A method for extracting beta-sitosterol

Abstract

The application discloses a method for extracting beta-sitosterol, comprising the following steps: collecting spore powder of aspergillus niger after fermentation by using wheat bran, and preparing the spore powder into fungus powder by using a spray dryer for standby; obtaining an aspergillus niger spore powder extract by reflux extraction with the fungus powder and acetone; performing vacuum concentration, petroleum ether extraction, and vacuum concentration again until the petroleum ether is dried to obtain a crude extract; performing silica gel column chromatography; performing silica gel column chromatography again; performing silica gel column chromatography again, observing color development of 5% concentrated sulfuric acid ethanol solution and 0.8% phosphomolybdic acid ethanol solution according to thin layer chromatography analysis, selecting fractions with the same point and Rf value, and combining the fractions; performing vacuum concentration until the organic solvent is dried; selecting fractions with Rf values of 0.3-0.5 under the development condition of petroleum ether: ethyl acetate = 5:1, and performing recrystallization in a mixed solvent of ethyl acetate:methanol = 1:50; and collecting crystals after crystallization to obtain beta-sitosterol. The substrate is cheap and easy to obtain, the extraction process is simple and easy to implement, the production process is environmentally friendly, solvent residue is small, pollution is low, and culture medium is easy to obtain.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a method for extracting β-sitosterol. Background Technology

[0002] β-Sitosterol is a commonly used medical raw material and health product, playing an important role in lowering cholesterol, inhibiting tumors, and preventing benign prostatic hyperplasia. Studies have shown that β-sitosterol can be isolated and purified from phytosterols derived from plant oils, and it plays an important role in the prevention and treatment of human diseases. Literature reports that the main purification methods for β-sitosterol involve esterification of phytosterols with acetic anhydride, followed by excess bromination, separation and purification, and recrystallization after debromination to obtain a mixture of sterols. High-speed countercurrent chromatography or multi-step extraction with special solvents followed by recrystallization can also be used. However, due to limitations in production processes and equipment, separation is difficult, and the reaction steps are numerous, resulting in high costs and low solvent recovery rates. These characteristics make large-scale production challenging, increasing product costs and reducing product value and competitiveness. Summary of the Invention

[0003] The purpose of this invention is to overcome the above-mentioned shortcomings and provide a method for extracting β-sitosterol with inexpensive and readily available substrates, simple and easy extraction process, environmentally friendly production process, low solvent residue, low pollution, and readily available culture medium.

[0004] The objective of this invention and the solution to its main technical problem are achieved by the following technical solution:

[0005] The present invention provides a method for extracting β-sitosterol, comprising the following steps:

[0006] (1) Preparation of crude extract

[0007] After collecting spores of Aspergillus niger through fermentation with wheat bran, the spores are spray-dried to obtain mycelial powder for later use. The mycelial powder and acetone are mixed at a mass ratio of 1:5 and refluxed three times at a standard atmosphere and a temperature of 40-60℃ for 3 hours each time to obtain an Aspergillus niger spore powder extract. The extract is concentrated under reduced pressure to 1 / 10-1 / 5 of the total volume and then dissolved in an equal volume of water. The aqueous solution is then extracted with an equal volume of petroleum ether to obtain a petroleum ether extract. The extract is then concentrated under reduced pressure until the petroleum ether evaporates to dryness to obtain a crude extract.

[0008] (2) Silica gel column chromatography

[0009] The crude extract was dissolved in an equal volume of ethyl acetate, then mixed with 40-80 mesh silica gel at a volume ratio of 1:1.2. The ethyl acetate was evaporated in a water bath, and the sample was then loaded onto a prepared 200-300 mesh silica gel column for elution at atmospheric pressure. The elution gradient was 100% petroleum ether, and petroleum ether:acetone ratios of 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, and 1:1, with equal proportions of 100% acetone. The flow rate was 10-20 ml / min. Thin-layer chromatography analysis was performed, observing the color development in 5% concentrated sulfuric acid ethanol solution and 0.8% phosphomolybdic acid ethanol solution. Fractions with similar spot formation and Rf values ​​were combined and concentrated under reduced pressure until the organic solvent evaporated, yielding different concentrated solutions.

[0010] (3) Second silica gel column chromatography

[0011] Collect the eluent of the petroleum ether:acetone = 10:1-2:1 system, concentrate under reduced pressure until the organic solvent evaporates to dryness, dissolve the concentrate in ethyl acetate, and mix with 40-mesh silica gel at a volume ratio of 1:1.2. Simultaneously, evaporate the solvent in a water bath, and load the sample onto a prepared silica gel column for elution at atmospheric pressure. The flow rate is 20-25 ml / min, and the elution system is petroleum ether:ethyl acetate = 10:1. Analyze the results by thin-layer chromatography, observing the color development of 5% concentrated sulfuric acid ethanol solution and 0.8% phosphomolybdic acid ethanol solution. Select fractions with the same spot characteristics and Rf values, combine them, and concentrate under reduced pressure until the organic solvent evaporates to dryness to obtain different concentrated fractions.

[0012] (4) Second silica gel column chromatography

[0013] Based on the analysis results of each concentrated component obtained in (3) on thin-layer chromatography, the fraction with an Rf value of 0.3-0.5 under the development condition of petroleum ether: ethyl acetate = 5:1 was selected for further silica gel column chromatography. After dissolving the concentrate in ethyl acetate, it was mixed with 40-mesh silica gel at a volume ratio of 1:1.2. At the same time, the solvent was evaporated in a water bath and the sample was loaded onto the prepared silica gel column for elution at normal pressure. The flow rate was 20-25 ml / min and the elution system was petroleum ether: ethyl acetate = 6:1. According to the thin-layer chromatography analysis, the color development of 5% concentrated sulfuric acid ethanol solution and 0.8% phosphomolybdic acid ethanol solution was observed. The fractions with the same spot formation and Rf value were selected and combined, and concentrated under reduced pressure until the organic solvent was evaporated.

[0014] (5) recrystallization

[0015] Fractions with Rf values ​​of 0.3-0.5 under petroleum ether:ethyl acetate = 5:1 development conditions were selected and recrystallized in a mixed solvent of ethyl acetate:methanol = 1:50. The collected crystals were β-sitosterol.

[0016] The above method for extracting β-sitosterol, wherein: Aspergillus niger is Aspergillus niger xj ( Aspergillus niger (xj), was deposited on March 7, 2006, at the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China), accession number: CCTCC NO: M206021.

[0017] Compared with existing technologies, this invention has significant advantages. As can be seen from the above technical solution: this invention extracts Aspergillus niger spore powder via acetone reflux, and then, through a targeted separation process in the petroleum ether extraction stage, analyzes the results of different thin-layer chromatography to separate β-sitosterol, providing a new route for the source of β-sitosterol raw materials. Furthermore, Aspergillus niger (… Aspergillus niger The strain (xj) is reliable in origin, has a wide range of adaptability to natural environmental conditions such as temperature and pH, and is easy to cultivate and preserve, making it suitable for extracting β-sitosterol. This technology offers low production costs, a simple and reproducible separation and extraction method, low solvent residue that is easily recovered, reduced energy consumption and waste gas emissions, and significantly lower environmental pollution. Attached Figure Description

[0018] Figure 1 This is the EI-MS mass spectrometry analysis chromatogram of β-sitosterol extracted in Example 1;

[0019] Figure 2 It is the β-sitosterol extracted in Example 2. 1 H NMR spectrum;

[0020] Figure 3 It is the β-sitosterol extracted in Example 3. 13 C-NMR spectrum; Detailed Implementation

[0021] Example 1:

[0022] A method for extracting β-sitosterol, comprising the following steps:

[0023] (1) Preparation of crude extract

[0024] Aspergillus niger xj ( Aspergillus niger xj), with accession number: CCTCC NO: M206021. After collecting spore powder through fermentation with wheat bran, the spore powder was spray-dried to prepare mycelial powder for later use. The mycelial powder and acetone were mixed at a mass ratio of 1:5 and refluxed three times at 60℃ under one standard atmosphere, for 3 hours each time, to obtain Aspergillus niger spore powder extract. The extract was concentrated under reduced pressure to 1 / 5 of the total volume and then dissolved in an equal volume of water. The aqueous solution was extracted with an equal volume of petroleum ether to obtain a petroleum ether extract. The extract was then concentrated under reduced pressure until the petroleum ether evaporated to dryness to obtain the crude extract.

[0025] (2) Silica gel column chromatography

[0026] The crude extract was dissolved in an equal volume of ethyl acetate, then mixed with 40-80 mesh silica gel at a volume ratio of 1:1.2. The ethyl acetate was evaporated in a water bath, and the sample was loaded onto a prepared 200-300 mesh silica gel column for elution at atmospheric pressure. The elution gradient was 100% petroleum ether, petroleum ether:acetone = 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 100% acetone in equal proportions. The flow rate was 15 ml / min. Thin-layer chromatography analysis was performed, observing the color development in 5% concentrated sulfuric acid ethanol solution and 0.8% phosphomolybdic acid ethanol solution. Fractions with the same spot formation and Rf value were combined and concentrated under reduced pressure until the organic solvent evaporated, yielding different concentrated solutions.

[0027] (3) Second silica gel column chromatography

[0028] Collect the eluent of the petroleum ether:acetone = 10:1-2:1 system, concentrate under reduced pressure until the organic solvent evaporates to dryness, dissolve the concentrate in ethyl acetate, mix with 40-mesh silica gel at a volume ratio of 1:1.2, and simultaneously evaporate the solvent in a water bath. Load the sample onto a prepared silica gel column for elution at atmospheric pressure; the flow rate is 20 ml / min, and the elution system is petroleum ether:ethyl acetate = 10:1. Analyze the results by thin-layer chromatography, observing the color development of 5% concentrated sulfuric acid ethanol solution and 0.8% phosphomolybdic acid ethanol solution. Select fractions with the same spot characteristics and Rf values, combine them, concentrate under reduced pressure until the organic solvent evaporates to dryness, and obtain different concentrated fractions.

[0029] (4) Second silica gel column chromatography

[0030] Based on the analysis results of each concentrated component obtained in (3) on thin-layer chromatography, the fraction with an Rf value of 0.3-0.5 under the development condition of petroleum ether: ethyl acetate = 5:1 was selected for further silica gel column chromatography. After dissolving the concentrate in ethyl acetate, it was mixed with 40-mesh silica gel at a volume ratio of 1:1.2. At the same time, the solvent was evaporated in a water bath and the sample was loaded onto the prepared silica gel column for elution at normal pressure. The flow rate was 20 ml / min and the elution system was petroleum ether: ethyl acetate = 6:1. According to the thin-layer chromatography analysis, the color development of 5% concentrated sulfuric acid ethanol solution and 0.8% phosphomolybdic acid ethanol solution was observed. The fractions with the same spot formation and Rf value were selected and combined, and concentrated under reduced pressure until the organic solvent was evaporated.

[0031] (5) recrystallization

[0032] Fractions with Rf values ​​of 0.3-0.5 under petroleum ether:ethyl acetate = 5:1 development conditions were selected and recrystallized in a mixed solvent of ethyl acetate:methanol = 1:50. The collected crystals were β-sitosterol.

[0033] Example 2:

[0034] A method for extracting β-sitosterol, comprising the following steps:

[0035] (1) Preparation of crude extract

[0036] Aspergillus niger xj ( Aspergillus niger xj), with accession number: CCTCC NO: M206021. After fermentation with wheat bran, the spore powder was collected and spray-dried to prepare mycelial powder for later use. The mycelial powder and acetone were mixed at a mass ratio of 1:5 and refluxed three times at 50°C for 3 hours each time to obtain Aspergillus niger spore powder extract. The extract was concentrated under reduced pressure to 1 / 6 of the total volume and then dissolved in an equal volume of water. The aqueous solution was extracted with an equal volume of petroleum ether to obtain a petroleum ether extract. The extract was then concentrated under reduced pressure until the petroleum ether evaporated to dryness to obtain the crude extract.

[0037] (2) Silica gel column chromatography

[0038] The crude extract was dissolved in an equal volume of ethyl acetate, then mixed with 40-80 mesh silica gel at a volume ratio of 1:1.2. The ethyl acetate and petroleum ether were evaporated in a water bath, and the mixture was then loaded onto a prepared 200-300 mesh silica gel column for elution at atmospheric pressure. The elution gradients were 100% petroleum ether, petroleum ether:acetone = 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 100% acetone in equal proportions. The flow rate was 18 ml / min. Thin-layer chromatography analysis was performed, observing the color development in 5% concentrated sulfuric acid ethanol solution and 0.8% phosphomolybdic acid ethanol solution. Fractions with the same spot formation and Rf value were combined and concentrated under reduced pressure until the organic solvent evaporated, yielding different concentrated solutions.

[0039] (3) Second silica gel column chromatography

[0040] The eluent of a petroleum ether:acetone system of 10:1-2:1 was collected, concentrated under reduced pressure until the organic solvent evaporated, dissolved in ethyl acetate, and mixed with 40-mesh silica gel at a volume ratio of 1:1.2. Simultaneously, the solvent was evaporated in a water bath, and the sample was loaded onto a prepared silica gel column for elution at atmospheric pressure. The flow rate was 22 ml / min, and the elution system was petroleum ether:ethyl acetate = 10:1. Thin-layer chromatography was performed, observing the color development of 5% concentrated sulfuric acid ethanol solution and 0.8% phosphomolybdic acid ethanol solution. Fractions with the same spot characteristics and Rf values ​​were combined and concentrated under reduced pressure until the organic solvent evaporated to obtain different concentrated fractions.

[0041] (4) Second silica gel column chromatography

[0042] Based on the analysis results of each concentrated component obtained in (3) on thin-layer chromatography, the fraction with an Rf value of 0.3-0.5 under the development condition of petroleum ether: ethyl acetate = 5:1 was selected for further silica gel column chromatography. After dissolving the concentrate in ethyl acetate, it was mixed with 40-mesh silica gel at a volume ratio of 1:1.2. At the same time, the solvent was evaporated in a water bath and the sample was loaded onto the prepared silica gel column for elution at normal pressure. The flow rate was 23 ml / min and the elution system was petroleum ether: ethyl acetate = 6:1. According to the thin-layer chromatography analysis, the color development of 5% concentrated sulfuric acid ethanol solution and 0.8% phosphomolybdic acid ethanol solution was observed. The fractions with the same spot formation and Rf value were selected and combined, and concentrated under reduced pressure until the organic solvent was evaporated.

[0043] (5) recrystallization

[0044] Fractions with Rf values ​​of 0.3-0.5 under petroleum ether:ethyl acetate = 5:1 development conditions were selected and recrystallized in a mixed solvent of ethyl acetate:methanol = 1:50. The collected crystals were β-sitosterol.

[0045] Example 3:

[0046] A method for extracting β-sitosterol, comprising the following steps:

[0047] (1) Preparation of crude extract

[0048] Aspergillus niger xj ( Aspergillus niger xj), with accession number: CCTCC NO: M206021. After collecting spore powder through fermentation with wheat bran, the spore powder was spray-dried to prepare mycelial powder for later use. The mycelial powder and acetone were mixed at a mass ratio of 1:5 and refluxed three times at 55℃ for 3 hours each time to obtain Aspergillus niger spore powder extract. The extract was concentrated under reduced pressure to 1 / 8 of the total volume and then dissolved in an equal volume of water. The aqueous solution was extracted with an equal volume of petroleum ether to obtain a petroleum ether extract. The extract was then concentrated under reduced pressure until the petroleum ether evaporated to dryness to obtain the crude extract.

[0049] (2) Silica gel column chromatography

[0050] The crude extract was dissolved in an equal volume of ethyl acetate, then mixed with 40-80 mesh silica gel at a volume ratio of 1:1.2. The ethyl acetate was evaporated in a water bath, and the sample was loaded onto a prepared 200-300 mesh silica gel column for elution at atmospheric pressure. The elution gradient was 100% petroleum ether, petroleum ether:acetone = 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 100% acetone in equal proportions. The flow rate was 16 ml / min. Thin-layer chromatography analysis was performed, observing the color development in 5% concentrated sulfuric acid ethanol solution and 0.8% phosphomolybdic acid ethanol solution. Fractions with the same spot formation and Rf value were combined and concentrated under reduced pressure until the organic solvent evaporated, yielding different concentrated solutions.

[0051] (3) Second silica gel column chromatography

[0052] The eluent of a petroleum ether:acetone system of 10:1-2:1 was collected, concentrated under reduced pressure until the organic solvent evaporated, dissolved in ethyl acetate, and mixed with 40-mesh silica gel at a volume ratio of 1:1.2. Simultaneously, the solvent was evaporated in a water bath, and the sample was loaded onto a prepared silica gel column for elution at atmospheric pressure. The flow rate was 21 ml / min, and the elution system was petroleum ether:ethyl acetate = 10:1. Thin-layer chromatography was performed, observing the color development of 5% concentrated sulfuric acid ethanol solution and 0.8% phosphomolybdic acid ethanol solution. Fractions with the same spot characteristics and Rf values ​​were combined, concentrated under reduced pressure until the organic solvent evaporated, yielding different concentrated fractions.

[0053] (4) Second silica gel column chromatography

[0054] Based on the analysis results of each concentrated component obtained in (3) on thin-layer chromatography, the fraction with an Rf value of 0.3-0.5 under the development condition of petroleum ether: ethyl acetate = 5:1 was selected for further silica gel column chromatography. After dissolving the concentrate in ethyl acetate, it was mixed with 40-mesh silica gel at a volume ratio of 1:1.2. At the same time, the solvent was evaporated in a water bath and the sample was loaded onto the prepared silica gel column for elution at normal pressure. The flow rate was 24 ml / min and the elution system was petroleum ether: ethyl acetate = 6:1. According to the thin-layer chromatography analysis, the color development of 5% concentrated sulfuric acid ethanol solution and 0.8% phosphomolybdic acid ethanol solution was observed. The fractions with the same spot formation and Rf value were selected and combined, and concentrated under reduced pressure until the organic solvent was evaporated.

[0055] (5) recrystallization

[0056] Fractions with Rf values ​​of 0.3-0.5 under petroleum ether:ethyl acetate = 5:1 development conditions were selected and recrystallized in a mixed solvent of ethyl acetate:methanol = 1:50. The collected crystals were β-sitosterol.

[0057] Experimental example: Identification of β-sitosterol:

[0058] ① Sample preparation

[0059] The substances prepared in Examples 1-3 were dissolved in chloroform and analyzed by EI-MS. 25 mg of the substances prepared in Examples 1-3 were dissolved in 0.5 ml of deuterated chloroform and transferred to an NMR tube for NMR scanning to obtain proton and carbon spectra.

[0060] ②EI-MS analysis conditions

[0061] Mass spectrometry conditions: Ion source: EI source; Ion source temperature: 230℃; Quadrupole temperature: 150℃; Electron energy: 70 eV; Emission current: 34.6 μA; Multiplier voltage: 1482 V; Interface temperature: 280℃; Mass range: 29–500 amu. Solvent delay time: 5 min.

[0062] ③NMR analysis conditions

[0063] MRI: ID-PFG probe used, scan width (sw) 16.0 ppm, center frequency (O1P): 4.9 ppm;

[0064] Pulse sequence s1pul; Time domain data points (td): 32 K; Measurement temperature: 303 K; Delay time (dl): 20 s; Number of samplings (ns): 32; Window function (lh): 0.3 Hz.

[0065] ④ Conclusion

[0066] The sample isolated from Aspergillus niger xj cells was analyzed by EI-MS and NMR. EI-MS analysis showed it to be β-sitosterol, and NMR spectroscopy confirmed it as β-sitosterol. The results are shown in the table below. Figure 1 , Figure 2 , Figure 3 .

[0067] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments without departing from the technical essence of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for extracting β-sitosterol, comprising the following steps: (1) Preparation of crude extract After collecting spores of Aspergillus niger through fermentation with wheat bran, the spores are spray-dried to obtain mycelial powder for later use. The mycelial powder and acetone are mixed at a mass ratio of 1:5 and refluxed three times at a standard atmosphere and a temperature of 40-60℃ for 3 hours each time to obtain an Aspergillus niger spore powder extract. The extract is concentrated under reduced pressure to 1 / 10-1 / 5 of the total volume and then dissolved in an equal volume of water. The aqueous solution is then extracted with an equal volume of petroleum ether to obtain a petroleum ether extract. The extract is then concentrated under reduced pressure until the petroleum ether evaporates to dryness to obtain a crude extract. (2) Silica gel column chromatography The crude extract was dissolved in an equal volume of ethyl acetate, then mixed with 40-80 mesh silica gel at a volume ratio of 1:1.

2. The ethyl acetate was evaporated in a water bath, and the sample was then loaded onto a prepared 200-300 mesh silica gel column for elution at atmospheric pressure. The elution gradient was 100% petroleum ether, and petroleum ether:acetone ratios of 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, and 1:1, with equal proportions of 100% acetone. The flow rate was 10-20 ml / min. Thin-layer chromatography was performed, observing the color development in 5% concentrated sulfuric acid ethanol solution and 0.8% phosphomolybdic acid ethanol solution. Fractions with similar spot formation and Rf values ​​were combined and concentrated under reduced pressure until the organic solvent evaporated, yielding different concentrated solutions. (3) Second silica gel column chromatography Collect the eluent of the petroleum ether:acetone = 10:1-2:1 system, concentrate under reduced pressure until the organic solvent evaporates, dissolve the concentrate in ethyl acetate, mix with 40-mesh silica gel at a volume ratio of 1:1.2, and simultaneously evaporate the solvent in a water bath. Load the sample onto a prepared silica gel column for elution at atmospheric pressure; the flow rate is 20-25 ml / min, and the elution system is petroleum ether:ethyl acetate = 10:

1. Analyze the results by thin-layer chromatography, observing the color development of 5% concentrated sulfuric acid ethanol solution and 0.8% phosphomolybdic acid ethanol solution. Select fractions with the same spot characteristics and Rf values, combine them, concentrate under reduced pressure until the organic solvent evaporates, and obtain different concentrated fractions. (4) Second silica gel column chromatography Based on the analysis results of each concentrated component obtained in (3) on thin-layer chromatography, the fraction with an Rf value of 0.3-0.5 under the development condition of petroleum ether: ethyl acetate = 5:1 was selected for further silica gel column chromatography. After dissolving the concentrate in ethyl acetate, it was mixed with 40-mesh silica gel at a volume ratio of 1:1.

2. At the same time, the solvent was evaporated in a water bath and the sample was loaded onto the prepared silica gel column for elution at atmospheric pressure. The flow rate was 20-25 ml / min and the elution system was petroleum ether: ethyl acetate = 6:

1. According to the thin-layer chromatography analysis, the color development of 5% concentrated sulfuric acid ethanol solution and 0.8% phosphomolybdic acid ethanol solution was observed. The fractions with the same spot formation and Rf value were selected and combined, and concentrated under reduced pressure until the organic solvent was evaporated. (5) recrystallization Fractions with Rf values ​​of 0.3-0.5 under the development condition of petroleum ether: ethyl acetate = 5:1 were selected and recrystallized in a mixed solvent of ethyl acetate: methanol = 1:

50. The collected crystals were β-sitosterol. The Aspergillus niger mentioned is Aspergillus niger xj, with accession number: CCTCC NO: M206021.

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