A8/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/29—Berberidaceae (Barberry family), e.g. barberry, cohosh or mayapple
    • A61K36/296—Epimedium
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/74—Rubiaceae (Madder family)
    • A61K36/746—Morinda
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

Definitions

  • the invention relates to a traditional Chinese medicine composition, a preparation method thereof and a quality control method, in particular to a traditional Chinese medicine composition for treating bronchial asthma, a preparation method thereof and a quality control method.
  • Low immune function is a symptom that can be manifested in patients with various diseases.
  • asthma is a common chronic respiratory disease, and its occurrence is related to low immune function.
  • Many people also cause respiratory, circulatory and digestive systems.
  • Secondary lesions; tumors are refractory, complex pathogenesis, and a variety of treatment methods, but surgical resection of tumors, radiotherapy and chemotherapy anti-tumor, anti-tumor treatment, patients generally have low immune function, poor quality of life, Tumors are prone to recurrence and other diseases; AIDS is also refractory and contagious.
  • the treatment methods are mainly combined drugs and anti-HIV. After drug treatment, patients are prone to further impaired immune function, poor quality of life, and HIV. Easy to relapse and other conditions ⁇
  • the literature reports mainly include four methods such as ultraviolet spectrophotometry, thin layer color method, thin layer chromatography-ultraviolet spectrophotometry, and high-performance liquid phase. , focusing on the determination of total flavonoids and icariin in different parts of Epimedium or Epimedium; but for the flavonoids of Epimedium The analysis of the color fingerprints of the points, the overall distribution characteristics of the active ingredients contained in this product, and the quality of monitoring, have not been observed. Guo Baolin et al.
  • the present invention aims to provide a pharmaceutical composition; the object of the present invention is to provide a method for preparing a pharmaceutical composition; and the object of the present invention is to provide the pharmaceutical composition for improving immune function and quality of life, treating bronchitis, asthma, tumor
  • the novel use of the present invention is also to provide a quality control method for preparing the pharmaceutical composition into an injection, which comprises identification, content determination and/or fingerprint measurement; and the object of the present invention is to provide a drug composition arrow of the pharmaceutical composition.
  • the pharmaceutical composition of the present invention is prepared from the following bulk parts of the drug substance:
  • the Epimedium is 15 to 40 parts by weight of Morinda, 40 to 110 parts by weight of Epimedium or 28 parts by weight of Morinda, and 75 parts by weight of Epimedium.
  • the Epimedium can be an arrow leaf Epimedium.
  • the above pharmaceutical composition is prepared by extracting an extract or a purified product in a conventional manner, and adding a conventional adjuvant or excipient to prepare a clinically acceptable dosage form, including an oral preparation or a parenterally administered dosage form.
  • the oral preparation is selected from the group consisting of a tablet, a capsule, a pill, a granule, a suspension, a dropping pill, and an oral liquid preparation;
  • the parenteral dosage form is selected from the group consisting of an injection and a gas.
  • the excipient may be a solvent, a disintegrant, a flavoring agent, a preservative, a coloring agent or the like.
  • the preparation method of the pharmaceutical composition of the invention is:
  • the Chinese herbal medicines, Basil, and the leaves of Epimedium are pre-treated, and the good medicines are selected from a large number of messy herbs, and then washed with water several times to remove sand and mud.
  • Epimedium cut into small pieces of about lcm, 50- 70 ° C or less dry; Morinda 5 0-70 ° C or less after dried, pulverized to 20 mesh coarse powder; then use this material for the second extraction step the net, bar
  • the raw materials of Haotian are extracted three times with 6-8 times of water, 1-2 hours each time, and the extract is concentrated to a relative density of 40-60 °C to 1. 1-1.
  • the above-described shield amount control method for the compound injection of the present invention includes the following methods for content determination, discrimination and/or fingerprinting.
  • the content determination method includes one or more of the following four methods:
  • Aspirate 10 ml of the compound injection solution of the present invention set a constant weight of the evaporated sub-medium, evaporate in a water bath, dry at 105 ° C for 3 hours, displace the silica gel drier for 30 minutes, weigh the weight, calculate, and obtain;
  • the total solids per 1 ml of the compound injection of the present invention shall not be less than 11. 2ra go B. Total flavonoids
  • the content of the reference substance stock solution under the item C is 1 ml, placed in a 10 ml volumetric flask, diluted with methanol to the mark, shaken, and used as a reference solution;
  • the absorbance is measured at 270nm wavelength, calculated, that is;
  • Icariin is determined by high performance liquid chromatography (Chinese Pharmacopoeia 2000 edition, an appendix VID);
  • the appropriate amount of the icariin reference substance dried overnight with phosphorus pentoxide was weighed and dissolved in methanol to prepare a solution containing 0.2 mg per 1 ml as a reference stock solution; 2 ml of the reference substance stock solution was taken and placed in a 10 ml volumetric flask. , and diluted with the mobile phase to the mark, shake well, filtered through a microporous membrane (0.45 ⁇ m), that is, the reference solution;
  • the compound injection of the present invention contains icariin (C 33 H 4 .0 15 ) per ml, and not less than 0.48 mg.
  • Another 60 mg of anhydrous glucose reference substance dried to constant weight at 105 °C was placed in a 50 ml volumetric flask, dissolved in water, and diluted to the mark to serve as a reference solution for glucose reference; respectively, 1 ml of glucose reference preparation solution and 2 ml were separately taken. Place in a 25ml volumetric flask, dilute to the mark with water, shake well, as a reference solution;
  • the total injection of the compound injection of the present invention is not less than 0.30 mg per lml.
  • the method for identifying the compound injection of the invention is:
  • the reference substance stock solution of icariin under the content determination is used as a reference solution; according to the thin layer chromatography (Chinese Pharmacopoeia 2000 edition, an appendix VIB) test, the test solution 10 ⁇ 1 and the reference substance are aspirated.
  • 1, 1 5 1, 1 layer of silica gel G with a layer of 0. lraol / L disodium hydrogen phosphate, 0.3% carboxymethyl fiber sodium as a binder, to 1.
  • 1: 1 1 The butyl acetate-formic acid-water is placed at a temperature below 10 ° C.
  • the layered upper layer solution is used as a developing solvent, unrolled, taken out, air-dried, sprayed with 5% aluminum trichloride in ethanol, and heated at 105 ° C for several minutes.
  • the ultraviolet light is detected at a wavelength of 365 nm; in the chromatogram of the test product, a fluorescent spot of the same color is displayed at a position corresponding to the color of the reference color.
  • the compound injection of the invention can also adopt the method of fingerprint image detection, the method is: chromatographic conditions and system suitability test: using diode array ultraviolet detector and LichrospherC 18 4 x 250mm, color i ⁇ column with a particle size of 5 ⁇ ⁇ ; detection wavelength is 270nm, column temperature is 40 ° C, flow rate is lml / min; the number of plates is not less than 200 according to icariin. 1000; in a ratio of 1000: 100: 4.
  • the gradient elution conditions are: 0 to 15 minutes, the mobile phase A volume ratio linearly decreases from 100% to 60%, and the mobile phase B volume ratio increases linearly from zero to 40%; 15 minutes to 35 minutes, the mobile phase A volume ratio is 60% linearly decreases to zero, and the mobile phase B volume ratio increases linearly from 40% to 100%;
  • the fingerprint of the test sample should be consistent with the fingerprint sharing pattern of the control (or the fingerprint of the accompanying control extract), that is, there should be 7 peaks in the section of about 24 min to 36 min, which are labeled as a, b, c, respectively.
  • d, e, f, g where e peak is the most prominent, and adjacent to it is very weak.
  • d, f, g peak (g peak is icariin) constitutes a cluster of peaks; a, b peaks are farther away from c ⁇ g peak clusters, forming the characteristics of this product; computer-aided similarity evaluation software calculation, similarity
  • the correlation coefficient is expressed as greater than 0.9.
  • the fingerprinting method of the extract of Epimedium sagittatum (intermediate product) of the present invention is as follows: Color condition and system suitability test: chromatographic conditions and system suitability test of the compound injection of the present invention;
  • the fingerprint of the test sample should be consistent with the fingerprint of the extract (or the fingerprint of the extract of the accompanying medicinal material), and the similarity should be greater than 0.9.
  • the fingerprint of the extract of Epimedium sagittatum of the present invention is very similar to the fingerprint of the compound injection, the similarity is above -0.9, the correlation between the two is very good, the identification of the chromatogram and the fingerprint of the extract i The same is true, so the fingerprint sharing pattern of the control is also the same as in Figure 1.
  • the method for determining the fingerprint of the leaves of Epimedium sagittatum used in the compound injection of the present invention is as follows: ' Chromatographic conditions and system suitability test, using diode, array ultraviolet detector and Lichrospher C 18 , 4 25 Omm > particle size 5 ' ⁇ m i police column, .
  • Detection wavelength is -270nm, column temperature is 4 (TC, flow rate is 1ml / min; the number of plates is not less than 200,000 according to icariin; the ratio is 1000: 100: 4 92g: 11 (usually the mobile phase of HPLC is a liquid solvent, only the ratio is not unit, but citric acid is a solid reagent, so the same as g, the same below) of water-acetonitrile-citric acid-methanol mobile phase A and ratio For 1000: 470: 50: 6. 08 g of water-acetonitrile-isopropanol-citric acid mobile phase B, eluted according to the following gradient elution conditions:
  • the gradient elution conditions are: 0 to 15 minutes, the mobile phase A volume ratio linearly decreases from 100% to 60%, and the mobile phase B volume ratio increases linearly from zero to 40%; 15 minutes to 35 minutes, the mobile phase A volume ratio is 60% linearly drops to zero, mobile phase B volume ratio is linear from 40% N2005/000999
  • the chromatograms of the original medicinal material Epimedium sagittatum are compared, and the characteristic peaks are mainly concentrated in the sputum area, and the other peaks are weak, that is, the peak of No. 21 is the most prominent, and the index component is icariin (peak 23) and 19 No. 20, the peak of No. 20 is very weak, and the peak of No. 22 is extremely weak, only visible. Therefore, a peak pattern (peak No. 21) is formed in the sputum section.
  • the peaks in the I and ⁇ regions are extremely weak, and the color peaks are not detected in the IV region.
  • the correlation coefficient is calculated by the similarity evaluation software. , to prove that the similarity is good.
  • the antibacterial, analgesic and the main components for the treatment of acute, chronic bronchitis, asthma and immune function are flavonoid compounds, oligosaccharides and the like, respectively, in the composition of the present invention and its combination preparation.
  • composition of the invention and the combination thereof have antibacterial, analgesic, anti-inflammatory, antitussive and immunomodulatory functions, have a good therapeutic effect on acute and chronic bronchitis and asthma, and can treat tumors and AIDS.
  • the preparation of the composition of the invention has the following pharmacological effects: the traditional Chinese medicine compound preparation has obvious antitussive effect on the ammonia water cough mice; the traditional Chinese medicine compound preparation has obvious sputum effect on the mouse trachea; the traditional Chinese medicine compound preparation has obvious guinea pig tracheal smooth muscle. Relaxation effect; traditional Chinese medicine compound preparation has obvious anti-passive skin allergy effect; Chinese medicine compound preparation has obvious enhancement of humoral cell immunity; Chinese medicine compound preparation inhibits inflammation caused by mouse ear xylene The traditional Chinese medicine compound preparation prolongs the anti-hypoxia effect and prolongs the swimming time of the mice; the traditional Chinese medicine compound preparation improves the immune function, inhibits the tumor cells, and has an immune activation effect to inhibit HIV.
  • the preparation of the composition of the invention is a traditional Chinese medicine preparation which is extracted and separated by modern scientific means and has high concentration of active ingredients, and the mechanism for treating asthma is: Wenyang Bushen method can exert multi-link regulation effect on the neuro-endocrine-immune network through the hypothalamus. Improves the body's secretion and immune function, improves the pituitary-adrenal Shield function of asthma patients, reduces the dependence of adrenocortical hormones in asthma patients; improves the inhibitory T cell function in asthma patients, and inhibits the seasonal increase of serum I g E Reduce asthma attacks.
  • the clinical study shows that: the compound injection of the present invention for treating advanced lung cancer, the tumor stability rate after the treatment is 82.5 %, and the distant metastasis rate after the treatment is 15.3%; the anti-tumor effect of the compound injection of the invention has Stable tumor, anti-recurrence, anti-metastasis.
  • the compound injection of the present invention was significantly more significant than the Huachansu group in the treatment of red blood cells, alanine aminotransferase and aspartate aminotransferase.
  • the difference indicates that the compound injection has a greater effect on improving anemia, liver and kidney function in patients with lung cancer than Huachansu injection.
  • the compound injection can improve the natural killing cells of the patient's body, improve the lymphocyte turnover rate and improve the immune function of the patient.
  • the compound injection of the invention can increase the maximum expiratory peak flow rate, the one-second force forced expiratory volume value (FEV1 value), and regulate the TM cell/Th2 cell imbalance in asthma patients, and is an effective and safe traditional Chinese medicine preparation for treating asthma.
  • FEV1 value the one-second force forced expiratory volume value
  • the fingerprint of the color of this invention was mainly studied with Epimedium, and an experiment was established. Method, and carried out the corresponding methodological investigation, respectively More than 10 batches of Epimedium sinensis, Epimedium extract and 10 batches of finished products, the knot proved to have good correlation, reproducibility and stability.
  • the fingerprinting method of the invention has a good overall separation effect, so that the quality of the preparation can be controlled more comprehensively and effectively.
  • Test drug (1) The composition preparation of the invention (combination injection), Zhuhai Jianxin Medicine Co., Ltd. product, 0. 9g/ml, 971205; (2), salt and sulphate injection, Wuxi seventh Pharmaceutical factory products. 50mg/ml, 960613; '(3), aspirin tablets, Beijing Pharmaceutical Factory products, 950825; (4), ginsenoside, provided by the Phytochemical Laboratory of Chinese Medicine Institute of Jilin Traditional Chinese Medicine Institute, 95%; (5), Pu Ermin Injection, Fuyang Pharmaceutical Factory, 960919;
  • the real animal Wi s tar rat, weight 180-250g, certificate number 970101018; Kunming mice, weight 18-20g, certificate number 970101017; mongrel, weight 10-20kg; Japanese big white rabbit , weight 2. 2kg; guinea pig, weight 250-300g; the above animals are male and female, both are provided by the experimental animal room of Jilin Provincial Institute of Traditional Chinese Medicine.
  • Ephedrine 8 8.0 ⁇ 3.9 0.5 ⁇ 2.4 7.5 ⁇ 1.5** 93.8
  • Compound injection 8 7.9 ⁇ 2.5 0.6 ⁇ 2.3 7.3 ⁇ 0.2** 93.4
  • mice Effect of Compound Injection on Humoral Immunity and Cellular Immunity in Chronic Bronchitis Model Animals Table 5 (1) Effect of compound injection on immunization of mice (X Shi SD) Group dose (ml / kg) Number of animals (only) HC50 Normal control group 10 294.66 ⁇ 38.43 Experimental control 10 203.54 + 66.43 Ginsenoside 20 mg / Kg 10 261.94 + 27.31** Compound injection 4 - 10 248.18 ⁇ 43.89* Compound injection 2 10 247.96 ⁇ 34 ⁇ 22* Compound injection 1 10 250.16 ⁇ 53.93*
  • mice X soil SD
  • Dose Number of animals (only) Swelling degree (mg) Swelling inhibition rate (%) Normal control 12.0 ⁇ 2.6
  • a Lewis lung cancer cell suspension of '( 1-2 ) X lOVml was prepared by homogenization under aseptic conditions, and the cell suspension was inoculated subcutaneously with 0.05 ml per mouse site. Sixty mice were randomly divided into 5 groups, 12 in each group. High, medium and low volume 3 groups. The injection was diluted with physiological saline so that 0.1 ml was equivalent to 50, 10, 2 g/kg, respectively, and intravenously administered 0. lml, 1 time/day for 7 consecutive days. CTX is 0. lml is equivalent to 30mg/kg. The control group was given an equal amount of physiological saline.
  • Test method for human sputum cells Ficol l routine method for the isolation of human peripheral blood lymphocytes, separation of T lymphocytes by rosette precipitation, co-culture with different doses of drugs, colorimetric colorimetric determination of cell proliferation by MTT assay Reaction (wavelength 570 nm).
  • mice were intraperitoneally administered daily for 4 days, and the spleen was taken for 3 days and 6 days after stopping the drug.
  • the solution (5 x 10 cells/ml) was cultured for 24 hours, and incubation was continued for 40 hours by adding H-TDR/uci/well, which was counted by liquid scintillation and expressed by CMP.
  • mice were injected intraperitoneally with different doses of drugs, 1 hour later, blood was taken to separate serum 2 times diluted 96-well plate L29 cells overnight, plus vesicular stomatitis virus (10 -3/0. 2 ml ), continue to culture for 48 hours, and observe under the microscope.
  • the standard interferon was used as a control.
  • the cell control cells were normal and the virus control group showed lesions, the serum dilution of 50% of the inhibitory lesions was 50% as the interferon titer.
  • Human T cell activation test When the dry powder amount of the culture combined with the compound injection was 62. 5- lOOOO/ml, the number of human T lymphocytes increased significantly. See the table below: Compound injection on human T cell activation compound injection (ug/ml) dry powder T cells (DD value)
  • 6 1 2 700 + 180 1800 ⁇ 600 2801 ⁇ 540
  • Example 11 Changes in blood routine and liver and kidney function before and after treatment in both groups
  • Table 14 Changes of aspartate aminotransferase (AST) before and after treatment in the two groups Group number of cases Treatment of gluten transaminase after treatment of aspartate aminotransferase Compound injection group 40 30. 9 + 10. 00 26. 31 + 8. 70
  • Table 15 Changes in alanine aminotransferase (ALT) before and after treatment in the two groups. Number of cases before treatment with alanine transaminase after treatment with alanine aminotransferase compound injection group 40 23. 60 + 9. 51 21. 90 + 8. 59 Hua Alizarin group 20 22. 00 ⁇ 8. 69 21. 22 ⁇ 9. 50 Note: After t test, there was a significant difference in the comparison of alanine aminotransferase before and after treatment (P ⁇ 0.01).
  • the CD 3 +, CD 4 + and CD / CD 8 + levels in the compound injection group and the Huachansu group were improved compared with those before treatment.
  • the compound injection group and the Huachansu group had 0 treatments before treatment. The decrease was significant, but the difference between the compound injection group and the treatment group was significant (P ⁇ 0.05), but there was no significant difference between the Huajing group (P>0.05).
  • the compound injection group Huahuasu group There was no significant difference between the two groups before treatment (P>0.05), but there was significant difference between the compound injection group and the Huachansu group after treatment (P ⁇ 0.05). The results suggest that compound injection has more effect on improving the immune function of patients than Huachansu injection.
  • Table 18 Changes in T cell subsets before and after treatment in the two groups of patients.
  • 62 patients with chronic asthma were enrolled.
  • the patients were divided into two groups according to the randomized parallel control method.
  • the former group received the compound injection (batch numbers 20011102 and 20011104), the specification was 2ml/branch, usage: intramuscular injection, each time. 4ml, once a day, The course of treatment is 4 weeks.
  • the latter placebo group received the same outpacked placebo, consistent usage. Results Of the 62 patients, 4 patients withdrew. One in Group A and three in Group B.
  • the number of breathering shots is 1.23 ⁇ 3.25 -1.87 ⁇ 2.98 >0.05
  • the ⁇ symptom score was 0.96 ⁇ 1.15 -0.59 ⁇ 0.92 ⁇ 0.01
  • the symptom score of the treatment group improved. 0. 13
  • the difference before and after treatment was not significant (P>0.05)
  • the placebo group improved 0. 10
  • the difference between the treatment before and after treatment was also meaningless, between the two groups The difference is meaningless (P>0.05).
  • the PEF of the treatment group increased by 18.25L/min, and the difference before and after treatment was significant (P ⁇ 0.01).
  • the placebo group decreased by 4.96 L/min.
  • the difference before and after treatment was meaningless. Meaningful, the treatment group was better than the placebo group (P ⁇ 0. ⁇ .
  • the treatment group FEV1 (-second forced expiratory volume) increased by 0.12L (P ⁇ 0.05), and the placebo group decreased by 0.07L (P>0.05).
  • the difference between the two groups was significant (P ⁇ 0: 05).
  • the number of Lenin sprays was significantly higher than that of the placebo group, but the difference was meaningless (P>0.05).
  • the symptom score of the treatment group improved by 0.96, the difference was significant before and after treatment (P>0.01), and the placebo group improved by 0.59. The difference before and after treatment was also meaningful. The symptom score of the treatment group The improvement was better than the placebo group, and the difference was significant (P ⁇ 0.01).
  • the PEF of the treatment group increased by 21. 54 L/min, and the difference was significant (P ⁇ 0.01).
  • the placebo group decreased by 1.97 L/min.
  • the difference was not significant before and after treatment (P>0.05). ), the difference between the two groups is significant (P ⁇ 0.01), the treatment group for PEF The improvement was better than the placebo group (P ⁇ 0.01).
  • the FEV1 in the treatment group was increased by 0.14L (P ⁇ 0.05), and the difference between the two groups in the placebo group was significant (P ⁇ 0.05), but the difference between the two groups was not meaningful (P> 0. 05).
  • the daily application of chuanling spray decreased the number of treatment groups by 2. 66 and the placebo group by 1.08.
  • the difference between the two groups was significant (P ⁇ 0.05).
  • the treatment group adjusted the Thl /Th2, IFN-YV IL-4 ratio from 17.64 to 32.98, and the difference was significant.
  • Experimental Example 17 A randomized, double-blind, placebo-controlled clinical study of a compound injection of the present invention in the treatment of bronchial asthma in children
  • the clinical study of the bed The median symptom score of the compound injection group was decreased from 14 (M25 - M75: 6. 75-34. 5) to 2 (M25 ⁇ M75: 0-21. 5). There is a significant difference between before and after.
  • the clinical symptom scores and VAS scores of the children are the most commonly used indicators in asthma clinical studies, mainly reflecting the subjective symptoms of asthma patients, and VAS has a good correlation with lung function.
  • the maximum peak expiratory flow rate (PEF) variability in the treatment group gradually decreased, which was significantly different from the placebo group. It shows that compound injection can significantly improve the ventilation status of asthma patients.
  • Experimental Example 18 A randomized, double-blind, placebo-controlled experimental study of a compound injection of the present invention in the treatment of bronchial asthma in children A single-center, randomized, double-blind, placebo-controlled study was performed in the same manner as in Experimental Example 7, and the subject was also examined in Experimental Example 7.
  • 57 patients received venous blood lml, heparin anticoagulation, PMA (20 g/ml), ionomycin (1 ⁇ g/ml) as stimulator, and protein transport inhibitor monens in ( 2 y g./ml), incubate at 37 ° C, 5 % C0 2 for 4-8 hours.
  • the surface antigen was labeled with cy-chrome-labeled mouse anti-human CD4, CD8, CD3 mAb, cell membrane fixation, perforation, intracellular anti-1L-4, IFN- ⁇ mAb labeling for 30 min.
  • the £-type fine monthly package is fully tested, and the data analysis uses the Lys is software.
  • Non-parametric analysis x 2 test, x 3.865, p ⁇ 0.05, with significant differences.
  • the expression of IFN- ⁇ in CD 4 +, CDa+ lymphocytes was increased (P ⁇ 0.05), and the ratio of Thl/Th2 was significantly increased.
  • oo was significantly different.
  • the results showed that the compound injection has a positive regulatory effect on the balance of Th1/Th2 cells, and promotes the superior expression of Th1 cells, which is beneficial to the improvement of asthma symptoms and long-term stability.
  • Experimental Example 19 Clinical study of the compound injection of the present invention for treating adult asthma In the respiratory department of the General Hospital of Chinese People's Liberation Army and the General Hospital of the Chinese People's Armed Police, 20 patients with acute episodes of mild to moderate asthma were selected according to certain criteria, and were divided into a treatment group and a control group. Observed. The treatment group was treated with the compound injection of the present invention; the control group was not given any antispasmodic and antiasthmatic treatment. The observed indicators included clinical observation indicators, blood routine, liver and kidney function, electrocardiogram and chest and lung function indicators. The results are as follows:
  • the efficacy of the two groups is shown in Table 25.
  • the total effective rate of the treatment group is 80% (8/10), effective disease In the case of asthma symptoms, the time to resolve was 1 to 5 days, with an average of 2.2 ⁇ 2.3 days.
  • the total effective rate of the control group was 30% (3/10), and the time to relieve asthma symptoms in effective cases was 2 to 5 days, with an average of 2.84 ⁇ 1.5 days.
  • the Epimedium medicinal materials used in the present invention are based on the provisions of the Chinese Pharmacopoeia 2000 version of Epimedium [traits], and the photos of the Chinese Pharmacopoeia Color Pharmacopoeia of the Chinese Pharmacopoeia and the New Chinese Medicine Journal. And description, identified as Episporium sagittatum Maxin (Epimcdiura sagittatum Maxin).
  • the medicinal part is the leaf; it is mainly purchased from Puning.
  • the medicinal ingredient of Epimedium medicinal herbs is mainly flavonoids, with a content of about 1.01 ⁇ 8.81%. More than 50 kinds have been isolated before, mainly 9 kinds, so the flavonoids are selected as the analysis object of chromatographic fingerprint.
  • the preparation of the reference solution is accurately weighed the appropriate amount of the icariin reference substance, and 30% of the methanol is added to make a solution containing 0.1 mg per 1 ml.
  • the test solution is prepared by taking Epimedium 3 ⁇ 4 material leaf powder (passing through No. 3 sieve) about 0. 2g (accurate to 0. Olg), precision adding 40ml of diluted ethanol, weighing, ultrasonic treatment for 1 hour, and weighing Filtration, the filtrate was evaporated to dryness, and the residue was made up to 10 mL with 30% methanol, shaken, placed in a refrigerator for 1 hour, filtered, and the filtrate was taken.
  • the measurement method accurately absorbs the reference solution and the test solution for each 20 ⁇ 1, and injects the liquid chromatograph to determine.
  • the present invention refers to the HPLC chromatographic conditions of ginkgo flavonoids, and after repeated comparison and optimization, finally selects the chromatographic conditions described in the technical scheme of the present invention.
  • the maximum absorption wavelength of each component is concentrated around 26 Onm and 320 nm, which reflects the two largest absorption regions of flavonoid glycoside ⁇ 3 ⁇ 4.
  • the maximum absorption of icariin is 270 nm.
  • the maximum absorption of the color Fu peak before 15 minutes is at 320 nm.
  • the above two wavelengths are selected to determine the color fingerprints of different varieties of Epimedium. In order to further find out the difference between them.
  • the same test solution was taken at different time points of 0 hours, 1 hour, 3 hours, 4 hours, 48 hours, 72 hours, and 96 hours.
  • the visual fingerprints showed no obvious changes, and the computer-aided fingerprints were similar.
  • Degree evaluation software calculation (software developed by the Central South University of Traditional Chinese Medicine Modernization Research Center, one of the two software recommended by the Pharmacopoeia, the same below), the correlation coefficient is greater than 0.9 (data shown in Table 28), indicating that during this time The test solution has good stability.
  • the correlation coefficient of the similarity evaluation of the fingerprint of the medicinal material is 0. 9971 0. 9997 0. 9990 0. 9998 0. 9997 0. 9997 0. 9990 0. 9997 0. 9990
  • the coincidence coefficient is 0. 9974 0. 9997 0. 9994 0. 9942 0. 9900 0. 9975 0. 9885 0. 9760 0. 9991
  • the coincidence coefficient is 0. 9980 0. 9994 0. 9872 0. 9831 0. 9969 0. 9959 0. 9751 0. 9990
  • the five kinds of Epimedium medicinal herbs contained in the Chinese Pharmacopoeia have different characteristics and can be distinguished.
  • the Pan Peaks are numbered, and then the whole chromatogram is divided. It is the four regions I, II, III and IV.
  • the I region includes peaks 1 to 9 (retention time is about Omin ⁇ 13min); the sputum region includes peaks 10 to 18 (about 13min-31min); the sputum region includes 19-28. Peak (about 31min - 44min); Zone IV Includes peaks 29 to 31 (approximately 44min ⁇ 60min).
  • the most important features of the fingerprints of five kinds of Epimedium medicinal herbs contained in the Chinese Pharmacopoeia are mainly in the sputum area, that is, a section with a retention time of about 31 min to 44 min, and 10 linguistic peaks with icariin as a reference.
  • the five main characteristic chromatograms such as 19, 20, 21, 22, 23 (icariin), which are called “characteristic segments”
  • the five characteristic peaks in the fingerprints of five Epimediums are different. And constitute their respective characteristics (Table 30).
  • the Chinese Pharmacopoeia contains the main differences between the five species of Epimedium
  • the raw material medicine arrow leaves Epimedium HPLC color fingerprint map establishment and similarity evaluation.
  • the color map is relatively simple, the characteristic peak It is necessary to concentrate on Zone III, and the other peaks are weak, that is, Peak 21 is the most prominent.
  • the indicator components are icariin (peak 23) and the peaks of 19 and 20 are weak, and the peak of 22 is extremely weak. see. Therefore, a peak pattern (peak No. 21) is formed in the II segment, and the peaks in the I region and the sputum region are extremely weak, and the peaks are not detected in the IV region.
  • Preparation of the reference solution Accurately weigh the appropriate amount of icariin reference substance, add 30% sterol to make a solution containing 0.1 mg per lml, that is, obtained.
  • Preparation of the test solution Accurately weigh the appropriate amount of Epimedium extract, add 30% sterol to make a solution containing lmg per lml, that is.
  • Determination method respectively, accurately draw the reference solution and the test solution for each 20 ⁇ 1, inject Liquid chromatograph, measurement, that is. After detection with 270 nm and 320 nm, there was no significant difference in the fingerprint of the extract, so only the detection wavelength was 270 dishes.
  • the fingerprint of the test sample should be consistent with the fingerprint pattern common mode attached to the quality standard (or the fingerprint of the accompanying reference drug extract), and the similarity should be greater than 0.9. .
  • test solution was taken at different time points of 0 hours, 1 hour, 3 hours, 4 hours, 48 hours, 72 hours, and 96 hours, and the visual fingerprints showed no significant changes in the whole picture.
  • the coefficients are all greater than 0.9 (Table 32), indicating that the test solution has good stability during this time.
  • the fingerprints of the extracts are basically identical to each other, but in the sputum area, the production process is separated by polyamide column and refined, so the extract II
  • the peak clusters corresponding to the peaks of No. 13 and No. 14 in the area are more concentrated than the herbs.
  • the original data of 10 batches of extracts of Epimedium sagittatum L. were analyzed by similarity evaluation software, and the correlation coefficients were all above 0.9 (Table 34), which proved that the similarity was good.
  • the fingerprint of the Epimedium extract and the fingerprint of the finished product used in the compound injection of the present invention are Several characteristic peaks are renumbered as a, b (in the sputum area of the original Epimedium medicinal herbs fingerprint), c, d, e, f, g are equivalent to the 19th to 23rd peaks of the original ffl region, g peak (ie The original No. 23 peak) is the indicator component icariin.
  • the fingerprint of the intermediate product should have 7 peaks in the section of about 24 min ⁇ 36 min, which are labeled as a, b, c, d, e, f, g, where e peak is the most prominent, Adjacent is a weak c, d, f, g peak (g peak is icariin) to form a peak cluster; a, b peak is farther away from the c ⁇ g peak cluster, forming the characteristics of this product.
  • Table 34 Epicures of Epimedium sinensis (intermediate product) Correlation coefficient of 10 batches of HPLC color fingerprint similarity evaluation results
  • the left-to-right sequence is 0 hours, 1 hour, 2 hours, 3 hours, 4 hours, 24 hours, 48 hours, 72 hours, 96 hours of compound injection for the similarity of the HPLC chromatogram of the test solution.
  • the correlation coefficient of the 10 batches of HPLC chromatographic fingerprint similarity evaluation results of the compound injection of the present invention is 0. 9953 0. 9970 0. 9984 0. 9992 0. 9990 0. 9968 0. 9976 0. 9991 0. 9997 0. 9997 (median position Number)
  • the coincidence coefficient is 0. 9964 0. 9949 0. 9990 0. 9992 0. 9985 0. 9947 0. 9976 0. 9988 0. 9992 0. 9997
  • the sample 4 ratio is 030201, 030202, 030203, 030204, 030205, 030206, 030207, 030208, 030209, 030210 from left to right. 4. Identification and evaluation of the fingerprint of the compound injection of the present invention:
  • the fingerprint of the compound injection of the present invention is very similar to the fingerprint of the extract, and the similarity is above 0.90, which proves that the correlation between the two is extremely good.
  • the chromatographic identification is identical to the extract fingerprint.
  • the a and b peaks belong to the range of the fingerprint of the raw material, and the two peaks in the fingerprint of Epimedium sinensis and the main characteristic peak area ( ⁇ )
  • the bee (the peak No. 21 in the medicinal material) is extremely weak. It is adsorbed on the polyamide column, and after elution and desorption, after the final refining step, the b peak is more obvious in the extract, that is, the finished product. improve.
  • Figure 1 Standard fingerprint of compound injection of the present invention
  • Fig. 2 Chromatogram of the original drug of the present invention, Epimedium sinensis.
  • the following examples can achieve the effects of the above experimental examples.
  • Example 1 Bayu Tian, Epimedium extraction
  • the extract obtained by the method described in Example 1 was prepared into a solution by using water, and the filter rod was coarsely filtered, filtered with a filter ball and filtered through a 0.45 filter, potted, sterilized at 115 ° C for 30 minutes, and leaked with color water. , light inspection, printing, and then packaged into the warehouse; where the ampoule treatment is prepared in advance: coarse washing with pure water, fine washing with water for injection, final sterilization and drying, for potting, that is.
  • Example 3 Making a muscle 'injection'
  • the extract obtained by the method described in Example 1 was prepared into a solution by using water, filtered, potted, and sterilized at 115 ° C for 30 minutes. Made of atomizing agent 1000, each 5ml, 1 time each time, 2 times each time.
  • the extract obtained by the method described in Example 1 was prepared into a solution by using water, filtered, divided, lyophilized, and sealed tightly. Make lyophilized powder injection 1000, 2 times a day, 2 times each time, intravenous injection.
  • Example 6 Making an intravenous drip
  • the extract obtained by the method described in Example 1 was prepared into a solution with water, and the filter rod was coarse. Filtration, filter ball and filter with 0. 45 filter, potting, sterilizing at 115 °C for 30 minutes, leaking with color water, lamp inspection, printing, and then packaging.
  • the ampoules are prepared in advance: pure Rough water washing, fine washing with water for injection, and final sterilization for drying, for potting. Make 1000 drops of intravenous drip, 2ml each, once a day, 2 drops each time.
  • Example 7 Making an oral tablet
  • the extract obtained by the method described in Example 1 was ground into a fine powder, passed through a 120 mesh sieve, and added with an appropriate amount of starch to prepare granules, and added with an appropriate amount of magnesium stearate, and mixed, and pressed into 1000 tablets of kouyueliang tablets. 2 times a day, 2 tablets at a time.
  • Example 8 Making a plasticizer '
  • the extract obtained by the method described in Example 1 was ground into a fine powder, passed through a 120 mesh sieve, and added with an appropriate amount of diluent, uniformly mixed, and filled into empty capsules. Made into a capsule of 1000 capsules, 2 times a day, 2 capsules at a time.
  • the content of the reference substance stock solution under the item C is 1 ml, placed in a 10 ml volumetric flask, diluted with methanol to the mark, shaken, and used as a reference solution;
  • the absorbance is measured at 270nm wavelength, calculated, that is;
  • C icariin C 33 H 4 .0 15
  • C icariin is determined by high performance liquid chromatography (Chinese Pharmacopoeia 2000 edition, an appendix VID);
  • the color and conditions of the system and the system suitability test using octadecylsilane bonded silica as a filler; in a ratio of 55: 45 methanol - 0.4% phosphoric acid as mobile phase; detection wavelength is 270nm;
  • the peak of icariin should be no less than 4000;
  • icariin reference substance dried overnight with phosphorus pentoxide was weighed and dissolved in methanol to prepare a solution containing 0.2 mg per 1 ml as a reference stock solution; 2 ml of the reference substance stock solution was taken and placed in a 10 ml volumetric flask. , and diluted with the mobile phase to the mark, shake well, filtered through a microporous membrane (0.45 ⁇ m), that is, the reference solution;
  • the compound injection of the present invention contains icariin (C 33 H 4fl 0 15 ) per ml, and not less than 0.48 mg.
  • Another 60 mg of anhydrous glucose reference substance dried at 105 ° C to a constant weight was placed in a 50 ml volumetric flask, dissolved in water, and diluted to the mark to serve as a reference solution for glucose reference; respectively, 1 ml of glucose reference stock solution and 2 ml were taken. Place in a 25ml volumetric flask, dilute to the mark with water, shake well, as a reference solution;
  • the compound injection of the present invention contains not less than 0. 30mg o per lml of total sugar.
  • Example 11 a reference substance stock solution of icariin under the content determination of Example 11 is used as a reference solution;
  • Expanding agent unrolling, taking out, drying, spraying with 5% aluminum trichloride in ethanol, 105 after heating for a few minutes, and setting it under ultraviolet light with a wavelength of 365 nm; in the chromatogram of the test sample, in the color of the reference ⁇ The corresponding position of the fluorescent spot of the same color.
  • Example 13 Quality Control Method of Compound Injection of the Invention (Identification and Content Determination) Content Determination:
  • Aspirate 10 ml of the compound injection of the present invention set a constant weight of evaporation i, evaporate in a water bath, dry at 105 ° C for 3 hours, displace the silica gel drier for 30 minutes, weigh the weight, calculate, that is, obtain the compound of the present invention 2mg ⁇
  • the total solids per 1ml of the injection should not be less than 11. 2mg.
  • the compound injection of the invention contains total flavonoids per 1 ml, and is not less than 1, 2 mg based on icariin (C 33 H 4 .0 15 ).
  • C icariin is determined by high performance liquid chromatography (Chinese Pharmacopoeia 2000 edition of an appendix VID);
  • the reference solution and the test solution are respectively taken up by 20 ⁇ ⁇ 1 , ⁇ into the liquid chromatograph, determined, calculated, and obtained;
  • the compound injection of the present invention contains icariin (C 33 H 4 .0 15 ) per ml, not less than 0. 48 mg 0
  • Another 60 mg of anhydrous glucose reference substance dried to constant weight at 105 °C was placed in a 50 ml volumetric flask, dissolved in water, and diluted to the mark as a reference solution for glucose reference; respectively, 1 ml of glucose reference stock solution and 2 ml were separately taken. Place in a 25ml volumetric flask, with water Dilute to the mark, shake the hook, as a reference solution;
  • the total injection of the compound injection of the present invention is not less than 0.30 mg per lml.
  • Another reference substance of icariin under the content determination as a reference solution; according to thin-layer chromatography (Chinese Pharmacopoeia 2000 edition of an Appendix VIB) test, draw the test solution 10 ⁇ 1 and the reference solution 5: 1: The ratio of the thin layer of the silica gel G with a molar ratio of 0.3 mol% of disodium hydrogen phosphate and 0.3% sodium carboxymethylcellulose as a binder.
  • test solution of the total flavonoids in the content determination of Example 13 was concentrated to dryness; the residue was added with methanol to dissolve lml as a test solution;
  • Example 13 a reference stock solution of icariin under the content determination of Example 13 is used as a reference solution;
  • Fingerprint High performance liquid chromatography (Chinese Pharmacopoeia 2000 edition an appendix VID) determination;
  • Chromatographic conditions and system suitability test detector is a diode array UV detector; color column: Lichrospherdg, 4 ⁇ 250mm, particle size 5 ⁇ ⁇ ; detection wavelength is 270nra, column temperature is 40 ° C, flow rate is lml / min; 3 ⁇ 4
  • the number of slabs should be no less than 200,000 according to icariin; the ratio of 1000:100: 4. 92g: 11 of water-acetonitrile- 4 linonic acid-mercaptool mobile phase A and the ratio of 1000: 470: 50: 6.
  • Each of the reference solution and the test solution are respectively taken up to 20 ⁇ l, and injected into the liquid phase color meter for measurement.
  • the fingerprint of the test sample should be consistent with the common pattern of the fingerprint used in the quality standard (or the fingerprint of the accompanying control extract), that is, the retention time is about 24 min ⁇
  • the peak is clustered.
  • the a and b peaks are farther away from the c-g peak cluster, and the characteristics of the product are formed.
  • the similarity should be greater than 0.9.
  • Example 15 Quality Control Method of Compound Injection of the Invention (Content Determination and Fingerprint Determination)
  • the content of the reference substance stock solution under the item C is 1 ml, placed in a 10 ml volumetric flask, diluted with methanol to the mark, and shaken as a reference solution;
  • the absorbance is measured at 270nm wavelength, calculated, that is;
  • C icariin is determined by high performance liquid chromatography (Chinese Pharmacopoeia 2000 edition of an appendix VID);
  • icariin reference substance dried overnight with phosphorus pentoxide was weighed and dissolved in methanol to prepare a solution containing 0.2 mg per 1 ml as a reference stock solution; 2 ml of the reference substance stock solution was taken and placed in a 10 ml volumetric flask. , and diluted with the mobile phase to the mark, shake well, filtered through a microporous membrane (0.45 ⁇ m), that is, the reference solution;
  • the compound injection of the present invention contains icariin (C 33 H 4 .0 15 ) per ml, and not less than 0.48 mg.
  • Another 60 mg of anhydrous glucose reference substance dried to constant weight at 105 °C was placed in a 50 ml volumetric flask, dissolved in water, and diluted to the mark to serve as a reference solution for glucose reference; respectively, 1 ml of glucose reference stock solution and 3 ⁇ 4nl were separately taken, respectively Place in a 25ml volumetric flask, dilute to the mark with water, shake well, as a reference solution;
  • the compound injection of the present invention contains not less than 0. 30rag o per lml of total sugar.
  • Chromatographic conditions and system suitability test detector is diode array UV detector; color error column: LichrospherCig, 4 250mm, particle size 5 ⁇ ⁇ ; , detection wavelength is 270nra, column temperature is 40 ° C, flow rate is 1ml / min; The number of slabs should not be less than 200,000 according to icariin; the ratio of water to acetonitrile to 4 citric acid-methanol in a ratio of 1000:100: 4. 92g: 11 is 1000 and the ratio is 1000: 470: 50: 6.
  • Pipette 5ml of the compound injection of the present invention dilute to 10 ml with water, shake well, and take 5 ml, and elute through 10 ml of water and methanol, respectively, through a C 18 column, collect the decyl alcohol eluting fraction, and evaporate to dryness, and use 30% of the residue.
  • the methanol is adjusted to a volume of 10 ml, that is, the test solution is obtained;
  • the fingerprint of the test sample should be consistent with the fingerprint pattern shared by the quality standard (or the fingerprint of the accompanying control extract) attached to the quality standard, that is, there should be 7 peaks in the 24 min ⁇ 36rain section, which are marked as a, b, c, d, e, f, g, wherein the e peak is the most prominent, and the adjacent c, d, f, g peaks (g peak is icariin) constitute a cluster; a 5 ⁇ The b peak is farther away from the c ⁇ g peak cluster, forming the characteristics of the product; calculated by computer-aided similarity evaluation software, the similarity should be greater than 0.9.
  • Chromatographic conditions and system suitability test detector is diode array UV detector; column: LichrospherC ls , 4 ⁇ 250mm 5 particle size 5 ⁇ ⁇ ; detection wavelength is 270nm, column temperature is 40 ° C, flow rate is lml / min; number plate according to perish icariin 1 ⁇ 4 1 should not be less than 200, 000; at a ratio of 1000: 100: 4.
  • the gradient elution conditions are: 0 to 15 minutes, the mobile phase A volume ratio linearly decreases from 100% to 60%, and the mobile phase B volume ratio increases linearly from zero to 40%; 15 minutes to 35 minutes, the mobile phase A volume ratio is 60% linearly decreases to zero, and the mobile phase B volume ratio increases linearly from 40% to 100%;
  • Pipette 5 ml of the compound injection of the present invention dilute to 10 ml with water, shake the hook, and pipet 5 ml, and elute with 10 ml of water and methanol, respectively, through a C 18 column, collect the methanol elution portion, and evaporate to dryness, and the residue is determined with 30% methanol. Capacitance to 10ml, that is, the test solution is obtained;
  • the fingerprint of the test sample should be consistent with the fingerprint pattern shared by the quality standard (or the fingerprint of the accompanying control extract), that is, there should be 7 peaks in the section of approximately 24 min to 36 min.
  • Marked as a, b, c, d, e, f, g, where e peak is most prominent, and adjacent to it is a weak c, d, f, g peak (g peak is icariin) constitutes a cluster 5 ⁇
  • the a, b peak is farther away from the c ⁇ g peak cluster, forming the characteristics of the product; calculated by computer-aided similarity evaluation software, the similarity should be greater than 0.9.
  • Example 17 Quality Control Method of Compound Injection of the Invention (Content Determination, Identification, Fingerprint) Determination of content:
  • Aspirate 10 ml of the compound injection of the present invention set a constant weight of evaporation i, evaporate in a water bath, dry at 105 ° C for 3 hours, displace the silica gel drier for 30 minutes, weigh the weight, calculate, that is, obtain the compound of the present invention 2mg ⁇
  • the total solids per 1ml of the injection should not be less than 11. 2mg.
  • the content of the reference substance stock solution under the item C is 1 ml, placed in a 1 ml ml volumetric flask, diluted with methanol to the mark, and shaken as a reference solution;
  • the absorbance is measured at 270nm wavelength, calculated, that is;
  • Icariin According to high performance liquid chromatography (Chinese Pharmacopoeia 2000 edition of an appendix VID);
  • the compound injection of the present invention contains icariin (C 33 iU) 15 per ml, which is not less than
  • the liquid is a developing agent, unrolled, taken out, air-dried, sprayed with 5% aluminum trichloride in ethanol, 105 ° €; after heating for a few minutes, the ultraviolet light is set at 365 nm; in the chromatogram of the test sample, A fluorescent spot of the same color is displayed at a position corresponding to the color term of the reference.
  • Fingerprint High-performance liquid color i-method (Chinese Pharmacopoeia 2000 edition an appendix VID) determination;
  • Chromatographic conditions and system suitability test detector is a diode array UV detector; Column: Li chros pherds, 4 ⁇ 250mm, particle size 5 ⁇ ⁇ ; detection wavelength is 270nm, column temperature is 40 °C, flow rate is lml / min; The number of plates should be no less than 200 000 according to icariin; the ratio of 1000: 100: 4. 92g: 11 of water-acetonitrile-citrate-methanol mobile phase A and the ratio of 1000: '470 : 50: 6.
  • Each of the reference solution and the test solution are respectively taken up to 20 ⁇ l, and injected into a liquid chromatograph, and measured, that is, obtained;
  • the fingerprint of the test sample should be consistent with the fingerprint pattern shared by the quality standard (or the fingerprint of the accompanying control extract), that is, there should be 7 peaks in the section of approximately 24 min to 36 min. It is a, b, c, d, e, f, g, where e peak is the most prominent, and adjacent to it is a weak c, d, f, g peak (g peak is icariin) 5 ⁇
  • the peaks are clustered; a, b peaks are farther away from the c ⁇ g peak clusters, forming the characteristics of the product; calculated by computer-aided similarity evaluation software, the similarity should be greater than 0.9.
  • Example 18 Fingerprint of the compound injection of Epimedium extract (intermediate product) of the present invention:
  • Chromatographic conditions and system suitability test chromatographic conditions and system suitability test of the compound injection of the present invention as described in Example 17;
  • the fingerprint of the test sample should be consistent with the fingerprint of the extract (or the fingerprint of the accompanying medicinal extract), and the similarity should be greater than 0.9.
  • Example 19 Method for determining the fingerprint of Epimedium sagittatum used in the compound injection of the present invention:
  • the gradient elution conditions are: 0 to 15 minutes, the mobile phase A volume ratio linearly decreases from 100% to 60%, and the mobile phase B volume ratio increases linearly from zero to 40%; 15 minutes to 35 minutes, the mobile phase A volume ratio is 60% linearly decreases to zero, and the mobile phase B volume ratio increases linearly from 40% to 100%; 2g (accurate to 0).
  • Take the lycopene reference solution add 30% methanol to make a solution of the reference solution containing 0. lrag per lml; Olg), add 40ml of diluted ethanol, weighed, sonicated for 1 hour, weighed, filtered, and the filtrate was evaporated to dryness.
  • the residue was made up to 10fflL with 30% methanol, shaken, placed in the refrigerator for 1 hour, filtered.
  • the filtrate is taken to obtain the test solution; the reference solution and the test solution are respectively taken up to 20 ⁇ l, and injected into a liquid chromatograph, and the high-performance liquid phase method (Chinese Pharmacopoeia 2000 edition, an appendix VID) is determined.
  • the chromatograms of the original medicinal material Epimedium sagittatum are compared, and the characteristic peaks are mainly concentrated in the sputum area, and the other peaks are weak, that is, the peak of No. 21 is the most prominent, and the index component is icariin (peak 23) and 19 No. 20, the peak of No. 20 is very weak, and the peak of No. 22 is extremely weak, only visible. Therefore, a peak pattern (peak No. 21) is formed in the II segment.
  • the peaks in the I and II regions are extremely weak, and the color region is not detected in the IV region.
  • the correlation coefficient is calculated by the similarity evaluation software. 9 or more, which proves that the similarity is good.

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Abstract

La présente invention concerne une composition pharmaceutique fabriquée à partir de Radix Morinda Officinalis et Herba Epimedii dans un rapport de 5-50 sur 30-120 en poids. Ladite composition est préparée sous des formes posologiques cliniquement acceptables, telles que l’injection, l’aérosol, le comprimé, la capsule, le liquide pour voie orale, etc. par formulation avec des excipients conventionnels après extraction des substances des herbes médicinales et/ou raffinage supplémentaire. Ladite composition à pour effet de réchauffer le Yang, de revigorer les reins, d’apaiser l’asthme, de soulager la toux et de réguler l’immunité. Elle peut être utilisée dans le traitement de l’asthme bronchitique, de l’asthénie rénale accompagnée d’accumulation de flegme, des tumeurs et du SIDA en tant que médicament auxiliaire. Parmi les procédés de contrôle de la qualité de l’injection de ladite composition, on peut citer l’identification, l’analyse quantitative et tout particulièrement la détermination par chromatogramme
PCT/CN2005/000999 2005-07-07 2005-07-07 Composition de médecine chinoise traditionnelle, préparation et procédé de contôle de la qualité de celle-ci WO2007006171A1 (fr)

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