CN110872337B - Method for simultaneously preparing different high-purity triterpenic acids from rosemary - Google Patents
Method for simultaneously preparing different high-purity triterpenic acids from rosemary Download PDFInfo
- Publication number
- CN110872337B CN110872337B CN201911241475.9A CN201911241475A CN110872337B CN 110872337 B CN110872337 B CN 110872337B CN 201911241475 A CN201911241475 A CN 201911241475A CN 110872337 B CN110872337 B CN 110872337B
- Authority
- CN
- China
- Prior art keywords
- acid
- solution
- methanol
- rosemary
- solid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
- C07J63/008—Expansion of ring D by one atom, e.g. D homo steroids
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Steroid Compounds (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention discloses a method for simultaneously preparing different high-purity triterpenic acids from rosemary, which comprises the following steps: extracting dry leaves of rosemary by refluxing with an ethanol solution, and concentrating and recovering alcohol from an extracting solution to obtain a solid substance A; washing the solid substance A with an ethanol solution to obtain a solid substance B; extracting and degreasing the solid substance B by using petroleum ether or n-hexane to obtain a solid substance C; adding the solid matter C into an ethanol solution, adjusting the pH value to 10-12, adding active carbon, stirring and decoloring to obtain a filtrate; adjusting the pH value of the filtrate to 2-4 to obtain a solid substance D; and (3) adding the solid matter D into methanol for dissolving, separating by modified reverse phase silica gel column chromatography, eluting and separating by adopting a gradient acidic methanol solution with the acid content of 0.05%, concentrating, drying, dissolving by adopting a high-concentration alcohol solution, standing, crystallizing, separating and drying to obtain three products of betulinic acid, oleanolic acid and ursolic acid with single purity of more than 95%.
Description
Technical Field
The invention belongs to the technical field of natural product extraction, and relates to a method for simultaneously preparing three different high-purity triterpenic acids from rosemary.
Background
Rosemary is native to Europe, Africa and Mediterranean coast, is a Labiatae sage plant, namely evergreen shrub, and has strong antioxidation due to active ingredients, so that the rosemary is planted in provinces such as Hunan, Guangxi, Yunnan and the like in China in recent years. The herba Rosmarini officinalis extract mainly contains terpenoids such as betulinic acid, oleanolic acid, ursolic acid, carnosol, rosemary dialdehyde, epi-rosmanol methyl ether, carnosic acid, methyl carnosic acid, rosmanol, etc., flavonoids such as apigenin, hesperetin, homoeosin, genkwanin, etc., and phenolic acids such as vanillic acid, caffeic acid, rosmarinic acid, ferulic acid, etc.
Triterpenic acids are a generic term for a class of biologically active compounds. The rosemary extract contains various triterpenic acids, triterpenic acid phenols and triterpenic acid esters. Researches show that triterpenic acid has anticancer and mutation resisting effects, so that natural preparation methods of triterpenic acid are widely concerned.
Betulinic acid has the function of resisting the growth of tumor cells, and has no harm to normal cells; it has broad-spectrum cytotoxicity on tumor cells such as neuroblastoma, glioma, malignant brain tumor, ovarian cancer, lung cancer, etc., and has inhibitory effect on HIV-1 infection; this makes betulinic acid a promising drug for development.
Betulinic acid currently has three main sources: firstly, extracting and separating from natural plants; secondly, obtaining betulinic acid by organic synthesis by taking betulin as a precursor; and thirdly, generating betulinic acid by microbial transformation by taking betulin as a precursor. The last two kinds of white birch bark are extracted from white birch bark, which has limited yield due to limited raw material of white birch bark.
CN109180772A discloses a preparation method of high-purity betulinic acid, which comprises the steps of obtaining betulinic acid sodium salt through extract treatment, purifying the sodium salt, reacidifying and recrystallizing to obtain the high-purity betulinic acid product. CN109666058A discloses a betulinic acid extraction and separation method, which adopts water chestnut of chufa of Cyperaceae of Gramineae as raw material, and realizes the separation of betulinic acid in water chestnut peel by crushing, subcritical water extraction, alkali-soluble acid precipitation and recrystallization.
Ursolic acid has effects of protecting liver, resisting hepatitis, resisting tumor, enhancing immunity, resisting inflammation, inhibiting bacteria, resisting HIV, and reducing blood lipid. In recent years, it has been found that it has anticancer, anti-carcinogenic, F9-inducing teratoma cell differentiation and anti-angiogenic effects, and ursolic acid has an obvious antioxidant function, and thus it is widely used as a raw material for medicines and cosmetics.
CN107325150A discloses a method for extracting ligustrum lucidum ursolic acid by supercritical carbon dioxide, which adopts supercritical equipment for extraction to obtain an ursolic acid product. CN106117303A discloses a method for extracting ursolic acid from loquat leaves, which comprises using loquat leaves as raw material, and adopting water as solvent to extract and separate ursolic acid from loquat leaves under subcritical condition.
Oleanolic acid is used as a triploid substance, and CN109010346A discloses a new application of oleanolic acid, which shows that oleanolic acid has the effect of inhibiting skin inflammation, and proves that oleanolic acid can effectively inhibit the release of inflammatory factors so as to achieve the purpose of diminishing inflammation, and oleanolic acid can be used for preparing a skin inflammation inhibitor. CN109223805A discloses the application of oleanolic acid in the preparation of drugs for treating autoimmune diseases, which shows that oleanolic acid has obvious function of inhibiting the activity of Th17 cells and provides a lead structure for the research and development of new drugs for treating autoimmune diseases.
CN106336440A discloses a method for extracting and separating oleanolic acid from olive leaves, which comprises the steps of extracting by using an ethanol solution with the volume concentration of 70-90%, dissolving a crude extract by using an ethanol solution with the volume concentration of 50-65%, loading the solution onto an AB-8 macroporous adsorption resin column, eluting by using a sodium bicarbonate solution, adjusting the pH value to the desired value, filtering, and drying a filter cake to obtain a high-content oleanolic acid product.
CN109498686A discloses a method for preparing triterpenic acid in jujube material, which prepares total triterpenic acid with a content of more than 90 percent by a liquid chromatography separation means, wherein the main component is a mixed product of betulinic acid, ursolic acid and oleanolic acid. CN107468761A discloses a method for preparing ligustrum lucidum total triterpenic acid and the use thereof, the method mainly adopts solvent extraction, the triterpenic acid prepared by the method is a mixture of oleanolic acid and ursolic acid, and the content only reaches more than 70%.
In summary, either a single high-content product is prepared from raw materials through equipment, or because the raw materials all contain a plurality of different triterpenic acid substances, and the prepared triterpenic acid is a mixed product thereof, based on the demands of different single high-purity triterpenic acid substances in the fields of medicine, cosmetics and the like, a method for simultaneously preparing different high-purity triterpenic acids is necessary to be developed.
Disclosure of Invention
In order to solve the problem that the existing triterpenic acid preparation process is a single product or all obtained products are mixed products, the invention aims to provide a method for simultaneously preparing different high-purity triterpenic acids from rosemary.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for simultaneously preparing different high-purity triterpenic acids from rosemary comprises the following steps:
(1) extracting dry leaves of rosemary by refluxing with a solution with the ethanol concentration of 60-85%, concentrating the extracting solution, recovering the ethanol, and carrying out solid-liquid separation to obtain a solid substance A;
(2) washing the solid substance A with 60-85% ethanol solution, and performing solid-liquid separation to obtain a solid substance B;
(3) extracting and degreasing the solid substance B by using petroleum ether or n-hexane, and then carrying out solid-liquid separation to obtain a solid substance C;
(4) adding the solid matter C into 75-85% ethanol solution, adjusting pH to 10-12, adding active carbon, stirring, decolorizing, and performing solid-liquid separation to obtain filtrate;
(5) adjusting the pH of the filtrate to 2-4, and performing solid-liquid separation to obtain a solid substance D;
(6) adding the solid matter D into methanol for dissolving, separating by modified reverse phase silica gel column chromatography, eluting and separating by adopting a gradient acidic methanol solution with the acid content of 0.05 percent to respectively obtain an eluent I containing the betulinic acid, an eluent II containing the oleanolic acid and an eluent III containing the ursolic acid, concentrating and drying, and respectively adopting a high-concentration alcohol solution for dissolving, standing for crystallization, separating and drying to obtain three products of the single high-purity betulinic acid, the oleanolic acid and the ursolic acid.
The dry rosemary leaves are purchased dry leaves or obtained by distilling fresh rosemary, then separating and falling off the obtained rosemary leaves, and drying the obtained rosemary leaves.
Further, in the step (1), the reflux extraction times are 2 times, the dosage of the ethanol solution at the 1 st time is 10mL of the ethanol solution/1 g of the dry rosemary leaves, the dosage of the ethanol solution at the 2 nd time is 8mL of the ethanol solution/1 g of the dry rosemary leaves, and the extraction time is 2 hours each time.
Further, in the step (2), the solid substance A is added into 60% -85% ethanol solution to be stirred and dissolved for 2 times, and each time lasts for 1-2 hours.
Further, in the step (3), the liquid-solid ratio of extraction is 25-30mL/g, and the extraction times are 2-3.
Further, in the step (4), the using amount of the 75-85% ethanol solution is 25-30mL ethanol solution/1 g solid matter C, the temperature is 60-75 ℃, activated carbon with the weight of 0.1% of the dry leaves of the rosemary is added, and the stirring and decoloring time is 2-3 h.
Further, in the step (5), after the pH is adjusted to 2-4, standing is carried out for at least 5h under the condition of room temperature.
Further, in the step (6), in the modified reverse phase silica gel column chromatography, the modified silica gel resin is a modified C18 reverse phase silica gel resin of octadecyl bonded silica gel. The modified silica gel resin is a commercial product, is a modified C18 reversed-phase silica gel resin of octadecyl bonded silica gel, can be obtained by acidizing the silica gel resin, performing alkylation reaction by using octadecyl trichlorosilane, and finally performing end capping treatment by using trimethylchlorosilane.
Further, in the step (6), the gradient arrangement of the gradient acidic methanol solution with the acid content of 0.05% is sequentially 60% methanol aqueous solution, 75% methanol aqueous solution, 90% methanol aqueous solution and 100% methanol, the flow rate is 6-10mL/min, and the acid is hydrochloric acid, phosphoric acid or acetic acid; performing high performance liquid chromatography, wherein the eluent I containing betulinic acid is obtained by eluting with 75% methanol aqueous solution, the eluent II containing oleanolic acid is obtained by eluting with 90% methanol aqueous solution, and the eluent III containing ursolic acid is obtained by eluting with 100% methanol.
Further, in the step (6), the high-concentration alcohol is ethanol or methanol with the concentration of not less than 95%, and the dissolving temperature is 70-80 ℃.
Further, in the step (6), the purity of the betulinic acid, the oleanolic acid and the ursolic acid is not lower than 95%.
According to the invention, rosemary is used as a raw material, and three betulinic acid, oleanolic acid and ursolic acid products with the content of more than 95% can be simultaneously prepared through the synergistic effect of the process steps of extraction, washing, degreasing, decoloring, modified reverse phase silica gel column chromatography separation, alcohol solution refining and the like. The inventors have found that changing part of the process parameters (e.g. using unmodified C18 reverse phase silica gel resin, eluting with acid-free aqueous methanol solution, etc.) or reducing part of the steps (e.g. degreasing process, etc.) affects the yield and purity of the final product, whereas strictly according to the process of the present invention, under the synergistic effect of each condition and process step, three products with betulinic acid, oleanolic acid and ursolic acid contents of more than 95% can be prepared simultaneously. .
The invention discloses a preferable method for simultaneously preparing different high-purity triterpenic acids from rosemary, which specifically comprises the following steps:
(1) extracting rosemary dry leaves with a solution with the ethanol concentration of 60-85% for 2 times by refluxing, wherein the dosage of the ethanol solution for the 1 st time is 10mL of ethanol solution per 1g of rosemary dry leaves, the dosage of the ethanol solution for the 2 nd time is 8mL of ethanol solution per 1g of rosemary dry leaves, the extraction time is 2 hours each time, after the extracting solution is concentrated and the ethanol is recovered until no ethanol smell exists, cooling and standing for 4-6 hours, and carrying out solid-liquid separation to obtain a solid substance A;
(2) adding the solid substance A into 60-85% ethanol solution, stirring for dissolving for 2 times, each time for 1-2h, and performing solid-liquid separation to obtain a solid substance B;
(3) extracting the solid substance B with petroleum ether or n-hexane for 2-3 times according to a liquid-solid ratio of 25-30mL/g, degreasing, and carrying out solid-liquid separation to obtain a solid substance C;
(4) adding the solid matter C into 75-85% ethanol solution according to the liquid-solid ratio of 25-30mL/g, adjusting the pH to 10-12, adding 0.1% active carbon of the weight of dry leaves at 60-75 ℃, stirring for 2-3h, decolorizing, and performing solid-liquid separation to obtain filtrate;
(5) adjusting the pH of the filtrate to 2-4, standing at room temperature for at least 5h, and performing solid-liquid separation to obtain a solid substance D;
(6) dissolving the solid D in methanol at a ratio of 1:1, separating by modified reverse phase silica gel column chromatography, eluting with 0.05% acid gradient acidic methanol solution at a flow rate of 6-10mL/min, and analyzing by high performance liquid chromatography, wherein, the eluent I containing betulinic acid is obtained by eluting with 75% methanol aqueous solution, the eluent II containing oleanolic acid is obtained by eluting with 90% methanol aqueous solution, the eluent III containing ursolic acid is obtained by eluting with 100% methanol, and after concentration and drying, the three products of betulinic acid, oleanolic acid and ursolic acid with single purity not lower than 95% are respectively obtained by dissolving with more than 95% ethanol or methanol solution, standing for crystallization, separating and drying.
Compared with the prior art, the invention has the following technical effects:
compared with the existing triterpenic acid preparation process, the method has the advantages that a single product or a mixed product is obtained, rosemary is used as a raw material, and three betulinic acid, oleanolic acid and ursolic acid products with the content of more than 95% can be simultaneously prepared through the synergistic effect of the process steps of extraction, washing, degreasing, decoloring, modified reverse phase silica gel column chromatography separation, alcohol solution refining and the like.
Drawings
FIG. 1 is a schematic process flow diagram of a method for simultaneously preparing different high-purity triterpenic acids from rosemary according to the present invention.
FIG. 2 is a chromatogram for detecting three products obtained in example 1 of the present invention.
FIG. 3 is a chromatogram for detecting three products prepared in comparative example 1 of the present invention.
Detailed description of the invention
The present invention is further illustrated by the following examples, which are not intended to be limiting but are to be construed in an illustrative manner, and unless otherwise specified, the percentages are by volume.
Example 1
Weighing 500g of dry rosemary leaves, carrying out reflux extraction for 2h by using 5000ml of 60% ethanol solution for the first time, filtering, continuously carrying out reflux extraction on filter residues for 2h by using 4000ml of 60% ethanol solution, filtering, and combining the two extracting solutions; concentrating the extractive solution under vacuum to remove alcohol, concentrating at 70 deg.C, cooling, standing for 5 hr, vacuum filtering to obtain filter cake, and air drying at 60 deg.C for 4 hr to obtain solid A60.6 g;
adding the solid substance A into 900ml of 60% ethanol solution, stirring for 1h at normal temperature, performing suction filtration to obtain a filter cake, continuously adding the filter cake into 900ml of 60% ethanol solution, and stirring for 1h at normal temperature; filtering to obtain a filter cake, and drying by blowing at 60 ℃ to obtain 15.4g of solid matter B;
extracting the solid substance B with n-hexane for three times, wherein the dosage is 450ml each time, separating, and carrying out forced air drying at 60 ℃ for 4h to obtain a total solid substance C of 14.2 g;
adding the solid matter C into 350ml of 85% ethanol solution, adjusting the pH value to 12, stirring and dissolving at 75 ℃, adding 0.5g of activated carbon, stirring and decoloring for 3h, and filtering while hot to obtain filtrate;
adjusting the pH value of the filtrate to 3, standing for 6 hours at room temperature, filtering to obtain a filter cake, and drying the filter cake to obtain 10.8g of solid matter D;
the modified C18 reversed phase silica gel resin was soaked in methanol, degassed, and then packed in a column, and the two column volumes were washed with 60% methanol in water. Dissolving the solid matter D in methanol at a ratio of 1:1, separating by modified reverse phase silica gel column chromatography, and eluting by gradient elution with 0.05% acidic methanol aqueous solution as eluting solvent, wherein the gradient is 60% methanol aqueous solution, 75% methanol aqueous solution, 90% methanol aqueous solution, and 100% methanol; controlling the flow rate to be 6-10mL/min, controlling the dosage of the eluent to be 3 column volumes, and analyzing the eluent by adopting high performance liquid chromatography to obtain a separated target eluent, wherein an eluent I containing betulinic acid is obtained by adopting 75% methanol aqueous solution for elution, an eluent II containing oleanolic acid is obtained by adopting 90% methanol aqueous solution for elution, and an eluent III containing ursolic acid is obtained by adopting 100% methanol for elution;
concentrating and drying the eluent I, the eluent II and the eluent III respectively; heating and dissolving the dried substance with 95% methanol solution, cooling, and standing in a refrigerating chamber for 12h for crystallization;
filtering and separating the crystallization solution respectively to obtain crystals, and drying to obtain 1.4g of betulinic acid with the purity of 97.3 percent respectively; 0.5g of oleanolic acid, the purity of which is 96.5 percent; 3.5g of ursolic acid, and the purity is 96.8%.
Example 2
Weighing 500g of dry rosemary leaves, carrying out reflux extraction for 2h by using 5000ml of 70% ethanol solution for the first time, filtering, continuously carrying out reflux extraction on filter residues for 2h by using 4000ml of 70% ethanol solution, filtering, and combining the two extracting solutions; concentrating the extractive solution under vacuum to remove alcohol, concentrating at 70 deg.C, cooling, standing for 5 hr, vacuum filtering to obtain filter cake, and air drying at 60 deg.C for 4 hr to obtain solid substance A (59.5 g);
adding the solid substance A into 900ml of 65% ethanol solution, stirring for 1h at normal temperature, performing suction filtration to obtain a filter cake, continuously adding the filter cake into 900ml of 65% ethanol solution, and stirring for 1h at normal temperature; filtering to obtain a filter cake, and drying by blowing at 60 ℃ to obtain 15.0g of solid matter B;
extracting the solid substance B with n-hexane for three times, wherein the dosage is 450ml each time, separating, and carrying out forced air drying at 60 ℃ for 4h to obtain 13.6g of the solid substance C;
adding the solid matter C into 350ml of 85% ethanol solution, adjusting the pH value to 11.5, stirring and dissolving at 75 ℃, adding 0.5g of activated carbon, stirring and decoloring for 2.5h, and filtering while hot to obtain a filtrate;
adjusting the pH value of the filtrate to 2, standing for 6 hours at room temperature, filtering to obtain a filter cake, and drying the filter cake to obtain 10.2g of solid matter D;
the modified C18 reversed phase silica gel resin was soaked in methanol, degassed, and then packed in a column, and the two column volumes were washed with 60% methanol in water. Dissolving the solid matter D in methanol at a ratio of 1:1, separating by modified reverse phase silica gel column chromatography, and eluting by gradient elution with 0.1% acidic methanol aqueous solution as eluting solvent, wherein the gradient is 60% methanol aqueous solution, 75% methanol aqueous solution, 90% methanol aqueous solution, and 100% methanol; controlling the flow rate to be 6-10mL/min, controlling the dosage of the eluent to be 3 column volumes, and analyzing the eluent by adopting high performance liquid chromatography to obtain a separated target eluent, wherein an eluent I containing betulinic acid is obtained by adopting 75% methanol aqueous solution for elution, an eluent II containing oleanolic acid is obtained by adopting 90% methanol aqueous solution for elution, and an eluent III containing ursolic acid is obtained by adopting 100% methanol for elution;
concentrating and drying the eluent I, the eluent II and the eluent III respectively; heating and dissolving the dried substance with 95% methanol solution, cooling, and standing in a refrigerating chamber for 12h for crystallization;
filtering and separating the crystallization solution respectively to obtain crystals, and drying to obtain 1.3g of betulinic acid with the purity of 96.8%; 0.4g of oleanolic acid, the purity of which is 96.6 percent; 3.4g of ursolic acid, and the purity is 97.2%.
Example 3
Weighing 500g of dry rosemary leaves, carrying out reflux extraction for 2h by using 5000ml of 80% ethanol solution for the first time, filtering, continuously carrying out reflux extraction on filter residues for 2h by using 4000ml of 80% ethanol solution, filtering, and combining the two extracting solutions; concentrating the extractive solution under vacuum to remove alcohol, concentrating at 70 deg.C, cooling and standing for 5 hr to obtain filter cake, and air drying at 60 deg.C for 4 hr to obtain solid A (58.9 g);
adding the solid substance A into 900ml of 70% ethanol solution, stirring for 1h at normal temperature, performing suction filtration to obtain a filter cake, continuously adding the filter cake into 900ml of 70% ethanol solution, and stirring for 1h at normal temperature; filtering to obtain a filter cake, and drying by blowing at 60 ℃ to obtain 14.6g of solid matter B;
extracting the solid substance B with n-hexane for three times, wherein the dosage is 450ml each time, separating, and carrying out forced air drying at 60 ℃ for 4h to obtain 13.2g of the solid substance C;
adding the solid matter C into 350ml of 85% ethanol solution, adjusting the pH value to 11, stirring and dissolving at 75 ℃, adding 0.5g of activated carbon, stirring and decoloring for 2h, and filtering while hot to obtain filtrate;
adjusting the pH value of the filtrate to 4, standing for 6 hours at room temperature, filtering to obtain a filter cake, and drying the filter cake to obtain 9.6g of solid matter D;
the modified C18 reversed phase silica gel resin was soaked in methanol, degassed, and then packed in a column, and the two column volumes were washed with 60% methanol in water. Dissolving the solid matter D in methanol at a ratio of 1:1, separating by modified reverse phase silica gel column chromatography, and eluting by gradient elution with 0.75% acidic methanol aqueous solution as eluting solvent, wherein the gradient is 60% methanol aqueous solution, 75% methanol aqueous solution, 90% methanol aqueous solution, and 100% methanol; controlling the flow rate to be 6-10mL/min, controlling the dosage of the eluent to be 3 column volumes, and analyzing the eluent by adopting high performance liquid chromatography to obtain a separated target eluent, wherein an eluent I containing betulinic acid is obtained by adopting 75% methanol aqueous solution for elution, an eluent II containing oleanolic acid is obtained by adopting 90% methanol aqueous solution for elution, and an eluent III containing ursolic acid is obtained by adopting 100% methanol for elution;
concentrating and drying the eluent I, the eluent II and the eluent III respectively; heating and dissolving the dried substance with methanol solution, cooling, and standing in a refrigerating chamber for 12h for crystallization;
filtering and separating the crystallization solution respectively to obtain crystals, and drying to obtain 1.5g of betulinic acid with the purity of 97.0 percent respectively; 0.4g of oleanolic acid, the purity of which is 96.5 percent; 3.3g of ursolic acid, and the purity is 96.2%.
Comparative example 1
Weighing 500g of dry rosemary leaves, carrying out reflux extraction for 2h by using 5000ml of 60% ethanol solution for the first time, filtering, continuously carrying out reflux extraction on filter residues for 2h by using 4000ml of 60% ethanol solution, filtering, and combining the two extracting solutions; concentrating the extractive solution under vacuum to remove alcohol, concentrating at 70 deg.C, cooling, standing for 5 hr, vacuum filtering to obtain filter cake, and air drying at 60 deg.C for 4 hr to obtain solid A60.2 g;
adding the solid substance A into 900ml of 60% ethanol solution, stirring for 1h at normal temperature, performing suction filtration to obtain a filter cake, continuously adding the filter cake into 900ml of 60% ethanol solution, and stirring for 1h at normal temperature; filtering to obtain a filter cake, and drying by blowing at 60 ℃ to obtain 15.2g of solid matter B;
adding the solid matter B into 350ml of 85% ethanol solution, adjusting the pH value to 12, stirring and dissolving at 75 ℃, adding 0.5g of activated carbon, stirring and decoloring for 3h, and filtering while hot to obtain filtrate;
adjusting the pH value of the filtrate to 3, standing for 6 hours at room temperature, filtering to obtain a filter cake, and drying the filter cake to obtain 11.9g of solid matter D;
the modified C18 reversed phase silica gel resin was soaked in methanol, degassed, and then packed in a column, and the two column volumes were washed with 60% methanol in water. Dissolving the solid matter D in methanol at a ratio of 1:1, separating by modified reverse phase silica gel column chromatography, and eluting by gradient elution with 0.05% acidic methanol aqueous solution as eluting solvent, wherein the gradient is 60% methanol aqueous solution, 75% methanol aqueous solution, 90% methanol aqueous solution, and 100% methanol; controlling the flow rate to be 6-10mL/min, controlling the dosage of the eluent to be 3 column volumes, and analyzing the eluent by adopting high performance liquid chromatography to obtain a separated target eluent, wherein an eluent I containing betulinic acid is obtained by adopting 75% methanol aqueous solution for elution, an eluent II containing oleanolic acid is obtained by adopting 90% methanol aqueous solution for elution, and an eluent III containing ursolic acid is obtained by adopting 100% methanol for elution;
concentrating and drying the eluent I, the eluent II and the eluent III respectively; heating and dissolving the dried substance with 95% methanol solution, cooling, and standing in a refrigerating chamber for 12h for crystallization;
filtering and separating the crystallization solution respectively to obtain crystals, and drying to obtain 1.7g of betulinic acid with purity of 87.3%; 0.6g of oleanolic acid, the purity of which is 85.5 percent; 3.8g of ursolic acid, and the purity is 84.8%.
In the comparative example, the solid matter B is not degreased, and the separation of products is influenced under the same condition, so that the purity of the obtained betulinic acid, oleanolic acid and ursolic acid is lower than 95%.
Comparative example 2
Weighing 500g of dry rosemary leaves, carrying out reflux extraction for 2h by using 5000ml of 70% ethanol solution for the first time, filtering, continuously carrying out reflux extraction on filter residues for 2h by using 4000ml of 70% ethanol solution, filtering, and combining the two extracting solutions; concentrating the extractive solution under vacuum to remove alcohol, concentrating at 70 deg.C, cooling, standing for 5 hr, vacuum filtering to obtain filter cake, and air drying at 60 deg.C for 4 hr to obtain solid substance A (59.4 g);
adding the solid substance A into 900ml of 65% ethanol solution, stirring for 1h at normal temperature, performing suction filtration to obtain a filter cake, continuously adding the filter cake into 900ml of 65% ethanol solution, and stirring for 1h at normal temperature; filtering to obtain a filter cake, and drying by blowing air at 60 ℃ to obtain 14.9g of solid matter B;
extracting the solid substance B with n-hexane for three times, wherein the dosage is 450ml each time, separating, and carrying out forced air drying at 60 ℃ for 4h to obtain 13.5g of the solid substance C;
adding the solid matter C into 350ml of 85% ethanol solution, adjusting the pH value to 11.5, stirring and dissolving at 75 ℃, adding 0.5g of activated carbon, stirring and decoloring for 2.5h, and filtering while hot to obtain a filtrate;
adjusting the pH value of the filtrate to 2, standing for 6 hours at room temperature, filtering to obtain a filter cake, and drying the filter cake to obtain 10.1g of solid matter D;
unmodified C18 reverse phase silica gel resin was soaked with methanol, degassed and loaded onto a column, and the two column volumes were washed with 60% methanol in water. Dissolving the solid matter D in methanol at a ratio of 1:1, separating by unmodified reverse phase silica gel column chromatography, and eluting by gradient elution, wherein the eluting solvent is 0.1% acidic methanol aqueous solution, and the gradient is 60% methanol aqueous solution, 75% methanol aqueous solution, 90% methanol aqueous solution and 100% methanol; controlling the flow rate to be 6-10mL/min, controlling the dosage of the eluent to be 3 column volumes, and analyzing the eluent by adopting high performance liquid chromatography to obtain a separated target eluent, wherein an eluent I containing betulinic acid is obtained by adopting 75% methanol aqueous solution for elution, an eluent II containing oleanolic acid is obtained by adopting 90% methanol aqueous solution for elution, and an eluent III containing ursolic acid is obtained by adopting 100% methanol for elution;
concentrating and drying the eluent I, the eluent II and the eluent III respectively; heating and dissolving the dried substance with 95% methanol solution, cooling, and standing in a refrigerating chamber for 12h for crystallization;
filtering and separating the crystallization solution respectively to obtain crystals, and drying to obtain 1.4g of betulinic acid with purity of 87.8%; 0.5g of oleanolic acid, the purity of which is 85.6 percent; 3.2g of ursolic acid, and the purity is 86.2%.
In the comparative example, the product is separated by adopting unmodified C18 reverse phase silica gel resin, and the separation of the product is influenced under the same condition, and the purity of the betulinic acid, oleanolic acid and ursolic acid is lower than 95 percent.
Comparative example 3
Weighing 500g of dry rosemary leaves, carrying out reflux extraction for 2h by using 5000ml of 80% ethanol solution for the first time, filtering, continuously carrying out reflux extraction on filter residues for 2h by using 4000ml of 80% ethanol solution, filtering, and combining the two extracting solutions; concentrating the extractive solution under vacuum to remove alcohol, concentrating at 70 deg.C, cooling and standing for 5 hr to obtain filter cake, and air drying at 60 deg.C for 4 hr to obtain solid A (58.7 g);
adding the solid substance A into 900ml of 70% ethanol solution, stirring for 1h at normal temperature, performing suction filtration to obtain a filter cake, continuously adding the filter cake into 900ml of 70% ethanol solution, and stirring for 1h at normal temperature; filtering to obtain a filter cake, and drying by blowing at 60 ℃ to obtain 14.5g of solid matter B;
extracting the solid substance B with n-hexane for three times, wherein the dosage is 450ml each time, separating, and carrying out forced air drying at 60 ℃ for 4h to obtain 13.3g of solid substance C;
adding the solid matter C into 350ml of 85% ethanol solution, adjusting the pH value to 11, stirring and dissolving at 75 ℃, adding 0.5g of activated carbon, stirring and decoloring for 2h, and filtering while hot to obtain filtrate;
adjusting the pH value of the filtrate to 4, standing for 6 hours at room temperature, filtering to obtain a filter cake, and drying the filter cake to obtain 9.7g of solid matter D;
the modified C18 reversed phase silica gel resin was soaked in methanol, degassed, and then packed in a column, and the two column volumes were washed with 60% methanol in water. Dissolving the solid matter D in methanol at a ratio of 1:1, separating by modified reverse phase silica gel column chromatography, and eluting by gradient elution with methanol water solution, wherein the gradient is 60% methanol water solution, 75% methanol water solution, 90% methanol water solution, and 100% methanol; controlling the flow rate to be 6-10mL/min, controlling the dosage of the eluent to be 3 column volumes, and analyzing the eluent by adopting high performance liquid chromatography to obtain a separated target eluent, wherein an eluent I containing betulinic acid is obtained by adopting 75% methanol aqueous solution for elution, an eluent II containing oleanolic acid is obtained by adopting 90% methanol aqueous solution for elution, and an eluent III containing ursolic acid is obtained by adopting 100% methanol for elution;
concentrating and drying the eluent I, the eluent II and the eluent III respectively; heating and dissolving the dried substance with methanol solution, cooling, and standing in a refrigerating chamber for 12h for crystallization;
filtering and separating the crystallization solution respectively to obtain crystals, and drying to obtain 1.4g of betulinic acid with the purity of 86.0%; 0.5g of oleanolic acid, the purity of which is 87.5 percent; 3.2g of ursolic acid, and the purity is 87.2%.
In the comparative example, the methanol aqueous solution without acid is adopted as the elution solvent, and under the same condition, the separation effect on the product is poor, and the purity of the betulinic acid, oleanolic acid and ursolic acid is lower than 95%.
Claims (9)
1. A method for simultaneously preparing triterpenic acid with different purities from rosemary is characterized by comprising the following steps:
(1) extracting dry leaves of rosemary by refluxing with a solution with the ethanol concentration of 60-85%, concentrating the extracting solution, recovering the ethanol, and carrying out solid-liquid separation to obtain a solid substance A;
(2) washing the solid substance A with 60-85% ethanol solution, and performing solid-liquid separation to obtain a solid substance B;
(3) extracting and degreasing the solid substance B by using petroleum ether or n-hexane, and then carrying out solid-liquid separation to obtain a solid substance C;
(4) adding the solid matter C into 75-85% ethanol solution, adjusting pH to 10-12, adding active carbon, stirring, decolorizing, and performing solid-liquid separation to obtain filtrate;
(5) adjusting the pH of the filtrate to 2-4, and performing solid-liquid separation to obtain a solid substance D;
(6) dissolving the solid matter D in methanol, separating by modified reverse phase silica gel column chromatography, wherein the modified silica gel resin is modified C18 reverse phase silica gel resin of octadecyl bonded silica gel, eluting with gradient acidic methanol solution with acid content of 0.05% to obtain eluent I containing betulinic acid, eluent II containing oleanolic acid and eluent III containing ursolic acid, wherein, the eluent I containing betulinic acid is obtained by eluting with 75% methanol aqueous solution, the eluent II containing oleanolic acid is obtained by eluting with 90% methanol aqueous solution, the eluent III containing ursolic acid is obtained by eluting with 100% methanol, ethanol or methanol with the concentration not lower than 95% is respectively adopted after concentration and drying, and three products of single high-purity betulinic acid, oleanolic acid and ursolic acid are obtained by dissolving, standing and crystallizing, separating and drying the solution.
2. The method for simultaneously preparing triterpenic acids with different purities from rosemary according to claim 1, wherein the triterpenic acids comprise: in the step (1), the reflux extraction times are 2 times, the dosage of the 1 st ethanol solution is 10mL of ethanol solution/1 g of dry rosemary leaves, the dosage of the 2 nd ethanol solution is 8mL of ethanol solution/1 g of dry rosemary leaves, and the extraction time is 2 hours each time.
3. The method for simultaneously preparing triterpenic acids with different purities from rosemary according to claim 1, wherein the triterpenic acids comprise: in the step (2), the solid substance A is added into 60-85% ethanol solution to be stirred and dissolved for 2 times, and each time lasts for 1-2 hours.
4. The method for simultaneously preparing triterpenic acids with different purities from rosemary according to claim 1, wherein the triterpenic acids comprise: in the step (3), the liquid-solid ratio of extraction is 25-30mL/g, and the extraction times are 2-3.
5. The method for simultaneously preparing triterpenic acids with different purities from rosemary according to claim 1, wherein the triterpenic acids comprise: in the step (4), the dosage of the 75-85% ethanol solution is 25-30mL ethanol solution/1 g solid matter C, the temperature is 60-75 ℃, activated carbon with the weight of 0.1% of the dry leaves of the rosemary is added, and the stirring and the decoloring time is 2-3 h.
6. The method for simultaneously preparing triterpenic acids with different purities from rosemary according to claim 1, wherein the triterpenic acids comprise: in the step (5), after the pH value is adjusted to 2-4, standing is carried out for at least 5h at room temperature.
7. The method for simultaneously preparing triterpenic acids with different purities from rosemary according to claim 1, wherein the triterpenic acids comprise: in the step (6), the gradient arrangement of the gradient acidic methanol solution with the acid content of 0.05% is sequentially 60% methanol aqueous solution, 75% methanol aqueous solution, 90% methanol aqueous solution and 100% methanol, the flow rate is 6-10mL/min, and the acid is hydrochloric acid, phosphoric acid or acetic acid; high performance liquid chromatography is adopted for analysis.
8. The method for simultaneously preparing triterpenic acids with different purities from rosemary according to claim 1, wherein the triterpenic acids comprise: in the step (6), the dissolving temperature is 70-80 ℃.
9. A process for the simultaneous preparation of triterpenic acids of different purity from rosemary according to any of claims 1 to 8, wherein: in the step (6), the purity of the betulinic acid, the oleanolic acid and the ursolic acid is not lower than 95%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911241475.9A CN110872337B (en) | 2019-12-06 | 2019-12-06 | Method for simultaneously preparing different high-purity triterpenic acids from rosemary |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911241475.9A CN110872337B (en) | 2019-12-06 | 2019-12-06 | Method for simultaneously preparing different high-purity triterpenic acids from rosemary |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110872337A CN110872337A (en) | 2020-03-10 |
| CN110872337B true CN110872337B (en) | 2022-04-19 |
Family
ID=69717582
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911241475.9A Active CN110872337B (en) | 2019-12-06 | 2019-12-06 | Method for simultaneously preparing different high-purity triterpenic acids from rosemary |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110872337B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112794872B (en) * | 2021-04-02 | 2022-07-15 | 湖南德诺贝莱健康产业有限公司 | Method for extracting luteolin-3' -glucuronide from rosemary |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1508149A (en) * | 2002-12-18 | 2004-06-30 | 中南大学 | Method for extracting total triterpenic acid, ursolic acid and oleanolic acid from Taiwan lectuce herb tea |
| CN101016328A (en) * | 2007-02-09 | 2007-08-15 | 南京工业大学 | Method of separating and purifying ursolic acid and oleanolic acid |
| CN101274012A (en) * | 2008-01-23 | 2008-10-01 | 上海海天医药科技开发有限公司 | Composition of Prunella plant extracts, preparation and pharmaceutical use thereof |
| CN103006824A (en) * | 2012-12-19 | 2013-04-03 | 新疆泽浦红农业发展股份有限公司 | Method for extracting tritepenoidic acid from jujube leaves |
-
2019
- 2019-12-06 CN CN201911241475.9A patent/CN110872337B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1508149A (en) * | 2002-12-18 | 2004-06-30 | 中南大学 | Method for extracting total triterpenic acid, ursolic acid and oleanolic acid from Taiwan lectuce herb tea |
| CN101016328A (en) * | 2007-02-09 | 2007-08-15 | 南京工业大学 | Method of separating and purifying ursolic acid and oleanolic acid |
| CN101274012A (en) * | 2008-01-23 | 2008-10-01 | 上海海天医药科技开发有限公司 | Composition of Prunella plant extracts, preparation and pharmaceutical use thereof |
| CN103006824A (en) * | 2012-12-19 | 2013-04-03 | 新疆泽浦红农业发展股份有限公司 | Method for extracting tritepenoidic acid from jujube leaves |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110872337A (en) | 2020-03-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102976909B (en) | Method for extracting and purifying 6-gingerol from ginger | |
| CN110845328A (en) | Method for preparing high-purity carnosic acid from rosemary oil paste by-product | |
| CN101274953B (en) | Method for extracting corosolic acid from plant | |
| CN101824018B (en) | Method for purifying dihydromyricetin | |
| US20190022159A1 (en) | Novel method for preparing purified extracts of harpagophytum procumbens | |
| CN101143887B (en) | Method for separating and preparing corosolicacid in loquat leaf | |
| CN112209979A (en) | Production process for combined separation of high-purity liquiritin, debitterized glycyrrhizin and licorice total flavone from glycyrrhizic acid monoammonium salt mother liquor paste | |
| CN110872337B (en) | Method for simultaneously preparing different high-purity triterpenic acids from rosemary | |
| CN109576048B (en) | Method for extracting volatile oil from usnea and simultaneously extracting usnea polysaccharide and usnic acid | |
| CN107298642B (en) | Extraction and purification method of 6-shogaol | |
| CN111171104B (en) | Method for preparing ursolic acid from rosemary oil paste by-product | |
| CN104987952B (en) | Method for extracting volatile oil and salidroside from rhodiola rosea whole plant | |
| CN112723997A (en) | Green process for extracting high-purity bakuchiol | |
| CN115010618B (en) | Separation and purification method of aureoyl amide alcohol ester capable of reducing uric acid and application thereof | |
| CN111440184B (en) | Method for preparing high-purity carnosol | |
| CN111171106A (en) | Preparation method of 24-hydroxystearyl glycyrrhetinate | |
| CN110627807A (en) | Bilobalide B raw material and preparation method thereof | |
| CN100427501C (en) | Method for separating and preparing ursolic acid and its derivative from persimmon leaf using counter current chromatography | |
| CN101985440A (en) | Method for producing piperine | |
| CN109970838B (en) | Preparation method of pedunculoside | |
| CN101327232A (en) | Method for preparing Hippophae rhamnoides flavones separated and purified by polyamide and uses thereof | |
| CN106632207B (en) | A kind of preparation method of high-purity GCG | |
| CN110627806A (en) | Bilobalide B compound and preparation method thereof | |
| CN105061458B (en) | Preparation method of nontoxic high-purity ginkgo total lactones and monomers thereof | |
| CN105753918B (en) | method for extracting apigenin-7-o- β -D-glucuronide from broussonetia papyrifera leaves |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20211208 Address after: 416117 interchange of Changji expressway, Wuxi Town, Luxi County, Xiangxi Tujia and Miao Autonomous Prefecture, Hunan Province (in Wuling Sunshine Biotechnology Co., Ltd.) Applicant after: Hunan Xianwei sunshine Biotechnology Co., Ltd Address before: No. 118, airport intersection, Huanghua Industrial Park, Changsha County, Changsha City, Hunan Province Applicant before: HUNAN SHINEWAY ENTERPRISE Co.,Ltd. |
|
| TA01 | Transfer of patent application right | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |