WO2020052196A1 - 蓝莓叶总黄酮的新应用 - Google Patents

蓝莓叶总黄酮的新应用 Download PDF

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WO2020052196A1
WO2020052196A1 PCT/CN2019/074553 CN2019074553W WO2020052196A1 WO 2020052196 A1 WO2020052196 A1 WO 2020052196A1 CN 2019074553 W CN2019074553 W CN 2019074553W WO 2020052196 A1 WO2020052196 A1 WO 2020052196A1
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blueberry leaves
liver
ethanol
liver injury
total flavones
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French (fr)
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汤庆发
焦豪妍
张伟
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南方医科大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/45—Ericaceae or Vacciniaceae (Heath or Blueberry family), e.g. blueberry, cranberry or bilberry
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/55—Liquid-liquid separation; Phase separation

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  • the invention belongs to the field of medicine, and particularly relates to a new application of total flavones in blueberry leaves.
  • Flavonoids are the main active ingredients in blueberry leaves. They are a class of compounds with a 2-phenylchromogenone structure in nature. They have a ketocarbonyl group in the molecule, and the first oxygen Atoms are basic and can form salts with strong acids. Most of the hydroxy derivatives are yellow, so they are also called flavonoids or flavones. Flavonoids are usually combined with sugars in plants to form glycosides. ) Form. Most plants contain flavonoids, which play an important role in plant growth, development, flowering, fruiting, and antibacterial and disease prevention.
  • Flavonoids are secondary metabolites of plants. Flavonoids are a class of yellow pigments derived from flavones (2-phenylchromogenone) as the mother nucleus, including the isomers of flavones and their hydrogenation and reduction products, that is, C6-C3-C6 as A series of compounds with a basic carbon frame. Flavonoids are widely distributed in the plant kingdom. Most of the flavonoids in plants are combined with sugars to form glycosides or carbon sugar groups, and some of them are in free form.
  • flavonoids have liver protection, liver detoxification, antifungal, treatment of acute and chronic hepatitis, cirrhosis, and anti-free radical and antioxidant effects.
  • flavonoids have the same effects as phytoestrogens.
  • the application of flavonoids can significantly improve animal production performance, improve animal disease resistance, and improve animal immune function.
  • One of the objects of the present invention is to provide an application of total flavonoids in blueberry leaves.
  • the liver injury is acute liver injury.
  • the liver injury is LPS / D-GalN-induced acute liver injury in mice.
  • the total flavones of blueberry leaves are obtained by the following preparation method:
  • step (1) Weigh blueberry leaves, add 20 ⁇ 2 times petroleum ether to reflux for 0.5 ⁇ 0.05 hours, filter, remove the filtrate, collect the filter residue, and add an equal volume of 70 ⁇ 5% Ethanol was refluxed for 1.5 ⁇ 0.5 hours, filtered, and the filtrate was discarded, and the filtrate was concentrated by rotary evaporation to a stream extract.
  • step (2) the macroporous resin is soaked in 95 ⁇ 1% hexanol for one day, and then loaded into a column, and then rinsed with 95 ⁇ 1% ethanol until no white, 5 ⁇ 0.5% hydrochloric acid and 2 Soak and wash to ⁇ 0.5% sodium hydroxide until neutral; wet-load the flow extract through a large-pore resin column, rinse the column with 70 ⁇ 1% ethanol and 95 ⁇ 1% ethanol, and collect 70 ⁇ 1% ethanol The liquid was collected to obtain the total flavonoids of blueberry leaves.
  • the inventors of the present invention used LPS / D-GalN to induce acute liver injury in mice as a model to study the preventive and protective effects of total flavones in blueberry leaves on acute liver injury.
  • the degree of liver cell damage was reduced, and serum ALT and AST levels were significantly lower than those in the model group.
  • serum ALT and AST levels were significantly lower than those in the model group.
  • the levels of TNF- ⁇ , IL-1 ⁇ and IL-6 in the liver of the model were significantly higher than those in the normal control group, and this phenomenon could be effectively inhibited by the total flavones of blueberry leaves, suggesting that the inhibition of liver inflammatory responses in blueberry leaves Total flavones play an important role in the hepatocyte protective activity.
  • the invention provides the total flavones of blueberry leaves which can protect liver cells and prevent liver damage.
  • the total flavonoids of blueberry leaves have a clear preventive and protective effect on acute liver injury induced by LPS / D-GalN in mice.
  • the mechanism may be to inhibit the occurrence of oxidative stress and inflammatory reactions, thereby preventing and preventing liver damage.
  • Figure 1 shows the effect of total flavonoids from blueberry leaves on liver histopathology in mice with acute liver injury (HE, ⁇ 200).
  • A normal control group
  • B model group
  • C bifendate group
  • E blueberry leaves total flavones medium-dose group
  • F blueberry leaves total flavones high-dose group
  • Figure 2 shows the effect of total flavonoids in blueberry leaves on serum ALT and AST levels in mice with acute liver injury induced by LPS / D-GalN;
  • Figure 3 shows the effect of total flavonoids from blueberry leaves on the levels of IL-1 ⁇ , IL-6, and TNF- ⁇ in liver tissue of mice with LPS / D-GalN-induced acute liver injury.
  • SPF Kunming mice male, weighing 18-22 g, were purchased from the Experimental Animal Research Center of Southern Medical University under license number SCKX (Guangdong) 2011-0015. The experimental animals were free to ingest deionized water and standard vocabulary daily, and were adaptively reared for one week before the experiment began.
  • Lipopolysaccharide was purchased from Guangzhou Saiguo Biotechnology Co., Ltd .; Aminogalactose (D-GalN) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo Chemical Industry);
  • TNF- ⁇ tumor necrosis factor- ⁇
  • IL-1 ⁇ interleukin-1 ⁇
  • IL-6 interleukin-6
  • High-speed refrigerated centrifuge (Zokia HC-3018R, Anhui Zhongke Zhongjia Scientific Instrument Co., Ltd.); electronic balance (PL602-S, Shanghai METTLER TOLEDO International Trade Co., Ltd.), rotary evaporator (GC03, Shanghai Shensheng Technology) Co., Ltd.), cooling water circulation device (CA-1111, Shanghai Ailang Instrument Co., Ltd.), water circulation vacuum pump (A-1000S, Shanghai Elon Technology International Trade Co., Ltd.), CNC ultrasonic cleaner (KH-500DB, Kunshan Hechuang Ultrasound Instrument Co., Ltd.), Thermo FC microplate reader (Thermo Fisher Scientific Company), paraffin embedder (JB-P5, Wuhan Junjie Electronics Co., Ltd.), microtome (RM2016, Shanghai Leica Instrument Co., Ltd.), tissue spreader (KD-P, Zhejiang Jinhua Kedi Instrument Equipment Co., Ltd.), an upright optical microscope (NIKON ECLIPSE
  • the macroporous resin was soaked with 95% hexanol for one day, and then loaded into a column, and then rinsed with 95% ethanol until it was white, and then immersed in 5% hydrochloric acid and 2% sodium hydroxide to neutrality.
  • the above-mentioned flow extract was wet-loaded through a large-pore resin column, and the columns were washed with 70% ethanol and 95% ethanol to collect solutions with different gradients of ethanol concentration.
  • the 70% ethanol collection solution was total flavonoids in blueberry leaves.
  • the total flavonoids in blueberry leaves were determined as follows:
  • the 70% ethanol collection solution was concentrated (total flavones of blueberry leaves), and dried at low temperature and vacuum (60 ° C), and then the dry powder was scraped and weighed.
  • Five different concentrations of medicinal solution were suspended in distilled water, respectively: 800 mg / mL (ultra high dose); 400 mg / mL (high dose); 200 mg / mL (medium dose); 100 mg / mL (low dose); 50 mg / mL (ultra-low dose).
  • mice Thirty-six mice were randomly divided into five groups, namely the blank saline group, the ultra-high-dose group, the high-dose group, the medium-dose group, the low-dose group, and the ultra-low-dose group. Suspensions of total flavonoids of blueberry leaves of different concentrations were administered to mice by gavage at a time, 1mL / 20g, and the death of mice was observed at 14 days.
  • mice Forty-eight mice were randomly divided into 6 groups, namely the blank control group, the model group, the positive group (bifendate), and the blueberry leaf total flavonoids 400, 200, and 100 mg / mL group (determined according to the results of acute toxicity experiments in mice).
  • the drug group was given 1 mL of total flavonoid suspension and bifendate dripping pills of 5 mg / kg per blueberry leaf, and the blank control group and model group were given equal volumes of normal saline once a day for 7 consecutive days.
  • mice in each group except the normal control group were injected intraperitoneally with LPS (8 mg / kg) and D-GalN (800 mg / kg) to prepare an acute liver injury model.
  • mice 2.3.1 Observe the behavior of mice, the number of survivors, and determine the effective dose.
  • mice When the mice were on the verge of death (10 h after surviving the mice), the eyeballs were removed for blood extraction, and the serum was separated. Serum AST and ALT levels were measured within 3 hours.
  • liver tissue sections were observed with an optical microscope.
  • Pruning and dipping Trim the fixed right leaf, rinse with running water first, make sure that the formalin is cleaned, and then dip in double distilled water for 2h.
  • Dipping wax First, configure a series of xylene and paraffin mixed solutions with a desired concentration gradient. Xylene is soluble in alcohol and paraffin. The purpose of this step is to gradually replace the xylene in the tissue block with paraffin to immerse the paraffin into the tissue space. First immerse the dehydrated tissue in a 50% xylene and paraffin mixed solution that has been heated and thawed in advance, the infiltration time is 3 minutes; then immerse each of the thawed paraffin I and paraffin II in advance for 30 minutes in sequence; and finally immerse in paraffin III (Need to maintain the temperature of 45 °C) 90min.
  • Embedding Fold a small carton, pour the melted paraffin into the carton, and then remove the cleared right tissue from the cup wax with warming tweezers, and quickly put it into the paraffin dissolved in the carton. In order to ensure that the paraffin can flood the tissue, if it is insufficient, it should be filled up. It is then left at room temperature to cool and solidify. At this point, the tissue is completely hardened, and you can slice it.
  • Roasted slices Place the sliced slices on a slide holder, and then dry them at room temperature. After 1 day, move to an oven at 40 ° C and increase the temperature by 5 ° C every 1h until it reaches 65 ° C and then maintain it for 1h. Finally, it is placed at room temperature to make the temperature equilibrate, then it can be taken out and placed in a slide box, marked with grouping and time, and stored at room temperature for later use.
  • Hematoxylin-eosin staining is a common method for pathological testing in animal experiments, and it is also a common method in paraffin section staining technology.
  • Hematoxylin stain is a basic stain, which can make the chromatin in the nucleus of cells and ribosomes in the cytoplasm appear purple blue; eosin is an acid stain, which can make the cytoplasm and extracellular matrix Red.
  • Staining Tissue staining according to the following steps: hematoxylin 10min; tap water wash for 5 seconds; 1% hydrochloric acid 70% alcohol to differentiate the tissue for 20 seconds; rinse under running water for 5-10 minutes; 0.5% eosin Dye for 90 seconds.
  • mice liver tissues were homogenized at low temperature, the supernatant was centrifuged, and the levels of TNF- ⁇ , IL-6, and IL-1 ⁇ were measured according to the instructions of the kit.
  • the experimental data were analyzed with SPSS statistical software. Two-tailed t test was used to compare and analyze the mean of the two groups of samples. One-way analysis of variance was used for comparison between groups. P ⁇ 0.05 was considered statistically significant.
  • livers of mice in the normal control group had pale appearance, smooth edges, and tough texture. Microscopically, the cells were uniform in size, arranged neatly, and intact in structure. The nuclei were located in the center of the liver cells. The liver of the model group was dark red in appearance, with obvious swelling and congestion. Microscopically, liver tissue was severely pathologically damaged, hepatocytes were degenerate, cytoplasm was filled with small vacuoles, and significant liver bleeding and inflammatory cell infiltration were observed. After administration of blueberry leaf total flavonoids, the appearance and color of the liver of the mice showed a marked improvement. Pathological examination showed that the clearance of hepatocytes decreased, and the congestion and inflammatory cell infiltration in the high-dose group significantly improved, and almost returned to normal. see picture 1.
  • the level of -6 was significantly reduced (P ⁇ 0.01; P ⁇ 0.01; P ⁇ 0.05), and the levels of TNF- ⁇ in the high, medium and low concentrations of total flavonoids in blueberry leaves were significantly reduced (P ⁇ 0.01; P ⁇ 0.05; P ⁇ 0.01 ).
  • the results of this experiment indicate that the total flavonoids of blueberry leaves can prolong the survival rate and survival time of experimental mice, and improve the appearance and tissue morphology of the liver.
  • the levels of AST and ALT in the serum of mice after intervention with total flavones of blueberry leaves And liver index were significantly reduced, and the levels of TNF- ⁇ , IL-1 ⁇ and IL-6 were significantly reduced.
  • Intraperitoneal injection of LPS and D-GalN is a more convenient method to construct a mouse model of acute liver injury.
  • LPS is the main component of endotoxins secreted by Gram-negative bacteria. It can stimulate immune cells, including macrophages, to release inflammatory factors, so that liver cells can undergo apoptosis and necrosis. D-GalN can inhibit the synthesis of biological macromolecules represented by RNA and protein by consuming uridine triphosphate in the liver, causing liver inflammation and diffuse necrosis of liver cells. With the synergistic effect of LPS and D-GalN, liver cells of experimental animals died in a large amount of time, and liver physiological functions were severely impaired. In this experiment, the 24-hour survival rate of mice in the model group was 0%. The 24-hour survival rate of mice with blueberry leaf total flavonoids was significantly improved, and the degree of liver cell damage was reduced. Serum ALT and AST levels were significantly lower than in the model group. The above results show that the total flavones of blueberry leaves can protect liver cells and prevent liver damage.
  • TNF- ⁇ , IL-1 ⁇ and IL-6 are inflammatory factors secreted by liver macrophages. Inflammatory factors such as TNF- ⁇ , IL-1 ⁇ , and IL-6 act on hepatocyte surface receptors on the one hand, causing necrosis of hepatocytes, and on the other hand, activate the intracellular NF- ⁇ B signaling pathway, which positively increases its release. Make liver cells worse.

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Abstract

本发明公开了蓝莓叶总黄酮在制备防治肝损伤的药物中的应用，特别是在急性肝损伤。本发明的发明人通过采用LPS/D-GalN诱导小鼠急性肝损伤作为模型，研究蓝莓叶总黄酮对急性肝损伤的预防与保护作用，发现蓝莓叶总黄酮组对小鼠24h生存率明显提高，肝细胞受损程度减少，血清ALT和AST水平显著低于模型组。此外，还显示模型肝组织中TNF-α、IL-1β及IL-6含量较正常对照组显著升高，而这一现象能被蓝莓叶总黄酮有效抑制，提示抑制肝脏炎性反应在蓝莓叶总黄酮的肝细胞保护活性中扮演了重要角色。

Description

蓝莓叶总黄酮的新应用 技术领域
本发明属于药物领域，具体是涉及一种蓝莓叶总黄酮的新应用。
背景技术
黄酮类化合物(flavonoids)是蓝莓叶中主要的活性成分，是一类存在于自然界的、具有2-苯基色原酮(结构的化合物。它们分子中有一个酮式羰基，第一位上的氧原子具碱性，能与强酸成盐，其羟基衍生物多具黄色，故又称黄碱素或黄酮。黄酮类化合物在植物体中通常与糖结合成苷类，小部分以游离态(苷元)的形式存在。绝大多数植物体内都含有黄酮类化合物，它在植物的生长、发育、开花、结果以及抗菌防病等方面起着重要的作用。
黄酮类化合物属植物次生代谢产物。黄酮类化合物是以黄酮(2-苯基色原酮)为母核而衍生的一类黄色色素，其中包括黄酮的同分异构体及其氢化和还原产物，也即以C6-C3-C6为基本碳架的一系列化合物。黄酮类化合物在植物界分布很广，在植物体内大部分与糖结合成苷类或碳糖基的形式存在，也有的以游离形式存在。
黄酮类化合物的作用非常广泛，例如有些黄酮类化合物具有护肝、解肝毒、抗真菌、治疗急、慢性肝炎、肝硬化及抗自由基和抗氧化作用。除此之外，黄酮类化合物还具有与植物雌激素相同的作用。在畜牧业动物生产上，黄酮类化合物的应用能显著提高动物生产性能，提高动物机体抗病力，改善动物机体免疫机能。
此外，机体的抗炎反应、受体识别和免疫反应等都与黄酮类化合物结构密切相关。内毒素所致的肝损伤在临床上尚缺乏有效的治疗手段，而氧化应激和内毒素介导的炎症反应与其有密切联系。目前有报道蓝莓对酒精引起的肝损伤，或者 脂肪肝等有一定的预防和保护作用。而蓝莓叶和蓝莓中的化学成分相似，并且含量远远超过蓝莓果实。但是蓝莓叶是否对LPS/D-GalN诱导的肝损伤起到保护作用未见报道，本申请采用LPS/D-GalN诱导小鼠急性肝损伤作为模型，研究蓝莓叶总黄酮对急性肝损伤的预防与保护作用并尝试探讨其机制。
发明内容
本发明的目的之一是提供一种蓝莓叶总黄酮的应用。
实现上述目的的技术方案如下。
蓝莓叶总黄酮在制备防治肝损伤的药物中的应用。
在其中一个实施例中，所述肝损伤为急性肝损伤。
在其中一个实施例中，所述肝损伤为LPS/D-GalN诱导小鼠的急性肝损伤。
在其中一个实施例中，所述蓝莓叶总黄酮由以下制备方法得到：
(1)将蓝莓叶打粉称重，加入石油醚回流提取，过滤，除去滤液，收集滤渣，加入70±5％乙醇回流提取，过滤，取滤液弃滤渣，将滤液旋转蒸发浓缩至流浸膏状；
(2)经大孔树脂柱纯化，即得。
在其中一个实施例中，步骤(1)：将蓝莓叶打粉称重，加入20±2倍的石油醚回流提取0.5±0.05小时，过滤，除去滤液，收集滤渣，加入相等体积的70±5％乙醇回流提取1.5±0.5小时，过滤，取滤液弃滤渣，将滤液旋转蒸发浓缩至流浸膏状。
在其中一个实施例中，步骤(2)：大孔树脂用95±1％己醇浸泡一天后装柱，再用95±1％的乙醇淋洗至无白色，分别5±0.5％盐酸和2±0.5％氢氧化钠浸泡洗至中性；将所述流浸膏湿法上样过大孔树脂柱，分别使用70±1％乙醇和95±1％乙 醇冲柱子，收集70±1％乙醇收集液，得蓝莓叶总黄酮。
本发明的发明人通过采用LPS/D-GalN诱导小鼠急性肝损伤作为模型，研究蓝莓叶总黄酮对急性肝损伤的预防与保护作用，发现蓝莓叶总黄酮组对小鼠24h生存率明显提高，肝细胞受损程度减少，血清ALT和AST水平显著低于模型组。此外，还显示模型肝组织中TNF-α、IL-1β及IL-6含量较正常对照组显著升高，而这一现象能被蓝莓叶总黄酮有效抑制，提示抑制肝脏炎性反应在蓝莓叶总黄酮的肝细胞保护活性中扮演了重要角色。
本发明提供了蓝莓叶总黄酮具有保护肝细胞、对抗肝损伤的作用。特别是蓝莓叶总黄酮对LPS/D-GalN诱导小鼠的急性肝损伤具有明确的预防与保护作用，其作用机制可能是抑制氧化应激反应及炎症反应的发生，从而预防和阻止肝损伤。
附图说明
图1为蓝莓叶总黄酮对急性肝损伤小鼠肝脏组织病理学的影响(HE，×200)，其中，A.正常对照组；B.模型组；C.联苯双酯组；D.蓝莓叶总黄酮低剂量组；E.蓝莓叶总黄酮中剂量组；F.蓝莓叶总黄酮高剂量组；
图2为蓝莓叶总黄酮对LPS/D-GalN致急性肝损伤小鼠血清ALT、AST水平的影响；
图3为蓝莓叶总黄酮对LPS/D-GalN致急性肝损伤小鼠肝组织中IL-1β、IL-6、TNF-α含量的影响。
具体实施方式
除非另有定义，本发明所使用的所有的技术和科学术语与属于本发明的技术 领域的技术人员通常理解的含义相同。本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不用于限制本发明。本发明所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
为了便于理解本发明，下面将参照相关附图对本发明进行更全面的描述。附图中给出了本发明的较佳实施例。但是，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
实施例1
1材料与仪器
1.1实验动物
SPF级昆明小鼠48只，雄性，体重18-22g，购自南方医科大学实验动物研究中心，许可证编号为SCKX(粤)2011-0015。实验动物每日自由摄取去离子水和标准词料，实验开始前适应性饲养一周。
1.2药物与试剂
脂多糖购自广州赛国生物科技有限责任公司；氨基半乳糖(D-GalN)购自东京化成工业株式会社(Tokyo Chemical Industry)；
小鼠肿瘤坏死因子-α(TNF-α)、白细胞介素1β(IL-1β)、白细胞介素-6(IL-6)含量测定试剂盒均购自上海源叶生物科技公司。
1.3主要仪器
高速冷冻离心机(Zokia HC-3018R，安徽中科中佳科学仪器公司)；电子天平(PL602-S，上海梅特勒-托利多国际贸易有限公司)，旋转蒸发器(GC03，上海申生科技有限公司)，冷却水循环装置(CA-1111，上海爱朗仪器有限公司)， 水循环真空泵(A-1000S，上海埃朗科技国际贸易有限公司)，数控超声波清洗器(KH-500DB，昆山禾创超声仪器有限公司)，Thermo FC酶标仪(美国Thermo Fisher Scientific公司)，石蜡包埋机(JB-P5，武汉俊杰电子有限公司)，切片机(RM2016，上海徕卡仪器有限公司)，组织摊片机(KD-P，浙江省金华市科迪仪器设备有限公司)，正置光学显微镜(NIKON ECLIPSE CI，日本尼康)，成像系统(NIKON DS-U3，日本尼康)。
2实验方法
2.1蓝莓叶总黄酮的制备
将蓝莓叶打粉称重，加入20倍的石油醚回流提取0.5小时，过滤，除去滤液，收集滤渣，加入相等体积的70％乙醇回流提取1.5小时，过滤，取滤液弃滤渣，将滤液旋转蒸发浓缩至流浸膏状。
大孔树脂用95％己醇浸泡一天后装柱，再用95％的乙醇淋洗至无白色，分别5％盐酸和2％氢氧化钠浸泡洗至中性。上述流浸膏湿法上样过大孔树脂柱，分别使用70％乙醇和95％乙醇冲柱子，收集不同乙醇浓度梯度的溶液。70％乙醇收集液为蓝莓叶总黄酮。
对蓝莓叶总黄酮的测定如下：
采用比色测定方法。蓝莓叶总黄酮与去离子水在试管中混匀，加入5％的亚硝酸钠溶液0.5mL，放置5min后加入10％的硝酸铝溶液0.5m L，摇匀，6min后再加入1mol/L的氢氧化钠溶液4mL，摇匀，15min后用分光光度计在波长510nm处进行测定。以芦丁对吸光度的标准曲线作为对照，测定除去空白后混合液的吸光度。得到蓝莓叶总黄酮的纯度为83.6％。
将70％乙醇收集液浓缩(蓝莓叶总黄酮)，低温真空烘干(60℃)后，刮取干燥粉末称重。分别用蒸馏水混悬五种不同浓度的药液，分别为：800mg/mL(超 高剂量)；400mg/mL(高剂量)；200mg/mL(中剂量)；100mg/mL(低剂量)；50mg/mL(超低剂量)。
将36只小鼠随机分成5组，即空白生理盐水组，超高剂量组，高剂量组，中剂量组，低剂量组，超低剂量组。不同浓度蓝莓叶总黄酮混悬液给一次性对小鼠进行灌胃，1mL/20g，观察小鼠在14d死亡情况。
2.3动物的给药及造模
48只小鼠随机分成6组，即空白对照组、模型组、阳性组(联苯双酯)及蓝莓叶总黄酮400、200、100mg/mL组(依照小鼠急性毒性实验结果确定)，灌胃给药，药物组给1mL每只蓝莓叶总黄酮混悬液和联苯双酯滴丸5mg/kg，空白对照组和模型组给予等体积生理盐水，每天一次，连续7天。最后一次给药两个小时以后，除正常对照组外其余的各组小鼠腹腔注射LPS(8mg/kg)和D-GalN(800mg/kg)制备急性肝损伤模型。
2.4指标测定
2.3.1观察小鼠行为，存活数量，确定有效剂量。
2.3.2血清肝功能活性检测
在小鼠濒临死亡状态下(存活小鼠10h后)，摘眼球取血，分离血清。3小时内检测肝功能血清AST与ALT水平。
2.3.3肝脏形态学观察
取小鼠肝脏右叶组织，放入10％甲醛中固定浸泡，常规石蜡包埋、切片、HE染色，光学显微镜观察肝组织切片病理学变化。
2.3.3.1肝组织石蜡切片制作
1)固定：小鼠处死后，将小鼠肝右叶剪下，用生理盐水冲洗干净后，使用滤纸把多余的水分吸干，再放入10％中性福尔马林溶液，固定24h以上。24h 后需更换新的10％中性福尔马林。
2)修剪与浸洗：将固定好的右叶修剪，先用流水冲洗，确保把福尔马林清洗干净后再放入双蒸水中浸洗2h。
3)脱水与透明：首先应配置一系列浓度的酒精。组织块在不同浓度的酒精内，其水分可被缓慢替换，最后组织块结构可变结实硬化，有利于组织的透明以及后续石蜡的浸润。把组织放入70％酒精浸泡12h，再放入80％酒精浸泡6h，85％酒精浸泡1h，90％酒精浸泡1h，95％酒精浸泡1h时。随即放入无水乙醇Ⅰ浸泡30min，换到无水乙醇Ⅱ浸泡30min，接着是无水乙醇Ⅲ浸泡30min。最后放入二甲苯Ⅰ浸泡20min和二甲苯Ⅱ20min。
4)浸蜡：首先配置一系列所需浓度梯度的二甲苯和石蜡混合溶液。二甲苯可溶于酒精，也可溶于石蜡。该步骤的目的是逐步将组织块中二甲苯用石蜡替换出来，使石蜡浸入到组织间隙中。首先将上述脱水后的组织浸泡于已经提前加热融化的50％二甲苯与石蜡混合液中，浸润时间为3min；随后依次浸入提前融化好的石蜡Ⅰ、石蜡Ⅱ中各30min；最后浸入石蜡Ⅲ中(需维持温度45℃)90min。
5)包埋：折叠一个小纸盒，将熔化了的石蜡倒入纸盒内，然后用加温的镊子将经过透明化的右组织从杯蜡中取出，迅速放入纸盒内溶解的石蜡中，保证石蜡可淹没组织，若不足应补满石蜡。随后放置在室温待其冷却凝固。此时组织硬化完全，则可切薄片。
6)切片：首先用锋利的刀片将含样本组织的蜡块切成方形，小心地切去多余石蜡。切修时注意，蜡块四边和要切的那一面要切平成矩形，否则，切片时切下的切片蜡带会发生卷曲。修整后的石蜡块置于切片机上，即可进行切片。注意切片不宜过厚，否则染色效果不佳，我们实验中的组织切片约厚4-5μm。
7)贴片：由于切片时容易出现褶皱，所以我们需要把切片放入温水池里， 使切片平整。用来贴片的载玻片在使用前必须保证使用多聚赖氨酸进行防脱片处理。贴片过程应在温水(38～42℃)中进行，将切下的薄片悬浮在温水池里，随后利用载玻片与薄片的张力将薄片贴附到载玻片上。
8)烤片：将切好的片子置于玻片架上，先放置于室温自然晾干。1天后，移至40℃的烘箱内，每隔1h后将温度调高5℃，直到65℃，然后维持1h。最后放置于室温内使其温度平衡，即可取出放入玻片盒中，标记好分组和时间，室温保存备用。
2.3.3.2HE染色
苏木精-伊红染色法(hematoxylin-eosin staining)，简称HE染色法，是动物实验病理检测的常用手段，同时也是石蜡切片染色技术中常用方法。苏木精染液是一种碱性染色剂，其可使细胞胞核内染色质与细胞质内的核糖体呈现紫蓝色；伊红是一种酸性染色剂，其可使细胞质和细胞外基质呈红色。
HE染色的步骤如下:
1)脱蜡至水：为了把组织间隙中的石蜡去除，首先将含有组织的载玻片分别浸入二甲苯Ⅰ、Ⅱ、Ⅲ中各5min。随后放入不同浓度的酒精当中，以便把组织间隙中的石蜡替换成水，具体如下：无水乙醇Ⅰ5min，无水乙醇Ⅱ5min，95％酒精5min，90％酒精5min，80％酒精5min，最后放入水中。
2)染色：按照以下的步骤进行组织着色：苏木精10min；自来水洗5秒钟；1％盐酸70％酒精对组织进行分化，20秒钟；在流水下冲洗5～10min；0.5％伊红染90秒钟。
3)脱水与透明：在上述操作中，组织块已被染色完毕。接着需要把组织块中的水分去除。具体步骤如下：置于80％酒精5min；90％酒精5min；95％酒精5min；无水乙醇Ⅰ5min；无水乙醇Ⅱ5min；无水乙醇Ⅲ5min；二甲苯Ⅰ5 min；二甲苯Ⅱ5min；二甲苯Ⅲ5min。
4)最后在每个载玻片上滴1滴中性树脂，小心地盖上盖玻片，在室温风干，待片子风干后则显微镜下观察并拍照记录。
2.3.4肝组织的生化分析
将取出的小鼠肝组织低温匀浆，离心取上清，按照试剂盒说明要求检测其TNF-α、IL-6、IL-1β水平。
2.5统计学分析
实验数据用SPSS统计软件进行分析。采用双尾t检验进行两组样本均数比较分析，组间比较采用单因素方差分析，以P＜0.05为差异有统计学意义。
3结果
表1 对D-GalN/LPS处理小鼠存活率的影响
Table 1 Effect on survival rate of D-GalN/LPS-treated mice
Figure PCTCN2019074553-appb-000001
蓝莓叶总黄酮对小鼠急性毒性实验结果见表1。结果表明，除了蓝莓叶总黄酮超高浓度(800mg/mL)对小鼠存在一定毒性反应外，其余剂量对小鼠均无明显毒性反应，故最终我们选择了400mg/mL作为蓝莓叶总黄酮高浓度进行接下来的实验，其中高、中、低蓝莓叶总黄酮剂量分别为400、200、100mg/mL。
3.2蓝莓叶总黄酮对LPS/D-GalN致急性肝损伤小鼠肝脏形态及组织病理学的影响
正常对照组小鼠肝脏外观色淡，边缘滑润，质韧；镜下见细胞大小均一，排列整齐，结构完整，细胞核位于肝细胞中央。模型组肝脏外观颜色暗红，有明显肿胀、淤血；镜下见肝组织病理损伤严重，肝细胞变性、胞浆内充满小空泡，且有明显的肝脏出血和炎症细胞浸润。经蓝莓叶总黄酮给药后，小鼠肝脏的外观及颜色呈现明显的好转，病理检查显示肝细胞间隙减小，高剂量组充血和炎细胞浸润状况明显改善，近于恢复正常。见图1。
3.3蓝莓叶总黄酮对LPS/D-GalN致急性肝损伤小鼠血清ALT、AST水平的影响
表2蓝莓叶总黄酮对LPS/D-GalN致急性肝损伤小鼠血清ALT、AST水平的影响(
Figure PCTCN2019074553-appb-000002
n＝10)
Table2 ALT,AST Levels and Liver Index of LPS/D-GalN-induced Acute Liver Injury in Blueberry Leaves(
Figure PCTCN2019074553-appb-000003
n＝10)
组别 浓度(mg/mL) AST(IU/L) ALT(IU/L)
空白组   61.6±3.28634 46.4±5.85662
模型组   692.4±66.3649** 454.6±46.48978**
联苯双酯 100 315.6±15.32319** 88.4±3.20936**
低剂量组 100 630.8±34.010293 424.6±8.82043
中剂量组 200 487.2±24.47856** 259.6±8.29457**
高剂量组 400 378.2±28.77846** 106.2±7.32803**
**P<0.01，和模型相比
结果如表2和图2所示，与空白组比较，模型组小鼠血清ALT、AST水平均显著升高(P＜0.01)；与模型组比较，阳性药组小鼠血清ALT、AST水平均显著降低(P＜0.01)，同时蓝莓叶总黄酮高浓度组(400mg/mL)和中浓度组(200mg/mL)ALT、AST水平均显著降低(P＜0.01)，而蓝莓叶总黄酮低浓度组(100mg/mL)对ALT和AST水平无明显影响。
3.4蓝莓叶总黄酮对LPS/D-GalN致急性肝损伤小鼠肝组织IL-1β、IL-6、TNF-α含量的影响结果
表3蓝莓叶总黄酮对LPS/D-GalN致急性肝损伤小鼠肝组织中IL-1β,IL-6,TNF-α含量的影响(
Figure PCTCN2019074553-appb-000004
n＝10)
Figure PCTCN2019074553-appb-000005
**P<0.01，*P<0.05，和模型组相比
如表3与图3所示，相比空白组，模型组小鼠肝组织IL-1β、IL-6、TNF-α含量均显著升高(P＜0.01；P＜0.01；P＜0.01)；与模型组比较，联苯双酯组小鼠肝组织IL-1β、IL-6、TNF-α水平均显著降低(P＜0.01；P＜0.01；P＜0.01)； 与模型组比较，蓝莓叶总黄酮高、中、低浓度组IL-1β水平均显著降低(P＜0.01；P＜0.01；P＜0.01)，且呈剂量依赖关系，同时，蓝莓叶总黄酮高、中、低浓度组IL-6水平均显著降低(P＜0.01；P＜0.01；P＜0.05)，蓝莓叶总黄酮高、中、低浓度组TNF-α水平均显著降低(P＜0.01；P＜0.05；P＜0.01)。
本实验结果表明，蓝莓叶总黄酮可延长实验小鼠的生存率和存活时间，改善肝脏的外观和组织形态；与模型组比较，经蓝莓叶总黄酮干预后，小鼠血清中AST、ALT水平和肝脏指数均显著降低，TNF-α、IL-1β和IL-6含量均显著降低。腹腔注射LPS以及D-GalN是构建小鼠急性肝损伤模型的较为便捷的方法。
LPS是革兰氏阴性细菌所分泌内毒素的主要成分，可通过刺激包括巨噬细胞在内的免疫细胞释放炎症因子，从而使肝细胞可发生凋亡与坏死。D-GalN可通过消耗肝脏中的三磷酸尿苷，抑制以RNA和蛋白质为代表的生物大分子合成，引起肝脏炎症反应和肝细胞弥漫性坏死。在LPS和D-GalN的协同作用下，实验动物的肝细胞在短时间内大量死亡，肝脏生理功能严重受损。在本实验中，模型组小鼠24h的生存率为0％，蓝莓叶总黄酮小鼠24h生存率明显提高，肝细胞受损程度减少，血清ALT和AST水平显著低于模型组。以上结果表明蓝莓叶总黄酮具有保护肝细胞、对抗肝损伤的作用。
TNF-α、IL-1β和IL-6是由肝脏巨噬细胞分泌的炎症因子。TNF-α、IL-1β、IL-6等炎症因子一方面作用于肝细胞表面受体，引起肝细胞坏死，另一方面激活细胞内NF-κB信号通路，正反馈地进一步增加其释放量，使肝细胞损伤加剧。本申请结果显示模型肝组织中TNF-α、IL-1β及IL-6含量较正常对照组显著升高，而这一现象能被蓝莓叶总黄酮有效抑制，提示抑制肝脏炎性反应在蓝莓叶总黄酮的肝细胞保护活性中扮演了重要角色。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上 述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (6)

  1. 蓝莓叶总黄酮在制备防治肝损伤的药物中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述肝损伤为急性肝损伤。
  3. 根据权利要求2所述的应用，其特征在于，所述肝损伤为LPS/D-GalN诱导小鼠的急性肝损伤。
  4. 根据权利要求1-3任一项所述的应用，其特征在于，所述蓝莓叶总黄酮由以下制备方法得到：
    (1)将蓝莓叶打粉称重，加入石油醚回流提取，过滤，除去滤液，收集滤渣，加入70±5％乙醇回流提取，过滤，取滤液弃滤渣；
    (2)将滤液收集旋转蒸发，得到的溶液在装有大孔树脂的过滤柱中过滤，用70％乙醇过柱，收集黄色溶液即为总黄酮与乙醇混合溶液；将收集到的混合溶液旋转蒸发得到液体总黄酮冷冻干燥24h得总黄酮粉末，粉末呈褐黄色。
  5. 根据权利要求4所述的应用，其特征在于，步骤(1)：将蓝莓叶打粉称重，加入20±2倍的石油醚回流提取0.5±0.05小时，过滤，除去滤液，收集滤渣，加入相等体积的70±5％乙醇回流提取1.5±0.5小时，过滤，取滤液弃滤渣，将滤液旋转蒸发浓缩至流浸膏状。
  6. 根据权利要求4所述的应用，其特征在于，步骤(2)：大孔树脂用95±1％己醇浸泡一天后装柱，再用95±1％的乙醇淋洗至无白色，分别5±0.5％盐酸和2±0.5％氢氧化钠浸泡洗至中性；将所述流浸膏湿法上样过大孔树脂柱，分别使用70±1％乙醇和95±1％乙醇冲柱子，收集70±1％乙醇收集液，得蓝莓叶总黄酮。
PCT/CN2019/074553 2018-09-11 2019-02-02 蓝莓叶总黄酮的新应用 WO2020052196A1 (zh)

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CN103211851A (zh) * 2013-05-09 2013-07-24 南京紫玉蓝莓科技有限公司 一种对酒精性肝损伤有保护作用的蓝莓叶提取物
AU2015101749A4 (en) * 2015-12-03 2016-01-14 Macau University Of Science And Technology Method of treating or preventing liver injury induced by acetaminophen
CN109078038A (zh) * 2018-09-11 2018-12-25 南方医科大学 蓝莓叶总黄酮的新应用

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CN103211851A (zh) * 2013-05-09 2013-07-24 南京紫玉蓝莓科技有限公司 一种对酒精性肝损伤有保护作用的蓝莓叶提取物
AU2015101749A4 (en) * 2015-12-03 2016-01-14 Macau University Of Science And Technology Method of treating or preventing liver injury induced by acetaminophen
CN109078038A (zh) * 2018-09-11 2018-12-25 南方医科大学 蓝莓叶总黄酮的新应用

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