Rheum moorcroftianum extract and method for preparing the same

Abstract

The application discloses a preparation method of a rhubarb extract, and comprises the following steps: S1, adding beta-glucosidase to rhubarb powder, then adding a eutectic solvent, stirring and enzymolysis to obtain an enzymolysis solution, wherein the eutectic solvent comprises a mixture of betaine, lactic acid and water; S2, filtering the enzymolysis solution to obtain filtrate and an enzymolysis solution; S3, sequentially immersing the filtrate in pure water and the eutectic solvent, and filtering to obtain an immersion solution; and S4, uniformly mixing the immersion solution and the enzymolysis solution, drying and sterilizing to obtain a rhubarb extract solid. The eutectic solvent composed of betaine and lactic acid and the beta-glucosidase are simultaneously added, so that the enzymolysis and extraction of the rhubarb can be realized, and the extraction rate of effective components, such as anthraquinones, stilbenes and tannin compounds, in the rhubarb is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of extraction of effective components of Tibetan medicine, and more particularly to a Tibetan rhubarb extract and a preparation method thereof. BACKGROUND

[0002] The chemical components of Tibetan rhubarb mainly include anthraquinones, stilbenes and tannins, wherein the anthraquinones such as emodin, emodin methyl ether and chrysophanol have obvious effects of antibacterial and anti-inflammatory, immune regulation, anti-aging and anti-tumor; the stilbenes have a high content, and a kind of substance having a stilbene nucleus or a polymer thereof is collectively referred to as stilbenes, which has a wide range of biological activities, in addition to the known antibacterial effect, in recent years, it has also been found to have effects of lowering blood lipids, lowering blood sugar, protecting liver, dilating capillaries to improve microcirculation, dilating coronary blood vessels and lowering blood pressure.

[0003] The prior art with the application number 201110124226.9 discloses a Tibetan rhubarb extract and a preparation method thereof, which is an extract of Tibetan rhubarb extracted by ethanol infiltration and elution in sequence, and has problems of long extraction time, low extraction rate, low content of effective components, etc., and the use of ethanol for extraction is flammable and explosive, which causes burden to the environment, and the method is performed in multiple steps, which is time-consuming and laborious, and increases the cost.

[0004] Therefore, how to design a preparation method of Tibetan rhubarb extract to solve the above technical defects is worth thinking about. SUMMARY

[0005] An object of the present application is to solve at least the above problems and to provide at least the advantages to be described later.

[0006] In order to achieve these objects and other advantages according to the present application, a preparation method of Tibetan rhubarb extract is provided, which comprises the following steps:

[0007] S1, adding β-glucosidase to Tibetan rhubarb powder, then adding a eutectic solvent, stirring and enzymolysis to obtain an enzymolysis solution, wherein the eutectic solvent comprises a mixture of betaine, lactic acid and water;

[0008] S2, filtering the enzymolysis solution to obtain a residue and an enzymolysis solution;

[0009] S3, sequentially immersing the residue in pure water and the eutectic solvent, and filtering to obtain an immersion solution;

[0010] S4, uniformly mixing the immersion solution and the enzymolysis solution, drying and sterilizing to obtain a Tibetan rhubarb extract solid.

[0011] Preferably, the pH value of the eutectic solvent in steps S1 and S3 is 5-6, and betaine and lactic acid in the eutectic solvent are mixed at a molar ratio of 1:1-10, wherein water is added to the betaine and lactic acid mixture to adjust the pH value of the eutectic solvent.

[0012] Preferably, the mass ratio of the beta-glucosidase to the rhubarb powder is 1-5:100.

[0013] Preferably, the solid-liquid mass ratio of the rhubarb powder to the eutectic solvent is 1:20-100.

[0014] Preferably, the stirring enzymolysis time is 0.5-1.5h.

[0015] Preferably, the stirring enzymolysis temperature is 35-50℃.

[0016] Preferably, the specific operation of the extraction in step S3 comprises:

[0017] The filtered residue is added with pure water at a volume ratio of 1-1.5 times, mixed and stirred for 10-30min, and then centrifuged to obtain an enzymolysis extraction solution, which is repeated twice and collected;

[0018] The dry residue after extraction is added with eutectic solvent at a volume ratio of 3-6 times for dry residue extraction again, stirred for 0.5-1h, repeated twice, and centrifuged and collected to obtain a dry residue extraction solution;

[0019] The collected enzymolysis extraction solution and dry residue extraction solution are combined to obtain an extraction solution.

[0020] Preferably, the dry residue extraction stirring temperature is 20-75℃.

[0021] Preferably, before step S1, it further comprises:

[0022] A1, screening fresh rhubarb crude drugs, cleaning with water, and drying;

[0023] A2, crushing the dried rhubarb to obtain rhubarb powder.

[0024] Provided is a rhubarb extract prepared by the method.

[0025] The present application at least includes the following beneficial effects: the present application simultaneously adds the eutectic solvent composed of betaine and lactic acid and beta-glucosidase, so as to realize the enzymolysis and extraction of rhubarb, simplify the process, and improve the extraction rate of effective components anthraquinones, stilbenes and tannin compounds in rhubarb.

[0026] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0027] Figure 1 This is a flowchart of the preparation process of the present invention;

[0028] Figure 2 This is the standard curve for the determination of anthraquinone compound content according to the present invention;

[0029] Figure 3 This is the standard curve for the determination of stilbene compounds in this invention;

[0030] Figure 4 This is the standard curve for the determination of tannin content in this invention. Detailed Implementation

[0031] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0032] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0033] Cellulase, pectinase, β-glucosidase and other drugs are available on the market. Among them, β-glucosidase is extracted from microorganisms.

[0034] I. Determination methods for anthraquinone compounds

[0035] For anthraquinone compounds, the main detection method is to determine the total anthraquinone content in the compound. The main detection methods are as follows:

[0036] This application employs ultraviolet spectrophotometry, using a methanol solution of magnesium acetate as the colorimetric reagent, to determine the content of anthraquinone compounds (total anthraquinones) by colorimetric determination.

[0037] 1.1 Preparation of color developer

[0038] Weigh 0.4g of magnesium acetate, dissolve it in methanol to prepare a 0.6% magnesium acetate methanol solution, and shake well before use.

[0039] 1.2 Preparation of the total anthraquinone standard curve

[0040] Accurately weigh 10.2 mg of 1,8-dihydroxyanthraquinone, dissolve it in methanol, and dilute to 25 mL with water to obtain a standard solution. Accurately pipette 20, 40, 80, 160, 200, 300, and 400 μL of the standard solution into 10 mL volumetric flasks, add 0.6% magnesium acetate methanol solution, and dilute to 10 mL. After thorough mixing, measure the absorbance at 510 nm. Use 0.6% magnesium acetate methanol solution as a blank control. The results are shown in Table 1 below.

[0041] Table 1 Standard curve of total anthraquinones

[0042] Standard solution (μL) 0 20 40 80 160 200 300 400 Concentration (mg / L) 0 0.816 1.632 3.264 6.528 8.160 12.240 16.320 Absorbance A 0 0.037 0.069 0.141 0.267 0.323 0.532 0.721

[0043] The concentration (mg / L) was plotted on the x-axis and the absorbance A on the y-axis to obtain the following... Figure 2 The standard curve shown has a linear regression equation of y = 0.043x - 0.008, R0. 2 =0.996.

[0044] II. Determination methods for stilbene compounds

[0045] The main determination of stilbene compounds is the content of stilbene glycosides. The main detection methods are as follows:

[0046] This application uses high performance liquid chromatography (HPLC) with 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside as a reference standard to determine the content of stilbene glycosides.

[0047] 2.1 Chromatographic conditions

[0048] C18 column, mobile phase acetonitrile-water (21:79), detection wavelength 320 nm, flow rate 1.0 mL / min -1 Column temperature 30℃.

[0049] 2.2 Preparation of stilbene glycoside standard curve

[0050] Accurately weigh 2.03 mg of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside reference standard and place it in a 25 mL volumetric flask. Dissolve and dilute to volume with 50% ethanol, shake well, and filter through a 0.45 μm microporous membrane to obtain the reference solution. Accurately inject 0 μL, 2.5 μL, 5.0 μL, 7.5 μL, 10.0 μL, and 12.5 μL of the reference solution into the chromatographic sample, respectively, and determine the peak area. Calculate the chromatographic peak area. The results are shown in Table 2 below.

[0051] Table 2 Standard curve of stilbene glycoside

[0052] Sample amount 0.0 μL 2.5 μL 5.0 μL 7.5 μL 10.0 μL 12.5 μL Peak area 0 45003 93214 139187 176325 216987

[0053] The sample amount was plotted on the x-axis and the peak area on the y-axis to obtain the following:Figure 3 The standard curve shown has a linear regression equation of y = 17427x + 2866, R0 2 =0.998.

[0054] III. Determination methods for tannins

[0055] The determination of tannins is mainly based on their content, and the main detection methods are as follows:

[0056] This application uses ultraviolet spectrophotometry, with gallic acid as a reference standard, and determines the content of tannin compounds by phosphomolybdic acid colorimetry.

[0057] 3.1 Preparation of reference solution

[0058] Accurately weigh 50 mg of gallic acid reference standard and place it in a 100 mL brown volumetric flask. Add aqueous solution and dilute to the mark. Accurately measure 2.5 mL of the solution and place it in a 25 mL brown volumetric flask. Dilute to the mark with water and shake well to obtain the reference solution (each 1 mL contains 0.05 mg of gallic acid).

[0059] 3.2 Preparation of gallic acid standard curve

[0060] Accurately measure 0.5 mL, 1.0 mL, 2.0 mL, 3.0 mL, 4.0 mL, and 5.0 mL of the reference solution and place them in separate 25 mL brown volumetric flasks. Add 1.0 mL of phosphomolybdic tungstate solution to each flask, and then add 11.5 mL, 11 mL, 10 mL, 9 mL, 8 mL, and 7 mL of water, respectively. Dilute to the mark with 29% sodium carbonate solution, shake well, and let stand for 30 min. Using the corresponding reagents as blanks, measure the absorbance at a wavelength of 760 nm using ultraviolet-visible spectrophotometry, as shown in Table 3 below.

[0061] Table 3 Standard curve of gallic acid

[0062] Concentration (mg / L) 0 1 2 4 6 8 10 Absorbance A 0.000 0.048 0.093 0.169 0.262 0.352 0.484

[0063] The concentration (mg / L) was plotted on the x-axis and the absorbance A on the y-axis to obtain the following... Figure 4 The standard curve shown has a linear regression equation of y = 0.046x - 0.005, R0. 2 =0.993.

[0064] <Example 1>

[0065] The preparation method of Tibetan rhubarb extract includes the following steps:

[0066] A1. Select fresh Tibetan rhubarb raw materials, wash them with water, and air dry them;

[0067] A2. Crush the dried Tibetan rhubarb into powder to obtain Tibetan rhubarb powder;

[0068] S1. Add 0.1g of β-glucosidase (200,000 U / g) to 10g of Tibetan rhubarb powder, then add 200g of betaine:lactic acid mixed in a molar ratio of 1:1 in a eutectic solvent with a pH of 6. Stir at 35°C for 1h to obtain an enzymatic hydrolysate. Water is added to the mixture of betaine and lactic acid to make the pH of the eutectic solvent 6.

[0069] S2. Filter the enzymatic hydrolysate to obtain filter residue and enzymatic hydrolysate solution;

[0070] S3. Add pure water at a volume ratio of 1 to the filtered residue, mix and stir for 20 minutes, then centrifuge to obtain the enzymatic hydrolysis solution. Repeat twice and collect the enzymatic hydrolysis solution.

[0071] Add a eutectic solvent at a volume ratio of 3 to the leached residue and leach again. Heat and stir at 50°C for 1 hour. Repeat twice. Centrifuge and collect the leaching residue liquid.

[0072] The collected enzymatic hydrolysis solution and the extracted residue were combined to obtain the extract.

[0073] S4. Mix the extract and the enzymatic hydrolysis solution evenly, dry and sterilize to obtain the solid extract of Tibetan rhubarb.

[0074] <Example 2>

[0075] The preparation method of Tibetan rhubarb extract includes the following steps:

[0076] A1. Select fresh Tibetan rhubarb raw materials, wash them with water, and air dry them;

[0077] A2. Crush the dried Tibetan rhubarb into powder to obtain Tibetan rhubarb powder;

[0078] S1. Add 0.3g of β-glucosidase (200,000 U / g) to 10g of Tibetan rhubarb powder, then add 600g of betaine:lactic acid mixed in a molar ratio of 1:5 in a eutectic solvent with a pH of 5.5. Stir at 45℃ for 1h to obtain an enzymatic hydrolysate. Water is added to the mixture of betaine and lactic acid to make the pH of the eutectic solvent 5.5.

[0079] S2. Filter the enzymatic hydrolysate to obtain filter residue and enzymatic hydrolysate solution;

[0080] S3. Add 1.2 times the volume of pure water to the filtered residue, mix and stir for 20 minutes, then centrifuge to obtain the enzymatic hydrolysis solution. Repeat twice and collect the enzymatic hydrolysis solution.

[0081] Add a eutectic solvent at a volume ratio of 4.5 to the leached residue and leach again. Heat and stir at 50°C for 1 hour. Repeat twice. Centrifuge and collect the leaching residue liquid.

[0082] The collected enzymatic hydrolysis solution and the extracted residue were combined to obtain the extract.

[0083] S4. Mix the extract and the enzymatic hydrolysis solution evenly, dry and sterilize to obtain the solid extract of Tibetan rhubarb.

[0084] <Example 3>

[0085] The preparation method of Tibetan rhubarb extract includes the following steps:

[0086] A1. Select fresh Tibetan rhubarb raw materials, wash them with water, and air dry them;

[0087] A2. Crush the dried Tibetan rhubarb into powder to obtain Tibetan rhubarb powder;

[0088] S1. Add 0.5g of β-glucosidase (200,000 U / g) to 10g of Tibetan rhubarb powder, then add 1000g of betaine:lactic acid mixed in a molar ratio of 1:10 in a eutectic solvent with a pH of 5. Stir at 50°C for 1h to obtain an enzymatic hydrolysate. Water is added to the mixture of betaine and lactic acid to make the pH of the eutectic solvent 5.

[0089] S2. Filter the enzymatic hydrolysate to obtain filter residue and enzymatic hydrolysate solution;

[0090] S3. Add 1.5 times the volume of pure water to the filtered residue, mix and stir for 20 minutes, then centrifuge to obtain the enzymatic hydrolysis solution. Repeat twice and collect the enzymatic hydrolysis solution.

[0091] Add a eutectic solvent at a volume ratio of 6 to the leached residue and leach again. Heat and stir at 50°C for 1 hour. Repeat twice. Centrifuge and collect the leaching residue liquid.

[0092] The collected enzymatic hydrolysis solution and the extracted residue were combined to obtain the extract.

[0093] S4. Mix the extract and the enzymatic hydrolysis solution evenly, dry and sterilize to obtain the solid extract of Tibetan rhubarb.

[0094] <Blank Group>

[0095] The preparation of Tibetan rhubarb extract using water extraction includes the following steps:

[0096] A1. Select fresh Tibetan rhubarb raw materials, wash them with water, and air dry them;

[0097] A2. Crush the dried Tibetan rhubarb into powder to obtain Tibetan rhubarb powder;

[0098] S1. Add 1000g of water to 10g of Tibetan rhubarb powder, stir and extract at 50℃ for 1h to obtain a mixed extract;

[0099] S2. Filter the mixed extract to obtain filter residue and filtrate;

[0100] S3. Add 1.5 times the volume of pure water to the filtered residue, mix and stir for 20 minutes, then centrifuge to obtain the extraction solution. Repeat twice and collect the extraction solution.

[0101] Add water at a volume ratio of 6 to the leached residue and leach again. Heat and stir at 50°C for 1 hour. Repeat twice. Centrifuge and collect the leaching residue liquid.

[0102] The collected extraction solution and the extracted residue were combined to obtain the extraction liquid;

[0103] S4. Mix the extract and filtrate evenly, dry and sterilize to obtain Tibetan rhubarb extract solid.

[0104] <Control Group>

[0105] The preparation of Tibetan rhubarb extract by ethanol extraction includes the following steps:

[0106] A1. Select fresh Tibetan rhubarb raw materials, wash them with water, and air dry them;

[0107] A2. Crush the dried Tibetan rhubarb into powder to obtain Tibetan rhubarb powder;

[0108] S1. Add 12 times the volume of percolation solvent to 10g of Tibetan rhubarb powder, soak for 30h, then pack into a percolator, add percolation solvent for percolation extraction, and collect the percolate.

[0109] Percolation extraction conditions: Percolation solvent: 80% ethanol; Percolation solvent flow rate: 7 mL / min -1 , room temperature;

[0110] S2. Percolate elution: The obtained percolate is slowly added to a macroporous adsorption resin separation column, and then eluted with 80% ethanol eluent. The eluate is collected, concentrated under reduced pressure, concentrated, dried and sterilized to obtain Tibetan rhubarb extract solid.

[0111] IV. Content Determination of Tibetan Rhubarb Extract

[0112] 4.1 Determination of anthraquinone compound content in rhubarb extract from Tibetan border areas

[0113] 10 mg of solid Rheum palmatum extract from Examples 1-3, the blank group, and the control group were weighed out respectively. Following method 1.2, the extracts were dissolved in methanol and diluted to 10 mL to prepare sample solutions of Rheum palmatum extract from Examples 1-3. 1 mL of each solution was transferred to a 10 mL volumetric flask, and 0.6% magnesium acetate methanol solution was added to dilute to 10 mL. The absorbance was measured at 510 nm, and the corresponding concentration was calculated based on the regression equation of the total anthraquinone standard curve. The mass of anthraquinone compounds was then calculated, and the content was determined by the mass ratio of anthraquinone compounds to Rheum palmatum extract. The results are shown in Table 4 below.

[0114] Table 4. Determination of anthraquinone content in Rheum palmatum extract from Tibetan border areas.

[0115]

[0116]

[0117] According to the data in Table 4, the anthraquinone compounds in the rhubarb prepared according to Examples 1-3 of the present invention are all higher than those in the blank group. The extraction method of the blank group is similar to the existing general water extraction method, indicating that the preparation method of the present invention can effectively improve the extraction of anthraquinone compounds in rhubarb.

[0118] 4.2 Determination of stilbene compounds in rhubarb extract from Tibetan border areas

[0119] 10 mg of solid Rheum palmatum extract from Examples 1-3, the blank group, and the control group were weighed out respectively. Following method 2.2, the extracts were dissolved in 50% ethanol and diluted to 10 mL to prepare sample solutions of Rheum palmatum extract from Examples 1-3. 10 μL of each solution was injected, the peak area was measured, and the sample amount was obtained according to the regression equation of the stilbene glycoside standard curve. The content of stilbene compounds in Examples 1-3 was then calculated. The results are shown in Table 5 below.

[0120] Table 5. Determination of stilbene compound content in Rheum palmatum extract from Tibetan border in each group.

[0121] Group Peak area Sample amount μL Stilbene compound content (%) Example 1 93779.000 5.217 52.170 Example 2 99259.000 5.531 55.310 Example 3 106051.000 5.921 59.210 Blank group 51873.000 2.807 28.070 Control group 53160.000 2.886 28.860

[0122] According to the data in Table 5, the content of stilbene compounds in the rhubarb prepared according to Examples 1-3 of the present invention is higher than that in the blank group. The extraction method of the blank group is similar to the existing general water extraction method, indicating that the preparation method of the present invention can effectively improve the extraction of stilbene compounds in rhubarb.

[0123] 4.3 Determination of Tannin Content in Rhubarb Extract from Tibetan Border Areas

[0124] Weigh 1g of the solid extract of Rheum palmatum from Examples 1-3, the blank group, and the control group into a 100mL brown volumetric flask, add 50mL of water, let stand overnight, sonicate for 10min, cool, dilute with water to the mark, shake well, let stand (to allow the solid to precipitate), filter, discard 50mL of the initial filtrate, accurately measure 20mL of the subsequent filtrate, put it into a 100mL brown volumetric flask, dilute with water to the mark, shake well, and the sample solution of Examples 1-3 is obtained.

[0125] 4.3.1 Determination of total phenols

[0126] Accurately measure 1 mL of the sample solution from Examples 1-3 and place it in a 25 mL brown volumetric flask. Following the method described under the preparation section of the standard curve, starting from "adding 1 mL of phosphomolybdic tungstic acid test solution", add 10 mL of water, dilute to the mark with 29% sodium carbonate solution, and shake well. Then measure the absorbance. Obtain the corresponding concentration according to the regression equation of the gallic acid standard curve, and calculate the total phenol content in Examples 1-3. The results are shown in Table 6 below:

[0127] Table 6. Determination of total phenolic content in rhubarb extract from Tibetan border regions for each group.

[0128] Group Absorbance A Concentration (mg / L) Total phenol content (%) Example 1 0.249 5.522 6.902 Example 2 0.262 5.804 7.255 Example 3 0.283 6.261 7.826 Blank group 0.146 3.283 4.103 Control group 0.175 3.913 4.891

[0129] 4.31 Determination of non-absorbable polyphenols and tannins

[0130] Accurately measure 1 mL of the sample solution from Examples 1-3 and add it to a 10 mL stoppered conical flask containing 0.6 g of casein. Seal the flask tightly and incubate it in a 30°C water bath for 1 hour, shaking occasionally. Remove the flask, cool it, shake it well, filter it, and discard the initial filtrate. Accurately measure 1 mL of the subsequent filtrate and place it in a 10 mL brown volumetric flask. Following the method under the preparation of the standard curve, starting from "add 1 mL of phosphomolybdic tungstic acid test solution", add 10 mL of water and dilute to the mark with 29% sodium carbonate solution. Shake well and then measure the absorbance. Obtain the corresponding concentration according to the regression equation of the gallic acid standard curve. Calculate the content of non-absorbed polyphenols and tannins in Examples 1-3. The tannin content is calculated using the formula: Tannin content = Total phenol content - Non-absorbed polyphenol content. The results are shown in Table 7 below.

[0131] Table 7. Determination of polyphenol and tannin content in rhubarb extracts from Tibetan border regions for each group.

[0132] Group Absorbance A Concentration (mg / L) Polyphenol content (%) Tannin compound content (%) Example 1 0.102 2.326 2.326 4.576 Example 2 0.109 2.478 2.478 4.777 Example 3 0.113 2.565 2.565 5.261 Blank group 0.059 1.391 1.391 2.712 Control group 0.0907 2.080 2.080 2.811

[0133] According to the data in Tables 6 and 7, the content of tannin compounds in the rhubarb prepared according to Examples 1 to 3 of the present invention is higher than that in the blank group. The extraction method of the blank group is similar to the existing general water extraction method, indicating that the preparation method of the present invention can effectively improve the extraction of tannin compounds in rhubarb.

[0134] To facilitate a direct analysis of the content of active ingredients in the rhubarb extract from Tibetan border areas, the data from Tables 4, 5, and 7 are summarized in Table 8 below:

[0135] Table 8. Content of effective components in Rheum palmatum extract from Tibetan border areas for each group.

[0136]

[0137] As shown in Table 8, the content of active ingredients in the rhubarb prepared according to Examples 1-3 of the present invention is higher than that in the blank group and the control group. This indicates that the preparation method of the present invention can effectively improve the extraction of active ingredients in rhubarb when the stirring and extraction time is similar.

[0138] <Comparative Example 1>

[0139] The preparation method of the Tibetan rhubarb extract is the same as in Example 3, except that β-glucosidase is not added.

[0140] <Comparative Example 2>

[0141] The preparation method of the Tibetan rhubarb extract is the same as that in Example 3, except that β-glucosidase is replaced with cellulase of the same specification.

[0142] <Comparative Example 3>

[0143] The preparation method of the Tibetan rhubarb extract is the same as that in Example 3, except that β-glucosidase is replaced with pectinase of the same specification.

[0144] The contents of anthraquinone compounds, stilbene compounds, and tannin compounds in Comparative Examples 1–3 were determined according to methods 4.1, 4.2, and 4.3, respectively. The results are shown in Table 9 below:

[0145] Table 9. Determination of effective component content in Rheum palmatum extract from Tibetan border in each group.

[0146] Group Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Anthraquinone compound content (%) 5.907 3.216 3.682 3.495 Stilbene compound content (%) 59.210 29.870 34.560 31.240 Tannin compound content (%) 5.261 2.904 3.293 3.143

[0147] According to the data in Table 9, the effective component content of Rheum palmatum prepared in Comparative Examples 1-3 was lower than that in Example 3, and the effective component content of Rheum palmatum prepared in Comparative Example 2 was higher than that in Comparative Example 3 and Comparative Example 1, respectively. This indicates that the addition of enzymes during the extraction of Rheum palmatum extract can improve the extraction of effective components from Rheum palmatum, and the effect of β-glucosidase is the best. This may be because the eutectic solvent composed of betaine and lactic acid can effectively maintain the activity of β-glucosidase, which helps to enzymatically hydrolyze Rheum palmatum and improve the extraction of effective components from Rheum palmatum.

[0148] <Comparative Example 4>

[0149] The preparation method of Tibetan rhubarb extract is the same as in Example 3, except that in step S1, the eutectic solvent is replaced with an equal amount of water.

[0150] <Comparative Example 5>

[0151] The preparation method of the Tibetan rhubarb extract is the same as that in Example 3, except that in step S3, the eutectic solvent is replaced with an equal amount of water.

[0152] The contents of anthraquinone compounds, stilbene compounds, and tannin compounds in Comparative Examples 4–5 were determined according to methods 4.1, 4.2, and 4.3, respectively. The results are shown in Table 10 below:

[0153] Table 10. Determination of effective component content in Rheum palmatum extract from Tibetan border in each group.

[0154] Group Example 3 Comparative Example 4 Comparative Example 5 Anthraquinone compound content (%) 5.907 5.258 5.421 Stilbene compound content (%) 59.210 47.250 57.920 Tannin compound content (%) 5.261 3.827 5.018

[0155] According to the data in Table 10, the content of active ingredients in Rheum palmatum prepared in Comparative Examples 4 and 5 was lower than that in Example 3, indicating that the eutectic solvent can effectively improve the extraction of active ingredients from Rheum palmatum. This may be because the eutectic solvent composed of betaine and lactic acid has a high affinity for the active ingredients (i.e., anthraquinone compounds, stilbene compounds, and tannins) in Rheum palmatum, increasing the solubility of the compounds and thus improving the extraction of active ingredients. The content of active ingredients in Rheum palmatum prepared in Comparative Example 5 was higher than that in Comparative Example 4, indicating that the eutectic solvent can effectively improve the extraction of active ingredients from Rheum palmatum, but its effect is not as good as the effect of eutectic solvent and β-glucosidase combined extraction. This may be because the eutectic solvent composed of betaine and lactic acid can effectively maintain the activity of β-glucosidase, which helps to enzymatically hydrolyze Rheum palmatum, and can also have a high affinity for the active ingredients in Rheum palmatum, increasing the solubility of the compounds, thus improving the extraction of active ingredients from Rheum palmatum in both directions.

[0156] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A method for preparing rhubarb extract from Tibetan border regions, characterized in that, Includes the following steps: S1. Add β-glucosidase to Tibetan rhubarb powder, then add a eutectic solvent, stir and hydrolyze to obtain an enzymatic hydrolysate, wherein the eutectic solvent is composed of betaine, lactic acid and water; S2. Filter the enzymatic hydrolysate to obtain filter residue and enzymatic hydrolysate solution; S3. Extract the filter residue sequentially with pure water and a eutectic solvent, and then filter to obtain the extract. S4. After mixing the extract and the enzymatic hydrolysis solution evenly, dry and sterilize to obtain the solid extract of Tibetan rhubarb. The specific operations of extraction in step S3 include: Add 1 to 1.5 times the volume of pure water to the filtered residue, mix and stir for 10 to 30 minutes, then centrifuge to obtain the enzymatic hydrolysis solution. Repeat twice and collect the enzymatic hydrolysis solution. Add a low-melting solvent with a volume ratio of 3 to 6 times to the leached residue and leach again. Stir for 0.5 to 1 h, repeat twice, centrifuge and collect the leaching residue liquid. The collected enzymatic hydrolysis solution and the extracted residue were combined to obtain the extract.

2. The method for preparing the rhubarb extract from Tibetan border regions as described in claim 1, characterized in that, The pH value of the eutectic solvent in steps S1 and S3 is 5-6, and the betaine and lactic acid in the eutectic solvent are mixed in a molar ratio of 1:1-10. Water is added to the mixture of betaine and lactic acid to adjust the pH value of the eutectic solvent.

3. The method for preparing the rhubarb extract from Tibetan border regions as described in claim 1, characterized in that, The mass ratio of the β-glucosidase to the Tibetan rhubarb powder is 1~5:

100.

4. The method for preparing the rhubarb extract from Tibetan border regions as described in claim 1, characterized in that, The solid-liquid mass ratio of the Tibetan rhubarb powder to the eutectic solvent is 1:20~100.

5. The method for preparing the rhubarb extract from Tibetan border regions as described in claim 1, characterized in that, The stirring and enzymatic hydrolysis time is 0.5~1.5 h.

6. The method for preparing the rhubarb extract from Tibetan border regions as described in claim 1, characterized in that, The temperature for the enzymatic hydrolysis with stirring is 35~50℃.

7. The Tibetan rhubarb extract and its preparation method as described in claim 1, characterized in that, The temperature for stirring the dry residue during extraction is 20~75℃.

8. The Tibetan rhubarb extract and its preparation method as described in claim 1, characterized in that, Before proceeding to step S1, the following is also included: A1. Select fresh Tibetan rhubarb raw materials, wash them with water, and air dry them; A2. Crush the dried Tibetan rhubarb into powder to obtain Tibetan rhubarb powder.

9. The Tibetan rhubarb extract prepared by the method according to any one of claims 1 to 8.

Images