Anti-liver cancer traditional Chinese medicine enzyme and preparation method thereof

Abstract

The invention relates to the field of pharmaceutical preparations, in particular to an anti-hepatoma traditional Chinese medicine enzyme and a preparation method thereof. The invention discloses a traditional Chinese medicine enzyme for treating liver cancer and a preparation method thereof. The traditional Chinese medicine enzyme is prepared from enzyme prepared by fermenting traditional Chinese medicine raw materials through compound bacteria, the traditional Chinese medicine raw materials comprise ginseng, radix salviae miltiorrhizae, radix bupleuri, fructus lycii, semen cassiae, rhizoma alismatis, radix gentianae, radix isatidis, schisandra chinensis, rhizoma curcumae longae, liquorice, curcuma zedoary, field melasma herb and centella asiatica; the compound bacteria comprise lactobacillus acidophilus, bifidobacterium longum, lactobacillus delbrueckii bulgaricus, streptococcus thermophilus and saccharomycetes. By introducing a multi-bacterium symbiosis technology, various traditional Chinese medicines are fermented to prepare the traditional Chinese medicine enzyme, the medication mode of the traditional Chinese medicines for treating rectum is improved to obtain the small-molecule traditional Chinese medicine enzyme, the stimulation effect of the traditional Chinese medicines on intestines and stomach is reduced, the absorptivity of beneficial components in the traditional Chinese medicines is improved, and the traditional Chinese medicine enzyme is unique in flavor and is more easily accepted by consumers as a health care product.

Description

[0001] This application claims the priority of the Chinese patent application filed with the China Patent Office on February 6, 2024, with application number 202410168265.6 and invention name “Anti-liver cancer Chinese medicinal enzyme and preparation method thereof”, all contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention relates to the field of pharmaceutical preparations, and in particular to an anti-liver cancer Chinese medicinal enzyme and a preparation method thereof. Background Art

[0003] At present, we have very limited effective treatments for liver cancer, mainly including surgery, ablation, interventional embolization, radiotherapy, chemotherapy, targeted therapy, immunotherapy, liver transplantation and other methods. At present, the low surgical resection rate of liver cancer is one of the main factors affecting prognosis. Therefore, preoperative neoadjuvant therapy or conversion therapy for liver cancer can maximize the surgical resection rate and improve prognosis. Secondly, anti-angiogenic drugs or targeted therapy drugs combined with immunotherapy have become an important treatment for unresectable or advanced liver cancer. However, the overall efficacy of liver cancer is still unsatisfactory. Chemotherapy has great side effects. Many liver cancer patients have cirrhosis with liver function damage, and the toxic side effects of chemotherapy promote further damage to liver function. In addition, after a period of time after radiotherapy, many patients quickly relapse and metastasize. There is also a lack of effective treatment for liver cancer recurrence and metastasis in clinical practice. Liver cancer progresses rapidly, and the overall treatment is not only expensive, but also painful and has a low quality of life.

[0004] Traditional Chinese medicine has its unique advantages in treating liver cancer, which runs through the entire process of liver cancer treatment, including: ① Traditional Chinese medicine treatment of liver cancer is applicable to the entire clinical treatment process. Combining traditional Chinese medicine treatment before surgery can improve the patient's immune function and enhance the patient's surgical tolerance; using it after surgery is beneficial to promote postoperative recovery and prevent postoperative complications or postoperative recurrence of liver cancer; ② Traditional Chinese medicine has the characteristics of strengthening the spleen and regulating qi, reducing swelling and removing blood stasis, softening and dispersing nodules, clearing away heat and detoxifying when treating liver cancer. It can significantly inhibit the growth of liver cancer cells and improve the body's immune ability. It is mainly suitable for liver diseases such as liver cancer, cirrhosis, and ascites. When used in combination with radiotherapy and chemotherapy, it can also significantly reduce the toxic and side effects caused by radiotherapy and chemotherapy, improve the patient's quality of life, and stabilize the condition. ③ Traditional Chinese medicine treatment of liver cancer can also be combined with radiotherapy, chemotherapy and immunotherapy, which can improve the long-term efficacy, reduce the toxic and side effects of radiotherapy and chemotherapy, and enhance the body's immune function; it can not only enhance the patient's immune function, but also reduce the side effects of radiotherapy and chemotherapy, and enhance the treatment effect. ④ For patients who are not suitable for surgery, radiotherapy or chemotherapy, Chinese medicine can be used alone as the main treatment for liver cancer. Through syndrome differentiation and treatment such as clearing away heat and detoxifying, invigorating qi and nourishing blood, strengthening the body and consolidating the foundation, it can achieve the goal of quickly relieving symptoms, alleviating pain, improving the patient's quality of life and prolonging life.

[0005] Enzyme refers to a product containing specific biologically active ingredients that is made from animals, plants, fungi, etc. through microbial fermentation. Chinese medicine enzymes are functional fermented products made from different herbal medicines through microbial fermentation (yeast, lactic acid bacteria, acetic acid bacteria, edible fungi, etc.). After a certain period of fermentation, the active ingredients and nutrients in the herbal medicines are decomposed and released by microorganisms and are more easily absorbed by the human body. Modern research has found that enzymes have many beneficial functions, including scavenging free radicals, anti-oxidation, antibacterial, alcohol detoxification and liver protection, laxative, blood purification, etc. These functions make enzymes an emerging active functional ingredient used in health care products and are favored by consumers. At present, there is a lack of anti-liver cancer Chinese medicine enzymes, while my country is rich in medicinal and edible materials with anti-liver cancer effects. Therefore, it is of great application value to develop an enzyme that can have a good auxiliary effect on liver cancer surgery. Summary of the invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide an anti-liver cancer Chinese medicinal enzyme and a preparation method thereof.

[0007] The present invention provides a method for preparing an anti-liver cancer Chinese medicinal enzyme, which comprises the following steps:

[0008] Step 1, extracting the Chinese medicinal raw materials to obtain an extract;

[0009] Step 2, the extract is fermented by composite bacteria to obtain the traditional Chinese medicine enzyme;

[0010] The Chinese medicine raw materials include the following raw materials by weight: 10-20 parts of ginseng, 15-25 parts of salvia miltiorrhiza, 15-25 parts of bupleurum, 10-20 parts of wolfberry, 5-15 parts of cassia seed, 15-25 parts of oriental rhizome, 20-30 parts of gentian, 15-25 parts of isatis root, 10-20 parts of schisandra chinensis, 10-20 parts of turmeric, 15-25 parts of liquorice and / or 10-25 parts of zedoaria;

[0011] The composite bacteria in the extract include: 1.0×10 8 ~2.0×10 8 CFU / mL Lactobacillus acidophilus, 1.0×10 8 ~2.0×10 8 CFU / mL Bifidobacterium longum, 1.0×10 8 ~2.0×10 8 CFU / mL Lactobacillus delbrueckii bulgaricus and / or 1.0×10 8 ~2.0×10 8 CFU / mL Streptococcus thermophilus.

[0012] Furthermore,

[0013] The Chinese medicine raw materials include the following raw materials by weight: 10-20 parts of ginseng, 15-25 parts of salvia miltiorrhiza, 15-25 parts of bupleurum, 10-20 parts of wolfberry, 5-15 parts of cassia seed, 15-25 parts of oriental rhizome, 20-30 parts of gentian, 15-25 parts of isatis root, 10-20 parts of schisandra, 10-20 parts of turmeric, 15-25 parts of liquorice, 10-25 parts of zedoaria, 12-25 parts of huaxuedan and / or 10-20 parts of centella asiatica;

[0014] The composite bacteria in the extract include: 1.0×10 8 ~2.0×10 8 CFU / mL Lactobacillus acidophilus, 1.0×10 8 ~2.0×10 8 CFU / mL Bifidobacterium longum, 1.0×10 8 ~2.0×10 8 CFU / mL Lactobacillus delbrueckii bulgaricus, 1.0×10 8 ~2.0×10 8 CFU / mL Streptococcus thermophilus and / or 1.0×10 8 ~2.0×10 8 CFU / mL Saccharomyces boulardii.

[0015] Furthermore,

[0016] The Chinese medicine raw materials include the following raw materials by weight: 12-18 parts of ginseng, 18-22 parts of salvia miltiorrhiza, 18-23 parts of bupleurum, 12-18 parts of wolfberry, 8-12 parts of cassia seed, 18-22 parts of oriental rhizome, 22-28 parts of gentian, 18-22 parts of isatis root, 12-18 parts of schisandra chinensis, 12-18 parts of turmeric, 18-22 parts of liquorice, 15-22 parts of zedoaria, 18-22 parts of huaxuedan and 12-18 parts of centella asiatica;

[0017] The composite bacteria in the extract include: 1.5×10 8 CFU / mL Lactobacillus acidophilus, 1.5×10 8 CFU / mL Bifidobacterium longum, 1.5×10 8 CFU / mL Lactobacillus delbrueckii bulgaricus, 1.0×10 8 CFU / mL Streptococcus thermophilus and 1.5×10 8 CFU / mL Saccharomyces boulardii.

[0018] Specifically,

[0019] The Chinese medicinal raw materials include the following raw materials in parts by weight: 15 parts of ginseng, 20 parts of salvia miltiorrhiza, 20 parts of bupleurum, 15 parts of wolfberry, 10 parts of cassia seed, 20 parts of oriental rhizome, 25 parts of gentian, 20 parts of isatis root, 15 parts of schisandra chinensis, 15 parts of turmeric, 20 parts of liquorice, 20 parts of zedoaria, 20 parts of huaxuedan and 15 parts of centella asiatica.

[0020] In the preparation method of the present invention,

[0021] The extraction is ultrasonic extraction, and the parameters of the ultrasonic extraction are: ultrasonic output power is 320W, and ultrasonic extraction is 6h.

[0022] The fermentation conditions are 14° C. to 36° C., 0.3 to 3 years, preferably 1 to 2 years.

[0023] The method further comprises a centrifugation step after the fermentation, wherein the centrifugation condition is 1000 g to 2000 g, 10 min to 20 min, preferably 1500 g, 15 min.

[0024] In a specific embodiment, the preparation method of the Chinese medicinal enzyme of the present invention comprises:

[0025] (1) The above Chinese medicine raw materials are dried and then crushed according to the proportion to obtain Chinese medicine powders, water is added to the Chinese medicine powders and stirred evenly, and then each Chinese medicine powder is separately extracted by ultrasonic extraction to obtain a Chinese medicine extract.

[0026] (2) Place the above Chinese herbal medicine extract in a fermentation tank, add composite bacteria, and stir thoroughly.

[0027] (3) The fermentation process is anaerobic fermentation, and the fermentation method is natural fermentation, that is, fermentation occurs automatically according to the changes in solar terms in the Tropic of Cancer region without human intervention.

[0028] (4) The fermentation liquid is centrifuged to remove the precipitate. The centrifugation conditions are preferably a speed of 1000-2000 g for 10-20 min; more preferably a speed of 1500 g for 15 min.

[0029] (5) The filtrate is taken and aseptically filled in a Class 10,000 aseptic workshop to obtain the anti-liver cancer Chinese medicine enzyme.

[0030] The fermentation period is 0.3 to 3 years, preferably 1 to 2 years.

[0031] The invention provides a traditional Chinese medicine enzyme prepared by the preparation method.

[0032] The present invention optimizes the Chinese medicine raw materials and fermentation bacteria of the Chinese medicine enzyme. The test results show that the Chinese medicine raw materials include the following raw materials by weight: 15 parts of ginseng, 20 parts of salvia miltiorrhiza, 20 parts of bupleurum, 15 parts of wolfberry, 10 parts of cassia seed, 20 parts of oriental rhizome, 25 parts of gentian, 20 parts of isatis root, 15 parts of schisandra, 15 parts of turmeric, 20 parts of liquorice, 20 parts of zedoaria, 20 parts of huaxuedan and 15 parts of centella asiatica; and the composite bacteria in the extract include: 1.5×10 8 CFU / mL Lactobacillus acidophilus, 1.5×10 8 CFU / mL Bifidobacterium longum, 1.5×10 8 CFU / mL Lactobacillus delbrueckii bulgaricus, 1.0×10 8 CFU / mL Streptococcus thermophilus and 1.5×10 8 The Chinese medicinal enzyme prepared when the CFU / mL of Saccharomyces boulardii has the best effect.

[0033] In view of the current situation of lack of anti-liver cancer Chinese medicine enzymes and the inadequacy of Chinese medicine enzyme production technology, the present invention provides a method for preparing a Chinese medicine enzyme with anti-liver cancer function. The method adopts compound probiotics and a fermentation technology combining traditional and modern bioengineering technologies. After sufficient fermentation, a Chinese medicine enzyme with stable quality, high safety and strong efficacy can be obtained. The enzyme can be used for nutritional support and auxiliary treatment of liver cancer patients before, during and after radiotherapy and chemotherapy, improve the quality of life of liver cancer patients, significantly improve the efficacy of radiotherapy and chemotherapy, and prolong the survival of liver cancer patients.

[0034] The anti-liver cancer Chinese medicine enzyme has the characteristics of stable quality and will not deteriorate within 3 years of storage at room temperature; the anti-liver cancer Chinese medicine enzyme has the characteristics of high safety and no obvious adverse reactions when taken orally 2000 mL per day; the anti-liver cancer Chinese medicine enzyme has the characteristics of rapid absorption and good efficacy, and related substances can be detected in plasma within 30 seconds after oral administration.

[0035] The present invention provides a traditional Chinese medicine enzyme prepared by the preparation method and / or the use of the traditional Chinese medicine enzyme of the present invention in the preparation of anti-liver cancer drugs.

[0036] The present invention provides an anti-liver cancer drug, the raw material of which is the traditional Chinese medicine enzyme prepared by the preparation method of the present invention and / or the traditional Chinese medicine enzyme of the present invention.

[0037] The present invention provides a method for preventing and / or treating liver cancer, which comprises taking the medicine of the present invention.

[0038] Specifically, it can be taken by liver cancer patients before, during and after radiotherapy and chemotherapy.

[0039] The present invention provides an anti-liver cancer Chinese medicine enzyme, which is obtained by fermenting ginseng, salvia miltiorrhiza, bupleurum, wolfberry, cassia seed, oriental rhizome, gentian, isatis root, schisandra chinensis, turmeric, liquorice, zedoaria, huaxuedan, and centella asiatica with composite bacteria consisting of lactobacillus acidophilus, bifidobacterium longum, lactobacillus delbrueckii bulgaricus, streptococcus thermophilus, and yeast. The anti-liver cancer Chinese medicine enzyme can be used for the prevention and treatment of liver cancer, and can also be used for body repair after liver cancer chemotherapy, and has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The toxic effects of the Chinese herbal enzymes described in Experimental Groups 1 to 4 on various organs and tissues in Kunming mice;

[0041] Figure 2 The liver scan and biochemical index changes of case 1 before treatment, where (A) is the CT scan of the liver before treatment, and the red circle indicates the size of the tumor; (B) is the CT scan of the liver after treatment, and the red circle indicates the size of the tumor; (C) is the hepatitis B virus DNA content in serum during treatment; (D) is the monitoring of blood cell changes during treatment; (E) is the alpha-fetoprotein content in serum during treatment; (F) is the change of biochemical indexes related to liver function; the horizontal axis in the bar graph is the date, and the vertical axis is the change of related indicators;

[0042] Figure 3 The liver scan and biochemical index changes of case 2 before treatment, where (A) is the MR scan of the liver before treatment, and the red circle indicates the size of the tumor; (B) is the MR scan of the liver after treatment, and the red circle indicates the size of the tumor; (C) is the serum thymidine kinase content during treatment; (D) is the monitoring of blood cell changes during treatment; (E) is the serum alpha-fetoprotein content during treatment; (F) is the change of biochemical indexes related to liver function; the horizontal axis in the bar graph is the date, and the vertical axis is the change of related indicators;

[0043] Figure 4 The liver scans and biochemical index changes of case 3 before treatment, where (A) is the CT scan of the liver before treatment, and the red circle indicates the size of the tumor; (B) is the CT scan of the liver after treatment, and the red circle indicates the size of the tumor; (C) is the thymidine kinase content in serum during treatment; (D) is the monitoring of changes in blood cells during treatment; (E) is the alpha-fetoprotein content in serum during treatment; (F) is the changes in biochemical indexes related to liver function; the horizontal axis in the bar graph is the date, and the vertical axis is the changes in related indicators;

[0044] Figure 5The liver scan and biochemical index changes of case 4 before treatment, where (A) is the MR scan of the liver before treatment, and the red circle indicates the size of the tumor; (B) is the MR scan of the liver after treatment, and the red circle indicates the size of the tumor; (C) is the hepatitis B virus DNA content in serum during treatment; (D) is the monitoring of blood cell changes during treatment; (E) is the alpha-fetoprotein content in serum during treatment; (F) is the change of biochemical indexes related to liver function; the horizontal axis in the bar graph is the date, and the vertical axis is the change of related indicators;

[0045] Figure 6 The liver scan and biochemical index changes of case 5 before treatment, where (A) is the CT scan of the liver before treatment, and the red circle indicates the size of the tumor; (B) is the CT scan of the liver after treatment, and the red circle indicates the size of the tumor; (C) is the monitoring of blood cell changes during treatment; (D) is the alpha-fetoprotein content in serum during treatment; (E) is the change of biochemical indexes related to liver function; the horizontal axis in the bar graph is the date, and the vertical axis is the change of related indexes;

[0046] Figure 7 The following are the changes in liver scans and biochemical indicators of control case 1 before treatment, where (A) is the MR scan of the liver before treatment, and the red circle indicates the size of the tumor; (B) is the MR scan of the liver after treatment, and the red circle indicates the size of the tumor; (C) is the hepatitis B virus DNA content in serum during treatment; (D) is the monitoring of changes in blood cells during treatment; (E) is the alpha-fetoprotein content in serum during treatment; (F) is the changes in biochemical indicators related to liver function; the horizontal axis in the bar graph is the date, and the vertical axis is the changes in related indicators;

[0047] Figure 8 The following are the changes in liver scans and biochemical indicators of control case 2 before treatment, where (A) is a CT scan of the liver before treatment, and the red circle indicates the size of the tumor; (B) is a CT scan of the liver after treatment, and the red circle indicates the size of the tumor; (C) is the level of alpha-fetoprotein in serum during treatment; (D) is the monitoring of changes in blood cells during treatment; (E) is the changes in biochemical indicators related to liver function; the horizontal axis in the bar graph is the date, and the vertical axis is the changes in related indicators;

[0048] Fig. 9The following are the changes in liver scans and biochemical indicators of control case 3 before treatment, where (A) is the MR scan of the liver before treatment, and the red circle indicates the size of the tumor; (B) is the MR scan of the liver after treatment, and the red circle indicates the size of the tumor; (C) is the serum thymidine kinase content during treatment; (D) is the monitoring of blood cell changes during treatment; (E) is the serum alpha-fetoprotein content during treatment; (F) is the changes in biochemical indicators related to liver function; the horizontal axis in the bar graph is the date, and the vertical axis is the changes in related indicators;

[0049] Fig.10 The following are the changes in liver scans and biochemical indicators of control case 4 before treatment, where (A) is the MR scan of the liver before treatment, and the red circle indicates the size of the tumor; (B) is the MR scan of the liver after treatment, and the red circle indicates the size of the tumor; (C) is the serum thymidine kinase content during treatment; (D) is the monitoring of blood cell changes during treatment; (E) is the serum alpha-fetoprotein content during treatment; (F) is the changes in biochemical indicators related to liver function; the horizontal axis in the bar graph is the date, and the vertical axis is the changes in related indicators;

[0050] Fig.11 The following are the changes in liver scans and biochemical indices of control case 5 before treatment, where (A) is the MR scan of the liver before treatment, and the red circle indicates the size of the tumor; (B) is the MR scan of the liver after treatment, and the red circle indicates the size of the tumor; (C) is the serum thymidine kinase level during treatment; (D) is the monitoring of blood cell changes during treatment; (E) is the serum alpha-fetoprotein level during treatment; (F) is the changes in biochemical indices related to liver function; the horizontal axis in the bar graph is the date, and the vertical axis is the changes in related indicators. DETAILED DESCRIPTION

[0051] The present invention provides an anti-liver cancer Chinese medicine enzyme and a preparation method thereof. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0052] Compared with the prior art, the Chinese medicinal enzyme of the present invention has the following advantages:

[0053] 1. The anti-liver cancer Chinese medicine enzyme provided by the present invention uses ginseng, salvia miltiorrhiza, bupleurum, wolfberry, cassia seed, oriental rhizome, gentian, isatis root, schisandra chinensis, turmeric, liquorice, zedoaria, huaxuedan, centella asiatica as fermentation raw materials, which are abundant in resources and easily available, and promotes the development of the deep processing industry of the above raw materials, improves economic benefits, and provides new ideas for the development of new products;

[0054] 2. The Chinese medicine enzyme of the present invention improves the use of Chinese medicine for treating liver cancer, obtains amino acid and polysaccharide small molecule Chinese medicine enzymes, improves the bitter taste of traditional Chinese medicine, reduces the irritation of Chinese medicine to the stomach and intestines, and increases the absorption rate of beneficial ingredients in Chinese medicine;

[0055] 3. The Chinese medicinal enzyme of the present invention is used to treat or prevent liver cancer, which is beneficial to improve the pain caused by liver cancer. It can improve the function of liver tissue and help liver cancer patients recover by nourishing and protecting the liver and soothing the liver and regulating qi.

[0056] 4. The Chinese medicinal enzyme described in the present invention can also be used in combination with Western medicines for the treatment of liver cancer. It can be used for the early and middle stage treatment of liver cancer, and can also be used for body repair after chemotherapy for liver cancer.

[0057] The reagents and consumables used in the present invention are all common commercial products and can be purchased in the market.

[0058] The present invention will be further described below in conjunction with embodiments:

[0059] Example 1 Preparation of Chinese medicine enzyme

[0060] 1. Different ratios and fermentation conditions

[0061] 1. Experimental Group 1

[0062] The raw material composition and weight proportions of the active ingredients of the Chinese herbal enzyme in this experimental group are as follows:

[0063] 10 parts of ginseng, 15 parts of salvia miltiorrhiza, 25 parts of bupleurum, 10 parts of wolfberry, 15 parts of cassia seed, 20 parts of oriental rhizome, 20 parts of gentian, 25 parts of isatis root, 20 parts of schisandra chinensis, 10 parts of turmeric, 15 parts of liquorice, and 15 parts of zedoaria.

[0064] The composition and concentration of the composite bacteria in this experimental group were 1.0×10 8 ~2.0×10 8 CFU / mL of Lactobacillus acidophilus (Shanghai Collection of Microorganisms, SHBCC D70932), with a concentration of 1.0×10 8 ~2.0×10 8 CFU / mL of Bifidobacterium longum (Shanghai Collection of Microorganisms, SHBCC D24449), with a concentration of 1.0×10 8 ~2.0×10 8CFU / mL of Lactobacillus delbrueckii bulgaricus (Shanghai Collection of Microbiological Products, SHBCC D50170), with a concentration of 1.0×10 8 ~2.0×10 8 CFU / mL of Streptococcus thermophilus (Shanghai Collection of Microorganisms, SHBCC D50184), and a concentration of 1.0×10 8 ~2.0×10 8 CFU / mL of Saccharomyces boulardii (Shanghai Collection of Microorganisms, SHBCC D24699).

[0065] The process steps of the preparation method of Chinese medicine enzyme in this experimental group are:

[0066] (1) The above Chinese medicine raw materials are dried and then crushed according to the proportion to obtain Chinese medicine powders, water is added to the Chinese medicine powders and stirred evenly, and then each Chinese medicine powder is separately extracted by ultrasonic extraction to obtain a Chinese medicine extract.

[0067] (2) The above Chinese medicine extract is placed in a fermentation tank, and composite bacteria are added and stirred thoroughly. The ratio of Lactobacillus acidophilus, Bifidobacterium longum, Lactobacillus delbrueckii bulgaricus, and Streptococcus thermophilus is 1.5:1.5:1:1.

[0068] (3) The fermentation process is anaerobic fermentation, and the fermentation method is natural fermentation. It ferments automatically (14-36° C.) according to the changes of the four seasons in the Tropic of Cancer region of Guangdong Province, without the need for human intervention, and the fermentation cycle is 0.3-3 years.

[0069] (4) The fermentation liquid is centrifuged to remove the precipitate. The centrifugation condition is preferably a speed of 1000-2000×g for 10-20 minutes.

[0070] (5) The filtrate is taken and aseptically filled in a Class 10,000 aseptic workshop to obtain the anti-liver cancer Chinese medicine enzyme.

[0071] 2. Experimental Group 2

[0072] The raw material composition and number of active ingredients of Chinese herbal enzymes in this experimental group are as follows:

[0073] 12 parts of ginseng, 18 parts of salvia miltiorrhiza, 23 parts of bupleurum, 15 parts of wolfberry, 12 parts of cassia seed, 20 parts of oriental rhizome, 20 parts of gentian, 18 parts of isatis root, 15 parts of schisandra chinensis, 15 parts of turmeric, 20 parts of liquorice, 22 parts of zedoaria, 18 parts of huaxuedan, and 12 parts of centella asiatica.

[0074] The composition and concentration of the composite bacteria in this experimental group were the same as those in experimental group 1.

[0075] The preparation method, process steps, usage and dosage of the Chinese medicine preparation in this experimental group are the same as those in experimental group 1.

[0076] 3. Experimental Group 3

[0077] The raw material composition and number of active ingredients of Chinese herbal enzymes in this experimental group are as follows:

[0078] 15 parts of ginseng, 20 parts of salvia miltiorrhiza, 20 parts of bupleurum, 15 parts of wolfberry, 10 parts of cassia seed, 20 parts of oriental rhizome, 25 parts of gentian, 20 parts of isatis root, 15 parts of schisandra chinensis, 15 parts of turmeric, 20 parts of liquorice, 20 parts of zedoaria, 20 parts of blood-transforming gall, and 15 parts of centella asiatica.

[0079] The composition and concentration of the composite bacteria in this experimental group were 1.0×10 8 ~2.0×10 8 CFU / mL of Lactobacillus acidophilus (Shanghai Collection of Microorganisms, SHBCC D70932), with a concentration of 1.0×10 8 ~2.0×10 8 CFU / mL of Bifidobacterium longum (Shanghai Collection of Microorganisms, SHBCC D24449), with a concentration of 1.0×10 8 ~2.0×10 8 CFU / mL of Lactobacillus delbrueckii bulgaricus (Shanghai Collection of Microbiological Materials, SHBCC D50170), with a concentration of 1.0×10 8 ~2.0×10 8 CFU / mL of Streptococcus thermophilus (Shanghai Collection of Microorganisms, SHBCC D50184) and 1.0×10 8 ~2.0×10 8 CFU / mL of yeast (Shanghai Collection of Microorganisms, SHBCC D24699).

[0080] The ratio of the compound bacteria added in the preparation method, process steps, usage and dosage of the traditional Chinese medicine preparation of this experimental group is 1.5:1.5:1:1:1:1.5. The fermentation liquid is centrifuged at a speed of 1500×g for 15 minutes. The remaining preparation method, process steps, usage and dosage are the same as those of experimental group 1.

[0081] 4. Experimental Group 4

[0082] The raw material composition and number of active ingredients of Chinese herbal enzymes in this experimental group are as follows:

[0083] 18 parts of ginseng, 25 parts of salvia miltiorrhiza, 25 parts of bupleurum, 18 parts of wolfberry, 10 parts of cassia seed, 20 parts of oriental rhizome, 25 parts of gentian, 25 parts of isatis root, 18 parts of schisandra chinensis, 18 parts of turmeric, 25 parts of liquorice, 25 parts of zedoaria, 25 parts of huaxuedan, and 18 parts of centella asiatica.

[0084] The composition and concentration of the composite bacteria in this experimental group were the same as those in experimental group 3.

[0085] The fermentation period in the process steps, usage and dosage of the Chinese medicine preparation of this experimental group is preferably 1 to 2 years. The remaining process steps, usage and dosage of the preparation method are the same as those of experimental group 3.

[0086] The application of the traditional Chinese medicine enzyme in the preparation of products with anti-liver cancer function is preferably edible by liver cancer patients before, during and after radiotherapy and chemotherapy.

[0087] The anti-liver cancer Chinese medicinal enzyme has the characteristic of stable quality and will not deteriorate within 3 years when stored at room temperature.

[0088] The anti-liver cancer Chinese medicinal enzyme has the characteristic of high safety, and there is no obvious adverse reaction when taking 2000 mL orally every day.

[0089] The anti-liver cancer Chinese medicinal enzyme has the characteristics of rapid absorption and good efficacy, and related substances can be detected in plasma within 30 seconds after oral administration.

[0090] 2. Effect Evaluation

[0091] 1. Acute toxicity test

[0092] (1) Oral acute toxicity test in mice: 50 Kunming mice, half male and half female, were selected and kept under artificial lighting for 12 hours and good ventilation for 3 days. They were then randomly divided into 4 experimental groups (corresponding to experimental groups 1 to 4) and 1 control group, with 10 mice in each group. The mice were fasted for 8 hours before and 4 hours after administration, but not water. The drug was administered orally by gavage at a dose of 0.5 mL / 20 g. The mice were observed for 10 consecutive days, and the symptoms of poisoning were recorded. The mice were dissected after death or after the end of the experiment to observe the changes in various tissues and organs. The heart, liver, spleen, kidney, lung and intestinal tissues were taken for pathological sections, and the median lethal dose (LD50) was calculated. 50 ).

[0093] After the Chinese medicine enzyme was orally injected with a gavage needle, some mice curled up in the corner and reduced their activities. However, on the third day, 28 mice were found to have returned to normal activities, drinking water and eating. After 10 consecutive days of observation, all 40 mice survived healthily, and no abnormal changes were observed in the spirit, appetite, skin color, secretions, breathing, appetite, muscle movement, etc. of all mice. After the end of the experiment, autopsy was performed and it was found that there was no obvious visual difference between the changes in the organs of the mice in the experimental groups and those in the control group. Figure 1 According to the toxicological safety evaluation standard, LD 50 >10000mg / kg, it is a non-toxic substance.

[0094] 2. Anti-tumor effect test

[0095] (1) Test materials:

[0096] ① BALB / c nude tumor-bearing mice, male, 4-5 weeks old, a total of 50 mice.

[0097] ②Huh7 cell suspension (1×10 7 / mL).

[0098] (2) Test method:

[0099] Establishment of Huh7 BALB / c nude tumor-bearing mouse transplant model: Huh7 cells in the logarithmic growth phase were taken, washed 3 times with PBS, resuspended with pre-cooled PBS, and added with Matrigel matrix gel at a ratio of 2:1 (PBS:Matrigel), and the final cell density was adjusted to 1×10 7 The nude mice were subcutaneously inoculated with 1 mL syringe into the right axilla of male BALB / c nu / nu mice, with 200 μL injected into each mouse. The nude mice were fed routinely, and the weight and tumor diameter of the nude mice were recorded regularly. The tumor volume V = 1 / 2 (ab 2 ), a and b represent the long and short diameters of the tumor, respectively.

[0100] After a lump formed in the armpits of mice 10 days after inoculation, they were randomly divided into four groups: the Chinese medicine enzyme was divided into four groups according to the experimental groups 1 to 4 (named as groups 1 to 4, with 10 mice in each group), and the drugs were administered by gavage once a day, with each mouse receiving a dose of 0.5 mL / 20 g; the normal saline group (10 mice) was administered with 0.5 mL / 20 g normal saline injection solution by gavage once a day; after 21 days of administration, the tumor mass of the mice in the control group had basically grown to more than 2000 mm 3 After that, the drug administration was stopped, the tumor-bearing mice were killed, the tumor mass and spleen were removed, weighed, recorded, and the mean and standard deviation were calculated and compared with those of the control group.

[0101] The experimental results were expressed as mean ± standard deviation, and SPSS16.0 statistical software was used for data processing, with P < 0.05 as a statistically significant difference. The experimental results are shown in Table 1 below.

[0102] Table 1. Antitumor effects of the Chinese herbal enzymes described in experimental groups 1 to 4 on BALB / c nude tumor-bearing mice

[0103]

[0104] ***P<0.001, compared with the control group

[0105] 3. Typical case examples:

[0106] (1) On January 9, 2023, the CT scan showed a 4.0 cm × 2.8 cm mass in the right lobe of the liver. Figure 2 A); received sintilimab injection (160 mg, once every three weeks during hospitalization, intravenous injection), donafenib tosylate tablets (Zepson) (20 mg, oral, twice a day) and 1-tiegalovirus (one tablet each time, twice a day, take for 14 days and stop for 7 days, 21 days as a treatment cycle) treatment, and zoledronic acid for anti-bone metastasis treatment, and oral administration of the traditional Chinese medicine enzyme described in experimental group 4 60 mL / time, 3 times a day. On April 17, 2023, the CT results showed a 4.0 cm × 2.6 cm tumor in the right lobe of the liver ( Figure 2 During the treatment, the hepatitis B virus level in the serum returned to normal, and the alpha-fetoprotein level remained within the normal range, proving the effectiveness of the treatment. Moreover, the results of blood cell analysis and liver function-related index analysis were all within the normal range, indicating that the bone marrow function and liver function were normal ( Figure 2 C, D, E, F).

[0107] (2) On January 8, 2021, the MRI results showed a 5.6 cm × 5.6 cm × 4.8 cm mass in the right lobe of the liver ( Figure 3 A); received sintilimab injection (160 mg, once every three weeks during hospitalization, intravenous injection), donafenib tosylate tablets (Zepson) (20 mg, oral, twice a day) and 1-tiegalovirus (one tablet each time, twice a day, take for 14 days and stop for 7 days, 21 days as a treatment cycle) treatment, and zoledronic acid for anti-bone metastasis treatment, and oral administration of the traditional Chinese medicine enzyme described in experimental group 3 60 mL / time, 3 times a day. On January 7, 2023, the MRI results showed a 5.1 cm × 4.6 cm × 4.3 cm tumor in the right lobe of the liver ( Figure 3 B); during the treatment, the serum thymidine kinase content returned to normal, and the alpha-fetoprotein content also decreased significantly, proving the effectiveness of the treatment. Moreover, the results of blood cell analysis and liver function-related index analysis were within the normal range, indicating that the bone marrow function and liver function were normal ( Figure 3 C, D, E, F).

[0108] (3) On January 26, 2022, the CT results showed a 5.8 cm × 3.9 cm × 3.8 cm mass in the right lobe of the liver. Figure 4A); received sintilimab injection (160 mg, once every three weeks during hospitalization, intravenous injection), donafenib tosylate tablets (Zepson) (20 mg, oral, twice a day) and 1-tiegalovirus (one tablet each time, twice a day, take for 14 days and stop for 7 days, 21 days as a treatment cycle) treatment, and zoledronic acid for anti-bone metastasis treatment, and oral administration of the traditional Chinese medicine enzyme described in experimental group 3 60 mL / time, 3 times a day. On February 7, 2023, the CT results showed a 4.2 cm × 3.4 cm × 2.1 cm tumor in the right lobe of the liver ( Figure 4 B); during the treatment, the levels of serum thymidine kinase and alpha-fetoprotein have been within the normal range, proving the effectiveness of the treatment. Moreover, the results of blood cell analysis and liver function-related index analysis are all within the normal range, indicating the normality of bone marrow function and liver function ( Figure 4 C, D, E, F).

[0109] (4) On June 3, 2022, the MRI results showed a tumor measuring 8.4 cm × 5.6 cm × 4.5 cm in the right lobe of the liver ( Figure 5 A); received sintilimab injection (160 mg, once every three weeks during hospitalization, intravenous injection), donafenib tosylate tablets (Zepson) (20 mg, oral, twice a day) and 1-tiegalovirus (one tablet each time, twice a day, take for 14 days and stop for 7 days, 21 days as a treatment cycle) treatment, and zoledronic acid for anti-bone metastasis treatment, and oral Chinese medicine enzymes described in experimental group 4 60 mL / time, 3 times a day. On February 13, 2023, MRI results showed a 1.6 cm × 1.5 cm × 1.2 cm tumor in the right lobe of the liver ( Figure 5 During the treatment, the hepatitis B virus level in the serum returned to normal, and the alpha-fetoprotein level also decreased significantly, proving the effectiveness of the treatment. Moreover, the results of blood cell analysis and liver function-related index analysis were all within the normal range, indicating that the bone marrow function and liver function were normal ( Figure 5 C, D, E, F).

[0110] (5) On December 31, 2022, the CT results showed a tumor measuring 7.1 cm × 6.8 cm × 6.4 cm in the right lobe of the liver. Figure 6 A); received sintilimab injection (160 mg, once every three weeks during hospitalization, intravenous injection), donafenib tosylate tablets (Zepson) (20 mg, oral, twice a day) and Tegafur (one tablet each time, twice a day, take for 14 days and stop for 7 days, 21 days as a treatment cycle) treatment, and zoledronic acid for anti-bone metastasis treatment, and oral administration of the traditional Chinese medicine enzyme described in experimental group 3 60 mL / time, 3 times a day. On May 4, 2023, the CT results showed a tumor of 7.0 cm × 6.4 cm × 5.6 cm in the right lobe of the liver ( Figure 6 B); during the treatment, the alpha-fetoprotein content has remained at a relatively low level, proving the effectiveness of the treatment. Moreover, the results of blood cell analysis and liver function-related index analysis are within the normal range, indicating that the bone marrow function and liver function are normal ( Figure 6 C, D, E).

[0111] 4. Examples of control cases:

[0112] (1) On August 18, 2022, the MRI results showed a 2.0 cm × 3.1 cm × 3.0 cm mass in the right lobe of the liver ( Figure 7 A); received sintilimab injection (160 mg, once every three weeks during hospitalization, intravenous injection), donafenib tosylate tablets (Zepson) (20 mg, oral, twice a day) and 1-tiegalovirus (one tablet each time, twice a day, take for 14 days and stop for 7 days, 21 days as a treatment cycle), and zoledronic acid for anti-bone metastasis treatment, and did not take Chinese medicine enzymes. On November 17, 2022, the MRI results showed a 2.8cm×4.0cm×4.5cm tumor in the right lobe of the liver ( Figure 7 At the same time, during the treatment, the hepatitis B virus content in the serum continued to rise, and the alpha-fetoprotein content remained high, proving that the treatment effect was poor. The results of blood cell analysis and liver function-related index analysis were all within the normal range, indicating that the bone marrow function and liver function were normal ( Figure 7 C, D, E, F).

[0113] (2) On January 14, 2023, the CT scan showed a 7.1 cm × 4.3 cm mass in the left lobe of the liver. Figure 8 A); received sintilimab injection (160 mg, once every three weeks during hospitalization, intravenous injection), donafenib tosylate tablets (Zepson) (20 mg, oral, twice a day) and 1-tiegalovirus (one tablet each time, twice a day, take for 14 days and stop for 7 days, 21 days as a treatment cycle), and zoledronic acid for anti-bone metastasis treatment, and did not take Chinese medicine enzymes. On April 11, 2023, the CT results showed a 7.4 cm × 4.3 cm tumor in the left lobe of the liver ( Figure 8 B); at the same time, during the treatment, the level of alpha-fetoprotein was still high, proving that the treatment was ineffective. The results of blood cell analysis and liver function-related index analysis were within the normal range, indicating that the bone marrow function and liver function were normal ( Figure 8 C, D, E, F).

[0114] (3) On June 1, 2021, the MRI results showed a 9.1 cm × 5.8 cm × 8.9 cm mass in the right lobe of the liver ( Fig. 9A); received sintilimab injection (160 mg, once every three weeks during hospitalization, intravenous injection), donafenib tosylate tablets (Zepson) (20 mg, oral, twice a day) and 1-tiegalovirus (one tablet each time, twice a day, take for 14 days and stop for 7 days, 21 days as a treatment cycle), and zoledronic acid for anti-bone metastasis treatment, and did not take Chinese medicine enzymes. On February 21, 2022, the MRI results showed a tumor of 13.0 cm × 9.0 cm × 10.5 cm in the right lobe of the liver ( Fig. 9 B); at the same time, during the treatment, the serum thymidine kinase concentration was relatively stable, but the alpha-fetoprotein content continued to increase, proving that the treatment effect was poor. The results of blood cell analysis and liver function-related index analysis were all within the normal range, indicating that the bone marrow function and liver function were normal ( Fig. 9 C, D, E, F).

[0115] (4) On June 16, 2022, the MRI results showed a 4.7 cm × 3.0 cm × 3.2 cm mass in the right lobe of the liver ( Fig.10 A); received sintilimab injection (160 mg, once every three weeks during hospitalization, intravenous injection), donafenib tosylate tablets (Zepson) (20 mg, oral, twice a day) and 1-tiegalovirus (one tablet each time, twice a day, take for 14 days and stop for 7 days, 21 days as a treatment cycle), and zoledronic acid for anti-bone metastasis treatment, and did not take Chinese medicine enzymes. On April 13, 2023, the MRI results showed a tumor of 7.3 cm × 5.8 cm × 9.5 cm in the right lobe of the liver ( Fig.10 B); at the same time, during the treatment, the serum thymidine kinase concentration was relatively stable, but the alpha-fetoprotein content continued to increase, proving that the treatment effect was poor. The results of blood cell analysis and liver function-related index analysis were all within the normal range, indicating that the bone marrow function and liver function were normal ( Fig.10 C, D, E, F).

[0116] (5) On March 6, 2021, the MRI results showed a 0.8 cm × 1.9 cm mass in the right lobe of the liver ( Fig.11 A); received sintilimab injection (160 mg, once every three weeks during hospitalization, intravenous injection), donafenib tosylate tablets (Zepson) (20 mg, oral, twice a day) and 1-tiegalovirus (one tablet each time, twice a day, take for 14 days and stop for 7 days, 21 days as a treatment cycle), and zoledronic acid for anti-bone metastasis treatment, and did not take Chinese medicine enzymes. On March 13, 2022, the MRI results showed a 4.2 cm × 2.7 cm tumor in the right lobe of the liver ( Fig.11B); at the same time, during the treatment, the serum thymidine kinase concentration and alpha-fetoprotein content were relatively stable, but the imaging report proved that the treatment effect was poor. The results of blood cell analysis and liver function-related index analysis were all within the normal range, indicating that the bone marrow function and liver function were normal ( Fig.11 C, D, E, F).

[0117] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing an anti-liver cancer Chinese medicinal enzyme, characterized in that: The steps include: Step 1, extracting the Chinese medicinal raw materials to obtain an extract; Step 2, the extract is fermented by composite bacteria to obtain the traditional Chinese medicine enzyme; The Chinese medicine raw materials include the following raw materials by weight: 10-20 parts of ginseng, 15-25 parts of salvia miltiorrhiza, 15-25 parts of bupleurum, 10-20 parts of wolfberry, 5-15 parts of cassia seed, 15-25 parts of oriental rhizome, 20-30 parts of gentian, 15-25 parts of isatis root, 10-20 parts of schisandra chinensis, 10-20 parts of turmeric, 15-25 parts of liquorice and / or 10-25 parts of zedoaria; The composite bacteria in the extract include: 1.0×10 8 ~2.0×10 8 CFU / mL Lactobacillus acidophilus, 1.0×10 8 ~2.0×10 8 CFU / mL Bifidobacterium longum, 1.0×10 8 ~2.0×10 8 CFU / mL Lactobacillus delbrueckii bulgaricus and / or 1.0×10 8 ~2.0×10 8 CFU / mL Streptococcus thermophilus.

2. The preparation method according to claim 1, characterized in that: The Chinese medicine raw materials include the following raw materials by weight: 10-20 parts of ginseng, 15-25 parts of salvia miltiorrhiza, 15-25 parts of bupleurum, 10-20 parts of wolfberry, 5-15 parts of cassia seed, 15-25 parts of oriental rhizome, 20-30 parts of gentian, 15-25 parts of isatis root, 10-20 parts of schisandra, 10-20 parts of turmeric, 15-25 parts of liquorice, 10-25 parts of zedoaria, 12-25 parts of huaxuedan and / or 10-20 parts of centella asiatica; The composite bacteria in the extract include: 1.0×10 8 ~2.0×10 8 CFU / mL Lactobacillus acidophilus, 1.0×10 8 ~2.0×10 8 CFU / mL Bifidobacterium longum, 1.0×10 8 ~2.0×10 8 CFU / mL Lactobacillus delbrueckii bulgaricus, 1.0×10 8 ~2.0×10 8 CFU / mL Streptococcus thermophilus and / or 1.0×10 8 ~2.0×10 8 CFU / mL Saccharomyces boulardii.

3. The preparation method according to claim 2, characterized in that: The Chinese medicine raw materials include the following raw materials by weight: 12-18 parts of ginseng, 18-22 parts of salvia miltiorrhiza, 18-23 parts of bupleurum, 12-18 parts of wolfberry, 8-12 parts of cassia seed, 18-22 parts of oriental rhizome, 22-28 parts of gentian, 18-22 parts of isatis root, 12-18 parts of schisandra chinensis, 12-18 parts of turmeric, 18-22 parts of liquorice, 15-22 parts of zedoaria, 18-22 parts of huaxuedan and 12-18 parts of centella asiatica; The composite bacteria in the extract include: 1.5×10 8 CFU / mL Lactobacillus acidophilus, 1.5×10 8 CFU / mL Bifidobacterium longum, 1.5×10 8 CFU / mL Lactobacillus delbrueckii bulgaricus, 1.0×10 8 CFU / mL Streptococcus thermophilus and 1.5×10 8 CFU / mL Saccharomyces boulardii.

4. The preparation method according to claim 3, characterized in that: The Chinese medicinal raw materials include the following raw materials in parts by weight: 15 parts of ginseng, 20 parts of salvia miltiorrhiza, 20 parts of bupleurum, 15 parts of wolfberry, 10 parts of cassia seed, 20 parts of oriental rhizome, 25 parts of gentian, 20 parts of isatis root, 15 parts of schisandra chinensis, 15 parts of turmeric, 20 parts of liquorice, 20 parts of zedoaria, 20 parts of huaxuedan and 15 parts of centella asiatica.

5. The preparation method according to any one of claims 1 to 4, characterized in that: The extraction is ultrasonic extraction, and the parameters of the ultrasonic extraction are: ultrasonic output power is 320W, and ultrasonic extraction is 6h.

6. The preparation method according to any one of claims 1 to 4, characterized in that: The fermentation conditions are 14° C. to 36° C. and 0.3 to 3 years.

7. The preparation method according to any one of claims 1 to 4, characterized in that: The method further comprises a centrifugation step after the fermentation, wherein the centrifugation condition is 1000 g to 2000 g for 10 min to 20 min.

8. The Chinese medicinal enzyme prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the traditional Chinese medicine enzyme obtained by the preparation method according to any one of claims 1 to 7 and / or the traditional Chinese medicine enzyme according to claim 8 in the preparation of anti-liver cancer drugs.

10. An anti-liver cancer drug, characterized in that: The raw materials include the traditional Chinese medicine enzyme prepared by the preparation method described in any one of claims 1 to 7 and / or the traditional Chinese medicine enzyme described in claim 8.

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