CN1923194B - Cortex fraxini coumarin composition - Google Patents

Abstract

The invention relates to a method for preparing ash bark coumarin compound. Wherein, it boils and extracts with water, uses porous resin to separate and purify ash bark coumarin compound; and said compound comprises aesculin, aesculetin, fraxin, and fraxetin, while the total coumarin content is higher than 50%, and the contents of aesculin, aesculetin, fraxin, and fraxetin are higher than 40%, while their ratios are 4-6:1-3:2-4:1. The invention also provides its application in medicine production.

Description

A kind of coumarin composition

technical field

The invention relates to a preparation method and application of a traditional Chinese medicine bark chinensis extract, more specifically a total coumarin mixture extracted and purified from bark bark chinensis, its preparation method and pharmaceutical use.

Background technique

Qinpi is the dry bark or branch bark of Fraxinus rhynchophylla Hance, Fraxinus chinensis Roxb., Fraxinus chinensis Roxb. var. acuminata Lingelsh. or Fraxinus stylosa Lingelsh. of Oleaceae plants, containing Aesculin (aesculin), aesculetin (aesculetin), aesculin (fraxin), and fraxetin (fraxetin) represent coumarin components, etc., although it has been reported in the literature that aesculin has anti-inflammatory and analgesic properties It is clinically used for hyperuricemia and acute attack of gouty arthritis. There are also literature reports on the anti-inflammatory, analgesic and uric acid pharmacological activities of aurocetine, aurocetine B, and urocetidin, but for quincetin There is no research report on the synergistic effect of various coumarin components in the drug.

For the extraction, separation and purification of coumarin components in Qinpi, the existing literature reports all use water extraction and alcohol precipitation method to obtain Qinpi extract, then dissolve it in hot water, refrigerate to form a precipitate, and filter the precipitate to obtain it. Chengdu This method is also adopted in the patent application of Dikang Pharmaceutical Institute. However, the so-called total coumarins obtained by this method are actually only alopecia, and the other several coumarins in the original medicinal materials are basically lost, and their pharmacological activity and clinical effect are significantly reduced.

Traditional Chinese medicine treats diseases, emphasizing the overall coordination of the active ingredients in the medicinal materials on the human body. Nowadays, a large number of studies have proved that the curative effect of the originally effective traditional Chinese medicine is greatly reduced after separation and purification. Through a large number of chemical and pharmacological screenings, the present inventors have proved the active contribution and synergistic effect of various coumarin components in Fructus Pectoris for the treatment of gout and hyperuricemia. Alopecia, Alopecin B, Alopecin, and Alopecin have dual effects of anti-inflammation and reducing uric acid, and are clinically effective in treating gout and hyperuricemia.

The object of the present invention is to obtain a kind of quincetrin coumarin composition represented by 4 kinds of components including quincetrin, quincetrin B, quincetrin and quincetine by studying the extraction and purification process of the coumarin components in quincetrum, The proportion of each component is basically the same as that in the original medicinal materials, so as to improve the clinical effect of treating gout and hyperuricemia.

Contents of the invention

The Qinpi coumarin composition of the present invention is obtained by the following method: take Qinpi, add water to decoct and extract 3 times, add 10 times the amount of water each time, extract for 1.5 hours, combine the extracts, filter, and concentrate the filtrate to a relative density of about 1.1 (heat measurement at 60°C), add ethanol to make the alcohol content reach 80%, let it stand, filter, and recover the ethanol from the filtrate until it has no alcohol smell, add an appropriate amount of water to dissolve it, and add it to the treated D101 macroporous adsorption On the resin column, rinse with water first, discard the washing solution, then elute with 50% ethanol until it is colorless, collect the ethanol eluate, recover the ethanol until it has no alcohol smell, and concentrate to a relative density of about 1.38 (60°C thermal measurement) , vacuum-dried, pulverized, passed through an 80-mesh sieve, and mixed uniformly.

The alopecia coumarin composition of the present invention is a mixture of total coumarins composed of a certain proportion of alopecia, alopecia B, aceticin, aceticine, etc., and is determined by ultraviolet absorption spectrometry, and contains 50% of total coumarins Above, measured by high-performance liquid chromatography, the total content of the 4 components of fenpetrin, fenpetrin, fenpetrin and fenpetine is more than 40%, and the ratio of the 4 components is sequentially 4-6:1-3:2 -4:1, this ratio is basically consistent with the ratio in the original medicinal materials.

Research has found that the ingredients in the fenugreek coumarin composition of the present invention have obvious synergistic effects, and have various pharmacological activities such as anti-gouty arthritis and lowering uric acid. From the extract, afrapertin was isolated and purified, and compared with that before purification, the activity was greatly reduced.

The fenugreek coumarin composition of the present invention can be made into various dosage forms for treating gout and hyperuricemia, including capsules, soft capsules, tablets, and granules, directly or in combination with common pharmaceutical methods. , dripping pills, injections and oral liquid dosage forms.

The medicament prepared by the fenugreek coumarin composition of the present invention is subjected to standardized animal experiments and clinical trials according to relevant requirements, and it is proved that the medicament has the following obvious advantages and remarkable effects.

1. Toxicology experiments show that the coumarin composition is non-toxic

The Qinpi coumarin composition of the present invention is extracted from the traditional Chinese medicine Qinpi, which is derived from natural resources. Long-term medicinal use proves that the long-term use of the Qinpi has no toxic or side effects. The fennel coumarin composition extracted from the fennel bark is enriched with active ingredients of the medicine, and has no obvious toxicity as proved by toxicological studies.

(1) The acute toxicity test in mice shows that with the extracted fenugreek coumarin composition, the maximum tolerated dose of oral administration to mice is 24g total coumarin/kg/24h, and the maximum tolerated multiple is 60kg body weight It is 4000 times of adult clinical oral daily dosage.

(2) The long-term toxicity test in rats shows that with the extracted fennel coumarin composition, the maximum tolerated dose of rat gavage administration is 18g total coumarin/kg/24h, and the maximum tolerance multiple is 60kg body weight It is 3000 times of adult clinical oral daily dosage. Rats were given the fenugreek coumarin composition and observed for 14 days. No rats died, and only the activity of the animals decreased 15 minutes to 18 hours after the administration, and no other abnormal symptoms were seen. After the rats were sacrificed, no abnormalities were found in the vital organs (heart, liver, spleen, lung, kidney, etc.) observed with the naked eye.

(3) The long-term toxicity test of Beagle dogs shows that the coumarin composition is given three doses of 300, 150 and 75 mg/kg, which are respectively equivalent to 50, 25 and 12.5 times of its clinical oral dose, and is administered orally to Beagle dogs continuously. 180 days of medicine and 30 days of drug withdrawal, the results are as follows: (1) There is no obvious effect on animal growth and behavior activities. (2) Routine urine examination values were within the normal range. (3) The values of liver function tests were all within the normal range, but there were effects of reducing ALT, AST, T-Bil, ALP and/or increasing ALT, which disappeared after stopping the drug. (3) The results of renal function tests were all within the normal range, but the high and middle dose groups had the effect of increasing creatinine and blood urea nitrogen, which disappeared after stopping the drug. (4) It has the effect of lowering serum cholesterol. (5) Electrocardiogram examination, the result values are all within the normal range, but the heart rate of the middle-dose group slows down when the drug is administered for 45 days; the Q-T interval time of the high-dose group decreases significantly when the drug is administered for 180 days; The R-wave voltages of the dose groups decreased significantly, significantly and extremely significantly, respectively. (6) The results of bone marrow examination of live animals were all within the normal range. However, after 180 days of administration, it can be seen that the promyelocytosis in the low-dose group significantly increased. After 30 days of drug withdrawal, the high-dose group significantly increased the rod-shaped cells of neutrophils; the middle- and low-dose groups significantly decreased lymphocytes. (7) Systematic autopsy and organ index examination showed no obvious abnormalities. (8) Histopathological examination, only found that there was mild focal inflammation pathological change of liver in 1 case animal in high-dose group and slight local focal inflammation change of kidney occurred in 1 case animal in administration 180 days, compared with the control group, there was no Significant difference (P>0.05). The rest were normal.

2. The main pharmacodynamics experimental research proves that the coumarin composition has the effect of lowering uric acid and anti-gouty arthritis

(1) The effect of coumarin composition on the formation of hypoxanthine type hyperuricemia in mice

When used alone, only aesperidin has a certain inhibitory effect on the increase of serum uric acid concentration in mice induced by Hypoxanthine (P>0.05), while aesperitin, aesperitin B or aesperitin did not significantly inhibit H. And 30,60 and 120mg/kg of the coumarin composition obtained by the above-mentioned technology can obviously, significantly and extremely significantly inhibit the increase of serum uric acid concentration in mice caused by Hypoxanthine (P＜0.05, P＜0.01 and P＜0.001 ), the maximum inhibition rate of uric acid production was 35.85%, and there was an obvious dose-effect relationship (P<0.05) within the dose range of 30-120 mg/kg. The experimental results are shown in Table 1.

Table 1 The effect of coumarin composition on the formation of hypoxanthine type hyperuricemia in mice

Note: ① Model control group and normal control group^^^P<0.001

②Comparison between the administration group and the model control group *P<0.05 **P<0.01 ***P<0.001

(2) Rat uric acid excretion test

Table 2 shows that the single use of aurocetine, afecortine B, aureticoside or auretine at 60-240 mg/kg has no obvious effect on promoting urinary uric acid excretion in rats (P>0.05). However, 60 and 120 mg/kg of coumarin compositions can significantly promote the excretion of uric acid in rats through urine (P<0.01).

Table 2 The effect of coumarin composition on rat urine uric acid excretion (x ± SD)

Note: Compared with the distilled water control group *P<0.05 **P<0.01 ***P<0.001

(3) Sodium urate-induced gouty arthritis model test in rats

Table 3 shows that the single use of aurecetine, afredicine B, afredetin or auvetine at 60-240 mg/kg has no obvious inhibitory effect on gouty joint swelling in rats induced by sodium urate (P>0.05). And the above four components are successively 4-6: 1-3: 2-4: 1 in proportion as the main component of the fennel coumarin composition can significantly inhibit the gouty joint swelling of rats caused by sodium urate (P＜0.01 ), there was a significant inhibitory effect 1 hour after administration (P<0.01), and it was maintained for 6 hours; 3 hours after 30mg/kg administration, there was still a significant inhibitory effect (P<0.01). There was an obvious dose-effect relationship (P<0.05) within the dose range of 120-30 mg/kg.

Table 3 The effect of coumarin composition on sodium urate-induced gouty arthritis in rats

Note: Compared with the control group *P<0.05 **P<0.01 ***P<0.001

(4) Rabbit gouty arthritis model test induced by sodium urate

Table 4 The effect of fennel coumarin composition on sodium urate-induced gouty arthritis in rabbits

Note: Compared with the model control group *P＜0.05 **P＜0.01

Table 4 shows that only the 240mg/kg dose group can significantly inhibit rabbit gouty joint swelling caused by sodium urate (P＜0.05), and 120～60mg/ There was no significant effect in the kg dose group (P>0.5). And the above four components are 4-6: 1-3: 2-4: 1 as the main component in the order of 4-6:1-3:2-4:1, and the 120mg/kg dosage group of the 120mg/kg dosage group of the main component appears to significantly inhibit the sodium urate. Rabbit gouty joint swelling effect (P<0.05), the effect is maintained for more than 5 hours; 30mg/kg still has obvious inhibitory effect (P<0.05), and there is an obvious dose-effect relationship in the dose range of 120-30mg/kg (P<0.05) 0.05).

The effect of table 5 coumarin composition inhibiting sodium urate to stimulate synovial fluid in rabbits

Note: ①Compared between the model control group and the normal control group^^^P<0.001

②Comparison between the administration group and the model control group *P<0.05 **P<0.01

Table 5 shows that only the 240mg/kg dose group can significantly inhibit the generation of sodium urate to stimulate the synovial fluid of rabbits (P＜0.05), and 120～60mg/kg/ There was no significant effect in the kg dose group (P>0.05). And the above four components are 4-6: 1-3: 2-4: 1 as the main component of the coumarin composition 120 and 60 mg/kg, respectively, which can significantly and obviously inhibit the stimulation of rabbit slippery by sodium urate. The formation of membrane fluid (P<0.01 and P<0.05), within the range of 30-120 mg/kg, showed a significant dose-effect relationship.

Table 6 coumarin composition inhibits sodium urate to stimulate the effect of WBC count increase in synovial fluid of rabbits (x ± SD)

Note: ①Compared between the model control group and the normal control group^^^P<0.001

②Comparison between each administration group and the model control group **P<0.01 ***P<0.001

Table 6 shows that only the 240mg/kg dose group can significantly inhibit the generation of sodium urate to stimulate the synovial fluid of rabbits (P＜0.05), and 120～60mg/kg/ There was no significant effect in the kg dose group (P>0.05). And the above four components are 4-6: 1-3: 2-4: 1 as the main components of the 120 and 60mg/kg groups of the coumarin composition of the above four ingredients, respectively, which can significantly and significantly inhibit the stimulation of sodium urate. The migration of WBC in rabbit synovial fluid (P<0.001 and P<0.05) showed a significant dose-effect relationship within the range of 30-120mg/kg.

Table 7 The effect of coumarin composition on rabbit synovium pathology caused by sodium urate

Note: Compared with the model control group *P＜0.05 **P＜0.01

Table 7 shows that only the 240mg/kg dose group can significantly inhibit the hyperemia, edema, crystal deposition and synovial fibrosis pathological changes of the rabbit knee joint synovial membrane caused by sodium urate when using teretine A, terperetin B, terperetin or terperetin alone Effect (P<0.05), 120～60mg/kg dose group has no obvious effect (P>0.05). And the above four components are 4-6: 1-3: 2-4: 1 in proportion successively, and the 120～60mg/kg dosage group of the fennel coumarin composition of the main component has significant inhibitory effect on rabbit knee joint caused by sodium urate. Synovial hyperemia, edema, crystal deposition and pathological changes of synovial fibrosis (P<0.01), in the range of 30-120 mg/kg, there is an obvious dose-effect relationship.

The best examples are given below to further illustrate the present invention but not to limit the present invention.

Example 1

Take 100 kg of bark of Qinpi branches, cut into filaments, add water to decoct and extract 3 times, add 1000 liters of water each time, decoct for 1.5 hours, combine the extracts, filter, combine the extracts three times, and concentrate to a relative density of about 1.1 (60 ℃ thermal measurement), add ethanol to make the alcohol content reach 80%, let it stand, filter, the filtrate decompresses and recovers ethanol until it has no alcohol smell, adds 100 liters of water to dissolve, and adds it to the D101 type macroporous adsorption resin column that has been treated First, rinse with 300 liters of water, discard the washing liquid, and then elute with 600 liters of 50% ethanol, collect the 50% ethanol eluate, recover the ethanol until it has no alcohol smell, and concentrate to a relative density of about 1.38 (60 ℃ heat test ), dried in a vacuum, pulverized, passed through a 80-mesh sieve, and mixed uniformly to obtain 7.12 kg of coumarin composition, calculated as raw medicinal materials, with a yield of 7%. The content of each component in the quincetrin composition is: total coumarin 57.5% (ultraviolet spectrophotometry), fretline A 22%, fenpetin B 8%, fretmanin 13%, fenpetin 4%, and The composition ratio is consistent with that in medicinal materials (see the accompanying drawing for the HPLC chromatogram).

Take 5217g of the above obtained fennel coumarin composition (containing 3000g of total coumarin), add appropriate amount of starch to dilute and adjust the total amount to 15000g, stir and mix, pass through an 80 mesh sieve, add talcum powder, mix evenly, pack into capsules, Make 50,000 capsules to obtain the capsule preparation of the coumarin composition. It has been proved by clinical research that the curative effect of treating gout and hyperuricemia is obviously better than that of acridine preparations.

Description of drawings:

Fig. 1 HPLC chart of the sample of fennel coumarin composition

Fig. 2 HPLC three-dimensional pattern of the sample of aurecetin composition (1. aurecetin; 2. aeretin; 3. aeretin B; 4. aeretin).

Claims (1)

1. a fennel coumarin composition, characterized in that: a, the aesculin composition is a total coumarin mixture composed of a certain proportion of aesculin (aesculin), aesculetin (aesculetin), fraxin (fraxin), aesculetin (fraxetin), etc. Determination by absorption spectrometry, containing more than 50% of total coumarin; b, the aesculin composition mainly contains four components of aesculin (aesculin), aesculetin (aesculetin), fraxin (fraxin), and aesculetin (fraxetin), which are determined by high performance liquid chromatography, 4 The total content of the first ingredient is more than 40%, and the ratio of the four ingredients is 4-6:1-3:2-4:1; The preparation method of the fennel coumarin composition comprises the following steps: take the fennel, decoct and extract 3 times, add 10 times the amount of water each time, extract for 1.5 hours, combine the extracts, filter, concentrate the filtrate to 60°C and heat Measure the relative density to 1.1, add ethanol to make the alcohol content reach 80%, let it stand, filter, and recover the ethanol from the filtrate until it has no alcohol smell, add an appropriate amount of water to dissolve it, and add it to the treated D101 macroporous adsorption resin column , wash with water first, discard the washing solution, then elute with 50% ethanol until it is colorless, collect the ethanol eluate, recover the ethanol until it has no alcohol smell, concentrate to 60°C with a thermal relative density of 1.38, vacuum dry, and pulverize, Pass through a 80-mesh sieve, mix well, and obtain.

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