CN111000946A

CN111000946A - Method for separating and purifying bamboo leaf flavone

Info

Publication number: CN111000946A
Application number: CN201911243348.2A
Authority: CN
Inventors: 陶文亮; 许子竞; 李洪庆
Original assignee: Guizhou Minzu University
Current assignee: Guizhou Minzu University
Priority date: 2019-12-06
Filing date: 2019-12-06
Publication date: 2020-04-14

Abstract

The invention discloses a method for separating bamboo leaf flavoneAn isolation and purification process comprising: dissolving or diluting folium Bambusae flavone extract or fluid extract in alcohol, precipitating with ethanol, and centrifuging; mixing the centrifugate with polyamide, performing column chromatography, performing gradient elution with water-alcohol system to obtain eluate a, and detecting total flavone content in eluate a with minimum value of b; performing silica gel column chromatography on the flavone with the content b, performing gradient elution by using petroleum ether-ethyl acetate and ethyl acetate-alcohol systems to obtain eluent of c groups, and detecting the flavone content in each eluent of the c groups, wherein the lowest value of the flavone content is d; selecting flavone with content d, performing silica gel or polyamide column chromatography twice to obtain eluent of group e, and detecting total flavone content in each eluent of group e, wherein the lowest value is f; using Al (N0)₃)₃、NaNO₂And NaOH is used for developing color, and the content of the total flavone is detected by an ultraviolet spectrum method. The method is suitable for industrial production.

Description

Method for separating and purifying bamboo leaf flavone

Technical Field

The invention relates to the field of separation and purification, in particular to a method for separating and purifying bamboo leaf flavone.

Background

The bamboo leaf flavone is extracted from bamboo leaves as a raw material. The functional factors contained in the bamboo leaves are mainly flavonoid glycoside and coumarin lactone, and the content and the biological activity of the effective components of the bamboo leaves are comparable to those of ginkgo leaf components. The flavonoids in the bamboo leaves have obvious effects of reducing blood fat and cholesterol. Pharmacological research shows that the bamboo leaf flavone has the function of resisting hypoxia of whole animals, can effectively expand coronary vessels, increase coronary flow, increase myocardial contractility, obviously improve myocardial ischemia, reduce myocardial infarction range, reduce platelet aggregation degree, effectively inhibit blood coagulation and thrombosis and has a certain protection effect on cerebral ischemia; researches show that the bamboo leaf extract rich in flavone has various biological effects, such as functions of removing free radicals, resisting oxidation, resisting aging, resisting bacteria and viruses and the like, and is widely applied to the aspects of health care products, cosmetics and food additives.

However, in the prior art, the extraction and purification process performed by taking bamboo leaf flavone as an object is less optimized, the purity is relatively low, and batch production is not realized. Therefore, it is required to provide a method for extracting bamboo leaf flavonoid which can realize high purity and is suitable for mass production.

Disclosure of Invention

Therefore, the present invention provides a method for separating and purifying bamboo leaf flavonoid to solve or at least alleviate the above problems.

According to one aspect of the present invention, there is provided a method for separating and purifying bamboo leaf flavonoids, comprising: heating and dissolving or diluting the bamboo leaf flavone extract or fluid extract with an ethanol solution with the concentration of more than or equal to 90%, standing, precipitating with ethanol, and centrifuging to obtain a centrifugate; mixing the centrifugate with polyamide, concentrating under reduced pressure, drying, separating with polyamide column chromatography, performing gradient elution with water-alcohol system mobile phase to obtain gradient eluents of group a, and detecting total flavone content in each eluent of group a, wherein the minimum content is b; separating the elution collection liquid with the total flavone content being more than or equal to the value b by a silica gel chromatographic column, performing gradient elution by adopting a petroleum ether-ethyl acetate system and an ethyl acetate-alcohol system to obtain c groups of eluents, and detecting the flavone content in each part of the eluents, wherein the lowest content is d; and selecting the eluent with the flavone content more than or equal to the value d, carrying out silica gel column chromatography or polyamide column chromatography separation for the second time, carrying out gradient elution by adopting a mobile phase corresponding to the selected chromatographic column to obtain e groups of eluent, and respectively detecting the flavone content in each eluent, wherein the lowest value of the flavone content of the e groups of eluent is f.

Optionally, in the separation and purification method according to the present invention, further comprising the steps of: separating the eluate with flavone content greater than or equal to f value with Sephadex LH-20 chromatographic column, and gradient eluting with water-methanol system as mobile phase to obtain single component bamboo leaf flavone.

Alternatively, in the separation and purification method according to the present invention, the bamboo leaves are any of bamboo genus bamboo leaves.

Optionally, in the separation and purification method according to the present invention, further comprising the steps of: and respectively concentrating and drying the eluent of the group a, the eluent of the group c and the eluent of the group e to obtain bamboo leaf flavone powder products with different purities.

Alternatively, in the separation and purification method according to the present invention, according to the original content of flavonoid in the extract, b is 20.02%, d is 50.24%, f is 80.11% in silica gel chromatography, and f is 78.92% in polyamide chromatography; the single-component bamboo leaf flavone comprises oxygen glycoside flavone, carbon glycoside flavone and flavone aglycone, and certainly, the b, d and f values are different when the raw extract is separated and purified due to different flavone contents.

Optionally, in the separation and purification method according to the present invention, the step of heating and dissolving or diluting the bamboo leaf flavonoid extract or the fluid extract with an ethanol solution of 90% or more, standing and precipitating with ethanol, and then centrifuging comprises: adding ethanol of 90% or more into the bamboo leaf flavone extract, heating to dissolve or dilute, adding purified water to adjust ethanol content to 80% or more; standing at 40-50 ℃, precipitating with ethanol, centrifuging, and reserving the centrifugate.

Alternatively, in the separation and purification method according to the present invention, the step of separating the polyamide column chromatography on the centrate-mixed polyamide comprises: adding polyamide into the centrifugate, concentrating the sample mixture under reduced pressure, drying the sample mixture until the sample mixture contains no ethanol, separating by polyamide column chromatography with purified water as the upper column solvent.

Alternatively, in the separation and purification method according to the present invention, the polyamide is mixed in a manner such that the mass ratio of the dry matter contained in the centrifugate to the polyamide is 1: (1-4) mixing the sample; the high ratio of the sample-mixed polyamide to the blank polyamide in the polyamide chromatographic column is (1-10): 1; the diameter range of the polyamide chromatographic column is 5-100 cm, and the particle size of the polyamide is 100-200 meshes.

Optionally, in the separation and purification method according to the invention, when gradient elution of polyamide column chromatography is carried out, the volume ratio of water to alcohol in the mobile phase is 10 (0-6), and when the elution color of one eluent becomes light, another alcohol solution with higher alcohol content is used for elution; wherein, the water-alcohol system in the water-alcohol system is a water-ethanol system or a water-methanol system.

Alternatively, in the separation and purification method according to the present invention, when gradient elution of polyamide column chromatography is performed, it is preferable that pure water, 10%, 20%, 30%, 60% by volume of an alcohol solution, or any volume ratio is used for the mobile phase in this order.

Alternatively, in the separation and purification method according to the present invention, the step of separating the eluate by silica gel column chromatography comprises: and (3) mixing the selected eluent and silica gel according to the mass ratio of dry substances to silica gel in the eluent of 1: (1-3) mixing the sample, and separating by silica gel column chromatography.

Optionally, in the separation and purification method according to the invention, the silica gel particle size is 200-400 meshes, the column bed height ratio of the blank silica gel and the sample-mixing silica gel in the silica gel chromatographic column is 1 (1-10), and the upper column solvent of the blank silica gel and the sample-mixing silica gel is petroleum ether or the volume ratio of petroleum ether to ethyl acetate is 10: (1-4), the height range of the column bed of the blank silica gel is 6-20 cm, and the diameter range of the silica gel chromatographic column is 5-100 cm.

Optionally, in the separation and purification method according to the present invention, when performing gradient elution on the silica gel chromatography column, the mobile phase sequentially performs gradient elution by using a petroleum ether-ethyl acetate system and an ethyl acetate-alcohol system, wherein the petroleum ether may have a boiling point in a range of 30 to 60 ℃, or 60 to 90 ℃, or 90 to 120 ℃.

Optionally, in the separation and purification method, when gradient elution of silica gel column chromatography is carried out, the volume of the mobile phase petroleum ether-ethyl acetate is (1-0): 1; the volume ratio of ethyl acetate to alcohol is 10 (0-4), and when the eluent with a certain volume ratio is eluted until the color is light yellow or nearly colorless, another mobile phase with higher alcohol content is used for elution.

Optionally, in the separation and purification method according to the invention, the polyamide is mixed according to the mass ratio of dry matter contained in the centrifugate to the polyamide of 1 (1-3) when the polyamide is mixed; when the silica gel is mixed, the sample is mixed according to the mass ratio of dry substances contained in the eluent collected by the upper column to the silica gel of 1 (2-3), and the granularity of the silica gel is 300-400 meshes.

Alternatively, in the separation and purification method according to the present invention, the total content of flavones in the eluate is passed through Al (N0)₃)₃、NaNO₂Developing with NaOH, and detecting at 510nm of ultraviolet spectrum; the single flavone component was detected by HPLC method.

Optionally, in the separation and purification method according to the present invention, the flavones in the bamboo leaf flavone extract and each eluent include oxygen glycoside flavones, carbon glycoside flavones and flavone aglycones, and the total flavones in each eluent are the total content of the flavonoids.

Optionally in accordance with the present inventionIn the separation and purification method of the invention, the step of detecting the total content of flavone in each group of eluent comprises the following steps: rutin is used as control, and Al (N0)₃)₃、NaNO₂Developing with NaOH, detecting the absorbance (A) of components with different concentrations at the ultraviolet 510nm, and drawing a standard curve; weighing the crude flavone product dried by each eluent, dissolving with 40% ethanol, diluting to 50mL, respectively taking each sample solution 10mL to 50mL volumetric flasks, supplementing to 25mL with 40% ethanol, adding 5mL NaNO₂Then 5mL of Al (N0)₃)₃And 5mL of NaOH, fixing the volume by 40% of ethanol, developing for 10 minutes, determining the absorbance (A) of each solution at 510nm by taking a blank reagent as a reference, and calculating the total content of flavone in each eluent by referring to a rutin standard curve.

The method for separating and purifying the bamboo leaf flavone is suitable for the bamboo leaf flavone extract extracted by any solvent. Removing polysaccharide, colloid, protein, pigment and other substances in the bamboo leaf flavone extract by an alcohol precipitation method for preliminary purification, separating by adopting polyamide column chromatography, performing gradient elution by using a water-alcohol system to obtain a group a of eluents, collecting the eluents in groups, and detecting the total flavone content of each part of the eluents by an ultraviolet method. Selecting total flavone greater than or equal to b from the eluent of the group a, performing silica gel column chromatography, performing gradient elution by a petroleum ether-ethyl acetate system and an ethyl acetate-alcohol system to obtain eluent of the group c, and detecting the content of the eluent to obtain the total flavone greater than or equal to d. Finally, selecting total flavone more than or equal to f, and purifying on SephadexLH-20 to obtain the flavone substance with single component. The method has the advantages of repeated reappearance, strong technical continuity, more byproducts, simple operation, environmental protection and low cost, can obtain the bamboo leaf flavonoid powder with different purity requirements, is suitable for industrial mass production, and has the characteristics of demonstration popularization and strong practicability.

The foregoing description is only an overview of the technical solutions of the present invention, and the embodiments of the present invention are described below in order to make the technical means of the present invention more clearly understood and to make the above and other objects, features, and advantages of the present invention more clearly understandable.

Drawings

To the accomplishment of the foregoing and related ends, certain illustrative aspects are described herein in connection with the following description and the annexed drawings, which are indicative of various ways in which the principles disclosed herein may be practiced, and all aspects and equivalents thereof are intended to be within the scope of the claimed subject matter. The above and other objects, features and advantages of the present disclosure will become more apparent from the following detailed description read in conjunction with the accompanying drawings. Throughout this disclosure, like reference numerals generally refer to like parts or elements.

Fig. 1 shows a flow chart of a method 100 for separating and purifying bamboo leaf flavonoid according to an embodiment of the present invention;

fig. 2 shows a flow chart of a method 200 for separating and purifying bamboo leaf flavonoid according to another embodiment of the present invention.

Detailed Description

Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

Fig. 1 shows a flow chart of a method 100 for separating and purifying bamboo leaf flavonoid according to an embodiment of the present invention. As shown in fig. 1, the method starts in step S110.

In step S110, the bamboo leaf flavonoid extract or fluid extract is dissolved or diluted by heating with an ethanol solution with a concentration of 90% or more, and then centrifuged after standing and alcohol precipitation to obtain a centrifugate.

The bamboo leaf may be any conventional bamboo leaf, and may include any bamboo leaf of the genus Phyllostachys, and may also include bamboo leaves of the genus Phyllostachys, Indocalamus, and the like. The bamboo leaf flavone extract can be any bamboo leaf flavone extract or fluid extract extracted by any solvent. According to one embodiment, the bamboo leaf flavone extract or fluid extract can be prepared according to the following method: crushing the dried bamboo leaf raw material into material fragments; putting the material fragments into an extraction tank, extracting for three times by using high-concentration alcohol as a solvent (such as methanol or ethanol with the concentration of more than or equal to 90%), and combining extracting solutions, wherein the extracting time is 1.5-2 h/time, the extracting temperature is 75-85 ℃, and the material-liquid ratio is 1 (8-10) g/mL; concentrating the combined extractive solutions into fluid paste or further drying into extract for use.

Specifically, the bamboo leaf raw materials that air-dry freshly can adopt the rubbing crusher that the sieve mesh diameter is 5 ~ 8mm to smash. The folium Bambusae material may also be selected from dried folium Sagittariae Sagittifoliae, dried folium Nandinae Domesticae or folium Bambusae. The extraction tank can be a pilot-scale extraction tank (with capacity of 100L or more) or a production-type extraction tank (with capacity of 2m or more)³). Each batch of materials can be extracted for three times, wherein the extraction time of the first two times of extraction is set to be 2 hours, and the extraction time of the third time of extraction is 1.5-2 hours. The ratio of the material to the liquid extracted for three times is respectively 10:1, 8:1 and 10:1, and the experimental process scheme can be pilot plant test or industrial production.

Mixing the extractive solutions, and concentrating. Specifically, the combined extracting solution is concentrated to 1/9-1/10 of the original volume at normal pressure, then is concentrated into a fluid paste under reduced pressure or is further dried into an extract for standby, and then the bamboo leaf flavone extract is pretreated and purified, and the method specifically comprises the following steps: adding ethanol (such as 95% but not limited thereto) of 90% or more into the bamboo leaf flavone extract or fluid extract, heating to dissolve or dilute, and adding purified water to adjust ethanol content to 80% or more. Standing at 40-50 ℃, precipitating with ethanol, and centrifuging to obtain a centrifugal liquid.

Subsequently, in step S120, the centrifugate is mixed with polyamide to obtain a mixed sample, the mixed sample is separated by polyamide column chromatography, gradient elution is performed by using a water-alcohol system as a mobile phase to obtain eluent of a group a, and the content of flavone in each eluent of the group a is detected, wherein the lowest value of the content is b.

According to one embodiment of the invention, the step of separating by polyamide column chromatography after adding polyamide into centrifugate and concentrating under reduced pressure comprises: adding polyamide into the centrifugate, concentrating the sample stirring liquid under reduced pressure, and drying the polyamide sample stirring substance to obtain an alcohol-free polyamide sample. And then, separating the polyamide-mixed sample and the blank polyamide by polyamide column chromatography.

Wherein, the mass ratio of dry matter contained in centrifugate to polyamide is 1: (1-4) carrying out sample mixing, preferably 1: (1-3). The high ratio of the sample-mixed polyamide to the blank polyamide in the polyamide chromatographic column is (1-10): 1, the diameter range of the polyamide chromatographic column is 5-100 cm, and the granularity of the polyamide is 100-200 meshes.

In addition, when gradient elution is performed on the polyamide column, the volume ratio of water to alcohol in the mobile phase is 10 (0-10), preferably 10: (0 to 6), the water-alcohol system in the water-alcohol system is a water-ethanol system or a water-methanol system. When the elution color of a certain volume proportion of the eluent is lighter, the elution is carried out by using another solution with higher alcohol content. Preferably, when the polyamide column chromatography gradient elution is carried out, the mobile phase is preferably subjected to content determination by sequentially adopting pure water, 10%, 20%, 30% and 60% alcohol solution in volume ratio or any alcohol-water volume ratio and collecting each component eluent.

Generally, the flavones in the bamboo leaf flavone extract and the eluent comprise oxygen glycoside flavone, carbon glycoside flavone and flavone aglycone, wherein the total content of the flavones is the total content of the three flavonoids. Wherein, the total content of flavone can be detected by adopting an ultraviolet spectroscopy, and can be obtained by the following steps:

rutin is used as control, and Al (N0)₃)₃、NaNO₂And developing with NaOH, detecting the absorbance (A) of the components with different concentrations at the ultraviolet 510nm, and drawing a standard curve. Weighing the crude flavone product dried by each eluent, dissolving with 40% ethanol, diluting to 50mL, respectively taking each sample solution 10mL to 50mL volumetric flasks, supplementing to 25mL with 40% ethanol, adding 5mL NaNO₂Then 5mL of Al (N0)₃)₃And 5mL of NaOH, fixing the volume by 40% of ethanol, developing for 10 minutes, determining the absorbance (A) of each solution at 510nm by taking a blank reagent as a reference, and calculating the total content of flavone in each eluent by referring to a rutin standard curve.

In addition, the invention can also detect the content of single flavone component in each eluent (such as a group of eluent, c group of eluent, e group of eluent, g group of eluent and the like in the following text), and the content of the component can be obtained by detecting through a High Performance Liquid Chromatography (HPLC) method, drawing a standard curve according to a reference substance and substituting the peak area of a certain single flavone component into the standard curve for calculation. Wherein the single flavone component comprises one or more of isoorientin, orientin, vitexin, isovitexin, etc.

After gradient elution of polyamide column, different water-alcohol ratios can obtain different total flavone contents, for example, the total flavone content of the eluent obtained by using 10% volume ratio of alcohol solution is greater than or equal to b, the total flavone content of the eluent obtained by using 20% volume ratio of alcohol solution is greater than or equal to d, and the eluent obtained by using 60% volume ratio of alcohol solution is mainly flavone aglycone.

Then, in step S130, the eluent with total flavone content greater than or equal to b is selected from the group a of eluents and is respectively separated by silica gel chromatography columns, gradient elution is successively carried out by adopting a petroleum ether-ethyl acetate system and an ethyl acetate-alcohol system to obtain a group c of eluents, and the flavone content in each gradient eluent is detected, wherein the lowest content is d.

It should be understood that one skilled in the art can select one or more eluates from the group a of eluates to separate and purify on silica gel column according to the target purity requirement, mainly based on the purification requirement. If 10 parts of the eluent in group a are collected, the minimum content b is 20.02%. The content of other eluent is 22.3%, 24.3%, 24.2%, 30.6%, etc., if the target content is more than 30%, the eluent with the content less than 30% can be combined and purified again to increase the content. Whereas 30.6% of the eluate can be purified separately to obtain a higher content requirement.

According to another embodiment, the step of separating the eluate on a silica gel column comprises: collecting the selected eluent and silica gel according to the mass ratio of dry matter in the eluent to the silica gel of 1: (1-3) stirring the sample, preferably stirring the sample according to the mass ratio of 1 (2-3). Then, the mixture is separated by a silica gel chromatographic column. Wherein, the particle size of the silica gel is 200-400 meshes, preferably 300 meshes. The column bed height ratio of the blank silica gel and the sample-mixing silica gel in the silica gel chromatographic column is 1 (1-10), and the upper column solvent of the blank silica gel is petroleum ether or the volume ratio of petroleum ether to ethyl acetate is 10: (1-4), the height range of the column bed of the blank silica gel is 6-20 cm, and the diameter range of the silica gel chromatographic column is 5-100 cm.

Furthermore, when gradient elution of the silica gel chromatographic column is carried out, a mobile phase petroleum ether-ethyl acetate system is adopted for elution at a volume ratio of (1-0): 1, then an ethyl acetate-alcohol system is adopted at a volume ratio of 10: (0-4), and the polarity of the solvent is changed from small to large. Preferably, when the mobile phase is a petroleum ether-ethyl acetate system, the volume ratio is 1:1, 1:3, 1:5 and 0:1 respectively; the petroleum ether can be selected from any one of the boiling point ranges of 30-60 ℃, 60-90 ℃ and 90-120 ℃. When the mobile phase is an ethyl acetate-alcohol system, the volume ratio of ethyl acetate to alcohol is 10 (0-4), the alcohol is methanol or ethanol, and when the eluent with a certain volume ratio is eluted to be yellowish in color or nearly colorless, the other mobile phase with higher alcohol content is used for elution. Preferably, the volume ratio of ethyl acetate to alcohol may be 10:0, 10:1, 10:2, 10: 3. And (3) separately collecting each eluent, and carrying out ultraviolet detection on each eluent to obtain and mark the total content of the flavone in the eluent. The method for detecting the total content of flavone is disclosed in detail in the description based on step S120, and is not described herein again.

Then, in step S140, the eluent with total flavone content greater than or equal to d is selected to be separated on a silica gel chromatographic column or a polyamide chromatographic column for two times, gradient elution is performed by using a mobile phase corresponding to the selected chromatographic column to obtain e groups of eluents, and the total flavone content in each eluent of e groups is respectively detected, wherein the lowest value is f.

That is, collecting fractions with total flavone content greater than or equal to d value from the c group of eluents as one or more fractions, and separating with polyamide chromatographic column twice or silica gel chromatographic column twice. Here again, based on the target purity requirement, the eluents are selected to have appropriate contents for purification, either individually or in combination. When a polyamide chromatographic column is used, the separation method can be implemented according to the contents described in step S120, such as the sample mixing ratio of the dry matter content in the eluate to the polyamide, the column bed height ratio of the sample-mixed polyamide to the blank polyamide, the diameter of the chromatographic column, the particle size of the polyamide, the ratio of the mobile phase component to the volume, and the detection of the flavone content in the eluate, and the like, and details thereof are not repeated herein. If silica gel column chromatography is adopted, the separation and detection method can be realized according to the content loaded in step S130, such as sample mixing ratio, column bed height ratio, column diameter, silica gel particle size, mobile phase component and volume ratio, and flavone content detection of eluent, and the like, and details are not repeated here.

In addition, the gradient eluents of group a after polyamide chromatography in step S120 can be referred to as first eluents, the gradient eluents of group c after silica gel chromatography column separation in step S130 can be referred to as second eluents, and the gradient eluents of group e after silica gel separation or secondary polyamide column separation in step S140 can be referred to as third eluents.

And c groups of eluent are obtained after purification, separation and purification, and the lowest concentration of the eluent is d. In order to meet the requirement of higher purity, one skilled in the art can select one or more parts of eluent from the c groups of eluent to perform secondary silica gel column chromatography or polyamide column chromatography separation and purification to obtain e groups of eluent. Wherein, one part of the collection liquid in the e group can be purified separately or a plurality of parts of the elution liquid can be combined and purified.

Optionally, the method 100 may further include the steps of: and respectively concentrating and drying the eluent of the group a, the eluent of the group c and the eluent of the group e to obtain bamboo leaf flavone powder products with different purities. Naturally, the selected eluent with the total flavone content of more than or equal to b, d or f can be concentrated and dried to obtain the bamboo leaf flavone powder product with corresponding purity.

Finally, the above-mentioned detection of flavones refers to the detection of total flavones.

Fig. 2 shows a flow chart of a method 200 for separating and purifying bamboo leaf flavonoid according to another embodiment of the present invention. The method comprises steps S210-S250, wherein steps S210-240 are the same as steps S110-S140 in the method 100, and are not described herein again.

After step S240, if higher purity or even single-component bamboo leaf flavonoid powder is desired, in step S250, from the e group of eluents, selecting fractions of the eluents with total flavone content of f or more, separating by Sephadex column chromatography (Sephadex LH-20), and performing gradient elution by using a water-methanol system as a mobile phase to obtain g groups of eluents, which can obtain single-component flavonoids. For each single flavone component, the flavone content can be detected by HPLC.

In another implementation manner of step S250, the e-group eluate can be further processed by combining a recrystallization method to obtain a single flavone component with high purity. It will also be appreciated that if only bamboo leaf flavonoid of content f is desired, the e eluate may be directly concentrated and dried without further application to a Sephadex LH-20 column or recrystallization.

Finally, it is noted that b and b corresponding to the eluent in the group a are 20.02%; d and d corresponding to the eluent in the group c are 50.24 percent; f for group e eluent (silica gel chromatography f 80.11%, polyamide chromatography f 78.92%). Of course, the values of b, d and f, which are based on the extract with different flavone contents, may be different in different experiments, but are not limited thereto, and may be other values, which is not limited by the present invention.

The method for separating and purifying bamboo leaf flavone from bamboo leaf extract of the present invention is illustrated by the following specific examples, which are only for the purpose of making those skilled in the art better understand the present invention, but not for the purpose of limiting the present invention in any way.

Example 1

Weighing a certain amount of folium Bambusae flavone extract (such as 4.0kg, total flavone content is 0.67%), adding 95% ethanol, heating for dissolving completely, adding purified water, adjusting ethanol content to about 85%, standing at 50 deg.C for precipitating with ethanol, centrifuging to remove polysaccharides, colloid and protein substances, concentrating the centrifugate, drying, weighing 2970.3g, and detecting total content by UV to be 0.88%.

Example 2

And (2) separating the centrifugate in the example 1 by using a polyamide column chromatography according to the mass ratio of the dry matter to the polyamide of 1:3, eluting with purified water until the eluent is pale, then eluting with 20% ethanol solution, collecting the eluent, concentrating and drying the eluent to obtain 106.59g, wherein the content of flavone is 22.56% by UV detection.

Example 3

And (3) mixing the dry matter obtained in the example 2 with 300-mesh silica gel according to the mass ratio of 1:2.5, performing column chromatography on blank petroleum ether, eluting with petroleum ether-ethyl acetate according to the volume ratio of 1-0: 1, and then eluting with ethyl acetate-methanol solution according to the volume ratio of 10: 2. Collecting eluate with volume ratio of 10:2, concentrating, and drying to obtain 25.23g with content of 81.04% by UV detection.

The method for separating and purifying the bamboo leaf flavone is suitable for the bamboo leaf flavone extract extracted by any solvent, and the bamboo leaves comprise any genus of bamboo leaves, and also comprise lophatherum and indocalamus. Removing polysaccharide, colloid, protein and other substances in the bamboo leaf flavone extract by an alcohol precipitation method, realizing primary purification, further separating by adopting polyamide column chromatography and silica gel column chromatography to obtain high-content bamboo leaf total flavone, and performing Sephadex LH-20 chromatography on the high-content bamboo leaf flavone to obtain the flavone substance with single component. The invention is implemented by small test and pilot test, has good effect and can be further produced industrially.

A11, the method of A10, wherein the silica gel particle size is 200-400 meshes, the column bed height ratio of blank silica gel and sample-mixing silica gel in the silica gel chromatographic column is 1 (1-10), the solvent of the upper column is petroleum ether or the volume ratio of petroleum ether to ethyl acetate is 10: (1-4), the height range of the column bed of the blank silica gel is 6-20 cm, and the diameter range of the silica gel chromatographic column is 5-100 cm. A12, the method is as described in A1-A11, wherein, when gradient elution of silica gel chromatographic column is carried out, the volume ratio of ethyl acetate and alcohol is 10 (0-4), the alcohol is methanol or ethanol, and when a certain volume ratio of eluent is eluted to light yellow or nearly colorless, another mobile phase with higher alcohol content is used for elution; the boiling point range of the petroleum ether can be 30-60 ℃, or 60-90 ℃ or 90-120 ℃, and the volume ratio of the petroleum ether to the ethyl acetate system is (1-0): 1;

a13, the method according to any one of A1-A11, wherein the gradient elution of the silica gel column is carried out by eluting with a petroleum ether-ethyl acetate system and then with an ethyl acetate-alcohol system.A14, the method A10, wherein the sample mixing is carried out according to the mass ratio of dry matter mass in the eluent collected by the upper column to the silica gel being 1 (1-3), and the particle size of the silica gel is 300-400 meshes. A15 method according to A1, wherein the total content of flavones in the eluate is measured by Al (N0)₃)₃、NaNO₂And NaOH, ultraviolet 510nm, single component by HPLC detection. A16, the method according to any one of A1-A15, wherein the flavones in the bamboo leaf flavone extract and each eluate include oxygen glycoside flavone, carbon glycoside flavone and flavone aglycone.

A17, the method according to A16, wherein the step of detecting the total content of flavones in each group of eluents comprises: rutin is used as control, and Al (N0)₃)₃、NaNO₂Developing with NaOH, detecting the absorbance (A) of components with different concentrations at the ultraviolet 510nm, and drawing a standard curve; detecting the content of the crude flavone, weighing the crude flavone dried by each eluent, dissolving with 40% ethanol, fixing the volume to 50mL, respectively taking each sample solution 10mL to 50mL volumetric flasks, supplementing to 25mL with 40% ethanol, adding 5mL NaNO₂Then 5mL of Al (N0)₃)₃And 5mL of NaOH, fixing the volume by 40% of ethanol, developing for 10 minutes, determining the absorbance (A) of each solution at 510nm by taking a blank reagent as a reference, and calculating the total content of flavone in each eluent by referring to a rutin standard curve.

In the description of the present specification, the terms "connected", "fixed", and the like are to be construed broadly unless otherwise explicitly specified or limited. Furthermore, the terms "upper", "lower", "inner", "outer", "top", "bottom", and the like, indicate orientations or positional relationships based on those shown in the drawings, and are only for convenience in describing the present invention and simplifying the description, but do not indicate or imply that the referred device or unit must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present invention.

In the description herein, the description of the terms "one embodiment," "some embodiments," "specific embodiments," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

In the description provided herein, numerous specific details are set forth. It is understood, however, that embodiments of the invention may be practiced without these specific details. In some instances, well-known methods, structures and techniques have not been shown in detail in order not to obscure an understanding of this description.

Claims

1. A method for separating and purifying bamboo leaf flavone comprises the following steps:

heating and dissolving or diluting the bamboo leaf flavone extract or fluid extract with an ethanol solution with the concentration of more than or equal to 90%, standing, precipitating with ethanol, and centrifuging to obtain a centrifugate;

mixing the centrifugate with polyamide to obtain a mixed sample, performing polyamide column chromatography separation, performing gradient elution by adopting a water-alcohol system mobile phase to obtain each gradient collection liquid of the group a, and detecting the total content of flavone in each gradient eluent, wherein the minimum content of the flavone is b;

separating the eluate with total flavone content greater than or equal to b by silica gel column chromatography, sequentially performing gradient elution with petroleum ether-ethyl acetate and ethyl acetate-alcohol system mobile phase to obtain gradient eluents of group c, and detecting flavone content in gradient eluents with minimum content of d; and

and (3) selecting the eluent with the total flavone content of more than or equal to d, carrying out silica gel column chromatography or polyamide column chromatography twice to carry out gradient elution separation to obtain e groups of gradient eluents, and detecting the flavone content in each gradient eluent, wherein the lowest value of the flavone content of the e groups of eluents is f.

2. The method of claim 1, further comprising the steps of:

and (3) selecting the eluent with the total flavone content more than or equal to f from the e groups of eluent, further separating and purifying the eluent by a Sephadex LH-20 column, and performing gradient elution by adopting a water-methanol system mobile phase to obtain the single-component bamboo leaf flavone.

3. The bamboo leaf flavonoid extract or fluid extract of claim 1, wherein the bamboo leaf is any bamboo leaf of the genus Phyllostachys.

4. The method of claim 2, wherein b is 20.02%, d is 50.24%, f is 80.11% for silica gel chromatography, and f is 78.92% for polyamide chromatography.

5. The method as claimed in any one of claims 1 to 4, wherein the step of heating and dissolving or diluting the bamboo leaf flavone extract or fluid extract with 90% or more ethanol solution, standing and precipitating with ethanol, and centrifuging comprises:

adding ethanol of 90% or more into folium Bambusae flavone extract or fluid extract, heating to dissolve or dilute, and adding purified water to adjust ethanol content to 80% or more;

standing at 40-50 ℃, precipitating with ethanol, centrifuging, and taking the centrifugate.

6. The method of any one of claims 1-5, wherein the step of subjecting the centrate to polyamide-mixed polyamide column chromatography comprises:

adding the polyamide into the centrifugate, concentrating under reduced pressure, and drying the polyamide sample until the polyamide sample does not contain ethanol to obtain a polyamide sample; and

and separating the polyamide-mixed sample from the blank polyamide by using a polyamide column chromatography.

7. The method of claim 6, wherein,

and (3) when the polyamide is stirred, according to the mass ratio of dry matter contained in the centrifugate to the polyamide of 1: (1-4) mixing the sample;

the high ratio of the polyamide-mixed sample to the blank polyamide in the polyamide chromatographic column is (1-10): 1;

the diameter range of the polyamide chromatographic column is 5-100 cm, and the particle size of the polyamide is 100-200 meshes.

8. The method of any one of claims 1-7,

when gradient elution of polyamide chromatography is carried out, the volume ratio of a water-alcohol system in a mobile phase is 10 (0-10), and when the color of one eluent gradually becomes lighter, another eluent with higher alcohol content is used for elution;

wherein the water-alcohol system is a water-ethanol system or a water-methanol system.

9. The method of any one of claims 1-7,

in the case of performing gradient elution of polyamide chromatography, it is preferable to use pure water, 10%, 20%, 30%, 60% by volume of an alcohol solution, or any alcohol-water volume ratio in this order as the mobile phase.

10. The process of any one of claims 1 to 9, wherein the step of subjecting the eluate to chromatographic separation on a silica gel column comprises:

and (3) mixing the selected elution collecting liquid and silica gel according to the mass ratio of dry substances to silica gel in the elution collecting liquid of 1: (2-3) mixing the sample, and separating by using a silica gel chromatographic column.