Rafael A. Burgos

Schisandra chinensis (Turcz.) Baill

Different aspects of the pharmacology of Schisandra chinensis fruit and dibenzo- wx a,c cyclooctene lignans from this plant are reviewed focusing in particular on the antihepa- totoxic, antioxidant and antitumoural activities, and on the effects on physical performance and on the central nervous system. Q 1999 Elsevier Science B.V. All rights reserved.

Andrographolide interferes with binding of nuclear factor-κB to DNA in HL-60-derived neutrophilic cells

1 Andrographolide, the major active component from Andrographis paniculata, has shown to possess anti‐inflammatory activity. Andrographolide inhibits the expression of several proinflammatory proteins that exhibit a nuclear factor kappa B (NF‐κB) binding site in their gene. 2 In the present study, we analyzed the effect of andrographolide on the activation of NF‐κB induced by platelet‐activating factor (PAF) and N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP) in HL‐60 cells differentiated to neutrophils. 3 PAF (100 nM) and fMLP (100 nM) induced activation of NF‐κB as determined by degradation of inhibitory factor B α (IκBα) using Western blotting in cytosolic extracts and by binding to DNA using electrophoretic mobility shift assay (EMSA) in nuclear extracts. 4 Andrographolide (5 and 50 μM) inhibited the NF‐κB‐luciferase activity induced by PAF. However, andrographolide did not reduce phosphorylation of p38 MAPK or ERK1/2 and did not change IκBα degradation induced by PAF and fMLP. 5 Andrographolide reduced the DNA binding of NF‐κB in whole cells and in nuclear extracts induced by PAF and fMLP. 6 Andrographolide reduced cyclooxygenase‐2 (COX‐2) expression induced by PAF and fMLP in HL‐60/neutrophils. 7 It is concluded that andrographolide exerts its anti‐inflammatory effects by inhibiting NF‐κB binding to DNA, and thus reducing the expression of proinflammatory proteins, such as COX‐2.

Use of visual analogue scale measurements (VAS) to asses the effectiveness of standardized Andrographis paniculata extract SHA-10 in reducing the symptoms of common cold. A randomized double blind-placebo study

The objective of our study was to measure the effectiveness of Andrographis paniculata SHA-10 extract in reducing the prevalence and intensity of symptoms and signs of common cold as compared with a placebo. A group of 158 adult patients of both sexes completed the randomized double blind study in Valdivia, Chile. The patients were divided in two equal size groups, one of which received Andrographis paniculata dried extract (1200 mg/day) and the other a placebo during a period of 5 days. Evaluations for efficacy were performed by the patient at day 0, 2, and 4 of the treatment; each completed a self-evaluation (VAS) sheet with the following parameters: headache, tiredness, earache, sleeplessness, sore throat, nasal secretion, phlegm, frequency and intensity of cough. In order to quantify the magnitude of the reduction in the prevalence and intensity of the signs and symptoms of common cold, the risk (Odds Ratio = OR) was calculated using a logistic regression model. At day 2 of treatment a significant decrease in the intensity of the symptoms of tiredness (OR = 1.28; 95% CI 1.07-1.53), sleeplessness (OR = 1.71; 95% CI 1.38-2.11), sore throat (OR = 2.3; 95% CI 1.69-3.14) and nasal secretion (OR = 2.51; 95% CI 1.82-3.46) was observed in the Andrographis SHA-10 group as compared with the placebo group. At day 4, a significant decrease in the intensity of all symptoms was observed for the Andrographis paniculata group. The higher OR values were for the following parameters: sore throat (OR = 3.59; 95% CI 2.04-5.35), nasal secretion (OR = 3.27; 95% CI 2.31-4.62) and earache (OR = 3.11; 95% CI 2.01-4.80) for Andrographis paniculata treatment over placebo, respectively. It is concluded that Andrographis paniculata had a high degree of effectiveness in reducing the prevalence and intensity of the symptoms in uncomplicated common cold beginning at day two of treatment. No adverse effects were observed or reported.

Activation of kinin B1 receptors induces chemotaxis of human neutrophils

Kinins are biologically active peptides that are powerful mediators of cellular inflammation. They mimic the cardinal signs of inflammation by inducing vasodilatation and by increasing vascular permeability and pain. Neutrophils are chemoattracted to sites of inflammation by several stimuli. However, the evidence concerning the chemotactic effect of kinin peptides has been contradictory. We analyzed the chemotactic effect of kinin B1 receptor agonists on neutrophils isolated from peripheral blood of human healthy subjects. Chemotaxis was performed using the migration under agarose technique. To test the effect of B1 receptor agonists, each assay was carried out overnight at 37°C in 5% CO2‐95% air on neutrophils primed with 1 ng/ml interleukin‐1β. Simultaneous experiments were performed using unprimed cells or cells challenged with formyl‐Met‐Leu‐Phe (fMLP). A clear chemotactic activity was observed when primed neutrophils were challenged with Lys‐des[Arg9]‐bradykinin (LDBK) or des[Arg9]‐bradykinin at 10−10 M but not when unprimed cells were used. A reduction in the chemotactic response was observed after priming of cells in the presence of 0.5 mM cycloheximide and 10 μg/ml brefeldin A, suggesting that some protein biosynthesis is required. Techniques such as reverse transcriptase‐polymerase chain reaction and in situ hybridization confirmed the expression of the B1 receptor mRNA, and immunocytochemistry and autoradiography demonstrated the expression of the B1 receptor protein. In contrast to other chemoattractants such as fMLP, cytosolic intracellular calcium did not increase in response to the B1 receptor agonist LDBK. A generation of kinin B1 receptor agonists during the early phase of acute inflammation may favor the recruitment of neutrophils to the inflammatory site.

Testicular toxicity assesment of Andrographis paniculata dried extract in rats

The possible testicular toxicity of Andrographis paniculata, Nees (Acanthaceae) standardized dried extract was evaluated in male Sprague Dawley rats for 60 days. No testicular toxicity was found with the treatment of 20, 200 and 1000 mg/kg during 60 days as evaluated by reproductive organ weight, testicular histology, ultrastructural analysis of Leydig cells and testosterone levels after 60 days of treatment. It is concluded that Andrographis paniculata dried extract did not produce subchronic testicular toxicity effect in male rats.

Kinin B1 receptor activation turns on exocytosis of matrix metalloprotease-9 and myeloperoxidase in human neutrophils: involvement of mitogen-activated protein kinase family

During neutrophil activation and degranulation, MMP‐9 and MPO are released into the extracellular space to propagate inflammatory disorders. As kinin peptides are major participants in acute inflammatory responses, and the G‐protein‐coupled B1R mediates the chemotaxis of human neutrophils, we examined the release of the neutrophil enzymes MMP‐9 and MPO by the B1R agonist LDBK and determined the signaling pathways that may regulate this cellular effect. Cytochalasin‐treated and ‐untreated neutrophils were suspended in HBSS and stimulated with a range concentration of LDBK for 5 min. Zymography and Western blotting revealed that LDBK induced the release of MMP‐9 and MPO. The use of specific signaling transduction inhibitors showed that release of MMP‐9 depended on ERK1/2 and p38 MAPKs, whereas release of MPO involved only the p38 cascade. Inhibition of the key steps in these pathways showed that the release of both enzymes depended on PKC and PI3K. Stimulation of neutrophils with LDBK produced phosphorylation of ERK1/2 and p38 MAPK, which was inhibited by B1R antagonists. The phosphorylated ERK1/2 MAPK translocated to the neutrophil nucleus, suggesting that transcription of new genes may follow activation of B1R. Our results demonstrate that in human neutrophils, activation of kinin B1R by LDBK initiates separate signaling cascades that trigger the release of MMP‐9 and MPO from tertiary and primary granules, respectively, suggesting that the B1R plays a pivotal role in inflammatory disorders.

Stimulation of the bradykinin B1 receptor induces the proliferation of estrogen-sensitive breast cancer cells and activates the ERK1/2 signaling pathway

Kinin peptides exert multiple biological effects by binding to two types of G protein-coupled receptors known as B1 (B1R) and B2 receptors. Expression of the B1R in human breast cancer was recently reported, but up to now the consequences of its stimulation are unknown. Our aims were (1) to investigate the capacity of B1R to trigger cell proliferation in breast cancer cells, (2) to explore some of the downstream events occurring after B1R stimulation that may be linked to cell proliferation, and (3) to determine whether human breast tumors express potentially active B1R assessed by the binding of a radiolabeled agonist. Breast cancer cells expressed both the mRNA and the immunoreactive protein of B1R that once stimulated triggered cell proliferation at nanomolar concentrations of the ligand. Inhibitor studies suggested that the proliferative effects depend on the activity of epidermal growth factor receptor and subsequent ERK1/2 mitogen-activated protein kinases phosphorylation. B1R binding sites, were detected in 3/4 fibroadenomas, in 4/4 ductal carcinomas in situ and in 11/13 invasive ductal carcinomas. The B1R-epidermal growth factor receptor crosstalk may be a key interaction that maintains tumor growth, and antagonism of B1R may be a valuable alternative for the treatment of breast cancer.

Hypoglycemic effect of lupin seed γ-conglutin in experimental animals and healthy human subjects

A lupin seed γ-conglutin-enriched preparation was tested in a glucose overload trial with both murine models and adult healthy volunteers. The results with rats showed a dose-dependent significant decrease of blood glucose concentration, which confirmed previous findings obtained with the purified protein. Moreover, three test-product doses equivalent to 630, 315, and 157.5 mg γ-conglutin, orally administered 30 min before the carbohydrate supply, showed a relevant hypoglycemic effect in human trials. Insulin concentrations were not significantly affected. The general hematic parameters did not change at all. This is the first report on the glucose-lowering effect of lupin γ-conglutin in human subjects.

Activation of kinin B1 receptor increases the release of metalloproteases-2 and -9 from both estrogen-sensitive and -insensitive breast cancer cells.

The kinin B(1) receptor (B(1)R) agonist Lys-des[Arg(9)]-bradykinin (LDBK) increases proliferation of estrogen-sensitive breast cancer cells by a process involving activation of the epidermal growth factor receptor (EGFR) and downstream signaling via the ERK1/2 mitogen-activated protein kinase pathway. Here, we investigated whether B(1)R stimulation induced release of the extracellular matrix metalloproteases MMP-2 and MMP-9 via ERK-dependent pathway in both estrogen-sensitive MCF-7 and -insensitive MDA-MB-231 breast cancer cells. Cells were stimulated with 1-100nM of the B(1)R agonist for variable time-points. Western blotting and gelatin zymography were used to evaluate the presence of MMP-2 and MMP-9 in the extracellular medium. Stimulation of B(1)R with as little as 1 nM LDBK induced the accumulation of these metalloproteases in the medium within 5-30min of stimulation. In parallel, immunocytochemistry revealed that metalloprotease levels in the breast cancer cells declined after stimulation. This effect was blocked either by pre-treating the cells with a B(1)R antagonist or by transfecting with B(1)R-specific siRNA. Activation of the ERK1/2 pathway and EGFR transactivation was required for release of metalloproteases because both the MEK1 inhibitor, PD98059, and AG1478, an inhibitor of the EGFR-tyrosine kinase activity, blocked this event. The importance of EGFR-dependent signaling was additionally confirmed since transfection of cells with the dominant negative EGFR mutant HERCD533 blocked the release of metalloproteases. Thus, activation of B(1)R is likely to enhance breast cancer cells invasiveness by releasing enzymes that degrade the extracellular matrix and thereby favor metastasis.

Andrographolide reduces IL-2 production in T-cells by interfering with NFAT and MAPK activation

The nuclear factor of activated T cells (NFAT) is a transcription factor essential for cytokine production during T-cell activation and is the target of several immunosuppressive drugs. Andrographolide is a diterpenic labdane that possesses anti-inflammatory and immunomodulatory effects. Several studies propose that andrographolide can reduce the immune response through inhibition of the nuclear factor kappa B (NF-kappaB) and mitogen-activated protein kinases (MAPK) such as extracellular signal regulated kinase 1/2 (ERK1/2) pathways. Moreover, andrographolide reduces IFN-gamma and IL-2 production induced by concanavalin A in murine T-cell. Nevertheless, the mechanisms involved in the decrease of cytokine production are unknown. In the present study, we determined that andrographolide reduced IL-2 production in Jurkat cells stimulated with phorbol myristate acetate and ionomycin (PMA/Ionomycin). We then showed that andrographolide reduced NFAT luciferase activity and interfered with its nuclear distribution, with these effects being linked to an increase in c-jun-N-terminal kinase (JNK) phosphorylation. Additionally, reduction of NF-kappaB activity in Jurkat cells treated with andrographolide was observed. Using Western blotting, we demonstrated that andrographolide decreased ERK1 and ERK5 phosphorylation induced by anti-CD3 or PMA/Ionomycin. Andrographolide did not affect cell viability at concentration of 10 and 50 muM; however, our results suggest that andrographolide increase early apoptosis at 100 muM. We concluded that andrographolide can exert immunomodulatory effects by interfering with NFAT activation and ERK1 and ERK5 phosphorylation in T-cells.