WO2023236626A1 - 一种降低药物肝毒性的甘草炭包衣材料及其制备方法和用途 - Google Patents

一种降低药物肝毒性的甘草炭包衣材料及其制备方法和用途 Download PDF

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WO2023236626A1
WO2023236626A1 PCT/CN2023/083073 CN2023083073W WO2023236626A1 WO 2023236626 A1 WO2023236626 A1 WO 2023236626A1 CN 2023083073 W CN2023083073 W CN 2023083073W WO 2023236626 A1 WO2023236626 A1 WO 2023236626A1
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licorice
charcoal
coating material
drug
preparation
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PCT/CN2023/083073
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English (en)
French (fr)
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汤迎湛
江涛
曾晨
苏锐辉
曾利杰
王川易
陈丽斯
彭红英
温德源
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广州白云山敬修堂药业股份有限公司
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Publication of WO2023236626A1 publication Critical patent/WO2023236626A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/28—Dragees; Coated pills or tablets, e.g. with film or compression coating
    • A61K9/2806—Coating materials
    • A61K9/288—Compounds of unknown constitution, e.g. material from plants or animals

Definitions

  • the invention belongs to the field of medicine, and specifically relates to a licorice charcoal coating material that reduces drug hepatotoxicity and its preparation method and use.
  • Traditional Chinese medicine charcoal medicine is a type of medicine with special processing characteristics.
  • the traditional charcoal making methods mainly include fried charcoal and calcined charcoal. That is, the items to be roasted are put into a pot and stir-fried until the surface is charred black and the interior is charred brown or heated to a specified level. , when making charcoal, it should be "preserving" and prevent ashing, and more importantly, avoid re-ignition.
  • Traditional Chinese medicine charcoal medicine has clinical functions of stopping bleeding, antidiarrheal, hypoglycemic, analgesic, liver protection, anti-inflammatory, anti-ulcer, etc. There are more than 70 types of charcoal medicine still in clinical use today.
  • Licorice charcoal is a processed product of the dried roots and rhizomes of leguminous plants licorice, licorice and licorice. Its essence is to partially carbonize licorice. It is widely used in traditional Chinese medicine preparations to cover the surface of Shui Pan Pills. 2020 edition of " The Zhuanggu joint pills and anti-bone hyperplasia pills recorded in the Pharmacopoeia of the People's Republic of China are both made by coating with licorice charcoal. The prepared Shuifan pills have the same color, shiny luster and are not easy to deteriorate; at the same time, licorice has the effect of harmonizing various medicines. , the color coating used to cover it will not affect the efficacy of the preparation.
  • the present invention provides a licorice charcoal coating material that reduces drug hepatotoxicity and its preparation method and use.
  • the present invention is implemented through the following technical solutions:
  • the present invention provides a method for preparing a licorice charcoal coating material that reduces drug hepatotoxicity, including the following steps:
  • the licorice slices are licorice slices that meet the quality standard requirements of the "Pharmacopoeia of the People's Republic of China".
  • the licorice slices are processed products of the dried roots and rhizomes of the leguminous plants Glycyrrhiza glabra, Glycyrrhiza glabra or Glycyrrhiza glabra.
  • the above preparation method includes the following steps:
  • the present invention provides a licorice charcoal coating material that reduces drug liver toxicity prepared by the preparation method described in the first aspect, and the licorice charcoal coating material is a black or gray-black powder, It has a slight odor, a light and slightly sweet taste, and has less than 3% impurities and less than 10% moisture in terms of weight percentage.
  • the present invention provides the use of the licorice charcoal coating material described in the second aspect as a cover color coating for water-flavored pills in traditional Chinese medicine preparations.
  • the water-flavored pills coated with the licorice charcoal coating material The pills are consistent in color, shiny and not easy to deteriorate.
  • the present invention provides the use of the licorice charcoal coating material described in the second aspect above in preparing coating materials that reduce drug hepatotoxicity.
  • the drug is a drug with drug-induced hepatotoxicity.
  • the drug with drug-induced hepatotoxicity is Polygonum multiflorum, Tripterygium wilfordii, Epimedium or Psoralen.
  • the present invention has the following beneficial effects:
  • the preparation method of the present invention increases the yield of licorice charcoal by about 10%.
  • the highest yield of the currently reported preparation method of licorice charcoal is 35%.
  • the yield of the preparation process is low, and there is still room for improvement (see CN201810717845.0) , and the yield of licorice charcoal prepared by the method of the present invention is about 45%, which is about 10% higher than the yield of existing reports.
  • the licorice charcoal prepared by the method of the present invention has the ability to reduce drug hepatotoxicity. It can be used as a coating for drugs with drug-induced hepatotoxicity to reduce liver damage in patients after taking the drug, and has broad application prospects.
  • test materials used in the following examples are all commercially available products unless otherwise specified.
  • the preparation process of the licorice charcoal coating material for reducing drug hepatotoxicity of the present invention is as follows: licorice slices are crushed and passed through a 1.6cm sieve, the starting temperature of the medicine frying machine is set to 150°C, the heating temperature is 300°C, and the heating time is 1.5h. Fry until the surface is burnt black, the inside is burnt yellow, and the smoke is thick. Stop heating. Spray a little water to extinguish the sparks. After the frying machine cools down to room temperature, take out the fried licorice, dry it, crush it, and pass it through No. 6 Sieve to obtain the licorice charcoal coating material.
  • Licorice charcoal is processed and produced with reference to the preparation method of the present invention.
  • the production situation is summarized as follows:
  • the prepared licorice charcoal should meet the following requirements:
  • Particle size All should pass No. 5 sieve, and ⁇ 98% should pass No. 6 sieve.
  • test sample Take an appropriate amount of the test sample, spread it out, observe it with the naked eye or with the help of a magnifying glass (5-10 times), and pick out the impurities; if there are impurities that can be screened, separate the impurities through an appropriate sieve. 2. Weigh each type of impurities separately and calculate their content (%) in the test sample.
  • the moisture content must not exceed 10.0% (Second Method 0832, General Chapter 2020 Edition of "Chinese Pharmacopoeia”). Oven-drying method: For measurement, take 25g of the test sample. If the diameter or length of the test sample exceeds 3mm, before weighing, it should be quickly made into particles or fragments with a diameter or length of no more than 3mm and laid flat in a flat shape that is dried to constant weight. In the weighing bottle, the thickness should not exceed 5mm, and the loose test sample should not exceed 10mm. Weigh it accurately. Open the bottle cap and dry it at 100-105°C for 5 hours. Cover the bottle cap, transfer it to a desiccator, and let it cool for 30 minutes. , weigh accurately, dry at the above temperature for 1 hour, let cool, and weigh until the difference between two consecutive weighings does not exceed 5 mg. Based on the weight loss, calculate the moisture content (%) in the test sample.
  • the licorice charcoal used in the following effect experiments is the licorice charcoal prepared in the "Preparation Examples" section of the present invention.
  • Effect example 1 Examining the effect of the compatibility of Polygonum multiflorum and licorice charcoal on the human normal liver cell line L02 cells
  • Cell source Human normal liver cell line L02 cells were purchased from Shanghai Meixuan Biotechnology Co., Ltd.
  • L02 cells are routinely cultured in RPMI-1640 culture medium containing 10% fetal bovine serum, placed in a saturated humidity cell incubator at 37°C, 5% CO2 , and the medium is changed once every 1-2 days, and the status is taken. Good, vigorously proliferating L02 cells in the logarithmic growth phase were used for subsequent experiments.
  • Polygonum multiflorum medicinal materials were purchased from Kangmei Pharmaceutical Co., Ltd. Polygonum multiflorum was extracted and concentrated with 70% ethanol, and the medicinal solution containing the corresponding crude drug concentration was prepared according to the experimental requirements for later use.
  • Blank control group i.e., normal saline group
  • Polygonum multiflorum group dose is 20 mg ⁇ mL -1
  • Polygonum multiflorum combined with high-dose licorice charcoal group Polygonum multiflorum dose is 20 mg ⁇ mL -1 , licorice charcoal dose is 15 mg ⁇ mL -1
  • Polygonum multiflorum combined with medium-dose licorice charcoal group Polygonum multiflorum dosage is 20 mg ⁇ mL -1 , licorice charcoal dosage is 10 mg ⁇ mL -1
  • Polygonum multiflorum combined with low-dose licorice charcoal polygonum multiflorum dosage is 20 mg ⁇ mL -1 , licorice charcoal dosage The dose is 5 mg ⁇ mL -1 ).
  • IR ( mean value of 1-A administration group / mean value of A negative control group ) ⁇ 100%.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • LDH lactate dehydrogenase
  • Cells were inoculated and divided into groups. After each group was added with drugs and cultured for 48 hours, the cell supernatants were collected, and the ALT, AST, and LDH activities in the supernatants were detected according to the instructions of the kit.
  • SPSS 17.0 software was used for statistical analysis, and data were measured between each group. means, two The t test was used for comparison between groups, the one-way analysis of variance was used for comparison between multiple groups, and the LSD test was used for comparison between groups. P ⁇ 0.05 means that the difference is statistically significant.
  • the Polygonum multiflorum group can significantly increase the activities of ALT, AST and LDH in the L02 cell culture supernatant (P ⁇ 0.01); compared with the administration of Polygonum multiflorum alone, high, medium and low doses of licorice charcoal Administration of Polygonum multiflorum combined with Polygonum multiflorum can significantly reduce the levels of ALT, AST, and LDH increased by Polygonum multiflorum (P ⁇ 0.05 or P ⁇ 0.01), and it has an obvious dose-effect relationship in reducing the levels of ALT, AST, and LDH.
  • Effect example 2 Examining the effect of the combination of Polygonum multiflorum and licorice charcoal on mouse liver
  • This effect example uses the levels of ALT and AST in mouse serum as important indicators of liver poisoning because During liver poisoning, damage to liver cells can lead to increased cell permeability, and a large amount of AST and ALT in cell mitochondria and liver cells are released into the blood, making their content in the blood higher than normal levels. This reflects The degree of liver damage; when the liver is damaged by poisoning, the main reaction that occurs is oxidative stress. In this process, superoxide dismutase (SOD) and malondialdehyde (MDA) indirectly reflect the ability to scavenge free radicals and Extent of free radical attack. In addition, the body will automatically repair itself due to organ damage caused by liver poisoning, causing cell tissue to proliferate, liver mass to increase, and liver body index to increase.
  • SOD superoxide dismutase
  • MDA malondialdehyde
  • mice Fifty SPF grade KM mice, half male and female, weighing 30.0 ⁇ 2.0g, were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. The mice were randomly divided into 5 groups according to body weight, and the blank control group (i.e., the normal saline group ), Polygonum multiflorum group (the dosage is 20g ⁇ kg -1 ), Polygonum multiflorum combined with high-dose licorice charcoal group (Polygonum multiflorum dosage is 20g ⁇ kg -1 , licorice charcoal dosage is 3g ⁇ kg -1 ), Polygonum multiflorum combined with medium-dose licorice charcoal group ( The dosage of Polygonum multiflorum is 20g ⁇ kg -1 and the dosage of Licorice charcoal is 2g ⁇ kg -1 ).
  • the blank control group i.e., the normal saline group
  • Polygonum multiflorum group the dosage is 20g ⁇ kg -1
  • Polygonum multiflorum is combined with low-dose licorice charcoal (the dosage of Polygonum multiflorum is 20g ⁇ kg -1 and the dosage of licorice charcoal is 1g ⁇ kg -1 ) . There are 10 animals in each group, and the males and females are kept in separate cages.
  • the intervention was continued for 15 days, with oral administration once a day. On the 16th day, after fasting for 12 hours without water, serum and liver samples were taken. Use a microplate reader to detect indicators of serum ALT, serum AST, liver tissue MDA, and liver tissue SOD. During the drug intervention period, the weight of the mice was measured every two days, the dosing volume was adjusted, and the physiological signs of the mice were closely monitored during the dosing period.
  • mice within 15 days of intragastric administration of mice, no mice in the five groups died. The weight, diet, and There were no obvious differences in physiological signs such as coat color and excretion.
  • the hepatosomatic index levels of Polygonum multiflorum combined with licorice charcoal were lower than those given Polygonum multiflorum alone, with significant differences (P ⁇ 0.01), and the average hepatosomatic index was dose-dependent on the concentration of licorice charcoal administered. It shows that licorice charcoal can effectively reduce the hepatotoxicity caused by Polygonum multiflorum.
  • the activity of SOD in the drug group was lower (P ⁇ 0.01 or P ⁇ 0.05); the activity of SOD administered by Polygonum multiflorum alone and Polygonum multiflorum combined with licorice charcoal was lower than that in the blank control group (P ⁇ 0.01), but the activity of SOD administered by Polygonum multiflorum combined with licorice charcoal was lower than that in the blank control group (P ⁇ 0.01). The activity was higher than that of Polygonum multiflorum administered alone (P ⁇ 0.01 or P ⁇ 0.05).
  • the microscopic oxidative stress response of the liver shows that licorice charcoal can effectively reduce the liver damage caused by Polygonum multiflorum.
  • Polygonum multiflorum has hepatotoxicity, and administration of a certain dose of Polygonum multiflorum can cause liver damage.
  • Polygonum multiflorum combined with licorice charcoal can effectively reduce liver damage caused by Polygonum multiflorum.
  • the main reason is why Polygonum multiflorum combined with licorice charcoal can reduce the liver damage caused by Polygonum multiflorum.
  • Increased body index levels, increased levels of AST and ALT, and increased MDA levels increase the activity of SOD. Therefore, licorice charcoal can effectively reduce the hepatotoxicity of Polygonum multiflorum, and the combination of licorice charcoal and Polygonum multiflorum can improve the medication safety of Polygonum multiflorum.
  • the licorice charcoal prepared by the method of the present invention can effectively reduce drug hepatotoxicity, can be used as a coating for drugs with drug-induced hepatotoxicity, and can reduce liver damage in patients after taking drugs, and has broad application prospects.

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Abstract

一种降低药物肝毒性的甘草炭包衣材料及其制备方法和用途。所述包衣材料为甘草炭，甘草炭的制备方法是将甘草饮片粉碎后，过筛，用武火炒制1-2h，喷淋清水，密封冷却，即得。该方法制备的甘草炭收率约45％，较现有报道的最高收率提高约10％，所得甘草炭为黑色或灰黑色粉末，气微，味淡微甘，杂质小于3％，水分小于10％。该方法制备的甘草炭可以有效的降低药物肝毒性，可作为具有药源性肝毒性药物的包衣，降低患者服药后的肝损伤，具有广泛的应用前景。

Description

一种降低药物肝毒性的甘草炭包衣材料及其制备方法和用途
相关申请的交叉引用
本申请要求2022年06月07日提交的中国申请号2022106390547的权益。所述申请号2022106390547据此全文以引用方式并入本文。
技术领域
本发明属于医药领域，具体涉及一种降低药物肝毒性的甘草炭包衣材料及其制备方法和用途。
背景技术
中药炭药作为一类非常具有炮制特色的药物，传统制炭方法主要有炒炭和煅炭两种，即取待炮炙品置锅内炒至表面焦黑色、内部焦褐色或加热至规定程度，制炭时应“存性”，并防止灰化，更要避免复燃。中药炭药临床具有止血、止泻、降糖、镇痛、保肝、抗炎、抗溃疡等功效，至今临床仍沿用的炭药种类达70余种。甘草炭为豆科植物甘草、胀果甘草或光果甘草的干燥根和根茎的炮制加工品，其本质是将甘草部分炭化，广泛用于中药制剂中水泛丸的盖面色衣，2020年版《中华人民共和国药典》中收载的壮骨关节丸与抗骨增生丸均用甘草炭包衣制得，所制得水泛丸色泽一致、光泽油亮、不易变质；同时甘草具有调和诸药的功效，用之盖包色衣不影响制剂的疗效。近年有研究从炭药中分离纯化得到的纳米类成分入手，并结合透射电子显微镜、傅里叶变换红外光谱等表征技术和药理活性实验对其进行鉴定与分析，结果表明炭纳米类成分为相应药材炭品药效的物质基础。在甘草炭中也发现了纳米类成分，通过小鼠急性酒精性胃溃疡模型证明纳米类成分具有抗溃疡作用(参见例如，朱雅凡，仲景“炙”法原貌考证及炙甘草抗溃疡作用的研究[D].北京中医药大学,2019.)。甘 草炭更多的药理活性仍需要深入挖掘。
随着中草药在国内外的广泛使用，近年来以肝损伤为代表的中药安全性问题/事件频被报道，特别是一些传统不认为有毒的中药有较多的肝损伤报道，其中以何首乌较为突出。我国药监部门多次发布了含何首乌制剂肝损伤警示和监管通告，包括养血生发胶囊、首乌延寿片、首乌延寿颗粒等。随着中医药更加广泛的应用以及人们对中医药认识的不断深入，新的中药安全性问题可能将不断被发现，只有在保障临床疗效的基础上，降低中药引起的肝毒性，趋利避害，最大程度降低临床用药风险才能有效解决中药安全性问题。目前，降低中药肝毒性的方法主要为炮制减毒和配伍减毒，比如，何首乌传统炮制方法“九蒸九晒”和现代主流的蒸制或加黑豆汁蒸制均能减毒何首乌的肝毒性，而临床上何首乌主要与茯苓、甘草等配伍减毒，有研究考察了何首乌与茯苓、甘草、三七的配伍减毒情况，发现茯苓、甘草、三七都有显著的减毒作用，且呈剂量依赖性，其中茯苓减毒的作用效果最明显。但是，中成药处方药味配伍和药材炮制是固定的，如果发生改变会影响临床药效。
因此，缺乏一种有效降低药物肝毒性，且通用于大多数中成药但不影响药效的技术。
发明内容
为了解决上述技术问题，本发明提供了一种降低药物肝毒性的甘草炭包衣材料及其制备方法和用途。
具体地，通过以下几个方面的技术方案实现了本发明：
在第一个方面中，本发明提供了一种降低药物肝毒性的甘草炭包衣材料的制备方法，包括以下步骤：
将甘草饮片粉碎过1.5-2.0cm筛，设置炒药机起始温度为120℃-160℃，加热温度为250-350℃，加热时间为1.0-2.0h，炒至表面焦黑色、内面焦黄色，并产生烟浓时结束加热，喷淋清水少许，熄灭火星，待炒药机冷却至室温后， 取出炒制后的甘草，晾干，粉碎，过六号筛，既得甘草炭包衣材料。
作为可选的方式，在上述制备方法中，所述甘草饮片为符合《中华人民共和国药典》质量标准要求的甘草饮片。
作为可选的方式，在上述制备方法中，所述甘草饮片为豆科植物甘草、胀果甘草或光果甘草的干燥根和根茎的炮制加工品。
作为可选的方式，在上述制备方法中，包括以下步骤：
将甘草饮片粉碎过1.6cm筛，设置炒药机起始温度为150℃，加热温度为300℃，加热时间为1.5h，炒至表面焦黑色、内面焦黄色，并产生烟浓时结束加热，喷淋清水少许，熄灭火星，待炒药机冷却至室温后，取出炒制后的甘草，晾干，粉碎，过六号筛，既得甘草炭包衣材料。
在第二个方面中，本发明提供了采用上述第一个方面所述的制备方法制备得到的降低药物肝毒性的甘草炭包衣材料，所述甘草炭包衣材料为黑色或灰黑色粉末，气微，味淡微甘，以重量百分比计，杂质小于3％，水分小于10％。
在第三个方面中，本发明提供了上述第二个方面所述的甘草炭包衣材料用于中药制剂中水泛丸的盖面色衣的用途，采用甘草炭包衣材料包衣的水泛丸色泽一致、光泽油亮、不易变质。
在第四个方面中，本发明提供了上述第二个方面所述的甘草炭包衣材料在制备降低药物肝毒性的包衣材料中的用途。
作为可选的方式，在上述用途中，所述药物为具有药源性肝毒性的药物。
作为可选的方式，在上述用途中，所述具有药源性肝毒性的药物为何首乌、雷公藤、淫羊藿或补骨脂。
本发明相对于现有技术，具有以下有益效果：
(1)本发明方法制备方法将甘草炭收率提高约10％，目前已报道的甘草炭制备方法收率最高为35％，制备工艺收率较低，尚有提升空间(参见CN201810717845.0)，而本发明方法制备的甘草炭收率约为45％，较现有报道的收率提高约10％。
(2)甘草炭药理活性研究较少，中药炭药作为一类非常具有炮制特色的药物，其药理活性有深入挖掘的潜力，本发明首次发现本发明方法制备的甘草炭具有降低药物肝毒性的作用，进而可将其作为具有药源性肝毒性药物的包衣，降低患者服药后的肝损伤，具有广泛的应用前景。
具体实施方式
下面参照具体的实施例对本发明做进一步说明。应当理解，此处所描述的具体实施例仅用于解释本发明，并不用于限定本发明的范围。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道购买得到的常规产品。
下面实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为市售产品。
制备实施例：
本发明降低药物肝毒性的甘草炭包衣材料的制备工艺如下所示：甘草饮片粉碎过1.6cm筛，设置炒药机起始温度为150℃，加热温度为300℃，加热时间为1.5h，炒至表面焦黑色、内面焦黄色，并产生烟浓时结束加热，喷淋清水少许，熄灭火星，待炒药机冷却至室温后，取出炒制后的甘草，晾干，粉碎，过六号筛，即得甘草炭包衣材料。
参照本发明制备方法进行甘草炭的炮制生产，生产情况汇总如下：
表1：甘草炭炮制记录表
上表数据表明，甘草炭炮制工艺成熟，已能满足生产需求。
制备所得的甘草炭应该符合以下要求：
粒度：应全部通过五号筛，通过六号筛应≥98％。
杂质不得过3％(《中国药典》2020年版通则2301)。检查方法：
1.取适量的供试品，摊开，用肉眼或借助放大镜(5-10倍)观察，将杂质拣出；如其中有可以筛分的杂质，则通过适当的筛，将杂质分出。2.将各类杂质分别称重，计算其在供试品中的含量(％)。
水分不得过10.0％(《中国药典》2020年版通则0832第二法)。烘干法：测定法取供试品25g，如果供试品的直径或长度超过3mm，在称取前应快速制成直径或长度不超过3mm的颗粒或碎片平铺于干燥至恒重的扁形称量瓶中，厚度不超过5mm，疏松供试品不超过10mm,精密称定，开启瓶盖在100-105℃干燥5小时，将瓶盖盖好，移置干燥器中，放冷30分钟，精密称定，再在上述温度干燥1小时，放冷，称重，至连续两次称重的差异不超过5mg为止。根据减失的重量，计算供试品中含水量(％)。
效果实施例：
以下效果实验中使用的甘草炭为本发明“制备实施例”部分制备得到的甘草炭。
效果例1：考察何首乌和甘草炭配伍对人正常肝细胞系L02细胞的影响
1.细胞来源以及培养条件：
细胞来源：人正常肝细胞系L02细胞购于上海美轩生物科技有限公司。
培养条件：L02细胞常规培养于含10％胎牛血清的RPMI-1640培养液中，置37℃，5％CO2的饱和湿度细胞培养箱中培养，1-2天换1次液，取状态良好，增殖旺盛的对数生长期的L02细胞用于后续实验。
药材来源：何首乌药材购于康美药业股份有限公司，何首乌70％乙醇提取浓缩，根据实验所需配制成含相应生药量浓度的药液备用。
2.实验分组：
空白对照组(即生理盐水组)，何首乌组(剂量为20mg·mL-1)，何首乌配伍高剂量甘草炭组(何首乌剂量为20mg·mL-1，甘草炭剂量为15mg·mL-1)，何首乌配伍中剂量甘草炭组(何首乌剂量为20mg·mL-1，甘草炭剂量为10mg·mL-1)，何首乌配伍低剂量甘草炭(何首乌给药剂量为20mg·mL-1，甘草炭给药剂量为5mg·mL-1)。
3.样品的制备：
将何首乌药材粉碎过1.5cm筛，备用。分别称取何首乌和何首乌与高低不同浓度甘草炭混合粉末，加入10倍量70％乙醇超声提取两次功率200W，频率40kHz)，每次1h，合并两次所得滤液，减压回收乙醇，50℃水浴锅上挥干浓缩成浸膏，用DMSO溶解，定容于25mL容量瓶中，4℃冰箱保存，用前以完全培养液配制所需的最高浓度后，再以完全培养液倍比稀释制得其余浓度，0.22μm微孔滤膜滤过除菌，即得。
4.CCK-8法测定各实验组对L02细胞的毒性：
取对数生长期的L02细胞，消化后重悬调整细胞密度为4×104个/mL，每孔200μL细胞接种于96孔板中，接种24h后换液，每孔分别加入200μL配好的何首乌以及何首乌和高低浓度甘草炭混合。实验设阴性对照组(加入等体积完全培养液)，每组5个复孔。加药48h后，每孔弃上清，PBS轻轻洗涤1遍，加入5％的CCK 8试剂，每孔100μL，于37℃培养箱中孵育1h，酶标仪于450nm波长处测定吸光度(A)。
按下式计算L02细胞的抑制率(IR):IR＝(1-A给药组均值/A阴性对照组均值)×100％。
5.培养上清液中谷丙转氨酶(ALT)、谷草转氨酶(AST)、乳酸脱氢酶(LDH)活力测定：
细胞接种和分组，各组加药培养48h后，收集细胞上清液，按试剂盒说明书检测上清液中ALT、AST、LDH活力。
6.数据分析：
采用SPSS 17.0软件进行统计分析，各组间计量检测数据表示，两 组间比较采用t检验，多组间比较采用单因素方差分析，组间比较采用LSD检验，P＜0.05即差异具统计学意义。
7.实验结果：
结果见表2，与空白对照组比较，何首乌组和何首乌配伍甘草炭组对L02细胞的生长抑制明显(P<0.01)，虽然不同剂量的甘草炭与何首乌配伍对L02细胞的仍然有不同程度的抑制作用，但中、高剂量甘草炭与何首乌配伍对L02细胞的抑制作用显著低何首乌组(P<0.05或P<0.01)，且其抑制作用具一定量效关系，说明甘草炭能显著降低何首乌对L02细胞的增殖抑制作用。
表2.对人正常肝细胞L02的增殖抑制率(x±s，n＝5)

注：与空白组比，*P<0.01；与何首乌组比较，##P<0.01。
结果见表3，与空白对照组比较，何首乌组能显著提高L02细胞培养上清液中ALT、AST和LDH活力(P＜0.01)；与单独何首乌给药比较，高、中、低剂量甘草炭配伍何首乌给药均能显著降低何首乌提高的ALT、AST、LDH的水平(P＜0.05或P＜0.01)，并且其降低ALT、AST、LDH的水平具有明显的量效关系。
表3.对L02细胞中ALT，AST，LDH的影响(x±s，n＝5)

注：与空白组比，*P<0.01，**P<0.05；与何首乌组比较，#P<0.05，##P<0.01。
效果例2：考察何首乌和甘草炭配伍对小鼠肝脏的影响
本效果例将小鼠血清中ALT、AST的含量作为肝中毒的重要指标，是因为 肝脏中毒时，肝细胞受损可以导致细胞通透性增大，细胞线粒体和肝细胞中的AST和ALT大量的释放到血液中，使其在血液中的含量高于正常水平，以此来反映肝脏的受损程度；肝脏中毒受损时，主要发生的反应是氧化应激，在此过程中超氧化物歧化酶(SOD)和丙二醛(MDA)分别间接地反映了清除自由基的能力和受自由基攻击的程度。此外肝脏中毒导致的器官损伤，机体会自动的进行自我修复，使得细胞组织增生，肝脏质量增大，肝体指数变大。
1.实验方法：
将50只SPF级KM小鼠，雌雄各半，体重30.0±2.0g，购于湖南斯莱克景达实验动物有限公司，小鼠按体质量随机分为5组，空白对照组(即生理盐水组)，何首乌组(剂量为20g·kg-1)，何首乌配伍高剂量甘草炭组(何首乌剂量为20g·kg-1，甘草炭剂量为3g·kg-1)，何首乌配伍中剂量甘草炭组(何首乌剂量为20g·kg-1，甘草炭剂量为2g·kg-1)，何首乌配伍低剂量甘草炭(何首乌给药剂量为20g·kg-1，甘草炭给药剂量为1g·kg-1)。每组10只，雌雄分笼饲养。
将何首乌药材粉碎过1.5cm筛，备用。分别称取何首乌和何首乌与高低不同浓度甘草炭混合粉末，加入10倍量70％乙醇超声提取两次功率200W，频率40kHz)，每次1h，合并两次所得滤液，减压回收乙醇，50℃水浴锅上挥干浓缩成浸膏，备用。根据实验所需加水配制成含相应生药量浓度的药液。
连续干预15天，每天灌胃一次，于第十六天不禁水禁食12h后，取血清以及肝脏样本。使用酶标仪，检测出血清ALT、血清AST、肝脏组织MDA、肝脏组织SOD的指标。药物干预期间，每隔两天测一次小鼠体重，调整给药体积，在给药期间密切关注小鼠的生理体征变化。
统计方法：本实验的计量数据均以表示。应用SPSS17.0统计软件进行分析，组间比较采用单因素方差分析(ANOVA)，多个样本均数之间的多重比较用Post-Hoc LSD法。P＜0.05为差异有统计学意义。
2.实验结果：
对小鼠实施灌胃的15天内，5组小鼠均没有死亡现象，体重、饮食情况、 毛色、排泄等生理体征均没有明显的差异。
通过观察表4中的数据，与空白对照组相比，何首乌组和何首乌配伍甘草炭组的肝体指数(肝体指数＝肝脏质量/体质量×100％)均比空白对照组高(P<0.05)，说明何首乌和何首乌与甘草炭配伍给药均有肝脏毒性，能引起肝损伤。而何首乌与甘草炭配伍给的药肝体指数水平均低于何首乌单独给药，具有显著差异(P<0.01)，且肝体指数平均与甘草炭的给药浓度呈剂量依赖性。说明甘草炭能有效降低何首乌引起的肝毒作用。
比较各组小鼠血清中AST和ALT的含量，何首乌组和何首乌配伍甘草炭组的AST、ALT的含量均比空白对照组高(P<0.01)，何首乌配伍甘草炭给药的AST、ALT水平均低于何首乌单独给药组(P<0.01或P<0.05)，且AST、ALT水平和甘草炭给药浓度剂量依赖性，从肝细胞受损角度表明，甘草炭可以有效降低何首乌对肝细胞的损害。
表4.小鼠肝体指数以及血清中AST、ALT的含量(X±SD，n＝10)

注：与空白组比，*P<0.01，**P<0.05；与何首乌组比较，#P<0.05，##P<0.01。
通过观察表5的数据，何首乌单独给药和何首乌配伍甘草炭给药的MDA含量比空白对照组高(P<0.01或P<0.05)，但何首乌配伍甘草炭给药的MDA均较何首乌单独给药组低(P<0.01或P<0.05)；何首乌单独给药和何首乌配伍甘草炭给药的SOD的活力均比空白对照组低(P<0.01)，但何首乌配伍甘草炭给药的SOD的活力较单独给药何首乌高(P<0.01或P<0.05)。从微观的肝脏的氧化应激反应表明，甘草炭可以有效减轻何首乌的肝损伤作用。
表5.何首乌组小鼠肝脏组织匀浆中SOD、MDA的含量(X±SD，n＝10)

注：与空白组比，*P<0.01，**P<0.05；与何首乌组比较，#P<0.05，##P<0.01。
综上所述，何首乌具有肝毒性，一定剂量的何首乌给药可引起肝损伤，而何首乌配伍甘草炭可以有效的减少何首乌所引起的肝损伤，主要表现为何首乌配伍甘草炭可下调何首乌引起的肝体指数水平升高，AST和ALT的含量升高以及MDA含量升高，上调SOD的活力。所以，甘草炭可以有效降低何首乌的肝毒性，甘草炭配伍何首乌可以提高何首乌的用药安全性。
由此可见，本发明方法制备的甘草炭可以有效的降低药物肝毒性，可作为具有药源性肝毒性药物的包衣，降低患者服药后的肝损伤，具有广泛的应用前景。
显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。

Claims (9)

  1. 一种降低药物肝毒性的甘草炭包衣材料的制备方法，其特征在于：包括以下步骤：
    将甘草饮片粉碎过1.5-2.0cm筛，设置炒药机起始温度为120℃-160℃，加热温度为250-350℃，加热时间为1.0-2.0h，炒至表面焦黑色、内面焦黄色，并产生烟浓时结束加热，喷淋清水少许，熄灭火星，待炒药机冷却至室温后，取出炒制后的甘草，晾干，粉碎，过六号筛，既得甘草炭包衣材料。
  2. 根据权利要求1所述的制备方法，其特征在于：所述甘草饮片为符合《中华人民共和国药典》质量标准要求的甘草饮片。
  3. 根据权利要求1或权利要求2所述的制备方法，其特征在于：所述甘草饮片为豆科植物甘草、胀果甘草或光果甘草的干燥根和根茎的炮制加工品。
  4. 根据权利要求1至3中任一项所述的制备方法，其特征在于：包括以下步骤：
    将甘草饮片粉碎过1.6cm筛，设置炒药机起始温度为150℃，加热温度为300℃，加热时间为1.5h，炒至表面焦黑色、内面焦黄色，并产生烟浓时结束加热，喷淋清水少许，熄灭火星，待炒药机冷却至室温后，取出炒制后的甘草，晾干，粉碎，过六号筛，既得甘草炭包衣材料。
  5. 采用权利要求1至4中任一项所述的制备方法制备得到的降低药物肝毒性的甘草炭包衣材料，其特征在于：所述甘草炭包衣材料为黑色或灰黑色粉末，气微，味淡微甘，以重量百分比计，杂质小于3％，水分小于10％。
  6. 权利要求5所述的甘草炭包衣材料用于中药制剂中水泛丸的盖面色衣的用途，其特征在于：采用甘草炭包衣材料包衣的水泛丸色泽一致、光泽油亮、不易变质。
  7. 权利要求5所述的甘草炭包衣材料在制备降低药物肝毒性的包衣材料中的用途。
  8. 根据权利要求7所述的用途，其特征在于：所述药物为具有药源性肝 毒性的药物。
  9. 根据权利要求8所述的用途，其特征在于：所述具有药源性肝毒性的药物为何首乌、雷公藤、淫羊藿或补骨脂。
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