CN107513094A - A kind of process of extraction purification oleanolic acid and ursolic acid from Sweet tea - Google Patents
A kind of process of extraction purification oleanolic acid and ursolic acid from Sweet tea Download PDFInfo
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- CN107513094A CN107513094A CN201710817875.4A CN201710817875A CN107513094A CN 107513094 A CN107513094 A CN 107513094A CN 201710817875 A CN201710817875 A CN 201710817875A CN 107513094 A CN107513094 A CN 107513094A
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- acid
- oleanolic acid
- ursolic acid
- oleanolic
- medicinal extract
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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
Abstract
The invention discloses a kind of process of extraction purification oleanolic acid and ursolic acid from Sweet tea, including using absolute ethyl alcohol ultrasonic wave extraction, suction filtration；Extract solution after concentration merges obtains medicinal extract, and the medicinal extract of gained is dissolved with chloroform, and brown medicinal extract is obtained after concentration and recovery chloroform；Medicinal extract obtains the oleanolic acid and ursolic acid of high-purity with the methods of 5% sodium hydroxide alkali soluble acid is heavy, sediment is separated with silica gel column chromatography, assay is carried out using high performance liquid chromatography, the purity of oleanolic acid and ursolic acid is more than 90%, the yield of oleanolic acid is in more than 20mg/100g, and the yield of ursolic acid is in more than 90mg/100g.Present invention process method, required equipment is simple and convenient to operate, extraction time is short, extraction efficiency is high, energy-conservation, saving raw material, mild condition, not the features such as not destroying active component, a kind of feasibility source is provided to extract oleanolic acid and ursolic acid from natural plants, the added value of Sweet tea is improved, there is important application value and economic and social benefits.
Description
Technical field
The present invention relates to effective ingredients in plant method for extraction and purification, is specifically one kind extraction purification oleanolic acid from Sweet tea
With the process of ursolic acid.
Background technology
Guangxi Folium hydrangeae strigosae（Rubus Suavissimus S.Lee）It is that the distinctive sweet taste in Guangxi is planted for rose family rubus
Thing, often it is used as tea-drinking among the people, containing main actives such as rubusoside, flavones, Tea Polyphenols, there is relieving heat and thirst, kidney tonifying, drop
Blood pressure and treatment diabetes and other effects.Document report is identified in Sweet tea by extracting separation and method of spectroscopy and contains oleanolic acid
And ursolic acid.Modern study shows that oleanolic acid has AntiHIV1 RT activity, antibacterial, anticancer, antiulcer, treatment osteoporosis etc. extensively
Pharmacological action and bioactivity, have been used to clinic as hepatic.Ursolic acid has antitumor, protect liver, angiocarpy, sugar
The multiple biological activities such as disease, anti-inflammatory, antiviral are urinated, at home and abroad receive extensive research.Oleanolic acid and ursolic acid market
Demand is big, artificial synthesized more complicated, still relies on to extract from natural plants at present and obtains.Therefore, to oleanolic acid in Sweet tea
And the research of ursolic acid extraction and purification process has great importance.Through Literature Consult, oleanolic acid and ursolic acid carry in Sweet tea
The research of purifying process is taken to have no report.The present invention is using ultrasonic wave extraction and silica gel column chromatography separation method to neat pier in Sweet tea
Tartaric acid and ursolic acid extraction and purifying process are studied, and are laid the foundation for the comprehensive utilization of Sweet tea.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of work of extraction purification oleanolic acid and ursolic acid from Sweet tea
Process.
Technical scheme provided by the invention is：
A kind of process of extraction purification oleanolic acid and ursolic acid from Sweet tea, comprises the following steps：
（1）Take Sweet tea sample to smash to pieces, weigh the sample after smashing to pieces, add the absolute ethyl alcohol of 2~4 times of amounts of its weight at 25~40 DEG C
10~20min of lower ultrasonic wave extraction, extract solution filter；
Filtered residue is washed with ethanol again, then is filtered, smoke filtrate merges with the extract solution of first time, carrying after concentration merging
Liquid is taken to obtain medicinal extract, the medicinal extract of gained is dissolved with chloroform, and brown medicinal extract is obtained after concentration and recovery chloroform；
（2）By step（1）Obtained medicinal extract is boiled with 5% sodium hydroxide solution, is filtered while hot, and filtrate is placed in into refrigerator 5~10
DEG C refrigeration 1h, filter, be washed with water to neutrality；
Filtered residue adds boiling to boil, and it is 1~2 to be adjusted to pH with hydrochloric acid, lets cool precipitation, filters, uses hot water washing sediment；
Sediment is separated with silica gel column chromatography, uses petroleum ether:Chloroform=1:2 elutions, TLC detections, discard eluent；Chlorine is used again
It is imitative:Ethyl acetate=18:1 elution, a, 30 parts of collection is collected per 100mL, TLC detections, merges 5-15 parts, concentrate eluant obtains
High purity oleanolic acid sample；
Merge 17-30 parts, concentrate eluant obtains high-purity ursolic acid sample；
By the oleanolic acid of gained and ursolic acid sample, dried respectively at 101 DEG C~105 DEG C to constant weight；
（3）By step（2）Dried oleanolic acid and ursolic acid sample is purified, is carried out using high performance liquid chromatography containing measurement
Fixed, more than 90%, the yield of oleanolic acid obtains the purity of oleanolic acid and ursolic acid in more than 20mg/100g, ursolic acid
Rate is in more than 90mg/100g.
The present invention and the B of Publication No. CN 105348364 patent of invention《One kind extracts oleanolic acid side from the fruit of glossy privet
Method》And master's thesis《The research of the chemical composition of Guangxi Folium hydrangeae strigosae leaf》Compare, there is the advantages of certain.《It is a kind of from glossy privet
Oleanolic acid method is extracted in son》Slightly purified using the separation elution of D101 macroreticular resins after ultrasonic wave extraction in patent, then
Be further purified using Sephadex LH-20 gel columns, purge process complex operation, take, consumption reagent, and because with the present invention
Raw material is different, can only extraction purification oleanolic acid, not including ursolic acid.Master's thesis《The chemical composition of Guangxi Folium hydrangeae strigosae leaf
Research》Middle use chloroform is heated to reflux mode and extracts oleanolic acid and ursolic acid, and extraction time is grown, high energy consumption, and efficiency is low.Carry
Do not purified slightly using straightforward procedure after taking, and silica gel column chromatography is used repeatedly with different organic solvent mixing eluent systems
Isolated and purified, same complex operation, taken, and consume a large amount of organic solvents, uneconomical environmental protection.The unjustified pier tartaric acid of the method
And the purity and extraction yield of ursolic acid are examined, the oleanolic acid and ursolic acid of purifying are only used for Components identification.The present invention
Extracted using ultrasonic wave, slightly purified using straightforward procedures such as the heavy, water washing and precipitatings of alkali soluble acid, pass through silica gel column chromatography point
Studied from the oleanolic acid and ursolic acid for respectively obtaining high-purity, and to purity and extraction yield, oleanolic acid and bear
The purity of tartaric acid more than 90%, the yield of oleanolic acid in more than 20mg/100g, the yield of ursolic acid 90mg/100g with
On.Present invention process is simple, easy to operate, purge process is simple, and extraction time is short, extraction efficiency is high, mild condition, does not destroy
Active component, it is easily operated.
Brief description of the drawings
Fig. 1 is the HPLC figures of embodiment oleanolic acid, ursolic acid standard liquid；
Fig. 2 is the HPLC figures of oleanolic acid sample after embodiment purifying is dried；
Fig. 3 is the HPLC figures of ursolic acid sample after embodiment purifying is dried.
In figure, 1. be that oleanolic acid 2. is ursolic acid.
Embodiment
Present invention is further elaborated with reference to embodiment and accompanying drawing, but not as the limit to the present invention
It is fixed.
Embodiment 1
The process of extraction purification oleanolic acid and ursolic acid from Sweet tea, comprises the following steps：
（1）Take Sweet tea sample to be smashed to pieces with bruisher, weigh sample 150g, add the absolute ethyl alcohol of 2 times of amounts of its weight at 25 DEG C
Ultrasonic wave extraction 10min, extract solution filter；Wash filtered residue 1 time with 300 mL ethanol again, then filter, smoke filtrate with
The extract solution of first time merges, and the extract solution after concentration merges obtains medicinal extract, and the medicinal extract of gained is dissolved with 30mL chloroforms, concentration and recovery
Brown medicinal extract is obtained after chloroform；
（2）Obtained medicinal extract adds 30mL5% sodium hydroxide solutions to boil 10min, filters while hot, by filtrate be placed in refrigerator 5 in~
10 DEG C of refrigeration 1h, filter, are washed with water to neutrality；Filtered residue adds 30 mL boilings to boil 10min, and it is 1 to be adjusted to pH with hydrochloric acid,
Precipitation is let cool, is filtered, with hot water washing sediment 3 times；Sediment is separated with silica gel column chromatography, uses petroleum ether:Chloroform=1:2
1000mL is eluted, TLC detections, discards eluent；Chloroform is used again:Ethyl acetate=18:1 elution, a, collection is collected per 100mL
30 parts, TLC detections, merge 5-15 parts, concentrate eluant obtains high purity oleanolic acid sample；Merge 17-30 parts, concentrate eluant
Obtain high-purity ursolic acid sample；By the oleanolic acid of gained and ursolic acid sample, dried respectively at 101 DEG C to constant weight；
（3）Dried oleanolic acid and ursolic acid sample is purified, assay is carried out using high performance liquid chromatography.
Chromatographic condition is as follows：
Chromatographic column：Sai Mo flies generation that Hypersil Gold C18（250 mm × 4.6 mm, 5 μm）, mobile phase is methanol -0.2%
Ammonium acetate solution（83：17）, flow velocity：0.6 mL/min, column temperature：30 DEG C, Detection wavelength：210 nm, sampling volume：20 μ L,
Spectral scanning range：The nm of 190 nm~600.
The g of standard substance oleanolic acid 0.1110 is accurately weighed respectively, the g of ursolic acid 0.0950 is placed in 50mL volumetric flasks,
Dissolved with ethanol and be settled to scale, concentration is respectively 2.220,1.900mg/mL.The mark of certain volume is accurately measured respectively
70% ethanol water of quasi- mixed liquor（Volume ratio）Be diluted to step by step various concentrations series, oleanolic acid concentration be 2.22,4.44,
11.1st, 22.2,44.4,111 μ g/mL, black bearberry acid concentration are 1.90,3.80,9.50,19.0,38.0,95.0 μ g/mL, are drawn
Standard curve.Embodiment 1 is accurately weighed respectively and purifies dried each 20mg of oleanolic acid and ursolic acid sample, uses absolute ethyl alcohol
Dissolving is settled to 25.0 mL, takes the solution to be measured after diluting 20 times with 70% ethanol water according to above-mentioned liquid phase chromatogram condition,
External standard method is used with its purity of calculated by peak area.
After testing, oleanolic acid, the linear relationship of ursolic acid are good, and the purity of oleanolic acid is 93.3%, and yield is
23.3mg/100g；The purity of ursolic acid is 94.2%, yield 94.9mg/100g.
Embodiment 2
The process of extraction purification oleanolic acid and ursolic acid from Sweet tea, comprises the following steps：
（1）Take Sweet tea sample to be smashed to pieces with bruisher, weigh sample 150g, the absolute ethyl alcohol for adding 3 times of amount weight surpasses at 35 DEG C
Sound wave extracts 15min, and extract solution filters, then wash filtered residue 1 time with 300 mL ethanol, then filters, smoke filtrate and the
Extract solution once merges, and the extract solution after concentration merges obtains medicinal extract, and the medicinal extract of gained is dissolved with 30mL chloroforms, concentration and recovery chlorine
Brown medicinal extract is obtained after imitative；
（2）Obtained medicinal extract adds 30mL5% sodium hydroxide solutions to boil 10min, filters while hot, by filtrate be placed in refrigerator 5 in~
10 DEG C of refrigeration 1h, filter, are washed with water to neutrality；Filtered residue adds the heating of 30 mL water to boil 10min, and pH is adjusted to hydrochloric acid
For 2, precipitation is let cool, is filtered, with hot water washing sediment 3 times；Sediment is separated with silica gel column chromatography, uses petroleum ether:Chloroform=
1:2 elution 1000mL, TLC detections, discard eluent；Chloroform is used again:Ethyl acetate=18:1 elution, portion is collected per 100mL,
30 parts are collected, TLC detections, merges 5-15 parts, concentrate eluant obtains high purity oleanolic acid sample；Merge 17-30 parts, concentration is washed
De- liquid obtains high-purity ursolic acid sample；By the oleanolic acid of gained and ursolic acid sample, dried respectively at 103 DEG C to constant weight；
（3）Dried oleanolic acid and ursolic acid sample is purified, content is carried out according to the high performance liquid chromatography in embodiment 1
Measure.
After testing, the purity of oleanolic acid is 93.1%, yield 24.1mg/100g；The purity of ursolic acid is 94.1%, is obtained
Rate is 94.0mg/100g.
Embodiment 3
The process of extraction purification oleanolic acid and ursolic acid from Sweet tea, comprises the following steps：
（1）Take Sweet tea sample to be smashed to pieces with bruisher, weigh sample 150g, the absolute ethyl alcohol for adding 4 times of amount weight surpasses at 40 DEG C
Sound wave extracts 20min, and extract solution filters, then wash filtered residue 1 time with 300 mL ethanol, then filters, smoke filtrate and the
Extract solution once merges, and the extract solution after concentration merges obtains medicinal extract, and the medicinal extract of gained is dissolved with 30mL chloroforms, concentration and recovery chlorine
Brown medicinal extract is obtained after imitative；·
（2）Obtained medicinal extract adds 30mL5% sodium hydroxide solutions to boil 10min, filters while hot, by filtrate be placed in refrigerator 5 in~
10 DEG C of refrigeration 1h, filter, are washed with water to neutrality；Filtered residue adds the heating of 30 mL water to boil 10min, and pH is adjusted to hydrochloric acid
For 2, precipitation is let cool, is filtered, with hot water washing sediment 3 times；Sediment is separated with silica gel column chromatography, uses petroleum ether:Chloroform
=1:2 elution 1000mL, TLC detections, discard eluent；Chloroform is used again:Ethyl acetate=18:1 elution, portion is collected per 100mL,
30 parts are collected, TLC detections, merges 5-15 parts, concentrate eluant obtains high purity oleanolic acid sample；Merge 17-30 parts, concentration is washed
De- liquid obtains high-purity ursolic acid sample；By the oleanolic acid of gained and ursolic acid sample, dried respectively 1 at 105 DEG C to perseverance
Weight；
（3）Dried oleanolic acid and ursolic acid sample is purified, content is carried out according to the high performance liquid chromatography in embodiment 1
Measure.
After testing, the purity of oleanolic acid is 93.0%, yield 22.8mg/100g；The purity of ursolic acid is 94.4%, is obtained
Rate is 93.6mg/100g.
Claims (1)
1. a kind of process of extraction purification oleanolic acid and ursolic acid from Sweet tea, it is characterised in that comprise the following steps：
（1）Take Sweet tea sample to smash to pieces, weigh the sample after smashing to pieces, add the absolute ethyl alcohol of 2~4 times of amounts of its weight at 25~40 DEG C
10~20min of lower ultrasonic wave extraction, extract solution filter；
Filtered residue is washed with ethanol again, then is filtered, smoke filtrate merges with the extract solution of first time, carrying after concentration merging
Liquid is taken to obtain medicinal extract, the medicinal extract of gained is dissolved with chloroform, and brown medicinal extract is obtained after concentration and recovery chloroform；
（2）By step（1）Obtained medicinal extract is boiled with 5% sodium hydroxide solution, is filtered while hot, and filtrate is placed in into refrigerator 5~10
DEG C refrigeration 1h, filter, be washed with water to neutrality；
Filtered residue adds boiling to boil, and it is 1~2 to be adjusted to pH with hydrochloric acid, lets cool precipitation, filters, uses hot water washing sediment；
Sediment is separated with silica gel column chromatography, uses petroleum ether:Chloroform=1:2 elutions, TLC detections, discard eluent；Chlorine is used again
It is imitative:Ethyl acetate=18:1 elution, a, 30 parts of collection is collected per 100mL, TLC detections, merges 5-15 parts, concentrate eluant obtains
High purity oleanolic acid sample；
Merge 17-30 parts, concentrate eluant obtains high-purity ursolic acid sample；
By the oleanolic acid of gained and ursolic acid sample, dried respectively at 101 DEG C~105 DEG C to constant weight；
（3）By step（2）Dried oleanolic acid and ursolic acid sample is purified, is carried out using high performance liquid chromatography containing measurement
Fixed, more than 90%, the yield of oleanolic acid obtains the purity of oleanolic acid and ursolic acid in more than 20mg/100g, ursolic acid
Rate is in more than 90mg/100g.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109503675A (en) * | 2018-12-07 | 2019-03-22 | 湖南食品药品职业学院 | The method of rubusoside and ursolic acid is extracted from Sweet tea |
| CN110508031A (en) * | 2019-07-30 | 2019-11-29 | 浙江工业大学 | A method of separating ursolic acid and oleanolic acid from plant |
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| CN105218616A (en) * | 2015-10-23 | 2016-01-06 | 山东省果树研究所 | A kind of method extracting ursolic acid and Oleanolic Acid from Blueberry |
| CN105232679A (en) * | 2015-11-11 | 2016-01-13 | 北京林业大学 | Method for simultaneously extracting ursolic acid and oleanolic acid in hawthorn by means of subcritical water |
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2017
- 2017-09-12 CN CN201710817875.4A patent/CN107513094B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105218616A (en) * | 2015-10-23 | 2016-01-06 | 山东省果树研究所 | A kind of method extracting ursolic acid and Oleanolic Acid from Blueberry |
| CN105232679A (en) * | 2015-11-11 | 2016-01-13 | 北京林业大学 | Method for simultaneously extracting ursolic acid and oleanolic acid in hawthorn by means of subcritical water |
Non-Patent Citations (3)
| Title |
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| 罗达伟: "甜茶果实化学成分的研究", 《广西师范大学硕士学位论文》 * |
| 谭冬明: "广西甜茶叶的化学成分研究", 《广西师范大学硕士学位论文》 * |
| 谭冬明等: "甜茶中齐墩果酸及熊果酸的检测方法的建立", 《食品研究与开发》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109503675A (en) * | 2018-12-07 | 2019-03-22 | 湖南食品药品职业学院 | The method of rubusoside and ursolic acid is extracted from Sweet tea |
| CN109503675B (en) * | 2018-12-07 | 2021-10-12 | 湖南食品药品职业学院 | Method for extracting rubusoside and ursolic acid from sweet tea |
| CN110508031A (en) * | 2019-07-30 | 2019-11-29 | 浙江工业大学 | A method of separating ursolic acid and oleanolic acid from plant |
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| CN107513094B (en) | 2019-05-28 |
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