Thesium chinense extracting solution as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of extraction of medicinal chemical components from plants, and discloses a thesium chinense extracting solution as well as a preparation method and application thereof, and the preparation method comprises the following steps: drying thesium chinense at low temperature, crushing and sieving to obtain thesium chinense powder; mixing the thesium chinense powder with an extracting agent, and extracting to obtain a crude extracting solution; and centrifuging and filtering the crude extracting solution to obtain the thesium chinense extracting solution. The extracting agent is a deep eutectic solvent or a deep eutectic solvent water solution. The deep eutectic solvent composed of betaine, caffeic acid and butanediol is used, the flavonoid glycoside compounds in thesium chinense can be selectively recognized, the flavonoid glycoside compounds in thesium chinense are effectively extracted, and the yield is increased. The betaine, the caffeic acid, the butanediol and the like are biocompatible reagents, so that the thesium chinense extracting solution can be directly prepared, the processes of concentration and redissolution are avoided, the sample preparation process and the use of organic solvents are simplified, and the thesium chinense extracting solution is more environment-friendly and has higher biological activity.

Description

Technical Field

[0001] The invention belongs to the technical field of extracting medicinal chemical components from plants, and specifically relates to a herba spatholobi extract, a preparation method and an application thereof. Background Art

[0002] Thesium chinense Turcz., also known as Jiuxiancao, Jiulongcao, Jiuxiancao, etc., is widely recorded in many Chinese pharmaceutical classics such as "Yunnan Chinese Herbal Medicine", "Kunming Folk Common Herbs" and "Chinese Materia Medica". As a traditional folk medicine, Thesium chinense Turcz. has shown significant efficacy in the treatment of mastitis and pneumonia. Its phytochemical components, pharmacological mechanisms and clinical applications have always attracted close attention from researchers. Studies have revealed that its main chemical components focus on flavonoid glycosides, including kaempferol-3-O-glucorhamnoside (thesium chinense I), isorhamnetin-3-O-glucoside, kaempferol-3-O-rutinoside, kaempferol-3-glucoside (thesium chinense II), kaempferol-7-O-glucoside, apigenin-7-O-β-D-pyranoglucopyranoside and naringenin-4-O-glucoside. It is worth noting that 3-glycosyl flavonoid glycosides not only show potential in the treatment of various cardiovascular diseases, but can also be used as selective immunosuppressants, and lack the common toxic side effects of conventional drugs, so the market application prospects are broad. However, given the current scarcity of effective methods for synthesizing flavonoid glycosides, and the fact that extracting natural plants under high temperature or high pressure conditions can easily lead to glycosidic bond breakage, and the low-temperature cold extraction method has a relatively low extraction efficiency, it is particularly important to develop a simple and efficient method for extracting flavonoid glycoside compounds. Summary of the invention

[0003] In view of the above technical problems, the main purpose of the present invention is to provide a method for preparing a herbaceous wormwood extract and to explore a natural deep eutectic solvent that can selectively identify flavonoid glycoside compounds in herbaceous wormwood. Compared with organic solvent extraction, the solvent can increase the content of extracted herbaceous wormwood flavonoid glycoside compounds and has similar or stronger biological activity.

[0004] To achieve this object, the present invention adopts the following technical solutions:

[0005] In a first aspect, the present invention provides a method for preparing a Herba Lycopodii extract, comprising the following steps:

[0006] Step S1, drying the herb at low temperature, crushing and sieving, to obtain herb powder;

[0007] Step S2, mixing the herbaceous spatholobi powder with an extractant for extraction to obtain a crude extract;

[0008] Step S3, centrifuging and filtering the crude extract to obtain a Herba Lycopodii extract;

[0009] Wherein, the extractant is a deep eutectic solvent or a deep eutectic solvent aqueous solution,

[0010] The deep eutectic solvent is composed of caffeic acid, betaine and butanediol in a molar ratio of 1:(1-15):(2-30).

[0011] Deep eutectic solvents (DES) are hydrogen bond acceptors and hydrogen bond donors that are efficiently screened based on AI structure prediction and molecular binding energy. The hydrogen bond acceptors and hydrogen bond donors of natural deep eutectic solvents in the cosmetic raw material catalog are screened through AIDD (artificial intelligence drug design); then small molecule docking is used to calculate the hydrogen bond binding force of hydrogen bond acceptors and hydrogen bond donors of natural deep eutectic solvents, and then hydrogen bond acceptors and hydrogen bond donors are screened according to the size of the binding energy; finally, the screened deep eutectic solvents are combined with flavonoid glycoside compounds to obtain a natural deep eutectic solvent that selectively recognizes flavonoid glycoside compounds.

[0012] The present invention screens hydrogen bond acceptors from L-proline, betaine and acetamide, and screens hydrogen bond donors from ethylene glycol, propylene glycol, glycerol, butanediol, urea, glycolic acid, citric acid or caffeic acid. A hydrogen bond acceptor: betaine, and two hydrogen bond donors: caffeic acid and butanediol are screened out.

[0013] The method for preparing a deep eutectic solvent is as follows: after stirring a hydrogen bond acceptor betaine and a hydrogen bond donor butanediol at high temperature to form a colorless transparent solution, another hydrogen bond donor caffeic acid is added, the temperature is lowered and heating and stirring are continued to form a colorless transparent solution.

[0014] Preferably, the mass percentage of water in the deep eutectic solvent aqueous solution is 10% to 95%. The amount of water added can be controlled according to actual conditions to achieve optimal solubility and viscosity and reduce costs.

[0015] Preferably, the ratio of the herbaceous spatholobi powder to the extractant is 1 g: (10-30) mL. The specific values ​​in (10-30) can be 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, etc. Other specific values ​​within the above numerical range can be selected, and they will not be repeated here.

[0016] Preferably, in step S1, the temperature of low-temperature drying is 30°C to 60°C, for example, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, etc., and other specific values ​​within the above numerical range can be selected, which will not be repeated here. The drying time is 30min to 90min, and 30min, 40min, 50min, 60min, 70min, 80min, 90min, etc. can be selected. Other specific values ​​within the above numerical range can be selected, which will not be repeated here.

[0017] Preferably, in step S1, after drying, the herb is crushed and passed through a 20-100 mesh sieve, such as a 20, 40, 60, 80, or 100 mesh sieve.

[0018] Preferably, in step S2, the powder of the herb is mixed with an extractant for ultrasonic extraction, the ultrasonic temperature is 30°C to 50°C, the time is 30min to 90min, the ultrasonic frequency is 20kHz to 40kHz, and the ultrasonic power is 100W to 400W. The temperature can be selected from 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, 42°C, 44°C, 46°C, 48°C, 50°C, etc., and the time can be selected from 30min, 40min, 50min, 60min, 70min, 80min, 90min, etc., and other specific point values ​​within the above numerical range can be selected, which will not be repeated here one by one.

[0019] Preferably, in step S3, the centrifugal force of the centrifuge is 3000g to 8000g, and the time is 10min to 30min. The centrifugal force can be selected from 3000g, 3500g, 4000g, 4500g, 5000g, 5500g, 6000g, 6500g, 7000g, 7500g, 8000g, etc., and the time can be selected from 5min, 8min, 10min, 12min, 15min, 18min, 20min, etc., and other specific point values ​​within the above numerical range can be selected, and will not be repeated here. The filter membrane for filtration can be 0.22μm to 0.45μm, for example, 0.22μm, 0.30μm, 0.45μm, etc., and other specific point values ​​within the above numerical range can be selected, and will not be repeated here.

[0020] In a second aspect, the present invention also provides a herbaceous grass extract prepared by a method for preparing a herbaceous grass extract. The herbaceous grass extract of the present invention contains three flavonoid glycoside active compounds, namely: herbaceous grass Ⅰ, isorhamnetin-3-O-glucoside and herbaceous grass Ⅱ.

[0021] Preferably, the peak area percentage of isorhamnetin-3-O-glucoside in the Herba Lysimachiae extract is 39.4% to 71.6%.

[0022] In a third aspect, the present invention also provides the use of the aforementioned Herba Lysimachiae extract in medicines or cosmetics with antioxidant and / or anti-aging effects.

[0023] In a fourth aspect, the present invention also provides the use of the aforementioned Herba Lysimachiae extract in hyaluronidase inhibitors or elastase inhibitors.

[0024] Beneficial effects:

[0025] (1) The present invention utilizes DES to form hydrogen bonds with analytes (such as flavonoid glycosides), and the protection of glycosidic bonds under low temperature and low pressure conditions can extract the properties of flavonoid glycoside compounds. The deep eutectic solvent composed of betaine, caffeic acid and butanediol can selectively identify flavonoid glycoside compounds in Herba Lycopersicum, thereby effectively extracting flavonoid glycoside compounds in Herba Lycopersicum and improving the yield. The peak area percentage of flavonoid glycosides in the Herba Lycopersicum extract obtained is as high as 79.0%, and the peak area percentage of the monomer compound isorhamnetin-3-O-glucoside (IR3G) is 71.6%. This shows that compared with organic solvent extraction, the solvent can not only increase the content of extracted Herba Lycopersicum flavonoid glycoside compounds, but also purify the monomer compounds.

[0026] (2) Betaine, caffeic acid, and butylene glycol are biocompatible reagents and can therefore be directly prepared into a Herba Lycopodii extract, avoiding the concentration and re-dissolution process, simplifying the sample preparation process and the use of organic solvents, making it more environmentally friendly and having stronger biological activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the interaction between the caffeic acid-betaine-butanediol deep eutectic solvent and the isorhamnetin-3-O-glucoside compound.

[0028] Figure 2 This is the appearance of deep eutectic solvents prepared with different molar ratios of caffeic acid-betaine-butanediol.

[0029] Figure 3 It is the liquid phase spectrum of isorhamnetin-3-O-glucoside (IR3G) in the Herba Lycopodii extracts (S1-S12) prepared by the methods of Examples 1-12.

[0030] Figure 4 It is the liquid phase spectrum of chelidonin I (I), isorhamnetin-3-O-glucoside (IR3G) and chelidonin II (II) in the chelidonin extract (S13-S18) prepared by Examples 13-18 and the chelidonin extract (D1-D2) prepared by the method of Comparative Examples 1-2.

[0031] Figure 5 It is a graph showing the clearance rate of DPPH by the Herba Lycopodii extract prepared in Example 14 and Comparative Example 2 and the positive control vitamin C (Vc).

[0032] Figure 6 This is a graph showing the inhibition rate of the Herba Lycopodii extract prepared in Example 14 and Comparative Example 2 on hyaluronidase and elastase.

[0033] Figure 7This is a graph showing the inhibitory effect of the Herba Lycopodii extract prepared in Example 14 and Comparative Example 2 on β-gal-induced human skin fibroblast (HFF) senescence. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0035] Unless otherwise specified, the raw materials and materials used in the examples of the present invention are purchased through general commercial channels.

[0036] The source information of the relevant raw materials and materials involved in the following examples or comparative examples are as follows:

[0037] Herba Lycopodii was purchased from Jianxin Chinese Medicinal Materials Business Department, Chenggong District, Kunming, Yunnan; caffeic acid (CA), CSA No.: 331-39-5, Shanghai Haohong Biotechnology Co., Ltd.; betaine (Bet), CSA No.: 107-43-7, Shanghai Yuanye Biotechnology Co., Ltd.; 1,3-Butanediol (BDO), CSA No.: 107-88-0, Shanghai Baihaobo Biotechnology Co., Ltd.; the raw materials used in other embodiments and comparative examples are also commercially available products.

[0038] Selection Example

[0039] A hydrogen bond acceptor is an atom or group in a molecule that can accept a hydrogen bond, while a hydrogen bond donor is an atom or group in a molecule that can provide a hydrogen bond. The strength of a hydrogen bond depends on the properties and structure of the hydrogen bond acceptor and donor, including the electronegativity, spatial arrangement, and structure of the atom or group. Therefore, this selected example uses the molecular simulation and drug molecule design software system AutoDockVina to calculate the binding energy of hydrogen bond acceptors and hydrogen bond donors, and screens out deep eutectic solvents with strong hydrogen bond binding energy in the used cosmetic raw materials catalog (2021 edition).

[0040] 1) Ligand pretreatment:

[0041] Screen out natural hydrogen bond acceptors and hydrogen bond donors from the used cosmetic raw material catalog, draw them on ChemDraw, save them as sdf, and then convert them with Open Babel. After obtaining the pdb file of the ligand, open it with Autocktools to adjust the charge and select the position of the rotatable bond of the ligand. The specific operations are as follows: ligand pretreatment requires hydrogenation, setting as ligand (automatic charge distribution), detecting torsion bonds, selecting torsion bonds, exporting to PDBQT, etc. The specific operations are as follows: open the pdb file of the ligand in the same way, add full hydrogen (if it has been added, this operation is not required), calculate the charge, then click the Ligand option, click Input→Choose→Select the ligand file→OK, determine the rotatable bond of the ligand, and the operation is as follows: click Ligand, click Torsion Tree→Detect Root; Torsion Tree→Choose Torsions→Done, click ligand→Output→Save aPDBQT→Save.

[0042] 2) Molecular docking for virtual screening:

[0043] The constructed small molecule essence library was imported into AutoDockTools, and the Dock module was used to set the molecular docking receptor. The hydrogen bond receptor was selected as the receptor, and CDOCKER was selected as the docking method. The molecular docking calculation was started, and the molecular docking results are shown in Table 1. Among them, the molecular docking program CDOCKER is based on a genetic algorithm and can dock flexible ligands into receptor binding sites. The Number of Dockings in the docking program is set to 10, that is, no more than 10 docking conformations are generated. From the results in Table 1, it can be seen that the formation of a deep eutectic solvent is affected by the properties and proportions of the components and the states of the components themselves. Therefore, the deep eutectic solvent betaine-caffeic acid formed by the hydrogen bond acceptor and hydrogen bond donor with the lowest binding energy is selected.

[0044] 3) Analysis of active molecular structure:

[0045] The three active compounds of the flavonoid glycosides of Herba Lycopersici (herba Lycopersici Ⅰ, isorhamnetin-3-O-glucoside and Herba Lycopersici Ⅱ) were docked into the deep eutectic solvent again to analyze the structural characteristics of the compounds and the interaction forces between small molecules. Figure 1 The binding of the caffeic acid-betaine-butanediol deep eutectic solvent and the isorhamnetin-3-O-glucoside target compound is demonstrated.

[0046] Table 1 Preliminary screening of binding energies of deep eutectic solvents

[0047]

[0048]

[0049] according to Figure 1 As shown in Table 1, the structural conformation of the deep eutectic model of isorhamnetin-3-O-glucoside was analyzed, and it was found that the compound isorhamnetin-3-O-glucoside had hydrogen bonds and π-π interactions with betaine-caffeic acid-butanediol. This binding mode has a good spatial fit, and the binding energy of betaine and caffeic acid is the strongest, reaching -9.98 kcal / mol.

[0050] Preparation Example 1

[0051] This preparation example provides a deep eutectic solvent, which is obtained by reacting caffeic acid, betaine and butanediol, and the molar ratio of caffeic acid, betaine and butanediol is 1:1:2.

[0052] The preparation method is as follows: betaine and butanediol are stirred at 80°C for 30 minutes to form a colorless transparent solution, caffeic acid is added, and the solution is heated and stirred at 70°C for 30 minutes to obtain the product.

[0053] Preparation Example 2

[0054] This preparation example provides a deep eutectic solvent, which is obtained by reacting caffeic acid, betaine and butanediol, and the molar ratio of caffeic acid, betaine and butanediol is 1:2:4.

[0055] The preparation method is the same as that of Preparation Example 1.

[0056] Preparation Example 3

[0057] This preparation example provides a deep eutectic solvent, which is obtained by reacting caffeic acid, betaine and butanediol, and the molar ratio of caffeic acid, betaine and butanediol is 1:3:6.

[0058] The preparation method is the same as that of Preparation Example 1.

[0059] Preparation Example 4

[0060] This preparation example provides a deep eutectic solvent, which is obtained by reacting caffeic acid, betaine and butanediol, and the molar ratio of caffeic acid, betaine and butanediol is 1:5:10.

[0061] The preparation method is the same as that of Preparation Example 1.

[0062] Preparation Example 5

[0063] This preparation example provides a deep eutectic solvent, which is obtained by reacting caffeic acid, betaine and butanediol, and the molar ratio of caffeic acid, betaine and butanediol is 1:8:16.

[0064] The preparation method is the same as that of Preparation Example 1.

[0065] Preparation Example 6

[0066] This preparation example provides a deep eutectic solvent, which is obtained by reacting caffeic acid, betaine and butanediol, and the molar ratio of caffeic acid, betaine and butanediol is 1:10:20.

[0067] The preparation method is the same as that of Preparation Example 1.

[0068] Preparation Example 7

[0069] This preparation example provides a deep eutectic solvent, which is obtained by reacting caffeic acid, betaine and butanediol, and the molar ratio of caffeic acid, betaine and butanediol is 1:15:30.

[0070] The preparation method is the same as that of Preparation Example 1.

[0071] Preparation Example 8

[0072] This preparation example provides an aqueous solution of a deep eutectic solvent, which includes the deep eutectic solvent described in Preparation Example 4 and water, and the volume ratio between the two is 9:1.

[0073] The preparation method thereof is as follows: a deep eutectic solvent and water are stirred and mixed to obtain the product.

[0074] Preparation Example 9

[0075] This preparation example provides an aqueous solution of a deep eutectic solvent, which includes the deep eutectic solvent described in Preparation Example 4 and water, and the volume ratio between the two is 7:3.

[0076] The preparation method is the same as Preparation Example 8.

[0077] Preparation Example 10

[0078] This preparation example provides an aqueous solution of a deep eutectic solvent, which includes the deep eutectic solvent described in Preparation Example 4 and water, and the volume ratio between the two is 1:1.

[0079] The preparation method is the same as Preparation Example 8.

[0080] Preparation Example 11

[0081] This preparation example provides an aqueous solution of a deep eutectic solvent, which includes the deep eutectic solvent described in Preparation Example 4 and water, and the volume ratio between the two is 3:7.

[0082] The preparation method is the same as Preparation Example 8.

[0083] Preparation Example 12

[0084] This preparation example provides an aqueous solution of a deep eutectic solvent, which includes the deep eutectic solvent described in Preparation Example 4 and water, and the volume ratio between the two is 1:9.

[0085] The preparation method is the same as Preparation Example 8.

[0086] Preparation Example 13

[0087] This preparation example provides an aqueous solution of a deep eutectic solvent, which includes the deep eutectic solvent described in Preparation Example 4 and water, and the volume ratio between the two is 1:19.

[0088] The preparation method is the same as Preparation Example 8.

[0089] Example 1

[0090] This embodiment provides a method for preparing a Herba Lycopodii extract, wherein the flavonoid glycoside compounds in the Herba Lycopodii are extracted using a deep eutectic solvent. The method comprises:

[0091] (1) drying the whole herb of Herba Lycoris Radiatae at 45° C. for 60 min, crushing it to obtain Herba Lycoris Radiatae powder, and passing it through a 60-mesh sieve;

[0092] (2) The herbaceous spatholobi powder was mixed with the deep eutectic solvent of Preparation Example 1 at a ratio of 1:20 g / mL, soaked at room temperature for 12 h, and ultrasonically extracted at 40 °C for 60 min, with an ultrasonic frequency of 30 kHz and an ultrasonic power of 200 W;

[0093] (3) centrifuging at 5000 g for 20 min, filtering through a 0.45 μm polytetrafluoroethylene microfilter to obtain a Herba Lycopodii extract, and collecting the filtrate.

[0094] Embodiment 2-7

[0095] Each embodiment provides a method for preparing a Herba Lycopodii extract, using a deep eutectic solvent to extract flavonoid glycoside compounds in the Herba Lycopodii. The only difference from Example 1 is that the deep eutectic solvent of Preparation Example 1 is replaced by the deep eutectic solvents of Preparation Examples 2-7 in equal amounts in sequence, and other operations remain unchanged.

[0096] Example 8

[0097] This embodiment provides a method for preparing a herbaceous spatholobi extract, wherein the flavonoid glycoside compounds in the herbaceous spatholobi are extracted using a deep eutectic solvent aqueous solution, the method comprising:

[0098] (1) drying the whole herb of Herba Lycopodii at 30° C. for 30 min, crushing it to obtain Herba Lycopodii powder, and passing it through a 100-mesh sieve;

[0099] (2) The herbaceous spatholobi powder was mixed with the deep eutectic solvent of Preparation Example 4 at a ratio of 1:20 g / mL, soaked at room temperature for 12 h, and ultrasonically extracted at 50 °C for 90 min, with an ultrasonic frequency of 40 kHz and an ultrasonic power of 400 W;

[0100] (3) centrifuging at 3000 g for 20 min, filtering through a 0.45 μm polytetrafluoroethylene microfilter to obtain a Herba Lycopodii extract, and collecting the filtrate.

[0101] Example 9

[0102] This embodiment provides a method for preparing a herbaceous spatholobi extract, wherein the flavonoid glycoside compounds in the herbaceous spatholobi are extracted using a deep eutectic solvent aqueous solution, the method comprising:

[0103] (1) drying the whole herb of Herba Lycopodii at 60° C. for 90 min, crushing it to obtain Herba Lycopodii powder, and passing it through a 20-mesh sieve;

[0104] (2) The herbaceous spatholobi powder was mixed with the deep eutectic solvent of Preparation Example 4 at a ratio of 1:10 g / mL, soaked at room temperature for 12 h, and ultrasonically extracted at 30 ° C for 30 min, with an ultrasonic frequency of 20 kHz and an ultrasonic power of 100 W;

[0105] (3) centrifuging at 5000 g for 30 min, filtering through a 0.22 μm polytetrafluoroethylene microfilter to obtain a Herba Lycopodii extract, and collecting the filtrate.

[0106] Example 10

[0107] This embodiment provides a method for preparing a herbaceous spatholobi extract, wherein the flavonoid glycoside compounds in the herbaceous spatholobi are extracted using a deep eutectic solvent aqueous solution, the method comprising:

[0108] (1) drying the whole herb of Herba Lycoris Radiatae at 45° C. for 60 min, crushing it to obtain Herba Lycoris Radiatae powder, and passing it through a 60-mesh sieve;

[0109] (2) The herbaceous spatholobi powder was mixed with the deep eutectic solvent of Preparation Example 4 at a ratio of 1:10 g / mL, soaked at room temperature for 12 h, and ultrasonically extracted at 40 °C for 60 min, with an ultrasonic frequency of 30 kHz and an ultrasonic power of 200 W;

[0110] (3) centrifuging at 8000 g for 10 min, filtering through a 0.22 μm polytetrafluoroethylene microfilter to obtain a Herba Lycopodii extract, and collecting the filtrate.

[0111] Embodiment 11

[0112] This embodiment provides a method for preparing a herbaceous spatholobi extract, wherein the flavonoid glycoside compounds in the herbaceous spatholobi are extracted using a deep eutectic solvent aqueous solution, the method comprising:

[0113] (1) drying the whole herb of Herba Lycopodii at 30° C. for 90 min, crushing it to obtain Herba Lycopodii powder, and passing it through a 100-mesh sieve;

[0114] (2) The herbaceous spatholobi powder was mixed with the deep eutectic solvent of Preparation Example 4 at a ratio of 1:30 g / mL, soaked at room temperature for 12 h, and ultrasonically extracted at 50 °C for 90 min, with an ultrasonic frequency of 40 kHz and an ultrasonic power of 400 W;

[0115] (3) centrifuging at 3000 g for 30 min, filtering through a 0.45 μm polytetrafluoroethylene microfilter to obtain a Herba Lycopodii extract, and collecting the filtrate.

[0116] Example 12

[0117] This embodiment provides a method for preparing a herbaceous spatholobi extract, wherein the flavonoid glycoside compounds in the herbaceous spatholobi are extracted using a deep eutectic solvent aqueous solution, the method comprising:

[0118] (1) drying the whole herb of Herba Lycoris Radiatae at 60° C. for 60 min, crushing the herb to obtain Herba Lycoris Radiatae powder, and passing the powder through a 20-mesh sieve;

[0119] (2) The herbaceous spatholobi powder was mixed with the deep eutectic solvent of Preparation Example 4 at a ratio of 1:30 g / mL, soaked at room temperature for 12 h, and ultrasonically extracted at 30 ° C for 30 min, with an ultrasonic frequency of 20 kHz and an ultrasonic power of 100 W;

[0120] (3) centrifuging at 8000 g for 20 min, filtering through a 0.22 μm polytetrafluoroethylene microfilter to obtain a Herba Lycopodii extract, and collecting the filtrate.

[0121] Embodiment 13

[0122] This embodiment provides a method for preparing a herbaceous spatholobi extract, wherein the flavonoid glycoside compounds in the herbaceous spatholobi are extracted using a deep eutectic solvent aqueous solution, the method comprising:

[0123] (1) drying the whole herb of Herba Lycoris Radiatae at 45° C. for 60 min, crushing it to obtain Herba Lycoris Radiatae powder, and passing it through a 60-mesh sieve;

[0124] (2) The herbaceous spatholobi powder was mixed with the deep eutectic solvent aqueous solution of Preparation Example 8 at a ratio of 1:20 g / mL, soaked at room temperature for 12 h, and ultrasonically extracted at 40 ° C for 60 min, with an ultrasonic frequency of 30 kHz and an ultrasonic power of 200 W;

[0125] (3) centrifuging at 5000 g for 20 min, filtering through a 0.45 μm polytetrafluoroethylene microfilter to obtain a Herba Lycopodii extract, and collecting the filtrate.

[0126] Examples 14-18

[0127] Each embodiment provides a method for preparing a herbaceous wormwood extract, using a deep eutectic solvent aqueous solution to extract flavonoid glycoside compounds in the herbaceous wormwood. The only difference from Example 13 is that the deep eutectic solvent aqueous solution of Preparation Example 8 is replaced in equal amounts by the deep eutectic solvent aqueous solutions of Preparation Examples 9-13 in sequence, and other operations remain unchanged.

[0128] Comparative Example 1

[0129] This comparative example provides a method for ethanol ultrasonic-assisted extraction of Herba Lycopodii, which is based on the document "Optimization of Extraction Process of Effective Components of Herba Lycopodii and Detection of Biological Activity" (Zhang Mei et al., Journal of Chongqing University of Technology (Natural Science), 2022, 36(10): 293-304.).

[0130] Comparative Example 2

[0131] This comparative example provides a method for preparing a flavonoid glycoside extract of Herba Lycopodii using a deep eutectic solvent, the method comprising:

[0132] (1) Dry the whole herb of Herba Lycopodii at 45°C for 60 min, grind it into Herba Lycopodii powder, and pass it through a 60-mesh sieve.

[0133] (2) The herbaceous lily powder and the extractant were mixed in a ratio of 1:20 g / mL, soaked at room temperature for 12 h, and ultrasonically extracted at 40°C for 60 min, with an ultrasonic frequency of 30 kHz and an ultrasonic power of 200 W; wherein the extractant was prepared by uniformly mixing betaine, caffeic acid and butanediol in a molar ratio of 1:5:10, and then uniformly mixing the mixture with water in a volume ratio / mass ratio of 7:3.

[0134] (3) centrifuging at 5000 g for 20 min, filtering through a 0.45 μm polytetrafluoroethylene microfilter to obtain a Herba Lycopodii extract, and collecting the filtrate.

[0135] The extractants and main parameters used in the above embodiments and comparative examples are shown in Table 2 below.

[0136]

[0137]

[0138] Test Example 1

[0139] The state and pH value of the deep eutectic solvents prepared in Preparation Examples 1-7 were tested. Figure 2 And see Table 3. Figure 2 The labels DES-1 to DES-7 on the bottle body represent Preparation Examples 1 to 7 respectively.

[0140] Table 3 State and pH value of deep eutectic solvents

[0141]

[0142] Test Example 2

[0143] Analysis of Compounds in Extract of Herba Lycopodii

[0144] The extracts of Herba Lysimachiae prepared in Examples 1-18 and Comparative Examples 1-2 were subjected to UPLC analysis using an Agilent 1290 InfinityⅡ system. The mobile phases were 0.1% formic acid solution (A) and acetonitrile (B), and the gradient elution time was 0-2 min, 5% B; 2-5 min, 5%→20% B; 5-30 min, 20%→25% B; 30-35 min, 25%→90% B; 35-40 min, 90%→5% B; 40-45 min, 5% B. The column temperature was maintained at 30°C, the injection volume was 5 μL, the flow rate was 0.3 mL / min, and the wavelength was 254 nm.

[0145] The results of Examples 1-12 are shown in Figure 3 The results of Examples 13-18 and Comparative Examples 1-2 are shown in Figure 4 .in, Figure 3 The arrow extending from outside the paper to inside the paper indicates the perspective direction; Figure 3 , 4 In the above, Examples 1-18 correspond to S1-S18, Comparative Examples 1-2 correspond to D1 and D2 respectively. The highest peak of each line is isorhamnetin-3-O-glucoside, with a retention time of 14.5 min, labeled as IR3G. In addition, the peak before isorhamnetin-3-O-glucoside, which appears at 8.8 min, is lycopodiol I, labeled as I; the peak after isorhamnetin-3-O-glucoside, which appears at 17.4 min, is lycopodiol II, labeled as II. According to Figure 3 , 4 The peak area percentage (dividing the area of ​​the peak by the total area of ​​all peaks and multiplying by 100) was used to calculate the peak area percentage of flavonoid glycosides in the herbaceous spatholobi extracts prepared in Examples 1-18 and Comparative Examples 1-2. The results are shown in Table 4.

[0146] Table 4 Isorhamnetin-3-O-glucoside content extracted from spatholobi powder

[0147]

[0148]

[0149] from Figure 3As shown in Table 4, the peak area percentage of flavonoid glycosides in the Herba Lycopodii extract provided by the present invention is significantly improved, such as in Example 14, which can reach up to 79.0%, while the maximum in the comparative example is only 21.9%. The Herba Lycopodii extract obtained by implementing the extraction scheme 14 not only has the highest peak area percentage of flavonoid glycosides, but also has a purer single compound, and the peak area percentage of isorhamnetin-3-O-glucoside reaches 71.6%.

[0150] Test Example 3

[0151] Evaluation of the activity of extracts from Herba Lycopodii in scavenging DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) free radicals

[0152] Test method: Divide the 96-well plate into a sample group, a control group and a control group, with 2 replicate wells in each group; set up blank controls for both the sample group and the control group, and add DPPH alcohol solution (prepared with anhydrous ethanol to 0.1 mg / mL) in an equal volume of 1:1; Vitamin C solution (Vc) is used as the positive control sample group; the amount added to each well and the distribution of the 96-well plate are as follows (pay attention to avoid light operation, after loading the plate, react at room temperature in the dark for 30 minutes, shake well, and measure the absorbance at 517nm):

[0153] Sample: 150 μL sample solution + 150 μL DPPH alcohol solution;

[0154] Blank group: 150 μL sample solution + 150 μL anhydrous ethanol;

[0155] Control group: 150 μL of DPPH alcohol solution + 150 μL of sample solvent.

[0156] The DPPH scavenging rates of the extracts of Herba Lysimachiae obtained in Example 14 and Comparative Example 2 were measured, and a curve was drawn with vitamin C (Vc) as a positive control to calculate the half inhibitory concentration (IC) of the two extracts. 50 ) and compare them. Figure 5 As shown, the extract of Herba Lycopodii has strong antioxidant activity and can effectively scavenge free radicals. Example 14 has a stronger DPPH inhibition effect than Comparative Example 2.

[0157] Test Example 4

[0158] Determination of hyaluronidase and elastase inhibition rate

[0159] Hyaluronidase activity detection: Hyaluronidase uses sodium hyaluronate as substrate, and the activity of different extracts is determined according to the Elson-Morgan method. The Elson-Morgan method is completely based on the literature: Zhao Fengli et al. Study on the anti-allergic activity of different solvent extracts of Phellodendron chinense leaves [J]. Food Industry Science and Technology, 2009 (01): 110-112+115.

[0160] Elastase inhibition experiment: Prepare porcine pancreatic elastase stock solution (0.015625 mg / mL) in sterile water. Use 0.1mM N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide buffer as substrate. Add 70μL of 0.015625mg / mL elastase solution, 10μL of samples of different concentrations (4, 2, 1mg / mL) and positive drug (sodium sivelestat) solution to each well. After incubation at 37°C for 10 minutes, add 100μL of 100μmoL / L substrate. The total reaction volume is 230μL. At the same time, set up a 100% enzyme activity control. The blank control 130μL is replaced by an equal volume of PBS. Incubate at 37°C for 15 minutes and measure the absorbance at 405nm. See the results. Figure 6 .

[0161] As Figure 6 As shown, the in vitro inhibitory activity of hyaluronidase and elastase was evaluated using different extracts, and the results showed that the inhibitory activity of the Herba Lycopodii extract prepared in Example 14 on hyaluronidase and elastase was stronger than that of the Herba Lycopodii extract prepared in Comparative Example 2, indicating that the Herba Lycopodii extract prepared in Example 14 has stronger enzyme inhibitory activity.

[0162] Test Example 5

[0163] Anti-aging efficacy evaluation

[0164] After incubating skin fibroblasts with the extract of hyacinthus annuus and 20 mg / mL D-gal for 72 hours, the cells were washed with PBS, fixed with 4% paraformaldehyde for 15 minutes, and then washed with PBS and incubated with a mixture of SA-β-GAL staining solution at 37°C for 12 hours. The in vitro cell anti-aging model was established by staining and observing the expression of the aging marker β-gal (blue-purple after staining) in cells (Mei Zaoju et al. Components of oregano essential oil from Tengchong, Yunnan and its anti-inflammatory and anti-aging activity characteristics [J]. Chinese Food Additives, 2024, 35(04): 192-200.).

[0165] The results are as follows Figure 7 As shown, the Herba Lysimachiae extract can effectively inhibit the expression of SA-β-gal, and the inhibitory effect of the Herba Lysimachiae extract prepared in Example 14 is more obvious than that of the Herba Lysimachiae extract in Comparative Example 1 (SA-β-gal positive cells are blue).

[0166] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for preparing a Herba Lysimachiae extract, characterized in that: The following steps are involved: Step S1, drying the herb at low temperature, crushing and sieving, to obtain herb powder; Step S2, mixing the herbaceous spatholobi powder with an extractant for extraction to obtain a crude extract; Step S3, centrifuging and filtering the crude extract to obtain a Herba Lycopodii extract; Wherein, the extractant is a deep eutectic solvent or a deep eutectic solvent aqueous solution, The deep eutectic solvent is composed of caffeic acid, betaine and butanediol in a molar ratio of 1:(1-15):(2-30).

2. The method for preparing the Herba Lysimachiae extract according to claim 1, characterized in that: The mass percentage of water in the deep eutectic solvent aqueous solution is 10% to 95%.

3. The method for preparing the Herba Lysimachiae extract according to claim 1, characterized in that: The ratio of the herbaceous lily powder to the extractant is 1 g: (10-30) mL.

4. The method for preparing the Herba Lysimachiae extract according to claim 1, characterized in that: In step S1, the temperature of low-temperature drying is 30°C to 60°C, the drying time is 30min to 90min, and the mesh size of sieving is 20 to 100 meshes.

5. The method for preparing the Herba Lysimachiae extract according to claim 1, characterized in that: In step S2, the herbaceous spatholobi powder is mixed with an extractant for ultrasonic extraction, wherein the ultrasonic temperature is 30°C to 50°C, the time is 30min to 90min, the ultrasonic frequency is 20kHz to 40kHz, and the ultrasonic power is 100W to 400W.

6. The method for preparing the Herba Lysimachiae extract according to claim 1, characterized in that: In step S3, the centrifugal force of the centrifugation is 3000 g to 8000 g, the time is 10 min to 30 min, and the filter membrane of the filtration is 0.22 μm to 0.45 μm.

7. A Herba Lysimachiae extract, characterized in that: The extract is prepared by the method for preparing the herbaceous spatholobi extract according to any one of claims 1 to 6.

8. The Herba Lysimachiae extract according to claim 7, characterized in that The peak area percentage of isorhamnetin-3-O-glucoside in the herbaceous lily extract is 39.4% to 71.6%.

9. Use of the Herba Lysimachiae extract according to claim 7 in medicines or cosmetics with anti-oxidation and / or anti-aging effects.

10. Use of the Herba Lysimachiae extract according to claim 7 in hyaluronidase inhibitors or elastase inhibitors.

Images