Compound bezoar composition, decoction piece type compound bezoar and preparation method of decoction piece type compound bezoar

Abstract

The invention belongs to the technical field of biochemical pharmacy, and particularly relates to a compound bezoar composition, decoction piece type compound bezoar and a preparation method of the decoction piece type compound bezoar. The compound calculus bovis composition provided by the invention comprises 94-96% by mass of main agents and 8 kinds of calculus bovis intermediates, the mass content of the auxiliary agent is 4-6%, and the auxiliary agent is six commercially available finished product combined cholic acid. The bilirubin content of the bezoar intermediate used in the invention is increased by more than 30% and the yield is increased by more than 25% compared with the existing biochemical reaction process of oxgall. The DPPH free radical scavenging rate of the compound bezoar composition reaches 89.6% + / -2.1% and is increased by 25.7-36.9% compared with 58.3% + / -3.5% of a product prepared from ox bile, and through t inspection, p is smaller than 0.01 (n is equal to 5). Free cholic acid intervention is strictly prohibited, lithocholic acid addition is completely eradicated, and the composition has the advantages of being high in bilirubin content, high in oxidation resistance and high in biological activity and adapts to the physiological characteristics of the human body.

Description

Technical Field

[0001] This invention belongs to the field of biochemical pharmaceutical technology, specifically relating to compound bezoar compositions and decoction-type compound bezoar and their preparation methods. Background Technology

[0002] Natural bezoar is the dried gallstone of cattle, a species of bovine animal, and its formation process takes several years. It is a mandatory ingredient in 650 kinds of traditional Chinese medicine. However, the source of natural bezoar is scarce and cannot meet clinical needs.

[0003] Calculus bezoar is a traditional and precious animal-derived medicine in my country. It has the effects of clearing heat and detoxifying, resolving phlegm and calming the nerves. It is used for delirium due to phlegm-heat, coma and aphasia, infantile febrile convulsions, sore throat, mouth and tongue sores, carbuncles and boils.

[0004] The bezoar listed in the Chinese Pharmacopoeia includes two types: natural bezoar and artificial bezoar. Artificial bezoar, as a substitute for natural bezoar, alleviated the long-term shortage of bezoar and ensured the urgent need for clinical medication. Artificial bezoar is mainly processed from bovine bile powder, cholic acid, porcine deoxycholic acid, taurine, bilirubin, cholesterol, and trace elements. It lacks the conjugated bilirubin and conjugated bile acid components found in natural bezoar; its total bilirubin content is extremely low, only 0.63%, and it consists entirely of toxic free bilirubin. The free bile acid content is as high as 13%, and it largely mimics the appearance of lower-grade natural bezoar.

[0005] Thirdly, although in vitro cultured bezoar contains 25% conjugated bilirubin and 4% conjugated bile acids, the content of free bile acids is still as high as 13% (refer to the standard under "In Vitro Cultured Bezoar" in Part I of the 2025 edition of the Chinese Pharmacopoeia). Its composition is far different from that of high-grade bezoar, and it is characterized by low yield, endotoxin degradation of bilirubin, poor antioxidant properties, and low biological activity.

[0006] Existing technologies (such as CN103690563A) use bovine bile as a biochemical reaction solution to prepare bovine gallstone intermediates. However, these technologies have drawbacks such as the presence of lithocholic acid in bovine bile, endotoxin degradation of bilirubin, low product yield (55%–70%), and conjugated bilirubin content of less than 60%, which cannot meet the needs of mid- to high-end pharmaceutical applications. Summary of the Invention

[0007] The purpose of this invention is to provide a compound bezoar composition, a processed compound bezoar, and a method for preparing the same. The compound bezoar composition provided by this invention eliminates free cholic acid and conjugated lithocholic acid, which are the most threatening to the human body; it has high bilirubin content, high antioxidant capacity, and high biological activity.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] This invention provides a compound bezoar composition, comprising a main agent and excipients. The main agent has a mass content of 94-96%, and the excipients have a mass content of 4-6%. The excipients include taurocholic acid, glycocholic acid, taurodeoxycholic acid, glycodeoxycholic acid, taurochenodeoxycholic acid, and glycochenodeoxycholic acid. The main agent is a bezoar intermediate, which includes bilirubin calcium, biliverdin calcium, bilirubin magnesium, biliverdin magnesium, bilirubin zinc, biliverdin zinc, covalent bilirubin, and bilirubin glucuronide.

[0010] Preferably, the preparation method of the bezoar intermediate includes the following steps:

[0011] The raw materials for preparing bezoar intermediates are mixed with a biochemical reaction solution to obtain a mixed raw material solution; the mixed raw material solution is subjected to a biochemical reaction to obtain bezoar intermediates; the biochemical reaction solution is sterile purified water, and the indicators of the sterile purified water include: total microbial colony count ≤10 CFU / 100mL, endotoxin content ≤0.25 EU / mL; the raw materials for preparing glucuronide bilirubin include vitamin C, glucuronic acid, bilirubin, and glucuronidase; the raw materials for preparing covalent bilirubin include vitamin C, glycine, Serum albumin and bilirubin, wherein the raw materials for preparing bilirubin calcium include vitamin C, amino acid chelated calcium and bilirubin, the raw materials for preparing biliverdin calcium include vitamin C, amino acid chelated calcium and biliverdin, the raw materials for preparing bilirubin magnesium include vitamin C, magnesium taurate and bilirubin, the raw materials for preparing biliverdin magnesium include vitamin C, magnesium taurate and biliverdin, the raw materials for preparing bilirubin zinc include vitamin C, zinc gluconate and bilirubin, and the raw materials for preparing biliverdin zinc include vitamin C, zinc gluconate and biliverdin.

[0012] Preferably, the sterile purified water is obtained by a combination of online ultraviolet treatment and in-tank ozone treatment. The conditions for the online ultraviolet treatment include: wavelength of 254 nm and intensity ≥90 μW / cm². 2 The irradiation time is ≥30 min, and the conditions for the ozone treatment in the tank include: the ozone concentration is 0.5~1.0 mg / L, and the treatment time is ≥60 min; the indicators of the sterile purified water also include: Escherichia coli and Salmonella not detected, and it meets the pharmaceutical water standard of General Chapter 0216 of Part IV of the 2025 edition of the Pharmacopoeia of the People's Republic of China.

[0013] Preferably, the bilirubin content in the bilirubin calcium, bilirubin magnesium, bilirubin zinc, covalent bilirubin and glucuronide bilirubin is 75% to 85%, the absorbance of free bilirubin is <0.7, and the water content is less than 9%.

[0014] The biliverdin content in the biliverdin calcium, biliverdin magnesium, and biliverdin zinc is 73%–83%, the absorbance of free biliverdin is <0.5, and the water content is less than 9%.

[0015] Preferably, the main agent comprises the following components in mass content:

[0016] Bilirubin calcium 65%, biliverdin calcium 5%, bilirubin magnesium 18%, biliverdin magnesium 3%, covalent bilirubin 5%, bilirubin zinc 1%, biliverdin zinc 1%, and glucuronide bilirubin 2%.

[0017] Preferably, the excipient comprises the following components in the indicated mass percentages: 12.5% ​​taurocholic acid, 25.5% glycocholic acid, 8.5% taurodeoxycholic acid, 16.5% glycodeoxycholic acid, 12.5% ​​taurochenodeoxycholic acid, and 24.5% glycodeoxycholic acid.

[0018] Preferably, the raw materials for preparing glucuronide bilirubin include the following components in weight percentage: 1%–3% vitamin C, 20%–25% glucuronic acid, 67%–78% bilirubin, and 1%–5% glucuronidase;

[0019] The raw materials for preparing the covalent bilirubin include the following components in weight percentage: 1%–3% vitamin C, 10%–15% glycine, 5%–10% serum albumin, and 72%–84% bilirubin;

[0020] The raw materials for preparing the bilirubin calcium include the following components in weight percentage: 1%–3% vitamin C, 10%–25% amino acid chelated calcium, and 72%–89% bilirubin;

[0021] The raw materials for preparing the biliverdin calcium include the following components in weight percentage: 1%–3% vitamin C, 10%–25% amino acid chelated calcium, and 72%–89% biliverdin;

[0022] The raw materials for preparing bilirubin magnesium include the following components in weight percentage: 1%–3% vitamin C, 10%–25% magnesium taurate, and 72%–89% bilirubin;

[0023] The raw materials for preparing the biliverdin magnesium include the following components in weight percentage: 1%–3% vitamin C, 10%–25% magnesium taurate, and 72%–89% biliverdin;

[0024] The raw materials for preparing bilirubin zinc include the following components in weight percentage: 1%–3% vitamin C, 10%–25% zinc gluconate and 72%–89% bilirubin;

[0025] The raw materials for preparing the zinc biliverdin include the following components in weight percentage: 1%–3% vitamin C, 10%–25% zinc gluconate, and 72%–89% biliverdin.

[0026] Preferably, the temperature of the biochemical reaction is 35℃~40℃.

[0027] This invention provides a compound bezoar extract in the form of medicinal slices, which is made from the compound bezoar extract composition described in the above technical solution.

[0028] This invention provides a method for preparing the compound bezoar in the form of medicinal slices as described above, comprising the following steps:

[0029] The main agent and excipients are mixed to obtain powdered compound bezoar;

[0030] The powdered compound bezoar is dry-processed into pills to obtain the processed compound bezoar.

[0031] This invention provides a compound bezoar composition, comprising a main agent and excipients. The main agent has a mass content of 94-96%, and the excipients have a mass content of 4-6%. The excipients include taurocholic acid, glycocholic acid, taurodeoxycholic acid, glycodeoxycholic acid, taurochenodeoxycholic acid, and glycochenodeoxycholic acid, totaling six types. The main agent is a bezoar intermediate, which includes bilirubin calcium, biliverdin calcium, bilirubin magnesium, biliverdin magnesium, bilirubin zinc, biliverdin zinc, covalent bilirubin, and glucuronide bilirubin, totaling eight types. This invention uses these eight bezoar intermediates as the main agent. Covalent bilirubin is a stabilizer for bezoar. Accelerated stability testing (40℃, 75% RH) shows that the compound bezoar composition containing covalent bilirubin shows a decrease rate of <3% in bound bilirubin content after 6 months, while the control group without covalent bilirubin shows a decrease rate of 18%. Bilirubin zinc, biliverdin zinc, and glucuronide bilirubin are the ingredients in the compound bezoar. This invention uses six conjugated bile acids as excipients; it precisely controls the mass ratio of eight bezoar intermediates (main agents) to six conjugated bile acids (excipients) to obtain a compound bezoar with high bilirubin content, high antioxidant properties, high bioactivity, and suitability for human physiology. Based on the results of the examples, the compound bezoar composition provided by this invention has the following characteristics: bilirubin content >60%, free bilirubin absorbance <0.7, bile acid content 4%–6%, water content <9%, and total heavy metal (lead + cadmium + arsenic + mercury + chromium) content ≤30 ppm, meeting the requirements of the Chinese Pharmacopoeia.

[0032] The compound bezoar composition provided by this invention is a natural bezoar substitute raw material prepared by biochemical pharmaceutical methods. Its core components are "complex conjugated bilirubin, biliverdin + complex conjugated bile acid"; it solves the problems of scarcity of natural bezoar and insufficient efficacy of existing substitutes.

[0033] The compound bezoar composition provided by this invention is a high-quality substitute raw material for Chinese medicinal materials (natural bezoar), which can be directly used in the production of traditional Chinese medicine (such as Xihuang Pill and Angong Niuhuang Pill) or processed into decoction pieces.

[0034] Furthermore, the present invention defines a method for preparing bezoar intermediates comprising the following steps: mixing raw materials for preparing bezoar intermediates with a biochemical reaction solution to obtain a mixed raw material solution; subjecting the mixed raw material solution to a biochemical reaction to obtain bezoar intermediates; wherein the biochemical reaction solution is sterile purified water, and the indicators of the sterile purified water include: total microbial colony count ≤10 CFU / 100mL, endotoxin content ≤0.25 EU / mL.

[0035] The method for preparing bezoar intermediates provided by this invention effectively eliminates the toxic and destructive effects of mycotoxins in the biochemical reaction solution on the content, yield, biological activity, and antioxidant properties of bezoar intermediates.

[0036] The bezoar intermediate prepared by this invention has a bilirubin / biliverdin content that is more than 30% higher than that obtained by existing biochemical reaction processes using bovine bile, and a yield that is more than 25% higher. The compound bezoar DPPH free radical scavenging rate reaches 89.6% ± 2.1%, which is 25.7%–36.9% higher than the comparative example (58.3% ± 3.5% from bovine bile processing). A t-test showed p < 0.01 (n = 5).

[0037] The preparation method provided by this invention optimizes the preparation method of bezoar intermediates, resulting in bezoar intermediates with a bilirubin / biliverdin content of 73%–85% and a free bilirubin / biliverdin absorbance of <0.7. This invention, through optimizing the preparation method of bezoar intermediates, yields high bilirubin or biliverdin content, high antioxidant properties, and high bioactivity, making it suitable for the human physiological environment. Detailed Implementation

[0038] This invention provides a compound bezoar composition, comprising a main agent and excipients. The main agent has a mass content of 94-96%, and the excipients have a mass content of 4-6%. The excipients include taurocholic acid, glycocholic acid, taurodeoxycholic acid, glycodeoxycholic acid, taurochenodeoxycholic acid, and glycochenodeoxycholic acid. The main agent is a bezoar intermediate, which includes bilirubin calcium, biliverdin calcium, bilirubin magnesium, biliverdin magnesium, bilirubin zinc, biliverdin zinc, covalent bilirubin, and bilirubin glucuronide.

[0039] In this invention, unless otherwise specified, all raw materials / components are commercially available products well-known to those skilled in the art. Percentages in this invention, unless otherwise specified, refer to mass percentages. Solutions in this invention, unless otherwise specified, are aqueous solutions with water as the solvent; for example, hydrochloric acid solution is an aqueous solution of hydrochloric acid. Room temperature in this invention generally refers to a temperature between 15°C and 25°C, typically defined as 25°C. Unless otherwise specified, the percentage content of each raw material in the mixed raw material solution in this invention is calculated based on the total mass of the raw materials excluding sterile purified water. The organic active raw materials used in this invention are medical-grade and / or food-grade substances. The reagents used to adjust the pH value of the solution in this invention include sodium carbonate or sodium hydroxide. The water used in this invention is sterile purified water. The sterile purified water is preferably used after degassing (deoxygenation).

[0040] The compound bezoar composition provided by this invention includes a main agent. In this invention, the main agent is a bezoar intermediate. In this invention, the main agent preferably includes the following components in the indicated mass percentages: 65% bilirubin calcium, 5% biliverdin calcium, 18% bilirubin magnesium, 3% biliverdin magnesium, 5% covalent bilirubin, 1% bilirubin zinc, 1% biliverdin zinc, and 2% glucuronide bilirubin.

[0041] In this invention, the method for preparing the bezoar intermediate preferably includes the following steps:

[0042] The raw materials for preparing bezoar intermediates are mixed with a biochemical reaction solution to obtain a mixed raw material solution; the mixed raw material solution is subjected to a biochemical reaction to obtain bezoar intermediates; the biochemical reaction solution is sterile purified water, and the indicators of the sterile purified water include: total microbial colony count ≤10 CFU / 100mL, endotoxin content ≤0.25 EU / mL; the raw materials for preparing glucuronide bilirubin include vitamin C, glucuronic acid, bilirubin, and glucuronidase; the raw materials for preparing covalent bilirubin include vitamin C, glycine, Serum albumin and bilirubin, wherein the raw materials for preparing bilirubin calcium include vitamin C, amino acid chelated calcium and bilirubin, the raw materials for preparing biliverdin calcium include vitamin C, amino acid chelated calcium and biliverdin, the raw materials for preparing bilirubin magnesium include vitamin C, magnesium taurate and bilirubin, the raw materials for preparing biliverdin magnesium include vitamin C, magnesium taurate and biliverdin, the raw materials for preparing bilirubin zinc include vitamin C, zinc gluconate and bilirubin, and the raw materials for preparing biliverdin zinc include vitamin C, zinc gluconate and biliverdin.

[0043] This invention involves mixing raw materials for preparing bezoar intermediates with a biochemical reaction solution to obtain a mixed raw material solution. The mixing is carried out in a reaction vessel. The biochemical reaction vessel is preferably a glass and / or enamel-lined reaction vessel. The mixing preferably includes: drawing sterile purified water into the biochemical reaction vessel, degassing the sterile purified water under vacuum while stirring, and then drawing in the raw materials for preparing bezoar intermediates. The stirring speed is preferably 1000–1500 r / min, and the vacuum degree is preferably -0.08–-0.09 MPa. The degassing is preferably the removal of oxygen from the sterile purified water. When the bezoar intermediate is bilirubin glucuronide, covalent bilirubin, or zinc bilirubin, the bilirubin in the raw materials is preferably bilirubin with an absorbance <1.5. When the bezoar intermediate is zinc biliverdin, the biliverdin in the raw materials is preferably biliverdin with an absorbance <1.0. In this invention, the bilirubin and bilirubin with absorbance <1.5 are preferably bovine and / or porcine, with a purity ≥95%, conforming to the standards under the "Bezoar" section of the 2020 edition of the Pharmacopoeia of the People's Republic of China, and aligning with the revision direction of the 2025 edition of the Pharmacopoeia. The biliverdin and biliverdin with absorbance <1.0 are preferably sheep, with a purity ≥95% (HPLC method, purity ≥95% at a specific absorption peak of 640nm).

[0044] In this invention, the glucuronidase is preferably activated by magnesium chloride. Preferably, the magnesium chloride used to activate the glucuronidase accounts for 1% of the mass percentage of the glucuronidase, and the activation time is preferably 15 minutes. The serum albumin preferably includes bovine serum albumin and / or porcine serum albumin. In a specific embodiment of this invention, the amino acid chelated calcium can be calcium aspartate. The calcium content of the calcium aspartate is preferably 12.0% to 14.0%, and in the embodiment it can be 13.2 wt%, conforming to the national standard GB29226-2012.

[0045] In this invention, the sterile purified water is preferably obtained by a combination of online ultraviolet (UV) treatment and in-tank ozone treatment. The preferred conditions for the online UV treatment include: wavelength of 254 nm and intensity ≥90 μW / cm². 2 The irradiation time is ≥30 min. The preferred conditions for the ozone treatment in the tank include: an ozone concentration of 0.5–1.0 mg / L and a treatment time ≥60 min. In a specific embodiment of the invention, the biochemical reaction solution is preferably sterile purified water that has undergone dual microbial sterilization using both online ultraviolet light and tank-based ozone.

[0046] In this invention, the preferred indicators of the sterile purified water include: total microbial colony count ≤10 CFU / 100mL, endotoxin content ≤0.25 EU / mL. Escherichia coli and Salmonella were not detected, meeting the pharmaceutical water standard of General Chapter 0216 in the 2025 edition of the Pharmacopoeia of the People's Republic of China.

[0047] In this invention, the raw materials for preparing bilirubin calcium include the following components in weight percentage: 1%–3% vitamin C, 10%–25% amino acid chelated calcium, and 72%–89% bilirubin. The raw materials for preparing biliverdin calcium include the following components in weight percentage: 1%–3% vitamin C, 10%–25% amino acid chelated calcium, and 72%–89% biliverdin. The raw materials for preparing bilirubin magnesium include the following components in weight percentage: 1%–3% vitamin C, 10%–25% magnesium taurate, and 72%–89% bilirubin. The raw materials for preparing biliverdin magnesium include the following components in weight percentage: 1%–3% vitamin C, 10%–25% magnesium taurate, and 72%–89% biliverdin. The raw materials for preparing bilirubin zinc include the following components by weight percentage: 1%–3% vitamin C, 10%–25% zinc gluconate, and 72%–89% bilirubin with absorbance <1.5. The raw materials for preparing biliverdin zinc include the following components by weight percentage: 1%–3% vitamin C, 10%–25% zinc gluconate, and 72%–89% biliverdin with absorbance <1.5. The raw materials for preparing covalent bilirubin include the following components by weight percentage: 1%–3% vitamin C, 10%–15% glycine, 5%–10% serum albumin, and 72%–84% bilirubin with absorbance <1.5. The raw materials for preparing glucuronide bilirubin include the following components by weight percentage: 1%–3% vitamin C, 20%–25% glucuronic acid, 67%–78% bilirubin with absorbance <1.5, and 1%–5% glucuronidase.

[0048] After obtaining the mixed raw material solution, the present invention subjects the mixed raw material solution to a biochemical reaction to obtain bezoar intermediate. In the present invention, the temperature of the biochemical reaction is preferably 35℃~40℃, and the biochemical reaction is preferably carried out under stirring conditions.

[0049] In this invention, when the intermediate bezoar is glucuronide bilirubin, the temperature of the biochemical reaction is 35℃~40℃, the pH value is 8~9, and the biochemical reaction is carried out under stirring at a speed of 100~150 r / min. In this invention, the biochemical reaction is preferably detected by absorbance method. The reaction ends when the absorbance of the reaction product is <1.0. In an embodiment of this invention, the specific method for absorbance detection preferably includes: sampling every 30 min, centrifuging the sample solution at high speed, rapidly drying the centrifuged material by microwave for 15 s and grinding it into a fine powder, accurately weighing 10 mg and dissolving it in 5 mL of chloroform, sonicating for 2 min, filtering it through a 0.33 μm needle filter into a cuvette, and measuring the absorbance at 533 nm using a UV spectrophotometer; an absorbance <1.0 indicates the endpoint of the enzymatic hydrolysis reaction. In this invention, after the biochemical reaction is completed, a glucuronide bilirubin reaction solution is obtained. Preferably, the invention further includes: concentrating the glucuronide bilirubin reaction solution using a reverse osmosis membrane to obtain a concentrated solution; adding sodium chloride to the concentrated solution, and finally adding an ethanol-water system for crystallization precipitation to obtain a solid product; drying the solid product under vacuum at -0.08 to -0.09 MPa for 8 to 12 hours at room temperature until the water content is ≤9%, to obtain the glucuronide bilirubin. In this invention, the molecular weight cutoff of the reverse osmosis membrane is preferably 100 Da. The volume ratio of the concentrated solution before and after is preferably 10:1. The mass percentage of sodium chloride in the concentrated solution is preferably 1%. The volume content of ethanol in the ethanol-water system is preferably 95%. The volume of the ethanol-water system is preferably 1.5 to 2 times the volume of the concentrated solution. After crystallization precipitation, the lower crystallization precipitate is preferably separated into solid and liquid components to obtain a solid product. The solid-liquid separation is preferably performed using a plate-mounted centrifuge. After obtaining the solid product, before drying, the solid product is preferably pressed.

[0050] In this invention, when the bezoar intermediate is covalent bilirubin, the preferred temperature for the biochemical reaction is 35℃~40℃. The preferred pH value is 8~9. The biochemical reaction is carried out under stirring conditions, with a preferred rotation speed of 50~100 r / min. In this invention, the biochemical reaction is preferably detected by absorbance method. The reaction ends when the absorbance of the reaction product is <1.0. In an embodiment of this invention, the specific method for absorbance detection preferably includes: taking samples every 30 min, and measuring the absorbance at 433 nm as <1.0 as the incubation endpoint. In this invention, after the biochemical reaction is completed, a covalent bilirubin reaction solution is obtained. This invention preferably further includes: concentrating the covalent bilirubin reaction solution using a reverse osmosis membrane to obtain a concentrate; adding sodium chloride to the concentrate, and then adding an ethanol-water system for crystallization precipitation to obtain a solid product; drying the solid product under vacuum at -0.08~-0.09 MPa for 8~12 h at room temperature until the water content is ≤9%, to obtain the covalent bilirubin. In this invention, the molecular weight cutoff of the reverse osmosis membrane is preferably 100 Da. The volume ratio of the concentrate before and after is preferably 10:1. The mass percentage of sodium chloride in the concentrate is preferably 1%. The volume content of ethanol in the ethanol-water system is preferably 95%. The volume of the ethanol-water system is preferably 1.5 times the volume of the concentrate. After crystallization and precipitation, the lower crystallization precipitate is preferably separated into solid and liquid components to obtain a solid product. The solid-liquid separation is preferably performed using a plate-mounted centrifuge. After obtaining the solid product, before drying, the solid product is preferably pressed. The drying is preferably vacuum drying, and the vacuum drying temperature is preferably room temperature, and the pressure is preferably -0.08 to -0.09 MPa.

[0051] In this invention, when the bezoar intermediate is bilirubin calcium, bilirubin magnesium, biliverdin calcium, or biliverdin magnesium: the biochemical reaction is carried out at a temperature of 35℃~40℃, a pH value of 9~12, and under stirring conditions at a rotation speed of 500~550 r / min. In this invention, the biochemical reaction is preferably detected by absorbance measurement. The reaction ends when the absorbance of the reaction product is <1.0. In an embodiment of this invention, the specific method for absorbance measurement preferably includes: sampling every 10 minutes, and measuring the absorbance at 453 nm; a value <1.0 is considered the critical reaction endpoint.

[0052] In this invention, when the bezoar intermediate is bilirubin zinc or biliverdin zinc: the biochemical reaction is carried out at a temperature of 35℃~40℃, a pH value of 9~12, and under stirring conditions at a rotation speed of 500~550 r / min. In this invention, the biochemical reaction is preferably detected by absorbance measurement. The reaction ends when the absorbance of the reaction product is <1.0. In an embodiment of this invention, the specific method for absorbance measurement preferably includes: sampling every 10 minutes, and measuring the absorbance at 453 nm; a value <1.0 is considered the critical reaction endpoint.

[0053] In this invention, when the bezoar intermediate is bilirubin calcium, bilirubin zinc, biliverdin calcium, or biliverdin zinc: after the biochemical reaction is completed, a reaction solution of bilirubin calcium, bilirubin zinc, biliverdin calcium, or biliverdin zinc is obtained; preferably, this invention further includes: centrifuging the bilirubin calcium, bilirubin zinc, biliverdin calcium, or biliverdin zinc reaction solution, washing the centrifuged material with degassed sterile purified water to remove unbound matter, and balancing the pH to 7.0 to obtain a solid product; drying the solid product under vacuum at -0.08 to -0.09 MPa for 8 to 12 hours at room temperature until the water content is ≤9%, to obtain the bilirubin calcium, bilirubin zinc, biliverdin calcium, or biliverdin zinc. The drying is preferably vacuum drying, the vacuum drying temperature is preferably room temperature, and the pressure is preferably -0.08 to -0.09 MPa, to obtain bilirubin calcium, bilirubin zinc, biliverdin calcium, or biliverdin zinc.

[0054] In this invention, when the bezoar intermediate is bilirubin magnesium or biliverdin magnesium: after the biochemical reaction is completed, a reaction solution of bilirubin magnesium or biliverdin magnesium is obtained; preferably, this invention further includes: concentrating the reaction solution of bilirubin magnesium or biliverdin magnesium using a reverse osmosis membrane and balancing the pH to 7.0 to obtain a concentrated solution; separating the concentrated solution into solid and liquid components using a dew-point low-temperature spray pre-dryer to obtain a solid product; drying the solid product under vacuum at -0.08 to -0.09 MPa for 8 to 12 hours at room temperature until the water content is ≤9% to obtain the bilirubin magnesium and biliverdin magnesium. In this invention, the molecular weight cutoff of the reverse osmosis membrane is preferably 100 Da. The volume ratio of the concentrated solution before and after is preferably 10:1. In this invention, unbound substances are concentrated and washed away using degassed sterile purified water and the pH value is balanced. Before the drying, this invention preferably uses degassed sterile purified water to concentrate and wash away unbound substances and balance the pH value, the balancing pH value being preferably 7.0. The drying process is preferably vacuum drying, and the vacuum drying temperature is preferably room temperature, and the pressure is preferably -0.08 to -0.09 MPa.

[0055] This invention is based on the "Quality Control Standard for Intermediates of Natural Drugs" (T / CACM 1055-2023):

[0056] In this invention, the bilirubin content in the bilirubin calcium, bilirubin magnesium, bilirubin zinc, covalent bilirubin and glucuronide bilirubin is 75% to 85% (HPLC method, detection wavelength 453nm), the absorbance of free bilirubin in the bilirubin intermediate is <0.7 (453nm), and the water content is less than 9%.

[0057] In this invention, the biliverdin content in the biliverdin calcium, biliverdin magnesium and biliverdin zinc is 73% to 83% (HPLC method, detection wavelength 640 nm), the absorbance of free biliverdin in the biliverdin intermediate is <0.5 (640 nm), the water content is less than 9%, and the microbial detection is ≤102 cfu / g.

[0058] In this invention, each type of bilirubin and biliverdin undergoes independent biochemical reactions, stabilizing and ensuring the quality of bezoar. The bilirubin / biliverdin content in the bezoar intermediate is increased by more than 30% compared to existing biochemical reaction processes using bovine bile, and the yield is increased by more than 25%. The compound bezoar exhibits a DPPH free radical scavenging rate of 89.6% ± 2.1%, which is 25.7%–36.9% higher than the comparative example (58.3% ± 3.5% from bovine bile processing). A t-test showed p < 0.01 (n = 5).

[0059] The fermentation-free, independent reaction process provided by this invention differs from traditional in vitro culture techniques in the prior art. It establishes core indicators for bezoar with high content, high activity, high yield, and high antioxidant properties.

[0060] In summary, the preparation methods for bezoar intermediates and bezoar provided by this invention employ a non-fermentation biochemical extraction and purification process, using sterile purified water as the biochemical reaction solution. This avoids the damage of the intermediates to bacterial toxins, completely circumventing the inherent defects of bacterial fermentation processes such as bacterial toxin residues and activity attenuation, while restoring the core quality of the ancient formula Xihuang Wan. Its high antioxidant properties also confirm the record in Tang Dynasty Su Jing's "Newly Revised Materia Medica" that bezoar "prolongs life and lightens the body with long-term use." This opens up a completely new path for the high-end medicinal and health care applications of bezoar.

[0061] The compound bezoar composition provided by this invention includes excipients. In this invention, the excipients preferably include the following components in the indicated mass percentages: 12.5% ​​taurocholic acid, 25.5% glycocholic acid, 8.5% taurodeoxycholic acid, 16.5% glycodeoxycholic acid, 12.5% ​​taurochenodeoxycholic acid, and 24.5% glycodeoxycholic acid.

[0062] In this invention, the excipient is a commercially available product, specifically a conjugated bile acid product that meets the requirements of the Chinese Pharmacopoeia. The excipient contains ≥95% bile acid, and in the examples, this can be 95%.

[0063] The mass content of the main agent in the compound bezoar composition provided by the present invention can be 94%, 95% or 96%; the mass content of the excipient can be 6%, 5% or 4%.

[0064] This invention provides a compound bezoar extract in the form of medicinal slices, which is made from the compound bezoar extract composition described in the above technical solution.

[0065] This invention provides a method for preparing the compound bezoar in the form of medicinal slices as described above, comprising the following steps:

[0066] The main agent and excipients are mixed to obtain powdered compound bezoar;

[0067] The powdered compound bezoar is dry-processed into pills to obtain the processed compound bezoar.

[0068] In a specific embodiment of the present invention, the preparation method of the compound bezoar in the form of medicinal slices preferably includes:

[0069] The main agent and excipients are mixed in a multidimensional mixer for 10-20 minutes at a stirring speed of 100-300 rpm, and the mixing uniformity RSD is ≤3% to obtain powdered compound bezoar.

[0070] The powdered compound bezoar is processed into pellets using a dry pelleting machine with operating parameters including: pressure 15-20 MPa and rotation speed 30-50 r / min, to obtain spherical medicinal slices with a diameter of 6 mm.

[0071] This invention mixes the main agent and excipients to obtain powdered compound bezoar. In this invention, the mixing is carried out in a multidimensional mixer. The mixing time is preferably 10-20 minutes, and in the examples, it can be 15 minutes. The mixing is carried out under stirring conditions, and the stirring speed is preferably 100-300 rpm, and in the examples, it can be 200 rpm. The uniformity of the mixture is RSD ≤ 3%. In this invention, the test results of the powdered compound bezoar are: bilirubin content > 60%, free bilirubin absorbance < 0.7, bile acid content 4%-6%, water content < 9%, and total heavy metal content ≤ 30 ppm, which indicates that it is a qualified product of powdered compound bezoar.

[0072] After obtaining the powdered compound bezoar, the present invention performs dry pelleting to obtain a processed compound bezoar. The dry pelleting is carried out in a dry pelleting machine. The processed compound bezoar is spherical in shape with a diameter of 6 mm.

[0073] In this invention, the specifications of the compound bezoar in the form of medicinal slices are: containing bilirubin >100mg / pill and weighing 0.16g / pill.

[0074] The present invention comprises eight bezoar intermediates in a ratio of complex conjugated bilirubin and complex conjugated biliverdin; six commercially available conjugated bile acids are then combined to form a complex conjugated bile acid composition; finally, these three major complexes (complex conjugated bilirubin, complex conjugated biliverdin, and complex conjugated bile acid) are compounded to obtain a compound bezoar composition. The composition exhibits the following characteristics: bilirubin content >60%, free bilirubin absorbance <0.7, bile acid content 4%–6%, water content <9%, and total heavy metal (lead + cadmium + arsenic + mercury + chromium) content ≤30 ppm, meeting the requirements of the Chinese Pharmacopoeia.

[0075] The compound bezoar composition provided by this invention strictly prohibits the introduction of free bile acids and eliminates the addition of conjugated lithocholic acids. It precisely controls the ratio of 8 bezoar intermediates (main agents) and 6 conjugated bile acids (excipients) in the compound bezoar, thereby obtaining a compound bezoar with high bilirubin content, high antioxidant properties, high biological activity, and suitability for human physiology.

[0076] Traditional processes (such as in vitro cultured bezoar) rely on microbial fermentation, which results in: conjugated bilirubin being broken down into fecal bilirubin by the microorganisms, reducing its activity; the product carrying live bacteria and bacterial toxins, requiring high-temperature sterilization at 100°C, which further destroys conjugated bilirubin (sensitive to high temperatures); and the final product retaining only anti-inflammatory activity, losing its core antioxidant value.

[0077] This invention prepares bezoar intermediates using a "biochemical method": directly purifying highly active conjugated bilirubin intermediates to avoid microbial fermentation; using aseptic production processes, eliminating the need for high-temperature sterilization and preserving the activity of conjugated bilirubin; and compounding conjugated bile acids according to human physiological ratios to match liver metabolic mechanisms, resulting in higher bioavailability; in principle, this ensures that the product is "highly active, high in content, and non-toxic," superior to the "naturally formed (containing impurities)" bezoar and the "fermentation degradation (inactivation)" of in vitro cultured bezoar.

[0078] The compound bezoar composition provided by this invention screens 14 core active ingredients (8 conjugated bilirubin / biliverdin + 6 conjugated bile acids), discarding toxic and ineffective components in natural bezoar (such as free bile acids, lithocholic acid, and mucin), achieving "retaining efficacy while removing toxicity"; this invention achieves standardized production: the reaction cycle for each batch is only 2 hours, and the conjugated bilirubin content deviation is ≤3%, far superior to the content fluctuation (25%~70%) of natural bezoar caused by origin and age of cattle; this invention achieves stable production capacity: the daily production capacity can reach 500kg~1000kg, which is 5~10 times the annual production capacity of natural bezoar (less than 100kg), which can meet the raw material needs of 650 kinds of bezoar-containing traditional Chinese medicines, filling the gap in clinical drug use.

[0079] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0080] All raw materials used in the following examples are medical-grade and / or food-grade. In the following examples and comparative examples, the absorbance of free bilirubin was measured as follows: Referring to Chapter 0401 of the 2025 edition of the Chinese Pharmacopoeia, 10 mg of sample was accurately weighed, 5 mL of chloroform was added, and the sample was sonicated for 2 min. The sample was then filtered through a 0.33 μm filter membrane, and the absorbance was measured at 453 nm. Commercially available in vitro cultured bezoar used in all examples of this invention was purchased from pharmacies, hospitals, and e-commerce platforms. The bezoar was prepared in slice form and packaged in 0.15 g / bottle and 0.35 g / bottle specifications. It was produced by Wuhan Dapeng Pharmaceutical Co., Ltd., Hubei Province.

[0081] Example 1: Preparation of sterile purified water:

[0082] Tap water or deep well water first passes through a pipeline ultraviolet sterilizer before entering a 50-ton capacity plastic storage tank, where it is sterilized using online ultraviolet light (wavelength 254nm, intensity ≥90μW / cm²). 2 Ensure irradiation intensity ≥90μW / cm 2 The sterile purified water already in the plastic storage tank is irradiated again by a pump for a time ≥30 minutes, ensuring that the irradiation intensity is ≥90μW / cm2 and the time is ≥30 minutes. Combined with the treatment of stationary ozone (ozone bubbles are injected from the stationary nozzle of the ozone generator, with an ozone concentration of 0.5~1.0mg / L and an action time ≥60 minutes), sterile purified water is obtained. In this invention, sterile purified water is used as the biochemical reaction liquid in the following embodiments.

[0083] The sterile purified water prepared in this embodiment was tested and found to have a total microbial count ≤10 CFU / 100mL and a bacterial endotoxin level (≤0.25 EU / mL), which meets the pharmaceutical water standard of the 2025 edition of the Pharmacopoeia of the People's Republic of China, Part IV, General Chapter 0216.

[0084] Example 2: Preparation of bilirubin with absorbance <1.5 and biliverdin with absorbance <1.0:

[0085] Sterile purified water is introduced into a double-walled glass reactor and dispersed at high speed with magnetic stirring at 1000–1500 rpm. Residual ozone and free oxygen in the sterile purified water are removed at -0.08–-0.09 MPa, while eliminating saturated oxygen from the outside air. The feed port is slowly opened to introduce 1% vitamin C, 1.5% calcium chloride (chemically pure), and 97.5% bilirubin or biliverdin under negative pressure. The stirring speed is reduced to 100 rpm to suspend the reaction solution. The reaction temperature is maintained at 35–40°C, and the pH is adjusted to 11.5–12.0. Every 1… Take 1 mL of the reaction solution at 0 min, add 4 mL of chloroform, sonicate for 2 min, filter through a 0.33 μm filter membrane, and measure the absorbance at 453 nm wavelength. The critical reaction endpoint is <2.0. For the biliverdin raw material reaction solution, the critical reaction endpoint is <1.5 at 640 nm. Centrifuge to separate the solid and liquid. Wash the centrifuged material with degassed sterile purified water to remove unbound matter and equilibrate the pH to 7.0. Press the centrifuged material and vacuum dry it at room temperature (-0.08 to -0.09 MPa) until the water content is less than 9%, obtaining a dry powder. The powder has a bilirubin content of 76% and a biliverdin content of 78%, with a yield of 80%–85%. The absorbance of free bilirubin is <1.5, and the absorbance of free biliverdin is <1.0.

[0086] Vitamin C, conforming to GB 14754-2010.

[0087] Calcium chloride conforms to GB 1886.45-2016.

[0088] Note: The absorbance was measured at 640 nm, which is the maximum absorption peak of biliverdin in chloroform solution.

[0089] Example 3 This example provides a method for preparing glucuronide bilirubin, specifically including the following steps:

[0090] Sterile purified water is introduced into a double-walled glass reactor and dispersed at high speed with magnetic stirring at 1000–1500 rpm. Residual ozone and free oxygen in the sterile purified water are removed at -0.08–-0.09 MPa, and saturated oxygen from the outside air is eliminated. The feed port is slowly opened, and the raw materials (based on the total mass of the raw materials, 100%) are drawn in under negative pressure: 1% vitamin C, 25% glucuronic acid, 69% bilirubin with absorbance <1.5, and 5% magnesium chloride to activate glucuronidase (the zymogen of glucuronidase is activated with 1% magnesium chloride by weight of the enzyme for 15 minutes to obtain magnesium chloride-activated glucuronidase). The reaction is suspended at 100 rpm, and the reaction temperature is maintained at 35–40℃. The pH is adjusted to 8.5–9.0. Samples are taken every 30 minutes, centrifuged at high speed, and the centrifuged material is rapidly dried using a 700W microwave for 15 seconds. Grind the sample until it passes through a No. 6 sieve (100 mesh). Accurately weigh 10 mg of the centrifuged sample, add 5 mL of chloroform, sonicate for 2 min, filter through a 0.33 μm needle filter into a cuvette, and measure the absorbance at 533 nm using a UV spectrophotometer. The absorbance is <1.0, which is the endpoint of the enzymatic hydrolysis reaction. Concentrate the sample to 1 / 10 of its original volume using a reverse osmosis membrane with a molecular weight cutoff of 100 Da. Balance the pH to 7.0 using sterile purified water that has been de-ozoned and free oxygen removed. Add 1 wt% sodium chloride to the concentrate to ensure ionic strength, and then add 1.5 times the volume of 95% (v / v) ethanol aqueous solution. Utilize the difference in solubility of bilirubin in ethanol (the solubility of bilirubin in 95% ethanol at 25℃ is <0.1 g / 100 mL) to induce crystallization through solvent. After standing for 2 h, a layered precipitate is formed, increasing the purity to 75% (HPLC method).

[0091] After the boundary of the precipitate layer is clear, the bottom valve is opened to release the lower layer of crystallized precipitate into a plate-type centrifuge for solid-liquid separation. The clear liquid is retained and distilled to recover the ethanol. The solid is wrapped in polyester filter cloth and pressed to reduce the liquid content. It is then placed in a vacuum drying oven and dried at room temperature (-0.08 to -0.09 MPa) until the water content is less than 9%, yielding glucuronide bilirubin with a bilirubin content of 75% and a yield of 83% to 87%. The absorbance of free bilirubin is <0.7.

[0092] Vitamin C conforms to GB 14754-2010, and glucuronic acid conforms to the industry standard Q / CZHG 0005S-2021.

[0093] Note: The absorbance measured at 533 nm is the maximum absorption peak of glucuronide bilirubin in chloroform solution.

[0094] Comparative Example 1

[0095] The preparation method is basically the same as in Example 3, except that: In the prior art, patent CN103690563A discloses a method for preparing bovine gallstone intermediates using bovine bile as a biochemical reaction solution, but it has drawbacks such as low bilirubin content and insufficient yield. This invention improves upon this by using fresh or quick-frozen bovine bile as the biochemical reaction solution, replacing it with sterile purified water as in Example 1, to prepare a mixed raw material solution. Glucuronite bilirubin was obtained, with a bilirubin content of 53% and a yield of 55%.

[0096] Example 4 This example provides a method for preparing covalent bilirubin, specifically including the following steps:

[0097] Sterile purified water is introduced into a double-walled glass reactor and dispersed at high speed with magnetic stirring at 1000–1500 rpm. Residual ozone and free oxygen in the sterile purified water are removed at -0.08–-0.09 MPa, while eliminating saturated oxygen from the outside air. The feed port is slowly opened, and the raw materials (based on the total mass of the raw materials, 100%) are drawn in under negative pressure: a mixture of 1% vitamin C, 15% glycine, 5% bovine serum albumin, and 79% bilirubin with absorbance <1.5. The mixture is incubated at 35–40°C at 50 rpm, and the pH is adjusted to 8.0–9.0 every 3 minutes. Sampling was performed at 0 min. An absorbance of <1.0 at 433 nm was considered the incubation endpoint. The solution was concentrated to 1 / 10 of its original volume using a reverse osmosis membrane with a molecular weight cutoff of 100 Da. Sodium chloride (1 wt%) was added to the concentrate, followed by a 95% (v / v) ethanol aqueous solution (twice the volume of the concentrate). Crystallization was carried out at room temperature. Utilizing the difference in bilirubin solubility in ethanol (bilirubin solubility in 95% ethanol at 25°C <0.1 g / 100 mL), solvent-induced crystallization was performed. After standing for 2 h, a layered precipitate formed, increasing the purity to 76% (HPLC method). Centrifugation and solid-liquid separation were performed. The pressed solid was vacuum dried at -0.08 to -0.09 MPa at room temperature until the water content was below 9%, yielding covalent bilirubin. The bilirubin content was 76%, the yield was 85%, and the absorbance of free bilirubin was <0.7.

[0098] Vitamin C, conforming to GB 14754-2010; glycine, conforming to GB 25542-2010; bovine serum albumin, conforming to GBW(E)100151.

[0099] Note: The absorbance measured at 433 nm is the maximum absorption peak of covalent bilirubin in chloroform solution.

[0100] Comparative Example 2

[0101] The preparation method is basically the same as that in Example 4, except that: in the prior art, patent CN103690563A discloses a method for preparing bovine gallstone intermediates using bovine bile as a biochemical reaction solution, but it has defects such as low bilirubin content and insufficient yield. This invention improves upon this by using fresh or quick-frozen bovine bile as the biochemical reaction solution instead of the sterile purified water in Example 4 to prepare a mixed raw material solution, thereby obtaining covalent bilirubin with a bilirubin content of 48.3% and a yield of 57%.

[0102] Example 5 This example provides a method for preparing bilirubin calcium, specifically including the following steps:

[0103] Sterile purified water is introduced into a double-walled glass reactor and dispersed at high speed with magnetic stirring at 1000-1500 rpm. Residual ozone and free oxygen in the sterile purified water are removed at -0.08 to -0.09 MPa, while saturated oxygen from the outside air is eliminated. The feed port is slowly opened, and a mixture of raw materials (total mass of raw materials is 100%) is drawn in under negative pressure: 1% vitamin C, 15% amino acid chelated calcium, and 84% bilirubin. The reaction is carried out at 35℃-40℃ at 500 rpm, with a pH of 11.0-12.0. Samples are taken every 10 minutes, and the critical reaction endpoint is reached when the absorbance at 453 nm is <1.0. The mixture is centrifuged, and the centrifuged material is washed with degassed sterile purified water to remove unbound matter and balance the pH of the solid to 7.0. The pressed solid is then vacuum dried at room temperature at -0.08 to -0.09 MPa until the moisture content is below 9%, yielding bilirubin calcium. 85% bilirubin content, 87% yield, and absorbance of free bilirubin <0.7.

[0104] Vitamin C, conforming to GB 14754-2010.

[0105] Amino acid chelated calcium (calcium aspartate) conforms to the national standard GB 29226-2012.

[0106] Comparative Example 3

[0107] The preparation method is basically the same as that in Example 5, except that: in the prior art, patent CN103690563A discloses a method for preparing bovine gallstone intermediates using bovine bile as a biochemical reaction solution, but it has defects such as low bilirubin content and insufficient yield. This invention improves upon this by using fresh or quick-frozen bovine bile as the biochemical reaction solution instead of the sterile purified water in Example 5, and preparing a mixed raw material solution to obtain bilirubin calcium with a bilirubin content of 58.9% and a yield of 70%.

[0108] Example 6 This example provides a method for preparing bilirubin magnesium, specifically including the following steps:

[0109] Sterile purified water is introduced into a double-walled glass reactor and dispersed at high speed with magnetic stirring at 1000-1500 rpm. Residual ozone and free oxygen in the sterile purified water are removed at -0.08 to -0.09 MPa, while saturated oxygen from the outside air is eliminated. The feed port is slowly opened, and a mixture of 1% vitamin C, 20% magnesium taurate, and 79% bilirubin (based on the total mass of the raw materials) is drawn in under negative pressure. The reaction is carried out at 500 rpm, 35℃-40℃, and pH = 11.0-12.0. Samples are taken every 10 minutes, and the absorbance at 453 nm is measured. A value <1.0 indicates the critical reaction endpoint. The mixture is concentrated using a reverse osmosis (RO) membrane. The concentrate is then concentrated with degassed sterile purified water to remove unbound substances and balance the pH to 7.0. The concentrate is then placed in a dew-point low-temperature spray pre-dryer to obtain moist granules. The moist granules are vacuum dried at -0.08 to -0.09 MPa at room temperature until the moisture content is below 9%, yielding magnesium bilirubin. 76% bilirubin content, 85% yield, and absorbance of free bilirubin <0.7.

[0110] Vitamin C, conforming to GB 14754-2010; magnesium taurine purity ≥98%, conforming to industry standards.

[0111] Comparative Example 4

[0112] The preparation method is basically the same as that in Example 6, except that: in the prior art, patent CN103690563A discloses a method for preparing bovine gallstone intermediates using bovine bile as a biochemical reaction solution, but it has defects such as low bilirubin content and insufficient yield. This invention improves upon this by using fresh or quick-frozen bovine bile as the biochemical reaction solution instead of the sterile purified water in Example 6, and preparing a mixed raw material solution for reaction to obtain bilirubin magnesium with a bilirubin content of 50.3% and a yield of 66%.

[0113] Example 7 This example provides a method for preparing bilirubin zinc, specifically including the following steps:

[0114] Sterile purified water is introduced into a double-walled glass reactor and dispersed at high speed with magnetic stirring at 1000-1500 rpm. Residual ozone and free oxygen in the sterile purified water are removed at -0.08 to -0.09 MPa, while saturated oxygen from the outside air is eliminated. The feed port is slowly opened, and the raw materials (based on the total mass of the raw materials as 100%) are drawn in under negative pressure: 1% vitamin C, 20% zinc gluconate, and 79% bilirubin with absorbance <1.5. The reaction is carried out at 500 rpm, 35℃-40℃, and pH = 9.0-10.0. Samples are taken every 10 minutes, and the critical reaction endpoint is defined as absorbance <1.0 at 453 nm. The mixture is centrifuged, and the centrifuged material is washed with degassed sterile purified water to remove unbound matter. The pH is then equilibrated to 7.0. The pressed solid is vacuum dried at room temperature at -0.08 to -0.09 MPa until the moisture content is below 9%, yielding bilirubin zinc. 75% bilirubin content, yield 83%–87%, free bilirubin absorbance <0.7.

[0115] Vitamin C, conforming to GB 14754-2010. Zinc gluconate purity (on anhydrous basis) 97.0%–102.0%, pH value between 5.5 and 7.5 (conforming to GB8820-2010).

[0116] Comparative Example 5

[0117] The preparation method is basically the same as that in Example 7, except that: in the prior art, patent CN103690563A discloses a method for preparing bovine gallstone intermediates using bovine bile as a biochemical reaction solution, but it has defects such as low bilirubin content and insufficient yield. This invention improves upon this by using fresh or quick-frozen bovine bile as the biochemical reaction solution instead of the sterile purified water in Example 7, and preparing a mixed raw material solution for reaction to obtain bilirubin zinc with a bilirubin content of 45% and a yield of 63%.

[0118] Example 8 This example provides a method for preparing biliverdin calcium, specifically including the following steps:

[0119] Sterile purified water is introduced into a double-walled glass reactor and dispersed at high speed with magnetic stirring at 1000-1500 rpm. Residual ozone and free oxygen in the sterile purified water are removed at -0.08 to -0.09 MPa, while saturated oxygen from the outside air is eliminated. The feed port is slowly opened, and a mixture of raw materials (total mass of raw materials is 100%) is drawn in under negative pressure: 1% vitamin C, 25% amino acid chelated calcium, and 74% biliverdin (sheep-derived). The reaction is carried out at 500 rpm, 35℃-40℃, and pH = 11.0-12.0. Samples are taken every 10 minutes, and the absorbance at 640 nm is measured. A value <1.0 indicates the critical reaction endpoint. The mixture is centrifuged, and the centrifuged material is rinsed with degassed sterile purified water to remove unbound matter and balance the pH to 7.0. The solids are pressed and dried in a vacuum drying oven at -0.08 to -0.09 MPa at room temperature until the moisture content is below 9%, yielding biliverdin calcium. 83% biliverdin content, yield 83%–87%, free biliverdin absorbance <0.5.

[0120] Vitamin C, conforming to GB 14754-2010. Biliverdin (sheep-derived, purity ≥95%, HPLC method, specific absorption peak at 640nm, purity ≥95%, conforming to the "Quality Control Standard for Natural Drug Intermediates" T / CACM 1055-2023). Amino acid chelated calcium (calcium aspartate), conforming to the national standard GB 29226-2012.

[0121] Comparative Example 6

[0122] The preparation method is basically the same as that in Example 8, except that: in the prior art, patent CN103690563A discloses a method for preparing bovine gallstone intermediates using bovine bile as a biochemical reaction solution, but it has defects such as low bilirubin content and insufficient yield. This invention improves upon this by using fresh or quick-frozen bovine bile as the biochemical reaction solution instead of the sterile purified water in Example 8; the result is a 58% biliverdin content and a yield of 65%.

[0123] Example 9 This example provides a method for preparing biliverdin magnesium, specifically including the following steps:

[0124] Sterile purified water is introduced into a double-walled glass reactor and dispersed at high speed with magnetic stirring at 1000–1500 rpm. Residual ozone and free oxygen in the sterile purified water are removed at -0.08–-0.09 MPa, while eliminating saturated oxygen from the outside air. The feed port is slowly opened, and a mixture of the preparation raw materials (based on the total mass of the raw materials being 100%) is drawn in under negative pressure at 500 rpm and 35–40°C. The pH was set at 11.0–12.0. Samples were taken every 10 minutes, and the absorbance at 640 nm was measured to be <0.8, indicating the critical reaction endpoint. The solution was concentrated to 1 / 10 of its original volume using a reverse osmosis membrane with a molecular weight cutoff of 100 Da. Unbound substances were removed by concentrating the concentrate with sterile purified water, and the pH was equilibrated to 7.0. The solution was then pre-dried in a dew-point low-temperature spray dryer to obtain moist granules. The moist granules were vacuum dried at room temperature (-0.08–-0.09 MPa) until the moisture content was below 9%, yielding biliverdin magnesium. The biliverdin content was 83%, the yield was 86%, and the absorbance of free biliverdin was <0.5.

[0125] Vitamin C, conforming to GB 14754-2010. Magnesium taurine purity ≥98%, conforming to industry standards.

[0126] Comparative Example 7

[0127] The preparation method is basically the same as that in Example 9, except that: in the prior art, patent CN103690563A discloses a method for preparing bezoar intermediates using bovine bile as a biochemical reaction solution, but it has defects such as low bilirubin content and insufficient yield. This invention improves upon this by using fresh or quick-frozen bovine bile as the biochemical reaction solution instead of the sterile purified water in Example 9; the result is a 57% biliverdin content and a yield of 65%.

[0128] Example 10 This example provides a method for preparing zinc biliverdin, specifically including the following steps:

[0129] Sterile purified water is introduced into a double-walled glass reactor and dispersed at high speed with magnetic stirring at 1000-1500 rpm. Residual ozone and free oxygen in the sterile purified water are removed at -0.08 to -0.09 MPa, while saturated oxygen from the outside air is eliminated. The feed port is slowly opened, and the raw materials (total mass of raw materials is 100%) are drawn in under negative pressure: 1% vitamin C, 20% zinc gluconate, and 79% biliverdin with absorbance <1.0. The reaction is carried out at 500 rpm, 35℃-40℃, and pH = 9.0-10.0. Samples are taken every 10 minutes, and the critical reaction endpoint is reached when the absorbance at 640 nm is <0.7. The mixture is centrifuged, and solid-liquid separation is performed. The centrifuged material is centrifuged and sprayed with degassed sterile purified water to remove unbound matter, and the pH is equilibrated to 7.0. The pressed solid is vacuum dried at room temperature at -0.08 to -0.09 MPa until the moisture content is below 9%, yielding zinc biliverdin. 75% biliverdin content, yield 83%–87%, free biliverdin absorbance <0.5.

[0130] Vitamin C, conforming to GB 14754-2010.

[0131] Zinc gluconate, purity (on anhydrous basis) 97.0%–102.0%, pH value between 5.5 and 7.5 (compliant with GB8820-2010).

[0132] Biliverdin (sheep-derived, purity ≥95%, HPLC method, purity ≥95% at a specific absorption peak of 640nm, conforming to the "Quality Control Standard for Natural Drug Intermediates" T / CACM 1055-2023).

[0133] Comparative Example 8

[0134] The preparation method is basically the same as that in Example 10, except that: in the prior art, patent CN103690563A discloses a method for preparing bezoar intermediates using bovine bile as a biochemical reaction solution, but it has defects such as low bilirubin content and insufficient yield. This invention improves upon this by using fresh or quick-frozen bovine bile as the biochemical reaction solution instead of the sterile purified water in Example 10; the result is a 50% biliverdin content and a yield of 61%.

[0135] Example 11

[0136] This embodiment provides a compound bezoar composition, including a main agent and an excipient, wherein the main agent has a mass content of 94% and the excipient has a mass content of 6%.

[0137] The main component comprises the following components by mass percentage: 65% bilirubin calcium, 5% biliverdin calcium, 18% bilirubin magnesium, 3% biliverdin magnesium, 5% covalent bilirubin, 1% bilirubin zinc, 1% biliverdin zinc, and 2% glucuronide bilirubin; the total mass percentage is 100%, and the total bilirubin content is >70%. It is mixed in a multi-dimensional mixer for 15 minutes (200 rpm), with a mixing uniformity RSD ≤3%. The excipients comprise the following components by mass percentage: 12.5% ​​taurocholic acid, 25.5% glycocholic acid, 8.5% taurodeoxycholic acid, 16.5% glycodeoxycholic acid, 12.5% ​​taurochenodeoxycholic acid, and 24.5% glycodeoxycholic acid. They are mixed in a multi-dimensional mixer for 15 minutes (200 rpm), with a mixing uniformity RSD ≤3%, a total mass percentage of 100%, and a total bile acid content >95%.

[0138] Take 6g of excipients and then add 94g of main agent to make the total mass of the compound bezoar composition reach 100g (total ratio of excipients and main agent 100%). Mix in a multi-dimensional mixer for 15 minutes (speed 200rpm). The mixing uniformity RSD ≤3%, the bilirubin content is >60%, the absorbance of free bilirubin is <0.7, the bile acid content is 4%~6%, the water content is <9%, and the total heavy metal content is ≤30ppm. This is a qualified product of powdered compound bezoar.

[0139] Powdered compound bezoar is fed into a dry pelleting machine to form small balls with a diameter of 6 mm, yielding a decoction-type compound bezoar containing bilirubin >100 mg / pill and weighing 0.16 g / pill.

[0140] Actual test results: bilirubin content 63.9%, free bilirubin absorbance 0.62, bile acid content 5.7%, water content 7.2%, total heavy metal content was detected by ICP-MS method (compliant with Pharmacopoeia General Chapter 0821), lead 2.1ppm, cadmium 0.3ppm, arsenic 0.8ppm, mercury 0.1ppm, chromium 5.0ppm, all of which meet the limit requirements.

[0141] The microbial content was 3 CFU / g, which meets the quality control standards of this invention and the microbial limit standards for non-sterile drugs in General Chapters 1105, 1106, and 1107 of the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part IV.

[0142] As can be seen from the above embodiments, the compound bezoar composition provided by the present invention completely eliminates free bile acids and conjugated lithocholic acids, which are the most threatening to the human body. It has a high bilirubin content, high antioxidant properties, and high biological activity; it not only conforms to the actual quality of natural first-grade bezoar but also aligns with corresponding human physiology. It abandons the superficial, ineffective, and pharmacologically ineffective oval-shaped processes such as solid rolling coating to form concentric layers and the magnesium cholate production process (Note: This abandons the existing forming process where magnesium cholate is used as an excipient; the bilirubin calcium and bilirubin magnesium in the main components of this invention are in an active bound state, not forming excipients), as well as the enzymatic hydrolysis of bovine gallbladder skin mucoprotein. This significantly reduces production costs, saves energy and labor, improves production efficiency, and further expands the scale and capacity of industrial production. Most notably, the production process produces no waste liquid, waste gas, or waste residue; only a small amount of ethanol is recovered (compliant with the "Technical Specification for Industrial Alcohol Recovery and Utilization" GB / T 32122-2015), and there is no noise or odor pollution.

[0143] Experimental studies have revealed that free bile acids are toxic, easily causing loose stools, and long-term use can irritate and toxicize the tubular glands of the colon, leading to lesions and potentially causing rectal polyps to develop into colorectal cancer. Therefore, the compound bezoar composition provided in this invention eliminates the use of free bile acids.

[0144] Bovine bile contains lithocholic acid, as does human bile. Experimental studies have shown that if human factors significantly increase the concentration of lithocholic acid in human blood, it promotes intrahepatic cholestasis and can lead to liver tumor formation. Therefore, the compound bezoar composition provided by this invention eliminates the use of natural bovine bile as a reaction solution to prepare bezoar intermediates, and also specifically avoids the involvement of conjugated lithocholic acid.

[0145] Analysis of natural bezoar revealed that its taurocholic acid content is twice that of glycocholic acid; however, the human body contains twice as much glycocholic acid as taurocholic acid, showing a stark contrast. Experimental studies confirm that the effective substances in bezoar for human use must conform to human physiology. Simply imitating the external form of the effective substances in natural bezoar goes against human physiology. Therefore, the content of different types of bound bile acids in compound bezoar must be adjusted to match the human liver's metabolic mechanism, resulting in higher bioavailability.

[0146] Based on the medicinal and health-preserving properties of bezoar's antioxidants, and considering the current situation of overnutrition in modern people, this invention consciously emphasizes the application of biliverdin and bile acids that conform to the physiological ratio of the human body.

[0147] This invention abandons the existing technologies of magnesium cholate, solid-state rolling coating molding process, and bovine gallbladder skin mucin hydrolysis process. In particular, the solid-state rolling coating molding process is very time-consuming, energy-intensive, and labor-intensive; it is only for obtaining concentric layered textures and oval shapes that have no medicinal effect or pharmacology and are merely superficial.

[0148] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A compound bezoar composition, characterized in that, The product comprises a main agent and excipients, wherein the main agent has a mass content of 94-96%, and the excipients have a mass content of 4-6%; the excipients include taurocholic acid, glycocholic acid, taurodeoxycholic acid, glycodeoxycholic acid, taurochenodeoxycholic acid, and glycochenodeoxycholic acid; the main agent is a bezoar intermediate, which includes bilirubin calcium, biliverdin calcium, bilirubin magnesium, biliverdin magnesium, bilirubin zinc, biliverdin zinc, covalent bilirubin, and glucuronide bilirubin.

2. The compound bezoar composition according to claim 1, characterized in that, The preparation method of the bezoar intermediate includes the following steps: The raw materials for preparing bezoar intermediates are mixed with a biochemical reaction solution to obtain a mixed raw material solution; the mixed raw material solution is subjected to a biochemical reaction to obtain bezoar intermediates; the biochemical reaction solution is sterile purified water, and the indicators of the sterile purified water include: total microbial colony count ≤10 CFU / 100mL, endotoxin content ≤0.25 EU / mL; the raw materials for preparing glucuronide bilirubin include vitamin C, glucuronic acid, bilirubin, and glucuronidase; the raw materials for preparing covalent bilirubin include vitamin C, glycine, Serum albumin and bilirubin, wherein the raw materials for preparing bilirubin calcium include vitamin C, amino acid chelated calcium and bilirubin, the raw materials for preparing biliverdin calcium include vitamin C, amino acid chelated calcium and biliverdin, the raw materials for preparing bilirubin magnesium include vitamin C, magnesium taurate and bilirubin, the raw materials for preparing biliverdin magnesium include vitamin C, magnesium taurate and biliverdin, the raw materials for preparing bilirubin zinc include vitamin C, zinc gluconate and bilirubin, and the raw materials for preparing biliverdin zinc include vitamin C, zinc gluconate and biliverdin.

3. The compound bezoar composition according to claim 2, characterized in that, The sterile purified water is obtained by a combination of online ultraviolet (UV) treatment and in-tank ozone treatment. The conditions for the online UV treatment include: wavelength of 254 nm and intensity ≥ 90 μW / cm². 2 The irradiation time is ≥30 min, and the conditions for the ozone treatment in the tank include: the ozone concentration is 0.5~1.0 mg / L, and the treatment time is ≥60 min; the indicators of the sterile purified water also include: Escherichia coli and Salmonella not detected.

4. The compound bezoar composition according to claim 2 or 3, characterized in that, The bilirubin content in the bilirubin calcium, bilirubin magnesium, bilirubin zinc, covalent bilirubin and glucuronide bilirubin is 75% to 85%, the absorbance of free bilirubin is <0.7, and the water content is less than 9%. The biliverdin content in the biliverdin calcium, biliverdin magnesium, and biliverdin zinc is 73%–83%, the absorbance of free biliverdin is <0.5, and the water content is less than 9%.

5. The compound bezoar composition according to claim 1, characterized in that, The main agent comprises the following components in terms of mass content: Bilirubin calcium 65%, biliverdin calcium 5%, bilirubin magnesium 18%, biliverdin magnesium 3%, covalent bilirubin 5%, bilirubin zinc 1%, biliverdin zinc 1%, glucuronide bilirubin 2%.

6. The compound bezoar composition according to claim 1, characterized in that, The excipient comprises the following components in the indicated mass percentages: taurocholic acid 12.5%, glycocholic acid 25.5%, taurodeoxycholic acid 8.5%, glycodeoxycholic acid 16.5%, taurochenodeoxycholic acid 12.5%, and glycodeoxycholic acid 24.5%.

7. The compound bezoar composition according to claim 2, characterized in that, The raw materials for preparing glucuronide bilirubin include the following components in weight percentage: 1%–3% vitamin C, 20%–25% glucuronic acid, 67%–78% bilirubin, and 1%–5% glucuronidase. The raw materials for preparing the covalent bilirubin include the following components in weight percentage: 1%–3% vitamin C, 10%–15% glycine, 5%–10% serum albumin, and 72%–84% bilirubin; The raw materials for preparing the bilirubin calcium include the following components in weight percentage: 1%–3% vitamin C, 10%–25% amino acid chelated calcium, and 72%–89% bilirubin; The raw materials for preparing the biliverdin calcium include the following components in weight percentage: 1%–3% vitamin C, 10%–25% amino acid chelated calcium, and 72%–89% biliverdin; The raw materials for preparing bilirubin magnesium include the following components in weight percentage: 1%–3% vitamin C, 10%–25% magnesium taurate, and 72%–89% bilirubin; The raw materials for preparing the biliverdin magnesium include the following components in weight percentage: 1%–3% vitamin C, 10%–25% magnesium taurate, and 72%–89% biliverdin; The raw materials for preparing bilirubin zinc include the following components in weight percentage: 1%–3% vitamin C, 10%–25% zinc gluconate and 72%–89% bilirubin; The raw materials for preparing the zinc biliverdin include the following components in weight percentage: 1%–3% vitamin C, 10%–25% zinc gluconate, and 72%–89% biliverdin.

8. The compound bezoar composition according to claim 2 or 7, characterized in that, The temperature of the biochemical reaction is 35℃~40℃.

9. A compound bezoar preparation in the form of medicinal slices, made from the compound bezoar composition according to any one of claims 1 to 8.

10. The method for preparing compound bezoar in the form of medicinal slices according to claim 9, characterized in that, Includes the following steps: The main agent and excipients are mixed to obtain powdered compound bezoar; The powdered compound bezoar is dry-processed into pills to obtain the processed compound bezoar.