CN107582621B - Medicine for relaxing vascular smooth muscle and preparation method thereof - Google Patents

Abstract

The invention discloses a medicine for relaxing vascular smooth muscle and a preparation method thereof, the medicine is an extract of a mixture of cynomorium songaricum and Chinese chestnut peel, the preparation method comprises various procedures of ultrafine grinding, dipping extraction, precipitation, enzymolysis and the like, the cynomorium songaricum and Chinese chestnut peel extract prepared by the preparation method can effectively relax vascular smooth muscle contraction, can be used as a novel vascular smooth muscle relaxing medicine, is used for preparing a medicine for treating hypertension, and can provide a new scheme for clinically treating hypertension.

Description

Medicine for relaxing vascular smooth muscle and preparation method thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a medicine for relaxing vascular smooth muscle and a preparation method thereof.

Background

Cynomorium songaricum (Cynomorium songaricum; Cynomorium) is a single herb plant of a single genus of a single family of the genus Cynomorium of the family Cynomoriaceae, and is mostly parasitized on the roots of the plants of the genus Nitraria of the family Tribulus. The cynomorium songaricum can grow in northwest areas of China, is common in grassland desert zones, and is an important medicinal material frequently used in Chinese medicinal materials. The cynomorium songaricum is sweet in taste and warm in nature, can calm the liver and tonify the kidney, has the functions of relaxing bowel, replenishing vital essence and nourishing blood, tonifying kidney yang and the like, and is mainly used for treating diseases such as impotence, spermatorrhea, insufficiency of kidney yang, soreness and weakness of waist and knees, constipation due to intestinal dryness, deficiency of essence and blood and the like.

The chestnut skin contains a large amount of carbohydrase substances (commonly called chestnut skin extract sugar), and simultaneously contains high-quality a-amylase and a-glucose active blocking factors, can delay the digestion and absorption of sugar by a digestive tract system in the digestive tract, can inhibit the increase of blood sugar of a human body, has peculiar efficacy for preventing and treating diabetes, and also has good efficacy for preventing and treating hyperglycemia and obesity, however, no research has found the pharmaceutical value of the chestnut skin for relaxing vascular smooth muscle.

Hypertension is the most common disease in cardiovascular diseases, whose causes are unknown, and the clinical syndrome is manifested by the continuous rise of arterial blood pressure in the circulation of body fluid, and has become the main cause of death in cardiovascular diseases. Along with the improvement of the substance living standard of people in modern society and the rapid work rhythm, the number of hypertension patients also rises year by year. However, although the currently available blood pressure lowering drugs can lower the blood pressure of most of patients with hypertension to normal, the control time is short, the patients are easy to relapse, and some adverse reactions occur, so that the development of safe, novel and effective blood vessel relaxing drugs is an important task for treating and improving hypertension.

Disclosure of Invention

Aiming at the problems in the prior art, the invention develops a brand new medicine for relaxing vascular smooth muscle and a preparation method thereof, the medicine is prepared by extracting cynomorium songaricum and Chinese chestnut peel in nature by a specific extraction process, and the specific extraction process ensures that the extraction rate of polysaccharide and flavone in the original medicine is more than 95 percent and the purity is more than 95 percent.

In one aspect, the invention provides a medicament for relaxing vascular smooth muscle, which is an extract of a mixture of cynomorium songaricum and Chinese chestnut peel.

Specifically, the weight ratio of the cynomorium songaricum to the chestnut skin is 1-30: 1.

specifically, the weight ratio of the cynomorium songaricum to the chestnut skin is 5-10: 1.

in another aspect, the present invention provides a method for preparing a medicament for relaxing vascular smooth muscle, comprising the steps of:

s1 crushing raw medicine: cleaning a certain proportion of cynomorium songaricum original drug and Chinese chestnut peel, removing impurities, drying, coarsely crushing, and sieving with a 100-mesh sieve to obtain original drug coarse powder; the coarse powder enters a jet mill for ultrafine powder to obtain fine powder with 5000-;

s2 immersion extraction: adding water into the fine powder prepared in the step S1, heating to 30-40 ℃, stirring for 3-4h, and then standing for 1-2 h; filtering to obtain filtrate A and filter residue B; adding the extracting solution into the filtrate A, standing at normal temperature for 3-5h, and leaching the effective components to obtain a leaching solution C;

s3 precipitation: adding chitosan solution into the leachate C obtained in the step S2, adjusting the pH of the solution to 5.5-6 by using a pH regulator, stirring for 10-30min under the condition of water bath at 40-50 ℃, stopping adding, standing at normal temperature for 1-2 days, and centrifuging to obtain supernatant D and precipitate E;

s4 enzymolysis: adding a certain amount of distilled water into the filter residue B in the step S2 and the precipitate E in the step S3, then heating to 60-80 ℃, and stirring for 1-2 hours at the temperature; cooling to room temperature, adding an enzyme preparation, stirring for 4-8h, and carrying out enzymolysis reaction;

s5 enzyme deactivation reaction: heating the solution reacted in the step S4 to 90-95 ℃, stirring for 0.5-1.5h at the temperature of 1000-2000rad/min, and filtering to obtain a filtrate F;

s6 freeze-drying at low temperature, mixing the filtrate F in the step S5 with the supernatant D in the step S3, distilling under reduced pressure, and freeze-drying at low temperature to obtain the cynomorium songaricum original drug and the chestnut peel extract.

Specifically, the extracting solution is a sodium chloride solution with the concentration of 2mol/L prepared by deionized water;

specifically, the enzyme preparation is one or more of cellulase, hemicellulase, pectinase and neutral protease.

Specifically, the adding amount of the enzyme preparation is 1-10% of the mass of the fine powder.

One or more technical schemes provided by the scheme of the invention at least have the following technical effects or advantages:

1. the cynomorium songaricum and Chinese chestnut peel extract prepared by the invention has the advantages that the cynomorium songaricum and Chinese chestnut peel extract can effectively relax vascular smooth muscle, can be used as a novel vascular smooth muscle relaxing medicine, is used for preparing a medicine for treating hypertension, and can provide a new scheme for clinically treating hypertension.

2. According to the invention, the single cynomorium songaricum extract can relax vascular smooth muscle to a certain extent, the single Chinese chestnut skin extract has no obvious relaxing effect on the vascular smooth muscle of a rat, however, the relaxing effect of the extract on the vascular smooth muscle can be greatly increased by adding a small amount of Chinese chestnut skin into cynomorium songaricum, namely, the effective components in the cynomorium songaricum and Chinese chestnut skin extracts extracted by the extraction process can synergistically relax the vascular smooth muscle.

3. According to the preparation method of the medicine, the sodium chloride solution is selected for leaching, the chitosan solution is added for flocculation and precipitation, no organic solvent is added in the whole extraction process, the extraction process is green and environment-friendly, and the safety of the extract in use is greatly improved.

4. The preparation method combines various procedures of ultrafine grinding, dipping extraction, precipitation, enzymolysis and the like, adopts a jet mill to carry out ultrafine powder in the process of crushing the raw medicine, so that the particle size of the raw medicine particles meets the requirement of aqueous solution extraction, breaks cell walls formed by cellulose in cynomorium songaricum and Chinese chestnut peels through the enzymolysis procedure on one hand, accelerates the leaching of polysaccharide substances in cells, and effectively decomposes a chitosan flocculating agent added in the S3 precipitation step on the other hand, so that effective components in the precipitate E in the S3 can be extracted, the extraction rate of the effective components of the raw medicine is further improved, the extraction rate of the polysaccharide and the flavone in the raw medicine is more than 95 percent, and the purity of the polysaccharide and the flavone is more than 95 percent.

Detailed Description

Aiming at the problems in the prior art, the invention develops a brand new medicine for relaxing vascular smooth muscle and a preparation method thereof, the medicine is prepared by extracting cynomorium songaricum and Chinese chestnut peel in nature by a specific extraction process, and the specific extraction process ensures that the extraction rate of polysaccharide and flavone in the original medicine is more than 95 percent and the purity is more than 95 percent.

In order to better understand the technical solution of the present invention, the technical solutions will be described in detail with reference to the specific embodiments, and it should be understood that the specific features in the embodiments and examples of the present invention are detailed descriptions of the technical solution of the present invention, but not limitations of the technical solution of the present invention, and the technical features in the embodiments and examples of the present invention may be combined with each other without conflict.

First, an embodiment

The drugs of examples 1 to 5 of the present invention and the drugs of comparative examples 1 and 2 were prepared according to the components and ratios of table 1, respectively.

TABLE 1 examples and comparative examples the pharmaceutical compositions and ratios

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION	Cynomorium (g)	Chinese chestnut skin (g)
			Example 1	0.50	0.50
Example 2	0.83	0.17
			Example 3	0.89	0.11
Example 4	0.91	0.09
			Example 5	0.97	0.03
Comparative example 1	1.00	0
			Comparative example 2	0	1.00

The extracts of examples 1 to 5 of the present invention and comparative examples 1 to 2 were obtained by the following steps:

s1 crushing raw medicine: cleaning a certain proportion of cynomorium songaricum original drug and Chinese chestnut peel, removing impurities, drying, coarsely crushing, and sieving with a 100-mesh sieve to obtain original drug coarse powder; the coarse powder enters a jet mill for ultrafine powder to obtain fine powder with 5000-;

s2 immersion extraction: adding water into the fine powder prepared in the step S1, heating to 30 ℃, stirring for 4 hours, and then standing for 2 hours; filtering to obtain filtrate A and filter residue B; adding the extracting solution into the filtrate A, standing at normal temperature for 5h, and leaching the effective components to obtain a leaching solution C; wherein the extracting solution is a sodium chloride solution with the concentration of 2mol/L prepared by deionized water, and the weight ratio of the original medicine fine powder to the sodium chloride solution is 1: 2;

s3 precipitation: adding 1.2g/L chitosan solution into the leaching solution C obtained in the step S2, adjusting the pH of the solution to 5.5 by using a pH regulator, stirring for 30min under the condition of 50 ℃ water bath, stopping adding, standing for 1 day at normal temperature, and centrifuging to obtain supernatant D and precipitate E;

s4 enzymolysis: taking the filter residue B in the step S2 and the precipitate E in the step S3, adding a certain amount of distilled water, wherein the amount of the distilled water is 3 times of the total mass of the filter residue B and the precipitate E, heating to 80 ℃, and stirring for 2 hours at the temperature; cooling to room temperature, adding an enzyme preparation, stirring for 8h, and carrying out enzymolysis reaction; the enzyme preparation is cellulase, and in other embodiments, the enzyme preparation can also be one or a mixture of more of hemicellulase, pectinase and neutral protease; in this example, the amount of the enzyme preparation added was 1% by mass of the fine powder obtained in the step of pulverizing the crude drug of S1.

S5 enzyme deactivation reaction: heating the solution reacted in the step S4 to 95 ℃, stirring for 1h at the temperature of 1000rad/min, and filtering to obtain filtrate F;

s6 freeze-drying at low temperature, mixing the filtrate F in the step S5 with the supernatant D in the step S3, distilling under reduced pressure, and freeze-drying at low temperature to obtain the cynomorium songaricum original drug and the chestnut peel extract.

In the extraction process, a sodium chloride solution is selected for leaching, a chitosan solution is added for flocculation and precipitation, no organic solvent is added in the whole extraction process, and the extraction process is green and environment-friendly and greatly improves the medication safety of the extract. In addition, in the process, various procedures such as ultrafine grinding, dipping extraction, precipitation, enzymolysis and the like are combined, an airflow pulverizer is adopted to carry out ultrafine powder in the process of crushing the raw medicines, so that the particle size of the raw medicines meets the requirement of aqueous solution extraction, cell walls formed by cellulose in cynomorium songaricum and Chinese chestnut peels are broken through the enzymolysis procedure on one hand, the leaching of polysaccharide substances in cells is accelerated, and on the other hand, a chitosan flocculating agent added in the S3 precipitation step is effectively decomposed, so that effective ingredients in the precipitate E in the S3 can be extracted, the extraction rate of the effective ingredients of the raw medicines is further improved, and the specific extraction process enables the extraction rate of polysaccharide and flavone in the raw medicines to be 97%, and the purity to be more than 96%. Wherein the extraction rate is the percentage of the target substance extracted from the unit mass of the raw medicines in the total mass of the target substance in the unit mass of the raw medicines.

Second, Experimental example

1 test reagent

1.1 preparation of high calcium solutions (PSS)

Taking 135mmol NaCl, 5mmol KCl and 2mmol CaCl₂10mmol of glucose, 10mmol of 4-hydroxyethylpiperazine ethanesulfonic acid and 1mmol of MgCl 2.6H2O were dissolved in 800mL of sterile water, the pH of the solution was adjusted to 7.40 with 1mol/L of NaOH, and the volume was adjusted to 1000 mL.

1.1 preparation of high Potassium solution

Taking 135mmol NaCl, 140mmol KCl and 2mmol CaCl₂10mmol of glucose, 10mmol of 4-hydroxyethylpiperazineethanesulfonic acid and 1mmol of MgCl 2.6H2O were dissolved in 800mL of sterile water, and the solution pH was adjusted to 7.40 with 1mol/L of NaOH and made to 1000 mL.

1.3 preparation of the drug solution

0.3g of each of the drugs of examples 1 to 5 was dissolved in 3mL of a high calcium solution, and the pH was adjusted to 7.40 to prepare solutions of the drugs of examples.

0.3g of each of the drugs of comparative examples 1 and 2 was dissolved in 3mL of a high calcium solution, and the pH was adjusted to 7.40 to prepare solutions of the drugs of comparative examples.

2, preparation and pretreatment of blood vessels:

rats were purchased and housed for 2 days to acclimatize. Selecting a rat with normal physiological state, and killing the rat for experiment by using a cervical dislocation method. After the rats were fixed, all ventral surfaces were sprayed with sterile alcohol. Then, the abdomen of the rat was cut with sterile surgical scissors, and after the thoracic cavity and the abdomen of the rat were cut, the visceral part of the rat was slowly opened with forceps, so that the thoracic aorta of the rat was located above the spine, and the aorta in the thoracic cavity was cut from the junction of the heart and the aorta with the intestinal artery, respectively, and then placed in the PSS solution.

The computer and oxygen supply valve required for the experiment were opened and then the bubbles were adjusted to maintain the rate at 3-4/sec. Before the experiment begins, the tension energy conversion system should be started to preheat for half an hour. Then, the BL-420 biofunctional system software is turned on, and all the measurement items of the four channels are adjusted to be tension, and the sampling rate is adjusted to be 100Hz, and then the device is standby. Then the thermostatic waterbath device is opened, and the water temperature is adjusted to 37 ℃. And preheating a proper amount of the PSS solution for standby.

The isolated aorta was fixed under a dissecting microscope, and then peripheral tissues of the isolated aorta were removed by Venus scissors, and blood remaining in the blood vessel was washed out using a 1000mL pipette. After the isolated aorta is treated, the aorta is cut into segments each about 6mm long, and 4 segments are cut. Then, the small section of the blood vessel is connected with the triangular hook, then is connected to the tension transducer, and is slowly placed in a 6mL PSS solution (preheated at 37 ℃) water bath which is kept at a constant temperature and keeps a proper amount of oxygen supply, and the position of the hook is slowly adjusted by rotating a fine adjustment knob, so that the blood vessel ring is in a resting state. The above steps are repeated to load four channels of tissues into the water bath. After the position of the hook and the size of the air bubble are adjusted again, the front loads of the four tissues are adjusted to 300mg, and then timing is started for balancing. During the equilibration period, the PSS solution was changed every 15 minutes for a total of 4 changes, for a total of 1 hour of equilibration. After the balance is finished, pre-stimulation treatment is carried out on isolated aorta by using 140mmol/L high potassium solution, and after tissue activity is fully stimulated, solution exchange elution is carried out by using PSS solution until the baseline is recovered.

After connecting the treated rat vascular ring with the triangular hook, connecting the sample to a tension transducer, then placing the tension transducer into a constant-temperature water bath with 6ml PSS, changing the PSS solution in the water bath into the preheated PSS solution, adjusting the oxygen in the water bath to be proper, and adjusting the front load of the channel to 300 mg. After adjustment of the bubbles and preload, equilibration can begin with a change of the PSS solution every 15 minutes for a total of four changes. After the time for balancing is enough for one hour, pre-stimulation treatment is carried out by using a high potassium solution with the concentration of 140mmol/L, after the muscle tension of the blood vessel is in the optimal contraction state, the high potassium solution is eluted by using a PSS solution, after the muscle tension of the blood vessel is recovered to a base line of 300mg, the high potassium solution is added to pre-contract the blood vessel, the tension is again in the optimal contraction state, a blood vessel smooth muscle model of a hypertensive is simulated, and a dosing test is started.

3 testing the vasodilating action of drugs on vascular smooth muscle

First, preshrinking treatment was performed on isolated vascular smooth muscle with high potassium solution, and after reaching a steady state, 50. mu.L of solutions of the drugs of comparative examples 1 and 2 and examples 1 to 5 were added to the bath, respectively, and changes in vascular tone were detected.

To compare the vasodilation of the drug solutions on rat vascular smooth muscle, two more control experiments were set up: the first is a blank control group, namely, the vascular smooth muscle of the rat is not treated; the other is a high potassium solution group, namely only adding the high potassium solution to stimulate the contraction of vascular smooth muscle.

The results of the experiments on the vasodilation of vascular smooth muscle in rats from different experimental groups are shown in table 2 below.

TABLE 2 Experimental results of different experimental groups on vasodilation of vascular smooth muscle of rat a

Experimental group	Vascular tension (mN) of rat	Diastolic ratio (%)
			Blank group	0.4	-
High potassium solution group	9.8	-
			Comparative example 1	6.2	38.3
Comparative example 2	10.1	-3.0
			Example 1	4.6	55.3
Example 2	1.9	84.0
			Example 3	0.5	98.9
Example 4	0.9	94.7
			Example 5	1.6	87.2

a rat vascular tension (mN) is vascular smooth muscle tension per unit weight;

the relaxation rate (rat vascular smooth muscle tension before drug addition-rat vascular smooth muscle tension after drug addition)/(rat vascular smooth muscle tension after high potassium solution addition-rat vascular smooth muscle tension before high potassium solution addition) × 100%, wherein rat vascular smooth muscle tension after drug addition is rat vascular smooth muscle tension after 10min drug addition.

From table 2 the following conclusions can be drawn:

1. the blank experiment group does not process the air pipe at all, and the tension of the air pipe is stabilized at 0.50 mN; the addition of only high potassium solution stimulated vascular smooth muscle contraction, the ex vivo vascular tone increased from 0.4mN to 9.8mN, and vascular tone was effectively maintained for 60 minutes without the addition of the drug of comparative example or example.

2. Using the drugs of comparative example 1 and comparative example 2 for vasodilation of vascular smooth muscle, the vasodilation rates of vascular smooth muscle in rats were 38.3% and-3.0%, respectively. From this result, it can be seen that cynomorium songaricum extract alone can relax vascular smooth muscle to some extent, whereas chestnut peel extract alone has no significant relaxing effect on rat vascular smooth muscle, even stimulating further contraction of rat vascular smooth muscle to some extent.

3. The extract of the mixture of cynomorium songaricum and chestnut peels of examples 1-5 has a diastolic rate for relaxing vascular smooth muscle of 55.3 at minimum and 98.9 at maximum. Compared with the method for extracting the cynomorium songaricum and the Chinese chestnut peel by using the cynomorium songaricum extract alone, the addition of the Chinese chestnut peel in the original medicine greatly promotes the vasodilation effect of the medicine on vascular smooth muscle, and the result shows that the active ingredients in the cynomorium songaricum and Chinese chestnut peel extract extracted by the extraction process can synergistically relax the vascular smooth muscle.

4. In the examples 1 to 5, cynomorium songaricum and Chinese chestnut peels in different proportions are selected, the inhibition effect on the contraction of vascular smooth muscle caused by a high potassium solution is different, the vasodilation rate of the medicine prepared from the extract of the cynomorium songaricum and the Chinese chestnut peels in the weight ratio of 8:1 in the example 3 is the maximum, and is 98.9%, which indicates that the optimal proportion of the cynomorium songaricum to the Chinese chestnut peels is 8: 1.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is intended to include such modifications and variations.

Claims (3)

1. An application of an extract prepared from a cynomorium songaricum original drug and chestnut peels in preparing a medicine for relaxing vascular smooth muscle is characterized in that the preparation method of the extract comprises the following steps:

s1 crushing raw medicine: cleaning a cynomorium songaricum original drug and Chinese chestnut peel in a weight ratio of 1-30: 1, removing impurities, drying, coarsely crushing, and sieving with a 100-mesh sieve to obtain original drug coarse powder; the coarse powder enters a jet mill for ultrafine powder to obtain fine powder with 5000-8000 meshes;

s2 immersion extraction: adding water into the fine powder prepared in the step S1, heating to 30-40 ℃, stirring for 3-4 hours, and then standing for 1-2 hours; filtering to obtain filtrate A and filter residue B; adding the extracting solution into the filtrate A, standing at normal temperature for 3-5 hours, and leaching effective components to obtain a leaching solution C; the extracting solution is a sodium chloride solution with the concentration of 2mol/L prepared by deionized water;

s3 precipitation: adding a chitosan solution into the leachate C obtained in the step S2, adjusting the pH of the solution to 5.5-6 by using a pH regulator, stirring for 10-30min under the condition of 40-50 ℃ water bath, stopping adding, standing at normal temperature for 1-2 days, and centrifuging to obtain a supernatant D and a precipitate E;

s4 enzymolysis: adding a certain amount of distilled water into the filter residue B in the step S2 and the precipitate E in the step S3, heating to 60-80 ℃, and stirring for 1-2 hours at the temperature; cooling to room temperature, adding an enzyme preparation, stirring for 8 hours, and carrying out enzymolysis reaction; the enzyme preparation is one or more of cellulase, hemicellulase, pectinase and neutral protease;

s5 enzyme deactivation reaction: heating the solution reacted in the step S4 to 90-95 ℃, stirring for 0.5-1.5h at the temperature of 1000-2000rad/min, and filtering to obtain a filtrate F;

s6 freeze-drying at low temperature, mixing the filtrate F in the step S5 with the supernatant D in the step S3, distilling under reduced pressure, and freeze-drying at low temperature to obtain the cynomorium songaricum original drug and the chestnut peel extract.

2. The use of claim 1, wherein the enzyme preparation is added in an amount of 1% to 10% by mass of the fine powder.

3. The use according to claim 1, wherein the weight ratio of cynomorium songaricum to chestnut skin is 5-10: 1.