Purification process of glabridin with low glabriol

Abstract

The invention relates to a glabridin purification process for low-glabridin, which comprises the following steps: removing glabridin in a glabridin product by using a metal organic framework material (MOFs); the purification process comprises the following steps: extracting glycyrrhiza glabra roots with ethanol or methanol, and filtering to remove medicine residues to obtain an extracting solution; concentrating the extracting solution under reduced pressure to a certain volume, and then adsorbing and removing the glabriol through a filler column filled with an MOFs material; or enabling the extract liquor in the purification process of the glabridin to flow through a filler column filled with the MOFs material to adsorb and remove the glabriol; or adding the MOFs material into other fillers in a glabridin column chromatography purification process to adsorb and remove the glabridin, so as to obtain a glabridin product with low glabridin content. According to the method disclosed by the invention, the glabriol in the glabridin is simply and effectively removed, the content of the glabriol in the prepared glabridin product is lower than 0.1%, the safety of the product is improved, and the method has a strong market competitive advantage. The adsorption material can be recycled after being heated and desorbed, and has potential application prospect and wide application value.

Description

Technical Field

[0001] The invention relates to the technical field of chemical purification, and in particular to a purification process of glabridin with low-glabricinol. Technical Background

[0002] Glabridin is a special isoflavanoid compound derived from the root and rhizome of Glycyrrhiza glabra L. It has anti-inflammatory, antioxidant, whitening, anti-atherosclerosis, neuroprotection, anti-osteoporosis, memory improvement and other pharmacological activities. In skin care, glabridin is mainly used to effectively inhibit the activity of tyrosinase and reduce the production of melanin to achieve the effect of skin freckle removal and whitening. At the same time, glabridin can play an anti-inflammatory role by inhibiting the activity of lipoxygenase and cyclooxygenase, reducing the occurrence of skin inflammation and the production of inflammatory red pigment in the skin, and preventing pigmentation caused by skin inflammation. In addition, its excellent antioxidant effect can remove oxygen free radicals produced by light and other conditions in the skin, reduce skin irritation and damage caused by free radicals, and slow down the aging of the skin. Due to its high safety and extremely strong tyrosinase activity inhibition, it is widely used as a whitening and freckle removal agent in the cosmetics industry.

[0003] Adverse reactions to licorice extract are also common. When licorice extract acts on human skin, it may cause allergic skin reactions due to unknown impurities. Nishioka K. [1] A personal case of a 43-year-old female with redness and itching on her face for more than one month was reported. Patch testing of seven ingredients present in three cosmetics revealed positive reactions to 0.5%, 1%, and 5% oil-soluble liquorice extract.

[0004] Studies have shown [2]、[3] The safety evaluation of glabridin from different manufacturers was conducted using zebrafish models and cell models. It was found that there were differences in biosafety between commercial glabridin products with similar contents. More glabrizone and glabriquinone were identified in highly toxic samples, and the experiment verified that the toxicity screening results of glabridin from different manufacturers on zebrafish embryos were basically consistent with the screening results of in vitro cell models. Further experimental verification showed that glabriquinone aggravated the toxicity of glabridin to zebrafish embryos, while glabriquinone had no toxic effect on zebrafish embryos at the maximum solubility concentration in 1% DMSO. Therefore, glabriquinone is considered to be one of the main key risk impurities that increase the acute toxicity of glabridin.

[0005] The group standard for cosmetic raw material Glabridin (T / HPCIA-010-2024) issued by the Huangpu Cosmetics Industry Association of Guangzhou Development Zone requires that the content of glabridinol in glabridin used as a cosmetic raw material be ≤1.0%.

[0006] The content of glabridin in the root of Glycyrrhiza glabra is about 0.1~0.15%, and the content of glabridin is 0.1~0.2%; the two have similar polarity and can be extracted simultaneously.

[0007] There is no technology specifically for removing the impurity glabridin in the existing glabridin production process. The traditional technologies such as extraction, column separation, dissolution and crystallization can purify glabridin and remove related impurities, but there is a possibility of introducing toxic and harmful ingredients such as methanol, dichloromethane, chloroform, acetone, toluene, etc. that are banned in cosmetics. At the same time, the complex production process will greatly prolong the process time, reduce the yield, and increase the production cost.

[0008] Therefore, it is necessary to develop a targeted process for removing glycyrrhizin.

[0009] There are significant structural differences between glabricinol and glabridin, in that glabricinol has two exocyclic double bonds, while glabridin has none.

[0010] Metal organic frameworks (MOFs) are a class of organic-inorganic hybrid materials that have attracted much attention. They have highly regular spatial network structures, large specific surface area and pore volume, good chemical stability, and modifiable ligands. They have good application prospects in industrial catalysis, selective adsorption, and gas separation. Some transition metal elements can form π bonds with olefin double bonds, and the coordination effect on the double bonds outside the ring is stronger than that on the double bonds inside the ring. Summary of the invention

[0011] In view of the above problems, the present invention provides a method for removing glabridin from glabridin, thereby obtaining a glabridin extract with low glabridin, improving the biosafety of the glabridin product and reducing the purification cost.

[0012] The technical problem to be solved by the present invention is achieved by adopting the following technical solution.

[0013] A process for purifying glabridin with low glabridin phenol, using a metal organic framework material to remove glabridin phenol from the glabridin product; the metal organic framework material comprises MIL-101 (Fe), ZIF-7 (Zn), ZIF-9 (Co), ZIF-67 (Co), C8H8N4Cu.F6Si (SIFSIX-3-Cu) transition metal element coordination materials; the purification process steps are as follows: (1) Slice or crush the root of Glycyrrhiza glabra and add 5-20 times the amount of ethanol or methanol with a concentration of 50-95%, extract at room temperature or with heating and stirring or with liquid circulation, and filter to remove the residue to obtain the Glycyrrhiza glabra extract; (2) The root extract of Glycyrrhiza glabra is concentrated under reduced pressure to a certain volume and then passed through a filler column separately filled with a metal organic framework material to adsorb and remove glycyrrhizin; the effluent is concentrated and dried to obtain a low-glycyrrhizin glabridin extract.

[0014] Step (2) further comprises: after the root extract of Glycyrrhiza glabra is concentrated under reduced pressure and extracted, the extract is passed through a filler column separately filled with a metal organic framework material to adsorb and remove glabridin; the specific method is: the extract of Glycyrrhiza glabra is concentrated under reduced pressure to 1 / 5 to 1 / 10 of the original volume, an equal amount of petroleum ether and ethyl acetate in a ratio of 5:1 to 1:5 is used for extraction, the organic layer is separated, and the organic layer is passed through a previously prepared filler column filled with a metal organic framework material, and the effluent is concentrated and dried to obtain a low-glabridin glabridin extract.

[0015] Step (2) further comprises: concentrating the licorice extract to obtain a paste and then performing chromatography separation, mixing the chromatography filler with the metal frame material; separating according to the corresponding column separation process, intercepting the glabridin segment chromatography liquid, and concentrating and drying to obtain the low-glabricinol glabridin extract.

[0016] During the column chromatography purification process, the chromatography filler is mixed with the metal frame material, and the filler is polyamide for chromatography, silica gel for chromatography or macroporous resin.

[0017] The mixing ratio of the metal frame material to the chromatography filler during column chromatography purification is 1:100~1:1.

[0018] The metal organic framework material is reused after being regenerated.

[0019] The present invention is a process for separating glabridin from low-glabridin using metal organic framework materials (MOFs-Metal organic Framework). The main purpose is to control and remove the glabridin component with certain cytotoxicity brought into glabridin, so as to improve the safety of the product and reduce or avoid the potential skin allergy problems of terminal cosmetics caused by glabridin. The main feature of the present invention is that the extract containing glabridin is adsorbed and removed by using the strong coordination between the transition metal group elements and the two distal olefins in glabridin by using the metal organic framework material as a filler or adding the metal framework material to other chromatography fillers. The glabridin in the glabridin extract is removed. The method adopted by the present invention can simply and effectively remove the glabridin from glabridin. The glabridin content in the glabridin product prepared by the present invention is less than 0.1%, which is suitable for the demand for high-safety glabridin in the skin care product market and has a strong market competitive advantage. The adsorption material can be recycled after heating and decomposition, which is convenient for large-scale use and has potential application prospects and wide application value. DETAILED DESCRIPTION

[0020] The present invention is further described below in conjunction with specific examples, but the present invention is not limited thereto. Those skilled in the art should recognize that the present invention is not limited to the above embodiments, and any changes and modifications to the present invention fall within the protection scope of the present invention.

[0021] The experimental methods described in the following examples are conventional methods unless otherwise specified. The experimental materials and reagents described can be obtained from commercial sources unless otherwise specified. In the following examples and comparative examples, the raw material is Glycyrrhiza glabra, produced in Kashgar, Xinjiang, and purchased by itself. Metal organic framework materials: MIL-101 (Fe), ZIF-7 (Zn), ZIF-9 (Co), ZIF-67 (Co) were purchased from Beijing Beike New Materials Technology Co., Ltd.; C8H8N4Cu.F6Si (SIFSIX-3-Cu) was purchased from Shanghai Kaishu Chemical Technology Co., Ltd.

[0022] Example 1 1 kg of Glycyrrhiza glabra root was crushed and put into a 20L glass kettle, and 10 times the amount of 75% ethanol was added for reflux extraction for 2h, filtered, and the extraction residue was washed with 2L75% ethanol; the residue of the first extraction was added with 10L75% ethanol for secondary extraction, the secondary extraction time was 1h, filtered, and the extraction residue was washed with 2L75% ethanol. The filtrate and washing liquid were combined to obtain the Glycyrrhiza glabra extract, with a volume of about 20L. The extract was concentrated to 2L and loaded onto a pre-prepared 5cm diameter SIFSIX-3-Cu packed column with a column volume of 500ml, a flow rate of 0.2BV / h, and washed with 50% ethanol 1BV, with a washing flow rate of 1BV / h. The combined liquid was concentrated under reduced pressure and evaporated to dryness to obtain 186.8g of glabridin extract. The measurement results showed that the content of glabridin was 1.3% and the content of glabridin was 0.09%.

[0023] Example 2 The same as in Example 1, 20L of extract was obtained, which was concentrated under reduced pressure to 2L, and extracted with equal amounts of petroleum ether and ethyl acetate in a ratio of 2:3, and the organic layer was separated and loaded onto a pre-prepared 5cm diameter packed column filled with MIL-101 (Fe), with a column volume of 500ml and a flow rate of 2BV / h. The packed column was washed with 2BV of 2:3 petroleum ether and ethyl acetate, and the liquid part was combined to obtain the glabridin effluent, which was monitored by TLC during the elution process. Concentrated and evaporated under reduced pressure to obtain 34.4g of glabridin extract, and the measurement results showed that the glabridin content was 7.4% and the glabricinol content was 0.08%.

[0024] Example 3 The same as in Example 1, 20L of extract was obtained, and it was concentrated under reduced pressure to 2L, and loaded onto a 10cm diameter chromatography column prepared in advance, which was filled with D101 macroporous resin and ZIF-7 (Zn) 100:1 (m:m) mixed evenly, and the column volume was 5L. It was gradient eluted with 30% ethanol 2BV and 70% ethanol 2BV, and the flow rate was controlled to 1BV / h to obtain the glabridin eluate. The elution process was monitored by TLC, and the glabridin-containing part was collected. The glabridin eluate was concentrated under reduced pressure to obtain 8.5g of glabridin extract, and the measurement results showed that the glabridin content was 24.2% and the glabricinol content was 0.06%.

[0025] Example 4 The same as in Example 1, 20L of extract was obtained, and the extract was concentrated under reduced pressure to obtain 202.8g of paste, 500ml of ethyl acetate was added to dissolve, 500g of chromatographic silica gel was taken, the sample was adsorbed, the ethyl acetate was evaporated under reduced pressure, and the sample was dry-loaded to a pre-prepared 10cm diameter chromatography column filled with silica gel and ZIF-67 (Co) 50:1 (m:m) mixed evenly, the column volume was 5L, and the mixture was eluted with a mixed solvent of petroleum ether-ethyl acetate 3:1, and the flow rate was controlled to 1BV / h to obtain the glabridin effluent, and the elution process was monitored by TLC. The glabridin eluate was concentrated under reduced pressure to obtain 4.5g of glabridin extract, and the measurement results showed that the glabridin content was 45.1% and the glabridin phenol was 0.05%.

[0026] Example 5 The same as in Example 1, 20L of extract was obtained, and the extract was concentrated under reduced pressure to obtain 208.6g of paste, which was dissolved in 500ml of methanol, and 500g of 100-mesh polyamide was taken for chromatography, mixed with samples for adsorption, and the methanol was evaporated under reduced pressure, and the sample was loaded onto a pre-prepared 10cm diameter chromatography column filled with polyamide macroporous resin and ZIF-9 (Co) 1:1 (m:m) mixed evenly, with a column volume of 1L, a flow rate of 1BV / h, and eluted with petroleum ether-ethyl acetate 4:1 to obtain glabridin effluent, and the elution process was monitored by TLC. The glabridin eluate was concentrated under reduced pressure to obtain 5.1g of glabridin extract, and the measurement results showed that the glabridin content was 40.3% and the glabricinol content was 0.02%.

[0027] Example 6 The same as in Example 1, 20L of extract was obtained, and it was concentrated under reduced pressure to 2L, and loaded onto a 10cm diameter column prepared in advance, which was filled with 5L of D101 macroporous resin and ZIF-9 (Co) 100:1 (m:m), and gradient eluted with 30% ethanol 2BV, 70% ethanol 2BV, and the flow rate was controlled to be 1BV / h, and the glabridin effluent was obtained. The elution process was monitored by TLC, and the part containing glabridin was collected. The primary purified product 30g was obtained by concentration and evaporation under reduced pressure, and 50ml of methanol was added to dissolve it, 60g of 100 mesh polyamide was taken for chromatography, and the sample was adsorbed, and the methanol was evaporated under reduced pressure, and the sample was loaded onto a 10cm diameter column prepared in advance, which was filled with polyamide and SIFSIX-3-Cu 2:1 (m:m) mixed evenly, and the column volume was 5L, and the flow rate was controlled to be 1BV / h, and it was eluted with petroleum ether-ethyl acetate 6:1 to obtain the glabridin effluent, and the elution process was monitored by TLC. The glabridin eluate was concentrated under reduced pressure to obtain 1.6 g of glabridin extract. The measurement results showed that the content of glabridin was 95.6% and the content of glabridin was 0.01%.

[0028] Example 7, the regeneration method of the metal organic framework material MOFs of the present invention For MOFs in Example 1-2, use 90-95% ethanol, heat to 60-70°C, rinse the packed column, 5-7BV, flow rate 1BV / h, control the rinse liquid, place the packed column in a vacuum drying oven, dry under reduced pressure at 70-80°C, and reuse it.

[0029] For MOFs in Example 3, use 90-95% ethanol, heat to 60-70°C, flush the packed column, 5-7BV, flow rate 1BV / h, and then use 3BV of pure water to push out the ethanol and reuse it.

[0030] For MOFs in Example 4-6: Use ethyl acetate, heat to 60-70°C, flush the packed column, 5-7BV, flow rate 1BV / h, then use petroleum ether-ethyl acetate at the corresponding elution ratio of 3BV and reuse.

[0031] Comparative Example 1 The extract was obtained in the same manner as in Example 1, and concentrated and evaporated to dryness under reduced pressure to obtain 206.2 g of glabridin extract. The measurement results showed that the content of glabridin was 1.2% and the content of glabricinol was 0.82%.

[0032] Comparative Example 2 The same as Example 1, to obtain 20L of extract, under reduced pressure concentrated to 2L, with equal amounts of petroleum ether and ethyl acetate 2:3 for extraction, separated the organic layer, the organic layer was concentrated and evaporated to dryness to obtain 33.8g of glabridin extract, the measurement results showed that the glabridin content was 7.2%, glabriquinone 4.3%.

[0033] Comparative Example 3 The chromatography filler D101 macroporous resin was not added with a metal frame material, and the remaining steps were the same as those in Example 3, to obtain 8.9 g of glabridin extract. The measurement results showed that the content of glabridin was 23.6% and the content of glabricinol was 11.8%.

[0034] Comparative Example 4 No metal frame material was added to the chromatography filler silica gel, and the remaining steps were the same as those in Example 4. 4.6 g of glabridin extract was prepared. The measurement results showed that the glabridin content was 43.6% and the glabriquinone content was 7.3%.

[0035] Comparative Example 5 No metal frame material was added to the chromatographic filler polyamide, and the remaining steps were the same as those in Example 5. The glycyrrhizin extract was 5.7 g. The measurement results showed that the content of glabridin was 38.5% and that of glabriquinone was 5.6%.

[0036] Comparative Example 6 No metal frame material was added to the two-step purification chromatography filler, and the remaining steps were the same as those in Example 6. 1.7 g of glycyrrhizin extract was obtained. The measurement results showed that the content of glabridin was 90.3% and that of glabriquinone was 0.26%.

[0037] Experimental Results Comparative Examples 1 to 5 show that glabricinol can be extracted and enriched simultaneously with glabridin. Comparative Example 6 shows that after one macroporous resin column and one silica gel column separation and purification, the glabricinol content can be reduced while increasing the glabridin content, but the removal effect of adding the metal framework material in Example 6 is more obvious. Through the glabricinol purification process test of low glabricinol in Examples 1 to 6 and Comparative Examples 1 to 6, and taking the glabricinol content and the residue of glabricinol in the extract as evaluation indicators, the test results are shown in Table 1, indicating that the metal organic framework material does have the effect of removing glabricinol and can improve the safety of glabricinol used in cosmetics.

[0038] Table 1 Test results of glabridin extracts obtained in various examples and comparative examples The detection method of glabridin and glabricinol in the present invention 1 Chromatographic conditions Octadecylsilane bonded silica gel was used as filler; acetic acid (2%, v / v): acetonitrile (2:3, v / v) was used as mobile phase; detection wavelength was 282 nm; column: 4.6 mm × 25 cm; column temperature: 40 °C; flow rate: 1.000 mL / min.

[0039] 2. Preparation of reference solution Take 20 mg of glabratadine reference substance, weigh it accurately, put it in a 100 mL volumetric flask, add 95% ethanol to dissolve and dilute to the mark, shake well, and set aside. Take 10 mg of glabratadine reference substance, weigh it accurately, put it in a 100 mL volumetric flask, add 95% ethanol to dissolve and dilute to the mark, shake well, and set aside.

[0040] 3 Preparation of test solution Preparation of the test solution of glabridin extract: Take 20 mg of the test sample, accurately weigh it, place it in a 100 mL volumetric flask, add 95% ethanol to dissolve and dilute to the scale, shake well, filter, discard the initial filtrate, and take the subsequent filtrate as the test solution.

[0041] 4. Determination method Accurately measure 20 μL of the reference solution and the test solution, respectively, and inject them into the liquid chromatograph to calculate the contents of glycyrrhizin and glycyrrhizinol based on their respective peak areas.

[0042] The sources of the standard products for this test are: Shanghai Yuanye Biotechnology's licorice product number B20474, CAS#59870-68-7, batch number C12A10M94485, HPLC≥98%; licorice phenol product number B28338, CAS#59870-65-4, batch number M10GB141122, HPLC≥98%.

[0043] References: [1] Nishioka K, Seguchi T. Contact allergy due to oil-solublelicoriceextracts in cosmetic products[J]. Contact Dermatitis, 1999, 40(1): 56. [2]AQG,ASW,BWL,et al.Glabrol impurity exacerbates glabridintoxicity inzebrafish embryos by increasing myofibril disorganization[J]. 2021. [3] Wu Shaojuan. Study on the biosafety evaluation and toxicity mechanism of glabridin[D]. Guangdong University of Technology, 2022. DOI: 10.27029 / d.cnki.ggdgu.2022.001787.

Claims

1. A process for purifying glabridin from low-light licorice phenol, characterized in that: Metal organic framework materials are used to remove glabridin from glabridin products; the metal organic framework materials include MIL-101 (Fe), ZIF-7 (Zn), ZIF-9 (Co), ZIF-67 (Co), C8H8N4Cu.F6Si (SIFSIX-3-Cu) transition metal element coordination materials; the purification process steps are as follows: Slice or crush the root of Glycyrrhiza glabra and add 5-20 times the amount of 50-95% concentration of ethanol or methanol, extract at room temperature or with heating and stirring or liquid circulation, and filter to remove the residue to obtain the Glycyrrhiza glabra extract; The root extract of Glycyrrhiza glabra is concentrated under reduced pressure to a certain volume and then flows through a filler column separately loaded with metal organic framework materials to adsorb and remove glabridin; the effluent is concentrated and dried to obtain a low-glabridin glabridin extract.

2. The process for purifying glabridin from low-light licorice phenol according to claim 1, characterized in that: Step (2): after the root extract of Glycyrrhiza glabra is concentrated under reduced pressure and extracted, the extract is passed through a filler column separately filled with a metal organic framework material to adsorb and remove glabridin. The specific method is: the extract of Glycyrrhiza glabra is concentrated under reduced pressure to 1 / 5 to 1 / 10 of the original volume, an equal amount of petroleum ether and ethyl acetate in a ratio of 5:1 to 1:5 is used for extraction, an organic layer is separated, and the organic layer is passed through a previously prepared filler column filled with a metal organic framework material, and the effluent is concentrated and dried to obtain a low-glabridin glabridin extract.

3. The process for purifying glabridin from low-light licorice phenol according to claim 1, characterized in that: Step (2): after the licorice extract is concentrated to a paste, it is subjected to chromatography separation, and the chromatography filler is mixed with the metal frame material; separation is carried out according to the corresponding column separation process, the glabridin segment chromatography liquid is intercepted, and the glabridin segment chromatography liquid is concentrated and dried to obtain the low-glabridin phenol glabridin extract.

4. A process for purifying glabridin from low-light licorice phenol according to claim 3, characterized in that: During the column chromatography purification process, the chromatography filler is mixed with the metal frame material, and the filler is polyamide for chromatography, silica gel for chromatography or macroporous resin.

5. A process for purifying glabridin from low-light licorice phenol according to claim 4, characterized in that: The mixing ratio of the metal frame material to the chromatography filler during column chromatography purification is 1:100~1:

1.

6. A process for purifying glabridin from low-light licorice phenol according to claim 5, characterized in that: The metal organic framework material is reused after being regenerated.

Images