CN114796305B

CN114796305B - Preparation method and application of licorice extract

Info

Publication number: CN114796305B
Application number: CN202210498704.0A
Authority: CN
Inventors: 李霞
Original assignee: Xinjiang Fuwo Pharmaceutical Co ltd
Current assignee: Xinjiang Fuwo Pharmaceutical Co ltd
Priority date: 2022-05-09
Filing date: 2022-05-09
Publication date: 2023-05-02
Anticipated expiration: 2042-05-09
Also published as: CN114796305A

Abstract

The invention provides a preparation method and application of licorice extract, and relates to the technical field of active ingredient extraction. The method comprises the following steps: raw material pretreatment: pulverizing dried Glycyrrhrizae radix, soaking in organic solvent and surfactant, filtering, and retaining residue; (2) extraction: extracting the filter residue obtained in the step (1) with water, filtering, discarding the filter residue, and reserving the filtrate; (3) Concentrating the filtrate obtained in the step (2) into paste, and then drying to obtain the licorice extract. According to the preparation method, the licorice powder is soaked in the mode of soaking in the organic solvent and the surfactant in the pretreatment step, so that the solubility of invalid components with different solubility in the organic solvent is increased, the content of the effective components in the licorice extract is further increased, and the content of insoluble matters in water and the total gray level of the obtained extract are obviously reduced.

Description

Preparation method and application of licorice extract

Technical Field

The invention relates to the technical field of extraction of effective components of traditional Chinese medicines, in particular to a preparation method and application of licorice extract.

Background

Glycyrrhrizae radix is dry root and rhizome of Glycyrrhrizae radix Glycyrrhiza uralensis Fisch of Leguminosae. Licorice root, radix Glycyrrhizae is sweet in nature and flat in taste, and enters heart, lung, spleen and stomach meridians. Has effects of invigorating spleen, replenishing qi, clearing heat, detoxicating, eliminating phlegm, relieving cough, relieving spasm, relieving pain, and harmonizing the medicines. Can be used for treating weakness of spleen and stomach, listlessness, debilitation, palpitation, short breath, cough with excessive phlegm, abdominal pain, limb spasm, carbuncle, swelling, sore and toxic materials, and relieving drug toxicity and intensity. Licorice is used as a common clinical traditional Chinese medicine, and is recorded in a plurality of prescriptions. According to the theory of 'monarch, minister, assistant and guide' of traditional Chinese medicine, liquorice is often used as a guiding drug for harmonizing various drugs. Chemical composition researches show that licorice mainly contains triterpenes, flavonoids, polysaccharides, coumarins, volatile oils, amino acids and the like, wherein the triterpenes and the flavonoids are main components. Modern pharmacological studies show that licorice has wide pharmacological actions of resisting tumor, inflammation, bacteria, virus, nerve protection, liver protection and the like. For example, flavonoids in licorice can play an antioxidant role, scavenge oxygen free radicals, protect cell membranes, activate macrophages to produce cytotoxic factors, and induce killing cancer cells; the glycyrrhizic acid compound can inhibit the activities of DNA synthesis rate-limiting enzyme and nucleotide reductase, and tumor cells can not pass from the pre-synthesis stage to the synthesis stage, so that the tumor cells are finally differentiated, and proliferation of the tumor cells is inhibited. The polysaccharide compound can inhibit the growth of tumor cells and induce apoptosis; for example, glycyrrhizic acid and glycyrrhetinic acid compounds are main substances in hay which exert anti-inflammatory activity, and the mechanism is to inhibit the activities of phospholipase A2 and lipase, so as to inhibit the synthesis of inflammatory mediators such as prostaglandin E2 and leukotriene, and glycyrrhizic acid has extremely strong antiviral activity, and glycyrrhizic acid exerts antiviral effect by inhibiting the synthesis of viral DNA and RNA, so as to inhibit the replication of viruses; in addition, the glycyrrhiza polysaccharide is an immunomodulator, can stimulate T lymphocyte proliferation, activate endothelial system, promote generation of immunoglobulin, and play a role in regulating human immune system.

Since licorice has many beneficial effects, more researches are carried out on the licorice, as disclosed in Chinese patent application 2020110129262. X, a preparation method of an extract containing the glycyrrhizin is disclosed, the used extraction solvent is a mixed solution of ethanol, isopropanol and acetone, and the volume ratio of the ethanol, the isopropanol and the acetone is controlled to be 85-95:4-10:1-5, so that the extract containing the glycyrrhizin is comprehensively extracted twice under different conditions, and the content of the glycyrrhizin in the extract containing the glycyrrhizin is more than 35.82%; meanwhile, the gradient heating mode is adopted in the first ultrasonic treatment process, so that the content of the liquiritin in the extract is further improved.

As further disclosed in chinese patent application 202110901300.7, a licorice extract, and a preparation method and application thereof, the licorice extract is prepared by the following method: extracting Glycyrrhrizae radix Chinese medicinal material slice with water and polar organic solvent to obtain extract, and treating the extract by alkali dissolution and acid precipitation to obtain Glycyrrhrizae radix extractive solution; concentrating the obtained extractive solution to a certain volume, removing liposoluble components with nonpolar organic solvent, and concentrating to obtain extract; stirring the obtained extract with alkali water solution, suspending, removing insoluble substances, adjusting pH with acid water to precipitate, filtering, and drying to obtain Glycyrrhrizae radix extract; experiments prove that the licorice extract has remarkable effect of inhibiting clostridium difficile in vitro, the minimum antibacterial concentration is 8 mug/ml, and the minimum antibacterial concentration is 16 mug/ml.

Although the prior art discloses a preparation method of licorice extract and related antibacterial performance, the insoluble substances in water and grey scale of the obtained extract are not detected, and a plurality of organic solvents are used for extraction in the extraction process, so that organic solvent residues are easily caused, and the safety is affected; therefore, there is a need to provide a preparation method of licorice extract with high content of active ingredient, low content of insoluble substances in water, low total gray scale and high safety.

Disclosure of Invention

Based on the defects of the prior art, the invention provides a preparation method and application of licorice extract. In order to achieve the above purpose, the present invention provides the following technical solutions:

a preparation method of licorice extract comprises the following steps:

(1) Pretreatment of raw materials: pulverizing dried Glycyrrhrizae radix, soaking in organic solvent and surfactant, filtering, and retaining residue;

(2) Extracting: extracting the filter residue obtained in the step (1) with water, filtering, discarding the filter residue, and reserving the filtrate;

(3) Concentrating the filtrate obtained in the step (2) into paste, and then drying to obtain the licorice extract.

Further, the grain diameter of the crushed liquorice in the step (1) is 10-20 meshes; preferably 20 mesh.

The organic solvent in the step (1) is a mixed solution of ethyl acetate, ethanol and isopropanol;

the volume ratio of the ethyl acetate to the ethanol to the isopropanol is 1-2:3-5:1; preferably, the volume ratio of the ethyl acetate to the ethanol to the isopropanol is 1.5-2:2-5:1; still preferably, the volume ratio of ethyl acetate, ethanol and isopropanol is 2:5:1.

The ethanol used in the invention is food grade ethanol with the volume fraction of 80 percent, and the ethyl acetate and the isopropanol are all commercially purchased food grade chemical purity.

The surfactant is one or more selected from sodium dodecyl sulfate, tritonX-114, tween-60 and Tween-20;

preferably, the surfactant is selected from tween-20.

The mass volume ratio of the liquorice raw material to the organic solvent is 1:3-6 (g/mL); preferably, the mass-volume ratio of the liquorice raw material to the organic solvent is 1:4 (g/mL).

The addition amount of the surfactant is 5-10% of the total mass of the liquorice raw material; preferably, the addition amount of the surfactant is 8% of the total mass of the liquorice raw material.

The soaking time is 5-10 hours; preferably 8 hours; the soaking temperature is 10-20 ℃; preferably 15 ℃.

The step (1) further comprises ultrasonic treatment, wherein the ultrasonic power is 500-600W, the frequency is 20-25KHz, and the time is 20-30min;

preferably, the ultrasonic power is 550W, the frequency is 20KHz, and the time is 20min.

The water extraction times in the step (2) are 3 times; adding 6-8 times of water for 1 st time, and decocting for 2-3 hours; adding 4-5 times of water for the second time, and decocting for 1-2 hours; adding 3-4 times of water for the third time, and decocting for 1-2 hours;

preferably, the number of water extractions in the step (2) is 3; adding 8 times of water for 1 st time, and decocting for 3 hours; adding 5 times of water for the second time, and decocting for 2 hours; adding 3 times of water for the third time, and decocting for 2 hours.

The relative density of the condensed paste in the step (3) is 18-20 Baume (60-70 ℃); the drying temperature is 55-70 ℃; the drying time is 5-8 hours.

The invention also provides application of the licorice extract prepared by the method in preparation of medicinal raw materials.

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

(1) The liquorice powder is soaked in the mode of soaking in the organic solvent and the surfactant in the pretreatment step, so that the purpose of better swelling cells and achieving the purpose of cell rupture is achieved, and the dissolution of active ingredients in the extraction process is facilitated;

(2) The invention adopts the mixed solution of ethyl acetate, ethanol and isopropanol as the soaking solution, and on the one hand, the invention aims to dissolve out the ineffective components as much as possible and remove the ineffective components along with the organic solvent; on the other hand, the method aims to ensure that more active ingredients are reserved in filter residues, so that the content of the active ingredients such as glycyrrhizic acid and glycyrrhizin in the product is improved;

(3) In the implementation process, the surfactant and the organic solvent are mixed and soaked accidentally, so that the solubility of invalid components with different solubility in the organic solvent can be increased, the content of the effective components in the licorice extract is further increased, and the content of insoluble matters in water and the total gray level of the obtained extract are obviously reduced;

(4) According to the invention, the licorice extract is extracted by adopting a water reflux extraction mode after being soaked in the organic solvent, so that the extraction rate of active ingredients in the licorice extract is improved, the organic solvent residue in the obtained licorice extract is less, the safety of the licorice extract can be ensured to a greater extent, and the performance meets the pharmacopoeia requirement.

Drawings

Fig. 1 is a detection chart of licorice extract prepared in example 1 of the present application.

Detailed Description

The above-mentioned features of the invention, or of the embodiments, may be combined in any desired manner. All of the features explained in this specification can be used in combination with any form of method, and each feature disclosed in this specification can be replaced by any alternative feature serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the disclosed features are merely general examples of equivalent or similar features.

The invention will be further illustrated with reference to specific examples. These examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. The following examples are presented to illustrate specific conditions, generally according to conventional conditions or according to manufacturer's recommended conditions. All percentages and fractions are by weight unless specifically indicated.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are exemplary only.

The licorice used in this application is Glycyrrhiza uralensis produced from Sinkiang karst.

The raw materials used in the invention are all common commercial products, and the effect detection result keeps 2 positions after decimal point.

Example 1 preparation method of Glycyrrhiza extract

The method comprises the following steps:

(1) Pretreatment of raw materials: pulverizing 200g of dried Glycyrrhrizae radix raw material to 20 mesh, adding mixed solution of ethyl acetate (120 mL), ethanol (360 mL) and isopropanol (120 mL) with volume ratio of 1:3:1, and tween-60, soaking for 5 hr, performing ultrasonic treatment at power of 600W and frequency of 25KHz for 20min for 2 times, filtering, and retaining residue;

(2) Extracting: carrying out water extraction on the filter residue obtained in the step (1), wherein the water extraction times are 3 times; adding 6 times of water for 1 st time, and decocting for 2 hours; adding 4 times of water for the second time, and decocting for 1 hour; adding 3 times of water for the third time, decocting for 1 hr, filtering, discarding residue, and retaining filtrate;

(3) Concentrating the filtrate obtained in the step (2) to obtain a paste with a relative density of 18.8 Baume (60-70 ℃), and drying at 50 ℃ for 8 hours to obtain 60.0g of licorice extract.

The mass-volume ratio of the liquorice raw material to the organic solvent in the step (1) is 1:3 (g/mL);

the addition amount of the tween-60 is 5 percent of the total mass of the liquorice raw materials.

Example 2 preparation method of Glycyrrhrizae radix extract

The method comprises the following steps:

(1) Pretreatment of raw materials: pulverizing 200g of dried Glycyrrhrizae radix raw material to 20 mesh, adding mixed solution of ethyl acetate (300 mL), ethanol (750 mL) and isopropanol (150 mL) with volume ratio of 2:5:1, and sodium dodecyl sulfate, soaking for 10 hr, performing ultrasonic treatment at power of 500W and frequency of 20KHz for 30min, performing ultrasonic treatment for 2 times, filtering, and retaining residue;

(2) Extracting: extracting the filter residue obtained in the step (1) with water for 3 times, adding 8 times of water for 1 st time, and decocting for 3 hours; adding 5 times of water for the second time, and decocting for 2 hours; adding 4 times of water for the third time, decocting for 2 hours, filtering, discarding filter residues, and retaining the filtrate;

(3) Concentrating the filtrate obtained in the step (2) to obtain a paste with a relative density of 18.5 Baume (60-70 ℃), and then drying at 70 ℃ for 5 hours to obtain 62.5g of licorice extract.

The mass-volume ratio of the liquorice raw material to the organic solvent in the step (1) is 1:6 (g/mL);

the adding amount of the sodium dodecyl sulfate is 10% of the total mass of the liquorice raw material.

Example 3 preparation method of Glycyrrhiza extract

The method comprises the following steps:

(1) Pretreatment of raw materials: pulverizing 200g of dried Glycyrrhrizae radix raw material to 20 mesh, adding mixed solution of ethyl acetate (200 mL), ethanol (500 mL) and isopropanol (100 mL) with volume ratio of 2:5:1, and tween-20, soaking for 8 hr, wherein the soaking process requires ultrasonic treatment under the condition of 550W power and 20KHz frequency for 20min, ultrasonic treatment for 2 times, filtering, and retaining filter residue;

(2) Extracting: extracting the filter residue obtained in the step (1) with water for 3 times, adding 8 times of water for 1 st time, and decocting for 3 hours; adding 5 times of water for the second time, and decocting for 2 hours; adding 3 times of water for the third time, decocting for 2 hours, filtering, discarding filter residues, and retaining the filtrate;

(3) Concentrating the filtrate obtained in the step (2) to obtain a paste with a relative density of 19.2 Baume (60-70 ℃), and then drying at 60 ℃ for 6 hours to obtain 65.4g of licorice extract.

The mass-volume ratio of the liquorice raw material to the organic solvent in the step (1) is 1:4 (g/mL);

the addition amount of the tween-20 is 8 percent of the total mass of the liquorice raw materials.

Comparative example 1

The difference from example 3 is that: without pretreatment, the licorice material was directly crushed to 20 mesh, and then extracted by the same method as in steps (2) - (3), to obtain 60.5g licorice extract.

Comparative example 2

The difference from example 3 is that: 62.4g of licorice extract was obtained by using a mixture of ethyl acetate, ethanol and isopropanol in a volume ratio of 1:1:1, and the other steps and operations were the same as in example 3.

Comparative example 3

The difference from example 3 is that: soaking was performed using only ethanol, and the other steps and operations were the same as in example 3, to obtain 60.5g of licorice extract.

Comparative example 4

The difference from example 3 is that: without adding surfactant, 58.2g of licorice extract was obtained in the same manner as in example 3.

Comparative example 5

The difference from example 3 is that: the volume fraction of ethanol used was 40%, and the other steps and operations were the same as in example 3, to obtain 60.8g of licorice extract.

Effect detection

Performance tests were performed on the licorice extracts prepared in examples 1 to 3 and comparative examples 1 to 5, and the glycyrrhizic acid content, the water insoluble content and the ash content of the obtained licorice extracts were tested.

1. Extract yield and glycyrrhizic acid and glycyrrhizin content detection

Detecting glycyrrhizic acid and glycyrrhizin content according to the method disclosed in the quality standard of Glycyrrhrizae radix extract in China pharmacopoeia of 2020 edition, wherein figure 1 is a Glycyrrhrizae radix extract detection map prepared in example 1, the integral result is shown in the following table 1, and the glycyrrhizic acid and glycyrrhizin content detection result in Glycyrrhrizae radix extract is shown in the following table 2; licorice extract yield = weight of glycyrrhizic acid/weight of licorice x 100%.

TABLE 1 integration results

TABLE 2 Glycyrrhiza extract yield and glycyrrhizic acid and glycyrrhizin content

	Licorice extract yield	Glycyrrhizic acid content%	Glycyrrhizin content%
				Example 1	30.00％	8.63％	0.68％
Example 2	31.25％	8.76％	0.62％
				Example 3	32.70％	8.85％	0.72％
Comparative example 1	30.25％	7.86％	0.50％
				Comparative example 2	31.20％	8.55％	0.63％
Comparative example 3	30.25％	8.32％	0.52％
				Comparative example 4	29.10％	8.40％	0.55％
Comparative example 5	30.40％	8.58％	0.62％

As can be seen from the detection data in the table 2, the glycyrrhizic acid content in the licorice extract prepared in the embodiments 1-3 can reach more than 8.6%, the glycyrrhizin content can reach more than 0.65%, and the glycyrrhizic acid and the glycyrrhizin content in the licorice extract are obviously improved; in comparative example 1, only water was used for reflux extraction, and the cell wall was not broken well, so that the contents of glycyrrhizic acid and glycyrrhizin were significantly reduced; the glycyrrhizic acid content of the licorice extract obtained by adopting the organic solvents with different proportions in the comparative example 2 is reduced to a certain extent compared with that of the example 3, but the glycyrrhizic acid content is not greatly different; the content of glycyrrhizic acid and glycyrrhizin in the licorice extract is affected by the comparative examples 3-4 by soaking in ethanol only or without adding surfactant, and the content of glycyrrhizic acid and glycyrrhizin in the licorice extract obtained by soaking in 40% ethanol in comparative example 5 is not much different from that in comparative example 2, which means that the ratio and concentration of the organic solvent both affect the extraction efficiency of the effective components.

2. Detection of content of insoluble substances in water

The content of insoluble substances in water is detected according to the method disclosed in the quality standard of licorice extract in Chinese pharmacopoeia of 2020 edition, and the detection result is shown in the following table 3.

TABLE 3 content of insoluble matter in Water in Glycyrrhiza paste

As can be seen from the detection data in the table 3, the content of insoluble matters in the licorice extractum prepared in the embodiments 1-3 can reach below 3%, which indicates that the content of effective components in the prepared licorice extractum is higher; in comparative example 1, only water is used for reflux extraction, and invalid components are not removed, so that the content of insoluble matters in the obtained extract is obviously improved; although the glycyrrhizic acid content in the licorice extract obtained by adopting the organic solvents with different proportions in comparative example 2 is not greatly different from that in example 3, the water insoluble content is obviously improved compared with example 3; the comparative examples 3 to 4 were only immersed in ethanol or without adding a surfactant to raise the content of insoluble matters in water in licorice extract, and the comparative example 5 was immersed in 40% ethanol to obtain licorice extract having a glycyrrhizic acid content which is not much different from that of example 3, but the content of insoluble matters in water was significantly improved as compared with example 3.

3. Ash content detection

Ash content was detected according to the method disclosed in the quality standard of licorice extract in chinese pharmacopoeia of 2020 edition, and the detection result is shown in table 4 below.

TABLE 4 ash content in Glycyrrhiza extract

	Ash content
		Example 1	2.53％
Example 2	2.45％
		Example 3	2.42％
Comparative example 1	3.26％
		Comparative example 2	3.48％
Comparative example 3	3.30％
		Comparative example 4	3.32％
Comparative example 5	3.45％

As can be seen from the detection data of the above Table 4, ash content in the licorice extractum prepared in examples 1-3 of the present invention is below 3%, further illustrating that the prepared licorice extractum has higher content of effective components; the ash content of the extracts prepared in comparative examples 1-5 is higher than that of the extracts prepared in examples, and the change of the solvent used in pretreatment or the omission of the pretreatment step can obviously affect the content of the effective components in the licorice extract and the extraction efficiency.

The invention has been further described above in connection with specific embodiments, which are exemplary only and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.

Claims

1. A preparation method of licorice extract is characterized by comprising the following steps: the method comprises the following steps:

(3) Concentrating the filtrate obtained in the step (2) into paste, and then drying to obtain the licorice extract;

the organic solvent in the step (1) is a mixed solution of ethyl acetate, ethanol and isopropanol; the volume ratio of the ethyl acetate to the ethanol to the isopropanol is 1-2:3-5:1;

the surfactant is selected from sodium dodecyl sulfate, tween-60 and tween-20; the mass volume ratio of the liquorice raw material to the organic solvent is 1:3-6 g/mL; the addition amount of the surfactant is 5-10% of the total mass of the liquorice raw material;

the ethanol is ethanol with the volume fraction of 80 percent.

2. The method of manufacturing according to claim 1, characterized in that: the volume ratio of the ethyl acetate to the ethanol to the isopropanol is 2:5:1.

3. The method of manufacturing according to claim 1, characterized in that: the water extraction times in the step (2) are 3 times; adding 6-8 times of water for 1 st time, and decocting for 2-3 hours; adding 4-5 times of water for the second time, and decocting for 1-2 hours; adding 3-4 times of water for the third time, and decocting for 1-2 hr.