Preparation method, decoction pieces and application of Arisaema cum Bile

Abstract

The present invention relates to a preparation method, a prepared slice and an application of Arisaema cum Bile, and belongs to the technical field of processing of prepared slices of traditional Chinese medicine. The preparation method comprises the following preparation steps: (1) pulverizing Pueraria lobata and / or Arisaema heterophyllum to obtain medicinal powder; (2) mixing bile and the medicinal powder in a mass ratio of 4-6:1, adding fermentation bacteria for fermentation to obtain a fermentation broth; (3) filling the fermentation broth into a bovine gallbladder and suspending it for fermentation to obtain a fermented product; (4) drying the fermented product at a low temperature and pulverizing it to obtain the Arisaema cum Bile prepared slice, wherein the fermentation bacteria are selected from at least one of Proteus mirabilis, Bacillus velezensis, Kazachstania servazzii and Penicillium. The prepared slice is prepared by using all of Pueraria lobata or adding Arisaema heterophyllum and fermenting with a mixed strain, and the content of active free cholic acid in the obtained fermented product is high, and it has preventive and therapeutic effects on various diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine decoction pieces processing, and particularly relates to a preparation method, a decoction piece and an application of Guangdanxing decoction pieces. Background Art

[0002] Arisaema cum Bile is an essential and emergency medicine in ancient pharmacies, and it is a fermented traditional Chinese medicine decoction piece with a long medicinal history and outstanding clinical efficacy. Arisaema cum Bile is bitter, slightly pungent, and cool in nature, and it belongs to the heart, liver, and lung meridians. It has the effects of clearing heat and resolving phlegm, calming endogenous wind and relieving convulsions. It is used to treat phlegm-heat cough, thick yellow phlegm, stroke with phlegm obstruction, mania and epilepsy. It is an important medicine for treating "phlegm" syndrome, including both the tangible phlegm in the lung meridian and the intangible phlegm in the heart and liver meridians, as well as phlegm stasis in the blood. The Chinese Pharmacopoeia (2020 Edition) includes 29 Chinese patent medicines containing Arisaema cum Bile, and 131 prescriptions in the standards of ready-made preparations use Arisaema cum Bile. The annual market demand reaches more than 2,600 tons, and it is one of the most widely used traditional Chinese medicine decoction pieces.

[0003] The key points of the preparation of the ancient method of nine-turn Arisaema cum Bile are summarized as follows: First, the raw materials are (December) ox bile, rarely sheep bile, and no pig bile; raw Arisaema powder is used. Second, the time is "December", "hang it to dry", "one hundred days", and "Medical Compendium of Golden Mirror" states that "repeated collection, nine times like this, the nine-turn one is better". Third, the brewing place is "avoiding sunlight", "ventilated", and "dried in the shade". Fourth, the production method clearly indicates "brewing", that is, the fermentation method.

[0004] By consulting the "Processing Specifications of Traditional Chinese Medicine Decoction Pieces" in 21 provinces such as Jiangxi and Guizhou, a total of 6 of them have Arisaema cum Bile decoction pieces. The processing techniques mainly include the fermentation method, the mixed steaming method, and the replication method; the bile used is from multiple sources, including bovine, ovine, and porcine bile; the ratio of the Arisaema powder to the bile used varies; there is no clear regulation on the fermentation time, and only Jiangxi Province and Guizhou Province stipulate that the fermentation is about 2 weeks; in Jilin, Shanghai, Jiangxi, Hunan, and Zhejiang, the fermentation conditions require placing it in a warm place or exposing it to the sun during the day and dew at night, which is completely opposite to the low-temperature and shady environment of the ancient method. The regulations on the fermentation degree are also all described in terms of the appearance, and it is difficult to determine whether the medicinal effect requirements are met. The Chinese Pharmacopoeia (2020 Edition) stipulates that Arisaema cum Bile is made from fine powder of processed Arisaema and bovine, ovine, or porcine bile through processing, or from fine powder of raw Arisaema and bovine, ovine, or porcine bile through fermentation processing. Two completely different preparation methods are stipulated: the mixing method and the fermentation method.

[0005] To sum up, the processing of Arisaema cum Bile varies from place to place and method to method, and the decoction piece standards are not unified, which has deviated from the traditional production techniques, resulting in large quality differences and difficult to guarantee the clinical efficacy.

[0006] To address the issues of unclear processing principles, simple standards, inconsistent specifications, and unclear efficacy and mechanism of action, our team conducted a systematic study of Arisaema consanguineum. This study, for the first time, clarified the processing principle of Arisaema consanguineum: "Arisaema powder and ox bile create a fermentation medium. Under the anaerobic conditions created by the gallbladder skin, bile salt-tolerant microorganisms ferment to achieve biotransformation of bile acids and produce highly active free bile acids." Based on traditional processing principles, this team has developed an innovative preparation for Guangdanxing (also known as Jiuzhuan Danxing) slices. Summary of the Invention

[0007] To address the above-mentioned technical problems, the inventors have innovatively discovered that kudzu root facilitates the production of active free bile acids and can replace or partially replace the toxic Chinese herbal medicine, arisaema, as a fermentation medium. Consequently, they have developed a method for preparing Guangdanxing (also known as Jiuzhuan Danxing) decoction slices. By innovatively replacing arisaema with kudzu root or adding it to arisaema in a specific proportion, the mixture is fermented with bile in a fermentation tank, first undergoing stirring fermentation and then suspension fermentation. The inventors have also specifically defined the relevant process parameters, identified beneficial bacteria species that promote the conversion of active bile acids, and developed a standardized process flow. While inheriting the traditional arisaema preparation techniques, the inventors have also developed a Guangdanxing (also known as Jiuzhuan Danxing) decoction based on modern bioengineering technology that aligns with traditional arisaema processing. This novel Guangdanxing production process, resulting from this method, establishes a new standard for Guangdanxing decoction slices, realizes a modern production model for Guangdanxing, changes the previous backward and rudimentary production model, and effectively ensures product quality, which is of great significance.

[0008] The first aspect of the present invention provides a method for preparing Guangdanxing decoction pieces, comprising the following preparation steps:

[0009] (1) Grinding Puerariae Radix and / or Arisaema Concha to obtain medicinal powder;

[0010] (2) Mix bile and drug powder in a mass ratio of 4-6:1, add fermentation bacteria and ferment to obtain fermentation liquid;

[0011] (3) The fermentation liquid is placed in the cattle gallbladder and suspended for fermentation to obtain the fermentation product;

[0012] (4) The fermented product is dried at low temperature, crushed, and pressed into tablets or pellets to obtain Guangdanxing decoction pieces;

[0013] Wherein the fermentation bacteria is selected from Proteus mirabilis ( Proteus mirabilis strain ), vegetable Bacillus ( Bacillus oleronii ), Kazakh yeast Saccharomyces sergeni ( Kazachstania servazzii ) and Penicillium ( Penicillium sp. ) at least one of the following.

[0014] Preferably, the medicinal powder in step (1) is prepared by crushing Pueraria lobata medicinal material and Arisaema consanguineum medicinal material into 80 meshes, and the mass ratio of Arisaema consanguineum medicinal powder to Pueraria lobata medicinal powder is 0-1:0.25-1.

[0015] Preferably, the weight ratio of the bile to the medicinal powder in step (2) is 4-6:1.

[0016] Preferably, the addition amount of the fermenting bacteria in step (2) accounts for 3-6% of the mass of the medicinal powder.

[0017] Preferably, the temperature of the fermentation in step (2) is 25-28 °C, and the fermentation time is 7-15 days.

[0018] Preferably, the temperature of the hanging fermentation in step (3) is lower than 28 °C, the relative humidity is lower than 80%, and the fermentation time is about 30 days.

[0019] Preferably, the temperature of the low-temperature drying in step (4) is not higher than 80 °C, the drying time is 12-24 hours, and it is pulverized into fine powder of 80-100 meshes.

[0020] More preferably, the fine powder is wetted and granulated, and pressed into a cylindrical shape, pill shape or tablet.

[0021] Even more preferably, the prepared Guangdanxing decoction pieces are coated with a film coating for easy administration.

[0022] The second aspect of the present invention relates to a kind of Guangdanxing (also known as Jiuzhuan Danxing) decoction pieces, and the Guangdanxing decoction pieces are prepared by using the above preparation method.

[0023] Preferably, the content of active free cholic acid in the Guangdanxing decoction pieces is ≥6.5%, and the content of deoxycholic acid is ≥2%.

[0024] The third aspect of the present invention relates to the use of the Guangdanxing (also known as Jiuzhuan Danxing) decoction pieces prepared by the above preparation method in the preparation of drugs for clearing heat-phlegm in the lung meridian, respiratory tract infection, sore throat, phlegm-heat cough, lung injury, high fever convulsion, clearing phlegm stasis in blood, protecting liver and reducing lipid, promoting metabolism, protecting nerves, preventing and treating liver injury, stroke, vertigo, Parkinson's disease, epilepsy, etc.

[0025] Preferably, the Guangdanxing (also known as Jiuzhuan Danxing) decoction pieces can be used directly or used as a raw material drug for preparing other compound preparations.

[0026] Compared with the prior art, the present invention has the following beneficial effects: (1) The preparation method provided by the present invention covers the connotation goal of preparing Jiuzhuan Dan Nanxing by the traditional ancient nine-turn process (9 fermentations), and establishes a content determination standard with free bile acid and deoxycholic acid as quality control indicators; (2) The fermentation is carried out in a process-controllable fermentation tank to investigate the effects of raw material ratio, fermentation temperature and fermentation time on the production of free bile acid and deoxycholic acid; (3) Pueraria lobata is innovatively screened out as a starch-rich culture medium and as a promoter for the production of active free bile acid, which can partially or completely replace the toxic Arisaema consanguineum medicinal material, making the medicine safer; (4) Fermentation strains that are beneficial to the production of active bile acid are innovatively screened out; (5) The present invention combines the use of the traditional hanging gall drying method for drying, and the prepared new medicinal piece "Guangdanxing" contains the same substances as the traditional Jiuzhuan Dan Nanxing fermented 9 times by the ancient method, and the preparation time is shorter than the ancient method, the conversion rate of the target product is high, the process is controllable, and the product quality is stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : Pathological sections of lung tissues of lung injury treated with Guangdanxing (HE staining, 200×);

[0028] Figure 2 : Effects of Guangdanxing on the levels of TNF-α, IL-6, IL-1β and IL-18 in lung tissue of mice with lung injury;

[0029] Figure 3 :Effects of Guangdanxing on the pathomorphology of liver tissue in NASH mice (HE, 200×);

[0030] Figure 4 : Effects of guandanxin on serum IL-1β, TNF-α and substantia nigra IL-1β, TNF-α, COX-2 and iNOS levels in MPTP parkinsonism mouse model;

[0031] Figure 5 : Effects of glaucoma on the relative expression of PKA, GPX4 and FTH1 in the substantia nigra of MPTP Parkinson's disease mouse model. DETAILED DESCRIPTION

[0032] The following non-limiting examples are provided to enable those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way. The following contents are merely illustrative of the scope of the present invention, and those skilled in the art may make various changes and modifications to the present invention based on the disclosed contents, which should also fall within the scope of the present invention.

[0033] In the following examples and experimental examples, the raw materials are: bile from bovine animals Bos niaTaurus domestic Gmelin's fresh bile; Pueraria lobata is a plant of the Leguminosae family. Pueraria thomsonii The dried root of Benth. Arisaema erubescens (Wall.) Schott, Arisaema heterophyllum Arisaema heterophyllum Bl., Arisaema confusa Arisaema amurense Maxim. or Aconite ( Brushes cut into pieces Schott . ) of dried tubers. The fermentation bacteria are selected from Proteus mirabilis ( Miraculous Proteus strain ), vegetable Bacillus ( Bacillus oleronii ), Kazakh yeast Saccharomyces sergeni ( Kazakhstan servazzii ) and Penicillium ( Penicillium sp. ) are all commercially available strains.

[0034] Take the dried Pueraria lobata medicinal material and Arisaema consanguineum medicinal material and grind them into 80-mesh fine powder for later use.

[0035] Experimental Example 1:

[0036] Prepare the traditional method of nine-time fermentation of Arisaema, the preparation steps are as follows:

[0037] Fresh raw ox bile and raw arisaema are added as raw materials, with the ratio of raw arisaema to bile being 1:1. Traditional fermentation conditions are followed, with a temperature of 19-28°C (varies day and night, seasonally), ventilation, and the addition time is in the twelfth lunar month (i.e. the eleventh lunar month). The first fermentation is carried out, and the fermentation end point is considered after the fermentation becomes dry. The fermentation time for one turn is calculated to be 28-32 days. The fermentation is taken out and crushed, and fermented again with new bile. A total of 9 fermentations are performed to obtain the product.

[0038] The bile-to-raw material ratio was adjusted based on gallbladder capacity, and the average bile volume was calculated. A total of 15 batches were prepared. Based on literature, the final weight ratio of raw arisaema to bile after nine fermentations was determined to be 1:4-6.

[0039] Chemical research was conducted on Arisaema cinerea after 9 fermentations, and the content of the main active markers free bile acid, deoxycholic acid, and low-activity taurocholic acid was determined. The determination method is as follows:

[0040] (1) Test solution: Take an appropriate amount of the test sample, accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL of 70% methanol to make a 10 mg / mL solution, weigh it, ultrasonicate it for 30 min, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, centrifuge it (speed: 8000 rpm, time: 10 min), filter it, and take the filtrate.

[0041] (2) Reference solution: Weigh an appropriate amount of the reference substance accurately, dissolve it in methanol and dilute to 1 mL in a volumetric flask to obtain a solution containing 2 mg per 1 mL.

[0042] (3) Detection method

[0043] Chromatographic column: Waters Symmetry C 18 (4.6×250 mm, 5 μm); Mobile phase: acetonitrile (A) - 0.2% acetic acid in water (B), gradient elution; Flow rate: 1.0 mL·min -1 ; Column temperature: 30 °C. Flow rate of carrier gas for evaporative light scattering detector: 2.2 mL / min; Drift tube temperature: 55 °C. Chromatographic elution conditions are shown in Table 1, and test results are shown in Table 2.

[0044] Table 1: Gradient elution program

[0045]

[0046] Table 2: Contents of related substances in 9 batches of fermented Arisaema cum Bile prepared by traditional method for 15 times

[0047]

[0048] The determination results of traditional method for nine-turn fermented Arisaema cum Bile show that the average content of taurocholic acid is 48.02%, which is a bile acid with relatively low binding activity; the average content of cholic acid is 3.47%, and the average content of deoxycholic acid is 1.29%.

[0049] According to the traditional process for preparing Arisaema cum Bile fermented nine times by traditional method, it is determined that cholic acid and deoxycholic acid are the active bile acid components after fermentation and transformation. The present invention takes the contents of cholic acid and deoxycholic acid as the criteria for screening process conditions.

[0050] Experimental example 2: Optimization of the ratio of Pueraria lobata and Arisaema heterophyllum

[0051] Take fresh bovine bile and the powder of Pueraria lobata medicinal material and Arisaema heterophyllum medicinal material crushed to 80 meshes, and mix them in the ratios of 0.25:1, 0.5:1, 1:1, 2:1, 3:1, 4:1, and all Pueraria lobata to obtain mixed medicinal powders with different weight ratios; mix them evenly according to the weight ratio of bile:medicinal powder of 4:1 respectively, and add 4% of Proteus mirabilis ( Miraculous Proteus strain ), Bacillus velezensis ( Bacillus oleronii ), Kazachstania servazzii ( Kazakhstan servazzii ), and Penicillium ( Penicillium sp.), (the four kinds of bacteria are composed in a mass ratio of 1:1:1:1). Under the condition of 25°C, stir and ferment for 15 days, with a stirring rate of 150 revolutions per minute. Sampling is carried out on the 15th day of fermentation to measure the contents of two free bile acids, deoxycholic acid and cholic acid, and the content of conjugated taurocholic acid in the fermentation broth. The measurement method is the same as that in Experimental Example 1, and the results are shown in Table 3.

[0052] Table 3: Contents of related substances in fermented products with different mass ratios of Pueraria lobata - Arisaema heterophyllum

[0053]

[0054] The results show that Pueraria lobata can significantly increase the production amounts of two free bile acids, active deoxycholic acid and cholic acid. The mass ratio of Arisaema heterophyllum powder to Pueraria lobata powder has good conversion within the range of 0 - 1:0.25 - 1. Arisaema heterophyllum has the efficacy of resolving phlegm and dissipating nodules. As a fermentation substrate, it can enhance the effect of "resolving phlegm and dissipating nodules" of Guangdansheng.

[0055] Experimental Example 3: Investigation on the raw material ratio of bile - powder

[0056] Take fresh bovine bile and Pueraria lobata medicinal material powder and Arisaema heterophyllum medicinal material powder crushed to 80 meshes, and mix them evenly according to the weight ratios of bile:powder (Pueraria lobata:Arisaema heterophyllum are mixed in a weight ratio of 1:1) of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1 respectively. Ferment in the same method as above (Experimental Example 2), and measure the contents of two free bile acids, deoxycholic acid and cholic acid, and the content of conjugated taurocholic acid in the fermentation broth. The measurement method is the same as that in Experimental Example 1, and the measurement results are shown in Table 4.

[0057] Table 4: Influence of fermentation with different bile - powder ratios on the contents of bile acids

[0058]

[0059] The results show that when the weight ratio of bile:powder is 4 - 6:1, the contents of cholic acid and deoxycholic acid produced by fermentation are relatively high. Under a high bile - powder ratio, although the content of conjugated taurocholic acid is higher, the amounts of its conversion products, cholic acid and deoxycholic acid, are not high. Therefore, the preferred weight ratio of bile:powder is determined to be 4 - 6:1.

[0060] Experimental Example 4: Investigation on the stirring fermentation time

[0061] Take fresh bovine bile and Pueraria lobata powder and Arisaema heterophyllum medicinal material powder crushed to 80 meshes, and mix them evenly according to the weight ratio of bile:powder (Pueraria lobata:Arisaema heterophyllum are mixed in a weight ratio of 1:1) of 4:1. Add Proteus mirabilis ([ Proteus mirabilis strain ), Bacillus cereus ([ Bacillus oleronii ), Kazachstania servazzii ( Kazachstania servazzii ), and Penicillium sp. ( Penicillium sp. The fermentation was carried out under stirring at 25°C and a stirring rate of 150 rpm. Samples were taken on fermentation days 0, 3, 7, 10, 15, 19, 23, 27, and 30 to determine the contents of two free bile acids, deoxycholic acid and cholic acid, and the content of conjugated taurocholic acid in the fermentation broth. The determination method was the same as that of Experimental Example 1. The results are shown in Table 5.

[0062] Table 5: Effects of different fermentation times on bile acid content

[0063]

[0064] The results showed that on the 15th day of fermentation, the levels of bile acid and deoxycholic acid produced by fermentation were the highest. As the fermentation time increased, the levels of conjugated taurocholic acid, bile acid, and deoxycholic acid decreased, possibly due to further conversion to other metabolites. Therefore, the fermentation time was determined to be 7-15 days, with 15 days being the preferred time.

[0065] Experimental Example 5: Investigation of stirring fermentation temperature

[0066] Take fresh ox bile and crush into 80 mesh arisaema powder and kudzu powder, mix them evenly in the ratio of bile: powder (kudzu: arisaema in a weight ratio of 1:1) by weight, add bile and powder weight and 4% Proteus mirabilis ( Proteus mirabilis strain ), vegetable Bacillus ( Bacillus oleronii ), Kazakh yeast Saccharomyces sergeni ( Kazachstania servazzii ) and Penicillium ( Penicillium sp. (the four strains were composed in a mass ratio of 1:1:1:1) and fermented at a stirring rate of 150 rpm for 15 days at fermentation temperatures of 20°C, 23°C, 25°C, 28°C, and 30°C. The contents of free bile acids (deoxycholic acid and cholic acid) and conjugated taurocholic acid in the fermentation broth were determined. The determination method was the same as in Experimental Example 1. The results are shown in Table 6.

[0067] Table 6: Effects of different fermentation temperatures on bile acid content

[0068]

[0069] The results showed that the content of free bile acid and deoxycholic acid was the highest on the 15th day of fermentation at 25°C; the fermentation results at 28°C were also good, slightly lower than 25°C. Therefore, the optimal fermentation temperature was determined to be 25°C-28°C.

[0070] Experimental Example 6:

[0071] Sample 1: A preparation method of Guangdanxing decoction pieces

[0072] The following steps are involved:

[0073] (1) Fermentation in a fermentation tank: Take the Radix Puerariae and Radix Arisaemae, crush them through an 80-mesh sieve, take fresh ox bile, mix them in a ratio of bile to medicinal powder (Radix Puerariae and Radix Arisaemae are mixed in a mass ratio of 1:1) 4:1, add the weight of bile and medicinal powder and 4% of Proteus mirabilis ( Proteus mirabilis strain ), vegetable Bacillus ( Bacillus oleronii ), Kazakh yeast Saccharomyces sergeni ( Kazachstania servazzii ) and Penicillium ( Penicillium sp. ) (the four bacteria were composed of a mass ratio of 1:1:1:1), and fermented at 25°C with stirring at a stirring rate of 150 rpm for 15 days;

[0074] (2) The mixed fermentation liquid obtained in the first stage is placed in the cattle gallbladder skin and suspended for fermentation. The temperature is controlled below 28°C and the relative humidity is controlled below 80%. The gallbladder is hung and fermented for 30 days to complete the second stage of fermentation.

[0075] (3) Molding process: Cut open the cow gallbladder skin, take out the fermented product, bake it at 70℃ for 20 hours, crush it into 80 mesh fine powder, wet granulate it, and press it into a cylindrical shape to obtain Guangdanxing decoction pieces.

[0076] Fifteen batches of pilot samples were prepared according to the above method, and the contents of two free bile acids, deoxycholic acid and cholic acid, and conjugated taurocholic acid in the slices were determined. The determination method was the same as that of Experimental Example 1, and the results are shown in Table 7.

[0077] Table 7: Content determination indicators of 15 batches of pilot products

[0078]

[0079] As shown in Table 7, the bile acid content of the 15 batches of pilot products ranged from 6.43% to 7.26%, with an average content of 6.89%, and the deoxycholic acid content ranged from 2.70% to 2.97%, with an average content of 2.81%.

[0080] Sample 2:

[0081] The only difference from Sample 1 is that Pueraria lobata was not added during the fermentation in the fermentation tank in step (1). The other procedures were the same as Sample 1. The contents of two free bile acids, deoxycholic acid and cholic acid, and the content of bound taurocholic acid in the prepared Arisaema confusa slices were determined. The determination method was the same as that in Experimental Example 1. The results of the six batches are shown in Table 8. At the same time, the relevant test data of Sample 1 and the traditional method of preparing Arisaema confusa by nine fermentations are supplemented in Table 8.

[0082] Table 8: Content determination results of products prepared by different preparation methods

[0083]

[0084] The results showed that when fermented without kudzu root, the average content of cholic acid was 3.80%, the content of deoxycholic acid was 1.46%, and the conversion level of free cholic acid was equivalent to the result of traditional fermentation for 9 times. However, the fermentation time was significantly shortened. The fermentation effect with the addition of kudzu root was significantly better than that without kudzu root. It can be seen that kudzu root is of great significance for the generation of active bile acids.

[0085] In the Arisaema cum Bile sample 1 obtained by using the preferred method of the present invention, the contents of cholic acid and deoxycholic acid were higher than those of sample 2 and traditional nine-turn Arisaema cum Bile, improving the activity of the cut crude drug. The preparation method of the present invention can promote the conversion of conjugated bile acids with lower activity in bile into free bile acids with higher activity. Especially with the addition of kudzu root, compared with traditional nine-turn fermented Arisaema cum Bile, the content of cholic acid in the Arisaema cum Bile obtained by the method of the present invention increased by 97.4%, and the content of deoxycholic acid increased by 117.83%.

[0086] The production process conditions of the preparation method of the Arisaema cum Bile obtained by the present invention are reasonable and feasible, can meet the requirements of industrial production, and the contents of cholic acid and deoxycholic acid in the obtained cut crude drug are high.

[0087] Experimental Example 7: Identification of the fermentation strain of Arisaema cum Bile

[0088] (1) Strain isolation: Appropriate amount of Arisaema cum Bile cut crude drug (batch number 20220904) was taken for streak plate culture to isolate single strains.

[0089] (2) Classical identification: After observing the colony characteristics and performing Gram staining (for bacteria) and lactophenol cotton blue staining (for fungi), it was observed under a microscope. Biochemical reaction identification included glucose fermentation experiment, methyl red experiment, urease experiment, indole experiment, gelatin experiment, and H2S experiment using 18 - 24 h young seeds.

[0090] (3) Molecular biology identification

[0091] The purified single strain was used to extract and amplify DNA with the TSINGKE plant DNA extraction kit. The PCR bands on 1% agarose gel electrophoresis were purified and recovered, and Sanger sequencing was performed; the software ContigExpress was used for splicing, removing the inaccurate parts at both ends to obtain the gene sequence of the strain, and a phylogenetic tree was constructed.

[0092] (4) Identification results: It was identified as Proteus mirabilis ( Proteus mirabilis strain ), Bacillus velezensis ( Bacillus oleronii ), Kazachstania servazzii ( Kazachstania servazzii ), and Penicillium sp. ( Penicillium sp. .).

[0093] Experimental Example 8: Pharmacodynamic experiment of Arisaema cum Bile against acute lung injury

[0094] (1) Experimental method: SPF-grade male BALB / c mice were purchased from the Guangdong Provincial Medical Experimental Animal Center, with the production license number: SCXK(Yue)2018-0002. They were divided into 9 groups according to the random number table method: blank group (CON), model group (MODEL), dexamethasone positive control group (Positive), low-dose group of Guangdanxing (batch number: 20220902) (TD, 1 g / kg), high-dose group (TG, 2 g / kg), low-dose group of Guangdanxing (batch number: 20220904) (HD, 1 g / kg), high-dose group (HG, 2 g / kg), cholic acid group (CA, 50 mg / kg), deoxycholic acid group (DCA, 50 mg / kg), with 10 mice in each group. Except for the blank group, mice in each group were intraperitoneally injected with LPS (5 mg / kg) to establish an acute lung injury model, and the intraperitoneal injection volume was 0.1 mL / 20 g. The blank group was injected with an equal volume of normal saline. 24 hours after modeling, the corresponding drugs were administered by gavage, and the gavage dosage was 0.2 mL / 20 g, once a day for 7 consecutive days.

[0095] (2) Results:

[0096] Pathological observation of lung tissue showed that: the positive drug group and each drug administration group were milder than the model group, with less inflammatory cell infiltration and mild congestion of capillaries. In the low-dose group, there were still alveolar stenosis and alveolar wall thickening. The improvement effects of the monomer cholic acid group and the high-dose group were better than those of the low-dose group, indicating that Guangdanxing and CA, DCA can all improve LPS-induced acute mouse lung injury. See attached Figure 1 .

[0097] The levels of TNF-α, IL-6, IL-1β, and IL-18 in the lung tissue of mice in the model group were extremely significantly increased (P<0.01), indicating a relatively high level of lung inflammation in mice. The low-dose group of Guangdanxing TD (batch number: 20220902) could significantly reduce the levels of TNF-α, IL-6, and IL-18 (P<0.05), and there was a tendency to reduce the level of IL-1β but no statistical significance (P>0.05); the high-dose group of Guangdanxing TG (batch number: 20220902) could significantly reduce the level of IL-6 (P<0.05), and extremely significantly reduce the levels of TNF-α, IL-1β, and IL-18 (P<0.01); the low-dose group of Guangdanxing (batch number: 20220904) could significantly reduce the levels of IL-1β and IL-18 (P<0.05), and extremely significantly reduce the levels of TNF-α and IL-6 (P<0.01); the high-dose group of Guangdanxing (batch number: 20220904), CA, and DCA groups could all extremely significantly reduce the levels of TNF-α, IL-6, IL-1β, and IL-18 (P<0.01). See attached Figure 2 .

[0098] Conclusion: Guangdanxing can alleviate pulmonary edema, improve the pathological morphology of lung tissue, reduce the levels of inflammatory factors TNF-α, IL-6, IL-1β and IL-18, inhibit the excessive inflammatory response in vivo, and thus play an anti-inflammatory and anti-acute lung injury role.

[0099] Experimental Example 9: Antipyretic effect of Guangdanxing

[0100] (1) Experimental method: SPF-grade male SD rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (No.: SCXK(Jing)2021-00011). All animal experiments were approved by the Ethics Committee of Heilongjiang University of Chinese Medicine (Approval No. 2023092207).

[0101] The rats with stable body temperature were randomly divided into a control group (Control), a model group (Model), a paracetamol (200 mg / kg) group, and a Guangdanxing (batch number: 20220904) group (1.4 g / kg), with 8 rats in each group. The rats in the control group were subcutaneously injected with normal saline (10 mL / kg) on the back, and the remaining rats were subcutaneously injected with 20% dry yeast suspension to establish a fever model. The rats in the paracetamol group and the Guangdanxing group were intragastrically administered once 30 min before modeling and 3.5 h after modeling, respectively. The control group and the model group were intragastrically administered an equal volume of normal saline (10 mL / kg). Starting from the 4th hour after modeling, the body temperature of the rats was measured every 1 h until the 8th hour ended, and the body temperature change value (ΔT, that is, the real-time body temperature value - the basal body temperature value) of the rats in each group at each time point was calculated.

[0102] (2) Results:

[0103] Effect on the body temperature of rats with fever induced by dry yeast: After injecting the dry yeast suspension at the 4th hour, the rectal temperature of the model rats increased significantly (P<0.01). After taking Guangdanxing, the rectal temperature of the rats decreased significantly (P<0.01) and continued until the 8th hour. The antipyretic time was similar to that of paracetamol. The results are shown in the following table:

[0104] Table 9: Changes in the anal temperature of rats in each group (`x, n = 8)

[0105]

[0106] Compared with the control group, ## P<0.01; compared with the model group, **P<0.01.

[0107] Effects on the levels of inflammatory factors in rats with dry yeast-induced fever: After dorsal injection of dry yeast suspension, the levels of TNF-α, IL-1β, IL-6 in the rat serum and PGE2 and cAMP in the hypothalamus were significantly increased (P<0.01). Compared with the model group, guandanxin significantly reduced the levels of TNF-α, IL-1β, IL-6 in the rat serum and PGE2 and cAMP in the hypothalamus (P<0.01). The results are shown in the table below:

[0108] Table 10: Effects on the levels of inflammatory factors in rat serum (`x, n=8)

[0109]

[0110] Compared with the control group, ## P<0.01; **P<0.01 compared with the model group.

[0111] Conclusion: Guangdanxing can significantly reduce the rectal temperature of febrile rats and last for a long time. The expression levels of pyrogenic factors (PGE2, cAMP, CRH, TNF-α, IL-1β and IL-6) in the rats in the Guangdanxing group are significantly reduced.

[0112] Experimental Example 10: Therapeutic Effect of Guangdanxing on Mice with Non-alcoholic Steatohepatitis

[0113] (1) Experimental methods: SPF male C57BL / 6 mice were purchased from the Guangdong Medical Laboratory Animal Center with the animal production certificate number SCXK (Yue) 2018-0002. The mice were randomly divided into 7 groups, including the normal group (ControL), the model group (ModeL), the positive control group of Yishanfu capsule (EssentiaLe, 0.178 g / kg), the low-dose group (TD, 0.78 g / kg) and high-dose group (TG, 2.34 g / kg) of Guangdanxing (batch: 20220902), the low-dose group (HD, 0.78 g / kg) and high-dose group (HG, 2.34 g / kg) of Guangdanxing (batch: 20220904), with 10 mice in each group. The mice in the normal group were fed MCS feed, while the mice in the model group and the drug-treated group were fed MCD feed three times a day. The weight of the feed consumed by the mice and the body weight of the mice were recorded. The success of the model was confirmed by liver function indexes and blood lipid indexes four weeks after modeling. Starting from the fifth week, the normal group and the model group were gavaged with 0.5% sodium carboxymethylcellulose solution, and the mice in the other groups were gavaged with the corresponding drug solution. The gavage volume was 10 mL / kg, and the drug was administered once a day for 21 consecutive days. The dosage of the low-dose and high-dose groups was calculated based on the clinical drug dose and 3 times the clinical drug dose.

[0114] (2) Results:

[0115] Effect on the liver histopathology of NASH mice: Compared with the model group, cytoplasmic fatty degeneration and nuclear pyknosis of hepatocytes in mice of each dosing group of Guangdanxing were significantly alleviated, especially in the high-dose group of Guangdanxing (batch: 20220902). Oil Red O results: It can be seen that there was no obvious fatty degeneration in the hepatocytes of mice in the normal group; there were many red-stained lipid droplets of different sizes in the liver lobules of mice in the model group, showing typical fatty degeneration and vacuolar degeneration; compared with the model group, the red-stained lipid droplets in the hepatocytes of mice in each dosing group of Guangdanxing were significantly reduced, especially in the high-dose group of TG Guangdanxing (batch: 20220902). See appendix Figure 3 。

[0116] Effect on serum liver function indexes and blood lipid indexes: The levels of ALT and AST in the serum of mice in the model group were significantly increased (P<0.01), and the levels of TC and TG were significantly increased (P<0.01); compared with the model group, the levels of ALT and AST in the serum of mice in the EssentiaLe group, TD group, TG group, HD group, and HG group were significantly decreased (P<0.01), and the levels of TC and TG were significantly decreased (P<0.01). The results are shown in the following table.

[0117] Table 11: Effect on serum liver function indexes and blood lipid indexes of NASH mice (`x±SD, n = 10)

[0118]

[0119] Compared with the Control group, ## P<0.01; compared with the Model group, **P<0.01.

[0120] Effect on serum inflammatory factors of NASH mice: The levels of IL-6, IL-1β, and TNF-α in the serum of mice in the model group were significantly increased (P<0.01); the levels of IL-6, IL-1β, and TNF-α in the serum of mice in the EssentiaLe group, TD group, TG group, HD group, and HG group were significantly decreased (P<0.01). The specific results are shown in the following table.

[0121] Table 12: Effect of Guangdanxing on serum inflammatory factors of NASH mice (`x±SD, n = 10)

[0122]

[0123] Compared with the Control group, ## P<0.01; compared with the Model group, **P<0.01.

[0124] Effects on oxidative stress in the liver tissue of NASH mice: MDA levels in the liver tissue of mice in the model group were significantly increased, while SOD levels were significantly decreased (P<0.01). MDA levels in the liver tissue of mice in the EssentiaLe, TD, TG, HD, and HG groups were significantly decreased, while SOD levels were significantly increased (P<0.01). The results are shown in the table below.

[0125] Table 13: Effects of guandanxin on oxidative stress in liver tissue of NASH mice (`x±SD, n=10)

[0126]

[0127] Compared with the Control group, ## P<0.01; **P<0.01 compared with the Model group.

[0128] Conclusion: Guangdanxing exerts significant anti-inflammatory, antioxidant, anti-steatosis effects in NASH, and regulates glucose and lipid metabolism and bile acid metabolism, reduces liver parenchymal cell damage, hepatic lipid accumulation and inflammatory cell infiltration in mice fed an MCD diet, and has the effect of treating non-alcoholic fatty liver disease.

[0129] Experimental Example 11: Study on the efficacy of Guangdanxing in MPTP Parkinson's disease mouse model

[0130] (1) Experimental methods:

[0131] SPF C57BL-6J male mice were purchased from the Guangdong Medical Laboratory Animal Center (animal license number: SCXK(粤)2018-0002). They were randomly divided according to body weight into a normal group (CON), a model group (MODLE), a levodopa tablet group (POSITIVE, 80 mg / kg), a low-dose group (TD, 0.39 g / kg) and a high-dose group (TG, 1.56 g / kg) of galactopyrin (batch number 20220902), a cholic acid group (CA, 10 mg / mL), and a deoxycholic acid group (DCA, 10 mg / mL), with 10 mice in each group. Except for the normal group, mice in all other groups received an intraperitoneal injection of methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) solution (35 mg / kg) once daily for 5 consecutive days to establish a subacute PD model. Mice in the normal group were simultaneously injected intraperitoneally with normal saline (10 g / kg). Starting from the sixth day of the experiment, mice in the normal group and the model group were gavaged with 0.5% sodium carboxymethylcellulose solution (10 g / kg) every day, and mice in the other groups were gavaged with the corresponding drug solution once a day for 7 consecutive days.

[0132] (2) Results:

[0133] Weight changes and behavioral observations: Within half an hour after intraperitoneal injection of MPTP, mice developed symptoms such as slow movement, crawling difficulties, erect hair, and tail stiffness. It can be seen that during the modeling period from day 1 to day 5, except for the blank group, which continued to gain weight, the weight of mice in the other groups decreased. From day 6, when the modeling was stopped and the corresponding therapeutic drug was given, the mice began to gain weight. However, when the modeling was repeated on day 12, the weight of the mice decreased again. This indicates that MPTP intraperitoneal injection modeling can induce behavioral disorders and physical stress in mice, affecting their feeding function, thereby leading to weight loss.

[0134] Results of the pole climbing test: After 5 days of intraperitoneal injection of MPTP, the climbing time of mice was significantly higher than that of CON (P<0.0001), indicating a severe decline in climbing ability and a stable modeling effect. After one week of treatment and the final modeling, the climbing time of MODEL mice was significantly prolonged (P<0.0001), indicating that the mice's behavioral ability was difficult to recover. Although POSITIVE and TD could shorten the climbing time of mice compared with MODEL, there was no significant difference. TG significantly shortened the climbing time of mice compared with MODEL (P<0.001). CA significantly shortened the climbing time compared with MODEL (P<0.05). Deoxy DCA significantly shortened the climbing time of mice compared with MODEL (P<0.01). The results of the pole climbing test showed that TG, CA, and DCA could effectively shorten the climbing time of MPTP mice and improve the motor ability of MPTP mice.

[0135] The results of TH protein immunofluorescence in the substantia nigra: after MPTP modeling, the number of TH-positive cells in the substantia nigra of MODEL decreased significantly compared with CON; compared with MODEL, the number of TH-positive cells in the substantia nigra of TD, TG, CA and DCA increased, among which the increase was particularly obvious in TG and CA.

[0136] ELISA detection of inflammatory levels in mouse serum and substantia nigra: The level of IL-1β in the serum of MODEL was extremely significantly increased (P<0.001); TD, TG, CA, and DCA all significantly decreased the level of IL-1β in mouse serum (P<0.001). The level of TNF-α in the serum of MODEL was significantly increased (P<0.01); TG and CA significantly decreased the level of TNF-α (P<0.05). The level of IL-1β in the substantia nigra of MODEL was extremely significantly high (P<0.001); TD and TG could significantly decrease the level of IL-1β in the substantia nigra (P<0.01). The level of TNF-α in the substantia nigra of MODEL was extremely significantly increased (P<0.001); TD and TG extremely significantly decreased the level of TNF-α in the substantia nigra (P<0.001), CA significantly decreased the level of TNF-α in the substantia nigra (P<0.01), and DCA significantly decreased the level of TNF-α in the substantia nigra (P<0.05). The level of iNOS in the substantia nigra of MODEL was significantly increased (P<0.05); TD and DCA significantly decreased the level of iNOS in the substantia nigra (P<0.05), and TG significantly decreased the level of iNOS in the substantia nigra (P<0.01). The level of COX-2 in the substantia nigra of MODEL was significantly increased (P<0.05); TD significantly decreased the level of COX-2 in the substantia nigra (P<0.05), and TG significantly decreased the level of COX-2 in the substantia nigra (P<0.01). See attachment Figure 4 , where A is the level of IL-1β in the serum of each group, B is the level of TNF-α in the serum of each group, C is the level of IL-1β in the substantia nigra of each group, D is the level of TNF-α in the substantia nigra of each group, E is the level of INOS in the substantia nigra of each group, and F is the level of COX-2 in the substantia nigra of each group; under the same index, compared with the CON group: # P < 0.05, ## P < 0.01, ### P < 0.001; compared with the MODEL group: *P < 0.05; **P < 0.01, ***P < 0.001; ns indicates P > 0.05.

[0137] WB detection of the levels of PKA, GPX4, and FTH1 in the substantia nigra of mice: After treatment with TG, CA, and DCA, the level of PKA in the TG group was significantly increased (P<0.05), and the levels of PKA in the CA group and DCA group were also extremely significantly increased (P<0.01); the levels of GPX4 in the TG and CA groups were significantly increased (P<0.05), and the level of GPX4 in the DCA group was extremely significantly increased (P<0.01); the level of FTH1 in the MODEL group was significantly decreased (P<0.05), and the levels of FTH1 in the TG, CA, and DCA groups were significantly increased (P<0.05). See attachment Figure 5, where A is the protein expression level of PKA, GPX4 and FTH1, B is the relative expression of PKA, C is the relative expression of GPX4, and D is the relative expression of FTH1; under the same indicators, compared with the CON group: # P<0.05; compared with the MODEL group: *P<0.05, **P<0.01.

[0138] Conclusion: Guangdanxing significantly shortened the pole climbing time in MPTP model mice, indicating significant improvement in behavioral impairment in MPTP model mice. TG significantly protected DA neurons from TH-positive neurons, demonstrating a neuroprotective effect. Regarding anti-inflammatory factors, TG significantly reduced IL-1β, TNF-α, iNOS, and COX-2 levels in MPTP model mice. Western blotting revealed that MPTP induced a decrease in GPX4 and FTH1 protein levels in mice, while TG inhibited ferroptosis in substantia nigra neurons by increasing PKA, GPX4, and FTH1 levels.

Claims

1. A method for preparing a Guangdanxing decoction piece, characterized in that: The preparation method comprises the following preparation steps: (1) Grinding Puerariae Radix and Arisaema Cone to obtain mixed powder; the mass ratio of Arisaema Cone powder to Puerariae Radix powder is 1:0.25-1; (2) Bile and mixed powder are mixed in a mass ratio of 4-6:1, and fermented with fermentation bacteria to obtain a fermentation liquid; the amount of the fermentation bacteria added is 3-6% of the total mass of the bile and the mixed powder; the fermentation temperature is 25-28°C, and the fermentation time is 7-15 days; (3) placing the fermentation liquid in a cattle gallbladder and performing suspension fermentation to obtain a fermented product; the suspension fermentation temperature is lower than 28° C., the relative humidity is lower than 80%, and the fermentation time is 30 days; (4) The fermented product is dried at low temperature, crushed, and tableted or pelleted to obtain Guangdanxing decoction pieces; the temperature of the low temperature drying is not higher than 80°C; Among them, the fermenting bacteria are Proteus mirabilis, Bacillus velezensis, Kazachstania servazzii, and Penicillium, with a mass ratio of 1:1:1:

1. Proteus mirabilis strain , Bacillus velezensis Bacillus oleronius , Kazachstania servazzii Kazachstania servazzii and Penicillium Penicillium sp .

2. The preparation method according to claim 1, characterized in that, The mixed medicinal powder in step (1) is prepared by crushing the Pueraria lobata medicinal material and the Arisaema consanguineum medicinal material into 80 mesh sizes.

3. The preparation method according to claim 1, characterized in that, The low-temperature drying in step (4) is carried out for 12-24 hours, and the product is crushed into 80-100 mesh fine powder.

4. A Guangdanxing decoction piece, characterized in that The method is described in any one of claims 1 to 3.

5. Use of the Guangdanxing decoction piece prepared according to the preparation method according to any one of claims 1 to 3 or the Guangdanxing decoction piece according to claim 4 in preparing drugs for acute lung injury, antipyretic, non-alcoholic fatty liver disease or Parkinson's disease.

6. The use according to claim 5, wherein the Guangdanxing decoction piece can be used directly or as a raw material for preparing other compound preparations.

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