A method for processing arisaema cum bile by regulating oxygen content in fermentation environment

Abstract

The application relates to a fermentation processing method of Arisaema cum Bile based on adjusting oxygen content in a fermentation environment. The application also relates to Arisaema cum Bile processed by the fermentation processing method. The fermentation process of Arisaema cum Bile is finely controlled, and the main feature is that the oxygen content in the environment during the fermentation process is controlled. The oxygen content in the fermentation environment is controlled by adding oxygen carriers and preheating air. The toxicity of the obtained Arisaema cum Bile is significantly reduced, and the tranquilizing and nerve-calming effects are improved.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine processing technology, specifically relating to a fermentation processing method for Arisaema cum Bile based on adjusting the oxygen content of the fermentation environment. Background Technology

[0002] Arisaema cum Bile is a traditional koji with a long history of application. It is made by processing fine powder of Arisaema cum Bile with bile from cattle, sheep or pigs, or by fermenting fine powder of raw Arisaema cum Bile with bile from cattle, sheep or pigs. It tastes bitter and slightly pungent, and is cool in nature. It has the functions of clearing heat and resolving phlegm, calming wind and relieving convulsions.

[0003] The 2020 edition of the Chinese Pharmacopoeia describes Arisaema cum Bile as being square or cylindrical in shape. It is brownish-yellow, grayish-brown, or brownish-black in color, hard in texture, with a slightly fishy odor and a bitter taste. The powder is pale yellowish-brown. The thin-walled cells are nearly round, filled with gelatinized starch granules. The calcium oxalate needle crystal bundles are 20–90 μm long. The spiral and annular vessels are 8–60 μm in diameter.

[0004] The earliest record of Arisaema cum Bile is found in the Song Dynasty text *Xiao'er Yaozheng Zhijue*, which states: "In the twelfth lunar month, it is brewed in ox gall and dried in the shade for one hundred days." The *Shengji Zonglu*, from the same period, records: "Soak in ox gall for three nights and then bake; boil ox gall for one fu (a period of time) and then dry in the sun." Although the methods of brewing ox gall and drying in the shade, and soaking and boiling ox gall followed by sun drying, differ in preparation time and drying method, both describe the relatively long period of "brewing" or "soaking" Arisaema cum Bile powder in ox gall, and can be considered the origin of later fermented Arisaema cum Bile preparation methods.

[0005] The "Formulary of the Bureau of Imperial Medical Services for the Benefit of the People" pioneered a method for preparing Arisaema bile cakes: "Wash with hot water, bake, grind into powder, mix with ox bile to make cakes, and bake dry." This method proposed pre-treatment of Arisaema, eliminating the need for long-term fermentation and improving production efficiency. The widely used method of preparing Arisaema bile powder by mixing it with bile to prepare Arisaema bile can be seen in its rudimentary form.

[0006] The "sheep bile processing" method was first mentioned in the *Puji Benshifang*. This indicates that in the Song Dynasty, the processing of Arisaema cum Bile was inconsistent, with varying degrees of fermentation and the use of either ox or sheep bile. After the Song Dynasty, different methods of processing Arisaema cum Bile were adopted, developed, and innovated upon.

[0007] Of the 17 historical records on the processing of Arisaema cum Bile from the Yuan Dynasty to the Republic of China period, 12 involved a long-term air-drying method using ox bile, which is classified as a fermentation method. The fermentation process during the Yuan and Ming Dynasties was basically the same as that of the Song Dynasty, with more detailed descriptions of the procedures. It was believed that extending the fermentation time and increasing the number of times ox bile was added could improve the quality of Arisaema cum Bile.

[0008] The *Huoyou Xinshu* records: "Crush it, brew it with the gall of a yellow ox in the twelfth lunar month, and use it after a summer." The *Danxi Xinfa* states: "It must use the gall of a yellow ox, powdered Arisaema in the twelfth lunar month, personally mix and air-dry, and use it after a year. The gall of the ox must be added three or four times for best results." The Ming Dynasty *Compendium of Materia Medica* records: "Method for preparing Arisaema: Grind raw Arisaema into powder, take the gall of a yellow ox in the twelfth lunar month, mix it with the powder, put it into the gall, and hang it in a windy place to dry; the longer it is dried, the better." In the late Ming Dynasty, more complex methods for processing Arisaema in gall were developed. The *Jingyue Quanshu* records that the Arisaema in gall used in the Baolong Wan formula is processed "nine times." The Qing Dynasty book "Youyou Jicheng" records in detail the method of preparing nine-times processed ox gall and arisaema: "Take an ox gall, pour out the bile into a bowl, mix it with arisaema powder, put it back into the gall, and hang it in a place with wind but no sun to dry in the shade. When the gall is ready, cut open the gall and take it out. Mix the arisaema powder with the bile again and put it back in and hang it up. If it can be filled with nine galls, it is truly a treasure, and no real bezoar can compare to it."

[0009] Nine-times-processed Arisaema requires nine ox gallbladders. It uses a large amount of bile, is complex to make, and has significant medicinal effects. However, there are different records about the time required for its production. Some say it takes two or three years, while others say it takes nine years to turn brown and that the best is the one that takes nine years to make.

[0010] The method of soaking in ox bile and then drying may have evolved into a method of mixing Arisaema powder with ox bile for fermentation and then steaming. Currently, many places across the country still use the bile-mixed fermentation and steaming method. During the Ming and Qing dynasties, it was common to process raw Arisaema with other auxiliary materials, then grind it into powder, add it to ox bile, and dry it in the shade. Methods for processing Arisaema included "soaking and stir-frying in ginger juice," "mixing with boiling alum or ginger juice and soaking," "grinding with peach kernels and drying in the sun," and "soaking Arisaema and Fritillaria cirrhosa in equal parts, grinding them into a very fine powder." Although all these methods involve drying in the shade inside ox bile, none of them specify the required time as in the aforementioned fermentation method, making it difficult to determine whether fermentation occurred during the drying process. Ancient methods often used ox bile, and many noted "ox bile from a yellow bull in the twelfth lunar month," believing its quality to be superior. The *Complete Collection of Experiences in Processing Chinese Medicine* records that Arisaema bile can be processed using pig bile.

[0011] The 1977 edition of the Chinese Pharmacopoeia first included Arisaema cum Bile, including its properties, microscopic characteristics, and physicochemical identification methods. The 1985 edition revised the dosage and indications of Arisaema cum Bile, and subsequent editions have followed the 1985 standard. Since its inclusion in the 1977 Pharmacopoeia, Arisaema cum Bile has been listed as an independent product. In the 1988 edition of the National Standards for Processing Traditional Chinese Medicine, Arisaema cum Bile was included as a processed product of Arisaema heterophyllum, specifying two processing methods: steaming processed Arisaema heterophyllum mixed with bile, or steaming raw Arisaema heterophyllum mixed with bile after fermentation. The standards quantified the ratio of Arisaema heterophyllum to bile, steaming time, fermentation time and number of fermentations, and specified that bovine (or pig, sheep) bile or bile powder could be used to prepare Arisaema cum Bile.

[0012] Due to historical reasons, the processing methods for Arisaema cum Bile have never been standardized across the country. However, the processing specifications for Arisaema cum Bile are gradually becoming more uniform and closer to the national standard, although differences still exist in processing techniques, bile dosage, and fermentation time. Of the 30 provincial and municipal processing specifications currently in place nationwide, 18 include Arisaema cum Bile, listing a total of 31 processing methods. Among these, the largest amount of bile used per 100kg of Arisaema cum Bile is 700kg, and the smallest is 64kg; 55% of the methods use a 1:4 ratio of Arisaema cum Bile to bile. Regarding the source of bile, Jiangxi specifies the use of bovine bile, Sichuan specifies the use of pig bile, Beijing, Inner Mongolia, and Zhejiang only specify bile, while other regions explicitly state that bovine (or pig, sheep) bile can be used. Hebei, Hubei, and other regions have removed the Arisaema cum Bile variety from their local specifications and directly implement the national standard. Beijing originally had four processing methods, but now only one is retained.

[0013] Ancient people believed that bile was not a commonly recognized auxiliary material in the preparation of Arisaema cum Bile, but should be considered one of the main raw materials. The composition of bile is an important direction in modern research on Arisaema cum Bile; the determination of bile acid components is frequently found in the literature, and in recent years, research reports have also emerged on the influence of bile source and dosage on the quality of Arisaema cum Bile. Traditionally, Arisaema cum Bile was processed mostly with ox bile, and rarely with sheep bile. Because pig bile is inexpensive and readily available, commercially available Arisaema cum Bile is mostly made with pig bile.

[0014] Traditional theory holds that the properties of bovine, porcine, and ovine bile are not entirely the same. However, Chen Jiangning et al., using high-performance liquid chromatography with electro-optical detector (HPLC-CAD), found differences in the bile acid composition of Arisaema cum Bile prepared from porcine, bovine, and ovine bile. The mass fraction of bile acids in Arisaema cum Bile prepared from bovine bile was higher than that in Arisaema cum Bile prepared from ovine and porcine bile. Furthermore, the effects of different bile-prepared Arisaema cum Bile on body temperature changes in a yeast-induced mouse fever model showed different trends over time.

[0015] Other studies have shown that Arisaema cum Bile prepared from bovine, ovine, and porcine bile has a protective effect against lipopolysaccharide-induced acute lung injury in rats. This may be achieved by inhibiting the release of inflammatory factors, thus improving the pathological morphology of lung tissue and the damage caused by oxidative stress. Bovine and ovine Arisaema cum Bile showed a certain advantage over porcine Arisaema cum Bile in protecting against acute lung injury. These studies suggest that all three types of bile-prepared Arisaema cum Bile have heat-clearing and phlegm-resolving effects, and that bovine, porcine, and ovine bile can be used to prepare Arisaema cum Bile, which is reasonable. However, pharmacodynamic studies of Arisaema cum Bile prepared from different bile preparations show differences, and its mechanism requires further in-depth research and repeated testing.

[0016] Li Zeyu et al. used ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) and negative ion multiple reaction monitoring (MRM) mode to determine the content of bile acid components in three ratios of raw materials (bile:arisaema 3:1, 1:1, and 1:3, respectively). The results showed that there were significant differences. A higher proportion of bile resulted in better antipyretic and expectorant effects of arisaema cum bile, while a lower proportion resulted in weaker effects, but had little impact on antitussive effects. They believed that the ratio of bile to arisaema should not be lower than 1:1.

[0017] In the 20th century, researchers improved the mixing and fermentation methods by using concentrated bile mixtures, increasing fermentation temperatures, and simmering techniques to shorten processing time and increase production efficiency. From the perspective of production efficiency alone, the mixing method is superior to the fermentation method. Modern research, when evaluating processing methods, places greater emphasis on the impact of the processing process on increasing the content of active ingredients and efficacy. Literature reports that Arisaema cum Bile prepared by the mixed steaming method mainly contains bound bile acids, while Arisaema cum Bile prepared by fermentation contains free bile acids. The fermentation process during the processing of Arisaema cum Bile can cause the bound bile acids in the bile to undergo chain breakage and decomposition, converting them into free bile acids. The bile acid components and their content in Arisaema cum Bile can be used as indicators for quality control. Zhu Yanfei et al., using principal component analysis (PCA) to investigate the overall impact of different process parameters on the thin-layer chromatograms of Arisaema cum Bile during mixed steaming, verified that the source of pig bile and different steaming times both affect the material basis of the finished Arisaema cum Bile, suggesting that the reference factors obtained by PCA can be used as a metrological means for quality control of Arisaema cum Bile. Tang Siyuan believes that different fermentation raw materials and conditions can affect the quality of Arisaema cum Bile. Under drying conditions of 37℃, Arisaema cum Bile prepared from bovine bile powder can obtain fermented Arisaema cum Bile products with high bile acid content. Li Yao studied the changes in composition of Arisaema cum Bile prepared by fermentation and mixed methods before and after processing, showing that after processing, compared with Arisaema cum Bile or processed Arisaema cum Bile, the number of calcium oxalate needle crystals decreased and the structure was destroyed; the contents of flavonoid components such as schaphtol, isochordol, and polysaccharides were all reduced. This indicates that both processing methods have the effect of reducing the toxicity of Arisaema cum Bile. Compared to the mixed-processed Arisaema cum Bile, the fermented Arisaema cum Bile shows a more significant decrease in flavonoid and polysaccharide content, and generates new glycoamines and other components. Conjugated bile acids are converted into free bile acids, while the mixed-processed Arisaema cum Bile mainly contains conjugated bile acids.

[0018] The effects of processed Arisaema cum Bile are recorded in numerous traditional Chinese medicine texts. For example, it is said that "it is not drying when processed with ox bile," and that "processing with ox bile reduces its toxicity, and the drying effect is also reduced; moreover, bile has beneficial effects on the liver and gallbladder." Raw Arisaema cum Bile has a strong numbing effect on the tongue; its water extract, when injected intraperitoneally or applied to the ears of mice, causes writhing and significant ear swelling, while processed Arisaema cum Bile does not exhibit these reactions. It is possible that processing with bile can indeed reduce or eliminate adverse toxic reactions. Experiments have also been conducted on fermented and synthetically prepared Arisaema cum Bile, both of which show a reduction in the adverse toxic reactions of Arisaema cum Bile.

[0019] Ancient and modern physicians agree that the efficacy of Arisaema cum Bile changes after processing, primarily clearing heat and phlegm, calming wind and relieving convulsions, and also possessing sedative and anticonvulsant effects. "Arisaema cum Bile effectively reduces phlegm due to its heat-induced properties, making it essential for treating infantile convulsions. Compared to raw Arisaema, it is bitter and cool in nature, thus better at resolving wind-phlegm-heat stagnation." Studies by Wang Wei et al. have confirmed that raw Arisaema has a certain degree of drying properties, which can reduce water intake, oxygen consumption, and Na+ in mice. + _K + - ATPase activity and total protein content in liver tissue increase, while the cooling properties of Arisaema cum Bile can reduce the levels of these indicators. This indicates that processing with bile can alter its medicinal properties. In modern clinical applications, Arisaema cum Bile is rarely used as a single herb, but rather in compound prescriptions. For example, Arisaema cum Bile combined with Bambusa textilis, Curcuma longa, Coptis chinensis, Acorus tatarinowii, and Polygala tenuifolia is used to treat heat-type stroke with coma and epilepsy; combined with Trichosanthes kirilowii, Scutellaria baicalensis, Platycodon grandiflorus, and Arctium lappa, it is used to treat lung congestion due to heat and thick yellow phlegm. When used with Scorpion (processed), Bombyx mori, Gastrodia elata, and Coptis chinensis, it has the effects of clearing heat and resolving phlegm, extinguishing wind and stopping spasms, and is used for all kinds of phlegm-induced asthma in children, acute and chronic infantile convulsions, and tetanus of the hands and feet, such as in Qianjin San.

[0020] The pharmacopoeia specifies a dosage of 3-6g for Arisaema cum Bile in clinical practice, while some believe 5-10g is generally appropriate. The pediatric dosage is 3-6g, which can be adjusted according to the patient's condition. Extensive clinical practice has shown that decocting raw Pinellia ternata and raw Arisaema cum Bile for 2 hours before mixing them with Poria cocos, Codonopsis pilosula, Astragalus membranaceus, and Bambusa textilis has proven effective in treating esophageal cancer. There are also clinical reports of using raw Arisaema cum Bile powder externally to treat chalazion in children. Applying Evodia rutaecarpa and Arisaema cum Bile in a 3:1 ratio to the Yongquan acupoint can also treat drooling in children.

[0021] Throughout history, physicians have frequently used different dosages of Arisaema cum Bile in combination with various types of Chinese herbal medicines to treat many diseases. However, the dosage conversion varied across different dynasties. For example, one liang (approximately 37.3g) in the Song Dynasty was about 41.4g, in the Ming Dynasty about 37g, and in the Qing Dynasty about 37.3g. For instance, in the Fritillaria and Trichosanthes Powder (Qing Dynasty, *Medical Insights*), Arisaema cum Bile (approximately 6.875g, five fen) clears heat and resolves phlegm. Combined with Trichosanthes kirilowii, it reduces lung fire and resolves heat-phlegm, treating stroke-like conditions with lung fire stagnation and lung heat with dryness. Similarly, in the Epilepsy-Stopping Pill (Qing Dynasty, *Medical Insights*), Arisaema cum Bile (18.75g, approximately 0.05g per dose, equivalent to about 0.2g in modern dosage) clears heat and resolves phlegm. Combined with Acorus tatarinowii, it resolves phlegm, opens the orifices, and awakens the mind, treating phlegm-heat epilepsy and also used for mania. For example, Xingzhu Powder (from Ming Dynasty's *One Hundred Questions for Infants and Children*) uses Arisaema cum Bile (one liang is 37g, each dose is about 0.12 qian, equivalent to a modern dose of 0.455g) to calm epilepsy and resolve phlegm. Combined with cinnabar, it clears the heart and calms the mind, treating phlegm-induced epilepsy. For example, Zhenjing Pill (from Ming Dynasty's *One Hundred Questions for Infants and Children*) uses Arisaema cum Bile (two and a half qian is about 9.25g, each dose is about 0.015 qian, equivalent to a modern dose of about 0.06g) to resolve phlegm and calm the mind. Combined with amber and Poria cocos, it calms the mind and stabilizes the spirit, treating all types of epilepsy. For example, Sanfeng Powder (from Ming Dynasty's *One Hundred Questions for Infants and Children*) uses Arisaema cum Bile (two qian is about 7.4g, each dose is about 0.05 qian, equivalent to a modern dose of 0.2g) to resolve phlegm and calm epilepsy. Combined with Saposhnikovia divaricata to dispel wind and relieve spasms, it treats wind-induced epilepsy. Summary of the Invention

[0022] The applicant has conducted in-depth and extensive research on the fermentation and processing technology of Arisaema cum Bile. The research found that by adjusting the oxygen content in the fermentation environment, the sedative effect of the prepared Arisaema cum Bile can be further improved, while the toxicity of Arisaema cum Bile can be further reduced.

[0023] This application first discloses a fermentation processing method for Arisaema cum Bile, the fermentation processing method comprising the following preparation steps:

[0024] Step 1) Soak the Arisaema heterophyllum in water, then remove it and boil it with ginger and alum until there is no dry core inside;

[0025] Step 2) Remove and air-dry or bake until semi-dry, cut into thin slices, and pre-treat the thin slices to obtain pre-made slices;

[0026] Step 3) Dry the pre-made sheets and then grind them into Arisaema powder;

[0027] Step 4) Add the prepared Arisaema powder, bile, and n-dodecane to purified water and mix well to obtain a suspension;

[0028] Step 5) Sterilize the suspension obtained in step 4) at high temperature;

[0029] Step 6) Take the sterilized suspension obtained in Step 5) and add compound fermentation fungi at a mass percentage of 0.2%-1.0%;

[0030] Step 7) Take the suspension obtained in Step 6) and ferment it under stirring conditions, keeping the temperature at 25-35℃ and the relative humidity at 65%-90%. Fermentation is completed after 5 days.

[0031] Step 8) Take the fermentation suspension obtained in step 7) and reduce the water content by low-temperature evaporation to obtain drug pellets of appropriate size. Then, make them into soft blocks of appropriate size.

[0032] Step 9) Take the soft block obtained in Step 8) and vacuum dry it to volatilize the n-dodecane in the soft block, and you will get the final product, Arisaema cum Bile.

[0033] As a preferred embodiment, the specific steps of soaking in step 1) are as follows: place Arisaema in a washing pool and soak it in water for 2-3 days, changing the water 2-3 times a day, adding 3wt% alum of Arisaema each time the water is changed, until there is no numbing sensation on the tongue when tasted.

[0034] In step 1), the mass ratio of Arisaema heterophyllum, ginger and alum when cooking together is 100:10:10.

[0035] Preferably, the thickness of the sheet in step 2) is 1~2mm;

[0036] The pretreatment process is as follows: the thin film is immersed in cellulase solution for 5-10 minutes, and then subjected to ultrasonic cell wall disruption treatment at 200-300W for 10-15 minutes together with the enzyme solution. The thin film is then filtered out and dried to obtain the pre-made film.

[0037] The amount of cellulase used is 200~300u / 1g of tablets.

[0038] Preferably, the drying temperature in step 3) is 50~70℃ and the drying time is 3~6h.

[0039] Preferably, in step 4), the mass ratio of Arisaema powder, bile, n-dodecane, and purified water is 100:(200~300):(5-30):100.

[0040] The bile in question is pig bile.

[0041] The role of bile is to neutralize the toxicity of Arisaema powder (i.e., Arisaema powder after preliminary processing).

[0042] Preferably, the temperature for high-temperature sterilization in step 5) is 110~121℃, and the sterilization time is 15~35min.

[0043] Preferably, the compound fermentation fungi in step 6) are composed of Lichtheimiaramosa and Hyphopichia burtonii in a mass ratio of 1:1.

[0044] Preferably, step 7) maintains a high oxygen level in the fermentation environment by introducing preheated fresh air.

[0045] Furthermore, in step 7), during the fermentation process, preheated fresh air is introduced to ensure that the oxygen content in the fermentation environment is not less than 15% until the fermentation is completed.

[0046] The beneficial effects of this invention are:

[0047] This invention, based on the traditional preparation method of Arisaema cum Bile, further detoxifies the Arisaema cum Bile slices. By reducing the toxicity of the alkaloids contained in Arisaema cum Bile through detoxification, pre-formed tablets are obtained. Then, the oxygen carrier n-dodecane is added to the fermentation suspension to increase the oxygen content of the fermentation broth, and the oxygen content above the fermentation broth is increased by introducing fresh air. This facilitates fermentation. The fermentation and processing method described in this application can effectively reduce the impact of Arisaema cum Bile's toxicity on the motor nervous system, i.e., accelerate the recovery of spontaneous motor ability after drug withdrawal, and effectively increase the duration of induced sleep, i.e., improve the sedative effect.

[0048] This invention provides precise control over the fermentation process of Arisaema cum Bile, primarily by controlling the fermentation environment and the oxygen content in the fermentation broth. This control is achieved through the introduction of preheated air and the addition of an oxygen carrier.

[0049] Finally, this application provides a type of Arisaema cum Bile, which is prepared by the aforementioned fermentation and processing process of Arisaema cum Bile. Detailed Implementation

[0050] The present invention will be further described in detail below through embodiments. These embodiments are illustrative of the invention, but do not limit the invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0051] Example 1: Preparation of Arisaema cum Bile

[0052] Step 1) Soak Arisaema in a washing pool for 2 days, changing the water 3 times a day. On the second day, add 3wt% alum of Arisaema to the water each time you change the water until there is no numbness on the tongue. Then, boil the Arisaema, ginger and alum in a mass ratio of 100:10:10 until there is no dry core inside.

[0053] Step 2) Take out the Arisaema obtained in Step 1) and air-dry or bake it until semi-dry. Cut it into thin slices with a thickness of 1 mm. Soak the thin slices in cellulase solution for 10 min. The ratio of the mass of the thin slices to the amount of cellulase in the enzyme solution is 1 g: 200 u. Then, together with the cellulase solution, perform ultrasonic cell wall disruption treatment at 200 W and 20 kHz for 15 min. Then filter out the thin slices and dry them to obtain the pre-made slices.

[0054] Step 3) Dry the preform at 50°C for 6 hours, then grind it into Arisaema powder;

[0055] Step 4) Mix the prepared Arisaema powder, pig bile, n-dodecane, and purified water in a mass ratio of 100:300:10:100 to obtain a suspension;

[0056] Step 5) Sterilize the suspension obtained in step 4) at 121°C for 15 minutes;

[0057] Step 6) Take the sterilized suspension obtained in Step 5) and add 0.2% by mass of a compound fermentation fungus consisting of Lichtheimia ramosa and Hyphopichia burtonii in a mass ratio of 1:1;

[0058] Step 7) Take the suspension obtained in Step 6) and ferment it under stirring conditions, keeping the temperature at 25-35℃ and the relative humidity at 65%-90%. During the fermentation process, preheated fresh air is introduced to ensure that the oxygen content in the fermentation environment is not less than 15%. After 5 days, the fermentation is completed.

[0059] Step 8) Take the fermentation suspension obtained in step 7) and reduce the water content by low-temperature evaporation to obtain drug pellets of appropriate size. Then, make them into soft blocks of appropriate size.

[0060] Step 9) Take the soft block obtained in Step 8) and vacuum dry it to evaporate the n-dodecane in the soft block, and you will get the final product, Arisaema cum Bile.

[0061] Example 2 Preparation of Arisaema cum Bile

[0062] Following the preparation steps of Example 1, the process parameters were modified to prepare Arisaema cum Bile, as shown in the table below. Process parameters not shown in the table are the same as those in Example 1:

[0063] Table 1 Preparation parameters of Arisaema cum Bile

[0064]

[0065] Comparative Example 1: Sample 1 and Sample 2 were prepared according to Example 1 of Chinese Patents CN 111407833 B and CN 113769000 B, respectively.

[0066] Example 3 Toxicity test of Arisaema cum Bile samples obtained in Examples 1 and 2 and Comparative Examples 1 and 2 (implemented with reference to Chinese Patent CN111407833 B)

[0067] The following toxicological tests were conducted on the Arisaema cum Bile prepared in Examples 1 and 2 and Comparative Examples 1 and 2 as test drugs. The experimental animals were male ICR strain mouse rats (hereinafter referred to as mouse rats) aged 4-6 weeks. The test environment conditions were as follows: temperature maintained at 22±1.0℃, humidity maintained at 50±5%, artificial lighting was simulated sunlight, and day and night changes were carried out in 12-hour units.

[0068] Before the test, the extract of each drug to be tested after soaking in deionized water for 7 days was taken as the test solution and numbered. The test solution numbers, their components and concentrations are shown in Table 2 below. The concentration of the drug to be tested is the ratio of the mass of the drug to the volume of deionized water during the soaking process.

[0069] Table 2: Comparison table of drug solution numbers, components and concentrations to be tested.

[0070]

[0071] Testing phase.

[0072] During testing: Ten mouse rats were used as a group, and each test drug solution was tested in one group. Mouse rats in the same group were fed the same numbered test drug solution via gastric tube. Therefore, the group number of the mouse rats was set to be the same as the number of the test drug solution used for clarity. The feeding amount of the test drug solution was 2cc / kg body weight (i.e., the dosage was based on the mouse's own weight; a 1kg mouse was given 2cc), administered three times a day at 9:00, 12:00, and 17:00. In addition, a control group (numbered S0) was set up, using the same experimental animals, but the test drug solution was replaced with physiological saline. All tests and data recordings before administration were recorded as day 0 data. Data during administration were recorded as day 1, day 2, and day 3. Administration was stopped from the fourth day after three days, and tests and data recordings were performed on the fourth (day 7), eleventh (day 14), eighteenth (day 21), twenty-fifth (day 28), and thirty-eighth (day 38) days after drug withdrawal. All recorded data are averaged. The test and data records are as follows.

[0073] Measurement of spontaneous mobilization in mice: This test was conducted according to the method described on pages 213-214 of the 2001, Vol. 28, No. 6 (CCMP97-RD-020) of the *Chinese Journal of Traditional Medicine*. Changes in various activities and behaviors of mice in each group were recorded, specifically including: horizontal locomotor, rest time, and number of jumps. Recording began 5 minutes after each administration and continued for 30 minutes.

[0074] Table 3. Toxicity test data of Arisaema cum Bile samples obtained in Examples 1, 2 and Comparative Example 1 in ICR strain mice.

[0075]

[0076] As can be clearly seen from the data in the table above, compared with the preparation processes of Comparative Example 1 and Comparative Example 2, the choleretic arisaema prepared by the present invention has lower toxicity and recovers its spontaneous movement much faster. The choleretic arisaema prepared by Comparative Example 1 and Comparative Example 2 could not fully recover its spontaneous movement until day 14 or even day 21, while the choleretic arisaema prepared by the present invention in Example 1 and Example 2 quickly recovered its spontaneous movement around day 7 (i.e. day 4 after drug withdrawal).

[0077] Example 4: Evaluation of the sedative effect of Arisaema cum Bile samples obtained in Examples 1, 2 and Comparative Example 1 (implemented with reference to Chinese Patent CN103417808 B).

[0078] Animal: Kunming mouse, male, SPF grade.

[0079] Equipment: Animal enclosure: 15cm wide, made of five-colored acrylic glass 25cm A 20cm rectangular box with sawdust padding at the bottom, and a stopwatch.

[0080] Drugs: Arisaema cum bile samples obtained in Examples 1, 2 and Comparative Example 1, and physiological saline. The preparation method of the drug administration samples was the same as in Example 3.

[0081] 4.2 Test Methods

[0082] Male mice weighing 18-22g were randomly divided into 8 groups of 10 mice each, for a total of 80 mice. The experimental groups included a saline group (0.2ml / mouse), Samples 1-4 of Example 1 and Example 2, and Samples 1 and 2 of the comparative examples (0.2ml / mouse), administered via gavage. The walking time and the number of times the mice raised their forelimbs within 2 minutes were used as indicators. Before administration, the mice were placed in a box for 5 minutes to acclimatize, and the activity values ​​for 2 minutes were recorded as normal values. Walking time and the number of times the mice raised their forelimbs were measured again at 30 and 60 minutes after administration, using the same method.

[0083] 1.3 Test Results

[0084] Table 4. Effects of Arisaema cum Bile samples obtained in Examples 1, 2 and Comparative Example 1 on walking time in mice (n=10)

[0085]

[0086] Arisaema cum Bile has a significant sedative effect, calming experimental animals and maintaining a relatively constant sedative effect. Experimental results show that after gavage administration of the Arisaema cum Bile samples prepared in Examples 1 and 2 of this application, mice exhibited reduced free movement and a significant sedative effect. Furthermore, the Arisaema cum Bile samples prepared according to the processes in Examples 1 and 2 of this application showed a more significant sedative effect compared to comparative samples 1 and 2, specifically manifested in fewer walking times and fewer forelimb raisings in mice 30 and 60 minutes after administration.

[0087] The above description of the invention and embodiments illustrates the basic principles, main features, and advantages of this patent application. Those skilled in the art should understand that this patent application is not limited to the above embodiments. The embodiments and descriptions in the specification are merely the optimal technical solutions of this patent application. Various changes and improvements can be made to this patent application without departing from the spirit and scope of this patent application. That is, the process of fermenting Arisaema cum Bile by appropriately controlling the oxygen content in the fermentation environment falls within the scope of this patent application. The scope of protection of this patent application is defined by the appended claims and their equivalents.

Claims

1. A fermentation processing method for Arisaema cum Bile, the fermentation processing method comprising the following preparation steps: Step 1) Soak the Arisaema heterophyllum in water, then remove it and boil it with ginger and alum until there is no dry core inside; Step 2) Remove and air-dry or bake until semi-dry, cut into thin slices, and pre-treat the thin slices to obtain pre-made slices; Step 3) Dry the pre-made sheets and then grind them into Arisaema powder; Step 4) Add the prepared Arisaema powder, bile, and n-dodecane to purified water and mix well to obtain a suspension; Step 5) Sterilize the suspension obtained in step 4) at high temperature; Step 6) Take the sterilized suspension obtained in Step 5) and add compound fermentation fungi at a mass percentage of 0.2%-1.0%; Step 7) Take the suspension obtained in Step 6) and ferment it under stirring conditions, keeping the temperature at 25-35℃ and the relative humidity at 65%-90%. Fermentation is completed after 5 days. Step 8) Take the fermentation suspension obtained in step 7) and reduce the water content by low-temperature evaporation to obtain drug pellets of appropriate size. Then, make them into soft blocks of appropriate size. Step 9) Take the soft block obtained in Step 8) and vacuum dry it to volatilize the n-dodecane in the soft block, thus obtaining the final product, Arisaema cum Bile; characterized in that, In step 1), the mass ratio of Arisaema, ginger and alum when cooking together is 100:10:

10. The pretreatment process in step 2) is as follows: the thin slices are immersed in cellulase solution for 5-10 minutes, and then subjected to ultrasonic cell wall disruption treatment at 200-300W for 10-15 minutes together with the enzyme solution. The thin slices are then filtered out and dried to obtain the pre-made slices. The amount of cellulase used is 200-300u / 1g of thin slices. In step 4), the mass ratio of prepared Arisaema powder, bile, n-dodecane, and purified water is 100:200~300:5-30:100, and the bile is pig bile. The compound fermentation fungi in step 6) are composed of *Pichia pastoris* and *Pichia bournei* in a mass ratio of 1:

1. Step 7) maintains the oxygen content in the fermentation environment at no less than 15% by introducing preheated fresh air until the fermentation is complete.

2. The processing method as described in claim 1, characterized in that, Step 1) The specific steps of soaking are as follows: Place Arisaema in a washing pool and soak it in water for 2-3 days, changing the water 2-3 times a day. Each time the water is changed, add 3wt% alum of Arisaema mass until there is no numbing sensation on the tongue.

3. The processing method as described in claim 1, characterized in that, In step 3), the drying temperature is 50~70℃ and the drying time is 3~6h.

4. The processing method as described in claim 1, characterized in that, The temperature for high-temperature sterilization in step 5) is 110~121℃, and the sterilization time is 15~35min.

5. Arisaema cum Bile prepared by the fermentation and processing method according to any one of claims 1-4.

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