阅读说明：本技术 一种基于亚临界水萃取技术提取甘草酸的方法 (Method for extracting glycyrrhizic acid based on subcritical water extraction technology ) 是由 朱荣耀 华梦云 于 2021-08-24 设计创作，主要内容包括：本发明公开了一种基于亚临界水萃取技术提取甘草酸的方法,包括如下步骤：(1)甘草的预处理；(2)取预处理的甘草,加入纯水或含食品级酸碱调节剂的纯水；(3)升温,加压,达到水亚临界状态；(4)在水亚临界状态下进行萃取；(5)萃取结束后,冷却,收集萃取液,即得。本发明提取了一种以水为溶剂,不添加任何辅料、任何有机试剂,使甘草酸提取率稳定在90％以上的绿色高效萃取工艺,有效解决了传统提取时甘草酸的提取率低和提取时大量使用有机溶剂的两大问题。(The invention discloses a method for extracting glycyrrhizic acid based on a subcritical water extraction technology, which comprises the following steps: (1) pretreating liquorice; (2) adding pure water or pure water containing food-grade acid-base regulator into pretreated Glycyrrhrizae radix; (3) heating and pressurizing to reach a subcritical water state; (4) extracting under the subcritical state of water; (5) and after extraction is finished, cooling, and collecting extract liquor to obtain the product. The invention extracts a green high-efficiency extraction process which takes water as a solvent and does not add any auxiliary materials or any organic reagent, so that the extraction rate of glycyrrhizic acid is stabilized to be more than 90 percent, and effectively solves the two problems of low extraction rate of glycyrrhizic acid in the traditional extraction and large use of organic solvent in the extraction.)

1. A method for extracting glycyrrhizic acid based on a subcritical water extraction technology is characterized by comprising the following steps:

(1) pretreating liquorice;

(2) adding pure water or pure water containing food-grade acid-base regulator into pretreated Glycyrrhrizae radix;

(3) heating and pressurizing to reach a subcritical water state;

(4) extracting under the subcritical state of water;

(5) and after extraction is finished, cooling, and collecting extract liquor to obtain the product.

2. The method for extracting glycyrrhizic acid based on subcritical water extraction technique according to claim 1, wherein in step (1), the pretreatment of licorice comprises: the licorice root is screened to remove silt and other impurities.

3. The method for extracting glycyrrhizic acid based on subcritical water extraction technique according to claim 1, wherein in step (2), the food grade pH adjusting agent comprises food grade sodium hydroxide, citric acid, lactic acid, malic acid and the like.

4. The method for extracting glycyrrhizic acid based on subcritical water extraction technology according to claim 1, wherein in step (2), the liquid-material ratio is 40: 1-80: 1 (mL/g).

5. The method for extracting glycyrrhizic acid based on subcritical water extraction technique according to claim 1, wherein in step (3), the temperature is raised to 105-170 ℃ and then kept; and (3) pressurizing, and stopping pressurizing when the pressure is 0.2-3 MPa.

6. The method for extracting glycyrrhizic acid based on subcritical water extraction technique according to claim 1, wherein: in the step (4), the extraction time is 20-50 min.

Technical Field

The invention belongs to the technical field of glycyrrhizic acid extraction, and particularly relates to a method for extracting glycyrrhizic acid based on a subcritical water extraction technology.

Background

Glycyrrhizic acid (Glycyrrhizic acid) is an effective active ingredient extracted from root and stem of Glycyrrhiza glabra L, is a very precious natural saponin, and has antioxidant and corticotropin-like activities. According to literature research, glycyrrhizic acid and series products thereof have the effects of inhibiting the growth of sarcoma and cancer cells, have the AIDS inhibition rate of up to 90 percent and have stronger effect of enhancing the human body immunity. And it has sweetness about 50 times that of sucrose, is a low calorie sweetener, and is a good food additive and flavor base. Therefore, the glycyrrhizic acid is widely applied to the industries of medicine, chemical industry, food and the like.

The liquorice is a common bulk medicinal material and a medicine and food dual-purpose variety, but the yield of high-quality products is not high. According to the introduction of documents, wild products are generally dug in spring and autumn, mainly in autumn and winter, but the yield is difficult to meet the market and medicinal research requirements; the liquorice grown by the family sexual propagation is harvested after 3-4 years of general growth, and the liquorice transplanted for seedling is harvested after 2-3 years of general growth; the wild quality of the liquorice has high elasticity, the liquorice variety of the family species is huge, the specification is disordered, and the production period is long. The glycyrrhizic acid in the glycyrrhizic acid can be extracted by ultrasonic method, supercritical extraction, alcohol extraction and the like. Patent CN201811404463.9 reports a method for extracting glycyrrhizic acid by a homogenization-ultrasonic method, wherein liquorice is dried in the shade, crushed, added with 50-60% ethanol which is 1-2 times of the raw material, and placed in a homogenizer for mixing and homogenizing; after homogenizing, adding 50-60% ethanol solution which is 3-5 times of the raw materials, performing ultrasonic extraction for 90-120 min, and performing reduced pressure concentration to obtain concentrated solution; eluting the obtained concentrated solution, concentrating, and drying to obtain glycyrrhizic acid; patent CN00105599.2 reports a supercritical CO₂Method for extracting and refining glycyrrhizic acid from Glycyrrhrizae radix (or crude glycyrrhizic acid) by supercritical CO₂Removing nonpolar oily impurities, controlling extraction pressure at 25 + -2 MPa and temperature at 55 deg.C, and supercritical CO₂Adding 95% ethanol modifier, and extracting glycyrrhizic acid with yield of 4% and refined glycyrrhizic acid purity of 98.2%; patent CN201410453575.9 reports a method for extracting glycyrrhizic acid from licorice, which mainly comprises the steps of drying and crushing fresh licorice, placing licorice particles in a container, adding an ethyl acetate-dichloromethane mixed solvent, standing for extraction, filtering, adding an ethanol aqueous solution for extraction, mixing with petroleum ether, standing for extraction, adding water, adjusting pH, adding activated carbon for decolorization, precipitating with acid, centrifuging, collecting precipitate, and drying to obtain glycyrrhizic acid. The traditional extraction method needs a large amount of organic reagents or complicated steps, and finally glycyrrhizic acid with lower extraction rate is obtained.

Disclosure of Invention

The purpose of the invention is as follows: in order to solve the technical problems, the invention provides a method for extracting glycyrrhizic acid based on a subcritical water extraction technology, which effectively solves the two problems of low extraction rate of glycyrrhizic acid in the traditional extraction process and large use of organic solvent in the extraction process.

The technical scheme is as follows: in order to achieve the purpose, the invention adopts the following technical scheme:

a method for extracting glycyrrhizic acid based on a subcritical water extraction technology comprises the following steps:

(1) pretreating liquorice;

(2) adding pure water or pure water containing food-grade acid-base regulator into pretreated Glycyrrhrizae radix;

(3) heating and pressurizing to reach a subcritical water state;

(4) extracting under the subcritical state of water;

(5) and after extraction is finished, cooling, and collecting extract liquor to obtain the product.

Preferably, in step (1), the pretreatment of licorice comprises: the licorice root is screened to remove silt and other impurities.

Preferably, in step (2), the pure water and the pure water containing the food-grade pH modifier are selected, and most preferably the pure water.

Preferably, in the step (2), the food-grade acid-base modifier is food-grade sodium hydroxide, citric acid, lactic acid, malic acid and the like.

Preferably, in the step (2), the liquid-to-material ratio is 40:1 to 80:1(mL/g), and most preferably 53:1 (mL/g).

Preferably, in the step (3), after the temperature is raised to 105-170 ℃, the temperature is maintained at 105 ℃ most preferably; and (3) pressurizing until the pressure is 0.2-3 MPa, most preferably 0.2MPa, and stopping pressurizing.

Preferably, in the step (4), the extraction time is 20-50 min, and most preferably 30 min.

As the most preferable scheme:

adding pure water into pretreated Glycyrrhrizae radix at a liquid-material ratio of 53:1(mL/g), heating to 105 deg.C, stopping heating, pressurizing, stopping pressurizing when the pressure reaches 0.2Mpa, extracting for 30min under heat preservation and pressure maintaining conditions, opening cooling water after extraction, condensing steam, further cooling, and collecting the extractive solution.

The subcritical water extraction method is an extraction technology which has low cost, high extraction rate of active ingredients and high extraction speed, and is particularly green, economic and environment-friendly. The principle is as follows: under subcritical conditions (temperature of 100-374 deg.C and pressure lower than 22MPa), the dielectric constant of water is changed, so that it has similar extraction effect to organic solvent such as methanol and ethanol, and can utilize this property to attain the goal of extracting polar or non-polar substance. The glycyrrhizic acid can be efficiently extracted by utilizing a green efficient extraction technology, and the quality and the supply of the effective components of the liquorice are ensured. At present, no report that only subcritical water is used for extracting glycyrrhizic acid in liquorice in the process is found.

Has the advantages that: compared with the prior art, the invention has the following advantages:

(1) the invention is based on the subcritical water extraction technology, takes water as an extraction medium, does not use any organic solvent, utilizes the preferred scheme process to stabilize the glycyrrhizic acid extraction rate to be more than 90 percent, and provides a green high-extraction-rate process.

(2) Glycyrrhizic acid is a natural sweetener extracted from liquorice, is 100-200 times sweeter than cane sugar, is widely applied to food, medicine and cosmetic industries at present, and has wide development and utilization prospects.

Drawings

FIG. 1 is a standard curve diagram of ammonium glycyrrhetate measured by HPLC;

FIG. 2 is a liquid chromatogram of ammonium glycyrrhizinate standard;

FIG. 3 is the effect of pressure on the extraction effect of glycyrrhizic acid in example 2;

FIG. 4 is the effect of the ratio of the feed to the liquid on the extraction of glycyrrhizic acid in example 3;

FIG. 5 is the effect of temperature on the extraction of glycyrrhizic acid in example 4;

FIG. 6 is a liquid chromatogram of the subcritical water extract in example 5;

FIG. 7 shows the effect of repeated extractions of the extraction process of example 5.

Detailed Description

The finishing procedures set by the invention are as follows:

1. pretreatment of liquorice: and screening silt and other impurities of the purchased liquorice.

2. And (3) detecting the total content of glycyrrhizic acid: the total content of glycyrrhizic acid in the purchased liquorice is detected according to a method of 'Chinese pharmacopoeia' 2020 edition.

3. The orthogonal test explores the optimal process: in subcritical water extraction, adding an extractant into pretreated liquorice in a liquid-material ratio of 40: 1-80: 1(mL/g), heating at an extraction temperature of 105-170 ℃, stopping heating after the preset temperature is reached, pressurizing at an extraction pressure of 0-3 MPa, stopping pressurizing after the pressure reaches the extraction pressure, performing heat preservation and pressure maintaining extraction for 20-50 min, opening cooling water after extraction is finished, condensing steam, further cooling, and finally collecting an extract.

4. And (3) testing the glycyrrhizic acid content: after the water extract is dehydrated by a freeze dryer, the content of the water extract is tested by HPLC, and the conditions of liquid chromatography are as follows: mobile phase: acetonitrile (A) -0.085% phosphoric acid water (B), gradient elution, detection wavelength 254 nm.

5. Referring to a detection method for liquorice in 'Chinese pharmacopoeia' 2020 edition, standard curves measured by ammonium glycyrrhizinate standard substances are taken as a reference (the weight of glycyrrhizic acid is equal to the weight of ammonium glycyrrhizinate/1.0207), and a relational expression of absorbance and concentration is obtained by fitting: the A (peak area) is 0.2474C (concentration) -0.3955, the retention time is about 31min, and the above formula is used for calculating the content of the extracted glycyrrhizic acid. The standard curve diagram of ammonium glycyrrhizinate is shown in FIG. 1, and the liquid chromatogram of the standard ammonium glycyrrhizinate is shown in FIG. 2.

Example 1

And (3) detecting the total content of glycyrrhizic acid: according to the method of 2020 edition of Chinese pharmacopoeia, 0.2g of licorice powder (sieved by a No. three sieve) is taken and placed in a conical flask, 100mL of 70% ethanol is precisely added, and the mixture is sealed and treated by ultrasonic for 30 min. And (4) passing the filtrate through a 0.45-micron filter membrane for liquid phase test, wherein the detection result is the total content of glycyrrhizic acid in the purchased liquorice. Comparing with standard liquid chromatogram to obtain peak at retention time of about 31min, and calculating to obtain glycyrrhizic acid total content of 17.85mg/g in Glycyrrhrizae radix.

Example 2

An orthogonal test is designed by adopting a factor alternation method, and the influence of pressure on the extraction effect is explored: taking 30g of pretreated liquorice, adding 1600mL of pure water, wherein the liquid-material ratio is 53:1(mL/g), heating, stopping heating after the temperature reaches 105 ℃, pressurizing to 0.2, 0.5, 1.5, 2.5 and 3Mpa respectively, preserving heat and maintaining pressure for extraction for 30min, opening cooling water after extraction is finished, condensing steam, further cooling, and finally collecting extract. Freeze drying the extractive solution for 3 days, removing water, dissolving in 70% ethanol, and testing with 0.45 μm filter membrane. The effect of pressure was measured using the extraction effect as an evaluation index, as shown in fig. 3.

Example 3

An orthogonal test is designed by adopting a factor alternation method, and the influence of liquid-material ratio on the extraction effect is explored: adding 1600mL of pure water, extracting at 105 ℃, and under the optimal pressure in example 1, namely pressurizing to 0.2Mpa, wherein the feed amount of liquorice is 20, 26, 30, 35 and 40g respectively, namely the liquid-material ratio is 40: 1-80: 1(mL/g), preserving heat and maintaining pressure for extraction for 30min, opening cooling water after extraction is finished, condensing steam, further cooling, and finally collecting the extract. Freeze drying the extractive solution for 3 days, removing water, dissolving in 70% ethanol, and testing with 0.45 μm filter membrane. The effect of the liquid-to-material ratio was measured by using the extraction effect as an evaluation index, as shown in fig. 4.

Example 4

An orthogonal test is designed by adopting a factor alternation method, and the influence of temperature on the extraction effect is explored: adding 1600mL of pure water, wherein the liquid-material ratio is 53:1(mL/g) which is the optimal liquid-material ratio in example 3, namely, the adding amount of the liquorice is 30g, the pressure is the optimal pressure in example 1, namely, the pressure is increased to 0.2Mpa, the temperature is respectively increased to 105 ℃, 110, 130, 150 and 170 ℃, the temperature and the pressure are kept for extraction for 30min, cooling water is opened after the extraction is finished, steam is condensed, further cooling is carried out, and finally the extract is collected. Freeze drying the extractive solution for 3 days, removing water, dissolving in 70% ethanol, and testing with 0.45 μm filter membrane. The effect of temperature was measured using the extraction effect as an evaluation index, as shown in fig. 5.

Example 5

And (3) exploring an optimal extraction process by adopting a factor alternation method through the orthogonal experimental design: taking 30g of pretreated liquorice, adding 1600mL of pure water, namely the liquid-material ratio is 53:1(mL/g), heating, stopping heating after the temperature reaches 105 ℃, pressurizing to 0.2Mpa, preserving heat and maintaining pressure for extraction for 30min, opening cooling water after extraction is finished, condensing steam, further cooling, and finally collecting extract. Freeze drying the extractive solution for 3 days, removing water, dissolving in 70% ethanol, and testing with 0.45 μm filter membrane. The above experiment was repeated three times.

As can be seen from fig. 7, the process was repeated three times under the conditions, the glycyrrhizic acid content was 16.12, 16.57, 17.64mg/g, and the extraction rate was all above 90%.