CN112641904A

CN112641904A - A medicinal injection for treating disturbance of consciousness and hyperpyrexia, and its preparation method

Info

Publication number: CN112641904A
Application number: CN202011558320.0A
Authority: CN
Inventors: 朱音; 於江华
Original assignee: Wuxi Jiyu Shanhe Pharmaceutical Co Ltd
Current assignee: Wuxi Jiyu Shanhe Pharmaceutical Co Ltd
Priority date: 2020-12-25
Filing date: 2020-12-25
Publication date: 2021-04-13

Abstract

The invention relates to a medicine injection for treating disturbance of consciousness and hyperpyrexia and a preparation method thereof, and the injectionThe food is prepared from the following components in parts by weight:

Description

A medicinal injection for treating disturbance of consciousness and hyperpyrexia, and its preparation method

Technical Field

The invention belongs to the field of medicines, relates to a traditional Chinese medicine preparation and a preparation method thereof, and particularly relates to a medicine injection for treating disturbance of consciousness and hyperpyrexia and a preparation method thereof.

Background

Xingnaojing injection is a medicine injection for treating disturbance of consciousness and high fever caused by various causes of disease, and is recorded in national medicine Standard, with the Standard number WS₃-B-3353-98-2003。

[ CHEM ] Moschus 7.5g, radix Curcumae 30g, Borneolum Syntheticum 1g, fructus Gardeniae 30g

[ PREPARATION METHOD ] adding 1500ml of water into radix Curcumae and fructus Gardeniae, distilling, and collecting 1000ml of distillate; adding Moschus into the above distillate, adding 250ml distilled water, distilling, and collecting 1000ml distillate; adding 5g of polysorbate 80 into Borneolum Syntheticum, grinding, adding into the distillate, mixing, adding injectable sodium chloride or glucose, stirring for dissolving, mixing, and standing; refrigerating overnight, filtering, bottling, and sterilizing.

[ PROPERTIES ] the product is a colorless clear liquid.

[ FUNCTIONS AND INDICATIONS ] can clear away heat and toxic materials, cool blood, promote blood circulation, induce resuscitation, and restore consciousness. Used for treating apoplexy and coma caused by reversed flow of qi and blood and brain blood stasis, hemiplegia; traumatic headache, coma; alcohol toxicity attacking heart, headache, nausea, vomiting, coma and convulsion. Cerebral embolism, cerebral hemorrhage in acute stage, craniocerebral trauma, acute alcoholism with the above symptoms.

[ DOSAGE AND ADMINISTRATION ] intramuscular injection, 2-4 ml at a time, 1-2 times a day. Dripping 10-20ml of the mixture into a vein once, diluting the mixture with 5-10% glucose injection or 250-500 ml of sodium chloride injection, and dripping the mixture or following the advice of a doctor.

[ Specification ] 2ml (2)5ml (3)10ml per bottle (1)

The injection for restoring consciousness and calming prepared by the prior art uses polysorbate-80 (commonly known as tween-80) as a solubilizer of liquid medicine. Polysorbate 80 as amphiphilic polyol nonionic surfactant has strong hydrophilicity, no dissociation, high resistance to strong electrolyte, high compatibility with Chinese medicine components, and low toxicity and hemolysis, and may be used widely in preparing injection for promoting the dissolution of insoluble effective components and raising the clarity and stability of injection. Polysorbate 80 has been loaded in the pharmacopoeia of many countries because of its relatively low toxicity. Polysorbate 80 is commonly used in chemical and biological products, especially in Chinese medicine injection. Along with the increasing reports of clinical adverse reactions in recent years, the influence of auxiliary materials on the safety of medicines is increasingly emphasized. Numerous studies currently show that polysorbate 80 may be one of the major causes of injection safety problems. The adverse reactions caused by intravenous injection of injection containing polysorbate 80 are mainly hypersensitivity reactions, especially anaphylactoid reactions. It is manifested as cutaneous symptoms such as erythema, urticaria, edema, and conjunctival congestion of the head, face, chest and limbs. It is also associated with symptoms of the digestive and respiratory circulatory system, such as nausea, vomiting, chest distress, palpitation, elevated blood pressure, dyspnea, etc., which can severely cause shock and even death. In addition, the Xingnaojing injection needs to be sterilized by circulating steam, and after the Xingnaojing injection is heated for a period of time at 100 ℃, the color of the liquid medicine is easy to yellow, the stability of the medicine is difficult to ensure, and the production, storage and transportation of the injection are limited.

To solve the above problems, improvements in formulation and preparation methods are required.

15, also known as

HS15, a novel nonionic solubilizer and emulsifier developed and marketed by Pasteur, Germany, polyethylene glycol 15-hydroxystearate prepared by reacting 15 moles of ethylene oxide with 1 mole of 12-hydroxystearic acid, is registered in the German, British, United states and European pharmacopoeias, wherein the chemical names in the United states and European pharmacopoeias are polyoxyl 15 hydroxide and macrogol 15 hydroxide, respectively.

HS15 is light yellow to white viscous at room temperature, becomes liquid at about 30 ℃, and has strong solubilizing capability for various insoluble drugs because the critical micelle concentration is only 0.005-0.02% (mass fraction). In recent years, HS15 has been widely used. Several formulations containing HS15 are already on the market or are undergoing clinical trials abroad. It shows good safety in the application of injection, and has been widely studied in various dosage forms such as oral capsules, emulsions for injection, ophthalmic preparations, and nasal drops. In addition to good solubilization capacity and safety, HS15 also represents a great advantage in promoting drug absorption and improving bioavailability.

The product is mainly characterized in that: firstly, the chemical stability is good, after long-time heating and cooling, the solid-liquid phase can be physically separated, and the solid-liquid phase can be recovered by homogenizing and stirring; the quality guarantee period is long, and the product can be stored in an unopened original container for at least 24 months at room temperature (less than or equal to 25 ℃); and thirdly, the HS15 aqueous solution can be heated to 121 ℃ for sterilization, phase separation can occur in the heating process, and the hot solution can be recovered only by stirring.

Products containing HS15 which are marketed abroad comprise injection, oral administration, subcutaneous administration and the like, wherein the injection Panitol

The content of medium HS15 is up to 50%. In addition, 5 preparations currently under clinical study in the united states contain HS15, 3 of which are administered intravenously, 1 of which is administered nasally, and 1 of which is administered orally. Clinical data indicate that the maximum tolerated dose for a single intravenous injection administration of HS15 in humans is 8 g.d^-1Better than poloxamer 188 tolerance (the maximum tolerance dose is 2960 mg kg)^-1·d^-1)。

The research finds that the hemolytic activity of HS15 is lower than that of polysorbate 80, the serum histamine release level and the possibility of causing biological allergy are far lower

In the latter case. In addition, it was found that the LD50 of the mouse was 3.16 g/kg when HS15 was intravenously injected into the mouse^-1. Results of guinea pig allergy experiments showed that histamine release was lower in the plasma of HS15 group compared to polysorbate 80 group (serum histamine level of 8 nmol. L in HS15 group after 60min intravenous injection)^-1The polysorbate 80 group is 247 mol.L^-1) The hemolysis is also lower (after intravenous injection with the mass concentration of the solubilizer being 1%, 1% of erythrocytes in the HS15 group are dissolved, and 4% of polysorbate 80 group); and because HS15 has low viscosity, the dissolved medicine has almost no influence on the viscosity of the solution, thereby greatly reducing the irritation of injection administration and ensuring that the injection content reaches 30 percent of HS15 without causing painPain becomes possible.

Because the anaphylaxis and the hemolytic property of HS15 are obviously superior to those of polysorbate 80, the HS15 can be applied to intramuscular injection, subcutaneous injection and intravenous injection, and HS15 is used as a solubilizer and is applied to the Xingnaojing injection. Stability investigation shows that the Xingnaojing injection prepared by HS15 is more stable than the Xingnaojing sample prepared by polysorbate, so that the problem existing in the prior art can be solved by adopting HS 15.

Disclosure of Invention

The injection is prepared by processing the following components in parts by weight:

preferably, the injection of the invention is prepared by processing the following components in parts by weight:

further preferably, the injection of the invention is prepared by processing the following components in parts by weight:

most preferably, the injection of the invention is prepared by processing the following components in parts by weight:

the weight portion of the invention is gram or kilogram.

The components are proportioned by weight, the weight can be increased or decreased, for example, the weight can be in kilogram unit in large-scale production, but the weight proportion of the components is not changed.

The invention also aims to provide a preparation method of the injection.

The preparation method comprises the following steps:

1) mixing the gardenia and the radix curcumae, adding purified water, and distilling and extracting to obtain a distillate I;

2) adding musk and water for injection into the distillate I, and distilling and extracting to obtain distillate II;

3) uniformly mixing solubilizer HS15, borneol and distillate II;

4) refrigerating overnight;

5) adding sodium chloride or glucose, filtering, bottling, and sterilizing.

Preferably, the preparation method comprises the following steps:

1) the gardenia and the radix curcumae are respectively taken and processed into traditional Chinese medicine decoction pieces according to the Chinese pharmacopoeia.

2) Mixing gardenia and radix curcumae according to a proportion, adding 20-30 times of purified water, soaking at 50-90 ℃ for 1-2 hours, and distilling and extracting for 20-24 hours to obtain 10-20 times of distillate I;

3) adding musk and 10 times of injection water into the distillate I according to the proportion, soaking for 2.5-4 hours at the temperature of 40-80 ℃, and distilling and extracting for 16-20 hours to obtain 10-20 times of distillate II;

4) the HS15 with the proportion is ground, crushed and then mixed with borneol passing through a 100-mesh sieve and part of distillate II, and then the rest of distillate II is added and mixed evenly.

5) Refrigerating for 9-12 hours;

6) adding sodium chloride or glucose at a certain ratio, filtering with 0.22um filter core, bottling, and sterilizing.

The invention finds application in

15 the clarity of the solution encountered during the preparation of the Xingnaojing injection. At the same time will solubilizeHS15 as an agent, crushed borneol and distillate II are mixed, turbidity is easy to occur due to different crushing effects, and the mixture can not be cleared again after being stirred for a period of time. To solve this difficulty, the present inventors have solved this problem by using the following process:

4) the borneol in the ratio is firstly crushed by adopting a grinding process, then HS15 and part of distillate are added in the ratio and mixed uniformly, and then the rest distillate is added in the mixture and mixed uniformly.

5) Refrigerating for 9-12 hours;

The inventors further aim at

15, the screening process is as follows:

according to the preparation method, after the borneol with the mixture ratio is crushed, 4-5 parts, 6-20 parts and 21-100 parts of HS15 are respectively added, and are firstly mixed with part of distillate II, and then are uniformly mixed in the rest of distillate II. By taking the solution property, pH and particle size as evaluation indexes, after HS15 is added in 4-5, 6-20 and 21-100 parts, the clarity of the solution meets the requirement, the pH basically does not change, and the particle size tends to be stable. Since HS15 is a solubilizer, for safety reasons, it is preferable to add HS 154-5 parts at the lowest amount if there is no difference in solubilization effect.

The inventor further screens the preparation method, and the screening process is as follows:

according to the process preparation method, the borneol with the mixture ratio is firstly ground by adopting a grinding process, and is respectively required to pass through a 50-mesh sieve, a 100-mesh sieve and a 200-mesh sieve, HS15 and part of distillate II with the mixture ratio are added and mixed uniformly, and then the rest distillate II is added and mixed uniformly. The characteristics, pH and clarification time of the solution are taken as evaluation indexes, the borneol passing through a 200-mesh sieve absorbs moisture after being sieved, the characteristics and pH of the solution of the refreshment quiet sample prepared from the borneol passing through a 50-mesh sieve and a 100-mesh sieve meet the requirements, but the clarification time of the refreshment quiet sample prepared from the borneol passing through a 100-mesh sieve is obviously short, and the process borneol is preferably ground and crushed and then passes through the 100-mesh sieve based on the consideration of saving the operation time in production.

The key innovation points are as follows:

1. innovatively adopt

15 can be used for preparing injection for refreshing brain.

2. Determine

15 the optimal dosage (4 per mill) for preparing the Xingnaojing injection is superior to the dosage (5 per mill) of the solubilizer in the prior Xingnaojing injection.

3. Determine

15 an optimal preparation method for preparing the Xingnaojing injection.

4. By using

15, the usage amount of the solubilizer is less, the risk of anaphylactoid reaction is lower, and the injection is possibly helpful for reducing the incidence rate of clinical adverse reactions of the injection.

1. According to example 1, HS15 is used as a solubilizer in the injection for refreshment, and the use amount of the solubilizer is 4% at the lowest, which is consistent with the solubilization effect of 5% of polysorbate 80 in the current prescription for refreshment. The HS15 has better solubilization effect by using the same concentration to solubilize the mind-refreshing target product; in order to clarify the target product of the brain calming, the use amount of HS15 is lower than that of polysorbate 80, and the safety risk is lower.

2. According to the embodiment 2, the same amount of HS15 and polysorbate 80 are respectively adopted to prepare the Xingnaojing injection, and the physicochemical indexes of the product are compared through accelerated stability and freeze-thaw tests, so that the Xingnaojing injection prepared by HS15 is more stable than the Xingnaojing sample prepared by polysorbate 80.

3. An in vitro hemolysis control experiment was performed according to example 3 using equal amounts of HS15 and polysorbate 80, respectively, to prepare a Xingnaojing injection. Judging according to the requirements of Chinese pharmacopoeia, judging by naked eyes of the Xingnaojing injection prepared from polysorbate 80 according to the requirements of the Chinese pharmacopoeia, considering that in vitro hemolytic reaction exists, but the hemolytic rate is 4.85 percent and is less than 5 percent, and judging that hemolysis occurs according to the reference evaluation standard hemolysis rate of more than 5 percent of the technical guideline for researching the immunotoxicity (anaphylaxis and light allergic reaction) of traditional Chinese medicines and natural medicines, but the occurrence of hemolytic reaction still needs to be noticed during clinical use; no hemolytic reaction was observed at this concentration for the Xingnaojing injection prepared from HS 15. When the two Xingnaojing injection solutions are used for intravascular administration such as intravenous injection, in-vitro hemolytic reaction is not found in the clinical maximum using concentration of the test object, and the test object is judged to be in accordance with the specification. According to the judgment of the dosage of 50 percent of hemolytic reaction caused by the Xingnaojing injection stock solution (0.17 ml of Xingnaojing injection prepared by polysorbate 80 and 0.22ml of Xingnaojing injection prepared by HS 15), the hemolytic properties of the two samples are as follows: the polysorbate 80 is used for preparing the Xingnaojing injection which is more than the Xingnaojing injection prepared by HS 15.

4. Rat allergy control experiments were performed according to example 4 using equal amounts of HS15 and polysorbate 80, respectively, to prepare a Xingnaojing injection. Under the clinical normal dosage, all batches of samples do not cause the mice to have obvious auricle blue staining reaction;

under the condition that the injection for restoring consciousness prepared by HS15 is 6.7 times of clinical dose, no obvious auricle blue staining reaction is caused to the mouse; from the above results, it is considered thatThe batch of samples has less risk of inducing anaphylactoid reaction; under the condition that the injection prepared from polysorbate 80 is 6.7 times of clinical dosage, the injection with the batch number of YF180604 has the suspicious anaphylactic reaction of 20 percent of mice, and the EB seepage amount is also higher than that of a control group; the injection with lot number YF180605 for refreshing brain, 33.3% mice had suspicious anaphylactic reaction, and EB exudation amount was also higher than that of control group; the injection with lot number YF180606 for refreshing brain and calming mind has suspicious anaphylactoid reaction in 33.3% of mice and obvious auricle blue staining reaction (P) in 10% of mice<0.01), and EB bleeding tends to increase. In conclusion, the risk of anaphylactoid reaction of the Xingnaojing injection prepared by adopting HS15 is obviously lower than that of the Xingnaojing injection prepared by adopting polysorbate 80. In conclusion, the method adopts

15, the comprehensive quality evaluation of the Xingnaojing injection prepared by the method is superior to that of the Xingnaojing injection prepared by polysorbate 80, and the Xingnaojing injection has obvious advantages in physical and chemical indexes such as dosage, stability, hemolysis, anaphylactoid and the like and safety indexes.

Drawings

FIG. 1, negative tube and positive tube

FIG. 2 shows the C100% concentration of Xingnaojing injection

FIG. 3 shows the C32% concentration of Xingnaojing injection

FIG. 4 shows the concentration of injection D100%

Fig. 5, D32% concentration of Xingnaojing injection

FIG. 6, saline control group

FIG. 7, histamine Positive control group

FIG. 8, YF180601 test group

FIG. 9, YF180602 test group

FIG. 10, YF180603 test group

FIG. 11, YF180604 test group

FIG. 12, YF180605 test group

FIG. 13, YF180606 test group

Detailed Description

The invention is further illustrated by the following examples.

Example 1

Evaluation of solubility of HS15

The results of solubility tests of polysorbate 80 and HS-15 provided by the present invention as solubilizers were compared:

TABLE 1HS15 comparison of the results as solubilizer with polysorbate 80 for the solubilised refreshment intermediate

As can be seen from the above table, HS15 is used as a solubilizer, 4mg/ml (4 ‰) can sufficiently dissolve the refreshment sample (borneol plus secondary distillate), and polysorbate 80 needs to reach 5mg/ml (5 ‰) to clarify the same solution and obtain stable pH and particle size. The HS15 has better solubilization effect by using the same concentration to solubilize the mind-refreshing target product; in order to clarify the target product of the brain tranquilization, the use amount of HS15 is lower than that of polysorbate 80, and the safety risk is lower.

Example 2 evaluation of stability of Xingnaojing solution

Accelerated stability test

Xingnaojing solution prepared from HS15

Batch number: YF180601, YF180602, YF180603

The preparation method comprises the following steps: the gardenia and the radix curcumae are respectively taken and processed into traditional Chinese medicine decoction pieces according to the Chinese pharmacopoeia. Mixing 3kg of cape jasmine and 3kg of radix curcumae, adding 25 times of purified water, soaking at 60 ℃ for 1.5 hours, and distilling and extracting for 20 hours to obtain 15 times of distillate I; adding musk and 10 times of injection water into the distillate I according to the proportion, soaking for 3 hours at 60 ℃, distilling and extracting for 18 hours to obtain 15 times of distillate II; adding 500gHS15, 100g borneol and part of distillate, grinding, mixing, adding the rest distillate, and mixing. Refrigerating for 10 hr, adding appropriate amount of sodium chloride, filtering with 0.22um filter core, bottling, and sterilizing.

Specification: 10ml of

Storage conditions are as follows: 40 +/-2 DEG C

Sampling detection time point: 0 month, 3 months and 6 months

TABLE 2 summary of the accelerated stability test results for 3 lots of samples of Xingnaojing solution prepared from HS15

A refreshing solution prepared from polysorbate 80

Batch number: YF180604, YF180605, YF180606

The preparation method comprises the following steps: the gardenia and the radix curcumae are respectively taken and processed into traditional Chinese medicine decoction pieces according to the Chinese pharmacopoeia. Mixing 3kg of cape jasmine and 3kg of radix curcumae, adding 25 times of purified water, soaking at 60 ℃ for 1.5 hours, and distilling and extracting for 20 hours to obtain 15 times of distillate I; adding musk and 10 times of injection water into the distillate I according to the proportion, soaking for 3 hours at 60 ℃, distilling and extracting for 18 hours to obtain 15 times of distillate II; adding 500g of polysorbate 80, 100g of borneol and part of distillate, grinding, uniformly mixing, adding the rest of distillate, and uniformly mixing. Refrigerating for 10 hr, adding appropriate amount of sodium chloride, filtering with 0.22um filter core, bottling, and sterilizing.

Specification: 10ml of

Storage conditions are as follows: 40 +/-2 DEG C

Sampling detection time point: 0 month, 3 months and 6 months

And (4) investigating items: property, identification, pH value, visible foreign matter, insoluble particles and content measurement.

TABLE 3 summary of the accelerated stability test results for 3 batches of the Xingnaojing solution prepared with polysorbate 80

From the table, the content of the Xingnaojing injection prepared by using the polysorbate 80 is gradually reduced after the injection is accelerated for 6 months at 40 ℃, and the Xingnaojing injection prepared by using the technology disclosed by the invention is basically unchanged and is more stable.

Freeze thaw test

Refreshment solution batch number prepared by HS 15: YF180601

A refreshment solution prepared from polysorbate 80: YF180604

Specification: 10ml of

The test method comprises the following steps: taking samples with lot numbers of YF180601 and YF180604, respectively performing freeze thawing test, placing in a low-temperature refrigerator at (-20 ℃) for 2 days, and then placing at 40 ℃ under constant temperature for 2 days; taking out, placing in a low temperature refrigerator at (-20 deg.C) for 2 days, and standing at constant temperature of 40 deg.C for 2 days; taking out, placing in a low temperature refrigerator at (-20 deg.C) for 2 days, placing at constant temperature of 40 deg.C for 2 days, sampling, and detecting.

The determination method comprises the following steps: national drug Standard WS₃-B-3353-98-2003

TABLE 4 summary of the results of the Xingnaojing solution prepared from HS15 and the Xingnaojing solution freeze-thaw test prepared from polysorbate 80

And (4) test conclusion: the product passes the freeze-thaw test, namely is placed at the temperature of 20 ℃ below zero for 2 days and then at the temperature of 40 ℃ for 2 days, and is repeatedly circulated for three times, the result shows that all indexes are basically unchanged, and the Xingnaojing injection prepared by adopting HS15 is stable under the condition and is consistent with the Xingnaojing injection prepared by polysorbate 80 in stability.

Example 3 in vitro hemolysis comparative assay

Dosage design

Basis of dosage design

According to the technical guidance principle of research on the immunotoxicity (anaphylaxis and light allergic reaction) of traditional Chinese medicines and natural medicines and the Chinese pharmacopoeia, the injection for clinical non-intravascular administration is diluted by normal saline at the ratio of 1:3 according to the clinical use concentration specified by the use instruction of each medicine to be used as a test solution; the injection for intravascular administration is a test solution at a clinical use concentration specified in each instruction manual.

According to the clinical application instruction of the Xingnaojing injection: the clinical dosage of intravenous drip is 10-20ml, and the intravenous drip is dissolved by normal saline or 250-500 ml of 5-10% glucose injection for use; intramuscular injection: 10-20ml, stock solution.

According to the data, the intramuscular injection concentration of the variety is the stock solution, and the ratio of 1: diluting by 3% to obtain 33%; the intravenous injection concentration is 8% -2%; for ease of dosing, the concentrations of the test substances were set at 2%, 4%, 8%, 16%, 32% and stock solutions, and these 6 doses covered the clinical use concentrations of all test substances.

Test dose

The tested doses are shown in table 5.

TABLE 5 Refreshment infusion dose grouping

Test article

Refreshment solution batch number prepared by HS 15: YF180601, experimental code: xingnaojing for restoring consciousness D

A refreshment solution prepared from polysorbate 80: YF180604, experimental code: xingnaojing C

Test method

Conventional in vitro test tube method

(1) Preparation of blood cell suspension: about 50ml of rabbit blood is taken and put into a triangular flask containing glass beads to shake for 10min, and fibrinogen is removed to obtain defibrinated blood. Adding 10 times of physiological saline, shaking, centrifuging at 1000r/min for 15 min, removing supernatant, and washing the precipitated red blood cells with physiological saline for 2-3 times until the supernatant is not red. The red blood cells were made up into 2% suspensions with physiological saline for testing.

(2) Preparation of a test substance: the Xingnaojing injection C, D is diluted by physiological saline for 3.125 times and then diluted by 6.25, 12.5, 25 and 50 times, wherein the concentration is 32%, 16%, 8%, 4% and 2%.

(3) The sample adding method of the test sample tube comprises the following steps: a plurality of clean test tubes are taken and numbered, wherein the tubes No. 1-5 are test sample tubes, the tubes No. 6 are negative tubes, and the tubes No. 7 are positive tubes. 2% erythrocyte suspension, 0.9% sodium chloride solution or distilled water or different concentration and different batches of Xingnaojing injection are added in sequence according to the table 6, and all the sample tubes are provided with parallel tubes.

(4) The sample adding method of the test sample control tube comprises the following steps: and (3) numbering a plurality of clean test tubes, wherein the tubes 11-15 are reference tubes of the test sample, the tubes 16 are negative reference tubes, and the tubes 17 are positive reference tubes. 0.9% sodium chloride solution or distilled water or different concentration and different batches of Xingnaojing injection are added in sequence as shown in Table 7.

(5) The incubation method comprises the following steps: the above test tube was incubated in a water bath at 37. + -. 0.5 ℃ for 3 hours, and the hemolysis and agglutination were observed as follows.

(6) And (4) observing results: if the solution in the test tube is clear red, no cells or a small amount of red blood cells remain at the bottom of the tube, indicating that hemolysis occurs; if the erythrocytes sink completely, the supernatant is colorless and clear, indicating no hemolysis. If the solution has a brownish red or reddish brown flocculent precipitate, the precipitate does not disperse after shaking, indicating that the erythrocyte coagulation occurs. If the red blood cells are aggregated, the true or false aggregation can be further determined by the following method: the aggregates can be dispersed uniformly after shaking in a test tube, or placed on a glass slide. 2 drops of 0.9% sodium chloride solution are dropped on the edge of the glass slide, and the coagulated erythrocytes are broken up into false coagulations when observed under a microscope, and the coagulated erythrocytes are broken up into true coagulations if the coagulations are not shaken or broken up on the glass slide. See table 8.

(7) And (5) judging a result: judging according to Chinese pharmacopoeia: when the negative tube has no hemolysis and coagulation and the positive tube has hemolysis, if the solution in the test tube has no hemolysis and coagulation within 3 hours, the test article is judged to be in accordance with the regulation; if the solution in the test tube is hemolyzed and/or agglutinated within 3 hours, the test tube is judged to be out of specification.

TABLE 6 hemolysis test sample tube sample loading summary (ml)

TABLE 7 sample addition summary of control tubes for hemolysis test (ml)

TABLE 8 criteria for determination of hemolytic reaction

Results	Performance of	Presentation method
			Total hemolysis	Clear red, no red cell residue at the tube bottom	++
Partial hemolysis	Clear red or brown, with a small amount of red blood cells remaining at the bottom of the tube	+
			Has no hemolysis	The red blood cells all sink, and the upper layer liquid is colorless and transparent	-
Agglutination	The blood is not dissolved, but the red blood cells agglutinate, and the shaking cannot be performed	##

Improved in vitro blood test method (spectrophotometry)

The tube solutions incubated at different time points in tables 6 and 7 were centrifuged at 1200r/min for 5min, the supernatant was aspirated, the absorbance (A) was measured at 545nm, and the degree of hemolysis was calculated according to the following formula.

Percent hemolysis ═ 100% [ (a sample tube-a sample control tube) - (a negative-a negative control tube) ]/[ (a positive tube-a positive control tube) - (a negative tube-a negative control tube) ].

And (5) judging a result: according to the technical guidance principle of research on immunotoxicity (anaphylaxis and light allergic reaction) of traditional Chinese medicines and natural medicines: a hemolysis rate of > 5% indicates hemolysis; if hemolysis occurs, the dosage with a hemolysis rate of 50% is further calculated using SPSS11.0 statistical software (if hemolysis occurs at multiple concentrations, the dosage at the highest concentration that can cause hemolysis is calculated).

Results

All samples were not agglutinated.

2 to 8 percent of Xingnaojing injection C, D0.1.1 to 0.5ml is incubated for 3 hours at 37 ℃, the red blood cells are observed by naked eyes to completely sink, the supernatant is colorless and clear, and the hemolysis rate is all less than 5 percent; no reddish-brown or reddish-brown flocculent precipitate was observed in the solution, indicating that no hemolysis and coagulation reactions were observed, consistent with the 0.9% NaCl solution control. The positive control group of distilled water had significant hemolytic effect.

Xingnaojing injection C group: adding 0.1-0.5ml of Xingnaojing injection C stock solution into a test tube, and causing 0.2-0.5ml of Xingnaojing injection C stock solution to generate total hemolysis, wherein the hemolysis rate is 68.15% -109.97%; 0.1ml was partially hemolyzed with a hemolysis rate of 8.63% and the dose for 50% hemolysis was 0.17 ml. Adding 0.1-0.5ml of Xingnaojing injection C32% into a test tube, and causing partial hemolysis in 0.3-0.5ml, wherein the hemolysis rate is 4.85% -49.15%; 0.1-0.2ml of the composition is not hemolyzed, and the hemolysis rate is less than 5%. 0.1-0.5ml of Xingnaojing injection C16% is added into the test tube, the hemolysis rate is less than 5%, and no hemolysis occurs.

Xingnaojing injection group D: adding 0.1-0.5ml of Xingnaojing injection D stock solution into a test tube, wherein 0.3-0.5ml of Xingnaojing injection D stock solution causes complete hemolysis, and the hemolysis rate is 83.51% -98.63%; 0.2ml of the solution is partially hemolyzed, and the hemolysis rate is 47.63%; 0.1ml was not hemolyzed, and the dose for 50% hemolysis was 0.22 ml. Adding 0.1-0.5ml of injection D32% into the test tube, and causing partial hemolysis in 0.4-0.5ml, wherein the hemolysis rate is 3.76-14.43%; 0.1-0.3ml was not hemolyzed. The test tube is added with 0.1-0.5ml of Xingnaojing injection D16% concentration, and no hemolysis occurs.

TABLE 9 in vitro hemolysis test results for Xingnaojing injection C

TABLE 10 results of in vitro hemolysis test for Xingnaojing injection D

In a hemolysis test, judging according to the requirements of Chinese pharmacopoeia, judging by naked eyes of Xingnaojing injection C according to the requirements of Chinese pharmacopoeia, considering that in vitro hemolysis reaction exists, but the hemolysis rate is 4.85 percent and is less than 5 percent, and according to the reference evaluation standard hemolysis rate of more than 5 percent of the technical guidance principle of research on immunotoxicity (anaphylaxis and light allergic reaction) of traditional Chinese medicines and natural medicines, the occurrence of hemolysis is indicated, but the occurrence of hemolysis reaction still needs to be noticed when the injection is clinically used; no hemolytic reaction was observed at this concentration for injection D. When the Xingnaojing injection C, D is used for intravascular administration such as intravenous injection, no in vitro hemolytic reaction is found in the clinical maximum use concentration of the test object, and the test object is judged to be in accordance with the regulations. According to the judgment of the dosage of 50% hemolytic reaction caused by the Xingnaojing injection stock solution (C: 0.17ml and D: 0.22ml), the hemolytic property of the two samples is C > D.

Example 4 rat anaphylactoid contrast test

4.1 test methods

According to the weight of each animal, the tail vein is injected with test substance liquid medicines with different concentrations according to 10ml/kg, and finally, the 10ml/kg of 0.85 percent Evans Blue (EB) solution is injected. 30min after administration, the general response of the animals, the number of auricle blue-stained animals was observed to calculate the response rate (%) (number of auricle blue-stained animals/total number of animals in the group x 100%); the blue stained areas of the ears were scored (table 11). Then, the mouse was sacrificed by removing the cervical vertebrae, ears were cut along the ear roots, cut into pieces, and soaked in formamide solution at room temperature in the dark for 48 hours, then the soak was filtered with a 200-mesh steel mesh screen, the filtrate was added to a 48-well culture plate, and absorbance was measured with an enzyme-labeling instrument (wavelength 610 nm). And calculating the EB content in the sample by taking EB as a standard curve.

TABLE 11 allergy class scores and results judgement

The result of the anaphylactoid test of the positive drug histamine by using the ICR mouse proves that after the ICR mouse is injected with histamine intravenously, the dose-dependent auricle blue staining is presented, and the ear blue staining degree and the EB exudation amount in the ear tissue are in good positive correlation with the histamine dose. At a minimum, a weak response of 1mg/kg histamine was detected by intravenous injection. However, at a histamine dose of 2mg/kg, staining of auricle blue was evident, and 10mg/kg gave a strong response.

4.2 test specimens

The Xingnaojing injection prepared by adopting HS15 has the following batch numbers: YF180601, YF180602 and YF180603 adopt a Xingnaojing injection prepared from polysorbate 80, and the batch number is as follows: YF180604, YF180605, YF180606

4.2 test results

A mouse anaphylactoid model is adopted to detect anaphylactoid reaction of different batches of products of the Xingnaojing injection, tail vein injection of Xingnaojing injection (stock solution) with different concentrations is carried out according to the weight of each animal and 3ml/kg (the dosage for clinical use is 0.33ml/kg, and the equivalent dosage converted into the mouse is 3ml/kg), and finally 5ml/kg of 1.6 percent Evans blue solution is injected.

(1) The batches of the Xingnaojing injection are YF180601, YF180602, YF180603, YF180604, YF180605 and YF180606 respectively, and all batches of samples do not cause obvious auricle blue staining reaction of mice under clinical normal dosage;

(2) the batches of the Xingnaojing injection are YF180601, YF180602 and YF180603 respectively, and under the condition that the dosage is 6.7 times of the clinical dosage, no obvious auricle blue staining reaction is caused to the mouse; from the above results, it is considered that the lot of the sample has a smaller risk of inducing the anaphylactoid reaction.

(3) Under the condition of 6.7 times of clinical dosage, the batch number of the Xingnaojing injection YF180604 causes suspicious anaphylactic reaction of 20 percent of mice, and the EB seepage amount is also higher than that of a control group; the injection with lot number YF180605 for refreshing brain, 33.3% mice had suspicious anaphylactic reaction, and EB exudation amount was also higher than that of control group; the injection lot number of YF180606 for refreshing brain, 33.3% of mice had suspicious anaphylactoid reaction, 10% of mice had obvious auricle blue staining reaction (P <0.01), and EB exudation had increasing tendency. According to the above results, it is considered that the above batches of samples may have a risk of anaphylactoid reaction if used clinically, and the risk of inducing anaphylactoid reaction in mice is obviously increased with the increase of dosage.

(4) The risk of anaphylactoid reaction of the Xingnaojing injection prepared by the HS15 is obviously lower than that of the Xingnaojing injection prepared by the polysorbate 80.

TABLE 12 results of anaphylactic reaction in mice induced by Xingnaojing injection (

n＝10)

Note: compared to the control group 1) P <0.05,2) P <0.01,3) P < 0.001.

Claims

1. A medicine injection for treating disturbance of consciousness and hyperpyrexia is prepared by processing the following components in parts by weight:

2. the injection according to claim 1, which is prepared from the following components in parts by weight:

3. the injection according to claim 1, which is prepared from the following components in parts by weight:

。

4. the injection according to claim 1, which is prepared from the following components in parts by weight:

5. a method of preparing the injectable solution of claim 1, comprising the steps of:

3) uniformly mixing solubilizer HS15, borneol and distillate II;

4) refrigerating overnight;

5) adding sodium chloride or glucose, filtering, bottling, and sterilizing.

6. The method of claim 5, comprising the steps of:

1) processing fructus Gardeniae and radix Curcumae into decoction pieces according to Chinese pharmacopoeia;

4) HS15, borneol after grinding and crushing and part of distillate II in the proportion are mixed uniformly, and then the rest of distillate II is added and mixed uniformly;

5) refrigerating for 9-12 hours;