CN116023519B - Application of nitraria polysaccharide in preparing antitumor drugs and extraction method - Google Patents
Application of nitraria polysaccharide in preparing antitumor drugs and extraction method Download PDFInfo
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Abstract
The invention discloses a method for extracting nitraria tangutorum polysaccharide by an ultrasonic enzymolysis method, and belongs to the technical field of extraction of effective components of traditional Chinese medicines. The invention aims to provide an optimized process for extracting the polysaccharide from the nitraria tangutorum bobr by a water extraction and alcohol precipitation method. According to the invention, a Box-Behnken response surface test is designed by taking the extraction time, the extraction temperature and the liquid-material ratio as influencing factors, and the optimal extraction condition is determined to be 10mL/g of liquid-material ratio; ultrasonic extraction time is 67min; the extraction temperature was 42 ℃. The polysaccharide of the nitraria tangutorum bobr obtained by the extraction process has good inhibitory activity on proliferation of hepatoma cells SMMC-7721 and HepG2 in vitro.
Description
Technical Field
The invention belongs to the technical field of extraction of effective components of traditional Chinese medicines, and relates to a preparation method and application of a nitraria tangutorum polysaccharide.
Background
The nitraria tangutorum bobr (Sophora davidii) is a plant of the genus Sophora of the family Leguminosae, takes roots, stems, leaves and flowers as medicines, is one of the common medicines for minority nationality, and is mostly grown in the shrubs beside hillside roads or grass slopes, and is widely distributed in the multiple provinces of Yunnan, guizhou, sichuan and the like in China. Nitraria sibirica has bitter taste and cool nature and is mainly used for clearing heat and detoxicating, and promoting diuresis and detumescence; the whole herb contains active ingredients such as alkaloid, flavonoid glycoside, amino acid, polysaccharide and the like.
Cancer is a disease which afflicts the world, the number of patients still has an increasing trend year by year, and the high death rate of the cancer seriously affects the life health safety of human beings. In recent years, the active ingredients of traditional Chinese medicines are paid attention to in the field of cancer treatment, and the development of new anticancer natural medicines becomes a research hotspot. Compared with the existing common anticancer methods such as surgery, radiotherapy and chemotherapy, the traditional Chinese medicine active ingredients have the advantages of both effectiveness and bioavailability, and can reduce the discomfort of the patient during the treatment process. At present, the active ingredients of the traditional Chinese medicine are widely accepted and become one of the cancer treatment means.
The current research shows that the active ingredient of the nitraria tangutorum bobr has good blood sugar regulation and anti-inflammatory effects, and also has good activities in the aspects of antioxidant activity, immunoregulation and the like. The invention provides an extraction method of nitraria tangutorum polysaccharide and application of the nitraria tangutorum polysaccharide in the field of liver cancer resistance, and provides a reference for further research in the field.
Disclosure of Invention
The invention aims at providing a method for extracting the polysaccharide from the nitraria tangutorum bobr by an ultrasonic enzymolysis method and provides a new application direction of the polysaccharide from the nitraria tangutorum bobr.
In order to achieve the purpose of the invention, the following technical means are specifically adopted:
the extraction method of the nitraria tangutorum polysaccharide comprises the following steps:
(1) Crushing: placing proper amount of flos Buddlejae in a traditional Chinese medicine pulverizer, pulverizing, and sieving to obtain flos Buddlejae coarse powder;
(2) Extracting: uniformly mixing the coarse powder of the nitraria tangutorum bobr obtained in the step (1) with purified water, adding 1% of complex enzyme, carrying out ultrasonic extraction for 2 times, filtering, and combining the two filtrates to obtain a crude extract of the nitraria tangutorum bobr;
(3) Concentrating: concentrating the crude extract of the polysaccharide of the nitraria tangutorum bobr obtained in the step (2) to be sticky under reduced pressure to obtain concentrated solution of the polysaccharide of the nitraria tangutorum bobr for standby;
(4) Alcohol precipitation: cooling the concentrated solution obtained in the step (3), adding an ethanol solution, uniformly stirring, and separating supernatant and lower-layer sediment in a centrifuge; taking supernatant after centrifugation once, repeating the step, collecting sediment twice, and recycling supernatant obtained by centrifugation;
(5) Refining: adding purified water into the precipitate obtained in the step (4) to stir and dissolve the precipitate to obtain a nitraria polysaccharide solution; repeating the step (4) to carry out secondary alcohol precipitation on the redissolved nitraria tangutorum polysaccharide solution;
(6) And (3) drying: transferring all the alcohol sediment obtained in the step (5) into an evaporation dish, and drying in an oven until the weight is constant to obtain the nitraria tangutorum polysaccharide.
Preferably, the nitraria tangutorum bobr obtained in the step (1) is crushed into coarse powder with the particle size of 15-80 meshes.
Preferably, the liquid-material ratio of the nitraria tangutorum bobr coarse powder and water in the step (2) is 5-15 mL/g; the ultrasonic extraction time is 30-90 min; the ultrasonic power is 300-500W; the extraction temperature is 20-70 ℃. The complex enzyme is one or more of cellulase, amylase, pectase, xylanase and beta-glucanase.
Preferably, the vacuum degree in the decompression concentration process in the step (3) is controlled to be between-0.07 and-0.1 Mpa; the temperature of the rotary steaming water bath is 40-60 ℃; concentrating to 1/5-1/15 of the volume of the added water.
Preferably, the concentration of the ethanol in the step (4) is 60-95%, the volume of the added ethanol is 3-5 times of the volume of the concentrated solution, and the centrifugal speed is 4500-5500 r/min.
Preferably, the drying temperature in the step (6) is 40-60 ℃; the drying time is 20-60 min.
Preferably, the polysaccharide of the nitraria tangutorum bobr obtained by the extraction process has an inhibition effect on cancer cell proliferation.
Preferably, the cancer cells are liver cancer cells SMMC-7721 and HepG2.
Advantageous effects
The invention adopts an ultrasonic enzymolysis auxiliary extraction method, and the complex enzyme can hydrolyze the cell wall of the nitraria tangutorum bobr and can better separate out the effective active ingredients by matching with ultrasonic extraction. The invention adopts a water extraction and alcohol precipitation method, and the ethanol in the step can be recycled, so that the energy consumption and pollution are reduced.
The polysaccharide extracted by the method has an inhibitory effect on the proliferation of liver cancer cells in vitro.
Drawings
FIG. 1A standard curve for detecting polysaccharide content by sulfuric acid-anthrone method.
FIG. 2 is a graph showing the single-factor effect of the liquid-to-material ratio of the Nitraria sibirica polysaccharide.
FIG. 3 is a graph showing the single-factor effect of the polysaccharide extraction time from Nitraria sibirica.
FIG. 4 is a graph showing the single-factor effect of the number of polysaccharide extractions from Nitraria tangutica.
FIG. 5 is a graph showing the single-factor effect of the temperature of the polysaccharide extraction from Nitraria tangutica.
FIG. 6 is a graph of the liquid-to-material ratio of the polysaccharide from the Nitraria sibirica versus the extraction time response surface.
FIG. 7 is a graph showing the liquid-to-material ratio of the polysaccharide from the Nitraria sibirica and the extraction temperature response surface.
FIG. 8 is a graph showing the time-temperature response of the extraction of the polysaccharide from Nitraria tangutica.
Detailed description of the preferred embodiments
The sulfuric acid-anthrone method establishes a polysaccharide standard curve.
Sulfuric acid solution (12 mol/L): a sulfuric acid solution of 12mol/L was prepared from analytically pure concentrated sulfuric acid (18 mol/L).
Anthrone solution: 50mg of finely ground anthrone was added to 50mL of sulfuric acid solution, and the anthrone was shaken off and left overnight to give a saturated solution. The solution was placed in a dark glass bottle with a stopper, stored in the dark, and filtered when used.
About 0.2g of dextran is taken, precisely weighed, placed in 30mL of sulfuric acid solution, left overnight, then diluted to 250mL with sulfuric acid solution, and uniformly mixed to prepare 0.8mg/mL dextran standard solution. And respectively precisely transferring 0.5mL, 1mL, 1.5mL, 2.0mL, 2.5mL and 3.0mL of dextran standard solution into a 50mL volumetric flask, adding 4mL of anthrone solution, shaking uniformly, carrying out water bath at 90 ℃ for 30 minutes, cooling to room temperature, diluting to a scale with sulfuric acid solution, and shaking uniformly to obtain a standard series solution. Absorbance values were measured at 625nm and a standard curve was drawn. See fig. 1.
The liquid-to-material ratio of the nitraria tangutorum polysaccharide affects the test by a single factor.
Weighing 10g of the nitraria tangutorum bobr coarse powder, uniformly mixing the nitraria tangutorum bobr coarse powder with purified water according to the liquid-to-material ratio of 6-12 mL/g, adding 1% complex enzyme, carrying out ultrasonic extraction for 2 times at 50 ℃ for 60min each time, filtering, combining the two filtrates, and determining the result, wherein the result is shown in figure 2.
The single factor of the extraction time of the polysaccharide from the nitraria tangutorum bobr influences the test.
Weighing 10g of the nitraria tangutorum bobr coarse powder, uniformly mixing the nitraria tangutorum bobr coarse powder with purified water according to the liquid-to-material ratio of 10mL/g, adding 1% of complex enzyme, carrying out ultrasonic extraction for 2 times at 50 ℃, respectively extracting for 30-90 min, filtering, combining the two filtrates, and determining the result, wherein the result is shown in figure 3.
The number of extraction times of the polysaccharide from the nitraria tangutorum bobr has a single factor to influence the test.
Weighing 10g of the nitraria tangutorum bobr coarse powder, uniformly mixing the nitraria tangutorum bobr coarse powder with purified water according to the liquid-to-material ratio of 10mL/g, adding 1% of complex enzyme, respectively carrying out ultrasonic extraction for 1-5 times at 50 ℃ for 60min each time, filtering, combining the two filtrates, and determining the result, wherein the result is shown in figure 4.
And (3) performing a single-factor influence test on the extraction temperature of the polysaccharide of the nitraria tangutorum bobr.
Weighing 10g of the coarse powder of the spiny flowers, uniformly mixing the coarse powder of the spiny flowers with purified water according to the liquid-to-material ratio of 10mL/g, adding 1% of compound enzyme, respectively carrying out ultrasonic extraction for 2 times at 30-70 ℃ for 60min each time, filtering, combining the filtrates, and determining the result, wherein the result is shown in figure 4.
According to the single-factor test result, the content of the polysaccharide of the nitraria tangutorum bobr is positively correlated with the extraction times, and the time, the energy consumption and other factors are comprehensively considered, so that the extraction efficiency is improved, the extraction times are determined to be 2 times, according to the better group in the single-factor test result, the liquid-material ratio, the extraction time and the extraction temperature are used as influencing factors, the response surface test of the 17 groups of the polysaccharide extraction method is designed through Design-Expert software, and the polysaccharide extraction rate is used as an investigation index.
Response surface design and results.
Fitting a Box-Behnken response surface model.
The table shows that the three models have extremely significant (P < 0.01) and the respective mismatch terms have no significant (P > 0.05), so that the equation has good fitting property, can be used for pre-test results, and the response surface test curved surface diagrams are shown in fig. 6, 7 and 8.
Polysaccharide extraction yield (％)＝-42.23303+4.22560×A+0.294582×B+0.718414×C-0.005105×A×B+0.000125×A×B-0.000052×B×C-0.199700×A2-0.001769×B2-0.007357×C2
According to response surface optimization, the method for optimizing and extracting the nitraria tangutorum polysaccharide comprises the following steps:
(1) Crushing: placing proper amount of flos Buddlejae in a traditional Chinese medicine pulverizer, pulverizing, and sieving with 20 mesh sieve to obtain flos Buddlejae coarse powder;
(2) Extracting: uniformly mixing the coarse powder of the nitraria tangutorum bobr obtained in the step (1) with purified water according to the liquid-to-material ratio of 10mL/g, adding 1% complex enzyme, carrying out ultrasonic extraction for 2 times at 42 ℃ for 67min each time, filtering, and combining the two filtrates to obtain a crude extract of the nitraria tangutorum bobr;
(3) Concentrating: concentrating the crude extract of the polysaccharide of the nitraria tangutorum bobr obtained in the step (2) to 1/10 of the volume of water to obtain a concentrated solution of the polysaccharide of the nitraria tangutorum bobr for standby;
(4) Alcohol precipitation: cooling the concentrated solution obtained in the step (3), adding three times of 95% ethanol solution, stirring uniformly, and separating supernatant and lower precipitate in a centrifuge; taking supernatant after centrifugation once, repeating the step, collecting sediment twice, and recycling supernatant obtained by centrifugation;
(5) Refining: adding purified water into the precipitate obtained in the step (4) to stir and dissolve the precipitate to obtain a nitraria polysaccharide solution; repeating the step (4) to carry out secondary alcohol precipitation on the redissolved nitraria tangutorum polysaccharide solution;
(6) And (3) drying: transferring all the alcohol sediment obtained in the step (5) into an evaporation dish, and drying in an oven until the weight is constant to obtain the nitraria tangutorum polysaccharide.
And 3 times of parallel tests are carried out according to the optimized extraction process, so that the average extraction rate of the polysaccharide of the nitraria tangutorum is 5.32%, the average extraction rate is close to a model predicted value, and the model optimizing effect is ideal.
MTT colorimetric method is used for detecting the influence of the nitraria polysaccharide on proliferation of liver cancer cells SMMC-7721.
SMMC-7721 cell culture
Thawing frozen SMMC-7721 cells in a water bath at 37 ℃, sucking a proper amount of cell suspension into a centrifuge tube, centrifuging at 800r/min for 6min, discarding supernatant, adding MEM culture solution containing 10% fetal bovine serum, and culturing in a culture box with 5% CO 2 at 37 ℃ for 24h until the cells adhere to the wall.
MTT assay
Cells in logarithmic growth phase were digested with 0.25% trypsin, and 1X 10 4 cells/mL of the cell suspension was inoculated into 96-well plates at 100. Mu.L per well. After adherence, adding different concentrations (100, 200, 400, 800 mug/mL) of the nitraria tangutorum polysaccharide solution into each hole, setting a blank group and cis-platinum positive control, setting 4 compound holes in each group, culturing for 24 hours in a culture box with 5% CO 2 at 37 ℃, adding 50 mug of 1mg/mLMTT solution, continuously culturing for 2 hours, sucking out supernatant, adding 100 mug LDMSO into each hole, oscillating for 10 minutes in a shaking table, and measuring OD value at 490nm of an enzyme-labeled instrument.
The inhibition rate calculation formula: cell growth inhibition = (1-average OD value of experimental group/average OD value of positive control group) ×100%.
MTT method results
MTT colorimetric method is used for detecting influence of the polysaccharide of the Nitraria tangutica on proliferation of hepatoma cells HepG 2.
HepG2 cell culture
Thawing frozen HepG2 cells in a water bath at 37 ℃, sucking a proper amount of cell suspension into a centrifuge tube, centrifuging at 800r/min for 6min, discarding supernatant, adding MEM culture solution containing 10% fetal bovine serum, and culturing in a 5% CO 2 incubator at 37 ℃ for 24h until the cells adhere to the wall.
MTT assay
Cells in logarithmic growth phase were digested with 0.25% trypsin, and 1X 10 4 cells/mL of the cell suspension was inoculated into 96-well plates at 100. Mu.L per well. After adherence, adding different concentrations (0, 200, 400 and 800 mug/mL) of the nitraria tangutorum polysaccharide solution into each hole, setting a blank group and cis-platinum positive control, setting 4 compound holes in each group, culturing for 24 hours in a culture box with 5% CO 2 at 37 ℃, adding 50 mug of 1mg/mLMTT solution, continuously culturing for 2 hours, sucking out supernatant, adding 100 mug LDMSO into each hole, oscillating for 10 minutes in a shaking table, and measuring OD value at 490nm of an enzyme-labeled instrument.
The inhibition rate calculation formula: cell growth inhibition = (1-average OD value of experimental group/average OD value of positive control group) ×100%.
MTT method results
Claims (9)
1. The application of the nitraria polysaccharide in preparing the anti-liver cancer medicament is characterized in that the preparation method of the nitraria polysaccharide comprises the following steps:
(1) Crushing: placing proper amount of flos Buddlejae in a traditional Chinese medicine pulverizer, pulverizing, and sieving to obtain flos Buddlejae coarse powder;
(2) Extracting: uniformly mixing the coarse powder of the nitraria tangutorum bobr obtained in the step (1) with water, adding compound enzyme, carrying out ultrasonic extraction for 2 times, filtering, and combining the two filtrates to obtain a crude extract of the nitraria tangutorum bobr;
(3) Concentrating: concentrating the crude extract of the nitraria tangutorum bobr obtained in the step (2) under reduced pressure to be sticky to obtain a nitraria tangutorum bobr polysaccharide concentrated solution for standby;
(4) Alcohol precipitation: cooling the concentrated solution obtained in the step (3), adding an ethanol solution, uniformly stirring, and separating supernatant and lower-layer sediment in a centrifuge; taking supernatant after centrifugation once, repeating the step, collecting sediment twice, and recycling supernatant obtained by centrifugation;
(5) Refining: adding purified water into the precipitate obtained in the step (4) to stir and dissolve the precipitate to obtain a nitraria polysaccharide solution; repeating the step (4) to carry out secondary alcohol precipitation on the redissolved nitraria tangutorum polysaccharide solution;
(6) And (3) drying: transferring all the alcohol sediment obtained in the step (5) into an evaporation dish, and drying in an oven until the weight is constant to obtain the nitraria tangutorum polysaccharide.
2. The use of the polysaccharide from Nitraria tangutica as claimed in claim 1 for preparing anti-hepatoma drugs, wherein said Nitraria tangutica is crushed to a coarse powder size of 15-80 mesh.
3. The application of the nitraria polysaccharide in preparing anti-liver cancer drugs according to claim 1, wherein the liquid-to-material ratio of the nitraria coarse powder to water is 5-15 mL/g; the ultrasonic extraction time is 30-90 min; the ultrasonic power is 300-500W; the extraction temperature is 20-70 ℃.
4. The application of the nitraria polysaccharide in preparing the anti-liver cancer medicament according to claim 1, wherein the complex enzyme is one or more of cellulase, amylase, pectinase, xylanase and beta-glucanase.
5. The application of the nitraria polysaccharide in preparing anti-liver cancer drugs according to claim 1, wherein the vacuum degree in the decompression concentration process is controlled between-0.07 and-0.1 Mpa; the temperature of the rotary steaming water bath is 40-60 ℃; concentrating to 1/5-1/15 of the volume of the added water.
6. The application of the nitraria polysaccharide in preparing the anti-liver cancer medicament according to claim 1, wherein the concentration of ethanol is 60-95%, the volume of added ethanol is 3-5 times of the volume of the concentrated solution, and the centrifugal speed is 4500-5500 r/min.
7. The use of the nitraria tangutorum bobr polysaccharide in preparing anti-hepatoma drugs according to claim 1, wherein the drying temperature is 40-60 ℃; the drying time is 20-60 min.
8. The application of the nitraria tangutorum bobr polysaccharide in preparing anti-hepatoma drugs according to claim 1, wherein the nitraria tangutorum bobr polysaccharide extracted by the process has an inhibitory effect on hepatoma cell proliferation.
9. The application of the nitraria polysaccharide in preparing anti-liver cancer drugs according to claim 8, wherein the cancer cells are liver cancer cells SMMC-7721 and HepG2.
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Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59104321A (en) * | 1983-10-12 | 1984-06-16 | T O C:Kk | Preparation of medicinal substance from leaf of sasa albo-marginata |
| WO2009031809A2 (en) * | 2007-09-05 | 2009-03-12 | Macrocare Tech., Ltd. | Method of preparation an inclusion-complex comprising hydrophobic physiological activation material including with cyclodextrin and its use |
| CN101555288A (en) * | 2008-04-08 | 2009-10-14 | 中国科学院西北高原生物研究所 | Extracting refining method of water soluble polysaccharide in berry of nitraria tangutorum bobr |
| CN104987428A (en) * | 2015-07-23 | 2015-10-21 | 中国科学院西北高原生物研究所 | Compound enzyme-microwave extraction method for optimizing Nitraria polysaccharide by response surface methodology |
| CN105077215A (en) * | 2015-08-20 | 2015-11-25 | 中国科学院西北高原生物研究所 | Cordyceps sinensis freshness-retaining product taking Nitraria tangutorun polysaccharide as natural bacteriostatic agent |
| CN105693878A (en) * | 2016-03-11 | 2016-06-22 | 李正梅 | Method for extracting lentinan from shiitake mushrooms and shiitake mushroom product |
| CN107556401A (en) * | 2017-10-20 | 2018-01-09 | 上海中医药大学附属曙光医院 | A kind of kuh-seng polysaccharide, its preparation method and liver protection and immunomodulation applications |
| CN110746517A (en) * | 2019-12-06 | 2020-02-04 | 中国科学院新疆理化技术研究所 | Method for optimizing ultrasonic-assisted extraction of Siberian nitraria polysaccharide based on response surface method and application |
| CN111574639A (en) * | 2020-07-08 | 2020-08-25 | 江苏汉邦科技有限公司 | Method for separating and purifying nitraria tangutorum bobr polysaccharide |
| CN112521522A (en) * | 2020-12-08 | 2021-03-19 | 中国科学院新疆理化技术研究所 | Preparation method and application of Siberian Nitraria polysaccharide effective part |
| CN112812197A (en) * | 2021-01-11 | 2021-05-18 | 中国药科大学 | Lanzhou lily polysaccharide and preparation method and application thereof |
| CN113603805A (en) * | 2021-09-03 | 2021-11-05 | 中国科学院西北高原生物研究所 | Preparation method of nitraria tangutorum bobr polysaccharide |
-
2023
- 2023-01-16 CN CN202310050975.4A patent/CN116023519B/en active Active
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59104321A (en) * | 1983-10-12 | 1984-06-16 | T O C:Kk | Preparation of medicinal substance from leaf of sasa albo-marginata |
| WO2009031809A2 (en) * | 2007-09-05 | 2009-03-12 | Macrocare Tech., Ltd. | Method of preparation an inclusion-complex comprising hydrophobic physiological activation material including with cyclodextrin and its use |
| CN101555288A (en) * | 2008-04-08 | 2009-10-14 | 中国科学院西北高原生物研究所 | Extracting refining method of water soluble polysaccharide in berry of nitraria tangutorum bobr |
| CN104987428A (en) * | 2015-07-23 | 2015-10-21 | 中国科学院西北高原生物研究所 | Compound enzyme-microwave extraction method for optimizing Nitraria polysaccharide by response surface methodology |
| CN105077215A (en) * | 2015-08-20 | 2015-11-25 | 中国科学院西北高原生物研究所 | Cordyceps sinensis freshness-retaining product taking Nitraria tangutorun polysaccharide as natural bacteriostatic agent |
| CN105693878A (en) * | 2016-03-11 | 2016-06-22 | 李正梅 | Method for extracting lentinan from shiitake mushrooms and shiitake mushroom product |
| CN107556401A (en) * | 2017-10-20 | 2018-01-09 | 上海中医药大学附属曙光医院 | A kind of kuh-seng polysaccharide, its preparation method and liver protection and immunomodulation applications |
| CN110746517A (en) * | 2019-12-06 | 2020-02-04 | 中国科学院新疆理化技术研究所 | Method for optimizing ultrasonic-assisted extraction of Siberian nitraria polysaccharide based on response surface method and application |
| CN111574639A (en) * | 2020-07-08 | 2020-08-25 | 江苏汉邦科技有限公司 | Method for separating and purifying nitraria tangutorum bobr polysaccharide |
| CN112521522A (en) * | 2020-12-08 | 2021-03-19 | 中国科学院新疆理化技术研究所 | Preparation method and application of Siberian Nitraria polysaccharide effective part |
| CN112812197A (en) * | 2021-01-11 | 2021-05-18 | 中国药科大学 | Lanzhou lily polysaccharide and preparation method and application thereof |
| CN113603805A (en) * | 2021-09-03 | 2021-11-05 | 中国科学院西北高原生物研究所 | Preparation method of nitraria tangutorum bobr polysaccharide |
Non-Patent Citations (3)
| Title |
|---|
| 响应面法优化白刺花总黄酮提取工艺研究;阳敬;兰慧;左强;;中国民族民间医药(第12期);16-23页 * |
| 我国槐属植物育种及种质资源开发利用进展;毛秀红;荀守华;孙居文;乔玉玲;康智;刘翠兰;燕丽萍;;植物生理学报(第04期);7-14页 * |
| 超声波在药用植物提取中的应用研究;何竞旻;王瑞侠;黄劲松;;池州学院学报(第03期);73-75页 * |
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