CN107184625A - The method and purposes of alkaloid valid target are extracted from catharanthus roseus, stem branch or herb - Google Patents

Abstract

The invention relates to a method and application for extracting effective parts of alkaloids from periwinkle flowers, stems or whole plants. Adding water-containing alcohol solution to cold soaking, percolation or heat reflux extraction of periwinkle flowers, stems or whole plants, and filtering After that, recover under reduced pressure until there is no alcohol smell, add water or alcohol solution to the concentrated extract, filter, and the filtrate is passed through an adsorption column filled with adsorption materials, and the adsorption column is washed with water before elution until the effluent is colorless ~75% alcohol solution was used for elution, the eluent was collected, and the solvent was recovered under reduced pressure to obtain a thick paste, which was then dried to obtain the active part of the alkaloid. The method does not use toxic organic solvents, does not pollute the environment, is less time-consuming, has high efficiency, simple extraction process, low cost, can carry out industrialized production, and meets the requirements of environmental protection. The effective parts of the extracted alkaloids can be used as drugs for treating or preventing neurodegenerative diseases such as senile dementia and cardiovascular and cerebrovascular diseases such as hypertension.

Description

Method and application of extracting effective parts of alkaloids from periwinkle, stems or whole plants

technical field

The invention belongs to the technical field of medicine, and relates to a method and application for extracting effective parts of alkaloids from periwinkle flowers, stems or whole plants, and in particular to a method for extracting effective parts of alkaloids from periwinkle flowers, stems and branches or whole plants. Use of its effective parts in the preparation of medicines for treating or preventing neurodegenerative diseases such as senile dementia and cardiovascular and cerebrovascular diseases such as hypertension.

Background technique

With the aging of the social population, Alzheimer's disease, cerebrovascular disease, and malignant tumors have been listed as the three major diseases of the elderly. Alzheimer's disease (AD) is a primary brain degenerative disease of unknown etiology, and it is a frequently-occurring and common disease in the elderly. The disease was first described by Alois Alzheimer in 1907. The etiology is still unclear. Clinically, it is characterized by the loss of progressive memory and acquired knowledge and the eventual loss of life ability. The incidence of AD accounts for about 40%-60% of dementia patients, and its pathological features are mainly the appearance of cortical neurofibrillary tangles (Neurofibrillary tangles, NFTs) and neuritic plaques (Senile plaques, SP) and the death of a large number of neurons. According to statistics, there are more than 50 million elderly people suffering from dementia in different degrees in the world today. The average survival time of AD after diagnosis is 7-8 years. Such patients in western countries cost about 15,000 US dollars per year. Although there is no statistics in this regard in my country, it is estimated that the total annual cost is about 20 billion yuan. It can be seen that AD has become a heavy burden on the family and society, not only a major medical problem, but also a serious social problem. The study of AD has made great progress in the past ten years. Foreign analysts predict that AD patients will increase to about 20% in the next ten years, and the growth rate of AD drug market will have a relatively large increase.

Hypertension refers to the increase of systemic arterial blood pressure, which is a common clinical syndrome. It can also be said that it is a systemic disease characterized by increased systolic and diastolic blood pressure, often accompanied by heart, brain, kidney and other organ functions or organic changes. It can be caused by a variety of pathogenic factors and complex pathogenesis. Central nervous system dysfunction, humoral endocrine inheritance, abnormal function of renal and cerebrovascular baroreceptors, and abnormal ion transport in cell membranes can all lead to hypertension. Hypertension is a common disease and the most important risk factor leading to major diseases such as coronary heart disease and cerebrovascular disease. The morbidity and mortality of cardiovascular and cerebrovascular diseases are closely related to the blood pressure level of patients. Therefore, the world All countries attach great importance to the prevention and research of hypertension. According to statistics from the national health department, there are more than 100 million hypertensive patients in my country, with an increase of more than 3 million people every year; there are more than 5 million stroke patients, with 1.5 million new cases and 200,000 deaths every year. Moreover, the incidence of hypertension worldwide is still on the rise, and my country has become the country with the most serious hazards of hypertension in the world.

Therefore, the research and development of drugs for the treatment or prevention of neurodegenerative diseases such as senile dementia and cardiovascular and cerebrovascular diseases such as hypertension have a huge market prospect.

Periwinkle was first recorded in the "Plant Names and Facts" in the Qing Dynasty. It tastes slightly bitter, and its cool nature returns to the liver and kidney meridians. It has the effects of cooling blood and lowering blood pressure, calming and calming the nerves. The 2010 edition of "Chinese Pharmacopoeia" stipulates that the traditional Chinese medicine periwinkle is the whole plant of Catharanthus roseus (L.) G.Don, a plant of the family Apocynaceae. It has been reported in the literature that the alkaloid components of vinca have sedative, antihypertensive, anticonvulsant, antiepileptic, protective nerve cells, anti-cerebral ischemia/reperfusion injury and other effects. Clinically, vinca total alkaloid tablets prepared from periwinkle can be used to treat high blood pressure; the combination of periwinkle and Gastrodia elata can be used to treat vascular dementia, which can significantly improve the cognitive function and behavior of patients, and can also treat hyperactivity of liver yang type hypertension, hypertensive cerebral hemorrhage embolism.

But at present, there is no oral medicine for the treatment or prevention of neurodegenerative diseases such as senile dementia and cardiovascular and cerebrovascular diseases such as high blood pressure with the effective parts of alkaloids extracted from periwinkle, stems and branches or whole plants with a total alkaloid content greater than 50% as the main raw material. Preparations are on the market.

Contents of the invention

The technical problem to be solved by the present invention is to provide a method and application for extracting effective parts of alkaloids from periwinkle, stems, or whole plants. Or hot reflux extraction, filtration, recovery under reduced pressure until there is no alcohol smell, add water or alcohol solution to the concentrated extract, filter, the filtrate is passed through the adsorption column filled with adsorption materials, and the adsorption column is washed with water before elution until the effluent is Colorless, elute with 20-75% alcohol solution, collect the eluent, recover the solvent under reduced pressure to obtain a thick paste, and obtain the active part of alkaloid after drying. The method does not use toxic organic solvents, does not pollute the environment, is less time-consuming, has high efficiency, simple extraction process, low cost, can be industrialized and meets the requirements of environmental protection. The effective parts of the extracted alkaloids can be used as drugs for treating or preventing neurodegenerative diseases such as senile dementia and cardiovascular and cerebrovascular diseases such as hypertension.

In order to solve the above problems, the specific technical scheme of the present invention is as follows: a method for extracting effective parts of alkaloids from periwinkle leaves, stems and branches or whole grass, characterized in that the extraction steps are as follows:

a. Add an alcohol concentration of 40-95% aqueous alcohol solution to the periwinkle leaves, stems or whole grass, extract by cold soaking, infiltration or heat reflux, and the amount of the aqueous alcohol solution is the weight of the periwinkle leaves, stems or whole grass 6 to 30 times as much, filtered, combined filtrates, and recovered under reduced pressure until there was no alcohol smell, to obtain concentrated extracts of periwinkle leaves, stems or whole plants;

b. Add 4 to 10 times the weight of periwinkle leaves, stems or whole grass in water or alcohol solution to the concentrated extract of periwinkle leaves, stems or whole grass, the alcohol concentration in the solution is 10 to 60%, filter, get the filtrate;

c. The filtrate is filled with an adsorption column of adsorption material;

d. Before elution, use water or 20-40% alcohol with a volume 4-8 times that of the adsorption material to rinse the attached column until the effluent is colorless;

e. Elute with an alcohol solution containing 40-90%, and the amount of eluent is 4-10 times that of the adsorption material, and collect the eluent;

f. The solvent is recovered under reduced pressure to obtain a thick paste, and the thick paste is dried to obtain effective parts of periwinkle leaves, stems or whole plant alkaloids.

The alcohol used in the alcoholic solution is ethanol or methanol.

The adsorption material in the step c is any one or both of macroporous adsorption resins or ion exchange resins, and the types of macroporous resins are AB-8, SP-70, D101, DM-130, S-8 , D3520, D4020 or HP-20.

In the step f, the total alkaloid content in the effective part of the alkaloid is 50-90%, wherein the sum of the contents of vinblastine, isodehydrovinblastine, dehydrovinblastine and isodehydrovinblastine is greater than 40%. .

5. The method for extracting effective parts of alkaloids from periwinkle leaves, stems or whole plants according to claim 1, characterized in that: the effective parts of alkaloids extracted in step f can be compared with pharmaceutically acceptable Carriers or excipients are combined to make tablets, capsules, dropping pills or granules.

The alkaloids are used to prevent neurodegenerative diseases such as senile dementia and cardiovascular and cerebrovascular diseases such as hypertension.

The beneficial effects brought by the present invention are as follows: add water-containing alcohol solution to the periwinkle, stems and branches or whole grass for cold soaking, percolation or hot reflux extraction, filter, recover under reduced pressure until there is no alcohol smell, add water to the concentrated extract Or alcohol solution, filter, the filtrate is passed through the adsorption column filled with adsorption materials, before elution, choose water to wash the adsorption column until the effluent is colorless, elute with 20-75% alcohol solution, collect the eluent, and recover under reduced pressure solvent to obtain a thick paste, and after drying to obtain the effective part of the alkaloid. The method does not use toxic organic solvents, does not pollute the environment, is less time-consuming, has high efficiency, simple extraction process, low cost, can carry out industrialized production, and meets the requirements of environmental protection. The effective parts of the extracted alkaloids can be used as drugs for treating or preventing neurodegenerative diseases such as senile dementia and cardiovascular and cerebrovascular diseases such as hypertension.

detailed description

A method for extracting effective parts of alkaloids from periwinkle leaves, stems or whole plants, the extraction steps are as follows:

a. Add an alcohol concentration of 40-95% aqueous alcohol solution to the periwinkle leaves, stems or whole grass, extract by cold soaking, infiltration or heat reflux, and the amount of the aqueous alcohol solution is the weight of the periwinkle leaves, stems or whole grass 6 to 30 times as much, filtered, combined filtrates, and recovered under reduced pressure until there was no alcohol smell, to obtain concentrated extracts of periwinkle leaves, stems or whole plants;

b. Add 4 to 10 times the weight of periwinkle leaves, stems or whole grass in water or alcohol solution to the concentrated extract of periwinkle leaves, stems or whole grass, the alcohol concentration in the solution is 10 to 60%, filter, get the filtrate;

c. The filtrate is filled with an adsorption column of adsorption material;

d. Before elution, use water or 20-40% alcohol with a volume 4-8 times that of the adsorption material to rinse the attached column until the effluent is colorless;

e. Elute with an alcohol solution containing 40-90%, and the amount of eluent is 4-10 times that of the adsorption material, and collect the eluent;

f. The solvent is recovered under reduced pressure to obtain a thick paste, and the thick paste is dried to obtain effective parts of periwinkle leaves, stems or whole plant alkaloids.

The alcohol used in the alcoholic solution is ethanol or methanol.

The adsorption material in the step c is any one or both of macroporous adsorption resins or ion exchange resins, and the types of macroporous resins are AB-8, SP-70, D101, DM-130, S-8 , D3520, D4020 or HP-20.

In the step f, the total alkaloid content in the effective part of the alkaloid is 50-90%, wherein the sum of the contents of vinblastine, isodehydrovinblastine, dehydrovinblastine and isodehydrovinblastine is greater than 40%. .

The extracted effective fraction of alkaloids in the step f can be combined with pharmaceutically acceptable carriers or excipients to form tablets, capsules, dropping pills or granules.

The alkaloids are used to prevent neurodegenerative diseases such as senile dementia and cardiovascular and cerebrovascular diseases such as hypertension.

Pharmacodynamics and preliminary toxicology studies of the effective parts of the alkaloids prepared by the invention:

1. Anti-microglial activation activity of the active parts of vinca alkaloids and main alkaloid monomer compounds

The chronic inflammatory response mediated by microglia is an important link in the occurrence and development of several major neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD). The pro-inflammatory cytokines and free radicals released by activation of microglia for various reasons initiate or aggravate neuron damage, and neuron damage will in turn further activate microglia, forming a vicious cycle of pathology process. Therefore, inhibiting microglial activation can effectively block or alleviate the consequences of this vicious cycle. In this experiment, the abnormal activation of microglia activated by lipopolysaccharide (LPS) in vitro was used as an index to evaluate the effective parts of vinca alkaloids and the main alkaloid components by inhibiting the abnormal activation of microglia. protection of the nervous system.

a. Culture of primary microglia

The cerebral cortex of neonatal rats (within 2 days after birth) was taken under aseptic conditions, placed in D-Hanks solution, and the meninges and blood vessels were peeled off. Digest with 0.25% trypsin for 10-15 minutes, stop the digestion with IMDM medium containing 10% fetal bovine serum, pipette with a Pasteur pipette, and mechanically filter with a 200-mesh sieve to prepare a mixed cell suspension. Count, adjust the cell density to (3~5)×10 ⁶ cells/mL, plant 5~10mL per bottle in a 75cm ² screw cap cell culture flask, medium composition: IMDM culture medium, 5% fetal bovine Serum was supplemented with antibiotics (penicillin 100 μg/mL, streptomycin 100 μg/mL). The loose cap was cultured statically in a CO ₂ incubator (37° C., 5% CO ₂ , 95% air), and after 48 hours, the medium was replaced once with an equal amount of medium to remove dead cell debris. Afterwards, the medium was changed once every 3-4 days, and the cells were collected until 11-14 days after culturing. Change the serum-free culture medium one day before harvesting, tighten the cap of the bottle the next day, and shake on a constant temperature shaker at 37°C for about 2 hours at 250 rev/min to collect the supernatant. At this time, the cell density of the supernatant is generally not lower than 5×10 ⁵ cells/mL. Transfer the collected cell suspension to a 75mL cell culture flask and culture it in a _CO2 incubator for about 4 hours, shake the flask gently at room temperature to remove components that are not firmly attached to the wall (mainly oligodendrocytes) cells and astrocytes), add fresh culture medium after aspiration.

b. Griess method to detect the inhibitory effect of active parts of vinca alkaloids and main alkaloid monomer compounds on LPS-activated microglia

Take primary rat microglial cells in the logarithmic growth phase, adjust the cell density to 5×10 ⁵ cells/mL with fresh IMDM medium containing 5% fetal bovine serum, inoculate in 96-well plate, 100 μL/well , cultured in an incubator at 37°C with 5% CO ₂ . After 24 hours of adherent culture, the cells were replaced with fresh serum-free culture medium, and drug treatment was performed at the same time to work together with LPS. Three parallel wells were set up for each concentration. A blank control and a positive control (resveratrol) were also set up. The final concentration of LPS in each administration group and positive control group was 1 μg/mL. After the cells were added with drugs and continued to culture for 48 hours, the supernatant was collected, and the NO ₂ ^- content in the supernatant was detected by Griess colorimetry. The NO release amount of the LPS stimulation group was set as 100%, and the NO release amount of the other groups was expressed as a percentage relative to the LPS stimulation group. The results are shown in Table 1.

Results: The half effective dose (IC ₅₀ ) of vinca alkaloids on LPS-induced NO release from microglial cells was 6.3 μg/mL, and the IC ₅₀ values of alkaloid monomer compounds ranged from 13.7 to 19.0 μM, all of which showed significant effects. inhibitory effect. However, the crude extracts of periwinkle or stems and branches failed to show obvious inhibitory effect. Therefore, the active part of the alkaloid obtained by using the macroporous resin is enriched with vinca alkaloid components, improves the pharmacological activity, reduces the dosage, and has medicinal value.

Table 1 Inhibitory effects of active parts of vinca alkaloids and main alkaloid monomer compounds on LPS-induced NO release from microglia

2. Acute toxicity test of the effective fraction of vinca alkaloids

50 mice, half male and half male, weighing 18-20g. According to body weight and sex, they were randomly divided into 5 groups, 10 in each group. After fasting without water for 16 hours, each group of animals was given different doses of effective parts of vinca alkaloids by intragastric administration, the initial dose was 2500mg/kg, the administration volume was 0.2mL/kg, and the dose ratio between adjacent groups was 1 : 0.75, then observed continuously for 7 days, recorded the acute toxic reaction and death number of mice, dissected dead mice and surviving mice, and observed whether there were any abnormalities in major organs and tissues. The LD ₅₀ value of intragastric administration of the effective parts of vinca alkaloids was calculated by comprehensive calculation method. The results are shown in Table 2.

The results showed that after intragastric administration of mice with the effective parts of vinca alkaloids for 5 minutes, some experimental animals with a dose of > 1054 mg/kg had reduced range of motion, shortness of breath, trembling, and finally struggled to death. The peak time of death was 60 minutes. However, the major organs and tissues of the dead mice and the surviving mice were normal. The LD ₅₀ value of the active fraction of vinca alkaloids administered to mice by intragastric administration was 1406.4±198.9mg/kg. Since the ED ₅₀ values of the antihypertensive activity and anti-cerebrovascular dementia activity of total alkaloids of vinca are 20.0 mg/kg and 15.0 mg/kg respectively, the effective part of the vinca alkaloids has a high therapeutic index and is safe for clinical use Larger range.

Table 2 The LD ₅₀ value of the effective fraction of vinca alkaloids given to mice by intragastric administration

3. Rapid screening test for cytotoxicity of vinca alkaloid monomer compounds

Neutral red uptake assay (NRU): Chinese hamster lung cells (CHL) and Chinese hamster ovary cells (CHO) were cultured in MEM medium containing 10% bovine serum in 5% CO ₂ , 37°C saturated humidity Routine cultivation in the incubator. After 24 hours, the cells grew close to a monolayer, and the test samples were added to the CHL and CHO cells respectively, and the culture was continued for 24 hours. 2 hours before the end of the culture, 50 μL of neutral red (NR) solution (250 μg/mL) was added to each well. After culturing, the cells were washed with PBS, 100 μL of extract solution (1% acetic acid-50% ethanol aqueous solution) was added to each well, and left at room temperature for 20 min to dissolve all NR in the cells. After fully shaking, use a microplate reader to measure the absorbance at a wavelength of 540 nm. The cell survival rate of the blank group was set as 100%, and the cell survival rates of the other groups were expressed as a percentage relative to the blank group. The results are shown in Table 3.

Table 3 Toxic effects of vinca alkaloid monomer compounds on CHL and CHO cells

The results showed that the alkaloid monomer compounds in the active parts of vinca alkaloids had no obvious toxicity to CHO cells, with IC ₅₀ >300μM; but vinblastine, isovinblastine, dehydrovinblastine, Hydrovinblastine had a greater effect on the survival rate of CHL cells, with IC ₅₀ values of 273, 10.9, 180, and 70.9 μM, respectively. Higoside had no obvious toxicity to CHL cells, with IC ₅₀ >300 μM. Conclusion: The components in the active parts of vinca alkaloids have no obvious toxicity to CHO cells at different doses, but only show certain cytotoxicity to CHL cells at higher doses, which belong to low toxicity substances.

4. Morris water maze experiment

a. Animals and models: 75 healthy female SD rats aged 12-24 months, weighing 340±37g, 70 rats with good learning and memory were screened by Morris water maze test, randomly divided into blank control group, operation group and mock surgical group.

b. Model making method: permanent ligation of bilateral common carotid arteries. The three groups of rats were fed under the same conditions, and the Morris water maze test was carried out one month after the operation. The observation index was the average escape latency, and the average value and standard deviation of the blank control group were obtained. Judged as learning and memory impairment. Rats with learning and memory impairment were screened out from the operation group, and then randomly divided into model group, drug treatment group and blank control group.

c. Medication method: The administration group was given 15 mg/kg of the effective part of vinca alkaloids by intragastric administration, once a day, and the dosage was changed to 5 mg/kg after 1 week. After 1.5 months and 2.5 months of medication (ie, 2.5 and 3.5 months after operation), the Morris water maze test was performed to detect the ability of learning and memory. In the blank control group, normal saline was used to replace the effective parts of vinca alkaloids, and the rest were the same as those in the administration group.

d. Morris water maze: The diameter of the pool in the water maze is 100cm, the depth is 50cm, the water depth is 30cm, the water temperature is 26±2°C, and the water surface is covered with a layer of foam plastic chips. Four equidistant points on the pool wall divide the pool into four quadrants, optional A quadrant places the platform in its center. The platform is colorless and transparent, with a diameter of 6cm and a height of 28cm. The platform is submerged 2cm below the water surface, and the reference objects around the pool remain unchanged.

e. Positioning navigation test: The test lasted 5 days, 4 times a day. The operator put the rats into the water from different quadrants facing the pool wall, and recorded the time for the rats to find the platform (escape latency). If the platform could not be found within 120s, the operator would help the rats to get on the platform. After the rats stood on the platform for 10s, the rats were taken off from the platform to rest for 60s, and then the next quadrant was tested in sequence.

f. Observation index: escape latency.

g. Data statistics: SPSS.10.0 statistical software was used to conduct one-way ANOVA (one-way ANOVA) and repeated measures (repeated measures) data analysis of variance, Dunnett-t test and LSD test on the average escape latency. The results are shown in Table 4.

h. Results: 3.5 months after operation (ie 2.5 months after medication), the average escape latency of the rats in the administration group was significantly shorter than that of the rats in the model group and the blank control group. The results showed that the active parts of vinca alkaloids could improve the learning and memory ability of rats with vascular dementia.

Table 4 Comparison of the average escape latency of each group of rats at different time periods (s)

5. Effects of effective fractions of vinca alkaloids on experimental hypertension in rats

a. Measure the normal blood pressure of rats: take rats with a healthy body weight of about 200 g, divide them into random groups, measure the normal blood pressure with a rat manometer, measure continuously for 3 days, and record.

b. Replicate the hypertension model: after measuring the blood pressure continuously for 3 days, inject testosterone propionate 4 mg subcutaneously every day for 14 days in total. When the blood pressure of rats is significantly increased to 1.3kPa (10mmHg), experimental treatment can be carried out.

Table 5 Effects of effective fractions of vinca alkaloids on experimental hypertension in rats

c. Experimental treatment: 20 mg/kg of the active part of vinca alkaloids was given to rats by intragastric administration every day for 16 consecutive days, and then blood pressure was measured every 4 days; the other two groups were given reserpine and distilled water respectively as controls. The results are shown in Table 5.

e. The results show that the effective parts of vinca alkaloids have obvious antihypertensive effect.

6. Effects of effective parts of vinca alkaloids on platelet aggregation

Animal SD rats, male, weighing 250-350 g, were divided into 4 groups. Different doses of effective fractions of vinca alkaloids, aspirin (ASA) or control solution were administered intravenously, and blood was collected 20 minutes later, anticoagulated with 3.8% sodium citrate (9:1), centrifuged, and platelet-rich plasma (PRP) was separated. ) and platelet-poor plasma (PPP), adjust the number of platelets in PRP to 4×10 ⁸ /mL, and measure the platelet aggregation reaction with PAM-2 PPP automatic balance platelet aggregation instrument (product of Shanghai Medical University) according to turbidimetry. The inducer was arachidonic acid (AA) 200 μmol/L, adenosine diphosphate sodium salt (ADP) 4.0 μmol/L and collagen 40 μg/mL.

Results: After the rats were intravenously injected with 10, 20 mg/kg of the effective fraction of vinca alkaloids or 30 mg/kg of aspirin, the platelet aggregation rate was significantly lower than that of the control group rats, as shown in Table 6. The results showed that the effective fraction of vinca alkaloids may become a clinically effective anti-platelet aggregation drug.

Table 6 Inhibition of effective fractions of vinca alkaloids on platelet aggregation induced by AA, ADP or collagen

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

With the advent of the world's aging society, the prevention and treatment of senile memory dysfunction such as senile memory loss and senile dementia is becoming increasingly important. A large number of research data at home and abroad show that one out of every ten elderly people shows different degrees of dementia symptoms, which seriously affects people's health and quality of life. In addition, hypertension is also the main disease that causes human death, such as coronary heart disease and cerebrovascular disease. It is a common and frequently-occurring disease that endangers human health. Therefore, the research and development of new drugs for the treatment or prevention of neurodegenerative diseases such as senile dementia and cardiovascular and cerebrovascular diseases such as antihypertension has a huge market prospect.

The preparation of the effective part does not use toxic organic solvents and does not pollute the environment. It is based on the conventional water-containing solvent extraction method and adopts the adsorption separation technology of adsorption materials. It takes less time and has high efficiency. Carry out industrialized production and meet the requirements of environmental protection. Moreover, ethanol can be recycled and reused, and macroporous resins and ion exchange resins can also be processed for reuse, which reduces production costs and is easy to promote. Therefore, it has strong competitiveness in domestic and foreign markets. Periwinkle flowers, stems or whole plants are taken from natural resources. The effective parts of the alkaloids extracted from periwinkle flowers, stems or whole plants have less toxic and side effects, are safe and reliable, and have a variety of biological activities. They have strong nerve cell protection and The antihypertensive effect is very beneficial to the treatment or prevention of neurodegenerative diseases such as senile dementia and cardiovascular and cerebrovascular diseases such as hypertension. Vinca flowers, stems or whole plants are very rich in natural resources in my country. Extracting alkaloid active ingredients from periwinkle flowers, stems or whole plants for the development of new drugs is a powerful measure for the effective use of natural resources. The invention proves that the effective parts of the alkaloids extracted from periwinkle flowers, stems or whole plants have protective and therapeutic effects on nerve cells and antihypertensive effects through drug efficacy tests, providing a theoretical basis for clinical popularization.

detailed description

Example 1

Add 75% ethanol solution 8 times the weight of periwinkle stems and branches to extract by percolation, filter, combine filtrates, recover under reduced pressure until there is no alcohol smell, and obtain concentrated periwinkle stems and branches extract. Dilute the concentrated extract with water to a medicinal solution of 6 times the amount of periwinkle stems and branches, and filter to obtain a filtrate. The filtrate was passed through an adsorption column filled with DM-130 macroporous resin, eluted with 6 times the amount of 80% ethanol, and the solvent was recovered under reduced pressure to obtain a thick paste, which was dried to obtain a total alkaloid content of 76.0% ( HPLC method measures) dry cream, wherein the content sum of vinblastine, isovinblastine, dehydrovinblastine and isodehydrovinblastine is 40.1% (HPLC method measures). The dry paste is pulverized by ultrafine grinding technology, take 1 part of dry paste powder and 0.1 part of microcrystalline cellulose, granulate with 70% ethanol solution, dry, granulate, add 1% magnesium stearate, mix well, and fill capsules , 15mg per capsule.

Example 2

Add 75% ethanol solution 10 times the weight of periwinkle coarse powder for percolation extraction, filter, combine filtrates, recover under reduced pressure until there is no alcohol smell, and obtain concentrated periwinkle extract. Dilute the concentrated extract with 10% ethanol to 8 times the medicinal solution of periwinkle, filter to obtain the filtrate. The filtrate was passed through an adsorption column filled with D101 macroporous resin, rinsed with 6 times the amount of 30% ethanol until the effluent was colorless, then eluted with 6 times the amount of 75% ethanol, and the solvent was recovered under reduced pressure. Get the thick paste, the dry paste that contains total alkaloids is 70.3% (HPLC method surveys) through drying, and wherein the content of vinblastine, isovinblastine, dehydrovinblastine and isodehydrovinblastine The sum is 42.4% (measured by HPLC method). Mix 1 part of crushed dry cream powder with 0.1 part of microcrystalline cellulose, granulate with 80% ethanol solution, dry, granulate, add 1% magnesium stearate, mix well, compress into tablets, and coat with film to obtain Film-coated tablets, 15mg each.

Example 3

Add 75% ethanol solution of 12 times the weight of periwinkle coarse powder to cold soak and extract for 12 hours, filter, combine the filtrates, recover under reduced pressure until there is no alcohol smell, and obtain concentrated periwinkle extract. Dilute the concentrated extract with 20% ethanol to a medicinal solution of 6 times the amount of periwinkle, and filter to obtain a filtrate. Pass the filtrate through the adsorption column filled with AB-8 macroporous resin, rinse with 6 times the amount of 30% ethanol until the effluent is colorless, then elute with 6 times the amount of 55% ethanol, and collect the 55% ethanol The eluent is recovered under reduced pressure to obtain a thick paste, and the thick paste is dried to obtain a dry paste containing 76.6% (HPLC method survey) of total alkaloids, wherein vinblastine, isovinblastine, dehydrovinblastine and The sum of the contents of isodehydrovinblastine is 41.5% (measured by HPLC method). After the dry paste is pulverized, put it in the melted PEG. After the drug is completely dispersed in the liquid carrier, put it in the dripping pill equipment, drop it into the liquid paraffin, and drop it into small pills.

Example 4

Add 75% ethanol solution 8 times the weight of whole periwinkle herb powder and heat reflux for extraction twice, each time of reflux for 1.5 hours, filter, combine the filtrates, recover under reduced pressure until there is no alcohol smell, and obtain the concentrated extract of periwinkle whole herb . Dilute the concentrated extract with 10% ethanol to a medicinal solution that is 6 times the amount of the whole periwinkle herb, and filter to obtain a filtrate. The filtrate was passed through a macroporous resin adsorption column filled with HP-20, rinsed with 4 times the amount of 20% ethanol until the effluent was colorless, then eluted with 6 times the amount of 75% ethanol, and recovered under reduced pressure solvent, to get a thick paste, and the thick paste is dried to obtain a dry paste containing total alkaloids of 69.8% (HPLC method), wherein vinblastine, isovinblastine, dehydrovinblastine and isodehydrovinblastine The sum of the contents is 40.3% (measured by HPLC method). The dry paste is crushed and dispersed in octyldodecanol, and processed into soft capsules, each 15mg.

Example 5

The dry paste was prepared by the preparation process in Example 2, the dry paste was crushed, 1 part of dry paste powder was mixed with 0.1 part of low-substituted hydroxypropyl cellulose, granulated with 80% ethanol solution, dried, and 1% hard Magnesium fatty acid, mixed evenly, compressed into tablets, film-coated to obtain dispersible tablets. 15mg per tablet.

What has been described above are only preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, some modifications and improvements can be made, which should also be considered as belonging to the protection scope of the present invention.

Claims (6)

1. A method for extracting effective parts of alkaloids from periwinkle flowers and leaves, stems and branches or whole grass, characterized in that its extraction steps are as follows: a. Add an alcohol concentration of 40-95% aqueous alcohol solution to the periwinkle leaves, stems or whole grass, extract by cold soaking, infiltration or heat reflux, and the amount of the aqueous alcohol solution is the weight of the periwinkle leaves, stems or whole grass 6 to 30 times as much, filtered, combined filtrates, and recovered under reduced pressure until there was no alcohol smell, to obtain concentrated extracts of periwinkle leaves, stems or whole plants; b. Add 4 to 10 times the weight of periwinkle leaves, stems or whole grass in water or alcohol solution to the concentrated extract of periwinkle leaves, stems or whole grass, the alcohol concentration in the solution is 10 to 60%, filter, get the filtrate; c. The filtrate is filled with an adsorption column of adsorption material; d. Before elution, use water or 20-40% alcohol with a volume 4-8 times that of the adsorption material to rinse the attached column until the effluent is colorless; e. Elute with an alcohol solution containing 40-90%, and the amount of eluent is 4-10 times that of the adsorption material, and collect the eluent; f. The solvent is recovered under reduced pressure to obtain a thick paste, and the thick paste is dried to obtain effective parts of periwinkle leaves, stems or whole plant alkaloids. 2. The method for extracting effective parts of alkaloids from periwinkle leaves, stems or whole plants according to claim 1, characterized in that: the alcohol used in the alcoholic solution is ethanol or methyl alcohol. 3. The method for extracting effective parts of alkaloids from periwinkle leaves, stems or whole plants according to claim 1, characterized in that: the adsorption material in the step c is macroporous adsorption resin or ion exchange resin Any one or both of them are used together. The models of macroporous resins are AB-8, SP-70, D101, DM-130, S-8, D3520, D4020 or HP-20. 4. The method for extracting effective parts of alkaloids from periwinkle leaves, stems or whole plants according to claim 1, characterized in that: in the step f, the total alkaloid content in the effective parts of alkaloids is 50-50%. 90%, wherein the sum of the contents of vinblastine, isovinblastine, dehydrovinblastine and isodehydrovinblastine is greater than 40%. 5. The method for extracting effective parts of alkaloids from periwinkle leaves, stems or whole plants according to claim 1, characterized in that: the effective parts of extracting alkaloids in the step f can be pharmaceutically acceptable Carriers or excipients are combined to make tablets, capsules, dropping pills or granules. 6. The effective part of alkaloids extracted from periwinkle leaves, stems or whole plants as claimed in any one of claims 1-5, characterized in that: said alkaloids are used to prevent neurodegenerative diseases such as senile dementia and hypertension and other cardiovascular and cerebrovascular diseases.