Yolanda Casali

In Vivo Immunomodulatory Effects of Aqueous Extracts of Larrea divaricata Cav

Several medicinal plants are considered immunomodulatory as they display a variety of anti-inflammatory, antimicrobial and antitumoral effects. Larrea divaricata Cav. (jarilla) (Zygophyllaceae) is a plant widely used in popular medicine to treat tumors, infections, and inflammatory diseases. So far, the immunostimulating activities of Larrea divaricata have not been studied in vivo. In this work, we used healthy mice to assess the immunomodulatory potential of aqueous extracts of Larrea divaricata Cav. We found that Decoction (D) and Infusion (I) from Larrea divaricata Cav showed any acute hepatotoxic activity. Only D at 0.5 mg/kg increased the carrageenan-induced inflammation. Macrophages harvested from treated mice showed no signs of apoptosis. These cells showed a significant increase in NO and TNF-α release and exhibited the strongest expression of iNOS. Decoction also increased the phagocytosis of zymosan and the binding of LPS-FITC. The expression of CD14, TLR4 and CR3 was lower in macrophages of mice treated than in controls. Thus, Larrea divaricata was able to prime Mφ in vivo and to induce full activation in vitro. Our finding contribute to characterize the biological activity of Larrea divaricata and to understand the ability of these extracts to enhance immune responses.

Larrea divaricata Cav (Jarilla): Production of Superoxide Anion, Hydrogen Peroxide and Expression of Zymosan Receptors

Larrea divaricata is a plant widely used in folk medicine in Argentina. This work aimed to study the mechanisms of decoction activity on the release of oxygen reactive species. Decoction increased the binding of zymosan-FITC and superoxide production. Cadmium decreased the superoxide production as well as malonate and barbital. Decoction decreased the release of hydrogen peroxide. Decoction increased the reduction of MTT but not when malonate and barbital were included. Together, decoction increased the expression of dectin-1 leading to increased superoxide production. It is possible that decoction increases the activity of peroxidase, and decreases the Cu, Zn-superoxide dismutase.

In vitro immunomodulatory effects of fractions obtained from aqueous extracts of Larrea divaricata Cav (Jarilla) on mouse peritoneal macrophages.

Background and aim: Larrea divaricata Cav. (Zygophyllaceae) is a plant widely used in Argentina. Material and methods: We isolated different fractions of L. divaricata aqueous extract containing minor amounts of NDGA, and we analyzed these fractions on mouse macrophages. Results: We showed that a fraction without NDGA was capableof activating macrophages, principally through the production of mitochondrial anion superoxide and H2O2. This could be important in the defense of infections. Moreover, this fraction decreased NO level suggesting an anti-inflammatory action. Conclusion: These results indicate that NDGA was not the compound responsible for the immunomodulatory action exerted by the aqueous extract from L. divaricata.

In vivo effect of three fractions of Larrea divaricata Cav. (jarilla) on the innate immune system: macrophage response against Candida albicans.

Larrea divaricata Cav. (jarilla) is a plant with well‐documented applications in folk medicine in Argentina. In this study, we aimed to evaluate functional parameters of peritoneal macrophages isolated from mice injected with three fractions (F1, F2 and F3) of L. divaricata. The response of macrophages against Candida albicans was evaluated. Cell viability was assessed using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) test, apoptosis was evaluated using Giemsa, acridine orange/ethidium bromide and ladder assay, oxidative burst was assayed using nitroblue tetrazolium test and nitrite production using Griess assay. Cell stimulation and their ability to kill C. albicans in vitro were measured. The number and cell viability were similar to controls. However, we found that F1 induces pre‐activation of macrophages, and this pre‐activation is enhanced by C. albicans. The effects exerted by F1 make it more important than F2 and F3 for the treatment of disseminated candidiasis in patients with immunodeficiency diseases such as AIDS and chronic granulomatous disease, among others.

Different activities of Schinus areira L.: anti-inflammatory or pro-inflammatory effect

The anti-inflammatory drugs possess many serious side effects at doses commonly prescribed. It is really important to discover novel regulators of inflammation from natural sources with minimal adverse effects. Schinus areira L. is a plant native from South America and is used in folk medicine as an anti-inflammatory herb. For this study, the activity of aqueous extracts on inflammation and the effect on superoxide anion production in mice macrophages were assayed. Aqueous extracts were prepared by soaking herbs in cold water (cold extract), boiling water (infusion), and simmering water (decoction). Cold extract possess an anti-inflammatory activity. Decoction and infusion showed pro-inflammatory activity. Cold extract increased the production of superoxide anion. It has been proposed to use diverse methods to obtain extracts of S. areira L. with different effects. Cold extract, decoction, and infusion could be utilized as extracts or as pharmacological preparations for topical application.

Cross-reaction between proteins of Larrea divaricata Cav. (jarilla) and proteins of Gram-negative bacteria.

Larrea divaricata is an abundant plant of northwest of Argentina used to treat different pathologies. We aimed to characterize the immunogenicity of proteins from a partially purified crude aqueous extract (JPCE) of jarilla. We evaluated the cross reaction between JPCE and whole cell-bacterial proteins (W-CBP) of Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, and Klebsiella pneumoniae using a mouse anti-JPCE serum. Protein profiles of JPCE and W-CBP were analyzed. For JPCE, 18 bands were observed in a 20–176 kDa range. Levels of IgG against JPCE and W-CBP were determined. Bacterial proteins showed a strong reaction with the anti-JPCE serum. Plant proteins could be used as immune stimulants.

Macrophages activation by a purified fraction, free of nordihydroguaiaretic acid (NDGA), from Larrea divaricata Cav. as a potential novel therapy against Candida albicans

Larrea divaricata Cav. (jarilla) is a plant with well-documented applications in Argentinean folk medicine. In order to determine if the treatment with a purified fraction named F1 was capable to maintain a state of priming of macrophages after 15 days of mice infection with Candida albicans. Infected and uninfected mice were used. The effect of F1 on: cytosolic protein levels, apoptosis, phagocytosis, reactive oxygen species production, nitric oxide (NO), cell activity, lysosomal activity and the tissue fungal burden were studied. The results showed that F1 increased macrophages yeast phagocytosis and reactive oxygen species and NO production. All these effects were related to a decrease of cell activity and possible apoptosis. In conclusion, it was observed that F1 could induce a state of long-term activation of macrophages, since we observed increased activity of macrophages 15 days after infection, and it could be related to the elimination of C. albicans. These data may suggest that F1 fraction could be useful against disseminated candidiasis in patients and further studies on this field are desirable.

Early Effects Triggered by Larrea divaricata Cav. on Murine Macrophages at Apoptotic Concentrations

Decoction and infusion of Larrea divaricata were tested at apoptotic concentrations (1 and 4 mg/ml) on peritoneal murine macrophages. Consistent changes were observed after incubation with 4 mg/ml decoction. Phagocytosis of zymosan, lysosomal enzyme activity, nitric oxide production, TNF-α release, and expression of CD14, TLR4, and CR3 increased significantly. Decoction at 1 and 4 mg/ml increased the binding of LPS-FITC. Apoptosis triggered by L. divaricata decoction is consequence of cell activation. The effects are independent of nordihydroguaiaretic acid. This “activation and death” could be the mechanism of L. divaricata to exert the antituberculosis effect known in folk medicine.

Cross reaction between proteins from Larrea divaricata Cav. (jarilla) and cellular and extracellular proteins of Pseudomonas aeruginosa.

Larrea divaricata is widely used in folk medicine to treat different pathologies, but little is known about its immunological properties. Pseudomonas aeruginosa is an opportunistic pathogen which causes several intrahospitalary infections. We aimed to assess the immunological relation between proteins from a crude extract of L. divaricata Cav. (JPCE) and cellular and extracellular proteins (EP) of P. aeruginosa, as well as to establish the cross reactivity between proteins of both species using a mouse anti-JPCE serum. Protein profiles of JPCE and P. aeruginosa were analyzed by SDS-PAGE. The percentage of similarity of protein bands between these two species was 43–57%. However, JPCE proteins were immunogenic. The reactivity of mouse anti-JPCE antibodies against different fractions was studied by western blot. The anti-JPCE serum detected several antigenic bands on different bacterial proteins. Several common immunoreactive bands were detected (27–100%) when bacterial proteins were incubated with anti-JPCE serum (heterologous reaction) and anti-bacterial proteins serum (homologous reaction). By enzyme-linked immunosorbant assay (ELISA) assays, high titers of anti-JPCE against different types of cellular bacterial fractions were observed (1/1280–1/2080). Our data clearly demonstrate that antibodies elicited with L. divaricata crude extract are able to cross-react with cellular and EP of P.aeruginosa. These findings could be relevant in the development of alternatives therapies for patients suffering intrahospitalary opportunistic infections with P.aeruginosa.

Neutralization of Pseudomonas aeruginosa enzymatic activity by antibodies elicited with proteins of Larrea divaricata Cav.

Larrea divaricata Cav. (Jarilla) is a bush widely used in folk therapy for the treatment of several pathologies. Partially purified proteins of crude extract (JPCE) cross-react with proteins of Gram-negative bacteria, including Pseudomonas aeruginosa, which is an opportunistic pathogen that causes several intrahospitalary infections. This bacterium produces many proteins with enzymatic activity, including hemolysins and proteases that play a major role in acute infection caused by this bacterium. The aim of our work was to investigate if antibodies against with L. divaricata neutralize the hemolytic and proteolytic activity of P. aeruginosa. The hemolytic activity of soluble cellular proteins was inhibited 100% and extracellular proteins (EP) showed an inhibition between 44 and 95% when both bacterial fractions were treated with anti-JPCE serum. Also, in EP the neutralization was directed towards the active site of the hemolysin. When protease activity of extracellular products was tested, bands of 217, 155, 121, 47 and 27 kDa were observed in native zymograms. Neutralization between 55 and 70% of the bands of 217, 155 and 121 kDa was observed when EP were treated with anti-JPCE serum. In conclusion, our data clearly demonstrate that antibodies elicited with L. divaricata’ proteins are able to neutralize the hemolytic and proteolytic activity of P. aeruginosa cellular and extracellular proteins. Our study constitutes the first report that associates the immunogenicity of plant proteins and bacterial proteins with enzymatic activity. These findings could be relevant in the development of alternatives therapies for patients suffering intrahospitalary opportunistic infections with P. aeruginosa.