Method for enriching flavonoids and / or saponins from plant materials and special extraction reagents

Abstract

The present invention discloses a method and a special extraction reagent for enriching flavonoids and / or saponins from plant materials. The present invention provides an extraction reagent for enriching flavonoids and / or saponins from plants or plant processed products, which is composed of water, ethanol, a low eutectic solvent and a surfactant; the raw material composition of the low eutectic solvent is choline chloride and ethylene glycol; the surfactant is AES; the plant material is a plant or a plant processed product. The present invention has the following beneficial effects: it can efficiently extract the insoluble secondary metabolite components flavonoids and saponins in plants, has the advantages of short time, high yield, simple operation, non-toxicity and low cost, has industrial value, and can be promoted and applied.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a method for extracting secondary metabolites from traditional Chinese medicines of plant origin and a special extraction reagent. Background Art

[0002] The chemical composition of plants is relatively complex, with primary metabolites represented by sugars, lipids, proteins, vitamins, cellulose, etc., and secondary metabolites represented by polysaccharides, alkaloids, flavonoids, saponins, cardiac glycosides, anthraquinones, volatile oils, organic acids, coumarins, etc. Plant secondary metabolites are usually physiologically active and are the core components of plant extract raw materials. They are also the material basis for natural plant extracts to play a relevant role when used in medicines, functional foods and cosmetics.

[0003] At present, the most commonly used method for extracting phytochemical components is still solvent extraction. Commonly used solvents are water, organic solvents (ethanol, methanol, acetone, etc.) or aqueous solutions of organic solvents. As the source of all things, water is non-toxic, harmless, and has low cost. It is one of the most commonly used solvents in the field of phytochemical component extraction. However, phytochemical components are often organic macromolecules with low solubility in water, and the extraction rate of water as an extraction solvent is very low. In order to increase the solubility of the target component when water is used as an extraction solvent, acid or alkali is usually added to the water, but this will produce a large amount of wastewater, which will bring pressure and cost to subsequent environmental protection. In addition, when the roots, stems, and fruits of plants are used as extraction raw materials, due to the large amount of starch and tanning components, the viscosity of the extract obtained when water is used as an extraction solvent is relatively large, which causes certain difficulties for industrial production filtration. In addition, the residue remaining after extraction with water as an extraction solvent is easy to rot and deteriorate, and is mostly used as fertilizer, and cannot be used as a more valuable livestock feed. Other valuable components in the residue cannot be fully utilized, resulting in a waste of plant resources. Compared with water, organic solvents as extraction solvents overcome the above disadvantages. However, the cost and toxicity of organic solvents are practical issues that R&D and production companies have to consider.

[0004] Since the concept of green chemistry was established in 1998, the demand for sustainable development has encouraged people to look for a non-toxic, low-cost, pollution-free and degradable green solvent as a substitute for organic solvents. The concept of deep eutectic solvents (DES) was first proposed by Abbott et al. in 2003. DES refers to a low eutectic mixture composed of a hydrogen bond acceptor (HBA) and a hydrogen bond donor (HBD) in a certain stoichiometric ratio. Compared with traditional solvents, DES has the advantages of low production cost, simple synthesis method, stable chemical properties, non-toxicity, harmlessness and easy degradation. It is considered to be an excellent substitute for traditional solvents and ionic solvents (IL). As a new type of green solvent, DES can be repeatedly extracted and still maintain stable physical and chemical properties, making it an ideal solvent system. However, most DES have high viscosity and are solid at room temperature. Usually, mass transfer does not occur well in solvents with higher viscosity. Therefore, the application of DES in the extraction of natural products is limited.

[0005] Flavonoids are a large class of natural phenolic compounds that are widely distributed in nature, especially in the plant kingdom, and are one of the main secondary metabolites in medicinal plants. Saponins, also known as alkali soap bodies, saponins, saponins, saponins, and saponins, have a variety of biological activities and medicinal values, such as regulating immunity, anti-inflammatory, anti-tumor, lowering blood lipids, and protecting cardiovascular health. Summary of the invention

[0006] The object of the present invention is to provide a method for enriching flavonoids and / or saponin from plant materials and a special extraction reagent.

[0007] The present invention provides an extraction reagent for enriching active ingredients from plants or plant preparations, which consists of water, ethanol, a deep eutectic solvent and a surfactant;

[0008] The raw materials of the deep eutectic solvent are choline chloride (hydrogen bond acceptor) and ethylene glycol (hydrogen bond donor);

[0009] The surfactant is AES;

[0010] The plant material is a plant or a plant preparation.

[0011] In the extraction reagent, the ratio of water, ethanol, deep eutectic solvent and surfactant is as follows:

[0012] 160-200ml water: 400-440ml ethanol: 580-620ml deep eutectic solvent: 2-4g surfactant.

[0013] Specifically, in the extraction reagent, the ratio of water, ethanol, deep eutectic solvent and surfactant is as follows:

[0014] 180ml water: 420ml ethanol: 600ml deep eutectic solvent: 3g surfactant.

[0015] The molar ratio of the raw materials of the low eutectic solvent is: choline chloride: ethylene glycol = 1:3.

[0016] AES: sodium lauryl polyoxyethylene ether sulfate (CAS registration number is 9004-82-4).

[0017] The present invention also protects the use of any of the above-mentioned extraction reagents in enriching active ingredients from plant materials; the plant materials are plants or plant processed products.

[0018] The present invention also protects a method for enriching active ingredients from plant materials, comprising the following steps: extracting the plant materials using any of the above-mentioned extraction reagents; the plant materials are plants or plant processed products.

[0019] The extraction conditions are: extraction at 40-60° C. for 20-40 minutes.

[0020] Specifically, the extraction conditions are: extraction at 50° C. for 30 minutes.

[0021] Specifically, the method further comprises the following steps: after the extraction is completed, filtering and collecting the filtrate, concentrating it to a specific gravity of 1.15-1.20, and then filtering to remove insoluble matter.

[0022] Specifically, the method further comprises the following steps: after the extraction is completed, filtering with filter paper and collecting the filtrate, concentrating it to a specific gravity of 1.15-1.20, and then filtering with filter cloth to remove insoluble matter.

[0023] Specifically, the method further comprises the following steps: after the extraction is completed, filtering with filter paper and collecting the filtrate, concentrating it to a specific gravity of 1.15-1.20, and then filtering with a 200-mesh filter cloth to remove insoluble matter.

[0024] Specifically, the method further comprises the following steps: after the extraction is completed, filtering with qualitative filter paper and collecting the filtrate, concentrating it to a specific gravity of 1.15-1.20, and then filtering with 200 mesh filter cloth to remove insoluble matter.

[0025] Specifically, the ratio of the plant material to the extraction reagent is: 100g plant material: 1000-1500ml extraction reagent. Specifically, the ratio of the plant material to the extraction reagent is: 100g plant material: 1200ml extraction reagent.

[0026] Any of the above active ingredients is A or B or C, wherein A is flavonoids and saponins, B is saponins, and C is flavonoids.

[0027] Adopting the method, the flavonoid yield per 100g of plant material is 1600mg or more and the saponin yield is 3500mg or more. Adopting the method, the flavonoid yield per 100g of plant material is 1600mg or more and the saponin yield is 4000mg or more. Adopting the method, the flavonoid yield per 100g of plant material is 1600mg or more and the saponin yield is 4500mg or more. Adopting the method, the flavonoid yield per 100g of plant material is 1800mg or more and the saponin yield is 4600mg or more. Adopting the method, the flavonoid yield per 100g of plant material is 2000mg or more and the saponin yield is 4700mg or more. Adopting the method, the flavonoid yield per 100g of plant material is 2050mg or more and the saponin yield is 4800mg or more. Adopting the method, the flavonoid yield per 100g of plant material is 2100mg or more and the saponin yield is 4900mg or more.

[0028] By adopting the method, the flavonoid yield per 100g of plant material is 1600mg or more. By adopting the method, the flavonoid yield per 100g of plant material is 1700mg or more. By adopting the method, the flavonoid yield per 100g of plant material is 1800mg or more. By adopting the method, the flavonoid yield per 100g of plant material is 1900mg or more. By adopting the method, the flavonoid yield per 100g of plant material is 2000mg or more. By adopting the method, the flavonoid yield per 100g of plant material is 2050mg or more. By adopting the method, the flavonoid yield per 100g of plant material is 2100mg.

[0029] By adopting the method, the saponin yield per 100g of plant material is 3500mg or more. By adopting the method, the saponin yield per 100g of plant material is 4000mg or more. By adopting the method, the saponin yield per 100g of plant material is 4500mg or more. By adopting the method, the saponin yield per 100g of plant material is 4600mg or more. By adopting the method, the saponin yield per 100g of plant material is 4700mg or more. By adopting the method, the saponin yield per 100g of plant material is 4800mg or more. By adopting the method, the saponin yield per 100g of plant material is 4900mg or more.

[0030] The present invention also provides a method for improving the enrichment of active ingredients from plant materials, named method A;

[0031] The plant material is a plant or a plant preparation;

[0032] The method A comprises the following steps: extracting the plant material using any of the above extraction reagents;

[0033] The yield of the active ingredient enriched from plant materials by using the method A is higher than that by using the method B;

[0034] The only difference between method A and method B is that different extraction reagents are used;

[0035] The extraction reagent used in the method B is ethanol aqueous solution (75% ethanol aqueous solution).

[0036] The present invention also provides a method for improving the enrichment of active ingredients from plant materials, named method A;

[0037] The plant material is a plant or a plant preparation;

[0038] The method A comprises the following steps: extracting the plant material using any of the above extraction reagents; the ratio of the plant material to the extraction reagent is: 100g plant material: 1200ml extraction reagent;

[0039] The yield of the active ingredient enriched from plant materials by using the method A is higher than that by using the method B;

[0040] The difference between method A and method B is only that different extraction reagents are used and the amount of extraction reagent added is different;

[0041] The extraction reagent used in method B is ethanol-water solution; the ratio of the plant material to the extraction reagent is: 100g plant material: 600ml extraction reagent (600ml extraction reagent: composed of 180ml water and 420ml ethanol).

[0042] Any of the above active ingredients is A or B or C, wherein A is flavonoids and saponins, B is saponins, and C is flavonoids.

[0043] The plant material may be a crushed plant material, for example, a powdered plant material crushed to 60 mesh.

[0044] The plant processed product can be a traditional Chinese medicine derived from plants.

[0045] Specifically, the plant is liquorice.

[0046] Specifically, the plant processed product is the traditional Chinese medicine licorice.

[0047] The present invention has the following beneficial effects: it can efficiently extract the insoluble secondary metabolites - flavonoids and saponins in plants, has the advantages of short time, high yield, simple operation, non-toxicity and low cost, has industrial value, and can be promoted and applied. DETAILED DESCRIPTION

[0048] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0049] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources. Unless otherwise specified, the quantitative tests in the following examples are set up for three repeated experiments, and the results are averaged. Unless otherwise specified, the concentration in the examples is concentrated under reduced pressure at 70°C. Preparation method of low eutectic solvent: Mix the raw materials and place them in a water bath at 80-100°C and stir to dissolve.

[0050] Flavonoids (also known as flavonoids) refer to a series of compounds formed by two benzene rings (ring A and ring B) with phenolic hydroxyl groups connected to each other through three central carbon atoms, and its basic parent nucleus is 2-phenylchromone.

[0051] Saponin (also known as saponin compounds): refers to a type of glycoside whose aglycone is a triterpene or spirostane compound. Saponin is divided into two categories according to the structure of the sapogenin ligand, namely steroidal saponin and triterpene saponin. Steroidal saponin: Its sapogenin ligand is a derivative of spirostane. Triterpene saponin: Its sapogenin ligand is a derivative of triterpene.

[0052] The Chinese medicinal liquorice is the dried root and rhizome of the leguminous plants Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat. or Glycyrrhiza glabra L. The raw materials used in the examples are all powders of Chinese medicinal liquorice, that is, the powders obtained by crushing the Chinese medicinal liquorice purchased from the pharmacy into 60 meshes.

[0053] Example 1: Optimization of adding a deep eutectic solvent to the extraction reagent

[0054] The addition of the deep eutectic solvent is mainly to increase the flavonoid yield, so the flavonoid yield is used as the screening index in this embodiment.

[0055] 1. Optimization of raw material composition of deep eutectic solvent

[0056] 1. Take 100g of raw material, add 1000ml of extraction reagent (consisting of 180ml of water, 420ml of ethanol and 400ml of low eutectic solvent) and stir evenly, extract at 50℃ for 30 minutes, filter with qualitative filter paper, and collect the filtrate.

[0057] The raw material compositions of the deep eutectic solvent are as follows:

[0058] ①Choline chloride:ethylene glycol = 1:3 (molar ratio);

[0059] ②Choline chloride: lactic acid = 1:3 (molar ratio);

[0060] ③Choline chloride: urea = 1:3 (molar ratio);

[0061] ④ Betaine: ethylene glycol = 1:3 (molar ratio);

[0062] ⑤Lactic acid:ethylene glycol = 1:3 (molar ratio).

[0063] 2. Take the filtrate obtained in step 1, concentrate it to a specific gravity of 1.15-1.20, and filter it with a 200-mesh filter cloth to remove insoluble matter, which is the extract (weigh, which is the extract yield, in g).

[0064] 3. Take the extract, test the flavonoid content (in mg / g, i.e. mg flavonoids / g extract), and calculate the flavonoid yield (in mg).

[0065] Five replicates were set up and the results were averaged. The results are shown in Table 1.

[0066]

[0067] According to the results in Table 1, the highest flavonoid yield was achieved by adding the deep eutectic solvent with composition ①. Therefore, the raw material composition of the deep eutectic solvent was determined as follows: choline chloride was used as a hydrogen bond acceptor and ethylene glycol was used as a hydrogen bond donor.

[0068] 2. Optimization of raw material ratio of deep eutectic solvent

[0069] Based on the raw material composition of the low eutectic solvent determined in step 1, the raw material ratio is optimized.

[0070] 1. Take 100g of raw material, add 1000ml of extraction reagent (consisting of 180ml of water, 420ml of ethanol and 400ml of low eutectic solvent) and stir evenly, extract at 50℃ for 30 minutes, filter with qualitative filter paper, and collect the filtrate.

[0071] The raw material compositions of the deep eutectic solvent are as follows:

[0072] ①Choline chloride:ethylene glycol = 3:1 (molar ratio);

[0073] ②Choline chloride:ethylene glycol = 2:1 (molar ratio);

[0074] ③Choline chloride:ethylene glycol = 1:1 (molar ratio);

[0075] ④Choline chloride:ethylene glycol = 1:2 (molar ratio);

[0076] ⑤Choline chloride:ethylene glycol = 1:3 (molar ratio);

[0077] ⑥Choline chloride:ethylene glycol = 1:4 (molar ratio).

[0078] 2. Same as step 1, step 2.

[0079] 3. Same as step 1, step 3.

[0080] Five replicates were set up and the results were averaged. The results are shown in Table 2.

[0081]

[0082] According to the results in Table 2, the addition of the deep eutectic solvent with composition ⑤ achieved the highest flavonoid yield. Therefore, the raw material ratio of the deep eutectic solvent was determined to be: choline chloride: ethylene glycol = 1:3 (molar ratio).

[0083] 3. Optimization of the amount of deep eutectic solvent added

[0084] Based on the raw material ratio of the low eutectic solvent determined in step 2, the addition amount is optimized.

[0085] 1. Take 100 g of raw material, add N ml of extraction reagent and stir evenly, extract at 50°C for 30 minutes, filter with qualitative filter paper, and collect the filtrate.

[0086] The compositions of the extraction reagents are as follows:

[0087] ① It is composed of 180 ml of water, 420 ml of ethanol and 900 ml of deep eutectic solvent, N=1500;

[0088] ② It is composed of 180 ml of water, 420 ml of ethanol and 600 ml of a low eutectic solvent, N = 1200;

[0089] ③ It is composed of 180 ml of water, 420 ml of ethanol and 400 ml of a low eutectic solvent, N = 1000;

[0090] ④ It is composed of 180 ml of water, 420 ml of ethanol and 257 ml of low eutectic solvent, N=857;

[0091] ⑤ Consists of 180 ml of water, 420 ml of ethanol and 150 ml of low eutectic solvent, N=750.

[0092] 2. Same as step 1, step 2.

[0093] 3. Same as step 1, step 3.

[0094] Five replicates were set up and the results were averaged. The results are shown in Table 3.

[0095]

[0096] According to the results in Table 3, the best effect is achieved when the amount of the low eutectic solvent added is 600 ml.

[0097] Example 2: Optimization of further adding surfactant to the extraction reagent

[0098] The addition of surfactant is mainly to increase the saponin yield, so the saponin yield is used as the screening index in this example.

[0099] The raw material ratio of the deep eutectic solvent used in this embodiment is: choline chloride: ethylene glycol = 1:3 (molar ratio).

[0100] 1. Choice of surfactant

[0101] 1. Take 100g of raw material, add 1200ml of extraction reagent (consisting of 180ml of water, 420ml of ethanol, 600ml of low eutectic solvent and 5g of surfactant) and stir evenly, extract at 50℃ for 30 minutes, filter with qualitative filter paper, and collect the filtrate.

[0102] The surfactants are as follows:

[0103] ① Tween 80 (polysorbate 80, CAS registration number is 9005-65-6);

[0104] ②SDS (sodium dodecyl sulfate, CAS registration number is 151-21-3);

[0105] ③ Hexadecyltrimethylammonium chloride (CAS registration number is 112-02-7);

[0106] ④ Sucrose stearate SE-15 (CAS registration number is 37318-31-3);

[0107] ⑤AES (sodium lauryl polyoxyethylene ether sulfate, CAS registration number is 9004-82-4);

[0108] ⑥PEG400 (polyethylene glycol 400, CAS registration number is 25322-68-3, average molecular weight is 380-430).

[0109] 2. Take the filtrate obtained in step 1, concentrate it to a specific gravity of 1.15-1.20, and filter it with a 200-mesh filter cloth to remove insoluble matter, which is the extract (weigh, which is the extract yield, in g).

[0110] 3. Take the extract, test the saponin content (in mg / g, i.e. mg saponin / g extract), and calculate the saponin yield (in mg).

[0111] Five replicates were set up and the results were averaged. The results are shown in Table 4.

[0112]

[0113] According to the results in Table 4, the addition of AES resulted in the highest saponin yield. Therefore, AES was determined to be the best surfactant.

[0114] 2. Optimization of the amount of surfactant added

[0115] Based on the determination of the optimal surfactant in step 1, the addition amount is optimized.

[0116] 1. Take 100g of raw material, add 1200ml of extraction reagent and stir evenly, extract at 50℃ for 30 minutes, filter with qualitative filter paper and collect the filtrate.

[0117] The compositions of the extraction reagents are as follows:

[0118] ① It is composed of 180 ml of water, 420 ml of ethanol, 600 ml of deep eutectic solvent and 5 g of AES;

[0119] ② It is composed of 180 ml of water, 420 ml of ethanol, 600 ml of a deep eutectic solvent and 3 g of AES;

[0120] ③ It is composed of 180 ml of water, 420 ml of ethanol, 600 ml of deep eutectic solvent and 1 g of AES;

[0121] ④ It is composed of 180 ml of water, 420 ml of ethanol, 600 ml of a low eutectic solvent and 0.5 g of AES.

[0122] 2. Same as step 1, step 2.

[0123] 3. Same as step 1, step 3.

[0124] Five replicates were set up and the results were averaged. The results are shown in Table 5.

[0125]

[0126] According to the results in Table 5, the best effect is achieved when the amount added is 3 g.

[0127] Example 3: Verification of the effect of extracting flavonoids and saponins using the optimized extraction reagent

[0128] 1. Perform parallel extraction in five groups (five replicates per group)

[0129] Group 1: Take 100g of raw material, add 1200ml of extraction reagent and stir evenly, extract at 50℃ for 30 minutes, filter with qualitative filter paper, and collect the filtrate. The raw material ratio of the low eutectic solvent is: choline chloride: ethylene glycol = 1:3 (molar ratio). 1200ml extraction reagent: composed of 180ml water, 420ml ethanol, 600ml low eutectic solvent and 3g AES.

[0130] Group 2: Take 100g of raw material, add 600ml of extraction reagent and stir evenly, extract at 50℃ for 30 minutes, filter with qualitative filter paper, and collect the filtrate. 600ml extraction reagent: composed of 180ml water and 420ml ethanol.

[0131] Group 3: Take 100g of raw material, add 600ml of extraction reagent and stir evenly, extract at 50℃ for 30 minutes, filter with qualitative filter paper, and collect the filtrate. 600ml extraction reagent: composed of 180ml water, 420ml ethanol and 3g AES.

[0132] Group 4: Take 100g of raw material, add 1020ml of extraction reagent and stir evenly, extract at 50℃ for 30 minutes, filter with qualitative filter paper, and collect the filtrate. The raw material ratio of the low eutectic solvent is: choline chloride: ethylene glycol = 1:3 (molar ratio). 1020ml extraction reagent: composed of 420ml ethanol, 600ml low eutectic solvent and 3g AES.

[0133] Group 5: Take 100g of raw material, add 780ml of extraction reagent and stir evenly, extract at 50℃ for 30 minutes, filter with qualitative filter paper, and collect the filtrate. The raw material ratio of the low eutectic solvent is: choline chloride: ethylene glycol = 1:3 (molar ratio). 780ml extraction reagent: composed of 180ml water, 600ml low eutectic solvent and 3g AES.

[0134] 2. Take the filtrate obtained in step 1, concentrate it to a specific gravity of 1.15-1.20, and filter it with a 200-mesh filter cloth to remove insoluble matter, which is the extract (weigh, which is the extract yield, in g).

[0135] 3. Take the extract, test the flavonoid content (in mg / g, i.e. mg flavonoids / g extract) and saponin content (in mg / g, i.e. mg saponin / g extract), and calculate the flavonoid yield (in mg) and saponin yield (in mg).

[0136] The results were averaged from five replicates and are shown in Table 6.

[0137]

[0138] Comparative ratio,

[0139] 1. Take 100g of raw material, add 2000ml of low eutectic solvent solution and 40g of Tween 80, mix well and then treat with ultrasound (temperature is 60℃, power is 150W) for 20min.

[0140] The raw material ratio of the low eutectic solvent is: choline chloride: glycerol = 1:2 (molar ratio).

[0141] The deep eutectic solvent solution consists of water and the deep eutectic solvent, and the volume percentage of water is 50%.

[0142] 2. After completing step 1, centrifuge at 100,000 rpm for 5 min, collect the supernatant and filter it with a 0.45 µm filter membrane.

[0143] 3. Collect the filtrate, detect the flavonoid content (in mg / g, i.e. mg flavonoids / g extract) and saponin content (in mg / g, i.e. mg saponin / g extract), and calculate the flavonoid yield (in mg) and saponin yield (in mg).

[0144] The results were averaged from five replicates and are shown in Table 7.

[0145]

[0146] Note 1: Method for detecting the content of flavonoids (total flavonoids) in the examples:

[0147] Weigh 20 mg (accurate to 0.1 mg) of rutin standard (CAS No. 153-18-4), dissolve it in 60% ethanol aqueous solution and dilute to 100 mL to obtain a 0.20 mg / mL rutin solution. Take 20 mL of 0.20 mg / mL rutin solution, add 5 mL of 60% ethanol aqueous solution, and then dilute to 50 mL with water to obtain a 0.08 mg / mL rutin solution, which is the standard solution.

[0148] Weigh an appropriate amount of extract (accurate to 0.1 mg), dissolve it in a 50 ml volumetric flask with 25 ml of 60% ethanol, cool it, and dilute it to the scale with distilled water to obtain the sample solution.

[0149] Take two 10mL volumetric flasks, add 1mL of standard solution to volumetric flask No. 1, add 1mL of sample solution to volumetric flask No. 2, then add 0.3mL of 5% sodium nitrite aqueous solution to each, shake well and let stand for 6 minutes, then add 0.3mL of 10% aluminum nitrate aqueous solution to each, shake well and let stand for 6 minutes, then add 4mL of 1mo / L sodium hydroxide aqueous solution to each, shake well, then dilute to the volumetric flask scale with 30% ethanol aqueous solution, let stand for 15 minutes, then sample into a 1cm cuvette, and measure the absorbance at a wavelength of 510nm.

[0150] Flavonoid content in 1mL sample solution = m1*A / A0;

[0151] m1—Rutin content in 1 mL standard solution (mg); A—Absorbance of sample solution obtained by the above steps; A0—Absorbance of standard solution obtained by the above steps.

[0152] The flavonoid content in each gram of extract was calculated based on the flavonoid content in 1 mL of sample solution.

[0153] Note 2: Method for detecting the content of saponin (total saponin) in the examples:

[0154] Accurately weigh 10 mg of ginsenoside Re standard (CAS number 51542-56-4), dissolve it in methanol and dilute to 10 ml to obtain a ginsenoside Re solution with a concentration of 1 mg / ml, which is the standard solution. Take a 10 ml colorimetric tube, add 200, 400, 600, 800 or 1000 μL of the standard solution, evaporate it in a water bath, then perform the color development step, and then measure the absorbance. Make a standard curve equation with the ginsenoside Re content and absorbance in the colorimetric tube as variables. There is a linear relationship between 0-167 μg / ml and the absorbance value, with a correlation coefficient (r) of 0.999.

[0155] Weigh about 1.0g of the extract accurately, dilute with water and make up to 10ml, then load it into a chromatographic column (15cm×1cm) filled with macroporous adsorption resin, incubate for 5-10 minutes, then wash with water (flow rate 1mL / min) until the effluent is colorless, then elute with 95% ethanol aqueous solution (flow rate 1mL / min) until the effluent is colorless, collect the post-column solution of the elution operation, make up to 50ml with 95% ethanol aqueous solution, then sample 0.1mL into a 10ml colorimetric tube, evaporate to dryness in a water bath, then perform the color development step, and then measure the absorbance. Substitute the absorbance into the standard curve equation to obtain the saponin content in the colorimetric tube. Calculate the saponin content per gram of extract based on the saponin content in the colorimetric tube.

[0156] Color development step: After completing the above-mentioned evaporation in a water bath, add 0.2 ml of 5% vanillin-glacial acetic acid solution (preparation method: weigh 5 g of vanillin, dissolve it with glacial acetic acid and make up to 100 ml) into the colorimetric tube, then add 0.8 ml of perchloric acid, plug the lid tightly, keep warm in a 50°C water bath for 20 minutes, then cool to room temperature, then add 5.0 ml of glacial acetic acid, shake well and sample into a 1.0 cm colorimetric dish, and measure the absorbance at a wavelength of 560 nm.

[0157] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. An extraction reagent for enriching flavonoids and saponins from plant materials, characterized in that: The extraction reagent is composed of water, ethanol, a low eutectic solvent and a surfactant; in the extraction reagent, the ratio of water, ethanol, a low eutectic solvent and a surfactant is as follows: 160-200 ml water: 400-440 ml ethanol: 580-620 ml low eutectic solvent: 2-4 g surfactant; The raw material composition of the deep eutectic solvent is choline chloride and ethylene glycol; the molar ratio of the raw materials of the deep eutectic solvent is: choline chloride: ethylene glycol = 1:3; The surfactant is AES; The plant material is liquorice or liquorice processed product.

2. Use of the extraction reagent according to claim 1 in enriching flavonoids and saponins from plant materials; The plant material is liquorice or liquorice processed product.

3. A method for enriching flavonoids and saponins from plant materials, comprising the following steps: The plant material is extracted using the extraction reagent described in claim 1; the extraction conditions are: extraction at 40-60° C. for 20-40 minutes; After the extraction is completed, the filtrate is filtered and collected, concentrated to a specific gravity of 1.15-1.20, and then filtered to remove insoluble matter; The plant material is liquorice or liquorice processed product.

4. The method according to claim 3, characterized in that: The ratio of the plant material to the extraction reagent is: 100g plant material: 1000-1500ml extraction reagent.

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