The Patterns of Secretory Structure and Their Relation to Hypericin Content in Hypericum

利用整体透明、石蜡制片和半薄切片法 ,对金丝桃属 (HypericumL .) 8组 2 0种 1变种植物的分泌结构进行了比较解剖研究 ,结果表明 :该属植物的分泌结构可分为分泌细胞团和分泌囊两种类型。但在不同植物种和不同器官内 ,分泌结构的类型和分布密度存在差异。对上述植物的提取物进行薄层层析和高效液相层析检测 ,结果表明 :具有分泌细胞团的植物器官含有金丝桃素 ;而无分泌细胞团的植物器官 ,则不含金丝桃素 ,从而证明金丝桃素由分泌细胞团合成和贮藏。在前者中 ,其金丝桃素的含量与其分泌细胞团密度成正相关。

Ultrastructure of the Multicellular Nodules in Hypericum perforatum Leaves

With the development of the multicellular nodules in the leaves of Hypericum per plastids increased both in number and volume, and some plastids degenerated and were surrounded by dark tubular elements and vesicles. Some vesicles fused with vacuoles and secreted substances into vacuoles. There are many multivesicles, multimembrane structures and osmiophilic droplets in vacuoles of nodule cells. Meanwhile, dictyosome secreted vesicles into the vacuoles. However, there is a large central vacuole completely filled with secretory materials (hypericin) in matured multicellular nodules. This suggests that the osmiophilic droplets may be a precursor of hypericin originated from the degenerated plastids. There were abundant endoplasmic reticulums and dictyosomes between the plastids and osmiophilic droplets, suggesting that they may be involved in the synthesis and/or transport of the precursor of hypericin.

Differential diagnosis of gallstones by using hypericin as a fluorescent optical imaging agent

AIM: To explore the feasibility of using hypericin as an optical imaging probe with affinity for cholesterol for differential fluorescent detection of human gallstones.METHODS: Cholesterol, mixed and pigment stones from cholecystectomy patients were incubated with hypericin or solvent. After 72 h, the stones were analysed for fluorescence(365 nm) and treated with 2-propanol/dimethyl sulfoxide for high performance liquid chromatography(HPLC) analysis. Rats with virtual gallbladder containing human cholesterol, mixed or pigment gallstones(VGHG) received 5 mg/kg hypericin or solvent and VGHG rats with cholesterol stones were given different hypericin doses(5-15 mg/kg). Twelve hours later, the stones were analysed at 365 nm. Biliary excretion and metabolites of hypericin were assessed in common bile duct(CBD) cannulated rats for 9 h using fluorospectrometry, HPLC and matrixassisted laser desorption/ionization-time-of-flight mass spectrometry(MALDI-TOF MS).RESULTS: Homogeneous high fluorescence was seen on cholesterol stones either pre-incubated with hypericin or extracted from VGHG rats receiving hypericin. Mixed stones showed a dotted fluorescent pattern, whereas pigment and solvent-treated ones lacked fluorescence. HPLC showed 7.68, 6.65 and 0.08 × 10^(-3) M of cholesterol in extracts from cholesterol, mixed, and pigment gallstones, respectively. Hypericin accounted for 2.0, 0.5 and 0.2 × 10-6 M in that order. On cholesterol stones from VGHG rats receiving different hypericin doses, a positive correlation was observed between dose and fluorescence. In the bile from CBD-cannulated rats, fluorescence represented 20% of the injected dose with two peaks in 9 h. HPLC analysis revealed that hypericin conjugates reached 60% of the peak area. By MALDI-TOF MS, hypericinglucuronide was detected. CONCLUSION: This study proves the potential use of hypericin for differential fluorescent detection of human gallstones regarding their chemical composition.

Evaluation of photodynamic efficiency of an endogenous enzyme-NQO1 activated hypericin

OBJECTIVE To investigate the therapeutic efficiency of an endogenous enzyme-NQO1 activated hypericin in photodynamic therapy.METHODS An endogenous enzyme-NQO1 responsive photo sensitizer was designed and synthesized by conjugating aquinonebased ligand to a natural photosensitizer derived from Chinese herb.The photophysical and photochemical properties were investigated through UV-visible and fluorescence spectrophotometer,and the photodynamic activity was evaluated with MTT assay.RESULTS An endogenous enzyme-NQO1 activated hypericin was prepared and fully characterized with various spectroscopic methods.The electronic absorption was almost the same with the free hypericin,indicating the introducing of the ligand to hypericin has little effect to its ground state,while there is almost no detected fluorescence and reactive oxygen species(ROS)generation in PBS solution indicating the introducing of the ligand can effectively quench the fluorescence emission and ROS generation.The in vitro study showed that both compounds have almost no dark toxicity,but they are highlyphotocytoxic with an IC50 less than 1μmol·L-1 against A549 cell lines indicating the modified compound can be activated in the intracellular environment.CONCLUSION A simple and efficient hypericin-based activated photosensitizer was prepared.The ROS generation was quenched in PBS solution and it would be activated inside A549 cell lines.It may be served as apromising tumor selective fluorescent probe and photosensitizer for targeted photodynamic therapy.

Topically applied hypericin exhibits skin penetrability on nude mice

OBJECTIVE Hypericin,a powerful naturally photosensitizer in photodynamic therapy(PDT),is suitable for treating skin diseases involving excess capillary proliferation.In the present study,we aimed to evaluate the skin penetrability of a topically applied hypericin,expecting reducing the risk of prolonged skin photosensitivity,which often occurs after systemic administration.METHODS The Franz diffusion cell assay was performed to evaluate different penetration enhancers.In vivo studies,fluorescence microscopy was performed to examine the distribution of hypericin in the skin,macroscopic and microscopic analyses were also carried out to detect pathological changes in the skin after topical hypericin-PDT treatment.Immunohistochemistry was used to determine the expression of PECAM-1 in the treated skin.RESULTS 5% menthol facilitated hypericin penetrate the skin of nude mice most.The results of in vivo assays revealed that hypericin penetrated nude mice skin,spread to the dermis,and resulted in obvious photosensitivity reaction on the dermal capillaries.Moreover,skin injured by the photosensitive reaction induced by hypericin was replaced by normal skin 7 d after hypericin-PDT treatment.CONCLUSION Topical hypericin could penetrate nude mouse skin well and be great potential in PDT treatment of skin diseases.

In vitro 3D malignant melanoma model for the evaluation of hypericin-loaded oil-in-water microemulsion in photodynamic therapy

Advances in biomimetic three-dimensional(3D) melanoma models have brought new prospects of drug screening and disease modeling, since their physiological relevancy for recapitulating in vivo tumor architectures is more accurate than traditional two-dimensional(2D) cell culture. Gelatin methacryloyl(GelMA) is widely used as a tissue-engineered scaffold hydrogel for 3D cell culture. In the present study, an in vitro 3D malignant melanoma model based on Gel MA was fabricated to evaluate the efficiency of hypericin(Hy)-loaded microemulsion(ME) in photodynamic therapy against melanoma. The ME was produced by the spontaneous emulsification method to enhance the bioavailability of Hy at tumor sites. Hy-loaded MEs were applied to a 3D malignant melanoma model made using 6% Gel MA and the co-culture of B16F10 and Balb/c 3T3 cells,followed by crosslinking using violet light(403 nm). The observation revealed excellent cell viability and the presence of F-actin cytoskeleton network. Hy-loaded MEs exhibited higher phototoxicity and cell accumulation(about threefold) than free Hy, and the cells cultured in the 3D system displayed lower susceptibility(about 2.5-fold) than those in 2D culture.These findings indicate that the developed MEs are potential delivery carriers for Hy;furthermore, Gel MA hydrogel-based modeling in polydimethylsiloxane(PDMS) molds is a user-friendly and cost-effective in vitro platform to investigate drug penetration and provide a basis for evaluating nanocarrier efficiency for skin cancer and other skin-related diseases.

Effects of Different Geographical Aspects and Ontogenetic Variability on Total Hypericin Content of Hypericum triquetrifolium Turra. and Hypericum scabrum L.

The present study was conducted to determine the total hypericin contents of Hypericum triquetrifolium Turra.and Hypericum scabrum L.species which are naturally distributed in the flora of Siirt province,Turkey.Hypericin contents of Hypericum species grown in different geographical aspects(North,South,East,and West),and it was measured at different harvest times(full bloom-ing and post blooming period).In the current study,it has been determined that total hypericin content varies considerably according to aspects,plant developmental stages(ontogenetic variance),and species.According to species x aspect interaction,the highest total hypericin content was recorded from the west aspect(3.13 mg/g)in Hypericum triquetrifolium,while,the lowest hypericin content was also obtained from the west aspect(1.22 mg/g)in Hypericum scabrum.When the highest total hypericin content was analyzed according to aspect x species x harvest time interaction,the highest total hypericin content was produced from Hypericum triquetrifolium at the harvest of west aspect with 5.28 mg/g,while the minimum amount of hypericin was obtained from the same aspect in Hypericum scabrum with 0.50 mg/g.In species x harvest time interaction,the highest total hypericin content was obtained from the full bloom(3.10 mg/g)harvest in Hypericum triquetrifolium,while the lowest hypericin was obtained from the full bloom(1.26 mg/g)harvest in Hypericum scabrum.The data suggest that the aver-age total hypericin content was 2.26 mg/g in Hypericum triquetrifolium and 1.28 mg/g in Hypericum scabrum.

Effect of Photoactivated Hypericin on Growth and Antibiotic Susceptibility of Hospital-Related Staphylococcus aureus and Enterococcus sp. Clinical Strains

Resistance against commonly used antibiotics is a serious clinical problem in recent medical practice. There exist several bacterial strains in which the possibilities of their inhibition are very limited due to multidrug resistance. Antimicrobial photodynamic therapy (aPDT) represents an option how to effectively suppress the growth of resistant pathogens. In this work we have studied interactions of potent photosensitizer hypericin (Hyp) with hospital-related gram positive (Gram+) and gram negative (Gram-) bacterial strains and the effects of photodynamic activated Hyp on bacterial susceptibility and/or resistance of these strains to antibiotics. We demonstrated a significant influence of photoactivated Hyp on growth of Staphylococcus aureus and Enterococcus sp. We have also shown that it is extremely important to use the effective concentrations of Hyp for aPDT, which completely inhibit the growth of microorganisms. Otherwise, there appears an increase in resistance, probably due to the activation of efflux mechanisms, which are involved in the efflux of Hyp and antibiotics as well.

Hypericin in hypericum:chemistry, botanical sources and biological activities

Hypericin, a secondary metabolite first reported in 1830, is a natural photosensitizing naphtodianthrone and is mainly from the natural plant sources of genus Hypericum. Hypericin exhibits a wide variety of biological and pharmacological activities, such as antiviral, antidepressant, antitumor, antimicrobial, and antioxidant activity. Hypericin can also be utilized in photodynamic diagnosis. Accumulating evidence is pointing to the effects of hypericin with potential pharmaceutical and clinical interests in the past decades. The present review gives a comprehensive outline of the chemistry, botanical occurrence and biological activities of this powerful compound.

Raman spectroscopic study on structure of human immunodeficiency virus (HIV) and hypericin-induced photosen- sitive damage of HIV

The first Raman spectra of HIV1- HIV2 in human sera and hypericin-induced pho- tosensitive damage of the virus have been obtained. The prominent Raman lines in the spectra are assigned respectively to the carbohydrates of viral glycoprotein, RNA, protein and lipid. The spectra are dominated by Raman scattering of the carbohydrates. The lines of D-Mannose and N-acetylglucosamine in carbohydrates are obvious and there is a β-configuration in the anomeric C1 position in D-Mannose. The viral RNA duplexes bound assumes an A-form geometry. The lines of backbone phosphate group, bases (involving interbase hydrogen bonding) and ribose of the RNA are complete and distinct. The secondary structure of the viral protein maintains α-helix, β-sheet, β-turn and random coil. Its side chains are rich and vary from tryptophan, phenylalanine and “buried” tyrosine; the stable conformation of the S-S bond of gauche-gauche-gauche; the two forms of C-S bonds of gauche and trans ; to sulfhydrl group and ionized and unionized carboxyl groups. The viral lipid bilayer molecules are probably in the liquid ordered phase or the gel phase. It was observed that the hypericin-induced photosensitive damage of HIV1-HIV2 in human sera changed various components of HIV1-HIV2 in different degrees : The orderly A-form viral RNA would become a disordered viral RNA. There were a breakage of interbase hydrogen bonds and disruption of vertical base-base stacking interactions. In addition, the groups of ribos and four bases were damaged obviously. A decrease in ordered structure (α-helix and β-sheet) of viral protein is accompanied by an increase in random coil. The Tyr buried in the three-dimensional structure of protein was damaged, but it was still “buried” and the damage of C-S bond of trans form was stronger. The groups of carbohydrates, including D-Mannos and N-acetyl glucosamine, in viral envelope glycoprotein had also been changed. The hydrophilic C-N bond of choline in viral lipid was damaged, which was the possible binding site to hypericin, whereas the viral lipids bilayers were still probably in the liquid ordered phase or the gel phase. So the space structure of HIV1-HIV2 was damaged under the experimental conditions, which might block viral infection and inhibit its growth and breeding. It is apparent that the laser Raman spectra have provided certain direct evidence at the molecular level for photosensitive damage of HIV1-HIV2.

Signal interaction between nitric oxide and hydrogen peroxide in heat shock-induced hypericin production of Hypericum perforatum suspension cells

Heat shock(HS, 40℃, 10 min) induces hypericin production, nitric oxide(NO) generation, and hydrogen peroxide(H2O2) accumulation of Hypericum perforatum suspension cells.Catalase(CAT) and NO spe-cific scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide(cPTIO) suppress not only the HS-induced H2O2 generation and NO burst, but also the HS-triggered hypericin produc-tion.Hypericin contents of the cells treated with both NO and H2O2 are significantly higher than those of the cells treated with NO alone, although H2O2 per se has no effects on hypericin production of the cells, which suggests the synergistic action between H2O2 and NO on hypericin production.NO treatment enhances H2O2 levels of H.perforatum cells, while external application of H2O2 induces NO generation of cells.Thus, the results reveal a mutually amplifying action between H2O2 and NO in H.perforatum cells.CAT treatment inhibits both HS-induced H2O2 accumulation and NO generation, while cPTIO can also suppress H2O2 levels of the heat shocked cells.The results imply that H2O2 and NO may enhance each other's levels by their mutually amplifying action in the heat shocked cells.Membrane NAD(P)H oxidase inhibitor diphenylene iodonium(DPI) and nitric oxide synthase(NOS) inhibitor S,S′-1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea(PBITU) not only inhibit the mutually amplifying action between H2O2 and NO but also abolish the synergistic effects of H2O2 and NO on hypericin production, showing that the synergism of H2O2 and NO on secondary metabolite biosynthesis might be dependent on their mutual amplification.Taken together, data of the present work demonstrate that both H2O2 and NO are essential for HS-induced hypericin production of H.perforatum suspension cells.Furthermore, the results reveal a special interaction between the two signal molecules in mediating HS-triggered secondary metabolite biosynthesis of the cells.

Hypericin: chemical synthesis and biosynthesis

Hypericin is one of the most important phenanthoperylene quinones extracted mainly from plants of the genus Hypericum belonging to the sections Euhypericum and Campylosporus of Keller's classification. Widespread attention to the antiviral and anti-tumor properties of hypericin has spurred investigations of the chemical synthesis and biosynthesis of this unique compound. However, the synthetic strategies are challenging for organic and biological chemists. In this review, specific significant advances in total synthesis, semi-synthesis, and biosynthesis in the past decades are summarized.

Variation of Bioactive Compounds in Hypericum perforatum Growing in Turkey During Its Phenological Cycle

The present study was conducted to determine phenologic and morphogeneUc variation of hyperlcln, chlorogenlc acid and flavonoids, as rutin, hyperoside, apigenin-7-O-glucoside, quercitrin, quercetin content of Hypericum perforatum L. growing in Turkey. Wild growing plants were harvested at vegetative, floral budding, full flowering, fresh frulUng and mature fruiting stages and dissected into stem, leaf and reproductive tissues and assayed for bioacUve compounds by the High performance liquid chromatography （HPLC） method. Hypericin concentration ranged between 0 and 2.73 mg/g DW, chlorogenic acid 0.00-3.64 mg/g DW, rutin 0.00-3.36 mg/g DW, hyperoside 0.04- 22.42 mg/g DW, quercitrin 0.03-3.46 mg/g DW and quercetin 0.04-1.02 mg/g DW depending on ontogenetic and morphogenetic sampling. Leaves were found to be superior to stems and reproductive parts with regard to phenolic accumulation for all compounds tested while flowers accumulated the highest levels of hypericln. Quercltrln, quercetln and hypericin content in all tissues increased with advancing of developmental stages and reached their highest level during flower ontogenesis. Similarly, chlorogenic acid, hyperoside and apigenin-7-O-glucoside content in different plant parts increased during plant development, however, the highest level was observed at different stages of plant phenology for each tissue. Chlorogenic acid was not detected in stems, leaves and reproductive parts in several stages of plant phenology and its variation during plant growth showed inconsistent manner. In contrast to the other compounds examined, rutin content of stems and leaves decreased with advanc- ing of plant development and the highest level for both tissues was observed at the vegetative stage. However, content of the same compound in reproductive parts was the highest at mature fruiting. The present findings might be useful to obtain increased concentration of these natural compounds.