青蒿素等4种中草药抑制出血热病毒的实验研究

提要: 就青篙素、甘草甜素、鱼腥草、柴胡4种中草药注射液对流行性出血热病毒(EHFV)的抑制作用进行了动物实验观察。结果显示：青蒿素及甘草甜素对EHFV具有较强的抑制作用，鱼腥草和柴胡对EHFV也具有一定的抑制作用。

青蒿，甘草，鱼腥草和柴胡均为常用的中草药。已有实验发现，青蒿的提取物青蒿素具有杀疟原虫、诱生干扰素[1]的作用；甘草的提取物甘草甜素也有免疫调节及诱生 干扰素[2]的作用；鱼腥草具有抗菌和利尿作用；柴胡具有解热作用。现已广泛应用上述中草药于临床治疗疟疾、肝炎[3]等疾病。甘草和柴胡试用于治疗艾滋病，并取得一定成效[4]。唯此等中草药或其提取物对流行性出血热病毒(EHFV)的抑制作用尚未见报道。本文就此进行了动物实验观察，现将结果报告如下。

RESEARCH ON FOUR CHINESE HERBAL MEDICINES, INCLUDING ARTEMETHER, TO EXAMINE THEIR INHIBITORY EFFECTS ON HEMORRHAGE FEVER VIRUS

Abstract: In order to study their inhibitory effects on epidemic hemorrhage fever virus (EHFV), animal experiments and observations were carried out on four Chinese herbal medicine injections: artemether, glycyrrhizin, houttuynin and bupleurum. Results: Artemether and glycyrrhizin showed fairly strong inhibitory effects on EHFV, while houttuynin and bupleurum only showed some inhibitory effects on EHFV.

Artemisia annua. L, Glycyrrhiza uralensis Fish, Houttuynia cordata Thunb and Bupleurum chinense DC are all common Chinese herbal medicines. Research has showed that: Artemether, the extract of A.annua. L, has plasmodicidal and interferon-

inducible[1] effects; glycyrrhizin, the extract of G.uralensis Fish has immunoregulation and interferon-inducible[2] effects; H.cordata Thunb has anti-biotic and diuretic effects; B.chinense DC has an antipyretic effect. The Chinese herbal medicines described above have been widely used for clinical treatments for diseases such as dysentery and hepatitis[3]. Positive results have been achieved in treatment trials for AIDS using G.uralensis Fish and B.chinense DC.[4] Only the inhibitory effects of these Chinese herbal medicines on epidemic hemorrhage fever virus (EHFV) have yet to be reported. Therefore, relevant animal experiments and observations were carried out and the results are reported as follows.

RGD-Functionalised Liposome for Tumour Targeting

Liposomal vectors have been widely studied for targeted gene and drug delivery to tumour cells. In vivo, these systems encounter many problems including serum stability, tumour localisation, cell internalisation, endosomal escape etc.

Modification or shielding of the liposome surface helps to inhibit non-specific interaction with blood components and enables an extended circulation of the vectors after systemic administration. This is achieved with a hydrophilic polymer of which polyethylene glycol (PEG) has been found to be best.

Targeting cell-surface receptors is a very attractive concept to achieve specific binding and internalisation of the liposome. To this end, cell-binding ligands are displayed from the surface of the liposome. The common denominator for these ligands is that their cell receptor targets are over-expressed in tumours.

We have developed a post-modification methodology for coupling cell or tissue targeting moieties onto liposomes via chemioselective oxime bond formation. Thus, liposomes comprising a highly nucleophilic aminoxy-functionalised lipid are formulated. The aminoxy lipid readily reacts with aldehyde functionalised targeting ligands in aqueous conditions to form a stable oxime bond conjugated targeted liposome.

A peptide displaying the Arginine-Glycine-Aspartic acid (RGD) sequence was selected as the targeting ligand to validate our post-coupling methodology. A cyclic RGD peptide conjugate has been successfully synthesised using a combination of solution- and solid-phase synthesis.

Additionally, control Arginine-Glycine-Glutamic acid (RGE) peptide and polyethylene glycol (PEG) linker conjugates have been prepared. All the ligands terminate in an aldehyde functional group separated from the targeting moiety by a PEG linker. The couplings between the RGD conjugate and aminoxy liposomes in water at pH 4 were shown to be efficient, simple and reproducible.

In vitro, Human Umbilical Vein Endothelial Cells (HUVEC) cell uptake studies show a definite targeted uptake of the RGD-targeted liposomes relative to appropriate control liposomes.

含RGD肽的肿瘤靶向脂质体

脂质体载体已被广泛应用于针对肿瘤细胞的基因及药物传递的研究。在体内，这类系统存在着血清稳定性、肿瘤靶向、细胞內化和核内体逃逸等一系列问题。

改性修改或保护脂质体表面有助于抑制其与血浆成分的非特异性相互作用，并延长载体在系统导入后的循环期。上述可通过使用亲水性聚合物而实现。其中，目前以聚乙二醇（PEG）为首选。

以细胞表面受体为靶向从而实现特异性结合和脂质体内化是一个极有吸引力的概念。为此，细胞结合配体被加置于脂质体表面。此类配体的共性是，其靶细胞受体在肿瘤表面呈过度表达。

我们研制了一种后期修饰的方法：通过具化学选择性的肟键形成反应，将细胞或组织的靶向成分加置于脂质体表面，由此配制成含高度亲核氨氧基类脂质的脂质体。 水相中，含氨氧基的类脂质迅速地与含醛基的靶向配体反应，形成一种含稳定的肟键共轭的靶向脂质体。

一种含精氨酸-甘氨酸-天冬氨酸（RGD）序列的肽被选定为靶向配体，以验证我们的后期偶合法。一种RGD环肽共轭物由液相及固相有机合成成功制成。

此外，精氨酸-甘氨酸-谷氨酸（RGE）肽对照物及聚乙二醇（PEG）连接共轭物也被制备而成。所有的配体均以一醛基宫能团为末端，并由一PEG连接部分接于靶向成分。

在pH值为4的水相中，RGD肽共轭物与含氨氧基脂质体的偶合反应被证明是高效、简便、易重复的。 人脐静脉血管内皮细胞（HUVEC）的体外摄取研究表明，相对于适当的对照脂质体， RGD肽靶向脂质体的摄取明确地显示靶向性。