CN110563793A - Method for extracting ursolic acid and oleanolic acid from loquat leaves by microwave - Google Patents
Method for extracting ursolic acid and oleanolic acid from loquat leaves by microwave Download PDFInfo
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Abstract
The invention discloses a method for extracting ursolic acid and oleanolic acid from loquat leaves by microwave, which comprises the following steps: step 1, placing loquat powder in a microwave reflux extraction device, and adding an ethanol solution to assist in microwave reflux extraction; step 2, carrying out secondary reflux extraction to obtain a second extracting solution; step 3, mixing the first extracting solution and the second extracting solution, adding a sodium hydroxide solution, adjusting the pH value, heating in a water bath, and filtering to obtain a first filtrate; step 4, adding a sulfuric acid solution into the first filtrate, adjusting the pH value, diluting with an acid aqueous solution with the same volume of pH value, and heating in a water bath to obtain a crude extract of ursolic acid and oleanolic acid; step 5, separating to obtain a high-purity ursolic acid sample and a high-purity oleanolic acid sample; and 6, drying, and performing content measurement by high performance liquid chromatography to obtain ursolic acid and oleanolic acid with purity of more than 96%, yield of ursolic acid of more than 3.1mg/g, and yield of oleanolic acid of more than 0.48 mg/g. The method effectively improves the purity and extraction rate of the ursolic acid and the oleanolic acid, saves time in operation and reduces cost consumption.
Description
Technical Field
The invention relates to the technical field of extraction of effective components of plants, in particular to a method for extracting ursolic acid and oleanolic acid from loquat leaves by microwave.
Background
The loquat leaves are leaves of the loquat of the Rosaceae family, are cultivated in most areas in China and can be harvested all the year round, have the functions of clearing away the lung-heat, relieving cough, calming the adverse-rising energy and preventing vomiting, and are traditional common traditional Chinese medicines. The active components in folium Eriobotryae include volatile oil, triterpenic acids, flavonoids, saponins, organic acids, polyphenols, etc. Pharmacological research make internal disorder or usurp shows that the main active component is triterpenic acid compound, and the triterpenic acid compound obtained from folium Eriobotryae at present mainly comprises ursolic acid, oleanolic acid and its parent derivatives.
Ursolic acid is a triterpenoid existing in natural plants, has various biological effects of sedation, anti-inflammation, antibiosis, antidiabetic, antiulcer, blood sugar reduction and the like, and also has an obvious antioxidant function, so the ursolic acid is widely used as a raw material of medicines and cosmetics. The clinical effect of the ursolic acid is to obviously and rapidly reduce the activity of glutamic-pyruvic transaminase and serum transaminase, eliminate jaundice, promote appetite, resist fibrosis and restore liver function, and has the characteristics of quick response, short treatment course and stable effect.
Oleanolic acid is a pentacyclic triterpenoid compound separated and extracted from swertia mileensis or ligustrum lucidum fruit of swertia of Gentianaceae, and exists in various plants as free body and glycoside. Oleanolic acid exists widely in plants, and the general content is 0.2-2%. The content of the hemsleya amabilis of the Cucurbitaceae plant is higher by 1.5 to 2 percent, and the content of the glossy privet fruit is 0.6 to 0.7 percent. Oleanolic acid is white needle crystal (ethanol), odorless, tasteless, unstable to acid and alkali, insoluble in water, and soluble in methanol, ethanol, diethyl ether, acetone and chloroform. Oleanolic acid is a broad-spectrum antibacterial drug, and can be used for protecting liver and lowering transaminase, and treating bronchitis, pneumonia, acute tonsillitis, periodontitis, bacillary dysentery, acute gastroenteritis, and urinary system infection. In addition, it is also used for treating acute hepatitis clinically.
The method for extracting ursolic acid and oleanolic acid from folium Eriobotryae mainly comprises cold soaking method, reflux extraction method, ultrasonic assisted extraction method, Soxhlet extraction method, etc., but the extraction rate is generally 92% -93.56%, the extraction time is long, and the solvent dosage is large. Therefore, the extraction purity and extraction rate of ursolic acid and oleanolic acid in loquat leaves are still to be improved to ensure the extraction efficiency.
Disclosure of Invention
an object of the present invention is to solve at least the above problems and to provide at least the advantages described later.
The invention also aims to provide a method for extracting ursolic acid and oleanolic acid from loquat leaves by microwave, which effectively improves the purity and extraction rate of the ursolic acid and the oleanolic acid, saves time for operation and reduces cost consumption.
To achieve these objects and other advantages in accordance with the present invention, there is provided a method for microwave extraction of ursolic acid and oleanolic acid from loquat leaves, comprising:
Step 1, drying and crushing loquat leaves, sieving the dried and crushed loquat leaves with a 30-50-mesh sieve, placing the loquat leaves into a microwave reflux extraction device, adding an ethanol solution with the mass concentration of 50-95%, and performing microwave-assisted reflux extraction to obtain a first extracting solution and a first filtrate; microwave extracting at 40-90 deg.C for 5-60 min; the microwave power is 200-800 w.
Step 2, adding the first filtrate into 50-95% ethanol solution again, and performing reflux extraction to obtain a second extract; the extraction temperature is 50-95 deg.C, and the extraction time is 20-50 min.
And 3, combining the first extracting solution and the second extracting solution to obtain a mixed extracting solution, adding a sodium hydroxide solution with the mass concentration of 5%, adjusting the pH value of the mixed extracting solution to 8-11, heating in a water bath for 10-15min, and filtering to obtain a first filtrate.
And 4, adding a sulfuric acid solution with the mass concentration of 5% into the first filtrate, adjusting the pH value to 2-4, diluting with an acid aqueous solution with the same volume and the pH value of 2-4, heating in a water bath for 10-15min, refrigerating, filtering and washing to obtain a crude extract of ursolic acid and oleanolic acid.
And 5, separating the crude extracts of the ursolic acid and the oleanolic acid obtained in the step 4 by silica gel column chromatography to respectively obtain a high-purity ursolic acid sample and a high-purity oleanolic acid sample.
And 6, drying the high-purity ursolic acid sample and the high-purity oleanolic acid sample obtained in the step 5, and performing content determination by adopting a high performance liquid chromatography to obtain the ursolic acid and the oleanolic acid with the purity of more than 96%, the yield of the ursolic acid of more than 3.1mg/g and the yield of the oleanolic acid of more than 0.48 mg/g.
Preferably, in the step 1, the mass concentration of the ethanol solution is 95%, the microwave extraction temperature is 60 ℃, the microwave extraction time is 15min, and the microwave power is 300 w.
Preferably, in the step 2, the liquid-solid ratio of the ethanol solution with the mass concentration of 50-95% to the first filtrate is 10-30: 1 mL/g.
Preferably, the microwave reflux extraction device in step 1 comprises:
The microwave oven comprises a shell, wherein an interlayer is arranged on the inner side of the shell, a microwave generator is arranged in the interlayer, and an opening is formed in the top end of the shell.
An extraction flask rotatably disposed within the housing, the extraction flask configured as a helical hollow tubular body, the extraction flask having a top end with an opening adapted to the top end of the housing:
And the reflux device comprises an air guide pipe and a condensation pipe which are arranged at the upper end of the opening of the shell.
Preferably, the rotatable arrangement mode of the extraction flask is as follows: the bottom end of the middle part of the shell is provided with a rotary table matched with the bottom end of the extraction flask, the outer side of the rotary table is symmetrically provided with clamping devices, each clamping device comprises a supporting rod and a clamping protrusion arranged at the top end of the supporting rod, and the clamping protrusions are matched with gaps between the spiral structures of the extraction flasks.
preferably, the inner diameter of the spiral structure tube of the extraction bottle is set to be 0.8-1.2 cm, and the gap between the adjacent tube bodies of the spiral hollow structure of the extraction bottle is set to be 0.5-0.8 cm.
The invention at least comprises the following beneficial effects:
The crushed loquat leaf raw material is placed in a microwave reflux extraction device and is mixed with 50-95% ethanol solution serving as a solvent to extract ursolic acid and oleanolic acid in the loquat leaves, the ursolic acid and the oleanolic acid are extracted under the assistance of microwaves, and meanwhile, reflux extraction is combined, so that the extraction efficiency of the ursolic acid and the oleanolic acid is improved, the sufficiency and the dissolution rate of the ursolic acid and the oleanolic acid are ensured by carrying out secondary reflux on a filtrate obtained after microwave reflux extraction, and the mixed solution is correspondingly treated by combining a first extracting solution and a second extracting solution, so that the effective accuracy of the yield of the ursolic acid and the oleanolic acid is ensured; adding sodium hydroxide into the mixed extracting solution, and adjusting the pH value of the obtained solution to 8-11, so that alkaloid and polyphenol can be effectively removed, and the purity of the crude extracts of ursolic acid and oleanolic acid can be improved; then adding a sulfuric acid solution into the first filtrate, adjusting the pH value of the solution to 2-4, effectively retaining ursolic acid and oleanolic acid, and removing impurities in the solution, thereby maximally extracting the ursolic acid and the oleanolic acid from the loquat leaf components and keeping the purity of the ursolic acid and the oleanolic acid, so that the purity of the ursolic acid and the oleanolic acid is over 96 percent, the yield of the ursolic acid is over 3.1mg/g, and the yield of the oleanolic acid is over 0.48mg/g, which are determined by high performance liquid chromatography, effectively improving the extraction rate of the ursolic acid and the oleanolic acid, reducing the dosage of an extraction solvent, and reducing the cost consumption.
additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
drawings
FIG. 1 is a block diagram of the structure of the method for microwave extraction of ursolic acid and oleanolic acid from loquat leaves according to the present invention;
FIG. 2 is a schematic structural diagram of the microwave reflux extraction apparatus according to the present invention;
Fig. 3 is a schematic structural diagram of the extraction flask of the present invention.
Detailed Description
The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
As shown in fig. 1, the present invention provides a method for microwave extraction of ursolic acid and oleanolic acid from loquat leaves, comprising:
Step 1, drying and crushing loquat leaves, sieving the dried and crushed loquat leaves with a 30-50-mesh sieve, placing the loquat leaves into a microwave reflux extraction device, adding an ethanol solution with the mass concentration of 50-95%, and performing microwave-assisted reflux extraction to obtain a first extracting solution and a first filtrate; microwave extracting at 40-90 deg.C for 5-60 min; the microwave power is 200-800 w.
Step 2, adding the first filtrate into 50-95% ethanol solution again, and performing reflux extraction to obtain a second extract; the extraction temperature is 50-95 deg.C, and the extraction time is 20-50 min.
And 3, combining the first extracting solution and the second extracting solution to obtain a mixed extracting solution, adding a sodium hydroxide solution with the mass concentration of 5%, adjusting the pH value of the mixed extracting solution to 8-11, heating in a water bath for 10-15min, and filtering to obtain a first filtrate.
and 4, adding a sulfuric acid solution with the mass concentration of 5% into the first filtrate, adjusting the pH value to 2-4, diluting with an acid aqueous solution with the same volume and the pH value of 2-4, heating in a water bath for 10-15min, refrigerating, filtering and washing to obtain a crude extract of ursolic acid and oleanolic acid.
and 5, separating the crude extracts of the ursolic acid and the oleanolic acid obtained in the step 4 by silica gel column chromatography to respectively obtain a high-purity ursolic acid sample and a high-purity oleanolic acid sample.
And 6, drying the high-purity ursolic acid sample and the high-purity oleanolic acid sample obtained in the step 5, and performing content determination by adopting a high performance liquid chromatography to obtain the ursolic acid and the oleanolic acid with the purity of more than 96%, the yield of the ursolic acid of more than 3.1mg/g and the yield of the oleanolic acid of more than 0.48 mg/g.
In the scheme, crushed loquat leaf raw materials are placed in a microwave reflux extraction device and are mixed with 50-95% ethanol solution serving as a solvent to extract ursolic acid and oleanolic acid in the loquat leaves, microwave-assisted extraction of the ursolic acid and the oleanolic acid is utilized, and reflux extraction is combined, so that the extraction efficiency of the ursolic acid and the oleanolic acid is improved, the sufficiency and the dissolution rate of the ursolic acid and the oleanolic acid are ensured by carrying out secondary reflux on a filtrate obtained after microwave reflux extraction, and the mixed liquid of the first extract and the second extract is correspondingly treated by combining the first extract and the second extract, so that the effective accuracy of the yield of the ursolic acid and the oleanolic acid is ensured; adding sodium hydroxide into the mixed extracting solution, and adjusting the pH value of the obtained solution to 8-11, so that alkaloid and polyphenol can be effectively removed, and the purity of the crude extracts of ursolic acid and oleanolic acid can be improved; then adding a sulfuric acid solution into the first filtrate, adjusting the pH value of the solution to 2-4, effectively retaining ursolic acid and oleanolic acid, and removing impurities in the solution, thereby maximally extracting the ursolic acid and the oleanolic acid from the loquat leaf components and keeping the purity of the ursolic acid and the oleanolic acid, so that the purity of the ursolic acid and the oleanolic acid is over 96 percent, the yield of the ursolic acid is over 3.1mg/g, and the yield of the oleanolic acid is over 0.48mg/g, which are determined by high performance liquid chromatography, effectively improving the extraction rate of the ursolic acid and the oleanolic acid, reducing the dosage of an extraction solvent, and reducing the cost consumption.
In a preferable scheme, in the step 1, the mass concentration of the ethanol solution is 95%, the microwave extraction temperature is 50 ℃, the microwave extraction time is 30min, and the microwave power is 300 w.
In the scheme, the fruit acid is a weak polar molecule, the volume of the ethanol is increased to be beneficial to the dissolution of the fruit acid, when the mass concentration of the ethanol reaches 95%, the dissolution amount of the ursolic acid and the oleanolic acid is larger, the mass concentration is higher than 95%, the extraction amount of the ursolic acid and the oleanolic acid is reduced on the contrary, because the protein denaturation is accelerated by the high-concentration ethanol, the permeation of the ethanol into cells is inhibited to a certain extent, and the dissolution of the ursolic acid and the oleanolic acid is hindered; under the condition of low microwave extraction temperature, the extraction amount is increased along with the rise of the temperature, when the microwave extraction temperature reaches 60 ℃, the extraction amount reaches a peak value, and after the microwave extraction temperature exceeds 60 ℃, the extraction amounts of ursolic acid and oleanolic acid are reduced on the contrary, because the volatilization of ethanol is accelerated due to the overhigh temperature, the volume fraction of the ethanol in the extracting solution is reduced, and the extraction amount is reduced; when the microwave extraction time is 15min, the extraction amount reaches the maximum, and when the microwave extraction time exceeds 15min, the extraction amount is slightly reduced, because the structure of ursolic acid and oleanolic acid is damaged due to overlong extraction time, the extraction amount is reduced; the extraction amount is close to balance and slowly decreases when the microwave power is more than 300w, and the temperature rise speed is too high, so that the bumping of the extracting solution is caused, the loss of the extracting solution is caused, and the energy consumption is increased when the power is increased; therefore, the ethanol solution preferably has a mass concentration of 95%, a microwave extraction temperature of 60 ℃, a microwave extraction time of 15min, and a microwave power of 300 w.
In a preferable embodiment, in step 2, the liquid-solid ratio of the ethanol solution with a mass concentration of 50-95% to the first filtrate is 10-30: 1 mL/g.
in the scheme, when the liquid-solid ratio reaches 15: 1mL/g, the extraction amount of the ursolic acid and the oleanolic acid reaches the maximum value, the liquid-solid ratio is continuously increased, the extraction amount of the ursolic acid and the oleanolic acid is reduced on the contrary, after the extraction solvent reaches a certain proportion, the ursolic acid and the oleanolic acid reach the dissolution balance, the increased dosage can increase the dissolution of other impurities in cells, the solvent is wasted, the concentration time is too long, the energy consumption is increased, and the recovery rate is reduced.
In a preferred embodiment, the microwave reflux extraction device in step 1 comprises:
The microwave oven comprises a shell, wherein an interlayer is arranged on the inner side of the shell, a microwave generator is arranged in the interlayer, and an opening is formed in the top end of the shell.
an extraction flask rotatably disposed within the housing, the extraction flask configured as a helical hollow tubular body, the extraction flask having a top end with an opening adapted to the top end of the housing:
And the reflux device comprises an air guide pipe and a condensation pipe which are arranged at the upper end of the opening of the shell.
In the above scheme, as shown in fig. 2-3, a microwave generator is arranged in the interlayer of the housing, so that microwave-assisted extraction is performed on the solution in the extraction bottle by microwaves emitted by the microwave generator, and meanwhile, the microwave extraction is not sufficient by combining a reflux extraction mode, and the advantages of rapidness and energy conservation of microwave extraction are utilized. The reflux extraction mode is communicated with the solution in the extraction bottle through a reflux device, namely a mode that the air duct and the condenser pipe are butted with an opening at the top end of the shell, so that microwave-assisted extraction and reflux extraction are carried out simultaneously, and the high-efficiency extraction of the ursolic acid and the oleanolic acid is realized in a relatively complementary mode.
In a preferred embodiment, the rotatable extraction flask is arranged in the following manner: the bottom end of the middle part of the shell is provided with a rotary table matched with the bottom end of the extraction flask, the outer side of the rotary table is symmetrically provided with clamping devices, each clamping device comprises a supporting rod and a clamping protrusion arranged at the top end of the supporting rod, and the clamping protrusions are matched with gaps between the spiral structures of the extraction flasks.
In the scheme, the cover body is arranged at the front end of the shell, so that an extraction bottle can be conveniently placed on the rotary disc from the cover body, the rotation of the rotary disc drives the extraction bottle to rotate, the spiral hollow structure of the extraction bottle is arranged, and the large-volume mixed solution can be uniformly dispersed in the spiral hollow structure of each layer, so that the solution can be uniformly and effectively dispersed in the space of the shell, the microwave can be conveniently projected, the microwave can uniformly act on each part in the solution in the same time, and the phenomenon of local overheating caused by non-uniform microwave projection effect is avoided; the clamping device outside the turntable fixes the bottom end of the extraction bottle body through the support rod to prevent the extraction bottle body from being inclined, and the clamping device is fixed in the layer gap of the spiral hollow structure by utilizing the clamping protrusion to reinforce the extraction bottle body, so that the extraction bottle is more stable in rotation.
In a preferable scheme, the inner diameter of the spiral structure tube of the extraction bottle is set to be 0.8-1.2 cm, and the gap between adjacent tube bodies of the spiral hollow structure of the extraction bottle is set to be 0.5-0.8 cm.
In the scheme, the inner diameter is set to be 0.8-1.2 cm, so that the solution is distributed more uniformly to ensure the uniformity of the microwave projection effect; the clearance, namely the clearance between each layer of spiral pipes is set to be 0.5-0.8 cm, so that the capacity of the whole extraction bottle in the shell is ensured.
Example 1
Step 1, drying and crushing loquat leaves, sieving the dried and crushed loquat leaves with a 30-mesh sieve, placing the loquat leaves into a microwave reflux extraction device, adding an ethanol solution with the mass concentration of 50%, and performing microwave-assisted reflux extraction to obtain a first extracting solution and a first filtrate; microwave extracting at 40 deg.C for 60 min; the microwave power is 200 w.
Step 2, adding the first filtrate into 50% ethanol solution again, and performing reflux extraction to obtain a second extract; the extraction temperature is 50 deg.C, and the extraction time is 50 min.
and 3, combining the first extracting solution and the second extracting solution to obtain a mixed extracting solution, adding a sodium hydroxide solution with the mass concentration of 5%, adjusting the pH value of the mixed extracting solution to 8, heating in a water bath for 10min, and filtering to obtain a first filtrate.
And 4, adding a sulfuric acid solution with the mass concentration of 5% into the first filtrate, adjusting the pH value to 2, diluting with an equal volume of an acidic aqueous solution with the pH value of 2, heating in a water bath for 10min, refrigerating, filtering and washing to obtain a crude extract of ursolic acid and oleanolic acid.
And 5, separating the crude extracts of the ursolic acid and the oleanolic acid obtained in the step 4 by silica gel column chromatography to respectively obtain a high-purity ursolic acid sample and a high-purity oleanolic acid sample.
And 6, drying the high-purity ursolic acid sample and the high-purity oleanolic acid sample obtained in the step 5, and performing content determination by adopting a high performance liquid chromatography to obtain the ursolic acid and the oleanolic acid with the purity of 96.1 percent and 96.5 percent respectively, the ursolic acid yield of 3.11mg/g and the oleanolic acid yield of 0.481 mg/g.
Example 2
Step 1, drying and crushing loquat leaves, sieving the dried and crushed loquat leaves with a 40-mesh sieve, placing the dried and crushed loquat leaves into a microwave reflux extraction device, adding an ethanol solution with the mass concentration of 85%, and performing microwave-assisted reflux extraction to obtain a first extracting solution and a first filtrate; microwave extracting at 90 deg.C for 15 min; the microwave power is 500 w.
Step 2, adding the first filtrate into an ethanol solution with the mass concentration of 85% again, and performing reflux extraction to obtain a second extracting solution; the extraction temperature is 95 deg.C, and the extraction time is 20 min.
And 3, combining the first extracting solution and the second extracting solution to obtain a mixed extracting solution, adding a sodium hydroxide solution with the mass concentration of 5%, adjusting the pH value of the mixed extracting solution to 9, heating in a water bath for 12min, and filtering to obtain a first filtrate.
And 4, adding a sulfuric acid solution with the mass concentration of 5% into the first filtrate, adjusting the pH value to 3, diluting with an equal volume of an acidic aqueous solution with the pH value of 3, heating in a water bath for 12min, refrigerating, filtering and washing to obtain a crude extract of ursolic acid and oleanolic acid.
and 5, separating the crude extracts of the ursolic acid and the oleanolic acid obtained in the step 4 by silica gel column chromatography to respectively obtain a high-purity ursolic acid sample and a high-purity oleanolic acid sample.
And 6, drying the high-purity ursolic acid sample and the high-purity oleanolic acid sample obtained in the step 5, and performing content determination by adopting a high performance liquid chromatography to obtain the purity of the ursolic acid and the purity of the oleanolic acid which are respectively more than 96.3% and 96.7%, wherein the yield of the ursolic acid is more than 3.15mg/g, and the yield of the oleanolic acid is more than 0.483 mg/g.
Example 3
Step 1, drying and crushing loquat leaves, sieving the dried and crushed loquat leaves with a 50-mesh sieve, placing the loquat leaves into a microwave reflux extraction device, adding an ethanol solution with the mass concentration of 95%, and performing microwave-assisted reflux extraction to obtain a first extracting solution and a first filtrate; microwave extracting at 60 deg.C for 15 min; the microwave power is 300 w.
step 2, adding the first filtrate into 95% ethanol solution again, and performing reflux extraction to obtain a second extract; the extraction temperature is 70 deg.C, and the extraction time is 40 min.
And 3, combining the first extracting solution and the second extracting solution to obtain a mixed extracting solution, adding a sodium hydroxide solution with the mass concentration of 5%, adjusting the pH value of the mixed extracting solution to 11, heating in a water bath for 15min, and filtering to obtain a first filtrate.
And 4, adding a sulfuric acid solution with the mass concentration of 5% into the first filtrate, adjusting the pH value to 4, diluting with an equal volume of an acidic aqueous solution with the pH value of 4, heating in a water bath for 15min, refrigerating, filtering and washing to obtain a crude extract of ursolic acid and oleanolic acid.
And 5, separating the crude extracts of the ursolic acid and the oleanolic acid obtained in the step 4 by silica gel column chromatography to respectively obtain a high-purity ursolic acid sample and a high-purity oleanolic acid sample.
And 6, drying the high-purity ursolic acid sample and the high-purity oleanolic acid sample obtained in the step 5, and performing content determination by adopting a high performance liquid chromatography to obtain that the purity of the ursolic acid and the purity of the oleanolic acid are respectively more than 97% and 97.3%, the yield of the ursolic acid is 3.3mg/g, and the yield of the oleanolic acid is 0.49 mg/g.
Example 4
Step 1, drying and crushing loquat leaves, sieving the dried and crushed loquat leaves with a 50-mesh sieve, placing the dried and crushed loquat leaves into a microwave synthesis extractor for extraction, adding an ethanol solution with the mass concentration of 95%, extracting and filtering to obtain a first extracting solution and a first filtrate; microwave extracting at 60 deg.C for 15 min; the microwave power is 300 w.
step 2, adding the first filtrate into 95% ethanol solution again, and performing reflux extraction to obtain a second extract; the extraction temperature is 70 deg.C, and the extraction time is 40 min.
And 3, combining the first extracting solution and the second extracting solution to obtain a mixed extracting solution, adding a sodium hydroxide solution with the mass concentration of 5%, adjusting the pH value of the mixed extracting solution to 11, heating in a water bath for 15min, and filtering to obtain a first filtrate.
And 4, adding a sulfuric acid solution with the mass concentration of 5% into the first filtrate, adjusting the pH value to 4, diluting with an equal volume of an acidic aqueous solution with the pH value of 4, heating in a water bath for 15min, refrigerating, filtering and washing to obtain a crude extract of ursolic acid and oleanolic acid.
And 5, separating the crude extracts of the ursolic acid and the oleanolic acid obtained in the step 4 by silica gel column chromatography to respectively obtain a high-purity ursolic acid sample and a high-purity oleanolic acid sample.
And 6, drying the high-purity ursolic acid sample and the high-purity oleanolic acid sample obtained in the step 5, and performing content determination by adopting a high performance liquid chromatography to obtain that the purity of the ursolic acid and the purity of the oleanolic acid are respectively over 93 percent and over 93.2 percent, the yield of the ursolic acid is 2.5mg/g, and the yield of the oleanolic acid is 0.36 mg/g.
As can be seen from examples 1-4, in examples 1-3, example 3 is the best solution of the present application, and the purity of ursolic acid and oleanolic acid obtained by extraction is 97% and 97.3% respectively, the yield of ursolic acid is 3.3mg/g, and the yield of oleanolic acid is 0.49mg/g, and example 4 adopts the best solution of the present application, but a common microwave synthesis extractor is adopted to perform microwave-assisted extraction, so that the purity of ursolic acid and oleanolic acid obtained by extraction is 923% and 93.2% respectively, the yield of ursolic acid is 2.5mg/g, and the yield of oleanolic acid is 0.36mg/g, and compared with examples 1-3, the purity and extraction rate of the extract are significantly reduced, thus, the method for extracting ursolic acid and oleanolic acid in leaves by microwave as claimed in the present application not only effectively improves the purity of ursolic acid and oleanolic acid, meanwhile, the extraction rate of the ursolic acid and the oleanolic acid is greatly improved, the steps are relatively simple, the operation is time-saving, and the cost consumption is reduced.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.
Claims (6)
1. A method for extracting ursolic acid and oleanolic acid from folium Eriobotryae by microwave comprises the following steps:
Step 1, drying and crushing loquat leaves, sieving the dried and crushed loquat leaves with a 30-50-mesh sieve, placing the loquat leaves into a microwave reflux extraction device, adding an ethanol solution with the mass concentration of 50-95%, and performing microwave-assisted reflux extraction to obtain a first extracting solution and a first filtrate; microwave extracting at 40-90 deg.C for 5-60 min; the microwave power is 200-800 w;
Step 2, adding the first filtrate into 50-95% ethanol solution again, and performing reflux extraction to obtain a second extract; the extraction temperature is 50-95 ℃, and the extraction time is 20-50 min;
step 3, combining the first extracting solution and the second extracting solution to obtain a mixed extracting solution, adding a sodium hydroxide solution with the mass concentration of 5%, adjusting the pH value of the mixed extracting solution to 8-11, heating in a water bath for 10-15min, and filtering to obtain a first filtrate;
step 4, adding a sulfuric acid solution with the mass concentration of 5% into the first filtrate, adjusting the pH value to 2-4, diluting with an acid aqueous solution with the pH value of 2-4 in the same volume, heating in a water bath for 10-15min, refrigerating, filtering and washing to obtain a crude extract of ursolic acid and oleanolic acid;
Step 5, separating the crude extracts of the ursolic acid and the oleanolic acid obtained in the step 4 by silica gel column chromatography to respectively obtain a high-purity ursolic acid sample and a high-purity oleanolic acid sample;
And 6, drying the high-purity ursolic acid sample and the high-purity oleanolic acid sample obtained in the step 5, and performing content determination by adopting a high performance liquid chromatography to obtain the ursolic acid and the oleanolic acid with the purity of more than 96%, the yield of the ursolic acid of more than 3.1mg/g and the yield of the oleanolic acid of more than 0.48 mg/g.
2. The method for microwave extraction of ursolic acid and oleanolic acid from loquat leaves as claimed in claim 1, wherein in step 1, the concentration of ethanol solution is 95% by mass, the microwave extraction temperature is 60 ℃, the microwave extraction time is 15min, and the microwave power is 300 w.
3. The method for microwave extraction of ursolic acid and oleanolic acid from loquat leaves as claimed in claim 1, wherein in step 2, the liquid-solid ratio of 50-95% by mass of said ethanol solution to said first filtrate is 10-30: 1 mL/g.
4. The method for microwave extraction of ursolic acid and oleanolic acid in loquat leaves as claimed in claim 1, wherein said microwave reflux extraction apparatus in step 1 comprises:
the microwave oven comprises a shell, a microwave generator and a heating device, wherein an interlayer is arranged on the inner side of the shell, the interlayer is internally provided with the microwave generator, and the top end of the shell is provided with an opening;
An extraction flask rotatably disposed within the housing, the extraction flask configured as a helical hollow tubular body, the extraction flask having a top end with an opening adapted to the top end of the housing:
And the reflux device comprises an air guide pipe and a condensation pipe which are arranged at the upper end of the opening of the shell.
5. The method for microwave extraction of ursolic acid and oleanolic acid from loquat leaves as claimed in claim 4, wherein said extraction flask is rotatably installed in such a manner that: the bottom end of the middle part of the shell is provided with a rotary table matched with the bottom end of the extraction flask, the outer side of the rotary table is symmetrically provided with clamping devices, each clamping device comprises a supporting rod and a clamping protrusion arranged at the top end of the supporting rod, and the clamping protrusions are matched with gaps between the spiral structures of the extraction flasks.
6. The method for microwave extraction of ursolic acid and oleanolic acid from loquat leaves as claimed in claim 4, wherein said inner diameter of said spiral structured tube of extraction flask is set to 0.8-1.2 cm, and the gap between adjacent tubes of said spiral hollow structure of extraction flask is set to 0.5-0.8 cm.
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| CN115607593A (en) * | 2021-07-13 | 2023-01-17 | 上海中医药大学 | Preparation method of loquat leaf extract rich in pentacyclic triterpenic acid |
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