Hypericin Research Articles

Comparison of phototoxic effects of hypericin-mediated photodynamictherapy in HT-29 and Caco-2 colon cancer cells

Hypericin (HYP) is a plant-derived photosensitizer. HYP is preferentially taken up by tumor cells. We designed this study to compare HYP-mediated photodynamic therapy (PDT) in HT-29 and Caco-2 colon cell lines. Cells were treated with 0.04, 0.08, and 0.15 μM HYP concentrations and irradiated. The effect of HYP on metabolic profiles, alterations in lactate dehydrogenase (LDH) leakage, and cell cycle progression was investigated for the first time. Changes in glucose consumption, lactate production, and LDH leakage were analyzed. HYP-induced cell death was quantified by double staining (acridine orange/propidium iodide) and alterations in cell cycle regulation were analyzed with flow cytometry (using propidium iodide). LDH leakage and the number of dead cells were elevated, and glucose consumption and lactate production decreased in a dose- and time-dependent manner. PDT resulted in an induction of apoptosis, mostly at the 0.08 μM HYP concentration. Apoptosis and/or necrosis were increased in the 0.15 μM HYP group. The accumulation of cells in the G2/M phase might account for the growth inhibition in HT-29 and Caco-2 cells with 0.08 μM HYP photoactivation. The observed G2/M arrest suggested that HYP may slow down the growth of colon cancer cells by regulating the cell cycle, leading either to growth inhibition or to initiation of apoptotic pathways.

Photoactivated hypericin is not genotoxic.

The study was designed to test the potential photogenotoxicity of hypericin (HYP) at three different levels: primary DNA damages, gene mutations and chromosome aberrations. Primary genetic changes were detected using the comet assay. The potential mutagenic activity of HYP was assessed using the Ames/Salmonella typhimurium assay. Finally, the ability of photoactivated HYP to induce chromosome aberrations was evaluated by the in vitro mammalian chromosome aberration test and compared to that of non-photoactivated HYP. The results have shown that photoactivated HYP can only induce primary DNA damages (single-strand DNA breaks), acting in a dose-response manner. This activity depended both on HYP concentrations and an intensity of the light energy needed for its photoactivation. However, mutagenic effect of photoactivated HYP evaluated in the Ames assay using three bacterial strains S. typhimurium (TA97, TA98 and TA100) was not confirmed. Moreover, photoactivated HYP in the range of concentrations (0.005-0.01 µg/ml) was not found to be clastogenic against HepG2 cells. Our findings from both the Ames assay and the chromosome aberrations test provide evidence that photoactivated HYP is not genotoxic, which might be of great importance mainly in terms of its use in the photodynamic therapy.

Evaluation of apoptotic cell death mechanisms induced by hypericin-mediated photodynamic therapy in colon cancer cells

Hypericin (HYP)-mediated photodynamic therapy (PDT) is a new alternative treatment strategy for colon cancer inducing various cell-death pathways. Apoptotic cell death is the desired cellular fate in cancer cells. Therefore, we investigated the apoptotic pathways by determining apoptosis-related proteins (survivin, caspase-3, caspase-9, Bcl-2, and Bax) at the mRNA level using real-time polymerase chain reaction (qPCR), and the percentage of survivin was determined by survivin ELISA in HT-29 and Caco-2 colon cancer cell lines. The downregulation of survivin was significant 16 h after PDT for both cells, while caspase-3 upregulation was apparent 24 h after PDT. Caspase-9 and caspase-3 upregulations were parallel to each other. There was no Bcl-2 expression in HT-29 cells, but we observed downregulation in Bcl-2 expression in Caco-2 cells after HYP photoactivation. The Bax expression downregulated significantly after 24 h incubation in HT-29 cells, while it was upregulated after 16 h incubation in Caco-2 cells. The present study provides evidence that HYP-induced alterations in apoptosis-related protein expression ended in different responses in HT-29 and Caco-2 colon cancer cells, and these may be used in the treatment of chemotherapy-resistant cancer types.

Polysaccharide elicitors enhance phenylpropanoid and naphtodianthrone production in cell suspension cultures of Hypericum perforatum

Hypericum perforatum cell suspensions were evaluated for their growth, phenylpropanoid and naphtodianthrone productions, and antioxidant activity after treatments with polysaccharide elicitors chitin (CHI), pectin (PEC) and dextran (DEX). Polysaccharide elicitation of cell cultures showed a considerable improving effect on phenylpropanoid production (phenolics, flavonoids, flavanols and anthocyanins) during the post-elicitation period. Even that enhanced phenylpropanoid production was related to growth suppression of CHI elicited cells, PEC and DEX were presented as effective elicitors without loss of cell biomass. Phenylpropanoid accumulation in elicited cells was correlated to markedly higher enzyme activities of phenylalanine ammonia lyase and chalcone-flavanone isomerase. Cell cultures were found to respond rapidly towards the applied elicitors through an early stimulation of both naphtodiantrones, hypericin (HYP) and pseudohypericin (PHYP). The CHI was proposed as a much more prominent elicitor in stimulation of HYP and PHYP levels in cell suspensions. All tested polysaccharide elicitors stimulated non-enzymatic antioxidant properties in cell extracts indicating the potential role of phenolic compounds as effective antioxidants. With regards to the antioxidant enzymes, an up-regulation of peroxidase and catalase activities was observed in PEC and DEX elicited cells, whereas CHI did not give any stimulatory effect on enzymatic activities. These findings suggest the involvement of an efficient antioxidant defense system in the adaptive response of cells to polysaccharide elicitation. Altogether, these results indicated that H. perforatum cells elicited with polysaccharides represent a promising experimental system for enhanced production of phenylpropanoids and naphtodianthrones.

LC–MS/MS based studies on the anti-depressant effect of hypericin in the chronic unpredictable mild stress rat model

Ethnopharmacological relevanceSt John׳s Wort (Hypericum perforatum, SJW) is a widely used herbal medicine in western countries but also an important Uygur drug in China. Hypericin (HY) is the main components in SJW extracts, which is used to treat fatigue, weakness, and mild depression. The aim of this study was to investigate the anti-depression effects of HY on chronic unpredictable mild stress (CUMS) model rats and identify the possible mechanisms. Materials and methodsIn this study, the protective effects of HY on CUMS-induced depression in rats were investigated by using a combination of behavioral assessments and urinary metabolites analysis. Urinary metabolites analyses were performed using LC–MS/MS in conjunction with principal components analysis (PCA) after oral administration of either HY or Venlafaxine (VF) for 27 days. During the procedure of experiment, food consumption, body weight, adrenal gland, thymus and spleen indices, behavior scores, sucrose consumption, and stress hormone levels were measured. ResultsChanges in the classic behavioral tests and pharmacological biochemical indices reflected that HY alleviated the symptoms of depression in a shorter period than VF, which was used as positive control for antidepression. Metabolites analysis of urine revealed that HY affected excitatory amino acids and monoamine neurotransmitter metabolites. Remarkably, urinary valine was increased remarkably by HY, even much higher than CUMS group. These results provide important mechanistic insights into the protective effects of HY against CUMS-induced depression and metabolic dysfunction. ConclusionAs the most important active ingredient in SJW extracts, HY possesses the better protective effect against CUMS-induced depression symptoms and metabolic disturbances.

Photodynamic efficiency of hypericin compared with chlorin and hematoporphyrin derivatives in HEp-2 and Vero epithelial cell lines

Hypericin (HY) is a photoactive aromatic dianthraquinone that is considered a potent photodynamic agent. In this study, hypericin and two other photosensitizers, a hematoporphyrin derivative (Photogem(®); PG) and a chlorin derivative (Photodithazine(®); PZ), were compared in terms of their phototoxicity toward two cell lines, HEp-2 and Vero. The median inhibitory concentration (IC(50)) of each of the photosensitizers was obtained after a 16.2J cm(-2) dose of irradiation at 630 ± 10 nm. The IC(50) values were 0.07 ± 0.01 (HY), 1.0 ± 0.2 (PZ), and 9 ± 1 μgmL(-1) (PG) in HEp-2 cells and 0.3 ± 0.1 (HY), 1.6 ± 0.2 (PZ) and 11 ± 1 μgmL(-1) (PG) in Vero cells, showing that HY is more phototoxic than the others when irradiated at 630 nm. If these results are analyzed, simultaneously, with the first-order constant for BSA tryptophan photooxidation, obtained by fluorescence decay (λ(excitation)=280 nm), which are 11×10(-3) min(-1)±1. 10(-3) min(-1) (HY), 10 × 10(-3) min(-1)±1 × 10(-3) min(-1) (PZ), and 6 × 10(-3)min(-1) ± 1×10(-3)min(-1) (PG), it is possible to infer that the photodynamic efficiency alone is not sufficient to explain the higher HY phototoxicity. The lipophilicity is also an important factor for an efficient target cell accumulation and was assessed for all sensitizers through the octanol-water partition coefficient (log P): 1.20 ± 0.02 (HY), -0.62 ± 0.03 (PZ), and -0.9 ± 0.2 (PG). The higher value for HY correlates well with its observed superior efficiency to promote damage at low concentrations and doses. As HY is used for the long-term treatment of mild depression, it is considered safe for humans. This fact and the present results reinforce the great potential of this photosensitizer to replace porphyrin derivatives, with the advantages that mean it could be used as photosensitizer in clinical photodynamic therapy.

Phototoxic effect of hypericin alone and in combination with acetylcysteine on Staphylococcus aureus biofilms

Resistance of bacteria against antibiotics and antimicrobials is arising worldwide and there is an urgent need for strategies that are capable of inactivating biofilm-state pathogens with less potential of developing resistance in pathogens. A promising approach could be photodynamic inactivation (PDI) which uses light in combination with a photosensitizer to induce a phototoxic reaction. In this study, we evaluated the in vitro phototoxic effect of hypericin (HYP) alone and in combination with acetylcysteine (AC) on Staphylococcus aureus biofilms. AC, a mucolytic agent, reduces the production of extracellular polysaccharide matrix while promoting the disruption of mature biofilm. In vitro phototoxic effect of HYP alone (0.5 μg/ml, light dose: 16 J/cm(2)), and in combination with AC (10 mg/ml) on ten clinical S. aureus isolates and S. aureus (ATCC 25923) biofilms was studied. Effect of HYP concentration (0.5 μg/ml) and light dose (8 J/cm(2)) on PDI of all eleven S. aureus strains in planktonic forms was also investigated. HYP-PDI did not result in a reduction in viable count for each of the strains when grown in biofilms. However, HYP-PDI applied on biofilms treated with AC was able to disrupt pre-formed biofilms (viable count reduction ranging from 5.2 to 6.3 log10-unit in comparison to controls in all tested strains). A 6.5 log killing was obtained for S. aureus (ATCC 25923) planktonic cells treated with 0.5 μg/ml at 8 J/cm(2). For this set of PDI parameters, ten clinical S. aureus isolates showed 5.5-6.7 log killing. HYP-PDI in combination with AC had significant ability to eradicate the pre-formed mature biofilms of S. aureus strains.

Antimicrobial photodynamic activity of hypericin against methicillin-susceptible and resistant Staphylococcus aureus biofilms.

To evaluate the effectiveness of the photodynamic therapy using hypericin (HYP) against both planktonic and biofilm-forming Staphylococcus aureus. HYP photoactivity was evaluated against methicillin-susceptible and resistant S. aureus. Bacterial suspension or biofilm were preincubated with HYP and subjected to LED illumination. Viable bacteria were determined by colony counting. Preincubation with HYP (5 min) plus light exposure (10 min) showed bactericidal effect against planktonic methicillin-susceptible S. aureus and methicillin-resistant S. aureus. Longer preincubation times (24 h) and time light exposure (30 min) were required to reach HYP-photoactivity against S. aureus biofilms. HYP-photoactivity was correlated to the biofilm production. HYP could be a potential photosensitizer for the inactivation of staphylococcal biofilms forming on the surfaces accessible to visible light.

A Review of the Photodynamic Application of 5-Aminolevulinic acid, Hypericin and Phthalocyanines in dermatology

Ultraviolet radiation can damage human skin leading to photo-aging and cancer. Treatment options for skin cancers are available. Amongst them, there is photodynamic therapy (PDT), the treatment modality that involves a photochemical reaction between a light sensitive compound, visible light and tissue oxygen. In PDT, a photosensitive compound also called photosensitizer (PS) is administered and allowed to accumulate in the cancerous tissue then irradiated with light corresponding to absorption wavelength of the PS, in the presence of molecular oxygen, to produce cytotoxic species that kill the cancerous tissue. PDT is increasingly used and studied globally for the treatment of different classes of cancers because of its selective destruction of diseased tissue or cancer. Newly developed non-invasive imaging technologies including photodynamic diagnosis, may assist with early identification of skin cancer to reduce the rates of morbidity and mortality following treatment. Photodynamic diagnosis (PDD) is a diagnostic modality defined from the PDT principle. It utilizes light and fluorescent PS to highlight tumour cells from normal cells. Excitation of PS by appropriate light source causes them to fluoresce over well-defined spectral regions. Therefore, the fluorescent properties of PSs can serve as an important diagnostic tool to highlight cancer at an early stage of development. In this review, our knowledge about PDT and PDD of skin cancers using 5-aminolevulinic acid (ALA), Hypericin and phthalocyanines as photosensitizers is presented.

The efficacy and mechanism of apoptosis induction by hypericin-mediated sonodynamic therapy in THP-1 macrophages.

PurposeTo investigate the sonoactivity of hypericin (HY), together with its sonodynamic effect on THP-1 macrophages and the underlying mechanism.Materials and methodsCCK-8 was used to examine cell viability. Confocal laser scanning microscopy was performed to assess the localization of HY in cells, reactive oxygen species (ROS) generation, and opening of the mitochondrial permeability transition pore (mPTP) after different treatments. Apoptosis was analyzed using Hoechst–propidium iodide and transmission electron microscopy. Mitochondrial membrane potential (ΔΨm) collapse was detected via fluorescence microscopy. Lipoprotein oxidation was determined in malondialdehyde (MDA) assays. Western blotting was conducted to determine the translocation of BAX and cytochrome C and the expression of apoptosis-related proteins.ResultsHY was sublocalized among the nuclei and the mitochondria, endoplasmic reticulum, Golgi apparatus, and lysosome in the cytosol of THP-1 macrophages. Under low-intensity ultrasound irradiation, HY significantly decreased cell viability and induced apoptosis. Furthermore, greater ROS generation, higher MDA levels, and greater ΔΨm loss were observed in the sonodynamic therapy (SDT) group. Both ROS generation and MDA levels were significantly reduced by the ROS scavenger N-acetyl cysteine (NAC) and the singlet oxygen scavenger sodium azide. Most of the loss of ΔΨm was inhibited by pretreatment with NAC, sodium azide, and the mPTP inhibitor cyclosporin A (CsA). mPTP opening was induced upon SDT but was reduced by pretreatment with bongkrekic acid, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid disodium, CsA, and NAC. Western blot analyses revealed translocation of BAX and cytochrome C, downregulated expression of Bcl-2, and upregulated expression of cleaved caspase-9, cleaved caspase-3, and cleaved poly(ADP-ribose) polymerase in the SDT group, which were reversed by NAC.ConclusionHY mediated SDT-induced apoptosis in THP-1 macrophages via ROS generation. Then, the proapoptotic factor BAX translocated from the cytosol to the mitochondria, increasing the ratio of BAX/Bcl-2, and the mPTP opened to release cytochrome C. This study demonstrated the great potential of HY-mediated SDT for treating atherosclerosis.

Hypericin damages the ectatic capillaries in a Roman cockscomb model and inhibits the growth of human endothelial cells more potently than hematoporphyrin does through induction of apoptosis.

Hypericin (HY) is a promising photosensitizer (PS) for use in photodynamic therapy (PDT). Port-wine stains (PWSs) are congenital superficial dermal capillary malformations. In this study, we evaluated the photocytotoxic effects of HY for PDT in human vascular endothelial cells and a chicken cockscomb model. HY significantly inhibited the growth of human umbilical vein endothelial cells (HUVECs), as determined by colorimetric assays and morphological observation, and flow cytometry assays indicated induction of apoptosis and collapse of the mitochondrial membrane potential. In addition, HY more effectively inhibited growth of and induced apoptosis in HUVECs compared with hematoporphyrin (HP). Further experiments performed in a Roman chicken cockscomb model also showed a clear photocytotoxic effect on the cockscomb dermal capillary upon intravenous injection of HY. This effect may be due to the role of HY in the induction of apoptosis. Transmission electron microscopical analysis showed mitochondrial morphological changes such as incomplete ridges and swelling, and immunohistochemical assays showed an increase in the release of cytochrome c. In conclusion, HY exhibited a greater photocytotoxic activity than did HP toward the growth of endothelial cells and may thus represent a potent PS for PWS PDT.

Radiopharmaceutical study on Iodine-131-labelled hypericin in a canine model of hepatic RFA-induced coagulative necrosis.

Hypericin (HYP) has been found avid to necrosis in small animal studies. We sought to evaluate the tissue distribution of (131)I-HYP in a large animal model and to explore the theranostic utilities of (131)I-HYP after radiofrequency ablation (RFA). This animal experiment was approved by the institutional ethics committee. Twenty-five male dogs were enrolled and subjected to transabdominal hepatic RFA. (131)I-HYP was prepared by an electrophilic substitution method and intravenously administered at 0.5 mCi/kg. Systemic and regional distributions of (131)I-HYP were monitored dynamically by single-photon emission computed tomography/computed tomography (SPECT-CT), gamma counting, autoradiography, and fluorescent and light microscopy at different time points up to 14 days. Experimental data were quantified and statistically analysed. Most of the tissues and organs retained (131)I-HYP only transiently. (131)I-HYP was mainly metabolised in the liver and excreted into the bile. (131)I-HYP gradually accumulated in the RFA-induced necrosis with a peak concentration occurring within 2 days and lasting over 2 weeks as visualised by in vivo SPECT-CT and ex vivo autoradiography and fluorescent microscopy, and quantified by radioactivity and fluorescence measurements. Accumulation of (131)I-HYP was low in both the necrosis centre and normal liver tissue. (131)I-HYP showed persistent high affinity to hepatic thermo-coagulative necrosis, but only a transient uptake by normal liver in dogs. Necrosis caused by RFA could be indicated by (131)I-HYP on nuclear imaging, which suggests a supplementary measure for tumour detection and therapy.

Hypericin suppresses osteoclast formation and wear particle-induced osteolysis via modulating ERK signalling pathway

Osteoclast-induced bone resorption and wear-particle-induced osteolysis leads to prosthetic loosening, one of the most common causes of joint implant failure, resulting in revision surgery. Thus, inhibition of osteoclastic bone resorption, which further prevents wear particle-induced osteolysis, is a potential treatment strategy for prosthetic loosening. Here, we examined the therapeutic effect of hypericin (HP), which was photosensitive, on osteoclastogenesis and wear particle-induced osteolysis in the absence of visible light. HP inhibited RANKL-induced osteoclast differentiation in bone marrow macrophages (BMMs) and RAW264.7 cell line without any evidence of cytotoxicity. The bone-resorbing activity of mature osteoclasts was significantly inhibited by HP. As HP has been previously reported to inhibit signalling pathway such as ERK and NF-κB in other cells, which is also important in osteoclast differentiation. We thus examined the molecular mechanism and showed that HP significantly inhibited the ERK/mitogen-activated protein kinase (MAPK) signalling pathway without affecting nuclear factor kappaB (NF-κB), c-Jun N-terminal kinase (JNK) and p38 signalling in RANKL-stimulated BMMs. Further in vivo studies revealed HP attenuated osteoclast formation and subsequently prevented wear particle-induced bone erosion. Taken together, the results suggest that HP inhibits RANKL-mediated osteoclastogenesis via affecting ERK signalling in vitro and suppresses wear particle-induced osteolysis in vivo. We therefore conclude that HP may be an innovative and safe alternative treatment for osteoclast-related prosthetic loosening.

Comparison of efficacy of topical phenytoin with hypericin in second-degree burn wound healing: An experimental study in rats

BackgroundThis experiment was performed to compare the effects of Phenytoin (PHT) and Hypericin (HP) cream on healing of burn wounds in rats.Material/MethodsTwenty rats were divided into 3 groups and second-degree burn wounds were created. The burn wounds in the first, second, and third groups were covered twice daily with PHT cream, HP cream, and saline (control), respectively. At the end of days 3, 7, 14, and 21, full-thickness skin biopsies were done for histopathologic and immunohistochemical analyses.ResultsHistopathologic evaluations at the 14th day showed that re-epithelialization scores were greater in the HP group than the PHT group, but on day 21, re-epithelialization scores were higher in the PHT group than the HP group. Collagen content on days 3 and 14 in the PHT group was found to be higher than in the HP group. Well-vascularized granulation tissue on day 7 in the PHT group was higher than in other groups. HP and PHT groups had a significant increase in VEGF and TGF-β expression in burn wound healing area compared to the control group on all days.ConclusionsTopical application of HP can promote re-epithelialization in burn wounds to shorten the wound healing time for superficial burns. Phenytoin, on the other hand, contributes to healing by increasing vascularized granulation tissue and collagen synthesis through re-epithelialization. The increased VEGF and TGF-β expression following PHT and HP treatment strongly indicate that PHT and HP treatment promotes VEGF and TGF-β production and action in the burn wound area.

Alterations in dysadherin expression and F-actin reorganization: a possible mechanism of hypericin-mediated photodynamic therapy in colon adenocarcinoma cells

Dysadherin is a recently found anti-adhesion molecule, therefore detection and down regulation of its expression is promising in cancer treatment. The up-regulation of dysadherin contributes to colon cancer recurrence and metastasis. Dysadherin also has connections with cytoskeletal proteins and it can cause alterations in the organisation of filamentous actin (F-actin) in metastatic cancers. In this study, hypericin (HYP)-mediated photodynamic therapy (PDT) was performed in two different grade colon adenocarcinoma cell lines HT-29 (Grade I) and Caco-2 (Grade II). Cells were treated with 0.04, 0.08 or 0.15μM HYP concentrations and irradiated with (4J/cm(2)) fluorescent lamps. The effects of HYP was examined 16 and 24h after the activation. We investigated for the first time the effect of HYP-mediated PDT on the expression of dysadherin and F-actin organisation. According to the results, HYP mediated PDT caused a decrease in gene expression and immunofluorescence staining of dysadherin and an increase in actin stress fibers and actin aggregates in HT-29 and Caco-2 cell lines. Besides, cytotoxicity, number of floating cells and apoptotic index changed depending on the cell type, HYP concentration and incubation time. We have demonstrated for the first time that dysadherin and F-actin could be target molecules for HYP-mediated PDT in HT-29 and Caco-2 colon cancer cell lines.

Unique Anti-Glioblastoma Activities of Hypericin Are at the Crossroad of Biochemical and Epigenetic Events and Culminate in Tumor Cell Differentiation

Failure of conventional therapies to alleviate glioblastoma (GBM) fosters search for novel therapeutic strategies. These include epigenetic modulators as histone deacetylase inhibitors (HDACi), which relax abnormally compact tumor cell chromatin organization, enabling cells to overcome blockage in differentiation. However, in clinical settings, HDACi efficacy is confined to subsets of hematologic malignancies. We reasoned that molecules targeting multiple epigenetic mechanisms may exhibit superior anti-cancer activities. We focused on the redox perylene-quinone Hypericin (HYP) and showed that HYP targets Hsp90 for polyubiquitination, degradation and inactivation. Hsp90 is implicated in mediating inheritable epigenetic modifications transferable to progeny. We therefore examined if HYP can induce epigenetic alterations in GBM cells and show here that HYP indeed, targets multiple mechanisms in human glioblastoma tumor cell lines via unique manners. These elicit major epigenetic signature changes in key developmentally regulated genes. HYP induces neuroglial tumor cell differentiation modulating the cytoarchitecture, neuroglial differentiation antigen expression and causes exit from cell proliferation cycles. Such activities characterize HDACi however HYP is not an HDAC inhibitor. Instead, HYP effectively down-regulates expression of Class-I HDACs, creating marked deficiencies in HDACs cellular contents, leading to histones H3 and H4 hyperacetylation. Expression of EZH2, the Polycomb repressor complex-2 catalytic subunit, which trimethylates histone H3K27 is also suppressed. The resulting histone hyperacetylation and diminished H3K27-trimethylation relax chromatin structure, activating gene transcription including differentiation-promoting genes. DNMT profiles are also modulated increasing global DNA methylation. HYP induces unique epigenetic down-regulations of HDACs, EZH2 and DNMTs, remodeling chromatin structure and culminating in tumor cell differentiation. These modulations generate clinically significant anti-GBM effects obtained in a clinical trial performed in patients with recurrent, progressive disease. Despite this advanced disease stage, patients responded to HYP, displaying stable disease and partial responses; patients on compassionate therapy survived for up to 34 months. Hypericin may constitute a novel anti-glioblastoma therapeutic paradigm.

Effect of glutathione on peroxyoxalate chemiluminescence of hypericin as the fluorophore

Herein, the effect of amino acid Glutathione (GSH) on Peroxyoxalate Chemiluminescence was studied for the first time. Hypericin (HYP) was employed as the efficient fluorophore. The investigated parameters included rise and fall rate constant for the chemiluminescence burst, theoretical and experimental maximum intensity, the time-needed to reach maximum intensity and the total light yield emission which theoretically was evaluated using the pooled intermediate model by a computerized non-linear least-squares curve fitting program (KINFIT). Furthermore, based on observed quenching effect of GSH, the Stern–Volmer plot in quencher concentration range of 2.8×10−6 to 3.4×10−5M with KQ value of 1.59×104 was calculated. The bimolecular quenching rate constant (Kq) was also estimated about 2.8×1012 and M−1S−1. Moreover the system was applied successfully to determine glutathione in biological samples.

Microendoscopy for hypericin fluorescence tumor diagnosis in a subcutaneous glioma mouse model

New treatment strategies for malignant gliomas are indispensible, due to the poor prognosis for patients. Fluorescence diagnosis (FD) and photodynamic therapy (PDT) are currently under intensive investigation and seem to improve the prognosis. Especially for deep seated malignant brain lesions and in order to optimize therapy new diagnostic tools are needed. In a syngeneic subcutaneous glioma mouse model we investigated the time dependent hypericin (HYP) uptake in malignant tumor tissue by microendoscopically fluorescence measurements. The HYP fluorescence in tumor was also detected by fluorescence microscopy (FM) and was compared to endoscopic data. Both methods, microendoscopy and FM, demonstrated time dependent HYP uptake in subcutaneously implanted mouse glioma. Maximum of HYP uptake was achieved after 6h, measured with both methods. FM reached a 10-fold increase in fluorescence intensity compared to the autofluorescence. Measured by microendoscopy a 2.2-fold HYP fluorescence intensity compared to the autofluorescence was detected. Microendoscopy enables visualization of small vessels even in healthy brain tissue by intravascular HYP fluorescence. The new developed microendoscope enables not only fluorescence based discrimination of tumor and healthy tissue, but also semiquantitative measurements of fluorescence intensities in vivo. Individual repetitive fluorescence diagnosis will become possible by this method and opens up new possibilities for determining optimal settings of light applications for PDT.

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.

Photodynamic fungicidal efficacy of hypericin and dimethyl methylene blue against azole‐resistant Candida albicans strains

Antimicrobial photodynamic therapy (aPDT) is an emerging alternative to treat infections based on the use of photosensitisers (PSs) and visible light. To investigate the fungicidal effect of PDT against azole-resistant Candida albicans strains using two PSs with a different mechanism of action, hypericin (HYP) and 1,9-dimethyl methylene blue (DMMB), comparing their efficacy and the reactive oxygen species (ROS) species involved in their cytotoxicity. Azole-resistant and the azole-susceptible C. albicans strains were used. Solutions of 0.5 and 4 McFarland inoculum of each Candida strain were treated with different concentrations of each PS, and exposed to two light-emitting diode light fluences (18 and 37 J cm⁻²). Mechanistic insight was gained using several ROS quenchers. The minimal fungicidal concentration of HYP for ≥3 log₁₀ CFU reduction (0.5 McFarland) was 0.62 μmol l⁻¹ for most strains, whereas for DMMB it ranged between 1.25 and 2.5 μmol l⁻¹. Increasing the fluence to 37 J cm⁻² allowed to reduce the DMMB concentration. Higher concentrations of both PSs were required to reach a 6 log₁₀ reduction (4 McFarland). H₂O₂ was the main phototoxic species involved in the fungicidal effect of HYP-aPDT whereas ¹O₂ was more important for DMMB-based treatments. aPDT with either HYP or DMMB is effective in killing of C. albicans strains independent of their azole resistance pattern. HYP was more efficient at low fungal concentration and DMMB at higher concentrations.