Improved self-assembled micelles based on supercritical fluid technology as a novel oral delivery system for enhancing germacrone oral bioavailability.

The main objective of this study was to develop a novel self-assembled micelle system to improve the oral bioavailability of the water-insoluble and thermal instable germacrone (GEM). Micelles were prepared with an improved supercritical reverse phase evaporation (ISCRPE) method, and the thin-film hydration (TFH) method was used for comparison. Physicochemical characterization confirmed the nanospherical morphology of the micelles prepared by the ISCRPE method (GEM@SDL-SCF Micelles) had smaller particle size and greater encapsulation efficiency than that of the micelles prepared by the TFH method (GEM@SDL-THF Micelles). Dilution resistance and stability experiments were performed to assess the structural integrity. In vitro GEM release was analyzed by the dialysis diffusion method, and the micelle system showed sustained and cumulative release. Furthermore, the fluorescence results from the cellular uptake study revealed improved drug absorption of GEM since the green fluorescence intensity of C6@SDL-SCF Micelles were stronger than that of C6@SDL-TFH Micelles. The transcellular transport study showed a distinct increase in the apparent permeability coefficient (AP → BL) in a Caco-2 cell transport model, and free GEM solution, GEM@SDL-TFH Micelles and GEM@SDL-SCF Micelles had permeability coefficients of 7.30 × 10-6, 8.01 × 10-6, and 10.57 × 10-6 cm·s-1, respectively. The pharmacokinetics study in Sprague-Dawley rats showed that the oral absorption capacity of the GEM@SDL-SCF Micelles was obviously enhanced, with a relative oral bioavailability of 298% and 147% compared with that of free GEM solution and GEM@SDL-TFH Micelles, respectively. The gastrointestinal safety assessment demonstrated that the micelles used in the experiment did not cause gastrointestinal toxicity. These results indicated that self-assembled micelles are promising nanocarriers aimed at enhancing the oral absorption of drugs with thermal sensitivity and low aqueous solubility, and micelles prepared by supercritical fluid (SCF) technology showed better performance than those prepared by the TFH method.

Gastroprotection of Calein D against Ethanol-Induced Gastric Lesions in Mice: Role of Prostaglandins, Nitric Oxide and Sulfhydryls.

Peptic ulcers are currently treated with various drugs, all having serious side effects. The aim of this study was to evaluate the gastroprotective activity of calein D (from Calea urticifolia), a sesquiterpene lactone with a germacrane skeleton. Gastric lesions were induced in mice by administering ethanol (0.2 mL) after oral treatment with calein D at 3, 10 and 30 mg/kg, resulting in 13.15 ± 3.44%, 77.65 ± 7.38% and 95.76 ± 2.18% gastroprotection, respectively, to be compared with that of the control group. The effect found for 30 mg/kg of calein D was not reversed by pretreatment with NG-nitro-l-arginine methyl ester (l-NAME, 70 mg/kg, ip), indomethacin (10 mg/kg, sc) or N-ethylmaleimide (NEM, 10 mg/kg, sc). Hence, the mechanism of action of calein D does not involve NO, prostaglandins or sulfhydryl compounds. Calein D was more potent than carbenoxolone, the reference drug. The findings for the latter are in agreement with previous reports.

Encapsulation of inuloxin A, a plant germacrane sesquiterpene with potential herbicidal activity, in ß-cyclodextrins.

Inuloxin A is a promising plant phytotoxic sesquiterpene that deserves further studies to evaluate its potential as a bioherbicide. However, its low solubility in water and its bioavailability could hamper its practical applications. For this reason, inuloxin A was complexed with ß-cyclodextrins by using three different methods, i.e., kneading, co-precipitation and grinding. The resulted complexes were fully characterized by different techniques such as 1H NMR, UV-vis, XRD, DSC and SEM, and they were biologically assayed in comparison with the pure compound in several biological systems. The efficacy of the kneading and grinding complexes was similar to that of inuloxin A and these complexes almost completely inhibit Phelipanche ramosa seed germination. The complete solubility in water and the preservation of the biological properties of these two complexes could allow further studies to develop a novel natural herbicide for parasitic plant management based on these formulations.

Assessment of sesquiterpene lactones isolated from Mikania plants species for their potential efficacy against Trypanosoma cruzi and Leishmania sp.

Four sesquiterpene lactones, mikanolide, deoxymikanolide, dihydromikanolide and scandenolide, were isolated by a bioassay-guided fractionation of Mikania variifolia and Mikania micrantha dichloromethane extracts. Mikanolide and deoxymikanolide were the major compounds in both extracts (2.2% and 0.4% for Mikania variifolia and 21.0% and 6.4% for Mikania micrantha respectively, calculated on extract dry weight). Mikanolide, deoxymikanolide and dihydromikanolide were active against Trypanosoma cruzi epimastigotes (50% inhibitory concentrations of 0.7, 0.08 and 2.5 µg/mL, for each compound respectively). These sesquiterpene lactones were also active against the bloodstream trypomastigotes (50% inhibitory concentrations for each compound were 2.1, 1.5 and 0.3 µg/mL, respectively) and against amastigotes (50% inhibitory concentrations for each compound were 4.5, 6.3 and 8.5 µg/mL, respectively). By contrast, scandenolide was not active on Trypanosoma cruzi. Besides, mikanolide and deoxymikanolide were also active on Leishmania braziliensis promastigotes (50% inhibitory concentrations of 5.1 and 11.5 µg/mL, respectively). The four sesquiterpene lactones were tested for their cytotoxicity on THP 1 cells. Deoxymikanolide presented the highest selectivity index for trypomastigotes (SI = 54) and amastigotes (SI = 12.5). In an in vivo model of Trypanosoma cruzi infection, deoxymikanolide was able to decrease the parasitemia and the weight loss associated to the acute phase of the parasite infection. More importantly, while 100% of control mice died by day 22 after receiving a lethal T. cruzi infection, 70% of deoxymikanolide-treated mice survived. We also observed that this compound increased TNF-α and IL-12 production by macrophages, which could contribute to control T. cruzi infection.

Germacrone Attenuates Hyperlipidemia and Improves Lipid Metabolism in High-Fat Diet-Induced Obese C57BL/6J Mice.

We previously showed that Aster spathulifolius Maxim extract (ASE) reduced body weight gain and serum and liver lipid levels and significantly suppressed serum insulin and leptin concentrations in high-fat diet (HFD)-induced obese rats. Germacrone (GM) was identified as a potent bioactive constituent of ASE. In this study, we hypothesized that GM can attenuate hyperlipidemia by alleviating fatty acid (FA) synthesis/uptake and improve lipid metabolism by stimulating FA ß-oxidation in HFD-induced obese C57BL/6J mice. To induce obesity, mice were fed an HFD for 6 weeks, while control mice were fed a commercial standard diet. The mice were allocated to six groups and fed either a normal diet, HFD, HFD with GM (5, 10, and 20 mg/kg), or HFD with 200 mg/kg Garcinia cambogia extract for 30 days. In the GM groups, body weight gain, visceral fat pad weight, fasting plasma glucose, serum insulin and leptin, and serum, as well as hepatic lipid, levels were attenuated. Transcriptional factors related to lipid metabolism, such as AMP-activated protein kinase α, sterol regulatory element-binding protein (SREBP) 1, SREBP 2, acetyl-CoA carboxylase, peroxisome proliferator-activated receptor (PPAR)-α, PPAR-γ, FA synthase, and carnitine palmitoyltransferase 1, showed higher expression in the GM groups. In summary, GM may help attenuate hyperlipidemia by suppressing FA synthesis and uptake by inhibiting SREBP signaling pathway activation and improve lipid metabolism by stimulating FA ß-oxidation by activating the AMPKα signaling pathway in HFD-induced obesity.

Development and application of an analytical method for curdione quantification in pregnant Sprague-Dawley rats by LC-MS/MS.

The vaginal administration route suffers from relatively low absorption efficiency, which may hinder the identification of the toxicokinetics of curdione in pregnant women. A sensitive analytical method for determining the plasma concentration of curdione was developed and applied in the determination of curdione in pregnant Sprague-Dawley rats as a simulated model. Glimepiride was used as an internal standard and chromatographic separation was achieved on a Capcell Pak C18 MGIII column. A gradient elution profile with 0.5% formic acid (A)-0.5% formic acid-acetonitrile (B) was selected as mobile phase. The selected reaction monitoring mode was used for quantification based on the target fragment ions m/z 237.2 to m/z 135.1 for curdione and m/z 491.3 to m/z 352.1 for the glimepiride. The standard curve was linear over the range of 0.5-500 ng/mL for curdione in rat plasma and yielded a consistent peak pattern, even at the lower limit of quantitation of 0.5 ng/mL. The retention times of curdione and IS were 6.55 and 6.59 min, respectively. The mean recovery of curdione in rat plasma was 95.5-101.1%. The intra-day and inter-day precisions were between 2.35 and 9.08%. This LC-MS/MS method provides a simple and sensitive means for determining the plasma concentration.

Germacrane sesquiterpenes isolated from the rhizome of Curcuma xanthorrhiza Roxb. inhibit UVB-induced upregulation of MMP-1, -2, and -3 expression in human keratinocytes.

Four sesquiterpenes were isolated from the rhizome of Curcuma xanthorrhiza Roxb.: furanodiene (1), germacrone (2), furanodienone (3), and 13-hydroxygermacrone (4). Importantly, this was the first time compounds 1 and 4 were isolated from this plant. The chemical structures of these compounds were determined using 1D- and 2D-nuclear magnetic resonance, infrared spectroscopy, and electron ionization mass spectrometry analyses. Among the isolated compounds, compounds 2 and 4 inhibited UVB-induced upregulation of the mRNA and protein expression levels of MMP-1, MMP-2, and MMP-3 in human keratinocytes (HaCaT). Moreover, this upregulation occurred in a dose-dependent manner over the range of 1-10 µM for each compound.

Germacrone inhibits early stages of influenza virus infection.

Highly pathogenic influenza viruses pose a serious public health threat to humans. Although vaccines are available, antivirals are needed to efficiently control disease progression and virus transmission due to the emergence of drug-resistant viral strains. In this study, germacrone, which is a major component of the essential oils extracted from Rhizoma Curcuma, was found to inhibit influenza virus replication. Germacrone showed antiviral activity against the H1N1 and H3N2 influenza A viruses and the influenza B virus in a dose-dependent manner. The viral protein expression, RNA synthesis and the production of infectious progeny viruses were decreased both in MDCK and A549 cells treated with germacrone. In a time-of-addition study, germacrone was found to exhibit an inhibitory effect on both the attachment/entry step and the early stages of the viral replication cycle. Germacrone also exhibited an effective protection of mice from lethal infection and reduced the virus titres in the lung. Furthermore, the combination of germacrone and oseltamivir exhibited an additive effect on the inhibition of influenza virus infection, both in vitro and in vivo. Our results suggest that germacrone may have the potential to be developed as a therapeutic agent alone or in combination with other agents for the treatment of influenza virus infection.

Germacrone induces apoptosis in human hepatoma HepG2 cells through inhibition of the JAK2/STAT3 signalling pathway.

Previous studies have shown that STAT3 plays a vital role in the genesis and progression of cancer. In this study, we investigated the relationship between the JAK2/STAT3 signalling pathway and germacrone-induced apoptosis in HepG2 cells. HepG2 cells were incubated with germacrone for 24 h, the protein expression of p-STAT3, STAT3, p-JAK2 and JAK2 was detected by Western Blotting, and RT-PCR was used to determine the expression of STAT3, p53, Bcl-2 and Bax at transcriptional levels. Besides that, HepG2 cells were pre-treated with AG490 or IL-6 for 2 h, and then incubated with germacrone for 24 h. The expression of p-JAK2, JAK2, p-STAT3, STAT3, p53, Bax and Bcl-2 was detected by Western blotting. The activity of HepG2 cells was tested by MTT assay. The apoptosis of HepG2 cells and levels of reactive oxygen species (ROS) were flow cytometrically measured. The results showed that germacrone exposure decreased p-STAT3 and p-JAK2 and regulated expression of p53 and Bcl-2 family members at the same time. Moreover, IL-6 enhanced the activation of the JAK2/STAT3 signalling pathway and therefore attenuated the germacrone-induced apoptosis. Suppression of JAK2/STAT3 signalling pathway by AG490, an inhibitor of JAK2, resulted in apoptosis and an increase in ROS in response to germacrone exposure. We therefore conclude that germacrone induces apoptosis through the JAK2/STAT3 signalling pathway.

Inhibition of platelet aggregation by curdione from Curcuma wenyujin essential Oil.

INTRODUCTION: Curdione, one of the major sesquiterpene compounds from Rhizoma Curcumae, has been shown to exhibit multiple bioactive properties. In this study, we investigated the anti-platelet aggregation and antithrombotic activities of curdione with different methods both in vitro and in vivo. The purpose of the study was to explore an inhibitor of platelet aggregation, which promised to be a preventive or therapeutic agent for various vascular diseases. MATERIALS AND METHODS: Curdione was isolated from the essential oil of Curcuma wenyujin using the silica gel column chromatography method. The effects of curdione on human platelet aggregation induced by thrombin (0.3 U/ml), platelet-activating factor (PAF, 0.375 µg/ml), adenosine diphosphate (ADP, 10 µM) and arachidonic acid (AA, 0.1mg/ml) were tested in vitro, and the potential mechanisms underlying such activities were investigated. We also tested the antithrombotic effect of curdione in a tail thrombosis model. RESULTS AND CONCLUSIONS: Curdione preferentially inhibited PAF- and thrombin- induced platelet aggregation in a concentration-dependent manner (IC(50): 60-80 µM), whereas much higher concentrations of curdione were required to inhibit platelet aggregation induced by ADP and AA. Curdione also inhibited P-selectin expression in PAF-activated platelets. Moreover, curdione caused an increase in cAMP levels and attenuated intracellular Ca(2+) mobilization in PAF-activated platelets. In vivo, we also found that curdione showed significant antithrombotic activity. Therefore, we conclude that the inhibitory mechanism of curdione on platelet aggregation may increase cAMP levels and subsequently inhibit intracellular Ca(2+) mobilization. Furthermore, the effect observed in the tail thrombosis model might be explained completely by increased vasodilation. These results indicate that curdione may be one of the main bioactive constituents in Rhizoma Curcumae that removes blood stasis.

Germacranolides from Anvillea radiata.

The aerial parts of Anvillea radiata yielded a new germacranolide, 8alpha,9alpha-epoxyparthenolide (3), together with two known compounds, 9alpha-hydroxyparthenolide (1) and parthenolid-9-one (2). The structures of the compounds were elucidated from IR, HRMS, 1H and 13C-NMR, COSY, HETCOR, HMBC and HOHAHA spectral data. The major component 1 was tested for its cytotoxicity and antibacterial activity.