Isoflavaspidic Acid PB Extracted from Dryopteris fragrans (L.) Schott Inhibits Trichophyton rubrum Growth via Membrane Permeability Alternation and Ergosterol Biosynthesis Disruption.

Isoflavaspidic acid PB (PB), a phloroglucinol derivative extracted from aerial parts of Dryopteris fragrans (L.) Schott, had antifungal activity against several dermatophytes. This study was aimed at exploring the antifungal mechanism of PB against Trichophyton rubrum (T. rubrum). The effectiveness of PB in inhibiting T. rubrum growth was detected by time-kill kinetics study and fungal biomass determination. Studies on the mechanism of action were investigated using scanning electron microscopy (SEM), transmission electron microscopy (TEM), sorbitol and ergosterol assay, nucleotide leakage measurement, and UPLC-based test and enzyme-linked immunosorbent assay. Fungicidal activity of PB was concentration- and time-dependent at 2 × MIC (MIC: 20 µg/mL) after 36 h. The total biomass of T. rubrum was reduced by 64.17%, 77.65%, and 84.71% in the presence of PB at 0.5 × MIC, 1 × MIC, and 2 × MIC, respectively. SEM analysis showed that PB changed mycelial morphology, such as shrinking, twisting, collapsing, and even flattening. TEM images of treated cells exhibited abnormal distributions of polysaccharide particles, plasmolysis, and cytoplasmic content degradation accompanied by plasmalemma disruption. There were no changes in the MIC of PB in the presence of sorbitol. However, the MIC values of PB were increased by 4-fold with exogenous ergosterol. At 4 h and 8 h, PB increased nucleotide leakage. Besides, ergosterol content in T. rubrum membrane treated with PB at 0.5 × MIC, 1 × MIC, and 2 × MIC was decreased by 9.58%, 15.31%, and 76.24%, respectively. There was a dose-dependent decrease in the squalene epoxidase (SE) activity. And the reduction in the sterol 14α-demethylase P450 (CYP51) activity was achieved after PB treatments at 1 × MIC and 2 × MIC. These results suggest that PB displays nonspecific action on the cell wall. The membrane damaging effects of PB were attributed to binding with ergosterol to increase membrane permeability and interfering ergosterol biosynthesis involved with the reduction of SE and CYP51 activities. Further study is needed to develop PB as a natural antifungal candidate for clinical use.

[Mechanism of Liangfu Pills in treatment of functional dyspepsia: based on network pharmacology and experimental verification].

This study aims to explore the potential mechanism of Liangfu Pills in the treatment of functional dyspepsia(FD) based on network pharmacology and molecular docking, and verify the mechanism by animal experiment. The active components of Liangfu Pills were screened from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP), and the targets of Liangfu Pills were predicted by SwissTargetPrediction. The targets of FD were retrieved from GeneCards. On this basis, the common targets of the disease and the pills were yielded and the protein interaction was retrieved based on STRING. The core targets were screened out, followed by Gene Oncology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis with DAVID. Finally, molecular docking was carried out with the help of AutoDock Tools to predict the binding degree between the effective components of Liangfu Pills and core targets. A total of 19 active components of Liangfu Pills and 591 FD-related targets were screened out by network pharmacology, of which 253 were common targets of the disease and the prescription. Liangfu Pills was mainly involved in the biological processes of response to drug, negative regulation of transcription, positive regulation of apoptotic process, and cell surface receptor signaling pathway, and the KEGG pathways of hypoxia-inducible factor-1(HIF-1) signaling pathway, serotonergic synapse, tumor necrosis factor(TNF) signaling pathway, cyclic adenosine monophosphate(cAMP) signaling pathway, calcium signal pathway, and inflammatory mediator regulation of transient receptor potential(TRP) channels. The results of molecular docking showed that the key active components of Liangfu Pills had certain binding activity to the targets mitogen-activated protein kinase 1(MAPK1), protein kinase B(AKT1), transient receptor potential cation channel subfamily V member 1(TRPV1), 5-hydroxytryptamine receptor 1 A(HTR1 A), and 5-hydroxytryptamine receptor 2 A(HTR2 A). FD was induced in rats, and then Liangfu Pills was given to FD rats for 7 days. The results showed that Liangfu Pills could significantly relieve the symptoms of FD rats, significantly increase the expression of 5-hydroxytryptamine(5-HT), and down-regulate the expression of TRPV1. Through network pharmacology, molecular docking, and experimental verification, this study proved that Liangfu Pills improved FD through multiple components and multiple targets. The result lays a basis for further research on the mechanism and clinical application of Liangfu Pills in the treatment of FD.

[A new phloroglucinol from Dryopteris fragrans and its antibacterial activity in vitro].

A new phloroglucinol was isolated from 50% ethanol extract of Dryopteris fragrans by silica gel column chromatography, Sephadex LH-20 gel column chromatography, thin-layer chromatography(TLC), and preparative liquid column chromatography. On the basis of MS, ~1H-NMR, ~(13)C-NMR, and reference materials, compound 1 was identified as 2,5-cyclohexadien-1-one, 2-{[2,6-dihydroxy-4-methoxy-3-methyl-5-(1-isobutyl)phenyl]methyl}-3,5-dihydroxy-4,4-dimethyl-6-(1-oxobutyl)(1), and named disaspidin BB. Compound 1 was evaluated for its antibacterial activity. The experimental results showed that compared with the commonly used topical antibiotics erythromycin or mupirocin, disaspidin BB exhibited significant antibacterial activities against Staphylococcus epidermidis(SEP), S. haemolyticus(SHA), and methicillin-resistant S. aureus(MRSA)(P<0.05). Additionally, disaspidin BB was sensitive to ceftazidime-resistant SEP1-SEP4, SHA5-SHA7, MRSA8, and MRSA9. The MIC values of disaspidin BB against SEP and SHA were 1.67-2.71 µg·mL~(-1) and 10.00-33.33 µg·mL~(-1) respectively. Disaspidin BB has good antibacterial activities and deserves development as a new anti-infective drug for external use.

Anoectochilus roxburghii flavonoids extract ameliorated the memory decline and reduced neuron apoptosis via modulating SIRT1 signaling pathway in senescent mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Anoectochilus roxburghii (A. roxburghii) is a precious herb and folk medicine in many Asian countries. It has been used traditionally to treat diabetes, etc., and also used as a dietary therapy to delay senescence. AIM OF THE STUDY: This study was to evaluate the neuroprotective effects of A. roxburghii flavonoids extract (ARF) and whether its effects were due to the regulation of SIRT1 signaling pathway in senescent mice and in D-galactose (D-gal) induced aging in SH-SY5Y cells. MATERIALS AND METHODS: 18-month-old mice were randomly divided into senescent model, low-dose ARF, high-dose ARF and vitamin E group. 2-Month-old mice were as a control group. After 8 weeks treatment, Morris water maze (MWM) was performed. The levels of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), monoamine oxidase (MAO) and acetylcholinesterase (ACh-E) in the cortex were determined. Hippocampus morphologic changes were observed with haematoxylin and eosin (H&E), Nissl, senescence-associated-galactosidase (SA-ß-gal) and terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) staining. Apoptosis-related molecular expressions in the hippocampus were performed by western blotting. Furthermore, after stimulated by EX527 (a SIRT1 inhibitor), the SIRT1-dependent neuroprotective effects of ARF were determined by measuring SRIT1 and p53 expression in SH-SY5Y aging cells induced by D-gal. RESULTS: ARF could significantly ameliorate memory decline in senescent mice and reduce the generations of ROS, MDA and the activities of MAO and ACh-E, while increasing SOD activities in the cortex of aging mice. ARF obviously improved hippocampus pathological alterations, increased the number of Nissl bodies, while reducing senescent and apoptotic cells in senescent mice hippocampus. Further, ARF positively regulated SIRT1 expression, and reduced apoptosis-related molecules p53, p21 and Caspase-3 expression, while increasing the ratio of Bcl-2/Bax. In D-gal-induced SH-SY5Y cells, the effects of ARF on SIRT1 and p53, and the ability of scavenging ROS were mostly abolished after incubation with the EX527. CONCLUSIONS: ARF, in a SIRT1-dependent manner, exerted neuroprotection via modulating SIRT1/p53 signaling pathway against memory decline and apoptosis due to age-induced oxidative stress damage in senescent mice.

Knockdown of LncRNA SNHG1 Suppresses Corneal Angiogenesis by the Regulation of miR-195-5p/VEGF-A.

LncRNA SNHG1 (SNHG1) has been widely studied as the causative factor of angiogenesis and proliferative agent in gastric, lung, cervical, and hepatocellular carcinomas. However, its significance of angiogenesis and progression of corneal neovascularization (CRNV) is least understood. This study focuses on the molecular mechanisms followed by SNHG1 to establish CRNV and its angiogenesis. Bioinformatics analysis to identify potential miRNA targets of SNHG1 and vascular endothelial growth factor A (VEGF-A) was conducted using StarBase and was subsequently confirmed by the luciferase reporter assay. Relative quantitative expression of SNHG1 in human umbilical vein endothelial cells (HUVECs) was detected through qRT-PCR and western blot analysis. Cell proliferation was detected through CCK-8 assay, whereas migratory abilities of the cells were determined with transwell assay. A capillary-like tube formation assay was performed to detect the tube formation ability of the cells. Following this, relative expression of miR-195-5p and VEGF-A was determined through qRT-PCR and western blot analysis. Results from the experiments manifested upregulated levels of SNHG1 and VEGF-A in HUVECs and CRNV tissues as compared with the control group, whereas downregulated levels of miR-195-5p were measured in the CRNV tissues and HUVECs, suggesting the negative correlation between lncRNA and miRNA. Overexpressed vascular endothelial growth factor promoted cell proliferation and tube formation; however, its silencing leads to inhibition in angiogenesis and proliferation. Potential binding sites of SNHG1 showed miR-195-5p as its direct target and SNHG1 as a sponge for this miRNA. Knockdown and downregulated levels of SNHG1 showed a notable decrease and inhibition in angiogenesis and migration of CRNV cells. The study showed that SNHG1 inhibition significantly reduced cell proliferation, migration, and tube formation in HUVECs transfect with lncRNA SNHG1. Mechanistic insights into the SNHG1 showed that SNHG1 acts as a sponge for miR-195-5p and upregulates the levels of VEGF-A.

The effect of isoflavaspidic acid PB extracted from Dryopteris fragrans (L.) Schott on planktonic and biofilm growth of dermatophytes and the possible mechanism of antibiofilm.

ETHNOPHARMACOLOGICAL RELEVANCE: Dryopteris fragrans (L.) Schott (D. fragrans), a deciduous perennial herb, has been traditionally used for treatment of various skin diseases in Heilongjiang province of China for many years. Phloroglucinol derivatives extracted from D. fragrans were the most effective fraction against dermatophytes. Isoflavaspidic acid PB is a typically phloroglucinol derivative which extracted from D. fragrans and has been reported to exert anti-fungal activities against several dermatophytes. AIM OF THE STUDY: This study aimed to evaluate anti-fungal and anti-biofilm activity of isoflavaspidic acid PB on planktonic and biofilm growth of dermatophytes and explore possible mechanisms of anti-biofilm. MATERIALS AND METHODS: Minimal inhibitory concentrations (MIC) and minimal fungicidal concentrations (MFC) of isoflavaspidic acid PB against 25 isolates of dermatophytes were determined by the Clinical and Laboratory Standards Institute (CLSI) M38-A2 method. The effects of isoflavaspidic acid PB on dermatophytes biofilm formation and pre-formed biofilm were assessed by 2.3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[carbonyl (phenylamino)]-2H-tetrazolium hydroxide (XTT) assay. Morphology of mature biofilm were observed by Scanning Electron Microscope (SEM). Biomass, exopolysaccharide and ergosterol content of mature biofilm were analyzed by gravimetric analysis, anthranone sulfuric acid method and Ultra Performance Liquid Chromatography (UPLC) assay respectively. RESULT: The MIC and MFC ranges of isoflavaspidic acid PB against 25 isolates of dermatophytes were 20-80 µg/mL and 40-80 µg/mL respectively. Isoflavaspidic acid PB (2 MIC) inhibited not only Trichophyton biofilm formation (54.8% ∼ 81.2%) but also the metabolic activity of mature biofilm (20.7% ∼ 44.2%). The result of SEM showed that isoflavaspidic acid PB (8 MIC) could destroy the morphology of hyphae seriously. Comparing with control group, biomass, exopolysaccharide and ergosterol content of the mature biofilm under isoflavaspidic acid PB (8 MIC) were significantly decreased (P < 0.01). CONCLUSION: Isoflavaspidic acid PB had anti-fungal and fungicidal activities against dermatophytes. Isoflavaspidic acid PB could inhibit the biofilm of Trichophyton. The mechanism might be related to the decline of the biofilm biomass, exopolysaccharide and ergosterol content. These results showed that isoflavaspidic acid PB could be explored for promising anti-biofilm drugs.

Analysis of chemical composition and in vitro antidermatophyte activity of ethanol extracts of Dryopteris fragrans (L.) Schott.

ETHNOPHARMACOLOGICAL RELEVANCE: Dryopteris fragrans (L.) Schott is a deciduous perennial herb, which has been used traditionally for treatment of ringworm infections and others skin diseases in the north of China. AIM OF THE STUDY: To characterize the chemical composition, evaluate the antifungal activity and explore possible mechanisms about action of ethanol extracts of D. fragrans. MATERIALS AND METHODS: The chemical components in the ethanol extracts of D. fragrans were detected by high-performance liquid chromatography coupled with electrospray ionization and quadruple time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS/MS). The minimal inhibitory concentrations (MIC) and minimal fungicidal concentrations (MFC) of the ethanol extracts of D. fragrans were determined by the Clinical and Laboratory Standards Institute (CLSI) M38-A2 method against 62 isolates of dermatophytes. The kinetics of fungal kill, synergy testing by checkerboard dilution and quantitation of sterol by ultra-performance liquid chromatography (UPLC) on Trichophyton rubrum and Trichophyton mentagrophytes were also investigated. RESULTS: Fourteen derivatives of phloroglucinol were identified in the ethanol extracts of D. fragrans. The MIC of the ethanol extracts of D. fragrans ranged from 0.059 to 3.780 mg/mL while MFC ranged from 0.118 to 3.780 mg/mL. The ethanol extracts of D. fragrans exerted fungicidal activity after 12 h of incubation against Trichophyton rubrum while it required 36 h of incubation against Trichophyton mentagrophytes at concentrations of 8 × MIC. In synergy testing, the interaction between miconazole (MCZ) and terbinafine (TBF) with the ethanol extracts of D. fragrans proved to be indifferent by testing fractional inhibitory concentration (FIC) values. Sterol in samples of fungal cells treated with the ethanol extracts of D. fragrans was significantly reduced. CONCLUSIONS: The ethanol extracts of D. fragrans had antifungal and fungicidal activity against dermatophytes and was likely a strain-dependent fungicidal agent. Interaction between drugs was indifferent on tested isolates. The inhibition of ergosterol biosynthesis was one of the antifungal mechanisms of the ethanol extracts of D. fragrans. These results showed that the ethanol extracts of D. fragrans could be explored for promising antifungal drugs. Dozens of phloroglucinol derivatives may contribute to high antifungal activity of the ethanol extracts of D. fragrans.

Germacrone derivatives: synthesis, biological activity, molecular docking studies and molecular dynamics simulations.

Germacrone is one of the major bioactive components in the Curcuma zedoaria oil product, which is extracted from Curcuma zedoaria Roscoe, known as zedoary. The present study designed some novel germacrone derivatives based on combination principles, synthesized these compounds, and investigated their inhibitions on Bel-7402, HepG2, A549 and HeLa cells. Meanwhile, the study evaluated inhibitions of these derivatives on c-Met kinase, which has been detected in a number of cancers. The results suggested that the majority of the compounds showed stronger inhibitory effect on cancers and c-Met kinase than germacrone. Furthermore, our docking experiments analyzed the results and explained the molecular mechanism. Molecular dynamics simulations were then applied to perform further evaluation of the binding stabilities between compounds and their receptors.

Antinociceptive and anti-inflammatory activities of Schefflera octophylla extracts.

ETHNOPHARMACOLOGICAL RELEVANCE: Schefflera octophylla (Lour.) Harms, a traditional Chinese herb mainly distributed in Southeast Asia, is extensively prescribed to alleviate pain and treat rheumatoid arthritis (RA), influenza, throat swelling, pain, etc. In this paper, the antinociceptive and anti-inflammatory activities of the ethanol extract and its five different polar fractions of this plant were evaluated. Furthermore, the anti-rheumatoid arthritis activity of the ethanol extract and its active fraction (CHCl3 fraction) were evaluated. And the chemical constituents of the CHCl3 active fraction displayed significant antinociceptive and anti-inflammatory activities were investigated. MATERIALS AND METHODS: Antinociceptive and anti-inflammatory activities were investigated by hot plate test, acetic acid-induced abdominal writhing test and formalin test, xylene-induced ear edema test. The anti-rheumatoid arthritis activity was evaluated through the model of adjuvant-induced arthritis (AA) in rats, paw swelling, pain response, arthritis index and histopathological changes of ankle, the levels of TNF-α, IL-1ß, IL-6 and rheumatoid factor (RF) of rats were detected. The chemical constituents of the CHCl3 fraction were isolated using chromatographic techniques. Their structures were elucidated by spectroscopic data analysis. RESULTS: The results showed that the ethanol extract of S. octophylla has significant dose-dependent anti-inflammatory and antinociceptive activities. And its five different polar fractions especially the CHCl3 fraction significantly inhibited the abdominal writhing induced by acetic acid and ear edema induced by xylene, also increased pain threshold in hot plate test in 120 min and reduced ticking times in formalin test. The ethanol extract of S. octophylla and the CHCl3 fraction demonstrated an anti-RA effect in a dose-dependent manner. The levels of TNF-α, IL-1ß and IL-6 in ethanol extract (600 mg/kg) and CHCl3 fraction (300 mg/kg) groups were significantly lower than those of the model group. The chemical constituents study of the CHCl3 fraction from S. octophylla led to six triterpenoids which were identified as taraxerone (1), 3-epi-taraxerol (2), aleuritolic acid (3), 3-oxofriedelan-28-oic acid (4), 3ß,19α -dihydroxy-urs-12-ene- 24,28-dioic acid (5) and asiatic acid (6). Compounds 1-5 were obtained from this plant for the first time. CONCLUSION: This study proved the antinociceptive, anti-inflammatory and anti-rheumatoid arthritis activities of S. octophylla. Triterpenoids obtained from its CHCl3 fraction may be responsible for those activities. These results could support the fact that S. octophylla is used traditionally to cure inflammatory and pain diseases.

[Study on antifungal susceptibility of different extract of Dryopteris fragrans].

OBJECTIVE: To examine the antifungal effect of different extract of Dryopteris fragrans (L.) Schott. in vitro, and screen the effective fraction from those extracts. METHODS: Separated the Dryopteris fragrans extract and got four parts by refluxing extraction,and determined the contents of total phloroglucinol. Disc agar diffusion method and solid agar dilution method were used to determine inhibitory effect. Minimum inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) of different parts of Dryopteris fragrans extract against four strains of common clinical dermatophytes were investigated. RESULTS: The data showed that the contents sequence of total phloroglucinol was in the following order: 95% -ethanol extract > water extract > diethyl ether extract > petroleum ether extract, and the antimicrobial activities against the four dermatophytes were as following order: 95% -ethanol extract > water extract > di-ethyl ether extract > petroleum ether extract. CONCLUSION: The contents of total phloroglucinol in 95% -ethanol extract of Dryopteris fragrans is the highest, and the antifungal activity against dermatophytes in vitro is the strongest. The effective fraction of Dryopteris fragrans is the 95%-ethanol extract.

[Studies on the chemical constituents of Dryopteris fragrans].

OBJECTIVE: To study the chemical constituents of Dryopteris fragrans. METHODS: The constituents of CHCl3-soluble portion and ethyl acetate-soluble portion from the alcohol extract were isolated and purified by means of chromatography. All the compounds were identified by their physical characteristics and spectral features. RESULTS: Five compounds were isolated and identified as beta-sitosterol (I), rutin (II), quercetin (III), quercetin-3-O-beta-D-pyranglucoside (IV) and 5,7-dihydroxy-2-hydroxymethyl chromone (V). CONCLUSION: Compounds II - V are isolated from this plant for the first time.

[Studies on phloroglucinol derivatives of Dryopteris fragrans L].

OBJECTIVE: To study the phloroglucinol derivatives of Dryopteris fragrans. METHODS: Isolation and purification were carried out on repeated silica gel, Sephadex LH-20 column chromatography and prepare HPLC. The structures of the compounds were determined by physicochemical properties and spectral analysis. RESULTS: Four compounds were isolated and identified as aspidin PB (I), dryofragin (II), aspidinol (III), aspidin BB (IV). CONCLUSION: Compounds IV is isolated from this plant for the first time.

[Study on terpene of Dryopteris fragrans L].

OBJECTIVE: To study the terpene of Dryopteris fragrans. METHODS: Isolation and purification were carried out on repeated silica gel, Sephadex LH-20 column chromatography and prepare HPLC. The structures of the compounds were determined by physicochemical properties and spectral analysis. RESULTS: Four compounds were isolated and identified as 10-hydroxyl-15-oxo-alpha-cadinol (I), albicany acetate (II), alpha-cadinene (III), albicanol (IV). CONCLUSION: Compounds I is isolated from this plant for the first time.