WO2004020427A1

WO2004020427A1 - The process for preparation of total coumarins from cortex fraxini and their use in medicine

Info

Publication number: WO2004020427A1
Application number: PCT/CN2003/000191
Authority: WO
Inventors: Andong Yang; Junning Zhao
Original assignee: Sichuan Dikang Industry Holdings Group Co., Ltd.
Priority date: 2002-08-29
Filing date: 2003-03-17
Publication date: 2004-03-11
Also published as: AU2003221296A1; CN1398861A; CN1186336C

Abstract

The present invention discloses a process for preparing total coumarins from Cortex Fraxini, which comprises: a. extracting Cortex Fraxini by the method of water-extracting and alcohol-depositing (50 %-90%) or alcohol-extracting and alcohol-depositing (50 %-90%) or alcohol-extracting (70 %-95%) to obtain a extract; b. subjecting the extract to the temperature between 4-12 °C for 8-24 hours to form a deposit; c. filtering the resulting deposit and drying, resulting the total coumarins from Cortex Fraxini . The invention also relates to the use of total coumarins in Cortex Fraxini in manufacturing medicines for treating acute or chronic gout diseases. The present invention also discloses a process for extracting esculin from total coumarins from Cortex Fraxini, and the use of esculin in manufacturing medicines for treating acute or chronic gout diseases.

Description

Preparation method of Qinpi total coumarin and its application in pharmaceutical technology

The invention relates to a method for preparing Qinpi extract and its application, in particular to a method for preparing total coumarin of Qinpi and its use in the field of pharmacy. Background technique:

Gout is caused by a disorder of purine metabolism or decreased uric acid excretion, and the continuous increase in blood uric acid is the basic cause, which causes various diseases. It is a metabolic rheumatism. The disease belongs to the categories of traditional Chinese medicine, such as paralysis, gout, calendar, etc., and refers to the symptoms of numbness, seriousness, and unfavorable flexion and extension of joint muscles caused by external evils blocking meridians, qi and blood stagnation, and liver and kidney deficiency. Hyperuricemia is the basis for the onset of gout. It is at different stages of the same disease as gout, and is a pre-stage state of gout. Gout is clinically characterized by hyperuricemia, recurrent episodes of acute and chronic arthritis, and goutstone sedimentation. Severe cases can lead to joint mobility disorders, deformities, renal uric acid stones, or gouty renal parenchymal disease. Its kidney disease, coronary heart disease, hypertension and other life-threatening complications are also higher.

Gout and hyperuricemia have become a common disease in the world today, especially in the elderly, with an incidence of 6% -13%. With the changes in diet structure and environmental factors, economic development and the extension of human lifespan, the incidence of gout and hyperuricemia is increasing. In China, due to the general improvement of people's living standards and the increase in animal food intake, the incidence of the disease has shown a straight upward trend, and its incidence has approached the level of developed countries (0.35%). Through outpatient visits, the disease has been observed in adolescents. The incidence of middle-incidence has also shown a straight upward trend.

Gout has the characteristics of latent onset and difficult to cure. There are two main purposes for clinical treatment of gout, timely control of the acute onset of gouty arthritis; long-term treatment of hyperuricemia, prevention of urate precipitation and acute recurrence of gout, and promotion of goutstones. absorb. The main drugs for controlling the onset of gouty arthritis are colchicine, non-steroidal anti-inflammatory drugs and adrenal corticosteroids. There are two main categories of drugs for the prevention and treatment of hyperuricemia: one is the drug that interferes with renal tubular excretion of uric acid, such as gouty elixirs, probenecid, thiazolone, etc .; the other is xanthine which can inhibit uric acid production Oxidase inhibitor, the representative drug is allopurinol. The above-mentioned chemical drugs all have the disadvantages of large toxic and side effects to varying degrees, especially the long-term medication is more obvious, and treatment of the disease often requires long-term medication.

In the past ten years, traditional Chinese medicine has achieved good results in treating gout, with few toxic and side effects, and is easily accepted by patients. It has achieved the purpose of long-term medication, prevention and treatment of diseases. However, the traditional Chinese medicines currently used are mainly compound formulas, and no single-traditional Chinese medicine extract has been reported as a preparation for treating gout. Due to the complexity of traditional Chinese medicines and the compound itself, it is difficult to determine the effective ingredients, so it is difficult to accurately evaluate them. At the same time, the clinical efficacy of traditional Chinese medicines is difficult to control because the content of traditional Chinese medicines varies according to the origin and batch. Disclosure of invention:

In view of the shortcomings of the compound Chinese patent medicine, the present invention provides a method for preparing a single Chinese herbal medicine Qinpi extract-Qinpi total coumarin and its application in pharmacy, and also provides a further extract of Qinpi total coumarin Qinpitin Methods and their application in pharmacy, both of them can obviously prevent the pain, anti-inflammatory and reduce the blood uric acid of patients, and have obvious therapeutic effects on acute and chronic gout. The total effective rate was 90%, and the effective rate was 56.7%. No obvious toxic and side effects were observed. The effective ingredients were clear, the effect was exact, and the toxic and side effects were small.

The invention also provides a simple method for preparing Qinpi total coumarin and Qinpidin A, specifically, a method of concentrating and purifying Qinpi total coumarin and Qinpidin A.

The technical scheme of the present invention is: cut the Qinpi medicinal material into granules, and use a. Boiled alcohol precipitation method (50% -90%) or alcohol extraction alcohol precipitation method (50% -90%) or alcohol extraction method (70% -95%) to extract the extract of Qinpi extract; b. Refrigerate the extract at 4-12 ° C for 8-24 hours to form a precipitate; c. Filter to obtain the precipitate and dry to obtain the total coumarin of Qinpi.

Another technical solution of the present invention is to dissolve the obtained Qinpi total coumarin with warm (40-50 ° C) methanol, recover a part of methanol, and concentrate it into a Qinpirin A methanol saturated solution, and leave it overnight to obtain a yellow color. Needle crystals were repeatedly recrystallized from methanol and water to obtain quincetin.

Qinpi total coumarin and Qinpidin are used as active ingredients, and pharmaceutically acceptable carriers or excipients can be used to prepare drugs for treating acute and chronic gout. The medicinal product has the advantages of clear active ingredients, precise curative effect, and no obvious toxic and side effects. The total effective rate is 90 ° / », and the effective rate is 56. VL

The following uses pharmacological experiments and results to explain the new uses of Qinpi Total Coumarin in the pharmaceutical field.

Take 100 kg of Qin peel, cut into granules, extract with water three times, concentrate the water extract into an extract, add 2.5 times the amount of ethanol, alcohol precipitation, filter, and concentrate the ethanol recovery solvent to a specific gravity of 1.0. 70 kg。 The paste, and then refrigerated at 4 ° C for 8 hours, filtered, the precipitate was collected, dried to obtain Qinpi total coumarin 1. 70 kg. The prepared Qinpi total coumarin is added with steam 7] to release it into an aqueous solution of various concentrations required, sterilize, and set aside.

1. Acute toxicity test

50ml Kunming mice of 18-21 g male and female were fasted for 12 hours without water, and were administered intragastrically with a concentration of 16.38% Qinpi total coumarin solution 0.4ml / 10gB. W., continuously Observe for 7 days, record the toxic reaction of the animal, and re-measure the body weight after 7 days. The maximum dose that does not cause death is the maximum tolerated amount (MTD) of one oral administration, and the calculated dose is equivalent to the clinical value. Multiples of the recommended daily dose. The above test results indicate that the maximum tolerated MTD of Qinpi total coumarin mice in a single gavage is> 6. 55g (total coumarin) I (equivalent to 655 times the amount of clinical daily recommended agent). There were no abnormalities in appearance, behavior, mental state, body weight, bowel movements and color, coat, skin color, breathing, nose, eyes, or oral secretions of the animals after administration, and no significant pathological changes were found in the anatomy at the end of the test. Judging from the amount of acute toxicity of Qinpi total coumarin and its post-administration performance, Qinpi total coumarin is a drug with no obvious toxicity.

2. Long-term toxicity test:

80 to 110 grams of male and female first-class Wistar rats were 260 experimental animals, randomly divided into 4 groups, including low, medium and high dose groups and a control group. Hours ~ 10:00 hours. The 8% Qinpi total coumarin solution was used for the high-dose group and diluted to 4% and 2 ° /. The medicinal solution was used for the middle dose group and the low dose group. Drugs of different concentrations and equal volumes were administered orally once a day, with an intragastric volume of 1 ml / 100 g body weight for 3 consecutive months. The control was given an equal volume of steamed water. During the experimental period, the patient was weighed once a week, and the dosage was adjusted according to the new weight. Three months after the administration, one third of the three dose groups and the control group were left without the test drug, and the observation was continued for two weeks to understand the reversibility of the toxic reaction and the possible delayed toxicity. Observation indicators include: general performance, such as animal intake, weight, physical signs, behavior, fecal traits, etc .; hematological indicators, blood biochemical indicators; systematic autopsy and pathological examination. The above test results show that no significant toxicity was seen in each dose group of the test drug.

3. Pharmacodynamic test—risk:

Wistar rats weighing 220 to 250 grams and New Zealand white rabbits weighing 2.5 to 3.0 kg were used as test animals. The maximum solubility of Qinpi total coumarin in cold water is 16.38%, but its solubility can be increased to more than 50% under heat preservation conditions. We used Qinpi total coumarin aqueous solution as the test drug. Tests have shown that different concentrations of Qinpi total coumarin administered intragastrically can significantly inhibit the acute injection of paw swelling in rats and the swelling of rabbits with acute gouty arthritis induced by local injection of microcrystalline sodium urate suspension. It can reduce the amount of effusion in the joint cavity of rabbits with acute gouty arthritis, and reduce pathological changes such as joint condyle tissue congestion, leeches, degeneration and necrosis, and infiltration of lymphocytes and neutrophils. The drug also significantly reduced blood uric acid in normal mice and high uric acid mice. Qinpi total coumarin has a significant reduction effect on the number of writhing reactions induced by acetic acid in mice, and also has a certain effect of extending the hot plate pain threshold of mice, indicating that the drug has a certain analgesic effect. Qinpi total coumarin p-xylene induced acute ear swelling in mice, and carrageenan induced injection of paw swelling in rats significantly counteracted the effects of reducing the weight of cotton ball stimulating granulations and preventing capillary permeability caused by inflammation Sexual increase and induced leukocyte migration indicate that the total coumarin of Qinpi has a certain anti-inflammatory effect, which can act in the early and late stages of inflammation. The above results show that the total coumarin of It can control the swelling of experimental gouty arthritis, reduce the pathological changes of the synovial tissue of the joints, reduce blood uric acid, and relieve the pain, anti-inflammatory, swelling and other aspects of pharmacological effects related to gout treatment. Table 1 Effect of Qinpi total coumarin on microcrystalline sodium urate-induced acute foot inflammation in rats

Table — 2 Effect of Qinpi total coumarin on joint bell swelling rate in rabbits with acute gouty arthritis induced by MSU (± s) Number of animals in group (only) Dose (mg / Kg * d) Swelling rate (%) Normal control 8 ¹ θ θ model comparison 9 ― 10. 89 ± 5. 86 Qin Qin Qin Qin Qin Qin 8 20x4 3. 30 ± 3. 16 Qiu Pippi Pipi ¾ Do 8 40x4 3. 12 ± 1. 73

Fragrant fragrant fragrant fragrant fragrant ¾ 9 80x4 2. 89 ± 2. 03 Soda beans Doudou beans Doudou Photool 9 160x4 4. 89 ± 2. 52 Susu Susu Susu 9 0. 4x4 4. 00 ± 1. 58

9 20x4 4. 67 + 2. 55 Table 3 Effect of Qinpi total coumarin on serum uric acid in hyperuricemia mice (X soil) Number of animals (only) Dose (mg / Kg * d) [ ^μ mol / L )

0-89. 39 ± 32. 75 0-173. 65 ± 46. 57

0 200x5 85. 60 ± 16. 32 0 400x5 68, 61 ± 10. 00 0 100x5 67. 16 ± 5. 71 The prepared Qinpi total coumarin is made into a capsule according to the conventional preparation method, and the specification is 200mg / Capsule, clinical observation of 30 patients with gout, the results are as follows: Table 4 Clinical observation table

Number of cases Dosage Dosing time Total effective rate (%) Total effective rate (%)

30 cases 1 capsule / time 30 days 90 56. 7

3 times / day

According to the determination, the content of total coumarin in Qinpi obtained exceeds 60%. In order to further study the pharmacological effects of Qinpidin, we use the following method to obtain Qinpirin: dissolve in warm methanol and recover a part of methanol 4%。 Concentrated to a saturated solution of Qinpioxin in methanol, left overnight, to obtain yellow needle crystals, repeated recrystallization through methanol, water to obtain Qinpipin A, purity 94.4%. The solubility of Qinpidin in water is similar to Qinpi total coumarin. An aqueous solution of Qinpi A is used as the test drug (the method is the same as that of Qinpi total coumarin), with a weight of 220-秦秦秦秋 ¾ 秦秦 ind <

Wis tar rats with 250 g skin ¾ ¾ ¾ indole suspension, and New Zealand white rabbits weighing 2.5 to 3.0 kg were used as test animals. The test results showed that different concentrations of quincetin were administered orally to microcrystalline uric acid sodium suspension, and local injection of alginin was induced by Zhao Zhaoxin to induce acute foot swelling in rats and acute rabbits. Gouty arthritis arthritis ^: Both have a significant inhibitory effect, and can reduce the amount of articular fluid in rabbits with acute gouty arthritis, reduce joint congestion, edema, degeneration and necrosis and lymphocytes, neutrophil infiltration and other pathologies change. Qinpi A also significantly reduced blood uric acid in normal mice and high uric acid in mice. Qinpi A has a significant reduction in the number of writhing reactions induced by acetic acid in mice, and also has a certain effect of extending the hot plate pain threshold of mice, indicating that the drug has a certain analgesic effect. Acute ear swelling in mice induced by p-xylene of Qinpiparin, and swelling of paw in rats induced by carrageenan local injection can significantly counteract the effects of swelling of granules on cotton balls. It can also counteract the increase of capillary permeability caused by inflammation. And induced leukocyte migration, indicating that Qinpidin has a certain anti-inflammatory effect, which can act in the early and late stages of inflammation. The above results show that Qinpi A has pharmacological effects related to gout treatment, such as inhibiting swelling of experimental gouty arthritis joints, reducing joint pathological changes in synovial tissue, reducing blood uric acid, and analgesic, anti-inflammatory, and swelling.

Table 5 Effect of Qinpioxin on MSU-induced articular fluid volume in rabbits with acute gouty arthritis ("Earth soil s" Number of animals (only) Dose ng / Kg * d) Joint cavity fluid volume

(ml)

8 0 ± 0 9-0. 309 ± 0. 106 8 20x4 0. 189 ± 0. 094 8 40x4 0. 170 ± 0. 055 9 80x4 0. 214 + 0. 108 9 160x4 0. 160 + 0. 060 9 0. 4x4 0. 241 ± 0. 137 9 20x4 0. 140 ± 0. 101 Table 6 Effect of Qinpiparin on serum uric acid in hyperuricemia mice (J soil) Number of animals in the group (only) Dose (mg / Kg * d) H ⁽ ) ^μ Normal control 10 ⁼ 69. 34 + 22. 35 Model control 10 ― 74. 25 ± 16. 57 Qin Qin Qin Qinbei 10 50x 192. 97 ± 55. 11 Pippi Pippi 10 100x5 63. 34 ± 10. 40 Purine formaldehyde 10 200x5 82. 23 ± 15. 42 Voxel Voxel 10 400x5 88. 51 ± 16. 20

10 100x5 65. 25 ± 8. 36 The following further describes the present invention with reference to the embodiments: The best way to implement the present invention Embodiment 1

Take 100 kg of Qin peel, cut into granules, extract with water three times, concentrate the water extract into an extract, add 3.5 times the amount of ethanol, alcohol precipitation, filter, and concentrate the ethanol recovery solvent to a specific gravity of 1.1. 15 kg。 The paste, and then refrigerated at 8 ° C for 10 hours, filtered, the precipitate was collected, dried, to obtain Qinpi total coumarin 2. 15 kg. Example 2

Take 100 kg of Qin peel, cut into granules, extract with water three times, concentrate the water extract into an extract, add 5 times the amount of ethanol, alcohol precipitation, filter, and concentrate the ethanol recovery solvent to a specific gravity of 1.2. 22 kg。 Then refrigerated at 12 ° C for 12 hours, filtered, collected the precipitate, and dried to obtain Qinpi total coumarin 2.22 kg. Example 3

2 kg of total coumarin obtained from Example 1 or Example 2 or other methods known to those skilled in the art, such as alcohol extraction and alcohol precipitation or alcohol extraction, are used to make an extract and concentrated by a macroporous resin adsorption method. Qinpi total coumarin, according to the conventional preparation method, is added with appropriate excipients to make a 200mg / capsule Qinpi total coumarin capsule. Example 4

According to the conventional preparation method, 2 kg of the total coumarin of Qinpi obtained was added with appropriate excipients to make a 200mg / tablet Qinpi total coumarin tablet. Example 5

According to the conventional preparation method, ² kg of total coumarin of Qinpi obtained was added with appropriate excipients to prepare 200mg / bag of Qinpi total coumarin granules. Example 6

Dissolve 2 kg of Qinpi total coumarin prepared in 3000ml of warm (40-50 ° C) methanol, recover part of the methanol, and concentrate to a methanol saturated solution of Qinpidin A, and leave it overnight to obtain yellow needle crystals. Repeated recrystallization from alcohol and water yielded 1.91 kg of Qinpidin, with a purity of 94.4%. Then, according to the methods of Examples 3, 4, and 5, 200 mg Qinpi A capsules, tablets and granules were prepared.

Claims

Rights request

1. A method for preparing Qinpi total coumarin, including:

a boiled alcohol precipitation method (50% -90%) or alcohol extraction alcohol precipitation method (50% -90%) or alcohol extraction method (70% -95%) extract Qinpi to obtain Qinpi extract extract;

b. Refrigerate the extract at 4-12 ° C for 8-24 hours to form a precipitate;

c The precipitate is obtained by filtration and dried to obtain the total coumarin of Qinpi.

^{2. The} method for preparing Qinpi total coumarin according to claim 1, further comprising dissolving Qinpi total coumarin obtained in step c with warm 45-50 ° C methanol, recovering a portion of methanol, and concentrating It is a step of obtaining a saturated solution of albinol in albinol and leaving it overnight to obtain yellow crystals, and recrystallizing it repeatedly by methanol and 7j to obtain albinol.

3. The application of Qinpi total coumarin in the preparation of medicines for treating acute and chronic gout.

4. Application of Qinpipioxin in the preparation of medicines for treating acute and chronic gout.