Licorice charcoal coating material for reducing drug hepatotoxicity, its manufacturing method, and applications.

Abstract

Disclosed are a licorice carbon coating material for reducing the hepatotoxicity of drugs, a method for producing the same, and uses thereof. The coating material is licorice carbon, and the method for producing licorice carbon is obtained by pulverizing licorice slices, sieving, stir-frying over high heat for 1-2 hours, spraying clear water, sealing and cooling. The yield of licorice carbon prepared by this method is about 45%, which is about 10% higher than the highest yield reported in the existing reports. The obtained licorice carbon is a black or grayish-black powder, with a slight fragrance, a light and slightly sweet taste, less than 3% impurities, and less than 10% moisture. The licorice carbon prepared by this method can effectively reduce the hepatotoxicity of drugs, be used as a coating for drug-induced hepatotoxic drugs, reduce liver damage after patients take medicine, and has the potential for wide application.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims the rights of Chinese application number 2022106390547, filed on June 7, 2022. The said application number 2022106390547 is hereby incorporated in its entirety by reference.

[0002] (Technical field) This invention belongs to the pharmaceutical field and, in particular, relates to a licorice charcoal coating material that reduces the hepatotoxicity of drugs, as well as its manufacturing method and applications. [Background technology]

[0003] Traditional Chinese medicine charcoal is one of the drugs with very distinctive preparation characteristics. There are two traditional methods of charcoal production: stir-frying and charring. These methods involve placing the prepared product in a pan and stir-frying it until the surface is charred black and the inside is dark brown, or heating it to a specified degree. During charcoal production, it is necessary to "preserve its properties," prevent ash formation, and avoid re-ignition. Traditional Chinese medicine charcoal has clinical effects such as hemostasis, antidiarrheal, hypoglycemia, analgesia, liver protection, anti-inflammatory, and anti-ulcer effects, and there are currently more than 70 types of charcoal used clinically. Licorice charcoal is a processed product of the dried roots and rhizomes of the legume plant licorice, licorice root, or licorice root. Its essence is carbonized licorice and it is widely used as a surface coating for traditional Chinese medicine formulas. Zhuanggu Jianzhen Wan and Antibacterial Zensheng Wan, listed in the 2020 edition of the "Pharmacopoeia of the People's Republic of China," are manufactured with licorice charcoal coating, and the resulting formulas have a uniform color, a glossy finish, and are resistant to deterioration. At the same time, licorice has a harmonizing effect with other drugs and does not affect the efficacy of the formulation when used in coating. Recent studies have identified and analyzed nano-components isolated and purified from charcoal drugs using a combination of representation techniques such as transmission electron microscopy and Fourier transform infrared spectroscopy, along with pharmacological activity experiments. The results have shown that charcoal nano-components are the material basis for the pharmacological effects of the corresponding charcoal medicinal materials. Nano-components have also been discovered in licorice charcoal, and their anti-ulcer activity has been demonstrated using a mouse model of acute alcoholic gastric ulcer (see, for example, Zhu Yafan, "A Study of the Original Form of Zhongjing's 'Roasted' Method and the Anti-Ulcer Activity of Roasted Licorice [D]. Beijing University of Traditional Chinese Medicine, 2019."). Further investigation into the pharmacological activity of licorice charcoal is still needed.

[0004] With the widespread use of traditional Chinese medicine (TCM) both domestically and internationally, safety issues and incidents related to TCM, particularly liver damage, have been frequently reported in recent years. Many TCMs traditionally considered non-toxic have been reported to cause liver damage, with Polygonum multiflorum (He Shou Wu) being a particularly prominent example. The Chinese Pharmacopoeia has repeatedly issued warnings and regulatory notices regarding liver damage from preparations containing Polygonum multiflorum, including Yangxue Shengfa capsules, Shou Wu Yanshou tablets, and Shou Wu Yanshou granules. As the application of TCM becomes more widespread and people's understanding of TCM deepens, new safety issues related to TCM are likely to be discovered one after another. By reducing hepatotoxicity from TCM while ensuring clinical efficacy, minimizing harm while prioritizing benefits, and minimizing the risks of clinical use, safety issues related to TCM can be effectively resolved. Currently, the main methods for reducing hepatotoxicity from TCM are detoxification through preparation and detoxification through combination. For example, the traditional preparation method of Polygonum multiflorum, "nine steaming and nine sun drying," and the modern mainstream methods of steaming or steaming with black bean paste, can both reduce the hepatotoxicity of Polygonum multiflorum. In clinical practice, Polygonum multiflorum is mainly used in combination with Poria cocos and Glycyrrhiza uralensis to reduce detoxification. Studies investigating the detoxification effects of combinations of Polygonum multiflorum, Poria cocos, Glycyrrhiza uralensis, and Panax notoginseng showed that Poria cocos, Glycyrrhiza uralensis, and Panax notoginseng all exhibited significant detoxification effects, and these effects were dose-dependent. Among these, Poria cocos showed the most pronounced detoxification effect. However, the formulations of traditional Chinese medicines, including the combination of herbs and the preparation of the ingredients, are fixed, and changing them will affect the clinical effect.

[0005] Therefore, there is a lack of technology that effectively reduces the hepatotoxicity of drugs, is applicable to the majority of conventional medicines, and does not affect their efficacy. [Overview of the Initiative]

[0006] To solve the above technical problems, the present invention provides a licorice charcoal coating material that reduces the hepatotoxicity of drugs, as well as a method for producing the same and its applications.

[0007] Specifically, the present invention was realized by the following technical means.

[0008] In its first aspect, the present invention provides a method for producing a licorice charcoal coated material that reduces the hepatotoxicity of drugs, comprising the following steps:

[0009] The licorice extract is crushed and sieved through a 1.5-2.0 cm sieve. The start temperature of the frying machine is set to 120°C-160°C, the heating temperature to 250-350°C, and the heating time to 1.0-2.0 hours. The licorice is fried until the surface turns black and the inside turns yellow. When the smoke becomes thick, the heating is stopped, a small amount of clean water is sprayed to extinguish the sparks, and after the frying machine has cooled to room temperature, the fried licorice is removed, dried, crushed, and sieved through a No. 6 sieve to obtain the licorice charcoal coating material.

[0010] As a selective method, in the above manufacturing method, the licorice extract is a licorice extract that conforms to the quality standards requirements of the "Pharmacopoeia of the People's Republic of China".

[0011] As a selective method, in the above manufacturing method, the licorice extract is a processed product of the dried roots and rhizomes of licorice, saccharomyces uralensis, or licorice root, all belonging to the legume family.

[0012] As a selective method, the above manufacturing method includes the following steps:

[0013] The licorice extract is crushed and sieved through a 1.6 cm sieve. The start temperature of the frying machine is set to 150°C, the heating temperature to 300°C, and the heating time to 1.5 hours. The licorice is fried until the surface turns black and the inside turns yellow. When the smoke becomes thick, the heating is stopped, a small amount of clean water is sprayed to extinguish the sparks, and after waiting for the frying machine to cool to room temperature, the fried licorice is removed, dried, crushed, and sieved through a No. 6 sieve to obtain the licorice charcoal coating material.

[0014] In a second aspect, the present invention provides a licorice charcoal coating material that reduces the hepatotoxicity of drugs obtained using the manufacturing method described in the first aspect above. This licorice charcoal coating material is a black or grayish-black powder with a faint aroma, a mild and slightly sweet taste, and contains less than 3% impurities and less than 10% moisture by weight.

[0015] In a third aspect, the present invention provides the use of the licorice carbon coating material described in the second aspect above for the surface coating of water pills of traditional Chinese medicine preparations. The water pills coated with the licorice carbon coating material have uniform color, luster, and are less likely to deteriorate.

[0016] In a fourth aspect, the present invention provides the use of the licorice carbon coating material described in the second aspect above for the production of a coating material that reduces the hepatotoxicity of drugs.

[0017] As a selective approach, in the above use, the drug is a drug having drug-induced hepatotoxicity.

[0018] As a selective approach, in the above use, the drug having drug-induced hepatotoxicity is Polygonum multiflorum, Tripterygium wilfordii, Aristolochia fangchi or Psoralea corylifolia.

[0019] The present invention has the following beneficial effects over the prior art.

[0020] (1) The production method of the present invention improves the yield of licorice carbon by about 10%. The currently reported yield of the production method of licorice carbon is at most 35%, and the yield of the production process is low, leaving room for improvement (see: CN201810717845.0). The yield of licorice carbon produced by the method of the present invention is about 45%, which is about 10% higher than the yield of the conventional reports.

[0021] (2) There is little research on the pharmacological activity of licorice carbon. As a very characteristic drug in the preparation of traditional Chinese medicine carbon drugs, its pharmacological activity has great potential for further exploration. The present invention discovers for the first time that the licorice carbon produced by the method of the present invention has the effect of reducing the hepatotoxicity of drugs. Furthermore, by using this as a coating for drugs having drug-induced hepatotoxicity, it can reduce liver damage after patients take the medicine and has a broad application prospect.

Embodiments for Carrying Out the Invention

[0022] The present invention will be further described based on specific examples below. It should be understood that the specific examples described here are only used for the description of the present invention and do not limit the scope of the present invention.

[0023] When specific techniques and conditions are not described in the examples, the techniques and conditions described in the literature of the relevant field or the instructions of the product shall be followed. When the manufacturer of the reagents and equipment used is not described, they are common products that can be purchased through regular channels.

[0024] The experimental methods in the following examples are all common methods unless otherwise specified. The test materials used in the following examples are commercially available products unless otherwise specified.

[0025] Manufacturing Example: The manufacturing process of the licorice carbon coating material for reducing the hepatotoxicity of the drug of the present invention is as follows. That is, licorice slices are pulverized, sieved through a 1.6 cm sieve, the starting temperature of the frying machine is set at 150 °C, the heating temperature is set at 300 °C, the heating time is set at 1.5 hours. When the surface becomes charred black and the inner surface becomes charred yellow and the smoke becomes thick, the heating is terminated, a small amount of clear water is sprayed to extinguish the sparks, and after waiting for the frying machine to cool to room temperature, the fried licorice is taken out, dried, pulverized, and sieved through a No. 6 sieve to obtain the licorice carbon coating material.

[0026] The production of licorice carbon was carried out according to the production method of the present invention, and the production situation is summarized below.

Table 1

[0027] The above data indicates that the preparation technology of licorice carbon is mature and can meet the production demand.

[0028] The produced licorice carbon should meet the following requirements. Particle size: All should be sieved through a No. 5 sieve, and those sieved through a No. 6 sieve should be 98% or more. Impurities must not exceed 3% (General Rule 2301, 2020 edition of the "Chinese Pharmacopoeia"). Testing method: 1. Take an appropriate amount of the test sample, spread it out, and observe it with the naked eye or using a magnifying glass (5-10x) to remove impurities. If there are sieveable impurities, separate them using an appropriate sieve. 2. Weigh each type of impurity and calculate its content (%) in the test sample. The moisture content must not exceed 10.0% (General Rule 0832, Method 2, 2020 Edition, Chinese Pharmacopoeia). Hot air drying method: Take 25g of the test sample. If the diameter or length of the test sample exceeds 3mm, quickly process it into particles or fragments no larger than 3mm in diameter or length before weighing. Spread it flat in a dry, constant-weight flat weighing bottle, ensuring the thickness does not exceed 5mm and any loose samples do not exceed 10mm. Weigh it precisely. Open the bottle lid and dry at 100-105°C for 5 hours. Close the bottle lid and transfer to a drying oven. Cool for 30 minutes and weigh precisely. Then, dry at the above temperature for 1 hour, cool, and weigh again, ensuring that the difference between two consecutive weighings does not exceed 5mg. Calculate the moisture content (%) in the test sample based on the weight loss.

[0029] Examples of effects: The licorice charcoal used in the following efficacy experiment is the licorice charcoal produced according to the "Manufacturing Examples" section of the present invention.

[0030] Example of effect 1: Investigation of the effects of a combination of Polygonum multiflorum and licorice charcoal on normal human hepatocyte LO2 cells. 1. Cell origin and culture conditions: Cell origin: Human normal hepatocyte L02 cells were purchased from Shanghai Meixuan Biotechnology Co., Ltd. Culture conditions: LO2 cells were cultured under normal conditions in RPMI-1640 culture medium containing 10% fetal bovine serum, in a saturated humidity cell culture box at 37°C and 5% CO2, with the culture medium changed every 1-2 days. LO2 cells in good condition and with vigorous growth during the logarithmic growth phase were used for subsequent experiments. Origin of the medicinal herb: He Shou Wu medicinal herb was purchased from Kangmei Pharmaceutical Co., Ltd., extracted and concentrated with 70% ethanol, and a reserve of the necessary amount and concentration of herbal medicine for the experiment was prepared.

[0031] 2. Experimental group: Blank control group (i.e., physiological saline group), He Shou Wu group (dose 20 mg / mL) -1 ), Heshuwura and high dose licorice charcoal combination group (Heshuwura dose 20 mg mL -1 , licorice charcoal dose 15mg·mL -1 ), Heshuwura and medium dose licorice charcoal combination group (Heshuwura dose 20 mg mL -1 , licorice charcoal dose 10mg·mL -1 ), Heshuwura and low-dose licorice charcoal combination group (Heshuwura dose 20 mg mL -1 , licorice charcoal dose 5mg·mL -1 ).

[0032] 3. Sample preparation: The Polygonum multiflorum herbs are crushed and sieved through a 1.5 cm sieve to set aside. Take the Polygonum multiflorum and the mixed powder of Polygonum multiflorum and licorice charcoal at high and low concentrations, and perform ultrasonic extraction (output 200 W, frequency 40 kHz) twice with 10 times the volume of 70% ethanol for 1 hour each time. Combine the two extracts, recover the ethanol under reduced pressure, and dry and concentrate in a 50°C water bath to obtain an infusion. Dissolve in DMSO, fill into 25 mL volume bottles, store in a refrigerator at 4°C, and before use, adjust to the required maximum concentration with complete culture medium, adjust other concentrations by ratio dilution with complete culture medium, and sterilize through a 0.22 μm micropore filter.

[0033] 4. Measurement of toxicity of each experimental group to L02 cells using the CCK-8 method: L02 cells in the logarithmic growth phase were taken, digested, and resuspended, and the cell density was increased to 4 × 10⁻⁶. 4 Prepare the cells / mL and inoculate 200 μL of cells into each well of a 96-well plate. After 24 hours, change the solution and add 200 μL of the prepared Polygonum multiflorum and a mixture of Polygonum multiflorum and high and low concentrations of licorice charcoal to each well. A negative control group (with an equal volume of complete culture medium) is provided for the experiment, and five double wells are prepared in each group. After 48 hours with the drug added, discard the supernatant from each well, wash lightly once with PBS, add 100 μL of 5% CCK-8 reagent to each well, incubate in a 37°C culture box for 1 hour, and measure the absorbance (A) at a wavelength of 450 nm using an enzyme labeler.

[0034] The inhibition rate (IR) of L02 cells is calculated by the following formula: IR = (1 - A 投薬群平均 / A 陰性対照群平均 ) × 100%.

[0035] 5. Measurement of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) activities in the culture supernatant:

[0036] Cells are seeded and grouped, drugs are added to each group, and after culturing for 48 hours, the cell supernatant is collected, and the activities of ALT, AST, and LDH in the supernatant are measured according to the instructions of the reagent kit.

[0037] 6. Data analysis: Statistical analysis is performed using SPSS 17.0 software. The measured data between groups are

Number

[0038] 7. Experimental results: The results are shown in Table 2. Compared with the blank control group, the polygonum multiflorum group and the polygonum multiflorum and carbonized licorice combination group significantly inhibited the growth of L02 cells (P < 0.01). The combinations of different doses of carbonized licorice and polygonum multiflorum still showed different degrees of inhibitory effects on L02 cells, but the combinations of medium and high doses of carbonized licorice and polygonum multiflorum showed a significantly lower inhibitory effect than the polygonum multiflorum group (P < 0.05 or P < 0.01). Also, there is a certain dose-response relationship in its inhibitory effect, indicating that carbonized licorice significantly reduces the inhibitory effect of polygonum multiflorum on the proliferation of L02 cells.

Table 2

[0039] The results are shown in Table 3. Compared to the blank control group, the He Shou Wu group significantly increased ALT, AST, and LDH activity in the L02 cell culture supernatant (P<0.01). Compared to He Shou Wu administration alone, high, medium, and low doses of He Shou Wu combined with licorice charcoal significantly reduced the levels of ALT, AST, and LDH increased by He Shou Wu (P<0.05 or P<0.01). Furthermore, there was a clear dose-response relationship in the reduction of ALT, AST, and LDH levels. [Table 3]

[0040] Example of effect 2: Examination of the effects of a combination of Polygonum multiflorum and licorice charcoal on the liver of mice. In this study, the levels of ALT and AST in mouse serum are used as important indicators of liver toxicity. This is because when liver cells are damaged during liver toxicity, cell permeability increases, and large amounts of AST and ALT from cellular mitochondria and liver cells are released into the bloodstream, causing their levels to exceed normal levels, thus reflecting the degree of liver damage. During liver toxicity, oxidative stress reactions mainly occur, and in this process, superoxide dismutase (SOD) and malondialdehyde (MDA) indirectly reflect the free radical scavenging ability and the degree of free radical attack, respectively. Furthermore, in response to organ damage caused by liver toxicity, the body automatically performs self-repair, cell tissue proliferates, liver mass increases, and the hepatosomal index increases.

[0041] 1. Experimental method: Fifty SPF-grade KM mice (half male, half female, body weight 30.0 ± 2.0 g) were purchased from Hunan Slyke Jingda Laboratory Animal Co., Ltd., and the mice were randomly divided into five groups based on body weight: a blank control group (i.e., the saline group), and a He Shou Wu group (dose 20 g / kg). -1 ), combination group of Heshuwura and high dose licorice charcoal (Heshuwura dose 20g·kg -1 , licorice charcoal dosage 3g·kg -1 ), a combination group of Heshouwura and medium-dose licorice charcoal (Heshuwura dose 20g·kg -1 , licorice charcoal dosage 2g·kg -1), combination group of Heshuwura and low dose licorice charcoal (Heshuwura dose 20g·kg -1 , licorice charcoal dosage 1g·kg -1 Each group will consist of 10 individuals, with males and females kept separately.

[0042] Crush the Polygonum multiflorum plant, sift it through a 1.5 cm sieve, and set it aside as a reserve. Take powders of Polygonum multiflorum mixed with licorice charcoal at different concentrations, and perform ultrasonic extraction twice with 10 times the volume of 70% ethanol (output 200W, frequency 40kHz, 1 hour each). Combine the two extracts, recover the ethanol under reduced pressure, and concentrate the extract in a 50°C water bath to obtain the extract, which is set aside as a reserve. Add the amount of water required for the experiment to prepare a medicinal solution with the appropriate concentration of herbal ingredients.

[0043] The intervention will be carried out for 15 consecutive days, with oral administration once daily. On the 16th day, serum and liver samples will be collected 12 hours later without any dietary restrictions. Serum ALT, serum AST, liver tissue MDA, and liver tissue SOD will be detected using an enzyme labeling device. During the drug intervention period, the body weight of the mice will be measured every two days to adjust the dosage volume, and changes in the physiological signs of the mice will be closely observed throughout the treatment period.

[0044] Statistical methods: All econometric data in this experiment are

number

[0045] 2. Experimental results: After oral administration to mice for 15 days, no deaths occurred in the five groups of mice, and there were no significant differences in physiological signs such as body weight, diet, coat color, or excretion.

[0046] Observing the data in Table 4, the hepatic body index (hepatic body index = liver mass / body mass × 100%) in the He Shou Wu group and the group treated with He Shou Wu and licorice charcoal was higher than in the blank control group (P<0.05). This indicates that He Shou Wu and the combined administration of He Shou Wu and licorice charcoal have hepatotoxic effects and cause liver damage. However, the hepatic body index for the combined administration of He Shou Wu and licorice charcoal was lower than that for He Shou Wu alone, showing a significant difference (P<0.01). Furthermore, the hepatic body index was shown to be dependent on the administered concentration of licorice charcoal. This indicates that licorice charcoal can effectively reduce the hepatotoxicity caused by He Shou Wu.

[0047] When comparing serum AST and ALT levels in mice from each group, the levels of AST and ALT in the He Shou Wu group and the group treated with a combination of He Shou Wu and licorice charcoal were higher than in the blank control group (P<0.01). The levels of AST and ALT in the He Shou Wu and licorice charcoal combination administration were both lower than in the He Shou Wu monotherapy group (P<0.01 or P<0.05). Furthermore, the levels of AST and ALT were shown to be dependent on the administered concentration of licorice charcoal. From the perspective of hepatocyte damage, licorice charcoal has been shown to effectively reduce hepatocyte damage caused by He Shou Wu. [Table 4]

[0048] Observing the data in Table 5, the MDA content of He Shou Wu administered alone and in combination with He Shou Wu and licorice charcoal was higher than that of the blank control group (P<0.01 or P<0.05), however, the MDA of the He Shou Wu and licorice charcoal combination was lower than that of the He Shou Wu administered alone group (P<0.01 or P<0.05); the SOD activity of He Shou Wu administered alone and in combination with He Shou Wu and licorice charcoal was lower than that of the blank control group (P<0.01), however, the SOD activity of the He Shou Wu and licorice charcoal combination was higher than that of He Shou Wu administered alone (P<0.01 or P<0.05). From the perspective of microscopic oxidative stress responses in the liver, licorice charcoal can effectively mitigate the liver-damaging effects of He Shou Wu. [Table 5]

[0049] In general, Polygonum multiflorum (He Shou Wu) is hepatotoxic, and administration of certain doses causes liver damage. However, the combination of Polygonum multiflorum and licorice charcoal can effectively reduce liver damage caused by Polygonum multiflorum. Specifically, the combination of Polygonum multiflorum and licorice charcoal has been shown to suppress the increase in hepatic body index, the increase in AST and ALT content, and the increase in MDA content caused by Polygonum multiflorum, and to increase the activity of SOD. Therefore, licorice charcoal effectively reduces the hepatotoxicity of Polygonum multiflorum, and the combination of licorice charcoal and Polygonum multiflorum can improve the drug safety of Polygonum multiflorum.

[0050] From this, it is clear that licorice charcoal produced by the method of the present invention can effectively reduce the hepatotoxicity of drugs, can be used as a coating for drugs with pharmacokinetic properties, can reduce liver damage after drug administration in patients, and has a wide range of application prospects.

[0051] Clearly, technicians in the art can make various modifications and variations to the present invention without departing from the spirit and scope of the invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and its equivalent art, the present invention is intended to encompass these modifications and variations as well.

Claims

1. A method for producing a licorice charcoal coating material for reducing the hepatotoxicity of drugs, characterized by comprising the following steps; The licorice extract is crushed and sieved through a 1.5-2.0 cm sieve. The frying machine is set to start at 120°C-160°C, heating at 250-350°C, and heating for 1.0-2.0 hours. The licorice is fried until the surface turns black and the inside turns yellow. When the smoke becomes thick, heating is stopped, a small amount of clean water is sprayed to extinguish the sparks, and after the frying machine has cooled to room temperature, the fried licorice is removed, dried, crushed, and sieved through a No. 6 sieve to obtain the licorice charcoal coating material. The aforementioned drug is Polygonum multiflorum, a drug that possesses pharmacokinetic properties.

2. A manufacturing method according to claim 1, characterized in that the licorice extract is a licorice extract that conforms to the quality standards requirements of the "Pharmacopoeia of the People's Republic of China".

3. A method for manufacturing according to claim 1 or claim 2, characterized in that the licorice extract is a processed product of the dried roots and rhizomes of licorice, licorice root, or licorice root, all of which are leguminous plants.

4. A manufacturing method according to any one of claims 1 to 3, characterized by comprising the following steps; The licorice extract is crushed and sieved through a 1.6 cm sieve. The start temperature of the frying machine is set to 150°C, the heating temperature to 300°C, and the heating time to 1.5 hours. The licorice is fried until the surface turns black and the inside turns yellow. When the smoke becomes thick, the heating is stopped, a small amount of clean water is sprayed to extinguish the sparks, and after waiting for the frying machine to cool to room temperature, the fried licorice is removed, dried, crushed, and sieved through a No. 6 sieve to obtain the licorice charcoal coating material.

5. The use of a licorice charcoal coating material for reducing the hepatotoxicity of drugs, manufactured by the manufacturing method described in claim 1, as a surface coloring coating for water pills in a Chinese medicine preparation, characterized in that the water pills coated with the licorice charcoal coating material have a uniform color, are glossy, and are resistant to deterioration.

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