How to make hypericin water-soluble

Hypericin, isolated from Hypericum perforatum, is an effective photodynamic substance as demonstrated by various studies. Practical forms of applications of hypericin solutions for systemic use and introduction into body cavities are, however, lacking. We developed an aqueous solution of hypericin non-covalently bound to polyvinylpyrrolidone (PVP). PVP is a poly-N-vinylamide of various degrees of polymerization and forms of intermolecular crosslinks suitable for diagnostic and therapeutic applications. We used PVP (molecular weights of PVP between 10 kD and 40 kD) as a complex forming agent to prepare hypericin for photodynamic therapy and diagnostics. In pure water, hypericin forms aggregates which are non-soluble and non-fluorescent. The hypericin-PVP complex binds more than 1000 mg of hypericin in presence of 100 g PVP or less and is soluble in 1 liter of pure water. Aqueous complex solutions of hypericin-PVP display a characteristic absorption spectrum and fluorescence emission band around 600 nm wavelength. Varying concentrations of hypericin do not cause a blue- or red-shift in the absorption maximum at 595 nm. Excitation at 200 nm to 500 nm leads to emission at 590 nm; a property conducive to diagnostic investigations both in vitro and in vivo. Furthermore, hypericin-PVP exhibits high photostability in the presence of oxygen and broad band light which ensures reproducible photodynamic therapy and diagnosis. CONCLUSION: Hypericin forms liquid molecular chromophore complexes in water when bound to PVP thus allowing investigations in biological media.     

金丝桃属植物中的金丝桃素:化学、植物来源和生物活性(英文)

金丝桃素是金丝桃属植物中一种重要的次生代谢产物,1830年首次被正式报道,它是一种天然光敏型的二蒽酮类化合物。金丝桃素具有抗病毒,抗抑郁、抗肿瘤、抗菌、抗氧化等广泛的生物和药理活性,并用于光动力治疗。在过去的几十年中越来越多的证据表明金丝桃素具有很大的药用潜力和临床价值。本篇综述了金丝桃素的化学、植物来源和生物活性。     

Hypericin lights up the way for the potential treatment of nasopharyngeal cancer by photodynamic therapy

Photodynamic therapy (PDT) involves the administration of a photosensitizer followed by light irradiation with a specific wavelength, giving rise to irreversible tissue destruction. Hypericin, a herbal extract derived from Hypericum perforatum or St. John's Wort, has minimal toxicity but exhibits potent photo-damaging effects in the presence of light. Hypericin is known to generate a high yield of singlet oxygen and other reactive oxygen species that are associated with photo-oxidative cellular damage. The application of PDT with hypericin for the treatment of cancers such as recurrent mesothelioma and skin cancer has been validated in clinical trials. This mini-review focuses on the investigative studies of hypericin as a potential photodynamic agent in the treatment of nasopharyngeal cancer (NPC) in in vitro and in vivo models. NPC is an enigmatic tumor with a multifactorial etiology and a high incidence in the populations of Southern China.     

Cellular mechanisms and prospective applications of hypericin in photodynamic therapy

During the last decades, Photodynamic Therapy (PDT) has been established as a powerful alternative approved by health agencies of several countries for treatment of various malignant and some non-malignant diseases. PDT makes use of the light-induced destruction of target cells by formation of cytotoxic products in the presence of a photosensitizing agent and oxygen. The light-dependent tumor destructive properties of Hypericin have drawn attention to its promising application as a photosensitizer in the frame of PDT. Hypericin is a naturally occurring secondary metabolite in plants of the Hypericum genus, with Hypericum perforatum (St. John's wort) as it is a commonly known representative. This review focuses on the cellular mechanisms of Hypericin-based phototoxicity and provides an outlook for future application of Hypericin as a fluorescing and photosensitizing agent for diagnosis and treatment of cancerous diseases, respectively.     

Hypericin--a new antiviral and antitumor photosensitizer: mechanism of action and interaction with biological macromolecules

Hypericin, a naturally occurring pigment, is found in certain species of plants from the genus Hypericum, the most common of which is Saint John's Wort (Hypericum perforatum). Recent interest in hypericin is provoked by the discovery that it possesses extremely high toxicity towards certain viruses notably the class of enveloped viruses that includes human immunodeficiency virus (HIV) and toward tumors, and that this toxicity absolutely requires light. Consequently, a detailed understanding of the interaction of hypericin with cellular components (membranes, proteins, nucleic acids) and with light is of fundamental biological importance. The antiviral and antineoplastic activities of hypericin and its derivatives and its mode of action have been widely studied, in the last two decades. This review is focused on the results obtained in the study of hypericin heteroassociations with biological macromolecules, DNA and human serum albumin in particular. An alternative type of the hypericin photosensitizing activity associated with its ability to produce a photogenerated pH drop is discussed that and discussed in connection with its potential application in photodynamic therapy. In the review, it is also presented how an interdisciplinary approach supported by sophisticated techniques of optical spectroscopy and molecular modeling can be effectively applied for the identification of the specific binding sites of the drug in some biomacromolecules as well as for the determination of the physico-chemical mechanism'of the biological activity of hypericin.     

金丝桃素合酶基因的快速克隆及功能鉴定

金丝桃素合酶能催化大黄素生成金丝桃素,根据已发表的金丝桃素合酶的基因序列,设计了6对引物,通过连续重叠PCR快速克隆得到了金丝桃素合酶基因hyp-1。构建含hyp-1基因的原核表达载体pET32ahyp,将该载体导入大肠杆菌进行诱导表达。SDS-PAGE结果表明,hyp-1基因在大肠杆菌中获得表达;Western blot结果表明,重组的Hyp-1蛋白具有特异的免疫活性,表明hyp-1基因在E.coli中得到了表达。酶促反应表明,Hyp-1确实能催化大黄素形成金丝桃素,上述结果表明,本研究克隆的hyp-1基因是金丝桃素合酶基因,具备催化大黄素形成金丝桃素的能力,从而为通过合成生物学技术制备金丝桃素奠定了物质基础。     

贯叶金丝桃药材中萘骈二蒽酮类成分的分光光度法和HPLC法定量分析

目的:建立测定贯叶金丝桃药材中萘骈二蒽酮类成分的紫外-可见分光光度法和HPLC法,比较两者测定结果的差异与定量可靠性。方法:紫外-可见分光光度法以金丝桃素为指标性成分,在590nm处测定。HPLC法以DiamonsilC18柱(150mm×4.6mm,5μm为色谱柱,甲醇-10mmol/L CH3COONH4水溶液(pH值为6.2)(90∶10)为流动相,流速1.0ml/min,检测波长590nm,柱温30℃。经紫外光谱与质谱确认其中主要色谱峰成分为伪金丝桃素和金丝桃素,并以伪金丝桃素、金丝桃素为对照品进行定量,萘骈二蒽酮大类成分含量以伪金丝桃素、金丝桃素含量之和来表征。结果:紫外-可见分光光度法定量金丝桃素在2.44～19.52μg/ml范围内线性关系良好(r=0.999 6,n=3),测得贯叶金丝桃药材中萘骈二蒽酮类成分以金丝桃素计为(0.990 2±0.006 0)μg/mg。HPLC法定量伪金丝桃素在0.402～8.032μg/ml范围内线性关系良好(r=0.999 5,n=3)),定量金丝桃素在0.488～9.760μg/ml范围内线性关系良好(r=0.999 7,n=3)),测得贯叶金丝桃药材中伪金丝桃素和金丝桃素含量分别为(0.387 2±0.001 4)、(0.220 2±0.000 7)μg/mg,两者的总量为(0.605 5±0.001 2)μg/mg。结论:对贯叶金丝桃药材中萘骈二蒽酮类成分的定量分析,紫外-可见分光光度法和HPLC法测定结果差异明显;即使化学结构与光谱特性相似的大类成分,其紫外-可见分光光度法的定量准确性仍需要验证与评价。     

高效液相色谱-荧光检测法测定贯叶连翘提取物中金丝桃素在小鼠体内的组织分布及药动学研究

目的建立测定贯叶连翘提取物中金丝桃素血浆浓度的高效液相色谱-荧光检测法,比较不同给药途径小鼠体内药动学特征及组织分布。方法采用甲醇：四氢呋喃：0.1mol/L磷酸盐缓冲液为流动相,激发波长315nm,检测波长590nm。对小鼠分别采用静脉注射和灌胃2种方法给予贯叶连翘提取物0.36mg/kg（以金丝桃素计）,测定给药后血浆药物浓度和各组织浓度,用3P97药动学软件对血药浓度-时间数据进行拟合。结果平均回收率≥90.5%,日内、日间相对标准差（RSD）均≤5.0%。2种方法给药后血药浓度-时间曲线均呈二室模型,消除半衰期较长,金丝桃素在肝脏浓度较高,且有一定程度的蓄积,在其余组织中药物浓度较低,消除迅速。结论金丝桃素在动物体内排泄或代谢缓慢,在肝脏的蓄积对肝炎治疗有一定意义。     

贯叶连翘中金丝桃素醇法提取工艺的改进

对现行醇法提取植物贯叶连翘中金丝桃素的生产工艺进行了研究和改进。研制出了价廉、无毒、新型的W．X．除杂剂，使生产工序大为简化。 一般经提取、除杂、调pH沉降三步，即可得到合格产品。经HPLC测定，产品中金丝桃素含量达到0．7％，远远大于国际市场要求的0．3％。同时，消除了原料中金丝桃素含量高低对产品质量的影响。     

不同方法除鞣质后贯叶金丝桃提取物中金丝桃素的含量变化

目的 考察用不同方法检测除鞣质后金丝桃素的含量变化和收率,建立贯叶金丝桃良好的除鞣质方法。方法采用明胶沉淀法、改良明胶沉淀法及碱性醇沉法除鞣质后,用高效液相色谱（HPLC）法测定贯叶金丝桃提取物中金丝桃素含量变化和收率;色谱柱为Phenomenex—C18柱（250mm×4．6mm,5μm）,流动相为甲醇-0．006mol／LNa214PO4（7：1V／V,H3P04调至pH=6．5）,流速为1．0mL／min,柱温30℃,检测波长590nm,外标法计算。结果金丝桃素的进样量线性范围为0．0194～0．7760μg（r=0．9999）,平均回收率为100．25％。RSD：1．29％（n=5）;明胶沉淀法、改良明胶沉淀法和碱性醇沉法除鞣质后金丝桃素含量分别为0．092％,0．098％和0．093％,收率分别为70．15％,85．21％和89．16％。结论改良明胶沉淀法具有鞣质去除完全、金丝桃素含量较高、损失较少的优点,且方法简便易行．可应用于贯叶金丝桃提取物除鞣质处理。     

Antagonist effect of pseudohypericin at CRF1 receptors

St. John's wort (Hypericum perforatum L.) is widely used for the treatment of mild to moderately severe depression. However, the nature of its active principles and the exact mode of antidepressant action are still unknown. It has been suggested repeatedly in preclinical and clinical studies that the content of the acylphloroglucinol hyperforin decisively contributes to the antidepressant efficacy of St. John's wort extracts. Experimental studies in vivo also indicate that the naphthodianthrone hypericin may reduce the activity of the hypothalamic-pituitary-adrenal axis. Exacerbated hypothalamic-pituitary-adrenal activity has often been associated with depressive states in patients. Corticotropin-releasing factor (CRF) seems to be a major determinant in the regulation of the hypothalamic-pituitary-adrenal activity via activation of CRF(1) receptors. In the present study, we investigated the CRF(1) receptor antagonist activity of three main constituents of St. John's wort (hypericin, pseudohypericin and hyperforin) by measuring their effect on CRF-stimulated cAMP formation in recombinant Chinese hamster ovary (CHO) cells. As a selectivity test, the compounds were also tested against calcitonin in the same cells. Of the three compounds tested, only pseudohypericin selectively antagonised CRF (K(B) 0.76 microM). Hypericin and hyperforin affected both CRF and calcitonin with similar potencies and the same type of behaviour (competitive antagonism for hypericin, noncompetitive for hyperforin). It is concluded that pseudohypericin is the only real CRF(1) receptor antagonist of the three constituents tested. In addition, evidence is provided that beside hyperforin, both pseudohypericin and hypericin are implicated in the antidepressant efficacy of St. John's wort.     

Stability of different formulations and ion pairs of hypericin.

Hypericin, solubilized in an instillation fluid consisting of an aqueous buffer supplemented with 1% plasma proteins, is currently used as a clinical diagnostic tool for the detection of superficial TCC (transitional cell carcinoma) tumors. However, the development of a sterile and stable hypericin stock formulation, excluding the presence of plasma constituents, would be an important factor in a more general clinical application of the method. Therefore, we investigated the stability of several heat sterilized hypericin formulations and ion pairs. Besides sodium hypericinate (in distilled water, in phosphate buffer, in polyethyleneglycol (PEG) 400), several other hypericinate salts (potassium, lysine, TRIS or hexylamine) were investigated. As to that, the physical appearance of different hypericin concentrates stored at 4 and 37 degrees C was investigated. Besides, after dilution into cell culture medium, the ability of hypericin remaining to accumulate in tumor cells and demonstrating photocytotoxic effects upon light irradiation was assessed. These findings suggest that PEG 400 is an excellent hypericin formulation, since it maintained the stability of the compound for at least 120 d when stored at either 4 or 37 degrees C. PEG 400 therefore is a suitable vehicle for the storage of hypericin prior to preparation of the bladder instillation solution.     

Antibiotic-free nanotherapeutics: Hypericin nanoparticles thereof for improved in vitro and in vivo antimicrobial photodynamic therapy and wound healing

Hypericin (HY) is a naturally-occurring, potent photosensitizer. However, its lipophilicity limits its therapeutic applications. Our attempt is, thus, to develop a biodegradable nanocarrier for hypericin capable of preserving its antibacterial photoactivity. Amphiphilic block copolymers were synthesized to prepare hypericin-laden nanoparticles (HY-NPs). The antimicrobial photoactivity of HY-NPs was assessed; in vitro against biofilm and planktonic cells of methicillin resistant Staphylococcus aureus (MRSA) clinical isolates and in vivo on infected wounds in rats. Nanoparticles of 45 nm in diameter ensured higher amounts of reactive oxygen species upon irradiation. HY-NPs demonstrated superior inhibition of biofilm over planktonic cells. In vivo wound healing studies in rats revealed faster healing, better epithelialization, keratinization and development of collagen fibers when HY-NPs were applied. Determination of growth factors and inflammatory mediators in the wound area confirmed superior healing potential of nanoencapsulated hypericin suggesting that hypericin can join the era of antibiotic-free antimicrobial therapy.     

Virucidal activity of hypericin against enveloped and non-enveloped DNA and RNA viruses.

Hypericin is a polycyclic anthrone first isolated from the plant St. Johnswort and was shown to have dramatic anti-retroviral activity against Friend leukemia virus and radiation leukemia virus in mice. Hypericin displayed marginal activity (IC50 = 6 micrograms/ml) against Moloney murine leukemia virus (Mo-MuLV) in vitro. Hypericin did not display selective antiviral activity against herpes simplex virus, influenza A, adenovirus, or poliovirus. The 50% cytotoxic concentration was approximately 25 micrograms/ml. When virus was incubated with hypericin before infecting cells, the drug was virucidal to all enveloped viruses tested (herpes simplex, influenza virus A, and Mo-MuLV) at concentrations of 1.56 micrograms/ml to 25 micrograms/ml. Hypericin was not virucidal to the non-enveloped viruses tested (adenovirus and poliovirus). These data indicate that the mechanism of viral inactivation for hypericin is dependent upon the presence of a viral lipid envelope. In vivo, hypericin (50 mg/ml) was effective against FLV or HSV-1 if incubated with the virus for 1 h at 37 degrees C before infecting mice, but was not effective if pre-incubated with virus for 1 h at 4 degrees C or if administered concurrently with virus.     

Radiopharmaceutical evaluation of 131I-protohypericin as a necrosis avid compound

Hypericin is a necrosis avid agent useful for nuclear imaging and tumor therapy. Protohypericin, with a similar structure to hypericin except poorer planarity, is the precursor of hypericin. In this study, we aimed to investigate the impact of this structural difference on self-assembly, and evaluate the necrosis affinity and metabolism in the rat model of reperfused hepatic infarction. Protohypericin appeared less aggregative in solution compared with hypericin by fluorescence analysis. Biodistribution data of ¹³¹I-protohypericin showed the percentage of injected dose per gram of tissues (%ID/g) increased with time and reached to the maximum of 7.03 at 24?h in necrotic liver by gamma counting. The maximum ratio of target/non-target tissues was 11.7-fold in necrotic liver at 72?h. Pharmacokinetic parameters revealed that the half-life of ¹³¹I-protohypericin was 14.9?h, enabling a long blood circulation and constant retention in necrotic regions. SPECT-CT, autoradiography, and histological staining showed high uptake of ¹³¹I-protohypericin in necrotic tissues. These results suggest that ¹³¹I-protohypericin is a promising necrosis avid compound with a weaker aggregation tendency compared with hypericin and it may have a broad application in imaging and oncotherapy.     

Hypericin induces both differentiation and apoptosis in human promyelocytic leukemia HL-60 cells

Hypericin is a unique photosensitizing plant pigment and has been separately reported to induce differentiation and apoptosis in neoplastic cells. In this study, we examined the relationship between activities to induce differentiation and apoptosis in human promyelocytic leukemia HL-60 cells, at a concentration range of 0.15 to 0.2 microM. When treated with hypericin, the cell ratio reducible of nitroblue tetrazolium was significantly increased and the cell size was enlarged by flow cytometry analysis. Hypericin also significantly increased the ratio of the cells, which were of positive alpha-naphthyl acetate esterase activity and phagocytic activity, whereas it hardly influenced the naphthol AS-D chloroacetate esterase activity in the cells, as well as 1 alpha, 25(OH)2D3 (10 nM). In addition, hypericin increased hypodiploid nuclei and caused a nucleosomal ladder. These results indicate that hypericin induces both differentiation toward monocyte/macrophage lineage and apoptosis in HL-60 cells.     

Hypericin prolongs action potential duration in hippocampal neurons by acting on K+ channels

Background and purpose:

Synaptic deficiency is generally accepted to be involved in major depression, and accordingly classic antidepressants exert their effects through enhancing synaptic efficiency. Hypericin is one of the major active constituents of extracts of St. John''s Wort (Hypericum perforatum L.) with antidepressive actions, but little is known about its therapeutic mechanisms. Our aim was to explore whether hypericin has a modulatory effect on neuronal action potential (AP) duration by acting on voltage-gated ion channels.

Experimental approach:

We used voltage-clamp and current-clamp techniques in a whole-cell configuration to study primary cultures of neonatal rat hippocampal neurones. We measured the effects of extracellularly applied hypericin on AP duration as well as on voltage-gated Na⁺, I_A and I_K currents.

Key results:

Extracellularly applied hypericin dose-dependently increased AP duration but barely affected its amplitude. Further analysis revealed that hypericin inhibited both transient I_A and delayed rectifier I_K potassium currents. In contrast, hypericin exerted no significant effect on both Na⁺ peak current and its decay kinetics.

Conclusions and implications:

Extracellularly applied hypericin increased AP duration, which might be ascribed to its effect on I_A and I_K currents. As a small increase in AP duration could lead to a dramatic increase in synaptic efficiency, our results imply that hypericin might exert its antidepressant effects by enhancing presynaptic efficiency.     

金丝桃素及其提取物对肺癌细胞SpcA1的体外杀伤效应

目的研究贯叶金丝桃中金丝桃素及其提取物对人肺癌细胞株SpcA1的体外杀伤效应。方法以发光二极管为光源，通过测定胞内荧光强度来确定细胞对金丝桃素的吸收，用显微镜观察、四甲基偶氮唑蓝（MTT）法和凋亡DNA电泳分析法研究金丝桃素及其提取物对细胞的杀伤作用。结果光活化的金丝桃素对SpcA1细胞有显著的体外杀伤效应，其抑制细胞生长的能力与金丝桃素的浓度及光照能量密切相关。结论金丝桃素及其提取物能明显抑制肺癌细胞的生长，提示金丝桃素在肿瘤治疗中应用前景良好。     

Evidence that total extract of Hypericum perforatum affects exploratory behavior and exerts anxiolytic effects in rats

Clinical trials have extensively reported the ability of Hypericum perforatum extracts to exert a significant antidepressant activity. Hypericin, the main constituent of H. perforatum extract, is no more regarded as the active principle of the antidepressant activity of the drug. Hence, the question of which constituents are involved in the basic activity of the total extract, is still waiting for an answer. In the present study we focused our attention on the potential anxiolytic activity of H. perforatum total extract, and of some pure components such as protohypericin and a fraction containing hypericin and pseudohypericin. Herein we report that the total extract of H. perforatum increases the locomotor activity in the open field and exerts anxiolytic activity in the light-dark test, whereas the single components did not show any effect. Interestingly, the anxiolytic activity of the total extract was blocked by pretreatment of rats with the benzodiazepine antagonist Flumazenil, hence suggesting an implication of benzodiazepine receptor activation in the anxiolytic effect of H. perforatum extract. Electrophysiological studies, performed to gain more information on the mechanism of action, showed that hypericin reduced the GABA-activated chloride currents, while pseudohypericin did an opposite effect. Furthermore, both hypericin and pseudohypericin inhibited the activation of NMDA receptors.