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Hypocrellins, a group of naturally occurring perylenequinone pigments produced by Shiraia bambusicola, are notable for their potential use in photodynamic therapy (PDT) for treating cancers and viruses. Traditionally, hypocrellins have been extracted from the fruiting bodies of S. bambusicola, a parasitic fungus on bamboo. However, the yield from wild Shiraia fruiting bodies is often insufficient, prompting a shift towards seeking other fungi with higher yields of hypocrellins as alternative sources. This review comprehensively examines the current research on the isolation, identification, and bioactivity of fungal perylenequinones from Shiraia isolates from ascostromata or fruiting bodies, Shiraia-like endophytes, and other endophytes from bamboos. Additionally, the review discusses the culture methods and conditions for solid-state and submerged fermentation of hypocrellin-producing fungi, including medium components, culture conditions, and optimisation of fermentation factors, as mycelium cultures have emerged as a promising alternative for the production of hypocrellins. Furthermore, novel elicitation strategies are presented to address the bottleneck of lower production of hypocrellins in mycelium cultures, focusing on the preparation, characterisation, and application of biotic and abiotic elicitors. This review aims to facilitate further exploration and utilisation of fungal resources and elicitation strategies for enhanced production of hypocrellins in mycelium cultures. [ABSTRACT FROM AUTHOR]

Shiraia-like fungi, which are rare parasitic fungi found around bamboo, play an important role in traditional medicine. Their main active component, hypocrellin, is widely used in medicine, food, and cosmetics. By comparing strains with different hypocrellin yields, we identified a transcription factor (SbTF) in the hypocrellin biosynthesis pathway. SbTF from high-yielding zzz816 and low-yielding CNUCC C72 differed in its protein structure. Subsequently, SbTF from high-yielding zzz816 was overexpressed in several strains. This stabilised the yield in zzz816 and significantly increased the yield in low-yielding CNUCC C72. Comparing downstream non-essential genes between wild type and SbTF-overexpressing CNUCC C72 showed that SbMNF was significantly up-regulated. Therefore, it was selected for further study. SbMNF overexpression increased the hypocrellin yield in low-yielding CNUCC C72 and altered the composition of compounds in high-yielding CNUCC 1353PR and zzz816. This involved an increased elsinochrome C yield in CNUCC 1353PR and an increased hypocrellin B yield in zzz816 (by 2 and 70.3 times that in the corresponding wild type, respectively). This study is the first to alter hypocrellin synthesis to alter the levels of one bioactive agent compared to another. The results provide new insights regarding genetic modification and will help to optimise fungal fermentation. [ABSTRACT FROM AUTHOR]

Hypocrellins are major bioactive perylenequinones from Shiraia fruiting bodies and have been developed as efficient photosensitizers for photodynamic therapy. Pseudomonas is the second dominant genus inside Shiraia fruiting bodies, but with less known actions on the host fungus. In this work, the effects of bacterial volatiles from the Shiraia-associated Pseudomonas on fungal hypocrellin production were investigated. Pseudomonas putida No.24 was the most active to promote significantly accumulation of Shiraia perylenequinones including hypocrellin A (HA), HC, elsinochrome A (EA) and EC. Headspace analysis of the emitted volatiles revealed dimethyl disulfide as one of active compounds to promote fungal hypocrellin production. The bacterial volatiles induced an apoptosis in Shiraia hyphal cell, which was associated with the generation of reactive oxygen species (ROS). ROS generation was proved to mediate the volatile-induced membrane permeability and up-regulation of gene expressions for hypocrellin biosynthesis. In the submerged volatile co-culture, the bacterial volatiles stimulated not only HA content in mycelia, but also HA secretion into the medium, leading to the enhanced HA production to 249.85 mg/L, about 2.07-fold over the control. This is the first report on the regulation of Pseudomonas volatiles on fungal perylenequinone production. These findings could be helpful to understand the roles of bacterial volatiles in fruiting bodies and also provide new elicitation method using bacterial volatiles to stimulate fungal secondary metabolite production.

Hypocrellins (HYPs), a kind of natural perylenequinones (PQs) with an oxidized pentacyclic core, are important natural compounds initially extracted from the stromata of Hypocrella bambusae and Shiraia bambusicola. They have been widely concerned for their use as anti-microbial, anti-cancers, and anti-viral photodynamic therapy agents in recent years. Considering the restrictions of natural stromal resources, submerged fermentation with Shiraia spp. has been viewed as a promising alternative biotechnology for HYP production, and great efforts have been made to improve HYP production over the past decade. This article reviews recent publications about the mycelium fermentation production of HYPs, and their bioactivities and potential applications, and especially summarizes the progresses toward manipulation of fermentation conditions. Also, their chemical structure and analytic methods are outlined. Herein, it is worth mentioning that the gene arrangement in HYP gene cluster is revised; previous unknown genes in HYP and CTB gene clusters with correct function annotation are deciphered; the homologous sequences of HYP, CTB, and elc are systematically aligned, and especially the biosynthetic pathway of HYPs is full-scale proposed. Key points: • The mycelial fermentation process and metabolic regulation of hypocrellins are reviewed. • The bioactivities and potential applications of hypocrellins are summarized. • The biosynthesis pathway and regulatory mechanisms of hypocrellins are outlined. [ABSTRACT FROM AUTHOR]

Hypocrellins are anthraquinone that can act as excellent photosensitizers for photodynamic therapy. In the present work, we found that high‐speed countercurrent chromatography using cupric chloride as a complexing agent effectively separated hypocrellins from Shiraia bambusicola extract. The optimal two‐phase solvent system consisted of petroleum ether/ethyl acetate/methanol/water (7:3:5.5:4.5, v/v/v/v), with 0.01 mol/L cupric chloride in the lower phase at pH of 2.45. This lower phase served as the mobile phase, whereas the upper phase acted as the stationary phase. Employing a continuous separation mode, three continuous injections were found to allow the purification of 1.2 g of crude extract in approximately 12 h. Hypocrellin B (10.8 mg), hypocrellin A (16.2 mg), and hypocrellin C (15.6 mg) were obtained from this process. Simulation of complexation of hypocrellin A with divalent copper ion by computational chemistry calculations indicated that three pairs of hydroxyl and carbonyl groups in hypocrellin A had similar binding energies, and demonstrated that hypocrellin A and B owned different metal‐to‐ligand ratios as compared to hypocrellin C. These factors could modify the partitioning of these compounds in two‐phase solvent system, and resulting in a suitable separation factor. This method would also be used to purify other anthraquinones from natural products. [ABSTRACT FROM AUTHOR]

Shiraia bambusicola, as a well-known medicinal and edible macro-fungus, is generally located on the branches of bamboo and plays an important role in herbal treatment. Furthermore, the natural products from its fruiting bodies, hypocrellins, are attracting increasing amounts of attention due to their notable bioactivities. In this report, we focused on constructing a fast and universal transgenic system for S. bambusicola, which could easily regulate the biosynthesis of hypocrellins by altering gene expression. By the consideration of the broadened application of the mycelial transformation, it was determined to implement the Agrobacterium tumefaciens-mediated transformation (ATMT) technique, and a transcription factor (SbTF1) was employed to activate the downstream genes in different producing strains. After real-time polymerase chain reaction analysis, gene SbTF1 was verified to be overexpressed in all of the transgenic strains, and the yield of hypocrellins was significantly improved in strain CNU103846. To confirm the regulated function, the transcript levels of the downstream genes and the corresponding products at different stages were screened. In this study, we developed a stable and easily available assay for genetic transformation of S. bambusicola and employed the method to demonstrate that one transcription factor SbTF1 could effectively increase the yield of the final products.

Shiraia bambusicola, as a well-known medicinal and edible macro-fungus, is generally located on the branches of bamboo and plays an important role in herbal treatment. Furthermore, the natural products from its fruiting bodies, hypocrellins, are attracting increasing amounts of attention due to their notable bioactivities. In this report, we focused on constructing a fast and universal transgenic system for S. bambusicola, which could easily regulate the biosynthesis of hypocrellins by altering gene expression. By the consideration of the broadened application of the mycelial transformation, it was determined to implement the Agrobacterium tumefaciens-mediated transformation (ATMT) technique, and a transcription factor (SbTF1) was employed to activate the downstream genes in different producing strains. After real-time polymerase chain reaction analysis, gene SbTF1 was verified to be overexpressed in all of the transgenic strains, and the yield of hypocrellins was significantly improved in strain CNU103846. To confirm the regulated function, the transcript levels of the downstream genes and the corresponding products at different stages were screened. In this study, we developed a stable and easily available assay for genetic transformation of S. bambusicola and employed the method to demonstrate that one transcription factor SbTF1 could effectively increase the yield of the final products. [ABSTRACT FROM AUTHOR]

Purpose: Rapid noninvasive delineation of necrotic myocardium in ischemic regions is very critical for risk stratification and clinical decision-making but still challenging. This study aimed to evaluate the necrosis avidity of radioiodinated hypocrellins and its potential for rapidly imaging necrotic myocardium.Procedures: The aggregation constants of four natural hypocrellins were analyzed by UV/vis spectroscopy. Then, they were radiolabeled with iodine-131 by iodogen oxidation method. Necrosis avidity of iodine-131-labeled hypocrellins was evaluated in rat models with reperfused liver infarction and muscular necrosis by gamma counting, autoradiography, and histopathology. Their pharmacokinetic properties were examined in normal rats. The potential of iodine-131-labeled hypomycin A ([131I]HD) for early imaging of necrotic myocardium was explored in rat models with reperfused myocardial infarction. Finally, the possible mechanism of necrosis avidity was investigated by in vitro DNA binding and in vivo blocking experiments.Results: The aggregation constants of four hypocrellins were all much smaller than that of hypericin, a most studied necrosis avid agent. The radiochemical purities of the four radiotracers after purification were all greater than 95 %, and more than 90 % of tracers remained intact after incubation in rat serum for 24 h. Among the four tracers, [131I]HD exhibited the highest necrotic to viable tissue uptake ratio and the fastest blood clearance. The necrotic myocardium could be clearly visualized 4 h after injection of [131I]HD by single-photon emission computed tomography/X-ray computed tomography (SPECT/CT). DNA binding studies suggested that HD could bind to DNA through intercalation. Blocking studies demonstrated that uptake of [131I]HD in necrotic muscle could be significantly blocked by excess unlabeled HD and ethidium bromide with 67 and 60 % decline at 6 h after coinjection, respectively.Conclusions: [131I]HD can be used to rapidly visualize necrotic myocardium. The necrosis avidity mechanism of [131I]HD may be attributed to its binding to the exposed DNA in necrotic tissues. [ABSTRACT FROM AUTHOR]

Photodynamic therapy (PDT) has been a routine treatment of tumors and some microvascular diseases, but clinically available photosensitizers are still scarce. Among all kinds of photosensitizers, hypocrellins possess the most characteristics of ideal photosensitizers, such as, high photo-activity but low dark toxicity, fast clearance from tissues. This review is focused on two main topics, drug-delivery problem of hypocrellins and how the environment-sensitive fluorescence of hypocrellins was used for recognition of various biomolecules. Drug-delivery of hypocrellins was mainly achieved in two strategies — preparing the drug-delivery vehicles and finding quantitatively amphiphilic derivatives. Hypocrellin fluorescence originated from the intramolecular proton transfer is very distinct from other kinds of photosensitizers. Recently, it was proved that quantitative hypocrellin fluorescence could not only recognize various biomolecules, including proteins, polysaccharides and lipids, but also distinguish the specific binding from nonspecific binding with some kind of biomolecules. Meantime, hypocrellin fluorescence was pH-sensitive. It is known that tumor cells or tissues have the features of a large amount of lipid, neonatal collagen, over-expression of polysaccharides, and lower pH values compared to normal tissues. According to the relative but not absolute specificity, further studies on quantitative recognition of various biomolecules at a cellular level, may find a new clue to treat tumors by joint usage of photodynamic diagnosis (PDD) and PDT.

Photodynamic therapy (PDT) has been a routine treatment of tumors and some microvascular diseases, but clinically available photosensitizers are still scarce. Among all kinds of photosensitizers, hypocrellins possess the most characteristics of ideal photosensitizers, such as, high photo-activity but low dark toxicity, fast clearance from tissues. This review is focused on two main topics, drug-delivery problem of hypocrellins and how the environment-sensitive fluorescence of hypocrellins was used for recognition of various biomolecules. Drug-delivery of hypocrellins was mainly achieved in two strategies - preparing the drug-delivery vehicles and finding quantitatively amphiphilic derivatives. Hypocrellin fluorescence originated from the intramolecular proton transfer is very distinct from other kinds of photosensitizers. Recently, it was proved that quantitative hypocrellin fluorescence could not only recognize various biomolecules, including proteins, polysaccharides and lipids, but also distinguish the specific binding from nonspecific binding with some kind of biomolecules. Meantime, hypocrellin fluorescence was pH-sensitive. It is known that tumor cells or tissues have the features of a large amount of lipid, neonatal collagen, over-expression of polysaccharides, and lower pH values compared to normal tissues. According to the relative but not absolute specificity, further studies on quantitative recognition of various biomolecules at a cellular level, may find a new clue to treat tumors by joint usage of photodynamic diagnosis (PDD) and PDT. [ABSTRACT FROM AUTHOR]

Abstract: Non-surfactant hypocrellins (Hys) is a kind of photosensitizer with strong and broad absorption in visible light region, obtained by isolating from the fungus sacs of hypocrella bambusae. Previous studies have not yet addressed the use of hypocrellins as non-surfactant template in the synthesis of photocatalyst CdS, let alone the achievement of higher efficient photocatalytic activity than Degussa P25. Herein Al doped CdS prepared by using hypocrellins as template (Al-CdS/Hys) can enhance the photocatalytic activity while Al doped CdS without template (Al-CdS) prepared by conventional solvothermal method reduced the photocatalytic activity. Al-CdS/Hys was synthesized and characterized by a combination of various physicochemical techniques, such as XRD, nitrogen adsorption/desorption, diffuse reflectance UV–vis and TEM. Al-CdS/Hys is even more efficient than Degussa P25 for the degradation of organic dye, basic fuchsin under artificial solar irradiation. Moreover, doping Al increased the surface area of CdS to169.1 m2 g–1. [Copyright &y& Elsevier]

Abstract: Hypocrellin B (HB), a naturally occurring photosensitizer, has been extensively and intensively studied as a promising photodynamic therapy (PDT) agent. In this work, a new Co(III) complex [Co2(HB)(tmp)4]4+ (tmp=3,4,7,8-tetramethyl-1,10-phenanthroline) was designed and synthesized with HB as bridging ligand and tmp as terminal ligand. [Co2HB(tmp)4]4+ exhibits improved water solubility, enhanced absorptivity in the phototherapeutic window, increased binding affinity and DNA photocleavage capability toward dsDNA with respect to HB. The photodynamic activity of [Co2(HB)(tmp)4]4+ stems from its 1O2 photosensitization ability, in sharp contrast to [Cu2(HB)(tmp)2]2+ which relies on superoxide anion radical (O2 - roxyl radical (·OH) to photocleave DNA, though the both complexes possess similar electrochemical properties. The remarkable difference between the photodynamic mechanisms of [Co2(HB)(tmp)4]4+ and [Cu2(HB)(tmp)2]2+ was discussed in detail. [Copyright &y& Elsevier]

Abstract: For making hypocrellins clinically applicable for phototherapy to vascular diseases, it is mainly focused onto finding a derivative which can be transported fluently in blood system but without serious loss of the inherent activity of its parents. Based on this consideration, a novel 17-3-amino-1-propane-sulfonic acid-HB Schiff-base (NSHB) was designed and synthesized in this work. As expected, NSHB is readily dissolved in phosphate buffered saline (PBS) or any other aqueous solvent in a concentration which is suitable for intravenous injection, while the quite higher partition coefficient (5:1) is beneficial to the affinity to biological targets. Based on EPR measurements, it is proved that the photosensitization activity of NSHB to photo-generate semiquinone anion radicals and superoxide anion radical () is even higher than its parent HB, while the ability to generate singlet oxygen () is not seriously reduced. In addition, nearly comparable PDT activity to A549 cells for NSHB and HB confirms that the molecular design is successful and NSHB is readily delivered into target tissues via blood circulation after intravenous injection. Furthermore, the quantum yield of for NSHB is as 12.5 times as that for HB under red light (600–700nm), which is beneficial to phototherapy to solid tumors. [Copyright &y& Elsevier]

Abstract: Previous work has proved that hypocrellin B (HB) binds to human serum albumin (HSA) at a specific site instead of distributed randomly on the surface of a protein. In the current work, further investigation by using bilirubin as a site I marker indicates that HB can compete for the same site with bilirubin, suggesting that the HB binding site is located at sub-domain IIA (site I) of HSA. Moreover, bound to HSA, the HB fluorescence was found to be pH sensitive in physiological range (pH 6.0–8.0). The increasing of binding constant of HB to HSA in the pH range 6–8 also indicates that the N↔B transition modulates the microenvironment changes of the binding site and influences considerably the binding between HB and HSA. Furthermore, picosecond time-resolved fluorescence spectra of HB–HSA complex in PBS indicate an additional short-lived component compared to that for HB in benzene, which may be assigned to the process of electron transfer from Trp-214 to HB. [Copyright &y& Elsevier]

In this paper, quantum chemical method RHF/6-31G(d,p)//AM1 was employed to investigate the photobleaching behaviors of hypocrellins. It is found that frontier charge rather than Mulliken charge is an applicable parameter to predict the 1O2- and O2ꔷ−-addition positions of the pigments; accordingly some experimental observations on the photobleaching mechanisms of hypocrellins were elucidated. In addition, some theoretical predictions that were not observed in the previous experiments will be helpful to direct the further study. [Copyright &y& Elsevier]

Two phenmethylamino hypocrellin B derivatives are novel photodynamic agents synthesized by a mild reaction between hypocrellin B and phenmethylamine. The red absorption of the photosensitizers is enlarged distinctly and the peri-hydroxylated perylenequinone structure of the parent HB is preserved. 9,10-diphenyl-anthracene (DPA) bleaching and electron paramagnetic resonance (EPR) spin trapping techniques were used to study the photodynamic activities of the phenmethylamino hypocrellin B derivatives in the presence of oxygen. Singlet oxygen (1O2) and superoxide anion radical (O2·–) generated in the process of illumination of the phenmethylamino hypocrellin B in aerobic solution were observed. The photodamage of PMAHBs to MGC803 cancer cells was investigated in vitro. The results in vitro reveal that the phenmethylamino hypocrellin B derivatives show a much less significant decrease in cytotoxicity than that of their parent HB. It exhibits higher selectivity of light-orientation, which can decrease the damage to normal tissues by irradiating the tumor tissues, and so increases the drug safety. [Copyright &y& Elsevier]

Mentions the advantages of hypocrellins over hematoporphyrin derivatives as phototherapeutic agents. The high-excited triplet state yield, high phototoxicity, low dark toxicity, and rapid metabolism of hypocrellins; Indication they show little absorption in the photodynamic window and are not water soluble, which limits their application in photodynamic therapy; Improved water solubility in the sulfonated and metal-ioned hypocrellins; Finding that the water-soluble derivatives have lower photodynamic activity than the parent hypocrellins; Synthesis of thiolated and aminated hypocrellins to enlarge their photoresponse; Indication that the authors have designed and synthesized some amphiphilic aminated hypocrellin derivatives that have enhanced photoresponse and water solubility; Outlook for experiments on the derivatives in vitro and in vivo.

The photophysical and photochemical properties of hypocrellins (HA and HB) are examined with two-photon excitations at 800 nm using femtosecond pulses from a Ti:sapphire laser. The two-photon excited fluorescence spectra of HA and HB are very similar to those obtained by one-photon excitation, which may indicate that the two-photon induced photodynamic processes of hypocrellins are similar to one-photon induced photodynamic processes. The two-photon excitation cross sections of HA and HB are measured at 800 nm as about 34.8×10−50 cm4 s/photon and 21.3×10−50 cm4 s/photon, respectively. The large two-photon cross sections of both HA and HB, suggest that the hypocrellins can be potential two-photon phototherapeutic agents. As an example for two-photon photodynamic therapy of hypocrellins, we also further examine the cell-damaging effects of HA upon two-photon illumination. Our preliminary results of cell viability test indicate hypocrellins can effectively damage the Hela cells under two-photon illumination. [Copyright &y& Elsevier]