Exopolysaccharides produced by Antrodia cinnamomea using microparticle-enhanced cultivation: Optimization, primary structure and antibacterial property.

Microparticle-enhanced cultivation was used to enhance the production of exopolysaccharides (EPSs) from Antrodia cinnamomea. The structure and antibacterial activity of two EPSs produced by A. cinnamomea treated with Al2O3 [EPS-Al (crude) and EPS-Al-p (purified)] and without Al2O3 [EPS-C (crude) and EPS-C-p (purified)] were compared. It was observed that the addition of 4 g/L Al2O3 at 0 h resulted in the highest EPS yield of 1.46 g/L, possible attributed to the enhanced permeability of the cell membrane. The structural analysis revealed that EPS-C-p and EPS-Al-p had different structures. EPS-C-p was hyperbranched and spherical with a Mw of 10.8 kDa, while EPS-Al-p was irregular and linear with a Mw of 12.5 kDa. The proportion of Man in EPS-Al-p decreased, while those of Gal and Glc increased when compared to EPS-C-p. The total molar ratios of 6-Glcp and 4-Glcp in EPS-Al-p are 1.45 times that of EPS-C-p. Moreover, EPSs could alter bacterial cell morphology, causing intracellular substance leakage and growth inhibition, with EPS-Al having a stronger antibacterial activity than EPS-C. In conclusion, A. cinnamomea treated with Al2O3 could produce more EPSs, changing monosaccharide composition and glycosidic linkage profile, which could exert stronger antibacterial activity than that produced by untreated A. cinnamomea.

Cancer immunotherapeutic effect of carboxymethylated ß-d-glucan coupled with iron oxide nanoparticles via reprogramming tumor-associated macrophages.

The cancer immunotherapeutic effect of a carboxymethylated ß-d-glucan (CMPTR)/iron oxide nanoparticles (IONPs) system (CMPTR/IONPs) were investigated by using cell culture of bone marrow-derived macrophages (BMDMs) and B16F10 melanoma skin cancer-bearing mouse model. When compared with that of control group, CMPTR/IONPs-treated M2-like BMDMs exhibited upregulated M1 biomarkers expression, significantly inhibited the migration of B16F10 cancer cells (p < 0.05), and had the highest apoptotic percentage of B16F10 cancer cells (80.39 ± 8.73 %) in co-culture system. Intratumoral administration of CMPTR/IONPs significantly (p < 0.05) suppressed tumor growth (46.58 % based on tumor weight) in mice and enhanced the M1/M2 ratio from 0.40 ± 0.09 (control group) to 6.64 ± 1.61 in tumor associated macrophages (TAMs) which was higher than that of in CMPTR (1.27 ± 0.38), IONPs (1.38 ± 0.17). CMPTR/IONPs treatment also promoted apoptosis in cancer cells and increased the infiltration of CD4 and CD8 T-lymphocytes in tumor tissues. These results could be due to the combined effects of CMPTR and IONPs in the CMPTR/IONPs system, possibly mediated by the activation of NF-κB and IRF5 pathways for inducing M1 macrophages polarization and had potential cancer immunotherapeutic applications.

A Tumor-Targeting Dual-Stimuli-Activatable Photodynamic Molecular Beacon for Precise Photodynamic Therapy.

A multifunctional photodynamic molecular beacon (PMB) has been designed and synthesized which contains an epidermal growth factor receptor (EGFR)-targeting cyclic peptide and a trimeric phthalocyanine skeleton in which the three zinc(II) phthalocyanine units are each substituted with a glutathione (GSH)-responsive 2,4-dinitrobenzenesulfonate (DNBS) quencher and are linked via two cathepsin B-cleavable GFLG peptide chains. This tailor-made conjugate is fully quenched in the native form due to the photoinduced electron transfer effect of the DNBS moieties and the self-quenching of the phthalocyanine units. It can target the EGFR overexpressed in cancer cells, and after receptor-mediated endocytosis, it can be activated selectively by the co-existence of intracellular GSH and cathepsin B, both of which are also overproduced in cancer cells, in terms of fluorescence emission and singlet oxygen generation. The cell-selective behavior of this PMB has been demonstrated using a range of cancer cells with different expression levels of EGFR, while the stimuli-responsive properties have been studied both in vitro and in various aqueous media. The overall results show that this advanced PMB, which exhibits several levels of control of the tumor specificity, is a promising photosensitizer for precise antitumoral photodynamic therapy.

Mushroom Carboxymethylated ß-d-Glucan Functions as a Macrophage-Targeting Carrier for Iron Oxide Nanoparticles and an Inducer of Proinflammatory Macrophage Polarization for Immunotherapy.

ß-d-glucans have the potential of serving as both macrophage-targeted carriers and immune stimulators via inducing trained immunity in macrophages. In this study, a carboxymethylated ß-glucan from mushroom sclerotium of Pleurotus tuber-regium (CMPTR) was combined with iron oxide nanoparticles (IONPs) to form nanocomplexes (CMPTR/IONPs) with particle size around 193 ± 7 nm, which could exert a concerted effect on inducing proinflammatory M1 phenotype macrophages for immunotherapy. This nanocomplex exhibited good stability and low cytotoxicity (over 80% cellular viability of RAW 264.7 and THP-1) and higher cellular uptake by murine macrophages compared with B16F10 cells (p < 0.05). CMPTR/IONPs could convert M2-like bone marrow-derived macrophages into M1 phenotypes with upregulated expression of pro-inflammatory cytokines (IL12 and TNF-α, p < 0.05) and reduced immune-suppressive cytokines (IL10 and TGF-ß, p < 0.05). Such polarization was mediated by the combined signaling regulatory factors, including IONP-stimulated IRF5 and CMPTR-triggered TLRs-NF-κB pathways (p < 0.05). Accordingly, CMPTR could have a dual function as a macrophage-targeting carrier for IONPs and an immunostimulant to induce inflammatory M1 macrophage polarization for immunotherapy.

Stimulating mechanism of corn oil on biomass and polysaccharide production of Pleurotus tuber-regium mycelium.

Hyperbranched polysaccharides (HBPSs) are the main components in cell wall and exopolysaccharide (EPS) of Pleurotus tuber-regium. To enhance the yield of these macromolecules, corn oil at 4% addition exhibited the best effect for production of mycelial biomass at 20.49 g/L and EPS at 0.59 g/L, which was 2.56 folds and 1.90 folds of the control, respectively. The treated hyphae were much thicker with smooth surface, while its cell wall content (43.81 ± 0.02%) was 1.96 times of the control (22.34 ± 0.01%). Moreover, a large number of lipid droplets could be visualized under the view of confocal laser scanning microscopy (CLSM). RNA-seq analysis revealed that corn oil could enter the cells and result in the up-regulation of genes on cell morphology and membrane permeability, as well as the down-regulation on expression level of polysaccharide hydrolase and genes involved in the MAPK pathway, all of which probably contribute to the increase of polysaccharides production.

Beta-d-glucan-based drug delivery system and its potential application in targeting tumor associated macrophages.

Use of polysaccharides as carriers in drug delivery system is a hot topic, especially those with specific recognition of immune cells, enabling them to be applied in targeting delivery system. ß-d-glucans are naturally occurring non-digestible polysaccharides with immunomodulatory activities that have attracted increasing attention to serve as therapeutic agents or immune-adjuvants. Being able to be specifically recognized by immune cells like macrophages, ß-d-glucans can be developed as promising carriers for targeting delivery with stability, biocompatibility and specificity when applied in immunotherapy. Targeting tumor associated macrophages (TAMs) is an emerging strategy for cancer immunotherapy since it exerts anti-cancer effects based on modulating body immunity in tumor microenvironment (TME). This new strategy does not require high concentration of drugs to kill cancer cells directly and lessen tumor recurrence by creating unique immune memory for malignant cells. In this review, construction strategies of polysaccharide-based drug delivery system of three types of ß-d-glucan including non-yeast and yeast ß-d-glucans as well as hyper-branched ß-d-glucan are discussed with reference to their branching characteristics and conformation. The applications of these ß-d-glucans as nano-carrier for drug delivery targeting TAMs are also discussed.

Research on a Specialty Mushroom (Pleurotus tuber-regium) as a Functional Food: Chemical Composition and Biological Activities.

Pleurotus tuber-regium (PTR) is an edible specialty mushroom that has attracted growing interest recently because of its sensory attributes, high nutritional values, and important medicinal properties. PTR is rich in bioactive polysaccharides, proteins with essential amino acids, essential fatty acids, dietary fiber, minerals, and vitamins. Current studies have shown that the nutrients and bioactive ingredients of PTR contribute to their antitumor, antihypercholesterolemic, antihypertensive, antiobesity, hepatic-protective, antimicrobial, antioxidant, and prebiotic activities, indicating that PTR is a promising functional food and nutraceutical. In this review, the chemical constituents and physiological functions of PTR are summarized, which provide the scientific basis to support the further research and development of its application in the food and pharmaceutical industries.

pH-sensitive PEG-coated hyper-branched ß-d-glucan derivative as carrier for CpG oligodeoxynucleotide delivery.

ß-d-glucan is a natural non-digestible polysaccharide that can be selectively recognized by recognition receptors such as Dectin-1 receptors, resulting in an emerging interest on exploring its capacity for carrying biological information to desired organs or cells. CpG oligodeoxynucleotide (ODN) has the potentiality to initiate an immune-stimulatory cascade via activating B cells inducing proinflammatory cytokines, which is conducive to immunotherapy and nucleic acid vaccine. Herein, we developed a pH-sensitive delivery system loading with CpG ODN by introducing poly-ethylenimine (PEI) to a hyperbranched ß-d-glucan (HBB) and coating with poly-ethylene glycol (PEG) shell via acidic liable Schiff bond. This delivery system exhibited a favorable biocompatibility and facilitated the cellular uptake of CpG ODN at pH 6.8 with the possibility of having higher accumulation in acidic cancer microenvironment. Furthermore, this carrier together with class B CpG ODN could enhance the secretion of cytokines including interleukin-6 and interferon-α as well as capable of interferon-α induction.

Immunomodulatory Effect of Structurally Characterized Mushroom Sclerotial Polysaccharides Isolated from Polyporus rhinocerus on Bone Marrow Dendritic Cells.

This study evaluated the immunomodulatory effects of two high-molecular-weight and structurally different mushroom polysaccharides, an alkali-soluble polysaccharide (mPRSon) and a water-soluble polysaccharide-protein complex (PRW), isolated previously from the sclerotia of Pleurotus rhinocerus, on the maturation of murine bone-marrow-derived dendritic cells (BMDCs). The effects of mPRSon and PRW on the expression of morphological change, surface molecules, phagocytic activity, and cytokine release in BMDCs were determined by flow cytometry and a mouse cytokine array. The results showed that both mPRSon and PRW could induce phenotypic and functional maturation of BMDCs. At the same time, mPRSon upregulated the expression of membrane phenotypic marker CD86 and PRW markedly upregulated CD40, CD80, and CD86. In addition, mPRSon could bind to the dectin-1 receptor and stimulate the release of MIP-1α, MIP-2, and IL-2, while PRW could bind to complement receptor 3 and toll-like receptor 2 with an upregulation of the expression of IL-2, IL-6, MIP-1α, MIP-2, RANTES, IL-12p40p70, IL-12p70, TIMP-1, IFN-γ, KC, MCP-1, and GCSF. The study provides additional information on how structural differences in sclerotial polysaccharides influence their immunomodulatory activities on BMDCs involving different PAMP receptors. It is anticipated that more understanding of the interactions between the sclerotial polysaccharides and their receptors in immune cells can facilitate their future application for cancer immunotherapy.

Anti-Inflammatory Activity of the Wild Mushroom, Echinodontium tinctorium, in RAW264.7 Macrophage Cells and Mouse Microcirculation.

The aim of this study was to investigate the anti-inflammatory activity of a previously un-studied wild mushroom, Echinodontium tinctorium, collected from the forests of north-central British Columbia. The lipopolysaccharide (LPS)-induced RAW264.7 macrophage model was used to study the in vitro anti-inflammatory activity. The crude alkaline extract demonstrated potent anti-inflammatory activity, and was further purified using a "bio-activity-guided-purification" approach. The size-exclusion and ion-exchange chromatography yielded a water-soluble anti-inflammatory polysaccharide (AIPetinc). AIPetinc has an average molecular weight of 5 kDa, and is a heteroglucan composed of mainly glucose (88.6%) with a small amount of galactose (4.0%), mannose (4.4%), fucose (0.7%), and xylose (2.3%). In in vivo settings, AIPetinc restored the histamine-induced inflammatory event in mouse gluteus maximus muscle, thus confirming its anti-inflammatory activity in an animal model. This study constitutes the first report on the bioactivity of Echinodontium tinctorium, and highlights the potential medicinal benefits of fungi from the wild forests of northern British Columbia. Furthermore, it also reiterates the need to explore natural resources for alternative treatment to modern world diseases.

Structure and Immunomodulatory Activity of Microparticulate Mushroom Sclerotial ß-Glucan Prepared from Polyporus rhinocerus.

In this study, an immunologically active novel microparticulate mushroom ß-glucan (PRA-1p) was prepared using an alkali-soluble glucan PRA-1 by an emulsification and cross-linking method. PRA-1 was a hyperbranched (1→3),(1→6)-ß-d-glucan with a degree of branching of 0.89, isolated from the sclerotia of Polyporus rhinocerus. PRA-1 had a rod-like conformation, while PRA-1p exhibited a monodisperse and homogeneous spherical conformation with a diameter ranging from 0.3 to 2.0 µm in water. PRA-1p significantly induced nitric oxide and reactive oxygen species production as well as morphological changes of murine macrophages (RAW 264.7 cells) and upregulated their phagocytic activity. Furthermore, PRA-1p treatment markedly enhanced the secretion of cytokines, including cutaneous T cell-attracting chemokine 27, granulocyte-colony-stimulating factor, monocyte chemoattractant protein 1, macrophage inflammatory protein 1α, macrophage inflammatory protein 2, regulated on activation, normal T cell expressed and secreted, soluble tumor necrosis factor receptor 1, and tissue inhibitors of metalloproteinases. Activation of RAW 264.7 cells triggered by PRA-1p was associated with activation of inducible nitric oxide synthase, nuclear factor κB, extracellular signal-regulated kinase, and protein kinase B. This work suggests that novel PRA-1p derived from the mushroom sclerotia of P. rhinocerus has potential application as an immunostimulatory agent.

Production of bioactive metabolites by submerged fermentation of the medicinal mushroom Antrodia cinnamomea: recent advances and future development.

Edible and medicinal mushrooms have usually been considered as a sustainable source of unique bioactive metabolites, which are valued as promising provisions for human health. Antrodia cinnamomea is a unique edible and medicinal fungus widespread in Taiwan, which has attracted much attention in recent years for its high value in both scientific research and commercial applications owing to its potent therapeutic effects, especially for its hepatic protection and anticancer activity. Due to the scarcity of the fruiting bodies, the cultivation of A. cinnamomea by submerged fermentation appears to be a promising substitute which possesses some unique advantages, such as short culture time period and its high feasibility for scale-up production. However, the amount of fungal bioactive metabolites derived from the cultured mycelia of A. cinnamomea grown by submerged fermentation is much less than those obtained from the wild fruiting bodies. Hence, there is an urgent need to bridge such a discrepancy on bioactive metabolites between the wild fruiting bodies and the cultured mycelia. The objective of this article is to review recent advances and the future development of the mycelial submerged fermentation of A. cinnamomea in terms of enhancement for the production of fungal bioactive components by the optimization of culture conditions and the regulation of fungal metabolism. This review provides valuable information for further biotechnological applications of A. cinnamomea as well as other mushrooms being the source of bioactive ingredients by submerged fermentation.

Mechanistic Study of the In Vitro and In Vivo Inhibitory Effects of Protocatechuic Acid and Syringic Acid on VEGF-Induced Angiogenesis.

The antiangiogenic activities of two structurally similar phenolics, protocatechuic acid (PA) and syringic acid (SA), were investigated. In vitro study using HUVECs demonstrated that both PA and SA (at 25 µM) significantly ( p < 0.05) inhibited VEGF-induced cell proliferation by 22.68 ± 5.6% and 21.93 ± 2.0%, respectively; cell migration by 50.04 ± 3.3% and 39.72 ± 4.7%, respectively; cell invasion by 44.16 ± 4.23% and 51.90 ± 2.73%, respectively; and cellular ROS generation by 11.48 ± 6.32% and 21.17 ± 9.10%, respectively. Our mechanistic study revealed that PA and SA blocked the VEGFR2-dependent Akt/ MMP2 and ERK pathways in HUVECs. These inhibitory effects were further confirmed by a decrease of endogenous alkaline phosphatase activity for PA and SA (21.47 ± 1.77% and 10.37 ± 1.27%, respectively) and the suppression of subintestinal vessel plexus formation in Tg (fli1a:EGFP) y1-type transgenic zebrafish embryos. PA and SA down-regulated the angiogenesis-related signal transduction pathway of VEGFα-VEGFR2 or Ang2- Tie2 in zebrafish. Moreover, it was also found that PA demonstrated a better inhibition on VEGF-induced migration of HUVEC and zebrafish vasculature. This might be due to the different number of hydroxyl and methoxy substituents possessed by PA and SA. Taken together, these findings indicate that phenolics PA and SA have potent antiangiogenic activities and are potential targets for the design and development of anticancer agents.

In Vitro and In Vivo Photothermal Cancer Therapeutic Effects of Gold Nanorods Modified with Mushroom ß-Glucan.

The photothermal cancer therapeutic effect of the AuNR-Glu nanohybrids produced by coating native gold nanorods (AuNRs) with a natural mushroom biopolymer from the Pleurotus tuber-regium sclerotia (Glu) were studied in the second near-infrared window (NIR-II). The AuNR-Glu exhibited low cytotoxicity and high biocompatibility due to the surface modification of Glu when compared with the native AuNRs. AuNR-Glu nanohybrids had a high photothermal transduction efficiency (η) of 43.12%, causing effective in vitro cell ablation in both HT-29 (94.2 ± 0.8% cell death) and SW480 (94.8 ± 1.1% cell death) colon cancer cells under 1064 nm NIR-II laser irradiation at 1.0 W/cm2. Intravenous injection of AuNR-Glu nanohybrids followed by irradiation from a NIR-II laser at a safe dose (1.0 W/cm2 for 5 min) in nude mice implanted with HT-29 tumors was effective in significantly reducing the tumor growth, with no obvious harmful side effects, as evidenced by histological analysis of major organs. The present results have shown that AuNRs modified by natural biopolymers from mushroom ß-glucans are novel nanomaterials with low cytotoxicity and effective photothermal anticancer agents with potential biomedical applications.

Anti-proliferative activity of a purified polysaccharide isolated from the basidiomycete fungus Paxillus involutus.

A growth-inhibitory polysaccharide (GIPinv) was purified using size-exclusion and ion-exchange chromatography from the fourth sodium hydroxide extraction step of a fungus found in British Columbia. The fungus was genetically identified as a member of the Paxillus involutus complex. GIPinv has an average molecular weight of 229kDa and is a heteroglycan composed of glucose (65.9%), galactose (20.8%), mannose (7.8%), fucose (3.2%) and xylose (2.3%). GC-MS methylation analysis suggests that GIPinv has mixed linkages in the backbone containing (1→6)-Gal (25.5%), (1→4)-Glc (18.3%), (1→6)-Glc (8.3%), (1→3)-Glc (5.3%) and (1→2)-Xyl (4.5%). GIPinv has branching points at (1→2, 6)-Man (8.6%) and (1→3, 6)-Man (4.9%) having unsubstituted fucose (8.3%) and glucose (16.3%) as terminal sugars. GIPinv had growth-inhibitory activity against several cancer cell lines and triggered apoptosis. GIPinv should be further explored as a potential anti-cancer agent and a unique polysaccharide.

Formation and characterization of polyelectrolyte complex synthesized by chitosan and carboxylic curdlan for 5-fluorouracil delivery.

In this study, negatively charged carboxylic curdlan (Cc) bearing a ß-1,3-polyglucuronic acid structure was employed to fabricate nanosized polyelectrolyte complexes (PECs) with positively charged chitosan (CS) in aqueous solution as potential carriers for 5-fluorouracil (5Fu) delivery. Nanosized CS/Cc PECs were formed by the addition of 0.5mg/mL solutions of CS and Cc with a mixing ratio of 1:1 (w/w) at pH 3.0. Under optimized conditions, the prepared CS/Cc PECs showed spherical morphology with an average size of about 180nm and a zeta potential of around 41mV. The 5Fu drug was incorporated into the nanosized CS/Cc PECs and showed excellent encapsulation efficiency (86.47%) and loading content (10.81%). The drug release data in vitro indicated that the nanosized CS/Cc PECs are promising carriers for the sustained release of 5Fu with an anomalous transport mechanism following the Ritger-Peppas model. Besides, the CS/Cc PECs exhibited low cytotoxic activity against SPCA-1 and HeLa cell lines in vitro. This finding suggested that the development of the nanosized CS/Cc PECs offered great promise as an antitumor drug platform.

Stable and Biocompatible Mushroom ß-Glucan Modified Gold Nanorods for Cancer Photothermal Therapy.

Naturally occurring ß-glucans have been widely regarded as a natural source for functional foods and pharmaceuticals due to their immunomodulatory property and antitumor activity. However, physicochemically stable and biocompatible ß-glucans are rarely explored as a carrier for nanomaterials to overcome the problems of aggregation and nanotoxicity. Here, we developed highly stable and biocompatible mushroom ß-glucan coated gold nanorods (AuNR-Glu) for cancer photothermal therapy by integrating Pleurotus tuber-regium sclerotial ß-glucan (Glu) and plasmonic gold nanorods (AuNRs) possessing photothermal property in the second near-infrared (NIR-II) window. AuNR-Glu showed high colloidal stability in various biological media, even in simulated gastric fluid. Moreover, AuNR-Glu had low cytotoxicity and high photothermal stability, which are excellent characteristics for photothermal agents for cancer therapy. In vitro experiments showed that AuNR-Glu nanohybrid was effective against MCF-7 (only 4.5 ± 0.9% viability) at a low dose of 20 µg/mL under NIR-II at a safe laser power density (0.75 W/cm2). Natural mushroom ß-glucans are potential functional polymers that can be used to fabricate nanohybrids for biomedical applications.

Immunomodulatory Activity of Polysaccharide-Protein Complex from the Mushroom Sclerotia of Polyporus rhinocerus in Murine Macrophages.

A novel water-soluble polysaccharide-protein complex (PRW1) isolated from the sclerotia of an edible mushroom Polyporus rhinocerus which was purified by membrane ultrafiltration could significantly activate murine macrophages RAW264.7 in vitro. PRW1 had a molecular weight of less than 50 kDa and was found to be a highly branched heteropolysaccharide-protein complex composed of 45.7 ± 0.97% polysaccharide and 44.2 ± 0.41% protein. Based on the results of total acid hydrolysis, methylation analysis, and Fourier transform infrared spectroscopy, the carbohydrate moiety of PRW1 was found to be a ß-d-mannoglucan with its backbone containing →1)-d-Glcp-(4→, →1)-d-Glcp-(6→, and →1)-d-Manp-(2→ residues (molar ratio of 5:4:6) and having terminal d-Glcp as side chain (degree of branching of 0.62). In vitro studies showed that PRW1 significantly induced NO production and enhanced the release of a variety of cytokines including G-CSF, GM-CSF, IL-6, IL12p40/70, MCP-1, MCP-5, MIP-1-α, MIP-2, RANTES, sTNFRI, and TNF-α. Mechanistically, PRW1 treatment triggered ERK phosphorylation to activate macrophages within 15 min and significantly increased the expression level of inducible NOS after 6 h. In summary, this study indicates that PRW1 derived from the sclerotia of P. rhinocerus is a potential immunomodulatory agent for cancer immunotherapy.

Antioxidant and antiangiogenic properties of phenolic extract from Pleurotus tuber-regium.

Pleurotus tuber-regium (Fries) Singer (PTR), both an edible and a medicinal mushroom also known as tiger milk mushroom, has experienced growing popularity and economic importance due to its flavor, nutritive value, and medicinal effects. In this study, the antioxidant and antiangiogenic activities of a 60% ethanol extract (EE) obtained from the sclerotium of PTR were investigated. Typical phenolic compounds including protocatechuic, chlorogenic, syringic, ferulic, and folic acid were identified and quantified in EE by the HPLC-UV-ESI/MS analyses. EE possessed strong antioxidant activity and could dose-dependently inhibit vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cells (HUVEC) migration and tube formation. qPCR results showed that VEGF-induced FGF, ANG-Tie, and MMP gene expression as well as VEGFR were down-regulated at the mRNA level after treated with EE, suggesting that multiple molecular targets related to angiogenesis was involved. Furthermore, EE also inhibited the formation of subintestinal vessel plexus (SIVs) in zebrafish embryos in vivo. All of these suggested that EE of PTR could be the source of potential inhibitors to target angiogenesis.