Inhibition of TNF-alpha synthesis in LPS-stimulated primary human monocytes by Harpagophytum extract SteiHap 69.

Harpagophytum procumbens (Devil's Claw) is often used in the supportive treatment of inflammatory and degenerative diseases of the skeletal system. Here we studied the anti-inflammatory properties of the Harpagophytum extract SteiHap 69 (Steiner Harpagophytum procumbens extract 69) on primary human monocytes, a useful model of peripheral inflammation. After eliminating lipopolysaccharides of bacterial origin, SteiHap 69 prevented the LPS-induced synthesis of tumour necrosis factor alpha (TNFalpha) in stimulated primary human monocytes in a dose-dependent manner. Harpagide and harpagoside had no effect on LPS-induced TNFalpha-release. Our data provides evidence that the Harpagophytum extract SteiHap 69 has anti-inflammatory properties. Further studies are required in order to elucidate the molecular mechanism of Devil's claw anti-inflammatory effects.     

High anti-inflammatory activity of harpagoside-enriched extracts obtained from solvent-modified super- and subcritical carbon dioxide extractions of the roots of Harpagophytum procumbens

Solvent-modified carbon dioxide extractions of the roots of Harpagophytum procumbens have been investigated with respect to extraction efficiency and content of harpagoside, and compared with a conventional extract. The effects of pressure, temperature, type and concentration of the modifier have been examined. Two extraction steps were necessary in order to achievehigh anti-inflammatory harpagoside-enriched extracts. The first extraction step was carried out in the supercritical state using carbon dioxide modified with n-propanol to remove undesired lipophilic substances. The main extraction was performed either in the supercritical or in the subcritical state with carbon dioxide modified with ethanol. The supercritical fluid extraction resulted in extracts containing up to 30% harpagoside. The subcritical extracts showed a harpagoside content of ca. 20%, but the extraction yield was nearly three times greater compared with supercritical conditions. The total harpagoside recovery resulting from the sum of the extract and the crude drug residue was greater than 99% in all experiments. The conventional extract and two carbon dioxide extracts were tested for in-vitro inhibition of 5-lipoxygenase or cyclooxygenase-2 biosynthesis. Both carbon dioxide extracts showed total inhibition on 5-lipoxygenase biosynthesis at a concentration of 51.8 mg/L. In contrast, the conventional extract failed to show any inhibition of 5-lipoxygenase biosynthesis.     

Physicochemical properties of harpagoside and its in vitro release from Harpagophytum procumbens extract tablets.

The objective of this investigation was to characterize the active-component harpagoside of Harpagophytum extract from a physico-chemical perspective and to determine its in-vitro release from tablets according to DAB 1996. It was found that both pure harpagoside and harpagoside in Harpagophytum extract have an octanol-water distribution coefficient of approximately 4 which is neither dependent on temperature nor on pH. The mean harpagoside content in Harpagophytum tablets of Batch 9102 was 16.4 mg (S.D. 0.2; S.E. 0.03). Related to a tablet weight of 365 mg (100%), this corresponds to a haragoside content of 4.5% (S.D. 0.049; S.E. 0.006). On average the tablets disintegrate after 18 +/- 3 minutes (mean +/- SD). The tablets taken from Batch 9102 released the active component harpagoside well, with a t50 of 13.5 min, a t90 of 23 min and a t95 of 25 min in relation to 16.5 mg of harpagoside per dose. Harpagoside content decreased by about 10% in artificial gastric fluid within a period of 3 hours and remained stable in artificial intestinal fluid for a period of 6 hours.     

Production of Iridoids and Phenolics by Transformed Harpagophytum procumbens Root Cultures

Harpagophytum procumbens (Devil's claw) is an important medicinal plant, which tubers are used for the treatment of different inflammatory diseases. In this study, the time courses of growth of Devil's claw hairy roots and accumulation of intracellular total iridoids and phenolics were investigated during cultivation under submerged conditions. After 21 days of growth in liquid hormone‐free MS medium, a growth index of 81.3 was achieved and the accumulated biomass reached 0.58 g/flask. It was found that the time courses of total iridoids and phenolics production showed almost the same patterns with a maximum on day 21 from the beginning of cultivation (15.93 mg harpagoside equivalents/L and 261 mg gallic acid equivalents/L, respectively). The methanolic extracts possessed relatively high DPPH‐radical scavenging properties (IC₅₀ 4.08 mg/mL), while for the culture media no antiradical activity was detected. According to our best knowledge, the present report is the first one dealing with the investigation of transformed root cultures from Harpagophytum procumbens plants.     

RANKL stimulates inducible nitric-oxide synthase expression and nitric oxide production in developing osteoclasts. An autocrine negative feedback mechanism triggered by RANKL-induced interferon-beta via NF-kappaB that restrains osteoclastogenesis and bone resorption

Nitric oxide (NO) is a multifunctional signaling molecule and a key vasculoprotective and potential osteoprotective factor. NO regulates normal bone remodeling and pathological bone loss in part through affecting the recruitment, formation, and activity of bone-resorbing osteoclasts. Using murine RAW 264.7 and primary bone marrow cells or osteoclasts formed from them by receptor activator of NF-kappaB ligand (RANKL) differentiation, we found that inducible nitric-oxide synthase (iNOS) expression and NO generation were stimulated by interferon (IFN)-gamma or lipopolysaccharide, but not by interleukin-1 or tumor necrosis factor-alpha. Surprisingly, iNOS expression and NO release were also triggered by RANKL. This response was time- and dose-dependent, required NF-kappaB activation and new protein synthesis, and was specifically blocked by the RANKL decoy receptor osteoprotegerin. Preventing RANKL-induced NO (via iNOS-selective inhibition or use of marrow cells from iNOS-/- mice) increased osteoclast formation and bone pit resorption, indicating that such NO normally restrains RANKL-mediated osteoclastogenesis. Additional studies suggested that RANKL-induced NO inhibition of osteoclast formation does not occur via NO activation of a cGMP pathway. Because IFN-beta is also a RANKL-induced autocrine negative feedback inhibitor that limits osteoclastogenesis, we investigated whether IFN-beta is involved in this novel RANKL/iNOS/NO autoregulatory pathway. IFN-beta was induced by RANKL and stimulated iNOS expression and NO release, and a neutralizing antibody to IFN-beta inhibited iNOS/NO elevation in response to RANKL, thereby enhancing osteoclast formation. Thus, RANKL-induced IFN-beta triggers iNOS/NO as an important negative feedback signal during osteoclastogenesis. Specifically targeting this novel autoregulatory pathway may provide new therapeutic approaches to combat various osteolytic bone diseases.     

Involvement of protein kinase C in the inhibition of lipopolysaccharide-induced nitric oxide production by thapsigargin in RAW 264.7 macrophages

This study explored the effects of inhibition of endoplasmic reticulum (ER) Ca²⁺-ATPase on lipopolysaccharide (LPS)-induced protein kinase C (PKC) activation, nuclear factor-κB (NF-κB) translocation, inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in RAW 264.7 macrophages. Thapsigargin (TG) irreversibly inhibits ER Ca²⁺-ATPase and LPS-induced NO production is reduced even after washout. TG also attenuated LPS-stimulated iNOS expression by using immunoblot analysis. However, another distinct fully reversible ER Ca²⁺-ATPase inhibitor, 2,5-di-tert-butylhydroquinone (DBHQ), ionophore A23187 and ionomycin could exert a similar effect to TG in increasing intracellular calcium concentration; however, these agents could not mimic TG in reducing iNOS expression and NO production. LPS increased PKC- and -β activation, and TG pretreatment attenuated LPS-stimulated PKC activation. Not did pretreatment with DBHQ, A23187 and ionomycin reduce LPS-stimulated PKC activation. Furthermore, NF-κB-specific DNA–protein-binding activity in the nuclear extracts was enhanced by treatment with LPS, and TG pretreatment attenuated LPS-stimulated NF-κB activation. None of DBHQ, A23187 and ionomycin pretreatment reduced LPS-stimulated NF-κB activation. These data suggest that persistent inhibition of ER Ca²⁺-ATPase by TG would influence calcium release from ER Ca²⁺ pools that was stimulated by the LPS activated signal processes, and might be the main mechanism for attenuating PKC and NF-κB activation that induces iNOS expression and NO production.     

Methanol extracts of Stewartia koreana inhibit cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) gene expression by blocking NF-kappaB transactivation in LPS-activated RAW 264.7 cells

Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) are involved in various pathophysiological processes such as inflammation and carcinogenesis. In a search for inhibitors of COX-2 and iNOS production we found that extracts of Stewartia koreana strongly inhibited NO and PGE2 production in LPS-treated macrophage RAW 264.7 cells. We have now shown that the mRNA and protein levels of iNOS and COX-2 are reduced by the Stewartia koreana extract (SKE). SKE inhibited expression of an NF-kappaB reporter gene in response to LPS, and gel mobility shift assays revealed that SKE reduced NF-kappaB DNA-binding activity. The extract also inhibited LPS-induced phosphorylation of IkappaB-alpha and nuclear translocation of p65. Administration of the extract reduced the symptoms of arthritis in a collagen-induced arthritic mouse model. These results indicate that Stewartia extracts contain potentially useful agents for preventing and treating inflammatory diseases.     

Comparative phytochemical analysis of wild and in vitro-derived greenhouse-grown tubers, in vitro shoots and callus-like basal tissues of Harpagophytum procumbens

Comparative phytochemical analysis of wild and in vitro-derived greenhouse-grown tubers, in vitro shoots and callus-like basal tissues of Harpagophytum procumbens was done. Dried samples were ground to fine powders and their total iridoid (colorimetric method), phenolic [Folin-Ciocalteu (Folin C) method] and gallotannin (Rhodanine assay) contents as well as anti-inflammatory activity [cyclooxygenase assays (COX-1 and COX-2)] were determined. The tissue culture-derived tubers had the highest total iridoid content which was significantly higher than that of the tubers collected from the wild and other tissue cultured materials evaluated. This suggests that cultivated plants can be a viable alternative source of the active principle(s). The total phenolic and gallotannin contents of the wild tubers were significantly higher than the tissue culture-derived tubers and other in vitro-derived plant materials. The presence of phenolic compounds including gallotannins in the tissue cultured materials is of interest from a pharmacological point of view given the pharmacological role of phenolics. In general, extracts from wild tubers demonstrated better inhibitory activities in both COX-1 and COX-2 assays when compared to the tissue culture-derived tubers. All the petroleum ether (PE) and dichloromethane (DCM) extracts showed moderate (50-70%) to good (> 70%) inhibitory activities whereas the ethanol (EtOH) extracts showed poor or no inhibition in both assays. Based on previous reports indicating weak inhibition of COX-2 enzyme by harpagoside, the inhibitory activities of both COX enzymes exhibited by PE and DCM extracts in the current study could be due to the presence of other constituents in the extracts. This points towards the need to identify other active constituents and evaluate their role and mode of action in relation to harpagoside — the main active principle.     

Inducible nitric oxide synthase aggresome formation is mediated by nitric oxide

Nitric oxide (NO) generated by inducible NO synthase (iNOS) contributes critically to inflammatory injury and host defense. While previously thought as a soluble protein, iNOS was recently reported to form aggresomes inside cells. But what causes iNOS aggresome formation is unknown. Here we provide evidence demonstrating that iNOS aggresome formation is mediated by its own product NO. Exposure to inflammatory stimuli (lipopolysaccharide and interferon-γ) induced robust iNOS expression in mouse macrophages. While initially existing as a soluble protein, iNOS progressively formed protein aggregates as a function of time. Aggregated iNOS was inactive. Treating the cells with the NOS inhibitor N-nitro-l-arginine methyl ester (L-NAME) blocked NO production from iNOS without affecting iNOS expression. However, iNOS aggregation in cells was prevented by L-NAME. The preventing effect of NO blockade on iNOS aggresome formation was directly observed in GFP-iNOS-transfected cells by fluorescence imaging. Moreover, iNOS aggresome formation could be recaptured by adding exogenous NO to L-NAME-treated cells. These studies demonstrate that iNOS aggresome formation is caused by NO. The finding that NO induces iNOS aggregation and inactivation suggests aggresome formation as a feedback inhibition mechanism in iNOS regulation.     

β-Glucan from Lentinus edodes inhibits nitric oxide and tumor necrosis factor-α production and phosphorylation of mitogen-activated protein kinases in lipopolysaccharide-stimulated murine RAW 264.7 macrophages

Lentinan (LNT), a β-glucan from the fruiting bodies of Lentinus edodes, is well known to have immunomodulatory activity. NO and TNF-α are associated with many inflammatory diseases. In this study, we investigated the effects of LNT extracted by sonication (LNT-S) on the NO and TNF-α production in LPS-stimulated murine RAW 264.7 macrophages. The results suggested that treatment with LNT-S not only resulted in the striking inhibition of TNF-α and NO production in LPS-activated macrophage RAW 264.7 cells, but also the protein expression of inducible NOS (iNOS) and the gene expression of iNOS mRNA and TNF-α mRNA. It is surprising that LNT-S enhanced LPS-induced NF-κB p65 nuclear translocation and NF-κB luciferase activity, but severely inhibited the phosphorylation of JNK1/2 and ERK1/2. The neutralizing antibodies of anti-Dectin-1 and anti-TLR2 hardly affected the inhibition of NO production. All of these results suggested that the suppression of LPS-induced NO and TNF-α production was at least partially attributable to the inhibition of JNK1/2 and ERK1/2 activation. This work discovered a promising molecule to control the diseases associated with overproduction of NO and TNF-α.