CN110128562B - An antitumor fructus Psoraleae polysaccharide, its extraction and separation method, and its application in preparing antitumor drugs - Google Patents
An antitumor fructus Psoraleae polysaccharide, its extraction and separation method, and its application in preparing antitumor drugs Download PDFInfo
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- CN110128562B CN110128562B CN201910456385.5A CN201910456385A CN110128562B CN 110128562 B CN110128562 B CN 110128562B CN 201910456385 A CN201910456385 A CN 201910456385A CN 110128562 B CN110128562 B CN 110128562B
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Images
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
The invention relates to a preparation method of anti-tumor psoralea fruit polysaccharide, which specifically comprises the following steps: 1) crushing fructus Psoraleae, sequentially extracting with petroleum ether and 70 + -5% ethanol at room temperature, heating with ultrapure water, concentrating the filtrate, and precipitating with ethanol to obtain fructus Psoraleae crude polysaccharide; 2) deproteinizing fructus Psoraleae crude polysaccharide by Sevag method, concentrating the obtained supernatant under reduced pressure, precipitating with ethanol, and drying the precipitate to obtain deproteinized fructus Psoraleae crude polysaccharide; 3) taking deproteinized fructus Psoraleae crude polysaccharide, purifying with DEAE-52 cellulose chromatographic column, sequentially performing gradient elution with ultrapure water, 0.05, 0.1, 0.2, 0.3mol/L NaCl solution, wherein the elution peak of distilled water is component PC-1, and the elution peak of 0.2mol/L NaCl solution is component PC-4; 4) polysaccharide components, namely PCp-1 and PCp-4, are respectively obtained by Sephadex G-100 Sephadex column chromatography purification of the components PC-1 and PC-4; PCp-1 and/or PCp-4 are anti-tumor psoralea fruit polysaccharide. The test shows that: the fructus Psoraleae polysaccharide can effectively inhibit activity of A549 cell, and can be used for preparing anti-tumor, especially anti-lung cancer medicine.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine extraction, and particularly relates to an anti-tumor active fructus psoraleae polysaccharide, an extraction and separation method and application in preparation of anti-tumor medicines.
Background
With the aggravation of aging of population, the damage to ecological environment, unhealthy life style and food safety problems, the incidence of tumor and death rate are continuously increased, and serious harm is brought to life, health and society of people. Although common tumor drugs and radiotherapy, chemotherapy and the like have certain treatment effects, the side effects are large. The traditional Chinese medicine is a medical treasury in China, the traditional Chinese medicine often has the characteristics of multiple target points, low toxicity, lasting effect and the like, and the traditional Chinese medicine alone or with the assistance of western medicines for treating tumors has positive significance in the aspects of improving the immunity of organisms, prolonging the life cycle, reducing the recurrence and metastasis rate, relieving side effects and the like. Therefore, research and development of novel natural antitumor drugs from traditional Chinese medicines is a key direction for research of antitumor drugs.
The fructus Psoraleae is Leguminosae plant fructus PsoraleaePsoralea corylifoliaL. the dried ripe fruit, is the traditional Chinese medicine. Notification of further standardization of health food Material management by Ministry of health (Law of defense) [2002]51), fructus psoraleae is an article which can be used for health-care food. Modern researches show that the fructus psoraleae contains various components such as coumarins, flavonoids, monoterpenes and the like, and has various effects of resisting inflammation, resisting bacteria, resisting oxidation, reducing blood sugar and the like. Regarding the research of fructus psoraleae polysaccharide, only several scholars in China carry out preliminary research on the content and the immunological activity of fructus psoraleae crude polysaccharide, and further separation and purification of fructus psoraleae crude polysaccharide are not seen to obtain a single polysaccharide component, and structural analysis and anti-tumor activity research are carried out on the single polysaccharide component.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a psoralea polysaccharide which is extracted and separated from psoralea and has anti-tumor activity.
The invention also provides an extraction and separation method of the psoralea corylifolia polysaccharide and application of the psoralea corylifolia polysaccharide in preparation of antitumor drugs.
In order to achieve the purpose, the invention adopts the following technical scheme:
a preparation method of anti-tumor psoralea corylifolia polysaccharide comprises the following steps:
1) preparing crude polysaccharide: crushing fructus Psoraleae, extracting with petroleum ether at room temperature for degreasing, volatilizing petroleum ether from the residue, extracting with 70 + -5% ethanol at room temperature to remove pigment and small molecular components, volatilizing the residue until no ethanol smell exists, heating and extracting with ultrapure water at 85 + -5 deg.C, hot filtering with Buchner funnel, concentrating the filtrate to obtain concentrated solution, adding anhydrous ethanol into the concentrated solution to make the final concentration be 70-75%, standing, collecting precipitate, sequentially washing with anhydrous ethanol and acetone, and volatilizing solvent to obtain fructus Psoraleae crude polysaccharide;
2) preparing deproteinized fructus psoraleae crude polysaccharide: completely dissolving fructus Psoraleae crude polysaccharide with ultrapure water to obtain fructus Psoraleae crude polysaccharide solution, deproteinizing by Sevag method, concentrating the obtained supernatant under reduced pressure, adding anhydrous ethanol to final concentration of 75 + -2%, standing, centrifuging, collecting precipitate, and drying to obtain deproteinized fructus Psoraleae crude polysaccharide;
3) dissolving deproteinized fructus Psoraleae crude polysaccharide with ultrapure water, centrifuging to remove insoluble substances, loading onto DEAE-52 cellulose chromatographic column, gradient eluting with ultrapure water, 0.05, 0.1, 0.2, and 0.3mol/L NaCl solution in sequence, detecting eluate by phenol-sulfuric acid method, measuring absorbance at 490 nm, drawing polysaccharide elution curve with number of elution tubes as abscissa and absorbance as ordinate, mixing polysaccharide samples of the same elution peak, concentrating, dialyzing, and freeze drying; wherein, the elution peak of the distilled water is a component PC-1, and the elution peak of the 0.2mol/L NaCl solution is a component PC-4;
4) dissolving the component PC-1 with ultrapure water, loading the sample to Sephadex G-100 Sephadex column chromatography, eluting with ultrapure water, detecting by a phenol-sulfuric acid method, measuring the absorbance at 490 nm, drawing a polysaccharide elution curve by taking the number of elution tubes as a horizontal coordinate and the absorbance as a vertical coordinate, combining polysaccharide samples of the same elution peak, and concentrating, dialyzing and freeze-drying the obtained polysaccharide component to be named as PCp-1;
eluting the component PC-4 by the same method as the component PC-1, combining polysaccharide samples with the same elution peak, concentrating, dialyzing, and freeze-drying to obtain a polysaccharide component named PCp-4;
PCp-1 and/or PCp-4 are anti-tumor psoralea fruit polysaccharide.
Specifically, in the step 1), petroleum ether is used for leaching and degreasing for 3-5 times at room temperature, and each time lasts for 2-4 d; extracting with 70 + -5% ethanol at room temperature for 3-5 times, each for 2-4 days; heating with ultrapure water for extraction for 2-4 times, each for 3-5 hr.
Specifically, in the step 2), when the Sevag method is used for deproteinization, the Sevag reagent is composed of chloroform and n-butanol with the volume ratio of 4: 1; the Sevag reagent is added in an amount of 1/4 to 1/2, preferably 1/3, based on the volume of the crude psoralea polysaccharide solution.
The invention also provides the anti-tumor psoralea corylifolia polysaccharide prepared by the method.
The invention also provides the application of the psoralea corylifolia polysaccharide in preparing antitumor drugs or foods.
Furthermore, the application of the psoralea corylifolia polysaccharide in preparing anti-lung cancer medicines or foods is preferred.
Compared with the prior art, the invention has the beneficial effects that:
the invention adopts a special extraction and separation method to separate polysaccharide components PCp-1 and PCp-4 from fructus psoraleae, and takes non-small cell lung cancer A549 cells as an example to investigate the anti-tumor effect of the polysaccharide components, and the test result shows that: the fructus Psoraleae polysaccharide can effectively inhibit the activity of A549 cells, and has good antitumor effect, and can be used for preparing antitumor drugs, especially lung cancer resisting drugs or foods.
Drawings
FIG. 1 shows the elution curve of a chromatographic column of crude psoralea fruit polysaccharide DEAE-52 cellulose;
FIG. 2 shows the elution curves of the components PC-1 and PC-4 by SephadexG-100 gel column chromatography;
FIG. 3 GC chromatograms of standard monosaccharide mixtures;
FIG. 4 GC chromatogram of hydrolysate of PCp-1;
FIG. 5 GC chromatogram of hydrolysate of PCp-4.
Detailed Description
The technical solution of the present invention is further described in detail with reference to the following examples, but the scope of the present invention is not limited thereto.
Example 1
1. A preparation method of anti-tumor psoralea corylifolia polysaccharide specifically comprises the following steps:
1) preparing crude polysaccharide: fructus Psoraleae (4.4 kg) is pulverized, extracted with petroleum ether at room temperature for 3 times (3 days each time), and degreased. Volatilizing petroleum ether from the extracted residue, extracting with 70% ethanol at room temperature for 3 d for 4 times, and volatilizing the extracted residue until no ethanol smell is generated (preferably soaking fructus Psoraleae with petroleum ether and 70% ethanol). Then heating and extracting for 2 times (the material-liquid ratio is about 1 g: 20 mL) with ultrapure water at 85 deg.C, each time for 5 h; and after extraction, carrying out suction filtration on the hot mixture by using a Buchner funnel, combining filtrates, and carrying out rotary evaporation and concentration on the filtrate to about 1/4 of the original volume to obtain a concentrated solution. Adding anhydrous ethanol into the concentrated solution to make the final concentration 70% (volume percentage, the same below), standing at room temperature for 12h, centrifuging to remove supernatant, collecting precipitate, sequentially washing the precipitate with anhydrous ethanol and acetone, and volatilizing solvent to obtain fructus Psoraleae crude polysaccharide.
2) The crude psoralea fruit polysaccharide prepared by the method is completely dissolved by ultrapure water to obtain a psoralea fruit crude polysaccharide solution, and deproteinization is carried out by adopting a Sevag method, which specifically comprises the following steps: adding a Sevag reagent according to 1/3 of the volume of the crude polysaccharide solution of the fructus psoraleae, magnetically stirring and shaking for 30 min, centrifuging at 4000 r/min for 5 min, removing middle denatured protein and lower organic solvent, and continuously repeating the operation on the supernatant until no denatured protein layer appears; the Sevag reagent consists of chloroform and n-butanol in a volume ratio of 4: 1. Concentrating the final supernatant under reduced pressure, adding anhydrous ethanol until the final ethanol concentration is 70%, standing at room temperature for 12 hr, centrifuging, collecting precipitate, and drying to obtain deproteinized fructus Psoraleae crude polysaccharide.
3) Taking 1.0g of the crude polysaccharide of the fructus psoraleae after deproteinization, dissolving the crude polysaccharide with 10 mL of ultrapure water, removing insoluble substances by high-speed centrifugation, loading the crude polysaccharide to a DEAE-52 cellulose chromatographic column, performing gradient elution by using distilled water, 0.05, 0.1, 0.2 and 0.3mol/L NaCl solution in sequence, controlling the flow rate at 1.0 mL/min, collecting one tube every 5 min, detecting the eluent by using a phenol-sulfuric acid method, measuring the absorbance at 490 nm by using a microplate reader, and drawing a polysaccharide elution curve by taking the number of elution tubes as a horizontal coordinate and the absorbance as a vertical coordinate (the elution curve is shown in figure 1). Combining polysaccharide solutions with the same elution peak, concentrating under reduced pressure at 50 ℃, dialyzing (room temperature, molecular weight cutoff of 8000-.
4) Dissolving 200 mg of component PC-1 with 2 mL of ultrapure water, loading the component PC-1 to a Sephadex G-100 Sephadex chromatographic column (1.5 multiplied by 100 cm) for further separation and purification, eluting with ultrapure water, controlling the flow rate at 0.8 mL/min, collecting one tube every 5 min, detecting by a phenol-sulfuric acid method, measuring the absorbance at 490 nm, drawing a polysaccharide elution curve (the elution curve is shown in figure 2) by taking the number of elution tubes as a horizontal coordinate and the absorbance as a vertical coordinate, merging polysaccharide samples of the same elution peak, concentrating under reduced pressure (50 ℃), dialyzing (room temperature, molecular weight cut-off 8000-;
eluting the component PC-4 by the same method as the component PC-1 (the elution curve is shown in figure 2), combining the polysaccharide samples with the same elution peak, concentrating, dialyzing, and freeze-drying to obtain a polysaccharide component named as PCp-4;
PCp-1 and/or PCp-4 are anti-tumor psoralea fruit polysaccharide.
And (3) determining the molecular weight of the psoralea fruit polysaccharide:
the preparation method comprises the following specific steps: taking polysaccharide components PCp-1 and PCp-4, sending to Beijing market for physical and chemical analysisThe molecular weight was determined by size exclusion chromatography as described in Chinese pharmacopoeia (2015). The results showed that the molecular weights of PCp-1 and PCp-4 were 2.721X 10, respectively4And 2.850 × 104g/mol, see Table 1 for details.
TABLE 1 molecular weights of PCp-1 and PCp-4
And determining the components of fructus psoraleae polysaccharide monosaccharide.
3.1 hydrolysis of polysaccharides
Polysaccharide PCp-1 and PCp-4 are weighed accurately, 10 mg of each is added into a5 mL ampoule bottle, 3 mL of 4 mol/L trifluoroacetic acid is added, and a tube is sealed by nitrogen. Hydrolyzing at 110 deg.C for 3h, rotary evaporating to remove trifluoroacetic acid solution, adding small amount of methanol to dissolve residue, rotary evaporating to dry, and repeating for 3 times to obtain hydrolysate.
3.2 derivatization of monosaccharides
Adding 10 mg of hydroxylamine hydrochloride into the hydrolysate, adding 0.5 mL of pyridine, oscillating, mixing uniformly, and placing into a water bath at 90 ℃ for heating reaction for 30 min. Taking out, cooling to room temperature, adding 0.5 mL of acetic anhydride, continuing to react at 90 ℃ for 30 min for acetylation, filtering the reaction product by a 0.22 mu m filter membrane, and injecting the filtered reaction product into a gas chromatograph for analysis; standard monosaccharides were treated in the same manner and a standard monosaccharide derivative mixture was prepared.
3.3 gas chromatography conditions
A chromatographic column: HP (30 m.times.0.35 mm, 0.25 μm); sample introduction temperature: 250 ℃; detector temperature: 280 ℃; temperature program of chromatographic column: maintaining the initial temperature at 100 deg.C for 1 min, and then increasing the temperature from 100 deg.C to 240 deg.C at 4 deg.C/min for 10 min; carrier gas: high-purity nitrogen with the flow rate of 2 mL/min; the sample size is 2 muL.
3.4 monosaccharide composition analysis results
The GC chromatogram of the standard monosaccharide mixture is shown in FIG. 3 (1. L-rhamnose; 2. L-arabinose 3. D-xylose; 4. D-mannose; 5. D-glucose; 6. D-galactose), the GC chromatograms of the monosaccharides after hydrolysis of PCp-1 and PCp-4 are shown in FIG. 4 and FIG. 5, respectively, and the monosaccharide composition of the sample can be determined by comparing the retention time with that in the standard monosaccharide chromatogram, and the results are shown in Table 2.
TABLE 2 monosaccharide composition of PCp-1 and PCp-4
As can be seen from table 2: both the PCp-1 and the PCp-4 are heteropolysaccharides and at least consist of rhamnose, arabinose, xylose, mannose, glucose and galactose, and the molar ratios are different; PCp-1 contains high galactose and arabinose, and PCP-4 mainly consists of rhamnose, xylose and galactose.
Activity analysis of anti-tumor psoralea corylifolia polysaccharides PCp-1 and PCp-4
Non-small cell lung cancer A549 cells in logarithmic growth phase are paved on a 96 micropore plate (2 multiplied by 10)4) After 24h of culture, adding medicines, respectively adding PCp-1 and PCp-4 with different concentrations and positive control cisplatin, acting for 48h, and then detecting the survival condition of cells by adopting an MTT method. The SPSS19.0 software One-Way ANOVA (One-Way ANOVA) is used for comparing the significance difference of the numerical statistics. The results are shown in tables 3 and 4.
TABLE 3 cell viability 48h after treatment of A549 cells with different concentrations of PCp-1
TABLE 4 cell viability 48h after treatment of A549 cells with different concentrations of PCp-4
As can be seen from tables 3 and 4, the survival rate of A549 cells tended to decrease with the increase of the concentrations of PCp-1 and PCp-4 in the medium, and there was a certain difference in the antitumor activities of the two polysaccharides, which was probably related to the composition and content of monosaccharides in the two polysaccharide fractions. When in useWhen the concentration of the PCp-1 and the PCp-4 reaches 100 mu M, the average cell survival rate is only 48.77 percent and 51.87 percent in turn, which shows that the PCp-1 and the PCp-4 have obvious inhibition effect on the survival of A549 cells (IC)50=64.84 and 126.3 μ M), in contrast, its activity is lower than that of the positive control cisplatin (IC)50=11 μM）。
The above test results show that: the psoralea corylifolia polysaccharide can effectively inhibit the activity of A549 cells, so that the psoralea corylifolia polysaccharide has good anti-tumor effect, can be used for preparing anti-tumor medicines, and particularly can be used for preparing anti-lung cancer medicines or foods.
Claims (3)
1. The application of the psoralea corylifolia polysaccharide in preparing the anti-lung cancer medicine is characterized in that the psoralea corylifolia polysaccharide is prepared by the following steps:
1) preparing crude polysaccharide: crushing fructus Psoraleae, extracting with petroleum ether at room temperature for degreasing, volatilizing petroleum ether from the residue, extracting with 70 + -5% ethanol at room temperature, volatilizing the residue until no ethanol smell exists, extracting with ultrapure water at 85 + -5 deg.C under heating, hot-filtering after extraction, concentrating the filtrate to obtain concentrated solution, adding anhydrous ethanol into the concentrated solution to make the final concentration 70-75%, standing, and collecting precipitate to obtain fructus Psoraleae crude polysaccharide;
2) preparing deproteinized fructus psoraleae crude polysaccharide: completely dissolving fructus Psoraleae crude polysaccharide with ultrapure water to obtain fructus Psoraleae crude polysaccharide solution, deproteinizing by Sevag method, concentrating the obtained supernatant under reduced pressure, adding anhydrous ethanol to final concentration of 75 + -2%, standing, centrifuging, collecting precipitate, and drying to obtain deproteinized fructus Psoraleae crude polysaccharide;
3) dissolving deproteinized fructus Psoraleae crude polysaccharide with ultrapure water, centrifuging to remove insoluble substances, loading onto DEAE-52 cellulose chromatographic column, gradient eluting with ultrapure water, 0.05, 0.1, 0.2, and 0.3mol/L NaCl solution in sequence, detecting eluate by phenol-sulfuric acid method, measuring absorbance at 490 nm, drawing polysaccharide elution curve with number of elution tubes as abscissa and absorbance as ordinate, mixing polysaccharide samples of the same elution peak, concentrating, dialyzing, and freeze drying; wherein, the elution peak of the distilled water is a component PC-1, and the elution peak of the 0.2mol/L NaCl solution is a component PC-4;
4) dissolving the component PC-1 with ultrapure water, loading the sample to Sephadex G-100 Sephadex column chromatography, eluting with ultrapure water, detecting by a phenol-sulfuric acid method, measuring the absorbance at 490 nm, drawing a polysaccharide elution curve by taking the number of elution tubes as a horizontal coordinate and the absorbance as a vertical coordinate, combining polysaccharide samples of the same elution peak, and concentrating, dialyzing and freeze-drying the obtained polysaccharide component to be named as PCp-1;
eluting the component PC-4 by the same method as the component PC-1, combining polysaccharide samples with the same elution peak, concentrating, dialyzing, and freeze-drying to obtain a polysaccharide component named PCp-4;
PCp-1 and/or PCp-4 are anti-tumor psoralea fruit polysaccharide.
2. The use of the psoralea corylifolia polysaccharide in the preparation of an anti-lung cancer medicament according to claim 1, wherein in step 1), petroleum ether is used for leaching and degreasing for 3-5 times at room temperature, and each time lasts for 2-4 days; extracting with 70 + -5% ethanol at room temperature for 3-5 times, each for 2-4 days; heating with ultrapure water for extraction for 2-4 times, each for 3-5 hr.
3. The use of psoralen polysaccharides in the preparation of anti-lung cancer drugs according to claim 1, wherein in step 2), the Sevag reagent used in the deproteinization by the Sevag method consists of chloroform and n-butanol in a volume ratio of 4: 1; sevag reagent is added in an amount of 1/4 to 1/2 parts by volume of the crude psoralea seed polysaccharide solution.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106244384A (en) * | 2016-10-14 | 2016-12-21 | 常州亚环环保科技有限公司 | A kind of preparation method of the agreeable to the taste fig wine of Luzhou-flavor |
| CN109422819A (en) * | 2017-08-25 | 2019-03-05 | 南京泽朗医药技术有限公司 | A kind of preparation method of Psoralen lipopolysaccharides |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2017525702A (en) * | 2014-08-18 | 2017-09-07 | ファーマジェネシス， インコーポレイテッド | Polygalacturonan rhamnogalacturonan (PGRG1) composition |
-
2019
- 2019-05-29 CN CN201910456385.5A patent/CN110128562B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106244384A (en) * | 2016-10-14 | 2016-12-21 | 常州亚环环保科技有限公司 | A kind of preparation method of the agreeable to the taste fig wine of Luzhou-flavor |
| CN109422819A (en) * | 2017-08-25 | 2019-03-05 | 南京泽朗医药技术有限公司 | A kind of preparation method of Psoralen lipopolysaccharides |
Non-Patent Citations (6)
| Title |
|---|
| "Two Novel Polysaccharides in Psoralea corylifolia L and anti-A549 Lung Cancer Cells Activity In Vitro";Zhenhua Yin等;《Molecules》;20191016;第24卷;第1-16页 * |
| "丹贝益肺方对人胚成纤维细胞（MRC-5）增殖及胶原蛋白合成的影响";杨天一;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20180215(第02期);E057-237 * |
| "补骨脂多糖的分离纯化、结构鉴定及其对RAW264.7巨噬细胞活力的影响";尹震花等;《食品科学》;20190104;第40卷(第24期);第27-32页 * |
| "补骨脂多糖的提取及免疫活性的研究";李发胜等;《中国药师》;20080215;第11卷(第2期);第140-142页 * |
| 尹震花等."补骨脂多糖的分离纯化、结构鉴定及其对RAW264.7巨噬细胞活力的影响".《食品科学》.2019,第40卷(第24期),第27-32页. * |
| 李发胜等."补骨脂多糖的提取及免疫活性的研究".《中国药师》.2008,第11卷(第2期), * |
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