阅读说明：本技术 一种从洋甘菊中同时提取芹菜素和木犀草素的方法 (Method for simultaneously extracting apigenin and luteolin from chamomile ) 是由 刘青 贺玉婷 易宇阳 曹慧璋 于 2021-07-16 设计创作，主要内容包括：本发明公开了一种从洋甘菊中同时提取芹菜素和木犀草素的方法,包括以下步骤：洋甘菊粉末采用乙醇提取后,固液分离,收集液相；液相脱除乙醇得到固相；在固相中加入碱性水溶液混匀,固液分离,收集液相,将液相过阴离子交换树脂,采用酸性乙醇溶液洗脱,收集洗脱液,洗脱液中加入阳离子交换树脂,固液分离,收集固相,所得固相用水淋洗至中性后醇沉,固液分离,所得液相即为木犀草素提取物,所得固相即为芹菜素。本发明方法简单,成本低、工艺周期短,所用试剂对环境污染小,通过本发明方法有效地提高了洋甘菊中芹菜素和木犀草素的得率和利用率,可进行工业化生产。(The invention discloses a method for simultaneously extracting apigenin and luteolin from chamomile, which comprises the following steps: extracting flos Matricariae Chamomillae powder with ethanol, performing solid-liquid separation, and collecting liquid phase; removing ethanol from the liquid phase to obtain a solid phase; adding an alkaline aqueous solution into the solid phase, uniformly mixing, carrying out solid-liquid separation, collecting the liquid phase, passing the liquid phase through anion exchange resin, eluting by adopting an acidic ethanol solution, collecting eluent, adding cation exchange resin into the eluent, carrying out solid-liquid separation, collecting the solid phase, leaching the obtained solid phase with water to be neutral, carrying out alcohol precipitation, carrying out solid-liquid separation, obtaining the liquid phase which is the luteolin extract, and obtaining the solid phase which is the apigenin. The method has the advantages of simplicity, low cost, short process period and small environmental pollution of the used reagent, effectively improves the yield and the utilization rate of the apigenin and the luteolin in the chamomile, and can carry out industrial production.)

1. A method for simultaneously extracting apigenin and luteolin from chamomile is characterized by comprising the following steps:

s1, extracting chamomile with ethanol, performing solid-liquid separation, and collecting a liquid phase;

s2, removing ethanol from the liquid phase obtained in the step S1 to obtain a solid phase;

s3, adding the solid phase obtained in the step S2 into an alkaline aqueous solution, uniformly mixing, carrying out solid-liquid separation, and collecting a liquid phase;

s4, purifying the liquid phase obtained in the step S3 by using anion exchange resin, eluting by using water and acidic ethanol solution in sequence, and collecting the acidic ethanol eluent;

s5, adding the acidic ethanol eluent obtained in the step S4 into cation exchange resin, heating and refluxing, taking a liquid phase, concentrating, separating solid from liquid of the concentrated solution, and collecting a solid phase;

s6, leaching the solid phase obtained in the step S5 to be neutral by water to obtain a mixed extract of apigenin and luteolin;

s7, precipitating the mixed extract obtained in the step S6 with ethanol, and then carrying out solid-liquid separation to obtain a liquid phase which is the luteolin extract and a solid phase which is the apigenin.

2. The method according to claim 1, wherein in step S1, the feed-to-liquid ratio of the chamomile to the ethanol is 1 g: 15-25 mL.

3. The method according to claim 1, wherein in step S1, the volume concentration of ethanol is 50-90%; preferably, the volume concentration of the ethanol is 60-80%; the extraction is heating reflux extraction, and the extraction temperature is 70-85 ℃.

4. The method of claim 1, wherein in step S3, the alkaline aqueous solution is one of sodium hydroxide solution, potassium hydroxide solution, sodium carbonate solution and potassium carbonate solution; preferably, the aqueous alkaline solution is a potassium hydroxide solution.

5. The method according to claim 1, wherein in step S3, the alkaline aqueous solution is added in an amount of 2-3 BV and the solution concentration is 5-10%.

6. The method according to claim 1, wherein in step S4, the pH of the acidic ethanol solution is 4-6, and the volume concentration is 70-85%.

7. The method according to claim 1, wherein in step S4, the volume of the acidic ethanol solution is 3-5 BV, and the elution speed is 1-2 BV/h.

8. The method according to claim 1, wherein in step S5, the cation exchange resin is a strong acid cation exchange resin.

9. The method according to claim 1, wherein in step S5, the mass ratio of the acidic ethanol eluent to the cation exchange resin is 1: 5 to 10.

10. The method according to claim 1, wherein in step S7, the concentration of the ethanol solution is 90-95%, and the addition amount is 3-5 BV.

Technical Field

The invention belongs to the technical field of biological extraction, and particularly relates to a method for simultaneously extracting apigenin and luteolin from chamomile.

Background

Chamomile, also known as chamomile, is an inflorescence or a whole plant of feverfew genus chamomile of the family Compositae, an annual or biennial herb, is a spice crop and a medicinal plant which are widely used in Europe, Japan, America and the like, has pharmacological actions of diminishing inflammation, relieving spasm, inhibiting fungi and the like, and is mechanically produced in a large area in many countries. The chamomile contains abundant flavonoid compounds, the beneficial effects of the chamomile are related to several flavonoid components, the core structure of the chamomile is flavone (apigenin and luteolin) or a flavonol derivative (quercetin), the flavonoid components are in various forms such as glucoside, glucoside and (or) acyl derivatives, wherein luteolin-7-O-beta-D-glucoside and apigenin-7-O-beta-D-glucoside are main flavonoid glycoside compounds in the chamomile. The structural formulas of apigenin, apigenin and luteolin are as follows:

apigenin has carcinogenic activity inhibiting effect; as antiviral drugs for treating HIV and other viral infections, MAP kinase inhibitors, various inflammations, antioxidants, anti-vascular proliferation, anti-inflammatory, antihypertensive, etc.; compared with other flavonoids (quercetin), the flavonoid has the characteristics of low toxicity, no mutagenicity and the like. Luteolin has anticancer, antibacterial, antiinflammatory, phlegm eliminating, spasmolytic, antiallergic and immunity enhancing effects.

Because the content ratio of apigenin to luteolin in chamomile is relatively low, the related technology shows that the content of total flavonoids in chamomile is about 6.95%, wherein the content of apigenin is about 0.37%, and the content of luteolin is about 0.34%, but a large amount of apigenin and luteolin exist in the form of glucoside, glucoside and (or) acyl derivatives in the total flavonoids, so that the purity and yield of the apigenin and the luteolin obtained from chamomile by conventional extraction are low.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the method for simultaneously extracting the apigenin and the luteolin from the chamomile is simple and efficient, and the obtained apigenin and the luteolin have high purity and high yield.

According to one aspect of the present invention, a method for simultaneously extracting apigenin and luteolin from chamomile is provided, which comprises the following steps:

s1, extracting chamomile with ethanol, performing solid-liquid separation, and collecting a liquid phase;

s2, removing ethanol from the liquid phase obtained in the step S1 to obtain a solid phase;

s3, adding the solid phase obtained in the step S2 into an alkaline aqueous solution, uniformly mixing, carrying out solid-liquid separation, and collecting a liquid phase;

s4, purifying the liquid phase obtained in the step S3 by using anion exchange resin, eluting by using water and acidic ethanol solution in sequence, and collecting the acidic ethanol eluent;

s5, adding the acidic ethanol eluent obtained in the step S4 into cation exchange resin, heating and refluxing, taking a liquid phase, concentrating, separating solid from liquid of the concentrated solution, and collecting a solid phase;

s6, leaching the solid phase obtained in the step S5 to be neutral by water to obtain a mixed extract of apigenin and luteolin;

s7, precipitating the mixed extract obtained in the step S6 with ethanol, and then carrying out solid-liquid separation to obtain a liquid phase which is the luteolin extract and a solid phase which is the apigenin.

In some embodiments of the present invention, in step S1, the feed-to-liquid ratio of chamomile to ethanol is 1 g: 15-25 mL.

In some embodiments of the present invention, in the step S1, the volume concentration of ethanol is 50 to 90%, and preferably, the volume concentration of ethanol is 60 to 80%.

In some embodiments of the present invention, in the step S1, the extraction is heating reflux extraction, the extraction temperature is 70 ℃ to 85 ℃, the extraction times are 2 to 3 times, and each extraction time is 1 to 3 hours.

In some embodiments of the present invention, in step S1, the solid-liquid separation is filtration.

In some embodiments of the invention, in step S2, the ethanol removal process is performed by vacuum concentration at 60-85 ℃, and the solution is concentrated to form an extract.

In some embodiments of the present invention, in step S3, the solid-liquid separation is centrifugation.

In some embodiments of the present invention, in the step S3, the alkaline aqueous solution is one of a sodium hydroxide solution, a potassium hydroxide solution, a sodium carbonate solution, and a potassium carbonate solution; preferably, the alkaline aqueous solution is a potassium hydroxide solution, more preferably, the addition amount of the alkaline aqueous solution is 2-3 BV, and the solution concentration is 5% -10%.

In some embodiments of the present invention, in the step S4, the anion exchange resin is a weak base anion exchange resin.

In some embodiments of the invention, the weakly basic anion exchange resin is type 703.

In some embodiments of the invention, in the step S4, the acidic ethanol solution has a pH of 4 to 6, a volume concentration of 70 to 85%, an eluent volume of 3 to 5BV, and an elution speed of 1 to 2 BV/h.

In some embodiments of the present invention, the pH of the acidic ethanol solution is adjusted by using one of hydrochloric acid, sulfuric acid and nitric acid, preferably, the acid is hydrochloric acid.

In some embodiments of the invention, the cation exchange resin is a strong acid cation exchange resin.

In some embodiments of the invention, the strong acid cation exchange resin is model number 732.

In some embodiments of the invention, the mass ratio of the acidic ethanol eluent to the cation exchange resin is 1: 5-10 ℃, wherein the heating reflux treatment temperature is 80-100 ℃, and the reaction time is 4-6 h.

In some embodiments of the present invention, the step S5 further includes the steps of: and standing the concentrated solution, cooling to room temperature, and performing solid-liquid separation.

In some embodiments of the present invention, in step S5, the solid-liquid separation is centrifugation.

In some embodiments of the present invention, in the step S7, the concentration of the ethanol solution is 90 to 95%, and the solution is added in an amount of 3 to 5 BV.

In some embodiments of the present invention, the step S7 further comprises a heating reflux step of heating reflux of the mixed extract with ethanol at 70-90 ℃ for 5-10min, and cooling to 15-25 ℃.

In some embodiments of the present invention, in step S7, the solid-liquid separation is filtration.

In some embodiments of the present invention, in the step S7, the temperature of the concentration under reduced pressure is 60 to 70 ℃.

In some embodiments of the invention, in the step S7, the solid phase processing step further includes rinsing with pure water for 2-3 times, and then drying at 60-70 ℃.

In some embodiments of the present invention, in step S7, the step of treating the liquid phase further includes steps of removing ethanol by concentration under reduced pressure, washing with water, and drying.

According to the embodiment of the invention, at least the following beneficial effects are achieved:

1. the chamomile is low in apigenin and luteolin content, but contains abundant apigenin and luteolin substances, wherein apigenin and luteolin are glycoside compounds formed by combining apigenin and luteolin with sugar respectively.

2. The scheme of the invention utilizes the characteristic that flavonoids are easily dissolved in alkaline aqueous solution, adopts alkaline anion exchange resin for purification, and elutes the extract through acidic ethanol solution, thereby effectively improving the separation and purification effects of target substances in the chamomile extract.

3. The technical process of the scheme of the invention has strong operability, the two selected resins and the recovered ethanol can be reused, the process cost is low, the industrial production is easy to realize, and no other organic solvent is used, so that the method is environment-friendly.

Drawings

The invention is further described with reference to the following figures and examples, in which:

FIG. 1 is a high performance liquid chromatogram of an apigenin sample in a test example of the present invention;

FIG. 2 is a high performance liquid chromatogram of a luteolin sample in a test example of the present invention.

Detailed Description

The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.

Example 1

In the embodiment, apigenin and luteolin are extracted from chamomile simultaneously, and the specific process comprises the following steps:

(1) taking 0.5kg of dried chamomile, crushing, sieving with a 20-mesh sieve, heating and reflux-extracting the powder for 2 times by using 80% ethanol, wherein the extraction temperature is 80 ℃, the material-liquid ratio is 1:15, filtering while the powder is hot, and collecting all extracting solutions;

(2) concentrating the extractive solution at 65 deg.C under reduced pressure, recovering ethanol, and concentrating at 75 deg.C to obtain extract, i.e. flos Matricariae Chamomillae total flavone;

(3) adding 3BV of 6% KOH solution, stirring, standing, and filtering;

(4) passing the filtrate through 703 anion exchange resin, eluting with pure water to colorless, eluting with 4BV of 85% ethanol water solution with pH 4 at an elution speed of 1.5BV/h, and collecting all acidic ethanol water solution eluates;

(5) adding 732 cation exchange resin into the eluate at a mass ratio of resin to solute of 8:1, heating in water bath at 90 deg.C under reflux for 4 hr, filtering while hot, concentrating until no alcohol smell exists, standing the concentrated solution, cooling to normal temperature, and centrifuging;

(6) leaching the centrifugal residue with pure water to neutrality to obtain mixed extract containing apigenin and luteolin with high content;

(7) adding 4BV 95% ethanol solution into the mixture, heating in 80 deg.C water bath for 10min, cooling to 20 deg.C, precipitating, and filtering;

(8) concentrating the filtrate at 65 deg.C under reduced pressure, recovering ethanol, washing with water, and drying to obtain high-content luteolin (1.85g), rinsing the residue with pure water for 3 times, and drying in 65 deg.C oven to obtain high-content apigenin (12.15 g).

According to the determination of high performance liquid chromatography, the content of apigenin is 92.76%, and the content of luteolin is 82.21%.

Example 2

In the embodiment, apigenin and luteolin are extracted from chamomile simultaneously, and the specific process comprises the following steps:

(1) taking 0.5kg of dried chamomile, crushing, sieving with a 30-mesh sieve, heating and reflux-extracting the powder for 2 times by using 70% ethanol, wherein the extraction temperature is 80 ℃, the material-liquid ratio is 1:20, filtering while the powder is hot, and collecting all extracting solutions;

(2) concentrating the extractive solution at 65 deg.C under reduced pressure, recovering ethanol, and concentrating at 75 deg.C to obtain extract, i.e. flos Matricariae Chamomillae total flavone;

(3) adding 3BV of 8% KOH solution into the filter residue, fully stirring, standing and filtering;

(4) passing the filtrate through 703 anion exchange resin, eluting with pure water to colorless, eluting with 4BV 85% ethanol water solution with pH of 5 at an elution speed of 1.5BV/h, and collecting all acidic ethanol water solution eluates;

(5) adding 732 cation exchange resin into the eluate at a mass ratio of resin to solute of 8:1, heating in water bath at 90 deg.C under reflux for 5 hr, filtering while hot, concentrating until no alcohol smell exists, standing the concentrated solution, cooling to normal temperature, and centrifuging;

(6) leaching the centrifugal residue with pure water to neutrality to obtain mixed extract containing apigenin and luteolin with high content;

(7) adding 4BV 95% ethanol solution into the mixture, heating in 80 deg.C water bath for 8min, cooling to 25 deg.C, precipitating, and filtering;

(8) concentrating the filtrate at 65 deg.C under reduced pressure, recovering ethanol, washing with water, and drying to obtain high-content luteolin (1.95g), rinsing the residue with pure water for 3 times, and drying in 65 deg.C oven to obtain high-content apigenin (11.95 g).

The content of apigenin and luteolin is 93.71% and 81.36%, respectively, by high performance liquid chromatography.

Example 3

In the embodiment, apigenin and luteolin are extracted from chamomile simultaneously, and the specific process comprises the following steps:

(1) taking 0.5kg of dried chamomile, crushing, sieving with a 40-mesh sieve, heating and refluxing the powder by using 60% ethanol for 3 times, wherein the extraction temperature is 85 ℃, the material-liquid ratio is 1:25, filtering while the powder is hot, and collecting all extracting solutions;

(2) concentrating the extractive solution at 65 deg.C under reduced pressure, recovering ethanol, and concentrating at 75 deg.C to obtain extract, i.e. flos Matricariae Chamomillae total flavone.

(3) Adding 3BV of 10% NaOH solution into the filter residue, fully stirring, standing and filtering;

(4) passing the filtrate through 703 anion exchange resin, eluting with pure water to colorless, eluting with 4BV 85% ethanol water solution with pH 6 at an elution speed of 1.5BV/h, and collecting all acidic ethanol water solution eluates;

(5) adding 732 cation exchange resin into the eluate at a mass ratio of resin to solute of 10:1, heating in water bath at 90 deg.C under reflux for 6 hr, filtering while hot, concentrating until no alcohol smell exists, standing the concentrated solution, cooling to normal temperature, and centrifuging;

(6) leaching the centrifugal residue with pure water to neutrality to obtain mixed extract containing apigenin and luteolin with high content;

(7) adding 4BV 95% ethanol solution into the mixture, heating in 80 deg.C water bath for 10min, cooling to 15 deg.C, precipitating, and filtering;

(8) concentrating the filtrate at 65 deg.C under reduced pressure, recovering ethanol, crystallizing to obtain high-content luteolin (1.90g), rinsing the residue with pure water for 3 times, and drying in 65 deg.C oven to obtain high-content apigenin (12.45 g).

The content of apigenin and luteolin is 91.70% and 79.26% respectively according to high performance liquid chromatography.

Example 4

This example simultaneously extracts apigenin and luteolin from chamomile, and the preparation method differs from example 1 in that 50% ethanol is used to extract chamomile powder in step (1).

The obtained apigenin content is 80.17% (12.95g) and luteolin content is 70.52% (2.0g) determined by high performance liquid chromatography.

Example 5

In this example, apigenin and luteolin were extracted from chamomile simultaneously, and the preparation method is different from example 1 in that chamomile powder in step (1) was extracted with 90% ethanol.

The determination by high performance liquid chromatography shows that the content of apigenin is 81.75% (12.60g) and the content of luteolin is 75.71% (1.90 g).

Example 6

In this example, apigenin and luteolin are simultaneously extracted from chamomile, and the preparation method is different from example 1 in that after water elution is adopted in step (4), 85% ethanol aqueous solution is adopted for elution.

The determination by high performance liquid chromatography shows that the content of apigenin is 79.99% (13.10g), and the content of luteolin is 69.51% (2.05g)

Comparative example 1

The preparation method is different from the preparation method of the example 1 only in that step 5 is not included, 732 strong-acid cation exchange resin is added into eluent, the mass ratio of the resin to solute is 8:1, heating reflux treatment is carried out in 90 ℃ water bath, treatment is carried out for 4 hours, hot filtration is carried out, concentration is carried out until no alcohol smell exists, the concentrated solution is kept stand and cooled to the normal temperature, centrifugation operation is carried out, and replacement is carried out, namely 20% hydrochloric acid is used for adjusting the pH of the eluent to 2, heating reflux treatment is carried out in 90 ℃ water bath, treatment is carried out for 4 hours, concentration is carried out until no alcohol smell exists, and the concentrated solution is kept stand and cooled to the normal temperature and centrifuged.

The apigenin and luteolin prepared by the comparative example are measured by high performance liquid chromatography, and the apigenin content is 72.68% (13.45g) and the luteolin content is 65.74% (1.85 g).

Test examples

1. Determination of apigenin content

Apigenin prepared in examples 1-6 and comparative example 1 was measured and detected by high performance liquid chromatography. The HPLC adopts Agilent 1260ll, and the chromatographic test method is shown in Table 1:

TABLE 1 conditions for the liquid phase assay of apigenin

Chromatographic column	WONDASILTMC18 column (250 mm. times.4.6 mm, 5 μm)
		Sample volume	20μL
Flow rate of flow	1.0mL/min
		Column temperature	25℃
Wavelength of light	350nm
		Mobile phase	Acetonitrile: water (V/V) ═ 38:62(V: V)
Time of rest	20min

(1) Preparation of control solutions

Precisely weighing 2-3mg of apigenin reference substance, placing in a 25mL volumetric flask, adding 15mL of methanol, ultrasonically dissolving, cooling, and diluting to constant volume with methanol to obtain the final product.

(2) Preparation of sample solution

Accurately weighing 10-20mg of chamomile extract, placing in a 50mL volumetric flask, adding a proper amount of methanol for ultrasonic dissolution, cooling to room temperature, diluting with methanol to scale, shaking up, and filtering with a 0.45 μm filter membrane for later use.

(3) And (3) sample content determination:

measured according to the chromatographic conditions described above.

(4) And (3) calculating the content:

wherein A is_{Sample (A)}: peak area of apigenin in the sample solution;

A_{to pair}: peak area of apigenin in the control solution;

W_{sample (A)}: sample weighing (mg);

W_{to pair}: sample weight (mg) of control;

V_{sample (A)}: volume of sample (mL);

V_{to pair}: volume of control (mL);

k: purity of the reference;

sample content results the average of the contents of two parallel samples was taken.

2. Content detection of luteolin

Luteolin prepared in examples 1-6 and comparative example 1 was measured and detected by high performance liquid chromatography. The HPLC adopts Agilent 1260ll, and the chromatographic test method is shown in Table 1:

TABLE 2 luteolin liquid phase assay conditions

(1) Preparation of control solutions

Accurately weighing 2-3mg of luteolin control, placing in a 25mL volumetric flask, adding 15mL of methanol, ultrasonically dissolving, cooling, and diluting to desired volume with methanol.

(2) Preparation of sample solution

Accurately weighing 10-20mg of chamomile extract, placing in a 50mL volumetric flask, adding a proper amount of methanol for ultrasonic dissolution, cooling to room temperature, diluting with methanol to scale, shaking up, and filtering with a 0.45 μm filter membrane for later use.

(3) And (3) sample content determination:

measured according to the chromatographic conditions described above.

And (3) calculating the content:

wherein A is as follows: peak area of apigenin in the sample solution;

a pair: peak area of apigenin in the control solution;

and (2) W sample: sample weighing (mg);

w pairs: sample weight (mg) of control;

and V sample: volume of sample (mL);

v pair: volume of control (mL);

k: purity of the reference;

sample content results the average of the contents of two parallel samples was taken.

The experimental results are shown in figures 1-2, and it can be seen from the figures that apigenin and luteolin are successfully prepared by the scheme of the invention, and the prepared apigenin and luteolin have high purity and low impurity content.

The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.