CN1514242A - A kind of quality control method of injection for treating apoplexy - Google Patents

Abstract

The invention discloses a traditional Chinese medicine composition for treating apoplexy, a preparation method and a quality control method thereof. The composition mainly consists of 150-220 parts by weight of gardenia and 3-9 parts by weight of total saponins of notoginseng. When the traditional Chinese medicine composition is prepared, Gardeniae is percolated with ethanol, concentrated to a clear paste, and activated carbon column is applied to obtain Gardenia semi-finished product; the Gardenia semi-finished product is dissolved with an appropriate amount of water for injection, and the prescription amount of Panax notoginseng saponins is taken separately, and an appropriate amount is added. Dissolve in water for injection, filter, mix notoginseng medicinal liquid and gardenia medicinal liquid, adjust the pH value, filter through a filter membrane, and sterilize by autoclaving to obtain an injection. Simultaneously, the invention also provides a quality control method for component identification, content determination and fingerprint spectrum of the composition.

Description

A kind of quality control method of injection for treating apoplexy

field of invention

The invention relates to a quality control method, in particular to a quality control method for an injection for treating apoplexy.

Background technique

Stroke belongs to the category of cerebrovascular diseases in modern medicine. It is a disease with high morbidity, mortality and disability. It is a common disease among middle-aged and elderly people. Moreover, the age of onset tends to be younger. In stroke, the proportion of ischemic stroke is relatively high. According to epidemiological survey, 18% of cerebrovascular diseases are cerebral hemorrhage, and 82% are cerebral infarction. Relevant survey data show that the annual incidence of stroke in my country is 115.8/100,000, especially mental retardation and limb disability after stroke, which have become serious social and medical problems; , 73%-86% have hemiplegia, 71%-77% have mobility difficulties, 47% cannot sit and stand alone, 44% have proprioception disturbance, and the disability rate of stroke is very high. It is generally believed that reducing the scope and degree of brain tissue damage in the acute stage is an important part of reducing the disability rate.

For the clinical treatment of ischemic stroke, Chinese medicine currently mainly uses prescriptions such as calming the liver and quenching wind, resolving phlegm and dredging collaterals, promoting qi and blood circulation, and resuscitating the mind. Due to the limitations of the route of administration and other aspects, it is difficult to give full play to the therapeutic advantages in the acute phase. Even if there are some injections such as Mailuoning, Qingkailing, etc., due to their complex components, the mechanism of action is not yet clear, and the purpose of clinical application is relatively poor; Xueshuantong (Xuesaitong) injection and other injections have better effects of promoting blood circulation and removing blood stasis, but It has little effect on the factors leading to the blockage of brain collaterals, and the clinical curative effect has not been significantly improved. There are still many unsolved problems in the drugs and treatment schemes created by western medicine based on the research results of the mechanism of cerebral ischemic injury in recent years. For example, drugs such as NMDA receptor antagonist (MK-801) and calcium ion channel antagonist (nimodipine), which are currently believed to prevent the ischemic injury process, can reduce the extent of ischemic lesions, but indiscriminately Blocking EAA-mediated neurotransmission and affecting some normal physiological functions, including neuroplasticity necessary for repair, may have adverse effects on prognosis and recovery. Ischemic cerebral edema in the acute phase is an important pathological process of stroke. Due to the mixture of two types of factors, cytotoxicity and vascularity, the former is aimed at membrane ion pump failure, and the treatment should be to reverse energy depletion, that is, to provide oxygenated blood to restore pump function. At this time, the effect of hypertonic agents is limited; after several hours of ischemia For vasogenic edema caused by the collapse of the blood-brain barrier, hyperosmotic agents should be effective, but CT scans show that hyperosmotic agents have no significant effect on cerebral infarct swelling, and mannitol has side effects of edema and rebound; try dexamethasone It is difficult to evaluate the curative effect. Steroid hormones seem to have no benefit even if they are used in large quantities, and may even be harmful to ischemic neurons. Glycerol has a certain effect in survivors of cerebrovascular disease, but it makes it difficult to control diabetes.

Therefore, there is a clinical need for a modern traditional Chinese medicine compound injection preparation that inhibits the ischemic cascade reaction injury, improves the microperfusion state of cerebral ischemia, thereby effectively protecting nerve cells, and has a relatively clear pharmacodynamic substance and mechanism of action.

Contents of the invention

One object of the present invention is to disclose a new Chinese medicine composition for treating stroke; the purpose of the present invention is also to disclose a new quality control method of the Chinese medicine composition.

The crude drug composition and proportioning of pharmaceutical composition of the present invention are as follows (by weight):

Plan A:

Gardeniae 150-220 parts by weight Three Seven Seven 70-110 parts by weight.

Preferred:

Gardeniae 170-200 parts by weight Three Seven Seven 80-100 parts by weight.

Plan B

Gardeniae 150-220 parts by weight Total Panax notoginseng saponins 3-9 parts by weight

Preferred:

Gardenia 170-200 parts by weight Total Panax notoginseng saponins 5-7 parts by weight

Plan C

Geniposide 3.8-11.5 parts by weight Total Panax notoginseng saponins 3-9 parts by weight.

Preferred:

Geniposide 6.3-8.9 parts by weight Total Panax notoginseng saponins 5-7 parts by weight.

According to the pharmaceutical method, the above-mentioned pharmaceutical composition of the present invention can be prepared into various clinical pharmaceutical dosage forms, including oral preparations or parenteral administration dosage forms. Said oral preparations are selected from one of tablets, capsules, pills, granules, suspensions, drop pills, and oral liquid preparations; said parenteral dosage forms are selected from injections, aerosol One of the dosage forms for bolus, suppository or subcutaneous administration.

The medicine of the present invention can also add conventional pharmaceutical excipients, such as solvents, disintegrating agents, flavoring agents, preservatives, coloring agents and the like.

The preparation method that B scheme is made into injection is:

Take the prescribed amount of gardenia, crush it into coarse powder, percolate with 7-9 times the amount of 65-80% ethanol, collect the percolation liquid, recover ethanol, concentrate to 55-70°C to a clear paste with a relative density of 1.00-1.20 , add 3-5 times the amount of water to dilute, stir well, refrigerate at 0-4°C for 45-50 hours, filter, and concentrate the filtrate under reduced pressure to a clear paste with a relative density of 1.00-1.20 at 55-70°C, put it on an activated carbon column, first Elute with distilled water until the eluent is colorless, then elute with 9-11 times the amount of 65-80% ethanol, collect the eluent, recover the ethanol under reduced pressure and concentrate, dry to obtain the semi-finished product of Gardenia; take the semi-finished product of Gardenia Dissolve with an appropriate amount of water for injection, and filter; take another prescribed amount of Panax notoginseng total saponins, add an appropriate amount of water for injection to dissolve, filter, mix the notoginseng medicinal liquid with the gardenia medicinal liquid, and adjust the pH value to 5.5-7.5, 0 Refrigerate at -4°C for 20-30 hours, filter through a 0.45μm filter membrane, add water for injection to the filtrate to the full amount, filter through a 0.22μm filter membrane, fill the filtrate into 10ml ampoule injection, and autoclave at 110-120°C for 20- 40 minutes and serve.

The quality control method for preparing the injection from the composition includes identification and/or content determination and/or fingerprint.

Identification methods include one and/or both of the following methods:

a. Take 5ml of this product, evaporate to dryness in a water bath, dissolve the residue with ethanol and quantitatively transfer it to 5ml of a volumetric flask, take another geniposide reference substance, add ethanol to make a solution containing 4mg per 1ml, and use it as the reference substance solution; Layer chromatography (Appendix VIB of Chinese Pharmacopoeia 2000 Edition) test; draw 2 μl of each of the above two solutions, and place them on the same silica gel G thin-layer plate respectively, with 8-11: 6-9: 1-3: 0.3-0.6 ethyl acetate Ester-acetone-formic acid-water as developing agent, develop, take out, dry in the air, spray with 5% vanillin sulfuric acid solution, and heat; in the chromatogram of the test product, at the position corresponding to the chromatogram of the reference product, spots of the same color appear ; b. Take 10ml of this product, add water-saturated n-butanol and shake and extract 2-4 times, 10ml each time, combine the n-butanol extract, wash 2-4 times with n-butanol-saturated water, each time 100ml; take the n-butanol layer and recover it under reduced pressure to dryness, add 0.5ml of methanol to the residue to dissolve it, and use it as the test solution; take ginsenoside Rb ₁ , ginsenoside Rg ₁ and notoginseng saponin R ₁ reference substances, add methanol to prepare Make a mixed solution containing 6 mg per ml as the reference substance solution; test according to thin-layer chromatography (Appendix IIB of Chinese Pharmacopoeia 2000 edition), draw 2 ul of each of the above two solutions, and place them on the same silica gel G thin-layer plate respectively , at ambient temperature below 15-25°C, use 11-15:6-9:1-3 chloroform-methanol-water below 10°C as developing agent, develop, take out, dry in the air, spray with 1→10 Sulfuric acid ethanol solution, heated at 105-115°C until the spots are clear in color; in the chromatogram of the test product, the spots of the same color appear at the position corresponding to the chromatogram of the reference product;

Content determination method comprises one and/or two kinds in following method: a. Geniposide, measure according to high performance liquid chromatography (" Chinese Pharmacopoeia " 2000 edition an appendix VID); Chromatographic condition and system suitability test, Use octadecylsilane bonded silica gel as filler; 13-17:80-90 acetonitrile-water as mobile phase; detection wavelength is 238nm; theoretical plate number should not be less than 1500 based on geniposide peak; reference substance For the preparation of the solution, accurately weigh an appropriate amount of geniposide reference substance, add an appropriate amount of methanol to dissolve it, and make a solution containing 0.14-0.15mg per 1ml, and obtain it; for the preparation of the test solution, accurately absorb 1ml of this product, steam in a water bath Dry, add absolute ethanol to dissolve the residue, transfer the ethanol solution to another crucible, evaporate to dryness in a water bath, dissolve the residue with methanol and transfer it to a 100ml measuring bottle, add methanol to the mark, shake well, and get it; Each 10 μl of the reference substance solution and the test solution is injected into a high-performance liquid chromatograph, measured, and obtained; this product contains no less than 4.6-4.9 mg of geniposide (C ₁₇ H ₂₄ O ₁₀ ) per ml; b. Heptasaponin was determined according to high performance liquid chromatography (Appendix VID of "Chinese Pharmacopoeia" 2000 edition); chromatographic conditions and system suitability test, using octadecylsilane bonded silica gel as filler; acetonitrile-water binary The gradient is the mobile phase (see Table 1); the detection wavelength is 203nm; the number of theoretical plates calculated based on the peak of ginsenoside Rg ₁ should not be less than 2500; the reference solution is prepared by accurately weighing notoginsenoside R1, ginsenoside Rg ₁ , An appropriate amount of ginsenoside Rb ₁ was dissolved in methanol to make a mixed reference solution containing 1.5 mg of notoginsenoside R ₁ , 2.8 mg of ginsenoside Rg ₁ , and 4.1 mg of ginsenoside Rb ₁ per 1 ml; 5ml, shake and extract three times with water-saturated n-butanol, 10ml each time; combine the n-butanol layers, wash once with 10ml n-butanol-saturated water, discard the water layer, recover the n-butanol layer under reduced pressure until dry, and wash the residue with methanol Dissolve to 5ml, shake well, and filter with a microporous membrane of 0.45 μm to obtain. Assay method, accurately draw 3 μl of the reference solution, 7 μl, and 10 μl of the test solution into the high performance liquid chromatography, and use the external standard two-point method Calculate and get it; this product contains not less than 0.18-1.25mg of notoginsenoside _R1 per ml; not less than 0.75-0.85mg of ginsenoside _Rg1 ; not less than 1.8-2.5mg of ginsenoside _Rb1 ;

Table 1: Acetonitrile-water binary gradient elution table

Time (min) Flow rate (ml/min) A(water) B(acetonitrile)

0 1.0 80 20

20 1.0 60 40

The injection quality control method of the present invention can also adopt the method of fingerprint, and this method comprises one and or both in the following methods:

a. Preparation of test solution: Accurately draw 5ml of injection solution, shake and extract twice with water-saturated n-butanol, 10ml for the first time; combine n-butanol extract with 5ml for the second time; Backwash once with 10ml of water, discard the water layer, recover the n-butanol extract to dryness under reduced pressure, dissolve the residue with methanol and dilute it to a 5ml volumetric flask, shake well, and filter through a 0.45um filter membrane to obtain the reference substance Preparation of the solution: take the geniposide reference substance, and use methanol to make a 0.5 mg/ml solution as the reference substance solution; measurement method: detection wavelength 238nm, acetonitrile-water gradient elution, see Table 2; theoretical plate number calculation, It should not be lower than 30000; set the sample volume of the test solution to be 10l, record the chromatogram for 60 minutes, and get it; take the retention time of the chromatographic peak (S peak) of geniposide and the area of the peak as 1 to calculate the relative retention Time and peak area ratio; Fingerprint and technical parameters; Recording time is 60 minutes; Common fingerprint peak calibration (relative retention time): according to the detection results of 10 batches of test products, the common fingerprint peak calibration is as follows: 1 (0.404) (0.462) 3(0.510) 4(0.589) 5(0.618) 6(0.666) 7(0.722) 8(0.798)S(1.000) 9(1.085) 10(1.110) 11(1.197) 12(1.238) 13(1.277 ); The ratio of common fingerprint peak area: 1: S=1: (0.060-0.113); Partial peak shape description: when separating badly, peak 4 is the shoulder peak of peak 5; Non-common peak area: test sample collection of illustrative plates and Comparison of control fingerprints; the total area of non-common peaks shall not be greater than 5% of the peak area;

Instrument reagents HPLC: Waters2695 pump; Waters 2996 UV detector; Millcnnium32 chromatography workstation; Diamonsil C18 column (5μm, 4.6mm×150mm); acetonitrile is chromatographically pure (J.T.Baker, USA); Wahaha purified water; Pure (Beijing Changhua Fine Chemical Factory);

Table 2 Mobile phase gradient elution table

Time (min) Flow rate (ml/min) Water% Acetonitrile%

0 0.8 95 5

8 0.8 92 8

21 0.8 88 12

35 0.8 88 12

40 0.8 70 30

45 0.8 40 60

50 0.8 0 100

60 0.8 0 100

b. Preparation of the test solution: Accurately draw 10ml of injection solution, extract by shaking twice with 20ml of n-butanol saturated with water, and combine the n-butanol extract; backwash three times with 300ml of n-butanol-saturated water, discard Remove the water layer, recover the n-butanol layer to dryness under reduced pressure, dissolve the residue with methanol and set the volume to a 2ml measuring bottle, shake well, and filter through a 0.45um filter membrane to obtain the product; the preparation of the substance: take ginsenoside Rg ₁ Reference substance (purchased from China National Institute for the Control of Pharmaceutical and Biological Products), a solution of 2mg/ml made with methanol is used as reference substance solution; assay method: detection wavelength 210nm, sensitivity 0.1AUFS: acetonitrile-water gradient elution, (see Table 3) The number of theoretical plates is calculated according to ginsenoside Rg ₁ , which should not be lower than 30000; precision draws 25 μ l of the test solution, injects it into a high-performance liquid chromatograph, and records the chromatogram for 70 minutes to obtain final product; the chromatographic peak of ginsenoside Rg ₁ The retention time of (S peak) and the area of peak are 1 to calculate relative retention time and peak area ratio; Fingerprint spectrum and technical parameter; Recording time is 70 minutes; The demarcation (relative retention time) of total fingerprint peak: according to 10 batches supply The common fingerprint peaks of the calibration results of the test product are as follows: 1(0.877) S(1.000) 2(1.027) 3(1.555) 4(1.592) 5(1.652) 6(1.675) 7(1.708) 8(1.744) 9(1.799 ) 10(2.005) 11(2.042) 12(2.074) 13(2.1111) 14(2.150) 15(2.212) 16(2.235) 17(2.364) 18(2.391) 19(2.420) 20(2.440) 21(2.771); The ratio of the common fingerprint peak area: S: 3: 8: 21 = 1: (0.222-0.412): (0.405-0.735): (1.428-2.380); non-shared peak area: comparison of the test sample spectrum and the control fingerprint spectrum; The total area of non-common peaks shall not be greater than 5% of the total area;

Instrument reagents: Waters 2695 Waters 2996 UV detector Millcnnium32 chromatographic workstation Symmetryshild ^TM RP ₁₈ (5μm, 4.6mm × 250mm); Fisher acetonitrile (US chromatographically pure) Fisher methanol (US chromatographically pure) Wahaha purified water Others are analytically pure

Table 3 Mobile phase gradient elution table

Time (min) Flow Rate (ml/min) A-Water B-Acetonitrile

0 1.0 83 17

20 1.0 76 24

40 1.0 60 40

50 1.0 50 50

55 1.0 0 100

60 1.0 0 100

70 1.0 0 100

The composition preparation of the present invention (Tongluo Jiunao Injection) is clinically used for acute ischemic stroke. It has the effects of cooling blood and detoxifying, removing blood stasis and dredging collaterals. Microperfusion to achieve the therapeutic purpose of anti-neuron damage;

The pharmacodynamics experiment research related to the detoxification efficacy of Tongluo Jiunao Injection, using ferric chloride-induced middle cerebral artery thrombosis model of cerebral ischemia in rats, studied the effect of Tongluo Jiunao Injection on this model. Neurological symptoms, brain water content, morphology of infarcted brain tissue, content of SOD and MDA in brain tissue, expression of Glu and NMDA in cerebral infarction lesions, and the mechanism of action of delayed rectifier potassium current ( _IN ); the experimental results show that Tongluo Jiunao Injection Can significantly reduce the content of MDA in the brain tissue of MCAT rats, increase the content of SOD, reduce the expression of Glu and NMDA in the infarcted brain tissue, increase the delayed rectifier potassium current ( _IN ), reduce the water content of the rat's surgical side brain, and improve neurological symptoms ;

The pharmacodynamics experiment research related to the efficacy of Tongluo Jiunao Injection for dredging collaterals, using ferric chloride-induced cerebral ischemic injury model of middle cerebral artery thrombosis in rats, studied the effect of Tongluo Jiunao Injection on the model. Cerebral blood flow in rats, cerebral blood flow in anesthetized dogs, blood rheology in rats with blood stasis, and angiogenesis of chicken embryo allantoic membrane; the results show that Tongluo Jiunao injection can promote the formation of new blood vessels and significantly reduce blood stasis The whole blood viscosity and plasma viscosity of rats can enhance the deformability of red blood cells and reduce the aggregation, and improve the cerebral blood flow of anesthetized dogs and MCAT rats, so as to realize their collateral dredging function;

The efficacy of Tongluo Jiunao Injection was compared with that of Xueshuantong. In the experiment of measuring the water content in the brain tissue of MCAT rats, the small dose of Tongluo Jiunao Injection group (compared with the model group, P<0.01) was obviously due to thrombosis. Tongluo Jiunao injection group (no statistical significance compared with model group); SOD of Tongluo Jiunao injection group increased significantly, compared with Xueshuantong group (P<0.05); Tongluo Jiunao injection small dose group significantly increased red blood cell degeneration (compared with the model group P<0.05, P<0.01), Xueshuantong had no obvious effect on the deformation of red blood cells; the high-dose Tongluo Jiunao injection group had significant angiogenesis (compared with the model group P<0.05, P<0.01), while The effect of Xueshuantong is weak; Xueshuantong can increase the delayed rectifier potassium current ( _IN ), but the effect of Tongluo Jiunao injection is not obvious. The current research has confirmed that the enhancement of delayed rectifier potassium current ( _IN ) can promote ischemia. In other drug efficacy experiments, Tongluo Jiunao Injection was superior to Xueshuantong; Tongluo Jiunao Injection obviously had the effect of promoting cerebral blood flow in anesthetized dogs; in short, Tongluo Jiunao Injection Compared with Xueshuantong, it can reduce the dosage, so as to achieve the purpose of reducing toxicity and increasing efficiency;

The summary of the experimental results is as follows:

1. Effect of Tongluo Jiunao Injection on Neurological Symptoms of MCAT Rats

The experiment used the rat cerebral ischemia model of middle cerebral artery thrombosis induced by ferric chloride. After the model was established, the results of the neurological symptom evaluation of MCAT rats showed that: Tongluo Jiunao injection 83.2, 41.6, 20.8mg/kg group ( Respectively equivalent to 20, 10, and 5 times of clinical human dosage) rats had different degrees of improvement in their neurological symptoms at 12h, 24h, and 48h after surgery (P<0.01, P<0.05); 2. Tongluojiunao injection The effect of liquid on the water content of the brain tissue of MCAT rats

48 hours after modeling, the brain water content of rats in the Tongluo Jiunao injection 83.2, 41.6, 20.8mg/kg group was significantly lower than that of the model group, and the difference was significant compared with the model group (P<0.01, p< 0.05);

3. Effect of Tongluo Jiunao injection on brain histomorphology of MCAT rats

HE and Nissl staining of brain histopathology showed that the number of cerebral ischemic cells in the model group was significantly reduced, the cell bodies were atrophied, degenerated, and lightly colored; Compared with the model group, the number was significantly increased, and the atrophy and degeneration of cells was significantly reduced compared with the model group; image analysis showed that the surface density of positive cells in the Tongluo Jiunao injection group was statistically significant compared with the model group (P<0.01), suggesting that Tongluo Jiunao injection The solution has the effect of reducing the damage of nerve cells caused by cerebral ischemia;

4. Effect of Tongluo Jiunao injection on SOD and MDA in brain tissue of MCAT rats

Tongluo Jiunao Injection 83.2, 41.6mg/kg group can significantly reduce the content of MDA in the brain tissue of MCAT rats, and increase the content of SOD (P<0.01); it suggests that Tongluo Jiunao Injection can resist free radical damage and protect the brain. the role of the organization;

5. Effect of Tongluo Jiunao Injection on Glu and NMDA Expression in Infarcted Brain Tissue of MCAT Rats

Using the rat cerebral ischemia model of middle cerebral artery thrombosis induced by ferric chloride and immunohistochemical methods, the results showed that Tongluo Jiunao injection could significantly reduce the expression of Glu and NMDA in the infarct focus;

6. Effect of Tongluo Jiunao Injection on Delayed Rectifier Potassium Current

The effect of Tongluo Jiunao Injection on the delayed rectifier potassium current of cardiomyocytes was detected by patch clamp electrophysiological technique _.

7. Effect of Tongluo Jiunao injection on cerebral blood flow in MCAT rats

The blood flow in the brain tissue of MCAT rats was significantly reduced, and the blood flow in the brain tissue of the Tongluo Jiunao injection 83.2 and 41.6 mg/kg groups after modeling for 48 hours was significantly higher than that of the model group (P<0.01), indicating that this drug has a positive effect on the brain after ischemia. Tissue blood flow can be significantly improved; Tongluo Jiunao Injection can significantly improve rat blood stasis caused by acute stress, and the effect of small doses is the most obvious;

8. Effect of Tongluo Jiunao injection on cerebral blood flow in anesthetized dogs

The experiment was carried out on 33 healthy mixed-breed dogs. The test showed that Tongluo Jiunao injection can significantly improve the cerebral blood flow of anesthetized dogs (P<0.05 or P<0.01); Maintained until 50 minutes after administration; Tongluo Jiunao Injection 83.2, 41.6 mg/kg groups had no significant difference compared with positive drug Xueshuantong; Tongluo Jiunao injection low dose group compared with positive drug Xueshuantong, the There was a significant difference between 45 minutes and 50 minutes after the drug (P<0.05);

9. Effect of Tongluo Jiunao Injection on hemorrheology in rats with acute blood stasis

Medium dose (41.6 mg/kg) and small dose (20.8 mg/kg) of Tongluo Jiunao injection can significantly reduce the whole blood viscosity of rats with blood stasis (P<0.01, P<0.001, P<0.0001 compared with the model group) ), the plasma viscosity showed a downward trend (but there was no statistical difference), and the low-dose group significantly reduced the hematocrit (compared with the model group, P<0.0001); 0.01, P<0.001);

10. Effect of Tongluo Jiunao Injection on Angiogenesis of Chicken Embryo Allantoic Membrane

Tongluo Jiunao Injection 83.2, 41.6, 20.8 mg/kg groups all have the effect of promoting angiogenesis of chicken embryo allantoic membrane;

The following is a detailed description of some of the above experiments:

Experimental Example 1 Effect of Tongluo Jiunao Injection on Neurological Symptoms of MCAT Rats

Experimental Materials

1. Drugs and reagents

Test drug: Tongluo Jiunao Injection provided by Inner Mongolia Kangyuan Pharmaceutical Co., Ltd., color: light yellow in appearance, content: 10.39mg/ml, has the functions of cooling blood and detoxification, removing blood stasis and dredging collaterals, and is mainly used for acute ischemia cerebral apoplexy; batch number: 2001042322; proposed clinical dosage: 4.16mg/kg, intravenous injection;

Positive control drug: Xueshuantong injection is provided by Inner Mongolia Kangyuan Pharmaceutical Co., Ltd., color: white, content: 50mg/ml, has the function of promoting blood circulation and removing blood stasis, and is mainly used for acute ischemic stroke; batch number: Neiwei Drug Accreditation (1996) No. 001945; clinical dosage: 10mg/kg, intravenous injection 1-2 times/day, 5ml each time;

Reagents and medicines: FeCl ₃ 6H ₂ O(AR), product of Beijing Chemical Plant, prepared with 1mol/L hydrochloric acid;

2. Animals

Wistar rats, both male and female, weighing 180-200g, provided by the breeding farm of Animal Center, Chinese Academy of Medical Sciences, certificate number: Yidongzi No. 01-3008;

3. Instrument

XTT dissecting microscope, product of Beijing Electro-optical Scientific Instrument Factory; SHZ-22 constant temperature water bath oscillator, product of Jiangsu Taicang Medical Instrument Factory; AEG-220 electronic analytical balance, product of Shimadzu Instrument Company;

Method and Results

1. Grouping and administration

60 rats were randomly divided into six groups, namely, sham operation group, MCAT model group, Tongluo Jiunao injection 83.2mg/kg group, Tongluo Jiunao injection 41.6mg/kg group, Tongluo Jiunao injection group 20.8mg/kg group (respectively equivalent to 20, 10, and 5 times of clinical human dosage), Xueshuantong 50mg/kg group (equal to 5 times of human dosage), 10 rats in each group; The daily dose was given in two divided doses for a total of five doses;

2. Modeling method

Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate solution (350 mg/kg); according to the method of Tamura ^[4] , with slight improvements; the rats were fixed in the right lateral position, and a point was made at the midpoint between the outer canthus of the eye and the external auditory canal. An arc-shaped incision, about 1.5 cm in length, was used to pinch off the temporalis muscle and resect it to expose the temporal bone. A 2.5 mm diameter bone window was made at the junction of the zygomatic bone and the temporal squamous bone with a dental drill at 1 mm to the side of the mouth to clean up the residue and expose the middle of the brain. Artery (located between the olfactory tract and the inferior cerebral vein); put a small piece of hollow plastic film to protect the tissue around the blood vessel; apply a small piece of quantitative filter paper soaked with 10 μl of 50% ferric chloride solution on this segment of the middle cerebral artery ⁽⁶⁾ , After 30 minutes, the filter paper was removed, the local tissue was washed with normal saline, sutured layer by layer, and returned to the cage for feeding; in the sham operation group, except that the ferric chloride solution was not added, the rest of the operation steps were the same as the model group;

3. Assessment of neurological symptoms in MCAT rats

At different times (12h, 24h, 48h) after the operation, according to the method of Bederson et al. ⁽⁷⁾ and improve it, the behavior of the animals was scored; 1. Lift the tail of the mouse about one foot from the ground, and observe the flexion of the forelimbs; Symmetrical extension to the ground is scored as 0 points; if the contralateral forelimb has shoulder flexion, elbow flexion, shoulder internal rotation or both wrist and elbow flexion and internal rotation, it is scored as 1, 2, 3 and 4 points; 2 .Place the animal on a smooth ground, push both shoulders to move to the opposite side respectively, and check the resistance; if the resistance on both sides is equal and strong, score 0; if the resistance decreases when pushing to the opposite side of the operation, score according to the degree of decline There are 3 degrees of light, medium and heavy, which are recorded as 1, 2 and 3 points respectively. 3. Put the two forelimbs of the animal on a metal net, and observe the muscle tension of the two forelimbs; if the muscle tension on both sides is equal and strong, the score is 0; the same 1, 2, and 3 points according to the degree of muscle tension decrease in the forelimb on the opposite side of the operation; 4. If the tail of the rat is lifted about one foot from the ground, and the animal keeps rotating to the opposite side of the operation, it is recorded as 1 point; according to the above criteria Score, the full score is 11 points, the higher the score, the more serious the behavioral disorder of the animal; the behavior detection scoring value is compared between groups, t test; the results are shown in Table 1;

Table 1 Effect of Tongluo Jiunao injection on neurological symptoms of MCAT rats ( X±SD)

Psychiatric Score

Group Dose mg/kg N

|

Sham group - 10 0 0 0 0

MCAT model group - 10 5.5±0.55 4.67±0.52 4±0.63

Tongluo rescue group 83.2 10 4.33±0.82** 3.83±0.41** 3.17±0.75*

41.6 10 4.4±0.55** 3.83±0.75** 3.33±0.82*

20.8 10 4.67±0.55 4±0.63* 3.33±1.03

Xueshuantong group 50 10 3.67±0.52** 4±0.63* 3.33±0.52*

Note: Compared with the model group, *P<0.05, **P<0.01;

The results showed that, except for the sham operation group, there was no abnormal behavior change, and the rats in the MCAT model group showed hemiplegia-like symptoms at 12h, 24h, and 48h after operation, mainly manifested in adduction of the contralateral forelimb, internal rotation of the shoulder, and decreased muscle tension of the forelimb , shoulder resistance decreased; Tongluo Jiunao Injection 20.8mg/kg group had no significant improvement in neurological symptoms 48 hours after operation, rats in each dose group of Tongluo Jiunao Injection and Xueshuantong group were at 12h, 24h after operation, At 48 hours, the neurological symptoms were improved to varying degrees (P<0.01, P<0.05);

Experimental Example 2 Effect of Tongluo Jiunao Injection on Brain Tissue Water Content of MCAT Rats

Experimental Materials

1. Drugs and reagents

Test drug and positive control drug are the same as test 20.1;

2. Animals

Wistar rats, both male and female, weighing 180-200g, 71 rats, provided by the breeding farm of Animal Center, Chinese Academy of Medical Sciences, certificate number: Yidongzi No. 01-3008;

3. Instrument

XTT dissecting microscope, product of Beijing Electro-optic Scientific Instrument Factory; AEG-220 electronic analytical balance, product of Shimadzu Instrument Co., Ltd.; Df-206 blast drying oven, product of Beijing Xicheng Medical Instrument Factory; methods and results

The grouping, administration, and operation were the same as in Experiment 20.1; the brain was decapitated 48 hours after the operation, and the middle part of the brain was cut out (removing 3 mm from the front and back), separated from the left and right, and the surface moisture was blotted with filter paper, and the wet weight of the left and right brain slices were weighed respectively; Then put it in an oven, bake at 105°C for 48 hours to constant weight, accurately weigh the dry weight, calculate the water content ^[8] , compare with the control side of the same group, and compare between groups, and perform t-test; the results are shown in Table 2;

Table 2 The effect of Tongluo Jiunao injection on the water content of brain tissue in MCAT rats ( X±SD)

Dosage Moisture content (%)

group

N

                                                                                                                                             

Sham group - 10 77.30±0.79 77.09±0.57 ^△△

MCAT model group - 10 77.04±0.48** 78.900.96

Tongluo rescue brain group 83.2 10 76.55±1.33** 78.09±1.03

                                                                                                                                  

20.8 10 77.51±2.16** 75.11±2.03 ^△△

Xueshuantong group 50 10 77.04±0.48** 78.90±0.96

Note: Compared with the model group, ^△△ P<0.01; compared with the water content of contralateral brain tissue in the same group, **P<0.01;

The results showed that 48 hours after operation, there was no abnormal change in the water content of the left and right sides of the brain in the sham operation group, and the water content in the brain tissue of the rats in the model group increased significantly. The brain water content of the operation side was significantly less than that of the model group, and there was a significant difference compared with the model group (P<0.01), but the water content of the brain tissue of the operation side in each administration group was significantly higher than that of the left brain (P<0.01);

Experimental Example 3 Effect of Tongluo Jiunao Injection on SOD and MDA in Brain Tissue of MCAT Rats

Experimental Materials

1. Drugs and reagents

Test drug: Xueshuantong injection and Tongluo Jiunao injection are the same as in Experiment 20.1;

MDA and SOD reagents were jointly purchased from Nanjing Jiancheng Institute of Bioengineering; batch number: 20011031;

2. Animals

Wistar rats, male, weighing 190-210g, 60 rats, provided by the breeding farm of the Animal Center of the Chinese Academy of Medical Sciences, certificate number: Yidongzi No. 01-3008;

3. Instrument

Model 721 spectrophotometer, product of Shanghai Third Analytical Instrument Factory;

Method and Results

The grouping, administration, and modeling methods were the same as those in Experiment 20.1; the brain was decapitated and taken 24 hours after modeling and administration, the cerebellum, olfactory bulb, and brainstem were removed, and 9 times normal saline was added to prepare a 10% brain homogenate for later use;

1. Determination of SOD

According to the instructions of the SOD reagent, the SOD activity was determined, and the results are shown in Table 3;

2. Determination of MDA

Determination of MDA content according to MDA reagent and instructions, the results are shown in Table 3;

Table 3 Effect of Tongluo Jiunao injection on SOD and MDA in brain tissue of MCAT rats ( X±SD)

Group Dose mg/kg N MDAmmol/g brain tissue SODnu/mg brain tissue

Sham group - 10 0.54±0.11** 144±20.20**

Model group - 10 0.87±0.21 92.17±21.54

83.2 10 0.56±0.1** 127.67±20.55**

Tongluo rescue brain group 41.6 10 0.59±0.12** 138.33±27.27**

20.8 10 0.79±0.21 98.5±29.04

Xueshuantong group 50 10 0.57±0.07** 114.5±21.86**

Note: Compared with the model group, **P<0.01 in each group;

The results showed that the superoxide dismutase (SOD) and MDA of the rats were significantly changed after coagulation of the middle cerebral artery. Luojiunao injection can significantly reduce the content of MDA and increase the activity of SOD (P<0.01), suggesting that the drug has the effect of resisting free radical damage and protecting brain tissue;

Experimental Example 4 Effects of Tongluo Jiunao Injection on Glu and NMD Expressions in Cerebral Cortical Infarction in Rats with Cerebral Ischemia

Experimental Materials

1. Drugs and reagents

Test drug: Tongluo Jiunao Injection provided by Inner Mongolia Kangyuan Pharmaceutical Co., Ltd., mainly for acute ischemic stroke;

Positive control drug: Xueshuantong injection was provided by Inner Mongolia Kangyuan Pharmaceutical Co., Ltd., mainly for acute ischemic stroke, intravenous injection, 2 times/day, 5ml each time; batch number: 2000091112;

Reagents and medicines: FeCl ₃ 6H ₂ O(AR), product of Beijing Chemical Plant, prepared with 1mol/L hydrochloric acid;

Immunohistochemical reagents: rabbit anti-Glu, NMDA (1:200, Santa Cruz Company), biotinylated goat anti-rabbit IgG and ABC complex (1:200, Histostatn-sp kit, ZYMED Company);

2. Animals

Wistar rats, both male and female, weighing 180-200g, provided by the breeding farm of Animal Center, Chinese Academy of Medical Sciences, certificate number: Yidongzi No. 01-3008;

3. Instrument

POLYVAR universal microscope, a product of the United States; C ₈ true color pathological image analyzer, a product of Beihang University;

experiment method

1. Grouping and administration

60 rats were randomly divided into six groups, namely, sham operation group, MCAT model group, Tongluo Jiunao injection 83.2mg/kg group, Tongluo Jiunao injection 41.6mg/kg group, Tongluo Jiunao injection group 20.8mg/kg group (respectively equivalent to 20, 10, and 5 times of clinical human dosage), Xueshuantong 50mg/kg group (equal to 5 times of human dosage), 10 rats in each group; The daily dose was given in two divided doses for a total of five doses;

2. Modeling method

Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate solution (350 mg/kg); according to the method of Tamura ^[3] , slightly improved; the rats were fixed in the right lateral position, and a point was made at the midpoint between the outer canthus of the eye and the external auditory canal. An arc-shaped incision, about 1.5 cm in length, was used to pinch off the temporalis muscle and resect it to expose the temporal bone. A 2.5 mm diameter bone window was made at the junction of the zygomatic bone and the temporal squamous bone with a dental drill at 1 mm to the side of the mouth to clean up the residue and expose the middle of the brain. Artery (located between the olfactory tract and the inferior cerebral vein); put a small piece of hollow plastic film to protect the tissue around the blood vessel; apply a small piece of quantitative filter paper soaked with 10 μl of 50% ferric chloride solution on this segment of the middle cerebral artery ⁽⁴⁾ , After 30 minutes, the filter paper was removed, the local tissue was washed with normal saline, sutured layer by layer, and returned to the cage for feeding; in the sham operation group, except that the ferric chloride solution was not added, the rest of the operation steps were the same as the model group;

3. Animal Handling

One hour after the last administration (that is, 48 hours after the operation), the brain was decapitated, fixed with 10% neutral formaldehyde, embedded in paraffin, and sectioned (brain slices before and after the optic chiasm) were used for immunohistochemical staining. Observe the changes in tissue morphology and perform image analysis;

4. The immunohistochemical staining method adopts the ABC method, and the steps are as follows: ①. Slice into 1% methanol H ₂ O ₂ , room temperature for 30 minutes ②. Proteinase K digestion, 37 ℃ for 30 minutes ③. Normal sheep serum, room temperature for 30 minutes ④. Rabbit anti-Glu , NMDA (1:200, Santa Cruz Company), 4°C overnight ⑤. Biotinylated goat anti-rabbit IgG and ABC complex (1:200, Histostatn-sp kit, ZYMED Company), 37°C 2h ⑥.0.05% DAB-0.1% H ₂ O ₂ solution for color development; before steps ①, ②, ③, ⑤, and ⑥, wash with 0.01MPBS 3 times for 5 minutes each time; for negative control, omit the primary antibody or replace it with normal sheep serum Antibody;

5. Image analysis and statistical processing

The results of Glu and NMDA immunohistochemistry were analyzed with the CMIAS8 true color pathological image analysis system (Image Analysis Center of China University of Aeronautics and Astronautics), and the surface density of each field was counted; the statistics between groups were t-tested;

Experimental results

1. Effect of Tongluo Jiunao Injection on the expression of Glu in cerebral cortex infarction in rats with cerebral ischemia

In the normal rat brain cortex, Glu is expressed in the cytoplasm of the cells, the nuclear area is negative, and the cell shape is mostly pyramid-shaped, with protrusions, and the cell layers are clear; the number of Glu cells in the cerebral cortex infarction of the rats in the model group is larger than normal The number of mice was significantly increased, and the expression of Glu was also significantly enhanced, and the cell morphology was atrophic and degenerated cone-shaped; the expression of Glu in the brain of the positive drug group, the high-dose group, and the middle-dose group was significantly weaker than that of the model group, and the expression of Glu in the middle-dose group was the weakest , and the number of cells is the least; the expression of Glu in the low-dose group is the same as that in the model group; the statistical results of image analysis are shown in Table 4;

Table 4 Effect of Tongluo Jiunao Injection on Glu expression in cerebral cortex infarction of MCAT rats ( X±SD)

Dosage

Group (mg/kg) N N Positive cell surface density

Sham operation group - 9 0.09±0.02**

MCAT model group - 9 0.13±0.02

Tongluo Jiunao injection group 83.2 9 0.09±0.02**

                                                                            

                                                                                

Xueshuantong group 50 9 0.10±0.10**

Note: Compared with the model group **P<0.01

2. Effect of Tongluo Jiunao Injection on NMDA Expression in Cerebral Cortical Infarction in Rats with Cerebral Ischemia

The expression of NMDA in the cerebral cortex of normal rats is in the cytoplasm of the cells, and the nuclear area is negative. , the cell morphology showed swelling and atrophy deformation; the expression of NMDA in the positive drug group and the high-dose group, middle-dose group, and low-dose group was weaker than that in the model group, and the expression of NMDA in the high-dose group and the middle-dose group was the weakest. The NMDA cells in the group were pyramid-shaped, and atrophied and degenerated; the NMDA-positive cells in the middle dose group were mostly small round; the statistical results are shown in Table 5;

Table 5 The effect of Tongluo Jiunao injection on the expression of NMDA in the cerebral cortex of MCAT rats ( X±SD)

Group Dose (mg/kg) N N Positive cell surface density

Sham operation group - 9 0.08±0.02*

MCAT model group - 9 0.11±0.02

Tongluo Jiunao injection group 83.2 9 0.09±0.02**

41.6 9 0.07±0.02**

                                                                                                                                  

Xueshuantong group 50 9 0.07±0.01*

Note: Compared with the model group, *P<0.05, **P<0.01

Conclusion: Tongluo Jiunao Injection can reduce the expression of Glu and NMDA in the infarct, and reverse the swelling and degeneration of cells, so as to achieve the therapeutic purpose;

Experimental Example 5 Effect of Tongluo Jiunao Injection on Cerebral Blood Flow in Anesthetized Dogs Experimental Materials

1. Animals

33 healthy mongrel dogs, both male and female, weighing 12-15kg, provided by Tongli Experimental Animal Farm, Haidian District, Beijing, animal certificate number: No. 024-069;

2. Drugs

Tongluo Jiunao injection: produced by Inner Mongolia Kangyuan Pharmaceutical Co., Ltd. (Inner Mongolia Ganqika Pharmaceutical Factory); batch number: 2001042322; specification: 20ml/bottle; 263mg ginsenosides/20ml;

Blank control drug: normal saline;

Xueshuantong injection (positive drug): produced by Inner Mongolia Kangyuan Pharmaceutical Co., Ltd. (Inner Mongolia Ganqika Pharmaceutical Factory); batch number: 2000091112; specification: 250mg/5ml

Store the drug stock solution in a refrigerator at 4°C;

3. Drug preparation

At the beginning of each experiment, drug formulation was performed based on animal body weight:

Xueshuantong injection: 0.5ml stock solution/kg; (25mg/kg)

Low dose group of Tongluo Jiunao injection: 0.394ml stock solution/kg (5.18mg ginsenoside/kg);

Medium dose group of Tongluo Jiunao injection: 0.788ml stock solution/kg (10.4mg ginsenoside/kg);

Tongluo Jiunao injection high dose group: 1.577ml stock solution/kg (20.7mg ginsenoside/kg);

Take the corresponding milliliters of the original solution, mix it with an appropriate amount of normal saline to form a solution for intravenous infusion with a total volume of 100ml, and heat it in a water bath to 37°C for intravenous infusion;

4. Experimental instruments and equipment

TA-4000 multi-channel physiological recorder (product of Gould Inc, USA), MF-27 electromagnetic flowmeter (product of Nihon Kohden Corporation), a set of animal surgical instruments;

5. Experimental Procedures for Experimental Animal Surgery

After the animals were weighed, 5% pentobarbital sodium 30 mg/kg was injected intraperitoneally. Record the blood pressure of the carotid artery; separate the left common carotid artery, separate and ligate the external carotid artery, embed the common carotid artery with an electromagnetic flowmeter probe (φ=2mm), fix it perpendicular to the common carotid artery, and measure the blood flow of the internal carotid artery; in order to prevent The probe is dry, and the normal saline is added dropwise to keep it moist; the electrocardiogram electrode is installed, and the standard limb II lead electrocardiogram is recorded; the breathing probe is placed at the nostril to record the breathing rate and breathing depth;

6. Administration of experimental animals

Stable for 30 minutes after the operation, record various indicators, as a control before administration; at the beginning of the experiment, prepare the medicinal solution according to the aforementioned method; the dog was uniformly infused with the corresponding research drug through the left femoral vein, and the infusion start time was recorded. Control the drug to be dripped at a uniform speed within 20 minutes;

7. Observation indicators

Measure immediately when the infusion starts (0 minute), record once every 5 minutes, and record for 15 to 30 seconds each time. The paper feeding speed is 10mm/s during recording, and the paper feeding speed is 50mm/h between recordings. The monitor of the physiological recorder monitors synchronously; observe and record the drug effect until 60 minutes after the infusion starts; and record the following physiological indicators synchronously:

1) Internal carotid artery blood flow

2) Arterial blood pressure

Experimental results

The TA-4000 multi-channel physiological recorder records the experimental data on a special recording paper, converts the recorded graphics into numbers through manual counting, and attaches them to each experimental record in the form of a table;

The experimental results use SAS statistical software to conduct t-tests on the indicators between each group to determine whether there are significant differences;

1. See Table 6 for the effect of Tongluo Jiunao Injection on cerebral blood flow in anesthetized dogs;

Table 6 Effect of Tongluo Jiunao injection on cerebral blood flow in anesthetized dogs (ml/min)

After administration, dredging collaterals and saving brain

Xueshuantong (n＝6)

Normal saline (n=7)

Between (minutes)

High dose (n=7) Medium dose (n=7) Low dose (n=6)

0 110.7±2.2 109.1±2.6 106.6±6.1 110.8±3.4 111.9±2.0

5 117.5±6.6 116.7±5.0 112.0±7.3 116.7±5.2 111.8±2.9

10 124.2±8.8 ^** 121.7±7.3 ^** 119.4±9.4 ^* 120.8±8.6 ^* 110.3±5.9

15 126.7±10.8 ^** 128.3±16.7 ^** 122.7±11.3 ^** 122.2±7.1 ^** 110.3±4.5

20 128.3±13.7 ^** 131.4±16.0 ^** 125.4±12.7 ^** 123.8±8.6 ^** 111.3±6.6

25 130.8±16.6 ^** 130.7±12.1 ^** 127.6±11.7 ^** 124.0±7.9 ^** 110.9±6.6

30 115.3±53.5 131.4±8.9 ^** 126.9±10.9 ^** 125.5±7.4 ^** 110.6±5.6

35 132.2±14.5 ^** 130.4±8.1 ^** 122.9±15.5 123.8±6.0 ^** 111.3±8.3

40 129.2±10.2 ^** 126.9±6.9 ^** 126.1±8.5 ^** 120.2±2.6 113.3±8.7

45 130.0±8.4 ^** 126.1±8.7 ^** 126.7±6.9 ^** 115.8±4.9 ^Δ 113.0±7.9

50 130.2±7.8 ^** 126.1±12.8 ^** 125.6±6.8 ^** 115.3±11.9 ^Δ 110.9±7.9

55 122.5±13.6 119.3±13.7 122.3±10.2 112.8±7.5 ^** 110.3±6.1

60 119.2±17.4 118.4±14.6 106.4±39.7 108.7±11.9 110.9±7.2

^* P<0.05 compared with the control group; ^** P<0.01 compared with the control group; ^ΔP <0.05 compared with Xueshuantong;

Experiments show that Tongluo Jiunao Injection can significantly increase the cerebral blood flow of anesthetized dogs (P<0.05 or P<0.01); it begins to take effect 10 minutes after the injection of the drug, and the drug effect is maintained until 50 minutes after the administration; There was no significant difference between the high-dose and middle-dose groups of Luojiunao injection and the positive drug Xueshuantong; there was a significant difference between the low-dose group of Tongluo Jiunao injection and the positive drug Xueshuantong at 45 minutes and 50 minutes after injection. Sex difference (P<0.05);

2. Effect of Tongluo Jiunao injection on blood pressure of anesthetized dogs

See Table 7 for the effect of Tongluo Jiunao injection on the systolic blood pressure of anesthetized dogs;

Table 7 Effect of Tongluo Jiunao Injection on Systolic Blood Pressure of Anesthetized Dogs (mmHg)

Time after administration dredging collaterals to save brain dredging collaterals to rescue brain normal saline

Xueshuantong (n=6) (points) High dose (n=7) Medium dose (n=7) Low dose (n=6) (n=7)

0 148.3±12.5 149.6±12.5 140.6±16.8 146.8±19.6 137.2±17.0

5 146.3±13.6 152.1±11.8 138.0±11.7 147.9±18.9 138.0±18.4

10 149.8±16.7 153.0±13.3 139.011.7 149.3±18.1 137.4±17.3

15 148.7±15.2 154.7±13.5 140.0±10.8 149.3±18.8 136.9±16.6

20 147.7±16.5 152.7±12.6 140.6±10.7 149.3±19.5 137.1±15.9

25 148.2±16.0 151.0±13.1 139.1±10.9 149.5±18.3 136.7±18.3

30 144.3±11.2 151.9±12.4 139.1±10.4 148.8±18.6 137.9±16.6

35 144.3±11.5 150.4±13.1 139.9±9.5 150.3±19.3 138.1±17.6

40 147.2±12.6 147.6±12.6 140.0±13.7 148.2±19.1 138.4±16.6

45 146.7±13.8 147.4±13.0 141.7±10.7 144.6±22.3 138.7±16.3

50 144.0±10.4 146.7±14.7 141.4±12.2 147.3±19.9 138.8±17.0

55 146.7±16.9 145.4±16.3 140.6±12.9 143.5±17.2 137.8±17.0

60 148.2±12.1 146.8±14.9 141.1±13.2 145.4±18.6 138.2±17.1

Experiments show that there is no significant difference in the effect of Tongluo Jiunao injection on the systolic blood pressure of anesthetized dogs (P>0.05)

See Table 8 for the effect of Tongluo Jiunao injection on the diastolic blood pressure of anesthetized dogs;

Table 8 Effect of Tongluo Jiunao Injection on Diastolic Blood Pressure of Anesthetized Dogs (mmHg)

Time after administration dredging collaterals to save brain dredging collaterals to save brain normal saline

Xueshuantong (n＝6)

(points) High dose (n=7) Medium dose (n=7) Low dose (n=6) (n=7)

0 116.3±10.2 121.3±10.5 112.9±14.2 119.7±13.2 109.6±13.1

5 115.0±8.6 119.7±11.6 111.2±10.4 120.0±12.9 110.8±14.7

10 119.5±14.4 120.0±10.9 112.1±10.6 121.3±13.5 110.3±13.5

15 116.0±7.9 119.4±13.9 112.4±10.0 120.5±12.7 110.3±13.5

20 116.8±10.6 118.1±12.3 112.8±10.6 119.7±13.1 109.9±13.1

25 117.7±9.8 117.0±12.8 112.0±10.7 120.8±12.4 110.7±14.1

30 116.0±8.0 117.6±13.0 112.1±10.6 119.7±13.0 110.3±14.0

35 115.7±8.0 118.4±12.6 113.0±9.4 122.8±11.9 110.4±14.7

40 116.7±8.4 115.6±12.8 112.1±12.1 121.8±12.8 110.9±13.2

45 117.2±9.2 117.0±12.2 113.7±10.5 117.8±16.7 111.1±12.2

50 114.8±8.5 116.0±14.9 113.0±12.1 118.7±17.0 111.1±13.5

55 118.7±13.3 115.5±16.0 112.6±13.5 117.7±15.5 110.7±13.7

60 117.3±10.1 117.0±13.9 112.9±13.4 116.3±15.8 110.7±13.7

Experiments show that there is no significant difference in the diastolic blood pressure of anesthetized dogs in high, medium and low dose groups of Tongluo Jiunao injection (P>0.05)

Experimental Example 6 Effect of Tongluo Jiunao Injection on Angiogenesis of Chicken Embryo Allantoic Membrane

Experimental materials and methods

1. Drugs:

Injections of traditional Chinese medicine Xueshuantong and Tongluojiunao were provided by Professor Niu Xin, with a concentration of 50 mg Panax notoginsenosides/ml solution; this medicinal solution was divided into large (50 mg Panax notoginseng saponins/ml), medium (25 mg Panax notoginseng saponins /ml), small (12.5mg Panax notoginseng saponins/ml) three concentrations;

2. Incubation:

Fresh white-skinned eggs were purchased from Changping Breeding Poultry Company. Selected eggs with no damage and cracks, which were similar in size and had air chambers, wiped and disinfected the surface with 75% ethanol, put the large head upwards, and placed them in a sterilized incubator at 37.8°C. , place a water tray in the incubator to maintain a certain humidity in the box; in order to prevent embryo adhesion and promote amniotic membrane movement, transfer eggs 4-5 times a day ^[3] ;

3. Open the window:

Refer to methods such as the laying method ^[4] and improve them; take out the eggs when they are incubated until the fifth day, and check the blood vessel development on the canister; select eggs with clear and well-developed blood vessels, and there are two large blood vessels at the lower right of the embryo head. Draw a window position of 1cm×1cm in between, and draw a mark on the air chamber; move it into the ultra-clean table, wipe and disinfect the shell of the egg at the drawn position with 75% ethanol, and first use a dental drill to clean the air of the egg. Drill a small hole in the chamber for decompression, then use a dental drill to carefully grind and cut the shell at the position of the window, use ophthalmic tweezers to gently remove the egg shell at the window, revealing the white shell, drip a little saline and then carefully Remove the eggshell membrane to expose the allantoic membrane;

4. Dosing:

According to the research results of Zhang Shucheng et al ^{. [5]} , we used gelatin sponge as the carrier of the drug; cut the sterile gelatin sponge (purchased from the China-Japan Friendship Hospital) into small pieces of 5mm×2mm×2mm, added 35μl and used a 0.22μm sterile filter After filtering the medicinal solution, place it on the allantoic membrane at the central position of the window, seal the small holes at the window and the air chamber with sterile scotch tape, put it back into the incubator and continue to incubate for 48 hours; and set the control group ( Sterile normal saline, 35 μl/one chicken embryo), Xueshuantong positive group, Tongluo Jiunao injection 83.2mg/kg, 41.6mg/kg, 20.8mg/kg groups; each group made 20 chicken embryos;

5. Take the film:

Uncover the transparent tape on the small window of the chicken embryo, drill a small hole in the lower part of the egg, and after about 1ml of egg white flows out, add a fixative solution (methanol:acetone=1:1) to the window, and fix it at room temperature for 15- After 20 minutes, break the eggshell to both sides, pour all the contents into a petri dish, carefully remove the allantoic membrane with ophthalmic scissors and forceps, put it into a petri dish filled with water, and apply Paste on filter paper and store in the shade;

6. Counting:

Observe and count the growth of blood vessels on the allantoic membrane under a dissecting microscope; counting range: within the range of 1.5 cm in diameter centered on administration, divide the blood vessels into large, medium and small blood vessels according to their diameters and count them;

7. Accounting processing:

We used the t test to carry out statistical processing with the counting results and the normal saline group;

Experimental results

The statistical results of the data are shown in Table 9:

Table 9 The effect of Tongluo Jiunao injection on the angiogenesis of chicken embryo allantoic membrane

The number of allantoic membrane vessels/embryo (x±s)

group

         mg/kg                                                                                                             

Control group 2.33±1.53 11.00±1.00 43.67±6.35

Tongluo rescue brain group 83.2 2.00±0.82 8.86±2.27 35.43±6.60

                                                                

20.8 2.29±1.11 9.43±2.30 36.71±3.86*

Xueshuantong group 50 2.01±0.55 6.80±3.11* 30.60±6.77**

Note: n=20, compared with the control group: *P<0.05 **P<0.01

The results showed that each dosage group of Tongluo Jiunao Injection, especially the high dosage group, had the effect of promoting angiogenesis compared with Xueshuantong;

The following embodiments can all achieve the effects of the above experimental examples.

Embodiment 1 injection

Gardenia 175g Panax notoginseng saponins 5.5g

Get the Gardenia Fructus of prescription quantity, be ground into coarse powder, according to the percolation method ("Chinese Pharmacopoeia" 2000 edition one appendix 10) under the liquid extract item, percolate with 70% ethanol of 8 times of amount, collect percolation liquid , recover ethanol, concentrate to a clear paste with a relative density of 1.10 (measured at 60°C), add 4 times the amount of water to dilute, stir well, refrigerate at 0-4°C for 48 hours, filter, and concentrate the filtrate under reduced pressure to a relative density of 1.10 ( 60 DEG C) clear paste, put on activated carbon column, first elute with distilled water until the eluate is colorless, then elute with 10 times the amount of 70% ethanol, collect the eluate, recover ethanol under reduced pressure and concentrate, dry, Get the semi-finished product of Gardenia; get the semi-finished product of Gardenia and dissolve it with an appropriate amount of water for injection, and filter; take another prescribed amount of Panax notoginseng saponins, add an appropriate amount of water for injection to dissolve, filter, and mix the Panax notoginseng liquid with the gardenia liquid, Adjust the pH value to 6.0-7.0, refrigerate at 0-4°C for 24 hours, filter through a 0.45μm filter membrane, add water for injection to the full amount of the filtrate, filter through a 0.22μm filter membrane, fill the filtrate into 10ml ampoule injection, heat at 110°C Sterilize by pressure for 30 minutes. Functions and indications: cool blood and detoxify, remove blood stasis and dredge collaterals. Indications for ischemic stroke. For stroke patients with hemiplegia, numbness in one side of the body, dizziness and headache, strong tongue, slurred or silent speech, crooked mouth and tongue, delirium; worse, suddenly falling down, delirium, snoring called phlegm, dark red tongue, Stringy and slippery pulse, etc. are evidences of wind-fire disturbance and blood stasis blocking the veins.

Usage and dosage: once a day, 154mg once, a course of treatment for two weeks.

Specifications: 10ml/bottle, 154mg (based on the sum of geniposide, ginsenoside Rg ₁ , ginsenoside Rb ₁ , and ginsenoside R ₁ ).

Embodiment 2 injection

Gardenia 190g Panax notoginseng saponins 7g

Take the Gardenia Fructus of prescribed quantity, crush into coarse powder, percolate with 75% ethanol of 7 times the quantity, collect percolation liquid, recycle ethanol, concentrate to 65 ℃ clear ointment with relative density of 1.10, add 5 times of quantity of water to dilute, Stir well, refrigerate at 0-4°C for 49 hours, filter, concentrate the filtrate under reduced pressure to a clear paste with a relative density of 1.10 at 60°C, put it on an activated carbon column, and elute with distilled water until the eluent is colorless, then use 10 times Measure 70% ethanol for elution, collect the eluate, recover the ethanol under reduced pressure and concentrate, dry to obtain the semi-finished product of Gardenia jasminoides; dissolve the semi-finished product of Gardenia jasminoides with an appropriate amount of water for injection, and filter; take another prescribed amount of Panax notoginseng saponins, add Dissolve an appropriate amount of water for injection, filter, mix the Panax notoginseng medicinal liquid and Gardenia medicinal liquid, adjust the pH value to 6.0-7.0, refrigerate at 0-4°C for 24 hours, filter through a 0.45μm filter membrane, add water for injection to the filtrate to the full amount , filtered with a 0.22 μm filter membrane, filled with the filtrate into 10ml ampoule injection, and sterilized by autoclaving at 115°C for 30 minutes to obtain the injection. Usage and dosage: once a day, 154mg once, a course of treatment for two weeks. Specifications: 10ml/bottle, 154mg (based on the sum of geniposide, ginsenoside Rg ₁ , ginsenoside Rb ₁ , and ginsenoside R ₁ ).

Example 3 tablet

Gardenia 175g Sanqi 90g.

Take the prescribed amount of Gardenia and Panax notoginseng, crush them into coarse powder, use 7 times the amount of 75% ethanol for percolation, collect the percolation liquid, recover the ethanol, concentrate to 65°C and a clear paste with a relative density of 1.10, add 5 times the amount Dilute with water, stir well, refrigerate at 0-4°C for 49 hours, filter, and concentrate the filtrate under reduced pressure to obtain a clear paste with a relative density of 1.10 at 60°C, granulate 100 grams of granules by conventional granulation, add excipients, and press into tablets ( The granulation tablet press machine completes at one time). Each piece weighs 0.5 grams.

Embodiment 4 capsules

Geniposide 70g Panax notoginseng saponins 60g

Usually made into capsules.

The quality control method of embodiment 5 composition injections of the present invention

Identification method: a. Take 5ml of this product, evaporate to dryness in a water bath, dissolve the residue with ethanol and quantitatively transfer it to a 5ml volumetric flask, take another geniposide reference substance, add ethanol to make a solution containing 4mg per 1ml, and use it as a reference substance solution ; test according to thin-layer chromatography (Appendix VIB of Chinese Pharmacopoeia 2000 edition); draw 2 μl of each of the above two solutions, point them on the same silica gel G thin-layer plate, and use ethyl acetate-acetone-formic acid in a ratio of 10:7:2:0.5 —Water is the developer, develop, take out, dry, spray with 5% vanillin sulfuric acid solution, heat; in the chromatogram of the test product, on the position corresponding to the chromatogram of the reference substance, the spots of the same color are shown; b. Extract 10ml, add water-saturated n-butanol and shake and extract 3 times, 10ml each time, combine the n-butanol extract, wash 3 times with n-butanol-saturated water, 100ml each time; take the n-butanol layer and decompress Recover to dryness, add methanol 0.5ml to the residue to dissolve, and use it as the test solution; take ginsenoside Rb ₁ , ginsenoside Rg ₁ and notoginsenoside R ₁ reference substances, add methanol to make a mixed solution containing 6 mg per ml , as a reference substance solution; test according to thin-layer chromatography (Appendix IIB of Chinese Pharmacopoeia in 2000), draw each 2ul of the above two solutions, and place them on the same silica gel G thin-layer plate respectively. 13:7:2 Chloroform-methanol-water The lower layer solution placed below 10°C is the developing agent, develop, take out, dry in the air, spray with 1→10 sulfuric acid ethanol solution, heat at 110°C until the spots are clearly colored; the test sample In the chromatogram, spots of the same color appear at positions corresponding to the chromatogram of the reference substance;

Content determination method: a. Geniposide, determined according to high performance liquid chromatography (Appendix VID of "Chinese Pharmacopoeia" 2000 edition); chromatographic conditions and system suitability test, using octadecylsilane bonded silica gel as filler 15:85 acetonitrile-water is the mobile phase; the detection wavelength is 238nm; the theoretical plate number should not be less than 1500 according to the geniposide peak; the preparation of the reference solution accurately weighs an appropriate amount of the geniposide reference substance, and adds an appropriate amount of methanol Make it dissolve, and make a solution containing 0.144mg per 1ml, and you will get it; for the preparation of the test solution, accurately absorb 1ml of this product, evaporate to dryness in a water bath, dissolve the residue with absolute ethanol, transfer the ethanol solution to another crucible, and evaporate it in a water bath. Dry, dissolve the residue with methanol and transfer it to a 100ml measuring bottle, add methanol to the scale, shake well, and get it; Assay method, respectively accurately draw 10 μl of the reference solution and the test solution, inject it into a high-performance liquid chromatograph, and measure , that is; this product contains geniposide (C ₁₇ H ₂₄ O ₁₀ ) per ml not less than 4.7mg; b. Notoginseng saponin, according to high performance liquid chromatography (Appendix VID of "Chinese Pharmacopoeia" 2000 edition) Determination; chromatographic conditions and system suitability test, with octadecylsilane bonded silica gel as filler; with acetonitrile-water binary gradient as mobile phase (see Table 1); detection wavelength is 203nm; theoretical plate number is based on ginseng The peak calculation of saponin Rg ₁ should not be less than 2500; to prepare the reference solution, accurately weigh notoginsenoside R ₁ , ginsenoside Rg ₁ , and ginsenoside Rb ₁ and dissolve them in methanol to make a solution containing notoginseng saponin R ₁ 1.5 mg, ginsenoside Rg ₁ 2.8mg, ginsenoside Rb ₁ 4.1mg mixed reference substance solution; for the preparation of the test solution, accurately draw 5ml of the sample, shake and extract three times with water-saturated n-butanol, 10ml each time; n-butanol layer Combine, wash once with 10ml of n-butanol saturated water, discard the water layer, recover the n-butanol layer under reduced pressure until dry, dissolve the residue with methanol to 5ml, shake well, and filter through a microporous membrane of 0.45 μm to obtain final product; Determination method, accurately draw 3 μ l of the reference substance solution, 7 μ l, and 10 μ l of the test solution into the high-performance liquid chromatograph, and use the external standard two-point method to calculate, that is; this product contains not less than 0.2 mg of notoginseng saponin _R1 per ml ; Ginsenoside Rg ₁ shall not be less than 0.8mg; Ginsenoside Rb ₁ shall not be less than 2.0mg;

Table 1: Acetonitrile-water binary gradient elution table

Time (min) Flow rate (ml/min) A(water) B(acetonitrile)

0 1.0 80 20

20 1.0 60 40

Injection quality control method of the present invention can also adopt the method for fingerprint collection:

a. Preparation of the test solution: Accurately draw 5ml of injection solution, shake and extract twice with water-saturated n-butanol, 10ml for the first time; 5nl for the second time, combine the n-butanol extract; Backwash once with 10ml of water, discard the water layer, recover the n-butanol extract to dryness under reduced pressure, dissolve the residue with methanol and dilute it to a 5ml volumetric flask, shake well, and filter through a 0.45um filter membrane to obtain the reference substance Preparation of the solution: take the geniposide reference substance, and use methanol to make a 0.5 mg/ml solution as the reference substance solution; measurement method: detection wavelength 238nm, acetonitrile-water gradient elution, see Table 2; theoretical plate number calculation, It should not be lower than 30000; set the sample volume of the test solution to be 10l, record the chromatogram for 60 minutes, and get it; take the retention time of the chromatographic peak (S peak) of geniposide and the area of the peak as 1 to calculate the relative retention Time and peak area ratio; Fingerprint spectrum and technical parameters; Recording time is 60 minutes; The calibration (relative retention time) of common fingerprint peak: according to the detection result calibration of 10 batches of test samples, the common fingerprint peak is as follows: 1 (0.404) 2 (0.462)3(0.510) 4(0.589) 5(0.618) 6(0.666) 7(0.722) 8(0.798) S(1.000)9(1.085) 10(1.110) 11(1.197) 12(1.238) 13(1.277 ); The ratio of common fingerprint peak area: 1: S=1: (0.060-0.113); Partial peak shape description: when separating badly, peak 4 is the shoulder peak of peak 5; Non-common peak area: test sample collection of illustrative plates and Comparison of control fingerprints; the total area of non-common peaks shall not be greater than 5% of the peak area;

Instrument reagents HPLC: Waters2695 pump; Waters 2996 UV detector; Millcnnium32 chromatography workstation; Diamonsil C18 column (5μm, 4.6mm×150mm); acetonitrile is chromatographically pure (J.T.Baker, USA); Wahaha purified water; Pure (Beijing Changhua Fine Chemical Factory);

Table 2 Mobile phase gradient elution table

Time (min) Flow Rate (ml/min) Water % Acetonitrile %

0 0.8 95 5

8 0.8 92 8

21 0.8 88 12

35 0.8 88 12

40 0.8 70 30

45 0.8 40 60

50 0.8 0 100

60 0.8 0 100

b. Preparation of the test solution: Accurately draw 10ml of injection solution, extract by shaking twice with 20ml of n-butanol saturated with water, and combine the n-butanol extract; backwash three times with 300ml of n-butanol-saturated water, discard Remove the water layer, recover the n-butanol layer to dryness under reduced pressure, dissolve the residue with methanol and set the volume to a 2ml measuring bottle, shake well, and filter through a 0.45um filter membrane to obtain the product; the preparation of the substance: take ginsenoside Rg ₁ Reference substance (purchased from China National Institute for the Control of Pharmaceutical and Biological Products), made of 2mg/ml solution with methanol as reference substance solution; assay method: detection wavelength 210nm, sensitivity 0.1AUFS: acetonitrile-water gradient elution, see Table 3; theory Plate number is calculated according to ginsenoside Rg ₁ , should not be low 30000 _; Precision draws 25 μ l of need testing solution, injects high-performance liquid chromatograph, records the chromatogram of 70 minutes, obtains; With the chromatographic peak (S The retention time of peak) and the area of peak are 1 to calculate relative retention time and peak area ratio; Fingerprint chromatogram and technical parameter; Recording time is 70 minutes; The demarcation (relative retention time) of common fingerprint peak: according to 10 batches of test samples The detection results of the calibration common fingerprint peaks are as follows: 1 (0.877) S (1.000) 2 (1.027) 3 (1.555) 4 (1.592) 5 (1.652) 6 (1.675) 7 (1.708) 8 (1.744) 9 (1.799) 10 (2.005) 11(2.042) 12(2.074) 13(2.111) 14(2.150) 15(2.212) 16(2.235) 17(2.364) 18(2.391) 19(2.420) 20(2.440) 21(2.771); Ratio of peak area: S: 3: 8: 21 = 1: (0.222-0.412): (0.405-0.735): (1.428-2.380); non-common peak area: comparison of test sample spectrum with control fingerprint; non-common The total peak area shall not be greater than 5% of the total area;

Instrument reagents: Waters 2695 Waters 2996 UV detector Millcnnium32 chromatographic workstation Symmetryshild ^TM RP ₁₈ (5μm, 4.6mm × 250mm); Fisher acetonitrile (US chromatographically pure) Fisher methanol (US chromatographically pure) Wahaha purified water Others are analytically pure

Table 3 Mobile phase gradient elution table

Time (min) Flow Rate (ml/min) A-Water B-Acetonitrile

0 1.0 83 17

20 1.0 76 24

40 1.0 60 40

50 1.0 50 50

55 1.0 0 100

60 1.0 0 100

70 1.0 0 100

Claims (6)

1. A method for quality control of an injection prepared from gardenia and Panax notoginseng for the treatment of apoplexy, characterized in that the identification method in the method includes one or more of the following identifications: a. Take 5ml of this product, evaporate to dryness in a water bath, dissolve the residue with ethanol and quantitatively transfer it to 5ml of a volumetric flask, take another geniposide reference substance, add ethanol to make a solution containing 4mg per 1ml, and use it as the reference substance solution; Layer chromatography test: draw 2 μl of each of the above two solutions, and place them on the same silica gel G thin-layer plate respectively, with 8-11:6-9:1-3:0.3-0.6 ethyl acetate-acetone-formic acid-water as Developing agent, develop, take out, dry, spray with 5% vanillin sulfuric acid solution, and heat; in the chromatogram of the test product, on the position corresponding to the chromatogram of the reference product, the spots of the same color appear; b. Take 10ml of this product, Add water-saturated n-butanol and shake and extract 2-4 times, 10ml each time, combine the n-butanol extracts, wash 2-4 times with n-butanol-saturated water, 100ml each time; take the n-butanol layer and subtract Pressure recovery to dryness, add methanol 0.5ml to the residue to dissolve, as the test solution; take ginsenoside Rb ₁ , ginsenoside Rg ₁ and notoginsenoside R ₁ reference substance, add methanol to make a mixed mixture containing 6 mg per m1 solution, as the reference solution; according to the thin-layer chromatography test, draw 2ul of each of the above two solutions, and place them on the same silica gel G thin-layer plate respectively, and use 11-15:6-9 : 1-3 chloroform-methanol-water The lower layer solution placed below 10°C is the developing agent, develop, take out, dry in the air, spray with 1→10 sulfuric acid ethanol solution, heat at 105-115°C until the spots are clearly colored; for testing In the chromatogram of the reference product, spots of the same color appear at the positions corresponding to the chromatogram of the reference product. 2. The quality control method of injection as claimed in claim 1, characterized in that the identification method in the method includes one or more of the following identifications: a. Take 5ml of this product, evaporate to dryness in a water bath, dissolve the residue with ethanol and quantitatively transfer it to 5ml of a volumetric flask, take another geniposide reference substance, add ethanol to make a solution containing 4mg per 1ml, and use it as the reference substance solution; Layer chromatography test: draw 2 μl of each of the above two solutions, respectively spot on the same silica gel G thin-layer plate, use 10:7:2:0.5 ethyl acetate-acetone-formic acid-water as the developer, develop, take out, and dry dry, spray with 5% vanillin sulfuric acid solution, and heat; in the chromatogram of the test product, on the position corresponding to the chromatogram of the reference substance, spots of the same color are evident; b. take 10ml of this product, add water-saturated n-butanol to shake Shake and extract 3 times, 10ml each time, combine the n-butanol extracts, wash 3 times with n-butanol-saturated water, 100ml each time; take the n-butanol layer and recover it under reduced pressure to dryness, add 0.5ml methanol to the residue to dissolve , as the test solution; take another ginsenoside Rb ₁ , ginsenoside Rg ₁ and notoginsenoside R ₁ reference substance, add methanol to make a mixed solution containing 6 mg per ml, as the reference substance solution; according to thin-layer chromatography For the test, absorb 2 ul of each of the above two solutions, and place them on the same silica gel G thin-layer plate, and use the lower layer solution of 13:7:2 chloroform-methanol-water below 10°C as the developing agent at an ambient temperature below 20°C. Unfold, take out, dry in the air, spray with 1→10 sulfuric acid ethanol solution, heat at 110°C until the spots are clear in color; in the chromatogram of the test product, the spots of the same color appear on the corresponding position of the chromatogram of the reference product. 3. A method for quality control of injections prepared from gardenia and notoginseng, characterized in that the assay method in the method includes one or more of the following assays: a. Geniposide, determined according to high performance liquid chromatography; chromatographic conditions and system suitability test, using octadecylsilane bonded silica gel as filler; 13-17: 80-90 acetonitrile-water as mobile phase; detection The wavelength is 238nm; the number of theoretical plates calculated according to the geniposide peak should not be less than 1500; for the preparation of the reference substance solution, accurately weigh an appropriate amount of the geniposide reference substance, add an appropriate amount of methanol to dissolve, and make each 1ml contain 0.14-0.15 mg solution, that is, ready; for the preparation of the test solution, accurately absorb 1ml of this product, evaporate to dryness in a water bath, dissolve the residue with absolute ethanol, transfer the ethanol solution to another crucible, evaporate to dryness in a water bath, dissolve the residue with methanol and transfer to In a 100ml measuring bottle, add methanol to the mark, shake well, and you get it; for the determination method, draw 10 μl each of the reference solution and the test solution, inject it into a high-performance liquid chromatograph, and measure it to get it; this product contains Gardenia Glycosides should not be less than 4.6-4.9 mg per ml; b. Notoginseng saponins, determined by high performance liquid chromatography; chromatographic conditions and system suitability tests, using octadecylsilane bonded silica gel as filler; acetonitrile-water The binary gradient is the mobile phase; the detection wavelength is 203nm; the number of theoretical plates calculated based on the peak of ginsenoside Rg ₁ should not be less than 2500; the reference solution is prepared by accurately weighing notoginsenoside R1, ginsenoside Rg ₁ and ginsenoside Rb ₁ Appropriate amount of methanol was added to dissolve to make a mixed reference solution containing 1.5 mg of notoginsenoside _R1 , 2.8 mg of ginsenoside _Rg1 and 4.1 mg of ginsenoside _Rb1 per 1 ml; Shake and extract saturated n-butanol three times, 10ml each time; combine the n-butanol layers, wash once with 10ml n-butanol saturated water, discard the water layer, recover the n-butanol layer under reduced pressure until dry, and dissolve the residue to 5ml with methanol , shake well, and filter with a microporous membrane of 0.45 μm to obtain the final product; for the determination method, 3 μl of the reference solution, 7 μl, and 10 μl of the test solution are injected into the high-performance liquid chromatograph, and the external standard two-point method is used for calculation, namely Each ml of this product contains not less than 0.18-1.25mg of notoginsenoside _R1 ; not less than 0.75-0.85mg of ginsenoside _Rg1 ; not less than 1.8-2.5mg of ginsenoside _Rb1 . 4. The quality control method for injections as claimed in claim 3, characterized in that the content determination method in the method includes one or more of the following content determinations: a. Geniposide, determined according to high performance liquid chromatography; chromatographic conditions and system suitability test, using octadecylsilane bonded silica gel as filler; 15:85 acetonitrile-water as mobile phase; detection wavelength is 238nm; Number of theoretical plates a. Geniposide, determined according to high performance liquid chromatography; chromatographic conditions and system suitability test, using octadecylsilane bonded silica gel as filler; 15:85 acetonitrile-water as mobile phase; detection The wavelength is 238nm; the number of theoretical plates calculated according to the geniposide peak should not be less than 1500; the preparation of the reference substance solution Accurately weigh an appropriate amount of the geniposide reference substance, add an appropriate amount of methanol to dissolve, and make a solution containing 0.144 mg per 1 ml , that is; for the preparation of the test solution, accurately absorb 1ml of this product, evaporate to dryness in a water bath, dissolve the residue with absolute ethanol, transfer the ethanol solution to another crucible, evaporate to dryness in a water bath, dissolve the residue with methanol and transfer it to a 100ml measuring bottle In the medium, add methanol to the scale, shake well, and you get it; for the assay method, respectively accurately draw 10 μl of each of the reference solution and the test solution, inject it into a high-performance liquid chromatograph, measure it, and get it; this product contains geniposide per ml Not less than 4.7 mg; b. Notoginseng saponin, determined according to high performance liquid chromatography; chromatographic conditions and system suitability test, using octadecylsilane bonded silica gel as filler; using acetonitrile-water binary gradient as the flow phase, see the table below; the detection wavelength is 203nm; the number of theoretical plates calculated based on the peak of ginsenoside Rg ₁ should not be less than 2500; for reference solution preparation, accurately weigh notoginsenoside R1, ginsenoside Rg ₁ and ginsenoside Rb ₁ Add appropriate amount of methanol to dissolve, and make a mixed reference solution containing 1.5 mg of notoginsenoside R ₁ , 2.8 mg of ginsenoside Rg ₁ , and 4.1 mg of ginsenoside Rb ₁ per 1 ml; to prepare the test solution, accurately draw 5 ml of the sample, and saturate it with water Shake and extract n-butanol three times, 10ml each time; combine the n-butanol layers, wash once with 10ml n-butanol saturated water, discard the water layer, recover the n-butanol layer under reduced pressure until dry, dissolve the residue to 5ml with methanol, Shake well, filter with a microporous membrane of 0.45 μm to obtain the product; for the determination method, precisely draw 3 μl of the reference solution, 7 μl, and 10 μl of the test solution into the high-performance liquid chromatograph, and calculate with the external standard two-point method to obtain the product ;This product contains not less than 0.2mg of notoginsenoside _R1 per ml; not less than 0.8mg of ginsenoside _Rg1 ; not less than 2.0mg of ginsenoside _Rb1 ; Acetonitrile-water binary gradient elution table Time (min) Flow rate (ml/min) A(water) B(acetonitrile) 0 1.0 80 20 20 1.0 60 40 5. A quality control method for injections prepared from Gardenia and Panax notoginseng, characterized in that one or more of the fingerprint methods can be used in the method: a. Preparation of test solution: Accurately draw 5ml of injection solution, shake and extract twice with water-saturated n-butanol, 10ml for the first time; combine n-butanol extract with 5ml for the second time; Backwash once with 10ml of water, discard the water layer, recover the n-butanol extract to dryness under reduced pressure, dissolve the residue with methanol and dilute it to a 5ml volumetric flask, shake well, and filter through a 0.45um filter membrane to obtain the reference substance Preparation of the solution: take the geniposide reference substance and make a 0.5 mg/ml solution with methanol as the reference solution; measurement method: detection wavelength 238nm, acetonitrile-water gradient elution, see the table below; theoretical plate number calculation, Should not be low 30000; Setting need testing solution sample volume is 10l, record the chromatogram of 60 minutes, promptly get; With the retention time of the chromatographic peak S peak of geniposide and the area of peak be 1 to calculate relative retention time and Peak area ratio; fingerprints and technical parameters; recording time is 60 minutes; the common fingerprint peaks are calibrated with relative retention time, and the common fingerprint peaks are as follows: 1(0.404) 2(0.462) 3(0.510) 4(0.589)5(0.618 ) 6(0.666) 7(0.722) 8(0.798) S(1.000) 9(1.085)10 (1.110)11(1.197) 12(1.238) 13(1.277); the ratio of the peak area of the common fingerprint: 1:S=1 : (0.060-0.113); non-shared peak area: the total area of the non-shared peak must not be greater than 5% of the peak area; Mobile Phase Gradient Elution Table Time (min) Flow rate (ml/min) Water% Acetonitrile% 0 0.8 95 5 8 0.8 92 8 21 0.8 88 12 35 0.8 88 12 40 0.8 70 30 45 0.8 40 60 50 0.8 0 100 60 0.8 0 100 b. Preparation of the test solution: Accurately draw 10ml of injection solution, extract by shaking twice with 20ml of n-butanol saturated with water, and combine the n-butanol extract; backwash three times with 300ml of n-butanol-saturated water, discard Remove the water layer, recover the n-butanol layer to dryness under reduced pressure, dissolve the residue with methanol and set the volume to a 2ml measuring bottle, shake well, and filter through a 0.45um filter membrane to obtain the product; the preparation of the substance: take ginsenoside Rg ₁ Reference substance, a solution of 2mg/ml made of methanol is used as a reference substance solution; determination method: detection wavelength 210nm, sensitivity 0.1AUFS: acetonitrile-water gradient elution, see the table below; the number of theoretical plates is calculated according to ginsenoside Rg ₁ , Should not be low 30000; Precisely draw need testing solution 25 μ l, inject high-performance liquid chromatograph, record the chromatogram of 70 minutes, promptly get; With the retention time of the chromatographic peak (S peak) of ginsenoside Rg ₁ and the area of peak as 1 Calculation of relative retention time and peak area ratio; fingerprint spectrum and technical parameters; recording time is 70 minutes; calibration of common fingerprint peaks, common fingerprint peaks are as follows: 1(0.877) S(1.000) 2(1.027) 3(1.555) 4 (1.592) 5(1.652) 6(1.675) 7(1.708) 8(1.744) 9(1.799) 10(2.005) 11(2.042) 12(2.074) 13(2.111) 14(2.150) 15(2.212) 16(2.235) ) 17(2.364) 18(2.391) 19(2.420) 20(2.440) 21(2.771); the ratio of the total fingerprint peak area: S: 3: 8: 21 = 1: (0.222-0.412): (0.405-0.735) : (1.428-2.380); non-shared peak area: the total area of non-shared peaks shall not be greater than 5% of the total area; Time (min) Flow rate (ml/min) A-water B-acetonitrile 0 1.0 83 17 20 1.0 76 24 40 1.0 60 40 50 1.0 50 50 55 1.0 0 100 60 1.0 0 100 70 1.0 0 100 6. A method for quality control of injections according to any one of claims 1-5, characterized in that the method comprises the following steps: Identification: a. Take 5ml of this product, evaporate to dryness in a water bath, dissolve the residue with ethanol and quantitatively transfer it to 5ml of a volumetric flask, take another geniposide reference substance, add ethanol to make a solution containing 4mg per 1ml, and use it as the reference substance solution; Test according to thin-layer chromatography; draw 2 μl of each of the above two solutions, and place them on the same silica gel G thin-layer plate respectively, and use 8-11:6-9:1-3:0.3-0.6 ethyl acetate-acetone-formic acid- Water is the developing agent, develop, take out, dry, spray with 5% vanillin sulfuric acid solution, and heat; in the chromatogram of the test product, spots of the same color appear on the position corresponding to the chromatogram of the reference product; b. Take 10ml of this product, add water-saturated n-butanol to shake and extract 2-4 times, 10ml each time, combine the n-butanol extract, wash 2-4 times with n-butanol-saturated water, 100ml each time Take the n-butanol layer and recover it under reduced pressure to dryness, add 0.5ml of methanol to the residue to dissolve it, and use it as the test solution; take ginsenoside Rb ₁ , ginsenoside Rg ₁ and notoginsenoside R ₁ reference substance, add methanol to make Each ml contains 6mg of the mixed solution as the reference solution; according to the thin layer chromatography test, draw 2ul of each of the above two solutions, and spot them on the same silica gel G thin-layer plate respectively. 11-15: 6-9: 1-3 Chloroform-methanol-water The lower layer solution placed below 10°C is used as developing agent, developed, taken out, dried in the air, sprayed with 1→10 sulfuric acid ethanol solution, heated at 105-115°C to The color of the spot is clear; in the chromatogram of the test product, at the position corresponding to the chromatogram of the reference substance, the spot of the same color appears; Content determination: a. Geniposide, determined according to high performance liquid chromatography; chromatographic conditions and system suitability test, using octadecylsilane bonded silica gel as filler; 15:85 acetonitrile-water as mobile phase; detection wavelength 238nm; theoretical plate number a. geniposide, determined according to high performance liquid chromatography; chromatographic conditions and system suitability test, using octadecylsilane bonded silica gel as filler; 15:85 acetonitrile-water as mobile phase; the detection wavelength is 238nm; the number of theoretical plates calculated according to the geniposide peak should not be less than 1500; the preparation of the reference substance solution accurately weighs an appropriate amount of the geniposide reference substance, adds an appropriate amount of methanol to dissolve, and makes every 1ml containing 0.144 mg solution, that is, ready; for the preparation of the test solution, accurately absorb 1ml of this product, evaporate to dryness in a water bath, dissolve the residue with absolute ethanol, transfer the ethanol solution to another crucible, evaporate to dryness in a water bath, dissolve the residue with methanol and transfer to In a 100ml measuring bottle, add methanol to the mark, shake well, and you get it; for the determination method, draw 10 μl each of the reference solution and the test solution, inject it into a high-performance liquid chromatograph, and measure it to get it; this product contains Gardenia Glycosides should not be less than 4.7 mg per ml; b. Notoginseng saponins, determined according to high performance liquid chromatography; chromatographic conditions and system suitability tests, using octadecylsilane bonded silica gel as filler; acetonitrile-water binary The gradient is the mobile phase, see the table below; the detection wavelength is 203nm; the theoretical plate number should not be less than 2500 based on the peak of ginsenoside Rg ₁ ; for the preparation of the reference solution, accurately weigh notoginsenoside R1, ginsenoside Rg ₁ , ginseng An appropriate amount of saponin Rb ₁ was dissolved in methanol to make a mixed reference solution containing 1.5 mg of notoginsenoside R ₁ , 2.8 mg of ginsenoside Rg ₁ , and 4.1 mg of ginsenoside Rb ₁ per 1 ml; , extracted by shaking with saturated n-butanol three times, 10ml each time; the n-butanol layers were combined, washed once with 10ml n-butanol saturated water, the water layer was discarded, the n-butanol layer was recovered under reduced pressure until dry, and the residue was dissolved in methanol to 5ml, shake well, and filter with a microporous membrane of 0.45 μm to obtain; Assay method, accurately draw 3 μl of the reference solution, 7 μl, and 10 μl of the test solution into the high-performance liquid chromatograph, and calculate with the external standard two-point method , to be obtained; each ml of this product contains not less than 0.2mg of notoginsenoside _R1 ; not less than 0.8mg of ginsenoside _Rg1 ; not less than 2.0mg of ginsenoside _Rb1 ; Acetonitrile-water binary gradient elution table Time (min) Flow rate (ml/min) A(water) B(acetonitrile) 0 1.0 80 20 20 1.0 60 40 Fingerprints: a. Preparation of the test solution: Accurately draw 5ml of the injection solution, shake and extract twice with n-butanol saturated with water, 10ml for the first time; 5ml for the second time, combine the n-butanol extract Backwash once with 10ml of water saturated with n-butanol, discard the water layer, recover the n-butanol extract to dryness under reduced pressure, dissolve the residue with methanol and set the volume to a 5ml volumetric flask, shake well, and filter through a 0.45um membrane filter The preparation of the reference substance solution: take the geniposide reference substance and make a 0.5mg/ml solution with methanol as the reference substance solution; determination method: detection wavelength 238nm, acetonitrile-water gradient elution, see the table below Theoretical plate number calculation should not be low 30000; Setting need testing solution sample volume is 10l, record the chromatogram of 60 minutes, promptly get; With the retention time of the chromatographic peak S peak of geniposide and the area of peak Calculate relative retention time and peak area ratio for 1; fingerprint spectrum and technical parameters; recording time is 60 minutes; total fingerprint peak is demarcated with relative retention time, and total fingerprint peak is as follows: 1(0.404) 2(0.462) 3(0.510) 4(0.589)5(0.618) 6(0.666) 7(0.722) 8(0.798) S(1.000) 9(1.085) 10(1.110)11(1.197) 12(1.238) 13(1.277); Ratio: 1:S=1:(0.060-0.113); non-shared peak area: the total area of non-shared peaks shall not be greater than 5% of the peak area; Mobile Phase Gradient Elution Table Time (min) Flow rate (ml/min) Water% Acetonitrile% 0 0.8 95 5 8 0.8 92 8 21 0.8 88 12 35 0.8 88 12 40 0.8 70 30 45 0.8 40 60 50 0.8 0 100 60 0.8 0 100 b. Preparation of the test solution: Accurately draw 10ml of injection solution, extract by shaking twice with 20ml of n-butanol saturated with water, and combine the n-butanol extract; backwash three times with 300ml of n-butanol-saturated water, discard Remove the water layer, recover the n-butanol layer to dryness under reduced pressure, dissolve the residue with methanol and set the volume to a 2ml measuring bottle, shake well, and filter through a 0.45um filter membrane to obtain the product; the preparation of the substance: take ginsenoside Rg ₁ Reference substance, a solution of 2mg/ml made of methanol is used as a reference substance solution; determination method: detection wavelength 210nm, sensitivity 0.1AUFS: acetonitrile-water gradient elution, see the table below; the number of theoretical plates is calculated according to ginsenoside Rg ₁ , Should not be low 30000; Precisely draw need testing solution 25 μ l, inject high-performance liquid chromatograph, record the chromatogram of 70 minutes, promptly get; With the retention time of the chromatographic peak (S peak) of ginsenoside Rg ₁ and the area of peak as 1 Calculation of relative retention time and peak area ratio; fingerprint spectrum and technical parameters; recording time is 70 minutes; calibration of common fingerprint peaks, common fingerprint peaks are as follows: 1(0.877) S(1.000) 2(1.027) 3(1.555) 4 (1.592) 5(1.652) 6(1.675) 7(1.708) 8(1.744) 9(1.799) 10(2.005) 11(2.042) 12(2.074) 13(2.111) 14(2.150) 15(2.212) 16(2.235) ) 17(2.364) 18(2.391) 19(2.420) 20(2.440) 21(2.771); the ratio of the total fingerprint peak area: S: 3: 8: 21 = 1: (0.222-0.412): (0.405-0.735) : (1.428-2.380); non-shared peak area: the total area of non-shared peaks shall not be greater than 5% of the total area; Time (min) Flow Rate (ml/min) A-Water B-Acetonitrile 0 1.0 83 17 20 1.0 76 24 40 1.0 60 40 50 1.0 50 50 55 1.0 0 100 60 1.0 0 100 70 1.0 0 100