CN114344252B - Catalpol nasal drops and preparation method and application thereof - Google Patents
Catalpol nasal drops and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses catalpol nasal drops, a preparation method and application thereof, and a formula of the catalpol nasal drops comprises the following components: catalpol, thickener, absorption promoter and pH regulator, wherein the components in the formula comprise the following components: catalpol 10mg/ml, thickener 0.1 wt%, absorption promoter 2.0 wt% and pH 6.5-7.0; the preparation has stable storage property at normal temperature and in dark condition; the nasal administration is faster in absorption, and the concentration of the drug in brain tissue is obviously higher than that of intravenous administration, so that the drug has brain targeting property; compared with oral administration and injection administration, the catalpol nasal administration dosage form can also reduce the infarct volume of brain tissues of patients suffering from acute cerebral ischemia, and can reduce the quantity of neuronal apoptosis in cortical areas caused by cerebral ischemia injury, which shows that the neuroprotective effect on cerebral cortical areas is remarkable; the catalpol nasal drops have low toxicity to cilia, no obvious nasal irritation is found, and the nasal administration safety is good.
Description
Technical Field
The invention relates to the field of traditional Chinese medicine preparations, in particular to a catalpa-containing nose drop with an active ingredient, and also relates to a preparation method and application of the catalpa-containing nose drop.
Background
Catalpol is one of the effective components of traditional Chinese medicine rehmannia root, is an iridoid glycoside compound, and modern researches prove that the catalpol has neuroprotective effect on cerebral ischemic injury, alzheimer Disease (AD) and Parkinson Disease (PD). However, because catalpol has good water solubility, it is difficult to penetrate the blood brain barrier in general administration routes (such as oral administration and injection), and the bioavailability is not very high, so that the application and development of catalpol in brain disease treatment are limited.
The nasal delivery system refers to a preparation which is absorbed by nasal mucosa after nasal delivery and can exert local or systemic treatment or prevention effect, and can avoid first pass effect. The preparation type for nasal administration mainly comprises nasal drops, spray, aerosol, gel, emulsion, etc. The nasal drop is one of common nasal preparations, and the nasal drop is prepared with medicine solution and through dropping into nasal cavity. Wu Lina et al have studied the oral kinetics of catalpol by gavage and found that it takes 1.3 hours for catalpol to reach its maximum concentration. Researches indicate that catalpol can only exert the effect of reducing blood sugar of rats after being orally taken by 50mg/kg, so that the treatment effect of catalpol can be presumed to be influenced or a certain time is required for exerting the treatment effect after oral taking. The concentration of catalpol in artificial gastric juice is obviously reduced by simulating the pH value of gastrointestinal physicochemical environment in the early stage of the subject group. The oral administration and injection can reach brain only through systemic blood circulation, the speed is slow, and the bioavailability is limited through the first pass effect, so that the treatment effect of the medicine is improved by changing the catalpol administration mode, the stimulation of the medicine to the gastrointestinal tract and the first pass metabolism of the medicine by the liver-gastrointestinal tract can be avoided by intranasal administration, and the bioavailability is improved.
The thrombolytic treatment time window of ischemic apoplexy is only 4-6h, and early administration is the key for affecting the curative effect of the disease. And during this time most patients cannot arrive at the hospital in time. Therefore, a convenient, quick, efficient and safe nasal administration dosage form is also urgently needed to be searched, the catalpa is quickly and timely delivered to the brain, the curative effect is improved, the brain injury caused by ischemic stroke is further reduced, the time is strived for further delivering to a hospital for treatment, and the opportunity is brought for saving patients such as stroke.
Disclosure of Invention
Therefore, one of the purposes of the invention is to provide catalpol nasal drops; the second purpose of the invention is to provide a preparation method of the catalpol nasal drops; the invention further aims to provide an application of the catalpol nasal drops in preparing a medicine for treating acute cerebral hemorrhage.
In order to achieve the above purpose, the present invention provides the following technical solutions:
1. catalpol nasal drop for acute cerebral hemorrhage disease
The formula comprises the following components: catalpol, thickener, absorption promoter and pH regulator.
Preferably, the thickener is one or more of carbomer, deacetylated chitin, hyaluronic acid, sodium carboxymethyl cellulose and cyclodextrin derivatives.
Preferably, the absorption promoting agent is one or a composition of more than one of water-soluble azone, glycyrrhizin, water-soluble azone, ethylenediamine tetraacetic acid, citrate and salicylate.
Preferably, the pH regulator is one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium bicarbonate, sodium carbonate and sodium hydroxide.
Further preferred in accordance with the invention, the formulation comprises the following components in amounts: catalpol 10mg/ml, carbomer mass concentration 0.1%, water-soluble azone volume concentration 2.0%, pH 6.5-7.0.
2. The preparation method of the catalpol nasal drops comprises the following steps:
weighing appropriate amount of catalpol extract, and preparing catalpol solution with normal saline to obtain solution A; weighing a proper amount of carbomer, adding physiological saline, heating for dissolution, and standing at room temperature for swelling to obtain a solution B; adding water-soluble azone into the solution B, stirring thoroughly, adding the solution A, mixing thoroughly again, adjusting pH to 6.5-7.0 with phosphate buffer solution, fixing volume, and sterilizing at 121deg.C under high pressure for 20 min to obtain catalpol nasal drop.
3. The catalpol nasal drops are applied to preparing medicines for treating acute cerebral hemorrhage.
Preferably, the catalpol nasal drops can increase the drug concentration in olfactory bulb, medulla oblongata, cerebellum, cortex and hippocampal tissues.
Preferably, the catalpol nasal drop can protect neurons in the brain outer cortex area caused by acute cerebral hemorrhage from damage and improve the number of surviving neurons in the brain outer cortex area.
Preferably, the catalpol nasal drop can reduce the number of apoptosis in a cerebral cortex area caused by cerebral ischemia injury.
The invention has the beneficial effects that:
the catalpol nasal drops prepared by the invention have stable storage property under the condition of normal temperature and light shading; the nasal administration is faster in absorption, and the concentration of the drug in brain tissue is obviously higher than that of intravenous administration, so that the drug has brain targeting property; compared with oral administration and injection administration, the catalpol nasal administration dosage form can also reduce the infarct volume of brain tissues of patients suffering from acute cerebral ischemia, and can reduce the quantity of neuronal apoptosis in cortical areas caused by cerebral ischemia injury, which shows that the neuroprotective effect on cerebral cortical areas is remarkable; meanwhile, the catalpol nasal drops have low toxicity to cilia, do not influence the capacity of nasal cilia movement, do not find obvious nasal irritation, and have good safety in nasal administration.
Drawings
In order to make the objects, technical solutions and advantageous effects of the present invention more clear, the present invention provides the following drawings for description:
FIG. 1 is a schematic diagram of the nasal cavity of a rat in systemic circulation;
FIG. 2 is a solution clarity observation after 3 days of sample placement;
FIG. 3 shows the catalpol concentration variation (15d, n=3) at different pH values;
FIG. 4 shows the concentration of catalpol in plasma after nasal and intravenous administration (n=3, i.n. nasal administration, i.v. intravenous administration);
FIG. 5 shows catalpol concentration in each tissue partition after nasal and intravenous administration (n=3, i.n. nasal administration, i.v. intravenous administration, A olfactory bulb, B hippocampus, C medulla, D cerebellum, E cortex);
fig. 6 shows the effect of different routes of administration on neuronal damage in the cortical areas with nikov staining (400×, n=6);
wherein, dark color cuts the head and indicates the neuron that is fuzzy, and light color cuts the head and indicates the nuclear dyeing deepens, A: a control group; b: a model group; c: gavage dosing group (i.g.); d: intraperitoneal administration group (i.p.); e: nasal administration group (i.n.); f: comparison of the number of neurons surviving cortical areas following different routes of administration.
Fig. 7 shows the effect of different routes of administration on damage to neurons in the CA1 region of the hippocampus on nisetum staining (400×, n=6);
Wherein, dark color cuts the head and indicates the neuron that is fuzzy, and light color cuts the head and indicates the nuclear dyeing deepens, A: a control group; b: a model group; c: gavage dosing group (i.g.); d: intraperitoneal administration group (i.p.); e: nasal administration group (i.n.); f: comparison of neuronal numbers surviving the CA1 region of the hippocampus following different routes of administration.
Fig. 8 shows the effect of different routes of administration on damage to neurons in the CA3 region of the hippocampus on nisetum staining (400×, n=6);
wherein, dark color cuts the head and indicates the neuron that is fuzzy, and light color cuts the head and indicates the nuclear dyeing deepens, A: a control group; b: a model group; c: gavage dosing group (i.g.); d: intraperitoneal administration group (i.p.); e: nasal administration group (i.n.); f: comparison of neuronal numbers surviving the CA3 region of the hippocampus following different routes of administration.
Fig. 9 shows the effect of different routes of administration on damage to neurons in the DG region of the hippocampus on nissen staining (400×, n=6);
wherein, dark color cuts the head and indicates the neuron that is fuzzy, and light color cuts the head and indicates the nuclear dyeing deepens, A: a control group; b: a model group; c: gavage dosing group (i.g.); d: intraperitoneal administration group (i.p.); e: nasal administration group (i.n.); f: comparison of neuronal numbers surviving hippocampal DG regions following different routes of administration.
Fig. 10 shows the effect of different routes of administration on apoptosis in the cortical areas of the brain, tunnel staining results (400×, n=6);
Wherein, clipping indicates apoptotic cells, a: a control group; b: a model group; c: gavage dosing group (i.g.); d: an abdominal cavity dosing group (i.p.); e: nasal administration group (i.n.); f: comparison of neuronal apoptosis numbers in cortical areas following different routes of administration, p < 0.01, p < 0.001 compared to model group.
Fig. 11 is a graph showing injury of catalpol nasal drops to rat nasal mucosa (400×, n=3);
wherein dark arrows indicate ciliated status and light arrows indicate epithelial cells; A-D, physiological saline group; E-H.1% deoxycholate sodium group; I-L catalpol nasal drop group.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to limit the invention, so that those skilled in the art may better understand the invention and practice it.
The main medicines and the reagents used in the invention are as follows: catalpol is purchased from Shijia Litsea Bobai biotechnology Co., ltd (cat No. 20161121105), catalpol is purchased from Chengdu Keloma biotechnology Co., ltd (cat No. CHB 171010), carbomer 934 is purchased from Mei Lun biotechnology Co., ltd (cat No. MB 3456), and medical water-soluble azone is purchased from Tianjin North refinement engineering Co., ltd (cat No. 20180518).
Experimental animals: SPF-grade healthy SD male rats (220+ -10 g) purchased from laboratory animal research institute of Chongqing city, the laboratory animals are fed in separate cages at room temperature of 18-25 ℃ and humidity of 50-60%, and the rats eat drinking water freely.
EXAMPLE 1 catalpa pure nasal drops prescription screening
The nasal cavity has abundant vascular system and high permeability structure in nasal membrane, so that the intranasal administration can achieve the effect of quick effect. However, intranasal absorption of drugs has two major obstacles, namely low membrane permeability of polar drugs and rapid removal of drugs due to mucociliary removal, so that the problem can be solved to a great extent by adding safe and effective thickening agents and absorption promoters.
The pH of nasal mucus can affect the action of the solution enzymes and ciliary movement. The pH value of the normal nasal secretion of an adult is 5.5-6.5, when the normal nasal secretion is alkaline, the normal nasal secretion is easy to cause the pathological changes of acute non-infectious inflammation, and when the pH value is up to 9, the normal movement of the solution bacterial enzyme and the nasal cilia is easy to be influenced, and bacterial infection is caused. In addition, the previous experiments of the subject group show that catalpol has acid-base instability, so that the partial study also screens the pH value of proper nasal drops.
1. Determination of the pure concentration of catalpa
The literature reports that the catalpol (5 mg/kg) has better neuron protection effect at high (10 mg/kg) dosage, and the preparation concentration of catalpol nasal drops is set to be 10mg/mL in the study comprehensively.
2. Screening of thickener concentration
The carbomer has good bioadhesion and no irritation to nasal mucosa, so the carbomer is used as a thickener in the study, and the catalpol concentration, the pH value and the clarity of the sample are used as investigation indexes.
Carbomers with mass of 0.1g, 0.2g and 0.3g were weighed and dissolved in 50mL of water, respectively. Placing in a water bath kettle at 40deg.C to make swelling complete, and standing at room temperature for 10 hr until no white core is observed for light inspection, and swelling is completed. And adding 1mL of catalpol solution with the concentration of 10mg/mL, supplementing physiological saline to 100mL, fully mixing the solution, and autoclaving at 121 ℃ for 20 minutes to obtain sample solutions with the carbomer concentrations of 0.1%,0.2% and 0.3% respectively. 3 samples were prepared in parallel for each concentration group, and catalpol concentration, pH value and clarity in the samples were measured and observed after 3 days of standing.
The experimental results are shown in tables 1, 2 and 3. When the carbomer concentration is in the range of 0.1% -0.3%, the solution is clear, the pH value is between 5.42-5.47, but the 0.3% group of solution is gelatinous in texture. After 3 days of standing, the concentration of 3 groups of nasal drops is still clear (see figure 2), the pH value is between 5.40 and 5.45, the texture of the solution is not changed significantly, and the RSD value of each group of catalpol concentration before and after standing is less than 5%. In combination, carbomers were selected as thickeners at a final concentration of 0.1%.
Table 1, 0 day sample physicochemical index (n=3)
Sample physicochemical index (n=3) in table 2, 3 days
TABLE 3 catalpol concentration variation before and after sample placement (n=3, mean.+ -. SD)
3. Absorbent-promoting concentration selection
The water-soluble azone is a novel absorption-promoting agent, can enlarge gaps among biomembrane cells, has stronger effect on hydrophilic drugs than lipophilic drugs, and is widely applied to the absorption promotion of various biomembranes. Therefore, the study contemplates the use of water-soluble azone as an absorption enhancer, and the concentration of water-soluble azone added was screened by in vivo nasal circulation experiments.
Preparing a circulating liquid: the catalpol solution of 20mg/mL is prepared by physiological saline, 100 mu L is put into a 5mL measuring cylinder, and diluted to 5mL by physiological saline. The solution was used to prepare water-soluble azone having a volume concentration of 1%, 2% and 3% and used as a circulating fluid.
In vivo nasal circulation experiments in rats: healthy SD male rats (220.+ -.10 g) were selected and anesthetized by intraperitoneal injection of 3.5% chloral hydrate. The patient was supine, the limbs were fixed, the neck skin of the rat was cut with surgical scissors, and the trachea and esophagus were separated with bending exposure. A small opening is cut at the rear part of the trachea, because the tidal volume of the rat is small, in order to reduce the ineffective cavity volume after the catheter is inserted, a polyethylene pipe with the diameter of 3mm is selected for heating and drawing into a thin pipe with the tip diameter of 2mm and the length of 2cm, a small opening is scalded at the junction of the thickness, and the polyethylene pipe is inserted into the incision at the rear part of the trachea for normal breathing of the rat. Then a polyethylene tube is taken and inserted into the front incision of the esophagus, and is gently pushed forward to the nasal cavity. One end of a polyethylene pipe inserted into the esophagus and exposed outside is connected with a peristaltic pump silicone pipe, and the other end of the peristaltic pump silicone pipe is inserted into circulating liquid, so that the filling circulating liquid circulates through the nasal cavity by means of the peristaltic pump. The schematic diagram of the experimental device is shown in fig. 1.
After the experimental device is fixed, the physiological saline is firstly used for circulating for 10min, the volumes of the physiological saline before and after circulation are compared, and whether the circulating loop has liquid leakage is checked. Draining physiological saline, placing 5mL catalpol-containing circulating liquid into a 5mL measuring cylinder, inserting a silicone tube, and starting a peristaltic pump, wherein the flow rate of the circulating liquid is 2.0mL/min. When the circulating liquid is dripped from the nostril of the rat, the volume V of the circulating liquid in the measuring cylinder is respectively read at 0, 15, 30, 60 and 90min n After sampling 0.2mL, 0.2mL of the circulating solution was added. Centrifuging the sample 10000r/min for 10min, sucking supernatant, sampling, and determining the residual drug concentration C at each time n 。
Absorption constant (K) calculation:
nasal cavity circulation dead volume of V 0 ＝(5－V 1 ). Residual medicine Q n The calculation is performed by a volume correction method.
Wherein Q is n : residual amount (μg) at the nth time point;
(V n +V 0 ): actual volume of circulating fluid (mL) at the nth time point;
C n : drug mass concentration (μg/mL) measured at the nth sampling point;
C 0 : the mass concentration (mug/mL) of the added circulating liquid medicine;
finally, the residual medicine quantity Q passing through different time points n Calculating the percentage X of the residual medicine amount at each time point, taking the logarithm, and carrying out linear regression with the corresponding time t, wherein the slope of the regression line is the absorption rate constant K, and the magnitude of the K value can reflect the absorption condition of catalpol through the nasal cavity.
X＝Q n /Q 0
㏑ X＝-Kt
Data statistics: experimental data samples IBM SPSS20.0 (SPSS Inc, chicago, USA) were analyzed for one-way variance and statistics were expressed in Mean SD. Statistics were performed using graphpadprsm5.0 (Graphpad Software, USA) and a result map was generated.
As a result, as shown in Table 4, the absorption rate constant K value was increased when the concentration of the water-soluble azone in the circulating liquid was changed from 1% to 2%, and was decreased when the concentration of the water-soluble azone was increased to 3%. The above results suggest that 2% water-soluble azone has a better permeation promoting effect on catalpol, and when the concentration is 3%, catalpol absorption is inhibited.
TABLE 4 influence of different doses of Water-soluble azone on catalpol nasal absorption (n=6, mean.+ -. SD)
Note that: in comparison with the group of water-insoluble azones, *** p is less than 0.001; in comparison with the 1% group, ### p is less than 0.001; in comparison with the 3% group, &&& p＜0.001.
4. nasal drop pH value screening
Preparing catalpol solution with physiological saline at a concentration of 0.1mg/mL, adjusting pH with phosphate buffer, storing at 4deg.C, detecting catalpol content by high performance liquid chromatography at 1, 3, 5, 7, 9, 11, 13, and 15 days, and determining catalpol concentration change.
The chromatographic conditions are as follows: chromatographic column Agilent 20RBAX Bonus-RP (4.6X105 mm,5 μm), detector SPD-20A, mobile phase acetonitrile-water (0.5-99.5), detection wavelength 210nm, flow rate 1.0mL/min, column temperature 30 ℃ and sample injection volume 10 μl.
As can be seen from the results of fig. 3 and table 5: the catalpol concentration at 15 days is small when the pH value is 6.5 and 7.0; and when the pH value is 6.0 or 7.5, the catalpol concentration is obviously reduced. The experimental result shows that catalpol is unstable under weak acid or weak alkaline condition, and is stable at pH value of 6.5-7.0. Therefore, the prescription is intended to control the pH value range of catalpol nasal drops to be between 6.5 and 7.0.
TABLE 5 catalpol concentration variation under different pH values (n=3, mean.+ -. SD)
Example 2 preparation Process and formulation of catalpa pure nose drops
According to the study of example 1, the final concentration of each component of catalpa pure nasal drops is as follows, and the specific concentrations are shown in table 6:
table 6, catalpol nasal drop formulation
The specific preparation process comprises the following steps: and precisely weighing a proper amount of catalpol extract, and preparing catalpol solution with normal saline to obtain solution A for later use. Precisely weighing 0.1g of carbomer, adding 50mL of normal saline, dissolving in a water bath kettle at 40 ℃, standing at room temperature for several hours until no white core exists in the carbomer solution, and finishing swelling to obtain solution B. Adding water-soluble azone into the solution B, stirring thoroughly, adding the solution A, mixing thoroughly again, adjusting pH to 6.5-7.0 with phosphate buffer solution, constant volume to 100mL, sterilizing at 121deg.C under high pressure for 20 min, and obtaining catalpol nasal drop sample solution.
When the catalpol concentration is 10mg/ml, the thickening agent is deacetylated chitin, hyaluronic acid, sodium carboxymethyl cellulose and cyclodextrin derivatives, the absorption promoting agent is glycyrrhizin, water-soluble azone, ethylenediamine tetraacetic acid, citrate and salicylate, and the pH regulator is sodium bicarbonate, sodium carbonate and sodium hydroxide, the expected effect of the invention can be achieved.
EXAMPLE 3 stability evaluation of catalpol nasal drops
Catalpol nasal drop sample solution is prepared according to the prescription of example 2, divided into 4 parts, sealed in test tubes, respectively stored at normal temperature (4 ℃ C., 25+/-2 ℃ C.), high temperature (40 ℃ C., high temperature) and strong light irradiation (illuminance + 5000Lx fluorescent lamp), and diluted to 1mg/mL at week 1, week 2, week 3 and week 4, respectively, catalpol concentration change is measured according to the measurement method of example 1, and solution clarification and pH change are observed.
Experimental data samples IBM SPSS20.0 (SPSS Inc, chicago, USA) were analyzed for one-way variance and statistics were expressed in Mean SD. Statistics were performed using graphpadprsm5.0 (Graphpad Software, USA) and a result map was generated.
The stability test results of the catalpol nasal drops are shown in Table 7 and Table 8, the nasal drops are placed at the temperature of 4 ℃ and the temperature of 25+/-2 ℃ for 4 weeks, the pH value is stable, and the solution is clarified by naked eyes, so that the catalpol nasal drops have small concentration change, and the nasal drops are stable at the temperature of 4 ℃ and the temperature of 25+/-2 ℃. The nasal drops are placed at a high temperature of 40 ℃ for 3 weeks, the apparent color is unchanged, the solution is clear, the catalpol concentration is basically unchanged, the solution is slightly turbid after the nasal drops are placed for 4 weeks, but the catalpol concentration is basically unchanged, the pH value is still stable, and the possibility that the catalpol nasal drops are unstable in solution state when being stored at a high temperature is indicated. The nasal drops are stored until all indexes of the nasal drops at week 2 are unchanged under strong light conditions, but the nasal drops are slightly turbid under naked eye observation at week 3, the turbidity degree is not obviously deepened until week 4, the catalpol concentration and the pH value are stable, and the phenomenon that the catalpol nasal drops are unstable in solution texture state under strong light conditions is suggested. The RSD value of the sample solution before and after catalpol concentration placement under the conditions of low temperature, normal temperature, high temperature and strong light is less than 5%.
Table 7 physicochemical property change of nasal drops at normal temperature, high temperature and strong light (n=3)
TABLE 8 catalpol concentration variation at Normal temperature, high temperature, and high light over 4 weeks (n=3, mean.+ -. SD)
Therefore, the experimental result shows that the catalpol nasal drops are more stable to store under the normal temperature and light-proof conditions. The catalpol nasal drop has good stability when being administered through nose, and can be used as a test drug for further pharmacological experiments of the preparation.
Example 4 catalpol nasal drops brain targeting evaluation
The brain targeting property of the catalpol nasal drops is evaluated by observing the distribution of the medicines in blood and brain tissues after the nasal administration of the catalpol nasal drops.
1. Animal grouping and administration method
30 healthy male SD rats (220+/-10 g) are selected, the rats are fasted for 12 hours before operation without water control, and are fed in separate cages in the environment with the room temperature of 18-25 ℃ and the humidity of 50-60%, and the rats eat water freely. The animals were randomized into intravenous (i.v.) and nasal (i.n.) administration groups, each group having 15 doses of 10mg/kg. After anesthetizing the rats with 3.5% chloral hydrate, the supine state was maintained and the neck was slightly raised. Catalpol nasal drops are alternately administered to the nasal cavities at two sides, and are absorbed by nostrils at one side and then administered to the other side, wherein each administration is 10 mu L, the interval is 2min, and the whole administration process lasts for 30min. The catalpol solution is prepared by using normal saline in the intravenous administration group, and after anesthesia is performed according to the same method, the catalpol solution with the same dosage is injected into the tail vein at one time.
2. Tissue specimen collection
Rats were sacrificed 15, 30, 60, 120, 180min after dosing, 3 rats were sacrificed at each time point of each group. The abdominal aorta is bled. The rat broken head completely removes brain tissue, removes vascular tissue on the surface of the brain tissue, cleans the brain tissue by PBS, and absorbs water by filter paper to quickly separate olfactory bulb, cerebellum, medulla oblongata, cortex and Hippocampus parts, and stores at-80 ℃. The blood sample is kept stand for 30min, centrifuged for 10min at 3000r/min, and the blood plasma is reserved for preservation at-80 ℃.
3. Plasma sample processing
Precisely sucking 0.5mL of a plasma sample, placing the plasma sample into a 10mL centrifuge tube, precisely adding 50uL of an internal standard solution of aucubin solution, swirling for 30s, fully and uniformly mixing, adding 2.5mL of acetonitrile, precipitating protein, swirling for 3min, fully and uniformly mixing, centrifuging for 10min at 3500r/min, sucking 2.5mL of supernatant, blowing nitrogen in a 37 ℃ water bath for drying, adding 200 mu L of mobile phase (acetonitrile: water=0.5:99.5) into residues for dissolution, swirling for 1min, centrifuging for 10min at 10000r/min at high speed, taking 10 mu L of supernatant for HPLC analysis, and calculating the catalpol concentration in the plasma sample according to an internal standard method.
4. Brain tissue sample processing
Precisely weighing 0.1g of brain tissue, placing the brain tissue into a 10mL centrifuge tube, and mixing the brain tissue with 1:5 adding mobile phase (acetonitrile: water=0.5:99.5), precisely adding 50 mu L of an internal standard solution of aucubin solution, swirling for 30s, fully mixing, adding 2.5mL of acetonitrile, precipitating protein, homogenizing by a homogenizer, mixing, swirling for 2min, fully mixing, centrifuging for 10min at 3500r/min, sucking 2.5mL of supernatant, blowing nitrogen in a water bath at 37 ℃, adding 200 mu L of mobile phase into residues for dissolution, centrifuging for 10min at 10000r/min after swirling for 1min, taking 10 mu L of supernatant for HPLC analysis, and calculating the concentration of catalpol in each brain tissue sample according to an internal standard method.
5. Evaluation criteria
The local bioavailability (F) of catalpol nasal drops in blood and brain tissues after nasal administration was evaluated using the following formula, and the brain targeting of catalpol nasal drops was evaluated using brain targeting index (DTI):
F％＝AUC i.n. /AUC i.v. ×100％
DTI＝(AUC brain i.n. /AUC Blood i.n. )/(AUC Brain i.v. /AUC Blood i.v. )
Wherein AUC refers to the area under the curve at the time of drug administration; i.n. means nasal route of administration; i.v. refers to intravenous route of administration.
When DTI > 1, the drug is indicated to have brain targeting in brain tissue partitions.
6. Catalpol blood concentration after intravenous and nasal administration
The concentration distribution results of catalpol in blood plasma after nasal cavity and vein administration are shown in figure 4, the concentration of the drug in blood plasma after nasal cavity administration of catalpol nasal drop is far lower than that of catalpol after vein administration, but absorption after nasal cavity administration is faster, the drug can be detected in blood plasma at 15min, and the drug is maintained at a certain concentration for a certain period of time.
7. Catalpol concentration distribution characteristics in brain tissue partitions after intravenous and nasal administration
Catalpol was detected in each region of brain tissue 15min after intravenous and nasal administration. The distribution results of the extract in olfactory bulb, medulla oblongata, cerebellum, cortex and hippocampus are shown in FIG. 5. The concentration of catalpol in each region of brain tissue is higher than that of intravenous administration, and the concentration of catalpol in the olfactory bulb is obviously higher than that of other brain tissue regions. Further adopting DAS software to calculate the pharmacokinetic parameters of each region of brain tissue under different administration routes. The results are shown in Table 9, and the AUC of catalpol in olfactory bulb, medulla oblongata, cerebellum, cortex and sea horse after nasal administration is significantly higher than that after intravenous administration. The above results suggest that catalpol absorption is faster by nasal administration and its concentration in brain tissue is significantly higher than by intravenous administration.
TABLE 9 pharmacokinetic parameters following nasal and intravenous administration
8. Brain targeting evaluation results
The brain targeting index of catalpol in rat brain tissue after nasal administration was calculated according to the formula in the evaluation standard, and the results are shown in table 10. The targeting indexes (DTI) of catalpol in olfactory bulb, medulla oblongata, cerebellum, cortex and hippocampus are respectively 7.99, 9.10, 28.65, 13.15 and 14.50 and are all more than 1, so that the catalpol nasal drops are suggested to have brain targeting, the distribution of concentration in brain is improved, the concentration accumulation of catalpol in the brain is hopefully realized through a nasal administration route, and the catalpol in the preparation can enter brain tissues through a direct communication path between the nasal cavity and the brain tissues. Meanwhile, the bioavailability F value in each brain tissue partition is 1.00-3.58%, which is larger than the bioavailability (0.12%) in blood plasma. Therefore, the preparation has brain targeting property, and the catalpol administration through nose is feasible.
TABLE 10 catalpol nasal drops brain targeting index and bioavailability
Example 5 pharmacodynamics evaluation of catalpol nasal drops on acute cerebral ischemia rat model
The treatment effect of catalpol nasal drops on central nervous system diseases is observed from the perspective of pharmacodynamics after nasal administration, permanent focal middle artery blocking cerebral ischemia (Permanent middle cerebral arter occlusion, pMCAO) of rats is taken as a model, the protection effect of catalpol nasal drops on neurons after nasal administration is observed, and the purpose of providing a basis for developing catalpol nasal administration systems is achieved.
1. Grouping animals
Healthy male SD rats (220+/-10 g) are selected, 60 rats are fasted for 12 hours before operation without water inhibition, and are fed in separate cages in an environment with the room temperature of 18-25 ℃ and the humidity of 50-60%, and the rats eat water freely. Randomly divided into a control group, a model group, a nasal administration group, an abdominal cavity administration group and a stomach irrigation administration group, and each group comprises 12 animals.
2. Method for preparing focal permanent cerebral ischemia model by electrocoagulation method and administration
The experimental rats are fasted before operation without water inhibition for 12 hours, after anesthesia by injecting 3.5% chloral hydrate (1 mL/100 g) into the abdominal cavity is successful, the prone position of the rats is fixed on a brain stereotactic apparatus, the right temporal side is used for craniotomy after shaving and iodophor disinfection, a surgical knife is used for cutting the scalp of the rats along the right side top temporal ridge (namely the osseous boundary line between the top bone and the temporal bone), the cut is an arc-shaped small opening, the mouth side end is propped against 0.2cm behind the outer canthus of the right eye, and the tail side end is up to 0.2cm in front of the right external auditory meatus. Then the temporomyomembrane is incised, the temporomyomembrane is separated by using a sharp-blunt combination method, a small bone drill is used for lightly drilling a bone window at the front lower part of the joint of the zygomatic arch and the temporo-osseous part, the dura mater is protected, a cotton ball is filled to ensure clear vision, the frontal arch is gently stripped by a glass minute hand until the vein and the olfactory tract under the right brain are clearly presented, the tiny blood vessel which vertically runs across the vein under the brain to the brain surface is the middle cerebral artery on the right side, an electrocoagulator (30W, time 2 s) coagulates the middle cerebral artery, the blood vessel is instantly whitened to finish the electrocoagulation, and the incision is sutured after the non-active bleeding is determined, and the iodine is disinfected.
The administration after 1h of ischemia in the model is carried out, the nasal administration method is the same as 4.1, and after the rats are anesthetized by 3.5% chloral hydrate, the supine state is maintained, and the neck is slightly raised. Catalpol nasal drops are alternately administered to the nasal cavities at two sides, and are absorbed by nostrils at one side and then administered to the other side, wherein each administration is 10 mu L, the interval is 2min, and the whole administration process lasts for 30min. The catalpol solution is prepared by physiological saline in the intraperitoneal administration group and the lavage administration group, and the catalpol solution with the same dosage is administered at one time. The control group was given the same volume of physiological saline.
3. Model inclusion criteria
Model scoring was performed by an experimenter without knowledge of the purpose of the experiment after the animals were fully awake. Referring to Longa scoring method, animals were scored after being anesthetized and awake. The scoring criteria are as follows:
(1) No symptom of neurological deficit, 0 score;
(2) Lifting the tail, reversely suspending the left forelimb to bend for 1 minute;
(3) Spontaneously inclining leftwards for 2 minutes when walking;
(4) When walking, the user spontaneously turns left for 3 minutes;
(5) The person cannot walk by himself, the consciousness level is lowered by 4 minutes;
(6) Animal death, 5 minutes;
post-operative observations follow-up experiments were performed with models scoring 1-3 points incorporated into the statistics.
4. Dyeing of 2,3,5-triphenyltetrazolium chloride (2, 3,5-Triphenyltetrazolium chloride, TTC)
Cerebral infarction volumes were assessed using a 2,3, 5-triphenyltetrazolium chloride (2, 3,5-tripheny ltetrazolium chloride, TTC) staining method. The principle is that TTC has a reduced form and an oxidized form 2 states, the reduced form is red, and the oxidized form is colorless. The normal brain tissue contains reduced nicotinamide adenine dinucleotide phosphate (nicotinamide adenine dinucleotide phosphate, NADPH) which can reduce colorless oxidized TTC to red, namely reduced TTC, so that the region is red; after cerebral ischemia, nerve cells in the infarcted area die due to ischemia, NADPH is lost, TTC cannot be reduced, and brain tissue in the area is gray, so that the infarct area and the normal area can be distinguished.
Rats were sacrificed at 24h of reperfusion by deep anesthesia with 3.5% chloral hydrate followed by decapitation. After craniotomy, completely taking out brain tissue, placing into brain mould, and quick freezing for 10min, and continuously making 5 pieces of coronal slices with thickness of 2mm with mouse brain slice bed. Rapidly placing the brain slice into 1% TTC dye solution, and dyeing in a incubator at 37deg.C for 30min in dark, and lightly turning the brain slice every 10min to make dyeing uniform.
After photographing and preservation, the photographed Image is analyzed by Image J software, and the cerebral infarction area = non-ischemic side hemisphere area-ischemic side hemisphere non-infarct area, each area multiplied by thickness (2 mm) is the volume, and the calculation is carried out according to the formula: infarct volume is a percentage of brain tissue volume= [ (non-ischemic side hemisphere volume mm 3-ischemic side hemisphere non-infarct volume mm 3)/non-ischemic side hemisphere volume mm ] ×100%.
5. Brain tissue material
After 24 hours of administration, rats are anesthetized by 3.5% chloral hydrate, and are perfused by 0.9% normal saline through the heart chamber until colorless liquid flows out of the right atrium, and then are perfused by 4% paraformaldehyde continuously, the infusion is fast and slow until the neck and the two upper limbs are stiff, the fixation is completed, and brain tissues are taken out after head breaking and are placed in the 4% paraformaldehyde for fixation for more than 48 hours. Alcohol dehydration and paraffin embedding to prepare slices.
6. Nib staining
The brain tissue paraffin sections were stained with toluidine blue stained hair. Nissl bodies or Nissl bodies are small triangular or oval masses distributed in the cytoplasm of nerve cells and can be dyed into purplish blue by basic dyes such as thionine, methylene blue, toluidine blue, tar violet and the like. Various nerve cells contain Nib's bodies, but the shape, number, and distribution are often different. Nii can change due to the change of physiological state, and nii is an important part of synthesis of protein in neurons, so that nii can be obviously reduced in vivo after the neurons are stimulated. The presence and disappearance of Nib is an important indicator of whether nerve cells are damaged, and Nib will dissolve or even disappear when encephalitis, cerebral ischemia, etc. occur. Therefore, the protection effect of catalpol nasal drops on neurons after nasal administration is observed by using Nile's staining.
7. TUNEL staining
The incorporation of tetramethylrhodamine-deoxyuridine triphosphate (TMR red-dUTP) was catalyzed at the 3' -OH end of the apoptotic cell-disrupted DNA using a terminal deoxyribonucleotide transferase (Terminal Deoxynucleotidyl Transferase, tdT) using the TUNEL (Tdt mediated DUTP Nick End Labeling) method. TMR red-dUTP specific and accurately labeled DNA can be used for directly observing apoptotic cells by a microscope.
8. Data statistics
Experimental data samples IBM SPSS20.0 (SPSS Inc, chicago, USA) for one-way analysis of variance, statistics expressed in Mean SD, and differences expressed in p < 0.05 were significant. Statistics were performed using graphpadprsm5.0 (Graphpad Software, USA) and a result map was generated.
9. Effects of different routes of administration on infarct volume in cerebral ischemic rat models
As shown in Table 11, the infarct volume of the model group was (33.46.+ -. 2.77)%, and the infarct volume was reduced to (27.66.+ -. 4.03)%, and (28.01.+ -. 7.01)%, and (24.08.+ -. 4.93)%, respectively, in the intragastric administration group, the intraperitoneal administration group, and the nasal administration group, and the infarct volume was reduced by a larger proportion than in the intragastric administration group and the intraperitoneal administration group.
Table 11 effect of different routes of administration on infarct volume indicated by TTC in cerebral ischemic rat model (n=4, mean±sd)
10. Influence of different routes of administration on the morphology of the neurons of the outer cortex of a rat model of cerebral ischemia
As can be seen from FIG. 6, the neurons of the outer cortex of the rat brain in the normal group (FIG. 6, A) are oval or round, the cell morphology is normal, the cell body is full, the nucleolus is clear, the nuclear membrane is complete, and the cell nucleus is bluish. Compared with the normal group, the model group (fig. 6, b) has the phenomenon that a large number of nucleolus blurs appear on the neurons of the brain exodermis of the rat, the cell nucleus is stained deeply, and most of neuron cell bodies are contracted, so that the neurons of the model group are damaged. Compared with the model group, the three administration routes of the stomach-filling administration group (i.g., fig. 6 and c), the abdominal administration group (i.p., fig. 6,D) and the nasal administration group (i.n., fig. 6,E) can reduce the phenomenon of neuron nucleolus blurring caused by cerebral ischemia of rats, and the i.g., i.p., i.n. administration routes are suggested to have a protective effect on neuron damage caused by cerebral ischemia of rats. The nasal administration group showed significant differences in the protective effect on injured neurons of the outer cortex compared to the model group (fig. 6, f and table 12).
Table 12 comparison of the number of surviving neurons in the cerebral cortex area following different routes of administration (n=6, mean±sd)
Note that: in comparison with the set of models, ** p＜0.01
11. effect of different routes of administration on the morphology of neurons in the hippocampal CA1 region of a rat model of cerebral ischemia
As shown in FIG. 7 and Table 13, the neurons in the CA1 region of the rat brain hippocampus of the normal group (FIG. 7, A) were orderly arranged, the cell structure was complete, most of the cell structure was clear, the nuclear membrane was complete, the Nib's body was uniformly stained, and the cell nuclei were bluish. Compared with the normal group, the model group (figure 7, B) has the advantages that the neurons in the CA1 region of the rat brain hippocampus have the phenomena of nucleolus blurring and cytokinesis, the cell gap is increased, the arrangement of the neurons is disordered, the cell nucleus is stained deeply, the cell distribution is sparse, the contour is blurred, and the neurons in the model group are seriously damaged. Compared with the model group, the three administration routes of the stomach-filling administration group (i.g., fig. 7 and c), the abdominal administration group (i.p., fig. 7 and D) and the nasal administration group (i.n., fig. 7 and e) have the protection effect on the neuronal damage caused by cerebral ischemia rats and reduce the phenomenon of neuronal nucleolus blurring caused by cerebral ischemia of rats. The protection effect on the damage of neurons is most obvious in the nasal administration group.
Table 13 comparison of the number of surviving neurons in the CA1 region of the hippocampus following different routes of administration (n=6, mean±sd)
12. Effect of different routes of administration on the morphology of neurons in the hippocampal CA3 region of a rat model of cerebral ischemia
From FIG. 8, table 14 shows that, compared with the normal group (FIG. 8, A), the model group (FIG. 8, B) showed a large number of abnormal arrangement of neurons in the CA3 region of the rat brain hippocampus, a phenomenon of nucleolus blurring occurred, the cell gap was increased, the Neisseria staining was uneven, and the neurons were significantly damaged; compared with the model group, the effects of the gastric administration group (i.g., fig. 8, c), the intraperitoneal administration group (i.p., fig. 8, d) and the nasal administration group (i.n., fig. 8, E) on the disorder of the arrangement of neurons in the CA3 region of the hippocampus are alleviated, the effects of the gastric administration group (fig. 8, c) are weaker, the nasal administration group (fig. 8, E) is most obvious, and the effects of the deepening of the nuclear staining of neurons in the three administration route groups are eased, and the protective effect is provided for the damage of neurons in the CA3 region of the brain sea horse of a cerebral ischemia rat, wherein the effects of the nasal administration group (fig. 8, E) on relieving the blur of the nuclei of neurons caused by the cerebral ischemia of the rat are stronger than those of the gastric administration group and the abdominal cavity administration group.
Table 14 comparison of the number of surviving neurons in the CA3 region of the hippocampus following different routes of administration (n=6, mean±sd)
13. Effect of different routes of administration on the morphology of neurons in the hippocampal DG region of a rat model of cerebral ischemia
As can be seen from fig. 9 and table 15, the neurons in the rat brain hippocampus DG region of the model group (fig. 9, b) had a phenomenon of deepening a small number of nuclei and blurring nucleoli, and a part of the neurons had a cell body contracted, suggesting that the neurons in the hippocampus DG region were slightly damaged; compared with the model group, the neuron arrangement of the three administration routes is tidy, the phenomena of deepening of cell nuclei and blurring of nucleolus are not obvious, and the phenomena of deepening of cell nuclei are not obvious in the gastric lavage administration group (i.g., fig. 9 and c), and are improved in the abdominal cavity administration group (i.p., fig. 9,D) and the nasal cavity administration group (i.n., fig. 9 and e); the phenomenon of damage to neurons in the DG region of the hippocampus is not serious, and the three administration routes have no obvious protective effect on the phenomenon of damage to neurons in the DG region of the hippocampus.
Table 15 comparison of the number of surviving neurons in hippocampal DG area following different routes of administration (n=6, mean±sd)
Effect of different routes of administration on apoptosis in the cerebral cortex region of a rat model of cerebral ischemia
TUNEL staining was used for hippocampal neuronal apoptosis detection. The nuclei of positive cells are stained dark brown, the nuclei shrink round or irregular, and the nuclei disintegrate. As shown in FIG. 10 and Table 16, the control group (FIG. 10, A) showed no positive cells in the infarct side cortex and the other groups, the model group (FIG. 10, B) showed a large number of positive cells in the periinfarct zone, and the negative cells scattered in the periinfarct zone, and the positive cells were decreased in the stomach-lavage group, the abdominal-cavity group and the nasal-cavity group. The positive cell count of the staining in the 10×10cm region of the cortex region of the hippocampal, the intraperitoneal and nasal administration groups was (20.5±5.9) (13.3±5.1) (9.0±5.5) in the model group (29.8±8.5), respectively, and the positive cell count was significantly decreased in the intraperitoneal administration group and the nasal administration group, respectively, compared with the model group (fig. 10, b), and the intraperitoneal administration group had a significant difference (p < 0.01, p < 0.001).
Table 16 comparison of neuronal apoptosis numbers in cortical areas following different routes of administration (n=6, mean±sd)
Note that: in comparison with the set of models, ** p＜0.01， *** p＜0.001
in summary, the study adopts a preparation method of a focal permanent cerebral ischemia rat model, and the states of neurons in cerebral ischemia infarct volume and ischemia semi-dark zone cortex tissues after three different administration routes of nasal administration, gastric lavage and abdominal cavity administration are observed. The experimental results show that the catalpol nasal drops can obviously reduce the infarct volume of an acute cerebral ischemia rat model, the neuron staining number of three different administration route groups at corresponding positions is superior to that of the model group, and the three different administration routes are indicated to have a protective effect on the neuron damage caused by the cerebral ischemia rat. However, in the DG region of the hippocampus, the neuronal damage phenomenon of each group is not obvious, and the apoptosis of the cortex region caused by cerebral ischemia damage of rats can be obviously reduced by the nasal administration of catalpol, which suggests that the neuroprotective effect of the catalpol in the cerebral cortex region is better than that of other two groups of administration routes after the nasal administration. Research shows that different administration modes of catalpol have different protective effects on nerves, and research and development of different administration modes of catalpol has important significance in clinically treating stroke.
Example 6 catalpol nasal drops safety evaluation
The invention adopts the bullfrog's palate cilium movement model and the rat nasal cavity administration model, and uses the continuous cilium swing time, the rat nasal mucosa tissue structure and the morphological change to examine the irritation of catalpol to the nasal mucosa, and evaluate the safety of catalpol nasal drops.
1. Effects on cilia clearance
The model is built according to the isolated toad palate method. 9 bullfrogs (35+ -5 g) are selected and randomly divided into a negative control group (physiological saline), a positive control group (1% deoxycholate sodium solution) and 3 catalpol nasal drops, wherein each group comprises 3 bullfrog nasal drops. The bullfrog is fixed on a hard plate in a supine way by ropes, the bullfrog is pulled by hemostats to open the oral cavity, the upper jaw mucous membrane is separated by surgical scissors, blood clots and sundries are washed by normal saline, mucous membranes with the thickness of about 3mm multiplied by 3mm are spread on a glass slide, 0.2mL of medicine solution is dropped on the surface of the mucous membranes, the mucous membranes are completely covered by the medicine solution, glass sheets are covered, the swinging condition of cilia of the mucous membranes is observed under a 40-time optical microscope, and then the bullfrog is placed in a chromatographic cylinder with a small amount of distilled water, and is sealed, so that the vapor is in a nearly saturated state. And taking out the specimens every 30min, and observing whether cilia swing or not under a microscope by adopting a single-blind method, wherein the swing frequency and the later period are shortened. If the cilia continue to move, the device is put back into the chromatographic cylinder until the cilia stop moving. The time required from the start of administration to complete cessation of cilia movement was recorded. Physiological saline was used as a negative control at the same time, and literature-recognized 1% sodium deoxycholate with cilia toxicity was used as a positive control. The continuous movement time of cilia of the bufo gargarizans in catalpol group is divided by the continuous movement time of cilia of bufo garizans in normal saline group to obtain the relative percentage (Per%) of the continuous movement time of cilia. The higher the Per%, the less ciliated toxicity of catalpol.
The relative percentages of continuous cilia movement for each group are shown in table 17. The results showed that the average time of cilia swing was only 6min for the 1% sodium deoxycholate solution group, the relative movement percentage was only 1.2%, and there was a very significant difference compared to the normal saline group (p < 0.001, table 17), suggesting that 1% sodium deoxycholate was severely toxic to cilia movement. The relative movement percentage of the cilia of the palate of the catalpol nasal drop group reaches 83%, which indicates that the catalpol nasal drop has less influence on the cilia movement. The toxicity of the drug was evaluated with the cilia of the frog class, with higher percentage of cilia relative movement indicating lower toxicity of the drug. Typically, cilia continue to move for a percentage of more than 50%, i.e., the ability of the drug to do not substantially affect cilia movement. The experimental result shows that the catalpol nasal drops have smaller toxicity to cilia and do not affect the cilia movement capacity of the nasal cavity.
Table 17 results of bullfrog cilia toxicity experiments (n=3, mean±sd)
Note that: in comparison with the group of physiological saline, *** p＜0.001
2. rat mucosa irritation test
The investigation of changes in surface cilia morphology and mucosal tissue structure following nasal administration of the drug is the most direct evaluation method for nasal mucosal toxicity. The study adopts rat nasal mucosa, and the pathological morphological change of catalpol nasal drops on the nasal mucosa after administration is examined.
Grouping and administration of animals
Healthy male rats (200+ -20 g) were selected and randomly divided into a negative control group (physiological saline), a positive control group (1% deoxycholate sodium solution), and catalpol nasal drop group 3, each group of 12.
After anesthetizing the rats with 3.5% chloral hydrate, the supine state was maintained and the neck was slightly raised to 45 °. The administration dose is 10mg/kg, the administration method is that nasal cavities on both sides are alternately administered, one side of nasal cavity is administered, and after the nasal cavity is absorbed, the other side of nasal cavity is administered, each time of administration is 10 mu L, the interval is 2min, and the whole administration process lasts for 30min. And the observation is recovered after stopping the nasal administration for one week after 7 consecutive days. Nasal mucosa was fixed in 4% paraformaldehyde on day 1, day 4, day 7, and day 7 of drug withdrawal, and tissue paraffin embedded sections were prepared and observed by HE staining.
In the experimental process, compared with a normal saline group, the observation of the administered rats shows that the rats with 1% of deoxysodium cholate group have obvious nasal scratching phenomenon, the nose position is red and swelling, the nasal scratching times are increased along with the prolonged administration time, the red and swelling phenomenon is obvious, and the nose has bleeding phenomenon, which indicates that 1% of deoxysodium cholate has obvious irritation to the nasal cavity of the rats, the rats with catalpol nasal drops group begin to have few nasal scratching phenomena at the later period of administration, the nose is slightly red and swelling, but no bleeding phenomenon exists, the nasal scratching and swelling phenomenon disappears after drug withdrawal, and the catalpol nasal drops are suggested to have slight irritation to the nasal cavity of the rats, and drug withdrawal can be quickly recovered.
The results of HE staining of the nasal mucosa of the rat are shown in FIG. 11, and the pseudomultilayer columnar epithelium of the nasal mucosa of the rat is complete after one week of administration of the physiological saline group (FIGS. 11, A, B, C). Cilia are consistent, compact and orderly, mucosal epithelial cells are orderly arranged, cell nucleus is consistent in size, no shedding phenomenon is seen in cilia and cells, mucous gland structure is normal, and mucous membrane has no abnormal structures such as congestion, edema necrosis and the like. The pseudo-double layer columnar epithelial structure on the 4 th day (figure 11, F) of 1% deoxycholate sodium group administration has obvious residual defect phenomenon, cilia basically drop off, most of the epithelial cells in the area are necrotic, the arrangement is sparse, and mucous gland atrophy is serious; on day 7 of administration (fig. 11, g) the pseudo-lamellar columnar epithelium is completely destroyed, cilia and epithelial cells completely fall off, whereas after 7 days of withdrawal (fig. 11, h), the pseudo-lamellar columnar epithelium is significantly recovered, but still has structural defects; the sodium deoxycholate proved to have serious toxicity to nasal mucosa. After catalpol nasal drop group is dosed for one week (fig. 11, i and J, K), the pseudo-multi-layer columnar epithelial structures are arranged more tightly, compared with a physiological saline group, the thickness of the epithelial layers is not obviously changed, cilia are distributed uniformly, the morphology of cilia is not changed abnormally, but the compactness of the epithelial cell arrangement is not as good as that of the physiological saline group in 7 days of dosing (fig. 11 and k), and the epithelial cell peeling phenomenon exists, so that other abnormal pathological structures do not appear in the dosing period; after 7 times of drug withdrawal and recovery (fig. 11, L), the epithelial cell arrangement was significantly improved, suggesting that the catalpol nasal drop group was reversible in stimulation of the nasal mucosa and could be regenerated and repaired.
In conclusion, the catalpol nasal drops have low toxicity to cilia, do not influence the capacity of nasal cavity cilia movement, do not find obvious nasal cavity irritation, and have good safety in nasal administration.
The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and these are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.
Claims (6)
1. Catalpol nasal drops for acute cerebral ischemia diseases are characterized in that: the formula comprises the following components in percentage by weight: catalpol 10mg/ml, carbomer 0.1% by weight, water-soluble azone 2.0% by volume, and pH 6.5-7.0.
2. The catalpol nasal drop preparation method of claim 1, comprising the following steps: weighing appropriate amount of catalpol extract, and preparing catalpol solution with normal saline to obtain solution A; weighing a proper amount of carbomer, adding normal saline, heating for dissolution, and standing for swelling at room temperature to obtain a solution B; adding water-soluble azone into the solution B, stirring thoroughly, adding the solution A, mixing thoroughly again, adjusting pH to 6.5-7.0 with phosphate buffer solution, fixing volume, and sterilizing at 121deg.C under high pressure for 20 min to obtain catalpol nasal drop.
3. The use of catalpol nasal drop as claimed in claim 1 in the preparation of a medicament for treating acute cerebral ischemia.
4. The use according to claim 3, wherein the catalpol nasal drops increase the concentration of the drug in olfactory bulb, medulla oblongata, cerebellum, cortex, hippocampal tissue.
5. The use according to claim 3, wherein the catalpol nasal drops can protect neurons in the brain outer cortex area caused by acute cerebral ischemia from damage and increase the number of surviving neurons in the brain outer cortex area.
6. The use according to claim 3, wherein the catalpol nasal drops can reduce the number of apoptosis in the cerebral cortex area caused by acute cerebral ischemia injury.
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