Nitric oxide production by murine peritoneal macrophages in vitro and in vivo treated with Phyllanthus tenellus extracts

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Abstract

Phyllanthus spp. are used traditionally for the treatment of viral, bacterial and parasitic infections. Macrophages may play a central role in innate and adaptive response against several infections. Nitric oxide (NO) can be induced during macrophage activation and may exert antimicrobial activity inhibiting the replication of several viruses or parasites. In the present study, we investigated the immunomodulatory role, both in vitro and in vivo, of aqueous extracts of fresh and dried Phyllanthus tenellus as well as an acetone/water extract of the dried plant. NO production by mouse peritoneal macrophages was detected in culture supernatants. Our results demonstrated that: (1) in vitro, a concentration of 100 μg/ml fresh extract stimulated significantly (P≤0.05) NO production in all assays and the optimal production was achieved at 48-h incubation; (2) 10 and 50 mg/kg fresh extract injected twice intraperitonealy primed macrophages in vivo. Priming was detected by in vitro addition of a second stimulus with 100 μg/ml extract of the fresh plant. Thus, P. tenellus was able to pre-activate macrophages in vivo, and induce full activation in vitro. Further studies should be carried out to better evaluate the optimal dose schedules in terms of time/response for obtaining antiviral or other antimicrobial activity without host damage.

Introduction

Nitric oxide (NO) plays a dual role in the inflammatory process. The NO secreted at high levels by activated macrophages and neutrophils is an important cytotoxic effector molecule in the defense against tumor cells, parasitic fungi, protozoa, helminthes, mycobacteria and virus (Croen, 1993, Macmicking et al., 1997). On the other hand, proinflammatory effects of NO seem to be mediated by its exacerbated production and have been associated with a range of inflammatory diseases including arteriosclerosis, ischemic reperfusion, hypertension and septic shock (Moncada et al., 1991). In viral hepatitis, NO has a protector function cooperating in viral clearance (Pinto et al., 2000).

Since ancient times, plants of the genus Phyllanthus (family Euphorbiaceae) have commonly been used in treatments for liver diseases (Thyagarajan and Jayaram, 1992). Reports about jaundice and other liver abnormalities describe Phyllanthus administration in India, China, Burma, Pakistan, Philippines, Guam, West Indies, South America, East and West Africa and elsewhere in tropic and subtropic areas (Blumberg et al., 1989). Phyllanthus tenellus has been used in Brazilian traditional medicine for the treatment of kidney and bladder calculi, diabetics, hepatitis, jaundice and asthma (Martins et al., 1995, Menezes, 1996). Crude extracts of Phyllanthus amarus were found to inhibit Hepatitis B virus (HBV) polymerase activity and clear Hepatitis B antigen (HbsAg) in chronic HBV carriers (Venkateswaran et al., 1987, Thyagarajan et al., 1988).

As a part of our search for understanding immunological mechanisms involved in host defense, we show, in this paper, that an aqueous extract of P. tenellus activated murine peritoneal macrophages both in vitro and in vivo resulting in NO triggering.

Section snippets

Plant material

Leaves of P. tenellus were collected from experimental fields in Far-Manguinhos, FIOCRUZ, Rio de Janeiro, Brazil in June 1994 and May 1995. The voucher specimen (no. 339, 186) is deposited in the Herbarium of the Botanic Garden, Rio de Janeiro.

Aqueous extracts of fresh leaves (fresh extract)

Freshly collected leaves (105 g) were macerated in 750 ml distilled water and heated at 55°C for 60 min. The aqueous extract was filtered through Whatman no. 1 filter paper and lyophilized (3.34 g, 3.18%).

Aqueous extracts of dried leaves (dried extract)

Collected leaves were left for drying during 15

In vitro cytotoxic effect by P. tenellus

During experimentation in the 72-h treatment of macrophage cultures using all three extracts, no cytotoxicity was observed at the 100 μg/ml concentration by Trypan blue exclusion method but higher concentrations were cytotoxic in most extracts. Acetone extracts of the dried plant were slightly more toxic than aqueous extracts after 24-h incubation but on longer incubation the toxicity levels became the same (Table 1). The macrophage viability was always successfully tested in each experiment

Discussion

It is well-known that Phyllanthus spp. display antiviral activity against hepatitis B virus (Unander and Blumberg, 1991) and is used popularly worldwide in liver affections. Furthermore, it is believed that induction of nitric oxide plays a role in the protective activity during viral hepatitis (Pinto et al., 2000). However, there were no previous reports describing NO production by macrophages treated with Phyllanthus spp. The three plant extracts used in this work were able to stimulate NO

Acknowledgements

To Drs Benjamin Gilbert and José Paulo Leite for continuous encouragement. Financial support: Fiocruz and CNPq, Brazil.

References (14)

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