CN109096112B

CN109096112B - Preparation method of tussilagone

Info

Publication number: CN109096112B
Application number: CN201811099346.6A
Authority: CN
Inventors: 张田勇; 杨双名; 营雪; 钱勇; 谢天培
Original assignee: SHANGHAI BAINIAN SHIDANDE INSPECTION TECHNOLOGY CO LTD; Shanghai Nature Standard R&d And Biotech Co ltd; Shanghai Standard Technology Co ltd
Current assignee: SHANGHAI BAINIAN SHIDANDE INSPECTION TECHNOLOGY CO LTD; Shanghai Nature Standard R&d And Biotech Co ltd; Shanghai Standard Technology Co ltd
Priority date: 2018-09-20
Filing date: 2018-09-20
Publication date: 2021-06-18
Anticipated expiration: 2038-09-20
Also published as: CN109096112A

Abstract

The invention provides a method for efficiently preparing tussilagone from tussilago farfara, which comprises the following steps: soaking and extracting flos Farfarae with petroleum ether, filtering, concentrating, subjecting the concentrated solution to silica gel column chromatography, and recrystallizing for multiple times to obtain tussilagone with purity of above 99%. Each index of the product meets the raw material requirements of a chemical reference substance of traditional Chinese medicine, and can be used as the reference substance, and the yield is obviously improved compared with the prior art; the method does not depend on special equipment such as ultrasonic equipment, a high-speed counter-current chromatograph, supercritical extraction and the like, has low equipment cost, is easy to put into production and convenient to use and operate, and can recycle the solvent in the process; and the single batch processing sample amount is large, thus obtaining a hectogram product; the process is stable and reliable, has good reproducibility and is beneficial to large-scale industrial production.

Description

Preparation method of tussilagone

Technical Field

The invention relates to the field of medicines, in particular to a method for efficiently preparing coltsfoot ketone from traditional Chinese medicine flos farfarae.

Background

Flos Farfarae (Farfarae Flos) is dried bud of Farfarae (Tussilago farfara L.) belonging to Compositae. Flos Farfarae is warm in nature, pungent and slightly bitter in taste, enters lung channel, has effects of moistening lung, descending qi, relieving cough, eliminating phlegm, etc., and can be used for treating chronic cough, cough with dyspnea, excessive phlegm, and cough with hemoptysis. Chemical components reported in flos farfarae include sesquiterpenes, triterpenes, flavonoids, phenolic acids, volatile oils, alkaloids, etc. Among them, sesquiterpenes are characteristic components of flos farfarae, and are the most intensive components in chemical and pharmacological research. The farfarinone is a sesquiterpene component which is characteristic in the farfarinone, and is a characteristic index related to quality control of the farinone in 2015 edition of Chinese pharmacopoeia.

The preparation method of tussilagone in the prior art is reported as follows:

CN 102659585A discloses a method for simultaneously preparing coltsfoot flower ketone and new coltsfoot flower lactone, which comprises the following steps: ultrasonic extraction, activated carbon decoloration, silica gel column chromatography and high-speed countercurrent chromatography separation to obtain the coltsfoot ketone. The disadvantages of this method are: 1) pure coltsone is obtained without separation, and the new coltsin lactone in the product is also a sesquiterpene compound, has a similar structure and is difficult to separate and remove; 2) no industrial-grade equipment exists for ultrasonic extraction; 3) the high-speed counter-current chromatograph has high cost, no industrial equipment and small sample handling capacity, and the handling capacity of analytical equipment is microgram grade, so that the high-speed counter-current chromatograph is not beneficial to large-scale industrial production; 4) the preparation process has high requirements on equipment, high-speed countercurrent chromatographic separation is not popularized, the equipment investment is large, the cost is high, the energy consumption is high, and the yield is low.

CN 103524342A discloses a method for rapidly separating tussilagone by adopting an adjustable ternary solvent system, which comprises the following steps: petroleum ether is repeatedly extracted by ultrasonic to prepare raw material extract, and the raw material extract is extracted by a ternary solvent system high-speed countercurrent chromatography to obtain the coltsfoot ketone. The disadvantages of this method are: 1) the yield is low, and only 97mg of coltsfoot ketone is prepared from 500g of raw materials; 2) no industrial-grade equipment exists in ultrasonic extraction: 3) the high-speed counter-current chromatograph has high cost, no industrial equipment and small sample handling capacity, and the handling capacity of analytical equipment is microgram level, so that large-scale and industrial production cannot be realized; 4) the preparation process has high requirement on equipment, a high-speed counter-current chromatograph is not popularized, the equipment investment is large, the cost is high, and the product purity is low (98.7%).

CN 102633636 a discloses a method for extracting tussilagone by supercritical fluid, which comprises the following steps: soaking the raw materials in ethanol, and supercritical CO₂Extracting with equipment, performing silica gel, aluminum oxide or magnesium oxide column chromatography, and eluting with multiple organic reagents to obtain coltsfoot ketone. The disadvantages of this method are: 1) the yield is low, and only 20-28 mg of coltsfoot ketone is prepared from 10kg of raw materials; 2) the supercritical fluid extraction has high requirements on equipment; 3) the mixed elution of various organic reagents is not beneficial to recovery and environmental pollution, has high cost and is not beneficial to industrial production.

The current technical status of the preparation of tussilagone is as follows: 1. expensive equipment such as a supercritical fluid chromatograph and a high-speed counter-current chromatograph is relied on, and the equipment has low preparation flux and low production efficiency; 2. the purity of the prepared product is not high, impurities of other sesquiterpenes compounds with similar structures are difficult to effectively remove, the purity of HPLC is generally lower than 99%, the purity of residual solvent, roasted residues, moisture residue and TLC is not controlled, and the purity requirement of China food and drug testing research institute on raw materials of traditional Chinese medicine chemical reference substances (used as quality control of medicinal materials) is difficult to meet; 3. the existing method has low preparation yield of only 20-32%, wherein the yield of about 32% can be achieved by adopting equipment such as high-speed counter-current chromatographic extraction and the like, but the preparation flux is low, and the yield of about 20% is achieved by adopting conventional column chromatography (extraction, silica gel, preparation and the like).

Disclosure of Invention

The technical problem to be solved by the invention is to provide a preparation method of tussilagone, which has the advantages of high yield, high purity, large single-batch sample processing capacity, low equipment requirement and easiness in industrial large-scale production.

In order to solve the technical problems, the preparation method of the tussilagone provided by the invention comprises the following steps:

(a) taking a dried flos farfarae medicinal material, crushing, adding 8-30L/kg of petroleum ether according to the weight of the medicinal material, soaking at room temperature for 12-72 hours, and extracting to obtain an extracting solution; filtering, and concentrating under reduced pressure to obtain extract A; the reagent can be recycled.

(b) And (3) performing silica gel column chromatography on the extract A, eluting with an eluent of petroleum ether and a polar solvent, wherein the volume ratio of the petroleum ether to the polar solvent is 100: 0-100: 30, the polar solvent is selected from ethyl acetate or acetone, detecting the eluent by HPLC, collecting the eluent with the content of the tussilagone more than 40% in the HPLC, and concentrating under reduced pressure to obtain a thick paste to obtain the tussilagone extract B.

(c) Recrystallizing the extract B, wherein the recrystallization solvent is petroleum ether, ethanol and water in a volume ratio of (0-100): (7-99): (0.2-1.3), wherein the dosage of the recrystallization solvent is 3-10L/kg according to the weight of the extract B, the recrystallization temperature is 0-minus 25 ℃, and the coltsfoot ketone product is obtained through 2-4 times of recrystallization. The product is white crystal with the coltsfoot ketone content of more than 99 percent.

Specifically, in the step (a), the dosage of the petroleum ether is preferably 10-20L/kg, and the room-temperature soaking time is preferably 24-48 h.

Specifically, in the step (b), gradient elution is adopted, the volume ratio of petroleum ether to a polar solvent is adjusted, elution is performed by using an eluent with low polarity, and then the polarity of the eluent is gradually increased in a gradient elution mode. Preferably, the elution is performed with petroleum ether and polar solvent at a ratio of 100:0, and then the ratio of the polar solvent is gradually increased to 100: 30.

Specifically, in the step (c), the ratio of the mixed recrystallization solvent petroleum ether to ethanol to water is preferably (0-100): (20-99): (0.7 to 1.3). Preferably, in the step (c), the ratio of petroleum ether to ethanol to water is (10-60): (20-99): (1-1.2).

Specifically, the number of recrystallization is preferably 3.

Specifically, the amount of the recrystallization solvent is preferably 5 to 7L/kg.

Specifically, the recrystallization temperature is preferably from-10 to-20 ℃.

The invention also provides the coltsfoot ketone prepared by the method, and the purity of the coltsfoot ketone is more than 99%.

The coltsfoot flower in the invention refers to the dry flower bud of Tussilago farfar L. of Compositae, and the content of the coltsfoot ketone in the dry coltsfoot flower medicinal material adopted in the invention is detected to be 0.060 percent according to the content measurement method of the coltsfoot flower in 2015 edition of Chinese pharmacopoeia.

The preparation method of the tussilagone provided by the invention has high yield, 3.05-3.65 g of the tussilagone can be prepared per 10kg of dried tussilagone, the yield can reach more than 50% by calculating the content of the tussilagone in the dried medicinal materials to be 0.060%, and the preparation method is remarkably improved compared with the prior art.

The purity of the prepared tussilagone is up to more than 99%, the product quality of the tussilagone is improved, and the prepared tussilagone can be used as a reference substance.

According to the preparation method of the coltsfoot ketone, silica gel column chromatography and recrystallization purification are adopted, special equipment such as ultrasonic equipment, a high-speed counter-current chromatograph and supercritical extraction is not relied on, the equipment cost is low, the operation is easy, the use and the operation are convenient, and the solvent in the process can be recycled; and the single batch processing sample amount is large, thus obtaining a hectogram product; the process is stable and reliable, has good reproducibility and is beneficial to large-scale industrial production.

The preparation method of the invention solves the technical problem that the coltsfoot ketone is difficult to produce in a large scale and realize industrialization, provides a material basis for the related research and quality control of the coltsfoot flower, and is beneficial to the implementation of the 'Chinese pharmacopoeia' of 2015 edition.

The invention adopts room temperature extract, silica gel column chromatography is carried out after concentration, and the coltsfoot ketone with the content of more than 99 percent can be obtained through repeated recrystallization, and each index meets the raw material requirement of the chemical reference substance of the traditional Chinese medicine, and the yield is more than 50 percent; the method has the advantages of low equipment requirement, low energy consumption, simple operation, short period, stable process and low cost, can produce hectogram products, is easy for industrialized and large-scale production, and solves the technical problem that the tussilagone is difficult to produce on a large scale and is difficult to industrialize; the product has high purity, each index meets the raw material requirement of a chemical reference substance of the traditional Chinese medicine, the yield is high, a material basis is provided for the related research and quality control of the coltsfoot flower, and the implementation of Chinese pharmacopoeia is facilitated.

Drawings

FIG. 1 is a high performance liquid chromatography chromatogram of the assay of tussilagone prepared in example 1, wherein the HPLC content of tussilagone is 99.5%.

FIG. 2 is a high performance liquid chromatography chromatogram of the extract of flos Farfarae of example 1.

FIG. 3 is a high performance liquid chromatography chromatogram of the coltsfoot extract B obtained in example 1, wherein the content of coltsfoot is 46%.

FIG. 4 is a high performance liquid chromatography chromatogram of coltsfoot ketone prepared in comparative example 1, wherein the content of coltsfoot ketone is 78%.

FIG. 5 is a high performance liquid chromatography chromatogram of coltsfoot obtained in comparative example 2, wherein the content of coltsfoot is 85%.

FIG. 6 is a high performance liquid chromatography chromatogram of coltsfoot prepared in comparative example 3, wherein the content of coltsfoot is 90%

FIG. 7 is a high performance liquid chromatography chromatogram of coltsfoot prepared in comparative example 4, wherein the coltsfoot content is 87%

FIG. 8 is a Thin Layer (TLC) detection chromatogram of colatone prepared in example 1, showing only the target spot and no impurity spot.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

Example 1

Taking 20kg of dried flos farfarae medicinal material, crushing, adding 20 times of petroleum ether according to the weight of flos farfarae, soaking and extracting for 16 h. Filtering the extracting solution, concentrating to obtain an extract A, and sampling and detecting the extracting solution. Mixing the extract A with silica gel, drying, performing silica gel column chromatography, performing gradient elution by using petroleum ether and ethyl acetate in a volume ratio of 100: 0-100: 30, detecting by using HPLC, collecting an elution part with the content of the coltsfoot ketone in the HPLC being more than 40%, and performing reduced pressure concentration to obtain an extract B, wherein the content of the coltsfoot ketone in the extract B is 46% by using HPLC. And recrystallizing the extract B by using a mixed solvent of 10 times of petroleum ether, ethanol and water in a ratio of 60:20:1.2, wherein the crystallization temperature is-10 to-25 ℃, and recrystallizing for 3 times to obtain coltsfoot powder, wherein the weight of the dried extract B is 7.3g, the HPLC content is 99.5%, and the yield is 60.8% calculated by taking the content of the coltsfoot in the raw material dry medicinal material as 0.060%.

Comparative example 1

Taking 20kg of dried flos farfarae medicinal material, crushing, adding 20 times of petroleum ether according to the weight of flos farfarae, soaking and extracting for 16 h. Filtering the extracting solution, and concentrating to obtain an extract A. Mixing the extract A with silica gel, drying, performing silica gel column chromatography, performing gradient elution by using petroleum ether and ethyl acetate in a volume ratio of 100: 0-100: 30, detecting by using HPLC, collecting an elution part with the content of the tussilagone more than 40% in the HPLC, and performing reduced pressure concentration to obtain an extract B. And recrystallizing the extract B by using a mixed solvent of 10 times of petroleum ether, ethanol and water in a ratio of 100:99:0, wherein the crystallization temperature is-10 to-25 ℃, and the recrystallization is performed for 3 times to obtain the coltsfoot, the weight of the dried extract B is only 3.3g, the content of the coltsfoot is at most 78%, and the yield is 27.5% calculated by the content of the coltsfoot in the raw material dried medicinal materials being 0.060%.

Example 2

Taking 30kg of dried flos farfarae medicinal material, crushing, adding petroleum ether with the volume 15 times of the weight of the flos farfarae, soaking and extracting for 48 hours. Filtering the extracting solution, and concentrating to obtain an extract A. Mixing the extract A with silica gel, drying, performing silica gel column chromatography, performing gradient elution by using petroleum ether and ethyl acetate in a volume ratio of 100: 0-100: 30, detecting by using HPLC, collecting an elution part with the content of the tussilagone more than 40% in the HPLC, and performing reduced pressure concentration to obtain an extract B. And recrystallizing the extract B by using a mixed solvent of 5 times of petroleum ether, ethanol and water in a ratio of 100:99:0.2, wherein the crystallization temperature is-10 to-25 ℃, and recrystallizing for 2 times to obtain 10.2g of coltsfoot powder with the content of more than 99 percent after drying, wherein the yield is 56.7 percent by calculating the content of the coltsfoot in the raw material dry medicinal materials to be 0.060 percent.

Comparative example 2

Taking 30kg of dried flos farfarae medicinal material, crushing, adding petroleum ether with the volume 15 times of the weight of the flos farfarae, soaking and extracting for 48 hours. Filtering the extracting solution, and concentrating to obtain an extract A. Mixing the extract A with silica gel, drying, performing silica gel column chromatography, performing gradient elution by using petroleum ether and ethyl acetate in a volume ratio of 100: 0-100: 30, detecting by using HPLC, collecting an elution part with the content of the tussilagone more than 40% in the HPLC, and performing reduced pressure concentration to obtain an extract B. And recrystallizing the extract C by using a mixed solvent of 5 times of petroleum ether, ethanol and water in a ratio of 0:7:2, wherein the crystallization temperature is-10 to-25 ℃, and recrystallizing for 2 times to obtain the coltsfoot, wherein the weight of the dried extract C is only 4.8g, the content of the coltsfoot is 85 percent at most, and the yield is 26.7 percent calculated by taking the content of the coltsfoot in the raw material dry medicinal materials as 0.060 percent.

Example 3

Taking 30kg of dried flos farfarae medicinal material, crushing, adding petroleum ether with the volume of 10 times of the weight of the flos farfarae, soaking and extracting for 24 h. Filtering the extracting solution, concentrating to obtain an extract A, and sampling and detecting the extracting solution. Mixing the extract A with silica gel, drying, performing silica gel column chromatography, performing gradient elution by using petroleum ether and ethyl acetate in a volume ratio of 100: 0-100: 30, detecting by using HPLC, collecting an elution part with the content of the tussilagone more than 40% in the HPLC, and performing reduced pressure concentration to obtain an extract B. And recrystallizing the extract B by using a mixed solvent of 10 times of petroleum ether, ethanol and water in a ratio of 0:99:0.2, wherein the crystallization temperature is-10 to-25 ℃, and recrystallizing for 3 times to obtain the coltsfoot, wherein the weight of the dried extract B is 9.8g, the content of the coltsfoot is more than 99%, and the yield is 54.4% calculated by using the content of the coltsfoot in the raw material dry medicinal materials as 0.060%.

Comparative example 3

Taking 30kg of dried flos farfarae medicinal material, crushing, adding petroleum ether with the volume of 10 times of the weight of the flos farfarae, soaking and extracting for 24 h. Filtering the extracting solution, concentrating to obtain an extract A, and sampling and detecting the extracting solution. Mixing the extract A with silica gel, drying, performing silica gel column chromatography, performing gradient elution by using petroleum ether and ethyl acetate in a volume ratio of 100: 0-100: 30, detecting by using HPLC, collecting an elution part with the content of the tussilagone more than 40% in the HPLC, and performing reduced pressure concentration to obtain an extract B. And recrystallizing the extract B by using a mixed solvent of 10 times of petroleum ether, ethanol and water in a ratio of 100:30:0, wherein the crystallization temperature is-10 to-25 ℃, and the recrystallization is performed for 3 times to obtain coltsfoot powder, the weight of the dried extract B is 3.2g, the HPLC content is 90%, the yield is 17.7% calculated by the content of the coltsfoot in the raw material dry medicinal materials being 0.060%.

Example 4

80kg of dried flos farfarae medicinal material is taken, crushed, added with petroleum ether with the volume of 20 times of the weight of the flos farfarae, soaked and extracted for 48 hours. Filtering the extracting solution, concentrating to obtain an extract A, and sampling and detecting the extracting solution. Mixing the extract A with silica gel, drying, performing silica gel column chromatography, performing gradient elution by using petroleum ether and ethyl acetate in a volume ratio of 100: 0-100: 30, detecting by using HPLC, collecting an elution part with the content of the tussilagone more than 40% in the HPLC, and performing reduced pressure concentration to obtain an extract B. And recrystallizing the extract B by using a mixed solvent of 10 times of petroleum ether, ethanol and water in a ratio of 0:7:1.3, wherein the crystallization temperature is-10 to-25 ℃, and recrystallizing for 3 times to obtain coltsfoot powder, wherein the weight of the dried extract B is 25.5g, the HPLC content is 99.5%, and the yield is 53.2% calculated by the content of the coltsfoot in the raw material dry medicinal material being 0.060%.

Comparative example 4

80kg of dried flos farfarae medicinal material is taken, crushed, added with petroleum ether with the volume of 20 times of the weight of the flos farfarae, soaked and extracted for 48 hours. Filtering the extracting solution, concentrating to obtain an extract A, and sampling and detecting the extracting solution. Mixing the extract A with silica gel, drying, performing silica gel column chromatography, performing gradient elution by using petroleum ether and ethyl acetate in a volume ratio of 100: 0-100: 30, detecting by using HPLC, and collecting an elution part with the content of the tussilagone more than 40% in the HPLC. And recrystallizing the extract B by using a mixed solvent of 10 times of petroleum ether, ethanol and water in a ratio of 0:7:1.3, wherein the crystallization temperature is 4 ℃, and the recrystallization is performed for 3 times to obtain coltsfoot powder, the weight of the dried coltsfoot powder is 8.6g, the HPLC content is 87%, and the yield is 17.9% calculated by taking the coltsfoot content in the raw material dry medicinal material as 0.060%.

Example 5

Taking 15kg of dried flos farfarae medicinal material, crushing, adding 20 times of petroleum ether according to the weight of the flos farfarae, soaking and extracting for 48 h. Filtering the extracting solution, concentrating to obtain an extract A, and sampling and detecting the extracting solution. Mixing the extract A with silica gel, drying, performing silica gel column chromatography, performing gradient elution by using petroleum ether and ethyl acetate in a volume ratio of 100: 0-100: 30, detecting by using HPLC, collecting an elution part with the content of the tussilagone more than 40% in the HPLC, and performing reduced pressure concentration to obtain an extract B. And recrystallizing the extract B by using a mixed solvent of 10 times of petroleum ether, ethanol and water in a ratio of 0:7:1.3, wherein the crystallization temperature is 0-minus 10 ℃, and recrystallizing for 3 times to obtain coltsfoot powder, wherein the weight of the dried extract B is 4.7g, the HPLC content is 99.5%, and the yield is 52.2% calculated by the content of the coltsfoot in the raw material dry medicinal material being 0.060%.

Example 6 detection of technical indicators of the product tussilagone

The tussilagone products prepared in examples 1-5 were tested according to the technical index requirements of the raw materials of the chemical reference substances of traditional Chinese medicine. The tussilagone product used as a reference raw material meets the following technical index requirements: 1. HPLC content > 98.5%; 2. no obvious impurity spots are generated on TLC under 100 mu g; 3. the water content is less than 2 percent; 4. residual solvent is less than 0.2 percent; 5. the residue after roasting is less than 0.1 percent.

The detection method of the technical indexes of the raw materials of the traditional Chinese medicine chemical reference substance comprises the following steps:

1. HPLC content detection adopts a method of [ content determination ] under the item of ' tussilago farfara ' in ' Chinese pharmacopoeia of 2015 edition.

2. TLC detection adopts methods of [ identification ] and general rule 0502 'under the item of' tussilago farfara 'in' Chinese pharmacopoeia of 2015 edition.

3. The water content was measured by the method of "general guidelines 0832 second method" in "Chinese pharmacopoeia" 2015 edition.

4. The residual solvent was detected by the method of general rule 0861 in "Chinese pharmacopoeia" 2015 edition.

5. The detection of the residue after roasting adopts the method of general rule 0841 in the Chinese pharmacopoeia of 2015 edition.

The detection shows that the colatone products prepared in the examples 1-5 all meet the technical index requirements. Taking example 1 as an example, the specific detection result is: 1. and (3) detection of HPLC content: 99.5 percent; 2. detection by TLC: no impurity spots (see fig. 8); 3. and (3) detection of moisture: 0.20 percent; 4. detection of residual solvent: 0.003%; 5. detection of roasting and burning residues: 0.06 percent.

In summary, the above embodiments and drawings are only preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. A preparation method of a coltsfoot reference substance is characterized by comprising the following steps:

(a) taking 20kg of dried flos farfarae medicinal material, crushing, adding 20 times of petroleum ether according to the weight of flos farfarae, soaking for 16h, and extracting to obtain an extracting solution; filtering, and concentrating under reduced pressure to obtain extract A;

(b) mixing the extract A with silica gel, drying, performing silica gel column chromatography, performing gradient elution by using an eluent with the volume ratio of petroleum ether to ethyl acetate being 100: 0-100: 30, collecting an eluent with the content of the tussilagone being more than 40% in HPLC, and performing reduced pressure concentration to obtain an extract B;

(c) recrystallizing the extract B by using a mixed solvent of 10 times of petroleum ether, ethanol and water with the ratio of 60:20:1.2, wherein the recrystallization temperature is-10 to-25 ℃, and recrystallizing for 3 times to obtain the colatone reference substance.