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

Abstract

The present invention relates to the field of pharmaceutical preparations, and more specifically to an anti-liver cancer Chinese medicine enzyme and its preparation method. The present invention discloses a Chinese medicine enzyme for treating liver cancer and its preparation method. The Chinese medicine enzyme is made from an enzyme fermented from Chinese medicinal raw materials by a composite bacteria; the Chinese medicinal raw materials include: ginseng, salvia miltiorrhiza, bupleurum, wolfberry fruit, cassia seed, rhizome of oriental Chinese medicine, gentian, isatis root, schisandra chinensis, turmeric, liquorice, zedoaria, huaxuedan, and centella asiatica; the composite bacteria include Lactobacillus acidophilus, Bifidobacterium longum, Lactobacillus delbrueckii bulgaricus, Streptococcus thermophilus, and yeast. The present invention introduces a multi-bacteria symbiotic technology to ferment multiple Chinese medicinal herbs to produce a Chinese medicine enzyme, improve the dosage of Chinese medicine for treating rectal problems, obtain a small molecule Chinese medicine enzyme, reduce the irritation of Chinese medicine to the gastrointestinal tract, improve the absorption rate of beneficial ingredients in Chinese medicine, and have a unique flavor, making it more easily accepted by consumers as a health product.

Description

[0001] This application claims priority to 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 herbal enzyme and its preparation method”, the entire 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 traditional Chinese medicine enzyme and a preparation method thereof. Background Art

[0003] Currently, effective treatment options for liver cancer are extremely limited, primarily including surgery, ablation, interventional embolization, radiotherapy, chemotherapy, targeted therapy, immunotherapy, and liver transplantation. Low surgical resection rates for liver cancer are a major factor affecting prognosis. Therefore, preoperative neoadjuvant therapy or conversion therapy for liver cancer can maximize surgical resection rates and improve prognosis. Furthermore, anti-angiogenic drugs or targeted therapies combined with immunotherapy have become important treatment options for unresectable or advanced liver cancer. However, overall efficacy remains unsatisfactory. Chemotherapy has significant side effects, and many liver cancer patients have cirrhosis and impaired liver function, which are further compromised by the toxic side effects of chemotherapy. Furthermore, many patients experience recurrence and metastasis soon after radiotherapy, leading to a lack of effective treatment options for recurrent and metastatic liver cancer. Liver cancer progresses rapidly, and overall treatment is not only expensive but also extremely painful and leads to a low quality of life.

[0004] Traditional Chinese Medicine (TCM) has unique advantages in treating liver cancer, and it is used throughout the entire treatment process. These advantages include: ① TCM treatment of liver cancer is applicable throughout the entire clinical treatment process. Preoperative use of TCM treatment can improve the patient's immune function and enhance surgical tolerance; postoperative use can help promote postoperative recovery and prevent postoperative complications or recurrence of liver cancer. ② TCM treatment of liver cancer has the advantages of strengthening the spleen and regulating Qi, reducing swelling and stasis, softening and dispersing nodules, and clearing away heat and detoxifying. It can significantly inhibit the growth of liver cancer cells and improve the body's immune capacity. It is primarily suitable for liver diseases such as liver cancer, cirrhosis, and ascites. Combined with radiotherapy and chemotherapy, it can significantly reduce the toxic side effects of radiotherapy and chemotherapy, improve the patient's quality of life, and stabilize the condition. ③ TCM treatment of liver cancer can also be combined with radiotherapy, chemotherapy, and immunotherapy to improve long-term efficacy, reduce the toxic and side effects of radiotherapy and chemotherapy, and enhance the body's immune function. It can both enhance the patient's immune function and reduce the side effects of radiotherapy and chemotherapy, enhancing 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 method for liver cancer. Through syndrome differentiation and treatment such as clearing away heat and detoxification, invigorating qi and nourishing blood, strengthening the body and consolidating the foundation, it can achieve the purpose of quickly relieving symptoms, alleviating pain, improving the quality of life of patients and prolonging life.

[0005] Enzymes refer to products containing specific biologically active ingredients that are made from animals, plants, fungi, etc. through microbial fermentation. Traditional Chinese medicine enzymes are functional fermentation products made from different herbal medicinal materials through fermentation by microorganisms (yeast, lactic acid bacteria, acetic acid bacteria, edible fungi, etc.). After a certain period of fermentation, the active ingredients and nutrients in the herbal medicinal materials are decomposed and released by microorganisms, making them more easily absorbed by the human body. Modern research has found that enzymes have many beneficial functions, including scavenging free radicals, anti-oxidation, antibacterial, hangover and liver protection, laxative, blood purification, etc. These functions make enzymes an emerging active functional ingredient used in health products and are favored by consumers. At present, there is a lack of traditional Chinese medicine enzymes for liver cancer treatment, while my country is rich in medicinal and edible herbs with anti-liver cancer effects. Therefore, developing an enzyme that can have a good auxiliary effect after liver cancer surgery has important application value. 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 medicinal 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 of Streptococcus thermophilus.

[0012] Further,

[0013] The Chinese medicinal raw materials include the following raw materials in parts 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, 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 medicinal 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 blood-transforming gall 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 conditions are 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 into Chinese medicine powders according to the proportions, water is added to the Chinese medicine powders and stirred evenly, and then each Chinese medicine powder is individually extracted by ultrasonic extraction to obtain Chinese medicine extracts.

[0026] (2) Place the above-mentioned 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 broth 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 sterile 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 present invention provides a traditional Chinese medicine enzyme prepared by the preparation method.

[0032] The present invention optimizes the Chinese medicinal raw materials and fermentation microbial community of the Chinese medicinal enzyme. The test results show that the Chinese medicinal 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 response to the current shortage of anti-liver cancer Chinese herbal enzymes and the shortcomings of Chinese herbal enzyme production technology, the present invention provides a method for preparing a Chinese herbal enzyme with anti-liver cancer function. This method uses a composite probiotic and a fermentation technology that combines traditional and modern bioengineering technologies. After sufficient fermentation, a Chinese herbal enzyme with stable quality, high safety, and strong efficacy can be obtained. The enzyme can be used for nutritional support and adjuvant treatment of liver cancer patients before, during, and after radiotherapy and chemotherapy, improving the quality of life of liver cancer patients, significantly enhancing the efficacy of radiotherapy and chemotherapy, and prolonging 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 after oral administration of 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 of 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 materials of which are 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 a traditional Chinese medicine enzyme for treating liver cancer. The enzyme is obtained by fermenting ginseng, salvia miltiorrhiza, bupleurum, wolfberry fruit, cassia seed, rhizome of oriental herbs, gentian, isatis root, schisandra chinensis, turmeric, liquorice, zedoaria, huaxuedan, and centella asiatica using a composite bacterial strain consisting of Lactobacillus acidophilus, Bifidobacterium longum, Lactobacillus delbrueckii bulgaricus, Streptococcus thermophilus, and yeast. The enzyme can be used for the prevention and treatment of liver cancer, and can also be used for body repair after chemotherapy for liver cancer, thus having important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The toxic effects of the traditional Chinese medicine enzymes described in experimental groups 1 to 4 on various organs and tissues in Kunming mice;

[0041] Figure 2 The following are the liver scans and biochemical index changes of Case 1 before treatment. (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 the serum during treatment; (D) is the monitoring of blood cell changes during treatment; (E) is the alpha-fetoprotein content in the serum during treatment; (F) is the change of biochemical indexes related to liver function; the horizontal axis of the bar graph is the date, and the vertical axis is the change of related indexes;

[0042] Figure 3 The following are the liver scans and biochemical index changes of Case 2 before treatment. (A) is the MR scan of the liver before treatment, with the red circle indicating the size of the tumor; (B) is the MR scan of the liver after treatment, with the red circle indicating 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 change of biochemical indexes related to liver function; the horizontal axis of the bar graph is the date, and the vertical axis is the change of related indexes;

[0043] Figure 4 The following are the liver scans and biochemical index changes of Case 3 before treatment. (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 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 change of biochemical indexes related to liver function; the horizontal axis of the bar graph is the date, and the vertical axis is the change of related indexes;

[0044] Figure 5The following are the changes in liver scans and biochemical indicators of Case 4 before treatment. (A) is the MR scan of the liver before treatment, with the red circle indicating the size of the tumor; (B) is the MR scan of the liver after treatment, with the red circle indicating the size of the tumor; (C) is the level of hepatitis B virus DNA in serum during treatment; (D) is the monitoring of changes in blood cells during treatment; (E) is the level of alpha-fetoprotein in serum during treatment; and (F) is the change in biochemical indicators related to liver function. The horizontal axis of the bar graph represents the date, and the vertical axis represents the change in related indicators.

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

[0046] Figure 7 The following are the changes in liver scans and biochemical indicators of control case 1 before treatment. (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 level of hepatitis B virus DNA in serum during treatment; (D) is the monitoring of changes in blood cells during treatment; (E) is the level of alpha-fetoprotein 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 change in related indicators;

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

[0048] Figure 9The following are the changes in liver scans and biochemical indicators of control case 3 before treatment: (A) is the MR scan of the liver before treatment, with the red circle indicating the size of the tumor; (B) is the MR scan of the liver after treatment, with the red circle indicating 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 change in biochemical indicators related to liver function; the horizontal axis of the bar graph is the date, and the vertical axis is the change in related indicators;

[0049] Figure 10 The following are the changes in liver scans and biochemical indicators of control case 4 before treatment. (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 change in liver function-related biochemical indicators; the horizontal axis in the bar graph is the date, and the vertical axis is the change in related indicators;

[0050] Figure 11 The following are the changes in liver scans and biochemical indicators of control case 5 before treatment, among which (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 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. 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 contents of this article and appropriately improve the process parameters to achieve the desired effect. It should be noted that all similar replacements and modifications are obvious to those skilled in the art and are 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 herein 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 medicinal enzyme provided by the present invention uses abundant and easily available resources such as ginseng, salvia miltiorrhiza, bupleurum, wolfberry, cassia seed, oriental rhizome, gentian, isatis root, schisandra chinensis, turmeric, liquorice, zedoaria, huaxuedan, and centella asiatica as fermentation raw materials, which promotes the development of the deep processing industry of the above raw materials, improves economic benefits, and provides new ideas for new product development;

[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 gastrointestinal tract, 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 for alleviating liver cancer pain. By nourishing and protecting the liver, soothing the liver and regulating Qi, it can improve liver tissue function and help liver cancer patients recover.

[0056] 4. The Chinese medicinal enzymes described in the present invention can also be used in combination with Western medicines for the treatment of liver cancer. They can be used for the early and middle stage treatment of liver cancer, or for the repair of the body 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 the 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 enzymes 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 fruit, 15 parts of cassia seed, 20 parts of oriental rhizome, 20 parts of gentian, 25 parts of isatis root, 20 parts of schisandra fruit, 10 parts of turmeric, 15 parts of liquorice, 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 Microorganisms, 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), with 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 traditional Chinese medicine enzyme in this experimental group are as follows:

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

[0067] (2) The above-mentioned Chinese herbal extract was placed in a fermentation tank, and composite bacteria were added and stirred thoroughly. The ratio of Lactobacillus acidophilus, Bifidobacterium longum, Lactobacillus delbrueckii bulgaricus, and Streptococcus thermophilus was 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 area in 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 conditions are preferably at a speed of 1000-2000×g for 10-20 minutes.

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

[0071] 2. Experimental Group 2

[0072] The raw material composition and quantity of the active ingredients of the 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 fruit, 12 parts of cassia seed, 20 parts of oriental rhizome, 20 parts of gentian, 18 parts of isatis root, 15 parts of schisandra fruit, 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 traditional 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 quantity of the active ingredients of the 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 fruit, 10 parts of cassia seed, 20 parts of oriental rhizome, 25 parts of gentian, 20 parts of isatis root, 15 parts of schisandra fruit, 15 parts of turmeric, 20 parts of liquorice, 20 parts of zedoaria, 20 parts of blood 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 Microorganisms, 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 preparation method, process steps, and dosages for this experimental group's TCM preparation included a composite bacterial strain of Lactobacillus acidophilus, Bifidobacterium longum, Lactobacillus delbrueckii bulgaricus, Streptococcus thermophilus, and yeast in a ratio of 1.5:1.5:1:1:1.5. The fermentation broth was centrifuged at 1500 × g for 15 minutes. The remaining preparation steps, process steps, and dosages were the same as those for Experimental Group 1.

[0081] 4. Experimental Group 4

[0082] The raw material composition and quantity of the active ingredients of the 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 fruit, 18 parts of turmeric, 25 parts of liquorice, 25 parts of zedoaria, 25 parts of blood gall, 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 cycle of the traditional Chinese medicine preparation in this experimental group is preferably 1 to 2 years. The remaining preparation steps, usage and dosage are the same as those of experimental group 3.

[0086] The application of the traditional Chinese medicine enzyme in the preparation of anti-liver cancer products 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 is highly safe and has no obvious adverse reactions when taken orally at a rate of 2000 mL per 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 well-ventilated conditions for observation 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 were not allowed to drink 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 administered 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 were healthy and alive, 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, the autopsy found that there was no obvious difference in the changes in the organs of the mice in the experimental groups compared with the control group. Figure 1 According to the toxicological safety evaluation standards, LD 50 >10000mg / kg, it is a non-toxic substance.

[0094] 2. Anti-tumor effect test

[0095] (1) Test materials:

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

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

[0098] (2) Test method:

[0099] Establishment of Huh7 BALB / c nude tumor-bearing mouse xenograft model: Huh7 cells in logarithmic growth phase were obtained, washed 3 times with PBS, resuspended in pre-cooled PBS, and added with Matrigel at a ratio of 2:1 (PBS:Matrigel). The final cell density was adjusted to 1×10 7 The tumor volume was 1 / 2 (ab 1 / 2) of the nude mice. 2 ), a and b represent the long diameter and short diameter of the tumor, respectively.

[0100] After lumps 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 dosing groups according to experimental groups 1 to 4 (named dosing groups 1 to 4, with 10 mice in each group), and was 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 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 are expressed as mean ± standard deviation, and data were processed using SPSS 16.0 statistical software. P < 0.05 was considered statistically significant. The experimental results are shown in Table 1 below.

[0102] Table 1. Antitumor effects of the traditional Chinese medicine 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. Examples of typical cases:

[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 Tegafur (one tablet each time, twice a day, take for 14 days and rest for 7 days, 21 days as a treatment cycle), and zoledronic acid for anti-bone metastasis treatment, and took the traditional Chinese medicine enzyme described in experimental group 4 orally 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 in the figure).

[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 Tegafur (one tablet each time, twice a day, take for 14 days and rest for 7 days, 21 days as a treatment cycle), and zoledronic acid for anti-bone metastasis treatment, and took the traditional Chinese medicine enzyme described in experimental group 3 orally 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 During the treatment, the serum thymidine kinase level 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 3 C, D, E, F in the figure).

[0108] (3) On January 26, 2022, the CT scan 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 Tegafur (one tablet each time, twice a day, take for 14 days and rest for 7 days, 21 days as a treatment cycle), and zoledronic acid for anti-bone metastasis treatment, and took the traditional Chinese medicine enzyme described in experimental group 3 orally 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 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 the normal function of bone marrow and liver function ( Figure 4 C, D, E, F in the figure).

[0109] (4) On June 3, 2022, the MRI results showed a tumor of 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 Tegafur (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 took the traditional Chinese medicine enzyme described in experimental group 4 orally 60 mL / time, 3 times a day. On February 13, 2023, the 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 in the figure).

[0110] (5) On December 31, 2022, the CT scan showed a 7.1cm×6.8cm×6.4cm mass 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 rest for 7 days, 21 days as a treatment cycle), and zoledronic acid for anti-bone metastasis treatment, and took the Chinese medicine enzyme described in experimental group 3 orally 60 mL / time, 3 times a day. On May 4, 2023, the CT results showed a 7.0 cm × 6.4 cm × 5.6 cm tumor in the right lobe of the liver ( Figure 6 During the treatment, the alpha-fetoprotein level 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 were within the normal range, indicating that bone marrow function and liver function were normal ( Figure 6 C, D, E in the figure).

[0111] 4. Examples of control cases:

[0112] (1) On August 18, 2022, 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-tioxetine (one tablet each time, twice a day, take for 14 days and stop for 7 days, 21 days as a treatment cycle), and was given zoledronic acid for anti-bone metastasis treatment. He did not take Chinese herbal enzymes. On November 17, 2022, the MRI results showed a 2.8 cm × 4.0 cm × 4.5 cm tumor in the right lobe of the liver ( Figure 7 At the same time, during the treatment, the hepatitis B virus level in the serum continued to rise, and the alpha-fetoprotein level remained high, proving that the treatment was ineffective. 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 in the figure).

[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 Tegafur (one tablet each time, twice a day, take for 14 days and rest for 7 days, 21 days as a treatment cycle) treatment, and zoledronic acid for anti-bone metastasis treatment, and did not take traditional 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 in the figure); At the same time, during the treatment, the level of alpha-fetoprotein remained high, proving that the treatment was ineffective. 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 8 C, D, E, F in the figure).

[0114] (3) MRI results on June 1, 2021, showed a 9.1cm×5.8cm×8.9cm mass in the right lobe of the liver ( Figure 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 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 did not take traditional 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 ( Figure 9 B); At the same time, during the treatment process, 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 ( Figure 9 C, D, E, F in the figure).

[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 ( Figure 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-tioxetine (one tablet each time, twice a day, take for 14 days and rest for 7 days, 21 days as a treatment cycle), and was given zoledronic acid for anti-bone metastasis treatment. He did not take Chinese herbal 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 ( Figure 10 B); At the same time, during the treatment process, 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 ( Figure 10 C, D, E, F in the figure).

[0116] (5) On March 6, 2021, the MRI results showed a 0.8 cm × 1.9 cm tumor in the right lobe of the liver ( Figure 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 Tegafur (one tablet each time, twice a day, take for 14 days and rest for 7 days, 21 days as a treatment cycle), and was given zoledronic acid for anti-bone metastasis treatment. He did not take Chinese herbal enzymes. On March 13, 2022, the MRI results showed a 4.2 cm × 2.7 cm tumor in the right lobe of the liver ( Figure 11B); At the same time, during the treatment process, the serum thymidine kinase concentration and alpha-fetoprotein content were relatively stable, but the imaging report showed 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 11 C, D, E, F in the figure).

[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 medicinal raw materials are prepared from the following raw materials in parts 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 10-25 parts of zedoaria; The composite bacteria in the extract are: 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 Saccharomyces boulardii and 1.0×10 8 ~2.0×10 8 CFU / mL of Streptococcus thermophilus.

2. A method for preparing an anti-liver cancer Chinese medicinal enzyme, characterized in that: 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 medicinal raw materials are prepared from the following raw materials in parts 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, 10-25 parts of zedoaria, 12-25 parts of huaxuedan and 10-20 parts of centella asiatica; The composite bacteria in the extract are: 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 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 medicinal materials are composed of the following raw materials in parts 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 are: 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 are composed of 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 conditions are 1000g-2000g, 10min-20min.

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 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 according to any one of claims 1 to 7.

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