CN110615824A - Method for separating triterpene alcohol and sterol from crude sterol of rice bran - Google Patents
Method for separating triterpene alcohol and sterol from crude sterol of rice bran Download PDFInfo
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Abstract
A method for separating triterpene alcohol and sterol from crude rice bran sterol relates to crude rice bran sterol. 1) Mixing the crude sterol of the rice bran with alcohol and alkali to obtain a mixed solution A, and performing saponification reaction to obtain a saponified solution B; 2) evaporating the saponified solution B, adding water after evaporation and concentration, extracting with an organic solvent, and combining organic solvent layers to obtain an extract liquid; 3) adding acid into the extract, neutralizing the pH value, washing with water, spin-drying, and recovering the organic solvent layer to obtain unsaponifiable matter C; 4) separating the unsaponifiable C obtained in the step 3) by passing through a silica gel column, eluting by using a mobile phase, collecting an eluent, detecting by using a thin layer, combining a sterol eluent and a triterpenoid alcohol eluent, and performing spin drying to obtain the sterol and the triterpenoid alcohol. The sterol and the triterpene alcohol can be directly separated from the waste rice bran crude sterol, so that the waste utilization is realized, and the pollution of the rice bran crude sterol solid waste to the environment is avoided; the method has the advantages of few process steps, realization of industrialization, mild operation conditions, low solvent toxicity, high product yield and high product purity of 99 percent.
Description
Technical Field
The invention relates to crude rice bran sterol, in particular to a method for separating triterpene alcohol and sterol from crude rice bran sterol.
Background
With the increasing importance of people on health, the triterpene and the phytosterol have increasingly large effects in the fields of food, medicine, health care, feed addition, beauty treatment, skin care and the like. The triterpene is a terpenoid compound with a basic mother nucleus composed of 30 carbon atoms, and has effects of reducing cholesterol, caring skin, protecting liver, resisting tumor, regulating immunity, resisting inflammation, relieving pain, tranquilizing mind, resisting aging, and resisting oxidation. The sterol is a sterol compound which is widely distributed in the nature, has good anti-inflammatory and antioxidant effects, can inhibit the absorption of human body to cholesterol, prevent and treat coronary atherosclerotic heart disease, promote wound healing, promote muscle proliferation, enhance capillary circulation, maintain skin surface moisture, promote skin metabolism, can be used as production raw materials of important steroid medicines and vitamin D3, and has the effects of hair growth, hair nourishing and the like.
At present, triterpenes are mainly and directly obtained from plant precursors or fungi such as ganoderma lucidum, antrodia camphorata and the like, and are also directly obtained by hydrolysis of oryzanol. For example, in the Chinese patent CN109364119A, triterpenoids are obtained from cyclocarya paliurus leaves through alcohol extraction, polyamide column separation and macroporous resin column separation, but the cost is high, the yield is low and the operation is complex; for example, Chinese patent CN109265510A obtains triterpene from coarse powder of balsam pear leaf by steps of extraction, gradient elution, semi-preparative liquid chromatography purification and the like, but the method is difficult to industrialize; for example, Chinese patent CN1261448C is used for extracting ganoderma triterpenes from ganoderma by ultrasonic circulating extraction, chloroform dissolution, ultrasonic circulating alkali extraction, hydrochloric acid acidification, ultrasonic circulating extraction and reduced pressure evaporation, but the method has complex process and high solvent toxicity; for example, Chinese patent CN103080124A is to obtain triterpene alcohol from gamma-oryzanol through alkaline hydrolysis, extraction and cooling, and the triterpene alcohol of the invention is obtained by chemical reaction of gamma-oryzanol. Triterpene alcohol is also obtained from rice bran, for example, chinese patent CN106102746A is prepared by extracting rice, rice bran and its extract, vegetable oil, γ -oryzanol, etc. with ethanol, hydrolyzing, recrystallizing and purifying, and the triterpene alcohol is separated by silica gel chromatography and ODS chromatography to obtain two types of triterpenes, but on one hand, the method does not realize the separation of sterol and triterpene alcohol; on the other hand, the rice bran extract of the method can be directly used as a product, and the raw material has economic value.
The phytosterol is mainly obtained from various vegetable oil deodorized distillates through separation, extraction, recrystallization, purification and other procedures, and the operation is simple but the extraction rate is low. For example, Chinese patent CN109422792A uses deodorized distillate of vegetable oil to obtain sterol by esterification, crystallization and recrystallization. The yields of vegetable oil deodorizer distillates are limited, require large imports, and have very low sterol content. At present, sterol is obtained from by-products of vegetable oil refining, for example, Chinese patent CN102898491A uses rice oil alkali to refine Chinese honeylocust fruit, and the phytosterol is obtained by ether extraction, freezing crystallization, alcohol dissolving and activated carbon decoloration. For example, Chinese patent CN101643495A uses fatty acid or fatty acid methyl ester to distill black feet to obtain sterol through saponification, calcium saponification, extraction, recrystallization and other processes, but these inventions can not realize enrichment of triterpene alcohol while obtaining sterol.
The rice bran oil is oil obtained from rice bran which is a byproduct of rice processing, and the raw material of the rice bran oil is very rich in China. The rice bran oil contains dozens of natural bioactive components such as abundant B vitamins, vitamin E, ferulic acid, squalene, oryzanol, sterol, triterpene alcohol and the like, and is oil beneficial to patients with hyperlipidemia and cardiovascular and cerebrovascular diseases. At present, rice bran oil, oryzanol, ferulic acid, sterol, rice bran wax, fatty acid and the like are obtained from rice bran crude oil in factories through processes such as degumming, decoloring, deacidification and the like or through methods such as multi-stage molecular distillation and the like. The waste rice bran crude sterol is generated when the oryzanol, the rice bran wax and the fatty acid are extracted, and the rice bran crude sterol contains rich effective components such as phytosterol, triterpene alcohol and the like. According to investigation, the sources of the crude rice bran sterols are very rich, about 500 tons of waste crude rice bran sterols are generated in a rice bran oil plant which produces 10 ten thousand tons each year, and due to technical limitation, the crude rice bran sterols are not utilized at present, so that precious crude rice bran sterol resources are idle or discarded as solid wastes, and environmental pollution and huge resource waste are caused. As the function of the rice bran oil is known more and more, the sales volume of the rice bran oil is wider and wider, and the quantity of the waste rice bran crude sterol produced in the production process is more and more, so that how to realize the waste recycling is very necessary for the utilization of the rice bran crude sterol.
Disclosure of Invention
The invention aims to separate high-purity triterpene alcohol and sterol from crude sterol of rice bran; the invention can directly separate two useful substances from waste rice bran crude sterol: the sterol and the triterpene alcohol realize the waste utilization, and avoid the pollution of the crude sterol solid waste of the rice bran to the environment; the method has the advantages of few process steps, realization of industrialization, mild operation conditions, low solvent toxicity, high product yield and high product purity of 99 percent.
The invention comprises the following steps:
1) mixing the crude sterol of the rice bran with alcohol and alkali to obtain a mixed solution A, and performing saponification reaction to obtain a saponified solution B;
2) evaporating the saponified solution B, adding water after evaporation and concentration, extracting with an organic solvent, and combining organic solvent layers to obtain an extract liquid;
3) adding acid into the extract, neutralizing the pH value, washing with water, spin-drying, and recovering the organic solvent layer to obtain unsaponifiable matter C;
4) separating the unsaponifiable C obtained in the step 3) by passing through a silica gel column, eluting by using a mobile phase, collecting an eluent, detecting by using a thin layer, combining a sterol eluent and a triterpenoid alcohol eluent, and performing spin drying to obtain the sterol and the triterpenoid alcohol.
In the step 1), a rice bran oil layer and an organic solvent layer containing fatty acid, sterol, oryzanol and the like are obtained by separating rice bran crude oil, and the waste obtained after the oryzanol, rice bran wax and fatty acid are separated by the organic solvent layer is rice bran crude sterol; the alcohol can be selected from at least one of ethanol or methanol; the alkali can be at least one of sodium hydroxide, potassium hydroxide and the like; the ratio of the alkali to the crude rice bran sterol to the alcohol can be 1 g: 1.2-5 g: 50-1000 ml, wherein the alkali and the crude rice bran sterol are calculated by mass, and the alcohol is calculated by volume; the saponification reaction can be normal-temperature saponification or thermal saponification; the specific method for normal-temperature saponification comprises the following steps: adding alcohol and alkali into the mixed solution A, performing ultrasonic dispersion for 10-30 min under the conditions that the power is 250-300W and the Hertz is 20-50 KHz, and then standing at normal temperature for 12-48 h to obtain a saponified solution; the specific method of the thermal saponification can be as follows: adding alcohol and alkali into the mixed solution A, ultrasonically dispersing for 10-30 min at the power of 250-300W and the Hertz of 20-50 KHz, and then performing reflux saponification for 2-6 h at the temperature of 60-80 ℃ to obtain saponified solution B;
in the step 2), the evaporation can be carried out at 30-45 ℃ until half of the original volume; the volume ratio of the water to the saponified solution after evaporation concentration can be (0.8-100): 1; the organic solvent can be at least one selected from ethyl acetate, diethyl ether, petroleum ether, n-hexane, etc.; the extraction may be at least once.
In the step 3), the acid may be selected from dilute hydrochloric acid or dilute sulfuric acid, the mass percentage of the dilute hydrochloric acid may be 10% to 35%, and the mass percentage of the dilute sulfuric acid may be 10% to 80%.
In the step 4), the filler for the column passing can be 100-300 meshes of silica gel; the elution can be carried out by adopting a mixed solvent of ethyl acetate and petroleum ether or a mixed solvent of diethyl ether and petroleum ether, and the flow rate is 1 drop/s; the volume ratio of the ethyl acetate to the petroleum ether can be 1: 1-8, and the volume ratio of the diethyl ether to the petroleum ether can be 1: 1-8;
in the step 4), the silica gel column can also adopt a regenerated silica gel column; the regenerated silica gel column can be obtained by regeneration treatment of the used silica gel column; the concrete method for regenerating the silica gel column can be as follows: eluting the used silica gel column with a high-polarity solvent, and then eluting and balancing with a mobile phase to obtain a regenerated silica gel column; the high-polarity solvent can be pure ethyl acetate and pure ether solvent with 2-10 times of column volume; the mobile phase elution can adopt a mixed solvent of ethyl acetate and petroleum ether or a mixed solvent of diethyl ether and petroleum ether with the volume of 2-10 times of that of a column, the volume ratio of the ethyl acetate to the petroleum ether can be 1: 1-8, and the volume ratio of the diethyl ether to the petroleum ether can be 1: 1-8; the used silica gel column can be repeatedly regenerated for 3 times, and still can achieve a good separation effect.
Compared with the prior art, the invention has the following beneficial effects:
1) the invention provides a production process for directly separating crude sterol from rice bran to obtain triterpene alcohol and sterol.
2) Compared with the traditional sterol separation process, the process provided by the invention directly adopts the waste rice bran crude sterol obtained in the rice bran oil extraction process, so that the solid waste treatment problem of the waste rice bran crude sterol is solved, the waste recycling is realized, and considerable economic benefits are created.
3) The invention can separate triterpene alcohol and obtain refined sterol. Because the phytosterol and the triterpene are synthesized by a biological pathway of methyl glutaric dihydroxy acid, the structures are similar, and the phytosterol and the triterpene are both mixtures with similar physicochemical properties; in addition, phytosterol and triterpene alcohol can remain in a silica gel column in the separation process, and are difficult to completely elute and collect; meanwhile, impurities in the crude sterol of the rice bran are difficult to separate from sterol and triterpene alcohol, so that the sterol and triterpene alcohol are difficult to separate. In the invention, the content of sterol in the used rice bran crude sterol is 22.09%, the content of triterpene alcohol is 77.60%, the content of impurities is 0.31%, the purity of the separated sterol is up to 99%, and the yield is up to 27%; the purity of the triterpene alcohol is up to 99 percent, and the yield is up to 55 percent.
4) The invention also carries out regeneration treatment on the silica gel column and researches the separation effect after the regeneration of the silica gel column, and finds that the silica gel column can still achieve better separation effect after being repeatedly regenerated for 3 times, thereby saving the silica gel cost.
Drawings
FIG. 1 is a GC-MS graph of crude sterols from rice bran of example 1.
FIG. 2 is a GC-MS graph of sterols separated from crude rice bran sterols from a single use of silica gel in example 1.
FIG. 3 is mass spectral information of peak 1 in the GC-MS graph of sterols.
FIG. 4 is mass spectral information of sterol molecule 1 similar to peak 1 searched in GC-MS library.
FIG. 5 is mass spectral information of peak 2 in the GC-MS graph of sterols.
FIG. 6 is mass spectral information of sterol molecule 2 similar to peak 2 searched in GC-MS library.
FIG. 7 is a GC-MS graph of triterpene alcohol separated from crude sterol from rice bran when silica gel was used once in example 1.
FIG. 8 is mass spectrum information of peak 3 in the GC-MS graph of triterpene alcohol.
FIG. 9 shows mass spectra information of triterpene alcohol molecules 3 similar to peak 3 searched for in GC-MS library.
FIG. 10 is mass spectrum information of peak 4 in the GC-MS graph of triterpene alcohol.
FIG. 11 is a graph showing the mass spectrum information of triterpene alcohol molecule 4 similar to peak 4 searched for in the GC-MS library.
FIG. 12 is mass spectrum information of peak 5 in the GC-MS graph of triterpene alcohol.
FIG. 13 is a graph showing mass spectral information of triterpene alcohol molecules 5 similar to peak 5 searched for in a GC-MS library.
FIG. 14 is mass spectrum information of peak 6 in the GC-MS graph of triterpene alcohol.
FIG. 15 is a graph showing mass spectral information of triterpene alcohol molecules 6 similar to peak 6 searched for in a GC-MS library.
Comparing the mass spectrum signal of each peak with the library search result, it can be seen that peaks 1 and 2 are peaks of sterol substances, and peaks 3, 4, 5 and 6 are peaks of triterpene alcohol substances. As is clear from FIGS. 1, 2 and 7, the present invention can separate high-purity sterols and triterpene alcohols from crude rice bran sterols.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below.
The rice bran crude sterol used in the embodiment of the invention is waste obtained after rice bran oil, oryzanol, rice bran wax and fatty acid are extracted from rice bran crude oil in the refining process of the rice bran oil. Wherein, the content of sterol is 22.09 percent, the content of triterpenoid alcohol is 77.60 percent, and the content of other substances is 0.31 percent; the silica gel used in the examples of the present invention was purchased from Shanghai Tantake Technique, Inc.; the raw materials or chemical agents used in the present invention are obtained by ordinary commercial means unless otherwise specified.
In the embodiment of the invention, the content of each substance is detected by adopting a gas chromatography-mass spectrometer.
Example 1: a method for separating sterol and triterpene alcohol from crude sterol from rice bran comprises the following steps:
1) saponification: mixing 1g crude sterol from testa oryzae with 100ml ethanol and 1.4g potassium hydroxide, performing ultrasonic decomposition at 40KHZ for 10mi, placing into a flask, and heating under reflux under nitrogen protection for about 6 hr to obtain saponified solution.
2) And (3) extraction: after saponification, the reflux unit was immediately turned on and heating was continued to evaporate about half of the ethanol. Evaporating, concentrating, adding 100ml water, extracting with diethyl ether for several times, and mixing organic solvent layers to obtain extractive solution.
3) Neutralization and evaporation: adding 25% of dilute hydrochloric acid by mass into the extract liquor to make the pH value of 7 after neutralization, washing the mixture with water for separating liquid, and evaporating and spin-drying to obtain an unsaponifiable substance C.
4) And (3) column chromatography separation: adsorbing the unsaponifiable C obtained in the step 3) on a silica gel column (the silica gel has the particle size of 300-400 meshes, the mass of 50g and the column diameter-height ratio of 10: 1) by a wet method, eluting by using a mobile phase of diethyl ether and petroleum ether (the volume ratio of 1: 4), collecting eluent, detecting by using a thin layer, combining sterol eluent and triterpene alcohol eluent, and distilling under reduced pressure to obtain the high-purity sterol and triterpene alcohol.
5) Silica gel column regeneration: eluting the used silica gel column with pure diethyl ether for about 3 column volumes, then eluting with diethyl ether/petroleum ether (volume ratio of 1: 4) for balancing about 3 column volumes, and soaking the silica gel column with the eluent overnight to obtain the regenerated silica gel column.
6) And (3) column chromatography separation of the regenerated silica gel column: and (3) loading the obtained unsaponifiable matter C on a regenerated silica gel column, eluting with an ethyl ether-petroleum ether (volume ratio of 1: 4), collecting the eluate, detecting by a thin layer, mixing the sterol eluate and the triterpene alcohol eluate, and concentrating to obtain the high-purity sterol and triterpene alcohol.
The purity of sterol and triterpene alcohol obtained in example 1 was determined by gas chromatography-mass spectrometry. The sterol purity, triterpenoid alcohol purity determination results and library retrieval results obtained when the silica gel is used once are shown in the attached drawing of the specification.
FIG. 1 shows a GC-MS chart of crude rice bran sterol of example 1, which shows that the crude rice bran sterol contains 22.09% of sterol, 77.60% of triterpene alcohol and 0.31% of impurities.
FIG. 2 shows a GC-MS graph of sterols separated from crude rice bran sterols from a single use of silica gel in example 1, indicating that the sterols had a purity of 99.26%.
FIG. 3 gives mass spectral information of peak 1 in the GC-MS plot of sterols.
FIG. 4 shows mass spectral information of sterol molecule 1 similar to peak 1 searched in GC-MS library.
FIG. 5 gives mass spectral information of peak 2 in the GC-MS plot of sterols.
FIG. 6 shows mass spectral information of sterol molecule 2 similar to peak 2 searched in GC-MS library.
FIG. 7 shows a GC-MS graph of triterpene alcohol separated from crude sterol from rice bran in example 1 when silica gel was used once, indicating that the triterpene alcohol was 99.35% pure.
FIG. 8 gives the mass spectral information of peak 3 in the GC-MS graph of triterpene alcohol.
FIG. 9 shows mass spectral information of triterpene alcohol molecules 3 similar to peak 3 searched in GC-MS library.
FIG. 10 gives the mass spectral information of peak 4 in the GC-MS graph of triterpene alcohol.
FIG. 11 shows mass spectral information of triterpene alcohol molecules 4 similar to peak 4 searched in GC-MS library.
FIG. 12 gives the mass spectral information of peak 5 in the GC-MS graph of triterpene alcohol.
FIG. 13 shows mass spectral information of triterpene alcohol molecules 5 similar to peak 5 searched in GC-MS library.
FIG. 14 gives the mass spectral information of peak 6 in the GC-MS graph of triterpene alcohol.
FIG. 15 shows mass spectral information of triterpene alcohol molecules 6 similar to peak 6 searched in GC-MS library.
Comparing the mass spectrum signal of each peak with the library search result, it can be seen that peaks 1 and 2 are peaks of sterol substances, and peaks 3, 4, 5 and 6 are peaks of triterpene alcohol substances. As is clear from FIGS. 1, 2 and 7, the present invention can separate high-purity sterols and triterpene alcohols from crude rice bran sterols.
The results of the purity and yield measurements of sterols and triterpene alcohols obtained in example 1 are shown in Table 1.
TABLE 1
Example 2: a method for separating sterol and triterpene alcohol from crude sterol from rice bran comprises the following steps:
1) saponification: mixing 1g crude sterol from testa oryzae with 100ml ethanol and 1.4g potassium hydroxide, performing ultrasonic decomposition at 40KHz for 10min, and standing at room temperature for about 24 hr to obtain saponified solution.
2) And (3) extraction: after saponification is complete, the saponified solution is heated to evaporate about half of the ethanol. Evaporating, concentrating, adding 100ml water, extracting with ethyl acetate for several times, and mixing ethyl acetate layers to obtain extractive solution.
3) Neutralization and evaporation: adding 25% of dilute hydrochloric acid by mass into the extract liquor to make the pH value of 7 after neutralization, washing the mixture with water for separating liquid, and evaporating and spin-drying to obtain an unsaponifiable substance C.
4) And (3) column chromatography separation: adsorbing the unsaponifiable C obtained in the step 3) on a silica gel column (the silica gel has the particle size of 300-400 meshes, the mass of 50g and the column diameter-height ratio of 10: 1) by a wet method, eluting by using a mobile phase with the volume ratio of ethyl acetate to petroleum ether of 3: 7, collecting eluent at the flow rate of 1 drop/s, detecting by using a thin layer, combining sterol eluent and triterpene alcohol eluent, and distilling under reduced pressure to obtain the high-purity sterol and triterpene alcohol.
5) Silica gel column regeneration: eluting the used silica gel column with pure ethyl acetate for about 3 column volumes, then eluting with ethyl acetate/petroleum ether at a volume ratio of 3: 7 for about 3 column volumes, and soaking the silica gel column with the eluent overnight to obtain the regenerated silica gel column.
6) And (3) column chromatography separation of the regenerated silica gel column: and (3) loading the obtained unsaponifiable matter C on a regenerated silica gel column, eluting with ethyl acetate-petroleum ether at a volume ratio of 3: 7, collecting the eluent, detecting by a thin layer, combining the sterol eluent and the triterpenoid alcohol eluent, and concentrating to obtain the high-purity sterol and triterpenoid alcohol.
The results of measuring the purity and yield of sterol and triterpene alcohol obtained in example 2 are shown in Table 2, and the purity was measured by the same gas chromatography-mass spectrometry as in example 1.
TABLE 2
Example 3: a method for separating sterol and triterpene alcohol from crude sterol from rice bran comprises the following steps:
1) saponification: mixing 5g crude sterol from testa oryzae with 500ml ethanol and 9g potassium hydroxide, performing ultrasonic decomposition at 40KHz for 30min, and standing at room temperature for about 24 hr to obtain saponified solution.
2) And (3) extraction: after saponification is complete, the saponified solution is heated to evaporate about half of the ethanol. Evaporating, concentrating, adding 300ml water, extracting with ethyl acetate for several times, and mixing ethyl acetate layers to obtain extractive solution.
3) Neutralization and evaporation: adding 25% of dilute hydrochloric acid by mass into the extract liquor to make the pH value of 7 after neutralization, washing the mixture with water for separating liquid, and evaporating and spin-drying to obtain an unsaponifiable substance C.
4) And (3) column chromatography separation: adsorbing the unsaponifiable C obtained in the step 3) on a silica gel column (the silica gel has the particle size of 300-400 meshes, the mass of 200g and the column diameter-height ratio of 3: 2) by a wet method, eluting by using a mobile phase with the volume ratio of ethyl acetate to petroleum ether of 3: 7, collecting eluent at the flow rate of 1 drop/s, detecting by using a thin layer, combining sterol eluent and triterpene alcohol eluent, and distilling under reduced pressure to obtain the high-purity sterol and triterpene alcohol.
5) Silica gel column regeneration: eluting the used silica gel column with pure ethyl acetate for about 3 column volumes, then eluting with ethyl acetate/petroleum ether at a volume ratio of 3: 7 for about 3 column volumes, and soaking the silica gel column with the eluent overnight to obtain the regenerated silica gel column.
6) And (3) column chromatography separation of the regenerated silica gel column: and (3) loading the obtained unsaponifiable matter C on a regenerated silica gel column, eluting with ethyl acetate-petroleum ether at a volume ratio of 3: 7, collecting the eluent, detecting by a thin layer, combining the sterol eluent and the triterpenoid alcohol eluent, and concentrating to obtain the high-purity sterol and triterpenoid alcohol.
The results of purity and yield measurement of sterols and triterpene alcohols obtained in example 3 are shown in Table 3, and the purity was measured by the gas chromatography-mass spectrometry method in the same manner as in example 1.
TABLE 3
Example 4: a method for separating sterol and triterpene alcohol from crude sterol from rice bran comprises the following steps:
1) saponification: mixing 5g crude rice bran sterol with 500ml ethanol and 9g potassium hydroxide, performing ultrasonic decomposition at 40KHz for 30min, placing into a flask, and heating and refluxing under nitrogen protection for about 6h to obtain saponified solution.
2) And (3) extraction: after saponification, the reflux unit was immediately turned on and heating was continued to evaporate about half of the ethanol. Evaporating, concentrating, adding 300ml water, extracting with diethyl ether for several times, and mixing organic solvent layers to obtain extractive solution.
3) Neutralization and evaporation: adding 25% of dilute hydrochloric acid by mass into the extract liquor to make the pH value of 7 after neutralization, washing the mixture with water for separating liquid, and evaporating and spin-drying to obtain an unsaponifiable substance C.
4) And (3) column chromatography separation: adsorbing the unsaponifiable C obtained in the step (1) on a silica gel column (the silica gel has the particle size of 300-400 meshes, the mass of 200g and the column diameter-height ratio of 3: 2) by a wet method, eluting by using a mobile phase with the volume ratio of diethyl ether to petroleum ether of 1: 4, collecting eluent at the flow rate of 1 drop/s, detecting by using a thin layer, combining sterol eluent and triterpene alcohol eluent, and distilling under reduced pressure to obtain the high-purity sterol and triterpene alcohol.
5) Silica gel column regeneration: eluting the used silica gel column with pure ether for about 3 column volumes, then eluting with ether-petroleum ether at a volume ratio of 1: 4 for about 3 column volumes, and soaking the silica gel column with the eluent overnight to obtain the regenerated silica gel column.
6) And (3) column chromatography separation of the regenerated silica gel column: and (3) loading the obtained unsaponifiable matter C on a regenerated silica gel column, eluting with diethyl ether-petroleum ether at a volume ratio of 1: 4, collecting the eluent, detecting by a thin layer, combining the sterol eluent and the triterpenoid alcohol eluent, and concentrating to obtain the high-purity sterol and triterpenoid alcohol.
The results of purity and yield measurements of sterols and triterpene alcohols obtained in example 4 are shown in Table 4, and the purity measurements were carried out by the same gas chromatography-mass spectrometry as in example 1.
TABLE 4 determination of purity and yield of sterols and triterpene alcohols
According to the research on the determination of sterol compounds in Syngnathus and Starfish by GC-MS, a gas chromatography-mass spectrometry method is used for determining sterol substances, and according to the research on the determination of the content of triterpenoid compound friedelin in caulis bambusae by gas chromatography, the method is used for determining triterpenoids by using the gas chromatography-mass spectrometry method.
Experiments prove that while the triterpene alcohol is separated, the refined sterol can be obtained. Because the phytosterol and the triterpene are synthesized by a biological pathway of methyl glutaric dihydroxy acid, the structures are similar, and the phytosterol and the triterpene are both mixtures with similar physicochemical properties; in addition, phytosterol and triterpene alcohol can remain in a silica gel column in the separation process, and are difficult to completely elute and collect; meanwhile, impurities in the crude sterol of the rice bran are difficult to separate from sterol and triterpene alcohol, so that the sterol and triterpene alcohol are difficult to separate. In the invention, the content of sterol in the used rice bran crude sterol is 22.09%, the content of triterpene alcohol is 77.60%, the content of impurities is 0.31%, the purity of the separated sterol is up to 99%, and the yield is up to 27%; the purity of the triterpene alcohol is up to 99 percent, and the yield is up to 55 percent.
The method adopts the waste rice bran crude sterol in the rice bran oil refining process as the raw material, realizes the waste recycling, and avoids the environment pollution caused by the waste rice bran crude sterol; the method is green and environment-friendly, and has simple process; the method can obtain high-purity triterpene alcohol and high-purity phytosterol, and the sterol and triterpene alcohol can be applied in medicine, health product, cosmetic, feed additive, etc.; the invention also researches that the silica gel column still has good separation effect after being repeatedly used for three times, thereby saving the silica gel cost.
The foregoing is merely an illustrative embodiment of the present invention, and any equivalent changes and modifications made by those skilled in the art without departing from the spirit and principle of the present invention should fall within the protection scope of the present invention.
Claims (10)
1. A method for separating triterpene alcohol and sterol from crude sterol of rice bran is characterized by comprising the following steps:
1) mixing the crude sterol of the rice bran with alcohol and alkali to obtain a mixed solution A, and performing saponification reaction to obtain a saponified solution B;
2) evaporating the saponified solution B, adding water after evaporation and concentration, extracting with an organic solvent, and combining organic solvent layers to obtain an extract liquid;
3) adding acid into the extract, neutralizing the pH value, washing with water, spin-drying, and recovering the organic solvent layer to obtain unsaponifiable matter C;
4) separating the unsaponifiable C obtained in the step 3) by passing through a silica gel column, eluting by using a mobile phase, collecting an eluent, detecting by using a thin layer, combining a sterol eluent and a triterpenoid alcohol eluent, and performing spin drying to obtain the sterol and the triterpenoid alcohol.
2. The method for separating triterpene alcohol and sterol from crude rice bran sterol according to claim 1, wherein in step 1), a rice bran oil layer and an organic solvent layer containing fatty acid, sterol, oryzanol and the like are separated from crude rice bran oil, and the waste obtained after the separation of oryzanol, rice bran wax and fatty acid from the organic solvent layer is crude rice bran sterol.
3. The method for separating triterpene alcohols and sterols from rice bran crude sterols according to claim 1, wherein in step 1), said alcohols are selected from at least one of ethanol and methanol; the alkali is at least one of sodium hydroxide and potassium hydroxide; the ratio of the alkali to the crude rice bran sterol to the alcohol can be 1 g: 1.2-5 g: 50-1000 ml, wherein the alkali and the crude rice bran sterol are calculated by mass, and the alcohol is calculated by volume.
4. The method for separating triterpene alcohol and sterol from crude sterol from rice bran according to claim 1, wherein in step 1), the saponification reaction is selected from normal temperature saponification or thermal saponification; the specific method for normal-temperature saponification comprises the following steps: adding alcohol and alkali into the mixed solution A, performing ultrasonic dispersion for 10-30 min under the conditions that the power is 250-300W and the Hertz is 20-50 KHz, and then standing at normal temperature for 12-48 h to obtain a saponified solution; the specific method of the thermal saponification can be as follows: adding alcohol and alkali into the mixed solution A, performing ultrasonic dispersion for 10-30 min at the power of 250-300W and the Hertz of 20-50 KHz, and then performing reflux saponification for 2-6 h at the temperature of 60-80 ℃ to obtain saponified solution B.
5. The method for separating triterpene alcohol and sterol from crude sterol from rice bran according to claim 1, wherein in step 2), the evaporation is performed at 30-45 ℃ to half of the original volume; the volume ratio of the water to the saponified solution after evaporation and concentration can be (0.8-100): 1.
6. The method for separating triterpene alcohol and sterol from crude sterol from rice bran according to claim 1, wherein in the step 2), the organic solvent is at least one selected from the group consisting of ethyl acetate, diethyl ether, petroleum ether and n-hexane; the extraction may be at least once.
7. The method for separating triterpene alcohol and sterol from crude sterol from rice bran according to claim 1, wherein in step 3), the acid is selected from diluted hydrochloric acid or diluted sulfuric acid, the diluted hydrochloric acid is 10-35% by mass, and the diluted sulfuric acid is 10-80% by mass.
8. The method for separating triterpene alcohol and sterol from crude sterol from rice bran according to claim 1, wherein in step 4), the filler for column chromatography is silica gel of 100-300 mesh; the elution can be carried out by adopting a mixed solvent of ethyl acetate and petroleum ether or a mixed solvent of diethyl ether and petroleum ether, and the flow rate is 1 drop/s; the volume ratio of the ethyl acetate to the petroleum ether can be 1: 1-8, and the volume ratio of the diethyl ether to the petroleum ether can be 1: 1-8.
9. The method for separating triterpene alcohols and sterols from crude sterols from rice bran according to claim 1, wherein in step 4), said silica gel column is a regenerated silica gel column; the regenerated silica gel column can be obtained by regeneration treatment of the used silica gel column; the concrete method for the regeneration treatment of the silica gel column comprises the following steps: and eluting the used silica gel column with a high-polarity solvent, and then eluting and balancing with a mobile phase to obtain the regenerated silica gel column.
10. The method for separating triterpene alcohol and sterol from crude sterol from rice bran according to claim 9, wherein the high polar solvent is pure ethyl acetate and pure diethyl ether solvent with 2-10 column volumes; the mobile phase elution adopts a mixed solvent of ethyl acetate and petroleum ether or a mixed solvent of diethyl ether and petroleum ether with the volume 2-10 times of that of a column, the volume ratio of the ethyl acetate to the petroleum ether can be 1: 1-8, and the volume ratio of the diethyl ether to the petroleum ether can be 1: 1-8; the used silica gel column can be repeatedly regenerated for 3 times.
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