WO2007006171A1

WO2007006171A1 - A traditional chinese medicine composition, the preparation and quality controlling method thereof

Info

Publication number: WO2007006171A1
Application number: PCT/CN2005/000999
Authority: WO
Inventors: Sik Kwan To
Original assignee: Guangzhou Perfect Set Medicine Limited
Priority date: 2005-07-07
Filing date: 2005-07-07
Publication date: 2007-01-18
Also published as: WO2007006171A8

Abstract

A pharmaceutical composition, made from Radix Morinda Officinalis and Herba Epimedii in a ratio of 5-50 to 30-120 by weight, is prepared into clinical acceptable dosage forms such as injection, aerosol, tablet, capsule or oral liquid, etc., by formulating with conventional excipients after extracting the medicinal herb materials and/or further refining. Said composition has the effects of warming Yang, invigorating the kidney, appeasing asthma and relieving cough, and regulating immunity. It can be used in the treatment of bronchitic asthma, kidney asthenia accompanied with phlegm accumulation, as well as tumor and ADIS as auxiliary medicament. Methods for quality controlling of the injection made from said composition include identification, quantitative analysis and feature chromatogram determination.

Description

Traditional Chinese medicine composition, preparation method thereof and quality control method thereof

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. Background technique

Low immune function is a symptom that can be manifested in patients with various diseases. For example, 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. At present, 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 ^

At present, for these diseases related to low immune function, Western medicine is mainly based on symptomatic treatment. Although it has certain curative effect, it is not very ideal, and the recurrence rate is high. At the same time, chemical drugs have certain side effects. However, traditional Chinese medicine has outstanding therapeutic advantages in this respect. Therefore, it is of great significance to research and develop traditional Chinese medicine preparations with curative effects and anti-recurrence, which have low therapeutic and immunological functions, and further improve the quality of life of patients.

Regarding the research and analysis of the flavonoids of Epimedium in the injection of the present invention, 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. performed high-performance liquid chromatography analysis on the aboveground and underground parts of Epimedium, and there were multiple control comparisons, but it was not analyzed from the perspective of fingerprints. The chromatographic conditions and measured parameters did not meet the requirements of fingerprints. . Summary of the invention

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. A method for determining the fingerprint of Epimedium sagittatum.

The pharmaceutical composition of the present invention is prepared from the following bulk parts of the drug substance:

Bayu Tian 5 ~ 50 parts by weight, Epimedium 30 ~ 120 parts by weight.

Preferably, it 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. One of an aerosol, suppository or subcutaneous dosage form. 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. 3, ethanol is added to make the alcohol content 50-70%, ethanol is recovered, concentrated to a relative density of 1.1, fractionated on a polyamide column, eluted with water and a 25-35% macroporous solvent, and the eluent is mixed, recovered, dried, and pulverized. Elution, concentration, filtration and drying, dissolving and crystallizing with a solvent, drying under vacuum at 75-85 °C, to obtain the extract of Morinda officinalis; the net raw material of Epimedium sinensis is extracted with 10-16 times of water in sequence 2-4 Next, each time 1-2 hours, the extract is concentrated to a relative density of 40-60 ° C to 1. 1-1. 3, ethanol is added to make the alcohol content of 65-85%, ethanol is recovered, and concentrated to a relative density of 1 At 1 time, the polyamide column is adsorbed in stages, eluted with water and a 30-45% macropolar solvent, and the eluent is mixed and recovered for drying, then pulverized and eluted, concentrated and filtered to dry. Analytical crystals with vehicle solution, and dried at 75- 9 (TC hereinafter vacuo to give Epimedium extract; last step directly or after addition of a conventional pharmaceutically acceptable excipient made clinically acceptable dosage form.

Take the aforementioned pharmaceutical compositions of the invention, as described above preparation method of Epimedium extract, 'Morinda extract; taken Epimedium extract, Morinda filling amount of sodium chloride and the extract washed with water radio _t, activated carbon Appropriate amount, mixing, filtration, potting, sterilization to obtain the compound injection of the present invention.

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:

A. Total solids

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

1 ml of the compound injection of the present invention was taken, and it was transferred to a C ₁₈ pretreatment column which was eluted with 10 ml of each of sterol and 7j in advance, and then eluted with 10 ml of each of water and methanol, and the respective eluted fractions were separately collected and washed with water. Take off part of the spare; take the methanol elution part, put it in a 100ml volumetric flask, dilute to the mark with methanol, shake well, as the test solution;

In addition, 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;

According to the spectrophotometry (Chinese Pharmacopoeia 2000 edition of an appendix VA) test, the absorbance is measured at 270nm wavelength, calculated, that is;

2重量。 The compound injection of the total amount of flavonoids, icariin (C ₃₃ iU) ₁₅ ), not less than 1.2 mg.

C. Icariin is determined by high performance liquid chromatography (Chinese Pharmacopoeia 2000 edition, an appendix VID);

Chromatographic conditions and system suitability test, using octadecylsilane bonded silica as a filler; methanol in a ratio of 50 ~ 60: 50 ~ 40 - 0.4% phosphoric acid as mobile phase; detection wavelength is 270nm; theoretical tower The number of plates should be no less than 4000 according to the peak of icariin;

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 μm), that is, the reference solution;

Draw 1 ml of the compound injection of the present invention, place it in a 10 ml volumetric flask, dilute to the mark with a mobile phase, shake the hook, and filter through a microporous membrane (0.45 μαι) to obtain a test solution;

Aspirate the solution of the reference solution and the test solution for 20 μl each, and inject the liquid chromatograph into the liquid chromatograph, determine, calculate, and obtain;

The compound injection of the present invention contains icariin (C ₃₃ H ₄ .0 ₁₅ ) per ml, and not less than 0.48 mg. D. Total sugar

Determine the water elution fraction of the C ₁₈ column under the total flavonoids, place in a 10 ml volumetric flask, add water to the mark, shake well, as the test solution;

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;

Pipette 2 ml of each of the above test solution and reference solution, place 25 ml of colorimetric tube, add 1 ml of 4% phenylhydrazine solution, 7 ml of concentrated sulfuric acid, shake the hook, place in a 40 ° C water bath, keep for 30 minutes, remove, and then Place in the ice bath for 5 minutes, 'take out, according to spectrophotometry (Chinese Pharmacopoeia 2000 edition, an appendix VB) test 3⁄4r, with water as a blank, measure the absorbance at 490nm wavelength, calculate, that is;

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:

Take the test solution of total flavonoids under the content determination, and concentrate to dryness; the residue is added with sterol lml to dissolve, as the test solution;

In addition, 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 μ 1 and the reference substance are aspirated. 5: 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. 3: 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 瞽 column with a particle size of 5 μ π; 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. 92g: 11 (usually the mobile phase of HPLC is a liquid solvent, only the ratio of not writing units, but citric acid is a solid reagent, so g, the same below) of water-acetonitrile A mobile phase A of citric acid-methanol and a ratio of 1000: 470: 50: 6. 08 g of mobile phase B of water-acetonitrile-isopropanol-citric acid, 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 decreases to zero, and the mobile phase B volume ratio increases linearly from 40% to 100%;

Take the right amount of icariin reference substance, weighed, add 30% methanol to make a solution containing 0.1 mg per lml, that is, get the reference solution; draw 5ml of the compound injection of the invention, dilute to 10ml with water, shake, suction 5ml, through C ₁₈ cartridge, washed with water, methanol and eluting with 10ml each, were collected methanol fractions were evaporated to dryness, the residue treated with 30% methanol to 10ml, to obtain the test solution;,

Aspirate the reference solution and the test solution ^ 20 μl each, and inject into a liquid chromatograph, and determine by high performance liquid chromatography;

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.

See Figure 1 for the common pattern of the fingerprint pattern for the compound injection of the present invention.

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;

Weigh the appropriate amount of icariin, add 30% methanol to make a reference solution containing 0. lrag per lml; weigh the appropriate amount of Epimedium extract, add 30% methanol to make each lml containing lag The test solution; respectively, the reference solution and the test solution are each 20μ1, injected into the liquid phase colorimeter, and determined by high performance liquid chromatography (Chinese Pharmacopoeia 2000 edition an appendix VID), that is;

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.

Because 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 'μ 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

Rise to 100%;

2克 (Exact to 0). Take the lycopene reference substance solution, add 30% methanol to make a solution of 0. lmg of the control solution solution; take the arrow leaf Epimedium leaf powder (over the 3rd sieve) about 0. 2g (accurate to 0 01g), add 40ml of diluted ethanol, weighed, sonicated for 1 hour, weighed, filtered, the filtrate was evaporated to dryness, the residue was made up to 10 mL with 30% methanol, shaken, placed in the refrigerator for 1 hour, filtered , the filtrate is taken, that is, the test solution is obtained; respectively, the reference solution and the test solution are respectively taken up to 20 μl, and injected into a liquid crystal color meter, and the high performance liquid color method (Chinese Pharmacopoeia 2000 edition, an appendix VID) is determined, that is, Have

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 Π 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 chromatogram of the original drug of the present invention, Epimedium sagittatum, is shown in Figure 2.

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.

The 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.

By comparing the compound injection group and the Huachansu group in 60 patients with advanced primary bronchogenic carcinoma, 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 (P<0.05) indicates that the compound injection has a greater effect on improving anemia, liver and kidney function in patients with lung cancer than Huachansu injection.

By comparing the compound injection group with the Huachansu group, it indicated that 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.

At the same time, 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.

Since the main effective part of Epimedium is flavonoid glycosides, the effective part of Morinda officinalis is the total sugar mainly composed of oligosaccharides. Therefore, 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.

The following experimental examples and examples are intended to further illustrate but not limit the invention.

The following experimental materials and methods are applicable to Experimental Example 1-8.

Experimental materials and methods:

1. 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;

2, 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.

3. Experimental methods: In vivo experiments are performed by intramuscular injection, and the in vitro test drugs are dissolved in the medium or buffer according to the final concentration requirement.

Experimental Example 1 : Experimental study on antitussive effect of compound injection

H0 one Table 1 Effect of compound injection on ammonia-induced cough mice (: X soil SD) Group dose (ml / kg) Number of animals C (seconds) EDT50 R

Weight (only)

Normal control group 12 16.2 ±0.96 22.39

Ephedrine group 2 12 18.0± 0.84* 36.87 165 Compound injection 4 12 18· 8 ±1.08* 34.15 153 Compound injection 2 12 18.4 ±1.08* 31.62 141 Compound injection 1 12 17.8 ±0.84* 30.43 136 Conclusion: (* p<0.01) compound injection has obvious antitussive effect; compound injection contains 0.9 grams of crude drug per ml.

Experimental Example 2: Experimental study on the effect of compound injection

Table 2 Effect of compound injection on tracheal phenol red excretion in mice Group dose (ml / kg) body weight tracheal phenol red excretion (ug/ml) Normal control group 0.019 ± 0.0004 Ephedrine group 2 1.6 ± 0.6* Compound injection 4 1.7±0.5* Compound injection 2 1.5±0.4* Compound injection 1 1.4 ±0.8* Conclusion: (*p<0.01) Compound injection has the effect of enhancing phenol red excretion, which can be considered obvious痰 role.

Experimental Example 3: Experimental study on anti-asthmatic effect of compound injection

Table 3 (1) Effect of compound injection on wheeze in guinea pigs Drug dosage before administration

Ml//kg latency (seconds) Number of twitching animals Latency (seconds) Number of twitching animals

Normal control 88±17 10/10 86±19 10/10

Ephedrine 1.3 83±22 10/10 296 ±77* 0/10

Compound injection 2.6 91 ±19 10/10 294 ±84* 0/10

Compound injection 1.3 82 + 23 10/10 288 ±9* 1/10

Compound injection 0.7 79±20 10/10 231 ±5* 2/10 *p<0.01

Table 3 (2) Effect of compound injection on guinea pig isolated tracheal smooth muscle Curve height Dissolved percentage Drugs Specimen number

After administration of histamine, difference after administration (%) saline 8 7.8 ±2.5 7.8 ±2.5 0

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

Conclusion: ( **P<0.01 ) compound injection has obvious relaxation and smooth muscle effect, and the rate of decompression is 92. 4%.

Experimental Example 4: Effect of compound preparation on the same passive allergy in rats

Table 4 Effect of compound injection on the same passive allergy in rats (X soil SD)

Group Another '] dose amount of animals (only) blue spot eluent 0D value control group 10

Park Ermin 2.0 10 0.429 + 0.017 Compound Injection 3.0 10 0.333 ± 0.027** Compound Injection 4.5 10 0.354 ± 0.036* Compound Injection 0.8 10 0.386 ±0.081

Conclusion: ( *p<0.05; **P<0.01 ) compound injection has obvious anti-passive skin allergic effect.

Experimental Example 5: 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*

Conclusion: (*p<0.05; **P<0.01) Medium and high doses of compound injection have obvious effects on enhancing humoral immunity in animals. Table 5 (2) Effect of compound injection on lymphocyte transformation in mice with chronic bronchial model (X soil SD)

Group dose (ml / kg) Number of animals (only) Conversion index Normal control group 10. 18.05 + 3.06 Experimental control 10.96 ± 3.45 Ginsenoside 20rag/kg 10 17.33 + 3.06** Compound injection 4 10 15.55 + 3.85* Compound injection 2 10 14.69 ±2.31* Compound injection 1 10 12.58 ±4.12*

Conclusion: (*p<0.05; **P<0.01) The experimental results indicate that medium and high doses of compound injection can improve the cellular immune function of model animals.

Experimental Example 6: Antibacterial effect of compound injection And the effect of the anti-bacterial test of the compound injection (liquid test tube method)

Other strain compound injection blank strain positive

0.4 0.2 0.1 0.056 0.025 0.0125 0.00625 Control control control Staphylococcus aureus - 1111 - _{+ + + +} Consistent disease large intestine - 11 + 1 + + + + + + + - Salmonella 1111 - + +++ ― +++ ― Shigella 1111 + +++ ― +++ ― pneumococcal — one — one — ■ + ₊ +++ ― +++ ―

"one" aseptic growth; "+" a small amount of growth; "++, +" apparent growth

Conclusion: Compound injection has different inhibitory effects on the above pathogenic bacteria. Experimental Example 7: Experimental study on anti-inflammatory effects of compound injection:

Table 7 (1) Effect of compound injection on xylene-induced inflammation in mice (X soil SD) Dose Number of animals (only) Swelling degree (mg) Swelling inhibition rate (%) Normal control 12.0±2.6

Aspirin 0.2 7.3 ± 3.4** 39.2 Compound Injection 4 7.5 ± 1.9** 37.5 Compound Injection 2 8.0 ± 2.7* 33.3 Compound Injection 1

9.3 ± 3.0 22.2

Conclusion: **p<0.05; *P<0.01 The results indicate that the compound injection has obvious anti-inflammatory effects.

Table 7 (2) Effect of compound injection on granuloma caused by cotton ball

Group other dose (ml / kg body weight) Number of animals (only) Granuloma number (mg/100g body weight) Normal control 10 38.9 ±9.2 Aspirin 0.2g/kg 10 27.9 + 16.2* Compound injection 3.0 10 27.8 ±3.9* Compound injection Liquid L 5 10 29.2 ±2.9* Compound injection 0.8 10 27.7 + 8.2* Conclusion: *P<0.01 test results show that medium and high doses of compound injection have anti-cotton granuloma-induced granuloma.

Experimental Example 8: Effect of compound preparation on stress test Table 8 (1) Effect of compound injection on hypoxia in mice under normal pressure

Dosage (mg/kg) Number of animals (only) Anti-hypoxia time (tnin) Control group 10 59. 7 ± 11. 9 Ginsenoside 20rag/kg 10 80. 3 ± 12. 1** Compound injection 4. 10 75 5 + 13. 9* Compound injection 2 10 67. 8 Soil 11. 6* Compound injection 1 I ff 68. 2 soil 10. 7*

**p<0. 05; *P<0. 01 The results indicate that the compound injection has obvious anti-hypoxia effect. Table 8 (2) Effect of compound injection on swimming time in mice Group dose (nig/kg) Number of animals (only) Swimming duration control group 10 49. 6 ± 13. 9 Ginsenoside 20 mg/kg 10 70. 6 ± 6. 15** Compound injection - 4 10 79. 5 + 10. 1* Compound injection 2 10 70. 5 + 16. 8* Compound injection 1 10 65. 6 ± 17. 0* Conclusion: (* P < 0. 01 ) Compound injection can significantly prolong the swimming time of mice and has anti-fatigue effect.

The above experimental examples 1-8 test proved that the compound injection has obvious antitussive effect on cough caused by ammonia cough method; it has antiasthmatic effect on wheezing animal; it has obvious relaxation effect on bronchial smooth muscle; the injection also It has an anti-allergic effect; it has obvious functions of enhancing humoral and cellular immunity of the body in chronic bronchitis model animals; In the antibacterial experiment, the drug exhibits certain anti-inflammatory and antibacterial effects and enhances the body's ability to stress.

Example 9: Tumor-assisted treatment

1. Test materials

(1) Preparation of the invention (combination injection), Zhuhai Jianxin Medicine Co., Ltd., 0.9g/ral, 20021205

(2) Cyclophosphamide (CTX) powder injection, 0.2g/piece, Shanghai Hualian Pharmaceutical Group Co., Ltd. (3) 60 female mice, weighing (19±l) g, were provided by Guangzhou University of Traditional Chinese Medicine.

2. Method: 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.

3. Observed indicators: 10 days after inoculation of tumor cells, the quality of the toes was aseptically cut out, and the tumor inhibition rate was calculated according to the following formula: [(The quality of the tumor toe in the control group and the quality of the tumor-bearing foot in the administration group) The quality of the tumor foot site] χ ΐοο%.

4, the results

Table 9 Inhibitory effect of compound injection on the growth of Lewis lung carcinoma in mice Group Tumor foot mass (m/g) Tumor inhibition rate (%)

Compound injection (mB/g.kg- ¹ )

50 0.350 ± 0.05 57.1

10 0.402 ± 0.03 48.8

2 0.501 ± 0.02 38.9

CTX 0.120 ± 0.03 85.4 Control 0.820 ±0.09 0 The results showed that the high, medium and low doses of compound injection had a strong inhibitory effect on tumor growth.

实^:Example 10: AIDS adjuvant treatment

1. Materials: (1) Compound injection: Zhuhai Jianxin Pharmaceutical Co., Ltd., 0. 9g/ml, 20021205; (2) BALB7C mice: provided by Guangzhou University of Traditional Chinese Medicine. (3) People's Weekly Blood: Normal adult whole blood. (3) α-interferon, interleukin-II: purchased from the Chinese Academy of Military Medical Sciences.

2, method

(1) 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).

(2) Test method for inducing interleukin II in vivo: According to 5mg/kg and 30mg/kg, the 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.

(3) In vivo induced α-interferon effect test method: 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. When 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.

3, the results

(1) 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)

0 0.08 soil 0.01

62.5 0.15 ±0.01

125 0.18 ±0.01

250 0.28 ±0.01

500 0.45±0.01

1000 0.78 ±0.01

(2) In vivo induction of interleukin II (IL-2): Compound injection has a strong role in inducing interleukin 小鼠 in mice, see the following table:

Table 11 Effect of compound injection on the induction of IL- ² (CMP) Days after spleen cell supernatant (mg/kg)

Dilution multiple 0 5 20

1: 2 450 + 200 1025 + 300 1600 ± 400

3 1: 4 245 ± 60 1500 ± 500 5000 ± 250

6 1: 2 700 + 180 1800 ± 600 2801 ±540

1: 2 800 ± 50 1540 ± 270 1650 ± 215

(3) In vivo; induced interferon effect test: Compound injection has the effect of inducing interferon in mice, and the interferon titer can be increased with the increase of drug amount. See the table below:

Table 12 Effect of compound injection on interferon-inducing dose mg/kg 60 30 15 Interferon titer 180 50 20 Results: Compound injection has significant activation on human tau cells, interleukin II, interferon There is an inducing effect. The following experimental methods are applicable to Experimental Examples 11-15 Sixty patients with advanced primary bronchogenic carcinoma were randomly divided into treatment group (combined injection group) and control group (huahuasu group), including 40 cases in the treatment group and 20 cases in the control group. The course of treatment (6 weeks), comparing the quality of life, blood analysis, liver function and immune function of the treatment group and the control group.

Example 11: Changes in blood routine and liver and kidney function before and after treatment in both groups

Table 13 Changes in red blood cells before and after treatment in both groups

Group number of cases before treatment of red blood cells after treatment of red blood cells compound injection group 40 3. 70 ± 0. 52 4. 27 ± 0. 45

Hua Tuo Su Group 20 3. 82 ± 0. 56 3. 89 ± 0. 52

Note: After t test, there was a significant difference in red blood cell contrast between the two groups (P<0.05). There was a significant difference in red blood cell comparison before and after treatment in the compound injection group (P<0.01). There was no significant difference in red blood cell contrast (P>0.05).

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

Huachansu group 20 25. 60 ± 3. 50 30. 6 + 9. 83 Note: After t test, there was a significant difference in the comparison between the compound injection and the transglutaminase (P<0.01).

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).

By comparing the compound injection group and the Huachansu group, there were significant differences in red blood cells, alanine aminotransferase and aspartate aminotransferase compared with the Huachansu group (P<0.05). It indicates that compound injection has a greater effect on improving anemia, liver and kidney function of lung cancer patients than Huachansu injection.

Experimental Example 12: Changes in natural killer cells (NKC) before and after treatment in both groups

There was a significant difference between the natural killer cells after treatment in the compound injection group (P<0.05), but there was no significant difference between the natural killer cells after treatment with the Huachansu group (P>0. 05), indicating that the compound injection has more effect on the natural killer cells of the patient than the Huachansu injection.

Changes in natural killer cells (NKC) before and after treatment

Group number of patients before treatment with NJ C compound injection group 40 27. 07 ± 5. 27 31. 41 ± 4. 44 Huachansu group 20 28. 35 ± 3. 91 31. 45 ± 5. 56 Experiment Example 13: Changes in lymphocyte turnover rate (LBT) before and after treatment in both groups There was a significant difference in lymphocyte turnover rate between the two groups after treatment (P < 0.05), but after treatment with the Huachansu group There was no significant difference in lymphocyte conversion rate compared with that before treatment (P>0.05). There was a significant difference in the rate of lymphocyte turnover after treatment between the two groups (P < 0.05), indicating that the compound injection has a higher effect on the lymphocyte turnover rate of the patient than the Huachansu injection.

Table 17 Changes in lymphocyte turnover rate (LBT) before and after treatment Group number of cases before treatment (LBT) After treatment (LBT) Compound injection group 40 38. 87 + 12. 45 43. 13 ± 11. 78 Huachansu group 20 40. 7 + 14. 66 41. 03 + 14. 03 Experimental Example 14: Changes in T cell subsets before and after treatment in both groups

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. Time group CD^CD/CD/CD ₄ 7 CD

Before treatment 37.09 ± 4.76 29.77 ± 3.67 29.04 ± 3.59 1.038 ± 0.74 Compound injection 40 After treatment 41.40 ± 8.01 32.76 ± 4.82 23.64 ± 4.20 1.131 ±0.70 Liquid group

Before treatment 37.65 ± 4.99 25.71 + 4.11 25.141 ± 3.40 1.039 ± 0.064 Huachansu group 20 After treatment 37.9 ± 5.60 26.10 ±4.51 23.81 ± 5.81 1.063 ±0.065 Experimental example 15: Changes in quality of life after treatment in both groups

The changes in quality of life before and after treatment were significantly different between the compound injection group and the Huachansu group (P<0.05), indicating that the combination injection combined with the Huachansu injection can improve the survival of cancer patients. The role of quality.

Assessment of quality of life in patients after treatment

The number of group cases increased and the stability decreased. Compound injection group 40 21 (52.5%) 16 (40%) 3 (7.5%) Huachansu group 20 6 (30%) 9 (45%) 5 (25%) Note: Ridit analysis. There was a significant difference in the quality of life between the two groups after treatment.

(P<0.05)

Experimental Example 16: Therapeutic effect of the compound injection of the present invention on asthma and the regulation of Thl/Th2

1, method:

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.

Of the 58 patients who completed the clinical study, 29 were in the sputum group (treatment group) and 29 in the group B (placebo group). The results are shown in Tables 20, 21, and 11.

2. The result shows:

Table 20 Comparison of various indicators between the two groups of patients Group A Group B

Average asthma before treatment 5.12 ± 3.29 4.98 ± 3.64

Symptom score 2 weeks after treatment 4.99 ± 3.65 4.88 ± 4.15

4 weeks after treatment 4.16 + 3.92 4.39 + 3.74

PEF value before treatment 368.12 ± 287. 45 420.13 ± 359.56

(L/min) 2 weeks after treatment 386.37 ± 298. 14 415.17 + 297.49

4 weeks after treatment 389.66 ± 285. 49 422.1 ± 365.44

FEV1 (L) before treatment 2.06 ± 1.95 2.11 ± 1.86

2 weeks after treatment 2.18 ±1.58 2.04 + 2.01

4 weeks after treatment 2.20 + 1.67 2.23 + 1.92

Daily use of 嚆乐宁喷数 Before treatment 8.58 + 6.67 10.44 + 9.49

2 weeks after treatment 6.35 ± 4.26 8.57 ± 6.24

4 weeks after treatment 5.92 + 4.25 9.36 ±5.12

Comparison of changes in indicators between the two groups before and after treatment

Group A Group B P value

Symptom scores of each sputum 2 weeks after treatment -0.13 ±0.05 - 0.10 ±0.04 >0.05

Index change △ PEF 18.25 + 28.74 -4.96 ±26· 75 <0.05

AFEVl 0.12 ± 0.32 - 0.07 ± 0.28 <0.05

The number of breathering shots is 1.23 ±3.25 -1.87 ±2.98 >0.05

After 4 weeks of treatment, the △ symptom score was 0.96 ± 1.15 -0.59 ± 0.92 <0.01

Index change △ PEF 21.54± 30.58 1.97 + 40.5 <0.01

厶 FEV1 0.14 ±0.31 0.12 ± 0.32 >0.05

舒乐宁喷数 - 2.66 ±1.25 - 1.08 ±3· 24 <0.05 Each indicator is obtained after treatment - pre-treatment values

Comparison of Thl/Th2 (IFN-γ/IL-4) between the two groups

Group A Group B

IFN- Y IL-4 Th/Th2 1FN- γ 1L-4 Th/Th2 Before treatment 6. 04 + 4. 40 0. 44 ± 0. 29 17. 64 ± 11. 90 8. 88 ± 9. 32 0. 44 ± 0. 37 23. 58 ± 14. 99 After treatment 8. 53 ± 11. 06 0. 56 ± 0. 84 32. 98 + 31. 31 8. 31 + 8. 31 0. 48 + 0. 33 32 81 + 45. 97

T value -1. 09 -0. 70 2. 54 0. 23 -0. 41 0. 95

P value >0. 05 >0. 05 >0. 05 >0. 05 >0. 05 >0. 05

After 2 weeks of treatment, 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. ου.

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 daily application of Chuan Le Ning.. spray number, the treatment group decreased. ₄ 2. 23, the placebo group decreased by 1. 8.7, although the difference between the treatment before and after treatment was significant (P <0.05), the treatment group reduced asthma The number of Lenin sprays was significantly higher than that of the placebo group, but the difference was meaningless (P>0.05).

After 4 weeks of treatment, 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.

Multiple indicators in the treatment group improved, and were superior to the placebo group, with abnormalities in liver and kidney function, blood, urine, and electrocardiogram.

Conclusion: Compound injection can regulate Thl/Th2 imbalance in asthma patients, and it is an effective and safe traditional Chinese medicine preparation for treating asthma. '

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

According to the 1999 Asthma Clinic of the Pediatric Respiratory Group of the Chinese Medical Association, 57 children were enrolled in the study, and 54 patients completed the study. All children were treated according to GINA and ARIA, according to the condition, symptom score, lung function, asthma and allergic rhinitis.

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.

Main experimental material: cy-chrome labeled anti-CD ₄ mAb (RPA-T4 clone). Cy-chrome-labeled anti-CD ₈ mAb (RPA-T8 clone), PE-labeled murine IgG isotype control immunoglobulin (R3-34 clone) anti-IL-4 monoclonal antibody, anti-IFN-γ monoclonal antibody Purchased from PharMingen, other reagents such as PMA, Ionomycin, and Monens in were purchased from Sigma, IMDM medium, fetal bovine serum, etc., purchased from GIBC0BRL, and red blood cell lysate was purchased from PharMingen. the company.

Before treatment and at the end of treatment, 57 patients received venous blood lml, heparin anticoagulation, PMA (20 g/ml), ionomycin (1 μg/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-γ mAb labeling for 30 min. The £-type fine monthly package is fully tested, and the data analysis uses the Lys is software.

The results showed: ₄ +, CD ₈ + lymphocytes expressing IFN- gamma] No significant difference in the amount of, after treatment, the treatment group CD ₄ +, CD ₈ + lymphocytes was specifically IFN- treatment group and the placebo before treatment CD γ was significantly elevated (p < 0.05), and there was no significant increase in IFN-γ expression in CD ₄ +, -. CD ₈ + lymphocytes in the placebo group (Table 23).

Table 23 IFN-γ expression in CD4+, CD8+ lymphocytes before and after treatment in the treatment and placebo groups

CD4+IFH- γ + CD8+IFN- γ +

t t* p* before treatment before treatment t * p* treatment group 26 12. 02 ± 4. 80 17. 85 ± 4. 12 7. 062 <0. 01 20. 53 ± 9. 55 26. 77 ± 8. 20 3. 0308 <0. 05 Control group 26 13. 11 ± 5. 51 13. 94 + 5. 21 0. 5652 >0. 05 19. 60 ± 9. 53 24. 28 ± 8. 23 2. 3353 <0. 05 t# 0. 765 3. 030 0. 3494 1. 0910

p# 〉0. 05 <0. 01 >0. 05 >0. 05

* Group t-test before and after treatment in the group - insurance Group t test before and after treatment

According to the ratio of the expression rate of cytokine IFN-γ/IL-4 in lymphocytes to the ratio of Th1/Th2, CD IFN-γ + represents Th1, CD ₄ +IL-4+ represents Th2, and after treatment with compound injection In 21 patients, the proportion of Th1/Th2 increased, accounting for 80.77%. In the placebo group, 15 patients had an increase in Th1/Th2 ratio, accounting for 55.56 %. Nonparametric X ² test, x ² = 3.865 , p <0. 05 , with significant difference (Table 24) ₆ Changes in Th1/Th2 ratio after treatment in the treatment group and the control group

Thl/Th2 rise Thl/Th2 decrease Total compound injection group 21 5 '

Placebo group 15 12 27 (55. 56%) Total 36 17 52

Non-parametric analysis x ² test, x = 3.865, p < 0.05, with significant differences. After treatment with compound injection, the expression of IFN-γ in CD ₄ +, CDa+ lymphocytes was increased (P<0.05), and the ratio of Thl/Th2 was significantly increased. Compared with placebo, 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:

First, the overall efficacy evaluation

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 total effective rate of the treatment group was significantly higher than that of the control group (X ² =5.05 Ρ<0.05), and there was no significant difference in the response time between the two groups.

Table 25 Asthma symptom control

Group Cases Clinical cure Significant progress Progress Ineffective Effective treatment group 10 2 2 4 2

20% 20% 40% 20% 80% Control group 10 0 1 2 7

0% 10% 20% 70% 30% Second, pulmonary function blood gas measurement results

The results of the lung function and blood gas measurement before and after treatment in the treatment group are shown in Table 26. It can be seen that PEF was significantly higher than that before treatment after one week of treatment, and FEV 1.0% was significantly increased after two weeks compared with before treatment. There was no significant difference between PEF and FEV 1.0% one week and two weeks after treatment.

The results of lung function and blood gas measurement before and after treatment in the control group are shown in Table 27. It can be seen that there was no significant change in PEF and FEV 1.0% in the control group at one week, and PEF and FEV 1.0% were significantly higher than the treatment two weeks later. There was no significant difference between PEF and FEV 1.0% in one week and two weeks. .

- 'Because of the ineffective cases in the two groups (including 2 in the 3⁄4 group, 7 in the control group), 7 cases were treated with conventional asthma (mainly β ₂ receptor agonist and theophylline), these cases were not included in the treatment. After the week, two weeks of statistics.

Table 26 Results of pulmonary function blood gas measurement in the treatment group (results are expressed by X soil S) Indicators Before treatment (n=10) One week later (n=8) Two weeks later (n=8)

FEV1.0% 69.5 ±9.3* 81.8 ±6.9* 十85.9±7.5 ⁺

PEF 67.8 ±10. Γ 83.5 ±5. Γ 85.6 ±6.5+

Η 7.380 ±0.033 7.387 ±0.042 7.406 ±0.040

Pa02 79.3 ±7.9 83.4 ±7.8 87.1±5.9

PaC02 36.9± 3.2 37.8 ±2.4 39.1±3.3 Note: 1. FEV 1.0% and PEF are expressed as a percentage of the predicted value. Pa02 and PaC02 are both mmHg.

2. The same superscript symbol indicates that P>0.05 has no significant difference, and there is a significant difference between P<0.05, and no superscript symbol indicates no significant difference.

Table 27 Results of pulmonary function blood gas measurement in the control group (results are expressed as X soil s) Indicators Before treatment (n=10) One week later (n=3) Two weeks later (n=3)

FEV1.0% 72.6 + 10.5* 82.1 + 9.7* ⁺ 86.7 ±12.1 ⁺

PEF 68.7 + 8.9' 77.9 ±8.4' ⁺ 87.3 ±11.3+

PH 7.405 ±0.041 7.367 + 0.021 7.381 ±0.038

Pa02 76.8 + 12.3 84.41 ±10.8 , 86.9 ±9.7

PaC02 37.4 ±5.6 41.5 + 4.8 38.5 ±4.1

The clinical treatment of 20 patients with mild to moderate asthma attack showed that the clinical effective rate of intramuscular injection was 80% in asthmatic patients, which was significantly higher than that in the control group (30%). At the same time, the treatment group treated with FEV 1.0% and PEF increased significantly. It shows that compound injection has obvious curative effect on patients with mild to moderate asthma attack in adults, and has an improvement effect on FEV 1.0% and PEF. '

Experimental Example 20: Chromatinography of Epimedium Herbs

1. Variety identification;

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.

1. Main chemical composition:

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.

3, the real insurance method

Refer to the HPLC method (Chinese Pharmacopoeia 2000 edition, an appendix VID), combined with the requirements of the fingerprint.

Color 侮 condition and system suitability test: same technical solution.

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 20μ1, and injects the liquid chromatograph to determine.

4. Preparation method of test solution

Refer to the Chinese Pharmacopoeia 2000 edition of an epimedium [content determination] under the test sample preparation method, the volume of the solution after the refrigerator is placed for 1-hour, filtered through a microporous membrane, pay.

5, chromatographic conditions screening

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.

(1) Liquid color "": HP1100

(2) Selection of color pan column:

According to the "Guidelines for the Study of Chromatographic Fingerprints of Traditional Chinese Medicine Injections", the comparison test of the columns was carried out. The columns of 5 different manufacturers were tested, namely Merck Lichrospher C ₁₈ (4 χ 250mm 5μ m) and Agi lent Hypers il ODS. (4 x 250mm 5μ m), Polar is C18-A (4. 6 250mm 5μ m), Dalian Elite YWG ODS (4.6 x 250mm 10 μ m), Waters Spher i sorb 0DS2 (4. 6 x 250ram 5μ«ι ). The experimental results show that, except for the separation effect of Hypers il ODS (4 x 250 hidden 5μ m) column, the spectra obtained by the other four color columns are relatively close, due to the overall separation effect of Merck Lichrospher C18 (4 x 250mm 5 Mm). Ideal and cheap, so the Merck Lichrospher C18 column was chosen.

(3) Selection of measurement wavelength

From the three-dimensional map drawn by DAD, 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 ό3⁄4. The maximum absorption of icariin is 270 nm. The maximum absorption of the color Fu peak before 15 minutes is at 320 nm. In order to avoid the loss of information in the map, the full spectrum of the components is reflected as much as possible. 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.

(4) Selection of column temperature

The effects of column temperature of 15 °C, 25 °C, 30 °C, 40 °C on the chromatographic separation effect were investigated. The _3⁄4 knot indicates that the separation temperature of the chromatographic peak clusters with different retention time of 30 ~ 45 minutes The effect is best at 40 ° C, so the selected column temperature is 40 ° C.

6. Methodological verification of color "i" fingerprint test

(1) Stability test

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

(median)

Correlation coefficient 0. 9990 0. 9990 0. 9995 0. 9990 0. 9875 0. 9981 0. 9992 0. 9990 0. 9999

(mean)

o

The coincidence coefficient is 0. 9974 0. 9997 0. 9994 0. 9942 0. 9900 0. 9975 0. 9885 0. 9760 0. 9991

(median)

The coincidence coefficient is 0. 9980 0. 9994 0. 9872 0. 9831 0. 9969 0. 9959 0. 9751 0. 9990

(mean)

Note: From left to right, 0 hours, 1 hour, 2 hours, 3 hours: 4 hours, 24 hours, 48 hours, 1 hour, 96 hours of Epimedium test solution HPLC map Corresponding similarity.

(2) Repeatability test

Take 5 samples of the same medicinal material and test according to the preparation and detection methods of the test solution. The visual fingerprint has no obvious change. After the similarity evaluation, the correlation coefficient is greater than 0.9 (Table 29), indicating the method repeatability. good.

Table 29 Results of repeated similarity evaluation of fingerprints of medicinal materials

Correlation coefficient (median) 0. 9802 0. 9994 0. 9985 0. 9998 0. 9980 Correlation coefficient (mean) 0. 9968 . , 0. 984 0. 9981 0. 9982 0. 9993 Coincidence coefficient (median) 0. 9872 0. 9995 0. 9986 0. 9994 0. 9985 Coincidence coefficient (mean) 0. 9983 0. 9989 0. 9981 0. 9985 0, 9992

7. Identification and difference of chromatographic fingerprints of various medicinal materials

The five kinds of Epimedium medicinal herbs contained in the Chinese Pharmacopoeia have different characteristics and can be distinguished. In order to facilitate the identification and analysis of the fingerprints of various medicinal materials, firstly, taking the fingerprint of the Korean esculenta medicinal material with the highest chromaticity peak as an example, according to the peak time, 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. Among 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

8, the raw material medicine arrow leaves Epimedium HPLC color fingerprint map establishment and similarity evaluation. In view of the raw material of this product is the arrow leaf Epimedium, 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. Therefore, as a whole, the fingerprint of Epimedium sagittatum is relatively simple, which is easy to distinguish from other varieties. The original data of 10 batches of the leaves of Epimedium sinensis were analyzed by the similarity evaluation software, and the correlation coefficients were all above 0.9, which proved that the similarity was good (Table 31).

Table 31 Evaluation results of 10 chromatographic fingerprint image similarity evaluation results of 10 batches of raw materials

0. 9718 0. 8861 0. 9953 0. 9830 0. 9830 0. 9124 0. 9768 0. 9946 0. 9891 0. 9659 (median)

Correlation coefficient

0. 9813 0. 9080 0. 8956 0. 9743 0. 9743 0. 9390 0. 9814 0. 9921 0. 9795 0. 9693 (mean)

Coincidence coefficient

0. 9723 0. 8983 0. 9043 0. 9823 0. 9823 0, 9261 0. 9708 0. 9949 0. 9682 0. 9707 (median)

Coincidence coefficient

0. 9819 0. 9175 0. 9093 0. 9732 0. 9732 0. 9495 0. 9774 0. 9920 0. 9553 0. 9730 (mean)

Experimental Example 21: Study on the HPLC chromatographic fingerprint of the extract of Epimedium sagittatum (intermediate product) in the compound injection of the present invention

1. Experimental method '

Refer to High Performance Liquid Chromatography (Chinese Pharmacopoeia 2000 Edition, an appendix VID), combined with the requirements of fingerprints.

Chromatographic conditions and system suitability test color Fu column: same technical solution.

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 20μ1, 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. .

2. Methodological verification of color fingerprint test

(1) Stability test

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, 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.

Table 32 Similarity evaluation results of stability test of Epimedium extract

0. 9942 0. 9906 0. 9931 0. 9697 0. 9830 0. 9872 0. 9977 0. 9992 0. 9991 0. 9989 (median)

Correlation coefficient

0. 9926 0. 9872 0. 9954 0. 9783 0. 9880 0. 9919 0. 9956 0. 9974 0. 9973 0. 9968 (mean)

Coincidence coefficient

0. 9915 0. 9241 0. 9967 0. 9647 0.-9664 .0. 9660 0. 9972 0. 9993 0. 9985 0. 9981 (median)

Coincidence coefficient

0. 9912 0. 9141 0. 9867 0. 9748 0. 9742 0. 9738 0. 9968 0. 9975 0. 9960 0. 9952 (mean)

Note: From left to right, 0 hours, 1 hour, 2 hours, 3 hours, 4 hours, 24 hours, 48 hours, 72 hours, 96 hours of Epimedium extract for the test solution HPLC Figure i昝 corresponding Similarity.

(2) Repeatability test

Take the same sample of the test sample 5 parts, according to the preparation and detection method of the test solution, the visual fingerprint has no obvious change in the whole picture, the similarity evaluation, the correlation system The number is greater than 0.9 (Table 33), indicating that the method is reproducible. Table 33 Correlation test results of similarity evaluation results (median) 0. 9971 0. 9998 0. 9999 0. 9902 0. 9920 Correlation coefficient (mean) 0. 9958 0. 9984 0 9986 0. 9958 0. 9966 Coincidence coefficient (median) 0. 9943 0. 9998 0. 9999 0. 9866 0. 9878 Coincidence factor (mean) 0. 9909 0. 9982 0. 9983 0. 9938 0. 9942

3, extract of Epimedium sagittatum (intermediate product) HPLC chromatographic fingerprint and similarity evaluation,

Compared with the fingerprints of Epimedium sagittatum, 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. In order to avoid the inconvenience of the peak number of the fingerprint of Epimedium medicinal materials in describing the fingerprint of the compound injection of the present invention, 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 (extract) 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

0. 9827 0. 9972 0. 9974 0. 9978 0. 9980 0. 9992 0. 9990 0. 9990 0. 9994 0. 9994

(median)

Correlation coefficient

0. 9851 0. 9976 0. 9974 0. 9971 0. 9981 0. 9988 0. 9986 0. 9992 0. 9995 0. 9993 (Average)

Coincidence coefficient

0. 9793 0. 9946 0. 9978 0. 9951 0. 9983 0. 9983 0. 9990 0. 9986 0. 9992 0. 9995 (median)

Coincidence coefficient

0. 9821 0. 9954 0. 9977 0. 9942 0. 9983 0. 9976 0. 9984 0. 9989 0. 9994 0. 9994 (average)

Note: From left to right are 4 ratios 030201, 030202, 030203, 030204

Samples of 030205, 030206, 030207, 030208, 030209, 030210.

Experimental Example 22: Study on HPLC color error fingerprint of compound injection of the present invention

1. Real ^: Method: Same technical solution.

1. Methodological verification of chromatographic fingerprint test

(1) Stability test

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 significant changes in the whole picture, and the similarity was evaluated. The coefficients are greater than 0.9 (Table 35)', indicating that the test solution is well characterized during this time. ' ·'

Evaluation result of similarity evaluation of compound injection stability test of the present invention

0. 9936 0. 9917 0. 9948 0. 9969 0. 9994 0. 9992 0. 9915 0. 9831 0. 9713 0. 9767 (median)

Correlation coefficient

0. 9968 0. 9848 0. 9908 0. 9945 0. 9964 0. 9961 0. 9970 0. 9928 0. 9843 0. 9881 (Average)

Coincidence coefficient

0. 9919 0. 9931 0. 9956 0. 9845 0. 9989 0. 9849 0. 9795 0. 9511 0. 9626 (median)

Coincidence coefficient

0. 9924 0. 9870 0. 9919 0. 9788 0. 9953 0. 9955 0. 9922 0. 9671 0. 9764 (mean) Note: 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.

(2) Repeatability test

Take 5 samples of the same compound injection solution, according to the preparation and detection method of the test solution, the visual fingerprint has no obvious change. The correlation coefficient is greater than 0.9 (the data is shown in Table 36). , indicating that the method is reproducible.

Repeated test result of compound injection of the invention

Correlation coefficient (median) 0. 9883 0. 9700 0. 9975 0. 9982 0. 9990

Correlation coefficient (mean) 0. 9958 0. 9834 0. 9954 ' 0. 9938 0. 9957

Convergence coefficient (median) 0. 9767 0. 9566 0. 9970 0. 9982 0. 9988

Coincidence coefficient (mean) 0. 9896 0. 9757 0. 9924 0. 9923 0. 9938

3. Ten batches of the compound injection of the invention HPLC chromatographic fingerprint and similarity evaluation

The original data of 10 batches of the compound injection of the present invention by HPLC chromatographic fingerprint test were calculated by the similarity evaluation software, and the correlation coefficients were all above 0.9 (Table 37), which proved that the similarity was good.

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)

Correlation coefficient 0. 9964 0. 9973 0. 9990 0. 9991 0. 9988 0. 9966 0. 9985 0. 9969 0. 9993 0. 9997

(mean)

Coincidence coefficient 0. ?952 0. 9952 0. 9983 0. 9993 0. 9988 0. 9936 0. 9966 0. 9991 0. 9998

(median)

The coincidence coefficient is 0. 9964 0. 9949 0. 9990 0. 9992 0. 9985 0. 9947 0. 9976 0. 9988 0. 9992 0. 9997

(mean)

Note: 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. In the extract (intermediate product) and the fingerprint of the finished product, 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 (ΠΙ) 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. BRIEF DESCRIPTION OF THE DRAWINGS: '

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

- Chinese herbal medicines, 戟天天, 箭叶淫羊藿, pre-treatment process, picking up good medicinal materials from many messy medicinal materials, and then washing with water several times to remove sand and mud, cut the leaves of Epimedium into lcm Small section, less than 60 ° C dry; Bashu days below 60 ° C dry, crushed into 20 mesh coarse powder. The singularity of the extract is concentrated to a relative density of 1.2 (50). °C), ethanol is added to make the alcohol content up to 60%, ethanol is recovered, concentrated to a relative density of 1.1, fractionated on a polyamide column, eluted with water and a 30% macroporous solvent, eluent mixing the collected dry, pulverized elution, filtration and drying was concentrated, dissolved solvent parsing crystallized, dried in vacuo at below 80 ° C, to give an extract Morinda; Epimedium net material successively with I ^5, 10, 10 times the amount of water was extracted for ^1.5 , 1, and 1 hour, and the extract was concentrated to a relative density of 1.2 (50 ° C), and ethanol was added to make the alcohol content up to 70%, recovered ethanol, concentrated to a relative density of 1.1, fractionated on a polyamide column, eluted with water and a 35% macroporous solvent, the eluent was mixed and dried, then pulverized and eluted, concentrated The mixture was filtered and dried, dissolved in a solvent, and dried under vacuum at 80 ° C to obtain Epimedium extract.

Example 2: Preparation process of injection preparation

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'

Bayu day 370g Arrow leaf Epimedium l OOOg

1000 pieces of the intramuscular injection solution were prepared by the method described in Example 2, 2 ml each, twice a day, one at a time.

Example 4: Preparation of an atomizing agent

Bayu day 370g Arrow leaf Epimedium l OOOg

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.

Example 5: Preparation of lyophilized powder injection

Bayu day 370g Arrow leaf Epimedium l OOOg

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

Bayu Tian 370g Arrow Leaf Epimedium 1000g

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

Bayu Tian 200g Epimedium 500g

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 '

Bayu Tian 200g Epimedium 500g

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.

Example 9: Preparation of oral liquid preparation

Bayu Tian 300g Arrow Leaf Epimedium ll OOg

3⁄4 The extract obtained by the method described in Example 1 was dissolved in water to form a solution. Filtered, - filled, capped, sterilized at 115 ° C for 30 minutes, and labeled. Made into 1000 oral liquids, 2 times a day, 1 time each time.

Example 10: Compound injection of the invention

Arrow Leaf Epimedium 50Gg Bayu Tian 400g

Cut the leaves of Epimedium into small pieces of about 1 cm, place them in the extraction tank, add 15 times, 10 times, 10 times of water in turn, and decoct them for 1.5, 1 and 1 hour respectively, and combine the extracts and filter. The upper polyamide column, the filtrate is concentrated to a relative density of 1.2 (50 ° C), ethanol is added to make the alcohol content up to 70%, allowed to stand overnight, filtered, ethanol is recovered, concentrated to a relative density of 1.1, on the polyamide column, Wash with water first, discard the aqueous solution, elute with 35% ethanol, and recover the eluent. Ethanol, filtration, concentration of the filtrate, and drying under reduced pressure of 80 Torr to obtain Epimedium extract.

The smashed smashed into a 20-mesh coarse powder, placed in an extraction tank, and successively extracted with 1, 7, and 6 times of water, respectively, for 1.5, 1, and 1 hour, and the combined extracts were filtered, and the filtrate was concentrated to a relative density L 2 . At the time of (50 ° C), add ethanol to make the alcohol content up to 60%, filter, recover ethanol, concentrate to a relative density of 1. 1, on the polyamide column, first wash with water, discard the water, then use 30% Ethanol elution, the eluate was concentrated, and dried under reduced pressure at 80 Torr to obtain Morinda officinalis extract.

Add the above-mentioned Epimedium extract, Morinda officinalis extract and sodium chloride to 1000ml of water for injection, add appropriate amount of activated carbon, mix the hook, filter, potting, and sterilize. Example 11: Quality Control Method of Compound Injection of the Invention (Content Determination)

A, total solid

Draw 10ml of the compound injection of the present invention, set it in constant weight evaporation, dry it in a water bath, dry it at 105 °C for 3 hours, displace it in a silica gel drier for 30 minutes, weigh the weight, calculate, and obtain; 2mg。 The total solids per 1ml of the injection should not be less than 11. 2mg.

B, total flavonoids

1 ml of the compound injection of the present invention was taken, and it was transferred to a C ₁₈ pretreatment column which was eluted with 10 ml of methanol and water in advance, and sequentially eluted with 10 ml of each of water and methanol, and the fractions were separately collected. Standby; take the methanol elution part, place it in a 100ml volumetric flask, dilute to the mark with methanol, shake well, as the test solution;

2重量。 The compound injection of the total amount of flavonoids per liter, icariin (C ₃₃ H ₄ .0 ₁₅ ), not less than 1. 2mg. 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;

The appropriate amount of 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 μm), that is, the reference solution;

Draw 1 ml of the compound injection of the present invention, place it in a 10 ml volumetric flask, dilute to the mark with a mobile phase, shake the hook, and filter through a microporous membrane (0.54 μm) to obtain a test solution;

Pipette the reference solution and the test solution for 20 μl each, inject into the liquid chromatograph, determine, calculate, and obtain;

The compound injection of the present invention contains icariin (C ₃₃ H _4fl 0 ₁₅ ) per ml, and not less than 0.48 mg.

D, total sugar

Determine the water elution fraction of the C ₁₈ column under the total flavonoids, place it in a lOjnl volumetric flask, add water to the mark, and shake the hook to serve as the test solution;

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;

Pipette 2 ml of each of the above test solution and reference solution, place 25 ml of colorimetric tube, add 4 ml of 4% benzene face solution, 7 ml of concentrated sulfuric acid, shake well, place in a 40 ° C water bath, keep for 30 minutes, remove, and then Place in the ice bath for 5 minutes, take out, take the spectrophotometry (Chinese Pharmacopoeia 2000 edition, an appendix VB) test ^ r, with water as a blank, at a wavelength of 490nm Determine the absorbance, calculate, and get;

30mg _o The compound injection of the present invention contains not less than 0. 30mg _o per lml of total sugar.

Example 12: Quality Control Method of Compound Injection of the Invention (Identification Method)

Taking the test solution of the total flavonoids containing the hydrazine assay in Example 11 and concentrating to dryness; the residue was added with methanol to dissolve lml as a test solution;

Further, a reference substance stock solution of icariin under the content determination of Example 11 is used as a reference solution;

Lmol/L disodium hydrogen phosphate, 0. lmol / L disodium hydrogen phosphate, 0. lmol / L disodium hydrogen phosphate, 0. lmol / L disodium hydrogen phosphate, 0. lmol / L disodium hydrogen phosphate, 0. lmol / L disodium hydrogen phosphate, 0. lmol / L disodium hydrogen phosphate, 0. lmol / L disodium hydrogen phosphate, 0. A thin layer of silica gel G with 3% sodium carboxymethyl cellulose as a binder is applied in a ratio of 1. 3: 1: 1 of butyl acetate-formic acid-water at a temperature below 10 ° C. 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荧光 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:

A, total solid

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.

B, total flavonoids

Another sample of the reference substance stock solution under the condition of C is taken in a 10 ml volumetric flask. Dilute to the mark with decyl alcohol, shake well, as a reference solution;

According to the spectrophotometric method (Chinese Pharmacopoeia 2000 edition, an appendix VA) test, the absorbance is measured at 270mn wavelength, and the calculation is obtained;

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);

Chromatographic conditions and 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; theoretical plate number according to Epiglottis The peak of glycosides should not be 'below 4000;

That learn dried over phosphorus pentoxide overnight icariin amount of reference substance, dissolved in methanol, a solution containing 0. ² mg per lml of, as the reference stock solution; drawing reference stock solution 2ral, set 10ml volumetric flask Medium, and diluted with the mobile phase to the mark, shake well, filtered through a microporous membrane (0. 45 μ ηη), that is, the reference solution;

Draw 1ml of the compound injection of the present invention, put it into a 10ml volumetric flask, dilute to the mark with a mobile phase, shake it, and filter it with a microporous membrane (0.54 μm) to obtain a test solution;

The reference solution and the test solution are respectively taken up by 20 μ _Γ 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

D, total sugar

Take the content of the total flavonoids to determine the water elution part of the C _1S column, set the Oral volumetric flask, add water to the scale, shake the hook, as the test solution;

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;

Pipette 2 ml of each of the above test solution and reference solution, place 25 ml of colorimetric tube, add 1 ml of 4% phenol solution, 7 ml of concentrated sulfuric acid, shake the hook, place in a 40 ° C water bath, keep for 30 minutes, remove, and then set Place in the water bath for 5 minutes, take it out, and take the spectrophotometric method (Chinese Pharmacopoeia 2000 edition, an appendix VB) test, take the water as the blank, measure the absorbance at the wavelength of 490nm, calculate, that is;

Identification method:

Take the test solution of the total flavonoids under the content determination, and concentrate to a thousand; the residue and methanol lml to dissolve, as the test solution;

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 μ 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. 1 butyl acetate-citric acid-water 10 ° C below the layered upper layer solution as a developing agent, unrolled, taken out, air-dried, sprayed with 5% aluminum trichloride ethanol solution, heated at 105 ° C for several minutes , under the ultraviolet light with a wavelength of 365ηπι; in the chromatogram of the test sample, the fluorescent spot of the same color is displayed at the position corresponding to the color term of the reference product.

Example 14: Quality Control Method of Compound Injection of the Invention (Identification and Fingerprint Measurement)

The 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;

Further, a reference stock solution of icariin under the content determination of Example 13 is used as a reference solution;

Thin layer chromatography (China Pharmacopoeia ²⁰⁰⁰ Edition an appendix VIB) test, to learn the test solution and reference solution 10 μ 1 5 μ 1, respectively, at the same point 0. lmol / L phosphate a thin layer of silica gel G with disodium acid hydrogenate and 0.3% sodium carboxymethyl cellulose as a binder, in a ratio of 1. 3: 1: 1 of butyl acetate-capric acid-water below 10 ° C Place the layered upper layer solution as a developing agent, unroll it, remove it, dry it, spray it with 5% aluminum trichloride in ethanol, heat it at 105 °C for a few minutes, and then set it under UV light with a wavelength of 365 nm. In the chromatogram, a fluorescent spot of the same color is displayed at a position corresponding to the reference color.

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 χ 250mm, particle size 5 μ ιη; 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. 08g of water-acetonitrile-isopropanol-citric acid mobile phase B, eluted according to the following gradient elution conditions: Gradient elution conditions: 0 to 15 minutes mobile phase A volume ratio From 100% linearly to 60%, the mobile phase B volume ratio increases linearly from zero to 40%; from 15 minutes to 35 minutes, the mobile phase A volume ratio decreases linearly from 60% to zero, and the mobile phase B volume ratio is linear from 40%. Rise to 100%; . '- - ' ' Take the appropriate amount of icariin, weighed, add 30% methanol to make a solution containing 0. lmg per lml, that is, the reference solution;

Draw the compound injection 5 ιιι1 of the present invention, dilute to 10 ml with water, shake the hook, and take 5 ml, pass through a C ₁₈ column, elute with 10 ml each of water and methanol, collect the methanol eluting portion, evaporate, and use 30% methanol for the residue. Capacitance to 10ml, that is, the test solution is available;

Each of the reference solution and the test solution are respectively taken up to 20 μl, 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 ~ There should be 7 peaks in the 36min section, and the markings are labeled a, b, c, d, e, f, g, where the e peak is the most prominent, and the adjacent it is the weak c, d, f, g peak ( The g-peak is icariin. 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)

Determination of content:

A, total solid

Draw 10ml of the compound injection of the present invention, set a constant weight of the evaporation 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, that is, the compound of the present invention 2mg。 The total solids per 1ml of the injection should not be less than 11. 2mg.

B, total flavonoids

Pipette 1ml of the compound injection of the present invention, and transfer it to methanol in advance, each! i Omi was eluted in a spare C ₁₈ pretreatment cartridge, and then eluted with 10 ml of water and methanol, respectively, and each eluted fraction was collected, and the water eluted fraction was used; the methanol eluted fraction was placed in a 100 ml volumetric flask, and methanol was used. Dilute to the mark and shake the hook as the test solution;

In addition, 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;

2rag。 The complex injection of the compound containing flavonoids per ml, with icariin (C ₃₃ H ₄ .0 ₁₅ ), not less than 1. 2rag.

Chromatographic conditions and system suitability tests were filled with octadecylsilane bonded silica Agent; a ratio of 55: 45 sterol to 0. 4% phosphoric acid is the mobile phase; the detection wavelength is 270nm; the theoretical plate number should be not less than 4000 according to the peak of icariin;

Aspirate the solution of the reference solution and the solution of the test solution by 20 μl each, and inject the liquid phase into the instrument, determine, calculate, and obtain;

The compound injection of the present invention contains icariin (C ₃₃ H ₄ .0 ₁₅ ) per ml, and not less than 0.48 mg.

D, total sugar

Determine the water elution fraction of the C _1S column under the total flavonoids, place it in a 10 ml volumetric flask, add water to the mark, and shake the hook to serve as the test solution;

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;

Pipette 2ml of each of the above two solutions, place in ml colorimetric tube, add 4ml of 4% phenol solution, 7ml of 3⁄41 ^ acid, shake well, set in 40 ° C water bath, keep for 30 minutes, take out, then place in ice bath 5 minutes, take out, according to spectrophotometry (Chinese Pharmacopoeia 2000 edition of an appendix VB) test, with water as a blank, the absorbance at 490nm wavelength, calculate, that is;

30rag _o The compound injection of the present invention contains not less than 0. 30rag _o per lml of total sugar.

Fingerprint: High Performance Liquid Chromatography (Chinese Pharmacopoeia 2000 Edition, an appendix VID) Determination

Chromatographic conditions and system suitability test detector is diode array UV detector; color error column: LichrospherCig, 4 250mm, particle size 5 μ οι; , 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. 08g of water-acetonitrile-isopropanol-citric acid mobile phase B, eluted according to the following gradient elution conditions: Gradient elution conditions: 0 to 15 minutes mobile phase A volume ratio From 100% linear P to 60%, mobile phase B volume ratio linearly increased from zero to 40%; 15 minutes to 35 minutes, mobile phase A volume ratio linearly decreased from 60% to zero, mobile phase B volume ratio by 40 % linearly rises to 100%;

Take the appropriate amount of icariin reference substance, weighed, add 30% methanol to make a solution containing 0.1 mg per 1 ml, that is, a reference solution;

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;

Take 20 μ 1 of the reference solution and the test solution, respectively, and inject into the liquid chromatograph, and measure, that is, obtain;

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.

Example 16: Quality Control Method of Compound Injection of the Invention (Fingerprint)

High performance liquid chromatography (Chinese Pharmacopoeia 2000 edition of an appendix VID) determination; Chromatographic conditions and system suitability test detector is diode array UV detector; column: LichrospherC _ls , 4 χ 250mm ₅ particle size 5 μ ιη; detection wavelength is 270nm, column temperature is 40 ° C, flow rate is lml / min; number plate according to perish icariin ¼ ₁ should not be less than 200, 000; at a ratio of 1000: 100: 4. 92g (water, methanol, ethanol, propanol liquid, no marked units, hereinafter the same): 11 The mobile phase A of water-acetonitrile-citrate monodecyl alcohol and the ratio of 1000: 470: 50: 6. 08g of water-acetonitrile-isopropanol-citric acid mobile phase B, according to the following gradient elution conditions Elution:

Take the appropriate amount of icariin reference substance, weighed, add 30% methanol to make a solution containing 0.1 mg per lml, that is, the reference solution;

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:

A, total solid

B, total flavonoids

1 ml of the compound injection of the present invention was taken, and it was transferred to a C ₁₈ pretreatment column which was eluted with 10 ml of methanol and water in advance, and the body was eluted with 10 ml of water and methanol, respectively, and each eluted fraction was collected and eluted with water. Partially reserved; take the methanol elution part, place it in a 100ml volumetric flask, dilute to the mark with methanol, shake well, as a solution for the product;

In addition, 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;

2重量。 The compound injection of the total amount of flavonoids per liter, icariin (C ₃₃ IW) ₁₅ ), not less than 1. 2mg.

- C, Icariin _: According to high performance liquid chromatography (Chinese Pharmacopoeia 2000 edition of an appendix VID);

Chromatographic conditions and 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; theoretical plate number according to Epig The peak of glycosides should be no less than 4000;

Weigh the appropriate amount of icariin dried overnight with phosphorus pentoxide, dissolve it in methanol, make a solution containing 0.2 rag per lml, as a reference stock solution; draw 2 ml of the reference stock solution, and place it in a 10 ml volumetric flask. , and diluted with the mobile phase to the mark, shake well, filtered through a microporous membrane (0.45 μm), that is, the reference solution;

Draw 1 ml of the compound injection of the present invention, place it in a 10 ml volumetric flask, and dilute with mobile phase until Scale, shake the hook, filter with microporous membrane (0. 45 μ πι), then obtain the test solution; respectively, draw the reference solution and the test solution 20 μ 1 each, inject into the liquid chromatograph, measure, Calculate, that is,

The compound injection of the present invention contains icariin (C ₃₃ iU) ₁₅ per ml, which is not less than

0. 48mg.

D, total sugar. '

Another 60 g of anhydrous glucose referenced to 105 ° C dried to constant weight, placed in a 50 ml volumetric flask, dissolved in water, and diluted to the mark, as a glucose reference stock solution; separately absorb the glucose reference stock solution lml and 2ml, respectively Place in a 25ml volumetric flask, dilute with water to the mark, shake the hook, as a reference solution;

Pipette 2 ml of each of the above test solution and reference solution, place 25 ml of colorimetric tube, add 1 ml of 4% phenol solution, 7 ml of concentrated sulfuric acid, shake well, place in a 40 ° C water bath, keep for 30 minutes, remove, and then set Placed in an ice bath for 5 minutes, taken out, and tested by spectrophotometry (Chinese Pharmacopoeia 2000 edition, an appendix VB), using water as a blank, measuring the absorbance at a wavelength of 490 nm, and calculating, that is, obtaining the invention; The compound injection should not contain less than 0. 30fflg _o per lml of total sugar.

Ways to raise:

Take the test solution of the total flavonoids under the content determination, concentrate to a thousand; the residue and methanol lml to dissolve, as the test solution;

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 μ 1 and the reference solution 5: 1: 1 : 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1 butyl acetate-formic acid-water is placed at a temperature below 10 ° C and the upper layer is dissolved. 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 χ 250mm, particle size 5 μ ιιι; 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. 08g of water-acetonitrile-isopropanol-citric acid mobile phase B, eluted according to the following gradient elution conditions: Gradient elution conditions: 0 to 15 minutes mobile phase A volume ratio by 100% linearly decreased to 60%, mobile phase B volume ratio increased linearly from zero to 40%; 15 minutes to 35 minutes, mobile phase A volume ratio linearly decreased from 60% to zero, mobile phase B volume ratio increased linearly from 40% To 100°/. ;

Take the right amount of icariin reference substance, weigh it, add 30% methanol to make a solution containing 0. lrag per lml to get the reference solution; ^ < - take 5ml of the compound injection of the invention, dilute to 10ml with water, shake Evenly, 5 ml was taken, and the mixture was eluted with 10 ml of water and methanol, respectively, through a C ₁₈ column, and the methanol eluted fraction was collected, evaporated to dryness, and the residue was made up to 10 ml with 30% methanol to obtain a test solution;

Each of the reference solution and the test solution are respectively taken up to 20 μl, 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;

Weigh the appropriate amount of icariin, add 30% methanol to make a reference solution containing 0.1 mg per 1 ml; weigh the appropriate amount of Epimedium extract, add 30% methanol to make lmg per lml The test solution; respectively, the reference solution and the test solution are each 20μ1, injected into the liquid phase colorimeter, and determined by high performance liquid chromatography (Chinese Pharmacopoeia 2000 edition an appendix VID), that is;

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:

Chromatographic conditions and - system suitability test V 'Using a diode array UV detector and LichrospherC ₁₈ , 4 χ 250mm, particle size 5 μ ηι color i column, detection wavelength 270nm, column temperature 40 ° C, flow rate 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-citric acid-methanol mobile phase A and the ratio of 1000: 470: 50: 6. 08g 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 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 μl, and injected into a liquid chromatograph, and the high-performance liquid phase method (Chinese Pharmacopoeia 2000 edition, an appendix VID) is determined. Have

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.

Claims

Rights request

A pharmaceutical composition for treating bronchial asthma, characterized in that the pharmaceutical composition is made of the following raw materials by weight:

Bayu Tian 5 ~ 50 parts by weight, Epimedium 30 ~ 120 parts by weight.

The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is prepared from the following parts by weight of a drug substance:

Bayu Tian 15 ~ 40 parts by weight, Epimedium 40 ~ 110 parts by weight.

Bayu Tian 28 parts by weight, Epimedium 75 parts by weight.

4. A pharmaceutical composition according to claim 3, characterized in that the epimedium is referred to as Epimedium.

The pharmaceutical composition according to claim 1, 2, 3 or 4, characterized in that the 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 one. Dosage forms include oral formulations or parenteral dosage forms.

The pharmaceutical composition according to claim 5, wherein 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. A parenteral administration form selected from the group consisting of an injection, an aerosol, a suppository, or a subcutaneous administration form.

The pharmaceutical composition according to claim 5, wherein the composition is prepared by:

Chinese herbal medicines, Baqiantian and Epimedium, are pre-treated, and a good selection of medicinal materials is selected from a large number of messy medicinal materials, and then washed with water several times to remove sand and mud, and the epimedium is cut into small pieces of about 1 and 50-70. Drying below °C; after drying at 50-70 °C, Morinda is pulverized into 20 mesh coarse powder; then the second raw material is used for the second extraction process. The mixture is 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. 3, ethanol is added to make the alcohol content 50-70 %, recovering ethanol, concentrated to a relative density of 1.1, fractionally adsorbed on a polyamide column, eluted with water and a 25-35% macroporous solvent, and the eluent was mixed, recovered, dried, and then pulverized and eluted. Concentrated, filtered and dried, dissolved and precipitated in a solvent, dried under vacuum at 75-85 °C to obtain the extract of Morinda officinalis; the net raw material of Epimedium is extracted 2-4 times with 10-16 times of water, each time 1约分分分分分分分分分分分分分分分分分分分分分分分分分分分分Adsorption on a polyamide column, elution with water and a 30-45% macroporous solvent, mixing and recovering the eluent, pulverizing and eluting, concentrating and drying, and dissolving and crystallizing with a solvent, at 75-90 The extract of Epimedium is obtained by vacuum drying below °C; finally, a clinically acceptable dosage form is prepared by a conventional procedure directly or by adding a pharmaceutically acceptable excipient.

8. A pharmaceutical composition according to claim 7 wherein the composition is prepared as a monthly preparation by:

Morinda medicines, Epimedium pretreatment step, chosen from the large number of good clutter medicinal herbs, then washed repeatedly with water to remove sand, dirt, the _Epimedium; lcm cut into small pieces and right, 60 ° C below the dried Basil day 6Q ° C, after the following dry, pulverized into 20 mesh coarse powder; then use this net raw material for the second extraction process, the Bashutian net raw materials are extracted with 8, 7, 6 times of water respectively 1. 5, 1, 1 hour, the extract is concentrated to a relative density of 50 ° C to 1.2, ethanol is added to make the alcohol content up to 60%, ethanol is recovered, concentrated to a relative density of 1. 1 Adsorption by amide column, elution with water and a 30% macroporous solvent, the eluent is mixed and recovered, dried, pulverized and eluted, concentrated and filtered, dried, dissolved and precipitated in a solvent, and dried under vacuum at 8 (TC). The extract of Morinda citrifolia; the net raw material of Epimedium sinensis is extracted with 15, 10, and 10 times of water, respectively, for 1.5, 1, and 1 hour, and the extract is concentrated to a relative density of 50 ° C to 1.2. Alcohol content up to 70%, ethanol is recovered, concentrated to a relative density of 1. 1 Attached, 35% water and one large The polar solvent is eluted, the eluent is mixed and recovered, and then pulverized and eluted. The mixture is filtered and dried, dissolved and precipitated with a solvent, and dried under vacuum at 80 ° C to obtain extract of Epimedium; the extract is formulated with water. Solution, filter rod coarse filtration, filter ball and 0.45 filter filtration, potting, 115 ° C 30 minutes sterilization, color water leak detection, light inspection, printing, and then packaging into the warehouse; Ready: Rough washing with pure water, fine washing with water for injection, final sterilization and drying, for potting, that is.

9. Use of a pharmaceutical composition according to claim 1, 2, 3 or 4 in the manufacture of a medicament for the treatment and prevention of bronchitis or asthma conditions. '

10. Use of a pharmaceutical composition according to Claim 1, 2, 3 or 4 for the preparation of a medicament for the treatment of tumor or AIDS immune dysfunction.

11. Use according to claim 10, characterized in that said treating a tumor means inhibiting the growth of tumor cells.

The use according to claim 10, characterized in that said treatment of AIDS means significant activation of human T cells or induction of interleukin 11, interferon.

13. Use according to claim 9, characterized in that said treatment of asthma refers to raising the maximum peak expiratory flow rate, exerting a forced expiratory volume value for one second or regulating the Thl cell/Th2 cell imbalance.

14. Use according to claim 10, characterized in that said treating a tumor means treating primary bronchogenic lung cancer.

15. Use according to claim 14, characterized in that said treatment of primary bronchogenic carcinoma refers to an increase in the number of red blood cells or a decrease in the values of aspartate aminotransferase and alanine aminotransferase.

16. Use according to claim 14, characterized in that said treatment of primary bronchogenic lung cancer means increasing the body's natural killer cells.

17. Use according to claim 14, characterized in that said treatment of primary bronchogenic carcinoma means increasing the rate of lymphocyte turnover in the body.

18. Use according to claim 14, characterized in that said treatment of primary bronchogenic lung cancer means improving the body's immune function.

19. Use according to claim 18, wherein said enhancing the body immune function means altering the T cell subset.

.

20. Use according to claim 14, characterized in that said treatment of primary bronchogenic carcinoma means improving the quality of life of the patient.

21. A method of quality control of a pharmaceutical composition according to claim 5 for injection, characterized in that the method comprises one or more of the following methods for determining the content:

A, total solid

Aspirate 10 ml of the compound injection of the present invention, evaporate in a constant weight, 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; 2mg; The total solids per 1ml of liquid is not less than 11. 2mg;

B, total flavonoids

1 ml of the compound injection of the present invention was taken, and it was transferred to a C ₁₈ pretreatment column which was eluted with 10 ml of methanol and water in advance, and then eluted with 10 ml of each of water and methanol, respectively, and each eluted fraction was collected, and the water eluted fraction was separately collected. Standby; take the methanol elution part, put it in a 100ml volumetric flask, dilute it to the mark with a drunkenness, shake it well as the test solution; and take the content of the reference substance stock lr under the C item, and put it in a 10ml volumetric flask. And diluted with methanol to the mark, shake well, as a reference solution; according to the spectrophotometric test, the absorbance is measured at a wavelength of 270 nm, calculated, that is; the compound injection of the present invention contains total flavonoids per lml, based on icariin , not less than 1. 2mg;

C, icariin according to high performance liquid color i method;

Chromatographic conditions and system suitability test: using octadecylsilane bonded silica as a filler; methanol in a ratio of 50 ~ 60: 50 ~ 40 - 0.4% phosphoric acid as mobile phase; detection wavelength is 270nm; theoretical tower The number of plates should be no less than 4000 according to the peak of icariin; weigh the appropriate amount of icariin dried overnight with phosphorus pentoxide, dissolved in methanol, Each 1ml contains 0. 2mg of the solution, as a reference stock solution; draw the reference stock solution 2ffll, put it in a 10ml volumetric flask, and dilute to the mark with the mobile phase, shake it, filter it with a pore filter, and obtain the reference solution. Draw 1ml of the compound injection of the present invention, put it into a 10ml volumetric flask, dilute it to the mark with a mobile phase, shake the hook, filter it with a filter of L-hole, and obtain the test solution; respectively, draw the reference solution and the test sample The solution of each of the solutions containing the icariin is not less than 0. 48mg;

D, total sugar

Determine the water elution fraction of the C ₁₈ column under the total flavonoids, place it in a 10 ml volumetric flask, add water to the mark, shake well, as the test solution; take another anhydrous glucose control dried to constant weight at 105 °C. 60mg, placed in the S Oinl volumetric flask, 'dissolved in water, and diluted to the scale, as a glucose reference stock solution; separately absorb the glucose reference stock lral and 2ral, respectively, placed in a 25ml volumetric flask, diluted with water to the mark, Shake well, as a reference solution; Pipette 2 ml of each of the above test solution and reference solution, place 25 ml of colorimetric tube, add 4 ml of 4% phenol solution, 7 ml of concentrated acid, shake the hook, set 4 (TC water bath) Insulation for 30 minutes, take out, put it in the ice bath for 5 minutes, take it out, take the spectrophotometric test, take water as empty, white, measure the absorbance at the wavelength of 490nm, calculate, that is, get the compound injection of the invention The liquid containing no more than 0. 30mg per lml of total sugar. ..

The method of claim 21, wherein the icariin content is determined by a ratio of 55:45 methanol to 0.4% phosphoric acid as a mobile phase.

The quality control method according to claim 21 or 22, characterized in that the method further comprises the following authentication method:

Take the test solution of the total flavonoids under the content determination, and concentrate to a thousand; the residue is added with methanol 1ml to dissolve, as the test solution; and the reference stock solution of icariin under the content determination is used as the reference solution. The smear of the test solution is as follows: 0 μl/L disodium hydrogen phosphate, 0.3% carboxymethyl fiber sodium is used as the test solution, 10 μl of the test solution and 5 μl of the reference solution are respectively taken. The adhesive layer of the silica gel G is pulled up to 1. 3: 1: 1 vinegar Butyrate-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, heated at 105 °C for several minutes, and then placed at a wavelength. It is examined under the ultraviolet light of 365riffl; in the chromatogram of the test sample, the fluorescent spot of the same color is displayed at the position corresponding to the color of the reference substance.

24. A method of quality control of a pharmaceutical composition according to claim 5 for injection, characterized in that the method comprises the following fingerprint measurements:

Chromatographic conditions and system suitability test: Diode array UV detector and LichrospherCig, 4 250 ΙΜΙ, particle size 5 μ ηι i| "column; detection wavelength is 270 nm, column temperature is 40 ° C, flow rate is 1 ml / min; The number of plates should be no less than 200,000 according to icariin; the ratio of 1000: 100: 4. 92g: 1 ί of water-acetonitrile-citrate-methanol has a mobile phase ratio of 1000: 470: 50 : 6. 08g of water-acetonitrile-isopropanol- 4 citric acid mobile phase B, elution; the elution condition is 0 to 15 minutes, the mobile phase A volume ratio linearly decreases from 100% to 60%, flow The phase B volume ratio increases linearly from zero to 40%; from 15 minutes to 35 minutes, the mobile phase A volume ratio decreases linearly from 60% to zero, and the mobile phase B volume ratio increases linearly from 40% to 100%;

Take the right amount of icariin reference substance, weigh it, add 30% sterol to make a solution containing 0.1 mg per 1 ml, that is, get the reference solution; take 5ml of the compound injection of the invention, dilute to 10ml with water, shake up 5 ml was taken, and the mixture was eluted with 10 ml of water and methanol through a C ₁₈ column, and the methanol eluted fraction was collected, evaporated to dryness, and the residue was made up to 10 ml with 30% methanol to obtain a test solution;

Each of the reference solution and the test solution are respectively taken up by 20 μl and injected into a liquid chromatograph, and determined by high performance liquid chromatography, which is obtained;

The fingerprint of the test sample should be consistent with the fingerprint pattern of the control fingerprint or the fingerprint of the accompanying control extract, that is, there should be 7 peaks in the 24 min ~ 36 min period of retention time, labeled as a, b, c > d respectively. , e, f, g, wherein the e peak is most prominent, and the neighboring is a weak c, d, f, g peak to form a peak cluster, the g peak is icariin; a, b peak is farther away C. The peak of the cluster is greater than 0.9.

The pharmaceutical composition according to claim 8, wherein the fingerprint method of the extract of Epimedium sagittatum is prepared according to the preparation method, wherein the method is:

Chromatographic conditions and system suitability test: using diode array UV detector and LichrospherC ₁₈ > 4 25 Oram, color 5 粒度粒度柱 column; 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- 4 citric acid-methanol mobile phase A and the ratio of 1000: 470: 50: 6. 08g of water-acetonitrile-isopropanol" mobile phase B of citric acid, elution; elution conditions of 0 to 15 minutes, the mobile phase A volume ratio linearly decreased from 100% to 60 %, the mobile phase B volume ratio linearly rises from zero to 40%; from 15 minutes to 35 minutes, the mobile phase A volume ratio decreases linearly from 60% to zero, and the mobile phase B volume ratio linearly rises from 40% to 100%;

Weigh the appropriate amount of icariin, add 30% methanol to make a reference solution containing 0.1 mg per lml; weigh the appropriate amount of Epimedium extract, add 30% methanol to make lmg per lml The test solution; respectively, the reference solution and the test solution are each 20μ1, and injected into the liquid color "meter, determined by high performance liquid chromatography, '.

The fingerprint of the test sample should be consistent with the fingerprint of the extract - Fig. -: ί or the fingerprint of the extract of the accompanying medicinal material. The similarity should be greater than 0.9.

26. The method of claim 4, wherein the method is:

Chromatographic conditions and system suitability test, using diode array UV detector and Lichrospherds.4 250mm, particle size 5 μ ιη color column, detection wavelength is 270nm, column temperature is 4 (TC, flow rate is lral / min; The number of plates should be no less than 200,000 according to the 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. 08g of water-acetonitrile-isopropanol-pitric acid mobile phase B, elution; elution conditions: 0 to 15 minutes mobile phase The volume ratio of A decreases linearly from 100% to 60%, and the volume ratio of mobile phase B increases linearly from zero to 40%. From 15 minutes to 35 minutes, the volume ratio of mobile phase A decreases linearly from 60% to zero, and the volume ratio of mobile phase B is 40% linearly rises to 100%;

Weigh the appropriate amount of icariin, add 30% sterol to make a solution of the reference solution containing l. lmg per lral; take the leaf powder of Epimedium sinensis 0. 2g, add 40ml of diluted ethanol, weigh the weight, ultrasound After treatment for 1 hour, weighed the weight, filtered, and the filtrate was evaporated to dryness. The residue was made up to 10 mL with 30% methanol, shaken, placed in the refrigerator for 1 hour, filtered, and the filtrate was taken to obtain the test solution; The reference solution and the test solution are each 20 μl, injected into a liquid chromatograph, and determined by high performance liquid chromatography;

The chromatographic peaks of the original medicinal material Epimedium sagittatum are mainly concentrated in the sputum area, and the peaks of other sections are weak, that is, the peak of No. 21 is the most prominent, and the indicator components are icariin, peak 23 and peaks 19 and 20. Very weak, the 22nd peak is extremely weak, only visible; in the Π section, a peak is formed, that is, the peak of the 21st peak is unique. The peaks of the I and Π regions are extremely weak, and the peaks in the IV area are not detected, and the correlation coefficient 9以上以上。 Both are above 0.9.