阅读说明：本技术 一种抑菌的牡丹花蕾提取物及其制备方法 (Bacteriostatic peony bud extract and preparation method thereof ) 是由 刘瑞强 王汝芹 于 2021-02-02 设计创作，主要内容包括：公开了一种牡丹花蕾提取物的制备方法,包括：对牡丹花蕾干粉进行溶剂提取和超声波提取；然后使用DM130大孔吸附树脂和聚酰胺树脂纯化。此外,公开了上述制备方法得到的产物干粉。本发明牡丹花蕾提取物总黄酮纯度(含量)更高,达到70％以上。(Discloses a preparation method of a peony bud extract, which comprises the following steps: carrying out solvent extraction and ultrasonic extraction on the peony bud dry powder; then purified using DM130 macroporous adsorbent resin and polyamide resin. In addition, the product dry powder obtained by the preparation method is disclosed. The purity (content) of the general flavone of the peony bud extract is higher and reaches more than 70 percent.)

1. A preparation method of a peony bud extract comprises the following steps:

(1) firstly, carrying out solvent extraction on the peony bud dry powder, then carrying out ultrasonic extraction under the condition of heat preservation, and filtering to obtain filtrate; centrifuging the filtrate, taking supernatant, and freeze-drying to obtain dry powder of a crude extract;

(2) dissolving the dry powder of the crude extract in 50-70% ethanol solution to obtain a sample solution; loading the sample loading liquid on the pretreated DM130 macroporous adsorption resin; then eluting with 50-70% ethanol solution as eluent;

(3) collecting the eluent, and adjusting the content of the total flavonoids in the concentrated solution to 1-7mg/mL to obtain a sample loading solution of the polyamide resin; loading the sample loading liquid on the pretreated polyamide resin; then eluting with 50-70% ethanol solution as eluent; collecting the eluent, concentrating and evaporating; finally, freeze drying to obtain the product dry powder.

2. The preparation method according to claim 1, wherein in the step (1), the solvent extraction is: extracting with 50-70% ethanol solution as extractant at a ratio of 1: 10-20(g/mL), the extraction temperature is 60-80 ℃, and the extraction time is 1-3 h.

3. The preparation method according to claim 1, wherein in the step (1), the ultrasonic extraction is: the ultrasonic power is 200-300W, and the ultrasonic time is 10-30 min.

4. The preparation method according to claim 1, wherein in the step (2), the weight volume ratio of the dry powder of the crude extract to the 50-70% ethanol solution is 1-2 mg: 1mL, pH adjusted to 4-5.

5. The preparation method according to claim 1, wherein in the step (2), the DM130 macroporous absorption resin is soaked in 90-98% ethanol solution for pretreatment; loading the column by a wet method, and loading the sample on a balance chromatographic column for 8-16 h.

6. The preparation method according to claim 1, wherein in the step (2), the flow rate of the sample solution is 2-6 BV/h; the flow rate of the eluent is 2-6 BV/h.

7. The preparation method according to claim 1, wherein in the step (3), the content of total flavonoids in the concentrated solution is adjusted to 2-6 mg/mL; preferably 2-4 mg/mL.

8. The preparation method according to claim 1, wherein in the step (3), the flow rate of the sample solution is 1-3 BV/h; the flow rate of the eluent is 1-3 BV/h.

9. The production method according to claim 1, wherein in the step (3), the polyamide resin is pre-treated by soaking in a 90-98% ethanol solution; loading the column by a wet method, and loading the sample on a balance chromatographic column for 8-16 h.

10. Bacteriostatic peony bud extract, characterized in that it is obtained by the preparation method according to any one of claims 1-9.

Technical Field

The invention belongs to the technical field of biological medicine; relates to a bacteriostatic peony bud extract and a preparation method thereof.

Background

With the improvement of the living standard of the public in China, the health concept of green, nature and safety is gradually accepted by people. The research and development of natural products such as plants are receiving attention from the academic and industrial circles at home and abroad. China has abundant medicinal plant resources, and extracts of medicinal plants generally have peculiar functions or curative effects such as antivirus, antioxidation, blood fat regulation, bacteriostasis, sterilization, immunity enhancement and the like. Among them, the extraction of natural antibacterial substances from medicinal plants is becoming a popular research direction. The extracted antibacterial substance can be widely applied to various fields, such as cosmetics, food preservation, crop control and the like. Therefore, the research on the natural plant bacteriostatic components of medicinal plants has universal social demands and promising development prospect.

Peony is a woody plant of Paeonia of Paeoniaceae of Ranunculaceae. Peony is a treasure throughout the body. The peony root bark, also called cortex moutan, has better medicinal effect. The peony seed oil is rich in unsaturated fatty acid, and has high health promotion and nutritive value. The peony seed pod contains plant polysaccharide and has certain effects of reducing blood sugar and improving human immunity. The peony bud is rich in polyphenol, flavonoid and anthocyanin substances, and has antibacterial, bactericidal and anti-inflammatory effects.

Research shows that the peony buds mainly contain seven flavonoid substances, namely kaempferol monoglycoside, kaempferol diglycoside, apigenin monoglycoside, apigenin diglycoside, luteolin monoglycoside, luteolin diglycoside and luteolin malyl glucoside. In recent years, many studies have been made on methods for separating and extracting such flavonoids having antibacterial activity.

The method comprises the steps of extracting total flavonoids in peony leaf dry powder by using an ethanol solution, adsorbing the total flavonoids by macroporous adsorption resins HPD600 and D101, eluting the total flavonoids by using a 70% ethanol aqueous solution, concentrating an eluent by rotary evaporation, and separating the eluent by using an ultrafiltration membrane with the molecular weight cutoff of 1 ten thousand daltons, thereby improving the purity of the total flavonoids to 40%. The peony leaf total flavonoids have certain inhibition effects on 3 bacteria such as escherichia coli, bacillus subtilis, salmonella and the like, wherein the inhibition effect on the salmonella is most obvious. However, the purity of the total flavonoids obtained by the method is not satisfactory, and especially the antibacterial effect of the total flavonoids obtained after purification by macroporous adsorption resin on escherichia coli and bacillus subtilis is not even as good as that of the peony leaf total flavonoids extract.

Firstly, preparing ultrafiltration membranes with different apertures by different membrane preparation liquid ratios for pre-filtering a peony flavone crude extract by people in good use; then, two stages of nanofiltration membranes with different apertures are utilized to carry out graded filtration and concentrate the peony flavone extract so as to realize the refining of the peony flavone. Finally, the purity of the flavone obtained after the purification by the ultrafiltration-nanofiltration combined process is improved from 15.6 percent to 79 percent. The peony flavone has obvious inhibition effect on tyrosinase, the monophenolase is 4.8mg/mL, the diphenolase is 13.8mg/mL, and the inhibition effect is closer to that of the high-efficiency whitening agent phenethyl resorcinol. However, the ultrafiltration-nanofiltration combined process not only needs two expensive roll-type nanofiltration membrane components NWCO-1000 and NWCO-300, but also obtains a PES ultrafiltration membrane by self-making different casting solution ratios; the process and apparatus are complicated. In addition, whether ultrafiltration membranes or nanofiltration membranes are used for several times, macromolecular impurities can be accumulated on the membrane surface, thereby reducing the membrane flux and the membrane performance.

Therefore, there is an urgent need to provide an antibacterial peony bud extract with higher purity of total flavonoids and a preparation method thereof, aiming at the technical defects.

Disclosure of Invention

The invention aims to provide an antibacterial peony bud extract with higher purity of total flavonoids and a preparation method thereof.

In order to achieve the above object, in one aspect, the technical solution adopted by the present invention is as follows:

a preparation method of a peony bud extract comprises the following steps:

(1) firstly, carrying out solvent extraction on the peony bud dry powder, then carrying out ultrasonic extraction under the condition of heat preservation, and filtering to obtain filtrate; centrifuging the filtrate, taking supernatant, and freeze-drying to obtain dry powder of a crude extract;

(2) dissolving the dry powder of the crude extract in 50-70% ethanol solution to obtain a sample solution; loading the sample loading liquid on the pretreated DM130 macroporous adsorption resin; then eluting with 50-70% ethanol solution as eluent;

(3) collecting the eluent, and adjusting the content of the total flavonoids in the concentrated solution to 1-7mg/mL to obtain a sample loading solution of the polyamide resin; loading the sample loading liquid on the pretreated polyamide resin; then eluting with 50-70% ethanol solution as eluent; collecting the eluent, concentrating and evaporating; finally, freeze drying to obtain the product dry powder.

The preparation method according to the present invention, wherein in the step (1), the solvent extraction is: extracting with 50-70% ethanol solution as extractant at a ratio of 1: 10-20(g/mL), the extraction temperature is 60-80 ℃, and the extraction time is 1-3 h.

In a specific embodiment, in the step (1), the solvent extraction is: and (2) performing solvent extraction by taking a 60% ethanol solution as an extractant, wherein the material-liquid ratio is 1: 15(g/mL), the extraction temperature is 70 ℃, and the extraction time is 2 h.

The preparation method of the invention, wherein in the step (1), the ultrasonic extraction is as follows: the ultrasonic power is 200-300W, and the ultrasonic time is 10-30 min.

In a specific embodiment, in the step (1), the ultrasonic extraction is: the ultrasonic power is 240W, and the ultrasonic time is 20 min.

The preparation method provided by the invention is characterized in that in the step (2), the weight-volume ratio of the dry powder of the crude extract to the 50-70% ethanol solution is 1-2 mg: 1mL, pH adjusted to 4-5.

In a specific embodiment, in the step (2), the weight-to-volume ratio of the dry powder of the crude extract to the 50-70% ethanol solution is 1.5 mg: 1mL, pH adjusted to 4.5.

The preparation method comprises the following steps of (2), soaking the DM130 macroporous adsorption resin in 90-98% ethanol solution for pretreatment; loading the column by a wet method, and loading the sample on a balance chromatographic column for 8-16 h.

In a specific embodiment, in the step (2), the DM130 macroporous adsorbent resin is soaked in a 95% ethanol solution for pretreatment; and (5) loading the column by adopting a wet method, and loading the sample on the equilibrium chromatographic column for 12 hours.

According to the preparation method, in the step (2), the flow rate of the sample loading liquid is 2-6 BV/h; the flow rate of the eluent is 2-6 BV/h.

In a specific embodiment, in the step (2), the flow rate of the sample solution is 4 BV/h; the flow rate of the eluent was 4 BV/h.

According to the preparation method, in the step (3), the content of the total flavone in the concentrated solution is adjusted to be 2-6 mg/mL; preferably 2-4 mg/mL.

In a specific embodiment, in the step (3), the content of total flavonoids in the concentrated solution is adjusted to be 3 mg/mL.

According to the preparation method, in the step (3), the flow rate of the sample loading liquid is 1-3 BV/h; the flow rate of the eluent is 1-3 BV/h.

In a specific embodiment, in the step (3), the flow rate of the sample solution is 2 BV/h; the flow rate of the eluent was 2 BV/h.

The preparation method of the invention comprises the following steps of (3), soaking polyamide resin in 90-98% ethanol solution for pretreatment; loading the column by a wet method, and loading the sample on a balance chromatographic column for 8-16 h.

In a specific embodiment, in the step (3), the polyamide resin is soaked in a 95% ethanol solution for pretreatment; and (5) loading the column by adopting a wet method, and loading the sample on the equilibrium chromatographic column for 12 hours.

On the other hand, the invention also provides an antibacterial peony bud extract, which is characterized in that the extract is obtained by the preparation method.

The beneficial technical effects of the invention are as follows: the bacteriostatic peony bud extract has higher purity (content) of total flavonoids, and the purity can reach more than 70%.

Without wishing to be bound by any theory, the two resin purification steps of DM130 macroporous adsorbent resin and polyamide resin employed in the present invention bring about unexpected technical effects.

Detailed Description

It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include both one and more than one (i.e., two, including two) unless the context clearly dictates otherwise.

Unless otherwise indicated, the numerical ranges in this disclosure are approximate and thus may include values outside of the stated ranges. The numerical ranges may be stated herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the numerical ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

Reference in the specification and concluding claims to parts by weight of a particular element or component in a composition or article refers to the weight relationship between that element or component and any other elements or components in the composition or article, expressed as parts by weight.

In the present invention, unless specifically indicated to the contrary, or implied from the context or customary practice in the art, all solutions referred to herein are aqueous solutions; when the solute of the aqueous solution is a liquid, all fractions and percentages are by volume and the volume percentages of a component are based on the total volume of the composition or product in which it is contained; when the solute of the aqueous solution is a solid, all fractions and percentages are by weight, and the weight percentages of a component are based on the total weight of the composition or product in which the component is included.

References to "comprising," "including," "having," and similar terms in this specification are not intended to exclude the presence of any optional components, steps or procedures, whether or not any optional components, steps or procedures are specifically disclosed. In order to avoid any doubt, all methods claimed through use of the term "comprising" may include one or more additional steps, apparatus parts or components and/or materials unless stated to the contrary. In contrast, the term "consisting of … …" excludes any component, step, or procedure not specifically recited or recited. Unless otherwise specified, the term "or" refers to the listed members individually as well as in any combination.

Furthermore, the contents of any referenced patent or non-patent document in this application are incorporated by reference in their entirety, especially with respect to definitions disclosed in the art (where not inconsistent with any definitions specifically provided herein) and general knowledge.

In the present invention, parts are parts by weight unless otherwise indicated, temperatures are indicated in ° c or at ambient temperature, and pressures are at or near atmospheric. The room temperature means 20-30 ℃. There are many variations and combinations of reaction conditions (e.g., component concentrations, desired solvents, solvent mixtures, temperatures, pressures, and other reaction ranges) and conditions that can be used to optimize the purity and yield of the product obtained by the process. Only reasonable routine experimentation will be required to optimize such process conditions.

In the invention, the method for measuring the content of the total flavonoids comprises the following steps: accurately weighing 20mg of dried rutin, dissolving with 60% ethanol solution, and fixing the volume in a 100mL volumetric flask to obtain a rutin standard solution with the mass concentration of 0.2 mg/mL. Respectively taking 1mL, 2 mL, 3 mL, 4 mL, 5mL, 6mL and 7mL from the rutin standard solution, adding 5% NaNO₂Shaking the solution 0.6mL, standing for 6min, adding 10% Al (NO)₃)₃0.6mL of the solution is shaken up and then kept stand for 5min, 5mL of 10% NaOH solution is added, then deionized water is used for fixing the volume to 20mL, and the solution is shaken up and then kept stand for 20 min. And (3) taking distilled water blank liquid as a reference, measuring the absorbance value at 510nm, paralleling each group of experiments for three times, and taking an average value to obtain a linear regression equation of the concentration and the absorbance of the rutin. Dissolving the solid extract with 60% ethanol, measuring absorbance of the solid extract or extractive solution according to the above operation steps, and substituting into the linear regression equation to calculate total flavone concentration or content of the extractive solution/extract.

Example 1

Drying peony buds to constant weight at 60 ℃, and grinding into fine powder; firstly, using 60% ethanol solution as an extractant to perform solvent extraction, wherein the material-liquid ratio is 1: 15(g/mL), the extraction temperature is 70 ℃, and the extraction time is 2 h; and then carrying out ultrasonic extraction under the condition of heat preservation, wherein the ultrasonic power is 240W, the ultrasonic time is 20min, and filtering by using double-layer gauze to obtain filtrate. Centrifuging the filtrate at 6000rpm for 5min, collecting supernatant, and vacuum concentrating and evaporating at 45 deg.C to obtain concentrate. Standing the concentrate at 4 deg.C for 24 hr, and freeze drying the supernatant to obtain dry powder of crude extract. The total flavone content in the crude extract dry powder was determined to be 13.5%.

Example 2

DM130 macroporous adsorption resin (Tianjin Haoyao resin science and technology, Inc.) is soaked in 95% ethanol solution for pretreatment. After 24h, the resin was washed with distilled water until the effluent was clear and free of ethanol odor. And (5) loading the column by adopting a wet method, and loading the sample on the equilibrium chromatographic column for 12 hours. The loading solution was prepared as follows: the dry powder of the crude extract of example 1 was dissolved in a 60% ethanol solution in a weight to volume ratio of 1.5 mg: 1mL, pH adjusted to 4.5.

Loading the sample solution on the pretreated DM130 macroporous adsorption resin at the flow rate of 4BV (volume of resin bed)/h; then eluting with 60% ethanol solution as eluent at flow rate of 4 BV/h; collecting eluate, vacuum rotary concentrating and evaporating at 45 deg.C, measuring and adjusting total flavone content in the concentrated solution to 3mg/mL to obtain sample solution of polyamide resin.

Polyamide resin (Shanghai chemical reagent company, China medicine, Inc., 14-30 mesh) was immersed in a 95% ethanol solution for pretreatment. Washing the resin with distilled water after 24h until the effluent is clear and has no ethanol smell; then washing by using 3BV of 95 percent ethanol solution, 2BV of 5 percent sodium hydroxide solution, 1BV of distilled water and 2BV of 10 percent acetic acid solution in turn; finally, the resin was washed with distilled water until the effluent was neutral. And (5) loading the column by adopting a wet method, and loading the sample on the equilibrium chromatographic column for 12 hours.

Loading the sample liquid on the pretreated polyamide resin at the flow rate of 2 BV/h; then eluting with 60% ethanol solution as eluent at the flow rate of 2 BV/h; collecting eluate, vacuum rotary concentrating at 45 deg.C, and evaporating; finally, freeze drying to obtain the product dry powder. The total flavone content in the product dry powder was determined to be 76.2%.

Comparative example 1

DM130 macroporous absorption resin is soaked in 95 percent ethanol solution for pretreatment. After 24h, the resin was washed with distilled water until the effluent was clear and free of ethanol odor. And (5) loading the column by adopting a wet method, and loading the sample on the equilibrium chromatographic column for 12 hours. The loading solution was prepared as follows: the dry powder of the crude extract of example 1 was dissolved in a 60% ethanol solution in a weight to volume ratio of 1.5 mg: 1mL, pH adjusted to 4.5.

Loading the sample loading liquid on the pretreated DM130 macroporous adsorption resin at the flow rate of 4 BV/h; then eluting with 60% ethanol solution as eluent at flow rate of 4 BV/h; collecting eluate, vacuum rotary concentrating at 45 deg.C, and evaporating; finally, freeze drying to obtain the product dry powder. The total flavone content in the product dry powder was determined to be 52.7%.

Comparative example 2

DM130 macroporous absorption resin is soaked in 95 percent ethanol solution for pretreatment. After 24h, the resin was washed with distilled water until the effluent was clear and free of ethanol odor. And (5) loading the column by adopting a wet method, and loading the sample on the equilibrium chromatographic column for 12 hours. The loading solution was prepared as follows: the dry powder of the crude extract of example 1 was dissolved in a 60% ethanol solution in a weight to volume ratio of 1.5 mg: 1mL, pH adjusted to 4.5.

Loading the sample loading liquid on the pretreated DM130 macroporous adsorption resin at the flow rate of 4 BV/h; then eluting with 60% ethanol solution as eluent at flow rate of 4 BV/h; collecting eluate, vacuum rotary concentrating and evaporating at 45 deg.C, measuring and adjusting total flavone content in the concentrated solution to 0.5mg/mL to obtain sample solution of polyamide resin.

The polyamide resin is soaked in a 95% ethanol solution for pretreatment. Washing the resin with distilled water after 24h until the effluent is clear and has no ethanol smell; then washing by using 3BV of 95 percent ethanol solution, 2BV of 5 percent sodium hydroxide solution, 1BV of distilled water and 2BV of 10 percent acetic acid solution in turn; finally, the resin was washed with distilled water until the effluent was neutral. And (5) loading the column by adopting a wet method, and loading the sample on the equilibrium chromatographic column for 12 hours.

Loading the sample liquid on the pretreated polyamide resin at the flow rate of 2 BV/h; then eluting with 60% ethanol solution as eluent at the flow rate of 2 BV/h; collecting eluate, vacuum rotary concentrating at 45 deg.C, and evaporating; finally, freeze drying to obtain the product dry powder. The total flavone content in the product dry powder was determined to be 64.1%.

Comparative example 3

DM130 macroporous absorption resin is soaked in 95 percent ethanol solution for pretreatment. After 24h, the resin was washed with distilled water until the effluent was clear and free of ethanol odor. And (5) loading the column by adopting a wet method, and loading the sample on the equilibrium chromatographic column for 12 hours. The loading solution was prepared as follows: the dry powder of the crude extract of example 1 was dissolved in a 60% ethanol solution in a weight to volume ratio of 1.5 mg: 1mL, pH adjusted to 4.5.

Loading the sample loading liquid on the pretreated DM130 macroporous adsorption resin at the flow rate of 4 BV/h; then eluting with 60% ethanol solution as eluent at flow rate of 4 BV/h; collecting eluate, vacuum rotary concentrating and evaporating at 45 deg.C, measuring and adjusting total flavone content in the concentrated solution to 8mg/mL to obtain sample solution of polyamide resin.

The polyamide resin is soaked in a 95% ethanol solution for pretreatment. Washing the resin with distilled water after 24h until the effluent is clear and has no ethanol smell; then washing by using 3BV of 95 percent ethanol solution, 2BV of 5 percent sodium hydroxide solution, 1BV of distilled water and 2BV of 10 percent acetic acid solution in turn; finally, the resin was washed with distilled water until the effluent was neutral. And (5) loading the column by adopting a wet method, and loading the sample on the equilibrium chromatographic column for 12 hours.

Loading the sample liquid on the pretreated polyamide resin at the flow rate of 2 BV/h; then eluting with 60% ethanol solution as eluent at the flow rate of 2 BV/h; collecting eluate, vacuum rotary concentrating at 45 deg.C, and evaporating; finally, freeze drying to obtain the product dry powder. The total flavone content in the product dry powder was determined to be 59.8%.

As can be seen, the bacteriostatic peony bud extract provided by the embodiment of the invention has higher purity (content) of total flavonoids. Without wishing to be bound by any theory, the two resin purification steps of DM130 macroporous adsorbent resin and polyamide resin employed in the present invention bring about unexpected technical effects.

Furthermore, it should be understood that various changes, substitutions, deletions, modifications or adjustments may be made by those skilled in the art after reading the disclosure of the present invention, and such equivalents are also within the scope of the invention as defined in the appended claims.