阅读说明：本技术 青皮总黄酮和总生物碱的联合提取方法及其应用 (Combined extraction method and application of total flavonoids and total alkaloids in pericarpium citri reticulatae viride ) 是由 刘青 辛英祥 周利丹 王新亮 马毅刚 齐秋月 周艳 卢伊娜 于 2020-05-21 设计创作，主要内容包括：本发明公开了一种青皮总黄酮和总生物碱的联合提取方法及其应用；包括以下步骤：S1植物原料用酸水超声或加热提取,分别收集提取液和药渣；S2对步骤S1的提取液进行超滤纯化；S3将步骤S2的滤液浓缩至小体积,过滤,收集滤液,得青皮总生物碱；S4将步骤S1的药渣用醇溶剂超声或加热提取,收集提取液；S5对步骤S4的提取液进行超滤纯化；S6将步骤S5的滤液浓缩至小体积,并调节pH至2-4之间,过滤,收集固体,得青皮总黄酮。青皮总生物碱和总黄酮按照一定比例复配而成的青皮提取物,对介导皮肤疼痛、瘙痒的重要受体——瞬时感受器电位香草酸受体1有显著的拮抗作用,因而具有良好的镇痛功效,可用于抗过敏产品的制备。(The invention discloses a combined extraction method and application of total flavonoids and total alkaloids in pericarpium citri reticulatae viride; the method comprises the following steps: s1 ultrasonic or heating extracting the plant material with acid water, and collecting the extractive solution and residue respectively; s2, carrying out ultrafiltration purification on the extracting solution obtained in the step S1; s3, concentrating the filtrate obtained in the step S2 to a small volume, filtering, and collecting the filtrate to obtain pericarpium citri reticulatae viride total alkaloids; s4 extracting the residue of step S1 with alcohol solvent under ultrasound or heating, and collecting extractive solution; s5, carrying out ultrafiltration purification on the extracting solution obtained in the step S4; s6 concentrating the filtrate obtained in step S5 to small volume, adjusting pH to 2-4, filtering, and collecting solid to obtain pericarpium Citri Reticulatae viride total flavone. The green tangerine peel extract compounded by the green tangerine peel total alkaloids and the total flavonoids according to a certain proportion has obvious antagonism on an important receptor mediating skin pain and pruritus, namely an instantaneous receptor potential vanillic acid receptor 1, so that the green tangerine peel extract has good analgesic effect and can be used for preparing antiallergic products.)

1. A preparation method of a green tangerine peel extract is characterized by comprising the following steps:

s1, ultrasonically or thermally extracting the pericarpium citri reticulatae viride medicinal material by using acid water, and respectively collecting an extracting solution and dregs of a decoction;

s2, sequentially carrying out microfiltration and ultrafiltration on the extracting solution obtained in the step S1, and collecting ultrafiltration filtrate;

s3, concentrating the ultrafiltration filtrate obtained in the step S2 to a small volume, filtering, collecting the filtrate, and recovering the solvent from the filtrate to obtain total alkaloids;

s4, carrying out ultrasonic or heating extraction on the decoction dregs obtained in the step S1 by using an alcohol solvent, and collecting decoction dreg extracting solution;

s5, sequentially carrying out microfiltration and ultrafiltration on the decoction dreg extracting solution in the step S4, and collecting decoction dreg ultrafiltration filtrate;

s6, removing alcohol in the dregs ultrafiltration filtrate in the step S5 by evaporation, concentrating the solution to a small volume, adjusting the pH value to 2-4, filtering, collecting solids, washing with water, and drying to obtain pericarpium citri reticulatae viride total flavonoids;

s7, compounding the pericarpium citri reticulatae viride total alkaloids and the pericarpium citri reticulatae viride total flavonoids according to a certain proportion to obtain the pericarpium citri reticulatae viride extract.

2. The method of claim 1, wherein in step S1, the acid water is selected from aqueous solution of formic acid, acetic acid or hydrochloric acid with pH of 2-5; the amount of the acid water is 10-20 times of the dry weight of the medicinal materials.

3. The method for preparing an extract of pericarpium Citri Reticulatae viride as claimed in claim 1, wherein in step S1, the temperature of ultrasonic extraction is 30-50 ℃ and the extraction time is 10-40 minutes; the heating extraction temperature is 40-60 deg.C, and the extraction times are 1-2 times, each time for 1-2 hr.

4. The method for preparing green tangerine peel extract according to claim 1, wherein in steps S2 and S5, the microfiltration is ceramic membrane or 0.1 μm polypropylene membrane filtration; the molecular weight cut-off of the ultrafiltration membrane is 3K daltons; after ultrafiltration, the extract was concentrated to 1/2-1/3% of the original volume.

5. The method of claim 1, wherein the concentration to a small volume in steps S3 and S6 is 1/10-1/15.

6. The method for preparing an extract of green tangerine peel as claimed in claim 1, wherein in step S4, the alcoholic solvent is selected from an aqueous solution of methanol or ethanol, the volume fraction of the alcohol is 50-95%; the dosage of the alcohol solvent is 5-20 times of the dry weight of the medicinal materials.

7. The method for preparing an extract of pericarpium Citri Reticulatae viride as claimed in claim 1, wherein in step S4, the temperature of ultrasonic extraction is 30-50 ℃ and the extraction time is 10-30 minutes; the heating extraction temperature is 60-80 deg.C, and the extraction time is 1-2 times, each time for 0.5-2 hr.

8. The method for preparing pericarpium Citri Reticulatae viride extract as claimed in claim 1, wherein in step S7, the weight ratio of the total alkaloids and total flavonoids in pericarpium Citri Reticulatae viride is 0.1-5: 1.

9. A pericarpium citri reticulatae viride extract is characterized in that the weight ratio of pericarpium citri reticulatae viride total alkaloids to pericarpium citri reticulatae viride total flavonoids in the pericarpium citri reticulatae viride extract is 0.1-5: 1.

10. Use of the extract of Citrus reticulata Blanco of claim 9 for preparing a product with analgesic and antipruritic effects.

Technical Field

The invention belongs to the technical field of Chinese herbal medicine extraction, and relates to a combined extraction method of total flavonoids and total alkaloids in pericarpium citri reticulatae viride and application thereof; in particular to a combined extraction process of pericarpium citri reticulatae viride total alkaloids and total flavonoids and application thereof in preparing a product with an anti-allergic effect.

Background

Worldwide, 52% of consumers consider their skin to be sensitive skin. Sensitive skin is a common clinical problem skin: slight external stimulation activates the skin self-protective response, causing skin prickling, burning, itching, redness, and the like. While burning and stinging sensations are the result of the over-expression of transient receptor potential vanilloid receptor 1 (TRPV1, also known as capsaicin receptor), TRPV1 is a potent mediator of Ca²⁺The receptor can detect and integrate the chemical and thermal stimulation signals for inducing pain sensation, can convert the chemical, mechanical and thermal stimulation signals into action potentials, and uploads the information to the center to finally cause the body to generate pain sensation or uncomfortable sensation, and in recent years, TRPV1 channel protein has become an important target for developing new analgesic drugs by inhibiting Ca²⁺The internal flow can effectively relieve pain.

In recent years, analgesic and anti-inflammatory active ingredients of traditional Chinese medicines are one of hot spots in medicine research and development, and researchers have conducted a great deal of research on analgesic traditional Chinese medicines and found a plurality of effective analgesic ingredients of traditional Chinese medicines, which mainly comprise alkaloids, flavonoids, saponins, volatile oils and the like. The pericarpium Citri Reticulatae viride is pericarp of dried young fruit or immature fruit of Mandarin orange (Citrus reticulata Blanco), contains abundant alkaloids (synephrine and N-methyltyramine) and flavonoids (naringin, hesperidin and polymethoxyflavonoids), and has been reported to be effective Ca²⁺Antagonists (related to TRPV1 inhibition), therefore, the green tangerine orange peel extract has certain relieving effect on sensitive skin. The citrus in China has a wide planting area, and green tangerine peel resources are rich, so that the citrus is a raw material with industrial production value, and the research on the effective components of the green tangerine peel at present is usually the extraction and separation of single components, such as the extraction of total flavonoids in the green tangerine peel. Various components in the traditional Chinese medicine generally play a certain role in the whole efficacy, and one component is usually separated and researched separately while the synergistic effect among the components is ignored. Therefore, in the invention, the total alkaloids and the total flavonoids are acted on a TRPV1 cell analgesic model according to a certain ratio to obtain a composition with better analgesic effect than the single pericarpium citri reticulatae viride total flavonoids and the single total alkaloids.

The green tangerine peel medicinal material contains flavone and alkaloid components, and also contains substances such as protein, pectin, polysaccharide and the like, which are extracted and exist in the crude extract. In order to obtain relatively pure total flavonoids and total alkaloids, the impurities are removed. Most of the existing methods for extracting the flavones and the alkaloids of the green tangerine peel, the immature bitter orange and the bitter orange adopt ethanol extraction or water decoction, and then resin adsorption, extraction or ion exchange purification, for example, the method disclosed in the patent document CN01106994 adopts petroleum ether for degreasing, a dilute alkali solution for reflux extraction, then resin adsorption and step-by-step elution to obtain the flavones and the alkaloids. The prior method uses resin column chromatography, and consumes a large amount of organic solvents, acids and alkalis in the industrial production of resin column chromatography and resin regeneration treatment, thus being not environment-friendly and consuming working hours.

Disclosure of Invention

Aiming at the defects of the prior art, the invention firstly extracts alkaloid by acid water, removes macromolecular impurities by ultrafiltration, and then concentrates and filters to obtain alkaloid with higher purity; the medicine residues are extracted by using high-concentration alcohol, and the flavone is obtained by ultrafiltration, concentration and acid adjustment, so that the medicinal materials are fully utilized, and meanwhile, resin is prevented from being used in production, so that the process is simple and time-saving, and the environment is more protected. The total alkaloids and the total flavonoids are compounded according to a certain proportion and applied to a TRPV1 analgesic model to obtain the optimal analgesic combination, and the analgesic combination is applied to the development of antiallergic products.

The purpose of the invention is realized by the following technical scheme:

in a first aspect, the present invention relates to a method for preparing an extract of green tangerine orange peel, said method comprising the steps of:

s1, ultrasonically or thermally extracting the pericarpium citri reticulatae viride medicinal material by using acid water, and respectively collecting an extracting solution and dregs of a decoction;

s2, sequentially carrying out microfiltration and ultrafiltration on the extracting solution obtained in the step S1, and collecting ultrafiltration filtrate;

s3, concentrating the ultrafiltration filtrate obtained in the step S2 to a small volume, filtering, collecting the filtrate, and recovering the solvent from the filtrate to obtain total alkaloids;

s4, carrying out ultrasonic or heating extraction on the decoction dregs obtained in the step S1 by using an alcohol solvent, and collecting decoction dreg extracting solution;

s5, sequentially carrying out microfiltration and ultrafiltration on the decoction dreg extracting solution in the step S4, and collecting decoction dreg ultrafiltration filtrate;

s6, removing alcohol in the dregs ultrafiltration filtrate in the step S5 by evaporation, concentrating the solution to a small volume, adjusting the pH value to 2-4, filtering, collecting solids, washing with water, and drying to obtain pericarpium citri reticulatae viride total flavonoids;

s7, compounding the pericarpium citri reticulatae viride total alkaloids and the pericarpium citri reticulatae viride total flavonoids according to a certain proportion to obtain the pericarpium citri reticulatae viride extract.

As an embodiment of the present invention, in step S1, the acid water is selected from aqueous solutions of formic acid, acetic acid or hydrochloric acid at pH 2-5; the amount of the acid water is 10-20 times of the dry weight of the medicinal materials.

In step S1, the ultrasonic extraction temperature is 30-50 ℃ and the extraction time is 10-40 minutes.

As an embodiment of the present invention, in step S1, the heating extraction temperature is 40-60 deg.C, and the extraction times are 1-2 times, each for 1-2 hours.

In one embodiment of the present invention, the microfiltration and ultrafiltration are performed in steps S2 and S5 to remove macromolecular components such as pectin and protein from the extract. The ultrafiltration can improve the purity of the total alkaloids/flavones on the one hand and the stability of the product in the solution on the other hand. If macromolecular substances are not removed, trace precipitates can be separated out after the product solution is placed for 3-4 weeks, and the application of the product is influenced.

As an embodiment of the present invention, in steps S2 and S5, the microfiltration is ceramic membrane or 0.1 μm polypropylene membrane filtration; the molecular weight cut-off of the ultrafiltration membrane is 3K daltons; after ultrafiltration, the extract was concentrated to 1/2-1/3% of the original volume.

As an embodiment of the present invention, the concentration to a small volume in steps S3 and S6 means concentration to 1/10 to 1/15 of the original volume.

As an embodiment of the present invention, the concentration is performed at 60 ℃ to 65 ℃ under reduced pressure in steps S3 and S6.

In step S3, a small amount of flavonoids extracted from the acidic water is supersaturated and precipitated when the acidic water is concentrated to a small volume. A small amount of flavone can be extracted by acid water extraction, and if the acid water leaching liquor is directly concentrated to be dry, the content of alkaloid impurities is higher. According to the invention, the acid water extracting solution is concentrated to a small volume, the flavone is separated out due to supersaturation in the solution, the filtrate is concentrated to be dry after solid is filtered, and the flavone residual quantity in the alkaloid is greatly reduced.

As an embodiment of the present invention, in step S3, the method for recovering the solvent may be recovery by heating under reduced pressure, spray drying or freeze drying.

As an embodiment of the present invention, in step S4, the alcohol solvent is selected from an aqueous solution of methanol or ethanol, and the volume fraction of the alcohol is 50-95%; the dosage of the alcohol solvent is 5-20 times of the dry weight of the medicinal materials.

In step S4, the ultrasonic extraction temperature is 30-50 ℃ and the extraction time is 10-30 minutes.

As an embodiment of the present invention, in step S4, the heating extraction temperature is 60-80 deg.C, and the extraction times are 1-2 times, each for 0.5-2 hours.

In step S6, an embodiment of the present invention is to concentrate the mixture to a small volume, dissolve a small amount of alkaloid extracted from the alcohol solvent in the acid water, and precipitate flavone.

As an embodiment of the present invention, in step S6, the acid for adjusting pH is selected from inorganic or organic acids, including but not limited to formic acid, acetic acid, hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, malic acid.

As an embodiment of the present invention, in step S6, the drying is vacuum drying at 40 ℃ to 60 ℃ for 8 to 12 hours.

As an embodiment of the present invention, in step S6, the water washing refers to washing the acid remaining in the solid with water until the water washing liquid is neutral.

In an embodiment of the present invention, in step S7, the weight ratio of the pericarpium citri reticulatae viride total alkaloids to the pericarpium citri reticulatae viride total flavonoids is 0.1-5: 1.

In a second aspect, the invention relates to a pericarpium citri reticulatae viride extract, wherein the weight ratio of pericarpium citri reticulatae viride total alkaloids to pericarpium citri reticulatae viride total flavonoids in the pericarpium citri reticulatae viride extract is 0.1-5: 1. Preferably, the weight ratio is 0.5-2: 1.

The inventor finds that the compound of the total alkaloids and the total flavonoids can obviously improve the inhibition rate of calcium ion inflow for the first time. Preferably, the pericarpium citri reticulatae viride extract is prepared by the above preparation method, combining the advantages of the extraction method of the present invention.

In a third aspect, the invention relates to an application of the green tangerine peel extract in preparation of products with analgesic and antipruritic effects.

As one embodiment of the invention, the product with the functions of easing pain and relieving itching comprises an antiallergic product. The antiallergic product can be applied to the fields of cosmetics and medicines.

As an embodiment of the invention, the green tangerine peel extract is added in the anti-allergic cosmetic in a weight ratio of 0.2-1%.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention provides a preparation method of a green tangerine peel extract, which comprises the steps of extracting alkaloid in green tangerine peel medicinal materials by using acid water in the extraction process, and then extracting total flavone from dregs of a decoction by using high-concentration alcohol, so that the medicinal materials are fully utilized, and the waste of effective substances is avoided;

2. compared with the conventional method, the method has simple process, can realize industrial production, does not need to use resin which is troublesome to treat and regenerate for separation and purification, and is more time-saving and environment-friendly.

3. The total alkaloids and the total flavonoids are compounded according to a certain proportion and applied to a TRPV1 analgesic model to obtain the optimal analgesic composition through synergistic effect.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 is an ultraviolet spectrum of the total alkaloid fraction of the present invention;

FIG. 2 is a synephrine hydrochloride UV spectrum;

FIG. 3 is an ultraviolet spectrum of the total flavonoids of the present invention;

FIG. 4 is the hesperidin UV spectrum.

Detailed Description

The present invention will be described in detail with reference to examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be apparent to those skilled in the art that several modifications and improvements can be made without departing from the inventive concept. All falling within the scope of the present invention. In the present invention, all the equipment and materials are commercially available or commonly used in the art, and the methods in the following examples are conventional in the art unless otherwise specified.