A new kaurane diterpenoid from Isodon inflexus.

A new 7,20-epoxy kaurane diterpenoid, 15-acetyldemethylkamebacetal A (1) and six known kaurane diterpenoids (2-7) were isolated from the aerial parts of Isodon inflexus in nuclear transcription factor-κB (NF-κB)-dependent reporter gene assay-guided fractionation. Their chemical structures were determined on the basis of extensive spectroscopic analysis (UV, IR, MS, 1D- and 2D-NMR) and comparison with literature data. The isolated compounds were evaluated for their inhibitory effects on TNF-α-induced NF-κB activation, and all compounds exhibited NF-κB inhibitory activities with IC50 values ranging from 1.91 to 20.15 µM.

Cryptopleurine targets NF-κB pathway, leading to inhibition of gene products associated with cell survival, proliferation, invasion, and angiogenesis.

BACKGROUND: Cryptopleurine, a phenanthroquinolizidine alkaloid, was known to exhibit anticancer activity; however, the underlying mechanism is poorly understood. Because the nuclear factor-κB (NF-κB) transcription factors control many physiological processes including inflammation, immunity, and development and progression of cancer, we investigated the effects of cryptopleurine on tumor necrosis factor alpha (TNF-α)-induced NF-κB activation pathway and on the expression of NF-κB-regulated gene products associated with many pathophysiological processes. METHODOLOGY AND PRINCIPAL FINDING: MDA-MB231, MDA-MB435, MCF-7, HEK293, RAW264.7 and Hep3B cells were used to examine cryptopleurine's effect on the NF-κB activation pathway. Major assays were promoter-reporter gene assay, electrophoretic mobility shift assay (EMSA), in vitro immune complex kinase assay, real-time PCR, Western blot analysis, and Matrigel invasion assay. Experiments documenting cell proliferation and apoptosis were analyzed by MTT method and flow cytometry, respectively. The results indicated that cryptopleurine suppressed the NF-κB activation through the inhibition of IκB kinase (IKK) activation, thereby blocking the phosphorylation and degradation of the inhibitor of NF-κB alpha (IκBα) and the nuclear translocation and DNA-binding activity of p65. The suppression of NF-κB by cryptopleurine led to the down-regulation of gene products involved in inflammation, cell survival, proliferation, invasion, and angiogenesis. CONCLUSIONS AND SIGNIFICANCE: Our results show that cryptopleurine inhibited NF-κB activation pathway, which leads to inhibition of inflammation, proliferation, and invasion, as well as potentiation of apoptosis. Our findings provide a new insight into the molecular mechanisms and a potential application of cryptopleurine for inflammatory diseases as well as certain cancers associated with abnormal NF-κB activation.

Effects of astilbic acid on airway hyperresponsiveness and inflammation in a mouse model of allergic asthma.

Bronchial asthma is characterized by chronic lung inflammation, airway hyperresponsiveness (AHR), and airway remodeling. Astilbic acid, extracted from the medicinal herb Astilbe chinensis, is used as a headache remedy in traditional medicine and has anti-pyretic and analgesic effects. However, the effect of astilbic acid on asthma remains to be established. In the present study, we therefore examined the effect of astilbic acid in a mouse model in which asthma was established by sensitization and challenge with ovalbumin (OVA). Astilbic acid inhibited OVA-induced AHR to inhaled methacholine and significantly suppressed the levels of T-helper 2-type cytokines (including IL [interleukin]-4, IL-5, and IL-13) and inflammatory cells (including eosinophils) in bronchoalveolar lavage (BAL) fluid. Histochemical analysis revealed reduced goblet cell hyperplasia and mucus production, as well as attenuated eosinophil-rich leukocyte infiltration, in the astilbic acid-treated group, compared with OVA-challenged mice. Moreover, the compound significantly inhibited synthesis of IL-4-, IL-5-, IL-13-, IL-17-, and eotaxin-encoding mRNA following asthma induction in lung tissue, in addition to suppressing the immunoglobulin E (IgE) response to asthma in both BAL fluid and serum. Our results indicate that astilbic acid has great potential as a therapeutic candidate for the treatment of asthma.

Hepatoprotective effects of Sedum sarmentosum on D-galactosamine/lipopolysaccharide-induced murine fulminant hepatic failure.

The hepatoprotective effects of sarmentosin-containing extracts of Sedum sarmentosum (SS) in D-galactosamine (D-GalN) / lipopolysaccharide (LPS)-induced fulminant hepatic failure mouse model. Pretreatment with SS markedly protected mice from lethal liver injury, which has known to be associated with an abrupt elevation of serum tumor necrosis factor (TNF)-α level. Indeed, SS significantly blocked the elevation of TNF-α and alanine aminotransferase and aspartate aminotransferase as well. SS also remarkably reduced number of apoptotic hepatocytes and DNA fragmentation in the liver, which correlated with blockade of caspase-3 activation. In addition, SS suppressed the increased expression of toll-like receptor 4 (TLR4). The activation of c-Jun NH(2)-terminal kinase, extracellular signal-regulated kinase, and p38 induced by D-GalN/LPS was also significantly suppressed by SS treatment. Furthermore, SS significantly inhibited the activation of nuclear factor-κB. In RAW 264.7 cells stimulated with LPS, TNF-α release and TLR4 expression was suppressed by SS pretreatment, which was in line with in vivo results. These findings suggested that SS prevents D-GalN/LPS-induced fulminant hepatic failure, and this protection is likely associated with its anti-apoptotic activity and the down-regulation of mitogen activated protein kinase activity associated at least in part with suppressing the transcription of LPS receptors.

Attenuation of experimental murine colitis by acanthoic acid from Acanthopanax koreanum.

Acanthoic acid (AA) is a pimaradiene diterpene isolated from Acanthopanax koreanum. We examined the effect of AA in dextran sulfate sodium (DSS)-induced colitis. AA (100 mg/kg or 300 mg/kg) was administered p.o. daily for 7 days. AA significantly inhibited Disease Activity Index, histological score, and myeloperoxidase activity. Furthermore, AA markedly suppressed the protein expression of TNF-alpha, COX-2, NF-kappaB and chymase as well as the mRNA expression of TNF-alpha and COX-2. These results suggest that AA exerts beneficial effects in experimental colitis, and therefore we propose that this compound may have therapeutic implications for ulcerative colitis.

Oleanane-type triterpenoids from Aceriphyllum rossii and their cytotoxic activity.

The hexane-soluble fraction of the roots of Aceriphyllum rossii was used to isolate seven new oleanane-type triterpenoids, aceriphyllic acids C-I (1-7), together with seven known triterpenoids. The structures of aceriphyllic acids C-I were determined as 3alpha-hydroxyolean-12-en-23,29-dioic acid (1), 3beta-hydroxyolean-12-en-23,29-dioic acid (2), 3beta,23-dihydroxyolean-12-en-29-oic acid (3), 3alpha-O-acetylolean-12-en-23,27-dioic acid (4), 3alpha-O-caffeoylolean-12-en-27-oic acid (5), 3alpha-O-acetylolean-12-en-23,29-dioic acid (6), and 3alpha-hydroxyolean-12-en-23-al-27-oic acid (7) by spectroscopic analyses. In the evaluation of the in vitro cytotoxicity of these compounds against the MCF-7 and LLC cancer cell lines, compounds 10 and 13 exhibited cytotoxic activity against the LLC cancer cell line with IC(50) values of 7.63 and 6.56 microM, respectively.

Cytotoxic triterpenoids from the rhizomes of Astilbe chinensis.

Six new triterpenoids (1-6) with a carboxylic acid functionality at C-27 were isolated from the rhizomes of a Korean native perennial herb, Astilbe chinensis, along with nine known triterpenoids. The structures of 1-6 were elucidated on the basis of spectroscopic data interpretation. All compounds isolated were evaluated for cytotoxic effects against a small panel of human cancer lines.

Bisacurone inhibits adhesion of inflammatory monocytes or cancer cells to endothelial cells through down-regulation of VCAM-1 expression.

Bisacurone, one of the active compounds of the traditionally used indigenous herb Curcuma longa Linne (Zingiberaceae), has anti-oxidant, anti-inflammatory, and anti-metastatic activities. We studied how the level of vascular cell adhesion molecule-1 (VCAM-1), one of the key molecules in the development of atherosclerosis as well as carcinogenesis and metastasis, might be affected by bisacurone in tumor necrosis factor-alpha (TNF-alpha)-activated human umbilical vein endothelial cells (HUVECs). Bisacurone dose-dependently inhibited TNF-alpha-mediated expression of VCAM-1. It showed significant suppressive effect on ROS generation in response to TNF-alpha stimulation and it blocked nuclear factor-kappa B (NF-kappaB) p65 translocation into the nucleus and phosphorylation of inhibitory factor kappaBalpha (IkappaBalpha). It also inhibited phosphorylation of Akt and PKC, which are upstream in the regulation of VCAM-1 by TNF-alpha. Furthermore, bisacurone decreased U937 monocyte and human oral cancer cell (Hep-2, QLL-I, SCC-15) adhesion to HUVECs stimulated by TNF-alpha, suggesting that it may inhibit the binding of these cells by regulating the expression of critical adhesion molecules by TNF-alpha. Thus, bisacurone may be beneficial in the treatment of inflammatory diseases, such as atherosclerosis, where inflammatory monocytes are involved in their pathology, and, moreover, in the development of tumors.

Comparison of suppressive effects of demethoxycurcumin and bisdemethoxycurcumin on expressions of inflammatory mediators in vitro and in vivo.

Demethoxycurcumin and bisdemethoxycurcumin are the main active ingredients isolated from Curcumae Longae Radix. Recent studies demonstrated that both compounds exhibit antioxidative and anti-inflammatory effects as well as effects on cancer cell lines. In this study, we compared the activities of demethoxycurcumin and bisdemethoxycurcumin, and both compounds were evaluated on lipopolysaccharide (LPS)-induced nitric oxide (NO) production, inducible nitric oxide synthase (iNOS), cycloxygenase-2 (COX-2) and nuclear factor-kappaB (NF-kappaB) activity in a RAW 264.7 macrophage cell line. The evaluation:results suggested that the anti-inflammatory properties of demethoxycurcumin and bisdemethoxycurcumin were attributed to the inhibition of iNOS and COX-2 expression, as initiated by the inhibition of NF-kappaB activity. Additionally, both of them significantly inhibited carrageenan-induced paw edema in mice. Taken together, all of the results showed that the suppressive effect of demethoxycurcumin was stronger than that of bisdemethoxycurcumin, indicating that the methoxy group had enhanced demethoxycurcumin's anti-inflammation effects.

A diterpenoid acanthoic acid from Acanthopanax koreanum protects against D-galactosamine/lipopolysaccharide-induced fulminant hepatic failure in mice.

The hepatoprotective effects of a diterpenoid acanthoic acid isolated from Acanthopanax koreanum NAKAI were evaluated in a D-galactosamine/lipopolysaccharide-induced fulminant hepatic failure mouse model. Mice were pretreated orally with acanthoic acid 12 and 1 h before intraperitoneal injection of D-galactosamine and lipopolysaccharide. Pretreatment with the compound markedly reduced lethal liver injury in experimental animals. The effects were likely associated with a significant decrease in serum tumor necrosis factor (TNF)-alpha levels, which are correlated not only with those of alanine aminotransferase and aspartate aminotransferase but also with the reduced number of apoptotic hepatocytes in the liver as confirmed using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling method and DNA fragmentation assay. These results suggest that acanthoic acid protects against D-galactosamine/lipopolysaccharide-induced fulminant liver failure at least in part by a mechanism associated with the down-regulation of TNF-alpha secretion.

Hypoxia-inducible factor-1 and nuclear factor-kappaB inhibitory meroterpene analogues of bakuchiol, a constituent of the seeds of Psoralea corylifolia.

Two new meroterpenoids, 12,13-dihydro-12,13-dihydroxybakuchiol (2) and (12'S)-bisbakuchiol C (3), were isolated from the seeds of Psoralea corylifolia L. (Fabaceae). The structures of 2 and 3 were elucidated by spectroscopic and chemical methods. Six meroterpenoids isolated from P. corylifolia and three semi-synthetic analogues were evaluated for HIF-1 and NF-kappaB inhibition, and O-methyl and O-ethylbakuchiols (6 and 7) inhibited HIF-1 and NF-kappaB activation without significantly decreasing the viability of the AGS and HeLa cells, respectively.

Triterpenoids and diarylheptanoids from Alnus hirsuta inhibit HIF-1 in AGS cells.

Bioassay-guided fractionation and purification of the EtOAc soluble fraction from the MeOH extract of the stem bark of Alnus hirsuta (Betulaceae), using an in vitro HIF-1 assay, led to the isolation of four triterpenoids (1-4) and six diarylheptanoids (5-10). Their structures were determined by comparison with the physicochemical and spectroscopic data in the literature. These compounds were investigated for their effects on the hypoxia-induced HIF-1 activation using an HIF-1a mediated reporter gene assay in AGS cells. Among them, two diarylheptanoids, 2-oxatrycyclo[13.2.2.13,7]eicosa-3,5,7(20),15,17,18-hexaen-10-16-diol (6) and 2-oxatrycyclo [13.2.2.13,7]eicosa-3,5,7-(20),15,17,18-hexaen-10-one (7), inhibited HIF-1 activation dose-dependently with IC50 values of 11.2 microM and 12.3 microM, respectively. These two compounds had no significant cytotoxicity to the AGS cells at the effective concentration for the inhibition of HIF-1 activation.

Diarylheptanoids from Alnus hirsuta inhibit the NF-kB activation and NO and TNF-alpha production.

Six diarylheptanoids (1-6) from the stem bark of Alnus hirsuta were investigated for their inhibitory activity against LPS-induced NF-kB activation and NO and TNF-alpha production. Among them, compounds 2, 3, and 6 displayed inhibitory activity against NF-kB activation and NO and TNF-alpha production with IC50 values of 9.2-9.9 microM, 18.2-19.3 microM, and 22.3-23.7 microM, respectively, in RAW264.7 cells. Three active compounds had no significant cytotoxicity in RAW264.7 cells at their effective concentrations. This is the first report of NF-kB-inhibitory activity of these compounds and supports the pharmacological use of A. hirsuta, which has been employed as a herbal medicine for the treatment of inflammatory diseases.

In vitro and in vivo antiallergic effect of the fructus of Evodia rutaecarpa and its constituents.

The unripe fruit of Evodia rutaecarpa (JUSS) BENTH (ER, Family Rutaceae) has been used frequently as a traditional medicine against inflammatory diseases in Korea, China and Japan. To evaluate antiallergic effect of ER, we isolated its main constituents, evodiamine and rutaecarpine, and evaluated in vivo their inhibitory effects against passive cutaneous anaphylaxis (PCA) reaction induced by IgE-antigen complex and scratching behaviors by compound 48/80. ER and its constituents, evodiamine and rutaecarpine, potently inhibited PCA reaction and scratching behaviors in mice, although ER weakly inhibited scratching behaviors. Evodiamine and rutaecarpine inhibited TNF-alpha and IL-4 protein expression in RBL-2H3 cells induced by IgE-antigen complex, although these did not inhibit degranulation of RBL-2H3 cells induced by IgE-antigen complex and rat peritoneal mast cells induced by compound 48/80. These findings suggest that ER and its constituents, evodiamine and rutaecarpine, may be effective for IgE-induced allergic diseases such as atopic dermatitis and rhinitis.

Phenanthroquinolizidine alkaloids from the roots of Boehmeria pannosa potently inhibit hypoxia-inducible factor-1 in AGS human gastric cancer cells.

A bioassay-guided phytochemical investigation on the methanol extract of Boehmeria pannosa, using a HIF-1-mediated reporter gene assay, led to the isolation of two phenanthroquinolizidine alkaloids, (-)-cryptopleurine (1) and (-)-(15R)-hydroxycryptopleurine (2). The structure of the new compound 2 was determined by spectroscopic methods. Compounds 1 and 2 potently inhibited the hypoxia-induced expression of a reporter gene under the control of a hypoxia response element (HRE) with IC(50) values of 8.7 and 48.1 nM, respectively. Furthermore, 1 and 2 suppressed the accumulation of HIF-1alpha protein in a dose-dependent manner, but not the HIF-1beta protein and inhibited expression of vascular endothelial growth factor (VEGF) by hypoxia.

Anti-inflammatory principles from the fruits of Evodia rutaecarpa and their cellular action mechanisms.

The fruits of Evodia rutaecarpa Benth (Rutaceae) has long been used for inflammatory disorders and some anti-inflammatory actions of its constituents such as dehydroevodiamine, evodiamine and rutaecarpine were previously reported. Since the pharmacological data is not sufficient to clearly establish the scientific rationale of anti-inflammatory medicinal use of this plant material and the search for its active principles is limited so far, three major constituents (evodiamine, rutaecarpine, goshuyuamide II) were evaluated for their anti-inflammatory cellular action mechanisms in the present study. From the results, evodiamine and rutaecarpine were found to strongly inhibit prostaglandin E2 synthesis from lipopolysaccharide-treated RAW 264.7 cells at 1-10 microM. Evodiamine inhibited cyclooxygenase-2 induction and NF-kappaB activation, while rutaecarpine did not. On the other hand, goshuyuamide II inhibited 5-lipoxygenase from RBL-1 cells (IC50 = 6.6 microM), resulting in the reduced synthesis of leukotrienes. However, these three compounds were not inhibitory against inducible nitric oxide synthase-mediated nitric oxide production from RAW cells up to 50 micorM. These pharmacological properties may provide the additional scientific rationale for anti-inflammatory use of the fruits of E. rutaecarpa.

Inhibition of protein tyrosine phosphatase 1B by diterpenoids isolated from Acanthopanax koreanum.

Inhibition of protein tyrosine phosphatase 1B (PTP1B) has been proposed as a therapy to treat type 2 diabetes and obesity. In our preliminary screening study on the PTP1B inhibitory activity, a CH2Cl2-soluble extract of the roots of Acanthopanax koreanum (Araliaceae) was found to inhibit PTP1B activity at 30 microg/ml. Eight diterpenoids were isolated from the active fraction and were evaluated for their inhibitory effect on PTP1B. A kaurane-type diterpene, 16alphaH,17-isovaleryloxy-ent-kauran-19-oic acid (7), inhibited PTP1B with an IC50 value of 7.1+/-0.9 microM in a non-competitive manner. Acanthoic acid (2) and ent-kaur-16-en-19-oic acid (5) also inhibited PTP1B in dose-dependent manners. Either introduction of a hydroxyl group or reduction of a carboxyl group at C-19 in pimarane-type to alcohol abolished the inhibitory effects toward PTP1B.

Inhibition of trypsin-induced mast cell activation by acanthoic acid.

Acanthoic acid (AA) is a pimaradiene diterpene isolated from the Korean medicinal plant, Acanthopanax koreanum (Araliaceae). In the present study, we examined whether AA has the inhibitory effect on the production of inflammatory mediators and activating signals induced in trypsin-treated human leukemic mast cell-1 (HMC-1). HMC-1 cells were stimulated with trypsin (100 nM) in the presence or absence of AA (1, 10, and 100 microg/ml). We assessed the production of TNF-alpha and tryptase by enzyme-linked immunosorbent assay (ELISA) or reverse transcription-PCR, ERK activation by Western blot, and NF-kappaB activation by gel shift assay. AA (10 and 100 microg/ml) significantly inhibited production of both TNF-alpha and tryptase in a dose-dependent manner in trypsin-stimulated HMC-1. Furthermore, AA inhibited ERK phosphorylation and NF-kappaB activation induced by trypsin treatment without blocking of trypsin activity even with 100 microg/ml. These results suggest that AA may inhibit the production of inflammatory mediators through inhibition of ERK phosphorylation and NF-kappaB activation pathway in human mast cells. It supports the evidence that AA may be used to blocks the development of inflammation caused from mast cells.