Preparation method and application of gentiana scabra extract

Abstract

The application belongs to the technical field of gentian root extract, and discloses a preparation method and application of a gentian root extract, and the preparation method comprises the following steps: S1, adding gentian root powder and chitosan-nano calcium carbonate composite microspheres into a buffer solution, uniformly mixing, and obtaining a suspension 1; S2, in the suspension 1, firstly adding cellulase, and then adding beta-glucanase and flavor protease, and obtaining a suspension 2 after enzyme hydrolysis; S3, adding the suspension 2 into a PEG-400 / ionic liquid ([BMIM]BF) / water extraction medium, carrying out first extraction at 20-30 DEG C and second extraction at 55-65 DEG C, obtaining a crude extract, and then carrying out post-treatment and purification to obtain the gentian root extract. The gentian root extract prepared by the preparation method has high content of effective components, high stability of effective components and low content of impurities.

Description

Technical Field

[0001] This invention relates to the field of gentian root extract technology, and more specifically, to a method for preparing gentian root extract and its application. Background Technology

[0002] Gentian root extract is derived from the dried roots and rhizomes of plants in the Gentianaceae family (such as Gentiana, Gentiana stripe-leaved, and Gentiana triflora). The active ingredient is gentiopicrin, an iridoid glycoside, and it also contains swertiamarin, flavonoids, and phenolic compounds. It has hepatoprotective, choleretic, anti-inflammatory, antibacterial, digestive-promoting, antioxidant, hypoglycemic, and lipid-lowering effects, and is widely used in medicine, health care, and cosmetics.

[0003] Patent document CN 118717604 A discloses a method for preparing gentian root extract. First, gentian root powder, cellulase, flavor protease, and β-glucanase are added to water for enzymatic hydrolysis, followed by filtration. Then, the filtrate is adsorbed, eluted, washed with water, and dried using a D101 macroporous adsorption resin to obtain the gentian root extract. This preparation method has the following problems:

[0004] (1) Low content of active ingredients. Cellulase, flavor protease and β-glucanase act simultaneously. Some cellulase acts on the cell wall. After the cell wall is damaged or cracked, flavor protease and β-glucanase act on the intracellular components prematurely through the damage or cracks in the cell wall. This interferes with the binding of the remaining cellulase to the cell wall, resulting in the cell wall not being fully destroyed. As a result, the active ingredients will also be wrapped in the cell wall and cannot be completely released. The filtration step after enzymatic hydrolysis will cause the cell wall that wraps the residual active components to be trapped in the filter residue and cannot enter the filtrate, resulting in a low content of active ingredients in the obtained gentian root extract.

[0005] (2) Poor stability of active ingredients. Flavonoids can interact with gentiopicrin, inhibiting oxidation and improving its stability. This preparation method uses water as the only extraction medium, which can extract the highly polar gentiopicrin, but cannot effectively extract the fat-soluble components—flavonoids. Therefore, the active ingredients in the obtained gentian root extract have poor stability.

[0006] (3) High impurity content. "Enzymatic hydrolysis followed by filtration" can only remove solid residues, while soluble impurities in gentian root (such as cellulose, pectin and other polysaccharides, protein fragments, tannins, pigments, etc.) will enter the extraction medium in large quantities. During the subsequent purification process with D101 macroporous resin, these impurities will compete with the effective components for adsorption sites, resulting in an excessively high impurity content in the final gentian root extract. Summary of the Invention

[0007] This invention provides a method for preparing gentian root extract, wherein the obtained gentian root extract has a high content of effective components, or high stability of effective components, or low content of impurities.

[0008] Another object of the present invention is to provide the application of the gentian root extract prepared by the method of preparation of the gentian root extract in the preparation of cosmetics.

[0009] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0010] A method for preparing gentian root extract includes the following steps:

[0011] S1. Add gentian root powder and chitosan-nano calcium carbonate composite microspheres to buffer solution and mix evenly to obtain suspension 1;

[0012] S2. In suspension 1, cellulase is added first, followed by β-glucanase and flavor protease. After enzymatic hydrolysis, suspension 2 is obtained.

[0013] S3. Add suspension 2 to PEG-400 / ionic liquid ([BMIM]BF) / water extraction medium, and perform a first extraction at 20-30℃ and a second extraction at 55-65℃ to obtain a crude extract, which is then purified by post-processing to obtain the gentian root extract.

[0014] This invention provides a method for preparing the gentian root extract, which has the following technical effects:

[0015] (1) High content of active ingredients. In step S1 of this invention, chitosan-nano-calcium carbonate composite microspheres are added. The nano-calcium carbonate releases CO gas to form microbubbles, which use micro-impact to form microcracks on the cell wall. The microspheres expand the cracks through mechanical collision, initially destroying the cell wall structure. Furthermore, in step S2 of this invention, the three enzymes, cellulase, flavor protease, and β-glucanase, are not added simultaneously. Instead, cellulase is added first to fully destroy the cell wall structure, and then flavor protease and β-glucanase are added to specifically decompose intracellular polysaccharides and proteins, so that the active ingredients in the cell are fully released. This reduces the residual encapsulation of active ingredients by the cell wall, resulting in a high content of active ingredients in the obtained gentian root extract.

[0016] (2) High stability of active ingredients. In step S3 of this invention, PEG-400 / ionic liquid ([BMIM]BF4) / water is used as the extraction medium. First, the flavonoids (such as gentianin) are extracted at 20-30℃. Then, gentiopicrin is extracted a second time at 55-65℃. During the two extractions, the ionic liquid [BMIM]BF4 in the extraction medium can capture free radicals and inhibit the oxidation reaction of gentiopicrin. The steric hindrance effect of PEG-400 can encapsulate the active ingredients and reduce their contact with oxygen and light. The resulting gentian root extract has high stability of active ingredients.

[0017] (3) Low impurity content. In step S1 before enzymatic hydrolysis, chitosan-nano-calcium carbonate composite microspheres are added. The chitosan can adsorb impurities such as tannins, pigments, and protein fragments in gentian root in advance. In step S3 after enzymatic hydrolysis, PEG-400 preferentially forms hydrogen bonds with water in the extraction medium, while the ionic liquid [BMIM]BF4 repels polysaccharides with weak polarity. This inhibits the dissolution of polysaccharides such as cellulose and pectin in the crude extract. The extraction medium of this invention is more likely to selectively dissolve gentiopicrin, which is moderately polar and electrically neutral, as the target component. The protein fragments after enzymatic hydrolysis (such as small molecule peptides and amino acids) are positively / negatively charged and highly polar. They are not easily selected by the extraction medium and are not easy to enter the crude extract as impurities. Therefore, the gentian root extract obtained has a low impurity content.

[0018] Preferably, the buffer solution in S1 is at a temperature of 35–39°C and a pH of 4.0–6.0.

[0019] The buffer solution in S1 of the present invention can be selected from citrate-sodium citrate buffer, acetate-sodium acetate buffer or disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

[0020] In the present invention, the mass ratio of gentian root powder to chitosan-nano calcium carbonate composite microspheres in S1 is 1:(0.2-1.0).

[0021] The composite microspheres of this invention have a particle size of 1–10 μm.

[0022] Preferably, the buffer solution is a citrate-sodium citrate buffer solution with a concentration of 0.1 mol / L and a pH of 5.0–5.5.

[0023] The buffer solution of this invention is an acetate-sodium acetate buffer solution with a pH of 4 to 5.6.

[0024] The buffer solution of this invention, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, has a pH of 6.

[0025] Preferably, the cellulase is added to S2 at a temperature of 35–39°C and a pH of 5.0–6.0.

[0026] Preferably, β-glucanase and flavor protease are added to S2 at a temperature of 45–55°C and a pH of 6–6.5.

[0027] Preferably, the enzymatic hydrolysis time of cellulase added in S2 is 0.5 to 2 hours, and the enzymatic hydrolysis time of β-glucanase and flavor protease added is 4 to 6 hours.

[0028] Preferably, ascorbic acid is added after the enzymatic hydrolysis in S2, and the amount of ascorbic acid added is 0.2% to 1.0% w / v.

[0029] Preferably, the pH of the first extraction in S3 is 2.5–3.5, and the pH of the second extraction is 6.5–7.5.

[0030] Preferably, the mass ratio of PEG-400: ionic liquid ([BMIM]BF4): water in the extraction medium of S3 is (2-4):(1-3):(4-6).

[0031] Preferably, the post-processing purification in S3 is as follows: after centrifugation and filtration, 0.6% to 1.0% (w / v) of citric acid is added to the crude extract, and then the pH is adjusted to 4.5 to 5.0. Finally, the extract is subjected to adsorption, elution, vacuum concentration, and drying by XAD-7HP macroporous resin to obtain the gentian root extract.

[0032] The post-processing purification in S3 of this invention specifically involves adjusting the pH to 4.5-5.0 with citric acid or sodium carbonate, and drying specifically involves spray drying, freeze drying, or vacuum drying to obtain the gentian root extract.

[0033] The crude extract of this invention is centrifuged and filtered to remove a large number of chitosan-nano calcium carbonate composite microspheres; the addition of 0.5% to 1.2% w / v citric acid can effectively remove cellulase, β-glucanase, and flavor protease; XAD-7HP macroporous resin preferentially adsorbs the target components (gentiopicrin and flavonoids) of gentiopicrin extract.

[0034] To remove the chitosan-nano calcium carbonate composite microspheres, cellulase, β-glucanase, flavor protease, PEG-400 / ionic liquid ([BMIM]BF), and ascorbic acid added in the preceding steps, the post-treatment purification can be further specifically as follows:

[0035] (I) After centrifugation and filtration of the crude extract, a pressure filtration step of 0.22μm microporous membrane is added (pressure 0.1~0.15MPa). The particle size of the composite microspheres is 1~10μm. Centrifugation and filtration remove most of the composite microspheres. The 0.22μm filter membrane can retain all solid particles and remove the residual chitosan-nano calcium carbonate trace composite microspheres.

[0036] (II) Add 0.5%–1.0% (w / v) citric acid to the crude extract after removing the chitosan-nano-calcium carbonate composite microspheres, stir for 10 min, and then measure the pH of the crude extract using a pH meter.

[0037] If pH > 5.0: Add 10% citric acid aqueous solution, stir for 5 minutes and then retest the pH until the pH stabilizes at 4.5-5.0;

[0038] If pH < 4.5: adjust to 4.5–5.0 with a 5% sodium carbonate aqueous solution;

[0039] Then, let it stand at 4℃ for 60–90 minutes, followed by centrifugation (12000 r / min, 25 min) to collect the supernatant. This effectively removes cellulase, β-glucanase, and flavor protease. Citric acid adjusts the pH of the system to near the isoelectric point of the enzyme, reducing the solubility of the protein (the essence of the enzyme) and thus promoting its aggregation and precipitation.

[0040] (III) XAD-7HP macroporous resin is selected because it has stronger polarity and preferentially adsorbs gentiopicrin and flavonoids with moderate polarity. This can reduce the non-specific adsorption of hydrophilic impurities (PEG, ionic liquids) by the resin and reduce the elution burden in the subsequent process.

[0041] (IV) The elution stage first removes ascorbic acid, inorganic salts, residual PEG, and ionic liquids, and then elutes the target components in a targeted manner. Specifically, the removal of ascorbic acid, inorganic salts, residual PEG, and ionic liquids is as follows: First, elution is performed with 0.1% (w / v) sodium chloride solution (2 BV, flow rate 1.5 BV / h): utilizing the salting-out effect, PEG-400 and [BMIM]BF4 are promoted to elute from the resin gaps. Then, elution is performed with distilled water (3 BV, flow rate 2 BV / h): removing residual salts and ascorbic acid. Ascorbic acid is highly water-soluble and can be completely eluted.

[0042] The targeted elution of the target components was as follows: First, a small amount of residual PEG-400 and [BMIM]BF4 (both of which have higher solubility in low concentrations of ethanol) were eluted with 20% ethanol (1 BV, flow rate 1 BV / h); then, gentiopicrin and flavonoids were eluted with 40% ethanol (3 BV, flow rate 1.5 BV / h).

[0043] The present invention also provides the application of gentian root extract prepared by any of the above-described methods in the preparation of cosmetics. Detailed Implementation

[0044] The present invention will be further described in detail below with reference to specific embodiments.

[0045] The chitosan-nano calcium carbonate composite microspheres in the embodiments and comparative examples of this invention are existing materials. This invention provides one preparation method, as detailed below:

[0046] 1. Raw materials and reagents

[0047] Chitosan (degree of deacetylation ≥ 90%), calcium nitrate (Ca(NO)·4H₂O), sodium carbonate (Na₂CO₃), glacial acetic acid, sodium tripolyphosphate (TPP, crosslinking agent), and anhydrous ethanol (for washing) were all of analytical grade.

[0048] 2. Specific steps (1) Preparation of nano-calcium carbonate

[0049] Preparation of the oil phase: Mix Span-80 (surfactant) and liquid paraffin at a ratio of 1:4 (by mass) and stir until homogeneous to form the oil phase.

[0050] Aqueous phase preparation: Prepare 0.5 mol / L Ca(NO)₂ solution and 0.5 mol / L Na₂CO₃ solution as aqueous phases.

[0051] Microemulsion reaction: Two aqueous phases were added to the oil phase at a ratio of 1:5 (water phase: oil phase volume ratio) and stirred to form a stable microemulsion; then the Ca(NO₃)₂ microemulsion was slowly added dropwise to the Na₂CO₃ microemulsion and stirred at 30°C for 2 hours to generate nano-calcium carbonate particles.

[0052] Post-processing: After the reaction was completed, anhydrous ethanol was added to break the emulsion, and the precipitate was collected by centrifugation (3000 r / min, 10 min). The precipitate was washed three times alternately with distilled water and ethanol, and then dried under vacuum at 60℃ to obtain nano-calcium carbonate powder (particle size can be controlled between 50-200 nm).

[0053] (2) Preparation of chitosan solution

[0054] Weigh a certain amount of chitosan, add 1% (v / v) glacial acetic acid solution, stir magnetically until completely dissolved, prepare a 2% (w / v) chitosan acetic acid solution, let stand to remove bubbles and set aside.

[0055] (3) Preparation of chitosan-calcium carbonate nanocomposite microspheres

[0056] Mixing and dispersing: Add the above-mentioned nano-calcium carbonate powder to the chitosan acetate solution at 10%-30% of the chitosan mass, and ultrasonically disperse for 30 minutes (power 300W) to form a uniform suspension.

[0057] Cross-linking to form microspheres: The suspension was slowly dripped into a 1% (w / v) TPP solution (the mass ratio of chitosan to TPP was 5:1) through a syringe, and stirred at 30°C for 1 hour to allow chitosan to form microspheres through ionic cross-linking with TPP. At the same time, nano-calcium carbonate was encapsulated or adsorbed inside and on the surface of the microspheres.

[0058] Curing and washing: After the reaction is complete, let it stand for 2 hours to cure, collect the microspheres by centrifugation, wash with distilled water until neutral, and freeze-dry to obtain chitosan-nano calcium carbonate composite microspheres.

[0059] Example 1

[0060] A method for preparing gentian root extract includes the following steps:

[0061] S1. Gentian root powder and chitosan-nano calcium carbonate composite microspheres are added to a buffer solution and mixed evenly to obtain suspension 1; the buffer solution is a citrate-sodium citrate buffer solution with a concentration of 0.1 mol / L, a pH of 5.3, and a temperature of 37℃; the chitosan-nano calcium carbonate composite microspheres have a particle size of 5 μm, and the mass ratio of gentian root powder to chitosan-nano calcium carbonate composite microspheres is 1:0.6.

[0062] S2. In suspension 1, first heat to 37℃, adjust pH to 5.5, add cellulase, and enzymatically hydrolyze for 1 hour. The cellulase content should be ≥50U / g.

[0063] Then, the temperature was raised to 50℃, the pH was adjusted to 6.2, β-glucanase and flavor protease were added, and enzymatic hydrolysis was carried out for 5 hours. After the enzymatic hydrolysis was completed, 0.6% w / v ascorbic acid was added to obtain suspension 2; the β-glucanase should be ≥50U / g and the flavor protease should be ≥100U / g.

[0064] Gentian root extract was prepared by extraction and post-treatment using a S3.PEG-400 / ionic liquid ([BMIM]BF) / water system, as detailed below:

[0065] S31. Add suspension 2 to PEG-400 / ionic liquid ([BMIM]BF4) / water extraction medium, wherein the mass ratio of PEG-400:ionic liquid ([BMIM]BF4):water in the extraction medium is 3:2:5;

[0066] S32. Adjust pH=3, perform the first extraction at 25℃, and stir for 2 hours in the dark at a speed of 200r / min to avoid system stratification;

[0067] S33. Adjust pH to 7, perform a second extraction at 60℃, stir for 3 hours at 300 r / min to maintain phase stability, and obtain a crude extract. Further purification through post-processing yields the gentian root extract. The specific post-processing steps are as follows:

[0068] (I) After centrifugation and filtration of the crude extract, a 0.22 μm microporous membrane is added for pressurized filtration at a pressure of 0.1–0.15 MPa;

[0069] (II) Add 0.8% (w / v) citric acid to the crude extract after removing the chitosan-nano-calcium carbonate composite microspheres, stir for 10 min, and then measure the pH of the crude extract using a pH meter:

[0070] If pH > 5.0: Add 10% citric acid aqueous solution, stir for 5 minutes and then retest the pH until the pH stabilizes at 4.5-5.0;

[0071] If pH < 4.5: adjust to 4.5–5.0 with a 5% sodium carbonate aqueous solution;

[0072] Then let it stand at 4℃ for 70 minutes, and then centrifuge (12000r / min, 25min) to take the supernatant, which can effectively remove cellulase, β-glucanase and flavor protease.

[0073] (III) XAD-7HP type macroporous resin was selected for adsorption and separation;

[0074] (IV) First, elute with 0.1% (w / v) sodium chloride solution (2 BV, flow rate 1.5 BV / h), then elute with distilled water (3 BV, flow rate 2 BV / h);

[0075] Then, a small amount of residual PEG-400 and [BMIM]BF4 were eluted with 20% ethanol (1 BV, flow rate 1 BV / h) and then with 40% ethanol (3 BV, flow rate 1.5 BV / h).

[0076] Finally, the eluent was concentrated under reduced pressure and spray-dried to obtain the gentian root extract.

[0077] Example 2

[0078] This embodiment is the second embodiment of the present invention. The difference from embodiment 1 is that S1. buffer is an acetate-sodium acetate buffer with a pH of 4 and a temperature of 35°C; the particle size of chitosan-nano-calcium carbonate composite microspheres is 1 μm; and the mass ratio of gentian root powder to chitosan-nano-calcium carbonate composite microspheres is 1:0.1.

[0079] Example 3

[0080] This embodiment is the third embodiment of the present invention. The difference from embodiment 1 is that S1. buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, pH is 6, and temperature is 39°C; the particle size of chitosan-nano calcium carbonate composite microspheres is 10 μm, and the mass ratio of gentian root powder to chitosan-nano calcium carbonate composite microspheres is 1:1.

[0081] Example 4

[0082] This embodiment is the fourth embodiment of the present invention. The difference from embodiment 1 is that S2. Cellulase is added to suspension 1 at a temperature of 35°C, a pH of 5.0, and a hydrolysis time of 0.5.

[0083] Example 5

[0084] This embodiment is the fifth embodiment of the present invention. The difference from embodiment 1 is that S2. Cellulase is added to suspension 1 at a temperature of 39°C, a pH of 6.0, and an enzymatic hydrolysis time of 2 hours.

[0085] Example 6

[0086] This embodiment is the fourth embodiment of the present invention. The difference from embodiment 1 is that β-glucanase and flavor protease are added in S2 at a temperature of 45°C, a pH of 6, and an enzymatic hydrolysis time of 4 hours.

[0087] Example 7

[0088] This embodiment is the fifth embodiment of the present invention. The difference from embodiment 1 is that β-glucanase and flavor protease are added in S2 at a temperature of 55°C, a pH of 6.5, and an enzymatic hydrolysis time of 6 hours.

[0089] Example 8

[0090] This embodiment is the 8th embodiment of the present invention. The difference from embodiment 1 is that after the enzymatic hydrolysis of S2 is completed, the amount of ascorbic acid added is 1.0% w / v.

[0091] Example 9

[0092] This embodiment is the 9th embodiment of the present invention. The difference from embodiment 1 is that after the enzymatic hydrolysis of S2 is completed, the amount of ascorbic acid added is 0.2% w / v.

[0093] Example 10

[0094] This embodiment is the 10th embodiment of the present invention. Unlike embodiment 1, after the enzymatic hydrolysis of S2 is completed, ascorbic acid is not added, and suspension 2 is obtained.

[0095] Example 11

[0096] This embodiment is the 11th embodiment of the present invention. The difference from embodiment 1 is that...

[0097] S31. Add suspension 2 to PEG-400 / ionic liquid ([BMIM]BF) / water extraction medium, where the mass ratio of PEG-400:ionic liquid ([BMIM]BF4):water is 2:3:4;

[0098] S32. Adjust pH to 2.5 and perform the first extraction at 20℃. S33. Adjust pH to 6.5 and perform the second extraction at 55℃.

[0099] Example 12

[0100] This embodiment is the 12th embodiment of the present invention. The difference from embodiment 1 is that...

[0101] S31. Add suspension 2 to PEG-400 / ionic liquid ([BMIM]BF) / water extraction medium, where the mass ratio of PEG-400:ionic liquid ([BMIM]BF4):water is 4:1:4;

[0102] S32. Adjust pH to 3.5 and perform the first extraction at 30℃. S33. Adjust pH to 7.5 and perform the second extraction at 65℃.

[0103] Example 13

[0104] This embodiment is the 13th embodiment of the present invention. The difference from embodiment 1 is that in step S3. post-processing (II), 0.5% w / v of citric acid is added to the crude extract of chitosan-nano calcium carbonate composite microspheres.

[0105] Example 14

[0106] This embodiment is the 14th embodiment of the present invention. The difference from embodiment 1 is that in step S3. post-processing (II), 1.0% w / v of citric acid is added to the crude extract of chitosan-nano calcium carbonate composite microspheres.

[0107] Example 15

[0108] This embodiment is the 15th embodiment of the present invention. Unlike embodiment 1, citric acid is not added in step S3. Post-processing (II).

[0109] Comparative Example 1

[0110] This comparative example is the first comparative example of the present invention. Unlike Example 1, chitosan-nano-calcium carbonate composite microspheres are not added in step S1.

[0111] Comparative Example 2

[0112] This comparative example is the second comparative example of the present invention. Unlike Example 1, step S1 is omitted. Gentian root powder is directly added to water, cellulase is added first, followed by β-glucanase and flavor protease. After enzymatic hydrolysis, suspension 2 is obtained.

[0113] Comparative Example 3

[0114] This comparative example is the third comparative example of the present invention. Unlike Example 1, in S2, cellulase, β-glucanase and flavor protease are added together to suspension 1, and suspension 2 is obtained after enzymatic hydrolysis.

[0115] Comparative Example 4

[0116] This comparative example is the fourth comparative example of the present invention. Unlike Example 1, it only undergoes one extraction in S3.

[0117] Comparative Example 5

[0118] This comparative example is the fourth comparative example of the present invention. Unlike Example 1, the first extraction temperature in S3 is 15°C and the second extraction temperature is 70°C.

[0119] Comparative Example 6

[0120] This comparative example is the sixth embodiment of the present invention. The difference from embodiment 1 is that the first extraction temperature in S3 is 35°C and the second extraction temperature is 50°C.

[0121] Comparative Example 7

[0122] This comparative example is the 7th embodiment of the present invention. The difference from embodiment 1 is that the extraction medium in S3 is water.

[0123] Performance testing:

[0124] 1. Content of active ingredients in gentian root extract:

[0125] (1) Method for testing gentiopicrin content, including the following steps:

[0126] S1. Preparation of reference solution: Weigh 10 mg of gentiopicroside reference standard (purity ≥98%), place it in a 10 mL volumetric flask, dissolve it in methanol and dilute to the mark, shake well to obtain a stock solution with a concentration of 1.0 mg / mL. Accurately measure 0.1, 0.5, 1.0, 2.0, and 5.0 mL of the stock solution and place them in 10 mL volumetric flasks respectively, dilute to the mark with methanol to prepare a series of reference solutions with concentrations of 0.01, 0.05, 0.1, 0.2, and 0.5 mg / mL. Filter the solutions through a 0.22 μm microporous membrane for later use.

[0127] S2. Preparation of the test solution: Accurately weigh 0.1 g of the gentian root extract obtained in Example 1, place it in a 50 mL stoppered conical flask, add 25 mL of methanol, and extract ultrasonically for 30 min (300 W power, 40 °C). After cooling, transfer to a 50 mL volumetric flask, add methanol to the mark, and shake well. Centrifuge a portion of the solution (5000 r / min, 10 min), and filter the supernatant through a 0.22 μm microporous membrane to obtain the test solution.

[0128] S3. Chromatographic conditions:

[0129] Chromatographic column: C18 reversed-phase column (250mm × 4.6mm, 5μm);

[0130] Mobile phase: methanol-water (35:65, v / v);

[0131] Flow rate: 1.0 mL / min;

[0132] Detection wavelength: 270nm (characteristic absorption peak of gentiopicroside);

[0133] Column temperature: 30℃;

[0134] Injection volume: 10 μL.

[0135] S4. Inject the series of reference solutions and the test solution obtained in S2 respectively, record the peak area, plot the standard curve with the reference concentration as the abscissa (x) and the peak area as the ordinate (y), and calculate the content of gentiopicroside in the test sample by external standard method.

[0136] The gentiopicroside content in the gentian root extracts obtained in each embodiment and comparative example is shown in Table 1.

[0137] (2) Flavonoid content:

[0138] S1. Accurately weigh 10 mg of rutin reference standard (purity ≥98%), place it in a 100 mL volumetric flask, add 70% ethanol to dissolve and dilute to the mark, shake well to obtain a rutin reference standard solution with a concentration of 0.1 mg / mL.

[0139] S2. Accurately weigh 0.2g of the gentian root extract obtained in Example 1, place it in a 100mL stoppered conical flask, add 50mL of 70% ethanol, reflux in a water bath for 1h (temperature 80℃), cool, transfer to a 100mL volumetric flask, add 70% ethanol to the mark, shake well, filter, and take the filtrate as the test solution.

[0140] S3. Take 1 mL of the test solution and place it in a 25 mL volumetric flask. Add 1 mL of 5% sodium nitrite solution, shake well, and let stand for 6 min. Add 1 mL of 10% aluminum nitrate solution, shake well, and let stand for 6 min. Add 10 mL of 4% sodium hydroxide solution, and dilute to the mark with 70% ethanol. Shake well and let stand for 15 min. Using the corresponding reagent as a blank control, measure the absorbance at a wavelength of 510 nm. Simultaneously, take 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 mL of rutin reference solution, and operate according to the above colorimetric method to plot a standard curve and calculate the total flavonoid content (calculated as rutin).

[0141] The flavonoid content in the gentian root extracts obtained in each embodiment and comparative example is shown in Table 1.

[0142] 2. Impurity content in gentian root extract:

[0143] (1) Protein content test method: Coomassie Brilliant Blue G-250 method. The gentian root extract solution obtained in Example 1 was reacted with Coomassie Brilliant Blue reagent for color development, and the absorbance was measured at 595 nm. The content was calculated by comparing with the bovine serum albumin standard curve.

[0144] (2) Appearance of gentian root extract: The color of the 1% aqueous solution of the prepared gentian root extract was observed: light yellow, yellow, light brown, brown.

[0145] The impurity content in the gentian root extracts obtained in each embodiment and comparative example is shown in Table 1.

[0146] 3. Loss of active ingredients during extraction:

[0147] Based on the total effective components (gentiopicroside) in the raw materials, the loss amount in each key step was determined, and the total loss amount was calculated:

[0148] Initial total amount in raw materials: Take gentian root powder, extract and determine the total amount of active ingredients (denoted as M) according to the method in "1. Preparation of test solution for active ingredient content in gentian root extract".

[0149] Content of active ingredients in the obtained gentian root extract: The total amount of active ingredients (denoted as M4) was extracted and determined according to the method in "1. Preparation of test solution for content of active ingredients in gentian root extract";

[0150] Loss of active ingredient = (M - M) / M × 100%.

[0151] The loss of active ingredients in the gentian root extracts obtained in each embodiment and comparative example is shown in Table 1.

[0152] 4. Stability of Gentian Root Extract: The test method involved sealing the gentian root extract in a brown glass bottle and placing it under conditions of 40℃±2℃ / relative humidity 75%±5% and light (4500lx±500lx) for 3 months. The results were as follows:

[0153] (1) The content of active ingredients was detected by HPLC and the retention rate of gentiopicrin was calculated;

[0154] (2) Record the pH fluctuation values ​​of the obtained gentianin extract aqueous solution (1%, w / v) from the start of testing to 3 months later;

[0155] (3) Appearance: Whether there is obvious color deepening (ΔE≤3, measured by colorimeter), whether there is sedimentation or layering.

[0156] The stability test results of the gentian root extracts obtained in each embodiment and comparative example are shown in Table 2.

[0157] Table 1. Tests on component content and loss of active ingredients of gentian root extracts obtained in Examples 1-15 and Comparative Examples 1-7.

[0158]

[0159]

[0160] Table 2. Stability tests of gentian root extracts obtained in Examples 1-15 and Comparative Examples 1-7

[0161]

[0162]

[0163] The above description is merely an embodiment of the present invention and does not limit the scope of patent protection. Any non-substantial changes or substitutions made by those skilled in the art based on the present invention will still fall within the scope of patent protection.

Claims

1. A method for preparing a gentian root extract, characterized in that, Includes the following steps: S1. Gentian root powder and chitosan-nano calcium carbonate composite microspheres are added to a buffer solution and mixed evenly to obtain suspension 1; the buffer solution is selected from citrate-sodium citrate buffer, acetate-sodium acetate buffer or disodium hydrogen phosphate-sodium dihydrogen phosphate buffer; the temperature of the buffer solution is 35~39℃ and the pH is 4.0~6.

0. S2. In suspension 1, cellulase is first added, followed by β-glucanase and flavor protease. After enzymatic hydrolysis, suspension 2 is obtained. The amount of cellulase added is ≥50 U / g, the amount of β-glucanase added is ≥50 U / g, and the amount of flavor protease added is ≥100 U / g. The temperature for adding cellulase is 35~39℃, and the pH is 5.0~6.

0. The temperature for adding β-glucanase and flavor protease is 45~55℃, and the pH is 6~6.

5. The enzymatic hydrolysis time for adding cellulase is 0.5~2 h, and the enzymatic hydrolysis time for adding β-glucanase and flavor protease is 4~6 h. S3. Add suspension 2 to PEG-400 / ionic liquid [BMIM]BF4 / water extraction medium, and perform a first extraction at 20~30℃ and a second extraction at 55~65℃ to obtain a crude extract, which is then purified by post-treatment to obtain the gentian root extract; the pH of the first extraction is 2.5~3.5, and the pH of the second extraction is 6.5~7.5; the mass ratio of PEG-400: ionic liquid [BMIM]BF4: water in the extraction medium is (2~4):(1~3):(4~6).

2. The method for preparing the gentian root extract according to claim 1, characterized in that, In S2, ascorbic acid is added after enzymatic hydrolysis, with the amount of ascorbic acid added being 0.2%~1.0% w / v.

3. The method for preparing the gentian root extract according to claim 1, characterized in that, In S3, after centrifugation and filtration, 0.5%~1.0% w / v of citric acid is added to the crude extract, and the pH is adjusted to 4.5~5.

0. Finally, the extract is subjected to adsorption, elution, vacuum concentration, and drying using XAD-7HP macroporous resin to obtain the gentian root extract.

4. The use of a gentian root extract prepared by the method of any one of claims 1 to 3 in the preparation of cosmetics.

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