In-vitro cultivation of cow-bezoar enriched with curcumin and method of production thereof

Abstract

The present application relates to the in vitro cultivation of curcumin-rich bovine bezoar and its production method, comprising the following steps: under stirring conditions, adding tetrahydrocurcumin, curcumin, protease and hepatocyte extract to fresh animal bile to obtain reaction liquid A after digestion reaction; adding calcium hydroxide to reaction liquid A, precipitate appears after heating reaction, cooling and standing, removing supernatant to obtain cholegallus precipitate liquid; cholegallus precipitate liquid includes pig cholegallus precipitate liquid and bovine cholegallus precipitate liquid; the pig cholegallus precipitate liquid is sequentially subjected to first acidification, filtration, drying and crushing to obtain glue powder; adding bilirubin and glue powder to bovine cholegallus precipitate liquid, uniformly mixing, then second acidification, stirring reaction until the appearance of clumpy coagulum, separating the clumpy coagulum and drying to obtain the in vitro cultivation of curcumin-rich bovine bezoar. The properties, cholic acid content and bilirubin content of the bovine bezoar obtained by the present application meet the relevant standards of Chinese Pharmacopoeia, and the preparation method is simple.

Description

Technical Field

[0001] This invention relates to the field of in vitro cultured bezoar, specifically to in vitro cultured bezoar rich in curcumin and its production method. Background Technology

[0002] Bezoar is a precious traditional Chinese medicine. Among the thousands of traditional Chinese medicine formulas, about 600 require bezoar, including some extremely valuable ones like "Angong Niuhuang Wan". This has made bezoar one of the most precious and scarce traditional Chinese medicines. Currently, the market price of natural bezoar has reached 750,000 yuan / kg, and it is still in short supply.

[0003] To alleviate this situation, domestic researchers have long been engaged in research on in vitro production methods for bezoar. These methods mainly include in vitro fermentation for producing cultured bezoar and artificial formulations of bezoar using bilirubin and bile acids as the main components. However, these methods have the following drawbacks: 1. Artificially formulated bezoar may lack unknown components from bile, failing to fully exert the pharmacological effects of natural bezoar. 2. The production process requires repeated, high-dose additions of chemical reducing agents (sodium bisulfite or sodium borohydride), which, if left in the bezoar product, can pose certain health risks and hinder the application of the technology. 3. In vitro fermentation for bezoar production involves excessively long in vitro culture times, typically two to three days, and the growth of harmful bacteria during this process is highly likely, significantly reducing the success rate of production and impacting the health of production personnel. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned technical deficiencies and provide an in vitro cultured bezoar rich in curcumin and its production method, solving the technical problems in the prior art of in vitro bezoar production that require repeated addition of large doses of chemical reducing agents or excessively long fermentation time, which easily leads to the generation of harmful bacteria.

[0005] To achieve the above-mentioned technical objectives, the technical solution provided by this invention is as follows:

[0006] In a first aspect, the present invention provides a method for producing in vitro cultured bezoar rich in curcumin, comprising the following steps: under stirring conditions, adding tetrahydrocurcumin, curcumin, protease and hepatocyte extract to fresh animal bile, and obtaining reaction solution A through digestion reaction; adding calcium hydroxide to reaction solution A, heating the reaction to produce a precipitate, cooling and allowing it to stand, removing the supernatant to obtain bile precipitate; the bile precipitate includes porcine bile precipitate and bovine bile precipitate; subjecting the porcine bile precipitate to a first acidification, filtration, drying and pulverization in sequence to obtain a colloidal powder; adding bilirubin and colloidal powder to the bovine bile precipitate, wherein the ratio of bovine bile precipitate, bilirubin and colloidal powder is 100mL:(2-3)g:(1-2)g; mixing evenly and then subjecting to a second acidification, stirring the reaction until clumps appear, separating the clumps and drying to obtain in vitro cultured bezoar rich in curcumin.

[0007] Secondly, the present invention provides an in vitro cultured bezoar rich in curcumin obtained by the above-mentioned production method.

[0008] Compared with the prior art, the beneficial effects of the present invention include:

[0009] This invention prepares a gelling agent powder, then adds bilirubin and the gelling agent powder to bovine bile precipitate to obtain the desired in vitro cultured bezoar. The properties, bile acid content, and bilirubin content of the obtained bezoar meet the relevant standards of the Chinese Pharmacopoeia. The preparation method of this invention is simple and avoids the disadvantages of fermentation methods, such as the need for long fermentation time and the easy generation of harmful bacteria. The addition of tetrahydrocurcumin and curcumin during the cultivation process effectively protects the main active ingredient bilirubin of bezoar during processing and subsequent applications. At the same time, the curcumin works synergistically with bilirubin to enhance pharmacological functions, resulting in good and long-lasting heat-clearing and cooling effects, anti-immune stress, and anti-oxidative stress effects. Detailed Implementation

[0010] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0011] In a first aspect, the present invention provides a method for producing in vitro cultured bezoar rich in curcumin, comprising the following steps:

[0012] 1) Preparation of bezoar colloid powder:

[0013] ① Extraction of bile flavonoids from pig bile:

[0014] A. Reagent weighing and preparation: Weigh 0.01–1% of protease, 0.005–0.05% of tetrahydrocurcumin, 0.01–0.08% of curcumin and 0.2–0.6% of calcium hydroxide based on the weight (g) of each raw material and the total volume (mL) of bile.

[0015] Since the density of bile is close to 1, the total weight and total volume of bile are equal. To avoid redundancy, the following text uses percentages to indicate the amount of each ingredient added relative to bile. For example, 0.01 to 1 g of protease is added to 100 mL of bile.

[0016] Preferably, the amount of protease used is 0.05-0.5% of the total bile volume, and most preferably 0.1%, which saves costs while ensuring rapid digestion.

[0017] Preferably, the protease is an alkaline protease, trypsin, or bromelain.

[0018] In some specific embodiments, when trypsin is used as the protease, 0.001% to 0.05% of EDTA (ethylenediaminetetraacetic acid) of the total bile volume needs to be added, preferably 0.01% to 0.03%; EDTA complexes with metal ions such as calcium in bile to avoid affecting the activity of trypsin.

[0019] In some specific embodiments, the trypsin activity units are 1:250.

[0020] In some specific embodiments, since the amount of protease added is small, it is difficult to mix evenly if added directly in industrial production. The protease and EDTA can be mixed with 50 to 100 times the weight of water to form a concentrated solution, and then the concentrated solution can be added to the bile to facilitate even mixing.

[0021] Preferably, the amount of tetrahydrocurcumin is 0.01-0.03% of the total bile volume; the amount of curcumin is 0.02-0.04% of the total bile volume; more preferably, the amount of tetrahydrocurcumin is 0.02% of the total bile volume; and the amount of curcumin is 0.03% of the total bile volume.

[0022] Preferably, the amount of calcium hydroxide used is 0.2-0.5% of the total volume of bile; more preferably, it is 0.3%.

[0023] Preparation of hepatocyte extract: Take fresh animal liver, homogenize it in a homogenizer, filter it with gauze, and freeze and thaw the filtrate repeatedly more than 3 times. After the last freeze-thaw, add physiological saline to the frozen and thawed mixture and mix well. Centrifuge at 4000 rpm for 10 minutes and collect the supernatant. This is the hepatocyte extract.

[0024] Preferably, the animal liver includes the liver of a pig or a cow; more preferably, it is the liver of a newborn calf or a fetal calf.

[0025] Preferably, the volume of added physiological saline is 2 to 5 times the volume of the frozen and thawed material, and more preferably 2 to 4 times.

[0026] C. Digestion of porcine bile protein and addition of curcumin and other raw materials: Place the reaction vessel containing porcine bile in a water bath, and add tetrahydrocurcumin, curcumin and protease in sequence under stirring; then add liver cell extract and carry out the digestion reaction for more than 2 hours.

[0027] Preferably, the amount of hepatocyte extract used is 2% to 3% of the total mass of bile.

[0028] Preferably, the water bath temperature and the digestion reaction temperature are both 30-40°C, more preferably 35-37°C; the digestion reaction time is preferably 1-5 hours, more preferably 2-3 hours.

[0029] D. Formation and Extraction of Porcine Bile Yellow: Under stirring, add weighed calcium hydroxide to the above reaction system and heat the reaction system to above 90°C for at least 20 minutes. At this time, a yellow precipitate appears in the reaction system. Stop stirring, remove the reaction vessel from the water bath, and let it stand for at least 24 hours. Siphon off the supernatant, leaving the lower yellow part (remove as much liquid as possible from the top of the yellow part). The lower yellow part is the porcine bile yellow precipitate.

[0030] Preferably, the heating reaction temperature is 90–100°C, and the reaction time is 10–30 min.

[0031] ② Acidification of pig bile flavonoids

[0032] Hydrochloric acid solution is added dropwise to the prepared porcine bile precipitate while stirring rapidly until the pH drops below 2 (preferably pH 1-2). At this point, extremely viscous clumps appear in the porcine bile precipitate, and a clear liquid precipitates out.

[0033] ③Chloroform extraction: Discard the clear liquid, add 5-15% of the original bile volume of chloroform to the remaining viscous substance, and use two glass rods to continuously tear the clot to make it contact the chloroform as much as possible.

[0034] Filtration: After 3 minutes, filter with gauze (or other filter cloth that is not dissolved in chloroform) to obtain a viscous clot, which is then transferred to a dry container.

[0035] ④ Drying: Place the filtered viscous clump into a vacuum dryer and dry it at 50°C.

[0036] ⑤ Pulverization: The vacuum-dried lumps are pulverized into fine powder using an ultra-micro pulverizer. This is the bezoar stone aggregate powder.

[0037] Preferably, the concentration of hydrochloric acid solution used in this invention is 8-15 wt%. Too low a concentration will result in slow pH adjustment and an excessively large total volume of hydrochloric acid solution added, while too high a concentration is not conducive to pH control.

[0038] Preferably, in this invention, filtration is performed using a 60-100 mesh filter cloth, the material of which is cotton or other fibrous material that is not dissolved by chloroform; the material remaining on the sieve is collected.

[0039] Verification of the crosslinking powder: Take a small amount of crosslinking powder, add a few drops of water, and stir quickly with a glass rod. It will immediately agglomerate into a very viscous, thick paste-like mass. When there are other powdery substances (such as flour, bezoar powder, etc.) around the mass, it can tightly adhere these powdery substances to itself, proving that the crosslinking powder has been successfully prepared.

[0040] 2) Preparation of bovine bile flavonoids:

[0041] Following the method described above for preparing porcine bile precipitate, bovine bile precipitate was prepared by replacing porcine bile with bovine bile.

[0042] 3) Adding bilirubin and coagulating powder causes it to coagulate into a yellow color:

[0043] Weigh out bilirubin at a ratio of 2-4% (by weight / volume, i.e., bilirubin precipitate: bilirubin = 100mL: (2-4)g, hereinafter abbreviated as percentage) and weigh out the colloidal powder at a ratio of 1-5% (by weight / volume). Mix the weighed bilirubin and colloidal powder evenly and add them to the bilirubin precipitate. Stir the liquid at 500 rpm to disperse the additives as quickly as possible. Then, reduce the stirring speed to below 100 rpm and adjust the pH of the bilirubin precipitate to below 5 with hydrochloric acid solution. Continue stirring until clumps of varying sizes appear in the reaction system. Collect these clumps and vacuum dry them to obtain "biological bezoar" rich in curcumin.

[0044] Preferably, the pH of the bovine bile precipitate is adjusted to 4-5 using hydrochloric acid solution.

[0045] Preferably, after stirring for 6 to 12 hours, the clumps are collected.

[0046] Preferably, the adhesive powder is added at a ratio of 1-2% (by weight and volume); and bilirubin is added at a ratio of 2-3%.

[0047] Secondly, this invention provides an in vitro cultured bezoar rich in curcumin, containing 0.005% to 0.015% curcumin. Throughout the production, processing, and in vivo efficacy of bezoar, curcumin consistently plays a protective and mutually reinforcing role against the core active ingredient of bezoar—bilirubin.

[0048] The main mechanism of action and advantages of this invention:

[0049] (1) In this invention, tetrahydrocurcumin and curcumin are added during the in vitro culture of bezoar. On the one hand, tetrahydrocurcumin has a strong reducing property, which can better protect the main active ingredient of bezoar - bilirubin during the processing and subsequent application. On the other hand, curcumin has the same pharmacological effects as bilirubin (such as antioxidant function, liver protection function, etc.). The combination of these two drugs can not only play a mutual protective role, but also enhance their pharmacological functions.

[0050] (2) The present invention extracts the effective components from bovine bile, then prepares the colloidal powder from pig bile, and then adds bilirubin and colloidal powder to bovine bile precipitate. The resulting product has bovine bile-related characteristics.

[0051] (3) The overall preparation time of this invention is short, avoiding the disadvantages of fermentation method which requires a long fermentation time and is prone to producing harmful bacteria.

[0052] (4) The present invention uses a high rotation speed when adding raw materials to bile to make the raw materials mix quickly and evenly, and then immediately reduces the rotation speed to reduce aeration and avoid oxidation of effective components in bile.

[0053] The present invention will be further described in detail below through specific embodiments.

[0054] Example 1

[0055] (1) Preparation of the adhesive powder

[0056] 3000 ml of fresh pig bile was poured into a 4000 ml beaker. The beaker was placed in a 37°C water bath, and a stirrer was inserted. While stirring at 500 rpm, 0.02% tetrahydrocurcumin, 0.03% curcumin, 0.1% trypsin, 0.02% EDTA, and 2% calf liver cell extract were added to the pig bile in sequence. After the addition was complete, the stirring speed was reduced to 100 rpm. The mixture was kept warm and stirred for 2 hours. Add calcium hydroxide (0.3% of the total bile volume) to the bile, and while stirring, raise the water bath temperature to 70°C and hold for 30 minutes. Then, adjust the water bath temperature to 95°C. Start timing when the bile temperature exceeds 90°C. Stop stirring after 20 minutes, remove the beaker from the water bath, and let it stand overnight. A yellow precipitate will appear in the bile; this is the porcine bile precipitate. Siphon off the upper layer of liquid, and add 1:2 hydrochloric acid (37% hydrochloric acid diluted with twice the amount of water) dropwise to the remaining yellow liquid while stirring continuously and frequently measuring the pH value. When the pH value drops to around 3, small curd-like clumps will float on the surface of the liquid. Increase the stirring speed at this point. Increase the frequency of pH detection. When the pH value drops below 2, almost all solids in the bile participate in clot formation, resulting in large clots in the bile and the precipitation of clear liquid. Discard the clear liquid and add 10% (300 ml) of the original fresh pig bile volume (to facilitate drying and grinding into powder) to the remaining clots. Quickly tear the clots with a glass rod for 3 minutes and filter with gauze. The clots remain on the filter cloth. Collect the clots in a container and place them in a vacuum drying oven at about 50°C. After drying, grind them into powder in a mortar (or perform ultra-fine grinding). This is the colloidal powder (the filtrate can be used for distillation to recover chloroform and extract bilirubin).

[0057] On average, about 8 grams of adhesive powder are obtained from every 1000 ml of pig bile.

[0058] The preparation of calf liver cell extract includes:

[0059] After slaughter, the liver of newborn male calves is removed, weighed, and cut into small pieces. The pieces are then placed in a tissue homogenizer and homogenized for approximately 3 minutes until a paste-like consistency free of visible lumps is formed. The homogenate is then transferred to a glass beaker and subjected to a three-stage freeze-thaw cycle (each freeze-thaw cycle must be thorough). After the final thawing, physiological saline is added at three times the volume of the frozen-thawed mixture. The mixture is stirred thoroughly, allowed to stand for 5 minutes, and the supernatant is collected. This supernatant is the calf liver cell extract.

[0060] (2) Preparation of ox bile precipitate

[0061] 1000 ml of fresh ox bile was poured into a 2000 ml beaker and placed in a water bath at 37°C. Under rapid stirring (500 rpm), 0.02% (w / v) of tetrahydrocurcumin, 0.03% of curcumin, 0.1% of trypsin (activity unit 1:250), and 0.02% of EDTA were added sequentially. After all four reagents were added and thoroughly mixed, the stirrer speed was reduced to slow (100-150 rpm) and stirred continuously for 2 hours. Then add calcium hydroxide at 0.3% of the total bile volume, continue stirring, and adjust the water bath temperature to 70°C. Maintain the bile temperature at 70°C for 30 minutes, then raise the water bath temperature to above 95°C. Start timing when the bile temperature reaches above 90°C. The reaction will stop after 20 minutes. Remove the beaker from the water bath and allow it to cool naturally. Let it stand for at least 24 hours, then siphon off the supernatant. The lower yellow precipitate is the ox bile precipitate.

[0062] Approximately 200 ml of ox bile precipitate solution can be obtained from every 1000 ml.

[0063] (3) Preparation of in vitro cultured bezoar products rich in curcumin

[0064] Weigh out bilirubin at a ratio of 2% (weight / volume percentage) of the total amount of the yellow precipitate obtained in step (2), and weigh out the colloidal powder obtained in step (1) at a ratio of 3%. Mix the two evenly and add them to the bovine bile precipitate solution from step (2). At the beginning of the addition, stir the liquid at a speed of 500 rpm. After 1 minute of addition, reduce the stirring speed to below 100 rpm. Adjust the pH of the yellow precipitate solution to between 5 and 4 with hydrochloric acid solution and continue stirring for 8 hours. At this time, clumps of varying sizes appear in the reaction system. Collect these clumps and vacuum dry them to obtain in vitro cultured bovine bile rich in curcumin.

[0065] 13 grams of cultured bezoar were obtained from 1000 ml of ox bile (some sediment could not be incorporated into the cultured bezoar and could be recovered by centrifugation for other uses).

[0066] The obtained in vitro cultured bezoar was tested:

[0067] 1. Appearance: Smooth surface, irregular shape, dark yellow color, brittle texture, and layered cross-section.

[0068] 2. Properties: Tested according to the "In Vitro Cultured Bezoar" standard of the 2020 edition of the Chinese Pharmacopoeia, it meets the requirements of the standard.

[0069] 3. Bile acid content: According to the 2020 edition of the Chinese Pharmacopoeia for "in vitro cultured bezoar", its bile acid content reached 8.4%.

[0070] 4. Bilirubin content: According to the 2020 edition of the Chinese Pharmacopoeia for "in vitro cultured bezoar", its bilirubin content reached 36.24%.

[0071] 5. Curcumin content: According to the method of "National Food Safety Standard for Food Additives Curcumin GB 1886", the curcumin content is 0.01%.

[0072] Example 2

[0073] The operation was carried out according to step (2) of Example 1, except that the amount of curcumin was adjusted to 0 (blank group A, without curcumin and tetrahydrocurcumin), 0.01%, 0.02%, 0.03%, 0.04%, 0.05% and 0.1%, respectively, without adding tetrahydrocurcumin.

[0074] Meanwhile, a standard operating control group A (using only 1% NaHSO3) was set up, which added 1% sodium bisulfite but did not add curcumin or tetrahydrocurcumin.

[0075] The obtained bovine bile precipitate was centrifuged at 4000 rpm for 10 minutes, and the centrifuged product was vacuum dried at 50℃. The total amount of each group of harvested products and the bilirubin content were determined, and the bilirubin retention amount (the product of the total amount of harvested products and the bilirubin content) was calculated to observe the protective effect of curcumin on the inherent bilirubin in bile during the extraction of bovine bile. The results are shown in Table 1.

[0076] Table 1. Test results of the harvest obtained in Example 2

[0077]

[0078] Table 1 shows that adding curcumin has a certain protective effect on bilirubin during bovine bile extraction, with an addition of 0.03% being the most economical and effective. However, its protective effect is not as strong as that of 1% sodium bisulfite.

[0079] Example 3

[0080] The only difference from Example 2 is that the amount of curcumin added was fixed at 0.03%, and the amount of tetrahydrocurcumin was adjusted to 0 (blank group B, containing 0.03% curcumin, without tetrahydrocurcumin), 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, and 0.1%. At the same time, a control group B with 1% sodium bisulfite and no curcumin or tetrahydrocurcumin was set up. Other steps and conditions were the same as in Example 2. The bilirubin content in the harvest of each group was measured to observe the protective effect of the combined use of curcumin and tetrahydrocurcumin on the intrinsic bilirubin in bile. The results are shown in Table 2.

[0081] Table 2. Test results of the harvest obtained in Example 3

[0082]

[0083] Table 2 shows that adding tetrahydrocurcumin to curcumin enhances the protective effect of bilirubin during processing. The combination of 0.03% curcumin and 0.02% tetrahydrocurcumin provides the strongest bilirubin protection, even surpassing that of 1% sodium bisulfite.

[0084] Test Example 1

[0085] The same preparation steps and conditions as in Example 1 (0.03% curcumin + 0.02% tetrahydrocurcumin) were used as test group C; in addition to test group C, blank group C (without curcumin and tetrahydrocurcumin) and control group C (with 1% sodium bisulfite and without curcumin and tetrahydrocurcumin) were set up.

[0086] Equal weights (4 g) of exogenous bilirubin and equal weights of coagulating powder were added to the bilirubin precipitates obtained from the three groups to prepare in vitro cultured bezoar, in order to observe the protective effect of the combined addition of tetrahydrocurcumin and curcumin on total bilirubin during the production of in vitro cultured bezoar. The results are shown in Table 3.

[0087] Table 3. Effects of combined addition of tetrahydrocurcumin and curcumin on total bilirubin.

[0088]

[0089] Table 3 illustrates that when curcumin and tetrahydrocurcumin are added at ratios of 0.03% and 0.02% respectively in the in vitro cultured bezoar preparation using the technology of this invention, they not only have a good protective effect on the intrinsic bilirubin in bile, but also have a good protective effect on the added exogenous bilirubin, and their effect is stronger than that of 1% sodium bisulfite.

[0090] Furthermore, as can be seen from Example 1 and Test Group C, the test results of the in vitro cultured bezoar prepared under the same conditions in this invention are very similar, indicating that it has reproducibility and good batch stability.

[0091] When scaled up production under the same conditions as in Example 1, it was found that the product yield increased by about 7-8%. It was verified that the bilirubin precipitate in step (2) was significantly lost during the small-scale test, which shows that the present invention is beneficial for scaled-up production.

[0092] Test Example 2

[0093] Twelve weaned piglets weighing approximately 6 kg were purchased from a pig farm and randomly divided into four groups of three piglets each. Group 1 served as a blank control group with conventional feeding. On day 8 of the experiment, pigs in groups 2, 3, and 4 were injected intraperitoneally with LPS 100 ug / kg body weight (artificially creating an immune stress model). Timing was started after the injection, and body temperature was measured at 0, 3, 6, 9, 12, 15, 18, 21, and 24 hours. Six hours after the LPS injection, pigs in group 3 were administered 0.3 g of in vitro cultured bezoar prepared in Example 1 of this invention by gavage. Six hours after the LPS injection, pigs in group 4 were administered 0.3 g of in vitro cultured bezoar prepared by fermentation without curcumin by gavage. Twenty-four hours after the administration, blood was collected from all participating pigs (with anticoagulant added to the blood collection tubes). The plasma was immediately centrifuged and separated, and then biochemical indicators related to immune stress and oxidative stress in the plasma were detected. The results are shown in Tables 4 and 5.

[0094] Table 4. Body temperature (°C) of pigs in each experimental group

[0095]

[0096]

[0097] Table 4 illustrates that: 1. The in vitro cultured bezoar prepared using this invention has a good heat-clearing and fever-reducing effect. 2. Compared with in vitro cultured bezoar without curcumin, the bezoar prepared using this invention has a more prolonged heat-clearing and fever-reducing effect.

[0098] Table 5 Biochemical indicators of pigs in each experimental group

[0099]

[0100] Table 5 explains: 1. When piglets are stimulated by LPS, their interleukin-1β, interleukin-6, and tumor necrosis factor-α levels are significantly increased. ɑ The extremely significant increase in these levels indicates that piglets are under immune stress. Both types of in vitro cultured bezoar significantly restored the levels of these cytokines, indicating that both the in vitro cultured bezoar produced using the technology of this invention have good anti-immune stress and anti-oxidative stress effects. 2. When piglets are stimulated by LPS, the levels of glutathione peroxidase and superoxide dismutase in their plasma decrease significantly, while the level of malondialdehyde increases significantly, indicating that piglets are under oxidative stress. Gavage administration of both types of in vitro cultured bezoar significantly restored the levels of these substances, indicating that they both have good anti-oxidative stress effects. The in vitro cultured bezoar prepared by this invention has a stronger ability to restore these indicators, indicating that its anti-oxidative stress effect is stronger.

[0101] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for producing in vitro cultured bezoar rich in curcumin, characterized in that, Includes the following steps: Under stirring conditions, tetrahydrocurcumin, curcumin, protease, and hepatocyte extract were added to fresh animal bile, and reaction solution A was obtained after digestion. Calcium hydroxide was added to reaction solution A, and after heating, a precipitate appeared. After cooling and settling, the supernatant was removed to obtain bile precipitate. The bile precipitate included porcine bile precipitate and bovine bile precipitate. The porcine bile precipitate was subjected to a first acidification, filtration, drying and pulverization in sequence to obtain a colloidal powder; Bilirubin and coagulant powder were added to the bovine bile precipitate, wherein the ratio of bovine bile precipitate, bilirubin and coagulant powder was 100 mL: (2-4) g: (1-5) g; after mixing evenly, a second acidification was carried out, and the reaction was stirred until clumps appeared. The clumps were separated and dried to obtain in vitro cultured bovine gallstone rich in curcumin. The protease used is alkaline protease, trypsin, or bromelain; and when trypsin is used, ethylenediaminetetraacetic acid (EDTA) must be added simultaneously, and the ratio of fresh animal bile to EDTA is 100 mL: (0.001–0.05) g. The first acidification involves adding hydrochloric acid solution until the pH of the porcine bile precipitate is below 2, and then adding 5-15% of the volume of fresh animal bile in chloroform for extraction. The second acidification involves adding hydrochloric acid solution until the pH of the bovine bile precipitate drops below 5.

2. The method for producing in vitro cultured bezoar rich in curcumin according to claim 1, characterized in that, The specific steps for preparing hepatocyte extract include: homogenizing fresh animal liver, filtering, and repeatedly freezing and thawing. After the final freeze-thaw cycle, the supernatant is obtained by centrifugation to obtain the hepatocyte extract.

3. The method for producing in vitro cultured bezoar rich in curcumin according to claim 1, characterized in that, The ratio of fresh animal bile, tetrahydrocurcumin, curcumin, protease and hepatocyte extract was 100 mL: (0.01-0.03) g: (0.02-0.04) g: (0.01-1) g: (2-3) g; the ratio of fresh animal bile and calcium hydroxide was 100 mL: (0.2-0.6) g.

4. The method for producing in vitro cultured bezoar rich in curcumin according to claim 1, characterized in that, The digestion reaction is carried out at 30-40℃ for 1-5 hours.

5. The method for producing in vitro cultured bezoar rich in curcumin according to claim 1, characterized in that, The heating reaction is carried out at 90–100°C for 10–30 minutes.

6. The method for producing in vitro cultured bezoar rich in curcumin according to claim 1, characterized in that, The filter uses 60-100 mesh filter cloth.

7. The method for producing in vitro cultured bezoar rich in curcumin according to claim 1, characterized in that, After stirring and reacting for 6 to 12 hours, separate the clumps.

8. Curcumin-rich in vitro cultured bezoar obtained by the production method according to any one of claims 1-7.

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