A preparation method and application of DES four yellow extract

Abstract

The present invention provides a method for preparing a DES four-yellow extract and its application, relating to the technical field of traditional Chinese medicine extraction. The method for preparing the DES four-yellow extract comprises the following steps: S1: preparing a low eutectic solvent: using amino acids as HBA and alcohols or carboxylic acids as HBD, heating and stirring to obtain DES; S2: preparing a DES aqueous solution: mixing the DES prepared in S1 with water to obtain a DES aqueous solution; S3: extracting: taking Phellodendron chinense, Coptis chinensis, Rhubarb, and Scutellaria baicalensis, crushing and sieving, adding the DES aqueous solution prepared in S2, ultrasonicating at 55-65°C for 30-60 minutes, cooling to room temperature, centrifuging, and drying the supernatant to obtain the DES four-yellow extract. The present invention is a green, non-toxic new solvent method for extracting Scutellaria baicalensis, Phellodendron chinense, Rhubarb, and Coptis chinensis. The obtained DES four-yellow extract has stable components and good solubility.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine extraction, and in particular to a preparation method of a DES four yellow extract and application thereof. Background Art

[0002] Acne, pimples, and blackheads, collectively known as acne, is a common chronic disease of the sebaceous glands. Numerous acne treatment options exist, including cosmetics, topical and oral medications, chemical peels, and physical therapy. However, existing acne treatments are ineffective, highly addictive, and have significant side effects. Traditional Chinese medicine extracts, known for their mild and non-irritating properties, have garnered widespread attention.

[0003] Flavonoids in traditional Chinese medicines, such as Scutellaria baicalensis, Phellodendron chinense, Coptis chinensis, and Rhubarb, all possess heat-clearing and detoxifying properties, anti-inflammatory and antibacterial properties, and antioxidant properties. They also have acne-inducing and anti-acne effects, as well as skin repair and soothing properties. Zhang Zhongjing's "Golden Chamber" (Jin Kui Yao Lue) from the Han Dynasty states that the "Sanhuang Xiexin Decoction," composed of Coptis chinensis, Scutellaria baicalensis, and Rhubarb, is a classic formula for purging heat and detoxifying, treating symptoms such as internal heat and internal accumulation of stagnant heat. Furthermore, the Huanglian Jiedu Decoction, originating from the "Emergency Prescriptions for the Elbow," uses "3 liang of Coptis chinensis, 2 liang each of Phellodendron chinense and Scutellaria baicalensis, 14 Gardenia jasminoides, 6 liters of water, decoct to 2 liters, and divide into portions." It functions as a heat-purging and detoxifying agent, specifically treating all forms of heat and fire toxins, and is highly effective in the surgical treatment of carbuncles and furuncles. Based on this ancient recipe, the "Sihuang" extract, combining Phellodendron chinense, Coptis chinensis, Rhubarb, and Scutellaria baicalensis in a specific ratio, not only enhances acne-removing efficacy but also represents a unique and innovative fusion of Chinese herbal medicine and cosmetics.

[0004] Chinese patent CN 106420490 A discloses a traditional Chinese medicine composition with acne-removing efficacy, its preparation method, and application. The composition is made of the following components in weight percentage: 10-20% of Pulsatilla scabra; 10-20% of Gentiana scabra, 5-10% of Sophora flavescens, 5-10% of Herba Polygoni Multiflori, 5-10% of Patchouli, 5-10% of Taraxacum mongolicum, 5-10% of Artemisia capillaris, 5-10% of Rhubarb, 5-10% of Phellodendron amurense, 1-5% of Coptis chinensis, and 1-5% of Scutellaria baicalensis. Chinese patent CN 105232725A relates to an acne-removing formula comprising: 1.5-4.5% liquorice, 2-5% mulberry bark, 1-5% chamomile, 0.5-5% salvia miltiorrhiza, 1-5% scutellaria baicalensis, 1.5-5% phellodendron, 1-7% sophora flavescens, 0.5-3.5% honeysuckle, 1.5-6% forsythia suspensa, 1-4% coptis chinensis, 1-3% isatis indigotica, 0.5-5% rhubarb, 3-5% purslane, and a solvent.

[0005] However, traditional methods for extracting traditional Chinese medicines for use as cosmetic ingredients typically use water or organic solvents such as methanol and ethanol, as described in the aforementioned Chinese patent. Using water as the extraction solvent often leads to problems such as high levels of water-soluble impurities and difficulty in separation and purification. Organic solvents, on the other hand, have drawbacks such as flammability, volatility, toxicity, high solvent consumption, and the tendency to leave residues.

[0006] Deep eutectic solvents (DES) are binary or ternary liquid eutectic mixtures composed of hydrogen bond donors (HBDs) such as carboxylic acids and alcohols and hydrogen bond acceptors (HBAs) such as quaternary ammonium salts, combined in specific molar ratios. DES boasts low vapor pressure, high thermal stability, structural tunability, and high solubility for a wide range of solutes. Furthermore, they are inexpensive, biodegradable, compatible, and low toxicity. These characteristics and advantages have made DES a highly sought-after green solvent. Notably, water can form hydrogen bonds with DES components. Increased water content reduces DES viscosity, allowing DES to better penetrate plant tissues, increasing its surface tension and improving extraction efficiency. Furthermore, studies have shown that DES can be used as an adjuvant to improve the stability, oral bioavailability, and skin permeability of some traditional Chinese medicines, thereby enhancing their therapeutic efficacy.

[0007] Based on this, a novel acne-removing raw material based on four traditional Chinese medicine components, namely, scutellaria baicalensis, phellodendron chinense, coptis chinensis, and rhubarb, and its preparation method were developed, and its extraction method was optimized. Solving the drawbacks of traditional extraction in the existing technology is an urgent problem to be solved by researchers in this field. Summary of the Invention

[0008] To address these issues, the present invention provides a DES (Dehydrated Emulsifier) ​​Four-Huang Extract and its preparation method. This extract utilizes a ternary DES system composed of specific amino acids, alcohol / carboxylic acid, and water to extract Scutellaria baicalensis, Phellodendron chinense, Coptis chinensis, and Rhubarb, and optimizes the formulation ratio. The selected DES system achieves high extraction rates and concentrations of the active ingredients of these four traditional Chinese medicines. Compared to water and alcohol extractions, this extract is naturally non-toxic. Furthermore, the DES Four-Huang Extract exhibits excellent antioxidant activity, solubility, and anti-acne bacteria activity.

[0009] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0010] In one aspect, the present invention provides a method for preparing a DES four yellow extract, comprising the following steps:

[0011] S1: Prepare a deep eutectic solvent: use amino acid as HBA and alcohol or carboxylic acid as HBD, heat and stir to obtain DES;

[0012] S2: preparing DES aqueous solution: mixing the DES prepared in S1 with water to obtain a DES aqueous solution;

[0013] S3: Extraction: Take Phellodendron amurense, Coptis chinensis, Rhubarb and Scutellaria baicalensis, grind and sieve, add DES aqueous solution prepared in S2, ultrasonicate at 55-65℃ for 30-60min, cool to room temperature, centrifuge, take the supernatant and dry to obtain DES four-yellow extract.

[0014] Preferably, in S1, the amino acid is selected from at least one of lysine and arginine; further preferably, in S1, the amino acid is selected from lysine or arginine.

[0015] Preferably, in S1, the alcohol is selected from at least one of xylitol and 1,3-propylene glycol; further preferably, in S1, the alcohol is selected from at least one of xylitol and 1,3-propylene glycol.

[0016] Preferably, in S2, the water is selected from at least one of ultrapure water, deionized water, and distilled water; further preferably, in S2, the water is selected from ultrapure water.

[0017] Preferably, in S1, the molar ratio of the HBA to the HBD is 1:2 to 4. Further preferably, in S1, the molar ratio of the HBA to the HBD is 1:2 or 1:4.

[0018] Preferably, in S2, the mass of the water is 35%-65% of the total mass of the DES aqueous solution. Further preferably, in S2, the mass of the water is 40%-60% of the total mass of the DES aqueous solution. More preferably, in S2, the mass of the water is 40%-50% of the total mass of the DES aqueous solution.

[0019] Preferably, in S1, the heating and stirring is stirring at 65-75°C.

[0020] Preferably, in S2, the mixing is performed by stirring at 55-65°C.

[0021] Preferably, in S3, the DES aqueous solution needs to be cooled to room temperature before transfer and stored away from light.

[0022] Preferably, in S3, the mesh number of the sieve is 60-80 mesh; further preferably, in S3, the mesh number of the sieve is 70 mesh.

[0023] Preferably, in S3, the mass ratio of Phellodendron chinense, Coptis chinensis, Rhubarb and Scutellaria baicalensis is 1-2:1-2:1-2:1-2; further preferably, in S3, the mass ratio of Phellodendron chinense, Coptis chinensis, Rhubarb and Scutellaria baicalensis is 1:1:1:1-2; more preferably, in S3, the mass ratio of Phellodendron chinense, Coptis chinensis, Rhubarb and Scutellaria baicalensis is 1:1:1:2.

[0024] Preferably, in S3, the solid-liquid ratio of Phellodendron chinense, Coptis chinensis, Rhubarb and Scutellaria baicalensis to the DES aqueous solution is 1:20-40; further preferably, in S3, the solid-liquid ratio of Phellodendron chinense, Coptis chinensis, Rhubarb and Scutellaria baicalensis to the DES aqueous solution is 1:20 or 1:40.

[0025] Preferably, in S3, the temperature of the ultrasound is 55-60°C and the time is 30-40 minutes.

[0026] Preferably, in S3, the centrifugal speed is 5500-6500 r / min, and the time is 15-25 min; further preferably, in S3, the centrifugal speed is 5500-6000 r / min, and the time is 15-20 min.

[0027] Preferably, in S3, the specific parameters of the drying are: temperature of 55-65°C, vacuum degree of -0.3 to -1 MPa, and time of 10-14 hours. Further preferably, in S3, the specific parameters of the drying are: temperature of 55-60°C, vacuum degree of -0.5 to -1 MPa, and time of 10-12 hours.

[0028] The present invention provides a DES four yellow extract prepared by the above-mentioned preparation method.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] This invention uses a novel, green, non-toxic solvent method to extract Scutellaria baicalensis, Phellodendron amurense, Rhubarb, and Coptis chinensis. The resulting DES Four-Huang extract has stable components, good solubility, and certain anti-acne bacteria activity. This invention addresses the issues of low bioavailability and the use of harmful organic solvents faced by traditional Chinese medicines, making it of great significance in the field of Chinese medicine extraction. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a comparison chart of the extraction rates of Scutellaria baicalensis in Examples 1-4.

[0032] Figure 2 This is a comparison chart of the extraction rates of Phellodendron chinense in Examples 5-8.

[0033] Figure 3 This is a comparison chart of the extraction rates of Coptis chinensis from Examples 9-12.

[0034] Figure 4 This is a comparison chart of the rhubarb extraction rates of Examples 13-16.

[0035] Figure 5 Graphs of DES extracts of Examples 2, 5, 11, and 16, wherein:

[0036] (a) The transparent and uniform DES aqueous solutions of Examples 2, 5, 11 and 16;

[0037] (b) the DES extracts of Examples 2, 5, 11 and 16;

[0038] (c) The DES extracts of Examples 2, 5, 11 and 16.

[0039] Figure 6 1 and 2 are temperature-viscosity graphs of the DES extracts of Examples 2, 5, 11 and 16.

[0040] Figure 7 This is a comparison chart of the antioxidant effects of the DES four-yellow extracts prepared in Examples 17-20.

[0041] Figure 8 This is the dissolution diagram of the DES four yellow extract prepared in Example 20 and Comparative Examples 1-2. DETAILED DESCRIPTION

[0042] In order to make the technical means, creative features, purpose and effect of the present invention easy to understand, the present invention is further illustrated below in conjunction with specific embodiment, but the following embodiment is only a preferred embodiment of the present invention, not all. Based on the embodiment in the embodiment, other embodiments obtained by those skilled in the art without making creative work all fall within the protection scope of the present invention. It is worth noting that the raw materials used in the present invention are all common commercial products, and their source is not specifically limited. The technology and scientific terms used in the embodiment have the meaning commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0043] The reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0044] Example 1:

[0045] A DES scutellaria baicalensis extract (lysine:citric acid molar ratio of 1:2, water content 50%)

[0046] Weigh 0.1000g of lysine and 0.2628g of citric acid into a 10mL centrifuge tube and heat in a 70°C water bath with stirring at 700-900 rpm until a clear, stable DES solution is obtained. Weigh 0.3744g of ultrapure water into the same centrifuge tube and stir in a 60°C water bath at 700-900 rpm until a transparent, homogeneous DES aqueous solution is formed. Weigh 0.0370g of Scutellaria baicalensis powder into the DES aqueous solution (solid-to-liquid ratio 1:20) and shake thoroughly. Ultrasonic extraction is performed at 60°C for 40 minutes. After the DES solution cools to room temperature, centrifuge at 6000 rpm for 20 minutes. The supernatant is the corresponding Scutellaria baicalensis DES extract. Vacuum the solution for 12 hours to remove moisture, resulting in the DES Scutellaria baicalensis extract.

[0047] Example 2:

[0048] A DES scutellaria baicalensis extract (lysine:citric acid molar ratio of 1:4, water content 50%)

[0049] Weigh 0.1008g of lysine and 0.5251g of citric acid into a 10mL centrifuge tube. Heat and stir in a 70°C waterbath at 700-900 rpm until a clear, stable DES solution is obtained. Weigh 0.6272g of ultrapure water into the same centrifuge tube and stir in a 60°C waterbath at 700-900 rpm until a transparent, uniform DES aqueous solution is formed. Then, weigh 0.0625g of Scutellaria baicalensis powder into the DES aqueous solution (solid-to-liquid ratio 1:20) and shake until evenly combined. Subsequent steps are the same as in Example 1. This is the DES Scutellaria baicalensis extract.

[0050] Example 3:

[0051] A DES scutellaria baicalensis extract (lysine:citric acid molar ratio of 1:4, water content 60%)

[0052] Weigh 0.1003g of lysine and 0.5255g of citric acid into a 10mL centrifuge tube. Heat and stir in a 70°C waterbath at 700-900 rpm until a clear, stable DES solution is obtained. Weigh 0.9386g of ultrapure water into the same centrifuge tube and stir in a 60°C waterbath at 700-900 rpm until a transparent, uniform DES aqueous solution is formed. Then, weigh 0.0784g of Scutellaria baicalensis powder into the DES aqueous solution (solid-to-liquid ratio 1:20) and shake until evenly combined. Subsequent steps are the same as in Example 1. This is the DES Scutellaria baicalensis extract.

[0053] Example 4:

[0054] A DES scutellaria baicalensis extract (lysine:citric acid molar ratio of 1:4, water content 60%)

[0055] Weigh 0.1002g of lysine and 0.5257g of citric acid into a 10mL centrifuge tube. Heat and stir in a 70°C waterbath at 700-900 rpm until a clear, stable DES solution is obtained. Weigh 0.6252g of ultrapure water into the same centrifuge tube and stir in a 60°C waterbath at 700-900 rpm until a transparent, uniform DES aqueous solution is formed. Then, weigh 0.0311g of Scutellaria baicalensis powder and add it to the DES aqueous solution (solid-to-liquid ratio 1:40) and shake until evenly combined. Follow the same steps as in Example 1. This is the DES Scutellaria baicalensis extract.

[0056] Example 5:

[0057] A DES Phellodendron amurense extract (lysine:xylitol molar ratio of 1:2, water content 50%)

[0058] Weigh 0.1005g of lysine and 0.2081g of xylitol into a 10mL centrifuge tube. Heat and stir in a 70°C waterbath at 700-900 rpm until a clear, stable DES solution is obtained. Weigh 0.3483g of ultrapure water into the same centrifuge tube and stir in a 60°C waterbath at 700-900 rpm until a transparent, uniform DES aqueous solution is formed. Then, weigh 0.0328g of Phellodendron amurense powder into the DES aqueous solution (solid-to-liquid ratio 1:20) and shake until evenly combined. Subsequent steps are the same as in Example 1. This is the DES Phellodendron amurense extract.

[0059] Example 6:

[0060] A DES cork extract (lysine:xylitol molar ratio of 1:4, water content 50%)

[0061] Weigh 0.1005g of lysine and 0.4164g of xylitol into a 10mL centrifuge tube. Heat and stir in a 70°C waterbath at 700-900 rpm until a clear, stable DES solution is obtained. Weigh 0.5175g of ultrapure water into the same centrifuge tube and stir in a 60°C waterbath at 700-900 rpm until a transparent, uniform DES aqueous solution is formed. Then, weigh 0.0515g of Phellodendron amurense powder into the DES aqueous solution (solid-to-liquid ratio 1:20) and shake until evenly combined. Subsequent steps are the same as in Example 1. This is the DES Phellodendron amurense extract.

[0062] Example 7:

[0063] A DES Phellodendron amurense extract (lysine:xylitol molar ratio of 1:2, water content 60%)

[0064] Weigh 0.1001g of lysine and 0.2082g of xylitol into a 10mL centrifuge tube. Heat and stir in a 70°C waterbath at 700-900 rpm until a clear, stable DES solution is obtained. Weigh 0.4620g of ultrapure water into the same centrifuge tube and stir in a 60°C waterbath at 700-900 rpm until a transparent, uniform DES aqueous solution is formed. Then, weigh 0.0384g of Phellodendron amurense powder into the DES aqueous solution (solid-to-liquid ratio 1:20) and shake until evenly combined. Subsequent steps are the same as in Example 1. This is the DES Phellodendron amurense extract.

[0065] Example 8:

[0066] A DES Phellodendron amurense extract (lysine:xylitol molar ratio of 1:2, water content 50%)

[0067] Weigh 0.1002g of lysine and 0.2083g of xylitol into a 10mL centrifuge tube. Heat and stir in a 70°C waterbath at 700-900 rpm until a clear, stable DES solution is obtained. Weigh 0.3107g of ultrapure water into the same centrifuge tube and stir in a 60°C waterbath at 700-900 rpm until a transparent, uniform DES aqueous solution is formed. Then, weigh 0.0154g of Phellodendron amurense powder into the DES aqueous solution (solid-to-liquid ratio 1:40) and shake until evenly combined. Subsequent steps are the same as in Example 1. This is the DES Phellodendron amurense extract.

[0068] Example 9:

[0069] A DES coptis root extract (arginine:malic acid molar ratio of 1:2, water content 50%)

[0070] Weigh 0.1006g of arginine and 0.1540g of malic acid into a 10mL centrifuge tube. Heat and stir in a 70°C waterbath at 700-900 rpm until a clear, stable DES solution is obtained. Weigh 0.2973g of ultrapure water into the same centrifuge tube and stir in a 60°C waterbath at 700-900 rpm until a transparent, uniform DES aqueous solution is formed. Then, weigh 0.0280g of Coptis chinensis powder into the DES aqueous solution (solid-to-liquid ratio 1:20) and shake until evenly combined. Subsequent steps are the same as in Example 1. This is the DES Coptis chinensis extract.

[0071] Example 10:

[0072] A DES coptis root extract (arginine:malic acid molar ratio of 1:4, water content 50%)

[0073] Weigh 0.1004g of arginine and 0.3076g of malic acid into a 10mL centrifuge tube. Heat and stir in a 70°C waterbath at 700-900 rpm until a clear, stable DES solution is obtained. Weigh 0.4308g of ultrapure water into the same centrifuge tube and stir in a 60°C waterbath at 700-900 rpm until a transparent, uniform DES solution is formed. Then, weigh 0.0413g of Coptis chinensis powder into the DES solution (solid-to-liquid ratio 1:20) and shake until evenly combined. Subsequent steps are the same as in Example 1. This is the DES Coptis chinensis extract.

[0074] Example 11:

[0075] A DES coptis root extract (arginine:malic acid molar ratio of 1:4, water content 40%)

[0076] Weigh 0.1002g of arginine and 0.3078g of malic acid into a 10mL centrifuge tube. Heat and stir in a 70°C waterbath at 700-900 rpm until a clear, stable DES solution is obtained. Weigh 0.6115g of ultrapure water into the same centrifuge tube. Stir in a 60°C waterbath at 700-900 rpm until a transparent, uniform DES solution is formed. Then, weigh 0.0509g of Coptis chinensis powder into the DES solution (solid-to-liquid ratio 1:20) and shake until evenly combined. Subsequent steps are the same as in Example 1. This is the DES Coptis chinensis extract.

[0077] Example 12:

[0078] A DES coptis root extract (arginine:malic acid molar ratio of 1:4, water content 40%)

[0079] Weigh 0.1006g of arginine and 0.3077g of malic acid into a 10mL centrifuge tube. Heat and stir in a 70°C waterbath at 700-900 rpm until a clear, stable DES solution is obtained. Weigh 0.4096g of ultrapure water into the same centrifuge tube and stir in a 60°C waterbath at 700-900 rpm until a transparent, uniform DES solution is formed. Then, weigh 0.0204g of Coptis chinensis powder into the DES solution (solid-to-liquid ratio 1:40) and shake until evenly combined. Subsequent steps are the same as in Example 1. This is the DES Coptis chinensis extract.

[0080] Example 13:

[0081] A DES rhubarb extract (arginine:1,3-propylene glycol molar ratio of 1:2, water content 50%)

[0082] Weigh 0.1006g of arginine and 0.0889g of 1,3-propylene glycol into a 10mL centrifuge tube. Heat and stir in a 70°C water bath at 700-900 rpm until a clear, stable DES solution is obtained. Weigh 0.1922g of ultrapure water into the same centrifuge tube and stir in a 60°C water bath at 700-900 rpm until a transparent, uniform DES aqueous solution is formed. Then, weigh 0.0191g of rhubarb powder into the DES aqueous solution (solid-to-liquid ratio 1:20) and shake until evenly combined. Follow the same steps as in Example 1. This is the DES rhubarb extract.

[0083] Example 14:

[0084] A DES rhubarb extract (arginine:1,3-propylene glycol molar ratio of 1:4, water content 50%)

[0085] Weigh 0.1001g of arginine and 0.1797g of 1,3-propylene glycol into a 10mL centrifuge tube. Heat and stir in a 70°C water bath at 700-900 rpm until a clear, stable DES solution is obtained. Weigh 0.2786g of ultrapure water into the same centrifuge tube and stir in a 60°C water bath at 700-900 rpm until a transparent, uniform DES aqueous solution is formed. Then, weigh 0.0280g of rhubarb powder into the DES aqueous solution (solid-to-liquid ratio 1:20) and shake until evenly combined. Follow the same steps as in Example 1. This is the DES rhubarb extract.

[0086] Example 15:

[0087] A DES rhubarb extract (arginine:1,3-propylene glycol molar ratio of 1:2, water content 60%)

[0088] Weigh 0.1003g of arginine and 0.0881g of 1,3-propylene glycol into a 10mL centrifuge tube. Heat and stir in a 70°C water bath at 700-900 rpm until a clear, stable DES solution is obtained. Weigh 0.2817g of ultrapure water into the same centrifuge tube and stir in a 60°C water bath at 700-900 rpm until a transparent, uniform DES aqueous solution is formed. Then, weigh 0.0234g of rhubarb powder into the DES aqueous solution (solid-to-liquid ratio 1:20) and shake until evenly combined. Follow the same steps as in Example 1. This is the DES rhubarb extract.

[0089] Example 16:

[0090] A DES rhubarb extract (arginine:1,3-propylene glycol molar ratio of 1:2, water content 50%)

[0091] Weigh 0.1006g of arginine and 0.0874g of 1,3-propylene glycol into a 10mL centrifuge tube. Heat and stir in a 70°C water bath at 700-900 rpm until a clear, stable DES solution is obtained. Weigh 0.1922g of ultrapure water into the same centrifuge tube and stir in a 60°C water bath at 700-900 rpm until a transparent, uniform DES aqueous solution is formed. Then, weigh 0.0191g of rhubarb powder into the DES aqueous solution (solid-to-liquid ratio 1:40) and shake until evenly combined. Follow the same steps as in Example 1. This is the DES rhubarb extract.

[0092] Example 17:

[0093] DES Four-Huang Extract, a compound of Phellodendron chinense, Coptis chinensis, Rhubarb and Scutellaria baicalensis

[0094] Weigh 1g of DES Phellodendron amurense extract, 0.5g of DES Coptis chinensis extract, 0.5g of DES Rhubarb extract, and 0.5g of DES Scutellaria baicalensis extract into a beaker. Dissolve in 60% ethanol / water and transfer to a 25mL volumetric flask to obtain 100mg / mL (compound weight ratio of 2:1:1:1).

[0095] Example 18:

[0096] DES Four-Huang Extract, a compound of Phellodendron chinense, Coptis chinensis, Rhubarb and Scutellaria baicalensis

[0097] DES Phellodendron chinense, Coptis chinensis, Rhubarb and Scutellaria baicalensis extracts were weighed separately (compound weight ratio of 1:2:1:1), and the rest of the operations were the same as in Example 17.

[0098] Example 19:

[0099] DES Four-Huang Extract, a compound of Phellodendron chinense, Coptis chinensis, Rhubarb and Scutellaria baicalensis

[0100] DES Phellodendron amurense, Coptis chinensis, Rhubarb and Scutellaria baicalensis extracts were weighed separately (compound weight ratio of 1:1:2:1), and the rest of the operations were the same as in Example 17.

[0101] Example 20:

[0102] DES Four-Huang Extract, a compound of Phellodendron chinense, Coptis chinensis, Rhubarb and Scutellaria baicalensis

[0103] DES Phellodendron chinense, Coptis chinensis, Rhubarb and Scutellaria baicalensis extracts were weighed separately (compound weight ratio of 1:1:1:2), and the rest of the operations were the same as in Example 17.

[0104] Comparative Example 1:

[0105] A four-yellow composition, water extraction

[0106] Weigh 10g of coarse powder of Phellodendron chinense (Phellodendron amurense, Coptis chinensis, Rhubarb, and Scutellaria baicalensis) in a certain proportion (weight ratio of 1:1:1:2) into a 250mL three-necked flask. Add 100mL of distilled water (liquid-to-solid ratio of 10:1 mg / mL). Reflux extraction for 1 hour, extract twice in total, filter through gauze, and set aside the filtrate. Combine the two filtrates, centrifuge at 1000rpm for 10min, and filter. Pour the filtered sample into a petri dish, pre-cool in a -24°C refrigerator, and then freeze-dry under vacuum. After drying, weigh it.

[0107] Comparative Example 2:

[0108] A four-yellow composition, 60wt% ethanol extraction

[0109] Weigh 10g of coarse powder of four yellows (Phellodendron amurense, Coptis chinensis, Rhubarb, and Scutellaria baicalensis) in a certain proportion (weight ratio of 1:1:1:2) into a 250mL three-necked flask. Add 100mL of 60% ethanol (liquid-to-solid ratio of 10:1 mg / mL) and reflux for 1 hour. Extract twice in total, filter through gauze, and set aside the filtrate. Combine the two filtrates, centrifuge at 1000rpm for 10min, and filter. Pour the filtered sample into a petri dish, place it in a -24℃ refrigerator for pre-cooling, vacuum freeze-dry, and weigh after drying.

[0110] Test Example 1:

[0111] 1. Extraction rate of Scutellaria baicalensis radix from DES Scutellaria baicalensis radix extract of Example 1-4:

[0112] Baicalin was used as the analytical control for Scutellaria baicalensis. Weigh 5 mg of baicalin analytical control and prepare a concentration of 0.0025 mg / mL-0.015 mg / mL. Measure the absorbance at 278 nm. The linear equation obtained by linear fitting is y=50.35x-0.00905 (y: absorbance; x: concentration in mg / mL), R 2 =0.9990.

[0113] Weigh the supernatants of Example 1, Example 2, Example 3 and Example 4, measure the corresponding absorbance at 278 nm, substitute into the linear equation, and calculate the corresponding extraction rate. Figure 1 As shown, the extraction rate of Scutellaria baicalensis based on the DES system of lysine and citric acid in Example 2 was the highest under the conditions of a molar ratio of 1:4, a water content of 50%, and a solid-liquid ratio of 1:20.

[0114] 2. Extraction rate of Phellodendron chinense extract of DES Phellodendron chinense in Example 5-8:

[0115] The analytical control for Phellodendron chinense is berberine hydrochloride. Weigh 2.2 mg of berberine hydrochloride analytical control substance and prepare a concentration of 0.0022 mg / mL-0.0132 mg / mL. Measure the absorbance at 350 nm. The linear equation obtained by linear fitting is: y = 78.14x - 0.01754 (y: absorbance; x: concentration in mg / mL), R 2 =0.9997.

[0116] Weigh the supernatants of Example 5, Example 6, Example 7 and Example 8, measure the corresponding absorbance at 350nm, substitute it into the linear equation, and calculate the corresponding extraction rate. Figure 2 As shown, the extraction rate of Phellodendron chinense based on the DES system of lysine and xylitol in Example 5 was the highest under the conditions of a molar ratio of 1:2, a water content of 50%, and a solid-liquid ratio of 1:20.

[0117] 3. Extraction rate of Coptis chinensis of DES Coptis chinensis extracts in Examples 9-12:

[0118] The analytical control for Coptis chinensis is berberine hydrochloride. Weigh 2.2 mg of berberine hydrochloride analytical control and prepare a concentration of 0.0022 mg / mL-0.0132 mg / mL. Measure the absorbance at 350 nm. The linear equation obtained by linear fitting is: y = 78.14x - 0.01754 (y: absorbance; x: concentration in mg / mL), R 2 =0.9997.

[0119] Weigh the supernatants of Example 9, Example 10, Example 11 and Example 12, measure the corresponding absorbance under 350nm UV spectrometer, substitute into the linear equation, and calculate the corresponding extraction rate. Figure 3 As shown, in Example 11, the extraction rate of Coptis chinensis based on the arginine and malic acid DES system was the highest under the conditions of a molar ratio of 1:4, a water content of 40%, and a solid-liquid ratio of 1:20.

[0120] 4. Rhubarb extraction rate of DES rhubarb extracts of Examples 13-16:

[0121] Rhubarb was analyzed using 1,8-dihydroxyanthraquinone as a control. Weigh 5 mg of 1,8-dihydroxyanthraquinone analytical reference and prepare a concentration of 0.0025 mg / mL to 0.0125 mg / mL. Measure the absorbance at 252 nm. The linear equation obtained from the linear fit is y = 83.8x - 0.0009 (y: absorbance; x: concentration in mg / mL). 2 =0.9990.

[0122] The supernatants of Example 13, Example 14, Example 15 and Example 16 were weighed and the corresponding absorbance was measured at 252 nm by UV spectrometer. The corresponding extraction rate was calculated by substituting the linear equation. Figure 4 As shown, the extraction rate of rhubarb based on arginine and 1,3-propylene glycol DES system in Example 16 is the highest under the conditions of molar ratio of 1:4, water content of 50%, and solid-liquid ratio of 1:40.

[0123] Therefore, the best embodiments are Example 2, Example 5, Example 11 and Example 16, and subsequent tests are represented by the above four best embodiments.

[0124] Figure 5 Graphs of DES extracts of Examples 2, 5, 11, and 16, wherein:

[0125] (a) The transparent and uniform DES aqueous solutions of Examples 2, 5, 11 and 16;

[0126] (b) the DES extracts of Examples 2, 5, 11 and 16;

[0127] (c) The DES extracts of Examples 2, 5, 11 and 16.

[0128] Test Example 2:

[0129] The viscosity of the DES scutellaria baicalensis, phellodendron amurense, coptis chinensis and rhubarb extracts prepared in Examples 2, 5, 11 and 16 was tested using a rheometer. The viscosity was measured by increasing the temperature to 60°C at a rate of 2°C / 30s.

[0130] The results are as follows Figure 6 As shown, the viscosity of the DES extracts prepared in each example decreases with increasing temperature, with the rate of decrease being faster in the low-temperature region and gradually slowing down with increasing temperature. This is because the DES extracts prepared in each example form a stable liquid DES due to the formation of stable hydrogen bonds and van der Waals forces within the DES system.

[0131] Test Example 3:

[0132] The antioxidant activity was determined by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging method. Weigh DPPH to prepare a 0.1 mg / mL DPPH solution, store in the dark, and use it immediately. Prepare the DES four-yellow extract according to Examples 17-20, dissolve it in 60% ethanol, and prepare a 0.2 mg / mL-0.7 mg / mL stock solution. Take 2 mL of the stock solution in a 10 mL centrifuge tube, add 2 mL of DPPH solution, shake well, and react in the dark for 30 minutes at room temperature. Measure the absorbance A at a wavelength of 517 nm. i The absorbance measured after mixing 2 mL of 60% anhydrous ethanol solution and 2 mL of DPPH solution is A0. The DPPH clearance rate is calculated using the following formula.

[0133]

[0134] Where: A0 is the absorbance of the blank control group, A i is the absorbance of samples of different concentrations after reaction with DPPH.

[0135] The specific data are shown in Table 1 and Figure 7 As shown, the sample data were fitted linearly, a trend line was drawn and a linear regression equation was obtained, and the IC of each sample group was calculated according to the formula 50 : IC of Example 17 (the weight ratio of the four yellows is 2:1:1:1) 50The IC of Example 18 (the weight ratio of the four yellows is 1:2:1:1) is 0.52 mg / mL. 50 The IC of Example 19 (the weight ratio of the four yellows is 1:1:2:1) is 0.48 mg / mL. 50 is 0.50 mg / mL, and the IC of Example 20 (the weight ratio of the four yellows is 1:1:1:2) 50 According to the above results, Examples 17-20 all have good antioxidant properties, and Example 18 has more significant antioxidant properties.

[0136] Table 1. Clearance of Examples 17-20

[0137]

[0138]

[0139] Test Example 4:

[0140] The DES four-yellow extracts and four-yellow extracts prepared in Examples 17-20 and Comparative Examples 1-2 were prepared into 200 mg / mL stock solutions. 200 μL of the stock solution was added to column 1 of a 96-well plate. Using the culture medium half-dilution method, working concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, and 0.78125 mg / mL were prepared. The concentration of P. acnes was 1.5×10 5 . Among them, 100 μL of culture medium and 100 μL of bacterial solution were added to the control group, and 200 μL of culture medium was added to the blank group. After the gradient concentration was prepared, the 96-well plate was placed in a 37°C constant temperature incubator for 12 hours to observe the bacterial production. The results are shown in Table 2-3. Example 20 is the optimal ratio for P. acnes, and its minimum MIC is 3.125 mg / mL. Combined with the antioxidant results, Example 20 has good antioxidant effect and excellent anti-P. acnes ability. Therefore, the compound ratio of DES four yellow extracts used in the following experiments is 1:1:1:2.

[0141] Table 2. Comparative MIC of Examples 17-20

[0142]

[0143] (Note: "-" indicates that no bacteria were formed at this concentration, successfully inhibiting bacterial growth; "+": indicates that bacteria were formed at this concentration.)

[0144] Table 3. Comparison of MICs of water, DES, and 60% anhydrous ethanol extractions

[0145]

[0146]

[0147] (Note: “-” indicates that no bacteria were formed at this concentration, successfully inhibiting bacterial growth; “+” indicates that bacteria were formed at this concentration.)

[0148] As shown in Table 3, the antibacterial MICs of the DES four-yellow extract prepared in Example 20, the water-extracted four-yellow extract in Comparative Example 1, and the 60% alcohol-extracted four-yellow extract in Comparative Example 2 against P. acnes were 3.125 mg / mL, 25 mg / mL, and 25 mg / mL, respectively. This indicates that the DES four-yellow extract has significantly higher antibacterial activity than the water extract and the 60% alcohol extract.

[0149] Test Example 5:

[0150] The DES four-yellow extract and four-yellow extract prepared in Example 20 and Comparative Examples 1-2 were diluted to the same concentration of 2 mg / mL, and then dissolved in water and 60 wt % ethanol solution, respectively, and their dissolution was observed.

[0151] Results (such as Figure 8 ) shows that water extraction or alcohol extraction has poor solubility and contains more impurities, while DES extraction has good solubility and is free of precipitation and impurities.

[0152] The DES extracts formed by the present invention use specific amino acids, carboxylic acids / alcohols and water to extract Phellodendron amurense, Coptis chinensis, Rhubarb and Scutellaria baicalensis respectively. Compared with water extraction and alcohol extraction, the DES extracts have the advantages of being green and non-toxic, and have good antioxidant activity, solubility and anti-acne bacteria ability.

[0153] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing a DES four yellow extract, characterized in that: The following steps are involved: S1: Prepare a deep eutectic solvent: use amino acid as HBA and alcohol or carboxylic acid as HBD, heat and stir to obtain DES; When preparing the DES Scutellaria baicalensis extract, lysine was used as HBA, citric acid was used as HBD, and the molar ratio of lysine:citric acid was 1:4; When preparing the DES Phellodendron amurense extract, lysine was used as HBA, xylitol was used as HBD, and the molar ratio of lysine:xylitol was 1:2; When preparing DES Coptis chinensis extract, arginine was used as HBA, malic acid was used as HBD, and the molar ratio of arginine:malic acid was 1:4; When preparing the DES rhubarb extract, arginine was used as HBA, 1,3-propanediol was used as HBD, and the molar ratio of arginine:1,3-propanediol was 1:2; S2: preparing DES aqueous solution: mixing the DES prepared in S1 with water to obtain a DES aqueous solution; When preparing the DES scutellaria baicalensis extract, the mass of the water is 50% of the total mass of the DES aqueous solution; the solid-liquid ratio of scutellaria baicalensis to the DES aqueous solution is 1:20; When preparing the DES Phellodendron chinense extract, the mass of the water is 50% of the total mass of the DES aqueous solution; the solid-liquid ratio of Phellodendron chinense to the DES aqueous solution is 1:20; When preparing the DES coptis chinensis extract, the mass of the water is 40% of the total mass of the DES aqueous solution; the solid-liquid ratio of coptis chinensis to the DES aqueous solution is 1:20; When preparing the DES rhubarb extract, the mass of the water is 50% of the total mass of the DES aqueous solution; the solid-liquid ratio of rhubarb to the DES aqueous solution is 1:40; S3: Extraction: Phellodendron amurense, Coptis chinensis, Rhubarb and Scutellaria baicalensis are crushed and sieved, and the DES aqueous solution prepared in S2 is added respectively. The mixture is ultrasonicated at 55-65°C for 30-60 minutes, cooled to room temperature, centrifuged, and the supernatant is dried to obtain the DES four-yellow extract; the weight ratio of DES Phellodendron amurense extract, DES Coptis chinensis extract, DES Rhubarb extract and DES Scutellaria baicalensis extract in the DES four-yellow extract is 1:1:1:

2.

2. The preparation method according to claim 1, characterized in that In S3, The ultrasonic temperature is 55-60°C and the time is 30-40 minutes; The centrifugal speed is 5500-6500 r / min, and the time is 15-25 min; The specific parameters of the drying are: temperature of 55-65° C., vacuum degree of -0.3 to -1 MPa, and time of 10-14 h.

3. The DES four yellow extract prepared by the preparation method according to any one of claims 1-2.

4. Use of the DES four yellow extract prepared by the preparation method according to any one of claims 1-2 in the preparation of acne-removing products.

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