CN114195908A - Enzyme extraction and purification process of polysaccharide from leaves of litsea cubeba - Google Patents
Enzyme extraction and purification process of polysaccharide from leaves of litsea cubeba Download PDFInfo
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- 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
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- 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
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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)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/08—Drugs for disorders of the urinary system of the prostate
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/10—Antioedematous agents; Diuretics
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- A—HUMAN NECESSITIES
- 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
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
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- A—HUMAN NECESSITIES
- 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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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/10—Process efficiency
Abstract
The invention discloses an enzyme extraction and purification process of polysaccharide from leaves of litsea cubeba. On the basis of a single-factor test, the ultrasonic-assisted enzyme extraction process conditions of the polysaccharide of the leaves of the litsea cubeba are optimized by a response surface method, and the macroporous adsorption resin purification process of the polysaccharide of the leaves of the litsea cubeba is researched by an orthogonal test method. The best enzyme extraction process of the polysaccharide of the litsea cubeba leaves comprises the following steps: the enzyme concentration is 0.9 percent, and the feed-liquid ratio is 1:30 (g/mL), the ultrasonic temperature is 65 ℃, the extraction time is 25min, and the extraction rate is 6.580%; the best process for separating and purifying macroporous adsorption resin comprises the following steps: HPD400 macroporous adsorption resin, 50% ethanol eluent, diameter-height ratio 1.3: 15. the concentration of the sample is 30mg/mL, the flow rate is 0.5mL/min, the polysaccharide retention rate is 86.23%, the protein removal rate is 59.20%, the decolorization rate is 55.79%, and the comprehensive score is 68.99. The research process is reasonable, stable and feasible, has high extraction efficiency and good purification effect, and provides scientific support for further development and utilization of polysaccharide parts of the Siberian cocklebur leaves.
Description
Technical Field
The invention relates to the technical field of polysaccharide of leaves of litsea cubeba, in particular to an enzyme extraction and purification process of polysaccharide of leaves of litsea cubeba.
Background
The litsea cubeba is a plant of the genus litsea of the family dracaenaceae, which is also called as follows: caulis et folium piperis, sargentgloryvine stem, etc. Has curative effects on hyperuricemia, gout, frequent micturition, urgent micturition and the like, has a habit of picking the litsea cubeba as tea for drinking in folk, and has precious medicinal value. The macroporous adsorption resin has the advantages of good selectivity to polysaccharide, large adsorption capacity, simple operation and the like. At present, the existing research shows that the aqueous extract part of the litsea cubeba has the effects of promoting urination and treating liver injury, and the litsea cubeba also has certain treatment effect on prostate diseases when being used for treating gout, but the research on the extraction and utilization value of the litsea cubeba polysaccharide is still lacked.
The existing polysaccharide extract of the litsea cubeba has high impurity content and low polysaccharide content, and the main impurities are protein and pigment, which seriously hinders the further development of the polysaccharide extract and the preparation and makes contribution to human health. To eliminate the influence of other substances on the therapeutic effect and quality control, and to increase the content of polysaccharide. The experiment adopts an ultrasonic-assisted enzyme extraction method to extract the polysaccharide of the leaves of the litsea cubeba, optimizes the technological conditions of the ultrasonic-assisted enzyme extraction of the polysaccharide of the leaves of the litsea cubeba by combining a single-factor test with a response surface method, and researches the optimal process of the separation and purification of macroporous adsorption resin of the polysaccharide of the leaves of the litsea cubeba by combining an orthogonal test method, thereby providing references for the further research, development and utilization of the polysaccharide of the leaves of the litsea cubeba.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the technical defects and provide an enzyme extraction and purification process of polysaccharide from leaves of litsea cubeba.
In order to solve the problems, the technical scheme of the invention is as follows: an enzyme extraction process of polysaccharide from leaves of litsea cubeba comprises the following steps;
step one, taking the extraction rate of polysaccharide from the leaves of the litsea cubeba as an index, respectively drawing extraction curves by screening enzyme types and taking four factors of enzyme concentration, liquid-material ratio, enzymolysis temperature and enzymolysis time as investigation objects;
and secondly, selecting enzyme concentration, liquid-material ratio, enzymolysis temperature and enzymolysis time as investigation factors on the basis of single factor, selecting 3 levels of each factor, designing a response surface test by adopting Design-expert11.0, and optimizing an extraction process.
In the first step, the enzyme types adopt cellulase and pectinase, the ratio of the cellulase to the pectinase is (1: 1), the enzyme concentration adopts 0.9%, and the ratio of liquid to material is 1:30, the enzymolysis temperature is 65 ℃, and the enzymolysis time is 25 minutes.
A purification process of polysaccharide from leaves of litsea cubeba comprises the steps of screening macroporous adsorption resin, single-factor investigation and orthogonal test of the macroporous adsorption resin, and specifically comprises the following steps;
step one, adopting a static adsorption method, and screening AB-8, HPD-400, NKA-9, D101, S-8 and X-5 resins according to comprehensive scores by calculating polysaccharide retention rate, protein removal rate and decolorization rate of the litsea cubeba;
step two, adopting a dynamic adsorption method to investigate the dynamic adsorption of the AB-8, HPD-400, NKA-9, D101, S-8 and X-5 resins by comprehensive scoring;
and step three, taking the comprehensive score as an index, respectively inspecting the influence of the elution rate, the diameter-height ratio, the sample loading concentration and the ethanol concentration of the eluent, and drawing a leakage curve.
Step four, on the basis of single factor, taking the comprehensive score as an index, selecting 4 factors of elution rate, diameter-height ratio, sample loading concentration and eluent ethanol concentration, selecting 3 levels of each factor, and carrying out L9(34) And (4) performing orthogonal test.
In the first step and the second step, the resin type is HPD400 macroporous adsorption resin.
In the third step, the elution rate is 1 ml/min, the loading concentration is 30mg/ml, the eluent ethanol concentration is 45%, and the ratio of the diameter-height ratio is 1.5: 11.
Compared with the prior art, the invention has the advantages that: the research process is reasonable, stable and feasible, has high extraction efficiency and good purification effect, and provides scientific support for further development and utilization of polysaccharide parts of the Siberian cocklebur leaves.
Drawings
FIG. 1 is a table of the effect of different enzyme types of the present invention on polysaccharide extraction yield from leaves of litsea cubeba.
FIG. 2 is a line graph showing the effect of the concentration of the enzyme of the present invention on the extraction yield of polysaccharide from leaves of litsea cubeba.
FIG. 3 is a line graph showing the effect of the liquid material ratio of the present invention on the extraction rate of polysaccharide from leaves of litsea cubeba.
FIG. 4 is a line graph showing the effect of the enzymolysis temperature on the extraction rate of polysaccharide from leaves of litsea cubeba.
FIG. 5 is a line graph showing the effect of enzymolysis time on polysaccharide extraction rate of the leaves of the litsea cubeba.
FIG. 6 is a table of the present invention incorporating response surface testing using Design-Expert software.
FIG. 7 is a table of the response surface test analysis of variance of the present invention.
FIG. 8 is a bar graph of a static test of the macroporous adsorbent resin of the present invention.
FIG. 9 is a bar graph of dynamic experiments of macroporous adsorbent resins of the present invention.
FIG. 10 is a line graph showing the effect of flow rate on purified polysaccharide from leaves of litsea cubeba according to the present invention.
FIG. 11 is a line graph showing the effect of loading concentration on purified polysaccharide from leaves of litsea cubeba according to the present invention.
FIG. 12 is a line graph showing the effect of different ethanol concentrations of the present invention on the purification of polysaccharide from leaves of Litsea cubeba.
FIG. 13 is a line graph showing the effect of aspect ratio on purified polysaccharide from leaves of Siberian cocklebur according to the present invention.
FIG. 14 is a leakage plot of purified polysaccharide from leaves of litsea cubeba according to the invention.
FIG. 15 is a table of the results of orthogonal experiments on the purification of polysaccharide from leaves of litsea cubeba according to the present invention.
FIG. 16 is a table of the results of a three-factor ANOVA of elution rate, aspect ratio, and ethanol concentration of eluent in accordance with the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments; all other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
As shown in fig. 1 to 16, a specific method of an enzyme extraction and purification process of polysaccharide from leaves of litsea cubeba is as follows:
step one, preparing a standard curve of glucose and protein, and developing by adopting a phenol-sulfuric acid method[10]Absorbance was measured at 515 nm, and a glucose standard curve was plotted with Y1 as absorbance and X1 as concentration (mg/ml): y is1＝0.0164X1+ 0.0747(r 0.9996); using Coomassie brilliant blue reagent[11]The absorbance was measured at 595 nm as the protein concentration X2As abscissa, absorbance Y2As ordinate, protein standard curves were plotted: y is2＝0.0135X2－0.1086(r＝0.9996)。
Step two, measuring polysaccharide of the leaves of the litsea cubeba, weighing 5.0g of each leaf of the litsea cubeba by adopting an ultrasonic-assisted enzyme method, adding an enzyme solution, soaking for 30 minutes, carrying out ultrasonic extraction, carrying out suction filtration after enzyme deactivation at 90 ℃, repeatedly extracting for two times, combining filtrates, concentrating, adding a 75% ethanol solution, standing for 8 hours, centrifuging for 10 minutes, washing and precipitating by using acetone and ethanol, and carrying out freeze drying to obtain a polysaccharide crude product. The crude polysaccharide was dissolved in water and the absorbance was measured.
Step three, performing an enzyme extraction single-factor test on the polysaccharide of the leaves of the litsea cubeba, taking the extraction rate of the polysaccharide of the leaves of the litsea cubeba as an index, and respectively drawing extraction curves by screening enzyme types and taking four factors of enzyme concentration, liquid-material ratio, enzymolysis temperature and enzymolysis time as investigation objects; secondly, a response surface test method is adopted, on the basis of single factor, enzyme concentration, liquid-material ratio, enzymolysis temperature and enzymolysis time are selected as investigation factors, each factor is selected to be 3 levels, Design-expert11.0 is adopted to Design a response surface test, and an extraction process is optimized[14,15]。
Step four, the litsea cubebaThe purification process of the leaf polysaccharide comprises the steps of firstly, screening macroporous adsorption resin, including static experiment investigation and dynamic experiment investigation, the static experiment investigation, adopting a static adsorption method, and screening AB-8, HPD-400, NKA-9, D101, S-8 and X-5 resin by calculating polysaccharide retention rate, protein removal rate and decolorization rate of the Siberian cocklebur fruit to obtain a comprehensive score (M ═ polysaccharide retention rate ═ 0.4+ protein removal rate ++ 0.3+ decolorization rate ×. 0.3); dynamic experimental investigation, wherein dynamic adsorption of AB-8, HPD-400, NKA-9, D101, S-8 and X-5 resins is investigated by a dynamic adsorption method according to a comprehensive score (M ═ polysaccharide retention rate 0.4+ protein removal rate 0.3+ destaining rate 0.3); secondly, performing single-factor investigation on the macroporous adsorption resin, taking a comprehensive score (M ═ polysaccharide retention rate 0.4+ protein removal rate 0.3+ destaining rate 0.3) as an index, respectively investigating the influence of elution rate, diameter-height ratio, sample loading concentration and eluent ethanol concentration, and drawing a leakage curve; finally, performing orthogonal test, selecting 4 factors of elution rate, diameter-height ratio, sample loading concentration and eluent ethanol concentration on the basis of single factor by taking comprehensive score (M ═ polysaccharide retention rate 0.4+ protein removal rate 0.3+ decolorization rate 0.3) as an index, selecting 3 levels of each factor, and performing L9(34) And (4) performing orthogonal test.
Through the specific method for the enzyme extraction and purification process of the polysaccharide from the leaves of the litsea cubeba, the following conclusion is drawn:
as shown in figure 1, when the cellulase and the pectinase (1: 1) are used, the extraction rate of the polysaccharide from the leaves of the litsea cubeba is the highest, so the cellulase and the pectinase (1: 1) are selected for the technical research of the polysaccharide from the leaves of the litsea cubeba.
As shown in FIG. 2, the extraction rate of polysaccharide from leaves of litsea japonica is the highest when the enzyme concentration is 0.9%, so that the next experiment was performed with 0.9% enzyme concentration.
As shown in fig. 3, the liquid-to-material ratio is 1: when 30, the extraction rate of polysaccharide in the leaves of the litsea cubeba is the highest, so the liquid-material ratio of 1:30 is selected for carrying out the next experiment.
As shown in fig. 4, the extraction rate of polysaccharide from leaves of litsea cubeba is the highest when the enzymolysis temperature is 65 ℃, so the enzymolysis temperature of 65 ℃ is selected for the next experiment.
As shown in fig. 5, when the enzymolysis time is 25 minutes, the extraction rate of polysaccharide from the leaves of the litsea cubeba is the highest, so the experiment of the next time is performed by selecting the enzymolysis time of 25 minutes.
As shown in fig. 6, from the response surface test of the polysaccharide extraction process from the leaves of litsea cubeba, the regression equation is obtained as follows: y ═ 6.58+ 0.15A +0.087B +0.27C +0.11D +0.090 AB-0.17 AC +0.095 AD-0.035 BC +0.095BD + 0.055 CD-1.32A2－0.93B2－0.46C2－1.35D2。
As shown in FIG. 7, the model is very significant (P < 0.0001), indicating that the unknown factors interfere little with the results. R2-0.9317 shows that the model can explain 93.17% response value change, RAdj 2-0.8633 shows that the fitting degree is better, and the model can be used for analyzing and predicting the extraction rate of polysaccharide in the litsea cubeba leaves. The primary and secondary sequence of the influencing factors is as follows: c is more than A and more than D and more than B, namely, the enzymolysis temperature is more than the enzyme concentration, the enzymolysis time is more than the material-liquid ratio. The items A2, B2, C2 and D2 are significant, which shows that the four factors have significant influence on the extraction rate of the polysaccharide of the litsea cubeba and the interaction among the factors is not significant.
As shown in fig. 8, the combined score for HPD400 was highest in the static adsorption experiment.
As shown in fig. 9, the comprehensive score of HPD400 was highest, and HPD400 macroporous adsorbent resin was selected for the lower experiment.
As shown in fig. 10, the flow rate of 1 ml/min was the highest score, so the orthogonal test was performed at 1 ml/min.
As shown in FIG. 10A, the highest score was obtained for a loading concentration of 30mg/ml, so an orthogonal test was performed using a loading concentration of 30 mg/ml.
As shown in fig. 10 b, when the ethanol concentration of the eluent is 45%, the overall score is the highest score, so that the ethanol concentration of the eluent is 45% for the orthogonal test.
As shown in fig. 10 c, the overall score is the highest score when the aspect ratio is 1.5:11, so that the aspect ratio is 1.5:11 for the orthogonal test.
As shown in FIG. 10C, since the absorbance increased greatly from 10 ml, reached the maximum value at 25 ml, and decreased significantly at 40 ml, 40 ml was selected for the next experiment.
As shown in fig. 10, the sequence of the influence of each factor on the purification of the polysaccharide from the litsea cubeba leaves by the HPD400 macroporous adsorbent resin is as follows: c > A > D > B.
As shown in fig. 10 six, the effect of the ethanol concentration of the eluent on the purification of the polysaccharide from the litsea japonica leaves by the HPD400 macroporous adsorption resin is significant (P < 0.05).
In conclusion, the process for extracting and purifying the polysaccharide from the leaves of the litsea cubeba. In static and dynamic experiments, the HPD400 macroporous adsorption resin shows stronger polysaccharide retention capacity on polysaccharide of the litsea cubeba leaf, has better adsorbability on protein and pigment, is not available in other macroporous adsorption resins in the same group of experiments, and is selected. On the basis of single factor, the optimal extraction process is obtained through a response surface experiment: enzyme concentration: 0.9%, enzymolysis temperature: 65 ℃, liquid-material ratio: 1:30, time: for 25 minutes. Through verification experiments, the average value of the extraction rate of the polysaccharide from the leaves of the litsea cubeba is 6.580%. In the single-factor investigation experiment of the macroporous adsorption resin, through different flow rates, different sample loading concentrations, different diameter-to-height ratios, different elution concentrations and the influence of four single factors on the purification effect of the polysaccharide of the Siberian cocklebur leaf, the optimal purification process is obtained in an orthogonal test: the loading concentration is 30mg/ml, the eluent ethanol concentration is 50%, the flow rate is 0.5ml/min, and the diameter-height ratio is 1.3: 15. The experimental results of the verification experiments performed according to the optimal purification process were that the polysaccharide retention rate was 86.23%, the protein removal rate was 59.20%, the decolorization rate was 55.79%, and the overall score was 68.99.
The invention and its embodiments have been described above, without limitation, and the embodiments shown in the drawings are only one of the embodiments of the invention, and the actual structure is not limited thereto. In summary, those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (5)
1. An enzyme extraction process of polysaccharide from leaves of litsea cubeba is characterized in that: the enzyme extraction process of the polysaccharide from the leaves of the litsea cubeba comprises the following steps;
step one, taking the extraction rate of polysaccharide from the leaves of the litsea cubeba as an index, respectively drawing extraction curves by screening enzyme types and taking four factors of enzyme concentration, liquid-material ratio, enzymolysis temperature and enzymolysis time as investigation objects;
and secondly, selecting enzyme concentration, liquid-material ratio, enzymolysis temperature and enzymolysis time as investigation factors on the basis of single factor, selecting 3 levels of each factor, designing a response surface test by adopting Design-expert11.0, and optimizing an extraction process.
2. The enzymatic extraction process of polysaccharide from leaves of litsea cubeba as claimed in claim 1, wherein: in the first step, the enzyme types adopt cellulase and pectinase, the ratio of the cellulase to the pectinase is (1: 1), the enzyme concentration adopts 0.9%, and the ratio of liquid to material is 1:30, the enzymolysis temperature is 65 ℃, and the enzymolysis time is 25 minutes.
3. A purification process of polysaccharide of Siberian cocklebur leaf is characterized in that: the purification process of the polysaccharide from the leaves of the litsea cubeba comprises the steps of screening macroporous adsorption resin, single-factor investigation and orthogonal test of the macroporous adsorption resin, and specifically comprises the following steps;
step one, adopting a static adsorption method, and screening AB-8, HPD-400, NKA-9, D101, S-8 and X-5 resins according to comprehensive scores by calculating polysaccharide retention rate, protein removal rate and decolorization rate of the litsea cubeba;
step two, adopting a dynamic adsorption method to investigate the dynamic adsorption of the AB-8, HPD-400, NKA-9, D101, S-8 and X-5 resins by comprehensive scoring;
and step three, taking the comprehensive score as an index, respectively inspecting the influence of the elution rate, the diameter-height ratio, the sample loading concentration and the ethanol concentration of the eluent, and drawing a leakage curve.
Step four, on the basis of single factor, taking the comprehensive score as an index, selecting 4 factors of elution rate, diameter-height ratio, sample loading concentration and eluent ethanol concentration, selecting 3 levels of each factor, and carrying out L9(34) And (4) performing orthogonal test.
4. The purification process of polysaccharide from leaves of litsea cubeba as claimed in claim 7, wherein: in the first step and the second step, the resin type is HPD400 macroporous adsorption resin.
5. The purification process of polysaccharide from leaves of litsea cubeba as claimed in claim 7, wherein: in the third step, the elution rate is 1 ml/min, the loading concentration is 30mg/ml, the eluent ethanol concentration is 45%, and the ratio of the diameter-height ratio is 1.5: 11.
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| CN106581091A (en) * | 2016-11-25 | 2017-04-26 | 黄能 | Extraction method of erythropalum scandens and application as medicine treating gout |
| CN107951922A (en) * | 2017-12-08 | 2018-04-24 | 杨英珠 | Erythropalum Scandens Blume deacidification tea and preparation method thereof |
| CN112273503A (en) * | 2019-07-26 | 2021-01-29 | 蒋鹤 | Tea bag and preparation method and application method thereof |
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