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Immunomodulatory activity of Mollugo verticillata L.

Summary

This article describes the evaluation of immunomodulatory activity of Mollugo verticillata L. (Molluginaceae), a weed plant common in warm and/or wet regions of the American continent. Nitric oxide (NO) release was evaluated in mice peritoneal cell cultures treated in vivo using the ethanolic extract of M. verticillata with and without BCG. The plant extract showed immunostimulatory activity when peritoneal cells were stimulated in vitro with BCG antigen only. However, mice peritoneal cells treated with M. verticillata plus BCG showed a drastic reduction in NO production when they received the additional stimulus in vitro with BCG. Ethanolic extracts of M. verticillata could directly increase NO release by peritoneal cells, but suppress the immune response of these cells when treated with BCG antigen and Mycobacterium tuberculosis whole antigen (TB). Preliminary phytochemical tests allowed the detection of quercetin and triterpenoid glycosides in the ethanolic extract of M. verticillata, and those compounds a re probably responsible for the effect of this plant material on the immune system.

Key words: Mollugo verticillata L., NO release, immunomodulatory activity

Introduction

Mollugo verticillata L. (Molluginaceae) is an annual herb with prostrate growth habit. This is a weedy plant common in warm and/or wet regions of the American continent (Mabberley, 1997). Some species of Molluginaceae have been the targets of studies leading the discovery of antifungal, antiinflamatory, antiplasmodial, antitumour, immunocompetent and spermicidal activity (Rajasekaran et al., 1993; Sadique et al., 1987; Traore et al., 2000; Yang et al., 1997a; Yang et al., 1997b). The chemistry of these plants is distinguished mainly by the production of triterpene saponins and flavonoids (Soares and Kaplan, 1995; Soares et al., 1995).

It was observed that triterpene saponins and flavonoid derivatives possess a remarkable effect on the immune system. The immunomodulatory activity of triterpene saponins has been cited (Rao and Gurfinkel. 2000) and its capacity to stimulate nitric oxide (NO) production is known (Tanner et al., 1999). However, flavonoid aglycones, such as quercetin (3,5,7,3',4'-pentahydroxyflavone) and apigenin (5,7,4'-trihydroxyflavone), can be potent inhibitors of NO production (Raso et al., 2001). Substitution pattems may be involved in the action of flavonoid derivatives on immune system. Rutin (3-0-rutinosylquercetin) proved to be immunosuppressive but methoxy derivatives, as 7-methoxyflavanone, show immunostimulatory properties (Sharma et al. 1996).

Therefore, the chemical profile indicates species of Molluginaceae as good sources of immunomodulatory compounds. We describe here the effects of the oral administration of an ethanolic extract of M verticillata on nitric oxide (NO) production in mice infected with BCG. The in vitro effect of this extract on NO production was also evaluated.

Materials and Methods

Plant Material

Aerial parts of M. verticillata (including branches, leaves, flowers and fruits) were collected in a reef, located in Marica, Rio de Janeiro State, Brazil. A reference specimen has been deposited in the Herbarium of the Universidade Federal do Rio de Janeiro (R) under the number 189849.

Tested animals and BCG strain

In all experiments 6 to 8-week old female Balb/c mice were used. These animals were raised in plastic cages with unlimited access to food and water. Mycobacterium bovis BCG strain Monroe was obtained from Ataulpho de Paiva Institute, Rio de Janeiro, RI, Brazil.

Extraction Methodology

Collected plant material was dried in a stove (35 [degrees]C) and subsequently extracted with petroleum ether in a Soxhlet device for 24 h. This material, with a low level of almost apolar compounds (hydrocarbons, long-chain alcohols and other lipids) was then submitted to exhaustive extraction with ethanol in a Soxhlet device. The resulting crude ethanolic extract (CEE) was dried in vacuo and fractionated using crescent polarity solvents (hexane, dichloromethane, ethyl acetate and water). The hydroalcoholic fraction (EE) was dried in vacuc and lyophilized.

Detection of Triterpene and Flavonoid Derivatives in the Ethanolic Extract of M. verticillata

Samples of EE (10 mg) were submitted to acid hydrolysis using a mixture of 2N HC1 and methanol (1:1) under controlled heating. Original samples, as well as their hydrolyzed products, were submitted to paper chromatography (PC) and thin-layer chromatography (TLC), using chloroform-methanol-water (13:7:2, lower layer), butanol-acetic acid-water (BAW 4:1:5, upper layer) and ethyl acetate/acetic acid/formic acid/water (100:11:11:27) as eluents. Triterpene derivatives were detected by the interpereation of the color reaction with Liebermann-Burchard's reagent. Two standards of the flavonols quercetin (Sigma[R]) and rutin (Sigma[R]) were co-eluted with all samples. Quercetin itself and its derivatives were detected with NP/PEG and UV (365 nm) (Markham, 1982; Matos, 1997; Wagner et al., 1984).

In Vivo Assay

Mice were inoculated with BCG (0.1 mg in 0.5 ml PBS) by i.p. injection seven days before death. Ethanolic extract of M. verticillata (500 mg/g body wt.) was given orally to the mice over four consecutive days. Four groups of mice were studied: control -- three mice i.p. injected only with PBS; Mollugo -- three mice i.p injected with PBS and treated with EE of M. verticillata; BCG -- three mice i.p. injected with BCG; BCG + Mollugo -- three mice i.p. injected with BCG and treated with EE of M. verticillata.

NO release was quantified by measuring the accumulation of nitrite in the supematants of peritoneal cell cultures from each studied group after 48 h in culture with or without additional in vitro stimulation by BCG antigen (10 [micro]g/ml), using the standard Griess method (Ding et al., 1988). Griess reagent was made fresh prior to use and added 1:1 with supematant, then left 5 mm at room temperature. Standards were prepared using sodium nitrite (2-100 [micro]M) and were included on each assay plate. Absorbances at 570 nm were read on a spectrophotometer (Titertek Uniskan II, Flow, Inc., Lugano, Switzerland).

In vitro Assay

Total peritoneal cells or macrophage peritoneal cells isolated as described by Almeida and Lopes (2001) were collected and cultured in RPMI-1640 medium supplemented with 5% heat-inactivated fetal calf serum and 4mM L-glutamine. Cells were stimulated in vitro with BCG antigen (10 pg/ml), LPS (1 [micro]g/ml) and Mycobacteriurn tuberculosis whole antigen (TB-50 [micro]g/ml) with or without M. verticillata (25 [micro]g/ml) and maintained at 37 [degrees]C in a controlled atmosphere with 5% [CO.sub.2] for 48 h. Supernatants were collected and the Griess Method was used to evaluate NO production.

Statistical analysis

The Student's t-test was used to evaluate the presence of significant differences between the groups with the level of significance set at p < 0.05.

Results and Discussion

Low levels of NO production were detected in all peritoneal cell cultures without BCG stimulation. (Fig. lA, Table 1). Stimulation with BCG antigen resulted in the alteration of NO production by macrophages obtained from mice treated in vivo with EE of M. verticillata, BCG and BGC + M. verticillata (Fig. lB, Table 2). An increase of NO production was detected in the cell culture from mice treated in vivo with the plant extract (Mollugo group) and stimulated in vitro with BCG antigen. As expected, we detected a high level of NO production by macrophages obtained from mice infected with BCG (BCG group) stimulated with BCG antigen in vitro (Ferreira et al., 1999). However, macrophages of the BCG + Mollugo group presented a strong decrease in NO production as compared to the-BCG group (Fig. 1B).

The in vitro assay (Fig. 2) confirms the immunomodulatory effect of the EE of M. verticillata observed in vivo. The treatment of macrophages with the extract only induces an increase in NO production, but when these cells were cultured with BCG or TB plus EE, significant decreases in NO production were observed. These effects become clearer when compared with the results obtained with cells treated only with BCG or TB. Indeed, NO production by cells treated with BCG and EE was similar to that observed for the control cells (BCG = 8.30 [micro]M and BCG + Mollugo = 5.09 [micro]M). Even though the extract did not completely reverse the effect of TB, it was effective tin suppressing this stimulus. In this case the decrease oin NO production was about 35% (TB = 12.37 [micro]M and TB + Mollugo = 8.01 [micro]M). It is also interesting to note that the immunostimulation of macrophages treated with LPS was not reversed by the addition of the extract of M. verticillata.

Protection against intracellular pathogens operates in two stages, namely an early innate nonspecific response followed by acquired immunity with a strong DTH response (Yoshida et al., 1995). During the first week of infection, the early innate immune response is the main mechanism controlling Mycobacteria proliferation. (Yoshida et al., 1995; Pelletier et al., 1982). In inbred strains of mice, early innate resistance to M. bovis BCG infection is controlled by a single dominant gene designated Nramp (natural resistance-associated protein macrophage protein) which regulates the initial activation of macrophages for NO production (Vidal et al., 1993; Brown et al., 1995). Thus, the immune mechanisms associated with the regulation of resistance and susceptibility in the innate early phase of BCG infection clearly involve cytokines and NO production (Yoshida et al., 1995; Bala et al., 1998).

It was observed recently that the administration of Ascaris sun extract (Asc) in infected mice increased NADPH diaphorase activity and TNF-[alpha] levels in the early phase of BCG infection (Ferreira et al., 1999). However, the ethanolic extract of M. verticillata showed a more complex effect than Asc extract, which only inhibits the NO production by macrophages. M. verticillata treatment probably induces different effects in cellular activation, which is correlated with the chemical complexity of the ethanolic extract and the cellular activation stage.

The results of preliminary phytochemical analysis revealed a complex mixture of flavonoid glycosides and, probably, triterpene glycosides in EE of M. verticillata. (Table 3). Several flavonoid derivatives were developed with NP/PEG and molibidic acid in this extract. In original samples, we detected no compound with chromatographic behavior identical to the standards of quercetin and rutin. However, spots with chromatographic behavior identical to quercetin were observed in samples of EE submitted to acid hydrolysis. These results suggest the presence of quercetin glycosides in the crude ethanolic extract of M. verticillata. It well know that flavonoids and triterpene glycosides can possess immunostimulatory and/or immunosupressive effects (Rao and Gurfinkel, 2000; Raso et al., 2001; Sharma et al., 1996; Tanner et al., 1999), which could explain the results of immunological analysis of this plant extract.

The present study suggests that the ethanolic extract of M. verticillata can be a good source of immunomodulatory compounds. Aside from the obvious therapeutic importance, these compounds would be useful to understanding the mechanism of diseases with higher levels of cellular activation, suchas autoimmune and hypersensibility diseases.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]
Table 1

Spontaneous NO release ([mu]M) of peritoneal cell after in vivo
treatment with Ethanolic extract of M. verticillata and BCG

Groups Control Mollugo BCG Mollugo + BCG

Average * 1.09 (a) 3.15 (b) 5.86 (c) 4.86 (c)
SD 0.00 0.00 3.91 3.54
Error 0.00 0.00 2.25 2.04

* Values with same letter showed no statistical difference [student "t"
test (p < 0.05)].

Table 2

Stimulated NO release ([mu]M) of peritoneal cell after in vivo
treatment with ethanolic extract of M. verticillata and BCG.

Groups Control Mollugo BCG Mollugo + BCG

Average * 1.51 (a) 18.14 (b) 43.32 (c) 2.58 (a)
SD 0.00 0.00 34.60 1.61
Error 0.00 0.00 19.98 1.18

* Values with same letter showed no statistical difference [student "t"
test (p < 0.05)].

Table 3

Summary of chromatography profile of ethanolic extract (EE) of aerial
parts of M. verticillata.

Sample Q R Molibidic NP/ Liebermann-
 acid PEG Burchard's

EE nd nd + + +
EE (Hydrolyzed) + nd + + +

nd = not detected


Acknowledgments

The authors are grateful to FAPEMIG, Brazil, for financial support, and to Dr. Paulo Ricardo Pereira de Oliveira and Prof. Dr. Rosy Mary dos Santos Isaias for comments and suggestions.

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A.P. Ferreira, (1) G.L.G. Soares, (2) C.A. Salgado, (1) L.S. Goncalves, (1) F.M. Teixeira, (1) H.C. Teixeira, (1) and M.A.C. Kaplan (3)

(1) Departamento de Parasitologia, Microbiologia e Imunologia, Universidade Federal de Juiz de Fora, MG, Brazil

(2) Depauamento de Botanica, Universidade Federal de Juiz de Fora, MG, Brazil

(3) Nucleo de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro, RJ, Brazil

Address

G. L. G. Soares, Departamento de Botanica, Universidade Federal de Juiz de Fora, MG, Brazil, 36036-330 Tel./Fax: ++55-32-3229 3216; e-mail: gsoares@icb.ufjf.br
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Author:Ferreira, A.P.; Soares, G.L.G.; Salgado, C.A.; Goncalves, L.S.; Teixeira, F.M.; Teixeira, H.C.; Kapl
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:1USA
Date:Mar 1, 2003
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