Preparation method and application of DES four-yellow extract

Abstract

The invention provides a preparation method and application of a DES four-yellow extract, and relates to the technical field of traditional Chinese medicine extraction. The preparation method of the DES four-yellow extract comprises the following steps: S1, preparing a deep eutectic solvent: taking amino acid as HBA, taking alcohol or carboxylic acid as HBD, and heating and stirring to obtain DES; s2, preparing a DES aqueous solution: mixing the DES prepared in the S1 with water to obtain the DES aqueous solution; s3, extraction: taking cortex phellodendri, rhizoma coptidis, radix et rhizoma rhei and radix scutellariae, crushing, sieving, adding the DES aqueous solution prepared in S2, carrying out ultrasonic treatment for 30-60 minutes at 55-65 DEG C, cooling to room temperature, centrifuging, taking supernate, and drying to obtain the DES, rhizoma coptidis, radix et rhizoma rhei and radix scutellariae extract. Scutellaria baicalensis, golden cypress, rheum officinale and coptis chinensis are extracted based on a novel green and non-toxic solvent mode, and the obtained DES four-yellow extract is stable in component and good in solubility.

Description

Technical Field

[0001] The 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 an application thereof. Background Art

[0002] Acne, blackheads and pimples are collectively referred to as acne in medicine. It is a common chronic disease of the sebaceous glands. There are many kinds of acne removal methods, including various acne removal cosmetics, acne removal external medications, acne removal internal medications, and various chemical peels and physical treatment methods. However, the existing acne removal products have poor effects, strong dependence and large side effects. Chinese herbal extracts are mild and non-irritating and have received widespread attention.

[0003] Flavonoid Chinese medicines such as Scutellaria baicalensis, Phellodendron chinense, Coptis chinensis, and Rhubarb all have the effects of clearing away heat and detoxification, anti-inflammatory and antibacterial, and antioxidant, as well as inhibiting acne, removing acne, repairing and soothing the skin. According to the "Golden Chamber" by Zhang Zhongjing in the Han Dynasty, the Sanhuang Xiexin Decoction, which consists of three herbs, Coptis chinensis, Scutellaria baicalensis, and Rhubarb, is a classic prescription for purging fire and detoxification, which can treat symptoms such as internal evil fire and internal accumulation of blood stasis and heat. In addition, the Huanglian Jiedu Decoction from the "Emergency Prescriptions for the Elbow" is "3 liang of Coptis chinensis, 2 liang of Phellodendron chinense and Scutellaria baicalensis, 14 Gardenia jasminoides, 6 liters of water, decocted to 2 liters, and divided into two doses". It has the function of purging fire and detoxification, and is mainly used to treat all kinds of real heat and fire toxins. It has a significant effect in the surgical treatment of carbuncle and furuncle. According to the ancient prescription, the Sihuang extract, which is made by mixing Phellodendron chinense, Coptis chinensis, Rhubarb, and Scutellaria baicalensis in a certain ratio, can not only improve the effect of acne removal to a certain extent, but also form a unique and innovative combination of Chinese herbal medicine and cosmetics.

[0004] Chinese patent CN 106420490 A discloses a Chinese medicine composition with acne-removing effect, its preparation method and application, which 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 Radix schizonepetae, 5-10% of Pogostemon cablin, 5-10% of Taraxacum mongolicum, 5-10% of 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, which comprises: 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, in the traditional method of extracting Chinese medicine for use as cosmetic raw materials, water or organic solvents such as methanol and ethanol are usually used, such as the above-mentioned Chinese patent. Using water as the extraction solvent often leads to problems such as many water-soluble impurities and difficulty in separation and purification; using organic solvents has the disadvantages of being flammable, volatile, toxic, consuming a lot of solvents and easily leaving residues.

[0006] Deep eutectic solvent (DES) is a binary or ternary liquid eutectic mixture formed by hydrogen bond donors (HBD) such as carboxylic acids and alcohols and hydrogen bond acceptors (HBA) such as quaternary ammonium salts in a certain molar ratio. In addition to the characteristics of low vapor pressure, high thermal stability, structural adjustability and high solubility in many solutes, DES also has the advantages of low price, biodegradability, good compatibility and low toxicity. These characteristics and advantages make DES a green solvent that has attracted much attention. It is worth noting that water can form hydrogen bonds with DES components. Increased water content can reduce DES viscosity, allowing DES to better penetrate into plant tissues, enhance DES surface tension, and make extraction more efficient. In addition, studies have found that DES can be used as an auxiliary drug to improve the stability, oral bioavailability and skin permeability of some traditional Chinese medicines, thereby enhancing the therapeutic effects of these drugs.

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

[0008] In view of the above problems, the present invention provides a DES four-yellow extract and a preparation method thereof, wherein scutellaria baicalensis, phellodendron chinense, coptis chinensis and rhubarb are extracted through a ternary DES system formed by specific amino acids, alcohol / carboxylic acid and water, and the formula ratio thereof is optimized. The effective components of the four traditional Chinese medicines of the present invention have a high extraction rate content under the selected DES system. Compared with water extraction and alcohol extraction, it has the advantages of being natural and non-toxic. In addition, the DES four-yellow extract also has good antioxidant activity, solubility and anti-acne bacillus ability.

[0009] To achieve the above purpose, 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 low 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, crush and sieve, add the DES aqueous solution prepared in S2, ultrasonicate at 55-65°C for 30-60min, cool to room temperature, centrifuge, take the supernatant and dry it to obtain the 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 being transferred 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 min.

[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 h. 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 h.

[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] The present invention is a method for extracting scutellaria baicalensis, phellodendron amurense, rhubarb and coptis chinensis by a new green and non-toxic solvent method, and the obtained DES four-yellow extract has stable components, good solubility and certain anti-acne bacillus ability. The present invention can solve the problems of low bioavailability and use of harmful organic solvents faced by traditional Chinese medicine, making it of great significance in the field of Chinese medicine extraction. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

[0035] Figure 5 The DES extracts of Example 2, Example 5, Example 11 and Example 16 are shown in FIG. 1 , wherein:

[0036] (a) is a transparent and uniform DES aqueous solution of Example 2, Example 5, Example 11 and Example 16;

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

[0038] (c) is the DES extract of Example 2, Example 5, Example 11 and Example 16.

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

[0040] Figure 7 This is a comparison chart of the antioxidant effects of 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 explained below in conjunction with specific embodiments, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without making creative work all belong to 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 sources are not specifically limited. The technology and scientific terms used in the embodiments have the meanings 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 the technical field.

[0044] Embodiment 1:

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

[0046] Weigh 0.1000g of lysine and 0.2628g of citric acid in a 10mL centrifuge tube, heat and stir at 700-900rpm in a 70℃ water bath until a clear and stable DES is obtained. Weigh 0.3744g of ultrapure water in the above centrifuge tube, stir at 700-900rpm in a 60℃ water bath until a transparent and uniform DES aqueous solution is formed. Weigh 0.0370g of Scutellaria baicalensis powder in the DES aqueous solution (solid-liquid ratio 1:20) and shake evenly. Ultrasonic extraction for 40min at 60℃. After the DES solution is cooled to room temperature, centrifuge at 6000r / min for 20min, and take the supernatant, which is the corresponding Scutellaria baicalensis DES extract. Remove the water by vacuum for 12h, which is the DES Scutellaria baicalensis extract.

[0047] Embodiment 2:

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

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

[0050] Embodiment 3:

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

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

[0053] Embodiment 4:

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

[0055] Weigh 0.1002g of lysine and 0.5257g of citric acid in a 10mL centrifuge tube, heat and stir in a 70°C water bath at 700-900rpm until a clear and stable DES is obtained. Weigh 0.6252g of ultrapure water in the above centrifuge tube, stir in a 60°C water bath at 700-900rpm until a transparent and uniform DES aqueous solution is formed, then weigh 0.0311g of scutellaria baicalensis powder in the above DES aqueous solution (solid-liquid ratio 1:40), and shake evenly. The subsequent steps are the same as in Example 1. This is the DES scutellaria baicalensis extract.

[0056] Embodiment 5:

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

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

[0059] Embodiment 6:

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

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

[0062] Embodiment 7:

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

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

[0065] Embodiment 8:

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

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

[0068] Embodiment 9:

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

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

[0071] Embodiment 10:

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

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

[0074] Embodiment 11:

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

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

[0077] Embodiment 12:

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

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

[0080] Embodiment 13:

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

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

[0083] Embodiment 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 in a 10mL centrifuge tube, heat and stir in a 70°C water bath at 700-900rpm until a clear and stable DES is obtained. Weigh 0.2786g of ultrapure water in the above centrifuge tube, stir in a 60°C water bath at 700-900rpm until a transparent and uniform DES aqueous solution is formed, then weigh 0.0280g of rhubarb powder in the above DES aqueous solution (solid-liquid ratio 1:20), and shake evenly. The subsequent steps are the same as in Example 1. This is the DES rhubarb extract.

[0086] Embodiment 15:

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

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

[0089] Embodiment 16:

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

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

[0092] Embodiment 17:

[0093] DES Four Yellow Extract from Phellodendron chinense, Coptis chinensis, Rhubarb and Scutellaria baicalensis

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

[0095] Embodiment 18:

[0096] DES Four Yellow Extract from Phellodendron chinense, Coptis chinensis, Rhubarb and Scutellaria baicalensis

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

[0098] Embodiment 19:

[0099] DES Four Yellow Extract from Phellodendron chinense, Coptis chinensis, Rhubarb and Scutellaria baicalensis

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

[0101] Embodiment 20:

[0102] DES Four Yellow Extract from Phellodendron chinense, Coptis chinensis, Rhubarb and Scutellaria baicalensis

[0103] DES Phellodendron amurense, Coptis chinensis, Rheum officinale and Scutellaria baicalensis extracts were weighed separately (compound weight ratio of 1:1:1:2), and the rest of the operation was the same as Example 17.

[0104] Comparative Example 1:

[0105] A four-yellow composition, water extraction

[0106] Weigh 10g of coarse powder of four yellows in a certain proportion (Phellodendron, Coptis chinensis, Rhubarb, and Scutellaria baicalensis in a weight ratio of 1:1:1:2) into a 250mL three-necked flask, add 100mL of distilled water (liquid-to-solid ratio of 10:1mg / mL), reflux extraction for 1 hour, extract twice in total, filter with 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, put it in a -24℃ refrigerator for precooling, vacuum freeze-dry, and weigh after drying.

[0107] Comparative Example 2:

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

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

[0110] Test Example 1:

[0111] 1. Extraction rate of Scutellaria baicalensis radix of 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 substance, prepare a concentration of 0.0025 mg / mL-0.015 mg / mL, and measure the absorbance at 278 nm. According to the linear fitting, the linear equation is y=50.35x-0.00905 (y: absorbance; x: concentration mg / mL), R 2 =0.9990.

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

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

[0115] The analytical control of 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 mg / mL), R 2 =0.9997.

[0116] The supernatants of Example 5, Example 6, Example 7 and Example 8 were weighed, and the corresponding absorbance was measured at 350 nm. The corresponding extraction rate was calculated by substituting the linear equation. Figure 2 As shown, the extraction rate of Phellodendron chinense based on lysine and xylitol DES system in Example 5 is the highest under the conditions of molar ratio of 1:2, water content of 50%, and solid-liquid ratio of 1:20.

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

[0118] The analytical control of Coptis chinensis 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 mg / mL), R 2 =0.9997.

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

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

[0121] Rhubarb uses 1,8-dihydroxyanthraquinone as the analytical reference. Weigh 5 mg of 1,8-dihydroxyanthraquinone analytical reference and prepare a concentration of 0.0025 mg / mL-0.0125 mg / mL. Measure the absorbance at 252 nm. The linear equation obtained by linear fitting is y=83.8x-0.0009 (y: absorbance; x: concentration mg / mL), R 2 =0.9990.

[0122] The supernatants of Example 13, Example 14, Example 15 and Example 16 were weighed, and the corresponding absorbance was measured under a 252nm ultraviolet spectrometer, and the corresponding extraction rate was calculated by substituting into 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 Embodiment 2, Embodiment 5, Embodiment 11 and Embodiment 16, and subsequent tests are represented by the above four best embodiments.

[0124] Figure 5 The DES extracts of Example 2, Example 5, Example 11 and Example 16 are shown in FIG. 1 , wherein:

[0125] (a) is a transparent and uniform DES aqueous solution of Example 2, Example 5, Example 11 and Example 16;

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

[0127] (c) is the DES extract of Example 2, Example 5, Example 11 and Example 16.

[0128] Test Example 2:

[0129] The viscosity of the DES scutellaria baicalensis, phellodendron amurense, coptis chinensis and rheum officinale extracts prepared in Examples 2, 5, 11 and 16 was tested using a rheometer, and the temperature was raised to 60°C at 2°C / 30s in a programmed temperature ramp.

[0130] The results are as follows Figure 6 As shown in the figure, the viscosity of the DES extracts prepared in each embodiment decreases with increasing temperature, and the decreasing rate in the low temperature region is faster and gradually slows down with increasing temperature. This is because stable hydrogen bonds and van der Waals forces are formed between the DES systems, and the DES extracts prepared in each embodiment form a stable liquid DES form.

[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 and dissolve it in 60% ethanol to 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 the 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 , where the absorbance measured after mixing 2 mL of 60% anhydrous ethanol solution and 2 mL of DPPH solution is A0. The scavenging rate of DPPH is calculated by 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 reacting with DPPH.

[0135] The specific data are shown in Table 1 and Figure 7 As shown, the sample data was 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. Removal rates 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 the first column of a 96-well plate, and the medium half-dilution method was used to prepare working concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, and 0.78125 mg / mL. The concentration of Propionibacterium 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 configured, the 96-well plate was placed in a 37°C constant temperature incubator for 12 hours, and the bacterial production was observed. The results are shown in Table 2-3. Example 20 is the optimal ratio for Propionibacterium acnes, and its minimum MIC is 3.125 mg / mL. Combined with the antioxidant results, Example 20 has good antioxidant effects and excellent anti-Propionibacterium acnes ability. Therefore, the compound ratios of DES four yellow extracts used in the following experiments are all 1:1:1:2.

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

[0142]

[0143] (Note: “-” indicates that no bacteria are generated at this concentration, successfully inhibiting the growth of bacteria; “+” indicates that bacteria are generated at this concentration.)

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

[0145]

[0146]

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

[0148] As shown in Table 3, the anti-acne bacteria MICs of the DES four-yellow extract prepared in Example 20, the water-extracted four-yellow extract of Comparative Example 1, and the 60% alcohol-extracted four-yellow extract of Comparative Example 2 were 3.125 mg / mL, 25 mg / mL, and 25 mg / mL, respectively. It can be seen that the antibacterial ability of the DES four-yellow extract is significantly higher than that of the water extract and the 60% alcohol extract.

[0149] Test Example 5:

[0150] The DES four-yellow extract and four-yellow extract solution prepared in Example 20 and Comparative Example 1-2 were diluted to the same concentration, 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 no precipitation and impurities.

[0152] The DES extracts formed by the present invention use DES constructed with specific amino acids, carboxylic acids / alcohols and water to respectively extract Phellodendron amurense, Coptis chinensis, Rheum officinale and Scutellaria baicalensis. 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 low eutectic solvent: use amino acid as HBA and alcohol or carboxylic acid as HBD, heat and stir to obtain DES; S2: preparing DES aqueous solution: mixing the DES prepared in S1 with water to obtain a DES aqueous solution; S3: Extraction: Take Phellodendron amurense, Coptis chinensis, Rhubarb and Scutellaria baicalensis, crush and sieve, add the DES aqueous solution prepared in S2, ultrasonicate at 55-65°C for 30-60min, cool to room temperature, centrifuge, take the supernatant and dry it to obtain the DES four-yellow extract.

2. The preparation method according to claim 1, characterized in that: In S1, the amino acid is selected from at least one of lysine and arginine.

3. The preparation method according to claim 1, characterized in that: In S1, the alcohol is selected from at least one of xylitol and 1,3-propylene glycol.

4. The preparation method according to claim 1, characterized in that: In S1, the molar ratio of the HBA to the HBD is 1:2-4.

5. The preparation method according to claim 1, characterized in that: In S2, the mass of the water is 35%-65% of the total mass of the DES aqueous solution.

6. The preparation method according to claim 1, characterized in that: In S3, the mass ratio of Phellodendron amurense, Coptidis chinensis, Rheum officinale and Scutellaria baicalensis is 1-2:1-2:1-2:1-2.

7. The preparation method according to claim 1, characterized in that: In S3, the solid-to-liquid ratio of Phellodendron amurense, Coptis chinensis, Rheum officinale and Scutellaria baicalensis to the DES aqueous solution is 1:20-40.

8. The preparation method according to claim 1, characterized in that: In S3, The temperature of the ultrasound is 55-60°C and the time is 30-40min; The centrifugal speed is 5500-6500r / min, and the time is 15-25min; 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.

9. The DES four yellow extract prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the DES four yellow extract prepared by the preparation method according to any one of claims 1 to 8 in the preparation of anti-acne products.

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