CN107216365B - Method for separating ergosterol and asterosterol reference substances from traditional Chinese medicine grifola - Google Patents
Method for separating ergosterol and asterosterol reference substances from traditional Chinese medicine grifola Download PDFInfo
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Abstract
The invention discloses a method for separating ergosterol and asterosterol reference substances from traditional Chinese medicine Polyporus umbellatus, which takes dried sclerotia of Polyporus umbellatus (pers.) belonging to Polyporus medicinal fungi of Polyporus of Polyporaceae as raw materials, and obtains high-purity monomeric compounds of ergosterol (ergosterol) and asterosterol (Stellasterol) by alcohol solution extraction, petroleum ether impurity removal, dichloromethane extraction, sephadexLH-20 column chromatography, recrystallization and semi-preparation. The method is simple and reliable, has easily obtained raw materials and lower economic cost, and is suitable for large-scale preparation of ergosterol and asterosterol reference substances; and the ethanol or methanol is adopted during the extraction and crystallization, so that the pollution to the environment is small; the crystallization or preparation method adopted by the invention shortens the time for separating and purifying the sample, reduces the cost of manpower and material resources and improves the economic effect.
Description
Technical Field
The invention relates to the technical field of separation and purification of traditional Chinese medicines and natural products, in particular to a method for separating ergosterol and asterosterol reference substances from traditional Chinese medicine grifola.
Background
The traditional Chinese medicine Polyporus umbellatus is a dry sclerotium of Polyporus umbellatus (pers.) belonging to Polyporus of Polyporaceae, which is mainly distributed in most of China and mainly produced in Gansu, shaanxi, shanxi, henan, sichuan, hunan, yunnan, jilin, heilongjiang and other places, wherein the Shanxi is the best place. Polyporus umbellatus was originally recorded in Shen nong Ben Cao Jing, listed as a middle-quality product. The famous physicians' bibliography, the evidence-based materia Medica and the compendium of materia Medica are well documented. The traditional Chinese medicine considers that the traditional Chinese medicine has the effects of promoting diuresis and excreting dampness, mainly treats swelling, fullness and pain in abdomen, treats thirst, removes dampness, relieves typhoid fever, epidemic febrile disease and high fever, and purges bladder. The medicinal history of the polyporus umbellatus is long, the formula is wide, the polyporus umbellatus is one of the traditional common traditional Chinese medicines, and the traditional Chinese medicine clinical common formulas comprise polyporus umbellatus soup, polyporus umbellatus pills, polyporus umbellatus powder, poria cocos powder, oriental wormwood poria cocos powder, xiaoxiao soup and the like.
The medicinal fungi are important components of natural medicines, in recent years, in order to find medicine sources and find lead compounds, the medicinal value of the polyporus umbellatus is valued by medical researchers and obtains favorable progress, and a large number of chemical and pharmaceutical research reports provide experimental basis for the action substance basis and the modern medicinal development of the polyporus umbellatus. Polyporus umbellatus reported mainly contains polysaccharides, steroids, pentacyclic triterpenes, anthraquinones, pyrimidines, purines, fatty acids, amino acids, vitamins and trace inorganic elements. Pharmacological research shows that the polyporus umbellatus has the functions of promoting urination, regulating immunity, protecting liver, resisting tumor, resisting bacteria, resisting inflammation, resisting virus, etc. The research of carding literature indicates that the exact bioactive substances in the umbellate pore fungus are sterol, sterone and polysaccharide components, definitely, the types and structures of the compounds in the umbellate pore fungus are various, and the medicinal potential of the compounds is huge.
Ergosterol (Ergosterol), a compound isolated from Polyporus umbellatus, was isolated from Polyporus umbellatus by Yosioka et al in 1964, and starfish sterol (Stellasterol) was obtained from Polyporus umbellatus by Lu W in 1985. The structural formulas of two sterol compounds, ergosterol (Ergosterol) and asterosterol (Stellasterol) are as follows:
aiming at the research on diuresis and substance basis of polyporus umbellatus, zhao Yingyong et al, which considers that the effective component of polyporus umbellatus for diuresis is mainly ergosterol, and applied for the patent of application of ergosterol in preparing diuresis medicine (patent number CN 101596201A). P. Prichard proves that ergosterol, asterosterol and the like can reduce the transfer of cholesterol ester between High Density Lipoprotein (HDL) and Low Density Lipoprotein (LDL) mediated by in vivo cholesterol ester transfer protein (patent numbers: PCT/CA2002/001457 CN 1558766A), and provides a basis for preparing blood lipid regulating medicines by the structural sterol. Li Farong et al have demonstrated that ergosterol can effectively promote the accumulation of rhodamine 123 in SGCR7901/ADR cells, and when used in combination with chemotherapeutic adriamycin at a non-toxic dose, can promote the accumulation of chemotherapeutic drugs in cells to reverse the drug resistance of tumor cells (patent No. CN 102552284A). Kong Jilie, etc. discovered a new asteriscus asterol A, and the evaluation of a cell toxicity model proves that it has good anti-tumor bioactivity, and can be made into a drug to provide a new way for preventing and treating cancer (patent No. CN 1660885A). Because Ergosterol (Ergosterol) and asterosterol (Stellasterol) have wider research space and drug application prospect, the preparation of each reference substance has practical application value and market demand necessity.
Aiming at the method for simultaneously preparing Ergosterol (Ergosterol) and asterosterol (Stellasterol) reference substances which are not reported in documents at present, the method for separating two sterol monomers from the polyporus umbellatus medicinal material is mainly obtained by gel column chromatography, crystallization and semi-preparation, and the Ergosterol and asterosterol are obtained quickly and economically. For example, an ergosterol derivative is obtained by extracting, separating and purifying from traditional Chinese medicine ganoderma lucidum spore powder (patent number: CN 102070695A), and a preparation method for producing ergosterol by yeast (patent number: CN 1765916). The methods reported in the literature all use a plurality of organic solvents for extraction and a plurality of fillers for column chromatography for separation and purification, the operation is complex, the steps are tedious and tedious, the whole process consumes a long time, the purity of the obtained sample is low, and in addition, the starfish sterol is not referred to the reference substance preparation method in the literature. Meanwhile, a large number of high-purity two reference substances with similar structures are rapidly obtained, the purification and preparation conditions are harsh, and mainly the two reference substances have similar structures, small property difference and large separation difficulty.
Disclosure of Invention
The invention aims to provide a method for separating ergosterol and asterosterol reference substances from traditional Chinese medicine grifola so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for separating ergosterol and asterosterol reference substances from traditional Chinese medicine grifola comprises the following steps of taking an alcohol extract of a grifola medicinal material as a raw material, removing impurities by using petroleum ether, extracting by using dichloromethane, separating by column chromatography, recrystallizing and carrying out reverse-phase high-efficiency chromatography to obtain the reference substances with the purity of more than 98%, wherein the specific steps are as follows:
(1) Crushing polyporus umbellatus, sieving, adding 5-10 times of methanol or ethanol solution with volume concentration not lower than 95%, percolating or reflux extracting, and recovering solvent to obtain extractum; dispersing the obtained extract with distilled water, sequentially extracting with petroleum ether and dichloromethane, recovering and concentrating dichloromethane extractive solution to obtain soft extract;
(2) Washing the obtained thick paste with distilled water with the volume of 3-5 times to remove impurities, carrying out Sephadex LH-20 column chromatography on filter residues, and then carrying out dichloromethane or chloroform: isocratic elution with methanol (1:1), collecting the lighter fraction, and recovering the eluate to obtain crude sterol product;
(3) Dissolving the crude sterol product in anhydrous methanol or ethanol, and filtering; heating the filtrate sample, dissolving completely under ultrasonic condition, cooling to separate out white needle crystal as ergosterol and asterosterol mixed sample;
(4) Dissolving the mixed sample obtained in the step (3) in chromatographic methanol or chromatographic acetonitrile, and then purifying by using a reversed phase high performance liquid chromatography; reasonably collecting the preparation solution within the retention time corresponding to the main absorption peak, concentrating under reduced pressure, recovering, and lyophilizing to obtain ergosterol and asterosterol reference substances.
As a further scheme of the invention: heating a filtrate sample in the step (3) to 50-60 ℃; and after reaching the saturated state, the mixture is cooled at room temperature or 4 ℃.
As a further scheme of the invention: and (3) dissolving the mixed sample obtained in the step (4) in chromatographic methanol or chromatographic acetonitrile with the concentration of 0.2-0.5 g/mL.
As a further scheme of the invention: the chromatographic conditions of the reversed-phase high performance liquid chromatography in the step (4) are that a chromatographic column is kromasil C18,250 multiplied by 30mm and 5 mu m or YMC C18,250 multiplied by 20mm and 5 mu m, the wavelength of a UV detector is 283nm, the flow rate is 5-20 mL/min, the column temperature is 25-30 ℃, and the mobile phase is methanol-water or acid water, acetonitrile-water or acid water.
Compared with the prior art, the invention has the beneficial effects that:
1) The ergosterol and asterosterol compounds have similar structures, small property difference and large separation difficulty, and two reference substances of high-purity ergosterol and asterosterol can be obtained simultaneously through one-time extraction and preparation process; the obtained reference substance is gram-grade to ten-gram-grade in scale, and is very feasible no matter small-scale laboratory preparation or large-scale industrial popularization.
2) Ergosterol and asterosterol have the functions and the purposes of promoting urination, resisting tumors, regulating blood fat, synthesizing precursor substances by medicines and the like, and have great development and utilization prospects. The invention can rapidly prepare gram-grade ergosterol and asterosterol for biological activity research, chemical structure modification and new drug development;
3) Ergosterol and asterosterol are both main components of fat-soluble substances contained in grifola, but no literature reports a method for preparing a reference substance of the two compounds at the same time at present; the invention can provide a reference substance for content measurement for quality control of traditional Chinese medicine grifola, and is beneficial to establishing a quality control method of the grifola;
4) The organic solvent selected by the invention can be recycled, the pollution to the environment is small, 95% ethanol or methanol is adopted during extraction and crystallization, and the solvent is low in price; the crystallization or preparation method adopted by the invention shortens the time for separating and purifying the sample, reduces the cost of manpower and material resources and improves the economic effect;
5) The invention is a rapid separation and purification technology, adopts conventional technology and means, has simple method and steps, is easy to popularize, convenient to operate, and has good specificity, high repeatability and good stability.
Drawings
FIG. 1 is an HPLC chromatogram for the ergosterol purity measurement in example 1.
FIG. 2 is the ergosterol UV absorption spectrum of example 1.
FIG. 3 is an HPLC chromatogram for detecting the purity of starfish sterol in example 1.
FIG. 4 is the starfish sterol UV absorption spectrum of example 1.
FIG. 5 is an ergosterol HR-ESI-TOF-MS spectrum obtained in example 1.
FIG. 6 is a HR-ESI-TOF-MS spectrum of starfish sterol from example 1.
FIG. 7 is a schematic view ofErgosterol Nuclear magnetism in example 1 1 H NMR spectrum.
FIG. 8 ergosterol NMR as in example 1 13 C NMR spectrum.
FIG. 9 shows starfish sterol nuclear magnetism in example 1 1 H NMRR profile.
FIG. 10 shows starfish sterol nuclear magnetism in example 1 13 C NMR spectrum.
FIG. 11 shows the fingerprint of Grifola frondosa (A).
FIG. 12 shows the corresponding spectrum positions of the two chemical control peaks (B).
Detailed Description
The technical solution of the present patent will be further described in detail with reference to the following embodiments.
Referring to FIGS. 1-12, a method for separating ergosterol and asterosterol control from traditional Chinese medicine Polyporus comprises the following steps:
1. separating ergosterol and asterosterol, which comprises the following steps:
(1) Weighing 500g of dry polyporus umbellatus medicinal material, pulverizing, sieving with a No. four sieve, and percolating with 10 times of 95% ethanol (5L); recovering ethanol to obtain Polyporus umbellatus extract 20.3g, and adding 100mL distilled water to obtain dispersion solution; extracting the obtained product with petroleum ether for three times in equal volume, wherein 100mL of petroleum ether is used each time, and then extracting with dichloromethane for 3 times, wherein 100mL of dichloromethane is used each time; finally concentrating the dichloromethane extract to obtain an extract;
(2) Eluting the extract obtained in the previous step with 50mL of distilled water for 3 times, filtering to remove larger impurities, collecting filter residue, performing Sephadex LH-20 column chromatography (500 g of packed column volume) on the filter residue, and performing column chromatography with V (dichloromethane): v (methanol) =1:1, mobile phase isocratic elution, collecting fractions once per 50mL, combining the fractions from Fr.5 to Fr.16 by TLC analysis, and concentrating and recovering the combined fractions under reduced pressure; then adding ethanol into the combined flow parts for dissolving, heating (50-60 ℃) to form saturated solution under the ultrasonic condition, standing for 8-12h at room temperature, and separating out 7.6g of needle-like crystals;
(3) Dissolving the crystal obtained in the previous step with chromatographic methanol to obtain a solution with the concentration of 0.3g/mL, filtering the solution with a 0.45-micron microporous membrane, and performing separation and purification by reversed-phase high performance liquid chromatography; chromatographic conditions are as follows: the chromatographic column is YMC C18, 250X 20mm,5 μm; the detection wavelength is 283nm; the flow rate is 20mL/min; the column temperature is 30 ℃; the mobile phase is V (methanol): v (water) =95:5; the sample injection amount is 0.5mL; the retention time of the compound a is 30.3min, and the preparation liquid within 29.3-32.2 min is collected; the retention time of the compound b is 35.3min, the preparation liquid within 34.8-35.9 min is collected and recovered to obtain 3.55g of monomer compounds a and 1.74g of monomer compounds b;
2. HPLC purity analysis of the compounds a and b comprises the following specific steps:
accurately weighing the obtained compounds a and b, dissolving with chromatographic methanol completely to obtain stock solution with concentration of 1mg/mL, and accurately sucking 10 μ L with sample injector for purity detection by HPLC;
chromatographic conditions are as follows: the chromatographic column is (kromasil C18, 250X 4.6mm,5 μm); PDA detector, 283nm wavelength monitoring; the column temperature is 30 ℃; the flow rate is 1.0mL/min; the mobile phase is V (methanol): v (water) =95:5; the purity of the two compounds is calculated by using a peak area normalization method, the results show that the HPLC purity of the compounds a and b reaches 98%, and the purity detection results of the compounds a and b are respectively shown in figures 1-2 and 3-4;
3. the mass spectrum analysis of the compounds a and b comprises the following specific steps:
respectively preparing the compounds a and b into 100ng/mL solutions to be detected, and analyzing by adopting high-resolution electrospray flight time mass spectrum (HR-ESI-TOF-MS), wherein the compound a m/z:397.3456[ H ]] - (ii) a Suggesting that compound b m/z:443.3522[ HCOO ]] + ；
4. Nmr analysis of compounds a and b:
a compound a: white amorphous powder is prepared from (A) white amorphous powder, 1 HNMR and 13 c NMR spectrum gave 6 methyl groups, 6 double-bonded carbon. Delta C 136.9 133.4, 121.0, 117.7, 142.8, 131.2 and 1 oxocarbon delta C 71.8 signal; the molecular formula of the compound is determined to be C by combining mass spectrometry data analysis and supposing that the compound contains 28 carbons 28 H 44 O。 1 H NMR(400MHz，CDCl 3 )δ H 5.57(1H，m，7-H)，5.39(1H，m，6-H)，5.21(1H，dd，J＝15.2，8.2Hz，22-H)，5.17(1H，dd，J＝15.2，8.0Hz，H-23)，3.63(1H，m，H-3)，1.04(3H，d，J＝6.6Hz，H-21)，0.95(3H，s，H-19)，0.92(3H，d，J＝6.8Hz，H-28)，0.84(3H，d，J＝6.4Hz，H-27)，0.82(3H，d，J＝6.4Hz，H-26)，0.63(3H，s，H-18)。 13 C NMR(101MHz，CDCl 3 )δ142.8(C-8)，141.2(C-5)，136.9(C-22)，133.4(C-23)，121.0(C-6)，117.7(C-7)，71.8(C-3)，57.1(C-17)，55.9(C-14)，47.6(C-9)，44.2(C-13)，42.2(C-24)，41.8(C-4)，40.5(C-20)，39.8(C-12)，38.4(C-1)，34.5(C-10)，33.4(C-25)，31.1(C-2)，29.7(C-16)，24.4(C-15)，22.5(C-11，21)，21.3(C-26)，21.0(C-27)，19.0(C-28)，17.7(C-19)，13.4(C-18)；
By analyzing the signal characteristics and comparing with the data reported in the literature, the structure of the compound a is analyzed as Ergosterol (Ergosterol), and the structural formula of the compound a is as follows:
compound b: white amorphous powder is prepared from (A) white amorphous powder, 1 HNMR and 13 c NMR spectrum gave 6 methyl groups, 2 double-bonded carbon (. Delta.) C 139.6 135.8, 132.0, 117.6 and 1 oxocarbon delta C 71.8 signal; determining the molecular formula of the compound as C by combining mass spectrum data analysis 28 H 46 O。 1 H NMR(400MHz，CDCl 3 )δ H 5.34(2H，overlapped，H-7，22)，5.18(1H，dd，J＝15.2，8.8Hz，H-23)，3.59(1H，m，H-3)，1.02(3H，d，J＝6.6Hz，H-21)，0.92(3H，d，J＝6.0Hz，H-28)，0.79(9H，overlapped，H-19，26，27)，0.54(3H，s，H-18)。 13 C NMR(101MHz，CDCl 3 )δ C 139.6(C-8)，135.8(C-22)，132.0(C-23)，117.6(C-7)，71.2(C-3)，56.1(C-17)，55.2(C-14)49.6(C-9)，43.4(C-13)，42.9(C-24)，40.6(C-5)，40.4(C-20)，39.6(C-12)，38.1(C-4)，37.3(C-1)，34.3(C-10)，33.2(C-25)，31.6(C-2)，29.8(C-6)，28.26(C-16)，23.0(C-15)，21.7(C-11)，21.2(C-21)，20.1(C-26)，19.8(C-27)，17.7(C-28)，13.1(C-19)，12.2(C-18)；
After the above analysis of signal characteristics and comparison with literature data, the structure of compound b is analyzed as asterosterol (Stellasterol);
the structural formula is as follows:
example 2
A method for separating ergosterol and asterosterol reference substances from traditional Chinese medicine grifola comprises the following steps:
1. separation of ergosterol and asterosterol
Weighing 300g of dry polyporus umbellatus, pulverizing, sieving with a third sieve, extracting with 5 times of 98% methanol (1.5L) under reflux for 3 times, recovering methanol to obtain 15.2g of polyporus umbellatus extract, and adding 50mL of distilled water to obtain a dispersion solution; extracting the obtained substance with petroleum ether for three times in equal volume, wherein 50mL of petroleum ether is used each time, and extracting with chloroform for 3 times, wherein 50mL of chloroform is used each time; finally, concentrating the chloroform extract to obtain an extract;
(2) Eluting the extract obtained in the previous step with 30mL distilled water for 3 times, filtering to remove larger impurities, collecting the filter residue, subjecting the filter residue to Sephadex LH-20 column chromatography (300 g of packed column volume), eluting with V (chloroform): v (methanol) =1:1, eluting with mobile phase isocratic, collecting fractions every 30mL, combining the fractions from Fr.4 to Fr.13 by TLC analysis, and concentrating and recovering the combined fractions under reduced pressure; then adding methanol into the combined flow parts for dissolving, heating (50-60 ℃) to form saturated solution under the ultrasonic condition, standing for 8-12h under the condition of 4 ℃, and separating out 4.5g of needle-like crystals;
(3) Dissolving the crystal obtained in the previous step with chromatographic acetonitrile to obtain a solution with the concentration of 0.3g/mL, filtering with a 0.45-micron microporous membrane, and separating and purifying by reversed-phase high performance liquid chromatography; chromatographic conditions are as follows: the chromatographic column is kromasil C18, 250X 20mm,5 μm; the detection wavelength is 283nm; the flow rate is 15mL/min; the column temperature is 30 ℃; mobile phase V (acetonitrile): v (0.02% trifluoroacetic acid water) =95:5; the sample injection amount is 0.4mL; the retention time of the compound a is 32.6min, and the preparation liquid within 31.9-33.7 min is collected; the retention time of the compound b is 35.7min, the preparation liquid within 35.2-36.1 min is collected and recovered to obtain monomer compounds a 2.84g and b 1.03g;
2. the purity detection of the compounds a and b comprises the following specific steps:
accurately weighing certain amounts of the compounds a and b, preparing a stock solution with the concentration of 0.8mg/mL by using chromatographic methanol, accurately sucking 20 mu L of the stock solution, and detecting by using a high performance liquid chromatograph under the same chromatographic conditions as in example 1, wherein the detection result shows that the HPLC purity of the compounds a and b is more than 98 percent.
3. The structural identification of the compounds a and b comprises the following specific steps:
the above two compounds were verified by ultraviolet full wavelength scanning, HR-ESI-TOF-MS and NMR spectroscopy in the same manner as in example 1, and showed that compounds a and b were Ergosterol (Ergosterol) and asterosterol (Stellasterol), respectively.
Example 3
A method for separating ergosterol and asterosterol reference substances from traditional Chinese medicine grifola comprises the following steps:
1. separating ergosterol and asterosterol, which comprises the following steps:
weighing 300g of dried polyporus umbellatus, pulverizing, sieving with No. three sieve, and extracting with 8 times of 95% ethanol (2.4L) under reflux for 3 times; recovering methanol to obtain Polyporus umbellatus extract 14.7g, and adding 50mL distilled water to obtain dispersion solution; extracting the obtained substance with petroleum ether for three times in equal volume, wherein 50mL of petroleum ether is used each time, extracting with dichloromethane for 3 times, wherein 50mL of dichloromethane is used each time, and finally concentrating the chloroform extract to obtain an extract;
(2) Eluting the extract with 40mL distilled water for 3 times, filtering to remove large impurities, collecting the filter residue, subjecting the filter residue to Sephadex LH-20 column chromatography (300 g packed column volume), and purifying with V (dichloromethane): v (methanol) =1:1, mobile phase isocratic elution, collecting fractions every 25mL, combining the fractions from Fr.8 to Fr.21 by TLC analysis, and concentrating and recovering the combined fractions under reduced pressure; then adding absolute ethyl alcohol into the combined flow portions, heating (50-60 ℃) and dissolving under ultrasonic conditions to form saturated solution, standing for 8-12h under the conditions, and separating out 5.1g of needle-like crystals;
(3) Dissolving the crystal with chromatographic methanol to obtain solution with concentration of 0.2g/mL, filtering with 0.45 μm microporous membrane, and separating and purifying by reversed phase high performance liquid chromatography; chromatographic conditions are as follows: the chromatographic column is kromasil C18, 250X 20mm,5 μm; the detection wavelength is 283nm; the flow rate is 12mL/min; the column temperature is 25 ℃; the mobile phase is V (methanol): v (0.02% trifluoroacetic acid water) =96:5; the amount of the sample was 0.3mL. The retention time of the compound a is 39.7min, and the preparation liquid within 37.4-41.7 min is collected; the retention time of the compound b is 45.5min, preparation liquid within 44.6-46.8 min is collected, and monomer compounds a 3.11g and b 1.27g are obtained after recovery;
2. the purity detection of the compounds a and b comprises the following specific steps:
accurately weighing a certain amount of the compounds a and b, preparing a stock solution with the concentration of 2.0mg/mL by using chromatographic methanol, accurately sucking 10 mu L by using a sample injector, and detecting by using a high performance liquid chromatograph under the same chromatographic conditions as example 1, wherein the detection result shows that the HPLC purity of the compounds a and b is more than 98 percent;
3. the structural identification of the compounds a and b comprises the following specific steps:
the above two compounds were verified by ultraviolet full wavelength scanning, HR-ESI-MS and NMR spectroscopy in the same manner as in example 1, and showed that compounds a and b were Ergosterol (Ergosterol) and asterosterol (Stellasterol), respectively.
Example 4
A method for separating ergosterol and asterosterol reference substances from traditional Chinese medicine grifola comprises the following steps:
1. separating ergosterol and asterosterol, which comprises the following steps:
weighing 5000g of dry Polyporus umbellatus, pulverizing, sieving with No. two sieve, and cold extracting with 3 times of 98% methanol (15L) for 3 times; recovering methanol to obtain Polyporus umbellatus extract 101.7g, and adding 1000mL distilled water to obtain dispersion solution; extracting the obtained product with petroleum ether for three times in equal volume, wherein 1000mL of petroleum ether is used each time, and then extracting with dichloromethane for 3 times, wherein 1000mL of dichloromethane is used each time; finally, concentrating the chloroform extract to obtain an extract;
(2) Eluting the extract with 500mL distilled water for 3 times, filtering to remove large impurities, collecting the residue, subjecting the residue to Sephadex LH-20 column chromatography (1500 g packed column volume), and purifying with V (dichloromethane): v (methanol) =1:1, mobile phase isocratic elution, collecting fractions once per 100mL, combining the fractions from Fr.7 to Fr.30 by TLC analysis, and concentrating and recovering the combined fractions under reduced pressure; adding methanol into the combined flow parts, dissolving under heating (50-60 ℃) and ultrasonic conditions to form saturated solution, standing for 8-12h at room temperature, and separating out 37.9g of needle-like crystals;
(3) Dissolving the crystal obtained in the previous step with chromatographic methanol to obtain a solution with the concentration of 0.5g/mL, filtering with a 0.45 mu m microporous membrane, and separating and purifying by reversed-phase high performance liquid chromatography; chromatographic conditions are as follows: the chromatographic column is YMC C18, 250X 20mm,5 μm; the detection wavelength is 283nm; the flow rate is 18mL/min; the column temperature is 30 ℃; the mobile phase is V (methanol): v (0.1% phosphoric acid water) =96:5; the sample injection amount is 1.0mL; the retention time of the compound a is 32.9min, and the preparation liquid within 31.5-33.8 min is collected; the retention time of the compound b is 35.3min, the preparation liquid within 34.6-35.9 min is collected and recovered to obtain monomer compounds a 22.89g and b 10.63g;
2. the purity detection of the compounds a and b comprises the following specific steps:
accurately weighing certain amounts of the compounds a and b, preparing a stock solution with the concentration of 1.0mg/mL by using chromatographic methanol, accurately sucking 10 mu L of the stock solution by using a sample injector, and detecting by using a high performance liquid chromatograph under the same chromatographic conditions as in example 1, wherein the detection result shows that the HPLC purity of the compounds a and b is more than 98 percent.
3. The structural identification of the compounds a and b comprises the following specific steps:
the above two compounds were verified by UV full-wavelength scanning, HR-ESI-MS and NMR spectroscopy in the same manner as in example 1, and showed that compounds a and b were Ergosterol (Ergosterol) and asterosterol (Stellasterol), respectively.
Example 5 use of ergosterol and asterosterol obtained as reference substances
The ergosterol and asterosterol compounds obtained in the above examples are used as reference substances, and the content of each reference substance in the polyporus umbellatus medicinal material is determined, so that an experimental method basis is provided for establishing quality control of the polyporus umbellatus medicinal material.
Preparing a test article: precisely weighing 1.0g of Polyporus umbellatus medicinal material powder, placing into 100mL conical flask, adding 40mL of anhydrous ethanol, sealing, weighing, ultrasonically extracting for 0.5h, cooling, weighing, adding ethanol to balance weight, filtering with 0.45 μ M microporous membrane, automatically feeding 10 μ L of sample, and detecting content with high performance liquid chromatograph.
The chromatographic conditions are as follows: chromatography column Waters C18, 250X 4.6mm,5 μm; the detection wavelength is 283nm; the flow rate is 0.8mL/min; the column temperature is 30 ℃; mobile phase gradient methanol (a) - (0.1% formic acid) water (B) (0-5min, 98-96% a, 5-25min, 96-95% a, 3-30min 95% a).
The fingerprint chromatogram of Polyporus umbellatus by high performance liquid chromatography is shown in figure 11, and the reference substance can be used for evaluating the content of ergosterol and asterosterol in Polyporus umbellatus according to the measurement result, and can provide reference for quality evaluation of Polyporus umbellatus.
The method for separating the ergosterol and asterosterol reference substances from the traditional Chinese medicine grifola has the following advantages:
1) The ergosterol and asterosterol compounds have similar structures, small property difference and large separation difficulty, and two reference substances of high-purity ergosterol and asterosterol can be obtained simultaneously through one-time extraction and preparation process; the obtained reference substance is gram-grade to ten-gram-grade in scale, and is very feasible no matter small-scale laboratory preparation or large-scale industrial popularization.
2) Ergosterol and asterosterol have the functions and the purposes of promoting urination, resisting tumors, regulating blood fat, synthesizing precursor substances by medicines and the like, and have great development and utilization prospects. The invention can rapidly prepare gram-grade ergosterol and asterosterol for biological activity research, chemical structure modification and new drug development;
3) Ergosterol and asterosterol are both main components of fat-soluble substances contained in grifola, but no literature reports a method for preparing a reference substance of the two compounds at the same time at present; the invention can provide a reference substance for content measurement for quality control of traditional Chinese medicine grifola, and is beneficial to establishing a quality control method of the grifola;
4) The organic solvent selected by the invention can be recycled, the pollution to the environment is small, 95% ethanol or methanol is adopted during extraction and crystallization, and the solvent is low in price; the crystallization or preparation method adopted by the invention shortens the time for separating and purifying the sample, reduces the cost of manpower and material resources and improves the economic effect;
5) The invention is a rapid separation and purification technology, adopts conventional technology and means, has simple method and steps, is easy to popularize, convenient to operate, and has good specificity, high repeatability and good stability.
In the description of the present method for separating ergosterol and asterosterol reference substances from traditional Chinese medicine grifola, it is to be noted that, unless otherwise specifically defined and limited, the terms "set", "connected" and "connected" are to be understood broadly, and may be, for example, a fixed connection, a detachable connection, or an integral connection; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.
Although the preferred embodiments of the present patent have been described in detail, the present patent is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present patent within the knowledge of those skilled in the art.
Claims (3)
1. A method for separating ergosterol and asterosterol reference substances from traditional Chinese medicine grifola is characterized in that grifola medicinal material alcohol extract is used as a raw material, and the reference substances with the purity of more than 98 percent are prepared by petroleum ether impurity removal, dichloromethane extraction, column chromatography separation, recrystallization and reversed-phase high-efficiency chromatography, and the specific steps are as follows:
(1) Crushing polyporus umbellatus, sieving, adding 5-10 times of methanol or ethanol solution with volume concentration not lower than 95%, percolating or reflux extracting, and recovering solvent to obtain extractum; dispersing the obtained extract with distilled water, sequentially extracting with petroleum ether and dichloromethane, recovering and concentrating dichloromethane extractive solution to obtain soft extract;
(2) Washing the obtained thick paste with distilled water with the volume of 3-5 times to remove impurities, carrying out chromatography on filter residues by using a Sephadex LH-20 column, and then carrying out chromatography by using dichloromethane or chloroform: methanol 1:1 isocratic elution, collecting the fraction with lighter color, and recovering the eluent to obtain a crude sterol substance;
(3) Dissolving the crude sterol substance in anhydrous methanol or ethanol, and filtering; heating the filtrate sample, completely dissolving the filtrate sample under the ultrasonic condition, cooling the filtrate sample until the filtrate sample is in a saturated state, and separating out white needle-shaped crystals, namely an ergosterol and asterosterol mixed sample, wherein the heating temperature of the filtrate sample is 50-60 ℃; after reaching the saturated state, cooling at room temperature or 4 ℃;
(4) Dissolving the mixed sample obtained in the step (3) in chromatographic methanol or chromatographic acetonitrile, and then purifying by using a reversed phase high performance liquid chromatography; reasonably collecting the preparation solution within the retention time corresponding to the main absorption peak, concentrating under reduced pressure, recovering, and lyophilizing to obtain ergosterol and asterosterol reference substances.
2. The method for separating the ergosterol and asterosterol control from the traditional Chinese medicine polyporus umbellatus as claimed in claim 1, wherein the mixed sample obtained in step (4) is dissolved in chromatographic methanol or chromatographic acetonitrile at a concentration of 0.2-0.5 g/mL.
3. The method of claim 1, wherein the reversed-phase HPLC of step (4) is performed under conditions of kromasil C18, 250X 30mm,5 μm, or YMC C18, 250X 2 mm,5 μm, UV detector wavelength of 283nm, flow rate of 5-20 mL/min, column temperature of 25-30 deg.C, and mobile phase of methanol-water or acid water, acetonitrile-water or acid water.
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