CN109498686B - Preparation method of triterpenic acid in jujube material - Google Patents
Preparation method of triterpenic acid in jujube material Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/72—Rhamnaceae (Buckthorn family), e.g. buckthorn, chewstick or umbrella-tree
- A61K36/725—Ziziphus, e.g. jujube
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
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- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
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Abstract
The invention discloses a preparation method of triterpenic acid in jujube material, which comprises the steps of heating, refluxing and extracting alcohol serving as an extracting agent and alcohol-acid serving as an eluent, and adopting two separation means of macroporous adsorption resin column chromatography and preparative liquid chromatography to obtain the triterpenic acid with high purity, wherein the content of the triterpenic acid is over 90 percent, and the main components of the triterpenic acid are betulinic acid, ursolic acid and oleanolic acid. The organic solvent is recycled in the preparation process, and the preparation method is economical and environment-friendly. The invention adopts AB-8 or HPD100 adsorption resin, and the two are styrene type weak polar or non-polar polymers, so that the service life is longer, and the elution efficiency of the triterpenic acid is higher. The preparative liquid chromatography separation is the biggest technical characteristic different from the prior art, and has the advantages of on-line monitoring, targeted separation and high separation efficiency.
Description
Technical Field
The invention belongs to the technical field of natural product extraction, and particularly relates to a preparation method of triterpenic acid in a jujube material.
Background
The jujube tree is a jujube plant belonging to the Rhamnaceae family, and has a wide planting area in China. The fruit, jujube, is a dry fruit that people like to hear, and the total yield is the first in dry fruit. The Chinese dates are sweet and delicious in taste and rich in nutrition, can be eaten and used as medicines, have various medicinal functions of nourishing yin and tonifying kidney, building body, softening blood vessels, stimulating appetite and invigorating spleen and the like, and are called the King of all fruits.
In recent years, with the development of the jujube processing industry, a lot of production waste jujube residues are brought. The research finds that the jujube residues contain a certain amount of triterpenic acid components. Triterpenic acids are a generic term for a class of biologically active compounds. Jujube is reported to contain more than ten triterpenic acids 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.
Related documents already describe the process for extracting triterpenic acid from jujube. But the extraction rate is low and the separation purity of the effective components is low.
Disclosure of Invention
The invention aims to solve the technical problem of providing a preparation method of triterpenic acid in jujube materials, and aims to simply and efficiently extract the triterpenic acid in the jujube materials by adopting alcohol-acid as an extracting solution and two separation means of macroporous adsorption resin column chromatography and preparative liquid chromatography.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a preparation method of triterpenic acid in jujube material comprises the following steps:
(1) preparation of crude extract of jujube
Carrying out reflux extraction on the jujube material by using an 80-100% alcohol solution at the temperature of 80-90 ℃, extracting for 2-3 times after each extraction for 60-120 min, concentrating the extracting solution under reduced pressure, and recovering the alcohol solution to obtain a jujube crude extract;
(2) preparation of triterpene acid macroporous resin separation component
Separating the jujube crude extract by macroporous resin column chromatography, wherein the volume ratio of the sample loading amount to the separation material is 1: 100-500, and the separation steps are as follows: washing with pure water until a separation column is colorless, eluting with 1-3 column volumes of 60-70% alcohol solution containing 0.1% -1% formic acid or acetic acid to remove impurities, eluting with an eluent at a flow rate of 1-3 column volumes/hour, collecting the eluent, and recovering an organic solvent in the eluent to obtain a triterpenic acid macroporous resin separation component;
(3) preparation of triterpenic acid residue
Separating components and extractum by using triterpenic acid macroporous resin, adding pure water with the volume of 1-2 times of that of extractum, standing for 12-24 hours at 4-8 ℃, and centrifuging for 5-10 minutes at 3000-6000 r/min to obtain triterpenic acid residue;
(4) preparation of triterpenic acid extract
Dissolving the triterpenic acid residue with methanol, separating by preparative liquid chromatography, collecting the target component, and drying to obtain the triterpenic acid extract.
As a limitation of the invention, the jujube material in the step (1) comprises jujube powder and jujube residue.
As a further limitation of the invention, the preparation method of the jujube powder comprises the following steps: removing core from fructus Jujubae, and pulverizing to obtain fructus Jujubae powder.
The preparation method of the jujube residues comprises the following steps: removing the core of the jujube fruit, and crushing to obtain jujube powder, wherein the jujube powder is prepared by mixing the jujube powder with an extraction solvent according to the weight-volume ratio of 1: adding the raw materials into an extraction solvent according to the proportion of 6-10, heating and pressurizing for extraction for 15-30 min or heating and refluxing for extraction for 1-2 h at the extraction temperature of 70-90 ℃, repeatedly extracting for 2-3 times, combining the extracting solutions, concentrating under reduced pressure, and filtering to obtain the jujube residues.
As a further limitation of the invention, the extraction solvent in the preparation of the jujube residues is 0-30% ethanol or methanol water solution.
As a second limitation of the present invention, the eluent in step (2) is 90-100% alcoholic solution containing 0.1% -1% formic acid or acetic acid in 2-5 column volumes.
As a third limitation of the invention, the alcohol solution is a methanol or ethanol solution.
As a fourth limitation of the present invention, the macroporous resin in step (2) is AB-8 or HPD100 adsorbent resin.
As a fifth limitation of the invention, the separation conditions for preparing the liquid chromatogram in the step (4) are that C18 silica gel bonded stationary phase with the particle size of 5 or 10 μm is used as chromatographic packing, methanol and 0.1% formic acid aqueous solution are used as mobile phase, isocratic elution is carried out by using 70-90% methanol, the column effect of the chromatogram is 2000-10000 column plates/m, the length of the chromatogram is 100 mm-500 mm, and the inner diameter of the chromatogram is 20 mm-200 mm.
Due to the adoption of the technical scheme, compared with the prior art, the invention has the technical progress that:
(1) the invention takes alcohol as extracting solution, and adopts two separation means of macroporous resin and preparative liquid chromatography, and the content of triterpenic acid in the prepared components is high and exceeds 90 percent.
(2) The invention adopts AB-8 or HPD100 adsorption resin. The two are styrene type weak polar or non-polar polymers, the service life is long, and the elution efficiency of the triterpenic acid is high.
(3) The method adopts the preparative liquid chromatography separation for on-line monitoring, has targeted separation and high separation efficiency, and the content of the triterpenic acid component can reach more than 90 percent.
The present invention will be described in further detail with reference to specific examples.
Drawings
FIG. 1: HPLC chromatograms of betulinic acid (a), oleanolic acid (b), and ursolic acid (c) controls;
FIG. 2: ultraviolet absorption spectrogram of betulinic acid (a), oleanolic acid (b) and ursolic acid (c) reference substances;
FIG. 3: an HPLC chromatogram of the triterpenic acid preparation component;
FIG. 4: ultraviolet absorption spectra of 3 main components (a, b, c) in the triterpenic acid preparation component.
Detailed Description
The reagents used in the following examples were all the conventional commercially available reagents unless otherwise specified, and the test methods were the conventional test methods unless otherwise specified.
EXAMPLE 1 preparation of triterpene acid from jujube powder
The preparation method is carried out according to the following steps:
(1) preparation method of fructus Jujubae powder
Removing the core of the jujube, and crushing to obtain jujube powder;
(2) preparation of crude extract of jujube
Extracting fructus Jujubae powder with 80% ethanol solution at 85 deg.C under reflux for 2 hr for 2 times, concentrating the extractive solution under reduced pressure, and recovering ethanol solution to obtain fructus Jujubae crude extract;
(3) preparation of triterpene acid macroporous resin separation component
Separating the jujube crude extract by using an AB-8 macroporous adsorption resin column, wherein the volume ratio of the sample loading to the separation material is 1: 200, the separation steps are as follows: washing with pure water until a separation column is colorless, eluting with 2 column volumes of 70% ethanol solution containing 0.1% formic acid to remove impurities, eluting with 4 column volumes of 100% ethanol solution containing 0.1% formic acid as eluent at the flow rate of 1.5 column volumes/hour, collecting the eluent, and recovering the organic solvent to obtain a triterpenic acid macroporous resin separation component;
(4) preparation of triterpenic acid residue
Separating components of triterpenic acid by macroporous resin, extracting, adding 2 times of pure water, standing at 6 deg.C for 12 hr, and centrifuging at 4000r/min for 8min to obtain triterpenic acid residue;
(5) preparation of triterpenic acid extract
Dissolving the triterpenic acid residue with methanol, separating by preparative liquid chromatography under the conditions that a C18 silica gel bonded stationary phase with the particle size of 5 mu m is used as a chromatographic filler, methanol and 0.1% formic acid aqueous solution are used as a mobile phase, isocratic elution is carried out by 85% methanol, the column efficiency is 5000 plates/m, the length of a chromatographic column is 150 mm, the inner diameter of the chromatographic column is 21.2 mm, collecting a target component, and drying to obtain the triterpenic acid extract. The total content of triterpenic acid in the triterpenic acid preparation component is 92.1%. The triterpenic acid preparation component mainly comprises betulinic acid, ursolic acid and oleanolic acid.
Example 2A method for preparing triterpenic acid from jujube residue
The preparation method is carried out according to the following steps:
(1) preparing fructus Jujubae residue
Removing cores of the jujube fruits, crushing to obtain jujube powder, adding an extraction solvent, wherein the weight-volume ratio of the jujube powder to the extraction solvent is 1: 10, heating and pressurizing for extraction, wherein the extraction solvent is 15% ethanol water solution, the extraction temperature is 80 ℃, the extraction time is 20 min each time, the extraction is repeated for 2 times, and filtering is carried out to obtain jujube residues;
(2) preparation of crude extract of jujube
Extracting fructus Jujubae residue with anhydrous ethanol under reflux at 80 deg.C for 90 min for 2 times, concentrating the extractive solution under reduced pressure, and recovering ethanol to obtain fructus Jujubae crude extract;
(3) preparation of triterpene acid macroporous resin separation component
Separating the jujube crude extract by using an AB-8 macroporous adsorption resin column, wherein the volume ratio of the sample loading to the separation material is 1: 200, the separation steps are as follows: washing with pure water until the separation column is colorless, eluting with 2 column volumes of 70% ethanol solution containing 0.1% formic acid to remove impurities, eluting with 4 column volumes of 0.1% formic acid-containing ethanol solution as eluent at a flow rate of 1.5 column volumes/hour, collecting the eluent, and recovering the organic solvent to obtain a triterpenic acid macroporous resin separation component;
(4) preparation of triterpenic acid residue
Separating components of triterpenic acid by macroporous resin, extracting, adding 2 times of pure water, standing at 6 deg.C for 12 hr, and centrifuging at 4000r/min for 8min to obtain triterpenic acid residue;
(5) preparation of triterpenic acid extract
Dissolving the triterpenic acid residue with methanol, separating by preparative liquid chromatography under the conditions that a C18 silica gel bonded stationary phase with the particle size of 5 mu m is used as a chromatographic packing, methanol and 0.1% formic acid aqueous solution are used as a mobile phase, isocratic elution is carried out by 80% methanol, the column efficiency is 8000 column plates/m, the length of a chromatographic column is 150 mm, the inner diameter of the chromatographic column is 21.2 mm, collecting a target component, and drying to obtain the triterpenic acid extract. The total content of triterpenic acid in the triterpenic acid preparation component is 95%. The triterpenic acid preparation component mainly comprises betulinic acid, ursolic acid and oleanolic acid.
Example 3-8 preparation method of triterpenic acid in jujube Material
Examples 3 to 8 are methods for preparing triterpenic acid from a jujube material, the operation steps are the same as those in examples 1 and 2, and only the type and purity of alcohol and the control parameters such as reaction time and reaction temperature in the process are different, which are specifically shown in the following table:
example 9A method for preparing fructus Jujubae dregs
Removing cores of the jujube fruits, crushing to obtain jujube powder, adding an extraction solvent, wherein the weight-volume ratio of the jujube powder to the extraction solvent is 1: and 8, heating and pressurizing for extraction, wherein the extraction solvent is 15% ethanol water solution, the extraction temperature is 80 ℃, the extraction is carried out for 15 min each time, the extraction is repeated for 2 times, and the filtration is carried out to obtain the jujube residues.
Example 10-19 preparation method of jujube residue
Examples 10 to 19 are respectively a method for preparing jujube dregs, the operation steps are the same as those in example 9, and only parameters such as the type, volume, extraction temperature, extraction mode, extraction time, extraction times and the like of an extraction solvent are different, which are specifically shown in the following table:
EXAMPLE 20 characterization of the major component in the triterpenoic acid extract
In this example, the main components of the triterpenic acid extract prepared in any one of examples 1 to 8 were characterized as follows:
a detection instrument: high performance liquid chromatograph equipped with diode array detector
Detection conditions are as follows:
a chromatographic column: c18 chromatography columns (4.6 x 250 mm, 5 μm);
mobile phase: the volume ratio is 27: 55: 18 parts of acetonitrile, methanol and 10 mmol/L of ammonium acetate aqueous solution; flow rate: 0.8 mL/min;
detection wavelength: 210 nm. Scanning wavelength range: 210-400 nm.
Preparation of control solutions: accurately weighing appropriate amount of triterpenic acid reference substances (betulinic acid, oleanolic acid and ursolic acid), dissolving with methanol to obtain stock solutions of about 1mg/mL, and preparing into mixed reference substance solution containing betulinic acid, oleanolic acid and ursolic acid of about 0.1 mg/mL.
Preparation of sample solution: accurately weighing about 10 mg of triterpene acid preparation component powder, dissolving with methanol, metering to 50 mL, shaking up, filtering with a 0.45 mu m microporous membrane, and taking the filtrate as a sample solution.
Measurement of sample solution: and respectively taking 10 muL of reference substance solution and sample solution for sample introduction and measurement according to the detection conditions.
The experimental results are shown in fig. 1-4, wherein a, b, and c in fig. 1 are chromatographic peaks of betulinic acid, oleanolic acid, and ursolic acid control, respectively, and the retention times are 37.454 min, 43.853 min, and 45.221 min, respectively; in FIG. 2, a, b and c are the ultraviolet absorption spectra of the reference substances betulinic acid, oleanolic acid and ursolic acid, respectively, and it can be seen that the 3 compounds have no characteristic maximum ultraviolet absorption wavelength; retention times of chromatographic peaks a, b, c in the preparation fraction of fig. 3 were 37.404 min, 43.858 min and 45.204 min, respectively; FIG. 4 is a diagram of the ultraviolet absorption spectra corresponding to a, b, c 3 chromatographic peaks in the prepared component.
From fig. 1 and 3, it can be seen that the retention times of a, b, c 3 chromatographic peaks in the prepared components correspond well to the retention times of the reference betulinic acid, oleanolic acid and ursolic acid, respectively; as can be seen from fig. 2 and 4, the ultraviolet absorption spectrograms corresponding to the a, b and c 3 chromatographic peaks in the prepared component are respectively consistent with the ultraviolet absorption spectrograms of the reference substances betulinic acid, oleanolic acid and ursolic acid, and the three peaks are main peaks. Therefore, 3 peaks of a, b and c in the triterpenic acid preparation component respectively correspond to betulinic acid, oleanolic acid and ursolic acid, and the triterpenic acid preparation component is the main component of the preparation component.
Example 21 determination of Total triterpene acid content in triterpene acid extracts
In this example, the total content of triterpenic acid in the triterpenic acid preparation components prepared in any one of examples 1 to 8 was determined as follows:
a detection instrument: visible spectrophotometer
Preparing a reference substance solution: taking about 10 mg of oleanolic acid reference substance, precisely weighing, placing in a 100 mL volumetric flask, dissolving with methanol, fixing the volume to the scale, and shaking up to obtain oleanolic acid reference substance solution.
Drawing a standard working curve: accurately sucking 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0mL of the control solution into a 10 mL test tube with a plug, evaporating in a water bath, and standing at room temperature. Then accurately absorbing 0.5mL (prepared immediately after use) of 5% vanillin-glacial acetic acid and 1.0mL of perchloric acid respectively, mixing uniformly, covering a plug, placing in a 70 ℃ water bath for 15 min, taking out, immediately placing in the ice water bath to cool to room temperature, transferring to a 10 mL volumetric flask, adding glacial acetic acid to dilute to a scale, shaking up, and using as each reference substance working solution. The absorbance values were measured at 554 nm wavelength, respectively. And drawing a standard working curve by taking the concentration of the working solution of the reference substance as an abscissa and the absorbance value of the working solution as an ordinate.
Preparation of sample solution: about 10 mg of sample is taken, precisely weighed, placed in a 100 mL volumetric flask, dissolved by methanol and fixed to the volume to the scale, and shaken up. Accurately sucking 0.5mL of the sample solution into a 10 mL glass test tube with a plug, evaporating in a water bath to dryness, and performing the following operation in the same way as the preparation of a reference working solution as a sample solution. Meanwhile, 0.5mL of 5% vanillin-glacial acetic acid and 1.0mL of perchloric acid are directly added without adding a sample solution, then the glacial acetic acid is added to dilute to a scale, and the mixture is shaken up, and the solution is used as a blank control solution.
Measurement of sample solution: under the same experimental conditions of the measurement standard working curve, measuring the absorbance values of the blank solution and the sample measurement solution, calculating the mass concentration rho of the oleanolic acid in the sample measurement solution and the blank solution according to a linear regression equation, and then calculating the content of the triterpenic acid (in terms of the oleanolic acid) in the sample according to the following formula. The calculation of the analysis results is expressed as follows:
the content of triterpenic acid (in terms of oleanolic acid) in the sample was calculated according to formula (1):
results of the experiment
Claims (5)
1. A preparation method of triterpenic acid in jujube material is characterized in that: the preparation method is carried out according to the following steps:
(1) preparation of crude extract of jujube
Carrying out reflux extraction on the jujube material by using 90-100% alcohol solution at the temperature of 80-90 ℃, extracting for 2-3 times after each extraction for 60-120 min, concentrating the extracting solution under reduced pressure, and recovering the alcohol solution to obtain a jujube crude extract;
(2) preparation of triterpene acid macroporous resin separation component
Separating the jujube crude extract by macroporous resin column chromatography, wherein the volume ratio of the sample loading amount to the separation material is 1: 100-500, and the separation steps are as follows: washing with pure water until a separation column is colorless, eluting with 1-3 column volumes of 60-70% alcohol solution containing 0.1-1% formic acid or acetic acid to remove impurities, eluting with eluent at the flow rate of 1-3 column volumes/hour, collecting the eluent, and recovering an organic solvent in the eluent to obtain a triterpenic acid macroporous resin separation component;
the macroporous resin is AB-8 or HPD100 adsorption resin;
the eluent is 90-100% alcoholic solution containing 0.1% -1% formic acid or acetic acid and 2-5 column volumes;
the alcohol solution is methanol or ethanol solution;
(3) preparation of triterpenic acid residue
Separating components of triterpenic acid macroporous resin, adding pure water with the volume of 1-2 times of that of extractum, standing for 12-24 hours at 4-8 ℃, and keeping for 3000-3000
Centrifuging at 6000r/min for 5-10 min to obtain triterpenic acid residue;
(4) preparation of triterpenic acid extract
Dissolving the triterpenic acid residue with methanol, separating by preparative liquid chromatography, collecting target components, and drying to obtain the triterpenic acid extract;
the separation conditions for preparing the liquid chromatogram are that C18 silica gel bonded stationary phase with the particle size of 5 or 10 mu m is used as chromatographic packing, methanol and 0.1% formic acid aqueous solution are used as mobile phase, isocratic elution is carried out by using 70-90% methanol, the column efficiency of the chromatographic column is 2000-10000 tower plates/m, the length of the chromatographic column is 100-500 mm, and the inner diameter of the chromatographic column is 20-200 mm.
2. The method for preparing triterpenic acid in the jujube material as claimed in claim 1, wherein the triterpenic acid is: the jujube material in the step (1) comprises jujube powder and jujube residues.
3. The method for preparing triterpenic acid in the jujube material as claimed in claim 2, wherein: the preparation method of the jujube powder comprises the following steps: removing core from fructus Jujubae, and pulverizing to obtain fructus Jujubae powder.
4. The method for preparing triterpenic acid in the jujube material as claimed in claim 2, wherein: the preparation method of the jujube residues comprises the following steps: removing the core of the jujube fruit, and crushing to obtain jujube powder, wherein the jujube powder is prepared by mixing the jujube powder with an extraction solvent according to the weight-volume ratio of 1: adding the raw materials into an extraction solvent according to the proportion of 6-10, heating and pressurizing for extraction for 15-30 min or heating and refluxing for extraction for 1-2 h at the extraction temperature of 70-90 ℃, repeatedly extracting for 2-3 times, combining the extracting solutions, concentrating under reduced pressure, and filtering to obtain the jujube residues.
5. The method for preparing triterpenic acid in the jujube material as claimed in claim 4, wherein: the extraction solvent is 0-30% ethanol or methanol water solution.
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| CN101156914A (en) * | 2007-10-18 | 2008-04-09 | 西北大学 | Method for extracting triterpenic acid from Chinese date dregs |
| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101156914A (en) * | 2007-10-18 | 2008-04-09 | 西北大学 | Method for extracting triterpenic acid from Chinese date dregs |
| 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 |
Non-Patent Citations (2)
| Title |
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| 大孔吸附树脂分离纯化枣渣中三萜酸的研究;樊君;《离子交换与吸附》;20081231;第24卷(第5期);第426-433页 * |
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