CN105943606B - A pharmaceutical composition for treating cerebral ischemia and preparation method thereof - Google Patents

Abstract

The invention relates to a medicament for treating the brainThe pharmaceutical composition for ischemia comprises the following raw materials in parts by weight: 30-50 parts of salvia miltiorrhiza and safflower extract phospholipid complex, 70-90 parts of soybean oil, 160-180 parts of medium chain triglyceride, 10-20 parts of emulsified phospholipid, 7-13 parts of poloxamer-188, 7-13 parts of glycerol, 0.5-2 parts of oleic acid, 0.5-2 parts of VE, EDTA-Na₂0.03-0.1 part of water for injection and the balance of water for injection; the pharmaceutical composition can be made into nasal cavity submicron emulsion preparation, and has the advantages of strong brain targeting effect, high bioavailability and small side effect. The preparation method is scientific and reasonable, is suitable for industrialized mass production, and has wide application prospect.

Description

A pharmaceutical composition for treating cerebral ischemia and preparation method thereof

Technical Field

The invention relates to the technical field of new dosage forms of traditional Chinese medicines, in particular to a pharmaceutical composition for treating cerebral ischemia and a preparation method thereof.

Background

Cerebral ischemia is the ischemia of brain tissue caused by transient insufficient blood supply to the brain, thus leading to ischemic necrosis of the localized brain tissue and the appearance of corresponding symptoms and signs. The clinical manifestations of the disease are sudden dizziness, dim eyesight, tinnitus, vertigo and unstable walking, and in severe cases, the disease has the symptoms of blurred consciousness, binocular blindness or double vision, weakness and paresthesia of single-side and double-side limbs, rare fall, speaking fluency, etc., and patients with transient cerebral ischemia symptoms may have cerebral infarction generally within one to five years, while one third to two thirds of patients with cerebral infarction have transient cerebral ischemia. It is known from relevant clinical documents and medical history in hospital that ischemic brain injury is one of the main inducing factors of recurrent cerebral edema diseases, and becomes one of three diseases causing human death, in recent years, the incidence rate is increased year by year, the inducement is increased, the number of patients is increased, and the high importance of the medical industry at home and abroad is aroused.

Salvia miltiorrhiza is the dried root and rhizome of Salvia miltiorrhiza belonging to Labiatae, and is a commonly used medicine for promoting blood circulation and removing blood stasis. Pharmacological studies show that salvia miltiorrhiza can improve microcirculation, dilate blood vessels, reduce platelet aggregation and blood viscosity and improve hemorheology. Carthami flos is dried flower of Carthamus tinctorius L of Compositae, and is a traditional medicine for promoting blood circulation and removing blood stasis. The current clinically used medicaments comprise salvia miltiorrhiza injection, safflower injection and salvia miltiorrhiza injection for treating cardiovascular and cerebrovascular diseases. The existing main application forms for clinically treating ischemic brain injury comprise intravenous injection and oral administration, the bioavailability of oral drugs is low, blood brain barrier obstruction exists, potential clinical risks of intravenous injection and blood brain barrier obstruction exist, the drugs enter the brain seriously, the bioavailability is extremely low, and the treatment effect is influenced to a certain extent. The potential clinical risks of injections place certain limitations on their clinical use. Therefore, the research for developing the cardiovascular and cerebrovascular external administration alternative preparation has certain clinical value and can be used as a clinical effective complementary treatment mode.

Nasal administration is a new administration route, not only can be used for treating nasal diseases, but also can play a role in systemic treatment through various nasal passages. There are some connections between the nasal cavity and the cerebrospinal fluid, which are the connections between the administration of the nasal cavity and the brain diseases, and there are natural "nose-brain paths". Research suggests that nasal administration can bypass the blood brain barrier, significantly increase the distribution of the drug in brain tissue, and be used for treating central nervous system diseases. Nasal administration, a non-invasive method of administration, is simpler and safer than other methods currently in use. The advantages are more obvious for the medicines which act on the central nervous system and the curative effect is related to the brain function, such as the medicines for treating Parkinson's disease, Alzheimer's disease and the like, in particular for the medicines with extremely low intracerebral concentration under the conventional administration route.

The invention prepares the salvia miltiorrhiza and safflower extracts into the nasal administration submicron emulsion preparation, which can be selectively accumulated in the brain, so that the concentration of the treatment medicine in the target area of the brain exceeds the traditional preparation by several times to hundreds of times, and the curative effect is obviously improved. Meanwhile, the medicine is distributed in a small amount in normal tissues, so that adverse reactions can be reduced, and the effects of high efficiency and low toxicity are achieved. The preparation can improve absorption of medicine in vivo, overcome blood brain barrier by nasal administration, and treat ischemic brain injury.

Disclosure of Invention

In order to overcome the drawbacks of the prior art, one of the objects of the present invention is: in order to provide a medicine composition for treating cerebral ischemia; the second purpose is that: also provides a preparation method of the pharmaceutical composition; the third purpose is to provide the administration route of the pharmaceutical composition. Generally, the pharmaceutical composition has the advantages of strong brain targeting effect, high bioavailability and small side effect.

The technical scheme of the invention is as follows:

the pharmaceutical composition comprises the following active components in parts by weight: 0.5-2 parts of salvia extract and 0.5-2 parts of safflower extract.

Preferably, the pharmaceutical composition comprises the following active components in parts by weight: 1 part of salvia extract and 1 part of safflower extract.

Further, the preparation method of the active ingredients of the pharmaceutical composition of the invention is as follows:

⑴ the Saviae Miltiorrhizae radix extract is prepared by soaking Saviae Miltiorrhizae radix in water, extracting, precipitating with ethanol, removing impurities, concentrating, and drying;

⑵ the preparation method of the flos Carthami extract comprises soaking flos Carthami in water, extracting, concentrating the extractive solution, and drying.

Further preferably, the preparation method of the active ingredients of the pharmaceutical composition of the present invention is as follows:

⑴ the preparation method of the red sage root extract comprises the steps of taking red sage root, adding water with the weight 8-12 times of that of the medicinal material, extracting twice at 75-85 ℃, extracting for 1-3 h each time, merging the extracting solutions, filtering, concentrating the filtrate at 55-65 ℃ under reduced pressure to obtain clear paste with the relative density of 1.18-1.22, cooling, adding ethanol to ensure that the alcohol content is 65-75%, standing for 10-14 h, taking supernatant, recovering ethanol, concentrating and drying to obtain the red sage root extract;

⑵ the preparation method of the safflower extract comprises the steps of taking safflower medicinal materials, adding water with the weight of 3-7 times of the medicinal materials, extracting for 2 times at 85-95 ℃ with the warm temperature, extracting for 1-3 h each time, combining the extracting solutions, filtering, concentrating the filtrate, and drying to obtain the safflower extract.

Still more preferably, the pharmaceutical composition of the present invention, the active ingredients thereof, are prepared by the following method:

⑴ the preparation method comprises extracting Saviae Miltiorrhizae radix with 10 times of water at 80 deg.C for 2 hr twice, mixing extractive solutions, filtering, concentrating the filtrate at 60 deg.C under reduced pressure to obtain fluid extract with relative density of 1.18-1.22, cooling, adding ethanol to ethanol content of 70%, standing for 12 hr, collecting supernatant, recovering ethanol, concentrating, and drying to obtain Saviae Miltiorrhizae radix extract;

⑵ the preparation method of the Carthami flos extract comprises adding 5 times of water into Carthami flos, extracting at 90 deg.C for 2 times (each time for 2 hr), mixing extractive solutions, filtering, concentrating the filtrate, and drying to obtain Carthami flos extract.

In the above technical scheme, the active ingredient of the pharmaceutical composition of the present invention is a phospholipid complex prepared by adding pharmaceutical excipients into the extracts of red sage root and safflower, and is used as an active carrier. In this respect, the present invention is described as "phospholipid complex of extracts of red sage root and safflower" throughout the technical scheme.

Furthermore, the pharmaceutical composition for treating cerebral ischemia comprises the following raw materials in parts by weight: 30-50 parts of salvia miltiorrhiza and safflower extract phospholipid complex, 230-270 parts of oil phase, 10-20 parts of emulsifier, 7-13 parts of co-emulsifier, 7-13 parts of isotonic regulator, 0.5-2 parts of stabilizer, 0.5-2 parts of antioxidant and 0-0.1 part of metal ion complexing agent.

Preferably, the oil phase is soybean oil and medium chain triglyceride, the emulsifier is emulsified phospholipid, the co-emulsifier is poloxamer-188, the isotonic regulator is glycerol, the stabilizer is oleic acid, the antioxidant is VE, and the metal ion complexing agent is EDTA-Na₂。

Further preferably, the pharmaceutical composition of the present invention comprises the following raw materials by weight: 30-50 parts of salvia miltiorrhiza, safflower extract phospholipid complex and soybean oil70-90 parts of medium chain triglyceride 160-180 parts, emulsified phospholipid 10-20 parts, poloxamer-1887-13 parts, glycerol 7-13 parts, oleic acid 0.5-2 parts, VE 0.5-2 parts, EDTA-Na₂0.03 to 0.1 portion.

More preferably, the pharmaceutical composition of the present invention comprises the following raw materials by weight: 50 parts of salvia miltiorrhiza and safflower extract phospholipid compound, 90 parts of soybean oil, 180 parts of medium chain triglyceride, 20 parts of emulsified phospholipid, poloxamer-1887 parts, 13 parts of glycerol, 2 parts of oleic acid, 0.5 part of VE, EDTA-Na₂0.1 part.

Still further preferably, the pharmaceutical composition of the present invention comprises the following raw materials by weight: 30 parts of salvia miltiorrhiza and safflower extract phospholipid compound, 70 parts of soybean oil, 160 parts of medium chain triglyceride, 10 parts of emulsified phospholipid, poloxamer-18813 parts, 7 parts of glycerol, 0.5 part of oleic acid, 2 parts of VE, EDTA-Na₂0.03 part.

Still further preferably, the pharmaceutical composition of the present invention comprises the following raw materials by weight: 40 parts of salvia miltiorrhiza and safflower extract phospholipid compound, 80 parts of soybean oil, 170 parts of medium chain triglyceride, 15 parts of emulsified phospholipid, poloxamer-18810 parts, 10 parts of glycerol, 1 part of oleic acid, 1 part of VE, EDTA-Na₂0.06 part.

Further, the pharmaceutical composition of the present invention is in the form of: submicron emulsion formulation.

The invention relates to a pharmaceutical composition for treating cerebral ischemia, wherein the preparation method of the submicron emulsion preparation comprises the following steps:

⑴, glycerol, poloxamer-188, EDTA-Na₂Dispersing in water for injection, heating to 50-60 ℃, and stirring until the water is completely dissolved to obtain a water phase;

⑵, respectively weighing the salvia miltiorrhiza extract and the safflower extract, then weighing the soybean lecithin, putting the three into a magnetic stirrer, adding methanol, carrying out composite reaction in a water bath at 50-60 ℃ for 1-3 hours, and recovering the methanol until the residual volume is 20% to prepare a salvia miltiorrhiza and safflower extract phospholipid composite methanol solution;

⑶, adding soybean oil and medium chain triglyceride into the methanol liquid of the salvia miltiorrhiza and safflower extract phospholipid complex in the step (2), continuously recovering until no methanol smell exists, adding Ve, oleic acid and emulsified phospholipid, stirring at the rotating speed of a high-speed tissue triturator of 15000-25000 rpm, stirring the water phase in the step ⑴ for 8-12 minutes to prepare primary emulsion, diluting the primary emulsion with distilled water to a prescribed volume, transferring the primary emulsion to a high-pressure homogenizer at a homogenizing pressure of 800-1200 par for 6-11 times, controlling the homogenizing temperature at 30-50 ℃, and adjusting the pH value to 4-6 with alkali to obtain the composition.

Further preferably, the pharmaceutical composition for treating cerebral ischemia according to the present invention, the preparation method of the submicron emulsion formulation is as follows:

⑴, glycerol, poloxamer-188, EDTA-Na₂Dispersing in water for injection, heating to 55 deg.C, stirring to dissolve completely to obtain water phase;

⑵, weighing radix Salviae Miltiorrhizae extract and flos Carthami extract, weighing soybean lecithin, placing the above three in a magnetic stirrer, adding methanol, carrying out composite reaction in 55 deg.C water bath for 2 hr, and recovering methanol to obtain methanol solution of radix Salviae Miltiorrhizae and flos Carthami extract phospholipid complex;

⑶, adding soybean oil and medium chain triglyceride into the methanol solution of the phospholipid complex of the red sage root and safflower extracts in the step (2), continuously recovering until no methanol smell exists, adding Ve, oleic acid and emulsified phospholipid, stirring at the rotating speed of a high-speed tissue triturator of 20000rpm, stirring the water phase in the step ⑴ for 10 minutes to prepare primary emulsion, diluting the primary emulsion with distilled water to a prescribed volume, transferring the primary emulsion to a high-pressure homogenizer at a homogenizing pressure of 1000par for 9 times, controlling the homogenizing temperature at 40 ℃, and adjusting the pH value to 4-6 with alkali to obtain the final product, wherein the primary emulsion has the following beneficial effects:

⑴, the active ingredients of the traditional Chinese medicine of the pharmaceutical composition of the invention are extracts of red sage root and safflower, the red sage root mainly contains fat-soluble and water-soluble ingredients, and salvianolic acid B is the water-soluble ingredient with the highest content, modern pharmacological experiments show that the ingredients have good antithrombotic effect, the safflower has the highest content of hydroxysafflor yellow A and the most effective water-soluble part with pharmacological effect of safflower, which can inhibit platelet aggregation and release induced by platelet activating factor, and competitively inhibit the combination of platelet activating factor and platelet receptor, and is the effective component of safflower yellow for promoting blood circulation and removing blood stasis, in order to extract the effective components of the two medicinal materials to the maximum and achieve good therapeutic effect, the invention respectively examines the extraction temperature and extraction time of the two medicinal materials, finally determines the best process conditions, finally obtains the extract of red sage root, the content of salvianolic acid B in the extract prepared in laboratory is 16.119%, and finally obtains the safflower extract (the yield is 35%).

⑵, respectively carrying out in vitro antioxidant experiments on the red sage root extract and the safflower extract with different component ratios to obtain the best component ratio of the red sage root extract to the safflower extract with the best ratio of 1: 1. duplicating a focal ischemia model by adopting a wire-tying method, and carrying out pharmacological experiments on the preferred ratio of the red sage root extract to the safflower extract, wherein the ratio of the nerve function defect score, the crossbar score, the brain index, the infarct volume, the biochemical index and the pathology are used as observation indexes.

⑶, because the extracts of red sage root and safflower have poor lipophilicity, the medicine adopts conventional oral preparation or injection, after absorbed by human body, the effective components in the medicine hardly pass through blood brain barrier, if the extracts of red sage root and safflower are made into nasal brain targeting preparation, the preparation has good biological membrane permeability, can increase the bioavailability of the medicine, and improve the treatment effect to cerebral ischemia.

⑷ research on preparation process of submicron emulsion preparation of pharmaceutical composition

① the phospholipid complex can be prepared by mixing radix Salviae Miltiorrhizae and flos Carthami extracts with soybean lecithin 1:1, compounding rate 60.27%, soybean lecithin 1: 2, compounding rate 97.57%, soybean lecithin 1: 3, and compounding rate 95.73%, so the preparation method of the submicron emulsion adopts a phase inversion technique to dissolve and disperse the phospholipid complex in an oil phase solution, and adopts an orthogonal test method to optimize and screen, and the result shows that the phospholipid complex can be uniformly dispersed in the oil phase by using methanol as a composite solvent, and can achieve a good dissolving effect.

②, the oil phase, the drug-loading rate, the oleic acid, the isotonic regulator and the antioxidant in the prescription composition are inspected, the optimized prescription composition proportion is determined, the drug-loading rate is 4%, the emulsified phospholipid dosage is 1.5%, the poloxamer-188 dosage is 1%, the pH is adjusted to 5, three batches of submicron emulsion preparations prepared according to the process have the particle size (nm) of 210 nm-240 nm and the PDI of 0.246-0.321, the temperature, the shearing speed and the shearing time of the colostrum are inspected, the high-pressure homogenization method is adopted to prepare the nasal brain targeting submicron emulsion of the red sage root and safflower extracts, and the optimized submicron emulsion preparation is subjected to electron microscope observation and particle size measurement, and the result shows that the submicron emulsion preparation of the pharmaceutical composition has the advantages of fine particle size, uniform dispersion, particle size of 209.55-217.0 (nm), PDI of 0.216-0.246, zeta potential-31.51-34.46.

⑸ pharmacodynamic experiment of submicron emulsion preparation of pharmaceutical composition of the invention

The experimental result shows that each preparation group can prolong the survival time of the hypoxic mouse, and the sub-microemulsion group can improve the SOD activity in the brain tissue of the hypoxic mouse and the ischemia mouse, reduce the MDA content, improve the ATPase activity and reduce the LD level. And the submicron emulsion with high dosage can reduce the neurological function defect of brain tissue, and compared with a model group (P is less than 0.05), the submicron emulsion can obviously improve the biochemical levels of SOD, MDA, GSH-PX and NO in serum and brain tissue, and compared with the model group (P is less than 0.05).

1. The preparation process of the red sage root and safflower extract of the invention is optimized as follows:

① Process for extracting radix Salviae Miltiorrhizae

The extraction process of the water-soluble components of the salvia miltiorrhiza is researched, the water is used as a solvent, the content of the salvianolic acid B is used as an evaluation index, the dynamic extraction rate of the salvia miltiorrhiza under different temperatures and different time conditions is considered, and the optimal extraction process is determined.

Investigation of the extraction method: taking 15g of salvia miltiorrhiza medicinal material, weighing 3 parts in total, precisely weighing, placing in a round bottom flask, adding a condensing device, adding 20 times of water (the solvent amount is referred to related documents), placing in a water bath pot or an electric heating jacket, respectively extracting under reflux at 70 ℃, 75 ℃, 80 ℃, 85 ℃, 90 ℃ and slightly boiling conditions, respectively extracting each sample for 10min, 20min, 30min, 45min, 60min, 90min, 120min and 180min, taking 2mL of extract, simultaneously supplementing 2mL of water, and measuring the content of salvianolic acid B in the samples at different times.

TABLE 1 content (mg/ml) of extract in leach liquor at different temperatures and for different times

As can be seen from the data in Table 1, the content of salvianolic acid B in the aqueous extract is continuously increased with the increase of the leaching time, the content is rapidly increased within 120 minutes, the increasing trend is gradually reduced, and the content curve of salvianolic acid B is balanced after 120 minutes. Within 120 minutes, the higher the temperature, the higher the extraction rate, and the highest extraction rate under a slightly boiling environment, which indicates that the temperature has great influence on the extraction of the salvianolic acid B. The content measurement result is highest in the environment of 80 ℃ after the equilibration, probably because the salvianolic acid B is decomposed in the high-temperature environment for a long time. According to the content determination result, the optimum extraction condition is selected to be warm-soaking extraction at 80 ℃ for 120 min.

Preferably and confirms the preparation process of the safflower extract: taking a proper amount of salvia miltiorrhiza, adding 10 times of water, extracting twice at 80 ℃ for 2h each time, combining extracting solutions, filtering, concentrating the filtrate at 60 ℃ under reduced pressure to obtain clear paste with the relative density of 1.18-1.22, cooling, adding ethanol until the ethanol content is 70%, standing for 12h, taking supernatant, recovering ethanol under reduced pressure, concentrating to obtain thick paste, and drying in a vacuum drying oven to finally obtain the salvia miltiorrhiza extract.

② research on extraction process of Carthami flos

The research researches the extraction process of the safflower, takes the content of the hydroxysafflor yellow A as an index, examines the dynamic extraction rate under different temperature conditions, and preferably selects a better extraction process.

Investigation of the extraction method: taking 10g of safflower medicinal material, precisely weighing, placing in a round-bottom flask, adding a condensing device, adding 5 times of water (the solvent amount is referred to related documents), placing in a water bath kettle or an electric heating jacket, respectively performing reflux extraction at 50, 60, 70, 80 and 90 ℃ and under the condition of slight boiling, respectively sampling 1mL of each sample when extracting 5, 10, 15, 20, 30, 45, 60 and 90min, and simultaneously supplementing 1mL of solvent.

Preparation of a test solution: precisely measuring 2mL of leaching solution at different temperatures (50, 60, 70, 80, 90 deg.C and slightly boiling), placing in a 10mL measuring flask, diluting with 25% methanol to scale, shaking, filtering with 0.45 μm microporous membrane, and collecting filtrate.

TABLE 2 content of extract (mg/ml) in leach liquors at different temperatures for different times

According to the experimental results in table 2, it can be known that the content of hydroxysafflor yellow a in the safflower leach liquor gradually increases with the extension of the extraction time, and the curve tends to be balanced at 60 min. The extraction temperature is higher and higher with the same extraction time, and the extraction temperature has larger influence on the content of the hydroxysafflor yellow A. Due to the thermal instability of hydroxysafflor yellow A, the high temperature environment for a long time can cause the hydrolysis of hydroxysafflor yellow A in the leaching solution. Under the condition of higher temperature, the extraction content is reduced to different degrees after the equilibrium is reached for 60 min. According to the content determination result, the final extraction condition is selected as warm immersion extraction at 90 ℃ for 60 min.

Preferably and confirms the preparation process of the safflower extract: adding 5 times of water into a safflower medicinal material, extracting for 2 times at 90 ℃ with warm immersion, each time for 1 hour, combining extracting solutions, filtering, concentrating the filtrate at 60 ℃ under reduced pressure to obtain clear paste with the relative density of 1.18-1.2, and drying in a vacuum drying oven to finally obtain the safflower extract.

2 optimization and pharmacodynamics of the composition proportion of the red sage root extract and the safflower extract

In order to obtain the formula ratio of the pharmaceutical composition with better curative effect, different formula ratios of a plurality of salvia extracts and safflower extracts are selected, and in-vitro antioxidant experiment research is carried out to obtain the conclusion that: the red sage root extract has the strongest oxidation resistance, the safflower extract has the weakest in vitro oxidation resistance, and the red sage root and safflower mixed extract has the in vitro oxidation resistance far better than that of the safflower extract and is between the red sage root extract and the safflower extract.

In order to verify the curative effect of the formula proportion of the red sage root extract and the safflower extract, a focal ischemia model is copied by adopting a wire-tying method, and each component is tested by taking a neurological deficit score, a rhamnus score, a brain index, an infarction volume, biochemical indexes and pathology as evaluation means.

2.1 instruments and reagents:

an electronic balance; glass needle, ophthalmological forceps, surgical scissors, hemostatic forceps, suture needle and nylon threads with different specifications. Animals: 96 clean-grade healthy SD rats with 2 months age, the weight of about 250g, ginseng female and ginseng male, purchased from the center of laboratory animals of the department of medicine of Xian transportation university, and the qualification number: scxk (shan) 2012 and 003. Reagent testing: the red sage root extract and the safflower extract are both prepared by laboratories; nimodipine injection (chenxin pharmaceutical products, ltd., lot No. 1404262102); danhong injection (Shandong Danhong pharmaceutical Co., Ltd., production lot: 14111011); chloral hydrate (Shanghai Shanpu chemical Co., Ltd., lot No. 20140905); TTC (Sigma, usa); the kit comprises: nitric Oxide Synthase (NOS), Malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) (batch number: 20141211,20141212,2141213,20141210), all of which were purchased from the institute of bioengineering, established in Nanjing.

2.2 test methods

Animal grouping: the method comprises the following steps of randomly dividing 96 rats into 6 groups, namely a model group, a nimodipine group, a red sage injection group, a red sage root extract group, a red sage root compatibility group (the mass ratio of the red sage root extract to the red flower extract is 1:1) and a pseudo-operation group, and considering the death rate existing in a wire-tying method model, setting 16 experimental animals in each group and half male and female animals in each group.

2.3 pharmaceutical formulation and dosing regimens

Preparing a salvia miltiorrhiza extract solution: precisely weighing a proper amount of the salvia miltiorrhiza extract, dissolving the salvia miltiorrhiza extract in physiological saline, and preparing into a solution of 1.5 g/ml; preparing a Danhong compatible solution: according to the known Danhong injection process, the crude drug dosage ratio of the Danhong and the safflower is 3: 1; red sage root extract and safflower extract: the compatibility is carried out according to the mass ratio of 1: 1. Dissolving appropriate amount of mixed extract of Saviae Miltiorrhizae radix and Carthami flos in physiological saline to obtain 1g/ml solution.

2.4 dosing regimen: the preventive administration is carried out for 7 days, corresponding medicines are respectively administered to different groups at 7 am and seven points in the evening, and the administration is carried out twice every day; and (8) making a wire-tying model. The Saviae Miltiorrhizae radix extract group and Saviae Miltiorrhizae radix and Carthami flos compatibility group adopt nasal drop administration mode, and 0.1ml nasal drop administration; the model group and the sham operation group also adopt a nasal drip administration mode, and 0.1ml of normal saline is administered by nasal drip; the nimodipine group and the danhong injection group adopt an intraperitoneal injection mode, the nimodipine group is subjected to intraperitoneal injection according to the dosage of 2ml/kg, and the danhong injection is subjected to intraperitoneal injection according to the dosage of 0.5 ml/kg.

2.5 establishing a rat focal cerebral ischemia model by an intravascular embolus line blocking method:

rats were injected with 10% chloral hydrate (35mg/kg) under intraperitoneal anesthesia. Fix the patient in the supine position, wipe the patient with iodophor, sterilize the patient, and cut the skin along the midline of the ventral neck. The muscle and fascia are separated along the inner edge of the sternocleidomastoid muscle. The right Common Carotid Artery (CCA), External Carotid Artery (ECA) and Internal Carotid Artery (ICA) were isolated. And hanging wires at the distal end, the proximal end and the external carotid artery of the right common carotid artery for standby. The internal carotid artery was temporarily clamped with a arteriole clamp and then the right common carotid artery and external carotid artery were ligated at a segment near the heart. A small opening was cut 4mm from the bifurcation site of the right common carotid artery, and a prepared fishing line was inserted into the internal carotid artery. The fish line was then gently tied with a thin wire wrapped around the more distal end of the right common carotid artery. The fish wire is pushed to 18mm away from the bifurcation of the blood vessel by using an ophthalmic forceps, and the distal end of the right common carotid artery is tied softly to obtain a thin wire. The wound was sutured conventionally. The sham operation group only performed anesthesia and vessel dissection operations, and did not perform vessel ligation nor insert a fish line. After 2 hours the plug wire was pulled out 5mm to achieve reperfusion. The drugs were administered immediately after reperfusion of each drug group, and the sham surgery group and model group were given the same amount of physiological saline.

2.6 index determination:

2.6.1 rat neurological deficit score: rats were modeled for 2 hours prior to reperfusion, and 3 hours after reperfusion, 5-point neurological function scoring was performed. 0 point, no obvious nerve function defect symptom; 1, when the tail is lifted, the left forelimb adducts and flexes and can not fully extend, belonging to mild focal neurological deficit; 2, rotating and circling to the left during crawling, belonging to moderate focal neurological deficit; 3, the patient inclines to the left side when standing, and belongs to severe focal neurological deficit; and 4, the patient cannot walk spontaneously or is in a coma state.

2.6.2 horizontal wood walk score: the horizontal wood is 80cm long, 2.5cm wide and 10cm away from the ground. 0 minute: the balance beam can jump; 1 minute: the balance beam can be jumped, and the walking chance on the cross beam is below 50 percent; and 2, dividing: the balance beam can jump up, and the walking chance on the balance beam is more than 50 percent; and 3, dividing: the balance beam can jump up under the help of the hind limbs without embolism, but the forward movement cannot be helped due to the tired hind limbs with paralysis; and 4, dividing: the balance beam can not walk but can sit on the balance beam; and 5, dividing: can not stay on the balance beam and can fall off.

2.6.3 determination of cerebral infarction rate: the cerebral infarction rate is measured by a red tetrazolium (TTC) staining method. Taking out brain after 24 hours of reperfusion, removing olfactory bulb, medulla oblongata and cerebellum, removing impurities in tissues such as fat, blood silk and the like under the condition of normal saline at 4 ℃, wiping off water, accurately weighing the whole brain weight, freezing in a refrigerator at-4 ℃ for about 24 hours, continuously making 5-6 coronal slices at intervals of 2mm, adding 2% TTC solution, immersing the slices, putting in an oven at 37 ℃ for decomposition for 30min, and turning over brain slices every 5-10min to uniformly contact with a staining solution. TTC is reduced by mitochondrial catalase, normal brain tissue is stained red, and infarcted tissue is white. Separating the infarcted area (pale area) from the normal area (non-pale area) with the ophthalmic forceps, accurately weighing the areas, and calculating the percentage of infarction, i.e., the weight of pale area/(pale area weight + non-pale area weight) × 100%.

2.6.4 determination of Biochemical indicators: 5ml of blood is taken from the abdominal aorta of the rat, centrifuged (3500r/min) for 0min by an adjustable high-speed centrifuge, and the supernatant is taken and stored in a refrigerator at 4 ℃ for later use. Rat plasma is taken, and test operation is carried out according to the operation instruction of a kit (SOD, MDA, glutathione peroxidase and nitric oxide synthase) provided by Nanjing institute of bioengineering.

2.6.5 pathological lesion assessment: the rat whole brain was taken and placed in a vial containing neutral formalin for use. Slicing brain tissue, soaking in 10% neutral formic acid fixing solution (volume ratio of 1:9) prepared from formaldehyde saturated solution and PBS buffer solution with pH of 7.4 for 8 hours, draining off the fixing solution, soaking in 70%, 80%, 90%, 95% and absolute ethyl alcohol in sequence for dehydration, draining off, soaking in xylene for half an hour, draining off, soaking, embedding, slicing, spreading, baking, and finally HE dyeing, sealing and filing. The evaluation was performed according to the scoring criteria of table 3 based on morphological performance.

TABLE 3 evaluation of pathological injury in brain section

Statistical treatment: data acquisition with

The soil S is represented, and statistics are analyzed by adopting one-way variance analysis and t test, and are represented by P<A statistic of 0.05 is statistically significant. And performing statistical analysis on each group of indexes by using SPSS19.0 statistical software.

2.7 test results:

2.7.1 results of rat neurological deficit and Cross-shoot scoring

Table 4 results of scoring in rats of different administration groups (n ═ 10)

Note: p <0.05 compared to model group

Table 4 shows that compared with the model group, the rats in each administration group have significant differences in neurological deficit and crossbar score, indicating that the modeling of the experiment is successful. In each administration group, the neurological deficit score and the rhamnus davurica score of the danhong injection group are both lowest; the neurological deficit score for the nimodipine group was lower than for the two extract groups, but the crossbar score was higher than for the two extract groups; the scoring results of the salvia miltiorrhiza bunge red proportioning group and the salvia miltiorrhiza bunge extract group are different, which indicates that the advantages and disadvantages of the treatment effect cannot be judged by the two scores due to the complexity of the cerebral ischemia mechanism and the complexity of the drug treatment mechanism, and further experimental study is needed.

2.7.2 determination of cerebral infarction Rate

Table 5 rat cerebral infarction rate test results (n ═ 6)

Note: p <0.05 compared to model group

Table 5 shows that compared with the model group, the cerebral infarction rates of rats in the administration groups have no significant difference, and it can be seen from visual analysis that the administration groups have certain therapeutic effects, the nimodipine group has the best therapeutic effect, and the therapeutic effect of the danhong proportioning group is superior to that of the danhong injection group and the danshen extract group.

2.7.3 measurement of Biochemical indicators

TABLE 6 Biochemical index measurement results (n ═ 6)

Note: p <0.05 compared to model group.

From the experimental results in table 6, it can be seen that in SOD and GSH, the model group and the danhong ratio group have significant differences, and the data of other groups have no significant difference. The data are visually observed, and in the SOD determination result, the SOD content of the administration group is higher than that of the model group, the SOD content of the danhong proportioning group is highest, the danhong injection group is second, and the danshen extract group is between the two groups; in the MDA measurement results, the MDA content of the administration group is lower than that of the model group, the MDA content of the red preparation group is the lowest, and the measurement results of the red injection group are between the red proportioning group and the red sage root extract group; in GSH determination results, the GSH content of the administration group is higher than that of the model group, the content of the danhong proportioning group is the highest, and the content of the danshen extract group is higher than that of the danhong injection group; in the NOS group, the NOS content in each administration group was the second model group, and the Danhong ratio group had the lowest content. According to comprehensive analysis of the four evaluation indexes, the administration group can play a role in treating ischemic brain injury by increasing the contents of SOD and GSH in a body and reducing the contents of MDA and NOS, wherein the treatment effect of the danhong proportioning group is optimal.

2.7.4 pathological injury assessment: observed by a microscope, the following can be obtained: model group, more glia hyperplasia and severe tissue edema (20 x light microscopic photograph) were observed; and severe inflammatory cell infiltration can be observed (10 x light under-lens photograph). In the Danhong injection group, mild gliosis and mild tissue edema can be observed. In the nimodipine group, mild glioblastomas and mild tissue edema were observed; a small amount of gliosis, mild inflammatory cell infiltration and mild tissue edema can be observed; a small amount of gliosis and mild inflammatory infiltration and mild tissue edema were observed (20-fold photomicrographs). The salvia miltiorrhiza extract group (20 times light microscope photograph) can observe a small amount of gliosis and mild tissue edema. A small amount of gliosis and mild inflammatory infiltration and mild tissue edema were observed (20-fold optical microscopic photograph). Danhong proportioning group in FIG. 1-a small amount of gliosis and mild tissue edema can be observed. Table 7 was obtained from the pathological lesion evaluation of rat brain sections. Compared with a model group, the four administration groups have certain alleviation effect on pathological changes, wherein the inhibition effect of the group with the ratio of red to red is the best.

TABLE 7 pathological injury scoring table for brain slice of rat with brain injury

Table 7 shows that compared with the model group, each administration group has a certain treatment effect and can effectively relieve the conditions of edema, glial cell proliferation and inflammatory cell infiltration of brain tissue cells of rats. Wherein, the proportion of the red sage root extract and the safflower extract has the best treatment effect, which is superior to the red sage root injection and the red sage root extract.

3. Preference of dosage forms of the pharmaceutical composition of the invention

3.1 in vitro permeability test of animal nasal mucosa in different dosage forms

3.1.1 instruments and reagents

3.1.1.1 instruments

U3000 high performance liquid chromatograph (Daian, USA), chromatographic column BDS HYPERSILC-C18 column (4.6mm x 250mm), AR1140 electronic balance (Mettler-Torledo instruments Shanghai Co., Ltd.), horizontal diffusion cell (Shanghai Yu research scientific instruments Co., Ltd.), pH meter (Orilong pHS-3C), constant temperature magnetic stirrer (Changzhou national China electric apparatus Co., Ltd.), and pig nose mucosa (obtained from Suiyang city five-tomb Hill Garden slaughterhouse).

3.1.1.2 reagent

Salvianolic acid B water extract, safflower yellow water extract (self-made), soybean lecithin (pc is more than or equal to 50 percent, Shanghai Taiwei pharmaceutical industry), salvianolic acid B standard (Chinese food and drug testing institute, lot number: 111562-201213) and hydroxysafflor yellow A standard (Chinese food and drug testing institute, lot number: 11637-201208); soybean oil (soybean oil development ltd. in Tian, rain mountain, Longyou, prefecture, Zhejiang), methanol acetonitrile as chromatographic pure, water as purified water, and injection glycerol (Shanghai Daliang chemical ltd.).

3.1.2 preparation of the test formulations

3.1.2.1 aqueous solutions

Taking 5g of the phospholipid complex of the red sage root and the safflower extract, adding 50mL of phosphate buffer solution with the pH value of 5.8, grinding, centrifuging for 10 minutes at 4000 rpm, and taking the upper layer solution as an aqueous solution.

3.1.2.2 oil solution agent

Taking 50mL of soybean oil, adding 5g of phospholipid compound of the extracts of the red sage root and the safflower, grinding to uniformly disperse the phospholipid compound, centrifuging for 10 minutes at 4000 rpm, and taking supernatant as an oil solution agent.

3.1.2.3 microemulsion preparation

Taking 4g of phospholipid complex, 8020 g of Tween, 20g of glycerol and 1.6g of medium-chain oil, adding water to 100mL, uniformly mixing the Danhong phospholipid complex, the Tween-80, the glycerol and the soybean oil, adding water to l00mL, and obtaining the microemulsion which is a semitransparent liquid. The microemulsion preparation is prepared by phase inversion method, which comprises mixing emulsifier/co-emulsifier with oil phase, slowly adding water phase into oil phase, adding water while stirring, and stopping adding enough amount to obtain the desired microemulsion preparation.

3.1.2.4 submicron emulsion preparation

Taking 4g of phospholipid compound, 10g of soybean oil, 8020 g of tween-8020 g, 20g of glycerol, 1881.5 g of poloxamer and 0.6g of emulsified phospholipid. Heating the oil phase and the water phase to 55 deg.C, mixing and stirring to prepare primary emulsion, and preparing the submicron emulsion with high-pressure homogenizer.

3.1.2.5 in vitro permeation test procedure

Removing pig nose (2h for sacrifice), cutting off nasal cavity, peeling off nasal cavity membrane with tweezers, and taking out pig noseThe saline water is used for cleaning the residual bloodstain, and the bloodstain is spread and used immediately. A horizontal permeation diffusion pool is adopted, magnetic rotors are respectively placed in the pools on the two sides, and the mucous membrane of the ex-vivo pig nose is fixed between the two pools. 6mL of the solution was added to the dosing reservoir and 6mL of physiological saline was added to the receiving reservoir. The device is placed in an environment with the temperature of 37 ℃ (water bath is 37 +/-1 ℃), and a stirrer is started to stir at a constant speed. At 5 th, 10 th, 15 th, 30 th, 45 th, 60 th, 90 th, 120 th, 150 th, 180 th, 240 th and 300 th min after the start of the experiment, 1mL of physiological saline was aspirated from the receiving well by a pipette, and the sample solution was centrifuged (150000 r. min.) while 1mL of physiological saline was replenished^-1) l0min, taking supernatant to measure the concentration of the drug, and calculating the steady-state flow rate Vss and the apparent permeability coefficient Papp of the drug.

3.1.2.6 sample measurement and results

Taking 1mL of the original solution of each dosage form, adding an appropriate amount of methanol, performing ultrasonic treatment for 10min, fixing the volume to 10mL by using methanol, and determining the content of salvianolic acid B and hydroxysafflor yellow A according to the chromatographic conditions. The measured content was the initial content C of each formulation₀And salvianolic acid B (aqueous solution of 0.56mg/mL, oil solution of 0.67mg/mL, microemulsion of 1.04mg/mL, and submicroemulsion of 1.17mg/mL) hydroxysafflor yellow A (aqueous solution of 0.162mg/mL, oil solution of 0.182mg/mL, microemulsion of 0.297mg/mL, and submicroemulsion of 0.324mg/mL), collecting the permeate at each time point, measuring the peak area according to the method, and calculating the content of salvianolic acid B and hydroxysafflor yellow A. The cumulative amount of each formulation was passed.

Fitting the permeation time t by the accumulated permeation amount R of the nasal mucosa in vitro, and calculating the apparent permeability coefficient (Papp, cm) of the drug by the following formula^-2·s^-1) And steady state flow rate (Vss,. mu.g.cm)^-2·s^-1)：

C₀For the initial concentration of each dosage form, S is the permeation area of the permeation cell, and dR/dt is determined from the slope of the cumulative permeation-time fit curve. Calculating two parameters of apparent permeability coefficient Papp and steady state flow Vss according to formula, and drawing salvianolic acid B in each preparation with reference to figure2 and hydroxysafflor yellow A cumulative permeation are shown in FIG. 3.

The data and curve analysis can show that the cumulative permeation amount of the salvianolic acid B and the hydroxysafflor yellow A of each dosage form within 0-120 min has a good linear relation with time, the data within 0-120 min are respectively fitted by a zero-order kinetic equation, an Hguichi equation and a first-order kinetic equation, and the first-order kinetic equation has the best fitting degree according to the result analysis of correlation coefficients. The analysis result indicates that the medicine in the preparation diffuses through the nasal mucosa of the pig and belongs to passive diffusion taking the concentration difference at two sides of the membrane as the power. The visual analysis shows that the penetration of the submicron emulsion medicament is far greater than that of other dosage forms. Papp and Vss were calculated according to the above formula, and the transmembrane effect of each dosage form was further analyzed and predicted.

TABLE 8 determination results of salvianolic acid B permeable membrane in different dosage forms

TABLE 9 determination of safflor yellow A permeant film in different formulations

From the experimental data, the permeability coefficient of the four preparations in the in vitro porcine nasal mucosa permeability experiment is calculated, the apparent permeability coefficient and the steady-state flow of the submicron emulsion preparation are higher than those of other three groups of formulations, and the result shows that the submicron emulsion preparation is an ideal formulation for the administration of the danhong phospholipid complex through the nasal cavity.

3.2 evaluation of cilia irritation on palate of Bufo siccus in different dosage forms

Because the four different formulations for nasal administration have different auxiliary materials and may have certain difference on the stimulation of nasal mucosa, the stimulation of the four formulations on the cilia palatine of the toad is observed by taking the toad as a research object, and the evaluation index is the time for the cilia palatine of the toad to continuously move after administration. Sodium deoxycholate at 1% was used as a positive control, and physiological saline was used as a negative control.

Fixing the toad in a supine position, dripping equal amount of the four solutions at the cilia of the palate to enable the liquid medicine to completely immerse the palate, cleaning the mucous membrane between two eyes of the toad by using normal saline, cleaning the mucous membrane by using the normal saline, flatly paving the mucous membrane on a glass slide, adding 200 mu l of the normal saline on the surface of the glass slide, covering the glass slide, observing the movement condition of the cilia of the palate under a 10 x 40 times optical microscope, then placing the glass slide in a chromatographic cylinder with distilled water, and maintaining the temperature at 20-25 ℃. The time intervals between removal, observation under a microscope, and cessation of palatal ciliary movement were recorded and the results are shown in Table 10.

TABLE 10 results of different formulations on the irritativeness of cilia of the palate of toads

Note: p in comparison with the 1% sodium deoxycholate group^c<0.01，p^b<0.05，p^a>0.05

The results in Table 10 show that the submicroemulsion formulation is minimally irritating to the cilia of the toad palate.

3.3 prescription and preparation Process research of the pharmaceutical composition of the invention

3.3.1 study of Dispersion method of Complex and phase inversion mechanism

3.3.1.1 dissolution Dispersion method Studies

The common drug dissolving and dispersing methods comprise stirring, grinding, homogenizing and the like, the dissolving methods are optimized, and the evaluation indexes are the macro and micro states after dissolving and dispersing and the uniformity of the contents of the salvianolic acid B and the hydroxysafflor yellow A in samples obtained by different dissolving and dispersing methods.

TABLE 11 results of different dissolution dispersion methods

TABLE 12 results of hydroxysafflor yellow A content

TABLE 13 measurement results of salvianolic acid B content

3.3.1.2 study of the phase inversion mechanism

3.3.1.2.1 analysis of phase inversion process and mechanism

The preparation method of the salvia miltiorrhiza and safflower extract phospholipid compound adopts methanol as a compound solvent, and adopts a reduced pressure drying method to recover the methanol, and because the medium-chain oil can be dissolved in the methanol, experimental researches show that after the medicine is reacted and compounded with the phospholipid, the methanol is recovered in a rotary evaporator to a certain amount, the methanol recovery is stopped, the oil phase is added according to the prescription amount, the residual methanol is continuously recovered, and the compound can be uniformly dispersed into the oil phase by ultrafine particles along with the reduction of the methanol amount, so that a better dissolving effect can be achieved.

3.3.1.2.2 study of the timing of phase inversion

We examined the phase inversion starting when the residual amount after recovering methanol was 100%, 80%, 60%, 40%, 30%, 20%, and the results showed that the phase inversion started when recovering methanol to the residual amount of 100% to 30%, and the complex was uniformly dispersed in the oil phase. And when the methanol is recovered until the residual amount is 20%, phase inversion occurs, obvious precipitation appears in the methanol solution, and the phospholipid complex of the salvia miltiorrhiza and safflower extracts cannot be well dispersed in the oil phase. Therefore, the optimal time for phase inversion is finally determined when the residual quantity of the recovered methanol is up to 30%, the heating time of the oil phase can be reduced, and the stability of the preparation is ensured.

3.3.2 major factors affecting the formation of submicroemulsion formulations

Emulsifier and its amount: the emulsifier is selected by considering not only the emulsifying capacity and the physical and chemical properties of the emulsifier, but also the stability of an oil-water interface film after the emulsifier is prepared. The ionic emulsifier is usually used in combination with the nonionic emulsifier, the ionic emulsifier can enhance the potential and enhance the stability of the emulsion, and the nonionic emulsifier has strong emulsifying capacity and can reduce the particle size of emulsion droplets. Emulsified lecithin and poloxamer are respectively selected as an emulsifier and an auxiliary emulsifier in the research. Drug loading rate: the previous research on the drug loading rate shows that the drug loading rate of less than 6 percent can effectively realize the uniform dispersion of the drug in the oil phase, but the stability of the submicron emulsion preparation is influenced by overhigh drug loading rate, the curative effect of the drug is difficult to realize by overlow drug loading rate, and the drug loading rates of 4 percent, 5 percent and 6 percent are finally designed and investigated through the preliminary experiment result. pH value of the emulsion: the pH value not only affects the stability of the drug itself, but also affects the Zeta potential of the submicron emulsion formulation, thereby affecting the stability of the submicron emulsion formulation. Also, a suitable pH range for nasal tolerance drugs is (4.5-6.5).

3.3.2.1 oil phase dosage screening

The oil phase commonly used for preparing the submicron emulsion preparation comprises soybean oil (LCT), medium chain triglyceride oil (MCT), cottonseed oil, safflower oil, sesame oil, corn oil, fish oil, fur seal oil and the like, the medium chain triglyceride oil (MCT) has certain toxicity and irritation as the oil phase, and the ratio of MCT to LCT is set to be 2:1 by combining the experimental result and the experimental cost, so that the irritation is reduced while the drug-loading rate is ensured. The general concentration of the submicron emulsion oil phase is about 10-30%, the proportion of the oil phase is too small, the drug loading rate is small, but if the proportion of the oil phase is too large, the stability of the preparation is reduced.

Table 14 oil phase amount screening results (n ═ 3)

From the results in Table 14, it can be seen that the oil phase ratio is too large to meet the index requirements of the submicron emulsion formulation, and too small to affect the drug loading of the formulation, so we select 25% of the oil phase ratio as the oil phase dosage of the formulation.

3.3.2.2 preliminary examination of drug loading

The drug loading is a key factor for whether the medicine exerts the curative effect or not, the drug loading in the oil phase is inspected according to the phase inversion method and the result in the experiment, the drug loading is preliminarily inspected by taking the color property of the oil phase after phase inversion as an index, and the dosage of the soybean oil is designed to be 25mL (100mL of preparation).

TABLE 15 examination of different drug loadings

Table 15 shows that the drug loading can reach 6% by the phase inversion method, and the drug loading can be preliminarily determined to be 4% -6% by the process through repeated tests.

3.3.2.3 examination of oleic acid content

After the medicine is added into the formula, the two-phase interface of the submicroemulsion is changed, and a stabilizing agent is required to be added to increase intermolecular force so as to improve the stability of the membrane. The common stabilizer of the submicron emulsion comprises oleic acid and sodium oleate, wherein the capacity of reducing the oil-water interfacial tension of the sodium oleate is stronger than that of the oleic acid, but the sodium oleate has strong water solubility and is difficult to fix on an oil-water interfacial film, and the sodium oleate is suitable for neutral or alkalescent preparations and is not suitable for nasal administration, so the oleic acid is selected as the stabilizer. The oleic acid remaining on the emulsifier interfacial film negatively charges the emulsion droplets to generate electrostatic repulsion, increases the electric potential, prevents the aggregation of the emulsion droplets, and thus enhances the stability of the submicron emulsion. According to the literature dosage and the combination of pre-experiments, the influence of the oleic acid dosage of 0mL, 0.1mL and 0.2mL on the stability of the preparation is examined.

Table 16 examination of oleic acid content

3.3.2.4 examination of isotonic regulators

In order for the submicron emulsion formulation to be better absorbed by the nasal mucosa and reduce irritation, the formulation must be made isotonic or slightly more (280-320 mosmol/kg). Hypoosmotic preparations are prone to nasal mucosal edema, and both hypoosmotic and hypertonic solutions can reduce the ciliary movement frequency of the nasal mucosa. The preparation simultaneously considers the influence of 1%, 1.5% and 2% of glycerol on osmotic pressure, and the results show that the osmotic pressure of three concentrations are 435 +/-8.97, 357 +/-12.03 and 323 +/-5.72 respectively, which indicates that the dosage of 2% of glycerol can cause the preparation to be hypertonic, probably because certain osmotic pressure exists in the medicament and the auxiliary material, and the osmotic pressure of the preparation meets the requirement when the dosage of the glycerol is 1% through a plurality of tests.

3.3.2.5 examination of antioxidants

In order to prevent the oxidation of raw and auxiliary materials in the preparation, 0.1 percent of VE is directly added into the oil phase, and in order to prevent the catalysis of metal ions on the raw and auxiliary materials, 0.006 percent of metal ion complexing agent EDTA-Na2 is added into the water phase.

Through the optimized preliminary experiment of the prescription, and the orthogonal experiment, the drug-loading rate, the dosage of the emulsified lecithin, the auxiliary emulsifier poloxamer-188 and the pH value are comprehensively screened, and the evaluation indexes are normalized values of indexes such as submicron emulsion particle size and PDI.

Orthogonal analysis results show that the drug loading has extremely obvious influence on the size and the forming of the submicron emulsion, the pH value also has obvious influence on the forming of the submicron emulsion, and the other two influences are not obvious, and by combining the results, the cost in the preparation production is taken into consideration, and the characteristic that the preparation is relatively stable in a slightly acidic environment is taken into consideration, so that the finally obtained optimal level combination is A1B1C1D1, namely the drug loading is 4%, the dosage of emulsified phospholipid is 1.5%, the dosage of poloxamer-188 is 1%, and the pH is adjusted to 5.

3.3.2.6 prescription factor verification test

And (4) carrying out verification tests according to the optimized prescription factors, wherein the particle sizes and PDI indexes of the three groups of experiments meet the requirements of the submicron emulsion preparation. The optimized parameters are stable and feasible.

TABLE 17 three verification test results

3.4 preparation of colostrum

3.4.1 examination of colostrum temperature

Depending on the temperature of complexation of the phospholipid complex, 55 ℃ was chosen as the temperature for preparing the colostrum, taking into account the stability of the phospholipids. The prepared colostrum needs to be cooled rapidly to prevent coagulation of the emulsion droplets.

3.4.2 examination of shear time

By adopting the phase inversion method, after the methanol is recovered, adding emulsified lecithin S-75, oleic acid and vitamin E into an oil phase, adding the rest poloxamer-188, glycerol, EDTA-Na2 and the like into water for dissolving, adding a water phase into the oil phase, transferring the oil phase into a beaker, respectively shearing 8, 10 and 12min by using a high-speed shearing instrument with the shearing strength of 20000rpm, respectively, taking a proper amount of colostrum at a corresponding time point, rapidly cooling, observing the emulsion drop distribution condition of oil drops under an electron microscope, and taking a colostrum microscopic picture, wherein the picture is shown in attached figures 4 to 6.

3.4.3 investigation of shear rotation speed

Shearing for a certain time according to the method in the basic process flow, setting the rotation speed of high speed shearing instrument at 15000rpm, 20000rpm and 25000rpm, preparing one portion of primary emulsion, sampling and microscopic examination, and the microscopic examination picture is shown in figure 7-9.

3.4.4 investigation of the preparation Process of the submicroemulsion

According to the investigation result of the prescription factors and the result of the screening research of the colostrum preparation process, the brain-targeted submicron emulsion for the danhong nose is further prepared by adopting a high-pressure homogenization method. In the experiment, the star point design-effect surface is adopted to optimize various parameters in the homogenization process, the grain diameter before homogenization is shown in the attached drawing 10, and the change of the grain diameter after homogenization is shown in the attached drawing 11.

The factors influencing the effect of the homogenized submicron emulsion preparation include homogenization pressure, homogenization times and homogenization temperature, wherein the homogenization times are homogenization time, and the time for completing one time of the liquid medicine with the same volume under the specified pressure condition is determined, so the three factors are continuous variables and can be optimized by adopting a star point design effect surface. The evaluation indexes were the particle size and PDI of the submicron emulsion, and the OD value of the evaluation index was calculated for each sample to optimize the evaluation index.

3.4.5 Star Point design experiment

Software is used to design the star point design factor level code table, see table 18.

Table 18 star point design factor level coding table

3.4.5.1 evaluation index of preparation process

According to the optimization result of the prescription factors, the glycerol, the poloxamer-188 and the EDTA-Na2 are dispersed in a proper amount of water for injection according to the prescription amount, and the mixture is placed in a magnetic stirring device to be heated to 55 ℃ and stirred until the components are completely dissolved; respectively weighing 0.67g of salvianolic acid B extract and 0.67g of safflower water extract, then weighing 2.61g of soybean lecithin, uniformly mixing the three, placing the mixture in a magnetic stirrer, adding 400mL of methanol, carrying out composite reaction in a water bath at 55 ℃ for 2 hours, recovering the methanol until the residual methanol is about 120mL, adding an oil phase, continuously recovering until no methanol smell exists, adding a water phase into the oil phase under the stirring of a high-speed tissue triturator (20000rpm), and stirring for 10 minutes to prepare primary emulsion each time; diluting the primary emulsion with distilled water to a constant volume to a prescribed amount, transferring to a high-pressure homogenizer, preparing the submicroemulsion under the condition in a star point design factor level table, and adjusting the pH value to 5 by using 0.1mol/L sodium hydroxide to obtain the final product.

The research researches the technological conditions of the preparation of the submicron emulsion, the evaluation indexes are the particle size and the PDI size of the submicron emulsion, and the smaller the two indexes is, the better the result is, so the normalization value is calculated by adopting the above formulas 1 and 3, and the final OD value is calculated.

In the research, Design-Exper8.0 software is used for designing an experimental scheme for process screening, the experimental results are respectively subjected to normalization (0-1) treatment by a hassan method according to the measurement method of each index, the normalization value of each index result is calculated, the geometric mean of the three index results is calculated, and further, the total evaluation normalization value OD is calculated, wherein 15-20 tests are 6 center point tests and are used for investigating the error of the model, and the OD is (d 1d 2 d3 … dn) 1/n.

Analyzing the total evaluation normalization value by adopting Design-Exper8.0 software according to the variance result of the test, fitting the test on three factors by taking the OD value as a dependent variable, wherein the correlation coefficient R2 of quadratic fitting is 0.9762, and P is<The correlation coefficient R2 of the 0.0001 cubic fit is0.8882，P<0.0001 according to the table 19, the first term, the second term and the third term of each factor have significant differences, so that the binomial fitting model is a successful model, and the experimental value and the predicted value are basically consistent. The fitting equation is OD +0.56+0.28X₁+0.10X₂+0.037X₃-0.018X1X2-0.040X₁X_3-3.314E-003X₂X₃-0.012X₁ ²-0.056X₂ ²-0.023X₃ ²。

TABLE 19 experimental design of the surface of the star point effect and the results of the index measurement

Preferred regions preferred from the star point design factor contour map are: x₁(980～1000)，X₂(8-10); preferably selecting X from A, C two-factor contour map₁(995～1000)，X₂(30-50); preferably selecting X2 (9.22-10) from B, C two-factor contour map₃(39-50), wherein the most addition range obtained by the optimization is X1 (995-1000); x₂(9.22～10)；X₃(39～50)。

In consideration of the reasons of industrial production, convenience of operation and the like, the final prescription and preparation process are as follows: the drug loading rate is 4%, the dosage of the emulsified phospholipid is 1.5%, the dosage of the poloxamer-188 is 1%, and the pH value is 5. The homogenization pressure was 1000par, the homogenization time was 9 times, the homogenization temperature was controlled at 40 ℃ and the homogenization pressure was 1000 par.

3.4.5.2 verification experiment

Three batches of samples were prepared to verify the reliability of the preferred manufacturing process, following the above recipe and manufacturing process. The evaluation indexes are particle size, particle size distribution PDI and Zeta potential of the submicron emulsion, and the results are shown in Table 20, particle size measurement chart is shown in FIG. 12, and Zeta potential measurement chart is shown in FIG. 13.

TABLE 20 three batches of verification test results

4 preliminary pharmacodynamic study of the pharmaceutical composition submicroemulsion formulation of the invention

The protection effect of the submicron emulsion preparation on the brain tissue function of the mice with acute ischemia and anoxia and the brain tissue of the mice with ischemia is observed.

4.1 Effect on acute ischemia and hypoxia brain tissue function of mice

The research duplicates a mouse acute cerebral ischemia and hypoxia model, and the influence of the three preparations on the functions of mouse acute cerebral ischemia and hypoxia is researched by measuring biochemical indexes after the red sage root, the safflower extract aqueous solution, the phospholipid compound aqueous solution and the phospholipid compound submicron emulsion are respectively given.

4.1.1 animals, reagents and instruments

4.1.1.1 animals: kunming mouse, clean grade, male, weight 20 + -2 g, provided by Experimental animals center of the fourth military medical university of people liberation force of China, animal use license number: SCXK (military) 2012 and 0007.

4.1.1.2 reagent: salvianolic acid B extract (self-made) safflower yellow extract (self-made); danhong phospholipid complex (self-made) Danhong phospholipid complex sub-microemulsion (self-made) nimodipine injection (Chenxin pharmaceutical industry, Ltd., production lot: 1404262102); danhong injection: (Shandongdan red pharmaceuticals Co., Ltd., production lot: 14111011); soda lime (Shanghai WUTIV chemical reagent factory, production lot: 20140315); the kit comprises: coomassie brilliant blue protein, lactic acid (LD), Malondialdehyde (MDA), superoxide dismutase (SOD), ultra-trace Atpase (ATP): (batch nos.: 20150401, 20150330, 20150409, 20150408), the above assay kit: are all products of the Nanjing institute of bioengineering.

4.1.1.3 Instrument: UV-1700 model UV-visible spectrophotometer, BS-200S-WEI model electronic balance (Sidolis, Beijing); FLUKO F6/10-10G type ultra-fine homogenizer (Frauk Shanghai fluid mechanical manufacturing Co., Ltd.); finnpipette pipettor (Shanghai Spdansai Biotechnology Co., Ltd.).

4.1.2 Experimental methods

4.1.2.1 acute cerebral ischemic hypoxia experiment

72 mice, half of male and female, were divided into 6 groups, each group consisting of 12 mice, and the groups were blank control group, nimodipine positive control group, danhong injection positive control group, danhong phospholipid complex group (4g phospholipid complex homogenate method dissolved in 100mL aqueous solution), proportioning group of red sage root and safflower extract (ratio of red sage root extract to safflower extract 1:1, concentration 0.015g/mL), and danhong phospholipid complex submicron emulsion group. Nimodipine group is intraperitoneally injected with a dose of 4mL/kg, the nimodipine group is diluted with an equal amount of normal saline before injection, a red lead injection is intraperitoneally injected with a dose of 0.8mL/kg, the normal saline before injection is diluted by 10 times, a red lead-red phosphorus lipid compound sub-microemulsion group is nasally administered with 50ul, a red lead-red phosphorus lipid compound group (the nasally administered with 50ul, the red lead and the safflower extract are proportioned to be nasally administered with 50ul, the administration is performed once a day and is continuously performed for 10 days, after the administration is performed for 1 hour on the tenth day, the ear is rapidly broken at the connecting line of ears by scissors under the condition of ether shallow anesthesia, the times of mouth breathing after the head breaking of the mouse are recorded, and the brain of the mouse is taken and placed.

4.1.2.2 mouse survival time determination

The mice 72 are divided into 6 groups with half male and female, each group comprises 12 mice, and the mice are respectively a blank control group, a nimodipine positive control group, a red injection positive control group, a red phosphorus lipid complex group, a red water extract group and a red phosphorus lipid complex sub-microemulsion group. The dose and method of administration were the same as above, and each group of mice was placed in a closed jar containing soda lime at the same time and the survival time of the mice was recorded.

4.1.2.3 determination of brain tissue SOD, MDA, LD, ATP

Taking out the brain of the mouse after the head of the mouse is broken, accurately weighing the brain, and then adding physiological saline 1:9 preparing 10% brain homogenate in ice-water bath, and freezing and storing in a refrigerator at-80 ℃ for later use. Biochemical indicators of brain tissue were determined according to the instructions.

4.1.3 results

4.1.3.1 acute cerebral ischemic hypoxia test

The result of analysis by using SPSS19.0 statistical software shows that the submicroemulsion group and the phospholipid complex group can obviously prolong the breathing time of the acute cerebral ischemia and anoxia mouse and increase the mouth opening times, and compared with a blank control group, the P is less than 0.05. Compared with the blank control group, the danhong water extract proportion group has no statistical significance P >0.05, but the numerical value is obviously improved, and the specific result is shown in a table 21.

TABLE 21 respiratory time and number of times of mouth opening of acute cerebral ischemic hypoxic mice by each group of drugs: (

n＝6)

Note that P is less than 0.05 in a, and less than 0.01 in b

4.1.3.2 Biochemical index results of brain tissue of mice with acute cerebral ischemia and anoxia

TABLE 22 Biochemical index results of brain tissue of acute cerebral ischemia and hypoxia mice (

n＝6)

Note that P is less than 0.05 in a, and less than 0.01 in b

The results in table 22 show that the submicron emulsion group has significant difference (P <0.05) in the four selected biochemical indexes compared with the blank control group, and the danhong proportioning group has no significant difference (P >0.05) in the blank control group, but the SOD content is increased, the MDA content is reduced, and the atpase activity is increased. Compared with a blank control group, the phospholipid complex group has the advantages that the LD level and the ATP enzyme activity are obviously reduced and increased (P is less than 0.05), the SOD content and the MDA content have no obvious difference (P is more than 0.05) compared with the blank control group, but the SOD content is increased, and the MDA content is reduced.

4.1.3.3 results of constant pressure airtight anoxia-resistant test for each group of mice

TABLE 23 Effect of groups of drugs on survival time of normbaric closed hypoxia-resistant mice: (

n＝6)

Note that P is less than 0.05 in a, and less than 0.01 in b

The results in table 23 show that the sub-microemulsion group and the phospholipid complex group have significant difference (P <0.05) compared with the blank control group, the survival time of the mice is obviously improved, and the danhong proportioning group has no significant difference (P >0.05) compared with the blank control group, but the survival time is prolonged.

4.1.3.4 Biochemical index results of brain tissue of mice sealed at normal pressure

TABLE 24 Biochemical index results of brain tissue of each group of mice sealed at normal pressure: (

n＝6)

Note that P is less than 0.05 in a, and less than 0.01 in b

The results in table 24 show that the sub-microemulsion group and the phospholipid complex group have significant difference (P <0.05) compared with the blank control group, and the danhong proportioning group has no significant difference (P >0.05) compared with the blank control group in each biochemical index, but the SOD content is increased, the MDA content is reduced, the LD level is reduced, and the ATP enzyme activity is increased. The results show that the anti-cerebral anoxia ability of the phospholipid complex is stronger than that of the danhong proportioning group, and the anti-cerebral anoxia ability of the sub-microemulsion of the danhong phospholipid complex is stronger.

4.2 protective action against ischemic injury in rat

4.2.1 animals, reagents and instruments

4.2.1.1 animals: 128 clean-grade healthy SD rats with the age of 2 months, the weight of 250 +/-25 g and half of male and female are selected and purchased from the experimental animal center of the department of medicine of the Siam traffic university, and the qualification number is as follows: scxk (shan) 2012 and 003.

4.2.1.1 reagent: salvianolic acid B extract (self-made) safflower yellow extract (self-made) nimodipine injection: chenxin pharmaceutical industry gmbh, lot No. 1504262102, dan hong injection: shandongdan red pharmaceutical Co., Ltd, production lot number: 15111011, chloral hydrate: shanghai Shanpu chemical Co., Ltd., lot number 20140905, TTC: sigma, USA. The kit comprises: nitric Oxide (NO), Malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), coomassie brilliant blue protein, (lot nos.: 20151211, 20151212, 2151213, 20151210, 20151211), the above assay kit: are all products of the Nanjing institute of bioengineering.

4.2.1.3 instruments: UV-1700 type ultraviolet-visible spectrophotometer; BS-200S-WEI type electronic balance (Beijing Saedodus Corp.); FLUKO F6/10-10G model superfine homogenizer (Frauke fluid machinery manufacturing Co., Ltd.), multifunctional microplate reader (Megu molecular instruments Co., Ltd.), Finnpipette pipettor (Shanghai Spanish Biotechnology Co., Ltd.).

4.2.2 methods

4.2.2.1 animal grouping and administration

After the rats are adapted to be raised for 1 week, the rats are randomly divided into 8 groups, namely a model group, a nimodipine group, a danhong injection group, a submicron emulsion high dose group, a submicron emulsion low dose group, a sham operation group and a blank emulsion group, and 16 rats are selected in each group. Administration: each group was administered once daily for 7 days. 0.1mL of physiological saline is dripped into a model group and a pseudo-operation group, 2mL/kg of intraperitoneal injection is dripped into an abdominal cavity of a nimodipine group, 0.5mL/kg of intraperitoneal injection is dripped into a danhong injection solution, 0.8mL/kg of dosage is dripped into a nasal cavity of a submicron emulsion high-dosage group, 0.4mL/kg of dosage is dripped into a nasal cavity of a submicron emulsion low-dosage group, and 0.2mL/kg of dosage is dripped into a nasal cavity of a submicron emulsion low-dosage group. 4.2.2.2 intravascular Thrombus occlusion method for establishing local cerebral ischemia model of rat

Rats were anesthetized with a 10% chloral hydrate (3mL/kg) by intraperitoneal injection. After supine fixation, the patient was sterilized with iodophor and the ventral cervical midline skin was incised. The fascia and muscle were isolated, anterior tracheal muscle was isolated, and the anterior tracheal muscle was isolated down the right sternocleidomastoid tendon, and after visualization of the carotid sheath, a hook was pulled up, and the right Common Carotid Artery (CCA), External Carotid Artery (ECA) and Internal Carotid Artery (ICA) were isolated. And hanging wires at the CCA far-end, the CCA near-end and the ECA for standby. ICA was clamped with artery clamps and CCA, ECA were ligated proximal to the heart. A small incision was cut 4mm from the CCA bifurcation and the tether was inserted into the ICA. The tether was then lightly tied with a thin wire wound around the distal end of the CCA. The plug wire is pushed gently with the ophthalmic forceps, starting from the vessel bifurcation and gently firmly holding the thin CCA distal filament at an insertion depth of 18 mm. The wound was sutured conventionally. The sham group only performed anesthesia and vessel dissection without ligation of vessels and introduction of ligatures. Reperfusion was performed after 2 hours. The corresponding drugs were administered immediately after reperfusion of each drug group, and equal volumes of physiological saline were administered to the sham surgery group and the model group.

4.2.2.3 rat neurological deficit score

Rats were reperfusion for 3 hours after 2 hours of cerebral ischemia and 5-point neurological scoring was performed.

0 minute: no obvious symptom of neurological deficit; 1 minute: when the tail is lifted, the left forelimb is adducted and flexed and can not be fully extended, belonging to mild focal neurological deficit; and 2, dividing: when crawling, the robot rotates to the left and circles, and belongs to moderate focal neurological deficit; and 3, dividing: inclining to the left side when standing, belonging to severe focal neurological impairment; and 4, dividing: it is unable to walk spontaneously or out of a coma.

4.2.2.4 determination of cerebral infarction rate

The determination of cerebral infarction rate adopts red tetrazolium (TTC) staining method, taking out brain after 24 hours of reperfusion, removing olfactory bulb, medulla oblongata and cerebellum, removing impurities such as fat, blood silk and the like in tissues under the condition of normal saline at 4 ℃, wiping off water, accurately weighing the whole brain weight, freezing for about 24 hours in a refrigerator at-4 ℃, continuously making 5-6 coronal slices at intervals of 2mm, adding 2% TTC solution, immersing the slices, placing in an oven at 37 ℃ for incubation for 30min, turning over the brain slices at intervals of 5-10min, and enabling the staining to be uniform. Normal brain tissue is stained red and infarcted brain tissue is stained white. The pale area (infarct zone) and the non-pale area (normal zone) were separated with ophthalmic forceps, accurately weighed, and the infarct rate (infarct rate ═ weight of pale area/(weight of pale area + weight of non-pale area) X100%) was calculated.

4.2.2.5 determination of brain index

The rat head is broken, the brain is accurately weighed, and the brain index is calculated, wherein the brain index is the whole brain weight (g)/the body weight (g).

4.2.2.6 preparation of rat plasma

5mL of blood is taken from the abdominal aorta of the rat, the blood is centrifuged for l0min by an adjustable high-speed centrifuge (3500r/min), and the supernatant is taken and stored in a refrigerator at 4 ℃ for later use.

4.2.2.7 preparation of brain tissue slices

The rat whole brain was taken and placed in a vial containing neutral formalin for use. Brain tissue sections are soaked for 8 hours in 10% neutral formaldehyde fixing solution (volume ratio is 1:9) prepared by formaldehyde saturated solution and PBS buffer solution with pH value of 7.4, the fixing solution is drained, 70%, 80%, 90%, 95% and absolute ethyl alcohol are sequentially used for soaking and dewatering, the draining is carried out, then xylene is used for soaking for half an hour, the draining, the soaking and embedding are carried out, and then the sections, the spreading and the baking are carried out, and the sections are dyed, sealed and filed by an HE dyeing method.

4.2.2.8 Scoring Standard for histopathological morphology of brain

TABLE 25 brain histomorphometry scoring criteria

4.2.2.9 Effect of the formulations on rat brain histopathomorphology

FIGS. 14-21 are brain histopathological sections of rat focal cerebral ischemic injury, and in the model group shown in FIG. 14, more gliosis and severe tissue edema can be observed, and in other drug groups, a small amount of gliosis and mild tissue edema can be observed.

4.2.2.10 determination of Biochemical indicators

The rat plasma and brain homogenate were collected and operated according to the kit (SOD, MDA, glutathione peroxidase, nitric oxide synthase) provided by Nanjing institute of bioengineering.

4.2.3 results of the experiment

4.2.3.1 Effect on nerve function

TABLE 26 neurobehavioral scores for the groups of rats: (

n＝6)

Note: in comparison to the set of models,^aP<0.05；^bp<0.01

the research shows that: the nerve function scores of each drug group and the positive control group are smaller than those of the model group, and have significant difference (P is less than 0.01), which indicates that the danhong phospholipid composite submicron emulsion has protective effect on the cerebral nerve function after ischemia-reperfusion injury. The blank emulsion group had a neurobehavioral score greater than that of the model group (P > 0.05).

4.2.3.2 cerebral infarction Rate

TABLE 27 cerebral infarction Rate of rats

n＝6)

Note that compared to the model set,^ap<0.05；^bp<0.01

the data show that the numerical values of the danhong injection group and the nimodipine group are significantly different (P <0.05), the numerical values of the drug groups are not significantly different, but are significantly reduced, which indicates that the nasal administration of the danhong phospholipid compound submicron emulsion preparation has certain improvement effect on the rat focal cerebral ischemic injury, but the effect is not significant, and the cerebral infarction volume ratio of the blank emulsion group is close to that of the model group (P > 0.05).

4.2.3.3 Effect on rat brain index

TABLE 28 Effect of brain index in groups of rats: (

n＝6)

Note that compared to the model set,^aP<0.05；^bP<0.01

the experimental results show that the brain index of the sham operation group is smaller than that of the model group, and the significant difference (P <0.01) exists, which indicates that the brain edema is caused by the increase of the permeability of the blood brain barrier after cerebral ischemia reperfusion, and the modeling is successful; the brain indexes of the nimodipine group and the danhong injection group are smaller than those of the model group, and the significant difference (P <0.01) indicates that the nimodipine and the danhong injection can relieve the increase of the permeability of the blood brain barrier after cerebral ischemia reperfusion; although the ratio of the danhong and the salvianolic acid B are not significantly different from the model group (P is greater than 0.05), the numerical value is smaller than that of the model group, which indicates that the preparation has certain improvement effect on the cerebral edema caused by the focal cerebral ischemia of rats.

4.2.3.4 Effect on Biochemical indicators in rat brain tissue

TABLE 29 levels of SOD, MDA, GSH-PX, and NO in rat brain tissue: (

n＝6)

Note that compared to the model set,^aP<0.05；^bp<0.01

from the experimental results, the biochemical indexes in the brain tissue of the sham operation group have statistical significance (P <0.05 and P <0.01) compared with the model group, the modeling is successful, and the biochemical indexes in the brain tissue of the positive control group have significant difference (P <0.05 and P <0.01) compared with the model group, which indicates that the positive control nimodipine and the danhong injection have larger influence on the biochemical indexes. The SOD activity in the brain tissue of the preparation group is significantly different (P <0.01) compared with that of the model group, the levels of MDA, GSH-PX and NO in the high-dose preparation group are significantly different (P <0.05 and P <0.01) compared with that of the model group, the levels of MDA, GSH-PX and NO in the medium-dose and low-dose preparation groups are not significantly different (P >0.05) compared with that of the model group, but the numerical value is obviously changed, which shows that the activity of MDA, GSH-PX and NO is not greatly influenced, and each biochemical index in the blank emulsion group is close to that of the model group, which shows that the blank emulsion has NO influence on the activities of SOD, MDA, GSH-PX and NO in the brain tissue after the ischemic brain injury of the rat.

4.2.3.5 Effect on various Biochemical indicators in rat serum

TABLE 30 rat serum SOD, MDA, GSH-PX, and NO levels (II) ((III))

n＝6)

Note that compared to the model set,^aP<0.05；^bp<0.01

the indexes have significant difference (P <0.05) compared with the model group, which indicates that the molding is successful. The contents of SOD and GSH-PX enzyme levels MDA and NO in the nimodipine group and the danhong injection group have significant difference (P is less than 0.01 and P is less than 0.05) with the model group, and the GSH-PX enzyme activity of each medicine group has NO significant difference compared with the model group, but the numerical value is larger than that of the model group, which indicates that each medicine group can improve the GSH-PX enzyme activity in the serum of a rat with focal cerebral ischemia injury, but has NO significant effect; the levels of SOD, MDA and NO in brain tissues of the high-dose group and the medium-dose group have significant difference (P <0.05) compared with the levels of SOD, MDA and NO in brain tissues of the model group, and the levels of biochemical indexes in brain tissues of the low-dose group have NO significant difference (P >0.05) compared with the levels of the model group, but the numerical values have obvious change, and the numerical values of the biochemical indexes in the blank emulsion group are close to the numerical values of the model group, which indicates that the blank emulsion has NO influence on the activities of SOD, MDA, GSH-PX and NO in rat ischemic brain injury or serum.

Drawings

FIG. 1-rat brain tissue section micrograph of the Salvia miltiorrhiza and safflower extract group of the present invention;

FIG. 2 is a graph showing the kinetics of cumulative permeation of salvianolic acid B in four dosage forms versus time;

FIG. 3-kinetics of cumulative permeation of hydroxysafflor yellow A versus time for the four dosage forms;

FIG. 4-micrograph (x 400 times) of a sub-microemulsion formulation of the present invention sheared for 8 min;

FIG. 5-micrograph (x 400 times) of a sub-microemulsion formulation of the present invention sheared for 10 min;

FIG. 6-micrograph (x 400 times) of a 12min sheared submicroemulsion formulation of the present invention;

FIG. 7-micrograph at 15000rpm of sheared submicroemulsion formulation of the present invention (. times.400);

FIG. 8 micrograph at 20000rpm of shear (x 400 times) of a submicroemulsion formulation according to the invention;

FIG. 9-micrograph at 25000rpm of sheared submicroemulsion formulation of the present invention (. times.400);

FIG. 10-microscopic photograph (. times.400) before homogenization of a submicron emulsion formulation according to the invention;

FIG. 11-microscopic picture (x 400 times) after homogenization of a sub-microemulsion formulation of the present invention;

FIG. 12-measured graph of submicron emulsion formulation particles of the present invention;

FIG. 13-Zeta potential measurement chart of a submicroemulsion formulation according to the invention;

figure 14-effect of model group on rat brain histopathomorphology (x 100 fold);

figure 15-effect of nimodipine on histopathological morphology in rat brain (× 100-fold);

FIG. 16-Effect of Danhong injection group on rat brain histopathomorphology (× 100 times);

FIG. 17-Effect of the sub-microemulsion high dose group of the present invention on rat brain histopathomorphology (x 100 fold);

FIG. 18-Effect of the sub-microemulsion dose set of the present invention on rat brain histopathomorphology (x 100 fold);

FIG. 19-Effect of the sub-microemulsion low dose group of the present invention on rat brain histopathomorphology (x 100 fold);

figure 20-effect of blank emulsion group on rat brain histopathomorphology (x 100 fold);

figure 21-effect of sham-operated group on rat brain histopathomorphology (x 100-fold).

Detailed Description

The present invention is further illustrated by the following exemplary embodiments in order that the practice of the invention may be more fully understood. Unless defined otherwise, technical or scientific terms used herein in the specification and claims of the present patent application shall have the ordinary meaning as understood by those of ordinary skill in the art to which the present invention belongs.

Example 1

⑴ the preparation method comprises soaking 100g radix Salviae Miltiorrhizae in 10 times of water at 80 deg.C for 2 hr, mixing extractive solutions, filtering, concentrating the filtrate at 60 deg.C under reduced pressure to obtain fluid extract with relative density of 1.18-1.22, cooling, adding ethanol to ethanol content of 70%, standing for 12 hr, collecting supernatant, recovering ethanol under reduced pressure, concentrating to obtain soft extract, and drying in vacuum drying oven to obtain radix Salviae Miltiorrhizae extract;

⑵ the preparation method of the safflower extract comprises the steps of taking 100g of safflower, adding water with the weight 5 times of that of the medicinal material, extracting for 2 times at 90 ℃ with the warm temperature, combining the extracting solutions, filtering, concentrating the filtrate at 60 ℃ under reduced pressure to obtain clear paste with the relative density of 1.18-1.2, and drying in a vacuum drying oven to obtain the safflower extract.

Example 2

⑴ the preparation method comprises soaking 100g radix Salviae Miltiorrhizae in 8 times of water at 75 deg.C for two times, each time for 1 hr, mixing extractive solutions, filtering, concentrating the filtrate at 65 deg.C under reduced pressure to obtain fluid extract with relative density of 1.18-1.22, cooling, adding ethanol to ethanol content of 65%, standing for 14 hr, collecting supernatant, recovering ethanol under reduced pressure, concentrating to obtain soft extract, and drying in vacuum drying oven to obtain radix Salviae Miltiorrhizae extract;

⑵ the preparation method of the safflower extract comprises the steps of taking 50g of safflower, adding water with the weight 7 times of that of the medicinal material, extracting for 2 times at 85 ℃ with the warm temperature, extracting for 1h each time, combining the extracting solutions, filtering, concentrating the filtrate at 55 ℃ under reduced pressure to obtain clear paste with the relative density of 1.18-1.2, and drying in a vacuum drying oven to obtain the safflower extract finally.

Example 3

⑴ the preparation method comprises soaking 50g radix Salviae Miltiorrhizae in 12 times of water at 85 deg.C for 3 hr, mixing extractive solutions, filtering, concentrating the filtrate at 65 deg.C under reduced pressure to obtain fluid extract with relative density of 1.18-1.22, cooling, adding ethanol to reach ethanol content of 75%, standing for 10 hr, collecting supernatant, recovering ethanol under reduced pressure, concentrating to obtain soft extract, and drying in vacuum drying oven to obtain radix Salviae Miltiorrhizae extract;

⑵ the preparation method of the safflower extract comprises the steps of taking 100g of safflower, adding 3 times of water by weight of the medicinal material, extracting for 2 times at 95 ℃ with warm immersion, wherein the extraction time is 3h each time, combining the extracting solutions, filtering, concentrating the filtrate at 65 ℃ under reduced pressure to obtain clear paste with the relative density of 1.18-1.2, and drying in a vacuum drying oven to obtain the safflower extract finally.

Example 4

⑴, collecting glycerol 10g, poloxamer-188, 10g, and EDTA-Na₂0.06g of the aqueous solution is dispersed in water for injection, heated to 55 ℃, and stirred until the aqueous solution is completely dissolved to prepare a water phase;

⑵, weighing 6.67g of radix Salviae Miltiorrhizae extract and 6.67g of flos Carthami extract, weighing 26.67g of soybean lecithin, placing the above three into a magnetic stirrer, adding methanol, carrying out composite reaction in 55 deg.C water bath for 2 hr, and recovering methanol to the residual 20% volume to obtain methanol solution of radix Salviae Miltiorrhizae and flos Carthami extract phospholipid complex;

⑶, adding 80g of soybean oil and 170g of medium-chain triglyceride into the methanol solution of the phospholipid complex of the red sage root and safflower extracts in the step (2), continuously recovering until no methanol smell exists, adding VE1g, 1g of oleic acid and 15g of emulsified phospholipid, stirring at the rotating speed of a high-speed tissue triturator of 20000rpm, stirring the water phase in the step ⑴ for 10 minutes to prepare colostrum, diluting the colostrum with distilled water to a specified volume, transferring to a high-pressure homogenizer at the homogenization pressure of 1000par for 9 times, controlling the homogenization temperature at 40 ℃, and adjusting the pH value to 5 with 0.1mol/L of sodium hydroxide to obtain the composition.

Example 5

⑴, collecting glycerol 13g, poloxamer-188, 7g, and EDTA-Na₂0.1g of the aqueous phase is dispersed in water for injection, heated to 50 ℃, and stirred until the aqueous phase is completely dissolved to obtain an aqueous phase;

⑵ weighing radix Salviae Miltiorrhizae extract 16.67g and flos Carthami extract 8.33g, weighing soybean lecithin 25g, placing the above three in a magnetic stirrer, adding methanol, performing composite reaction in 50 deg.C water bath for 3 hr, and recovering methanol to the residual 20% volume to obtain radix Salviae Miltiorrhizae and flos Carthami extract phospholipid complex methanol solution;

⑶, adding 90g of soybean oil and 180g of medium-chain triglyceride into the methanol solution of the phospholipid complex of the salvia miltiorrhiza and safflower extracts in the step (2), continuously recovering until no methanol smell exists, adding 0.5g of VE, 2g of oleic acid and 20g of emulsified phospholipid, stirring at the rotating speed of a high-speed tissue triturator of 15000rpm, stirring the water phase in the step ⑴ for 12 minutes to prepare colostrum, diluting the colostrum with distilled water to a specified volume, transferring to a high-pressure homogenizer at the homogenizing pressure of 1200par for 6 times, controlling the homogenizing temperature at 30 ℃, and adjusting the pH value to 6 with disodium hydrogen phosphate to obtain the composition.

Example 6

⑴, collecting glycerol 7g, poloxamer-188, 13g, EDTA-Na₂0.03g of the aqueous phase is dispersed in water for injection, heated to 60 ℃, and stirred until the aqueous phase is completely dissolved to obtain an aqueous phase;

⑵, weighing 2g of radix Salviae Miltiorrhizae extract and 4g of flos Carthami extract, weighing 24g of soybean lecithin, placing the three into a magnetic stirrer, adding methanol, carrying out composite reaction in water bath at 60 deg.C for 1 hr, and recovering methanol to the residual 20% volume to obtain methanol solution of radix Salviae Miltiorrhizae and flos Carthami extract phospholipid complex;

⑶, adding 70g of soybean oil and 160g of medium-chain triglyceride into the methanol solution of the salvia miltiorrhiza and safflower extract phospholipid complex in the step (2), continuously recovering until no methanol smell exists, adding 2g of VE, 0.5g of oleic acid and 10g of emulsified phospholipid, stirring at 25000rpm of a high-speed tissue triturator, stirring the water phase in the step ⑴ for 8 minutes to prepare colostrum, diluting the colostrum with distilled water to a specified volume, transferring to a high-pressure homogenizer at a homogenization pressure of 800par for 11 times, controlling the homogenization temperature at 50 ℃, and adjusting the pH value to 4 with sodium bicarbonate to obtain the composition.

Example 7

⑴, collecting glycerol 9g, poloxamer-188, 9g, and EDTA-Na₂0.05g of the aqueous phase is dispersed in water for injection, heated to 50-60 ℃, and stirred until the aqueous phase is completely dissolved to prepare a water phase;

⑵ weighing radix Salviae Miltiorrhizae extract 8g and flos Carthami extract 12g, weighing soybean lecithin 20g, placing the three into a magnetic stirrer, adding methanol, carrying out composite reaction in 55 deg.C water bath for 1.5 hr, recovering methanol to the residual 20% volume, and making into phospholipid composite methanol solution of radix Salviae Miltiorrhizae and flos Carthami extracts;

⑶, adding 85g of soybean oil and 165g of medium-chain triglyceride into the methanol solution of the salvia miltiorrhiza and safflower extract phospholipid complex in the step (2), continuously recovering until no methanol smell exists, adding 0.8g of VE, 1.5g of oleic acid and 12g of emulsified phospholipid, stirring at the rotating speed of a high-speed tissue triturator of 22000rpm, stirring the water phase in the step ⑴ for 9 minutes to obtain colostrum, diluting the colostrum with distilled water to a specified volume, transferring to a high-pressure homogenizer at a homogenizing pressure of 900par for 8 times, controlling the homogenizing temperature at 35 ℃, and adjusting the pH value to 5 with alkali to obtain the composition.

Example 8

⑴, collecting glycerol 11g, poloxamer-188, 11g, and EDTA-Na₂0.08g of the water-soluble polymer is dispersed in water for injection, heated to 50-60 ℃, and stirred until the water-soluble polymer is completely dissolved to prepare a water phase;

⑵, weighing 8g of radix Salviae Miltiorrhizae extract and 12g of flos Carthami extract, weighing 20g of soybean lecithin, placing the three into a magnetic stirrer, adding methanol, carrying out composite reaction in water bath at 60 deg.C for 2.5 hr, and recovering methanol to the residual 20% volume to obtain methanol solution of radix Salviae Miltiorrhizae and flos Carthami extract phospholipid complex;

⑶, adding 75g of soybean oil and 175g of medium-chain triglyceride into the methanol solution of the phospholipid complex of the salvia miltiorrhiza and safflower extracts in the step (2), continuously recovering until no methanol smell exists, adding 1.5g of VE, 0.8g of oleic acid and 18g of emulsified phospholipid, stirring at the rotating speed of a high-speed tissue triturator of 17000rpm, stirring the water phase in the step ⑴ for 10 minutes to prepare colostrum, diluting the colostrum with distilled water to a specified volume, transferring to a high-pressure homogenizer at a homogenizing pressure of 800-1200 par for 6-11 times, controlling the homogenizing temperature at 30-50 ℃, and adjusting the pH value to 4-6 with alkali to obtain the final product.

The present invention is described in detail with reference to the above-mentioned embodiments. It should be noted that the above embodiments are only for illustrating the present invention. Numerous alternatives and modifications can be devised by those skilled in the art without departing from the spirit and scope of the invention, which should be construed as within the scope of the invention.

Claims (10)

1. A pharmaceutical composition for treating cerebral ischemia is characterized in that the pharmaceutical composition comprises the following active components in parts by weight: 1 part of salvia extract and 1 part of safflower extract; the preparation method of the salvia miltiorrhiza extract and the safflower extract comprises the following steps:

⑴ the preparation method of the red sage root extract comprises the steps of taking red sage root, adding water with the weight 8-12 times of that of the medicinal material, extracting twice at 75-85 ℃, extracting for 1-3 h each time, merging the extracting solutions, filtering, concentrating the filtrate at 55-65 ℃ under reduced pressure to obtain clear paste with the relative density of 1.18-1.22, cooling, adding ethanol to ensure that the alcohol content is 65-75%, standing for 10-14 h, taking supernatant, recovering ethanol, concentrating and drying to obtain the red sage root extract;

⑵ the preparation method of the safflower extract comprises soaking Carthami flos in 3-7 times of water at 85-95 deg.C for 2 times (each time for 1-3 hr), mixing extractive solutions, filtering, concentrating the filtrate, and drying to obtain Carthami flos extract;

the dosage form of the pharmaceutical composition is submicron emulsion; the submicron emulsion preparation is a phospholipid compound prepared by adding medicinal auxiliary materials into two extracts of salvia miltiorrhiza and safflower as an active carrier; the submicron emulsion preparation comprises the following raw materials in parts by weight: 30-50 parts of salvia miltiorrhiza and safflower extract phospholipid complex, 230-270 parts of oil phase, 10-20 parts of emulsifier, 7-13 parts of co-emulsifier, 7-13 parts of isotonic regulator, 0.5-2 parts of stabilizer, 0.5-2 parts of antioxidant and 0-0.1 part of metal ion complexing agent.

2. The pharmaceutical composition for treating cerebral ischemia according to claim 1, wherein the active ingredients in the composition are prepared by the following method:

⑴ the preparation method comprises extracting Saviae Miltiorrhizae radix with 10 times of water at 80 deg.C for 2 hr twice, mixing extractive solutions, filtering, concentrating the filtrate at 60 deg.C under reduced pressure to obtain fluid extract with relative density of 1.18-1.22, cooling, adding ethanol to ethanol content of 70%, standing for 12 hr, collecting supernatant, recovering ethanol, concentrating, and drying to obtain Saviae Miltiorrhizae radix extract;

⑵ the preparation method of the Carthami flos extract comprises adding 5 times of water into Carthami flos, extracting at 90 deg.C for 1 hr for 2 times, mixing extractive solutions, filtering, concentrating the filtrate, and drying to obtain Carthami flos extract.

3. The pharmaceutical composition for treating cerebral ischemia according to claim 2, wherein the oil phase is soybean oil and medium chain triglyceride, the emulsifier is emulsified phospholipid, the co-emulsifier is poloxamer-188, the isotonic regulator is glycerol, the stabilizer is oleic acid, the antioxidant is VE, and the metal ion complexing agent is EDTA-Na₂。

4. The pharmaceutical composition for treating cerebral ischemia according to claim 3, which isIs characterized in that the composition comprises the following raw materials in parts by weight: 30-50 parts of salvia miltiorrhiza and safflower extract phospholipid complex, 70-90 parts of soybean oil, 160-180 parts of medium chain triglyceride, 10-20 parts of emulsified phospholipid, 7-13 parts of poloxamer-1887, 7-13 parts of glycerol, 0.5-2 parts of oleic acid, 0.5-2 parts of VE, EDTA-Na₂0.03 to 0.1 portion.

5. The pharmaceutical composition for treating cerebral ischemia according to claim 4, wherein the composition comprises the following raw materials in parts by weight: 50 parts of salvia miltiorrhiza and safflower extract phospholipid compound, 90 parts of soybean oil, 180 parts of medium chain triglyceride, 20 parts of emulsified phospholipid, poloxamer-1887 parts, 13 parts of glycerol, 2 parts of oleic acid, 0.5 part of VE, EDTA-Na₂0.1 part.

6. The pharmaceutical composition for treating cerebral ischemia according to claim 4, wherein the composition comprises the following raw materials in parts by weight: 30 parts of salvia miltiorrhiza and safflower extract phospholipid compound, 70 parts of soybean oil, 160 parts of medium chain triglyceride, 10 parts of emulsified phospholipid, poloxamer-18813 parts, 7 parts of glycerol, 0.5 part of oleic acid, VE 2 parts, EDTA-Na₂0.03 part.

7. The pharmaceutical composition for treating cerebral ischemia according to claim 4, wherein the composition comprises the following raw materials in parts by weight: 40 parts of salvia miltiorrhiza and safflower extract phospholipid compound, 80 parts of soybean oil, 170 parts of medium chain triglyceride, 15 parts of emulsified phospholipid, poloxamer-18810 parts, 10 parts of glycerol, 1 part of oleic acid, 1 part of VE, EDTA-Na₂0.06 part.

8. The pharmaceutical composition for the treatment of cerebral ischemia according to any of claims 5 to 7, wherein said submicron emulsion formulation is prepared by the following process:

⑴, glycerol, poloxamer-188, EDTA-Na₂Dispersing in water for injection, heating to 50-60 ℃, and stirring until the water is completely dissolved to obtain a water phase;

⑵, respectively weighing the red sage root extract and the safflower extract, then weighing the soybean lecithin, putting the red sage root extract, the safflower extract and the soybean lecithin into a magnetic stirrer, adding methanol, carrying out composite reaction in a water bath at 50-60 ℃ for 1-3 hours, and recovering the methanol to prepare a methanol solution of the red sage root extract and the safflower extract phospholipid complex;

⑶, adding soybean oil and medium chain triglyceride into the methanol liquid of the salvia miltiorrhiza and safflower extract phospholipid complex in the step (2), continuously recovering until no methanol smell exists, adding Ve, oleic acid and emulsified phospholipid, stirring at the rotating speed of a high-speed tissue triturator of 15000-25000 rpm, stirring the water phase in the step ⑴ for 8-12 minutes to prepare primary emulsion, diluting the primary emulsion with distilled water to a prescribed volume, transferring the primary emulsion to a high-pressure homogenizer at a homogenizing pressure of 800-1200 par for 6-11 times, controlling the homogenizing temperature at 30-50 ℃, and adjusting the pH value to 4-6 with alkali to obtain the composition.

9. The pharmaceutical composition for treating cerebral ischemia according to claim 8, wherein said submicron emulsion is prepared by the following method:

⑴, glycerol, poloxamer-188, EDTA-Na₂Dispersing in water for injection, heating to 55 deg.C, stirring to dissolve completely to obtain water phase;

⑵, weighing radix Salviae Miltiorrhizae extract and flos Carthami extract, weighing soybean lecithin, placing the above three in a magnetic stirrer, adding methanol, carrying out composite reaction in 55 deg.C water bath for 2 hr, and recovering methanol to obtain methanol solution of radix Salviae Miltiorrhizae and flos Carthami extract phospholipid complex;

⑶, adding soybean oil and medium chain triglyceride into the methanol solution of the phospholipid complex of the red sage root and safflower extracts in the step (2), continuously recovering until no methanol smell exists, adding Ve, oleic acid and emulsified phospholipid, stirring at the rotating speed of a high-speed tissue pounder of 20000rpm, stirring the water phase in the step ⑴ for 10 minutes to prepare primary emulsion, diluting the primary emulsion with distilled water to a constant volume, transferring the primary emulsion to a high-pressure homogenizer at a homogenizing pressure of 1000par for 9 times, controlling the homogenizing temperature at 40 ℃, and adjusting the pH value to 4-6 with alkali to obtain the final product.

10. The pharmaceutical composition for treating cerebral ischemia according to claim 9, wherein the route of administration of the pharmaceutical composition is nasal spray.

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