Preparation method and application of rumex madaio extract

Abstract

The invention provides a preparation method and application of a rumex madaio extract. The preparation method comprises the following steps: (1) pre-treating nepal dock root: cleaning fresh nepal dock root, and grinding into paste; (2) fermenting with compound bacteria; (3) hydrochloric acid acidification; (4) flash extraction; (5) decolorizing with activated carbon; and (6) performing membrane concentration to obtain clear paste. The total anthraquinone content and the emodin content extracted by the method are relatively high, and the problems of high loss rate and low extraction rate of effective components in a traditional extraction method are solved. Pharmacological test results prove that the hepatitis B virus resisting effect of the extracted clear paste is not obviously different from that of a lamivudine group, so that the extract disclosed by the invention has relatively strong hepatitis B virus resisting capability.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine, and particularly relates to a preparation method and application of a rhubarb extract. Background Art

[0002] Rumex nepalensis is the fresh or dried root and rhizome of Rumex nepalensis Spreng., Rumex dentatus Linn., or Rumex crispus Linn., all members of the Polygonaceae family. It has the properties of clearing heat and detoxifying, cooling blood and stopping bleeding, killing insects, and promoting purgation. It is used for hepatitis and various inflammatory conditions, conjunctivitis, constipation, and stubborn ringworm. Rumex nepalensis is a medicinal herb used by ethnic minorities in Guizhou Province, and its standard is included in the 2003 edition of the "Quality Standards for Guizhou Traditional Chinese Medicines and Ethnic Medicinal Materials," page 24. Modern research indicates that anthraquinones are the primary active ingredients in Rumex nepalensis for treating hepatitis. However, due to their instability, anthraquinones are significantly lost during Rumex nepalensis harvesting and processing. Experiments conducted by our research team have shown that when fresh Rumex nepalensis is sliced and dried using traditional methods to make medicinal slices, the content of emodin, a key anthraquinone component, decreases by 42.3%. At the same time, due to the poor water solubility of anthraquinones, the extraction rate of anthraquinones using the traditional water decoction process is low. The extraction rate of emodin after water decoction is only 8.72%.

[0003] In view of the above reasons, it is necessary to explore a method to improve the extraction rate of anthraquinone active ingredients in Rheum officinale. After Rheum officinale is prepared into extracts and applied in clinical practice, on the one hand, it can improve the extraction rate of active ingredients and enhance the efficacy of drugs. On the other hand, it can improve the utilization rate of medicinal materials, avoid waste of medicinal materials, and save precious medicinal resources. Summary of the Invention

[0004] The purpose of the present invention is to provide a preparation method of Rheum officinale extract, improve the extraction rate of anthraquinone active ingredients in Rheum officinale, prepare Rheum officinale into extract and apply it in clinical practice, solve the problems of high loss rate of active ingredients and low extraction rate in traditional harvesting, processing and extraction of Rheum officinale, achieve the purpose of enhancing therapeutic effect and saving Rheum officinale medicinal resources.

[0005] Another object of the present invention is to provide the use of the Rheum officinale extract prepared by the preparation method of the present invention in the preparation of a pharmaceutical preparation for treating hepatitis B virus.

[0006] In order to achieve the above objectives, the present invention is implemented through the following technical solutions:

[0007] A method for preparing a rhubarb extract comprises the following steps:

[0008] S1, Rhubarb pretreatment: Wash fresh Rhubarb and grind it into a paste;

[0009] S2, compound fermentation: add 5% to 15% of the total amount of medicinal materials, stir well, inoculate Lactobacillus acidophilus and Paecilomyces hymenoptera, the inoculation amount of both is 5×10 6 CFU / mL, fermented at 35℃~40℃ for 5~9 days;

[0010] S3, acidification: add 60% to 90% ethanol at a concentration of 2 to 6 times the total amount of the medicinal materials, and adjust the pH to 3 to 5 with hydrochloric acid;

[0011] S4, flash extraction: extraction initial temperature 50-60°C, extraction voltage 120-160V, extraction time 10-20min, centrifugal filtration to obtain filtrate;

[0012] S5, decolorization: add 1% to 3% activated carbon to the filtrate for decolorization and filter;

[0013] S6, membrane concentration: using a polyethersulfone nanofiltration membrane with a relative molecular weight cutoff of 100, the filtrate is concentrated into a clear paste at a feed temperature of 50-60°C and a pressure of 0.4-0.6 MPa.

[0014] The step S2 composite fermentation of the present invention is as follows: adding 10% of the total amount of the medicinal material to starch, stirring evenly, inoculating Lactobacillus acidophilus and Paecilomyces avium, and the inoculation amount of both is 5×10 6 CFU / mL, fermented at 35 °C for 7 days.

[0015] The acidification step S3 of the present invention is as follows: adding 75% ethanol with a concentration twice the total amount of the medicinal materials, and adjusting the pH to 4 with hydrochloric acid.

[0016] The flash extraction step S4 of the present invention is as follows: extraction initial temperature 55° C., extraction voltage 140 V, extraction time 20 min, centrifugal filtration to obtain filtrate.

[0017] The decolorization step S5 of the present invention comprises: adding 2% activated carbon to the filtrate for decolorization, and filtering.

[0018] The membrane concentration step S6 of the present invention is as follows: using a polyethersulfone nanofiltration membrane with a relative molecular weight cutoff of 100, at a feed temperature of 50-55° C. and a pressure of 0.4-0.5 MPa, the filtrate is concentrated into a clear paste.

[0019] The membrane concentration step S6 of the present invention is as follows: using a polyethersulfone nanofiltration membrane with a relative molecular weight cutoff of 100, the filtrate is concentrated into a clear paste at a feed temperature of 50° C. and a pressure of 0.4 MPa.

[0020] The invention discloses an application of the clear paste prepared by the method for preparing the rhubarb extract in preparing a pharmaceutical preparation for treating hepatitis B virus.

[0021] The pharmaceutical preparation of the present invention is a solid preparation or a liquid preparation.

[0022] The solid preparations of the present invention include but are not limited to granules, capsules, tablets, and pills; the liquid preparations include but are not limited to mixtures.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. Avoid loss of active ingredients. During the traditional cutting and drying process of fresh rhubarb, the loss rate of the anthraquinone active ingredient, emodin, is 42.3%. However, the present invention reduces the loss of emodin to only 10% after fresh refining. Fresh refining maximizes the retention of active ingredients in the medicinal material and avoids loss of active ingredients during the cutting and drying process.

[0025] 2. Improve the extraction rate of effective ingredients. The present invention adopts a "composite fermentation + flash extraction + membrane concentration" extraction and purification process, which is carried out entirely at low temperature, resulting in a high extraction rate of effective ingredients. Testing shows that the extraction rate of emodin using the conventional water decoction process is only 8.72%, while the "composite fermentation + flash extraction" extraction process has an extraction rate of up to 59.62%.

[0026] 3. Saving medicinal resources. The "fresh composite fermentation and flash extraction membrane filtration method" of the present invention can effectively avoid the loss of active ingredients and improve the extraction rate of active ingredients, thereby saving medicinal resources.

[0027] 4. Enhance the efficacy of drugs. The results of pharmacological tests showed that the anti-HBV effect of the present invention group was not significantly different from that of the lamivudine group, indicating that the extract of the present invention has a strong anti-HBV ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 :Chromatogram of Rhein Reference Substance 01

[0029] Figure 2 :Chromatogram of Rhein Reference Substance 02

[0030] Figure 3 :Best process verification experiment 1 flavin content determination chromatogram

[0031] Figure 4 :Best process verification experiment 2 flavin content determination chromatogram

[0032] Figure 5 :Best process verification experiment 3 flavin content determination chromatogram DETAILED DESCRIPTION

[0033] Example 1 Preparation method of Rheum officinale extract

[0034] (1) Pretreatment of Rhubarb: Wash the fresh Rhubarb and grind it into a paste using a grinder;

[0035] (2) Compound fermentation: Add 10% of the total amount of the medicinal materials into the starch, stir well, and inoculate Lactobacillus acidophilus and Paecilomyces hymenoptera with the inoculation amount of both being 5×10 6 CFU / mL, fermented at 35°C for 7 days;

[0036] (3) Acidification: Add 75% ethanol (2 times the total amount of the medicinal material) and adjust the pH to 4 with hydrochloric acid;

[0037] (4) Flash extraction: extraction initial temperature 55 ° C, extraction voltage 140 V, extraction time 20 min, centrifugal filtration, and obtain the filtrate;

[0038] (5) Decolorization: Add 2% activated carbon to the filtrate for decolorization and filter;

[0039] (6) Membrane concentration: Using a polyethersulfone nanofiltration membrane with a relative molecular weight cutoff of 100, the decolorized filtrate was concentrated into a clear paste with a ratio of fresh medicinal materials to clear paste of 3:1 at a feed temperature of 50°C and a pressure of 0.4 MPa.

[0040] Example 2 Preparation method of Rheum officinale extract

[0041] (1) Pretreatment of Rhubarb: Wash the fresh Rhubarb and grind it into a paste using a grinder;

[0042] (2) Compound fermentation: Add 5% starch of the total amount of medicinal materials, stir well, and inoculate Lactobacillus acidophilus and Paecilomyces hymenoptera with the inoculation amount of both being 5×10 6 CFU / mL, fermented at 40°C for 5 days;

[0043] (3) Acidification: Add 60% ethanol (4 times the total amount of the medicinal material) and adjust the pH to 3 with hydrochloric acid;

[0044] (4) Flash extraction: extraction initial temperature 50 ° C, extraction voltage 120 V, extraction time 15 min, centrifugal filtration, and obtain the filtrate;

[0045] (5) Decolorization: Add 1% activated carbon to the filtrate for decolorization and filter;

[0046] (6) Membrane concentration: Using a polyethersulfone nanofiltration membrane with a relative molecular weight cutoff of 100, the decolorized filtrate was concentrated into a clear paste with a ratio of fresh medicinal materials to clear paste of 3:1 at a feed temperature of 50°C and a pressure of 0.5 MPa.

[0047] Example 3 Preparation method of Rheum officinale extract

[0048] (1) Pretreatment of Rhubarb: Wash the fresh Rhubarb and grind it into a paste using a grinder;

[0049] (2) Compound fermentation: Add 15% of the total amount of the medicinal materials into the starch, stir well, and inoculate Lactobacillus acidophilus and Paecilomyces hymenoptera with the inoculation amount of both being 5×10 6 CFU / mL, fermented at 40°C for 9 days;

[0050] (3) Acidification: Add 80% ethanol (6 times the total amount of the medicinal material) and adjust the pH to 5 with hydrochloric acid;

[0051] (4) Flash extraction: extraction initial temperature 60 ° C, extraction voltage 160 V, extraction time 10 min, centrifugal filtration, and obtain the filtrate;

[0052] (5) Decolorization: Add 3% activated carbon to the filtrate for decolorization and filter;

[0053] (6) Membrane concentration: Using a polyethersulfone nanofiltration membrane with a relative molecular weight cutoff of 100, the decolorized filtrate was concentrated into a clear paste with a ratio of fresh medicinal materials to clear paste of 3:1 at a feed temperature of 60°C and a pressure of 0.5 MPa.

[0054] Example 4 Preparation method of Rheum officinale extract

[0055] (1) Pretreatment of Rhubarb: Wash the fresh Rhubarb and grind it into a paste using a grinder;

[0056] (2) Compound fermentation: Add 10% of the total amount of the medicinal materials into the starch, stir well, and inoculate Lactobacillus acidophilus and Paecilomyces hymenoptera with the inoculation amount of both being 5×10 6 CFU / mL, fermented at 35°C for 5 days;

[0057] (3) Acidification: Add 70% ethanol (2 times the total amount of the medicinal material) and adjust the pH to 3 with hydrochloric acid;

[0058] (4) Flash extraction: extraction initial temperature 50 ° C, extraction voltage 120 V, extraction time 20 min, centrifugal filtration, and obtain the filtrate;

[0059] (5) Decolorization: Add 3% activated carbon to the filtrate for decolorization and filter;

[0060] (6) Membrane concentration: Using a polyethersulfone nanofiltration membrane with a relative molecular weight cutoff of 100, the decolorized filtrate was concentrated into a clear paste with a ratio of fresh medicinal materials to clear paste of 3:1 at a feed temperature of 55°C and a pressure of 0.5 MPa.

[0061] Example 5 Preparation method of Rheum officinale extract

[0062] (1) Pretreatment of Rhubarb: Wash the fresh Rhubarb and grind it into a paste using a grinder;

[0063] (2) Compound fermentation: Add 15% of the total amount of the medicinal materials into the starch, stir well, and inoculate Lactobacillus acidophilus and Paecilomyces hymenoptera with the inoculation amount of both being 5×10 6 CFU / mL, fermented at 40°C for 9 days;

[0064] (3) Acidification: Add 60% ethanol (4 times the total amount of the medicinal material) and adjust the pH to 4 with hydrochloric acid;

[0065] (4) Flash extraction: extraction initial temperature 60 ° C, extraction voltage 160 V, extraction time 10, centrifugal filtration, and obtain filtrate;

[0066] (5) Decolorization: Add 23% activated carbon to the filtrate for decolorization and filter;

[0067] (6) Membrane concentration: Using a polyethersulfone nanofiltration membrane with a relative molecular weight cutoff of 100, the decolorized filtrate was concentrated into a clear paste with a ratio of fresh medicinal materials to clear paste of 3:1 at a feed temperature of 50°C and a pressure of 0.4 MPa.

[0068] Example 6 Preparation method of Rheum officinale extract

[0069] (1) Pretreatment of Rhubarb: Wash the fresh Rhubarb and grind it into a paste using a grinder;

[0070] (2) Compound fermentation: Add 5% starch of the total amount of medicinal materials, stir well, and inoculate Lactobacillus acidophilus and Paecilomyces hymenoptera with the inoculation amount of both being 5×10 6 CFU / mL, fermented at 35°C for 9 days;

[0071] (3) Acidification: Add 75% ethanol (6 times the total amount of the medicinal material) and adjust the pH to 3 with hydrochloric acid;

[0072] (4) Flash extraction: extraction initial temperature 60 ° C, extraction voltage 140 V, extraction time 20 min, centrifugal filtration, and obtain the filtrate;

[0073] (5) Decolorization: Add 2% activated carbon to the filtrate for decolorization and filter;

[0074] (6) Membrane concentration: Using a polyethersulfone nanofiltration membrane with a relative molecular weight cutoff of 100, the decolorized filtrate was concentrated into a clear paste with a ratio of fresh medicinal materials to clear paste of 3:1 at a feed temperature of 55°C and a pressure of 0.5 MPa.

[0075] Example 7

[0076] Take 80 g of the clear paste prepared in any of Examples 1 to 6, add 20 g of monosaccharide syrup, and stir well to obtain the rhubarb mixture.

[0077] Example 8

[0078] Take any of the clear pastes prepared in Examples 1 to 6, add an appropriate amount of soluble starch, granulate, dry, and re-granulate to obtain Rhubarb granules.

[0079] Example 9

[0080] Take any clear paste prepared in Example 1 to Example 6, concentrate it into a thick paste, add appropriate amount of poly starch and dextrin, mix well, granulate, dry, granulate, and tablet, so as to obtain Rhubarb tablets.

[0081] Example 10

[0082] Take any of the clear pastes prepared in Examples 1 to 6 and concentrate them into a thick paste.

[0083] Add appropriate amount of polyethylene glycol 4000 to the thick paste and drip it into Tu Da Huang pills.

[0084] Example 11

[0085] Take any clear paste prepared in Example 1 to Example 6, concentrate it into a thick paste, add an appropriate amount of sodium carboxymethyl starch and mix evenly, then add a small amount of 0.5% sodium carboxymethyl cellulose solution to granulate, dry, and granulate, then add magnesium stearate and mix evenly, and fill into capsules to obtain Rhubarb capsules.

[0086] In order to further verify the feasibility and effectiveness of the present invention and screen out the best solution, the present invention conducted a series of experiments, some of which are excerpted as follows:

[0087] 1. Extraction process research

[0088] 1 Comparison of Rhubarb Processing Methods

[0089] (1) Emodin content determination method

[0090] Determined by high performance liquid chromatography.

[0091] The chromatographic conditions and system suitability test used octadecylsilane bonded silica gel as the filler, methanol-0.1% phosphoric acid solution (85:15) as the mobile phase, and a detection wavelength of 436 nm. The theoretical plate number calculated based on the rhein peak should be no less than 3000.

[0092] Preparation of reference solution: Accurately weigh an appropriate amount of emodin reference solution and add methanol to make a solution containing 80 μg per 1 ml.

[0093] Preparation of the test solution: Take approximately 1.5 g of the sample, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of methanol, weigh it, heat and reflux for 1 hour, let it cool, weigh it again, make up the lost weight with methanol, shake it well, and filter it. Accurately measure 5 ml of the filtrate, place it in a flask, evaporate the solvent, add 10 ml of 8% hydrochloric acid solution, sonicate for 2 minutes, add 10 ml of chloroform, heat and reflux for 1 hour, let it cool, place it in a separatory funnel, wash the container with a small amount of chloroform, and combine it in the separatory funnel. Separate the chloroform layer, extract the acid solution with chloroform three times, 10 ml each time, combine the chloroform solutions, recover the solvent under reduced pressure to dryness, dissolve the residue in methanol, transfer it to a 10 ml volumetric flask, add methanol to the mark, shake it well, filter it, and take the filtrate.

[0094] Determination method: Accurately aspirate 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0095] (2) Comparison of processing methods

[0096] Take fresh Rhubarb, wash it, and dry the surface moisture of the fresh material. The emodin content is measured to be 0.14% while it is fresh. Take 2 portions of fresh material, each 1000g, one portion is sliced and dried according to the traditional process, and the other portion is ground into pulp while it is fresh. The emodin content of each portion is measured. The results are shown in Table 1.

[0097] Table 1 Comparative test results of Rheum officinale processing methods

[0098]

[0099] The experimental results show that the loss rate of rheum officinale is as high as 42.3% when processed by the traditional slicing and drying method, while the loss rate is only 10.0% when the fresh grinding method is used, indicating that rheum officinale should be processed while it is fresh.

[0100] 2. Comparison of water extraction

[0101] Take 100 g of dried Rheum officinale and 100 g of fresh Rheum officinale respectively, add water and boil them separately. After the decoction is concentrated, the volume is fixed to 50 ml. The content of emodin is determined according to the above method. The results are shown in Table 2.

[0102] Table 2 Water extraction comparison test results

[0103]

[0104] The experimental results show that the extraction rate of emodin using water extraction process is low and the utilization rate of the effective ingredients of the medicinal materials is not high, so it is necessary to seek a better extraction method.

[0105] 3. Research on the extraction process of fresh Rheum officinale

[0106] Since the loss rate of effective ingredients in Rheum officinale is high after being processed into dry medicinal materials, this study used different extraction methods while it was fresh. The content of emodin and total anthraquinones in the extracts was used as indicators to compare ultrasonic extraction, flash extraction, fermentation and enzymatic extraction.

[0107] (1) Determination of total anthraquinone content

[0108] Using ultraviolet spectrophotometry.

[0109] Preparation of color developer: Weigh an appropriate amount of magnesium acetate, dissolve it in methanol to prepare a 0.5% magnesium acetate methanol solution, shake it thoroughly and set aside.

[0110] Preparation of reference solution: Weigh 13 mg of 1,8-dihydroxyanthraquinone standard, place in a 50 mL volumetric flask, add methanol to the mark, and shake well.

[0111] To create a standard curve, pipette 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, and 10.0 mL of the 1,8-dihydroxyanthraquinone standard solution into a 100 mL volumetric flask. Add 0.5% magnesium acetate methanol solution to 100 mL, shake well, and measure the absorbance at 523 nm using a spectrophotometer with the reagent blank solution as the reference. Plot a standard curve with the 1,8-dihydroxyanthraquinone concentration as the abscissa and the absorbance as the ordinate.

[0112] Preparation of test solution Take 20 mL of sample under each test item, place it in a conical flask, add 3.0 mL of 30% hydrogen peroxide solution and 0.5 mL of hydrochloric acid solution (1+1), reflux in a 90°C water bath for 30 min, cool, extract with ether 3 times, 20 mL each time, combine the ether solution, wash with water 2 times, 10 mL each time, evaporate the ether solution, dissolve the residue with magnesium acetate methanol solution and transfer it to a 10 mL volumetric flask, dilute to the scale with magnesium acetate methanol solution, mix well, and use as the test solution.

[0113] (2) Ultrasonic method

[0114] Take 100g of fresh rhubarb, wash it and grind it into pulp, add 4 times 75% ethanol, and ultrasonically extract it at an ultrasonic power of 250W for 30 minutes. Filter to obtain the filtrate, concentrate the filtrate and make the volume to 50ml, and determine the contents of total anthraquinone and rhein. The results are shown in Table 3.

[0115] (3) Enzymatic hydrolysis

[0116] Take 100g of fresh rhubarb, wash it and grind it into pulp, add 4 times water and 2% cellulase, and hydrolyze it at 50℃ and pH 5.0 for 24 hours. Filter it, concentrate the filtrate and make it up to 50ml. Determine the contents of total anthraquinone and rhein. The results are shown in Table 3.

[0117] (4) Flash Extraction

[0118] Take 100g of fresh rhubarb, wash it and grind it into pulp, add 4 times 75% ethanol, extract at an initial temperature of 60℃, extraction voltage of 140V, extraction time of 20min, centrifuge and filter, concentrate the filtrate and make the volume to 50ml, determine the contents of total anthraquinone and rhein, the results are shown in Table 3.

[0119] (5) Fermentation method

[0120] Take 100g of fresh rhubarb, wash it and grind it into pulp, add 10% of the total amount of the medicinal material starch, stir well, inoculate Lactobacillus acidophilus, and the inoculation amount is 5×10 6 CFU / mL, fermented at 35℃ for 6 days, centrifuged and filtered, the filtrate concentrated and fixed to 50ml, and the total anthraquinone and emodin contents were determined. The results are shown in Table 3.

[0121] Table 3 Optimization results of different extraction methods

[0122]

[0123] The test results show that the fermentation method has the highest content of total anthraquinone and emodin in the extracted medicinal liquid, followed by the flash extraction method, but the extraction rate of both methods does not exceed 40%. The two methods can be combined for extraction. At the same time, research data show that the extraction rate of anthraquinone will increase after acidification, so the rhubarb pulp can be acidified for further extraction.

[0124] (6) Composite extraction method

[0125] Take 100g of fresh rhubarb, wash it and grind it into pulp, add 10% of the total amount of the medicinal material starch, stir well, inoculate Lactobacillus acidophilus, and the inoculation amount is 5×10 6 CFU / mL, ferment at 35℃ for 6 days, add 70% ethanol (6 times the total amount of the medicinal materials), adjust the pH to 4 with hydrochloric acid, stir well, heat the material to 60℃ for flash extraction, extraction voltage 140V, extraction time 20min, centrifuge and filter, concentrate the filtrate and make up to 50ml, and determine the contents of total anthraquinone and emodin.

[0126] Results: The total anthraquinone content was 32.18 mg / ml, the emodin content was 1.121 mg / ml, and the emodin extraction rate was 40.0%. These results indicate that the mixed extraction method significantly improved the extraction rates of both total anthraquinones and emodin, and further experiments are warranted to determine the optimal extraction parameters.

[0127] 4. Optimization of process parameters for composite extraction method

[0128] (1) Optimization of fermentation strains

[0129] Traditional Chinese medicine fermentation strains mainly fall into two categories: bacteria and fungi. The main bacteria include Bifidobacterium, Bifidobacterium adolescentis, Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus acidophilus, etc. The main fungi include Saccharomyces cerevisiae, Saccharomyces cerevisiae, Paecilomyces pelargonifolia, and Monascus.

[0130] Based on the fermentation characteristics of bacteria and fungi and combined with literature search data, this study selected Bifidobacterium adolescentis (strain number ATCC15703, purchased from Beijing Biobowei Biotechnology Co., Ltd.), Lactobacillus acidophilus (strain number CICC6096, purchased from China Industrial Microbiological Culture Collection Center), Paecilomyces hymenoptera (strain number Bio-57623, purchased from Beijing Biobowei Biotechnology Co., Ltd.) and Monascus purpureus (strain number L807, purchased from Beijing Biobowei Biotechnology Co., Ltd.) as key screening strains to carry out the following experiments:

[0131] Take 800g of fresh rhubarb, wash it and grind it into pulp, divide it into 8 equal parts, add 10% of the total amount of medicinal materials into starch, stir well, and inoculate the strains according to Table 4, with the inoculation amount of 5×10 6 CFU / mL, fermented at 35℃ for 6 days, added 70% ethanol (6 times the total amount of the medicinal materials), adjusted the pH to 4 with hydrochloric acid, stirred well, heated the material to 60℃ for flash extraction, the extraction voltage was 140V, the extraction time was 20min, centrifuged and filtered, the filtrate was concentrated and the volume was made up to 50ml, and the contents of total anthraquinone and emodin were determined. The results are shown in Table 4.

[0132] Table 4 Fermentation strain optimization results

[0133]

[0134] The experimental results showed that after fermentation extraction with "Lactobacillus acidophilus + Paecilomyces aviculare", the content of total anthraquinone and emodin in the extract was the highest, so Lactobacillus acidophilus and Paecilomyces aviculare were selected as the fermentation strains for this process.

[0135] (2) Optimal amount of fermented starch

[0136] Take 300g of fresh rhubarb, wash it and grind it into pulp, divide it into 3 parts, add 5%, 10% and 15% of the total amount of the medicinal materials into starch respectively, stir well, inoculate Lactobacillus acidophilus and Paecilomyces aviculare strains, and inoculate 5×10 6 CFU / mL, fermented at 35℃ for 6 days, added 70% ethanol (6 times the total amount of the medicinal materials), adjusted the pH to 4 with hydrochloric acid, stirred well, heated the material to 60℃ for flash extraction, the extraction voltage was 140V, the extraction time was 20min, centrifuged and filtered, the filtrate was concentrated and the volume was made up to 50ml, and the contents of total anthraquinone and emodin were determined. The results are shown in Table 5.

[0137] Table 5 Optimization results of starch dosage

[0138]

[0139] The test results show that the extraction effects are similar when the starch dosage is 10% and 15%, and are significantly higher than that of 5%. 10% can be selected as the optimal starch dosage parameter.

[0140] (3) Optimal fermentation temperature

[0141] Take 300g of fresh rhubarb, wash it and grind it into pulp, divide it into 3 parts, add 10% of the total amount of the medicinal material into starch, stir it well, inoculate Lactobacillus acidophilus and Paecilomyces aviculare strains, and inoculate 5×10 6 CFU / mL, fermented at 35℃, 37℃ and 40℃ for 6 days, added 70% ethanol (6 times the total amount of the medicinal materials), adjusted the pH to 4 with hydrochloric acid, stirred well, heated the material to 60℃ for flash extraction, the extraction voltage was 140V, the extraction time was 20min, centrifuged and filtered, the filtrate was concentrated and the volume was made up to 50ml, and the contents of total anthraquinone and emodin were determined. The results are shown in Table 6.

[0142] Table 6 Fermentation temperature optimization results

[0143]

[0144] The experimental results show that the fermentation temperature has little effect on the extraction effect, and 35℃~40℃ can be selected.

[0145] (4) Optimization of fermentation time

[0146] Take 400g of fresh rhubarb, wash it and grind it into pulp, divide it into 4 parts, add 10% of the total amount of the medicinal material into starch, stir it well, inoculate Lactobacillus acidophilus and Paecilomyces aviculare strains, and inoculate 5×10 6 CFU / mL, fermented at 35℃ for 3 days, 5 days, 7 days and 9 days respectively, added 70% ethanol (6 times the total amount of the medicinal materials), adjusted the pH to 4 with hydrochloric acid, stirred well, heated to 60℃ for flash extraction, extraction voltage 140V, extraction time 20min, centrifuged and filtered, the filtrate was concentrated and the volume was made up to 50ml, and the contents of total anthraquinone and emodin were determined. The results are shown in Table 7.

[0147] Table 7 Fermentation time optimization results

[0148]

[0149] The test results showed that the increase in the contents of total anthraquinone and emodin was not significant after 5 days of fermentation, and 7 days could be selected as the optimal fermentation time.

[0150] (5) Optimal ethanol concentration

[0151] Take 300g of fresh rhubarb, wash it and grind it into pulp, add 10% of the total amount of the medicinal material starch, stir well, inoculate Lactobacillus acidophilus and Paecilomyces aviculare strains, and inoculate 5×10 6 CFU / mL, fermented at 35°C for 6 days, divided the fermentation material into 3 equal parts, added 4 times 60%, 75% and 90% ethanol respectively, adjusted the pH to 4 with hydrochloric acid, and then heated to 60°C for flash extraction. The extraction voltage was 140V and the extraction time was 20min. The material was centrifuged and filtered, and the filtrate was concentrated and fixed to 50ml. The contents of total anthraquinone and emodin were determined. The results are shown in Table 8.

[0152] Table 8 ethanol concentration optimization results

[0153]

[0154] The test results show that the extraction effects are similar when the ethanol concentration is 75% and 90%, both higher than 60% ethanol. Considering the cost factor, 75% can be selected as the optimal ethanol concentration.

[0155] (6) Adding alcohol twice as much is preferred

[0156] Take 300g of fresh rhubarb, wash it and grind it into pulp, add 10% of the total amount of the medicinal material starch, stir well, inoculate Lactobacillus acidophilus and Paecilomyces aviculare strains, and inoculate 5×10 6 CFU / mL, fermented at 35°C for 6 days, divided the fermentation material into 3 equal parts, added 2 times, 4 times and 6 times 75% ethanol respectively, adjusted the pH to 4 with hydrochloric acid, and then heated to 60°C for flash extraction, with an extraction voltage of 140V and an extraction time of 20min. The material was centrifuged and filtered, and the filtrate was concentrated and fixed to 50ml. The contents of total anthraquinone and emodin were determined. The results are shown in Table 9.

[0157] Table 9 Optimal results of alcohol addition times

[0158]

[0159] The experimental results show that the extraction effects of different alcohol dosages are not much different. In order to save production costs, 2 times the amount can be selected as the optimal ethanol dosage.

[0160] (7) pH

[0161] Take 300g of fresh rhubarb, wash it and grind it into pulp, add 10% of the total amount of the medicinal material starch, stir well, inoculate Lactobacillus acidophilus and Paecilomyces aviculare strains, and inoculate 5×10 6CFU / mL, fermented at 35°C for 6 days, divided the fermentation material into 3 equal parts, added 4 times 75% ethanol, adjusted the pH to 3, 4, and 5 respectively with hydrochloric acid, and then heated to 60°C for flash extraction. The extraction voltage was 140V, the extraction time was 20min, and the material was centrifuged and filtered. The filtrate was concentrated and the volume was made up to 50ml. The contents of total anthraquinone and emodin were determined. The results are shown in Table 10.

[0162] Table 10 pH optimization results

[0163]

[0164] The test results show that the lower the pH, the better the extraction effect. There is little difference between pH3 and pH4, but both are significantly higher than pH5. Therefore, pH4 can be selected as the optimal process parameter.

[0165] (8) Initial temperature of flash extraction

[0166] Take 300g of fresh rhubarb, wash it and grind it into pulp, add 10% of the total amount of the medicinal material starch, stir well, inoculate Lactobacillus acidophilus and Paecilomyces aviculare strains, and inoculate 5×10 6 CFU / mL, fermented at 35°C for 6 days, divided the fermentation material into 3 equal parts, added 4 times 75% ethanol, adjusted the pH to 4 with hydrochloric acid, and then heated to 50°C, 55°C and 60°C for flash extraction, with an extraction voltage of 140V and an extraction time of 20min. The material was centrifuged and filtered, and the filtrate was concentrated and fixed to 50ml. The contents of total anthraquinone and emodin were determined. The results are shown in Table 11.

[0167] Table 11 Optimization results of initial temperature of flash extraction

[0168]

[0169] The test results show that among the three initial temperatures, 55℃ has the best extraction effect, followed by 60℃, and the extraction effect at 50℃ is slightly worse, so 55℃ is selected as the optimal process parameter.

[0170] (9) Flash voltage

[0171] Take 300g of fresh rhubarb, wash it and grind it into pulp, add 10% of the total amount of the medicinal material starch, stir well, inoculate Lactobacillus acidophilus and Paecilomyces aviculare strains, and inoculate 5×10 6 CFU / mL, fermented at 35°C for 6 days, divided the fermentation material into 3 equal parts, added 4 times 75% ethanol, adjusted the pH to 4 with hydrochloric acid, and then heated to 60°C for flash extraction. The extraction voltage was set to 120V, 140V and 160V respectively, the extraction time was 20min, and the mixture was centrifuged and filtered. The filtrate was concentrated and the volume was made up to 50ml. The contents of total anthraquinone and emodin were determined. The results are shown in Table 12.

[0172] Table 12 Flash voltage optimization results

[0173]

[0174] The test results show that the extraction voltage has little effect on the extraction effect, so the voltage of 120V~160V is selected as the process parameter.

[0175] (10) Flash withdrawal time

[0176] Take 300g of fresh rhubarb, wash it and grind it into pulp, add 10% of the total amount of the medicinal material starch, stir well, inoculate Lactobacillus acidophilus and Paecilomyces aviculare strains, and inoculate 5×10 6 CFU / mL, fermented at 35°C for 6 days, divided the fermentation material into 3 equal parts, added 4 times 75% ethanol, adjusted the pH to 4 with hydrochloric acid, and then heated to 60°C for flash extraction. The extraction voltage was 160V, and the extraction times were 10min, 15min and 20min respectively. The filtrate was centrifuged and filtered, and the volume was adjusted to 50ml. The contents of total anthraquinone and emodin were determined. The results are shown in Table 13.

[0177] Table 13 Flash extraction time optimization results

[0178]

[0179] The test results show that there is little difference between flash extraction for 15 minutes and 20 minutes, but both are significantly higher than extraction for 10 minutes, so extraction for 20 minutes is selected as the optimal process parameter.

[0180] 5. Optimization of membrane concentration process parameters

[0181] Since anthraquinones are unstable when exposed to heat, the extract should be concentrated by low-temperature concentration method. Membrane concentration is a low-temperature concentration method that has been widely used in the pharmaceutical industry. Therefore, this study decided to use membrane concentration method to concentrate the rhubarb extract. According to the molecular characteristics of rhubarb anthraquinones, a polyethersulfone nanofiltration membrane with a cutoff relative molecular weight of 100 was selected. According to the characteristics of the membrane concentration process, the feed temperature and pressure of membrane concentration were studied.

[0182] (1) Material pretreatment

[0183] Take 600g of fresh rhubarb, wash it and grind it into pulp, add 10% of the total amount of the medicinal material starch, stir it evenly, inoculate Lactobacillus acidophilus and Paecilomyces aviculare strains, and inoculate 5×10 6 CFU / mL, ferment at 35℃ for 7 days, add 2 times 75% ethanol, adjust the pH to 4 with hydrochloric acid, then heat the material to 55℃ for flash extraction, extraction voltage 140V, extraction 20min, centrifugal filtration, decolorize the filtrate with 2% activated carbon and determine the total anthraquinone and emodin contents, and divide the filtrate into 6 parts for subsequent tests.

[0184] (2) Feed temperature

[0185] Three portions of the filtrate were taken and concentrated into a clear paste using a polyethersulfone nanofiltration membrane with a relative molecular weight cutoff of 100 at feed temperatures of 50°C, 55°C, and 60°C and a pressure of 0.5 MPa. The contents of total anthraquinone and emodin were determined, and the retention rates of total anthraquinone and emodin were calculated. The results are shown in Table 14.

[0186] Table 14 Feed temperature optimization results

[0187]

[0188] The test results showed that the retention rates of total anthraquinone and emodin decreased with increasing temperature, and 50℃~55℃ was finally selected as the optimal process parameters.

[0189] (3) Concentration pressure

[0190] Three portions of the filtrate were taken and concentrated into a clear paste using a polyethersulfone nanofiltration membrane with a relative molecular weight cutoff of 100 at a feed temperature of 55°C and pressures of 0.4 MPa, 0.5 MPa, and 0.6 MPa, respectively. The contents of total anthraquinone and emodin were determined, and the retention rates of total anthraquinone and emodin were calculated. The results are shown in Table 15.

[0191] Table 15 Concentration pressure optimization results

[0192]

[0193] The test results show that the retention rates of total anthraquinone and emodin decrease with increasing pressure, and the retention rate at 0.6 MPa is less than 90%. Therefore, the optimal concentration pressure is 0.4 MPa to 0.5 MPa.

[0194] 6. Best process verification test

[0195] Based on the above experimental research results, the optimal extraction process of Rheum officinale is determined to be:

[0196] Take fresh rhubarb, wash it and grind it into pulp, add 10% of the total amount of the medicinal material starch, stir it evenly, inoculate Lactobacillus acidophilus and Paecilomyces aviculare strains, the inoculation amount is 5×10 6 CFU / mL, ferment at 35°C for 7 days, add 2 times 75% ethanol, adjust the pH to 4 with hydrochloric acid, then heat the material to 55°C for flash extraction, the extraction voltage is 140V, the extraction time is 20min, and centrifugal filtration is performed; the filtrate is decolorized with 2% activated carbon and then filtered through a polyethersulfone nanofiltration membrane with a relative molecular weight cutoff of 100, at a feed temperature of 50°C and a pressure of 0.4MPa, the filtrate is concentrated into a clear paste.

[0197] In order to verify the stability and reproducibility of the process, three batches of validation tests were carried out in this study, and the validation results are shown in Table 16. Figures 1 to 5.

[0198] Table 16 Process verification results

[0199]

[0200] The test results show that the process has good stability and reproducibility, indicating that the process is reasonable and feasible.

[0201] 2. Drug Efficacy Verification

[0202] In order to verify the scientific nature and advancement of the extraction method of the present invention, as well as the effectiveness of the extract of the present invention, we conducted an anti-hepatitis B virus pharmacological test on the extract of the present invention.

[0203] 1. Experimental Animals

[0204] Fifty adult male rats of specific pathogen-free (SPF) grade, weighing (210±20) g, aged 4 to 6 weeks, were housed in a cage of 5 rats. The animal breeding environment temperature was (23±0.5)°C, relative humidity was 65%, and drinking water and food were available ad libitum. All rats were fed adaptively for 7 days before the experiment.

[0205] 2. Animal grouping, modeling, medication, and specimen collection

[0206] The rats were randomly divided into 5 groups: normal group, model group, positive control group, invention group and decoction group, with 10 rats in each group. Except for the normal group, 1×10 6 An AAV-HBV rat model was established by administering 10 ml / kg / day of HBV virus (diluted with Hanks' solution) once every 7 days. A positive result in the hepatitis B antigen test panel indicated successful modeling, and subsequent experiments were initiated.

[0207] The invention group received an extract of Rheum officinale obtained using the extraction process of the present invention, while the decoction group received an extract obtained by decocting Rheum officinale in water. Rats in the invention group and the decoction group were gavaged with extracts equivalent to 1.5g / ml and 3.0g / ml of the crude drug (the invention group received half the amount of the decoction group), with a dosing volume of 0.01ml / kg / day. The positive control group received 0.3mg / kg of lamivudine by gavage, while the remaining groups received the same dose of normal saline by gavage. All five groups of rats were treated for 28 days. One day after the last dose, the rats were anesthetized with sodium pentobarbital (40mg / kg, 1% concentration) and fixed. Six ml of blood was collected from the lower peritoneal vein, allowed to stand at 4°C for 4 hours, and centrifuged at 6000r / min for 10 minutes. The supernatant was collected and frozen for later use.

[0208] 3. Detection method

[0209] GPT and GOT were detected by colorimetry, IFN-γ, IL-2, and IL-4 levels of lymphocyte-related factors were detected by enzyme-linked immunosorbent assay, and hyaluronic acid (HA), laminin (LN), and type III procollagen (PCⅢ) levels of fibrosis indicators were detected by ELISA kits.

[0210] 4. Results

[0211] (1) Serum GPT and GOT levels of rats in each group

[0212] Table 17 Comparison of serum GPT and GOT levels in rats of each group

[0213]

[0214] Note: Compared with the normal group, *P<0.05; compared with the model group, + P<0.05; compared with the decoction group, △P<0.05.

[0215] (2) Levels of lymphocyte-related factors IFN-γ, IL-2, and IL-4 in rats of each group

[0216] Table 18 Comparison of the levels of lymphocyte-related factors IFN-γ, IL-2, and IL-4 in rats in each group

[0217]

[0218] Note: Compared with the normal group, *P<0.05; compared with the model group, + P<0.05; compared with the decoction group, △P<0.05.

[0219] (3) Fibrosis indicators HA, LN, and PCⅢ content in rats in each group

[0220] Table 19 Comparison of the levels of lymphocyte-related factors IFN-γ, IL-2, and IL-4 in rats in each group

[0221]

[0222] Note: Compared with the normal group, *P<0.05; compared with the model group, + P<0.05; compared with the decoction group, △P<0.05.

[0223] 5. Results Analysis

[0224] The results of the pharmacological tests showed that there were no significant differences in the indicators between the invention group and the positive control group, indicating that the extract of the invention has an anti-hepatitis B virus effect comparable to that of lamivudine, and has a strong anti-hepatitis B virus effect. However, there were significant differences between the invention group and the decoction group, with the anti-hepatitis B virus effect of the decoction group being significantly lower than that of the invention group, and the amount of crude drugs taken by the invention group was only half of that of the decoction group, further indicating that the invention can reduce the amount of medicinal materials used, confirming the innovation and advancement of the invention.

[0225] Although the present invention has been described in detail above using general explanations, specific embodiments, and experiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A method for preparing a rhubarb extract, characterized in that: The steps include: S1, Rhubarb pretreatment: Wash fresh Rhubarb and grind it into a paste; S2, compound fermentation: add 5% to 15% of the total amount of medicinal materials, stir well, inoculate Lactobacillus acidophilus and Paecilomyces hymenoptera, the inoculation amount of both is 5×10 6 CFU / mL, fermented at 35℃~40℃ for 5~9 days; S3, acidification: add 60% to 90% ethanol at a concentration of 2 to 6 times the total amount of the medicinal materials, and adjust the pH to 3 to 5 with hydrochloric acid; S4, flash extraction: extraction initial temperature 50-60°C, extraction voltage 120-160V, extraction time 10-20min, centrifugal filtration to obtain filtrate; S5, decolorization: add 1% to 3% activated carbon to the filtrate for decolorization and filter; S6, membrane concentration: using a polyethersulfone nanofiltration membrane with a relative molecular weight cutoff of 100, the filtrate is concentrated into a clear paste at a feed temperature of 50-60°C and a pressure of 0.4-0.6 MPa.

2. The method for preparing the Rheum officinale extract according to claim 1, wherein: The step S2 composite fermentation is as follows: adding 10% of the total amount of the medicinal materials into starch, stirring evenly, inoculating Lactobacillus acidophilus and Paecilomyces hymenoptera, and the inoculation amount of both is 5×10 6 CFU / mL, fermented at 35°C for 7 days.

3. The method for preparing the Rheum officinale extract according to claim 1, wherein: The acidification step S3 is as follows: adding 75% ethanol in an amount twice the total amount of the medicinal materials, and adjusting the pH to 4 with hydrochloric acid.

4. The method for preparing the Rheum officinale extract according to claim 1, wherein: The flash extraction in step S4 is as follows: the initial extraction temperature is 55° C., the extraction voltage is 140 V, the extraction time is 20 min, and centrifugal filtration is performed to obtain a filtrate.

5. The method for preparing the Rheum officinale extract according to claim 1, wherein: The decolorization step S5 is as follows: 2% activated carbon is added to the filtrate for decolorization, and then filtered.

6. The method for preparing the Rheum officinale extract according to claim 1, wherein: The membrane concentration in step S6 is as follows: using a polyethersulfone nanofiltration membrane with a relative molecular weight cutoff of 100, at a feed temperature of 50-55° C. and a pressure of 0.4-0.5 MPa, the filtrate is concentrated into a clear paste.

7. The method for preparing the Rheum officinale extract according to claim 6, characterized in that: The membrane concentration step S6 is as follows: using a polyethersulfone nanofiltration membrane with a relative molecular weight cutoff of 100, the filtrate is concentrated into a clear paste at a feed temperature of 50° C. and a pressure of 0.4 MPa.

8. Use of the paste prepared by the method for preparing the Rheum officinale extract according to any one of claims 1 to 7 in preparing a pharmaceutical preparation for treating hepatitis B virus.

9. The use according to claim 8, characterized in that The pharmaceutical preparation is a solid preparation or a liquid preparation.

10. The pharmaceutical preparation according to claim 9, characterized in that The solid preparations are granules, capsules, tablets, and pills; the liquid preparations are mixtures.

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