CN108276375A - A method of isolating Stilbene effective constituents from acanthopanax leucorrhizus Harms stem skin - Google Patents

Abstract

The present invention relates to a method for isolating active ingredients of stilbenes from the stem bark of Radix Acanthopanax. Concentrate under reduced pressure to obtain the crude extract; (2) the crude extract is sequentially extracted with petroleum ether and ethyl acetate until the extract becomes colorless, then the ethyl acetate extracts are combined and evaporated to dryness under reduced pressure to obtain the sample to be separated; (3) The sample solution is obtained by ultrasonically dissolving the sample to be separated with a mixed solvent; ⑷determine the preparative HSCCC two-phase solvent system; Inject the sample solution into the tube for purification and separation; (6) collect 7 chromatographic peak components in sequence according to the chromatogram, and concentrate the 7 components under reduced pressure and vacuum dry to obtain 7 kinds of stilbene compounds. The invention has the advantages of simple operation, large extraction amount and good reproducibility, and the purity of the obtained seven stilbene compounds is all higher than 98.0%.

Description

A method for isolating effective components of stilbenes from the stem bark of vine root

technical field

The invention relates to the technical field of extraction of effective components of traditional Chinese medicinal materials, in particular to a method for isolating effective components of stilbenes from stem bark of Acanthopanax vine.

Background technique

Acanthopanax leucorrhizus is a small perennial deciduous shrub of the genus Acanthopanax. It is a unique folk medicinal plant in China. It is mainly distributed in Shaanxi, Gansu, Yunnan, Guizhou, and the middle and lower reaches of the Yangtze River. Its medicinal parts are mainly root bark and stem bark, which have the functions of dispelling rheumatism, dredging meridian, strengthening scorpion, etc. Wet swelling embolism. The main medicinal components of the stem bark of vine root are stilbenes, flavonoids, phenylpropanoids, terpenoids, lignans, coumarins, polysaccharides and other components. The study found that part of the stilbene components isolated from the bark of the Radix vinescensus had inhibitory effects on SMMC-7721 (human liver cancer cells), HL-60 (human promyelocytic leukemia cells) and MCF-7 (human breast cancer cells). With different degrees of selective inhibition, a very small number of stilbene components can be used as the lead of promising antitumor drugs after structural modification. However, due to the low content of most stilbene compounds in nature, it is difficult to separate and identify their structures, which limits their systematic research and development.

At present, the separation methods for stilbene components are mainly traditional solvent-assisted various chromatography methods (such as silica gel column chromatography, polyamide column chromatography, preparative thin-layer chromatography, ODS, Sephadex LH-20 column chromatography and HPLC, etc.), but All have the disadvantages of complex process, long cycle, large solvent consumption, low extraction rate and low purity to varying degrees.

Contents of the invention

The technical problem to be solved by the present invention is to provide a quick and efficient method for isolating effective components of stilbenes from the stem bark of Radix Acanthopanax.

In order to solve the above problems, a method for isolating the active ingredients of stilbenes from the stem bark of vine five plus of the present invention, comprises the following steps:

(1) Dry the fresh vine root bark, crush it, pass through a 40-60 mesh sieve, extract it with microwaves, and concentrate it under reduced pressure to obtain a crude extract;

(2) The crude extract is extracted 3 to 4 times respectively with petroleum ether and ethyl acetate of 3 to 4 times the volume of its mass, until the extract becomes colorless, the ethyl acetate extracts are combined, and the extracts are decompressed. Evaporate to dryness to obtain the sample to be separated;

(3) Obtain a sample solution after ultrasonically dissolving the sample to be separated with a mixed solvent at a ratio of 1000 mg:50 mL;

(4) Screened by TLC and optimized for analytical HSCCC, add n-hexane, ethyl acetate, methanol and water into the separatory funnel according to the volume ratio of 5:4:7:5, oscillate fully and let it stand overnight until the two phases After the system is balanced, the upper phase is the stationary phase, and the lower phase is the mobile phase, and the preparative HSCCC two-phase solvent system is obtained;

(5) After the stationary phase and the mobile phase were ultrasonically degassed for 15 minutes, they were sequentially pumped into the multi-layer spiral tube of the preparative countercurrent chromatograph, and then injected into the sample solution for purification and separation;

(6) Collect 7 chromatographic peak components in sequence according to the chromatogram, and after the 7 components are concentrated under reduced pressure and dried in vacuum, 7 kinds of stilbene compounds are obtained; the 7 kinds of stilbene compounds are respectively 4-hydroxyl-11 ,12, 13-Trimethoxydibenzo[ b , f ]oxheptin, isotanthin, 3,4′,5-trimethoxy-( E )-stilbene, 2′,3, 4′,5-tetrahydroxy-( E )-stilbene, 3′-prenyl-( E )-resveratrol, 3,4′,5-trihydroxy-2′-methoxy- ( E )-stilbene and 2-(3′-hydroxy-5′-methoxybenzene)-4-hydroxybenzofuran.

The conditions of microwave extraction in the step (1) refer to the extraction solvent as a volume concentration of 75% to 95% ethanol, a liquid-solid ratio of 10 to 20 mL/g, a stirring speed of 600 to 700 rpm, and an extraction temperature of 55 to 65 °C , The microwave power is 650~750 W, the extraction time is 15~20 min, and the extraction times are 2~3 times.

The decompression concentration conditions in the step (1) refer to a pressure of 0.05-0.06 MPa, a temperature of 40-50 °C, and a rotation speed of 100-150 rpm.

The vacuum evaporation conditions in the step (2) refer to a pressure of 0.04-0.05 MPa, a temperature of 30-40 °C, and a rotation speed of 80-100 rpm.

The mixed solvent in the step (3) refers to adding n-hexane, ethyl acetate, methanol and water into the separatory funnel according to the volume ratio of 5:4:7:5, fully oscillating and standing overnight, and waiting for two After the phase system is balanced, it is divided into two phases, and then the upper phase solvent and the lower phase solvent are mixed in equal volumes.

The ultrasonic dissolution conditions in the step (3) refer to an ultrasonic frequency of 40 kHz, a temperature of 40 °C, and an ultrasonic time of 5 min.

The purification and separation conditions in step (5) refer to the temperature at 25 °C, the rotation speed of the spiral tube at 800-900 rpm, the detection wavelength at 325 nm, the injection flow rate at 1.5-2.0 mL/min, and the injection volume at one time of 160 mg.

The decompression concentration conditions in the step (6) refer to a pressure of 0.03-0.05 MPa, a temperature of 30-50 °C, and a rotation speed of 80-150 rpm.

The vacuum drying conditions in the step (6) refer to a pressure of 0.05-0.06 MPa, a temperature of 30-50 °C, and a drying time of 2-10 h.

Compared with the prior art, the present invention has the following advantages:

1. Compared with the traditional solvent extraction technology, the present invention: under the action of microwaves, the stilbene components can be selectively heated and exuded from the plant matrix, so that the separation of the stilbene components from the matrix can be realized quickly and efficiently. Effectively improve the extraction rate and purity.

2. Compared with the traditional chromatographic separation technology, the present invention adopts the HSCCC purification method, the same solvent system can be reused, the separation volume is large, and the reproducibility is good; at the same time, no solid carrier is required, and the separation caused by irreversible adsorption is avoided Problems such as material loss, inactivation, denaturation, and tailing have improved the recovery rate of active ingredients, and are very suitable for the preparation of reference substances for active ingredients in Chinese herbal medicines.

3. The present invention realizes the integration of extraction, separation, purification and preparation of stilbene components in the stem bark of Acanthopanax vine, and the MAE crude extract can directly enter HSCCC for separation and purification after concentration, which greatly reduces the intermediate operation links, reduces labor intensity and effective The loss of components shortens the separation time and improves the separation efficiency.

4. The present invention integrates microwave extraction and high-efficiency countercurrent chromatographic purification preparation. It is simple and easy to operate, with large extraction volume and good reproducibility. The purity of the obtained 7 kinds of stilbene compounds are all higher than 98.0%.

Detailed ways

Embodiment 1 A kind of method for isolating the active ingredient of stilbene class from the stem bark of Radix Acanthopanax, comprising the following steps:

(1) After drying, pulverizing and passing 40 mesh sieves of 300 g of fresh vine root bark, vine root bark powder is obtained, and the volume concentration of 10 mL/g is added into the vine root bark powder according to the liquid-solid ratio 75% ethanol, placed in a stirrer magnet, placed in a microwave extractor, at a stirring speed of 600 rpm, an extraction temperature of 60 °C, a microwave power of 700 W, an extraction time of 15 min, and extraction times of 2 Under the conditions of microwave extraction, the extract was obtained; the extract was concentrated under reduced pressure at a pressure of 0.05 MPa, a temperature of 40 °C, and a rotation speed of 100 rpm to obtain a crude extract.

(2) Extract the crude extract three times with petroleum ether and ethyl acetate in order of 3 times the volume of its mass, until the extract becomes colorless, combine the ethyl acetate extracts, and put them under pressure of 0.04 MPa and temperature of 30 Under the conditions of 80 rpm and 80 rpm, 4.20 g of the sample to be separated was obtained by evaporating under reduced pressure, and the yield was 1.40% of the mass of the dry stem bark of Tengshenus.

(3) The sample to be separated was ultrasonically dissolved with a mixed solvent at a ratio of 1000 mg:50 mL at an ultrasonic frequency of 40 kHz and a temperature of 40 °C, and a 20 mg/mL sample solution was obtained after 5 min.

Among them: the mixed solvent refers to adding n-hexane, ethyl acetate, methanol and water into the separatory funnel according to the volume ratio of 5:4:7:5, fully oscillating evenly, and standing overnight until the two-phase system is balanced , separated into two phases, and then obtained by mixing equal volumes of the upper phase solvent and the lower phase solvent.

(4) Screened by TLC and optimized for analytical HSCCC, add n-hexane, ethyl acetate, methanol and water into the separatory funnel according to the volume ratio of 5:4:7:5, oscillate fully and let it stand overnight until the two phases After the system is balanced, the upper phase is the stationary phase, and the lower phase is the mobile phase, and the preparative HSCCC two-phase solvent system is obtained.

(5) After degassing the stationary phase and the mobile phase by ultrasonic for 15 min, pump them into the multi-layer spiral tube of the preparative countercurrent chromatograph in turn, and then inject the sample solution for purification and separation.

Specific process: pump the stationary phase into the multi-layer spiral tube of the preparative countercurrent chromatograph at a flow rate of 6.0 mL/min. When the spiral column is completely filled, turn on the constant temperature circulation device to keep the temperature at 25 °C, start the detector and HSCCC main engine, slowly forward rotation (FWD) to adjust the rotation speed of the spiral tube to 900 rpm, and then pump the mobile phase at a flow rate of 2.0 mL/min until the mobile phase flows out from the end of the spiral tube. When the system reaches kinetic equilibrium (baseline is stable), the sample solution is injected into the spiral tube through the injection valve at a flow rate of 2.0 mL/min for separation. The detection wavelength is 325 nm, and the injection volume at one time is 160 mg.

(6) After the sample solution was injected into the separation spiral tube for about 45 minutes, the chromatographic peaks began to appear, and seven chromatographic peak components were collected in sequence according to the chromatogram, which took 3.0 hours. Concentrate the 7 components under reduced pressure at a pressure of 0.03 MPa, a temperature of 30 °C, and a rotation speed of 80 rpm to obtain a concentrate, which is vacuum-dried at a pressure of 0.05 MPa and a temperature of 30 °C After 10 h, a total of 7 stilbene compounds were obtained, including 6 solid powders and 1 yellow oil.

After NMR, MS, IR and UV analysis, it was confirmed that the seven stilbene compounds were 4-hydroxy-11,12,13-trimethoxydibenzo[ b , f ]oxepin (4-hydroxy-11,12 ,13-trimethoxyldibenz[ b , f ] oxepin), isorhapontigenin, 3,4′,5-trimethoxyl-( E )-stilbene (3,4′,5-trimethoxy-( E )-stilbene), 2′,3,4′,5-tetrahydroxy-( E )-stilbene (2′,3,4′,5-tetrahydroxy- ( E )-stilbene), 3′-iso Pentenyl-( E )-resveratrol (3′-isopentenyl-( E )-resveratrol), 3,4′,5-trihydroxy-2′-methoxy-( E )-stilbene ( 3,4′,5-trihydroxy-2′-methoxy-( E )-stilbene) and 2-(3′-hydroxy-5′-methoxybenzene)-4-hydroxybenzofuran (2-(3′ -hydroxy-5′-methoxyphenyl)-4-hydroxybenzofuran), and successively named ZCⅠ, ZCⅡ, ZCⅢ, ZCⅣ, ZCⅤ, ZCⅥ and ZCⅦ.

After 4 consecutive injections on the HSCCC separation column, the cumulative injection volume was 640 mg, and the total time was 15.5 h.

Purity was analyzed by HPLC: the quality and purity of the seven components were ZCⅠ (9.23 mg, 98.6%), ZCⅡ (23.01 mg, 99.2%), ZCⅢ (13.62 mg, 99.5%), ZCⅣ (6.18 mg, 99.3%) , ZCⅤ (7.14 mg, 98.1%), ZCⅥ (14.25 mg, 99.4%) and ZCⅦ (8.61 mg, 98.7%), all with a purity above 98.0%.

Embodiment 2 A kind of method for isolating the active ingredient of stilbene class from the stem bark of Radix Acanthopanax, comprising the following steps:

(1) After drying 500 g of fresh vine root bark, pulverizing and passing through a 50-mesh sieve, the vine root bark powder is obtained, and the volume concentration of 15 mL/g is added into the vine root bark powder according to the liquid-solid ratio 80% ethanol was put into a stirrer magnet, placed in a microwave extractor, and the stirring speed was 650 rpm, the extraction temperature was 60 °C, the microwave power was 650 W, the extraction time was 20 min, and the number of extractions was 3 times. Microwave extraction was carried out under certain conditions to obtain an extract; the extract was concentrated under reduced pressure at a pressure of 0.06 MPa, a temperature of 50 °C, and a rotation speed of 150 rpm to obtain a crude extract.

(2) Extract the crude extract three times with petroleum ether and ethyl acetate which are 4 times the volume of its mass, until the extract becomes colorless, then combine the ethyl acetate extracts and store them under pressure of 0.05 MPa and temperature of 40 ℃ and 100 rpm, the sample to be separated was evaporated to dryness under reduced pressure to obtain 7.10 g, and the yield was 1.42% of the mass of the dry stem bark of Tengshenus.

(3) The sample to be separated was ultrasonically dissolved with a mixed solvent at a ratio of 1000 mg:50 mL at an ultrasonic frequency of 40 kHz and a temperature of 40 °C, and the sample solution was obtained after 5 min.

Wherein: mixed solvent is with embodiment 1.

(4) The preparative HSCCC two-phase solvent system is the same as in Example 1.

(5) After degassing the stationary phase and the mobile phase by ultrasonic for 15 min, pump them into the multi-layer spiral tube of the preparative countercurrent chromatograph in turn, and then inject the sample solution for purification and separation.

Specific process: pump the stationary phase into the multi-layer spiral tube of the preparative countercurrent chromatograph at a flow rate of 6.0 mL/min. When the spiral column is completely filled, turn on the constant temperature circulation device to keep the temperature at 25 °C, start the detector and HSCCC main engine, slowly forward rotation (FWD) to adjust the rotation speed of the spiral tube to 900 rpm, and then pump the mobile phase at a flow rate of 2.0 mL/min until the mobile phase flows out from the end of the spiral tube. When the system reaches kinetic equilibrium (baseline is stable), the sample solution is injected into the spiral tube through the injection valve at a flow rate of 1.5 mL/min for separation. The detection wavelength is 325 nm, and the injection volume at one time is 160 mg.

(6) After the sample solution was injected into the separation spiral tube for about 45 minutes, the chromatographic peaks began to appear, and seven chromatographic peak components were collected in sequence according to the chromatogram, which took 3.5 hours. Concentrate the 7 components under reduced pressure at a pressure of 0.05 MPa, a temperature of 50 °C, and a rotation speed of 150 rpm to obtain a concentrate, which is vacuum-dried at a pressure of 0.06 MPa and a temperature of 50 °C After 2 h, a total of 7 stilbene compounds were obtained, including 6 solid powders and 1 yellow oil.

After 4 consecutive injections on the HSCCC separation column, the cumulative injection volume was 640 mg, and the total time was 17.5 h.

Purity was analyzed by HPLC: the quality and purity of the seven components were ZCⅠ (9.47 mg, 98.3%), ZCⅡ (23.46 mg, 98.6%), ZCⅢ (13.10 mg, 99.3%), ZCⅣ (6.25 mg, 99.6%) , ZCⅤ (7.31 mg, 98.5%), ZCⅥ (13.43 mg, 98.7%) and ZCⅦ (9.45 mg, 98.1%), all with a purity above 98.0%.

Embodiment 3 A kind of method for isolating the active ingredient of stilbene class from the stem bark of Radix Acanthopanax, comprising the following steps:

(1) After drying 400 g of fresh vine root bark, crushing and passing through a 60-mesh sieve, vine root bark powder is obtained, and the volume concentration of 20 mL/g is added into the vine root bark powder according to the liquid-solid ratio 95% ethanol was put into a stirrer magnet, placed in a microwave extractor, and the stirring speed was 700 rpm, the extraction temperature was 65 °C, the microwave power was 750 W, the extraction time was 20 min, and the number of extractions was 3 times. Under the conditions of microwave extraction, the extract was obtained; the extract was concentrated under reduced pressure at a pressure of 0.055 MPa, a temperature of 45 °C, and a rotation speed of 125 rpm to obtain a crude extract.

⑵The crude extract was sequentially extracted 4 times with petroleum ether and ethyl acetate of 3 times the volume of its mass until the extract became colorless, then the ethyl acetate extracts were combined, and the mixture was extracted at a pressure of 0.045 MPa and a temperature of 35 ℃ and 90 rpm, the sample to be separated was evaporated to dryness under reduced pressure to obtain 5.50 g.

(3) The sample to be separated was ultrasonically dissolved with a mixed solvent at a ratio of 1000 mg:50 mL at an ultrasonic frequency of 40 kHz and a temperature of 40 °C, and the sample solution was obtained after 5 min.

Wherein: mixed solvent is with embodiment 1.

(4) The preparative HSCCC two-phase solvent system is the same as in Example 1.

(5) After degassing the stationary phase and the mobile phase by ultrasonic for 15 min, pump them into the multi-layer spiral tube of the preparative countercurrent chromatograph in turn, and then inject the sample solution for purification and separation.

Specific process: pump the stationary phase into the multi-layer spiral tube of the preparative countercurrent chromatograph at a flow rate of 6.0 mL/min. When the spiral column is completely filled, turn on the constant temperature circulation device to keep the temperature at 25 °C, start the detector and HSCCC main engine, slowly forward rotation (FWD) to adjust the rotation speed of the spiral tube to 800 rpm, and then pump the mobile phase at a flow rate of 2.0 mL/min until the mobile phase flows out from the end of the spiral tube. When the system reaches kinetic equilibrium (baseline is stable), the sample solution is injected into the spiral tube through the injection valve at a flow rate of 2.0 mL/min for separation. The detection wavelength is 325 nm, and the injection volume at one time is 160 mg.

(6) After the sample solution was injected into the separation spiral tube for about 45 minutes, the chromatographic peaks began to appear, and seven chromatographic peak components were collected in sequence according to the chromatogram, which took 3.2 hours. Concentrate the 7 components under reduced pressure at a pressure of 0.04 MPa, a temperature of 40 °C, and a rotation speed of 100 rpm to obtain a concentrate, which is vacuum-dried at a pressure of 0.055 MPa and a temperature of 40 °C After 6 h, a total of 7 stilbene compounds were obtained, including 6 solid powders and 1 yellow oil.

After 4 consecutive injections to the HSCCC separation column, the cumulative injection volume was 640 mg, and the total time was 16.0 h.

Purity was analyzed by HPLC: the quality and purity of the seven components were ZCⅠ (9.21 mg, 99.1%), ZCⅡ (22.65 mg, 98.4%), ZCⅢ (14.02 mg, 98.6%), ZCⅣ (6.36 mg, 98.9%) , ZCⅤ (7.05 mg, 98.5%), ZCⅥ (14.32 mg, 98.6%) and ZCⅦ (8.51 mg, 99.2%), all with a purity above 98.0%.

Embodiment 4 A kind of method for isolating the active ingredient of stilbene class from the stem bark of Radix Acanthopanax, comprising the following steps:

(1) After drying, pulverizing and passing 40 mesh sieves of 350 g of fresh vine root bark, vine root bark powder is obtained, and the volume concentration of 10 mL/g is added into the vine root bark powder according to the liquid-solid ratio 90% ethanol was put into a stirrer magnet, placed in a microwave extractor, and the stirring speed was 650 rpm, the extraction temperature was 55 °C, the microwave power was 750 W, the extraction time was 15 min, and the number of extractions was 2 times. Under the conditions of microwave extraction, the extract was obtained; the extract was concentrated under reduced pressure at a pressure of 0.05 MPa, a temperature of 40 °C, and a rotation speed of 100 rpm to obtain a crude extract.

⑵The crude extract was extracted 4 times with petroleum ether and ethyl acetate which were 4 times the volume of its mass in sequence, until the extract became colorless, the ethyl acetate extract was combined, and the mixture was extracted at a pressure of 0.04 MPa and a temperature of 30 Under the conditions of 85 rpm and 85 rpm, 4.69 g of the sample to be separated was obtained by evaporating under reduced pressure, and the yield was 1.34% of the dry stem bark of Tengshenus.

(3) The sample to be separated was ultrasonically dissolved with a mixed solvent at a ratio of 1000 mg:50 mL at an ultrasonic frequency of 40 kHz and a temperature of 40 °C, and the sample solution was obtained after 5 min.

Wherein: mixed solvent is with embodiment 1.

(4) The preparative HSCCC two-phase solvent system is the same as in Example 1.

(5) After degassing the stationary phase and the mobile phase by ultrasonic for 15 min, pump them into the multi-layer spiral tube of the preparative countercurrent chromatograph in turn, and then inject the sample solution for purification and separation.

Specific process: pump the stationary phase into the multi-layer spiral tube of the preparative countercurrent chromatograph at a flow rate of 6.0 mL/min. When the spiral column is completely filled, turn on the constant temperature circulation device to keep the temperature at 25 °C, start the detector and HSCCC main engine, slowly forward rotation (FWD) to adjust the rotation speed of the spiral tube to 850 rpm, and then pump the mobile phase at a flow rate of 2.0 mL/min until the mobile phase flows out from the end of the spiral tube. When the system reaches kinetic equilibrium (baseline is stable), the sample solution is injected into the spiral tube through the injection valve at a flow rate of 1.8 mL/min for separation. The detection wavelength is 325 nm, and the injection volume at one time is 160 mg.

(6) After the sample solution was injected into the separation spiral tube for about 45 minutes, the chromatographic peaks began to appear, and seven chromatographic peak components were collected in sequence according to the chromatogram, which took 3.6 hours. Concentrate the 7 components under reduced pressure at a pressure of 0.03 MPa, a temperature of 30 °C, and a rotation speed of 90 rpm to obtain a concentrate, which is vacuum-dried at a pressure of 0.05 MPa and a temperature of 30 °C After 10 h, a total of 7 stilbene compounds were obtained, including 6 solid powders and 1 yellow oil.

After 4 consecutive injections to the HSCCC separation column, the cumulative injection volume was 640 mg, and the total time was 17.2 h.

Purity was analyzed by HPLC: the quality and purity of the seven components were ZCⅠ (9.18 mg, 98.6%), ZCⅡ (22.37 mg, 98.9%), ZCⅢ (13.96 mg, 98.5%), ZCⅣ (6.14 mg, 99.2%) , ZCⅤ (7.08 mg, 98.3%), ZCⅥ (14.22 mg, 98.7%) and ZCⅦ (8.24 mg, 99.1%), all with a purity above 98.0%.

Embodiment 5 A kind of method for isolating the active ingredient of stilbene class from the stem bark of Radix Acanthopanax, comprising the following steps:

(1) After drying, pulverizing and passing through 40 mesh sieves of 300 g of fresh vine root bark, the vine root bark powder is obtained, and the volume concentration of 15 mL/g is added into the vine root bark powder according to the liquid-solid ratio 80% ethanol was put into a stirrer magnet, placed in a microwave extractor, and the stirring speed was 700 rpm, the extraction temperature was 65 °C, the microwave power was 650 W, the extraction time was 15 min, and the number of extractions was 3 times. Microwave extraction was carried out under certain conditions to obtain an extract; the extract was concentrated under reduced pressure at a pressure of 0.06 MPa, a temperature of 50 °C, and a rotation speed of 150 rpm to obtain a crude extract.

⑵The crude extract was extracted 4 times with petroleum ether and ethyl acetate of 3 times the volume of its mass, until the extract became colorless, the ethyl acetate extract was combined, and the pressure was 0.05 MPa, the temperature was 40 ℃ and 100 rpm, the sample to be separated was evaporated to dryness under reduced pressure to obtain 4.23 g, and the yield was 1.41% of the mass of the dried bark of Tengshenus.

(3) The sample to be separated was ultrasonically dissolved with a mixed solvent at a ratio of 1000 mg:50 mL at an ultrasonic frequency of 40 kHz and a temperature of 40 °C, and the sample solution was obtained after 5 min.

Wherein: mixed solvent is with embodiment 1.

(4) The preparative HSCCC two-phase solvent system is the same as in Example 1.

(5) After degassing the stationary phase and the mobile phase by ultrasonic for 15 min, pump them into the multi-layer spiral tube of the preparative countercurrent chromatograph in turn, and then inject the sample solution for purification and separation.

Specific process: pump the stationary phase into the multi-layer spiral tube of the preparative countercurrent chromatograph at a flow rate of 6.0 mL/min. When the spiral column is completely filled, turn on the constant temperature circulation device to keep the temperature at 25 °C, start the detector and HSCCC main engine, slowly forward rotation (FWD) to adjust the rotation speed of the spiral tube to 900 rpm, and then pump the mobile phase at a flow rate of 2.0 mL/min until the mobile phase flows out from the end of the spiral tube. When the system reaches kinetic equilibrium (baseline is stable), the sample solution is injected into the spiral tube through the injection valve at a flow rate of 1.5 mL/min for separation. The detection wavelength is 325 nm, and the injection volume at one time is 160 mg.

(6) After the sample solution was injected into the separation spiral tube for about 45 minutes, the chromatographic peaks began to appear, and seven chromatographic peak components were collected in sequence according to the chromatogram, which took 3.5 hours. Concentrate the 7 components under reduced pressure at a pressure of 0.05 MPa, a temperature of 50 °C, and a rotation speed of 95 rpm to obtain a concentrate, which is vacuum-dried at a pressure of 0.06 MPa and a temperature of 50 °C After 2 h, a total of 7 stilbene compounds were obtained, including 6 solid powders and 1 yellow oil.

After 4 consecutive injections on the HSCCC separation column, the cumulative injection volume was 640 mg, and the total time was 17.0 h.

Purity was analyzed by HPLC: the quality and purity of the seven components were ZCⅠ (9.15 mg, 98.5%), ZCⅡ (22.41 mg, 99.2%), ZCⅢ (13.84 mg, 98.4%), ZCⅣ (6.09 mg, 99.0%) , ZCⅤ (7.12 mg, 98.5%), ZCⅥ (14.51 mg, 98.8%) and ZCⅦ (8.13 mg, 99.4%), all with a purity above 98.0%.

Embodiment 6 A method for isolating active ingredients of stilbenes from the bark of Radix Acanthopanax, comprising the following steps:

(1) After drying 400 g of fresh vine root bark, pulverizing and passing through a 50-mesh sieve, vine root bark powder is obtained, and the volume concentration of 20 mL/g is added in the vine root bark powder according to the liquid-solid ratio 80% ethanol was put into a stirrer magnet, placed in a microwave extractor, and the stirring speed was 700 rpm, the extraction temperature was 60 °C, the microwave power was 650 W, the extraction time was 20 min, and the number of extractions was 3 times. Under the conditions of microwave extraction, the extract was obtained; the extract was concentrated under reduced pressure at a pressure of 0.055 MPa, a temperature of 45 °C, and a rotation speed of 130 rpm to obtain a crude extract.

⑵The crude extract was extracted three times with petroleum ether and ethyl acetate which were 3 times the volume of its mass, until the extract became colorless, then the ethyl acetate extracts were combined, at a pressure of 0.045 MPa and a temperature of 35 ℃ and 90 rpm, the sample to be separated was evaporated to dryness under reduced pressure to obtain 5.54 g, and the yield was 1.39% of the mass of the dry bark of Tengshenus.

(3) The sample to be separated was ultrasonically dissolved with a mixed solvent at a ratio of 1000 mg:50 mL at an ultrasonic frequency of 40 kHz and a temperature of 40 °C, and the sample solution was obtained after 5 min.

Wherein: mixed solvent is with embodiment 1.

(4) The preparative HSCCC two-phase solvent system is the same as in Example 1.

(5) After degassing the stationary phase and the mobile phase by ultrasonic for 15 min, pump them into the multi-layer spiral tube of the preparative countercurrent chromatograph in turn, and then inject the sample solution for purification and separation.

Specific process: pump the stationary phase into the multi-layer spiral tube of the preparative countercurrent chromatograph at a flow rate of 6.0 mL/min. When the spiral column is completely filled, turn on the constant temperature circulation device to keep the temperature at 25 °C, start the detector and HSCCC main engine, slowly forward rotation (FWD) to adjust the rotation speed of the spiral tube to 900 rpm, and then pump the mobile phase at a flow rate of 2.0 mL/min until the mobile phase flows out from the end of the spiral tube. When the system reaches kinetic equilibrium (baseline is stable), the sample solution is injected into the spiral tube through the injection valve at a flow rate of 2.0 mL/min for separation. The detection wavelength is 325 nm, and the injection volume at one time is 160 mg.

(6) After the sample solution was injected into the separation spiral tube for about 45 minutes, the chromatographic peaks began to appear, and seven chromatographic peak components were collected in sequence according to the chromatogram, which took 3.1 hours. Concentrate the 7 components under reduced pressure at a pressure of 0.04 MPa, a temperature of 40 °C, and a rotation speed of 100 rpm to obtain a concentrate, which is vacuum-dried at a pressure of 0.055 MPa and a temperature of 40 °C After 6 h, a total of 7 stilbene compounds were obtained, including 6 solid powders and 1 yellow oil.

After 4 consecutive injections on the HSCCC separation column, the cumulative injection volume was 640 mg, and the total time was 15.0 h.

Purity was analyzed by HPLC: the quality and purity of the seven components were ZCⅠ (9.32 mg, 98.3%), ZCⅡ (22.45 mg, 99.0%), ZCⅢ (13.67 mg, 98.2%), ZCⅣ (6.05 mg, 98.9%) , ZCⅤ (7.24 mg, 98.7%), ZCⅥ (14.38 mg, 98.7%) and ZCⅦ (8.24 mg, 99.2%), all with a purity above 98.0%.

Example 7 A method for isolating active ingredients of stilbenes from the stem bark of Rattan Acanthopanax, comprising the following steps:

(1) After drying 500 g of fresh vine root bark, crushing, and passing through a 60-mesh sieve, the vine root bark powder is obtained, and the liquid-solid ratio of the vine root bark powder is added to a volume concentration of 20 mL/g 95% ethanol, placed in a stirrer magnet, placed in a microwave extractor, at a stirring speed of 700 rpm, an extraction temperature of 55 °C, a microwave power of 650 W, an extraction time of 15 min, and an extraction frequency of 3 times Under the conditions of microwave extraction, the extract was obtained; the extract was concentrated under reduced pressure at a pressure of 0.05 MPa, a temperature of 40 °C, and a rotation speed of 100 rpm to obtain a crude extract.

⑵The crude extract was extracted 4 times with petroleum ether and ethyl acetate which were 4 times the volume of its mass in sequence, until the extract became colorless, the ethyl acetate extract was combined, and the mixture was extracted at a pressure of 0.04 MPa and a temperature of 30 Under the conditions of 80 rpm and 80 rpm, 7.14 g of the sample to be separated was obtained by evaporating under reduced pressure, and the yield was 1.43% of the mass of the dried bark of Tengshenus.

(3) The sample to be separated was ultrasonically dissolved with a mixed solvent at a ratio of 1000 mg:50 mL at an ultrasonic frequency of 40 kHz and a temperature of 40 °C, and the sample solution was obtained after 5 min.

Wherein: mixed solvent is with embodiment 1.

(4) The preparative HSCCC two-phase solvent system is the same as in Example 1.

(5) After degassing the stationary phase and the mobile phase by ultrasonic for 15 min, pump them into the multi-layer spiral tube of the preparative countercurrent chromatograph in turn, and then inject the sample solution for purification and separation.

Specific process: pump the stationary phase into the multi-layer spiral tube of the preparative countercurrent chromatograph at a flow rate of 6.0 mL/min. When the spiral column is completely filled, turn on the constant temperature circulation device to keep the temperature at 25 °C, start the detector and HSCCC main engine, slowly forward rotation (FWD) to adjust the rotation speed of the spiral tube to 900 rpm, and then pump the mobile phase at a flow rate of 2.0 mL/min until the mobile phase flows out from the end of the spiral tube. When the system reaches kinetic equilibrium (baseline is stable), the sample solution is injected into the spiral tube through the injection valve at a flow rate of 2.0 mL/min for separation. The detection wavelength is 325 nm, and the injection volume at one time is 160 mg.

(6) After the sample solution was injected into the separation spiral tube for about 45 minutes, the chromatographic peaks began to appear, and seven chromatographic peak components were collected sequentially according to the chromatogram, which took 3.2 hours. Concentrate the 7 components under reduced pressure at a pressure of 0.03 MPa, a temperature of 30 °C, and a rotation speed of 80 rpm to obtain a concentrate, which is vacuum-dried at a pressure of 0.05 MPa and a temperature of 30 °C After 10 h, a total of 7 stilbene compounds were obtained, including 6 solid powders and 1 yellow oil.

After 4 consecutive injections on the HSCCC separation column, the cumulative injection volume was 640 mg, and the total time was 15.5 h.

Purity was analyzed by HPLC: the quality and purity of the seven components were ZCⅠ (9.45 mg, 98.5%), ZCⅡ (22.48 mg, 98.6%), ZCⅢ (13.71 mg, 98.1%), ZCⅣ (6.01 mg, 99.1%) , ZCⅤ (7.23 mg, 98.5%), ZCⅥ (14.24 mg, 99.0%) and ZCⅦ (8.17 mg, 99.3%), all with a purity above 98.0%.

Example 8 A method for isolating active ingredients of stilbenes from the stem bark of Radix Acanthopanax, comprising the following steps:

(1) After drying 300 g of fresh vine root bark, crushing, and passing through a 60-mesh sieve, vine root bark powder is obtained, and the volume concentration of 20 mL/g is added into the vine root bark powder according to the liquid-solid ratio 75% ethanol was extracted by microwave under the conditions of stirring speed of 700 rpm, extraction temperature of 60 °C, microwave power of 700 W, extraction time of 18 min, and extraction times of 23 times to obtain the extract; The pressure was 0.06 MPa, the temperature was 50 °C, and the rotation speed was 150 rpm to carry out concentration under reduced pressure to obtain a crude extract.

⑵The crude extract was extracted 4 times with petroleum ether and ethyl acetate of 3.5 times the volume of its mass, until the extract became colorless, the ethyl acetate extract was combined, and the pressure was 0.05 MPa, the temperature was 40 ℃ and 100 rpm, the sample to be separated was evaporated to dryness under reduced pressure to obtain 4.35 g, and the yield was 1.45% of the mass of the dry bark of Tengshenus.

(3) The sample to be separated was ultrasonically dissolved with a mixed solvent at a ratio of 1000 mg:50 mL at an ultrasonic frequency of 40 kHz and a temperature of 40 °C, and the sample solution was obtained after 5 min.

Wherein: mixed solvent is with embodiment 1.

(4) The preparative HSCCC two-phase solvent system is the same as in Example 1.

(5) After degassing the stationary phase and the mobile phase by ultrasonic for 15 min, pump them into the multi-layer spiral tube of the preparative countercurrent chromatograph in turn, and then inject the sample solution for purification and separation.

Specific process: pump the stationary phase into the multi-layer spiral tube of the preparative countercurrent chromatograph at a flow rate of 6.0 mL/min. When the spiral column is completely filled, turn on the constant temperature circulation device to keep the temperature at 25 °C, start the detector and HSCCC main engine, slowly forward rotation (FWD) to adjust the rotation speed of the spiral tube to 900 rpm, and then pump the mobile phase at a flow rate of 2.0 mL/min until the mobile phase flows out from the end of the spiral tube. When the system reaches kinetic equilibrium (baseline is stable), the sample solution is injected into the spiral tube through the injection valve at a flow rate of 2.0 mL/min for separation. The detection wavelength is 325 nm, and the injection volume at one time is 160 mg.

(6) After the sample solution was injected into the separation spiral tube for about 45 minutes, the chromatographic peaks began to appear, and seven chromatographic peak components were collected in sequence according to the chromatogram, which took 3.0 hours. Concentrate the 7 components under reduced pressure at a pressure of 0.05 MPa, a temperature of 50 °C, and a rotation speed of 150 rpm to obtain a concentrate, which is vacuum-dried at a pressure of 0.06 MPa and a temperature of 50 °C After 2 h, a total of 7 stilbene compounds were obtained, including 6 solid powders and 1 yellow oil.

After 4 consecutive injections on the HSCCC separation column, the cumulative injection volume was 640 mg, and the total time was 15.2 h.

Purity was analyzed by HPLC: the quality and purity of the seven components were ZCⅠ (9.44 mg, 98.6%), ZCⅡ (22.45 mg, 98.7%), ZCⅢ (13.68 mg, 98.3%), ZCⅣ (6.11 mg, 99.0%) , ZCⅤ (7.18 mg, 98.6%), ZCⅥ (14.27 mg, 98.9%) and ZCⅦ (8.22 mg, 99.8%), all with a purity above 98.0%.

Claims (9)

1. a method for isolating the active ingredient of stilbene class from the stem bark of Acanthopanax vine, comprising the following steps: (1) Dry the fresh vine root bark, crush it, pass through a 40-60 mesh sieve, extract it with microwaves, and concentrate it under reduced pressure to obtain a crude extract; (2) The crude extract is extracted 3 to 4 times respectively with petroleum ether and ethyl acetate of 3 to 4 times the volume of its mass, until the extract becomes colorless, the ethyl acetate extracts are combined, and the extracts are decompressed. Evaporate to dryness to obtain the sample to be separated; (3) Obtain a sample solution after ultrasonically dissolving the sample to be separated with a mixed solvent at a ratio of 1000 mg:50 mL; (4) Screened by TLC and optimized for analytical HSCCC, add n-hexane, ethyl acetate, methanol and water into the separatory funnel according to the volume ratio of 5:4:7:5, oscillate fully and let it stand overnight until the two phases After the system is balanced, the upper phase is the stationary phase, and the lower phase is the mobile phase, and the preparative HSCCC two-phase solvent system is obtained; (5) After the stationary phase and the mobile phase were ultrasonically degassed for 15 minutes, they were sequentially pumped into the multi-layer spiral tube of the preparative countercurrent chromatograph, and then injected into the sample solution for purification and separation; (6) Collect 7 chromatographic peak components in sequence according to the chromatogram, and after the 7 components are concentrated under reduced pressure and dried in vacuum, 7 kinds of stilbene compounds are obtained; the 7 kinds of stilbene compounds are respectively 4-hydroxyl-11 ,12, 13-Trimethoxydibenzo[ b , f ]oxheptin, isotanthin, 3,4′,5-trimethoxy-( E )-stilbene, 2′,3, 4′,5-tetrahydroxy-( E )-stilbene, 3′-prenyl-( E )-resveratrol, 3,4′,5-trihydroxy-2′-methoxy- ( E )-stilbene and 2-(3′-hydroxy-5′-methoxybenzene)-4-hydroxybenzofuran. 2. a kind of method as claimed in claim 1 isolating the active ingredient of stilbene class from the stem bark of Acanthopanax vine, is characterized in that: the condition of microwave extraction in the described step (1) refers to that the extraction solvent is that the volume concentration is 75% ~95% ethanol, liquid-solid ratio of 10-20mL/g, stirring speed of 600-700 rpm, extraction temperature of 55-65 ℃, microwave power of 650-750 W, extraction time of 15-20 min, and extraction times of 2~3 times. 3. a kind of method for isolating the active ingredient of stilbene class from the stem bark of Acanthopanax as claimed in claim 1, it is characterized in that: the decompression concentration condition in the described step (1) refers to that the pressure is 0.05 ~ 0.06 MPa, The temperature is 40~50°C, and the rotation speed is 100~150 rpm. 4. a kind of method for isolating the active ingredient of stilbene class from the stem bark of Radix Acanthopanax as claimed in claim 1, it is characterized in that: the vacuum evaporation condition in the described step (2) refers to that the pressure is 0.04 ~ 0.05 MPa , the temperature is 30~40 ℃, and the speed is 80~100rpm. 5. a kind of method isolating the active ingredient of stilbene class from the stem bark of Acanthopanax as claimed in claim 1, it is characterized in that: the mixed solvent in the described step (3) refers to normal hexane, ethyl acetate, methyl alcohol Add water and water into the separatory funnel according to the volume ratio of 5:4:7:5, shake it well and let it stand overnight. The volume is mixed. 6. a kind of method as claimed in claim 1 isolating the active ingredient of stilbene class from the stem bark of Radix Acanthopanax, it is characterized in that: the ultrasonic dissolution condition in the described step (3) refers to that the ultrasonic frequency is 40 kHz, and the temperature is 40°C, ultrasonic time is 5 min. 7. A method for isolating stilbene-like active ingredients from the stem bark of Rattan Acanthopanax as claimed in claim 1, characterized in that: the purification and separation conditions in the step (5) refer to that the temperature is 25 ° C, the spiral tube speed The speed is 800-900 rpm, the detection wavelength is 325 nm, the injection flow rate is 1.5-2.0 mL/min, and the injection volume at one time is 160 mg. 8. a kind of method for isolating the active ingredient of stilbene class from the stem bark of Radix Acanthopanax as claimed in claim 1, it is characterized in that: the decompression concentration condition in the described step (6) refers to that the pressure is 0.03 ~ 0.05 MPa, The temperature is 30~50℃, and the rotation speed is 80~150rpm. 9. A kind of method for isolating the active ingredients of stilbenes from the stem bark of Acanthopanax as claimed in claim 1, characterized in that: the vacuum drying condition in the step (6) refers to that the pressure is 0.05 ~ 0.06 MPa, and the temperature The temperature is 30~50 ℃, and the drying time is 2~10h.