阅读说明：本技术 一种提取高纯度补骨脂酚的绿色工艺 (Green process for extracting high-purity bakuchiol ) 是由 付书清 张�林 徐敏 曹怀塘 于 2021-01-18 设计创作，主要内容包括：本发明公开了一种从补骨脂中提取纯化补骨脂酚的绿色工艺,属于植物提取物的制备技术领域。该工艺采用二氧化碳超临界萃取技术提取补骨脂中的补骨脂酚,经过低极性有机溶剂溶解处理除去大极性不溶物的初步分离后,应用柱层析技术进行纯化,得到纯度大于98％的补骨脂酚,得率大于5.0％。本发明是一种高效环保工艺,样品得率高,纯度高。该工艺过程简单、安全、可控性好、成本低、生产周期短,适合大规模工业生产。(The invention discloses a green process for extracting and purifying bakuchiol from fructus psoraleae, and belongs to the technical field of preparation of plant extracts. The process adopts carbon dioxide supercritical extraction technology to extract bakuchiol from fructus Psoraleae, and adopts column chromatography technology to purify after preliminary separation of low-polarity organic solvent dissolution treatment to remove large-polarity insoluble substances, so as to obtain bakuchiol with purity of more than 98%, and yield of more than 5.0%. The invention is a high-efficiency environment-friendly process, and has high sample yield and high purity. The process is simple, safe, good in controllability, low in cost, short in production period and suitable for large-scale industrial production.)

1. A green process for extracting and purifying bakuchiol from fructus psoraleae is characterized by comprising the following steps:

(1) supercritical extraction: drying fructus psoraleae, crushing to 40-100 meshes, putting into an extraction kettle, and extracting by adopting a supercritical carbon dioxide extraction technology to obtain oily liquid containing bakuchiol; optionally, the supercritical carbon dioxide further comprises an entrainer ethyl acetate;

(2) primary separation: dissolving the oily liquid obtained in the step (1) by using a low-polarity organic solvent with the volume being 6-12 times that of the oily liquid, filtering insoluble substances, and concentrating the filtrate under reduced pressure to obtain a psoralen crude extract;

(3) and (3) column chromatography purification: purifying the crude extract of bakuchiol obtained in step (2) by column chromatography, eluting with mixed solvent, collecting eluate containing bakuchiol, and recovering solvent from the eluate under reduced pressure to obtain oily bakuchiol;

(4) optionally, decolorizing the obtained oily bakuchiol to obtain bakuchiol product.

2. The green process for extracting and purifying bakuchiol from psoralea fruit as claimed in claim 1, wherein:

the supercritical extraction conditions of the step (1) are as follows: drying fructus psoraleae and crushing to 60-80 meshes, wherein an extraction solvent is supercritical carbon dioxide, the extraction temperature is 20-60 ℃, the extraction pressure is 10-40 MPa, the flow rate of the extraction solvent is 20-60L/h, and the extraction time is 0.5-3 hours; the temperature of the resolution kettle A is 20-80 ℃, and the pressure of the resolution kettle A is 5-15 MPa; the temperature of the resolution kettle B is 15-50 ℃, and the pressure of the resolution kettle B is 2-10 MPa; optionally, the supercritical carbon dioxide further contains an entrainer ethyl acetate, and the weight ratio of the carbon dioxide to the ethyl acetate is 10-30: 1;

the low-polarity organic solvent used in the dissolving in the step (2) is at least one selected from petroleum ether, n-hexane and cyclohexane;

the filler in the chromatographic column in the step (3) is silica gel or alumina, the mixed solvent for elution is a mixed solvent of a solvent A and a solvent B, the solvent A is selected from petroleum ether, n-hexane or cyclohexane, and the solvent B is selected from ethyl acetate or acetone;

optionally, activated carbon is used for decoloring in the step (4).

3. The green process for extracting and purifying bakuchiol from psoralea fruit as claimed in claim 2, wherein:

the supercritical extraction conditions of the step (1) are as follows: drying fructus psoraleae and crushing to 60-80 meshes, wherein an extraction solvent is supercritical carbon dioxide and an entrainer ethyl acetate, and the weight ratio of the carbon dioxide to the ethyl acetate is 15-25: 1; the extraction temperature is 45-55 ℃, the extraction pressure is 20-30 MPa, the flow of the extraction solvent is 35-45L/h, and the extraction time is 1-2 hours; the temperature of the resolution kettle A is 40-60 ℃, and the pressure of the resolution kettle A is 8-12 MPa; the temperature of the resolving kettle B is 20-40 ℃, and the pressure of the resolving kettle B is 4-6 MPa.

The low-polarity organic solvent used for dissolving in the step (2) is selected from petroleum ether, and the dosage of the petroleum ether is 8-10 times of the volume of the oily liquid obtained in the step (1);

the filler in the chromatographic column in the step (3) is silica gel, the mixed solvent for elution is a mixed solvent composed of petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether to the ethyl acetate is 30: 1-5: 1.

Technical Field

The invention relates to a green process for extracting and purifying bakuchiol from fructus psoraleae, belonging to the technical field of preparation of plant extracts.

Background

The Chinese medicine Fructus Psoraleae (Psoralea Fructus) is dry mature fruit of Psoralea corylifolia L. of Leguminosae, also known as Fructus Psoraleae, and Fructus Psoraleae. Collecting the infructescence in autumn when the fruit is mature, drying in the sun, rubbing out the fruit, and removing impurities.

Psoralea (Psoralea Linn.) plants are about 120 species worldwide, mainly distributed in the south of africa, north and south america and australia, and a few in asia and temperate europe, with only 1 species in china. Psoralea corylifolia is an imported medicinal material, is widely cultivated in China later, is mainly produced in Yunnan, Sichuan, Henan and other places of China, and is cultivated in other places.

The bone fat is tonified, pungent, bitter and warm, and the bone fat enters kidney and spleen channels, and has the effects of warming kidney, tonifying yang, absorbing qi, relieving asthma, warming spleen and stopping diarrhea; dispel wind and remove spots for external use. Clinically, fructus psoraleae is commonly used for treating kidney-yang deficiency, yang-qi-dispersing spermatorrhea, enuresis, frequent micturition, cold pain in waist and knees, asthma due to kidney deficiency, and morning diarrhea; it can be used for treating vitiligo and alopecia areata, and Vietnam can be used for treating rheumatism with ethanol extract. 2015 edition "pharmacopoeia of the people's republic of China" first part records about 30 kinds of Chinese patent medicine compound containing fructus psoraleae, including four-god pill, bone-strengthening joint pill, Qing' e pill, lumbago pill (tablet), hair-growing liniment (hair-growing tincture), white erosion pill, etc., which shows that the clinical application of the Chinese medicine is very wide.

The chemical components in the fructus psoraleae are mainly divided into three types: coumarin compounds, flavonoid compounds and monoterpene phenol compounds, which have the common characteristic that most of them contain isopentenyl.

Bakuchiol (bakuchiol) belongs to monoterpene terpenoid phenolic compounds, and has a structural formula as follows:

the content of bakuchiol in the fructus Psoraleae is about 6%, and the content of bakuchiol in the fructus Psoraleae volatile oil is more than 60%.

Some preclinical studies have reported that bakuchiol has anti-tumor activity, which may be related to its structure close to that of resveratrol. A rat model research result shows that the ethanol extract of bakuchiol and Chinese medicine for supplementing bone fat has the effect of preventing bone loss.

The bakuchiol also has antioxidant, antiinflammatory, and antibacterial effects. The bakuchiol has antibacterial effect on various gram-positive and gram-negative oral pathogens; under the conditions of a certain sucrose concentration range and pH range and the presence of organic acid, the bakuchiol can inhibit the growth of streptococcus mutans and shows temperature dependence; bakuchiol also inhibits the growth of adherent cells.

Although bakuchiol has no structural similarity to retinol, bakuchiol has been found to have a similar effect on regulation of gene expression as retinol. In 2018, a result of a random double-blind experiment of 44 volunteers in 12 weeks shows that bakuchiol is equivalent to retinol in improving photoaging (wrinkles and pigmentation) and has better skin tolerance.

Bakuchiol has also been found to have antiandrogenic activity in prostate cancer cells, which inhibits cell proliferation.

CN101823940A discloses a preparation method of bakuchiol: pulverizing fructus Psoraleae, extracting with ethanol, recovering ethanol, concentrating, dissolving in water completely, standing, filtering, concentrating the filtrate, loading onto macroporous adsorbent resin, washing with water, removing water washing solution, eluting with 30-90% ethanol, discarding the eluate, eluting with 95% or anhydrous ethanol, recovering ethanol, concentrating, drying, extracting the dried product with organic solvent, discarding the extractive solution, and drying the residue to obtain bakuchiol.

CN101898938A discloses a method for preparing bakuchiol, which is prepared by extracting fructus psoraleae with ethanol, precipitating with water, purifying filtrate with macroporous resin, extracting with organic solvent, discarding the extract and washing residues with water. The content of the obtained product psoralea corylifoliol is 90 to 97 percent

CN105237361A discloses a method for extracting and preparing bakuchiol, which comprises the steps of crushing a bakuchiol medicinal material, extracting by cold soaking with petroleum ether, and recovering a solvent to obtain a bakuchiol extract; dissolving with petroleum ether, stirring with silica gel to obtain loose sample, volatilizing solvent, loading, performing silica gel column chromatography, eluting with petroleum ether, tracking by TLC, mixing the fractions containing bakuchiol, and recovering solvent to obtain crude bakuchiol; further purifying the crude product of the bakuchiol, dissolving by petroleum ether, stirring by silica gel to be loose, volatilizing the solvent, loading, performing silica gel column chromatography, eluting by petroleum ether-ethyl acetate, tracking by TLC, combining the flow parts containing the bakuchiol, and recovering the solvent to obtain the pure product of the bakuchiol, wherein the purity is more than or equal to 98 percent, and the yield is 1.0 percent.

CN108299453A discloses a method for separating psoralen, isopsoralen and bakuchiol from fructus psoraleae, comprising the following steps: a) pulverizing fructus Psoraleae, and extracting with petroleum ether to obtain petroleum ether crude extract of fructus Psoraleae; b) loading petroleum ether crude extract of fructus Psoraleae on polyamide column, gradient eluting with mixed solution of ethanol and water, collecting target fraction, and concentrating under reduced pressure to obtain bakuchiol and mixture of psoralen and isopsoralen, which comprises: ethanol with water according to 5: 95-30: 70, sequentially carrying out gradient elution on the mixed solution in the volume ratio, collecting target fractions, and carrying out reduced pressure concentration to obtain a mixture of psoralen and isopsoralen; ethanol with water according to 35: 65-55: and (4) carrying out gradient elution on the mixed solution formed by the volume ratio of 45 in sequence, collecting target fractions, and carrying out reduced pressure concentration to obtain the bakuchiol. The yield of the bakuchiol from the crude extract of the petroleum ether is 22 to 23 percent, and the yield is about 2 percent calculated from the bakuchiol medicinal material.

The preparation method of the bakuchiol has the advantages of complex process steps, high solvent consumption, low economy, environmental protection and low yield.

Disclosure of Invention

The invention aims to overcome the defects of the prior background art and provide a green new process for efficiently extracting and purifying bakuchiol by taking bakuchiol as a raw material.

The technical process provided by the invention comprises the following steps:

(1) supercritical extraction: drying fructus psoraleae, crushing to 40-100 meshes, putting into an extraction kettle, and extracting by adopting a supercritical carbon dioxide extraction technology to obtain oily liquid containing bakuchiol; optionally, the supercritical carbon dioxide further comprises an entrainer ethyl acetate;

(2) primary separation: dissolving the oily liquid obtained in the step (1) with a low-polarity organic solvent with the volume being 6-12 times that of the oily liquid, filtering to remove large-polarity insoluble substances, and concentrating the filtrate under reduced pressure to obtain a crude psoralen extract;

(3) and (3) column chromatography purification: purifying the crude extract of bakuchiol obtained in step (2) by column chromatography, eluting with mixed solvent, collecting eluate containing bakuchiol, and recovering solvent from the eluate under reduced pressure to obtain oily bakuchiol;

(4) optionally, decolorizing the obtained oily bakuchiol to obtain bakuchiol product.

In some embodiments, the supercritical extraction conditions of step (1) above are: drying fructus psoraleae and crushing to 60-80 meshes, wherein an extraction solvent is supercritical carbon dioxide, the extraction temperature is 20-60 ℃, the extraction pressure is 10-40 MPa, the flow rate of the extraction solvent is 20-60L/h, and the extraction time is 0.5-3 hours; the temperature of the resolution kettle A is 20-80 ℃, and the pressure of the resolution kettle A is 5-15 MPa; the temperature of the resolution kettle B is 15-50 ℃, and the pressure of the resolution kettle B is 2-10 MPa; optionally, the supercritical carbon dioxide further contains an entrainer ethyl acetate, and the weight ratio of the carbon dioxide to the ethyl acetate is 10-30: 1;

in some embodiments, the low polarity organic solvent used in the dissolving in the step (2) is at least one selected from petroleum ether, n-hexane and cyclohexane;

in some embodiments, the filler in the chromatography column in step (3) above is silica gel or alumina, and the mixed solvent for elution is a mixed solvent of a solvent a and a solvent B, wherein the solvent a is selected from petroleum ether, n-hexane or cyclohexane, and the solvent B is selected from ethyl acetate or acetone;

in some embodiments, the decoloring treatment using activated carbon is performed in the above step (4).

Further, the supercritical extraction conditions of the step (1) are as follows: drying fructus psoraleae and crushing to 60-80 meshes, wherein an extraction solvent is supercritical carbon dioxide and an entrainer ethyl acetate, and the weight ratio of the carbon dioxide to the ethyl acetate is 15-25: 1; the extraction temperature is 45-55 ℃, the extraction pressure is 20-30 MPa, the flow of the extraction solvent is 35-45L/h, and the extraction time is 1-2 hours; the temperature of the resolution kettle A is 40-60 ℃, and the pressure of the resolution kettle A is 8-12 MPa; the temperature of the resolving kettle B is 20-40 ℃, and the pressure of the resolving kettle B is 4-6 MPa.

In some embodiments, the low polarity organic solvent used for dissolving in step (2) above is selected from petroleum ether in an amount of 8 to 10 times the volume of the oily liquid obtained in step (1);

in some embodiments, the filler in the chromatography column in step (3) is silica gel, and the mixed solvent for elution is a mixed solvent composed of petroleum ether and ethyl acetate, and the volume ratio of the mixed solvent to the ethyl acetate is 30: 1-5: 1.

The technical process provided by the invention is based on the following principle: psoralen is a monoterpene phenol substance with strong fat solubility, and long-time organic solvent reflux extraction can cause chemical change of the psoralen, so that the yield is reduced. The invention adopts the carbon dioxide supercritical extraction technology to extract the bakuchiol in the fructus psoraleae, so that the bakuchiol can be efficiently extracted and cannot be changed due to long-time heating. In the purification process, the invention adopts column chromatography for purification, thereby improving the purity of the product and reducing the loss of the product.

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

(1) the process is an environment-friendly process: the carbon dioxide supercritical extraction technology is adopted instead of the organic solvent reflux extraction technology, so that the use amount of the organic solvent is reduced, and the energy consumption is reduced; the amount of the organic solvent used in other steps of the process is greatly reduced, the pollution is low, and the energy consumption is low; the chromatographic column can be repeatedly used after being treated by the filler. Therefore, the invention provides a green production process.

(2) The process has high yield: the method can thoroughly extract bakuchiol from raw materials by supercritical carbon dioxide extraction, and has low loss and final yield of more than 5.0%.

(3) The purity of the sample is high: purifying with chromatographic column to obtain bakuchiol sample with purity higher than 98%.

(4) The process is simple, safe, good in controllability, low in cost and short in production period, and is suitable for large-scale industrial production.

Detailed Description

The foregoing summary of the invention is described in further detail below with reference to specific embodiments. It should not be understood that the scope of the above-described subject matter of the present invention is limited to the following examples. Various substitutions and alterations can be made without departing from the technical idea of the invention as described above, according to the common technical knowledge and conventional means in the field, and the scope of the invention is covered.

EXAMPLE 1 preparation of bakuchiol

The method comprises the following steps:

(1) supercritical extraction: drying 5 kg of fructus psoraleae, crushing to 80 meshes, putting into an extraction kettle, and extracting by adopting supercritical carbon dioxide; the extraction temperature is 40 ℃, the extraction pressure is 30Mpa, the flow of carbon dioxide is 20L/h, and the flowing extraction is carried out after the balance is carried out for 20 minutes; extracting for 2 hours; the pressure of the resolution kettle A and the pressure of the resolution kettle B are respectively 8Mpa and 6Mpa, the temperature is respectively 45 ℃ and 40 ℃, and resolution is carried out to obtain 780mL of yellow oily liquid containing the bakuchiol.

(2) Primary separation: the oily liquid is dissolved by 8000mL petroleum ether, large-polarity insoluble substances are removed by filtration, and 418g of crude psoralen extract is obtained by concentrating the filtrate under reduced pressure.

(3) And (3) column chromatography purification: loading the crude extract of bakuchiol into a silica gel chromatographic column, eluting with a mixed solvent of petroleum ether and ethyl acetate of 8: 1, collecting eluent, and recovering the solvent under reduced pressure to obtain 254 g of oily bakuchiol with the yield of 5.1%; the content was 99.2% by HPLC normalization detection.

EXAMPLE 2 preparation of bakuchiol

The method comprises the following steps:

(1) supercritical extraction: drying 5 kg of fructus psoraleae, crushing to 100 meshes, putting into an extraction kettle, and extracting by adopting supercritical carbon dioxide; the extraction temperature is 45 ℃, the extraction pressure is 30Mpa, the flow of carbon dioxide is 20L/h, and the flow extraction is carried out after the balance is carried out for 20 minutes; extracting for 3 hours; the pressure of the resolution kettle A and the pressure of the resolution kettle B are respectively 10Mpa and 8Mpa, the temperature is respectively 30 ℃ and 25 ℃, and resolution is carried out to obtain 760mL of oily liquid containing the bakuchiol.

(2) Primary separation: dissolving the oily liquid with 5000mL of petroleum ether, filtering to remove large-polarity insoluble substances, and concentrating the filtrate under reduced pressure to obtain 433g of crude psoralen extract.

(3) And (3) column chromatography purification: loading the crude extract of the bakuchiol into a silica gel chromatographic column, eluting with a mixed solvent of petroleum ether and acetone which are 10:1, collecting eluent, and recovering the solvent under reduced pressure to obtain 260 g of oily bakuchiol with the yield of 5.2 percent; the purity of bakuchiol is 98.4% by HPLC normalization detection.

EXAMPLE 3 preparation of bakuchiol

The method comprises the following steps:

(1) supercritical extraction: drying 5 kg of fructus psoraleae, crushing to 40 meshes, putting into an extraction kettle, and extracting by adopting supercritical carbon dioxide; the extraction temperature is 45 ℃, the extraction pressure is 30Mpa, the flow of carbon dioxide is 25L/h, and the flow extraction is carried out after the balance is carried out for 20 minutes; extracting for 2 hours; the pressure of the resolution kettle A and the pressure of the resolution kettle B are respectively 15Mpa and 10Mpa, the temperature is respectively 50 ℃ and 30 ℃, and the resolution is carried out to obtain 740mL of oily liquid containing the bakuchiol.

(2) Primary separation: the oily liquid was dissolved in 9000mL of n-hexane, the polar insoluble material was removed by filtration, and the filtrate was concentrated under reduced pressure to give 420g of crude bakuchiol extract.

(3) And (3) column chromatography purification: loading the crude extract of bakuchiol onto an alumina chromatographic column, eluting with a mixed solvent of n-hexane and ethyl acetate of 6: 1, collecting the eluent, and recovering the solvent under reduced pressure to obtain oily bakuchiol.

(4) And (3) decoloring: dissolving oily bakuchiol in methanol, and decolorizing with active carbon to obtain 255 g of light yellow oily bakuchiol product with the yield of 5.1%; the content was 99.2% by HPLC normalization detection.

EXAMPLE 4 preparation of bakuchiol

The method comprises the following steps:

(1) supercritical extraction: drying 5 kg of fructus psoraleae, crushing to 80 meshes, putting into an extraction kettle, and extracting by using supercritical carbon dioxide, wherein an extraction solvent is supercritical carbon dioxide and entrainer ethyl acetate, and the weight ratio of the extraction solvent to the supercritical carbon dioxide is 10: 1; the extraction temperature is 45 ℃, the extraction pressure is 30Mpa, the flow of the extraction solvent is 35L/h, and the flow extraction is carried out after the balance is carried out for 20 minutes. Extracting for 2.5 hours; the pressure of the resolution kettle A and the pressure of the resolution kettle B are respectively 5Mpa and 2Mpa, the temperature is respectively 40 ℃ and 25 ℃, and resolution is carried out to obtain 790mL of oily liquid containing bakuchiol.

(2) Primary separation: the oily liquid is dissolved by 6500mL of cyclohexane, the large-polarity insoluble substances are removed by filtration, and the filtrate is concentrated under reduced pressure to obtain 453g of crude psoralen extract.

(3) And (3) column chromatography purification: loading the crude extract of bakuchiol onto an alumina chromatographic column, eluting with a mixed solvent of petroleum ether and ethyl acetate in a ratio of 6: 1, collecting eluent, and recovering the solvent under reduced pressure to obtain 272 g of oily bakuchiol with a yield of 5.4%; the content was 98.3% by HPLC normalization detection.

EXAMPLE 5 preparation of bakuchiol

The method comprises the following steps:

(1) supercritical extraction: drying 5 kg of fructus psoraleae, crushing to 80 meshes, putting into an extraction kettle, and extracting by using supercritical carbon dioxide, wherein an extraction solvent is supercritical carbon dioxide and entrainer ethyl acetate, and the weight ratio of the extraction solvent to the supercritical carbon dioxide is 20: 1; the extraction temperature is 50 ℃, the extraction pressure is 25Mpa, the flow of the extraction solvent is 40L/h, and the flow extraction is carried out after the balance is carried out for 20 minutes. Extracting for 1.5 hours; the pressure of the resolution kettle A and the pressure of the resolution kettle B are respectively 10Mpa and 5Mpa, the temperature is respectively 50 ℃ and 30 ℃, and resolution is carried out to obtain 812mL of oily liquid containing the bakuchiol.

(2) Primary separation: the oily liquid is dissolved by 8000mL petroleum ether, the large-polarity insoluble substances are removed by filtration, and 481g of crude psoralen extract is obtained by concentrating the filtrate under reduced pressure.

(3) And (3) column chromatography purification: loading the crude extract of bakuchiol onto silica gel chromatographic column, eluting with mixed solvent of petroleum ether and ethyl acetate at ratio of 10:1, collecting eluate, and recovering solvent under reduced pressure to obtain oily bakuchiol.

(4) And (3) decoloring: dissolving the oily bakuchiol in methanol, and decolorizing with active carbon to obtain 286 g of light yellow oily bakuchiol product with the yield of 5.7%; the content was 99.6% by HPLC normalization detection.

EXAMPLE 6 preparation of bakuchiol

The method comprises the following steps:

(1) supercritical extraction: drying 5 kg of fructus psoraleae, crushing to 70 meshes, putting into an extraction kettle, and extracting by using supercritical carbon dioxide, wherein an extraction solvent is supercritical carbon dioxide and entrainer ethyl acetate, and the weight ratio of the extraction solvent to the supercritical carbon dioxide is 30: 1; the extraction temperature is 55 ℃, the extraction pressure is 40Mpa, the flow rate of the extraction solvent is 45L/h, and the flow extraction is carried out after the balance is carried out for 20 minutes. Extracting for 3 hours; the pressure of the resolution kettle A and the pressure of the resolution kettle B are respectively 12Mpa and 6Mpa, the temperature is respectively 40 ℃ and 25 ℃, and resolution is carried out to obtain 741mL of oily liquid containing bakuchiol.

(2) Primary separation: the oily liquid is dissolved by 8800mL petroleum ether, the large-polarity insoluble substances are removed by filtration, and the filtrate is concentrated under reduced pressure to obtain 475g of the bakuchiol crude extract.

(3) And (3) column chromatography purification: loading the crude extract of the bakuchiol on a silica gel chromatographic column, eluting with a mixed solvent of petroleum ether and ethyl acetate of which the ratio is 30:1, collecting eluent, and recovering the solvent under reduced pressure to obtain 275 g of oily bakuchiol, wherein the yield is 5.5%; the content was 98.8% by HPLC normalization detection.