CN114805466A - Preparation method of ivy extract - Google Patents
Preparation method of ivy extract Download PDFInfo
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Abstract
The invention relates to a preparation method of a hedera helix extract, belonging to the technical field of extraction and processing of plants. The preparation method comprises the following steps: s1, mixing the ivy raw material with the extraction aid solution, and performing ultrasonic extraction to obtain an extracting solution; s2, concentrating the extracting solution, mixing with ethanol, and collecting a concentrated solution; s3, sequentially eluting the concentrated solution with water, ethanol water solution and acidic ethanol water solution by using an aluminum peroxide chromatographic column; collecting the eluent of the ethanol water solution to prepare a hederacoside C eluent; collecting the eluent of the acidic ethanol water solution to prepare an alpha-hederin eluent; s4, crystallizing the hederacoside C eluent to obtain a hederacoside C extract; crystallizing the alpha-hederin eluent to obtain an alpha-hederin extract; the extraction aid solution comprises the following preparation raw materials: surfactants and flocculants. The method can obtain high-purity hederacoside extract.
Description
Technical Field
The invention belongs to the technical field of plant extraction and processing, and particularly relates to a preparation method of an ivy extract.
Background
Hedera helix (Latin's name: Hedera nepalensis var. sinensis (Tobl.).) is the dry stem and leaf of Hedera helix of Araliaceae, called Tubuteng, ZUANTIANFENG and triangle Fengfeng, from Ben Cao gang mu Shi Yi (supplement to compendium of materia Medica), and has the effects of dispelling pathogenic wind and promoting diuresis, promoting blood circulation and detumescence, calming liver and detoxicating. The main components of ivy include triterpene saponin, carotene, tannin, saccharide, etc., wherein the stem contains hederacoside, tannin (about 12.01 wt%), resin, etc., and the leaf contains hederacoside, inositol, carotene, saccharide, and in addition, about 29.4 wt%. Saponins (such as triterpenoid saponins) are a kind of components which are found in hedera helix plants in a large variety and have the best activity, and have various pharmacological activities such as anti-inflammation, anti-tumor, anti-fungus and insecticidal action.
Modern pharmacological studies have shown that: the main active component of ivy is saponin. Alpha-hederin extracted from hedera helix, hederin C, hederin E and hederin F all have good anti-inflammatory effect. Hederacoside C (hederacoside C) and alpha-hederacoside with disaccharide chains are considered as main effective components in hedera helix, wherein the alpha-hederacoside can be formed by ester hydrolysis of the hederacoside C, and the content of the hederacoside C in the medicinal hedera helix is not less than 3 percent according to the European pharmacopoeia. Related technologies record that the content of ivy C in the stem and leaf of ivy is 1.30-13.65%, the content of alpha-ivy glycoside is 0.56-4.42%, and the content in the leaf is 3-4 times of the stem.
In the related technology, a crude extract (alcohol extract) of ivy is adopted to prepare a preparation for treating diseases such as cough, dyspnea and the like, and the main active ingredient of the preparation is ivy saponin. It has also been found in the related art that α -hederin is particularly useful for bronchial spasmolysis, by inhibiting receptor/ligand internalization, constantly activating adenylate cyclase system, relaxing smooth muscle (spasmolysis), whereas hederin C and hederin aglycone cannot inhibit the internalization process; but hederacoside C has the functions of dispelling wind, promoting diuresis, calming the liver, detoxifying and the like.
The related technology also provides a method for extracting and separating the total saponins of Chinese ivy, compares the extraction methods of solvent extraction, microwave extraction, biological enzyme and the like, and obtains the ivy saponin C with the purity of 87 percent by means of purification of different macroporous resins, preparative reverse phase high performance liquid chromatography separation and the like. The process uses a preparative chromatographic purification technology to separate the hederacoside C, has high cost and complex technology, and is not beneficial to industrial production.
The related art also provides a method for preparing an ivy leaf extract containing active ingredients of ivy glycoside C and alpha-ivy element, and the method mainly invents a high-efficiency extract prepared by mixing 4% of alpha-ivy element and 6.5% of ivy glycoside C. The method does not disclose how to separate hederacoside C and alpha-hederin, and the total content of the separated hederacoside C and alpha-hederin is low.
The related technology also provides a method for preparing hederacoside C from hedera helix, the hederacoside C monomer with the content of more than 98 percent is obtained by drying and crushing hedera helix stems and leaves, extracting with ethanol water, sequentially extracting with petroleum ether, ethyl acetate and n-butyl alcohol, dissolving the n-butyl alcohol phase with ethanol, precipitating with ethyl acetate, separating and purifying with a silica gel column. Although the invention can obtain the hederacoside C monomer with high purity, the process is complex, the cost is high, high-risk reagents such as petroleum ether, chloroform and the like are used, the production safety is low, and other components in the raw materials are not fully utilized, thereby causing resource waste.
The related technology also provides a preparation method of the hedera plant extract, which comprises the steps of soaking a hedera raw material, extracting with ethanol water solution, enriching with macroporous resin (eluting with ethanol water solution of more than or equal to 40 percent), and drying the enriched solution under reduced pressure to obtain the extract, wherein the total glycoside content of the extract is not more than 60 percent (colorimetric method), and the monomer content (hederin C or alpha-hedera extract) is not more than 25 percent. Namely, the purity of the extract obtained by the above method is low.
Therefore, it is required to develop a method for preparing an hederacoside C extract and an α -hederacoside extract at the same time, which are highly pure.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a preparation method of a hedera helix extract, and the preparation method can simultaneously prepare a high-purity hederacoside C extract and an alpha-hederacoside extract.
In order to solve the technical problems, the invention provides a preparation method of an ivy extract, which comprises the following steps:
s1, mixing the ivy raw material with the extraction aid solution, and performing ultrasonic extraction to obtain an extracting solution;
s2, concentrating the extracting solution, mixing with ethanol, and collecting a concentrated solution;
s3, sequentially eluting the concentrated solution with water, ethanol water solution and acidic ethanol water solution by using an aluminum peroxide chromatographic column;
collecting the eluent of the ethanol water solution to prepare a hederacoside C eluent;
collecting the eluent of the acidic ethanol water solution to prepare an alpha-hederin eluent;
s4, crystallizing the hederacoside C eluent to obtain a hederacoside C extract;
crystallizing the alpha-hederin eluent to obtain an alpha-hederin extract;
the extraction aid solution comprises the following preparation raw materials:
surfactants and flocculants.
According to at least one technical scheme in the technical scheme of the preparation method, the preparation method at least has the following beneficial effects:
the invention ensures that the liquid film thickness between the hederacoside compounds (alpha-hederacoside and hederacoside C) in the hederacoside raw material and the extraction aid solution is thin enough through the synergistic effect of the extraction aid and the ultrasonic extraction, reduces the resistance of the hederacoside in the process of diffusing from the surface of the raw material to the extraction aid solution, and greatly improves the extraction rate of effective components (the content of total glycosides in the extracting solution is more than 50%).
The extraction aid is used by matching the surfactant (reducing the surface tension of the solution of the extraction aid, increasing the permeability to cell tissues, desorption, dissolution, solubilization and the like of active ingredients) and the flocculant (flocculating with impurities such as tannin in the extracting solution to achieve the effect of removing impurities; improving the effect of the surfactant on assisting the dissolution of the active ingredients)), so that the extraction rate of the active ingredients (alpha-hederin and hederin C) is remarkably enhanced, the extraction time is shortened, and the effect of removing impurities is achieved.
The hederacoside compound contains hydroxyl and has good solubility in ethanol; ethanol is added into the extracting solution to prevent the hederacoside compound from being separated out due to the reduction of the solvent in the concentration process; so that the hederacoside compounds are fully dissolved in the concentrated solution.
The separation of alpha-hederin and hederin C is realized by adopting water, ethanol water solution and acidic ethanol water solution for elution.
The method realizes the simultaneous preparation of high-purity hederacoside C and alpha-hederacoside by using the extraction aid, namely ultrasonic extraction, alumina column chromatographic separation and crystallization, and has high yield; the whole process does not use other organic solvents except ethanol, is safe and environment-friendly, and is economical and efficient.
Hederacoside C has molecular formula of C 59 H 96 O 26 The molecular weight is 1221, and the compound is soluble in cold water, soluble in hot water, methanol, ethanol, acetonitrile, etc., and insoluble in petroleum ether, etc., which is an organic solvent with high polarity. The structural formula is shown as the following formula:
alpha-hederin has a molecular formula of C 41 H 66 O 12 Molecular weight is 750, its water solubility is poor, it is easy to dissolve in hot methanol and hot ethanol. The structural formula is shown as the following formula:
according to some embodiments of the invention, the ivy material is at least one of ivy leaves or ivy stems.
According to some embodiments of the invention, the ivy leaves are ivy dry leaves.
According to some embodiments of the invention, the ivy stem is a dried ivy stem.
According to some embodiments of the invention, the ivy raw material is pulverized to obtain a ivy raw material powder.
According to some embodiments of the invention, the ivy raw material powder has a particle size of 40 to 60 mesh.
The contact area of the raw material and the extraction aid solution is increased by crushing the raw material to a proper mesh number, so that the extraction efficiency is accelerated.
According to some embodiments of the invention, the surfactant is selected from at least one of tween 20, tween 40 and tween 80.
According to some embodiments of the invention, the flocculant is hydroxypropyl chitosan.
According to some embodiments of the invention, the hydroxypropyl chitosan has a degree of substitution above 80%.
The hederagenin compound has a plurality of hydroxyl groups, the electron cloud density is high, the compound is electronegative, and forms an ion pair with cationic high-molecular hydroxypropyl chitosan, compared with the polar form of a target compound (the hederagenin compound), the ion pair form is easier to be captured by non-ionic surfactants Tween (Tween 20, Tween 40 and Tween 80), so the extraction rate is obviously improved; while the extraction rate of effective components is improved, the dissolved impurities such as tannin and the like and hydroxypropyl chitosan are subjected to high-speed molecular collision under the assistance of ultrasonic waves, so that a good flocculation effect is achieved.
Hydroxyl in hydroxypropyl chitosan is introduced, so that a large amount of hydroxyl exists on the molecular chain of the whole flocculating agent, the molecular structure of chitosan and the molecular structure of tannic acid are more strongly fused, and a hydrogen bond can be formed with phenolic hydroxyl in the molecules of tannic acid colloidal particles to generate a characteristic adsorption effect; ionization thereof produces ions, and electrical neutralization thereof also promotes flocculation.
According to some embodiments of the invention, the mass ratio of the surfactant to the flocculant is 2.5-6: 1.
Under the condition of surfactant fixation, if the flocculating agent is excessive, excessive positive charges are neutralized with negative charges of hederacoside, and the flocculating agent is flocculated to cause more loss; too little, the impurity removal rate is low, the purity of the effective components is influenced, the subsequent purification effect is further influenced, and the synergistic effect of the surfactant in dissolving aid is reduced.
According to some embodiments of the invention, the mass fraction of the extraction aid in the extraction aid solution is between 0.3% and 0.6%.
When the concentration of the extraction aid is too high, a large amount of foam can be generated, the operation difficulty is increased, meanwhile, the dissolution of impurities can be increased, and the later separation is not facilitated. When the concentration is too low, the formed micelle concentration is insufficient, and the compatibilization effect is not obvious.
According to some embodiments of the invention, the mass fraction of the flocculant in the extraction aid solution is between 0.08 and 0.1 thousandths.
According to some embodiments of the invention, the pH of the extraction aid solution is between 7.5 and 8.5.
Because the alpha-hederin has poor water solubility, the extraction rate of the alpha-hederin can be effectively improved by adjusting the pH value; the pH value is too large, the flocculation effect of hydroxypropyl chitosan is influenced, and the synergistic effect leads to low extraction rate and low purity of alpha-hederin.
And due to the weak alkaline environment in the extraction process, the partially ionized hederacoside is easier to be adsorbed by the alumina column chromatography, and then a good separation effect is achieved by gradient elution through the polarity difference and the difficulty of the chromatographic adsorption of the target component and the alumina column.
According to some embodiments of the invention, the pH of the extraction aid solution is adjusted by the selection of an alkaline substance.
According to some embodiments of the invention, the alkaline material comprises an inorganic base, a carbonate, and a bicarbonate.
According to some embodiments of the invention, the inorganic base is at least one of sodium hydroxide, potassium hydroxide and cesium hydroxide.
According to some embodiments of the invention, the carbonate is at least one of sodium carbonate, potassium carbonate and cesium carbonate.
According to some embodiments of the invention, the bicarbonate is at least one of sodium bicarbonate, potassium bicarbonate and cesium bicarbonate.
According to some embodiments of the invention, the starting material for the preparation of the extraction aid solution further comprises water.
According to some embodiments of the invention, the mass ratio of the extraction aid solution to the ivy raw material is 1: 12-25.
According to some embodiments of the invention, the mass ratio of the extraction aid solution to the ivy raw material is 1: 15-20.
According to some embodiments of the invention, the parameters of the ultrasound extraction are as follows: the extraction time is 30min to 45min, the extraction temperature is 45 ℃ to 60 ℃, and the ultrasonic frequency is 20kHz to 25 kHz.
The excessive temperature can destroy the structure of the surfactant and influence the extraction efficiency, the degradation phenomenon exists in hederacoside C, and in a proper temperature range, the rising temperature can accelerate the movement speed of effective component molecules, accelerate the diffusion and increase the dissolving amount.
The structure of flocculation of the flocculating agent and the impurities can be broken due to overlong ultrasonic time, so that the flocculation effect is poor, and the collision of molecules can be improved by proper ultrasonic assistance, so that the formation of the linking acting force between the flocculating agent and the impurities is promoted, and the flocculation effect is improved; too high ultrasonic frequency can destroy the molecular structure formed by the flocculating agent and the impurity molecules, and reduce the flocculation effect.
According to some embodiments of the invention, the ultrasound extraction is batch ultrasound extraction.
According to some embodiments of the invention, the total duration of the intermittent ultrasound extraction consists of several adjacent ultrasound times and intervals.
According to some embodiments of the invention, each intermittent time of the intermittent ultrasonic extraction is 5s to 10 s.
According to some embodiments of the invention, each ultrasound time of the intermittent ultrasound extraction is 5min to 6 min.
According to some embodiments of the invention, the concentration is a concentration under reduced pressure.
According to some embodiments of the invention, the concentration is carried out to a solids content of 9% to 15%.
According to some embodiments of the invention, the volume fraction of ethanol in the concentrate is between 30% and 40%.
By controlling the volume fraction of the ethanol in the concentrated solution within the range, the alpha-hederacoside is ensured to be completely dissolved, and the aim of removing part of impurities with larger polarity is fulfilled.
According to some embodiments of the invention, the amount of water used in step S3 is 0.25BV to 0.5 BV.
A small amount of water removes a small amount of impurities with high polarity and difficult adsorption, and excessive water has no influence on resin adsorbates; but will affect the extraction time and also cause waste of water resources.
According to some embodiments of the invention, the flow rate of the water in step S3 is 1BV/h to 1.5 BV/h.
According to some embodiments of the invention, the volume fraction of ethanol in the ethanol aqueous solution in step S3 is 50% to 60%.
According to some embodiments of the present invention, the ethanol aqueous solution in step S3 is used in an amount of 2BV to 2.5 BV.
The dosage is less than the limited range, the elution is incomplete, and a small amount of alpha-hederin is washed off and the solvent is wasted when the dosage is higher than the limited range.
According to some embodiments of the invention, the flow rate of the ethanol aqueous solution in the step S3 is 1BV/h to 1.5 BV/h.
According to some embodiments of the invention, the volume fraction of ethanol in the acidic aqueous ethanol solution in step S3 is 70% to 80%.
According to some embodiments of the invention, the acidic aqueous ethanol solution in step S3 is used in an amount of 2BV to 3 BV.
The dosage is less than the limited range, the elution is incomplete, a small amount of impurities exist above the limited range to be eluted, and the solvent is wasted.
According to some embodiments of the invention, the flow rate of the aqueous acidic ethanol solution in step S3 is 1BV/h to 1.5 BV/h.
According to some embodiments of the invention, the pH of the acidic ethanol aqueous solution in the step S3 is 5-6.
The pH value is not in the range, the separation effect is poor, the impurities are peracid or alkaline, and the impurities are easy to elute, so that the purity of the alpha-hederacoside is influenced. And too high a pH may result in incomplete elution of alpha-hederin.
According to some embodiments of the invention, the pH of the aqueous acidic ethanol solution is adjusted with an acid.
According to some embodiments of the invention, the acid is at least one of hydrogen chloride or sulfuric acid.
According to some embodiments of the invention, the alumina chromatography column is a neutral alumina chromatography column or an acidic alumina chromatography column.
The hederacoside C and the alpha-hederacoside are effectively separated by neutral alumina and acidic alumina column chromatography, and the adsorption capacity and the resolution are high.
According to some embodiments of the invention, the temperature at which the hederacoside C eluate is crystallized is between 4 ℃ and 10 ℃.
According to some embodiments of the invention, the time for crystallization of the hederacoside C eluate is between 8h and 24 h.
The temperature and time of crystallization are controlled to fully crystallize hederacoside C, so that the yield of the hederacoside C is improved.
According to some embodiments of the invention, the crystallization of the hederacoside C eluate comprises the steps of:
and concentrating the hederacoside C eluent under reduced pressure until the eluent is saturated, crystallizing at the temperature of 4-10 ℃ for 8-24 h, collecting a solid phase, and drying to obtain the hederacoside C extract.
According to some embodiments of the invention, the temperature at which the alpha-hederacoside eluate is crystallized is between 4 ℃ and 10 ℃.
According to some embodiments of the invention, the time for crystallization of the alpha-hederin eluent is between 8h and 24 h.
The alpha-hederacoside is fully crystallized by controlling the temperature and the time of crystallization, so that the yield of the alpha-hederacoside is improved.
According to some embodiments of the invention, the crystallization of the alpha-hederin eluate comprises the steps of:
and (3) concentrating the alpha-hederin eluent under reduced pressure until the alpha-hederin eluent is saturated, crystallizing for 8-24 h at 4-10 ℃, collecting a solid phase, washing and drying to obtain the alpha-hederin extract.
According to some embodiments of the invention, the detergent for washing is water.
According to some embodiments of the invention, the number of washes is 1 to 2.
The term "saturation" as used herein means a state in which crystals are just precipitated.
Drawings
FIG. 1 is a high performance liquid chromatography assay of hederacoside C extract prepared in example 3 of the present invention.
FIG. 2 is a high performance liquid chromatography assay of the alpha-hederacoside extract prepared in example 3 of the present invention.
Detailed Description
The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.
In the description of the present invention, reference to the description of the terms "one embodiment," "some embodiments," "an illustrative embodiment," "an example," "a specific example," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
According to the embodiment of the invention, the mass content of hederacoside C in the selected hederacoside leaf dry raw material is 5.34%, and the mass content of alpha-hederacoside is 2.16%.
The content of hederacoside C and alpha-hederacoside in the embodiment of the invention is detected by high performance liquid chromatography.
The liquid chromatography detection conditions of hederacoside C are as follows:
a chromatographic column: WONDASILTMC18 column (250mm (length). times.4.6 mm (inner diameter), 5 μm (filler particle size));
flow rate: 1 mL/min;
detection wavelength: 203 nm;
sample introduction amount: 10 mu L of the solution;
sensitivity: 2.000 AUFS;
mobile phase: the volume ratio of acetonitrile to water is 28: 72;
column temperature: at 25 ℃.
The liquid chromatography detection conditions of the alpha-hederacoside are as follows:
a chromatographic column: WONDASILTMC18 column (250mm (length). times.4.6 mm (inner diameter), 5 μm (filler particle diameter)).
Flow rate: 0.8 mL/min;
detection wavelength: 203 nm;
sample introduction amount: 10 mu L of the solution;
sensitivity: 2.000 AUFS;
mobile phase: the volume ratio of acetonitrile to 0.1 percent phosphoric acid aqueous solution is 45: 55;
column temperature: at 25 ℃.
Example 1
This example is a method for preparing an ivy extract, comprising the following steps:
s1, ultrasonic-assisted extraction:
crushing the ivy leaf dry raw material to 40 meshes, adding 15 times of extraction aid solution by mass, and performing ultrasonic-assisted continuous countercurrent extraction at 45 ℃ for 30 min; solid-liquid separation; collecting liquid phase to obtain ultrasonic extractive solution (the sum of hederacoside C and alpha-hederacoside content is 53.97%).
The mass fraction of the extraction aid in the extraction aid solution is 0.3 per mill.
The extraction aid consists of Tween 20(CAS number: 9005-64-5) and hydroxypropyl chitosan (Shanghai Shi Fenghe Biotech limited, substitution degree is more than or equal to 80%, CAS number: 104673-29-2), wherein the mass ratio of Tween 20 to hydroxypropyl chitosan is 2.5: 1.
the pH of the extraction aid solution was 8.5 (pH at 25 ℃ C.; adjusted by addition of sodium hydroxide).
The conditions of ultrasound assistance were as follows: and (3) carrying out low-frequency intermittent ultrasound, wherein the ultrasound time is 5s/5min (namely, the ultrasound is stopped for 5s every 5 min), and the ultrasound frequency is 20 kHz.
S2, column chromatography:
concentrating the ultrasonic extracting solution obtained in the step S1 under reduced pressure (the solid mass concentration is 12% +/-3%), adding ethanol, controlling the volume concentration of the ethanol to be 30%, and carrying out solid-liquid separation; collecting the liquid phase to obtain a reduced pressure concentrated solution;
subjecting the vacuum concentrated solution to neutral alumina column chromatography, and sequentially eluting with 0.25BV water, 2BV ethanol water solution with volume fraction of 50% and 2BV acidic ethanol water solution with volume fraction of 70% (pH 5, adjusted by hydrochloric acid with mass concentration of 35%) (elution flow rate of 1 BV/h);
collecting 50% ethanol water solution eluent to obtain hederacoside C crude extract;
collecting 70% acidic ethanol water solution eluent to obtain alpha-hederin crude extract;
s3, crystallization:
concentrating the hederacoside C crude extract obtained in the step S2 under reduced pressure until just precipitation, refrigerating at 4 ℃ and standing for 8h, precipitating crystals, carrying out solid-liquid separation, and drying the crystals to obtain a hederacoside C extract;
and (4) concentrating the alpha-hederin crude extract obtained in the step (S2) under reduced pressure until just precipitation occurs, refrigerating at 4 ℃ and standing for 8h, precipitating crystals, carrying out solid-liquid separation, rinsing the crystals for 2 times by using water, and drying to obtain the alpha-hederin extract.
The detection of high performance liquid chromatography shows that the mass fraction of hederacoside C in the hederacoside C extract is 98.71%, and the yield is 84.17%.
The mass fraction of the alpha-hederin in the alpha-hederin extract is 98.41%, and the yield is 82.00%.
Example 2
This example is a method for preparing an ivy extract, comprising the following steps:
s1, ultrasonic-assisted extraction:
crushing the dried ivy leaf raw material to 60 meshes, adding an extraction aid solution with the mass of 20 times, and performing ultrasonic-assisted continuous countercurrent extraction at 60 ℃ for 45 min; solid-liquid separation; collecting liquid phase to obtain ultrasonic extractive solution (the sum of hederacoside C and alpha-hederacoside content is 52.79%).
The mass fraction of the extraction aid in the extraction aid solution is 0.6 per mill.
The extraction aid consists of Tween 20(CAS number: 9005-64-5) and hydroxypropyl chitosan (Shanghai Fenghe Biotechnology Limited, the substitution degree is more than or equal to 80 percent; CAS number: 104673-29-2), wherein the mass ratio of the Tween 20 to the hydroxypropyl chitosan is 6: 1.
the pH of the extraction aid solution was 7.5 (pH at 25 ℃ C.; adjusted by addition of sodium bicarbonate).
The conditions of ultrasound assistance were as follows: and (3) carrying out low-frequency intermittent ultrasound, wherein the ultrasound time is 10s/5min (namely, the ultrasound is stopped for 10s every 5 min), and the ultrasound frequency is 25 kHz.
S2, column chromatography:
concentrating the ultrasonic extracting solution obtained in the step S1 under reduced pressure (the solid mass concentration is 12% +/-3%), adding ethanol, controlling the volume concentration of the ethanol to be 40%, and carrying out solid-liquid separation; collecting the liquid phase to obtain a reduced pressure concentrated solution;
subjecting the vacuum concentrated solution to acidic alumina column chromatography, and sequentially eluting with 0.5BV water, 2.5BV ethanol water solution with volume fraction of 60% and 3BV acidic ethanol water solution with volume fraction of 80% (pH 6, adjusted by hydrochloric acid with mass concentration of 35%) (elution flow rate of 1.5 BV/h);
collecting 60% ethanol water solution eluent to obtain hederacoside C crude extract;
collecting 80% acidic ethanol water solution eluent to obtain alpha-hederin crude extract;
s3, crystallization:
concentrating the hederacoside C crude extract obtained in the step S2 under reduced pressure until just precipitation, refrigerating at 10 ℃ and standing for 24h, precipitating crystals, carrying out solid-liquid separation, and drying the crystals to obtain a hederacoside C extract;
and (4) concentrating the alpha-hederin crude extract obtained in the step (S2) under reduced pressure until just precipitation occurs, refrigerating at 10 ℃ and standing for 24h, precipitating crystals, carrying out solid-liquid separation, rinsing the crystals for 1 time by using water, and drying to obtain the alpha-hederin extract.
The detection of high performance liquid chromatography shows that the mass fraction of hederacoside C in the hederacoside C extract is 98.59%, and the yield is 84.80%.
The mass fraction of the alpha-hederin in the alpha-hederin extract is 98.20 percent, and the yield is 82.82 percent.
Example 3
This example is a method for preparing an ivy extract, comprising the following steps:
s1, ultrasonic-assisted extraction:
crushing the dried ivy leaf raw material to 60 meshes, adding an extraction aid solution with the mass being 18 times that of the ivy leaf raw material, and performing ultrasonic-assisted continuous countercurrent extraction at the temperature of 55 ℃ for 40 min; solid-liquid separation; collecting liquid phase to obtain ultrasonic extractive solution (the sum of hederacoside C and alpha-hederacoside content is 53.07%).
The mass fraction of the extraction aid in the extraction aid solution is 0.4 per mill.
The extraction aid consists of Tween 20(CAS number: 9005-64-5) and hydroxypropyl chitosan (Shanghai Shi Fenghe Biotech limited, substitution degree is more than or equal to 80%, CAS number: 104673-29-2), wherein the mass ratio of Tween 20 to hydroxypropyl chitosan is 4: 1.
the pH of the extraction aid solution was 8.0 (pH at 25 ℃ C.; adjusted by addition of potassium hydroxide).
The conditions of ultrasound assistance were as follows: and (3) carrying out low-frequency intermittent ultrasound, wherein the ultrasound time is 8s/5min (namely, the ultrasound is stopped for 8s every 5 min), and the ultrasound frequency is 20 kHz.
S2, column chromatography:
concentrating the ultrasonic extracting solution obtained in the step S1 under reduced pressure (the solid mass concentration is 12% +/-3%), adding ethanol, controlling the volume concentration of the ethanol to be 40%, and carrying out solid-liquid separation; collecting the liquid phase to obtain a reduced pressure concentrated solution;
subjecting the vacuum concentrated solution to neutral alumina column chromatography, and sequentially eluting with 0.25BV water, 2BV ethanol water solution with volume fraction of 60% and 2BV acidic ethanol water solution with volume fraction of 80% (pH is 5.5, and adjusted by 50% sulfuric acid solution) at an elution flow rate of 1 BV/h;
collecting 60% ethanol water solution eluent to obtain hederacoside C crude extract;
collecting 80% acidic ethanol water solution eluent to obtain alpha-hederin crude extract;
s3, crystallization:
concentrating the hederacoside C crude extract obtained in the step S2 under reduced pressure until just precipitation, refrigerating at 6 ℃ and standing for 14h, precipitating crystals, carrying out solid-liquid separation, and drying the crystals to obtain a hederacoside C extract;
and (4) concentrating the alpha-hederin crude extract obtained in the step (S2) under reduced pressure until the alpha-hederin crude extract is just separated out, refrigerating at 6 ℃ and standing for 14h, separating out crystals, carrying out solid-liquid separation, rinsing the crystals for 1 time by using water, and drying to obtain the alpha-hederin extract.
High performance liquid chromatography detection shows that the mass fraction of hederacoside C in the hederacoside C extract is 98.46%, and the yield is 83.45%; the detection pattern is shown in FIG. 1 (retention time is 15.233 min).
The mass fraction of the alpha-hederin in the alpha-hederin extract is 98.39%, and the yield is 81.90%; the detection pattern is shown in FIG. 2 (retention time 25.178 min).
Comparative example 1
The comparative example is a preparation method of an ivy extract, and the difference with the example 3 is as follows:
the extraction aid solution of example 3 was replaced equally with potassium hydroxide solution (pH 8.0).
The detection of high performance liquid chromatography shows that the mass fraction of hederacoside C in the hederacoside C extract is 86.76%, and the yield is 69.17%.
The mass fraction of the alpha-hederin in the alpha-hederin extract is 81.16%, and the yield is 60.04%.
Comparative example 2
The comparative example is a preparation method of an ivy extract, and the difference with the example 3 is as follows:
the extraction aid solution in the example 3 is replaced by a Tween 20 solution (with the mass fraction of 0.4 per mill and the pH value of 8.0, and the extraction aid solution is obtained by adding potassium hydroxide for adjustment).
The detection of high performance liquid chromatography shows that the mass fraction of hederacoside C in the hederacoside C extract is 86.93%, and the yield is 74.44%.
The mass fraction of the alpha-hederin in the alpha-hederin extract is 81.42%, and the yield is 69.89%.
Comparative example 3
The comparative example is a preparation method of an ivy extract, and the difference with the example 3 is as follows:
the ultrasound-assisted extraction in example 3 was replaced by the following operation:
continuously extracting at 55 deg.C under countercurrent for 40 min.
The detection of high performance liquid chromatography shows that the mass fraction of hederacoside C in the hederacoside C extract is 89.73%, and the yield is 74.64%.
The mass fraction of the alpha-hederin in the alpha-hederin extract is 83.94%, and the yield is 71.17%.
Comparative example 4
The comparative example is a preparation method of an ivy extract, and the difference with the example 3 is as follows:
the neutral alumina column of example 3 was replaced with macroporous resin HP 20.
The detection of high performance liquid chromatography shows that the mass fraction of hederacoside C in the hederacoside C extract is 85.49%, and the yield is 68.01%.
The mass fraction of the alpha-hederin in the alpha-hederin extract is 81.61%, and the yield is 66.62%.
Comparative example 5
The comparative example is a preparation method of an ivy extract, and the difference with the example 3 is as follows:
the mass fraction of the extraction aid in the extraction aid solution in example 3 was adjusted to 0.8 ‰.
The detection of high performance liquid chromatography shows that the mass fraction of hederacoside C in the hederacoside C extract is 88.89%, and the yield is 74.03%.
The mass fraction of the alpha-hederin in the alpha-hederin extract is 83.11%, and the yield is 71.25%.
Comparative example 6
The comparative example is a preparation method of an ivy extract, and the difference with the example 3 is as follows:
the pH of the booster solution of example 3 was not adjusted (pH of the booster solution was 6.8).
The detection of high performance liquid chromatography shows that the mass fraction of hederacoside C in the hederacoside C extract is 98.18%, and the yield is 80.68%.
The mass fraction of the alpha-hederin in the alpha-hederin extract is 98.02%, and the yield is 73.36%.
Comparative example 7
The comparative example is a preparation method of an ivy extract, and the difference with the example 3 is as follows:
the pH of the extraction aid solution of example 3 was adjusted to 9.
The detection of high performance liquid chromatography shows that the mass fraction of hederacoside C in the hederacoside C extract is 89.35%, and the yield is 74.57%.
The mass fraction of the alpha-hederin in the alpha-hederin extract is 84.50%, and the yield is 73.04%.
Comparative example 8
The comparative example is a preparation method of an ivy extract, and the difference with the example 3 is as follows:
the ultrasound-assisted extraction in example 3 was replaced by batch ultrasound with continuous ultrasound.
The detection of high performance liquid chromatography shows that the mass fraction of hederacoside C in the hederacoside C extract is 91.61%, and the yield is 72.99%.
The mass fraction of the alpha-hederin in the alpha-hederin extract is 86.05 percent, and the yield is 70.26 percent.
Comparative example 9
The comparative example is a preparation method of an ivy extract, and the difference with the example 3 is as follows:
the 80% by volume aqueous acidic ethanol solution (pH 5.5) in the elution of the column chromatography of example 3 was replaced by 80% by volume aqueous alkaline ethanol solution (pH 9, adjusted with sodium hydroxide).
The detection of high performance liquid chromatography shows that the mass fraction of hederacoside C in the hederacoside C extract is 98.17%, and the yield is 83.20%.
The mass fraction of the alpha-hederin in the alpha-hederin extract is 83.59%, and the yield is 75.24%.
Comparative example 10
The comparative example is a preparation method of an ivy extract, and the difference with the example 3 is as follows:
the 80% volume fraction of the acidic aqueous ethanol solution (pH 5.5) in the elution of the column chromatography of example 3 was replaced by 80% volume fraction of the aqueous ethanol solution.
The detection of high performance liquid chromatography shows that the mass fraction of hederacoside C in the hederacoside C extract is 98.63%, and the yield is 83.30%.
The mass fraction of the alpha-hederin in the alpha-hederin extract is 93.29 percent, and the yield is 65.59 percent.
The difference between comparative example 1 and example 3 is that: no surfactant and no flocculant are added; the extraction rate of the alpha-hederacoside with poor water solubility is low, and the extracted impurities such as tannins and the like cannot be effectively removed, so that the purity of the effective components in the extracting solution is low, the separation and purification effect and yield of the alumina resin are influenced, and the purity and yield of the crystallized target product are further influenced.
The difference between comparative example 2 and example 3 is that: no flocculant is added; the method has the advantages that the flocculant-free synergistic extraction is adopted, the extraction rate of the alpha-hederacoside is low, and extracted impurities such as tannin and the like cannot be removed, so that the purity of effective components in an extracting solution is low, the purification effect and yield of the alumina resin are influenced, and the purity and yield of a target product after crystallization are further influenced.
The difference between comparative example 3 and example 3 is that: ultrasonic-assisted extraction is not adopted: the extraction rate of the effective components is low and the flocculation effect is slightly poor, so that the purity of the effective components in the extracting solution is low, the separation and purification effect and yield of the alumina resin are influenced, and the purity and yield of the crystallized target product are further influenced.
The difference between comparative example 4 and example 3 is that: replacing the alumina column with macroporous resin; the difference between the macroporous resin and the alumina column chromatographic separation is mainly reflected in the separation effect, the macroporous resin cannot well separate the hedera helix C and the alpha-hederin in the test, and the recovery rate is low, so that the purity of the extract obtained by separation is low, and the crystallization yield is further influenced.
The difference between comparative example 5 and example 3 is that: the mass fraction of the extraction aid becomes high; the dissolution of the effective components reaches saturation, the dissolution of impurities is increased, the purity of the effective components in the extracting solution is influenced, the foaming is serious, the production operation difficulty is increased, the loss is caused, and the yield is influenced.
The difference between comparative example 6 and example 3 is that: the pH of the extraction aid solution is not adjusted; because the water solubility of the alpha-hedera helix is poor, the extraction rate of the alpha-hederacoside can be effectively improved by adjusting the pH, the pH is not adjusted, the extraction rate of effective components is influenced, the influence on the effective components in the extracting solution is small, and therefore, the purity of a target product is high, and the yield is low.
The difference between comparative example 7 and example 3 is that: the pH of the extraction aid solution is too high; the flocculation effect of the flocculating agent is influenced, so that the purity of the effective components in the extracting solution is low, the separation and purification effect and yield of the alumina resin are influenced, and the purity and yield of the crystallized target product are further influenced.
The difference between comparative example 8 and example 3 is that: adopting continuous ultrasonic treatment; the intermittent ultrasonic assistance can improve the collision among molecules, promote the formation of the linking acting force between the flocculating agent and impurities, improve the flocculation effect and promote the dissolution of effective components; the structure of flocculation of the flocculating agent and impurities can be broken by overlong ultrasonic time, the flocculation effect is poor, and the purity of effective components in the extracting solution is low, so that the separation and purification effect and yield of the alumina resin are influenced, and the purity and yield of a target product after crystallization are further influenced.
The difference between comparative example 9 and example 3 is that: replacing the acidic ethanol aqueous solution with an alkaline ethanol aqueous solution; impurities are easy to elute, which affects the purity of the alpha-hederacoside, and further causes the purity and yield of the crystals to be low.
The difference between comparative example 10 and example 3 is that: the acidic aqueous ethanol solution is replaced by an aqueous ethanol solution. The alpha-hederin can not be completely eluted, which affects the purity and yield of the alpha-hederin, and further causes the purity and yield of the crystal to be lower.
The structure of the surfactant can be damaged by overhigh temperature in the extraction process, the extraction efficiency is influenced, and in a proper temperature range, the movement speed of effective component molecules can be accelerated by raising the temperature, the diffusion is accelerated, and the dissolving amount is increased. And the temperature is too high, and the degradation phenomenon of hederacoside C exists.
The structure of flocculation of the flocculating agent and the impurities can be broken due to overlong ultrasonic time, so that the flocculation effect is poor, and the collision of molecules can be improved by proper ultrasonic assistance, so that the formation of the linking acting force between the flocculating agent and the impurities is promoted, and the flocculation effect is improved; too high ultrasonic frequency can destroy the molecular structure formed by the flocculating agent and the impurity molecules, and reduce the flocculation effect.
In conclusion, the preparation method comprises the steps of crushing the dried ivy leaf raw material, extracting the ivy leaf raw material with an extraction aid-ultrasonic-assisted continuous countercurrent extraction, and carrying out solid-liquid separation; concentrating the extract under reduced pressure, performing solid-liquid separation, performing aluminum peroxide column chromatography on the concentrated solution, and performing gradient elution; concentrating and crystallizing the target section eluent under reduced pressure to obtain hederacoside C and alpha-hederacoside extracts. The invention comprehensively utilizes the effective components in the ivy raw material, simultaneously prepares and obtains the high-content ivy glycoside C and alpha-ivy glycoside extract (more than or equal to 98 percent), can realize industrial production, has safe and reliable process and is environment-friendly.
While the embodiments of the present invention have been described in detail with reference to the specific embodiments, the present invention is not limited to the embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.
Claims (10)
1. A preparation method of an ivy extract is characterized by comprising the following steps:
s1, mixing the ivy raw material with the extraction aid solution, and performing ultrasonic extraction to obtain an extracting solution;
s2, concentrating the extracting solution, mixing with ethanol, and collecting a concentrated solution;
s3, sequentially eluting the concentrated solution through an aluminum peroxide chromatographic column by adopting water, an ethanol water solution and an acidic ethanol water solution;
collecting the eluent of the ethanol water solution to prepare a hederacoside C eluent;
collecting the eluent of the acidic ethanol water solution to prepare an alpha-hederin eluent;
s4, crystallizing the hederacoside C eluent to obtain a hederacoside C extract;
crystallizing the alpha-hederin eluent to obtain an alpha-hederin extract;
the extraction aid solution comprises the following preparation raw materials:
surfactants and flocculants.
2. The method for preparing an ivy extract according to claim 1, wherein: the surfactant is selected from at least one of Tween 20, Tween 40 and Tween 80.
3. The method for preparing an ivy extract according to claim 1, wherein: the flocculant is hydroxypropyl chitosan.
4. The method for preparing an ivy extract according to claim 1, wherein: the mass fraction of the extraction aid in the extraction aid solution is 0.3-0.6 per mill.
5. The method for preparing an ivy extract according to claim 1, wherein: the pH value of the extraction assistant solution is 7.5-8.5.
6. The method for preparing an ivy extract according to claim 1, wherein: the parameters of the ultrasound extraction are as follows: the extraction time is 30min to 45min, the extraction temperature is 45 ℃ to 60 ℃, and the ultrasonic frequency is 20kHz to 25 kHz.
7. The method for preparing an ivy extract according to claim 1, wherein: in the step S3, the volume fraction of ethanol in the ethanol aqueous solution is 50-60%; preferably, the ethanol aqueous solution used in step S3 is 2BV to 2.5 BV.
8. The method for preparing an ivy extract according to claim 1, wherein: the volume fraction of ethanol in the acidic ethanol aqueous solution in the step S3 is 70-80%; preferably, the dosage of the acidic ethanol aqueous solution in the step S3 is 2 BV-3 BV; preferably, the pH of the acidic ethanol aqueous solution in the step S3 is 5-6.
9. The method for preparing an ivy extract according to claim 1, wherein: the alumina chromatographic column is a neutral alumina chromatographic column or an acidic alumina chromatographic column.
10. The method for preparing an ivy extract according to claim 1, wherein: the crystallization temperature of the hederacoside C eluent is 4-10 ℃; preferably, the crystallization temperature of the alpha-hederacoside eluent is 4-10 ℃.
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