[Molecular mechanism of ligustilide attenuating OGD/R injury in PC12 cells by inhibiting ferroptosis].

The aim of this study is to explore the mechanism of ligustilide, the main active constituent of essential oils of traditional Chinese medicine Angelicae Sinensis Radix, on alleviating oxygen-glucose deprivation/reperfusion(OGD/R) injury in PC12 cells from the perspective of ferroptosis. OGD/R was induced in vitro, and 12 h after ligustilide addition during reperfusion, cell viability was detected by cell counting kit-8(CCK-8) assay. DCFH-DA staining was used to detect the level of intracellular reactive oxygen species(ROS). Western blot was employed to detect the expression of ferroptosis-related proteins, glutathione peroxidase 4(GPX4), transferrin receptor 1(TFR1), and solute carrier family 7 member 11(SLC7A11), and ferritinophagy-related proteins, nuclear receptor coactivator 4(NCOA4), ferritin heavy chain 1(FTH1), and microtubule-associated protein 1 light chain 3(LC3). The fluorescence intensity of LC3 protein was analyzed by immunofluorescence staining. The content of glutathione(GSH), malondialdehyde(MDA), and Fe was detected by chemiluminescent immunoassay. The effect of ligustilide on ferroptosis was observed by overexpression of NCOA4 gene. The results showed that ligustilide increased the viability of PC12 cells damaged by OGD/R, inhibited the release of ROS, reduced the content of Fe and MDA and the expression of TFR1, NCOA4, and LC3, and improved the content of GSH and the expression of GPX4, SLC7A11, and FTH1 compared with OGD/R group. After overexpression of the key protein NCOA4 in ferritinophagy, the inhibitory effect of ligustilide on ferroptosis was partially reversed, indicating that ligustilide may alleviate OGD/R injury of PC12 cells by blocking ferritinophagy and then inhibiting ferroptosis. The mechanism by which ligustilide reduced OGD/R injury in PC12 cells is that it suppressed the ferroptosis involved in ferritinophagy.

Unveiling the Disaccharide-Branched Glycosaminoglycan and Anticoagulant Potential of Its Derivatives.

Glycosaminoglycans (GAGs) are conserved polysaccharides composed of linear repeating disaccharides and play crucial roles in multiple biological processes in animal kingdom. However, saccharide-branched GAGs are rarely found, except the fucose-branched one from sea cucumbers. There was conjecture about the presence of disaccharide-branched GAG since 30 years ago, though not yet confirmed. Here, we report a GAG containing galactose-fucose branches from Thelenota ananas. This unique branch was confirmed as d-Gal4S(6S)-α1,2-l-Fuc3S by structural elucidation of oligosaccharides prepared from T. ananas GAG. Bioassays indicated that oligomers with a larger degree of polymerization exhibited a potent anticoagulation by targeting the intrinsic tenase. Heptasaccharide was proven as the minimum fragment retaining the anticoagulant potential and showed 92.6% inhibition of venous thrombosis in vivo at sc. of 8 mg/kg with no obvious bleeding risks. These results not only solve a long-standing question about the presence of disaccharide-branched GAG in Holothuroidea, but open up new opportunities to develop safer anticoagulants.

Oligosaccharides from fucosylated glycosaminoglycan prevent breast cancer metastasis in mice by inhibiting heparanase activity and angiogenesis.

The invasion and metastasis of tumor cells are the hallmarks of malignant diseases and the greatest obstacle to overcome. Heparanase-mediated degradation of heparan sulfate (HS) is the critical process for tumor angiogenesis and metastasis, therefore, heparanase become an attractive target for cancer research. Herein, we reported a native fucosylated glycosaminoglycan (nHG) extracted from sea cucumber Holothuria fuscopunctata and a depolymerized nHG (dHG) and its contained oligosaccharides (hs17, hs14, hs11, hs8 and hs5), acting as heparanase inhibitors. nHG and its derivatives have the ability to bind with heparanase directly, leading to significant inhibition of heparanase activity. Moreover, their apparent binding affinity to heparanase was comparable to their inhibitory effect, which was elevated along with the increase of chain length, similar to the effect of heparins. In addition, oligosaccharides inhibited the migration and invasion of 4T1 mammary carcinoma cells and human umbilical vein endothelial cells (HUVECs) and also suppressed tube formation in Matrigel matrix and angiogenesis in the chick chorioallantoic membrane (CAM) assay. In the metastatic mouse model, oligosaccharides exhibited practical antimetastatic effects on 4T1 mammary carcinoma cells. According to the reported anticoagulant activity and the low bleeding tendency of dHG and its oligosaccharides, the use of the oligosaccharides may lead to better effects on tumor patients with thrombosis tendency.

High-molecular-weight fucosylated glycosaminoglycan induces human platelet aggregation depending on αIIbß3 and platelet secretion.

Fucosylated glycosaminoglycan (FG) from sea cucumbers has been reported to have anticoagulant effects via targeting intrinsic tenase. However, FG from natural source also potentially poses risks due to its FXIIa activation and platelet aggregating effects. Here, we found that the effect of FG on human platelet aggregation depended on both the sulfation pattern and chain length. FGs with higher content of Fuc2S4S and larger molecular weight (≥14 kD) had stronger activity. Both platelet aggregation and P-selectin expression induced by TaFG (an FG from Thelenota ananas) were decreased as the molecular weight reduced. Ticagrelor, aspirin and wortmannin completely blocked the secretion (ADP) but only partially blocked the aggregation induced by TaFG. Tirofiban an αIIbß3 antagonist however potently inhibited both the secretion and aggregation, with IC50 of 6.01 ± 1.1.97 nM. Furthermore, TaFG could bind to human αIIbß3 with high affinity, and the affinities of two FGs were paralleled with their activity in platelet aggregation or activation. These results indicated that αIIbß3 played an important role in TaFG-induced platelet aggregation which was mediated by PI3K, and that platelet secretion was required for the amplification of aggregation.

Structural Characterization and Heparanase Inhibitory Activity of Fucosylated Glycosaminoglycan from Holothuria floridana.

Unique fucosylated glycosaminoglycans (FG) have attracted increasing attention for various bioactivities. However, the precise structures of FGs usually vary in a species-specific manner. In this study, HfFG was isolated from Holothuria floridana and purified by anion exchange chromatography with the yield of ~0.9%. HfFG was composed of GlcA, GalNAc and Fuc, its molecular weight was 47.3 kDa, and the -OSO3-/-COO- molar ratio was 3.756. HfFG was depolymerized by a partial deacetylation-deaminative cleavage method to obtain the low-molecular-weight HfFG (dHfFG). Three oligosaccharide fragments (Fr-1, Fr-2, Fr-3) with different molecular weights were isolated from the dHfFG, and their structures were revealed by 1D and 2D NMR spectroscopy. HfFG should be composed of repeating trisaccharide units -{(L-FucS-α1,3-)d-GlcA-ß1,3-d-GalNAc4S6S-ß1,4-}-, in which sulfated fucose (FucS) includes Fuc2S4S, Fuc3S4S and Fuc4S residues linked to O-3 of GlcA in a ratio of 45:35:20. Furthermore, the heparanase inhibitory activities of native HfFG and oligosaccharide fragments (Fr-1, Fr-2, Fr-3) were evaluated. The native HfFG and its oligosaccharides exhibited heparanase inhibitory activities, and the activities increased with the increase of molecular weight. Additionally, structural characteristics such as sulfation patterns, the terminal structure of oligosaccharides and the presence of fucosyl branches may be important factors affecting heparanase inhibiting activity.

Regimen Study of High Myopia-Partial Reduction Orthokeratology.

OBJECTIVE: This study aims to compare the increase in refractive error and axial length, variation of endothelium cells, and ratio of corneal staining between two regimens of high myopia-partial reduction orthokeratology (ortho-k) in children. METHODS: The present clinical prospective study recruited 102 high-myopia subjects (204 eyes). These subjects were randomly divided into three groups: (1) ortho-k group 1, subjects with a target myopia reduction of 6.00 D; (2) ortho-k group 2, subjects with a target myopia reduction of 4.00 D; and (3) control group, the refractive error of subjects was corrected using a pair of single-vision spectacles. Vision acuity, refractive error, and the cornea were examined at baseline, and at 2 days, 1 week, 1, 3, 6, and 12 months after commencing lens wear. The measurement of the axial length of the eyeball and a corneal endothelium examination were performed at baseline and at 12 months. RESULTS: The uncorrected vision acuities improved in subjects in these groups after treatment with ortho-k. Furthermore, the diopters of myopia and corneal curvature significantly decreased at 1 month, and the values continuously improved at 12 months, when compared with subjects at 1 month (P<0.05). Subjects in the control group had a significant increase in refractive error (0.565±0.313 D) and axial length (0.294±0.136 mm), when compared with subjects in the ortho-k-treated groups (P<0.05). However, there were no significant differences in changes in refractive error and axial length between ortho-k groups 1 (0.101±0.176 mm) and 2 (0.123±0.193 mm) at 12 months (P>0.05). Furthermore, subjects in group 1 (28.97%) had a higher rate of corneal staining, when compared with subjects in group 2 (13.06%) (P<0.05). CONCLUSION: The two ortho-k regimens, target reduction of 6.00 D and target of 4.00 D, had similar effects in controlling the increase in axial length and refractive error in high-myopia children. However, subjects with a target myopia reduction of 6.00 D had a higher rate of corneal staining than in subjects with a target myopia reduction of 4.00 D.

Oligosaccharides from depolymerized fucosylated glycosaminoglycan: Structures and minimum size for intrinsic factor Xase complex inhibition.

Fucosylated glycosaminoglycan (FG), a structurally complex glycosaminoglycan found up to now exclusively in sea cucumbers, has distinct anticoagulant properties, notably a strong inhibitory activity of intrinsic factor Xase complex (FXase). Knowledge of the FG structures could facilitate the development of a clinically effective intrinsic FXase inhibitor for anticoagulant drugs. Here, a new fucosylated glycosaminoglycan was obtained from the widely traded sea cucumber Bohadschia argus The precise structure was deduced as {→4)-[l-Fuc3S4S-α-(1→3)-]-d-GlcA-ß-(1→3)-d-GalNAc4S6S-ß-(1} through analysis of its chemical properties and homogeneous oligosaccharides purified from its ß-eliminative depolymerized products. The B. argus FG with mostly 3,4-di-O-sulfated fucoses expands our knowledge on FG structural types. This ß-elimination process, producing oligosaccharides with well-defined structures, is a powerful tool for analyzing the structure of complex FGs. Among these oligosaccharides, an octasaccharide displayed potent FXase inhibitory activity. Compared with oligosaccharides with various degrees of polymerization (3n and 3n - 1), our analyses reveal that the purified octasaccharide is the minimum structural unit responsible for the potent selective FXase inhibition, because the d-talitol in the nonsaccharide is unnecessary. The octasaccharide with 2,4-di-O-sulfated fucoses is more potent than that of one with 3,4-di-O-sulfated fucoses. Thus, sulfation patterns can play an important role in the inhibition of intrinsic factor Xase complex.

Precise Structure and Anticoagulant Activity of Fucosylated Glycosaminoglycan from Apostichopus japonicus: Analysis of Its Depolymerized Fragments.

Apostichopus japonicus is one of the most economically important species in sea cucumber aquaculture in China. Fucosylated glycosaminoglycan from A. japonicus (AjFG) has shown multiple pharmacological activities. However, results from studies on the structure of AjFG are still controversial. In this study, the deaminative depolymerization method that is glycosidic bond-selective was used to prepare the depolymerized products from AjFG (dAjFG), and then a series of purified oligosaccharide fragments such as tri-, hexa-, nona-, and dodecasaccharides were obtained from dAjFG by gel permeation chromatography. The 1D/2D NMR and ESI-MS spectrometry analyses showed that these oligosaccharides had the structural formula of l-FucS-α1,3-d-GlcA-ß1,3-{d-GalNAc4S6S-ß1,4-[l-FucS-α1,3-]d-GlcA-ß1,3-}n-d-anTal-diol4S6S (n = 0, 1, 2, 3; FucS represents Fuc2S4S, Fuc3S4S, or Fuc4S). Thus, the unambiguous structure of native AjFG can be rationally deduced: it had the backbone of {-4-d-GlcA-ß1,3-d-GalNAc4S6S-ß1-}n, which is similar to chondroitin sulfate E, and each d-GlcA residue in the backbone was branched with a l-FucS monosaccharide at O-3. Bioactivity assays confirmed that dAjFG and nonasaccharides and dodecasaccharides from AjFG had potent anticoagulant activity by intrinsic FXase inhibition while avoiding side effects such as FXII activation and platelet aggregation.

Physicochemical Characteristics and Anticoagulant Activities of the Polysaccharides from Sea Cucumber Pattalus mollis.

Sulfated polysaccharides from sea cucumbers possess distinct chemical structure and various biological activities. Herein, three types of polysaccharides were isolated and purified from Pattalus mollis, and their structures and bioactivities were analyzed. The fucosylated glycosaminoglycan (PmFG) had a CS-like backbone composed of the repeating units of {-4-d-GlcA-ß-1,3-d-GalNAc4S6S-ß-1-}, and branches of a sulfated α-l-Fuc (including Fuc2S4S, Fuc3S4S and Fuc4S with a molar ratio of 2:2.5:1) linked to O-3 of each d-GlcA. The fucan sulfate (PmFS) had a backbone consisting of a repetitively linked unit {-4-l-Fuc2S-α-1-}, and interestingly, every trisaccharide unit in its backbone was branched with a sulfated α-l-Fuc (Fuc4S or Fuc3S with a molar ratio of 4:1). Apart from the sulfated polysaccharides, two neutral glycans (PmNG-1 & -2) differing in molecular weight were also obtained and their structures were similar to animal glycogen. Anticoagulant assays indicated that PmFG and PmFS possessed strong APTT prolonging and intrinsic factor Xase inhibition activities, and the sulfated α-l-Fuc branches might contribute to the anticoagulant and anti-FXase activities of both PmFG and PmFS.

Plasma contact activation by a fucosylated chondroitin sulfate and its structure-activity relationship study.

Plasma contact system is the initial part of both the intrinsic coagulation pathway and kallikrein-kinin pathway, which mainly involves three proteins: coagulation factor XII (FXII), prekallikrein (PK) and high-molecular weight kininogen. Fucosylated chondroitin sulfate (FCS) is a unique sulfated glycosaminoglycan (GAG) composed of a chondroitin sulfate-like backbone and sulfated fucose branches. The native FCS was preliminary found to cause undesired activation of the plasma contact system. How this unusual GAG functions in this process remains to be clarified. Herein, the relationship between its structure, plasma contact activation and its effects on the PK-FXII reciprocal activation loop were studied. The recalcification time assay indicated that the FCS at high concentration could be procoagulant which may be attributed to its contact activation activity. The structure-activity relationship study indicated that its high molecular weight and distinct fucose side chains are required for contact activation by FCS, although the sulfate substitution types of its side chains have less impact. In human plasma, the native FCSs potently induced FXII-dependent contact activation. However, in purified systems FCS did not significantly activate FXII per se or induce its autoactivation, whereas FCS significantly promoted the activation of PK by factor XIIa. Polysaccharide-protein interaction assays showed that FCS bound to PK with higher affinity than other contact system proteins. These data suggested that potent contact activation by FCS requires the positive feedback loop between PK and FXII. These findings contribute to better understanding of contact activation by complex GAG.

Natamycin and Azithromycin Are Synergistic In Vitro against Ocular Pathogenic Aspergillus flavus Species Complex and Fusarium solani Species Complex Isolates.

The interaction of natamycin-azithromycin combination against 60 ocular fungal isolates was tested in vitro The combination produced 100% synergistic interactions when natamycin was added to azithromycin 20, 40, and 50 µg/ml against Aspergillus flavus species complex (AFSC) isolates and to azithromycin 50 µg/ml against Fusarium solani species complex isolates. Addition of 50 µg/ml azithromycin enhanced natamycin's effect against AFSC isolates by reducing natamycin MIC90 from 64 to 0.031 µg/ml. No antagonism was observed.

Discovery of an intrinsic tenase complex inhibitor: Pure nonasaccharide from fucosylated glycosaminoglycan.

Selective inhibition of the intrinsic coagulation pathway is a promising strategy for developing safer anticoagulants that do not cause serious bleeding. Intrinsic tenase, the final and rate-limiting enzyme complex in the intrinsic coagulation pathway, is an attractive but less explored target for anticoagulants due to the lack of a pure selective inhibitor. Fucosylated glycosaminoglycan (FG), which has a distinct but complicated and ill-defined structure, is a potent natural anticoagulant with nonselective and adverse activities. Herein we present a range of oligosaccharides prepared via the deacetylation-deaminative cleavage of FG. Analysis of these purified oligosaccharides reveals the precise structure of FG. Among these fragments, nonasaccharide is the minimum fragment that retains the potent selective inhibition of the intrinsic tenase while avoiding the adverse effects of native FG. In vivo, the nonasaccharide shows 97% inhibition of venous thrombus at a dose of 10 mg/kg in rats and has no obvious bleeding risk. This nonasaccharide may therefore serve as a novel promising anticoagulant.

Rifampin Enhances the Activity of Amphotericin B against Fusarium solani Species Complex and Aspergillus flavus Species Complex Isolates from Keratitis Patients.

The in vitro activities of amphotericin B in combination with rifampin were assessed against 95 ocular fungal isolates. The interactions between amphotericin B and rifampin at 4, 8, 16, and 32 µg/ml were synergistic for 11.8%, 51.0%, 90.2%, and 94.1%, respectively, of Fusarium solani species complex isolates and for 13.6%, 45.5%, 93.2%, and 95.5%, respectively, of Aspergillus flavus species complex isolates. Antagonism was never observed for the amphotericin B-rifampin combinations.

Effects of contact lens solution disinfectants against filamentous fungi.

PURPOSE: To assess and compare the antifungal activity of polyhexamethylene biguanide (PHMB), thimerosal, cetylpyridinium chloride, and chlorhexidine, which are disinfectants used in multipurpose disinfectant solutions (MPDSs) against ocular pathogenic Fusarium solani and Aspergillus flavus isolates in vitro. METHODS: The in vitro activity of PHMB, thimerosal, cetylpyridinium chloride, and chlorhexidine was assessed against 40 isolates of ocular pathogenic fungi that included 24 F. solani and 16 A. flavus isolates. The strains were tested by broth dilution antifungal susceptibility testing of filamentous fungi approved by the CLSI (Clinical and Laboratory Standards Institute) M38-A document. RESULTS: MIC90 (minimum inhibitory concentration for 90% of the organisms) values of PHMB were 4 and 16 µg/mL for F. solani and A. flavus, respectively. MIC90 values of thimerosal were 0.0313 and 0.0625 µg/mL for F. solani and A. flavus, respectively. MIC90 values of cetylpyridinium chloride were 2 and 2 µg/mL for F. solani and A. flavus, respectively. MIC90 values of chlorhexidine were 32 and 32 µg/mL for F. solani and A. flavus, respectively. CONCLUSIONS: As a disinfectant used in MPDSs, thimerosal showed the highest levels of antimicrobial activity against ocular pathogenic F. solani and A. flavus isolates. The concentrations of PHMB (0.0001%), cetylpyridinium chloride (0.00014%), and chlorhexidine (0.003%) in MPDSs are sublethal levels for ocular pathogenic F. solani and A. flavus isolates. Although multiple ingredients within MPDSs play a role in antimicrobial efficacy, antimicrobial activity may be significantly influenced by the disinfectants used in the solution formulations.

In vitro activity of phenylmercuric acetate against ocular pathogenic fungi.

OBJECTIVES: To determine the antifungal activity of phenylmercuric acetate against ocular pathogenic fungi in vitro and develop new antifungal eye drops to combat keratomycosis. METHODS: The in vitro activity of phenylmercuric acetate was assessed against 261 isolates of ocular pathogenic fungi that included 136 Fusarium spp. isolates, 98 Aspergillus spp. isolates, 10 Alternaria alternata isolates and 17 other pathogens. The activity of phenylmercuric acetate was compared with the activities of amphotericin B and natamycin. In vitro susceptibility testing was performed by broth microdilution assay, in accordance with the CLSI (formerly NCCLS) M38-A guidelines for filamentous fungi. RESULTS: MIC90s of phenylmercuric acetate were 0.0156, 0.0156, 0.0156 and 0.0156 mg/L for Fusarium spp., Aspergillus spp., A. alternata and other pathogens, respectively. MIC90s of amphotericin B were 2, 2, 1 and 1 mg/L for Fusarium spp., Aspergillus spp., A. alternata and other pathogens, respectively. MIC90s of natamycin were 8, 32, 4 and 4 mg/L for Fusarium spp., Aspergillus spp., A. alternata and other pathogens, respectively. CONCLUSIONS: Phenylmercuric acetate has promising antifungal activity, which is significantly superior to the activities of amphotericin B and natamycin against a wide variety of ocular pathogenic fungi based on comparative MIC values. Additional evaluation is required to determine its clinical utility.

A regular fucan sulfate from Stichopus herrmanni and its peroxide depolymerization: Structure and anticoagulant activity.

Marine sulfated polysaccharides have aroused widespread concern for their various structures and bioactivities. Peroxide depolymerization is a common strategy in analysis of structures and structure-activity relationships of polysaccharides. However, confirming the depolymerization process and exact structures of the degradation products is still a considerable challenge. This study reported the structures of a fucan sulfate (FS) from sea cucumber Stichopus herrmanni and its depolymerized products (dFS) prepared by peroxide degradation. The FS was elucidated with a highly regular structure, {-3)-L-Fuc2S-(α1-}n. Structure analysis of oligosaccharides purified from dFS suggested that peroxide degradation involved in cleavage of glycosidic bonds and oxidative modification of reducing end of sugar residue, while no break in sugar ring was observed. Both FS and series of dFSs exhibited significant anticoagulant activities due to their anti-thrombin effects in presence of heparin cofactor II and their potencies were related to their molecular sizes, dFS with ∼ 20 kDa showed the strongest activity.

Structural characterization of oligosaccharides from free radical depolymerized fucosylated glycosaminoglycan and suggested mechanism of depolymerization.

Free radical depolymerization is a common method in structural analysis of polysaccharides, the major challenge is the analysis of the cleavage site and characterization of newly formed ends in this reaction. Here, a fucosylated glycosaminoglycan from H. fuscopunctata (HfFG) was depolymerized by H2O2 and a series of oligosaccharides were purified and their structures were elucidated. For non-reducing ends of the trisaccharides were intact GalNAc4S6S, the cleavage site should mainly be the ß(1,3) linkages between GlcA and GalNAc in the backbone of FG. Meanwhile, the reducing ends of the disaccharides and trisaccharides were almost dicarboxylic acid derivatives of GlcA, possibly arising from oxidative breaking of the CC bond of GlcA at the reducing ends. In addition, glycosidic linkages in D-GalNAc-ß(1,4)-D-GlcA and L-FucS-α(1,3)-D-GlcA located at the reducing end could be cleaved, and the released GalNAc4S6S were oxidized to N-acetylgalactosaminic acid.

Structural characterization and anticoagulant analysis of the novel branched fucosylated glycosaminoglycan from sea cucumber Holothuria nobilis.

Glycosaminoglycan HnFG was extracted from sea cucumber Holothuria nobilis. Its chemical structure was characterized by analyzing the physicochemical properties, oligosaccharides from its mild acid hydrolysates and depolymerized products. The disaccharide d-GalNAc4S6S-α1,2-l-Fuc3S-ol found in its mild acid hydrolysates provided a clue for the presence of a unique disaccharide-branch in HnFG. Furthermore, it was confirmed by a series of oligosaccharides from the low-molecular weight HnFG prepared by ß-eliminative depolymerization. Combining with the analysis of its peroxide depolymerized products, the precise structure of HnFG was determined: A chondroitin sulfate E (CS-E)-like backbone branched with sulfated monofucoses (~67%) and disaccharides d-GalNAcS-α1,2-l-Fuc3S (~33%) at O-3 position of each GlcUA. This is the first report on the novel branches in glycosaminoglycan. Biologically, the native and depolymerized HnFG showed potent activities in prolonging the activated partial thrombin time (APTT) and inhibiting intrinsic coagulation Xase (iXase), whereas the oligosaccharides (degree of polymerization ≤6) had no obvious effects.

In vitro activity of thimerosal against ocular pathogenic fungi.

The in vitro activity of thimerosal versus those of amphotericin B and natamycin was assessed against 244 ocular fungal isolates. The activity of thimerosal against Fusarium spp., Aspergillus spp., and Alternaria alternata was 256 times, 512 times, and 128 times, respectively, greater than that of natamycin and 64 times, 32 times, and 32 times, respectively, greater than that of amphotericin B. Thimerosal's antifungal activity was significantly superior to those of amphotericin B and natamycin against ocular pathogenic fungi in vitro.

[Activity of butenafine against ocular pathogenic filamentous fungi in vitro].

OBJECTIVE: To investigate antifungal activity of butenafine in comparison with that of natamycin, amphotericin B and fluconazole against ocular pathogenic filamentous fungi in vitro. METHODS: It was an experimental study. Susceptibility tests were performed against 260 isolates of ocular pathogenic filamentous fungi by broth dilution antifungal susceptibility test of filamentous fungi approved by the Clinical and Laboratory Standards Institute (CLSI) M38-A document. The isolates included Fusarium spp. (136), Aspergillus spp. (98), Alternaria alternata (9), Curvularia lunata (3), and unusual ocular pathogens (14). Final concentration ranged from 0.008 to 16.000 mg/L for butenafine, from 0.031 to 16.000 mg/L for amphotericin B and natamycin, and from 0.5 to 256.0 mg/L for fluconazole. Following incubation at 35 degrees C for 48 h, minimal inhibitory concentration (MIC) was determined according to the CLSI M38-A document. For amphotericin B and natamycin, the MIC was defined as the lowest drug concentration that prevented any discernible growth. For butenafine and fluconazole, the MIC was defined as the lowest concentration in which an approximately 75% reduction compared to the growth of the control was observed. Candida parapsilosis ATCC22019 was used as quality control strains to validated the results. Mean MIC and MIC range, the MIC at which 50% of the isolates tested were inhibited (MIC(50)) and the MIC at which 90% of the isolates tested were inhibited (MIC(90)), were provided for all the isolates tested by using descriptive statistical analysis with the statistical SPSS package (version 13.0). RESULTS: MIC(90) of butenafine, natamycin, amphotericin B and fluconazole were 4, 8, 2 and 512 mg/L for Fusarium spp., respectively; 0.063, 32.000, 2.000 and 256.000 mg/L for Aspergillus spp., respectively; 0.5, 8.0, 2.0 and 128.0 mg/L for Alternaria alternate, respectively; 0.125, 2.000, 0.500 and 4.000 mg/L for Curvularia lunata, respectively; and 1, 4, 1 and 256 mg/L for unusual ocular pathogens, respectively. CONCLUSIONS: Butenafine exhibits potent antifungal activity against a wide variety of ocular pathogenic fungi, especially for Aspergillus spp., Alternaria alternata, Curvularia lunata, and some unusual ocular pathogens and may have a role in future studies of antifungal eye drops and treating fungal keratitis.