The Inhibition Activity of Natural Methoxyflavonoid from Inula britannica on Soluble Epoxide Hydrolase and NO Production in RAW264.7 Cells.

Soluble epoxide hydrolase (sEH) is an enzyme targeted for the treatment of inflammation and cardiovascular diseases. Activated inflammatory cells produce nitric oxide (NO), which induces oxidative stress and exacerbates inflammation. We identify an inhibitor able to suppress sEH and thus NO production. Five flavonoids 1-5 isolated from Inula britannica flowers were evaluated for their abilities to inhibit sEH with IC50 values of 12.1 ± 0.1 to 62.8 ± 1.8 µM and for their effects on enzyme kinetics. A simulation study using computational chemistry was conducted as well. Furthermore, five inhibitors (1-5) were confirmed to suppress NO levels at 10 µM. The results showed that flavonoids 1-5 exhibited inhibitory activity in all tests, with compound 3 exhibiting the most significant efficacy. Thus, in the development of anti-inflammatory inhibitors, compound 3 is a promising natural candidate.

Inhibiting NF-κB-S100A11 signaling and targeting S100A11 for anticancer effects of demethylzeylasteral in human colon cancer.

Colon cancer is a common and deadly malignancy of the gastrointestinal tract. Targeting proteins that inhibit tumor proliferation could lead to innovative treatment strategies for this disease. Demethylzeylasteral, extracted naturally from Tripterygium wilfordii Hook. f., demonstrates incredible anti-colon cancer activity. However, the molecular mechanism behind this requires further investigation. This study aims to identify crucial targets and mechanisms of demethylzeylasteral in treating colon cancer, making it a promising candidate for anti-tumor therapy. Through gene knockout, overexpression techniques, and double Luciferase experiments, we confirmed that demethylzeylasteral reduces S100A11 expression in HT29 cells and in vivo tumor models to anti-colon cancer. By conducting Surface Plasmon Resonance, immunofluorescence staining, and confocal laser microscopy observations, we verified the direct interaction between demethylzeylasteral and S100A11, and explored the impact of S100A11's subcellular localization on cell proliferation. Demethylzeylasteral inhibited S100A11 expression and exhibited anti-cancer activity in both in vitro and in vivo colon cancer models. Conversely, overexpression of S100A11 hindered apoptosis induced by demethylzeylasteral. Additionally, we found that knockdown or overexpression of NF-κB respectively decreased or increased S100A11 expression, subsequently affecting cell proliferation. The dual Luciferase reporting experiment revealed that NF-κB is an upstream transcription factor regulating S100A11 expression. And Surface plasmon resonance confirmed that S100A11 can directly interact with demethylzeylasteral, this interaction limited the transport of S100A11 from the cytoplasm to nucleus, attenuation S100A11 mediated cell proliferation effect.

Eupatin, a Flavonoid, Inhibits Coronavirus 3CL Protease and Replication.

The coronavirus disease 2019 (COVID-19) pandemic has caused more than six million deaths worldwide since 2019. Although vaccines are available, novel variants of coronavirus are expected to appear continuously, and there is a need for a more effective remedy for coronavirus disease. In this report, we isolated eupatin from Inula japonica flowers and showed that it inhibits the coronavirus 3 chymotrypsin-like (3CL) protease as well as viral replication. We showed that eupatin treatment inhibits SARS-CoV-2 3CL-protease, and computational modeling demonstrated that it interacts with key residues of 3CL-protease. Further, the treatment decreased the number of plaques formed by human coronavirus OC43 (HCoV-OC43) infection and decreased viral protein and RNA levels in the media. These results indicate that eupatin inhibits coronavirus replication.

The Effects of 1-O-Acetylbritannilactone Isolated from Inula britannica Flowers on Human Neutrophil Elastase and Inflammation of RAW 264.7 Cells and Zebrafish Larvae.

During a search for natural inflammatory inhibitors, 1-O-acetylbritannilactone (ABL), a sesquiterpene lactone, was isolated from the flowers of Inula britannica. ABL significantly inhibited human neutrophil elastase (HNE) with a half-maximal inhibitory concentration (IC50) of 3.2 ± 0.3 µM, thus did so more effectively than the positive control material (epigallocatechin gallate) (IC50 7.2 ± 0.5 µM). An enzyme kinetic study was performed. ABL noncompetitively inhibited HNE with an inhibition constant Ki of 2.4 µM. ABL inhibited lipopolysaccharide-induced nitric oxide and prostaglandin E2 production by RAW 264.7 cells in a dose-dependent manner, as well as the protein-level expression of inducible nitric oxide synthase and cyclooxygenase-2. The anti-inflammatory effect of ABL was confirmed using a transgenic Tg(mpx:EGFP) zebrafish larval model. The exposure of the larvae to ABL inhibited neutrophil recruitment to the site of injury after tail fin amputation.

Inhibition of Soluble Epoxide Hydrolase Activity by Components of Glycyrrhiza uralensis.

Soluble epoxide hydrolase (sEH) is a target enzyme for the treatment of inflammation and cardiovascular disease. A Glycyrrhiza uralensis extract exhibited ~50% inhibition of sEH at 100 µg/mL, and column chromatography yielded compounds 1-11. Inhibitors 1, 4-6, 9, and 11 were non-competitive; inhibitors 3, 7, 8, and 10 were competitive. The IC50 value of inhibitor 10 was below 2 µM. Molecular simulation was used to identify the sEH binding site. Glycycoumarin (10) requires further evaluation in cells and animals.

The inhibitory activity of methoxyl flavonoids derived from Inula britannica flowers on SARS-CoV-2 3CLpro.

In our ongoing efforts to identify effective natural antiviral agents, four methoxy flavonoids (1-4) were isolated from the Inula britannica flower extract. Their structures were elucidated using nuclear magnetic resonance. Flavonoids 1-4 exhibited inhibitory activity against SARS- CoV-2 3CLpro with IC50 values of 41.6 ± 2.5, 35.9 ± 0.9, 32.8 ± 1.2, and 96.6 ± 3.4 µM, respectively. Flavonoids 1-3 inhibited 3CLpro in a competitive manner. Based on molecular simulations, key amino acids that form hydrogen bond with inhibitor 3 were identified. Finally, we found that inhibitors (1-3) suppressed HCoV-OC43 coronavirus proliferation at micromole concentrations.

Inhibitory Activity of Quaternary Isoquinoline Alkaloids on Soluble Epoxide Hydrolase.

The quaternary isoquinoline alkaloids of palmatine (1), berberine (2), and jatrorrhizine (3) were evaluated in terms of their ability to inhibit soluble epoxide hydrolase (sEH). They had similar inhibitory activities, with IC50 values of 29.6 ± 0.5, 33.4 ± 0.8, and 27.3 ± 0.4 µM, respectively. Their respective Ki values of 26.9, 46.8, and 44.5 µM-determined by enzyme kinetics-indicated that they inhibited the catalytic reaction by binding noncompetitively with sEH. The application of computational chemistry to the in vitro results revealed the site of the receptor to which the ligand would likely bind. Accordingly, three alkaloids were identified as having a suitable basic skeleton for lead compound development of sEH inhibitors.

Cinnamomum cassia (L.) J.Presl Alleviates Allergic Responses in Asthmatic Mice via Suppression of MAPKs and MMP-9.

The prevalence of asthma is gradually increasing, and endangers human health. Many therapeutic agents have been developed to address this concern. Cinnamomum cassia (L.) J.Presl is a traditional herbal remedy in China, Japan, and Korea and used mainly to control common cold, cough, pneumonitis and fever in Donguibogam, a medical encyclopedia of Korea. Therefore, we investigated whether C. cassia (L.) J.Presl extract (CCE) confers protective effects on asthma model induced by ovalbumin (OVA). The animals were received intraperitoneal administration of OVA on day 1 and 14, and then subjected to OVA inhalation from day 21-23. They were orally treated CCE (30 and 100 mg/kg) from day 18-23. CCE administration decreased allergic responses, including airway hyperresponsiveness, eosinophilia, inflammatory cytokine production, and immunoglobulin E in OVA-exposed mice, along with the decline in inflammatory cell count and mucus secretion in respiratory tract. Additionally, CCE suppressed MAPK phosphorylation and MMP-9 expression in OVA-exposed mice. Overall, CCE treatment attenuated allergic responses induced by OVA exposure, which may be connected to the suppression of MAPK phosphorylation.

Microbial Transformation of Yakuchinone A and Cytotoxicity Evaluation of Its Metabolites.

Yakuchinone A (1) is a bioactive diarylheptanoid isolated from the dried fruits of Alpinia oxyphylla. Microbial transformation has been recognized as an efficient method to produce new biologically active derivatives from natural products. In the present study, microbial transformation of yakuchinone A was performed with the fungus Mucor hiemalis KCTC 26779, which led to the isolation of nine new metabolites (2, 3a, 3b, and 4-9). Their structures were elucidated as (3S)-oxyphyllacinol (2), (3S,7R)- and (3S,7S)-7-hydroxyoxyphyllacinol (3a and 3b), (3S)-oxyphyllacinol-4'-O-ß-d-glucopyranoside (4), (3S)-4″-hydroxyoxyphyllacinol (5), (3S)-3″-hydroxyoxyphyllacinol (6), (3S)-2″-hydroxyoxyphyllacinol (7), (3S)-2″-hydroxyoxyphyllacinol-2″-O-ß-d-glucopyranoside (8), and (3S)-oxyphyllacinol-3-O-ß-d-glucopyranoside (9) based on the comprehensive spectroscopic analyses and the application of modified Mosher's method. All compounds were evaluated for their cytotoxic activities against melanoma, as well as breast, lung, and colorectal cancer cell lines. Compound 9, which was O-glucosylated on the diarylheptanoid alkyl chain, exhibited the most selective cytotoxic activities against melanoma cell lines with the IC50 values ranging from 6.09 to 9.74 µM, indicating that it might be considered as a possible anti-cancer lead compound.

Microbial Transformation and Biological Activities of the Prenylated Aromatic Compounds from Broussonetia kazinoki.

Broussonetia kazinoki has been used as a traditional medicine for the treatment of burns and acne, and its extracts have been found to show tyrosinase inhibitory and anticancer activities. In this study, the tyrosinase inhibitory and cytotoxic activities of B. kazinoki were explored, leading to the isolation of kazinol C (1), kazinol E (2), kazinol F (3), broussonol N (4), and kazinol X (5), of which the compounds 4 and 5 have not been previously reported. Microbial transformation has been recognized as an efficient tool to generate more active metabolites. Microbial transformation of the major compounds 1 and 3 was conducted with Mucor hiemalis, where four glucosylated metabolites (6-9) were produced from 1, while one hydroxylated (10) and one glucosylated (11) metabolites were obtained from 3. Structures of the isolated metabolites were determined by extensive spectroscopic analyses. All compounds were evaluated for their tyrosinase inhibitory and cytotoxic activities. Compound 3 and its metabolites, kazinol Y (10) and kazinol F-4″-O-ß-d-glucopyranoside (11), exhibited the most potent tyrosinase inhibitory activities with the IC50 values ranging from 0.71 to 3.36 µM. Meanwhile, none of the metabolites, except for kazinol C-2',3″-di-O-ß-d-glucopyranoside (7), showed moderate cytotoxic activities (IC50 17.80 to 24.22 µM) against A375P, B16F10 and B16F1 cell lines.

Effects of Microbial Transformation on the Biological Activities of Prenylated Chalcones from Angelica keiskei.

Microbial transformation is an alternative method for structural modification. The current study aimed at application of microbial transformation for discovering new derivatives and investigating the structure-activity relationship of isobavachalcone (1), 4-hydroxyderricin (2), and xanthoangelol (3) isolated from the herb Angelica keiskei. In the initial screening process, 1-3 were incubated with microbes using a two-stage fermentation method and analyzed through TLC monitoring. The screening results showed that Rhizopus oryzae and Mucor hiemalis were able to transform 1 and 2, respectively. Additionally, M. hiemalis and Mortierella ramanniana var. angulispora were able to transform 3. Following scale-up fermentation, four new (4, 5, 7, and 10) and five known (6, 8, 9, 11, and 12) metabolites were produced. Cytotoxicity of all the compounds (1-12) was investigated using three human cancer cell lines including A375P, HT-29, and MCF-7 by MTT method. Meanwhile, the tyrosinase inhibitory activity of 1-12 was evaluated using l-tyrosine as a substrate. Overall, 1 and 3 displayed the highest cytotoxicity, and 5 and 7 exhibited the most potent tyrosinase inhibitory activity with relatively low cytotoxicity. This allowed us to postulate that the introduction of 4'-O-glucopyranosyl group led to the reduction in cytotoxicity and improvement in tyrosinase inhibitory activity.

Biotransformation of (-)-α-Bisabolol by Absidia coerulea.

(-)-α-Bisabolol, a bioactive monocyclic sesquiterpene alcohol, has been used in pharmaceutical and cosmetic products with anti-inflammatory, antibacterial and skin-caring properties. However, the poor water solubility of (-)-α-bisabolol limits its pharmaceutical applications. It has been recognized that microbial transformation is a very useful approach to generate more polar metabolites. Fifteen microorganisms were screened for their ability to metabolize (-)-α-bisabolol in order to obtain its more polar derivatives, and the filamentous fungus Absidia coerulea was selected for scale-up fermentation. Seven new and four known metabolites were obtained from biotransformation of (-)-α-bisabolol (1), and all the metabolites exhibited higher aqueous solubility than that of the parent compound 1. The structures of newly formed metabolites were established as (1R,5R,7S)- and (1R,5S,7S)-5-hydroxy-α-bisabolol (2 and 3), (1R,5R,7S,10S)-5-hydroxybisabolol oxide B (4), (1R,7S,10S)-1-hydroxybisabolol oxide B (5), 12-hydroxy-α-bisabolol (7), (1S,3R,4S,7S)- and (1S,3S,4S,7S)-3,4-dihydroxy-α-bisabolol (8 and 10) on the basis of spectroscopic analyses. These compounds could also be used as reference standards for the detection and identification of the metabolic products of 1 in the mammalian system.

Microbial transformation of icariin and its derivatives.

Microbial transformation is an important tool to perform selective conversion of compounds to derivatives which are difficult to produce synthetically. In order to obtain icariside II and icaritin, the active components in Herba Epimedii in vivo, biotransformation studies using microbes as biocatalysts were carried out. Icariside II (2) and icaritin (3) were produced through biotransformation of icariin (1) using the fungi Hormoconis resinae and Mortierella ramanniana var. angulispora in 98% and 92% yields, respectively. In the subsequent transformation studies, 2 was deglycosylated to form 3 by Gliocladium deliquescens, whereas 3 was further converted to a novel compound icaritin-3-O-ß-d-glucopyranoside (4) and previously known icaritin-3,7-O-ß-d-diglucopyranoside (5) by Mucor hiemalis. Biological evaluation of these compounds using MTT assay exhibited potent cytotoxic activities against human cancer cell lines A549, A375P, and MCF-7, with icariin being the most active, indicating that glycosylation plays a role in the cytotoxic activity.

Extract of Moutan radicis cortex and Cinnamomi ramulus ameliorates laser-induced choroidal neovascularization in Brown-Norway rats.

BACKGROUND: Moutan radicis cortex (MRC) and Cinnamomi ramulus (CR) are commonly used in eastern Asian traditional medicine to treat various diseases including cerebrovascular and cardiovascular, and have wide spectrum of pharmacological activities. However, the effect against laser-induced choroidal neovascularization (CNV) of extract of MRC and CR (1:1) (MRCCR) has not yet been studied. PURPOSE: Our aim was to investigate the inhibitory effect of MRCCR on pathological CNV in laser-treated Brown-Norway (BN) rats. METHODS: MRCCR (60, 90 mg/kg) was orally administered twice per day for 15 days from the day of CNV formation in laser-treated BN rats. Effects of MRCCR or its constituents on cell migration, tube formation, hyperpermeability and phosphorylation of FAK/p38 MAPK were confirmed in humane retinal microvascular endothelial cells or human retinal pigment epithelial cells. RESULTS: MRCCR significantly reduced the CNV lesions areas and the extent of fluorescein leakage. MRCCR and its constituents such as ellagic acid, paeonol or gallic acid decreased cell migration, tube formation or hyperpermeability. MRCCR inhibited the phosphorylation of FAK and p38 MAPK. CONCLUSION: Combining the oral MRCCR and intravitreal injection of anti-VEGF medicine may result in a more potent therapeutic effect and consequently bring the reduction in eye injection numbers for patients with wet AMD.

Antimelanogenic activity of patuletin from Inula japonica flowers in B16F10 melanoma cells and zebrafish embryos.

During the search for natural melanogenesis inhibitors, patuletin, a flavonoid, was isolated from Inula japonica flowers. We investigated the antimelanogenic effects of patuletin on B16F10 melanoma cells and zebrafish embryos. Patuletin dose-dependently reduced melanocyte-stimulating hormone-induced melanogenesis and L-DOPA oxidation in B16F10 cells. Western blot analysis showed that patuletin reduced cellular tyrosinase expression in a dose-dependent manner. Patuletin treatment significantly decreased melanin pigmentation in the embryo compared to the untreated controls.

Cimicifugae Rhizoma Extract Attenuates Oxidative Stress and Airway Inflammation via the Upregulation of Nrf2/HO-1/NQO1 and Downregulation of NF-κB Phosphorylation in Ovalbumin-Induced Asthma.

Cimicifugae Rhizoma has been used as a medicinal herb for fever, pain, and inflammation in East Asia. We conducted this study because the effect of Cimicifugae Rhizoma extract (CRE) on allergic asthma has not yet been evaluated. To induce allergic airway inflammation, we intraperitoneally injected ovalbumin (OVA) mixed with aluminum hydroxide into mice twice at intervals of 2 weeks (Days 0 and 14) and then inhaled them thrice with 1% OVA solution using a nebulizer (Days 21 to 23). CRE (30 and 100 mg/kg) was administered orally daily for 6 days (Days 18 to 23). The mice showed remarkable reduction in allergic inflammation at 100 mg/kg of CRE, as evidenced by decreased inflammatory cell counts, pro-inflammatory cytokine levels, OVA-specific immunoglobulin E level, airway hyperresponsiveness, and production of mucus. Additionally, these effects were involved with the enhancement of heme oxygenase-1 (HO-1), NAD(P)H: quinone oxidoreductase (NQO1), and nuclear factor erythroid 2-related factor 2 (Nrf2) expression and reduction of nuclear factor-κB (NF-κB) phosphorylation and matrix metalloproteinase-9 expression. Our findings indicated that CRE effectively protected against OVA-induced inflammation and oxidative stress via upregulation of the Nrf2/HO-1/NQO1 signaling and downregulation of NF-κB phosphorylation in asthma caused by OVA.

Biotransformation of the Phenolic Constituents from Licorice and Cytotoxicity Evaluation of Their Metabolites.

Biotransformation of four bioactive phenolic constituents from licorice, namely licoisoflavanone (1), glycyrrhisoflavone (2), echinatin (3), and isobavachalcone (4), was performed by the selected fungal strain Aspergillus niger KCCM 60332, leading to the isolation of seventeen metabolites (5-21). Structures of the isolated compounds were determined on the basis of extensive spectroscopic methods, twelve of which (5-7, 10-17 and 19) have been previously undescribed. A series of reactions including hydroxylation, hydrogenation, epoxidation, hydrolysis, reduction, cyclization, and alkylation was observed in the biotransformation process. All compounds were tested for their cytotoxic activities against three different human cancer cell lines including A375P, MCF-7, and HT-29. Compounds 1 and 12 exhibited most considerable cytotoxic activities against all the cell lines investigated, while compounds 2 and 4 were moderately cytotoxic. These findings will contribute to expanding the chemical diversity of phenolic compounds, and compounds 1 and 12 may serve as leads for the development of potential cancer chemopreventive agents.

Microbial Conjugation Studies of Licochalcones and Xanthohumol.

Microbial conjugation studies of licochalcones (1-4) and xanthohumol (5) were performed by using the fungi Mucor hiemalis and Absidia coerulea. As a result, one new glucosylated metabolite was produced by M. hiemalis whereas four new and three known sulfated metabolites were obtained by transformation with A. coerulea. Chemical structures of all the metabolites were elucidated on the basis of 1D-, 2D-NMR and mass spectroscopic data analyses. These results could contribute to a better understanding of the metabolic fates of licochalcones and xanthohumol in mammalian systems. Although licochalcone A 4'-sulfate (7) showed less cytotoxic activity against human cancer cell lines compared to its substrate licochalcone A, its activity was fairly retained with the IC50 values in the range of 27.35-43.07 µM.

Microbial Transformation of Broussochalcones A and B by Aspergillus niger.

Broussochalcones A (BCA, 1) and B (BCB, 2) are major bioactive constituents isolated from Broussonetia papyrifera, a polyphenol-rich plant belonging to the family Moraceae. Due to their low yields from natural sources, BCA (1) and BCB (2) were prepared synthetically by employing Claisen-Schmidt condensation, and these were used as substrates for microbial transformation to obtain novel derivatives. Microbial transformation of BCA (1) and BCB (2) with the endophytic fungus Aspergillus niger KCCM 60332 yielded 10 previously undescribed chalcones (1a-1e and 2a-2e). Their structures were established based on the spectroscopic methods. The cytotoxicity of BCA (1), BCB (2), and their metabolites (1a-1e and 2a-2e) was determined by human cancer cell lines A375P, A549, HT-29, MCF-7, and HepG2, with 1e shown to be most cytotoxic.

Anti-inflammatory isocoumarins from the bark of Fraxinus chinensis subsp. rhynchophylla.

A new isocoumarin (1) named fraxicoumarin was isolated from the bark of Fraxinus chinensis subsp. rhynchophylla along with three known compounds (2-4). The structure of the new compound was established by extensive spectroscopic studies and chemical evidence. The anti-inflammatory effects of the isolated compounds (1-4) on lipopolysaccharide (LPS)-induced RAW 264.7 macrophage cells were evaluated in vitro. Of the compounds tested, compounds 1 and 3 inhibited LPS-induced nitric oxide (NO) production in RAW 264.7 cells. Consistent with these findings, they also suppressed LPS-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the protein level in RAW 264.7 cells.