CN109010409B - Extraction and purification process and content detection method of total flavonoids in herminium leaves - Google Patents

Abstract

The invention provides a method for enriching and purifying total flavonoids in folium terminaliae immaturus. Extracting the human facefruit leaves by heating and refluxing with 60-90% ethanol, concentrating under reduced pressure to obtain a crude extract, and purifying by a polyamide column to obtain a purified sample. The invention adopts an ultraviolet visible spectrophotometry to determine the content of the total flavone of the folium fici, and adopts a high performance liquid chromatography to determine the content of the luteolin (fukugetin) and the fukugiside (fukugiside) in the total flavone. The invention adopts the raw materials of the Chinese prickly ash leaves, has low cost, stable content of the purified total flavone, can reach more than 50 percent by utilizing ultraviolet spectrophotometry and high performance liquid chromatography detection, and can meet the requirement of five types of new Chinese medicines on the content of effective parts.

Description

Extraction and purification process and content detection method of total flavonoids in herminium leaves

Technical Field

The invention relates to the technical field of plant medicines, in particular to a process for enriching and purifying total flavonoids in folium terminaliae immaturus and a method for detecting the content of flavonoids in corresponding extracts.

Background

Saposhnikovia Herminium (Garcinia xanthochymus hook. f.) is a plant of the genus Garcinia of the family Guttiferae. In Thailand and Indian traditional medicine, the fruits of Nippon pricklyash can treat diseases of the liver and gallbladder, diarrhea and dysentery. As a traditional Dai medicine, the fruits and leaves of the manyflower pricklyash fruit have the effects of expelling parasites, clearing heat and removing toxicity, relieving food poisoning and the like. Earlier researches on leaf phytochemistry of the plants show that main chemical components of the plants are flavonoid compounds, luteolin (fukuetin) and fukugenide (fukugenide) are representative compounds of the plants, the plants show good alpha amylase inhibitory activity and GSK-3 beta inhibitory activity, have various pharmacological activities of resisting diabetes, oxidation and inflammation, and have the potential of developing a novel diabetes-treating medicine by enriching and purifying total flavonoids in human facial leaves. The folium fici Praeparata is a medicinal and edible material, the leaf extract of the folium fici Praeparata can reduce the common side effects of the existing commercially available medicines such as digestive tract irritation, dizziness, palpitation and the like, and the folium fici Praeparata extract contains abundant total flavonoids with phenolic hydroxyl groups, can regulate blood sugar in multiple ways, has antioxidant activity, can obviously improve diabetic polyneuropathy, and relieves the pathological occurrence and development process of diabetes to a certain extent. The luteolin (fukugetin) in the leaves of the Chinese gooseberry has good anti-diabetic activity, antioxidant activity, anti-tumor activity and anti-diabetic activity.

The process research of the separation and purification of the total flavonoids in the Chinese gooseberry leaves is particularly important in the research and development of new medicines. The application and preparation method of the Chinese patent CN106074636A manyflower cherry leaf extract disclose a preparation method of the manyflower cherry leaf extract, which comprises the following steps: (1) crushing dried medicinal material of the folium terminaliae immaturus, extracting with 40-80% ethanol water solution by volume percentage concentration, filtering the extracting solution and recovering ethanol to obtain ethanol extract of the folium terminaliae immaturus; (2) adding water to the alcohol extract of the folium terminaliae immaturus to adjust the concentration, adjusting the pH value to 7-8, adsorbing the alcohol extract by using an HPD100 macroporous adsorption resin column, eluting with water, eluting with an ethanol water solution with the volume percentage concentration of 20-30%, eluting with an ethanol water solution with the volume percentage concentration of 50-70%, collecting the ethanol water solution eluent with the concentration of 50-70%, recovering under reduced pressure until no alcohol smell exists, eluting with the HPD100 resin, and drying to obtain the total flavone extract of the folium terminaliae immaturus. The composition contains 3 flavonoid compounds which are respectively: fukugetin, fukugisenide and bilobalide. And the mass sum of the 3 compounds is not less than 50% of the mass of the total flavone part. The total mass of the 3 compounds is 55% of the total flavone of the folium fici by high performance liquid chromatography. (3) Dissolving the total flavone extract of the folium terminaliae immaturus in water at 50-60 ℃, adjusting the concentration, loading the solution onto a polyamide column with 30-60 meshes, eluting with water, sequentially eluting with 20%, 35%, 60% and 75% ethanol in volume percentage, respectively collecting 35%, 60% and 75% ethanol eluates, recovering a small volume of the eluates under reduced pressure, and freeze-drying to respectively obtain compounds of fukuchiin, fukukoxanthin and bilobalide.

However, the above preparation method has the following disadvantages: the process adopts HPD100 macroporous adsorbent resin to enrich total flavone, and adopts 10% NaHCO as sample₃The pH value is adjusted to 8, the extracting solution is in an alkaline medium, and because biflavonoid compounds in the acorn have a flavanone structural unit, the flavanone is easy to open rings in alkaline liquor and is converted into a corresponding chalcone structural unit, so that the enriched total flavone is not the flavonoid compounds originally existing in plants but the compounds derived chemically.

Disclosure of Invention

The invention solves the defects in the prior art and provides a process for enriching and purifying total flavonoids in the leaves of the manyflower, a method for detecting the total flavonoids and a method for detecting the contents of luteolin and fukuoside. The method has simple process, obvious effect, high economic benefit and is suitable for the requirements of technological production.

The technical scheme adopted for realizing the above purpose of the invention is as follows:

a process for extracting and purifying total flavone substances in folium fici Tikouae comprises the following steps:

(1) taking a manyflower cherry leaf raw material, and heating, refluxing and extracting the manyflower cherry leaf raw material by using an ethanol solution with 6-8 times of volume amount and 60-90% of volume concentration to obtain an extracting solution;

(2) concentrating the extractive solution under reduced pressure until no alcohol smell exists to obtain alcohol extract;

(3) and (3) passing the alcohol extract obtained in the previous step through a 80-100-mesh polyamide column, and extracting the alcohol extract: the polyamide resin content is 1:2, the diameter-height ratio is 1:3, the flow rate is 0.5BV/h, 5-7 times of column volume is eluted by 25-35% of ethanol solution by volume concentration, 9-11 times of column volume is eluted by 45-55% of ethanol solution by volume concentration, 5-7 times of column volume is eluted by 65-75% of ethanol solution by volume concentration, third column volume eluent of 45-55% of ethanol solution by volume concentration and second column volume eluent of 65-75% of ethanol solution by volume concentration are collected, the two parts of eluates are combined, ethanol is recovered under reduced pressure, and the dry powder obtained after freeze drying is the total flavone extract, the content of the total flavone in the total flavone extract is detected, and the content of the total flavone exceeds 50%.

In the step (1), the heating reflux temperature is 80 ℃, and the time is 1 hour.

And (3) eluting by using 30% ethanol solution with volume concentration for 6 times of column volume, eluting by using 50% ethanol solution with volume concentration for 10 times of column volume, and eluting by using 70% ethanol solution with volume concentration for 6 times of column volume in sequence.

In the step (3), the content of the total flavonoids in the total flavonoid extract can be detected by adopting an ultraviolet-visible spectrophotometry, and the method comprises the following steps:

(1) preparing a reference substance solution: taking a luteolin reference substance, precisely weighing, dissolving with methanol, and preparing into reference substance solution containing luteolin reference substance 0.1mg per 1mL solution;

(2) preparation of a test solution: precisely weighing the total flavone extract, dissolving with ethanol, and preparing into test solution containing 0.25mg sample per 1mL solution;

(3) preparation of a standard curve: precisely measuring reference substance solutions 0.5mL, 1.0mL, 1.5mL, 2.0mL and 2.5mL, respectively placing in 10mL volumetric flasks, adding methanol to 3mL, adding 0.1mol/L aluminum chloride ethanol solution 2mL, adding 3mL of sodium acetate/acetic acid buffer solution with pH of 5.2, finally fixing the volume to a scale with ethanol, taking corresponding reagents as blanks, shaking up, placing at 35 ℃ for 15min, measuring absorbance at a wavelength of 415nm, taking the absorbance as a vertical coordinate and the mass as a horizontal coordinate, and drawing a standard curve;

(4) the determination method comprises the following steps: precisely measuring 1mL of the test solution, placing the test solution in a 10mL measuring flask, measuring the absorbance according to the measuring method in the step (3), and further calculating the amount of the luteolin in the test solution from the standard curve;

(5) and (3) measuring results: the total flavone content was calculated from the regression equation.

And (3) detecting the content of the total flavonoids in the total flavonoid extract by adopting a high performance liquid chromatography, wherein the method comprises the following steps:

(1) preparing a reference substance solution: because the active ingredients in the total flavone extract are mainly gamboge biflavone and fukuoside, firstly, 20mg of the gamboge biflavone reference substance and 10mg of the fukuoside reference substance are respectively precisely weighed, and are dissolved by adding 95% chromatographic methanol to prepare a reference substance solution containing 0.4mg of the gamboge biflavone and 0.2mg of the fukuoside per mL;

(2) preparation of a test solution: precisely weighing 20mg of total flavone extract, dissolving with 95% chromatographic methanol, and diluting to 50mL to obtain test solution with concentration of 0.4 mg/mL;

(3) and (3) sample determination: measuring reference substance and sample with high performance liquid chromatograph under the following chromatographic conditions: a chromatographic column: agilent C18 chromatography column (4.6 mm' 250mm, 5 μm); detection wavelength: 289 nm; flow rate: 1.0 mL/min; column temperature: 30 ℃; sample introduction amount: 10 mu L of the solution; mobile phase: acetonitrile (a), methanol (B), 0.5% aqueous phosphoric acid (C), gradient elution, the elution procedure was as follows:

time (minutes)	Mobile phase A (%)	Mobile phase B (%)	Mobile phase C (%)
				0	15	20	65
15	15	20	65
				20	20	25	55
40	20	25	55

(4) And (4) calculating a result: according to the chromatograms of the sample and the reference substance, recording peak areas, and calculating the contents of the luteolin and the fukuloside in the sample by adopting an external standard method.

Compared with the prior art, the technical scheme provided by the invention has the advantages that:

(1) the preparation method disclosed by the invention is simple in process, remarkable in effect and suitable for technological production requirements, compared with the patent CN106074636A, the preparation method avoids using an alkaline medium, and simultaneously determines the sampling quantity, the diameter-height ratio and the flow rate of the purified technological parameters.

(2) The preparation method of the invention adopts the ethanol water system as the solvent, is a common solvent in normal drug production, has good safety to human body, is easy to recycle and has high economic benefit.

(3) Under the premise of ensuring the content and yield of the total flavone, 50 percent and 70 percent are collected in sections, and 50 percent ethanol has the function of removing impurities, so that the content of a part of the total flavone in 50 percent and 70 percent of elution components respectively reaches more than 50 percent.

(4) Compared with the patent CN106074636A, the invention provides a method for measuring the total flavone content of the folium fici extract by using an ultraviolet-visible spectrophotometry, which has strong specificity, accuracy and simplicity, and can effectively detect the total flavone content of the folium fici compared with the traditional method for measuring the total flavone content by using rutin or quercetin as a reference substance. Meanwhile, a new chromatographic condition is developed through high performance liquid chromatography, and the contents of the antidiabetic active substances of gamboge biflavone and fukuoside in the total flavonoids are simultaneously determined.

(5) The preparation and detection method of the total flavone extract of the herminium europaeum leaves is established by researching the total flavone extract of the herminium europaeum leaves, and is beneficial to the development and research of relevant products of the herminium europaeum leaves.

Drawings

FIG. 1 is a flow diagram of an extraction process provided by the present invention;

FIG. 2 is a high performance liquid chromatogram of the total flavonoid extract prepared in the example of the present invention.

Detailed Description

The present invention will be described in detail with reference to specific examples.

Example 1

The preparation process flow of the total flavone sample is shown in figure 1, and the specific preparation method is as follows:

taking 2000g of dried and pulverized Japanese facial fruit leaves, adding 12L of 70% ethanol, extracting under reflux at 80 deg.C for 1 hr, extracting once, filtering under reduced pressure, concentrating the extractive solution until no alcohol smell, purifying with 4.0kg polyamide chromatographic column (column diameter 20cm, column height 60cm, 1BV 10L), flow rate 0.5 BV/h. The elution conditions were: eluting with 30% ethanol for 6 column volumes; eluting with 50% ethanol for 10 column volumes; 70% ethanol elutes 6 column volumes each. Collecting the fractions with maximum content in 50% and 70% ethanol eluate, recovering ethanol under reduced pressure, and freeze drying to obtain dried powder, to obtain 110.4g total flavone extract.

Example 2

The content of total flavone is determined by using a visible spectrophotometry (2015 edition of general rules 0401 of Chinese pharmacopoeia), using gamboge biflavone (self-made, purity 98%) as a reference substance, and reacting flavonoid compounds with aluminum salt to generate yellow complex, wherein the specific experimental method comprises the following steps:

1 preparation of control solution

Taking a proper amount of the gamboge biflavone Fukugetin reference substance, precisely weighing, dissolving with methanol, and preparing into 0.1mg of reference substance solution containing the gamboge biflavone Fukugetin reference substance per 1 mL.

2 preparation of test solution

Taking a proper amount of the dry sample powder in the example 1, precisely weighing, dissolving with ethanol, and preparing into a test solution containing 0.25mg of the sample per 1 mL.

3 Total Flavonoids assay

Precisely measuring reference substance solutions 0.5mL, 1.0mL, 1.5mL, 2.0mL and 2.5mL, respectively placing in 10mL volumetric flasks, adding methanol to 3mL, adding 0.1mol/L aluminum chloride ethanol solution 2mL, adding 3mL of sodium acetate/acetic acid buffer solution with pH of 5.2, finally adding ethanol to constant volume to scale, taking corresponding reagents as blank, shaking up, and placing at 35 deg.C for 15 min. The absorbance was measured at a wavelength of 415nm by UV-visible spectrophotometry (national pharmacopoeia 2015 edition, general rule 0401). The absorbance was plotted as the ordinate and the mass (mg) as the abscissa, to obtain a standard curve. 1mL of the test sample solution was precisely measured and placed in a 10mL measuring flask, and the absorbance was measured according to the method from "adding methanol to 3 mL" according to the method under the standard curve preparation. And (3) calculating the content of the luteolin in the test solution from the standard curve, and calculating to obtain the luteolin test solution.

As a result: the product contains 55.24% of total flavonoids (calculated as luteolin (C30H20O 11)) in terms of dry product.

Example 3

The HPLC method for measuring the content comprises the following specific experimental methods, considering that the active ingredients of the Chinese prickly ash fruit extract are luteolin (fukugetin) and glycoside fukugiensis glycoside (fukugisiide), and the high performance liquid chromatography is used for measuring the contents of the two compounds in the purified sample:

1 chromatographic conditions

A chromatographic column: agilent C18 chromatography column (4.6 mm. times.250 mm, 5 μm); detection wavelength: 289 nm; flow rate: 1.0 mL/min; column temperature: 30 ℃; sample introduction amount: 10 mu L of the solution; mobile phase: acetonitrile (A), methanol (B), 0.5% phosphoric acid aqueous solution (C), gradient elution, elution program see Table 1 below, sample chromatogram as shown in figure 2.

2 preparation of test article

About 20mg of the sample dried powder obtained in example 1 was precisely weighed, dissolved in 95% methanol for chromatography, and then diluted to 50mL to obtain a sample with a concentration of 0.4 mg/mL.

3 preparation of control

Accurately weighing 20mg of resina Garciniae biflavone reference substance and 10mg of fukuoside reference substance, respectively, adding 95% chromatographic methanol for dissolving, and preparing into reference substance solution containing 0.4mg of resina Garciniae biflavone and 0.2mg of fukuoside per mL.

4 content determination

Precisely sucking 1.0mL, 2.0mL, 3.0mL, 4.0mL and 5.0mL of the control mother liquor, placing in a 10mL volumetric flask, and diluting to constant volume with 95% methanol to obtain the final product. Then, 10. mu.L of each sample was aspirated and subjected to sample injection measurement under the following chromatographic conditions.

Time (minutes)	Mobile phase A (%)	Mobile phase B (%)	Mobile phase C (%)
				0	15	20	65
15	15	20	65
				20	20	25	55
40	20	25	55

TABLE 1

Linear regression was performed on the concentration X (mg/mL) using the peak area Y to obtain the regression equations of garcinia biflavone and formoside, Y being 460.22X-0.5378(r being 0.999) and Y being 308.64X-0.148(r being 0.999), respectively. And (3) carrying out sample injection detection on the test sample according to the same conditions, substituting the peak area into a regression equation to calculate the concentration, and further calculating the respective content. The HPLC chromatogram is shown in FIG. 2.

As a result: the content of luteolin is 33.32%, the content of formosan is 19.17%, and the sum of the content of luteolin and formosan is 52.49%.

The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and those skilled in the art should understand that the above embodiment does not limit the present invention in any way, and all technical solutions obtained by using equivalent substitution or equivalent transformation fall within the scope of the present invention.

Claims (5)

1. A process for extracting and purifying total flavone substances in the leaves of the manyflower cherry is characterized by comprising the following steps:

(1) taking a manyflower cherry leaf raw material, and heating, refluxing and extracting the manyflower cherry leaf raw material by using an ethanol solution with volume concentration of 60-90% and volume of 6-8 times to obtain an extracting solution;

(2) concentrating the extractive solution under reduced pressure until no alcohol smell exists to obtain alcohol extract;

(3) and (3) passing the alcohol extract obtained in the previous step through a 80-100-mesh polyamide column, and extracting the alcohol extract: polyamide resin weight =1:2, diameter-height ratio =1:3, flow rate =0.5BV/h, eluting 5-7 times of column volume with 25-35% ethanol solution, eluting 9-11 times of column volume with 45-55% ethanol solution, eluting 5-7 times of column volume with 65-75% ethanol solution, collecting the third column volume eluent with 45-55% ethanol solution and the second column volume eluent with 65-75% ethanol solution, merging the two eluents, recovering ethanol under reduced pressure, freeze-drying to obtain dry powder, namely the total flavone extract, and detecting the total flavone content in the total flavone extract, wherein the total flavone content exceeds 50%.

2. The process for extracting and purifying total flavonoids from human facial leaves as claimed in claim 1, wherein the process comprises the steps of: in the step (1), the heating reflux temperature is 80 ℃, and the time is 1 hour.

3. The process for extracting and purifying total flavonoids from human facial leaves as claimed in claim 1, wherein the process comprises the steps of: and (3) eluting by using 30% ethanol solution with volume concentration for 6 times of column volume, eluting by using 50% ethanol solution with volume concentration for 10 times of column volume, and eluting by using 70% ethanol solution with volume concentration for 6 times of column volume in sequence.

4. The process for extracting and purifying total flavonoids from human facial leaves as claimed in claim 1, wherein the process comprises the steps of: detecting the content of the total flavonoids in the total flavonoid extract by adopting an ultraviolet-visible spectrophotometry in the step (3), wherein the method comprises the following steps:

(1) preparing a reference substance solution: taking a luteolin reference substance, precisely weighing, dissolving with methanol, and preparing into reference substance solution containing luteolin reference substance 0.1mg per 1mL solution;

(2) preparation of a test solution: precisely weighing the total flavone extract, dissolving with ethanol, and preparing into test solution containing 0.25mg sample per 1mL solution;

(3) preparation of a standard curve: precisely measuring reference substance solutions 0.5mL, 1.0mL, 1.5mL, 2.0mL and 2.5mL, respectively placing in 10mL volumetric flasks, adding methanol to 3mL, adding 0.1mol/L aluminum chloride ethanol solution 2mL, adding 3mL of sodium acetate/acetic acid buffer solution with pH of 5.2, finally fixing the volume to a scale with ethanol, taking corresponding reagents as blanks, shaking up, placing at 35 ℃ for 15min, measuring absorbance at a wavelength of 415nm, taking the absorbance as a vertical coordinate and the mass as a horizontal coordinate, and drawing a standard curve;

(4) the determination method comprises the following steps: precisely measuring 1mL of the test solution, placing the test solution in a 10mL measuring flask, measuring the absorbance according to the measuring method in the step (3), and further calculating the amount of the luteolin in the test solution from the standard curve;

(5) and (3) measuring results: the total flavone content was calculated from the regression equation.

5. The process for extracting and purifying total flavonoids from human facial leaves as claimed in claim 1, wherein the process comprises the steps of: detecting the content of the total flavonoids in the total flavonoid extract by adopting a high performance liquid chromatography in the step (3), wherein the method comprises the following steps:

(1) preparing a reference substance solution: because the active ingredients in the total flavone extract are mainly gamboge biflavone and fukuoside, firstly, 20mg of the gamboge biflavone reference substance and 10mg of the fukuoside reference substance are respectively precisely weighed, and are dissolved by adding 95% chromatographic methanol to prepare a reference substance solution containing 0.4mg of the gamboge biflavone and 0.2mg of the fukuoside per mL;

(2) preparation of a test solution: precisely weighing 20mg of total flavone extract, dissolving with 95% chromatographic methanol, and diluting to 50mL to obtain test solution with concentration of 0.4 mg/mL;

(3) and (3) sample determination: measuring reference substance and sample with high performance liquid chromatograph under the following chromatographic conditions: a chromatographic column: agilent C18 chromatography column 4.6mm x 250mm, 5 μm; detection wavelength: 289 nm; flow rate: 1.0 mL/min; column temperature: 30 ℃; sample introduction amount: 10 mu L of the solution; mobile phase: acetonitrile A, methanol B, 0.5% phosphoric acid water solution C, and gradient elution, wherein the elution procedure is as follows:

(4) and (4) calculating a result: according to the chromatograms of the sample and the reference substance, recording peak areas, and calculating the contents of the luteolin and the fukuloside in the sample by adopting an external standard method.

Images