CN112521441B - Method for separating and purifying betulinic acid from lotus seedpod shells - Google Patents
Method for separating and purifying betulinic acid from lotus seedpod shells Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
- C07J63/008—Expansion of ring D by one atom, e.g. D homo steroids
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicines Containing Plant Substances (AREA)
- Steroid Compounds (AREA)
Abstract
The invention discloses a method for separating and purifying betulinic acid from lotus seedpod shells, which comprises the steps of extraction and concentration of the lotus seedpod shells, acid-soluble and alkali-soluble precipitation, degreasing and decoloration, crystallization and the like, wherein the extracting solution is concentrated and then subjected to acid adjustment treatment, the precipitate is obtained through centrifugation, and the supernatant is discarded; adding low-polarity petroleum ether into the precipitate for elution to remove fat-soluble substances in the precipitate; continuously adding methanol into the precipitate for thermal dissolution, adsorbing with active carbon, filtering, collecting filtrate, and concentrating the filtrate; and (4) adding water into the decolored concentrated solution while stirring until the solution is slightly turbid, carrying out crystallization, and centrifuging to collect crystals. The method solves the problems of long production period, low yield and inconvenience for industrial production of the prior art by repeatedly using a mixed solvent and column chromatography, quickly realizes the separation of active ingredients and impurities by acid dissolution and alkali precipitation, simplifies a process route, and can effectively improve the yield of betulinic acid.
Description
Technical Field
The invention belongs to the technical field of extraction of effective active ingredients of plants, and particularly relates to a method for separating and purifying betulinic acid from lotus seedpod shells.
Background
Nelumbo nucifera (Nelumbo nucifera), family genus: nymphaeaceae, Nelumbo, perennial aquatic plants and herbaceous plants have more than 2000 years of planting history in China. At present, the number of lotus varieties owned by China is more than 800, and the lotus varieties are mainly distributed in Fujian, Zhejiang, Hubei, Hunan, Jiangsu, Anhui, Jiangxi and the like. The lotus can be eaten, drunk, used in medicine and packaged besides having ornamental value. Lotus leaf can be used for treating vexation, summer heat, hematochezia, metrorrhagia, etc.; lotus plumule can be used for treating insomnia, dreaminess, hematemesis, etc.; semen Nelumbinis has effects in nourishing heart, tranquilizing mind, arresting seminal emission, and invigorating kidney; the lotus root is a daily edible aquatic vegetable. The lotus is rich in various active ingredients such as alkaloid, flavone, glycoside, triterpenes, polysaccharide, polyphenol, volatile oil and the like, and has physiological activities of resisting oxidation, diminishing inflammation, resisting bacteria, resisting arrhythmia, reducing blood sugar, relieving diarrhea, regulating immunity and the like. At present, the development and development of lotus products mostly stay on plant organ products such as lotus leaves, lotus seeds, lotus plumule and the like, and the research on waste lotus seedpod shells is less.
The lotus seedpod shell is a dry receptacle of lotus of Nymphaeaceae, also called lotus seedpod shell. The lotus seed feed is agricultural waste generated in the processing process of lotus seeds, is only used for animal feed at present, and even is randomly thrown on the roadside or beside a ditch as waste, is rotten and mildewed, or is directly buried and burned; even if the method is used, the method is more primary, the technical content is low, and therefore, not only is the environment pollution caused, but also the lotus resource is greatly wasted. At present, related researches on chemical components and medicinal effects of the lotus seedpod shells at home and abroad are only reported sporadically and have not been paid attention to all the time. The ingredients known to date for lotus seedpod are limited to alkaloids (alkaloids) and flavonoids (flavanoids), and the extraction and separation of triterpenoids, especially betulinic acid monomeric compounds, is still blank.
Aiming at the agricultural waste of the lotus seedpod shells, the invention adopts the steps of extraction and concentration, acid-soluble and alkali-soluble precipitation, degreasing and decoloration, crystallization and the like to obtain the high-purity betulinic acid.
Disclosure of Invention
In view of the current technical situation of the prior art for preparing betulinic acid, the mixed solvent is repeatedly used and purified by column chromatography and the like. Aiming at the raw material of the lotus seedpod, the invention provides a method for separating and purifying betulinic acid from the lotus seedpod, and the method has the advantages of cheap and easily-obtained lotus seedpod, convenient process operation, high product content, high recovery rate, small harm of used solvent and suitability for large-scale production operation.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows; a method for separating and purifying betulinic acid from lotus seedpod shells is characterized by comprising the following steps:
and (3) extraction and concentration: crushing a lotus seedpod shell raw material, adding 40-80% of organic reagent which is 5-20 times the weight of the raw material, pretreating in a flash extractor for 10-30min, performing reflux extraction for 1-2h, filtering to obtain an extracting solution, adding 5-20% of alkali liquor by mass into the extracting solution, adjusting the pH value to 8-10, and concentrating until the alcohol content is 10-30%;
2) acid dissolution and alkali precipitation: adjusting pH of the concentrated solution to 2-7 with acid water, heating and refluxing for 30-60min, cooling, filtering, adjusting pH of the filtrate to 10-12 with alkali solution, standing for 6-10 hr, centrifuging to obtain precipitate, and removing supernatant
3) Degreasing: adding a low-polarity solvent into the precipitate for elution, and removing fat-soluble substances in the precipitate;
4) and (3) decoloring: continuously adding 3-5 times of solvent into the precipitate for thermal dissolution, adding 5-20% of activated carbon into the dissolved solution, adsorbing at 60-80 deg.C for 1-3 hr, filtering, collecting filtrate, and concentrating the filtrate to specific gravity of 1.0-1.2;
5) and (3) crystallization: adding water, methanol, ethanol, isopropanol, n-butanol or pentanol into the decolorized concentrated solution under stirring until the solution is slightly turbid, keeping the temperature at 5-10 deg.C for more than 3h, crystallizing, centrifuging, and collecting crystals to obtain betulinic acid with purity of more than 98%.
Particularly, in the step 1), 70% ethanol or methanol which is 10 times of the mass of the raw materials is added, the mixture is pretreated in a flash extractor for 20min, reflux extraction is carried out for 1.5h, 5% -20% of alkali liquor which is sodium hydroxide, potassium hydroxide, sodium carbonate or sodium bicarbonate is added into the obtained extracting solution, the pH value is adjusted to 8, and then the mixture is concentrated until the alcohol content is 10%.
Specifically, in step 2), the acid solution is hydrochloric acid, sulfuric acid, formic acid, acetic acid or citric acid solution, the PH of the acid solution is adjusted to 2.5 by the acid solution, the acid solution is heated and refluxed for 30min, the acid solution is cooled and filtered, the PH of the filtrate is adjusted to 10.4 by alkali liquor, and the filtrate is kept stand for 10h and then centrifuged to obtain precipitate.
In particular, in step 3), the low-polarity reagent is any one of n-hexane, cyclohexane or petroleum ether.
Specifically, in step 4), methanol in an amount 4 times the mass of the precipitate was added to the precipitate to perform thermal dissolution, activated carbon in an amount of 10% by mass of the precipitate was added to the above dissolved solution, adsorption was performed at 60 ℃ for 1 hour, filtration was performed, the filtrate was collected, and the filtrate was concentrated to a specific gravity of 1.2.
In particular, in step 5), water is added with stirring until the solution is slightly turbid.
Due to the adoption of the technical scheme, the invention has the following advantages:
the method avoids the defects of repeated use of mixed organic solvents and column chromatography in the traditional separation and purification, adopts the lotus seedpod as the raw material for extracting the betulinic acid, has cheap and easily obtained lotus seedpod shell compared with the raw material of birch bark, and has relatively high betulinic acid content in the lotus seedpod shell. The extracting solution is protected by weak alkali liquor to prevent the oxidation of effective components, and in the step of acid-soluble alkali precipitation, the acid-soluble alkali precipitation is carried out in low-concentration alcohol solution, so that the betulinic acid glycoside can be increased and converted into betulinic acid aglycone, the specific gravity of the extracting solution can be effectively reduced, the use of subsequent reagents is reduced, and substances with larger polarity can be effectively removed by adopting alkali precipitation, thereby facilitating the subsequent refining. The organic reagent used in the preparation process is simple, nontoxic, green and environment-friendly, no column chromatographic packing is used, the time is greatly saved, the yield and the purity of the obtained betulinic acid are high, the whole process operation is simple, and the method is suitable for large-scale production.
Drawings
Fig. 1 is a hydrogen spectrum of betulinic acid according to the present invention.
Fig. 2 is a carbon spectrum of betulinic acid according to the present invention.
Detailed Description
The present invention will be further described in detail with reference to the following examples; however, the following examples are merely illustrative, and the present invention is not limited to these examples.
A method for separating and purifying betulinic acid from lotus seedpod shells comprises the following steps:
1) crushing the lotus seedpod shell raw material, adding 40-80% of organic reagent which is 5-20 times the weight of the raw material, pretreating in a flash extractor for 10-30min, performing reflux extraction for 1-2h, filtering to obtain an extracting solution, adding 5-20% of alkali liquor by mass into the extracting solution, adjusting the pH value to 8-10, and concentrating to the alcohol content of 10-30%;
2) adjusting pH of the concentrated solution to 2-7 with acid water, heating and refluxing for 30-60min, cooling, filtering, adjusting pH of the filtrate to 10-12 with alkali solution, standing for 6-10h, centrifuging to obtain precipitate, and removing supernatant;
3) adding a low-polarity solvent into the precipitate for elution, and removing fat-soluble substances in the precipitate; a
4) Continuously adding a solvent with the mass of 3-5 times of that of the precipitate into the precipitate for thermal dissolution, adding active carbon with the mass of 5-20% of that of the precipitate into the dissolved solution, adsorbing for 1-3h at the temperature of 60-80 ℃, filtering, collecting the filtrate, and concentrating the filtrate until the specific gravity is 1.0-1.2;
5) adding water, methanol, ethanol, isopropanol, n-butanol or pentanol into the decolorized concentrated solution under stirring until the solution is slightly turbid, keeping the temperature at 5-10 ℃ for more than 3h, performing crystallization, centrifuging, and collecting crystals to obtain betulinic acid with purity of more than 98%, wherein the yield is 1-3%.
Example 1
Crushing a lotus seedpod shell raw material, weighing 1kg, adding 10L of 70% ethanol water solution, pretreating in a flash extractor for 30min, performing reflux extraction for 2h, filtering to obtain an extracting solution, adding a 20% sodium hydroxide solution by mass fraction into the extracting solution, adjusting the pH value to 8, and concentrating to reach the alcohol content of 10%; adjusting the pH value of the concentrated solution to 2 with hydrochloric acid, heating and refluxing for 30min, cooling, filtering, adjusting the pH value of the filtrate to 10 with 20% sodium hydroxide solution, standing for 10h, centrifuging to obtain precipitate, and removing the supernatant; adding low-polarity petroleum ether into the precipitate for elution to remove fat-soluble substances in the precipitateQuality; continuously adding methanol with the mass of 3 times of the precipitate into the precipitate for thermal dissolution, adding activated carbon with the mass of 10% of the precipitate into the dissolved solution, adsorbing for 1h at 60 ℃, filtering, collecting filtrate, and concentrating the filtrate until the specific gravity is 1.0; adding water into the decolorized concentrated solution under stirring until the solution is slightly turbid, keeping the temperature at 5 deg.C for more than 3h, crystallizing, centrifuging, and collecting crystals to obtain betulinic acid with purity of more than 98%, wherein the obtained betulinic acid has a mass of 28.68 g. The nuclear magnetic hydrogen spectrum carbon spectrum of betulinic acid is shown in fig. 1 and fig. 2.1H-NMR(400MHz,CDCl3)δ:0.74(3H,s,27-CH3),0.81(3H,s,26-CH3),0.92(3H,s,25-CH3),0.95(3H,s,24-CH3),0.97(3H,s,23-CH3),1.68(3H,s,30-CH3),4.60(1H,s,H-29a),4.73(1H,s,H-29b)；13C-NMR(150MHz,CDCl3)δ:38.8(C-1),27.5(C-2),79.2(C-3),39.0(C-4),55.5(C-5),18.4(C-6),34.4(C-7),40.8(C-8),50.6(C-9),37.3(C-10),21.0(C-11),25.6(C-12),38.5(C-13),42.6(C-14),30.7(C-15),32.2(C-16),56.4(C-17),49.4(C-18),47.0(C-19),150.6(C-20),29.8(C-21),37.2(C-22),28.1(C-23),15.5(C-24),16.2(C-25),16.3(C-26),14.8(C-27),180.1(C-28),109.9(C-29),19.5(C-30)。
Example 2
Crushing a lotus seedpod shell raw material, weighing 1kg, adding 20L of 80% ethanol aqueous solution, pretreating in a flash extractor for 10min, performing reflux extraction for 1h, filtering to obtain an extracting solution, adding a 5% potassium hydroxide solution by mass fraction into the extracting solution, adjusting the pH value to 10, and concentrating to reach the alcohol content of 30%; adjusting pH of the concentrated solution to 3 with citric acid, heating and refluxing for 60min, cooling, filtering, adjusting pH of the filtrate to 10 with 10% potassium hydroxide solution, standing for 8 hr, centrifuging to obtain precipitate, and removing supernatant; adding low-polarity n-hexane into the precipitate for elution, and removing fat-soluble substances in the precipitate; continuously adding methanol with the mass of 3 times of the precipitate into the precipitate for thermal dissolution, adding activated carbon with the mass of 5% of the precipitate into the dissolved solution, adsorbing at 60 ℃ for 1h, filtering, collecting filtrate, and concentrating the filtrate until the specific gravity is 1.0; adding n-butanol into the decolorized concentrated solution under stirring until the solution is slightly turbid, keeping the temperature at 10 deg.C for more than 3h, crystallizing, centrifuging, and collecting crystals to obtain betulinic acid with purity of more than 98%, wherein the obtained betulinic acid has a mass of 23.12 g.
Example 3
Crushing a lotus seedpod shell raw material, weighing 1kg, adding 15L of 60% ethanol water solution, pretreating in a flash extractor for 20min, performing reflux extraction for 1.5h, filtering to obtain an extracting solution, adding a 15% sodium bicarbonate solution in mass fraction into the extracting solution, adjusting the pH value to 9, and concentrating to the alcohol content of 20%; adjusting pH of the concentrated solution to 4 with formic acid, heating and refluxing for 60min, cooling, filtering, adjusting pH of the filtrate to 11 with 15% sodium carbonate solution, standing for 10 hr, centrifuging to obtain precipitate, and removing supernatant; adding low-polarity cyclohexane into the precipitate for elution to remove fat-soluble substances in the precipitate; continuously adding methanol with the mass of 3 times of the precipitate into the precipitate for thermal dissolution, adding activated carbon with the mass of 10% of the precipitate into the dissolved solution, adsorbing for 1h at 60 ℃, filtering, collecting filtrate, and concentrating the filtrate until the specific gravity is 1.0; adding water into the decolorized concentrated solution under stirring until the solution is slightly turbid, keeping the temperature at 10 deg.C for more than 3h, crystallizing, centrifuging, and collecting crystals to obtain betulinic acid with purity of more than 98%, wherein the obtained betulinic acid has a mass of 12.65 g.
Example 4
Crushing a lotus seedpod shell raw material, weighing 1kg, adding 15L of 70% ethanol water solution, pretreating in a flash extractor for 30min, performing reflux extraction for 1h, filtering to obtain an extracting solution, adding a 20% sodium bicarbonate solution in mass fraction into the extracting solution, adjusting the pH to 9, and concentrating to reach the alcohol content of 15%; adjusting pH of the concentrated solution to 6 with sulfuric acid, heating and refluxing for 30min, cooling, filtering, adjusting pH of the filtrate to 10 with 20% sodium bicarbonate solution, standing for 10 hr, centrifuging to obtain precipitate, and removing supernatant; adding low-polarity petroleum ether into the precipitate for elution to remove fat-soluble substances in the precipitate; continuously adding methanol with the mass of 3 times of the precipitate into the precipitate for thermal dissolution, adding activated carbon with the mass of 5% of the precipitate into the dissolved solution, adsorbing at 60 ℃ for 1h, filtering, collecting filtrate, and concentrating the filtrate until the specific gravity is 1.0; adding isopropanol into the decolorized concentrated solution under stirring until the solution is slightly turbid, keeping the temperature at 5 ℃ for more than 3h, crystallizing, centrifuging, and collecting crystals to obtain betulinic acid with purity of more than 98%, wherein the obtained betulinic acid has a mass of 10.58 g.
Example 5
Crushing a lotus seedpod shell raw material, weighing 1kg, adding 20L of 40% ethanol water solution, pretreating in a flash extractor for 30min, performing reflux extraction for 1h, filtering to obtain an extracting solution, adding a 15% sodium hydroxide solution by mass fraction into the extracting solution, adjusting the pH to 9, and concentrating to reach the alcohol content of 30%; adjusting pH of the concentrated solution to 5 with acetic acid, heating and refluxing for 30min, cooling, filtering, adjusting pH of the filtrate to 10 with 15% sodium hydroxide solution, standing for 10 hr, centrifuging to obtain precipitate, and removing supernatant; adding low-polarity petroleum ether into the precipitate for elution to remove fat-soluble substances in the precipitate; continuously adding methanol with the mass of 3 times of the precipitate into the precipitate for thermal dissolution, adding activated carbon with the mass of 20% of the precipitate into the dissolved solution, adsorbing for 1h at 60 ℃, filtering, collecting filtrate, and concentrating the filtrate until the specific gravity is 1.0; adding water into the decolorized concentrated solution under stirring until the solution is slightly turbid, keeping the temperature at 10 deg.C for more than 3h, crystallizing, centrifuging, and collecting crystals to obtain betulinic acid with purity of more than 98%, wherein the obtained betulinic acid has a mass of 6.58 g.
The invention solves the problems of repeated use of mixed solvent, long production period of column chromatography, low yield and difficult industrial production in the prior art. The yield of the betulinic acid can be effectively improved; through acid dissolution and alkali precipitation, the separation of active ingredients and impurities is quickly realized, the process route is simplified, the production cost can be effectively reduced, and the production efficiency is improved.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (6)
1. A method for separating and purifying betulinic acid from lotus seedpod shells is characterized by comprising the following steps:
1) and (3) extraction and concentration: crushing the lotus seed pod raw material, adding 40-80% ethanol which is 5-20 times the weight of the raw material, pretreating in a flash extractor for 10-30min, performing reflux extraction for 1-2h, filtering to obtain an extracting solution, adding 5-20% of alkali liquor by mass into the extracting solution, adjusting the pH to be 8-10, and concentrating until the ethanol content is 10-30%;
2) acid dissolution and alkali precipitation: adjusting the acid of the concentrated solution obtained in the step (1), adjusting the pH of the solution to be 2-7 with acid water, heating and refluxing for 30-60min, cooling, filtering, adjusting the pH of the filtrate to be 10-12 with alkali liquor, standing for 6-10h, centrifuging to obtain a precipitate, and discarding the supernatant;
3) degreasing: adding a low-polarity solvent into the precipitate obtained in the step (2) for elution, and removing fat-soluble substances in the precipitate;
4) and (3) decoloring: continuously adding 3-5 times of solvent into the precipitate for thermal dissolution, adding 5-20% of activated carbon into the dissolved solution, adsorbing at 60-80 deg.C for 1-3 hr, filtering, collecting filtrate, and concentrating the filtrate to specific gravity of 1.0-1.2;
5) and (3) crystallization: adding water, methanol, ethanol, isopropanol, n-butanol or pentanol into the decolorized concentrated solution under stirring until the solution is slightly turbid, keeping the temperature at 5-10 deg.C for more than 3h, crystallizing, centrifuging, and collecting crystals to obtain betulinic acid with purity of more than 98%.
2. The method for separating and purifying betulinic acid from lotus seedpod as claimed in claim 1, wherein: in the step 1), 70% ethanol which is 10 times of the mass of the raw materials is added, the mixture is pretreated in a flash extractor for 20min, reflux extraction is carried out for 1.5h, 5% -20% of alkali liquor which is sodium hydroxide, potassium hydroxide, sodium carbonate or sodium bicarbonate is added into the obtained extracting solution, the pH =8 is adjusted, and the mixture is concentrated until the alcohol content is 10%.
3. The method for separating and purifying betulinic acid from lotus seedpod as claimed in claim 1, wherein: in the step 2), the acid water is hydrochloric acid, sulfuric acid, formic acid, acetic acid or citric acid solution, the pH of the acid water solution is adjusted to be 2.5, the acid water solution is heated and refluxed for 30min, the acid water solution is cooled and filtered, the pH of the filtrate is adjusted to be 10.4 by alkali liquor, and the filtrate is kept stand for 10h and then centrifuged to obtain precipitate.
4. The method for separating and purifying betulinic acid from lotus seedpod as claimed in claim 1, wherein: in the step 3), the low-polarity reagent is any one of n-hexane, cyclohexane or petroleum ether.
5. The method for separating and purifying betulinic acid from lotus seedpod as claimed in claim 1, wherein: and 4) adding methanol with the mass of 4 times of that of the precipitate into the precipitate for thermal dissolution, adding activated carbon with the mass of 10% of that of the precipitate into the dissolved solution, adsorbing for 1 hour at 60 ℃, filtering, collecting filtrate, and concentrating the filtrate until the specific gravity is 1.2.
6. The method for separating and purifying betulinic acid from lotus seedpod as claimed in claim 1, wherein: in step 5), water is added with stirring until the solution is slightly turbid.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003011891A1 (en) * | 2001-08-03 | 2003-02-13 | Council Of Scientific And Industrial Research | A process for the isolation of betulinic acid from nelumbo nucifera gaertn rhizomes |
| CN101768200A (en) * | 2009-12-30 | 2010-07-07 | 山东省中医药研究院 | Process for preparing 3-epibetulinic acid by extracting from rhizoma nelumbinis and charred rhizoma nelumbinis |
| CN102827241A (en) * | 2012-09-20 | 2012-12-19 | 南京泽朗农业发展有限公司 | Method for extracting betulinic acid from persimmon leaves |
| CN104961792A (en) * | 2015-06-16 | 2015-10-07 | 南京中医药大学 | Methods for extracting and measuring content of betulinic acid and polysaccharide in rhizoma nelumbinis |
| CN109666058A (en) * | 2018-12-25 | 2019-04-23 | 东南大学 | A kind of method that betulic acid extraction is isolated |
| CN109879927A (en) * | 2019-03-29 | 2019-06-14 | 上海欧润化妆品有限公司 | A kind of extraction process of white pond lily rhizome portion extract |
-
2020
- 2020-12-29 CN CN202011589140.9A patent/CN112521441B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003011891A1 (en) * | 2001-08-03 | 2003-02-13 | Council Of Scientific And Industrial Research | A process for the isolation of betulinic acid from nelumbo nucifera gaertn rhizomes |
| CN101768200A (en) * | 2009-12-30 | 2010-07-07 | 山东省中医药研究院 | Process for preparing 3-epibetulinic acid by extracting from rhizoma nelumbinis and charred rhizoma nelumbinis |
| CN102827241A (en) * | 2012-09-20 | 2012-12-19 | 南京泽朗农业发展有限公司 | Method for extracting betulinic acid from persimmon leaves |
| CN104961792A (en) * | 2015-06-16 | 2015-10-07 | 南京中医药大学 | Methods for extracting and measuring content of betulinic acid and polysaccharide in rhizoma nelumbinis |
| CN109666058A (en) * | 2018-12-25 | 2019-04-23 | 东南大学 | A kind of method that betulic acid extraction is isolated |
| CN109879927A (en) * | 2019-03-29 | 2019-06-14 | 上海欧润化妆品有限公司 | A kind of extraction process of white pond lily rhizome portion extract |
Non-Patent Citations (4)
| Title |
|---|
| "建莲"化学质量标准体系的研究;刘久;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20160115(第1期);第E057-63页 * |
| A Comprehensive Review on Chemical Profiling of Nelumbo Nucifera: Potential for Drug Development;Bhesh Raj Sharma et al.;《Phytother. Res.》;20160926;第31卷;第3-26页 * |
| 荷蒂和荷梗的化学成分研究;周传顺;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20190915(第9期);第E057-85页 * |
| 藕和藕节的化学成分研究;张朔;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20190915(第9期);第E057-81页 * |
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