A method for extracting and separating atractylodesin from atractylodes fibrous roots

Abstract

The invention discloses a method for extracting and separating atractylodesin from traditional Chinese medicine, wherein the method uses atractylodes fibrous roots as raw materials, and performs preparative liquid phase separation and purification after extraction. The invention can obtain a high-purity atractylodesin monomer, has high yield and yield, and also has the advantages of simple production process, short cycle, low cost, etc.

Description

Technical Field

[0001] The invention relates to the extraction and separation of traditional Chinese medicine monomers, and in particular to a method for extracting and separating atractylodesin from atractylodes fibrous roots. Background Art

[0002] Atractylodesin is one of the important active ingredients in Atractylodes lancea, with a molecular formula of C 13 H 10 O, molecular weight 182, CAS number 55290-63-6, has multiple pharmacological activities such as hypoglycemic, diuretic, anti-inflammatory, anti-tumor, etc. Atractylodesin has high lipophilicity, poor stability, and is easily degraded under strong light and high temperature. At present, it is mainly extracted and separated from the dried rhizomes of Atractylodes or Atractylodes macrocephala of the Asteraceae family, and mainly exists in volatile oils.

[0003] At present, the extraction and separation of atractylodesin mainly uses atractylodes rhizomes as raw materials, and the volatile oil is separated by supercritical CO 2 The extraction of atractylodes lancea by the method of extraction, reflux extraction or microwave-assisted extraction is carried out, but the purity of the product atractylodes lancea obtained by these methods is low, and it is essentially still a mixture of multiple components. There are also literature reports that the atractylodes lancea extract can be treated with extraction, silica gel column chromatography, crystallization, etc. to obtain atractylodes lancea monomer with a purity of more than 99%. However, these methods are relatively complicated to operate, silica gel chromatography and crystallization require a lot of time, and the product yield is very low, resulting in low extraction efficiency.

[0004] Preparative chromatography has long been widely used in the extraction and separation of Chinese medicine monomers. The technology is now relatively mature. Compared with column chromatography, preparative liquid chromatography has the advantages of short separation time, and preparative liquid chromatography usually has its own detector, which can accurately separate the target components. Therefore, it also has the advantages of simple operation and high degree of automation.

[0005] When extracting atractylodesin, the fibrous roots of Atractylodes lancea are often removed as impurities. It is generally believed in the art that different parts of Chinese herbal medicines have similar chemical compositions. However, Yu Junlin et al. compared the components of rhizomes and fibrous roots of Atractylodes lancea using HPLC and found that the components of the two were very different, and the similarity difference of the fingerprints was much greater than 20%. They believed that the fibrous roots of Atractylodes lancea should not replace the rhizomes as medicine. The applicant's research found that the content of atractylodesin in the fibrous roots of Atractylodes lancea was much higher than that in the rhizomes, while the content of interfering impurities was much lower than that in the rhizomes, thus creating extremely favorable conditions for the implementation of the preparative chromatographic method. Summary of the invention

[0006] The invention aims to provide a method for extracting and separating atractylodesin from atractylodes fibrous roots, aiming to improve the extraction efficiency of atractylodesin.

[0007] Those skilled in the art are aware that a prerequisite for preparative chromatography is that the peak of the separated target must have good separation and peak area to ensure the purity and yield of the product. The applicant compared the HPLC fingerprints of the rhizomes and fibrous roots of Atractylodes lancea and found that the contents of atractylodesin and interfering impurities in the two were very different, which has never been reported in previous literature. Since the content of atractylodesin in the fibrous roots of Atractylodes lancea is much higher than that in the rhizomes, while the content of interfering impurities is much lower than that in the rhizomes, this creates extremely favorable conditions for preparative chromatography. On this basis, the applicant used the following technical scheme to complete the present invention.

[0008] A method for extracting and separating atractylodesin from traditional Chinese medicine, comprising the following steps:

[0009] S1. Wash the Chinese medicinal materials with clean water to remove the sediment, drain, dry at 50-55℃ and crush into powder;

[0010] S2, extracting the Chinese herbal medicine powder with 75-85% ethanol, filtering, combining the extracts and concentrating them into a paste;

[0011] S3, adding petroleum ether to the paste extract, stirring evenly and filtering to remove insoluble matter, collecting the petroleum ether solution, filtering through a 0.45µm filter membrane and setting aside;

[0012] S4, the petroleum ether solution was loaded onto a DAC dynamic axial compression preparative column system, the chromatographic filler was 100-500 mesh silica gel, the mass ratio of atractylodesin in the column feed liquid to the filler was controlled to be 1:50-100, the mobile phase was a non-polar organic solvent containing petroleum ether, the flow rate was 100-500 mL / min, the detection wavelength of the UV detector was set to 340 nm, and the peak segment containing atractylodesin was collected;

[0013] S5. Concentrate the collected target peak segment to dryness under reduced pressure at 45-50°C to obtain a light yellow powder, which is atractylodesin.

[0014] Preferably, the Chinese medicinal material is the fibrous roots of Atractylodes lancea that are 2 to 4 years old.

[0015] Preferably, in step S2, the temperature of the warm immersion extraction is 50-70°C, the number of extractions is 1-3 times, the time of each extraction is 0.5-3 hours, and the amount of ethanol used in each extraction is 5-10 times the weight of the medicinal material.

[0016] Preferably, in step S3, the amount of petroleum ether used is 20-40 times the volume of the paste extract.

[0017] Preferably, the chromatographic filler is 300-400 mesh silica gel.

[0018] Preferably, the mass ratio of atractylodesin to filler in the column feed liquid is 1:60-70.

[0019] Preferably, the mobile phase is a mixed non-polar organic solvent consisting of petroleum ether and ethyl acetate or acetone or tetrahydrofuran.

[0020] Preferably, the mobile phase is petroleum ether and ethyl acetate, and gradient elution is used, which is divided into 4 time periods, and the volume ratio of the mobile phase in each time period is as follows:

[0021]

[0022] Preferably, the flow rate is 300 mL / min.

[0023] According to a specific embodiment of the present invention, a preferred method is as follows:

[0024] Wash the fibrous roots of Atractylodes lancea with clean water to remove the mud and sand, drain, dry at 50-55℃ and crush into powder, then add 85% ethanol to the powder and extract it twice at 50℃ for 2 hours each time, and the amount of ethanol used for each extraction is 6 times the weight of the medicinal material. After extraction, filter, combine the filtrate and concentrate it into a paste, then dissolve the thick paste with 25 times the volume of petroleum ether, stir evenly and filter to remove insoluble matter, collect the petroleum ether solution, filter with 0.45µm filter membrane and set aside;

[0025] The petroleum ether solution obtained above was purified on a DAC dynamic axial compression preparative column system. The chromatographic filler was 300-400 mesh silica gel with a pore size of 2-3 nm. The mass ratio of atractylodesin in the column feed liquid to the filler mass was controlled to be 1:60. The mobile phase was petroleum ether and ethyl acetate. The gradient elution was performed at a flow rate of 300 mL / min. The elution procedure was shown in the table above. The peak segments containing atractylodesin were detected and collected by ultraviolet light. Finally, the collected target peak segments were concentrated to dryness under reduced pressure to obtain pure atractylodesin.

[0026] The ethanol concentrations involved in the present invention are all volume concentrations, such as 85% ethanol means that every 100 ml of ethanol aqueous solution contains 85 ml of ethanol.

[0027] The beneficial effects of the present invention are:

[0028] (1) The present invention can obtain high-purity atractylodesin monomer by preparing the liquid phase once. The separation time for preparing 1g of atractylodesin is only 2 hours, which is much shorter than the 12-24 hours of silica gel column chromatography. In addition, the present invention does not require crystallization treatment after preparation, thereby shortening the preparation cycle. The present invention can reuse the chromatographic column 5-8 times, which has the advantage of low production cost.

[0029] (2) The present invention uses the fibrous roots of Atractylodes lancea as raw materials and combines the preparation of liquid phase to improve the yield of the product. The yield of atractylodes lancea monomer can reach more than 0.4%, and the transfer rate can reach more than 75%, both of which are higher than the known methods reported in the literature.

[0030] The present invention can obtain more products in a shorter time, thus greatly improving the extraction efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 : HPLC chromatogram of Atractylodes lancea (rhizome).

[0032] Figure 2 : HPLC chromatogram of Atractylodes lancea fibrous roots.

[0033] Figure 3 : The preparative liquid chromatogram of Example 2.

[0034] Figure 4 : HPLC detection spectrum of atractylodesin monomer. DETAILED DESCRIPTION

[0035] The present invention is described in detail below with reference to specific embodiments.

[0036] Instruments and reagents:

[0037] Glass reactor: YHGSF(EX)-50L, Gongyi Yuhua Instrument Co., Ltd.

[0038] Preparative liquid chromatography system: Model—DAC-50, DAC-150; Manufacturer—Jiangsu Hanbang Technology Co., Ltd.; Filler—silica gel (100-200 mesh, 200-300 mesh, 300-400 mesh, pore size 2-3 nm); Detector—NU3000C UV variable wavelength preparative detector;

[0039] Analytical balance: XSE205DV, Mettler Toledo;

[0040] Electronic balance: Model YP20002, Shanghai Guangzheng Medical Instrument Co., Ltd.;

[0041] Rotary evaporator: R-20, Yuhua Instrument, Gongyi City;

[0042] Atractylodes lancea (with fibrous roots): purchased from Yingshan County Qiangsheng Planting Professional Cooperative, produced in Yingshan County, Hubei Province;

[0043] Reagents: ethanol, petroleum ether, ethyl acetate, acetone, tetrahydrofuran, all of analytical grade;

[0044] Atractylodesin reference substance: purchased from Chengdu Pusi Biotechnology Co., Ltd., batch number PS012994.

[0045] Example 1 Comparison of atractylodesin content in Atractylodes lancea and Atractylodes lancea fibrous roots

[0046] Referring to the 2020 edition of the Chinese Pharmacopoeia, the specific methods are as follows:

[0047] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel is used as filler; methanol-water (79:21) is used as mobile phase; detection wavelength is 340nm. The theoretical plate number calculated based on the atractylodesin peak should be no less than 5000.

[0048] Preparation of reference solution: Take an appropriate amount of atractylodesin reference substance, weigh it accurately, and add methanol to make a solution containing 20 μg per 1 ml.

[0049] Preparation of test solution: Take about 0.2 g of test sample powder (passed through No. 3 sieve), accurately weigh, place in a stoppered conical flask, accurately add 50 ml of methanol, stopper, weigh, ultrasonically treat (power 250 W, frequency 40 kHz) for 1 hour, cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain.

[0050] Determination method: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0051] 2. Chromatogram: see Figure 1 , Figure 2 .

[0052] 3. Test results

[0053] The test results of atractylodesin content in three batches of medicinal materials are shown in the following table:

[0054]

[0055] The results showed that the content of atractylodesin in the fibrous roots of Atractylodes lancea was significantly higher than that in the raw materials of Atractylodes lancea. Figure 1 and Figure 2 It can also be seen from the chromatogram that the impurity peaks near the atractylodesin peak (24.195min) in the Atractylodes lancea rootlets are fewer and smaller in area than those near the atractylodesin peak (24.818min) in the Atractylodes lancea medicinal material, indicating that the types and contents of impurities are lower, thus creating extremely favorable conditions for the separation degree and peak area of ​​the preparation liquid phase.

[0056] Example 2 Preparation of Atractylodesin

[0057] Take the remaining fibrous root part of the production of Atractylodes lancea, which is the statutory non-medicinal part, wash the mud and sand with clean water, drain it, dry it at 50-55℃ (because atractylodesin is unstable, too high drying temperature can easily lead to component degradation) for 8-10 hours, crush it and pass it through a No. 2 sieve, then add 85% ethanol to the powder and extract it twice at 50℃, each time for 2 hours, and the amount of ethanol used for each extraction is 6 times the weight of the medicinal material. After extraction, filter, combine the filtrate and concentrate it into a paste, then dissolve the thick paste with 25 times the volume of petroleum ether, stir it evenly and filter it, remove the insoluble matter, collect the petroleum ether solution, the petroleum ether can be added twice, after the first filtration, add the remaining petroleum ether to the insoluble matter to dissolve and filter it again, the amount of petroleum ether added each time is not limited, and the total amount of organic solvent can be controlled, the collected petroleum ether solution is filtered with a 0.45µm filter membrane and the atractylodesin content is detected by HPLC for standby use.

[0058] The petroleum ether solution obtained above was purified on a DAC dynamic axial compression preparative column system. The chromatographic filler was 300-400 mesh silica gel with a pore size of 2-3 nm. The column volume ratio was 60:1 (filler: atractylodesin). The mobile phase was petroleum ether and ethyl acetate. The gradient elution was performed at a flow rate of 300 mL / min. The procedure was as follows:

[0059]

[0060] The eluted fractions were detected by an ultraviolet detector at 340 nm, and the peak segments containing atractylodesin were collected. Finally, the collected target peak segments were concentrated to dryness under reduced pressure at 45-50 ° C to obtain a light yellow powder, which was atractylodesin with a purity of 99.33% and a yield of 0.48% (0.48 g of product was extracted from every 100 g of medicinal material. The yield was related to the content of the medicinal material and the extraction method, and was jointly determined by the two). The transfer rate was 85.7% (referring to the proportion of the extracted atractylodesin monomer to the total amount of atractylodesin in the medicinal material, which is often used to evaluate the extraction ability of the method and has nothing to do with the amount of the medicinal material. The atractylodesin content in the medicinal material in this embodiment is 0.56%).

[0061] Example 3 Preparation of Atractylodesin

[0062] The extraction of medicinal materials in this embodiment is the same as that in Embodiment 2, the only difference being the conditions for preparing the liquid phase.

[0063] The petroleum ether solution obtained above was purified on a DAC dynamic axial compression preparative column system. The chromatographic filler was 300-400 mesh silica gel with a pore size of 2-3 nm. The column ratio was 60:1 (filler: atractylodes macrocephala), the mobile phase was petroleum ether and ethyl acetate (8:2), the elution was isocratic, and the flow rate was 300 mL / min.

[0064] Atractylodesin was obtained with a purity of 95.11%, a yield of 0.45%, and a transfer rate of 80.3% (the content of atractylodesin in the medicinal material was 0.56%). The impurity peaks in the preparation spectrum were included in the target peak or overlapped, and the target peak and the impurity peak could not be effectively separated, resulting in low product purity.

[0065] Example 4 Preparation of Atractylodesin

[0066] The extraction of medicinal materials in this embodiment is the same as that in Embodiment 2, the only difference being the conditions for preparing the liquid phase.

[0067] The petroleum ether solution obtained above was purified on a DAC dynamic axial compression preparative column system. The chromatographic filler was 300-400 mesh silica gel with a pore size of 2-3 nm. The column volume ratio was 60:1 (filler: atractylodesin). The mobile phase was petroleum ether and acetone. The gradient elution was performed at a flow rate of 300 mL / min. The procedure was as follows:

[0068]

[0069] Atractylodesin was obtained with a purity of 98.62%, a yield of 0.39%, and a transfer rate of 69.6% (the content of atractylodesin in the medicinal material was 0.56%). The elution ability of acetone was not as good as that of ethyl acetate, resulting in a low yield and transfer rate.

[0070] Example 5 Preparation of Atractylodesin

[0071] The extraction of medicinal materials in this embodiment is the same as that in Embodiment 2, the only difference being the conditions for preparing the liquid phase.

[0072] The petroleum ether solution obtained above was purified on a DAC dynamic axial compression preparative column system. The chromatographic filler was 100-200 mesh silica gel with a pore size of 2-3 nm. The column volume ratio was 80:1 (filler: atractylodes macrocephala), the mobile phase was petroleum ether and ethyl acetate, gradient elution, and a flow rate of 500 mL / min. The procedure was the same as in Example 2.

[0073] Atractylodesin was obtained with a purity of 99.52%, a yield of 0.41%, and a transfer rate of 73.2% (the content of atractylodesin in the medicinal material was 0.56%).

[0074] Example 6 Preparation of Atractylodesin

[0075] Take the remaining fibrous root part of the production Atractylodes lancea, wash the mud and sand with clean water, drain and dry at 50-55℃ and crush into powder, then add 75% ethanol to the powder and extract it at 65℃ for 3 times, each time for 1.5 hours. The amount of ethanol extracted each time is 8 times the weight of the medicinal material. Filter after extraction, combine the filtrate and concentrate it into a paste, then dissolve the thick paste with 35 times the volume of petroleum ether, stir evenly and filter to remove insoluble matter, collect the petroleum ether solution, filter with 0.45µm filter membrane and set aside.

[0076] The chromatographic conditions for the preparative liquid phase were the same as those in Example 2.

[0077] Atractylodesin was obtained with a purity of 99.28%, a yield of 0.40%, and a transfer rate of 71.4% (the content of atractylodesin in the medicinal material was 0.56%).

[0078] Comparative Example

[0079] Take the remaining fibrous root part of the production Atractylodes lancea, wash the mud and sand with clean water, drain, dry at 50-55℃ and crush into powder, then add 85% ethanol to the powder and extract twice at 50℃, 2 hours each time, and the amount of ethanol used for each extraction is 6 times that of the medicinal material. After extraction, filter, combine the filtrate and concentrate it into a paste. Then dissolve the extract with 25 times the volume of petroleum ether, stir evenly and filter, remove insoluble matter, collect the petroleum ether solution, and condense it into a thick paste by rotary evaporation.

[0080] The thick paste was stirred and mixed with a small amount of silica gel, dried and then dry-loaded for silica gel column chromatography. The silica gel particle size was 200-300 meshes, and the loading ratio was 1:30 (atractylodesin: silica gel). After loading, petroleum ether was used for elution. After high performance liquid chromatography detection, the elution segment containing atractylodesin was collected. The collected target segment was concentrated, crystallized, and filtered to obtain atractylodesin with a purity of 99.07%, a yield of 0.32%, and a transfer rate of 57.1% (the content of atractylodesin in the medicinal material was 0.56%).

[0081] The above results show that the present invention uses a preparative liquid phase and conducts experimental screening on the extraction conditions and chromatographic conditions, so that the prepared atractylodesin monomer has a high purity, which can ensure clinical efficacy. More importantly, the yield of the present invention is more than 0.4%, and the highest can reach 0.48%, while the yield of the conventional column chromatography method is only 0.32%. The yield of the present invention is increased by about 50% compared with the conventional method, which can greatly increase the output and reduce the cost of medicinal materials in actual production. The reason for this is that, on the one hand, the present invention uses a medicinal material with a higher content of atractylodesin, namely, the fibrous root of Atractylodes lancea, and on the other hand, it uses a preparative liquid phase method with a more powerful extraction ability.

Claims

1. A method for extracting and separating atractylodesin from atractylodes fibrous roots, characterized in that The following steps are involved: S1. Wash the fibrous roots of Atractylodes lancea with clean water to remove the sediment, drain, dry at 50-55℃ and crush into powder; S2, extracting the Atractylodes lancea root powder with 75-85% ethanol, filtering, combining the extracts and concentrating them into a paste; S3, adding petroleum ether to the paste extract, stirring evenly and filtering to remove insoluble matter, collecting the petroleum ether solution, filtering through a 0.45µm filter membrane and setting aside; S4, the petroleum ether solution was loaded onto a DAC dynamic axial compression preparative column system, the chromatographic filler was 100-500 mesh silica gel, the mass ratio of atractylodesin in the column feed liquid to the filler was controlled to be 1:50-100, the mobile phase was petroleum ether and ethyl acetate, gradient elution was used, the flow rate was 100-500 mL / min, the detection wavelength of the UV detector was set to 340 nm, and the peak containing atractylodesin was collected; S5. Concentrate the collected target peak at 45-50°C under reduced pressure to dryness to obtain a light yellow powder, which is atractylodesin. The gradient elution is divided into 4 time periods, and the mobile phase volume ratio in each time period is as follows:

2. The method for extracting and separating atractylodesin from the fibrous roots of Atractylodes lancea as claimed in claim 1, characterized in that: In step S2, the temperature of the warm immersion extraction is 50-70°C, the number of extractions is 1-3 times, the time of each extraction is 0.5-3 hours, and the amount of ethanol used in each extraction is 5-10 times the weight of the medicinal material.

3. The method for extracting and separating atractylodesin from the fibrous roots of Atractylodes lancea as claimed in claim 1, characterized in that: In step S3, the amount of petroleum ether used is 20-40 times the volume of the paste extract.

4. The method for extracting and separating atractylodesin from the fibrous roots of Atractylodes lancea as claimed in claim 1, characterized in that: The chromatographic filler is 300-400 mesh silica gel.

5. The method for extracting and separating atractylodesin from the fibrous roots of Atractylodes lancea as claimed in claim 1, characterized in that: The mass ratio of atractylodesin to filler in the column feed liquid is 1:60-70.

6. The method for extracting and separating atractylodesin from atractylodes fibrous roots as claimed in claim 1, characterized in that: The flow rate was 300 mL / min.

Images