CN112321578A - Method for preparing four monomers in bamboo leaf flavone by Prep-HPLC - Google Patents
Method for preparing four monomers in bamboo leaf flavone by Prep-HPLC Download PDFInfo
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- CN112321578A CN112321578A CN202011293363.0A CN202011293363A CN112321578A CN 112321578 A CN112321578 A CN 112321578A CN 202011293363 A CN202011293363 A CN 202011293363A CN 112321578 A CN112321578 A CN 112321578A
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- isoorientin
- isovitexin
- vitexin
- orientin
- bamboo leaf
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
- C07D407/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings
- C07D407/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
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- Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
Abstract
The invention discloses a method for preparing four monomers in bamboo leaf flavone by Prep-HPLC, which comprises five steps of extracting, drying, dissolving, preparing liquid phase purifying and distilling and separating, and finally orientin, isoorientin, vitexin and isovitexin powder is obtained. The method has the advantages of simple process, short time, high purity and the like.
Description
Technical Field
The invention relates to the technical field of biomass extraction, in particular to a method for preparing four monomers in bamboo leaf flavone by Prep-HPLC.
Background
Bamboo is a perennial evergreen plant of the subfamily Bambusoideae of the family Gramineae (Gramineae), and has a very abundant secondary metabolite. Bamboo leaves have a long history of medicinal and edible use in China, have the effects of clearing heat, relieving restlessness and promoting urination, and are mainly used for symptoms such as fever, polydipsia, mouth and tongue sores, dark urine, stranguria and the like. The herb action is recorded in detail in Ben Cao gang mu and Ben Jing Yuan.
The bamboo contains flavonoids and glycosides, active polysaccharides, special amino acids and their derivatives, which are related to human body life activity; contains manganese, zinc, selenium, germanium, silicon and other elements capable of activating human cells, and these substances form the basis of the wide physiological and pharmacological activities of bamboo leaf flavone.
Flavonoid compounds (flavonoids), also called flavonoids, are secondary metabolites of major plant polyphenols, widely existing in herbs, fruits, tea leaves, etc., and most of the flavonoid compounds are natural phenolic compounds. It is a plant active component which is needed by human body but can not be synthesized by itself, and is also a main functional factor of a plurality of Chinese herbal medicines such as scutellaria, ginkgo, sea backthern and the like. About 4000 different structures of flavonoids have been found.
Flavonoid compounds in bamboo leaves mainly exist in a glycoside form, most commonly in a flavone form, and mainly comprise carbon glycoside flavone. The content of the bamboo leaf flavone varies with the variety, the production place, the growth period, the picking season and other conditions of the bamboo leaves.
The bamboo leaf flavone has the obvious effects of resisting oxidation, eliminating free radicals, regulating blood fat, resisting tumors, blocking nitrosation reaction, resisting aging, resisting cardiovascular diseases, resisting inflammation and bacteria, enhancing immunity, resisting radiation and the like.
The bamboo leaf flavone is a novel plant flavone preparation which is separated from leaves of phyllostachys species represented by phyllostachys pubescens and has the characteristics of local resources and independent intellectual property rights in China, and has rich raw material sources, stable preparation quality and mild flavor. The bamboo leaf flavone has excellent biological effects of eliminating active oxygen free radicals, resisting lipid peroxidation, regulating blood lipid, resisting radiation, resisting fatigue, resisting tumor, inhibiting bacteria, diminishing inflammation, protecting heart and cerebral vessels, etc. The bamboo leaf antioxidant is a bamboo leaf phenolic part taking bamboo leaf flavone as a main component, is approved to be listed as the first national standard of the health standards for food additives of the people's republic of China in the year, and is approved to be used in eight major food systems of edible oil and fat, meat products, aquatic products, puffed food, fried food, baked food, instant grains, fruit and vegetable juice and meat beverages, tea beverages and the like at present, and the maximum use amount is five parts per million.
The extraction and separation of bamboo leaf flavone are also based on the principle of similar and compatible compounds. The flavonoid contained in bamboo leaf is flavone glycoside, has high polarity and hydrophilicity, and can be extracted with hot water, methanol, ethanol, n-butanol, acetone, etc. The water extraction has the disadvantages that the polarity of the solvent is too high, and impurities such as protein, saccharides and the like are easily brought out, so that the next separation and purification are influenced, and the methanol has high toxicity and is not beneficial to process control in industrial production, so that a safe and economic ethanol-water solution is usually selected as the extraction and elution solvent. The extraction method of the bamboo leaf flavone mainly comprises hot reflux extraction, ultrasonic-assisted extraction, microwave-assisted extraction and the like at present.
The typical compounds of the bamboo leaf carbon glycoside flavone are orientin, isoorientin, vitexin and isovitexin, few research reports about the separation, purification and preparation of bamboo leaf flavone monomers are reported, and only the researchers take the giant bamboo leaves of Vietnam as test materials and extract the bamboo leaves by ethanol extraction, so that the extract yield is 2.04%. Research reports that the purity of the four monomers in the bamboo leaf flavone powder is 76.939mg/g by processing the bamboo leaf extract by using macroporous resin and polyamide, namely the content of the bamboo leaf flavone in the four monomers is only 7.69%.
Disclosure of Invention
The invention aims to provide a method for preparing four high-purity monomers in bamboo leaf flavone by Prep-HPLC (preparative high performance liquid chromatography), and the method for preparing the four high-purity monomers in the bamboo leaf flavone has the advantages of simple process, short time, high purity and the like, and is very suitable for separating and preparing the four monomers in the bamboo leaf flavone.
In order to solve the technical problems, the invention adopts the following technical scheme:
a method for preparing four monomers in bamboo leaf flavone by Prep-HPLC comprises the following steps:
(1) extraction: pulverizing folium Bambusae, and extracting with 50% ethanol by volume under the following conditions: at 30 deg.C for 20 hr, at a ratio of 1:30, and filtering to obtain extractive solution.
(2) And (3) drying: concentrating the folium Bambusae extractive solution with rotary evaporator, and drying with freeze dryer to obtain folium Bambusae extract powder.
(3) Dissolving: weighing folium Bambusae extract powder, dissolving in 40% methanol, and filtering with microporous membrane to obtain clarified folium Bambusae extract solution.
(4) Preparation and liquid phase purification: the conditions of the preparative chromatography are as follows: c18 reverse phase chromatography column, mobile phase methanol (a): 0.5% acetic acid water, flow rate 15mL/min, detection wavelength 360nm, gradient elution.
(5) Orientin, isoorientin, vitexin, isovitexin: collecting orientin, isoorientin, vitexin and isovitexin fractions, and drying with a freeze dryer to obtain orientin, isoorientin, vitexin and isovitexin powder.
Preferably, the parameters of gradient elution in step (4) are: 0-10min25: 75; 10-20min35: 65; 20-30min 40: 60, adding a solvent to the mixture; 30-80min 50: 50.
Preferably, after preparing liquid phase and injecting sample, gradient elution is automatically carried out, and fractions with retention time of 50.806min, 52.586min, 57.878min and 64.712min are collected, namely orientin, isoorientin, vitexin and isovitexin respectively.
The invention has the following advantages:
the method is simple, and the production time is short: the separation and purification method of bamboo leaf flavone needs to firstly remove impurities, then uses polyamide and AB-8 resin for purification and separation, and then purifies the solution containing the extract to obtain a certain bamboo leaf flavone monomer. The invention directly utilizes Prep-HPLC to prepare liquid phase for separation and purification to obtain four monomers, and the method achieves the purpose of shortening the production time.
Secondly, high transfer rate: the conventional extraction, separation and purification process of the bamboo leaf flavone needs a plurality of working procedures, and the operation is complex, so that the yield of the bamboo leaf flavone monomer is reduced, and the invention directly utilizes Prep-HPLC to prepare liquid phase separation and purification after filtering the bamboo leaf extract, thereby reducing the loss of the flavone monomer and improving the transfer rate of the flavone monomer.
Thirdly, the purity of the monomer is high: the bamboo leaf extract is separated and purified by a Prep-HPLC method, has good effects of impurity removal, enrichment and elution, and can directly obtain bamboo leaf flavone monomers with higher purity.
Detailed Description
The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.
Example 1
A method for preparing four monomers in bamboo leaf flavone by Prep-HPLC comprises the following steps:
(1) extraction: weighing 10g of bamboo leaf raw medicinal materials, crushing into powder, adding ethanol water solution, extracting for 2 times at the extraction temperature of 30 ℃, adding 300mL of water for two times, and extracting for 20 hours.
(2) And (3) drying: concentrating the folium Bambusae extractive solution with rotary evaporator, and drying with freeze dryer to obtain folium Bambusae extract powder.
(3) Dissolving: weighing folium Bambusae extract powder, dissolving in 40% methanol, and filtering with microporous membrane to obtain clarified folium Bambusae extract solution.
(4) Preparation and liquid phase purification: the conditions of the preparative chromatography are as follows: c18 reverse phase chromatography column, mobile phase methanol (a): 0.5 percent acetic acid water, gradient elution, flow rate of 15mL/min and detection wavelength of 360 nm.
(5) Isoorientin, orientin, vitexin, isovitexin: collecting isoorientin, orientin, vitexin and isovitexin, and drying with a freeze dryer to obtain powder, wherein the content of orientin is 95%, and the content of other isoorientin, vitexin and isovitexin is more than 99%.
Example 2
A method for preparing four monomers in bamboo leaf flavone by Prep-HPLC comprises the following steps:
(1) extraction: weighing 20g of bamboo leaf raw medicinal materials, crushing into powder, adding ethanol water solution, extracting for 2 times at the extraction temperature of 30 ℃, adding 600mL of water for two times, and extracting for 20 hours.
(2) And (3) drying: concentrating the folium Bambusae extractive solution with rotary evaporator, and drying with freeze dryer to obtain folium Bambusae extract powder.
(3) Dissolving: weighing folium Bambusae extract powder, dissolving in 40% methanol, and filtering with microporous membrane to obtain clarified folium Bambusae extract solution.
(4) Preparation and liquid phase purification: the conditions of the preparative chromatography are as follows: c18 reverse phase chromatography column, mobile phase methanol (a): 0.5 percent acetic acid water, gradient elution, flow rate of 15mL/min and detection wavelength of 360 nm.
(5) Isoorientin, orientin, vitexin, isovitexin: collecting isoorientin, orientin, vitexin and isovitexin, and drying with a freeze dryer to obtain powder, wherein the content of orientin is 94%, and the content of other isoorientin, vitexin and isovitexin is more than 98%.
Example 3
A method for preparing four monomers in bamboo leaf flavone by Prep-HPLC comprises the following steps:
(1) extraction: weighing 50g of bamboo leaf raw medicinal materials, crushing into powder, adding ethanol water solution, extracting for 2 times at the extraction temperature of 30 ℃, adding 1500mL of water for two times, and extracting for 20 hours.
(2) And (3) drying: concentrating the folium Bambusae extractive solution with rotary evaporator, and drying with freeze dryer to obtain folium Bambusae extract powder.
(3) Dissolving: weighing folium Bambusae extract powder, dissolving in 40% methanol, and filtering with microporous membrane to obtain clarified folium Bambusae extract solution.
(4) Preparation and liquid phase purification: the conditions of the preparative chromatography are as follows: c18 reverse phase chromatography column, mobile phase methanol (a): 0.5 percent acetic acid water, gradient elution, flow rate of 15mL/min and detection wavelength of 360 nm.
| Time (min) | Methanol (%) | 0.5% acetic acid water (%) |
| 0 | 25 | 75 |
| 10 | 25 | 75 |
| 20 | 35 | 65 |
| 30 | 40 | 60 |
| 80 | 50 | 50 |
(5) Isoorientin, orientin, vitexin, isovitexin: collecting isoorientin, orientin, vitexin and isovitexin, and drying with a freeze dryer to obtain powder, wherein the content of isoorientin is 96%, the content of isoorientin is 97%, the content of vitexin is 99% and the content of isovitexin is 99%.
The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that are not thought of through the inventive work should be included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope defined by the claims.
Claims (3)
1. A method for preparing four monomers in bamboo leaf flavone by Prep-HPLC is characterized by comprising the following steps:
(1) extraction: pulverizing folium Bambusae, and extracting with 50% ethanol under the following conditions: filtering at 30 deg.C for 20 hr at a ratio of 1:30 to obtain extractive solution.
(2) And (3) drying: concentrating the folium Bambusae extractive solution with rotary evaporator, and drying with freeze dryer to obtain folium Bambusae extract powder.
(3) Dissolving: weighing folium Bambusae extract powder, dissolving in 40% methanol, and filtering with microporous membrane to obtain clarified folium Bambusae extract solution.
(4) Preparation and liquid phase purification: the conditions of the preparative chromatography are as follows: c18 reverse phase chromatography column, mobile phase methanol (a): 0.5% acetic acid water, flow rate 15mL/min, detection wavelength 360nm, gradient elution.
(5) Orientin, isoorientin, vitexin, isovitexin: collecting orientin, isoorientin, vitexin and isovitexin fractions, and drying with a freeze dryer to obtain orientin, isoorientin, vitexin and isovitexin powder.
2. The method according to claim 1, wherein the parameters of gradient elution in step (4) are: 0-10min25: 75; 35:65 after 10-20 min; 40-60 min for 20-30 min; 30-60min 50: 50.
3. The method according to claim 1, wherein in step (5), after the preparation of the liquid phase and the injection, gradient elution is automatically performed, and fractions with retention time of 50.806min, 52.586min, 57.878min and 64.712min, i.e., orientin, isoorientin, vitexin and isovitexin, are collected.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202011293363.0A CN112321578A (en) | 2020-11-18 | 2020-11-18 | Method for preparing four monomers in bamboo leaf flavone by Prep-HPLC |
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| CN202011293363.0A CN112321578A (en) | 2020-11-18 | 2020-11-18 | Method for preparing four monomers in bamboo leaf flavone by Prep-HPLC |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102285974A (en) * | 2011-09-27 | 2011-12-21 | 天津市尖峰天然产物研究开发有限公司 | Method for extracting orientin from bamboo leaf flavones |
| CN104370895A (en) * | 2014-08-22 | 2015-02-25 | 江苏汉邦科技有限公司 | Orientin and homoorientin preparation method |
| CN104557893A (en) * | 2014-12-19 | 2015-04-29 | 国际竹藤中心 | Extraction and purification technology of four kinds of flavone C-glycosides in moso bamboo leaves |
-
2020
- 2020-11-18 CN CN202011293363.0A patent/CN112321578A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102285974A (en) * | 2011-09-27 | 2011-12-21 | 天津市尖峰天然产物研究开发有限公司 | Method for extracting orientin from bamboo leaf flavones |
| CN104370895A (en) * | 2014-08-22 | 2015-02-25 | 江苏汉邦科技有限公司 | Orientin and homoorientin preparation method |
| CN104557893A (en) * | 2014-12-19 | 2015-04-29 | 国际竹藤中心 | Extraction and purification technology of four kinds of flavone C-glycosides in moso bamboo leaves |
Non-Patent Citations (1)
| Title |
|---|
| 袁珂 等: ""RP-HPLC同时测定淡竹叶中4种黄酮苷的含量"", 《中国中药杂志》 * |
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Application publication date: 20210205 |