Glaucocalyxin A

The herbs of Rabdosia rubescens

Inhibition by glaucocalyxin A of aggregation of rabbit platelets induced by ADP, arachidonic acid and platelet-activating factor, and inhibition of [3H]-PAF binding.[Pubmed:1631795]

Thromb Haemost. 1992 Apr 2;67(4):458-60.

Glaucocalyxin A is a new diterpenoid isolated from the ethereal extract of the leaves of Rabdosia japonica (Burm f) Hara var glaucocalyx (Maxim) Hara (Labiatae) collected in the northeastern China. When it was incubated with washed rabbit platelets, Glaucocalyxin A inhibited ADP- or arachidonic acid-induced platelet aggregation with IC50 values of 4.4 mumol/l, 14.1 mumol/l respectively. Glaucocalyxin A also inhibited PAF-induced aggregation of rabbit platelets which were refractory to ADP and arachidonic acid with an IC50 value of 13.7 mumol/l. Analysis of [3H]-PAF binding showed that Glaucocalyxin A prevented [3H]-PAF binding to intact washed rabbit platelets with an IC50 value of 8.16 mumol/l, which was consistent with its inhibition of PAF-induced platelet aggregation.

Glaucocalyxin A inhibits platelet activation and thrombus formation preferentially via GPVI signaling pathway.[Pubmed:24386454]

PLoS One. 2013 Dec 30;8(12):e85120.

Platelets play a pivotal role in atherothrombosis and the antiplatelet agents have been proved to be useful in preventing onset of acute clinical events including myocardial infarction and stroke. Increasing number of natural compounds has been identified to be potential antiplatelet agents. Here we report the antiplatelet effect of Glaucocalyxin A (GLA), an ent-diterpenoid that we isolated and purified from the aerial parts of Rabdosia japonica (Burm. f.) var. glaucocalyx (Maxim.) Hara, and investigate the molecular mechanisms by which GLA inhibits platelet activation and thrombus formation. The effect of GLA on platelet activation was measured using platelets freshly isolated from peripheral blood of healthy donors. Results showed that pretreatment of human platelets with lower concentrations of GLA (0.01 mug/ml, 0.1 mug/ml) significantly inhibited platelet aggregation induced by collagen (P<0.001) and CRP (P<0.01), a synthetic GPVI ligand, but not by ADP and U46619. Accordingly, GLA inhibited collagen-stimulated tyrosine phosphorylation of Syk, LAT, and phospholipase Cgamma2, the signaling events in collagen receptor GP pathway. GLA also inhibited platelet p-selectin secretion and integrin activation by convulxin, a GPVI selective ligand. Additionally, GLA was found to inhibit low-dose thrombin-induced platelet activation. Using a flow chamber device, GLA was found to attenuate platelet adhesion on collagen surfaces in high shear condition. In vivo studies showed that GLA administration increased the time for complete occlusion upon vascular injury in mice, but did not extend tail-bleeding time when mice were administered with relatively lower doses of GLA. Therefore, the present results provide the molecular basis for the inhibition effect of GLA on platelet activation and its in vivo effect on thrombus formation, suggesting that GLA could potentially be developed as an antiplatelet and antithrombotic agent.

Glaucocalyxin A, a negative Akt regulator, specifically induces apoptosis in human brain glioblastoma U87MG cells.[Pubmed:24041957]

Acta Biochim Biophys Sin (Shanghai). 2013 Nov;45(11):946-52.

Akt is becoming an attractive target in the development of anti-tumor agents. In the present study, we aimed to discover novel negative Akt regulators against malignant glioma. An Akt regulator screening platform performed in an Akt-GFP overexpression cell line was developed, and natural product library was screened and evaluated using this platform. In addition, the cytotoxic effect of the regulator was detected by MTT assay. Cell apoptosis was assayed by Hoechst 33342 staining and flow cytometry analysis. Afterwards, the apoptotic signaling pathway was investigated by western blot analysis. Glaucocalyxin A, isolated from Rabdosia japonica, was identified as a potent negative regulator of Akt. In human-derived malignant glioma U87MG cells, Glaucocalyxin A inhibited Akt phosphorylation, suppressed proliferation, and promoted apoptosis in a dose-dependent manner, but not in normal glial cells. Furthermore, Glaucocalyxin A activated caspase-3, decreased BAD phosphorylation, and reduced the expression of X-linked inhibitor of apoptosis protein. Taken together, these results indicated that Glaucocalyxin A may become a promising candidate in the treatment of malignant glioma.

Glaucocalyxin A activates FasL and induces apoptosis through activation of the JNK pathway in human breast cancer cells.[Pubmed:24289581]

Asian Pac J Cancer Prev. 2013;14(10):5805-10.

THis study was conducted to analyze the molecular mechanisms responsible for anti-proliferation effects of Glaucocalyxin A in cultured MCF-7 and Hs578T breast cancer cells. The concentration that reduced cell viability to 50% (IC50) after 72 h treatment was derived and potential molecular mechanisms of anti-proliferation using the IC50 were investigated as changes in cell cycle arrest and apoptosis. Gene and protein expression changes related to apoptosis were investigated by semi-quantitative RT-PCR and western blotting, respectively. Involvement of phosphorylated mitogen-activated protein kinases and JNK signaling in regulation of these molecules was characterized by western blotting. Cell viability decreased in a concentration-dependent manner and the IC50 was determined as 1 muM in MCF-7 and 4 muM in Hs578T cell. Subsequently, we demonstrated that the GLA-induced MCF-7 and Hst578T cell death was due to cell cycle arrest at the G2/M transition and was associated with activation of the c-jun N-terminal kinase (JNK) pathway. We conclude that GLA has the potential to inhibit the proliferation of human breast cancer cells through the JNK pathway and suggest its application forthe effective therapy for patients with breast cancer.

Glaucocalyxin A improves survival in bleomycin-induced pulmonary fibrosis in mice.[Pubmed:27816453]

Biochem Biophys Res Commun. 2017 Jan 1;482(1):147-153.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease with unclear etiology and poor prognosis. Despite numerous studies on the pathogenesis of IPF, only scant treatment options are available for the management of IPF. Glaucocalyxin A (GLA), an ent-Kaurane diterpenoid, has been demonstrated to exert anti-inflammatory, anti-neoplastic and anti-platelet activities. In this study, we evaluated the role of GLA as an anti-fibrotic agent in bleomycin-induced pulmonary fibrosis in mice and investigated the underlying mechanisms by which GLA attenuates lung fibrosis. Intraperitoneal administration of GLA (10 mg/kg) significantly reduced collagen deposition and hydroxyproline content in mouse lungs treated with bleomycin. Importantly, GLA significantly improved survival in bleomycin treated mice. In addition, GLA reduced weight loss in mice that reflects cachexia due to pulmonary fibrosis. Mechanistically, GLA alleviated the infiltration of macrophages and neutrophils in lungs, attenuated the increases of proinflammatory cytokines in lung tissue and bronchoalveolar lavage fluid, and inhibited the activation of NF-kappaB in fibrotic lungs induced by bleomycin. These results provide evidence that GLA can effectively ameliorate pulmonary fibrosis through the antagonism of leukocyte infiltration and proinflammatory cytokine production, suggesting that it may become a potential anti-fibrotic agent in IPF management.

Regulation of microglia activity by glaucocalyxin-A: attenuation of lipopolysaccharide-stimulated neuroinflammation through NF-kappaB and p38 MAPK signaling pathways.[Pubmed:23393601]

PLoS One. 2013;8(2):e55792.

Microglial cells are the resident macrophages and intrinsic arm of the central nervous system innate immune defense. Microglial cells become activated in response to injury, infection, environmental toxins, and other stimuli that threaten neuronal survival. Therefore, regulating microglial activation may have therapeutic benefits that lead to alleviating the progression of inflammatory-mediated neurodegeneration. In the present study, we investigated the effect of Glaucocalyxin A (GLA) isolated from Rabdosia japonica on the production of pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated primary microglia and BV-2 cells. GLA significantly inhibited LPS-induced production of nitric oxide and reversed the morphological changes in primary microglia. Further, GLA suppressed expression of inducible nitric oxide synthase and cyclooxygenase-2 dose-dependently at the mRNA and protein levels. The production of proinflammatory cytokines such as tumor necrosis factor-alpha, interleukin-1beta (IL)-1beta, and IL-6 were inhibited by suppressing their transcriptional activity. Furthermore, GLA suppressed nuclear factor-kappaB activation by blocking degradation of IkappaB-alpha and inhibited the induction of lipocalin-2 expression in LPS-stimulated BV-2 cells. Mechanistic study revealed that the inhibitory effects of GLA were accompanied by blocking the p38 mitogen activated protein kinase signaling pathway in activated microglia. In conclusion, given that microglial activation contributes to the pathogenesis of neurodegenerative diseases, GLA could be developed as a potential therapeutic agent for treating microglia-mediated neuroinflammatory diseases.

Preparation, characteristic and pharmacological study on inclusion complex of sulfobutylether-beta-cyclodextrin with glaucocalyxin A.[Pubmed:24697809]

J Pharm Pharmacol. 2014 Jul;66(7):927-34.

OBJECTIVES: The objective of this study was to improve the water solubility and solubility of Glaucocalyxin A (GLA) by producing its inclusion complex with sulfobutylether-beta-cyclodextrin (SBE-beta-CD). METHODS: The formation of its 1:1 complex with SBE-beta-CD in solution was confirmed by phase-solution and spectral-shift studies. The interaction of GLA and SBE-beta-CD was examined by differential scanning calorimetry, powder X-ray diffraction, Fourier transform infrared spectroscopy, proton nuclear magnetic resonance spectroscopy and ultraviolet-visible spectroscopy to determine the formation of the GLA-SBE-beta-CD inclusion complex. KEY FINDINGS: The solubilities of GLA and its complexes were 2.38 x 10(2) and 1.82 x 10(4) mug/ml, respectively, and the values of the inclusion complexes were significantly improved by 76-fold compared with the solubility of free GLA. Moreover, a higher area under the curve0-infinity after inclusion technique was observed in the pharmacokinetics study. CONCLUSIONS: The aforementioned results indicate that GLA-SBE-beta-CD could be useful with a better solubility and sustained function in drug delivery.

Synthesis and biological evaluation of glaucocalyxin A derivatives as potential anticancer agents.[Pubmed:25164762]

Eur J Med Chem. 2014 Oct 30;86:235-41.

A series of Mannich base type derivatives of Glaucocalyxin A (GLA) were designed and prepared. The cytotoxicity of these compounds was evaluated against six tumor cell lines (SMMC-7721, B16, SGC-7901, A549, KB, HL-60). Most compounds exhibited potent antiproliferative effects with low micromolar IC50 values. Compound 1 with para methyl benzyl amine moiety and compound 16 with cyclohexylamine moiety displayed the highest inhibition efficacy. Significantly, the cytotoxicity of compound 1 was much lower than GLA against the normal human liver cell (HL-7702). The in vitro stability assay revealed that transformation of GLA to Mannich base type derivatives improved the compound stability in rat plasma. Finally, decomposition product analysis supported that compound 1 could act as a prodrug and release GLA in the intracellular environment.

Glaucocalyxin A, an ent-kauranoid diterpene from Rabdosia japonica var., induces apoptosis in osteosarcoma by inhibiting nuclear translocation of Five-zinc finger Glis 1 (GLI1) via regulating PI3K/Akt signaling pathway. Glaucocalyxin A has antitumor effect.

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