CN109503675B - Method for extracting rubusoside and ursolic acid from sweet tea - Google Patents
Method for extracting rubusoside and ursolic acid from sweet tea Download PDFInfo
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- CN109503675B CN109503675B CN201811491679.3A CN201811491679A CN109503675B CN 109503675 B CN109503675 B CN 109503675B CN 201811491679 A CN201811491679 A CN 201811491679A CN 109503675 B CN109503675 B CN 109503675B
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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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- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/20—Carbocyclic rings
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Abstract
The invention provides a method for extracting rubusoside and ursolic acid from sweet tea, which comprises the steps of crushing, alcohol extraction, filtering, concentrating, water dissolving, alkali dissolving and filtering, wherein filter residues are subjected to aspergillus niger fermentation, alcohol extraction again, filtering, concentrating, water dissolving and alkali dissolving, column chromatography, elution, reduced pressure concentration and crystallization recrystallization are respectively carried out to obtain a pure rubusoside product and a pure ursolic acid product. The invention improves the dissolution and conversion efficiency of functional components through microbial conversion, and simultaneously, the aspergillus niger is used for producing acid and neutralizing alkali treatment substances, thereby being beneficial to the rapid growth of the aspergillus niger; in addition, the rubusoside and the ursolic acid are separated by alcohol extraction, water dissolution and alkali dissolution, and a high-purity product is obtained by column chromatography and crystallization, so that the rubusoside is effectively prevented from being damaged by an alkali reagent, and the yield of the rubusoside is improved.
Description
Technical Field
The invention belongs to the technical field of natural product chemistry, and mainly relates to a method for extracting rubusoside and ursolic acid from sweet tea leaves.
Background
The sweet tea is a perennial vine of Rosaceae, is a wild sweet plant unique to Guangxi, is mainly produced in Dayaoshan in China, and is named as sweet tea due to sweet taste. The sweet plants are called Guangxi three sweet plants together with fructus momordicae and stevia rebaudiana, have a long application history in our local folks, and can be drunk as tea for a long time by local people, used for processing foods instead of sugar and used as medicines. The Guangxi analysis and test research center carries out chemical component analysis on the sweet tea in 1979, and determines that the chemical structure of the sweet substance is consistent with that of the stevioside, the sweetness is 300 times that of the cane sugar, and the calorific value is only 5% of that of the cane sugar. The content of rubusoside (rubusoside) is up to 6-7% by identification. Pharmacological tests show that the compound sugar-reducing tea has the characteristics of reducing blood sugar, reducing blood pressure, promoting metabolism, inhibiting hyperacidity, clearing heat, moistening lung, promoting fluid production, quenching thirst, and having high sweetness and low calorific value, and can be used for the auxiliary treatment of diabetes and obesity. Has better curative effect on heat sensation and hot cough, has no toxic or side effect through toxicological tests, and is a novel high-grade sweet health drink in the world. Has fresh, sweet and tasty mouthfeel, and is suitable for both the old and the young. It can be drunk frequently to strengthen body constitution.
The separation and extraction of rubusoside and terpenoids, functional components and the like are researched at home and abroad, the extraction of rubusoside and terpenoids with high purity on a small scale is also reported, and a Chinese patent with the publication number of CN 102239983A introduces a method for extracting rubusoside from rubus suavissimus, and the rubusoside pure product is prepared on a large scale by means of column chromatography, crystallization, recrystallization and the like and is added as a food additive, so that the method is widely applied to health-care foods of patients with obesity, hypertension, diabetes, heart disease and the like. The specific process is that dried sweet tea leaves are extracted by ethanol solution, filtered, decompressed and concentrated, then resin adsorption and ethanol elution are carried out, and eluent is decompressed and concentrated, decolored, crystallized and recrystallized to prepare the pure sweet tea element. Chinese patent publication No. CN107513094A describes a process for extracting purified oleanolic acid and ursolic acid from folium hydrangeae strigosae, which comprises ultrasonic extracting with anhydrous ethanol, and vacuum filtering; concentrating the combined extractive solution to obtain extract, dissolving the obtained extract with chloroform, concentrating, and recovering chloroform to obtain brown extract; dissolving the extract with 5% sodium hydroxide, precipitating, separating precipitate with silica gel column chromatography to obtain high purity oleanolic acid and ursolic acid, and measuring content by high performance liquid chromatography, wherein the purity of oleanolic acid and ursolic acid is above 90%, the yield of oleanolic acid is above 20mg/100g, and the yield of ursolic acid is above 90mg/100 g.
According to the existing literature data, the utilization rate of the functional components of the sweet tea is not high, and the sweet tea is treated as waste, so that a large amount of sweet tea resources are wasted, and if the functional components of the sweet tea can be fully utilized, the production cost of the effective components of the sweet tea can be greatly reduced and the environment can be protected.
Disclosure of Invention
Aiming at low utilization rate of the sweet tea, the invention provides the preparation method of the rubusoside and the ursolic acid, which has the advantages of high utilization rate of the sweet tea, simple method, low cost and high purity of functional products.
Accordingly, the present invention provides a method for extracting rubusoside and ursolic acid from sweet tea, comprising the steps of:
(1) crushing and drying sweet tea, adding 70-85% ethanol water solution, performing reflux extraction at 70-80 ℃ for 1-3 h, and filtering to obtain filtrate I and filter residue I, wherein the volume L of the ethanol water solution and the weight kg of the sweet tea are 2-4: 1;
(2) concentrating the filtrate I under reduced pressure to obtain an extract, adding water to dissolve the extract, and filtering to obtain a filtrate II and a filter residue II, wherein the ratio of the volume L of the added water to the volume L of the filtrate I is 1-2: 10;
(3) adding 0.2-0.8 mol/L sodium hydroxide solution into the filter residue II to 7.2-7.8 mol/L, stirring for dissolving, and filtering to obtain a filtrate III and a filter residue III;
(4) mixing the filter residue III with the filter residue I, adding 4-10% of Aspergillus niger fermentation liquor, fermenting at normal temperature for 4-6 days, adding 70-85% of ethanol water solution, performing reflux extraction at 70-80 ℃ for 1-3 hours, and filtering to obtain a filtrate IV and a filter residue IV, wherein the volume L of the ethanol water solution and the weight kg of the sweet tea are 2-4: 1;
(5) concentrating the filtrate IV under reduced pressure to obtain an extract, adding water for dissolving, and filtering to obtain a filtrate V and a filter residue V, wherein the ratio of the volume L of the added water to the volume L of the filtrate IV is 1-2: 10;
(6) adding 0.2-0.8 mol/L sodium hydroxide solution into the filter residue V to 7.2-7.8 mol/L, stirring for dissolving, and filtering to obtain a filtrate VI;
(7) combining the filtrate II and the filtrate V, loading the filtrate II and the filtrate V on XD-5 resin, eluting the filtrate II and the filtrate V by using 40 to 60 percent ethanol with three to five times of the volume of the column, collecting eluent I, directly adding 95 percent ethanol to the final concentration of 75 to 85 percent, standing the eluent I in a refrigerator overnight, and crystallizing and recrystallizing the eluent once to obtain a pure rubusoside;
(8) and combining the filtrate III and the filtrate VI, adjusting the mixture to 5.5-6.5, adding two to four times of volume of butanol, extracting to obtain butanol extract, concentrating under reduced pressure to 1/5-1/15 of the original volume, directly loading the butanol extract on a D4006 macroporous resin column, eluting with 80-85% ethanol of four to six times of the column volume, concentrating under reduced pressure to 1/5-1/10 of the original volume, standing for 15-30 min, filtering for 1 time while hot, placing the obtained filtrate in a refrigerator overnight, and crystallizing and recrystallizing once to obtain a pure ursolic acid product.
The filtration refers to 1-time filtration with ceramic membrane.
The temperature of the reduced pressure concentration is 40-60 ℃, and the reduced pressure concentration is carried out by adopting a rotary evaporator.
The Aspergillus niger fermentation liquid can be purchased or obtained by self-production, namely, the Aspergillus niger fermentation liquid is inoculated to Aspergillus niger strains by adopting a conventional fungus liquid culture medium and is subjected to shake cultivation for 2-3 days at the temperature of 28-30 ℃, wherein the ratio of the amount L of the Aspergillus niger fermentation liquid to the weight kg of the sweet tea is 4-10: 100.
technical effects
1. The invention improves the dissolution and conversion efficiency of functional components through microbial conversion, and simultaneously, the aspergillus niger is used for producing acid and neutralizing alkali treatment substances, thereby being beneficial to the rapid growth of the aspergillus niger.
2. The invention adopts alcohol extraction, water dissolution and alkali dissolution to separate the rubusoside and the ursolic acid, and the high-purity product is obtained through column chromatography and crystallization, thereby effectively preventing the rubusoside from being damaged under an alkali reagent and improving the yield of the rubusoside.
3. The purification is carried out by adopting crystallization and recrystallization, the process is simple, the operation is easy, and the purification efficiency is high.
Detailed Description
The present invention will be further described with reference to examples, but it is not limited to any one of these examples or the like.
Example 1
Pulverizing 100kg of dried folium hydrangeae strigosae, adding 300L of 80% ethanol water solution, reflux-extracting at 80 deg.C for 2h, and filtering to obtain 260L of filtrate I and residue I; concentrating the filtrate I under reduced pressure to obtain extract, adding 30L of water for dissolving, and filtering to obtain filtrate II and filter residue II; and adding 0.5mol/L sodium hydroxide solution into the filter residue II to 7.5, stirring and dissolving, and filtering to obtain a filtrate III and a filter residue III.
Mixing the filter residue III with the filter residue I, adding 8L of Aspergillus niger fermentation liquor, fermenting at normal temperature for 4-6 days, adding 300L of 85% ethanol aqueous solution, performing reflux extraction at 80 ℃ for 2h, and filtering to obtain 280L of filtrate IV and filter residue IV; concentrating the filtrate IV under reduced pressure to obtain extract, adding 35L water for dissolving, and filtering to obtain filtrate V and residue V; and adding 0.5mol/L sodium hydroxide solution into the filter residue V to 7.5, stirring and dissolving, and filtering to obtain a filtrate VI.
Combining the filtrate II and the filtrate V, loading the filtrate II and the filtrate V on XD-5 resin, eluting with 55% ethanol with five times of column volume, collecting eluent I, directly adding 95% ethanol until the final concentration of the ethanol in the solution is 80%, standing overnight in a refrigerator, and crystallizing and recrystallizing once to obtain 3.26kg pure rubusoside; and combining the filtrate III and the filtrate VI, adjusting to 6, adding four times of volume of butanol for extraction to obtain butanol extract, concentrating under reduced pressure to 1/5-1/15 of the original volume, directly feeding the butanol extract to a D4006 macroporous resin column (Technology, Ltd.), eluting with 80-85% ethanol of six times of the column volume, concentrating under reduced pressure to 1/5-1/10 of the original volume, standing for 15-30 min, filtering for 1 time while hot, standing the obtained filtrate in a refrigerator overnight, and crystallizing and recrystallizing once to obtain 1.83kg pure ursolic acid. HPLC detection shows that the obtained pure rubusoside product has rubusoside content of 98.68% and ursolic acid content of 98.71%.
Comparative example 1
Pulverizing 100kg of dried folium hydrangeae strigosae, adding 300L of 80% ethanol water solution, reflux-extracting at 80 deg.C for 2h, and filtering to obtain 260L of filtrate I and residue I; adding 300L of 85% ethanol water solution into the filter residue I, performing reflux extraction at 80 ℃ for 2h, and filtering to obtain 280L of filtrate II.
Mixing the filtrate I and the filtrate II, concentrating under reduced pressure to obtain extract, adding 60L of water for dissolving, and filtering to obtain filtrate III and filter residue III; adding 0.5mol/L sodium hydroxide solution into the filter residue II to 7.5, stirring and dissolving, and filtering to obtain filtrate IV and filter residue IV; and adding 0.5mol/L sodium hydroxide solution into the filter residue IV to 7.5, stirring and dissolving, and filtering to obtain a filtrate V.
Loading the filtrate III on XD-5 resin, eluting with 55% ethanol with five times column volume, collecting eluate I, directly adding 95% ethanol until ethanol concentration is 80%, standing overnight in refrigerator, and crystallizing and recrystallizing once to obtain 2.75kg pure rubusoside; and combining the filtrate V, adjusting to 6, adding four times of volume of butanol, extracting to obtain butanol extract, concentrating under reduced pressure to 1/5-1/15 of the original volume, directly passing through a D4006 macroporous resin column, eluting with 80-85% ethanol of six times of column volume, concentrating under reduced pressure to 1/5-1/10 of the original volume, standing for 15-30 min, filtering for 1 time while hot, standing the obtained filtrate in a refrigerator overnight, and crystallizing and recrystallizing once to obtain 1.53kg pure ursolic acid. HPLC detection shows that the pure rubusoside product has rubusoside content of 98.52% and ursolic acid content of 98.48%.
Claims (3)
1. A method for extracting rubusoside and ursolic acid from sweet tea comprises the following steps:
(1) crushing and drying sweet tea, adding 70-85% ethanol water solution, performing reflux extraction at 70-80 ℃ for 1-3 h, and filtering to obtain filtrate I and filter residue I, wherein the volume L of the ethanol water solution and the weight kg of the sweet tea are 2-4: 1;
(2) concentrating the filtrate I under reduced pressure to obtain an extract, adding water to dissolve the extract, and filtering to obtain a filtrate II and a filter residue II, wherein the ratio of the volume L of the added water to the volume L of the filtrate I is 1-2: 10;
(3) adding 0.2-0.8 mol/L sodium hydroxide solution into the filter residue II until the pH value is 7.2-7.8, stirring for dissolving, and filtering to obtain a filtrate III and a filter residue III;
(4) mixing the filter residue III with the filter residue I, adding 4-10% of Aspergillus niger fermentation liquor, fermenting at normal temperature for 4-6 days, adding 70-85% of ethanol water solution, performing reflux extraction at 70-80 ℃ for 1-3 hours, and filtering to obtain a filtrate IV and a filter residue IV, wherein the volume L of the ethanol water solution and the weight kg of the sweet tea are 2-4: 1;
(5) concentrating the filtrate IV under reduced pressure to obtain an extract, adding water for dissolving, and filtering to obtain a filtrate V and a filter residue V, wherein the ratio of the volume L of the added water to the volume L of the filtrate IV is 1-2: 10;
(6) adding 0.2-0.8 mol/L sodium hydroxide solution into the filter residue V until the pH value is 7.2-7.8, stirring for dissolving, and filtering to obtain a filtrate VI;
(7) combining the filtrate II and the filtrate V, loading the filtrate II and the filtrate V on XD-5 resin, eluting the filtrate II and the filtrate V by using 40 to 60 percent ethanol with three to five times of the volume of the column, collecting eluent I, directly adding 95 percent ethanol to the final concentration of 75 to 85 percent, standing the eluent I in a refrigerator overnight, and crystallizing and recrystallizing the eluent once to obtain a pure rubusoside;
(8) and combining the filtrate III and the filtrate VI, adjusting the pH value to 5.5-6.5, adding two to four times of volume of butanol, extracting to obtain butanol extract, concentrating under reduced pressure to 1/5-1/15 of the original volume, directly loading on a D4006 macroporous resin column, eluting with 80-85% ethanol of four to six times of the column volume, concentrating under reduced pressure to 1/5-1/10 of the original volume, standing for 15-30 min, filtering for 1 time while hot, standing the obtained filtrate in a refrigerator overnight, and crystallizing and recrystallizing once to obtain a pure ursolic acid product.
2. The method according to claim 1, wherein the filtration is 1 ceramic membrane filtration.
3. The method according to claim 1, wherein the concentration under reduced pressure is carried out at a temperature of 40 to 60 ℃ by using a rotary evaporator.
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| CN201811491679.3A CN109503675B (en) | 2018-12-07 | 2018-12-07 | Method for extracting rubusoside and ursolic acid from sweet tea |
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| CN201811491679.3A CN109503675B (en) | 2018-12-07 | 2018-12-07 | Method for extracting rubusoside and ursolic acid from sweet tea |
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| CN109503675B true CN109503675B (en) | 2021-10-12 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102239983A (en) * | 2011-05-16 | 2011-11-16 | 向华 | Method for extracting rubusoside from sweet tea leaves |
| CN104370981A (en) * | 2014-09-30 | 2015-02-25 | 桂林市一峰食品有限公司 | Method for extraction of rubusoside from sweet tea |
| CN107513094A (en) * | 2017-09-12 | 2017-12-26 | 桂林市产品质量检验所 | A kind of process of extraction purification oleanolic acid and ursolic acid from Sweet tea |
| CN108178775A (en) * | 2017-12-28 | 2018-06-19 | 长沙湘资生物科技有限公司 | The method that cape jasmine extracts gardenoside and ursolic acid |
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2018
- 2018-12-07 CN CN201811491679.3A patent/CN109503675B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102239983A (en) * | 2011-05-16 | 2011-11-16 | 向华 | Method for extracting rubusoside from sweet tea leaves |
| CN104370981A (en) * | 2014-09-30 | 2015-02-25 | 桂林市一峰食品有限公司 | Method for extraction of rubusoside from sweet tea |
| CN107513094A (en) * | 2017-09-12 | 2017-12-26 | 桂林市产品质量检验所 | A kind of process of extraction purification oleanolic acid and ursolic acid from Sweet tea |
| CN108178775A (en) * | 2017-12-28 | 2018-06-19 | 长沙湘资生物科技有限公司 | The method that cape jasmine extracts gardenoside and ursolic acid |
Non-Patent Citations (4)
| Title |
|---|
| Triterpenoids from the Roots of Rubus parvifolius;Yoon Soo Choi,等;《Arch.Pharm.Res.》;19911231;第14卷(第3期);225-230 * |
| Ursolic acid analogues: non-phenolic functional food components in Jamaican raspberry fruits;Camille S. Bowen-Forbes，等;《Food Chemistry》;20091231;第116卷;633-637 * |
| 广西甜茶化学成分、提取工艺及生物活性研究进展;谭冬明,等;《中成药》;20170630;第39卷(第6期);1252-1255 * |
| 近5年悬钩子属植物化学成分研究进展;刘敬，等;《时珍国医国药》;20171231;第28卷(第5期);1195-1197 * |
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