A method for preparing gentian extract using deep eutectic solvent and its application

Abstract

The present invention belongs to the field of plant extracts, and relates to a method and application of preparing a gentian extract using a deep eutectic solvent, the method comprising the following steps: draining the surface moisture of the washed gentian, drying it in a drying oven to constant weight, and then grinding it into powder with a high-energy nano ball mill to obtain gentian powder; adding the gentian powder to a deep eutectic solvent and mixing it with water, intermittently ultrasonicating it, and cooling it to obtain a crude extract; centrifuging the crude extract to obtain a gentiopicroside extract; the deep eutectic solvent includes a hydrogen bond donor and a hydrogen bond acceptor; the hydrogen bond donor is betaine, and the hydrogen bond acceptor is 1,3-propylene glycol or glycerol. The method of the present invention has a green, safe and environmentally friendly extraction process, a simple preparation process, low cost, simple extraction equipment operation, no special requirements for samples, higher extraction efficiency than conventional methods, and the obtained product is natural and non-toxic, avoiding the use of organic solvents, long extraction process, and the disadvantages of easy deterioration of extracts.

Description

Technical Field

[0001] The invention belongs to the field of plant extracts and relates to a method for preparing a gentian extract by using a deep eutectic solvent and an application thereof. Background Art

[0002] Gentiana scabra Bunge is a perennial herbaceous plant of the Gentianaceae family and the Gentiana genus. Gentiopicroside is the main active medicinal ingredient in gentian, and it tastes very bitter. It belongs to the class of secoiridoid glycosides. Modern pharmacological studies have shown that gentiopicroside has significant anti-inflammatory, antioxidant, anti-tumor, liver-protecting, stomachic and choleretic effects, and is mainly used to treat liver and gallbladder diseases, chronic bronchitis, respiratory tract infections and other diseases.

[0003] In the related art, a preparation method and application of gentian extract gentianopicrin (application number: 202111436362.1) is to crush the rhizome of gentianopicrin, add pure water for extraction, filter the extract, concentrate it, and obtain a concentrated solution; filter the concentrated solution, pour the filtrate into a macroporous resin, add water for elution, discharge the eluate, and then pour ethanol for elution, collect the eluate, and recover the ethanol to obtain an extract; add silica gel to the extract, stir it evenly, put it on a silica gel column, elute it with a mixed mobile phase of ethyl acetate, ethanol and water, elute until there is no gentianopicrin in the thin layer detection, collect the eluate, concentrate it to 1 / 3 to 1 / 2 of the volume of the eluate, let it stand for crystallization, filter it, wash the filter cake with ethanol, and dry it to obtain gentianopicrin. This process does not identify the content and yield of gentianopicrin, and the separation method using silica gel column chromatography not only requires the use of a large amount of organic solvent (ethyl acetate), but also has low production capacity.

[0004] Extraction method of gentiopicroside (application number: 202311056150.X) discloses a method for extracting gentiopicroside, which comprises soaking fresh gentian with a pretreatment solution, draining the surface water, freezing, and then drying the pretreated gentian, adding a hydrophilic organic solvent and mixing, ultrasonic extraction, filtering, and reducing pressure to recover the hydrophilic organic solvent, concentrating the filtrate to obtain a crude extract, adding a hydrophilic organic solvent to the crude extract, stirring and re-dissolving, and then refrigerating and placing, filtering and recrystallizing to obtain gentiopicroside crude crystals, adding the gentiopicroside crude crystals to the hydrophilic organic solvent until the solution is saturated, and then pouring into the pretreated macroporous adsorption resin, adding water for elution, and then pouring into the hydrophilic organic solvent for elution, collecting the eluate, and reducing pressure to recover the hydrophilic organic solvent, and concentrating the eluate and drying to obtain gentiopicroside. This process uses a large amount of hydrophilic organic solvents in the extraction process, which does not conform to the principles of green chemistry.

[0005] In the prior art, organic solvents such as methanol, ethyl acetate, ethanol and water are often used to extract gentiopicroside from gentian, but the amount of reagents used is large and the use of volatile toxic reagents poses great safety and health risks. Therefore, it is of great practical significance to develop an efficient, rapid and low-cost method for extracting gentiopicroside. In recent years, natural deep eutectic solvent (NDES) has been widely used in the extraction of plant active ingredients as a green and efficient extractant. At present, there is no relevant report on the preparation of gentian extract picroside from gentian, which has good application value and development prospects. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides a method and application of preparing gentian extract using deep eutectic solvent. The method provided by the present invention uses natural deep eutectic solvent as extraction solvent and intermittent ultrasound-assisted heating extraction, which has the advantages of being green and environmentally friendly, simple preparation process, high extraction efficiency, low cost, and safe and non-toxic products.

[0007] The technical solution provided by the present invention is as follows:

[0008] A method for preparing a gentian extract using a deep eutectic solvent comprises the following steps: draining surface water from a cleaned gentian, drying it in a drying oven to constant weight, and then grinding it into powder using a high-energy nano ball mill to obtain gentian powder; adding the gentian powder to a deep eutectic solvent and water, mixing them, performing intermittent ultrasonic treatment, and standing to cool to obtain a crude extract; centrifuging the crude extract to obtain a gentiopicroside extract; the deep eutectic solvent comprises a hydrogen bond donor and a hydrogen bond acceptor; the hydrogen bond donor is betaine, and the hydrogen bond acceptor is 1,3-propylene glycol or glycerol.

[0009] Furthermore, the nano ball mill can grind for 1 to 2 hours, and the ball-to-material ratio (mass ratio) is 15:1;

[0010] Furthermore, the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 1:2 to 6;

[0011] Furthermore, the preparation method of the deep eutectic solvent includes: fully mixing the hydrogen bond donor and the hydrogen bond acceptor, and heating them in a water bath at a temperature of 80 to 90° C. to form a uniform, stable and transparent liquid.

[0012] Furthermore, in the mixed system of the deep eutectic solvent and water, the water content is 30-60 wt %.

[0013] Furthermore, the solid-liquid ratio of the mixed system of the gentian powder, the deep eutectic solvent and water is 1:10-25.

[0014] Furthermore, the intermittent ultrasound time is set to work for 8 minutes and stop for 2 minutes.

[0015] Furthermore, the temperature of the intermittent ultrasonic treatment is 40-70° C., the extraction power is 100-150 W, and the extraction time is 30-60 min.

[0016] Furthermore, the rotation speed during centrifugation is 6000-8000 rpm, and the centrifugation time is 5-10 min.

[0017] The present invention also provides gentiopicroside prepared by the above preparation method.

[0018] The present invention also provides the above-mentioned preparation method or the use of the above-mentioned gentiopicroside in preparing a product with soothing effect to help alleviate skin discomfort and redness and swelling.

[0019] The present invention also provides the above-mentioned preparation method or the use of the above-mentioned gentiopicroside in the preparation of a product for improving skin redness.

[0020] The present invention also provides the above-mentioned preparation method or the use of the above-mentioned gentiopicroside in the preparation of skin anti-inflammatory products.

[0021] Beneficial Effects

[0022] As an emerging green solvent, natural deep eutectic solvent has the advantages of strong designability, good biodegradability, and low toxicity. The natural deep eutectic solvent used in the present invention can efficiently extract gentiopicroside from gentian, the extraction process is green, safe and environmentally friendly, the preparation process is simple, the cost is low, the extraction equipment is simple to operate, and there is no special requirement for the sample. The extraction efficiency is higher than that of conventional methods, and the obtained product is natural and non-toxic, avoiding the disadvantages of using organic solvents, time-consuming extraction process, and easy deterioration of extracts. NDES is mainly composed of betaine, 1,3-propylene glycol and glycerol, which can not only efficiently extract the effective ingredients in gentian, but also can be directly used as a moisturizer as an added ingredient, so it has good application value and development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a graph showing the extraction yield of gentiopicroside using deep eutectic solvents obtained by mixing betaine and 1,3-propylene glycol in different molar ratios.

[0024] Figure 2 HPLC chromatogram of gentiopicroside standard.

[0025] Figure 3 is the HPLC chromatogram of NDES-1 extract.

[0026] Figure 4 is the HPLC chromatogram of NDES-2 extract.

[0027] Figure 5 HPLC chromatogram of the water extract.

[0028] Figure 6 HPLC chromatogram of methanol extract.

[0029] Figure 7 This is the effect of NDES-1 gentiopicroside extract on the survival rate of RAW 264.7 cells.

[0030] Figure 8 Effect of NDES-1 gentiopicroside extract on NO release from RAW 264.7 cells stimulated by LPS. DETAILED DESCRIPTION

[0031] The present invention provides a method for preparing a gentian extract using a deep eutectic solvent. In order to make the purpose, technical solution and effect of the present invention clearer and more specific, the present invention is further described in detail below. The specific embodiments described in the embodiments of the invention are only exemplary embodiments of the present invention, but are not intended to limit the present invention.

[0032] Example 1

[0033] The present invention provides a method for preparing a gentian extract using a deep eutectic solvent, the method comprising the following steps:

[0034] Step A: Preparation of a deep eutectic solvent (NDES-1) containing betaine-1,3-propylene glycol: Specifically, betaine and 1,3-propylene glycol in a molar ratio of 1:2, 1:3, 1:4, 1:5, 1:6 are mixed, and heated in a water bath at 80°C to form a uniform, stable, transparent liquid to obtain a deep eutectic solvent (NDES-1), which is then sealed and stored for later use.

[0035] Step B: drain the surface moisture of the washed gentian, dry it in a drying oven to constant weight, and then grind it in a high-energy nano ball mill at a ball-to-material ratio of 15:1 for 1 hour to obtain gentian powder; the deep eutectic solvent (NDES-1) obtained in step (A) is mixed with water to form a mixed system (water content is 40%), and then mixed with the gentian powder, the material-liquid ratio is 1:20, the unit is g / mL, and intermittent ultrasonic heating assisted extraction is performed in an ultrasonic extraction container (temperature 50°C, time 40min, extraction power 120W), and after standing and cooling, a crude extract is obtained;

[0036] Step C: Centrifuge the crude extract obtained in step (B), take the supernatant, and obtain the gentiopicroside extract. The sample solution was subjected to HPLC analysis using an EliteHPLC 1260 system (Agilent, USA) and a C18 column (4.6×250mm, 5μmS.No.1). The flow rate was 1.0mL per minute; the column temperature was 30°C; the detection wavelength was 270nm; and the mobile phase ratio was shown in Table 1. The results of the gentiopicroside content are as follows: Figure 1 shown.

[0037] Table 1 Gentiopicroside HPLC separation method

[0038]

[0039] Example 2

[0040] The same as Example 1, except that a deep eutectic solvent (NDES-2) containing betaine-glycerol was prepared: specifically, betaine and glycerol were mixed at a molar ratio of 1:3. The content of gentiopicroside therein was detected by high performance liquid chromatography (HPLC). The results of the content of gentiopicroside are shown in Tables 2 and Figure 3 shown.

[0041] Comparative Example 1

[0042] Gentiopicroside was extracted by intermittent ultrasonic extraction with pure water, and the steps were as follows: drain the surface water of the washed gentian, dry it in a drying oven to constant weight, and then grind it in a high-energy nano ball mill at a ball-to-liquid ratio of 15:1 for 1 hour to obtain gentian powder; add the gentian powder to a round-bottom flask, add ultrapure water at a material-liquid ratio of 1:20 (g / mL), extract it by intermittent ultrasonic heating (temperature 50°C, time 40min, extraction power 120W), and measure the crude extract after standing and cooling; centrifuge the crude extract, take the supernatant, and obtain the gentiopicroside extract. The content of gentiopicroside in the extract was detected by high performance liquid chromatography (HPLC), and the results of the content of gentiopicroside are shown in Table 2 and Figure 5 shown.

[0043] Comparative Example 2

[0044] Gentiopicroside was extracted by intermittent ultrasonic extraction using 80% methanol solution, and the steps were as follows: drain the surface water of the washed gentian, dry it in a drying oven to constant weight, and then grind it in a high-energy nano ball mill at a ball-to-material ratio of 15:1 for 1 hour to obtain gentian powder; add the gentian powder to a round-bottom flask, add 80% methanol solution at a material-liquid ratio of 1:20 (g / mL), extract it by intermittent ultrasonic heating (temperature 50°C, time 40min, extraction power 120W), stand and cool to obtain a crude extract; centrifuge the crude extract, take the supernatant, and obtain a gentiopicroside extract. The content of gentiopicroside in the extract was detected by high performance liquid chromatography (HPLC), and the results of the content of gentiopicroside are shown in Table 2 and Figure 6 shown.

[0045] Comparative Example 3

[0046] The same as Example 1, except that the extraction is performed by direct water bath heating, and intermittent ultrasonic heating is not used for auxiliary extraction. The content of gentiopicroside therein is detected by high performance liquid chromatography (HPLC).

[0047] Comparative Example 4

[0048] The same as Example 1, except that a deep eutectic solvent (NDES-1) containing betaine-1,3-propylene glycol was prepared in a molar ratio of 1:2: specifically, betaine and 1,3-propylene glycol were mixed in a molar ratio of 1:2. The content of gentiopicroside therein was detected by high performance liquid chromatography (HPLC).

[0049] Comparative Example 5

[0050] The same as Example 1, except that a deep eutectic solvent (NDES-1) containing betaine-1,3-propylene glycol was prepared in a molar ratio of 1:4: specifically, betaine and 1,3-propylene glycol were mixed in a molar ratio of 1:4. The content of gentiopicroside therein was detected by high performance liquid chromatography (HPLC). The extracts of Examples 1 to 2 and Comparative Examples 1 to 5 were detected and analyzed, and the specific results are shown in Table 2:

[0051] Table 2 Extraction rate of gentiopicroside in different solvents

[0052]

[0053] According to the HPLC chromatogram, it can be seen that the water-extracted gentian extract contains very few ingredients, and the extraction rate of gentiopicroside is low, while the gentian extract of NDES can extract more effective ingredients. The data show that the extraction rate of gentiopicroside by the four NDES is higher than that of pure water and methanol, among which NDES1 has the highest extraction rate of 36.52 mg / g.

[0054] Test Example 1

[0055] Anti-inflammatory activity studies

[0056] Model building

[0057] Macrophages, as key immune cells in the body, have the ability to regulate immune function through phagocytosis and release of inflammatory factors. Lipopolysaccharide (LPS), a highly active pathogen that is ubiquitous in nature, can activate the monocyte-macrophage system and cause these cells to release a large number of pro-inflammatory mediators. This process can trigger a systemic inflammatory response syndrome and may further develop into multiple organ dysfunction syndrome, which may eventually lead to multiple organ failure.

[0058] In this experiment, the anti-inflammatory activity of gentiopicroside extract was tested using the LPS-induced pro-inflammatory differentiation model of RAW264.7 macrophages.

[0059] Table 3 Experimental reagents and equipment

[0060]

[0061]

[0062] Cell viability assay

[0063] According to the MTT kit steps, the effect of the gentiopicroside solution of Example 1 on the activity of macrophages was detected. The sample preparation method was the same as that of Example 1. Macrophages in the logarithmic growth phase were taken and their cell concentration (1×10 6 The cells were inoculated into 96-well cell culture plates with 100 μL of cell suspension per well and placed in a 37°C, 5% CO 2 After culturing in an incubator for 24 hours, 100 μL of gentiopicroside extracts with different final concentrations were added (the concentration of gentiopicroside in the extract was 50, 100, 200, 400, 800, and 1600 μg / mL). Three replicate wells were set for each concentration group, and a blank culture medium control group was set up at 37°C and 5% CO. 2 After incubation in an incubator for 24 hours, 100 μL of freshly prepared MTT solution (0.5 mg / mL) was added to each well. After incubation for 4 hours under the same culture conditions, the supernatant was discarded, 200 μL of DMSO was added to each well, and the cells were shaken gently on a shaker at 37°C for 10 minutes. The optical density absorbance (OD) was measured at 570 nm using a microplate reader. Cell survival rate (%) = (OD 加药组 -OD 空白组 ) / (OD 对照组 -OD 空白组 )×100%, and the cell viability of the control group was set as 100%.

[0064] Induction and treatment of RAW264.7 inflammation model

[0065] According to the steps of the NO detection kit, mouse macrophages (RAW264.7) were used to detect the inhibitory effect of the compound on nitric oxide production. This model uses the inflammatory response of mouse macrophages induced by lipopolysaccharide (LPS). When the cells are stimulated by LPS, a large amount of nitric oxide synthase is generated, which generates NO for immune response. Therefore, inhibiting NO production is a direct indicator of the anti-inflammatory activity of the compound.

[0066] Take RAW264.7 macrophages in the logarithmic growth phase and adjust the cell density to 1×10 5 Each well was inoculated into a 96-well cell culture plate and placed at 37°C and 5% CO 2 After 24h of adherent culture in an incubator, LPS (1μg / mL) was added to induce differentiation into an inflammatory cell model for 24h, and PBS was set as a control group. 100μL of gentiopicroside extracts of different final concentrations were added to the inflammatory cell model (the concentration of gentiopicroside in the extract was 50, 100, 200, 400, and 800μg / mL) and continued to be cultured for 24h, then the supernatant culture solution was aspirated, and 50μL of NO reagent 1 and NO reagent 2 were added respectively. The optical density absorbance (OD) was measured at 490nm with an enzyme marker, and the secretion of inflammatory factor NO in different treatments was calculated by the standard curve. At the same time, the freshly prepared MTT solution was added to the 96-well plate after the supernatant was removed. After incubation in the incubator for 4h, the supernatant was discarded, DMSO 200μL / well was added, and the cells were gently shaken on a shaker at 37℃ for 10min. The OD value (A570 nm) was detected by an enzyme marker, and the relative cell viability of each group was calculated.

[0067] Calculation formula: Cell survival rate (%) = (OD administration / model-OD blank) / (OD control-OD blank)×100%.

[0068] The results are as follows Figure 7 As shown in (Effect of NDES-1 gentiopicroside extract on the survival rate of RAW 264.7 cells), the cell survival rate of NDES-1-gentiopicroside extract reached 100% below the concentration of 800 μg / mL, and it had no toxicity to macrophages, indicating that these concentrations can be used as safe doses for subsequent NO inflammation level detection.

[0069] The results are as follows Figure 8 As shown in (Effect of NDES-1 gentiopicroside extract on NO release from RAW 264.7 cells stimulated by LPS), NDES-1 gentiopicroside extract can significantly reduce NO inflammatory levels within the concentration range of 50-800 μg / mL, indicating that it has good anti-inflammatory effects and a dose-dependent effect.

[0070] Test Example 2

[0071] Skin redness analysis

[0072] Test sample preparation

[0073] A composition containing gentiopicroside extracts of different concentrations was prepared, the main ingredients of which were: gentiopicroside extracts, trehalose, erythritol, xanthan gum, EDTA-2Na, and deionized water. According to the different concentrations of the gentiopicroside extracts, they were labeled as sample 1 (50 μg / mL), sample 2 (100 μg / mL), sample 3 (200 μg / mL), sample 4 (400 μg / mL), sample 5 (800 μg / mL), and a composition without gentiopicroside (control).

[0074] Model building

[0075] The skin redness degree parameter is obtained by collecting facial images through Visia-CR, and then using Image-Pro-Plus image analysis software to analyze the a* value of the cheek area skin under the Standard2 light source. The smaller the value, the lighter the skin redness.

[0076] Subject screening criteria

[0077] Healthy female subjects aged between 20 and 50 years old; and the subjects' skin tested positive after the 10% lactic acid sting test (determined as sensitive skin); there was slight redness on the face; 85 people, 5 randomly selected for each sample.

[0078] Application area: face

[0079] Frequency of use: Use once in the morning and once in the evening for 28 consecutive days, 1.2g each time.

[0080] Test environment conditions: temperature 21±1℃, relative humidity 50±5%.

[0081] Experimental design: Single-blind, randomized, self-controlled before and after, face (the side opposite to the area where the lactic acid solution was applied was selected according to the random table).

[0082] Evaluation method: Analyze the changes in the redness level of facial skin.

[0083] Experimental Results

[0084] The results of skin redness analysis are shown in Table 4 below.

[0085]

[0086] The results show that the gentiopicroside extract has an improving effect on facial redness, and as the concentration of the gentiopicroside extract in the test samples 1-5 gradually increases, the improving effect on the degree of facial skin redness is also greater, indicating that the use of the extract of the present invention can have the effect of soothing skin discomfort.

[0087] It should be noted that the parameters of the embodiments of this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the conditions under which the present invention can be implemented. Any changes in the proportional relationship or adjustments in the components should still fall within the scope of the technical contents disclosed in the present invention without affecting the effects and purposes that can be achieved by the present invention.

Claims

1. A method and application of preparing a gentian extract using a deep eutectic solvent, characterized in that: The following steps are involved: The surface moisture of the washed gentian is drained, the gentian is dried in a drying oven to constant weight, and then ground into powder in a high-energy nano ball mill to obtain gentian powder; the gentian powder is added into a deep eutectic solvent and water for mixing, intermittent ultrasonic treatment is performed, and a crude extract is obtained after standing and cooling; the crude extract is centrifuged to obtain a gentiopicroside extract; the deep eutectic solvent includes a hydrogen bond donor and a hydrogen bond acceptor; the hydrogen bond donor is betaine, and the hydrogen bond acceptor is 1,3-propylene glycol or glycerol.

2. The method for preparing a gentian extract using a deep eutectic solvent according to claim 1, characterized in that: The molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 1:2-6.

3. The method for preparing a gentian extract using a deep eutectic solvent according to claim 1, characterized in that: The preparation method of the deep eutectic solvent comprises: fully mixing a hydrogen bond donor and a hydrogen bond acceptor, and heating in a water bath at a temperature of 80 to 90° C. to form a uniform, stable and transparent liquid.

4. The method for preparing a gentian extract using a deep eutectic solvent according to claim 1, characterized in that: The water content in the mixed system of the deep eutectic solvent and water is 30-60 wt %.

5. The method for preparing a gentian extract using a deep eutectic solvent according to claim 1, characterized in that: The solid-liquid ratio of the mixed system of the gentian powder, the deep eutectic solvent and water is 1:10-25.

6. The method for preparing a gentian extract using a deep eutectic solvent according to claim 1, characterized in that: The temperature of the ultrasonic treatment is 40-70° C., the extraction power is 100-150 W, and the extraction time is 30-60 min.

7. The method for preparing a gentian extract using a deep eutectic solvent according to claim 1, characterized in that: The rotation speed during centrifugation is 6000-8000 rpm, and the centrifugation time is 5-10 minutes.

8. A gentiopicroside, characterized in that: Prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the preparation method according to any one of claims 1 to 7 or the gentiopicroside according to claim 8 in the preparation of a skin care product for improving skin redness.

10. Use of the preparation method according to any one of claims 1 to 7 or the gentiopicroside according to claim 8 in the preparation of skin anti-inflammatory products.

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