Humulus japonicus ameliorates irritant contact dermatitis by suppressing NF­κB p65­dependent inflammatory responses in mice.

As a type of contact dermatitis (CD), irritant CD (ICD) is an acute skin inflammation caused by external irritants, such as soap, water and chemicals. Humulus japonicus (HJ) is a herbal medicine widely distributed in Asian countries and has anti-inflammatory, antimicrobial and antioxidant effects. The current study aimed to investigate the anti-dermatitis effect of HJ on ICD and determine the molecular basis of this effect using 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced dermatitis mice models and lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Mice were orally administered HJ and luteolin, the major compound in HJ, and topically administered TPA on the right ear to induce dermatitis. Topical application of TPA induced ear redness, oedema and increased infiltration of neutrophils and macrophages, which ameliorated following HJ and luteolin administration. The gene expression levels of inflammatory cell migrating chemokines, chemokine ligand 3 (CCL3) and chemokine (C-X-C motif) ligand 2 (CXCL2), and pro-inflammatory cytokine, IL-1ß, were reduced in the ears of HJ- and luteolin-treated mice. HJ and luteolin also inhibited the gene expression of chemokines, CCL3 and CXCL2, and pro-inflammatory cytokines, IL-1ß, IL-6 and TNF-α, in LPS-stimulated RAW264.7 cells. Moreover, HJ and luteolin decreased the expression levels of two key inflammatory enzymes, cyclooxygenase-2 (COX2) and inducible nitric oxide synthase (iNOS), and total and active phosphorylation of NF-κB p65. These results suggest that HJ could have a protective effect against ICD by suppressing inflammatory responses; therefore, HJ is a promising therapeutic strategy for ICD treatment.

Humulus japonicus rescues autistic­like behaviours in the BTBR T+ Itpr3tf/J mouse model of autism.

Humulus japonicus (HJ) is a traditional herbal medicine that exhibits anti­inflammatory, antimicrobial and anti­tumor effects that is used for the treatment of hypertension, pulmonary disease and leprosy. Recently, it has also been reported that HJ demonstrates neuroprotective properties in animal models of neurodegenerative diseases. The current study hypothesised that the administration of HJ would exhibit therapeutic effects in autism spectrum disorder (ASD), a neurodevelopmental disorder with lifelong consequences. The BTBR T+ Itpr3tf/J mouse model of ASD was used to investigate the anti­autistic like behavioural effects of HJ. Chronic oral administration of the ethanolic extract of HJ significantly increased social interaction, attenuated repetitive grooming behaviour and improved novel­object recognition in BTBR mice. Anti­inflammatory effects of HJ in the brain were analysed using immunohistochemistry and reverse­transcription quantitative PCR analysis. Microglia activation was markedly decreased in the striatum and hippocampus, and pro­inflammatory cytokines, including C­C Motif Chemokine Ligand 2, interleukin (IL)­1ß and IL­6, were significantly reduced in the hippocampus following HJ treatment. Moreover, HJ treatment normalised the phosphorylation levels of: N­methyl­D­aspartate receptor subtype 2B and calcium/calmodulin­dependent protein kinase type II subunit α in the hippocampus of BTBR mice. The results of the present study demonstrated that the administration of HJ may have beneficial potential for ameliorating behavioural deficits and neuroinflammation in ASD.

Glucose Uptake-Stimulating Galloyl Ester Triterpenoids from Castanopsis sieboldii.

Using molecular networking-guided isolation, three new galloyl ester triterpenoids (1-3), two new hexahydroxydiphenic acid-conjugated triterpenoids (6 and 7), and four known compounds (4, 5, 8, and 9) were isolated from the fruits and leaves of Castanopsis sieboldii. The chemical structures of 1-3, 6, and 7 were elucidated on the basis of interpreting their NMR, HRESIMS, and ECD spectra. All compounds (1-9) were evaluated for their glucose uptake-stimulating activities in differentiated adipocytes using 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-d-glucose as a fluorescent-tagged glucose probe. Compounds 2 and 9 resulted in a 1.5-fold increase in glucose uptake. Among them, compound 2 from the fruits showed an upregulation of p-AMPK/AMPK ratio in differentiated C2C12 myoblasts to support the mechanism proposed of glucose uptake stimulation.

Antiviral activity of furanocoumarins isolated from Angelica dahurica against influenza a viruses H1N1 and H9N2.

ETHNOPHARMACOLOGICAL RELEVANCE: Angelica dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav. (Umbelliferae family) is an herbaceous, perennial plant native to northern and eastern Asia. The root of A. dahurica has traditionally been used under the name "Bai Zhi" as a medicinal plant for colds, dizziness, ulcers, and rheumatism. Moreover, it is also an important ingredient of various prescriptions, such as Gumiganghwal-Tang, for the common cold and influenza. AIM OF THE STUDY: Even though various biological activities of the root of A. dahurica have been reported along with its chemical components, the detailed mechanism of how it exerts anti-influenza activity at the compound level has not been studied. Therefore, we investigated the anti-influenza properties of furanocoumarins purified by bioactivity-guided isolation. MATERIALS AND METHODS: Bioactivity-guided isolation from a 70% EtOH extract of the root of A. dahurica was performed to produce four active furanocoumarins. The inhibition of cytopathic effects (CPEs) was evaluated to ascertain the antiviral activity of these compounds against influenza A (H1N1 and H9N2) viruses. The most potent compound was subjected to detailed mechanistic studies such as the inhibition of viral protein synthesis, CPE inhibition in different phases of the viral replication cycle, neuraminidase (NA) inhibition, antiapoptotic activity using flow cytometry, and immunofluorescence. RESULTS: The bioactivity-guided isolation produced four active furanocoumarins, isoimperatorin (1), oxypeucedanin (2), oxypeucedanin hydrate (3) and imperatorin (4) from the n-BuOH fraction. Among them, compound 2 (followed by compounds 1, 4 and 3) showed a significant CPE inhibition effect, which was stronger than that of the positive control ribavirin, against both H1N1 and H9N2 with an EC50 (µM) of 5.98 ± 0.71 and 4.52 ± 0.39, respectively. Compound 2 inhibited the synthesis of NA and nucleoprotein (NP) in a dose-dependent manner. In the time course assays, the cytopathic effects of influenza A-infected MDCK cells were reduced by 80-90% when treated with compound 2 for 1 and 2 h after infection and declined drastically 3 h after infection. The level of viral NA and NP production was markedly reduced to less than 20% for both proteins in compound 2 (20 µM)-treated cells compared to untreated cells at 2 h after infection. In the molecular docking analysis, compound 2 showed a stronger binding affinity for the C-terminus of polymerase acidic protein (PAC; -36.28 kcal/mol) than the other two polymerase subunits. Compound 2 also exerted an antiapoptotic effect on virus infected cells and significantly inhibited the mRNA expression of caspase-3 and Bax. CONCLUSION: Our results suggest that compound 2 might exert anti-influenza A activity via the inhibition of the early phase of the viral replication cycle, not direct neutralization of surface proteins, such as hemagglutinin and NA, and abnormal apoptosis induced by virus infection. Taken together, these findings suggest that furanocoumarins predominant in A. dahurica play a pivotal role in its antiviral activity. These findings can also explain the reasons for the ethnopharmacological uses of this plant as an important ingredient in many antiviral prescriptions in traditional Chinese medicine (TCM).

Sicyos angulatus Prevents High-Fat Diet-Induced Obesity and Insulin Resistance in Mice.

Obesity is a medical condition in which excess body fat has accumulated to a serious extent. It is a chronic disease that can lead to dyslipidemia, insulin resistance, and type 2 diabetes. In the present study, we investigated the anti-obesity effects of Sicyos angulatus (SA) extract on a high-fat diet (HFD)-induced C57BL/6J obese mice. The mice were divided into vehicle and three SA groups (25, 50, and 100 mg/kg body weight). The mice were fed a HFD with or without SA for 12 weeks. The oral administration of SA reduced body and adipose tissue weight in HFD-fed mice compared to those in the vehicle group (p<0.05). Adipocyte size and inflammation significantly decreased in the SA-administered groups in a dose-dependent manner. In particular, adipocytes larger than 5000 µm2 were remarkably reduced by around 50% in the SA-treated groups (p<0.05). In addition, SA contributes towards reducing insulin resistance (measured as the HOMA-IR index) and glucose intolerance in HFD-induced obese mice (p<0.05; Vehicle 21.5±3.1 vs. SA100 4.7±0.4). These beneficial effects of SA on obesity may be linked to the suppression of lipogenesis and stimulating energy metabolism in white adipose tissue and muscle. In white adipose tissue and muscle, the administration of SA activated AMPK pathway, leading to the inhibition of the development of pathophysiological conditions associated with obesity, including lipogenesis and inflammation. These findings suggest that SA may prevent obesity through inhibiting fat accumulation in HFD-induced obese mice. Therefore, SA is able to exert metabolic benefits in the prevention of obesity and insulin resistance.

Development of a Building Block Strategy to Target the Classification, Identification, and Metabolite Profiling of Oleanane Triterpenoids in Gymnema sylvestre Using UHPLC-qTOF/MS.

The major class of bioactive metabolites in Gymnema sylvestre, a popular Ayurvedic medicinal plant for the treatment of diabetes mellitus, is oleanane triterpenoids. In this study, a targeted, biosynthesis-inspired approach using UHPLC-qTOF/MS was implemented to elucidate the whole chemical profile of this plant for the standardization of the Vietnamese G. sylvestre variety. The known compounds were first determined to identify the building blocks of the biosynthetic intermediates and the construction rules for synthesizing oleanane triterpenoids in the plant. These blocks were recombined to build a virtual library of all reasonable compounds consistent with the deduced construction rules. Various techniques, including relative mass defect filtering, multiple key ion analysis, mass fragmentation analysis, and comparison with standard references, were applied to determine the presence of these predicted compounds. Conventional isolation and structure elucidation of six of the new compounds were carried out to identify the new building blocks and validate the assignments. Consequently, 119 peaks were quickly assigned to oleanane triterpenoids, and among them, 77 peaks were predicted to be new compounds based on their molecular formulas and mass fragmentation patterns. All the identified metabolites were then classified into different layers to analyze their logical relationships, and a multilayered chemical profile of the oleanane triterpenoids was constructed. This new approach is expected to be practical for characterizing structures of modular secondary metabolites, such as triterpenoid saponins, and for proposing biosynthetic relationships among compounds of the same class of metabolites in medicinal plants.

Anti-hepatic steatosis activity of Sicyos angulatus extract in high-fat diet-fed mice and chemical profiling study using UHPLC-qTOF-MS/MS spectrometry.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a hepatic manifestation of metabolic syndrome. Recently, the inhibitory effects of flavone glycosides isolated from Sicyos angulatus extract on hepatic lipid accumulation in vitro were demonstrated. However, the effects of S. angulatus extract and its major flavonoid glycoside on in vivo hepatic steatosis induced by a high-fat diet have not yet been established. HYPOTHESIS/PURPOSE: The aim of this study was to investigate the effects of S. angulatus extract and its major flavonoid glycoside, kaempferol 3-O-[α-l-rhamnopyranosyl-(1→6)]-ß-d-glucopyranosyl-7-O-α-l-rhamnopyranoside, on hepatic steatosis in high-fat diet-fed mice, which serves as a model of NAFLD. In addition, attempts have been made to chemically profile the metabolites involved in the activity of the S. angulatus extract. METHODS: C57BL/6 J mice were divided into vehicle, total extract of S. angulatus (SA; 50, 100 and 200 mg/kg) and major active component (20 mg/kg) groups. The mice were fed a high-fat diet (HFD) with or without S. angulatus extract or its major single compound for 10 weeks. Chemical identification was carried out using ultra-high-pressure liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-qTOF-MS/MS) and then quantified by HPLC-DAD. RESULTS: Administration of S. angulatus extract significantly lowered plasma ALT and AST levels in HFD-fed mice compared to those of the vehicle group. The hepatic lipid content, as evidenced by oil-red O staining and quantification, was significantly lower in the S. angulatus-administered group, and the effect was dose dependent. These beneficial effects of S. angulatus extract were related to the decreased expression of hepatic genes involved in fatty acid (ACC1, FAS and SCD1) and triglyceride (DGAT) synthesis. The expression levels of two key transcription factors regulating lipogenesis, SREBP-1c and PPARγ, were significantly suppressed in the liver by administration of S. angulatus extract with HFD. Treatment of the HFD-fed mice with the major compound isolated from S. angulatus extract resulted in improved liver function along with an anti-steatotic effect similar to the results seen with S. angulatus extract. For the standardization of the S. angulatus extract, 23 compounds were identified based on MS/MS fragmentation and UV spectroscopy. Quantitative analysis of the major compound showed that the major component was present in 15.35 ± 0.01 mg/g of total extract. CONCLUSION: These findings suggest that S. angulatus extract and its major component have the potential to improve liver function and hepatic steatosis in diet-induced obese mice.

Dereplication of Components Coupled with HPLC-qTOF-MS in the Active Fraction of Humulus japonicus and It's Protective Effects against Parkinson's Disease Mouse Model.

Humulus japonicus is an annual plant belonging to the Cannabacea family, and it has been traditionally used to treat pulmonary tuberculosis, dysentery, chronic colitis, and hypertension. We investigated the active components against Parkinson's disease from H. japonicus fraction (HJF) using high performance liquid chromatography (HPLC) coupled with quadruple-time-of-flight mass spectroscopy (qTOF-MS) and NMR. Fourteen compounds were isolated from HJF, including one new compound, using HPLC-qTOF-MS and NMR. The major compounds of HJF were luteolin-7-O-glucoside and apigenin-7-O-glucoside, and there was approximately 12.57- and 9.68-folds increase in the contents of these flavonoids compared to those of the 70% EtOH extract. Apigenin and luteolin exhibited the strongest inhibitory effects on monoamine oxidase (MAO) B enzyme activity. In animal studies, limb-use behavior was significantly reduced by unilateral 6-OHDA lesion and ipsilateral rotations. These results indicated that oral administration of 300 mg/kg HJF resulted in the improvement of motor asymmetry and motor impairment in unilateral 6-OHDA-lesioned mice. HJF, including active components leads to an improvement of motor behavior in a Parkinson's disease mouse model.

Sicyos angulatus ameliorates acute liver injury by inhibiting oxidative stress via upregulation of anti-oxidant enzymes.

OBJECTIVE: We aimed to investigate the effect of Sicyos angulatus (SA) ethanolic extracts as antioxidants and potential treatments for liver disease. METHODS: To establish a mouse model of liver injury, C57BL/6 male mice were injected via the caudal vein with a single dose of concanavalin A (Con A, 15 mg kg-1). SA extracts were administered once by oral gavage 30 min before Con A injection. RESULTS: In vitro studies showed that SA decreased tert-butyl hydroperoxide (t-BHP)-induced reactive oxygen species (ROS) production. SA administration reduced plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, as well as hepatic ROS levels, in a dose-dependent manner. Moreover, SA increased the activities of the hepatic antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase in a dose-dependent manner. Furthermore, SA treatment reduced pro-apoptotic protein levels. Con A-mediated cytosolic release of Smac/DIABLO and apoptosis-inducing factor (AIF), which are markers of necrosis, were dramatically decreased in HepG2 cells treated with SA. CONCLUSION: SA ameliorated liver injury and might be a good strategy for the treatment of liver injury.

Insulin Mimetic Activity of 3,4- Seco and Hexanordammarane Triterpenoids Isolated from Gynostemma longipes.

As part of ongoing research to find new antidiabetic agents from medicinal plants, the chemical composition of Gynostemma longipes, an ethnomedicinal plant used to treat type 2 diabetes mellitus by local communities in Vietnam, was investigated. Ten new dammarane triterpenes, including two 3,4- seco-dammarane analogues, secolongipegenins S1 and S2 (1 and 2), a 3,4- seco-hexanordammarane, secolongipegenin S3 (3), two hexanordammarane glycosides, longipenosides ND1 and ND2 (4 and 5), and five other dammarane glycosides, longipenosides GL1-GL5 (6-10), were isolated from a 70% EtOH extract of the whole G. longipes plant. The structures of the new compounds were elucidated using diverse spectroscopic methods. All of the isolates were evaluated for their stimulatory activities on glucose uptake in differentiated 3T3-L1 adipocyte cells using 2-[ N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose as a fluorescent-tagged glucose probe. The stimulant activities on glucose uptake by the test compounds were mediated via the activation of the AMPK pathway using differentiated mouse C2C12 skeletal myoblasts. Consequently, compounds 1, 2, and 4 enhanced glucose uptake and GLUT4 translocation significantly by regulating the AMPK signaling pathway.

Protein Tyrosine Phosphatase 1B Inhibitors from the Stems of Akebia quinata.

PTP1B deficiency in mouse mammary tumor virus (MMTV)-NeuNT transgenic mice inhibited the onset of MMTV-NeuNT-evoked breast cancer, while its overexpression was observed in breast cancer. Thus, PTP1B inhibitors are considered chemopreventative agents for breast cancer. As part of our program to find PTP1B inhibitors, one new diterpene glycoside (1) and 13 known compounds (2-14) were isolated from the methanol extract of the stems of Akebia quinata. All isolates were identified based on extensive spectroscopic data analysis, including UV, IR, NMR and MS. Compounds 2, 3, 6, 8 and 11 showed significant inhibitory effects on the PTP1B enzyme, with IC50 values ranging from 4.08 ± 1.09 to 21.80 ± 4.74 µM. PTP1B inhibitors also had concentration-dependent cytotoxic effects on breast cancer cell lines, such as MCF7, MDA-MB-231 and tamoxifen-resistant MCF7 (MCF7/TAMR) (IC50 values ranging from 0.84 ± 0.04 to 7.91 ± 0.39 µM). These results indicate that compounds 6 and 8 from Akebia quinata may be lead compounds acting as anti-breast cancer agents.