Preparation method and application of a rhamnus utilis extract

Abstract

The application provides a preparation method and application of a Rumex madaio extract. The preparation method comprises the following steps: (1) Rumex madaio pretreatment: washing fresh Rumex madaio, and grinding into paste; (2) composite bacteria fermentation; (3) hydrochloric acid acidification; (4) flash extraction; (5) activated carbon decolorization; and (6) membrane concentration into clear paste. The method has high total anthraquinone content and high emodin content, and solves the problems of high effective component loss rate and low extraction rate in traditional extraction methods. The pharmacological test results prove that the anti-hepatitis B virus effect of the clear paste obtained by the application has no obvious difference from that of the lamivudine group, which indicates that the extract has strong anti-hepatitis B virus ability.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a method for preparing and applying an extract of Rheum palmatum. Background Technology

[0002] Rhubarb (Rubus idaeus) is the fresh or dried root and rhizome of *Rumex nepalensis* Spreng., *Rumex dentatus* Linn., or *Rumex crispus* Linn., belonging to the Polygonaceae family. It possesses the effects of clearing heat and detoxifying, cooling blood and stopping bleeding, killing parasites, and promoting bowel movements. It is used for hepatitis and various inflammations, conjunctivitis, constipation, and stubborn tinea. Rhubarb is a medicinal herb used by ethnic minorities in Guizhou Province, and its standard is included on page 24 of the 2003 edition of *Guizhou Traditional Chinese Medicine and Ethnic Medicine Quality Standards*. Modern research shows that anthraquinones are the main effective components of rhubarb in treating hepatitis. However, due to the instability of anthraquinones, significant losses occur during the harvesting and processing of rhubarb. Experiments conducted by our research team show that after fresh rhubarb is sliced ​​and dried using traditional processing methods to produce medicinal slices, the content of the anthraquinone indicator component, emodin, decreases by 42.3%. Meanwhile, due to the poor water solubility of anthraquinones, the extraction rate of anthraquinones using the traditional water decoction process is low, with the extraction rate of emodin after water decoction being only 8.72%.

[0003] For the reasons mentioned above, it is necessary to explore a method to improve the extraction rate of anthraquinone active ingredients in rhubarb. After preparing rhubarb into an extract for clinical application, it can improve the extraction rate of active ingredients and enhance the efficacy of the drug. On the other hand, it can improve the utilization rate of medicinal materials, avoid waste of medicinal materials, and save valuable medicinal resources. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing rhubarb extract, which improves the extraction rate of anthraquinone active ingredients in rhubarb, and allows the rhubarb to be prepared into an extract for clinical application. This solves the problems of high loss rate and low extraction rate of active ingredients in the traditional harvesting, processing and extraction of rhubarb, thereby enhancing the therapeutic effect and saving medicinal resources of rhubarb.

[0005] Another object of the present invention is to provide the application of the rhubarb extract prepared by the method of the present invention in the preparation of pharmaceutical formulations for treating hepatitis B virus.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution: A method for preparing an extract of Rheum palmatum includes the following steps: S1, Pre-treatment of Rhubarb: Wash fresh rhubarb and grind it into a paste; S2, Compound Fermentation: Add 5%–15% starch (based on the total amount of medicinal materials), stir well, and inoculate with *Lactobacillus acidophilus* and *Paecilomyces hepiali*, with an inoculum size of 5 × 10⁻⁶ for both. 6 CFU / mL, fermented at 35℃~40℃ for 5~9 days; S3, Acidification: Add 2 to 6 times the total amount of medicinal materials to 60% to 90% ethanol, and adjust the pH to 3 to 5 with hydrochloric acid; S4, flash extraction: initial extraction temperature 50-60℃, extraction voltage 120-160V, extraction time 10-20min, centrifugation and filtration to obtain filtrate; S5, Decolorization: Add 1% to 3% activated carbon to the filtrate for decolorization, then filter; S6, Membrane Concentration: Using a polyethersulfone nanofiltration membrane with a molecular weight cutoff of 100, the filtrate is concentrated into a clear paste at a feed temperature of 50-60℃ and a pressure of 0.4-0.6MPa.

[0007] The S2 compound fermentation step of this invention is as follows: 10% starch (based on the total amount of medicinal materials) is added, stirred well, and then inoculated with *Lactobacillus acidophilus* and *Paecilomyces hepiali*, with an inoculum quantity of 5 × 10⁻⁶ for both. 6 CFU / mL, fermented at 35℃ for 7 days.

[0008] The acidification step S3 of this invention is as follows: add ethanol with a concentration of 75% twice the total amount of medicinal materials, and adjust the pH to 4 with hydrochloric acid.

[0009] The flash extraction step S4 of this invention is as follows: initial extraction temperature 55℃, extraction voltage 140V, extraction time 20min, centrifugation and filtration to obtain filtrate.

[0010] The decolorization step S5 of this invention is as follows: 2% activated carbon is added to the filtrate for decolorization, followed by filtration.

[0011] The membrane concentration in step S6 of this invention is as follows: using a polyethersulfone nanofiltration membrane with a molecular weight cutoff of 100, the filtrate is concentrated into a clear paste under the conditions of feed temperature of 50-55°C and pressure of 0.4-0.5 MPa.

[0012] The membrane concentration step S6 of this invention involves using a polyethersulfone nanofiltration membrane with a molecular weight cutoff of 100 to concentrate the filtrate into a clear paste at a feed temperature of 50°C and a pressure of 0.4 MPa.

[0013] The application of the extract prepared by the method of Rheum palmatum extract described in this invention in the preparation of pharmaceutical preparations for treating hepatitis B virus.

[0014] The pharmaceutical preparations described in this invention are solid or liquid preparations.

[0015] The solid dosage forms of this invention include, but are not limited to, granules, capsules, tablets, and pills; the liquid dosage forms include, but are not limited to, mixtures.

[0016] Compared with the prior art, the present invention has the following advantages: 1. Avoid loss of active ingredients. In the traditional process of cutting and drying fresh rhubarb, the loss rate of emodin, an anthraquinone active ingredient, is 42.3%. In this invention, the loss rate of emodin is only 10% after grinding the fresh rhubarb into a pulp. Grinding the fresh rhubarb into a pulp maximizes the preservation of the active ingredients in the medicinal material and avoids the loss of active ingredients during the cutting and drying process.

[0017] 2. Improved extraction rate of active ingredients. This invention employs a "compound fermentation + flash extraction + membrane concentration" extraction and purification process, which is carried out entirely at low temperatures, resulting in a high extraction rate of active ingredients. Testing showed that the commonly used traditional water decoction process yielded only 8.72% of emodin, while the "compound fermentation + flash extraction" process achieved an extraction rate as high as 59.62%.

[0018] 3. Conservation of medicinal resources. The "fresh-while-fermentation flash extraction membrane filtration method" of this invention can effectively avoid the loss of active ingredients and improve the extraction rate of active ingredients, thereby conserving medicinal resources.

[0019] 4. Enhanced drug efficacy. Pharmacological test results showed that the anti-hepatitis B virus effect of the present invention group was not significantly different from that of the lamivudine group, suggesting that the extract of the present invention has a strong anti-hepatitis B virus ability. Attached Figure Description

[0020] Figure 1 Chromatogram 01 of emodin reference standard Figure 2 Chromatogram 02 of emodin reference standard Figure 3 Optimal process validation experiment 1: Chromatogram of flavin content determination Figure 4 Optimal process validation experiment 2: Chromatogram of flavin content determination Figure 5 Optimal process validation experiment 3: Chromatogram of flavin content determination Detailed Implementation

[0021] Example 1: Preparation method of Rheum palmatum extract (1) Pretreatment of Rhubarb: Wash the fresh rhubarb and grind it into a paste using a grinder; (2) Compound fermentation: Add starch equal to 10% of the total amount of medicinal materials, stir well, and inoculate with Lactobacillus acidophilus and Paecilomyces hepiali, with an inoculum size of 5×10⁻⁶ for both. 6 CFU / mL, fermented at 35℃ for 7 days; (3) Acidification: Add 75% ethanol at twice the total amount of medicinal materials, and adjust the pH to 4 with hydrochloric acid; (4) Flash extraction: The initial extraction temperature was 55℃, the extraction voltage was 140V, the extraction time was 20min, and the product was centrifuged and filtered to obtain the filtrate; (5) Decolorization: Add 2% activated carbon to the filtrate for decolorization, then filter; (6) Membrane concentration: Using a polyethersulfone nanofiltration membrane with a molecular weight cutoff of 100, the decolorized filtrate is concentrated into a clear extract with a fresh herb to clear extract ratio of 3:1 under the conditions of feed temperature of 50℃ and pressure of 0.4MPa.

[0022] Example 2: Preparation method of Rheum palmatum extract (1) Pretreatment of Rhubarb: Wash the fresh rhubarb and grind it into a paste using a grinder; (2) Compound fermentation: Add starch equal to 5% of the total amount of medicinal materials, stir well, and inoculate with Lactobacillus acidophilus and Paecilomyces hepiali, with an inoculum size of 5×10⁻⁶ for both. 6 CFU / mL, fermented at 40℃ for 5 days; (3) Acidification: Add 4 times the total amount of medicinal materials in 60% ethanol, and adjust the pH to 3 with hydrochloric acid; (4) Flash extraction: The initial extraction temperature is 50℃, the extraction voltage is 120V, the extraction time is 15min, centrifuge and filter to obtain the filtrate; (5) Decolorization: Add 1% activated carbon to the filtrate for decolorization, then filter; (6) Membrane concentration: Using a polyethersulfone nanofiltration membrane with a molecular weight cutoff of 100, the decolorized filtrate is concentrated into a clear extract with a fresh medicinal material to clear extract ratio of 3:1 under the conditions of feed temperature of 50℃ and pressure of 0.5MPa.

[0023] Example 3: Preparation method of Rheum palmatum extract (1) Pretreatment of Rhubarb: Wash the fresh rhubarb and grind it into a paste using a grinder; (2) Compound fermentation: Add starch equal to 15% of the total amount of medicinal materials, stir well, and inoculate with Lactobacillus acidophilus and Paecilomyces hepiali, with an inoculum size of 5×10⁻⁶ for both. 6 CFU / mL, fermented at 40℃ for 9 days; (3) Acidification: Add 6 times the total amount of medicinal materials in 80% ethanol, and adjust the pH to 5 with hydrochloric acid; (4) Flash extraction: The initial extraction temperature is 60℃, the extraction voltage is 160V, the extraction time is 10min, centrifuge and filter to obtain the filtrate; (5) Decolorization: Add 3% activated carbon to the filtrate for decolorization, then filter; (6) Membrane concentration: Using a polyethersulfone nanofiltration membrane with a molecular weight cutoff of 100, the decolorized filtrate is concentrated into a clear extract with a fresh medicinal material to clear extract ratio of 3:1 under the conditions of feed temperature of 60℃ and pressure of 0.5MPa.

[0024] Example 4: Preparation method of Rheum palmatum extract (1) Pretreatment of Rhubarb: Wash the fresh rhubarb and grind it into a paste using a grinder; (2) Compound fermentation: Add starch equal to 10% of the total amount of medicinal materials, stir well, and inoculate with Lactobacillus acidophilus and Paecilomyces hepiali, with an inoculum size of 5×10⁻⁶ for both. 6 CFU / mL, fermented at 35℃ for 5 days; (3) Acidification: Add 70% ethanol at twice the total amount of medicinal materials, and adjust the pH to 3 with hydrochloric acid; (4) Flash extraction: The initial extraction temperature is 50℃, the extraction voltage is 120V, the extraction time is 20min, centrifuge and filter to obtain the filtrate; (5) Decolorization: Add 3% activated carbon to the filtrate for decolorization, then filter; (6) Membrane concentration: Using a polyethersulfone nanofiltration membrane with a molecular weight cutoff of 100, the decolorized filtrate is concentrated into a clear extract with a fresh herb to clear extract ratio of 3:1 under the conditions of feed temperature of 55℃ and pressure of 0.5MPa.

[0025] Example 5: Preparation method of Rheum palmatum extract (1) Pretreatment of Rhubarb: Wash the fresh rhubarb and grind it into a paste using a grinder; (2) Compound fermentation: Add starch equal to 15% of the total amount of medicinal materials, stir well, and inoculate with Lactobacillus acidophilus and Paecilomyces hepiali, with an inoculum size of 5×10⁻⁶ for both. 6 CFU / mL, fermented at 40℃ for 9 days; (3) Acidification: Add 4 times the total amount of medicinal materials in 60% ethanol, and adjust the pH to 4 with hydrochloric acid; (4) Flash extraction: The initial extraction temperature is 60℃, the extraction voltage is 160V, the extraction time is 10 minutes, centrifuge and filter to obtain the filtrate; (5) Decolorization: Add 23% activated carbon to the filtrate for decolorization, then filter; (6) Membrane concentration: Using a polyethersulfone nanofiltration membrane with a molecular weight cutoff of 100, the decolorized filtrate is concentrated into a clear extract with a fresh herb to clear extract ratio of 3:1 under the conditions of feed temperature of 50℃ and pressure of 0.4MPa.

[0026] Example 6: Preparation method of Rheum palmatum extract (1) Pretreatment of Rhubarb: Wash the fresh rhubarb and grind it into a paste using a grinder; (2) Compound fermentation: Add starch equal to 5% of the total amount of medicinal materials, stir well, and inoculate with Lactobacillus acidophilus and Paecilomyces hepiali, with an inoculum size of 5×10⁻⁶ for both. 6 CFU / mL, fermented at 35℃ for 9 days; (3) Acidification: Add 6 times the total amount of medicinal materials in 75% ethanol, and adjust the pH to 3 with hydrochloric acid; (4) Flash extraction: The initial extraction temperature is 60℃, the extraction voltage is 140V, the extraction time is 20min, centrifuge and filter to obtain the filtrate; (5) Decolorization: Add 2% activated carbon to the filtrate for decolorization, then filter; (6) Membrane concentration: Using a polyethersulfone nanofiltration membrane with a molecular weight cutoff of 100, the decolorized filtrate is concentrated into a clear extract with a fresh herb to clear extract ratio of 3:1 under the conditions of feed temperature of 55℃ and pressure of 0.5MPa.

[0027] Example 7

[0028] Take 80g of the clear paste prepared in any of Examples 1 to 6, add 20g of simple sugar syrup, stir well, and you will get the rhubarb mixture.

[0029] Example 8

[0030] Take any of the prepared extracts from Examples 1 to 6, add an appropriate amount of soluble starch, granulate, dry, and granulate to obtain rhubarb granules.

[0031] Example 9

[0032] Take any of the clear extracts prepared in Examples 1 to 6, concentrate them into a thick paste, add an appropriate amount of starch and dextrin, mix well, granulate, dry, granulate, and compress into tablets to obtain rhubarb tablets.

[0033] Example 10

[0034] Take any of the clear pastes prepared in Examples 1 to 6, and concentrate them into a thick paste. Add an appropriate amount of polyethylene glycol 4000 to the thick paste and drip it to make rhubarb pills.

[0035] Example 11

[0036] Take any of the clear extracts prepared in Examples 1 to 6, concentrate them 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, granulate, then add magnesium stearate and mix evenly, and then fill into capsules to obtain rhubarb capsules.

[0037] To further verify the feasibility and effectiveness of the present invention and to select the optimal solution, a series of experiments were conducted. Some of these experiments are excerpted below: I. Research on Extraction Process 1. Comparison of processing methods for wild rhubarb (1) Method for determining the content of emodin The determination was performed using high performance liquid chromatography.

[0038] Chromatographic conditions and system suitability tests were conducted using octadecylsilane-bonded silica gel as the stationary phase; 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 emodin peak, should be no less than 3000.

[0039] Preparation of reference solutions: Accurately weigh an appropriate amount of rhein reference standard and add methanol to prepare solutions containing 80 μg per ml.

[0040] Preparation of the test solution: Accurately weigh approximately 1.5 g of sample and place it in a stoppered conical flask. Accurately add 25 ml of methanol, weigh, heat under reflux for 1 hour, cool, weigh again, replenish the lost weight with methanol, shake well, and filter. 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, then add 10 ml of chloroform, heat under reflux for 1 hour, cool, place in a separatory funnel, wash the container with a small amount of chloroform and add the wash to the separatory funnel, separate the chloroform layer, extract the acid solution three times with 10 ml of chloroform each time, combine the chloroform solutions, recover the solvent under reduced pressure to dryness, dissolve the residue in methanol, transfer to a 10 ml volumetric flask, add methanol to the mark, shake well, filter, and collect the filtrate.

[0041] The assay involves precisely pipetting 10 μl each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0042] (2) Comparison of processing methods Fresh rhubarb was taken, washed, and dried. The rhein content was measured to be 0.14% while fresh. Two portions of fresh rhubarb were taken, each weighing 1000g. One portion was sliced ​​and dried using traditional methods, while the other portion was ground into a paste while fresh. The rhein content of each portion was measured. The results are shown in Table 1.

[0043] ; The experimental results show that when processing rhubarb using the traditional slicing and sun-drying method, the loss rate of emodin is as high as 42.3%, while the loss rate is only 10.0% when using the fresh grinding method, indicating that rhubarb is best processed while fresh.

[0044] 2. Comparison of water extraction methods Take 100g of dried rhubarb and 100g of fresh rhubarb, add water and decoct them separately. After the decoction is concentrated, make up to 50ml. Determine the content of rhein according to the aforementioned method. The results are shown in Table 2.

[0045] ; The experimental results show that the extraction rate of emodin using water extraction is low, and the utilization rate of the effective components of the medicinal material is not high, so a better extraction method needs to be sought.

[0046] 3. Research on the extraction process of fresh rhubarb Since the effective components of rhubarb are lost relatively quickly after being processed into dried medicinal materials, this study used different extraction methods while it was still fresh. The contents of emodin and total anthraquinones in the extracts were used as indicators to compare ultrasonic extraction, flash extraction, fermentation and enzymatic extraction.

[0047] (1) Method for determining total anthraquinone content Ultraviolet spectrophotometry was used.

[0048] Preparation of the color developer: Weigh an appropriate amount of magnesium acetate, dissolve it in methanol to prepare a 0.5% magnesium acetate methanol solution, shake well and set aside.

[0049] Preparation of the reference solution: Weigh 13 mg of 1,8-dihydroxyanthraquinone standard, place it in a 50 mL volumetric flask, add methanol to the mark, and shake well.

[0050] To construct the standard curve, pipette 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, and 10.0 mL of 1,8-dihydroxyanthraquinone standard solution into separate 100 mL volumetric flasks. Add 0.5% magnesium acetate methanol solution to each flask, mix well, and measure the absorbance at 523 nm using a spectrophotometer with a reagent blank solution as a reference. Plot the standard curve with the concentration of 1,8-dihydroxyanthraquinone on the x-axis and the absorbance on the y-axis.

[0051] Preparation of the test solution: Take 20 mL of the sample from each test item and place it in an Erlenmeyer flask. Add 3.0 mL of 30% hydrogen peroxide solution and 0.5 mL of hydrochloric acid solution (1+1). Reflux in a 90℃ water bath for 30 min. Cool and extract with ether 3 times, 20 mL each time. Combine the ether extracts and wash with water twice, 10 mL each time. Evaporate the ether extract to dryness. Dissolve the residue in magnesium acetate methanol solution and transfer it to a 10 mL volumetric flask. Dilute to the mark with magnesium acetate methanol solution and mix well to obtain the test solution.

[0052] (2) Ultrasonic method Take 100g of fresh rhubarb, wash it and grind it into a pulp. Add 4 times the amount of 75% ethanol and extract it by ultrasonic extraction for 30 minutes under ultrasonic power of 250W. Filter the mixture to obtain the filtrate. Concentrate the filtrate and make up to 50ml. Determine the total anthraquinone and emodin content. The results are shown in Table 3.

[0053] (3) Enzymatic hydrolysis Take 100g of fresh rhubarb, wash it, grind it into a paste, add 4 times the amount of water and 2% cellulase, and enzymatically hydrolyze it for 24 hours at 50℃ and pH 5.0. Filter it, concentrate the filtrate and make up to 50ml, and determine the total anthraquinone and emodin content. The results are shown in Table 3.

[0054] (4) Flash Lifting Method Take 100g of fresh rhubarb, wash it and grind it into a pulp. Add 4 times the amount of 75% ethanol, extract at an initial temperature of 60℃, an extraction voltage of 140V, and an extraction time of 20min. Centrifuge and filter, concentrate the filtrate and make up to 50ml. Determine the total anthraquinone and emodin content. The results are shown in Table 3.

[0055] (5) Fermentation method Take 100g of fresh rhubarb, wash it, grind it into a paste, add starch (10% of the total weight of the medicinal material), stir well, and inoculate with Lactobacillus acidophilus at a rate of 5×10⁻⁶. 6 The total anthraquinone and emodin content was determined by fermenting at 35℃ for 6 days with CFU / mL, centrifuging and filtering, concentrating the filtrate and making up to 50 ml, and the results are shown in Table 3.

[0056] ; The experimental results showed that the fermentation method yielded the highest content of total anthraquinones and emodin in the extract, followed by the flash extraction method. However, the extraction rates of both methods did not exceed 40%, so they can be combined for extraction. In addition, research data indicated that acidification would increase the extraction rate of anthraquinones, so the extract of rhubarb pulp can be further extracted after acidification.

[0057] (6) Compound extraction method Take 100g of fresh rhubarb, wash it, grind it into a paste, add starch (10% of the total weight of the medicinal material), stir well, and inoculate with Lactobacillus acidophilus at a rate of 5×10⁻⁶. 6 The total anthraquinone and emodin content was determined by fermenting the material at 35℃ for 6 days with CFU / mL, adding 6 times the total amount of 70% ethanol, adjusting the pH to 4 with hydrochloric acid, stirring well, heating the material to 60℃ for flash extraction, using an extraction voltage of 140V and an extraction time of 20min, centrifuging and filtering, concentrating the filtrate and adjusting the volume to 50ml, and determining the total anthraquinone and emodin content.

[0058] 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%. The experimental results indicate that the mixed extraction method significantly improved the extraction rates of total anthraquinones and emodin, and further experiments can be conducted to determine the optimal extraction parameters.

[0059] 4. Optimization of process parameters for compound extraction method (1) Preferred fermentation strains The fermentation strains of traditional Chinese medicine mainly fall into two categories: bacteria and fungi. Bacteria mainly include Bifidobacterium, Bifidobacterium adolescentis, Streptococcus thermophilus, Lactobacillus bulgaricus, and Lactobacillus acidophilus, while fungi mainly include Saccharomyces cerevisiae, Saccharomyces cerevisiae, Paecilomyces hepiali, and Monascus purpureus.

[0060] Based on the fermentation characteristics of bacteria and fungi and in conjunction with literature review, this study selected *Bifidobacterium adolescentis* (strain number ATCC15703, purchased from Beijing BioBio Biotechnology Co., Ltd.), *Lactobacillus acidophilus* (strain number CICC6096, purchased from China Industrial Microbial Culture Collection Center), *Paecilomyces hepiali* (strain number Bio-57623, purchased from Beijing BioBio Biotechnology Co., Ltd.), and *Monascus purpureus* (strain number L807, purchased from Beijing BioBio Biotechnology Co., Ltd.) as the key strains for screening and conducted the following experiments: Take 800g of fresh rhubarb, wash it, grind it into a paste, divide it into 8 equal portions, add starch equal to 10% of the total amount of the medicinal materials, mix well, and inoculate each portion with the bacterial strains listed in Table 4, with an inoculation rate of 5×10⁻⁶. 6 The total anthraquinone and emodin content was determined by fermentation at 35℃ for 6 days, followed by the addition of 70% ethanol at 6 times the total amount of medicinal materials. The pH was adjusted to 4 with hydrochloric acid and stirred well. The material was then heated to 60℃ for flash extraction at an extraction voltage of 140V for 20 minutes. After centrifugation and filtration, the filtrate was concentrated and diluted to 50ml. The total anthraquinone and emodin content were determined, and the results are shown in Table 4.

[0061] ; The experimental results showed that the extract contained the highest total anthraquinone and emodin content after fermentation with "Lactobacillus acidophilus + Paecilomyces hepiali". Therefore, Lactobacillus acidophilus and Paecilomyces hepiali were selected as the fermentation strains for this process.

[0062] (2) Optimal amount of fermented starch Take 300g of fresh rhubarb, wash it, grind it into a paste, divide it into 3 equal portions, and add 5%, 10%, and 15% starch (based on the total amount of the medicinal material) to each portion respectively. Mix well, and inoculate with *Lactobacillus acidophilus* and *Paecilomyces hepiali* strains at a rate of 5 × 10⁻⁶. 6 The total anthraquinone and emodin content was determined by fermenting at 35℃ for 6 days with CFU / mL. Then, 6 times the total amount of the medicinal material was added with 70% ethanol, and the pH was adjusted to 4 with hydrochloric acid. The mixture was stirred well and then heated to 60℃ for flash extraction. The extraction voltage was 140V and the extraction time was 20min. The mixture was centrifuged and filtered, and the filtrate was concentrated and diluted to 50ml. The total anthraquinone and emodin content were determined. The results are shown in Table 5.

[0063] ; The experimental results show that the extraction effect is not much different when the starch dosage is 10% and 15%, both of which are significantly higher than that of 5%. Therefore, 10% can be selected as the optimal starch dosage parameter.

[0064] (3) Optimal fermentation temperature Take 300g of fresh rhubarb, wash it, grind it into a paste, divide it into 3 equal portions, add starch equal to 10% of the total amount of the medicinal materials, mix well, and inoculate with Lactobacillus acidophilus and Paecilomyces hepiali strain, with an inoculum size of 5×10⁻⁶. 6 The samples were fermented at 35℃, 37℃ and 40℃ for 6 days with CFU / mL. Six times the total amount of the medicinal materials were added with 70% ethanol, and the pH was adjusted to 4 with hydrochloric acid. The mixture was stirred and heated to 60℃ for flash extraction at 140V for 20 min. The mixture was centrifuged and filtered, and the filtrate was concentrated and diluted to 50 ml. The total anthraquinone and emodin contents were determined. The results are shown in Table 6.

[0065] ; The experimental results show that the fermentation temperature has little effect on the extraction effect, and 35℃~40℃ is acceptable.

[0066] (4) Optimal fermentation time Take 400g of fresh rhubarb, wash it, grind it into a paste, divide it into 4 equal portions, add starch equal to 10% of the total amount of the medicinal materials, mix well, and inoculate with Lactobacillus acidophilus and Paecilomyces hepiali strain, with an inoculum density of 5×10⁻⁶. 6 The samples were fermented at 35℃ for 3, 5, 7 and 9 days, respectively, with 6 times the total amount of 70% ethanol added. The pH was adjusted to 4 with hydrochloric acid and stirred well. The material was then heated to 60℃ for flash extraction at an extraction voltage of 140V for 20 minutes. After centrifugation and filtration, the filtrate was concentrated and diluted to 50 ml. The total anthraquinone and emodin contents were determined, and the results are shown in Table 7.

[0067] ; The experimental results showed that the increase in total anthraquinone and emodin content was not significant after 5 days of fermentation, so 7 days was selected as the optimal fermentation time.

[0068] (5) Optimal ethanol concentration Take 300g of fresh rhubarb, wash and grind it into a paste. Add starch (10% of the total weight of the medicinal material), mix well, and inoculate with *Lactobacillus acidophilus* and *Paecilomyces hepiali* strains at a rate of 5 × 10⁻⁶. 6 The total anthraquinone and emodin content was determined by fermenting at 35℃ for 6 days. The fermentation product was divided into 3 equal parts and 4 times the amount of 60%, 75% and 90% ethanol were added respectively. The pH was adjusted to 4 with hydrochloric acid, and then the material was heated to 60℃ for flash extraction. The extraction voltage was 140V and the extraction time was 20min. The product was centrifuged and filtered, and the filtrate was concentrated and diluted to 50ml. The total anthraquinone and emodin content were determined. The results are shown in Table 8.

[0069] ; The experimental results show that the extraction effect is not much different when the ethanol concentration is 75% and 90%, both of which are higher than that of 60% ethanol. Considering cost factors, 75% can be selected as the optimal ethanol concentration.

[0070] (6) Double the amount of alcohol is preferred Take 300g of fresh rhubarb, wash and grind it into a paste. Add starch (10% of the total weight of the medicinal material), mix well, and inoculate with *Lactobacillus acidophilus* and *Paecilomyces hepiali* strains at a rate of 5 × 10⁻⁶. 6 The total anthraquinone and emodin content was determined by fermenting at 35℃ for 6 days. The fermentation product was divided into 3 equal parts, and 2, 4, and 6 times 75% ethanol were added respectively. The pH was adjusted to 4 with hydrochloric acid, and then the material was heated to 60℃ for flash extraction. The extraction voltage was 140V and the extraction time was 20min. The product was centrifuged and filtered, and the filtrate was concentrated and diluted to 50ml. The total anthraquinone and emodin content were determined. The results are shown in Table 9.

[0071] ; The experimental results show that the extraction effect is not significantly different depending on the amount of alcohol added. To save production costs, 2 times the amount of alcohol can be selected as the optimal amount of ethanol.

[0072] (7) pH Take 300g of fresh rhubarb, wash and grind it into a paste. Add starch (10% of the total weight of the medicinal material), mix well, and inoculate with *Lactobacillus acidophilus* and *Paecilomyces hepiali* strains at a rate of 5 × 10⁻⁶. 6 The total anthraquinone and emodin content was determined by fermenting at 35℃ for 6 days with CFU / mL. The fermentation product was divided into 3 equal parts, and 4 times the amount of 75% ethanol was added. The pH was adjusted to 3, 4 and 5 with hydrochloric acid respectively. The material was then heated to 60℃ for flash extraction at 140V for 20 min. After centrifugation and filtration, the filtrate was concentrated and diluted to 50 ml. The total anthraquinone and emodin content were determined. The results are shown in Table 10.

[0073] ; The experimental results show that the lower the pH, the better the extraction effect. The pH 3 and pH 4 are not much different, but both are significantly higher than pH 5. Therefore, pH 4 can be selected as the optimal process parameter.

[0074] (8) Initial temperature of flash extraction Take 300g of fresh rhubarb, wash and grind it into a paste. Add starch (10% of the total weight of the medicinal material), mix well, and inoculate with *Lactobacillus acidophilus* and *Paecilomyces hepiali* strains at a rate of 5 × 10⁻⁶. 6 The total anthraquinone and emodin content was determined by fermenting at 35℃ for 6 days with CFU / mL. The fermentation product was divided into 3 equal parts, and 4 times the amount of 75% ethanol was added. The pH was adjusted to 4 with hydrochloric acid. The materials were then heated to 50℃, 55℃ and 60℃ for flash extraction. The extraction voltage was 140V and the extraction time was 20min. The samples were centrifuged and filtered. The filtrate was concentrated and diluted to 50ml. The total anthraquinone and emodin content were determined. The results are shown in Table 11.

[0075] ; The experimental results show that 55℃ has the best extraction effect among the three initial temperatures, followed by 60℃, while 50℃ has a slightly worse extraction effect. Therefore, 55℃ was selected as the optimal process parameter.

[0076] (9) Flash voltage Take 300g of fresh rhubarb, wash and grind it into a paste. Add starch (10% of the total weight of the medicinal material), mix well, and inoculate with *Lactobacillus acidophilus* and *Paecilomyces hepiali* strains at a rate of 5 × 10⁻⁶. 6 The total anthraquinone and emodin content was determined by fermenting at 35℃ for 6 days with CFU / mL. The fermentation product was divided into 3 equal parts, and 4 times the amount of 75% ethanol was added. The pH was adjusted to 4 with hydrochloric acid, and the material was heated to 60℃ for flash extraction. The extraction voltage was set to 120V, 140V and 160V respectively, and the extraction time was 20min. The product was centrifuged and filtered, and the filtrate was concentrated and diluted to 50ml. The total anthraquinone and emodin content were determined. The results are shown in Table 12.

[0077] ; The experimental results show that the extraction voltage has little effect on the extraction effect, so a voltage of 120V to 160V was selected as the process parameter.

[0078] (10) Flash time Take 300g of fresh rhubarb, wash and grind it into a paste. Add starch (10% of the total weight of the medicinal material), mix well, and inoculate with *Lactobacillus acidophilus* and *Paecilomyces hepiali* strains at a rate of 5 × 10⁻⁶. 6 The product was fermented at 35℃ for 6 days with CFU / mL. The fermentation product was divided into 3 equal parts, 4 times the amount of 75% ethanol was added, the pH was adjusted to 4 with hydrochloric acid, and then the material was heated to 60℃ for flash extraction. The extraction voltage was 160V, and the extraction time was 10 min, 15 min and 20 min respectively. The product was centrifuged and filtered, the filtrate was concentrated and the volume was adjusted to 50 ml. The total anthraquinone and emodin content were determined. The results are shown in Table 13.

[0079] ; The experimental results show that there is little difference between 15 minutes and 20 minutes of flash extraction, but both are significantly higher than that of 10 minutes. Therefore, 20 minutes of extraction is selected as the optimal process parameter.

[0080] 5. Optimization of membrane concentration process parameters Since anthraquinones are unstable when heated, the extract should be concentrated at low temperatures. Membrane concentration is a type of low-temperature concentration method that is widely used in the pharmaceutical industry. Therefore, this study decided to use membrane concentration to concentrate the extract of Rheum palmatum. Based on the molecular characteristics of anthraquinones in Rheum palmatum, a polyethersulfone nanofiltration membrane with a molecular weight cutoff of 100 was selected. The feed temperature and pressure of the membrane concentration process were studied according to the characteristics of the membrane concentration process.

[0081] (1) Material pretreatment Take 600g of fresh rhubarb, wash and grind it into a paste. Add starch (10% of the total weight of the medicinal material), mix well, and inoculate with *Lactobacillus acidophilus* and *Paecilomyces hepiali* strains at a rate of 5 × 10⁻⁶. 6 The material was fermented at 35℃ for 7 days with CFU / mL, then 2 times the amount of 75% ethanol was added, the pH was adjusted to 4 with hydrochloric acid, and then the material was heated to 55℃ for flash extraction. The extraction voltage was 140V and the extraction time was 20min. After centrifugation and filtration, the total anthraquinone and emodin content of the filtrate was determined after decolorization with 2% activated carbon. The filtrate was divided into 6 equal portions for subsequent experiments.

[0082] (2) Feed temperature Take three portions of the filtrate and concentrate them into a clear paste using a polyethersulfone nanofiltration membrane with a molecular weight cutoff of 100 at feed temperatures of 50℃, 55℃ and 60℃ and a pressure of 0.5MPa. Determine the total anthraquinone and emodin content and calculate the retention rates of total anthraquinone and emodin. The results are shown in Table 14.

[0083] ; The experimental results showed that the retention rates of total anthraquinones and emodin decreased with increasing temperature, and the optimal process parameters were finally selected as 50℃~55℃.

[0084] (3) Concentration pressure Take three portions of the filtrate and concentrate them into a clear extract using a polyethersulfone nanofiltration membrane with a molecular weight cutoff of 100 at a feed temperature of 55℃ and pressures of 0.4MPa, 0.5MPa and 0.6MPa, respectively. Determine the total anthraquinone and emodin content and calculate the retention rates of total anthraquinone and emodin. The results are shown in Table 15.

[0085] ; The experimental results show that the retention rates of total anthraquinones and emodin decrease with increasing pressure, with the retention rate below 90% at 0.6 MPa. Therefore, the optimal process parameters for concentration are 0.4 MPa to 0.5 MPa.

[0086] 6. Optimal process verification test Based on the results of the aforementioned experimental studies, the optimal extraction process for Rheum palmatum was determined to be: Take fresh rhubarb, wash it, grind it into a paste, add starch (10% of the total weight of the medicinal material), stir well, and inoculate with *Lactobacillus acidophilus* and *Paecilomyces hepiali* strains at a rate of 5 × 10⁻⁶. 6The material was fermented at 35℃ for 7 days with CFU / mL, then 2 times the amount of 75% ethanol was added, and the pH was adjusted to 4 with hydrochloric acid. The material was then heated to 55℃ for flash extraction at an extraction voltage of 140V for 20 minutes. After centrifugation and filtration, the filtrate was decolorized with 2% activated carbon and then concentrated into a clear paste using a polyethersulfone nanofiltration membrane with a molecular weight cutoff of 100 at a feed temperature of 50℃ and a pressure of 0.4MPa.

[0087] To verify the stability and reproducibility of the process, three batches of verification experiments were conducted. The verification results are shown in Table 16. Figures 1 to 5 .

[0088] ; The experimental results show that the process has good stability and reproducibility, indicating that the process is reasonable and feasible.

[0089] II. Efficacy Verification To verify the scientific validity and advancement of the extraction method of this invention, as well as the effectiveness of the extract, we conducted anti-hepatitis B virus pharmacological tests on the extract.

[0090] 1. Laboratory animals 50 adult male rats of specific pathogen-free (SPF) grade, weighing... Rats aged 4-6 weeks, 5 rats per cage, ambient temperature of the animal housing environment. The relative humidity was 65%, and the rats had free access to water and food. All rats were acclimatized for 7 days before the experiment.

[0091] 2. Animal grouping modeling, drug administration, and specimen collection Rats were randomly divided into five groups: normal group, model group, positive control group, invention group, and decoction group, with 10 rats in each group. Except for the normal group, all rats were injected with 1×10⁻⁶ ppm via the tail vein. 6 AAV-HBV rat models were established using the method of injecting 10 ml / kg / day of HBV virus (diluted with Hanks' solution) once every 7 days, for a total of 3 times. The model was considered successfully established using a hepatitis B antigen detection plate; a positive result in the model group indicated successful modeling, and subsequent experiments could begin.

[0092] The invention group consisted of an extract obtained from Rheum palmatum using the extraction process of this invention, while the decoction group consisted of an extract obtained from Rheum palmatum after decoction. Rats in the invention group and the decoction group were administered the extracts at doses equivalent to 1.5 g / ml and 3.0 g / ml of the raw drug, respectively, via gavage (half the dose in the invention group). The administration volume was 0.01 ml / kg / day. The positive control group was administered 0.3 mg / kg of lamivudine via gavage, while the other groups were administered the same dose of physiological saline via gavage. All five groups of rats were treated continuously for 28 days. One day after the last administration, rats were anesthetized and fixed with sodium pentobarbital (40 mg / kg, 1% concentration). 6 ml of blood was collected from the inferior abdominal vein, incubated at 4°C for 4 hours, centrifuged at 6000 rpm for 10 minutes, and the supernatant was collected and frozen for later use.

[0093] 3. Detection Method GPT and GOT were detected by colorimetric method, and the levels of lymphocyte-related factors IFN-γ, IL-2, and IL-4 were detected by enzyme-linked immunosorbent assay (ELISA). The levels of fibrosis markers hyaluronic acid (HA), laminin (LN), and type III procollagen (PCⅢ) were detected by ELISA kit.

[0094] 4. Results (1) Serum GPT and GOT levels in each group of rats

[0095] (2) Levels of lymphocyte-related factors IFN-γ, IL-2, and IL-4 in each group of rats

[0096] (3) Content of fibrosis indicators HA, LN, and PCⅢ in rats of each group

[0097] 5. Results Analysis Pharmacological test results 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 present invention has a similar anti-hepatitis B virus effect to lamivudine and has a strong anti-hepatitis B virus effect. However, there was a significant difference between the invention group and the decoction group. The anti-hepatitis B virus effect of the decoction group was significantly lower than that of the invention group. Moreover, the amount of raw herbs taken by the invention group was only half that of the decoction group, which further illustrates that the present invention can reduce the amount of medicinal materials used, thus confirming the innovation and advancement of the present invention.

[0098] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing an extract of Rheum palmatum, characterized in that, Includes the following steps: S1, Pre-treatment of Rhubarb: Wash fresh rhubarb and grind it into a paste; S2, Compound Fermentation: Add starch equal to 10% of the total amount of medicinal materials, stir well, and inoculate with *Lactobacillus acidophilus* and *Paecilomyces hepiali*, with an inoculum size of 5 × 10⁻⁶ for both. 6 CFU / mL, fermented at 35℃ for 7 days; S3, Acidification: Add 75% ethanol at twice the total amount of medicinal materials, and adjust the pH to 4 with hydrochloric acid; S4, flash extraction: initial extraction temperature 55℃, extraction voltage 140V, extraction time 20min, centrifugation and filtration to obtain filtrate; S5, Decolorization: Add 2% activated carbon to the filtrate for decolorization, then filter; S6, Membrane Concentration: Using a polyethersulfone nanofiltration membrane with a 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.

2. The application of the extract prepared by the method of preparing rhubarb extract according to claim 1 in the preparation of pharmaceutical preparations for treating hepatitis B.

3. The application according to claim 2, characterized in that, The pharmaceutical preparation is either a solid or liquid preparation.

4. The application according to claim 3, characterized in that, The solid dosage form is granules, capsules, tablets, or pills; the liquid dosage form is a mixture.

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