CN1589845A - Preparation technology and application of total phenolic acid fraction of hawthorn - Google Patents

Abstract

The invention provides a technology for extracting total phenolic acid parts of hawthorn from hawthorn leaves or hawthorn fruits. The part can be made into oral preparation and injection preparation alone or together with other pharmaceutically acceptable adjuvants, and is suitable for preventing and treating various ischemic cardiovascular and cerebrovascular diseases, senile dementia, and hyperlipidemia.

Description

Preparation technology and application of total phenolic acid fraction of hawthorn

Technical field:

The invention belongs to the field of medicine, in particular to the preparation process and preparation of the total phenolic acid parts isolated from hawthorn leaves or fruits and its potential application in medicine and health food.

Background technique:

Cardiovascular and cerebrovascular diseases are one of the diseases with higher incidence in the world. With the development of social economy and the improvement of people's living standards, the prevalence of cardiovascular and cerebrovascular diseases is also increasing year by year. According to relevant reports, in 2001, 56 million people died in the world. The two leading causes, coronary heart disease and stroke, are responsible for 7 million and 5.5 million deaths, respectively. Because of this, countries all over the world attach great importance to the research and prevention of cardiovascular and cerebrovascular diseases. But so far, although there are many cardiovascular and cerebrovascular drugs in clinical application, they have played a good role in the treatment of cardiovascular and cerebrovascular diseases, but they have not been able to control the growth of the prevalence of cardiovascular and cerebrovascular diseases. It reflects that continuous efforts are needed to develop more effective new drugs and put them into clinical application. At the same time, due to the unbalanced economic development, the current cost of cardiovascular and cerebrovascular treatment prevents many patients from receiving good treatment. It is also necessary to develop new drugs that can meet the needs of the majority of patients and have a lower price, so as to contribute to the prevention and treatment of cardiovascular and cerebrovascular diseases. .

Hawthorn is a plant of the genus Hawthorn in the family Rosaceae. There are more than 280 species of this genus in the world, and 19 species in China. Its fruit is a commonly used traditional Chinese medicine, and it is also a variety of Chinese Pharmacopoeia. According to the "Compendium of Materia Medica": This product is "sweet and sour, warm in taste, relieves food accumulation, and nourishes the spleen". Its fruit contains a variety of physiologically active ingredients, such as flavonoids, ursolic acid, oleanolic acid, chlorogenic acid, organic acids, minerals and trace elements, etc., which have a variety of pharmacological effects and are important biological resources in my country. , It is listed as a variety of medicine and food by the country. Since the 1950s, researchers have discovered that hawthorn leaves also have various physiological activities. The most researched is the total flavonoids of hawthorn leaves. At present, there are several extracts of total flavonoids from hawthorn leaves and preparations of total flavonoids from hawthorn leaves (such as Yixindone tablets and capsules), which are mainly used for the treatment of coronary heart disease and hyperlipidemia. treat.

At present, it is believed that the total flavonoids in hawthorn play a pharmacological role, and there are also some patents on the separation and extraction of total flavonoids in hawthorn (such as application number: 01103986.8 and application number: 01118628.3), but we have found through research that when the hawthorn After the flavonoid content in the total flavonoid extract was further increased, the activity decreased instead. This suggests that the pharmacological effects of hawthorn may be non-flavonoid components in hawthorn.

Invention content:

The invention provides a preparation method for the total phenolic acid part of hawthorn isolated from the leaves or fruits of the hawthorn plant, and the potential application value of the part in medicine and health food.

According to the following method, we obtained the total phenolic acid fraction of hawthorn. That is: the hawthorn leaves or fruits are extracted with water-containing alcohol, the extract is concentrated under reduced pressure, placed for precipitation, the supernatant is subjected to macroporous adsorption resin chromatography, and the pH value of the water-eluted part is adjusted to 1-2, then extracted with ethyl acetate , the extract was concentrated under reduced pressure and dissolved in water, then chromatographed on a macroporous adsorption resin, eluted with water, followed by elution with water-containing alcohol or water-containing acetone, the eluent was concentrated under reduced pressure, spray-dried to obtain a yellow powder, namely It is the total phenolic acid part of hawthorn.

The medicinal materials used in the present invention can be hawthorn leaves or hawthorn fruits, but the total phenolic acid content in hawthorn leaves is higher than that of hawthorn fruits, and because the sugar and starch contents in hawthorn fruits are high, the actual use of hawthorn leaves is better than that of hawthorn fruits. .

The water-containing alcohol used in the process can be methanol, ethanol and other C1-C5 lower aliphatic alcohols. The concentration of water-containing alcohol used for extraction is 50%-90%, and 20%-60% water-containing alcohol or water-containing acetone is used for eluting the macroporous adsorption resin.

The macroporous adsorption resin used in this process includes various non-polar, weak polar, medium polar and polar domestic or imported macroporous adsorption resins.

The above-mentioned effective parts contain phenolic acid components such as caffeic acid, caffeoylquinic acid compounds, and caffeoyltharanic acid compounds. The content of the chemical components recognized by HPLC accounts for more than 50% of the effective parts.

The effective parts can be used to make various clinical preparations: injections, injection powders, oral tablets, granules, capsules, dropping pills, oral liquids, etc.

The above preparation can be used in the prevention and treatment of various ischemic cardiovascular and cerebrovascular diseases, senile dementia and hyperlipidemia.

The following animal experiments have further illustrated the effective effect of the total phenolic acid parts of hawthorn of the present invention:

1. Acute toxicity test

40 healthy Kunming mice, half male and half female, weighing 18-22 g; 40 healthy SD rats, half male and half male, weighing about 150 g.

The total phenolic acid fraction of hawthorn was given orally, and the LD50 value could not be measured, so the maximum tolerated dose (MTD) to mice and rats was determined in this experiment. Rats and mice were administered by gavage according to the maximum administration volume, once in the morning and in the afternoon. Before the administration, the mice were fasted for 5 hours and the rats were fasted for 12 hours.

The results of the experiment showed that taking 2.2g/kg of the total phenolic acid part of hawthorn orally in mice once a day (divide into two doses), would cause the animal's autonomic activities to decrease, appetite to decrease, and body weight to grow slowly. The above reactions recovered within two weeks. No animals were killed. It shows that the maximum tolerated dose of total phenolic acid parts of hawthorn oral administration in mice is greater than 2.2g/kg (equivalent to 550 times of clinical dose).

Oral administration of 2.8g/kg of the total phenolic acid fraction of hawthorn to rats once a day (in two divided doses) will cause the animals to reduce their autonomous activities and appetite, and the above reactions will recover within two weeks without causing the animals to die. It shows that the maximum tolerated dose of total phenolic acid parts of hawthorn oral administration in mice is greater than 2.8g/kg (equivalent to 700 times of clinical dose). It shows that the total phenolic acid part of hawthorn has higher security.

2. Effects on focal cerebral ischemia in rats

Get 70 male SD rats, body weight 300～400g, divide them into 6 groups at random according to body weight, be respectively hawthorn total phenolic acid high-dose group (8mg/kg), middle-dose group (4mg/kg), low-dose group (2mg/kg). /kg), the positive control Nimo is the same group (1.2mg/kg), the sham operation group and the ischemia model control group (both give the same amount of 0.5% CMC-Na).

7 days before the experiment, ig once a day, and the last dose was given 60 minutes before ischemia on the 7th day. The rat model of middle cerebral artery occlusion (MCAO) was established by internal carotid artery suture method. Rats were anesthetized with 10% chloral hydrate (300 mg/kg) ip, and placed on the supine constant temperature operating table, a midline incision was made on the neck to expose the right common carotid artery, and the digastric and sternocleidomastoid muscles were pulled outward, Ligate and cut off the branches of the external carotid artery: the inferior occipital artery and the superior thyroid artery, ligate at the distal end of the external carotid artery, cut off the external carotid artery to free its main trunk, and then separate the internal carotid artery Arteries, loosen the root of the external carotid artery with a silk thread, and clamp the common carotid artery and the internal carotid artery. Pass the nylon thread through the main incision of the external carotid artery, and slowly push it toward the cranial direction of the internal carotid artery. With the bifurcation of the common carotid artery as a mark, resistance is felt when it is advanced about 20 mm, and it reaches the thinner anterior cerebral artery. All sources of blood supply to the MCA were blocked, and the loose buckle at the root of the external carotid artery was tightened. After 1 hour, the nylon thread was pulled out, and the arterial stump was tied tightly. The skin was sutured to complete the focal cerebral ischemia-reperfusion model induced by MCAO. After the rats in the sham operation group were anesthetized, only the bifurcation of the internal and external carotid arteries was exposed, and the middle cerebral artery was not occluded. 24 hours after operation, the behavioral deficits of the animals were graded and scored according to Bederson's method. After 24 hours of MCAO, the rats were sacrificed by decapitation, the whole brain was taken out, the left and right brains were cut separately, and weighed. Coronal slices were made at the optic chiasm of the right brain and 2 mm before and after it, and the brain slices were incubated in 1% TTC solution at 37°C for 25 minutes in the dark, and the pale area (infarction area) and non-pallid area (normal area) were separated, and the infarction was calculated. Percentage. The stained brain tissue was dried, and the water content of the brain was calculated according to the wet weight of the brain.

The experimental results showed that: the results showed that after rats ig hawthorn total phenolic acids 2, 4, 8 mg/kg, the changes in animal behavior and infarction range were significantly improved compared with those in the normal saline control group, and the behavioral scores were reduced by 43.25% after 24 hours (p <0.01), 60.70 (p<0.001), 63.88 (p<0.001); the average size of cerebral infarction was reduced by 30.2% (p<0.01), 47.3 (p<0.01), 50.9 (p<0.001). The high-dose group had the same effect strength as the same group of Nimo. 2) Effects on biochemical indicators of focal cerebral ischemia rats 70 male rats (300-400 g) were divided into groups for administration, and the operation method was the same as before. After 24 hours of MCAO, the rats were executed by decapitation, the brain was removed, and the cerebellum was removed. The brain was made into 10% homogenate with physiological saline, and the contents of superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA) and lactic acid (LA) in the brain tissue were determined. The experimental results show that the total phenolic acids of hawthorn can reduce the content of MDA and LA in the brain tissue of rats with reperfusion injury, indicating that the degree of tissue ischemia, hypoxia and peroxidation is significantly inhibited; at the same time, the content of SOD and GSH increases, reflecting the The drug improves the body's antioxidant capacity and ability to scavenge free radicals.

3. Therapeutic effect and hemodynamic effects on myocardial ischemia in dogs

30 dogs were divided into 6 groups. Left anterior descending coronary artery (LAD) sham operation normal saline group, LAD blocking normal saline group, LAD blocking propranolol group, LAD blocking hawthorn total phenolic acid 3 dose groups (2, 4, 8mg/kg) . iv pentobarbital sodium 30mg/kg anesthetized, tracheal intubation for artificial respiration, thoracotomy in the left fourth intercostal space, left circumflex coronary artery root was separated, electromagnetic flowmeter probe was placed, and blood flow was recorded. Common carotid artery cannulation was used to record common carotid blood pressure. Left ventricular pressure was measured from the apical cannula to the left ventricle, and the maximum rate of change of left ventricular end-diastole and left ventricular pressure was determined. The heart rate was measured with a heart rate meter, and the lead II electrocardiogram was observed at the same time. After 10 minutes of postoperative stabilization, the above indicators on the RM-6000 physiological recorder are the data before administration. After femoral vein injection, the LAD was ligated 5 minutes later, and the above-mentioned indicators (post-drug data) were recorded at 1, 2, 5, 10, 20, 30, 60, 90, 120, 240, and 360 minutes after the LAD was blocked. Before administration and 20, 40, 60, 90, 120 minutes after administration, blood was collected from coronary sinus and common carotid artery at the same time, and blood oxygen content was measured with CORNING178 blood gas analyzer. Calculate myocardial blood flow, coronary resistance, myocardial oxygen consumption, myocardial oxygen consumption index, and myocardial oxygen utilization.

The test results showed that infusion of 2 mg/kg of the total phenolic acid part of hawthorn had no significant effect on hemodynamic indicators; 4 and 8 mg/kg could significantly increase myocardial blood flow, reduce coronary resistance, and significantly reduce myocardial oxygen consumption and Myocardial oxygen consumption index, reducing myocardial oxygen utilization.

4. Effect on quail hyperlipidemia and atherosclerosis

Sixty healthy male quails were randomly divided into 6 groups, 10 in each group. Normal feed group; high-fat model group: fed high-fat feed (containing 79% basic feed, 1% cholesterol, 20% lard); hawthorn total phenolic acid parts small, medium and high dose groups 40, 80 and 160mg/kg .d; lovastatin positive control group 4mg/kg.d. Except that the normal control group was given normal feed and the high-fat model group was given high-fat feed, each group was given different drugs while being given high-fat feed. Animals in each group were housed in separate cages, fed quantitatively, and administered once a day. After continuous administration for 60 days, blood was collected from the jugular vein, and serum TC, TG, and HDL-C were measured respectively. LDLC-C was calculated according to the formula LDL-C=TC- (1/2.2TG+HDLC-C) was calculated, and then the animals were sacrificed, the aorta and liver were taken out, and the lesion of the arterial wall and the degree of fatty degeneration of the liver were observed.

The experimental results showed that: 1) Effects on blood lipids After the quails were fed with the total phenolic acid parts of hawthorn for 60 days, the middle dose group (80mg/kg.d) could significantly reduce the contents of serum TC, TG and LDL-C (p<0.05), The reduction percentages were 36.21%, 29.18% and 35.27%, respectively. The high-dose group (160mg/kg.d) can significantly reduce the content of serum TC, TG and LDL-C (p<0.01), and the percentage reductions are 47.29%, 43.38% and 45.87% respectively. At the same time, it can make TC/HDL-C The ratio decreased significantly (p<0.05), and the percentage decrease was 36.92%. 2) Effects on the formation of atherosclerotic plaques After quails were ingested with the total phenolic acids of hawthorn, the plaque area percentage and average gray level could be reduced by 7.3% to 29.8% and 8.4% to 21.9% in the three different dose groups, respectively. The effect of reducing plaque area percentage and average gray level in high dose group was very significant (p<0.01). Observation under the microscope also showed that the thickness of the aortic intima in the total phenolic acid part of hawthorn group was higher than that in the lipid model group, and the number of foam cells appeared was also less. 3) Effects on the liver After quails were given 80 mg/kg and 160 mg/kg of total phenolic acids from hawthorn for 60 days, the liver weight coefficient decreased significantly (p<0.01). Microscopic examination of liver tissue sections also showed that the degree of hepatic steatosis in the treatment group was significantly lower than that in the lipid model group. The above experimental results show that the total phenolic acid fraction of hawthorn has obvious effects of lowering blood fat and preventing atherosclerosis.

Compared with the prior art, the present invention has the following advantages or positive effects:

1. Compared with some other cardiovascular and cerebrovascular drugs, hawthorn leaves or fruits have sufficient sources of raw materials, cheap prices, and low cost.

2. In the prior art, it is generally believed that the active part of hawthorn is the total flavonoids therein. Our results showed that the activity of the total phenolic acid fraction of hawthorn was stronger than that of the total flavonoid fraction of hawthorn.

3. The existing hawthorn extracts and preparations are all aimed at the total flavonoids of hawthorn. The invention provides the preparation process, quality standard and application in medicine of the total phenolic acid fraction of hawthorn.

4. The quality standards of the existing hawthorn extracts and preparations are formulated by measuring the content of total flavonoids, and the determination methods are mostly determined by the ultraviolet method, which is inaccurate due to the large interference of the ultraviolet method. And the present invention uses HPLC method to measure, wherein the content of the active ingredient that can recognize reaches more than 50%.

5. Due to the difference in ingredients, the existing hawthorn preparations have poor water solubility, and generally can only be made into oral preparations, such as tablets, capsules, and dripping pills. However, the total phenolic acid fraction of hawthorn obtained in the present invention can be made into various preparations including injection forms.

Description of drawings :

Fig. 1 is the flow chart of the extraction process of the total phenolic acid parts of hawthorn according to the present invention.

Specific implementation methods :

Embodiment 1: Preparation of effective part

Take 100kg of dried hawthorn leaves, grind them into coarse powder, and extract with 10 times the amount of 60% ethanol under reflux for three times, combine the extracts, concentrate under reduced pressure to 50L, place for precipitation, centrifuge, and pass the supernatant through AB-8 macroporous adsorption resin Chromatography, the water eluted part adjusted the pH value to 1, extracted with ethyl acetate, the extract was concentrated under reduced pressure, dissolved in water, and then chromatographed by AB-8 macroporous adsorption resin, eluted with water until the pH value was 3 to 5, continue to elute with 60% diacetone, concentrate the eluent under reduced pressure, and spray dry to obtain 0.52 kg of light yellow powder, which is the total phenolic acid fraction of hawthorn (the yield is 0.52% based on the crude drug amount). Through assay, the purity is 84.79%.

Embodiment 2: Preparation of effective fraction

Take 100kg of dried hawthorn fruit, crush it, extract it by percolation with 20 times the amount of 80% methanol, combine the percolation solution, concentrate it under reduced pressure to 50L, place it for precipitation, centrifuge, and the supernatant is chromatographed by D ₁₀₁ macroporous adsorption resin. After adjusting the pH value of the water-eluted part to 2, extract it with ethyl acetate, concentrate the extract under reduced pressure and dissolve it in water, then go through AB-8 macroporous adsorption resin chromatography, and elute with water until the pH value is 3-5 , continue to elute with 20% dilute ethanol, the eluate is concentrated under reduced pressure, and spray-dried to obtain 0.42kg of light yellow powder, which is the total phenolic acid part of hawthorn (the yield is 0.42% according to the crude drug amount). Through content determination, the purity is 67.25%.

Embodiment three: the preparation of tablet

Take 100g of the part of the present invention, 80g of starch, and 5g of dextrin and mix evenly, add 10% starch slurry to make soft material, granulate with a 14-mesh nylon screen, ventilate and dry at 60-70°C, granulate with a 16-mesh sieve, add stearic acid 1.5g of magnesium and 5g of sodium carboxymethyl starch were mixed evenly, pressed into 1000 tablets, and coated. Each tablet contains 100 mg of the site of the present invention. Adults take 2 to 5 times a day, 1 to 10 tablets each time.

Embodiment four: the preparation of oral liquid

Get 20g of parts of the present invention, mix with honey 30g, sucrose 50g, sodium benzoate 2g and distilled water 300ml, heat to 85～90°C, stir to dissolve, keep warm for 30min, filter, add water to dilute the filtrate to 1000ml, stir well, potting ( 10ml per bottle), sterilized. Adults take 2 to 5 times a day, 1 to 5 sticks each time.

Embodiment five: the preparation of injection

Take 50g of the site of the present invention, add an appropriate amount of water for injection to dissolve, add a prepared amount of 0.02% activated carbon and stir for 5-10min, filter, dilute the filtrate to about 10L, add sodium chloride to adjust the osmotic pressure to isotonicity, and adjust the pH to 5.5- 7.5, ultrafiltration, potted into 1000 tubes (10ml/tube), sterilized at 100°C for 30 minutes. Intravenous or intramuscular injection for adults, 1 to 2 times a day, 1 to 5 tubes each time.

Embodiment six: the preparation of powder injection

Take 50g of the part of the present invention, add an appropriate amount of water for injection to dissolve, add the prepared amount of 0.02% activated carbon, stir for 5-10min, filter, dilute the filtrate to 1L, adjust the pH to 5.5-7.5, ultra-filter, spray dry, dry powder aseptically separate Just install it. Each tube is 50 mg, add an appropriate amount of water for injection to dissolve before use, dilute with 100-500 ml of sodium chloride infusion, and infuse slowly intravenously. Adults take 1-2 times a day, 1-5 sticks each time.

Claims (9)

1. A preparation process for extracting effective parts from hawthorn leaves or fruits, which is characterized in that the total phenolic acid parts thereof are extracted from hawthorn leaves or fruits. 2. The total phenolic acid part according to claim 1, characterized in that its extraction process can be obtained by the following extraction method: Hawthorn leaves or fruits are pulverized, extracted with water-containing alcohol, the extract is concentrated under reduced pressure, placed for precipitation, and the supernatant The liquid is subjected to macroporous adsorption resin chromatography, and the pH value of the water-eluted part is adjusted to 1-2, and then extracted with ethyl acetate. Elute with water-containing acetone, concentrate the eluent under reduced pressure, and spray dry to obtain a light yellow powder, which is the total phenolic acid fraction of hawthorn. 3. The total phenolic acid fraction of hawthorn according to claims 1 and 2, characterized in that the effective fraction contains caffeic acid, caffeoylquinic acid compounds, and caffeoyltharabinic acid compounds. 4. the extraction method of total phenolic acid part of hawthorn according to claim 2, is characterized in that said alcohol is methanol, ethanol and other C1～C5 lower fatty alcohols. 5. The method for extracting the total phenolic acid parts of hawthorn according to claim 2, characterized in that the concentration of the water-containing alcohol used when extracting the medicinal materials is 50% to 90%. 6. The method for extracting total phenolic acid parts of hawthorn according to claim 2, characterized in that the concentration of water-containing alcohol or water-containing acetone used when eluting the macroporous adsorption resin is 20% to 60%. 7. The total phenolic acid fraction of hawthorn according to claims 1 and 2, characterized in that the total phenolic acid content is above 50%. 8. The total phenolic acid part of hawthorn according to claims 1 and 2, characterized in that the part can be made into tablets, capsules, dripping pills, granules according to existing methods after the parts are used alone or in combination with other pharmaceutically approved adjuvants Various oral and injection dosage forms such as medicaments, oral liquids, and injections. 9. The application of the hawthorn total phenolic acid preparation according to claim 8 in the prevention and treatment of various ischemic cardiovascular and cerebrovascular diseases, senile dementia, and hyperlipidemia.