In vitro activities of Cedrela tubiflora aqueous leaf extracts on murine macrophages, polymorphonuclear leukocytes and complement

PHYTOTHERAPY RESEARCH, VOL. 10.37-41 (1996)

In vitro Activities of Cedrela tubijlora Aqueous Leaf Extracts on Murine Macrophages, Polymorphonuclear Leukocytes and-Complement F. Benencia,* M. C. Courri?ges and F. C. CoulombiC Laboratory of Virology, Faculty of Science, University of Buenos Aires, 1428 Buenos Aires, Argentina

Cedrela tubijlora aqueous leaf extracts are capable of inhibiting in uitro the activity of some components of the mouse immune system related to inflammatory responses. A significant reduction in the phagocytic capability and respiratory burst response (61.5y0 and 57.6%, respectively) of murine peritoneal macrophages was observed when these cells were incubated for 24 h with medium containing 1 mg/mL extract. On the other hand, at a concentration of 4 mg/mL, the extract reduced significantly the phagocytic activity of mouse polymorphonuclear leukocytes (87.5%) without altering the oxidative metabolism of these cells. Finally, a concentration of 2 mg/mL was required to inhibit the haemolytic activity of both pathways of mouse complement.

Keywords: antiinflammatory activity; Cedrela tubifiora aqueous extract; macrophages; complement;

neutrophils. INTRODUCTION Plants belonging to the family Meliaceae have been widely used in natural medicine. Antiviral, antihelmintic, antiinflammatory and antirheumatic activities have been reported (Bhakuni et al., 1969; Fujiwara et al., 1982; Patel, 1986; Andrei et al., 1990; Bray et al., 1990; CoulombiC et al., 1992). As the antiinflammatory and antirheumatic properties of some members of this family, such as Azadirachta indica, Mumromia pumila and Melia azedarach, have been explained by their action on the immune response (Labadie el al., 1989; Courri3ges et al., 1994), we began a survey of autochthonous members of this family searching for immunomodulatory activities. In this report we describe the effect of aqueous leaf extracts of the Meliaceae tree Cedrela rubifEora Bert. on the bioactivity of both phagocytic cells, murine macrophages and polymorphonuclear leukocytes and the activation of the complement system, elements that are strongly related to inflammatory processes.

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MATERIALS AND METHODS Animals. Inbred BALBk mice, 8-10 weeks old, reared

in our own animal house, were used in all experiments. Mice were housed five per cage with sterile wood chip bedding and provided with chow pellets and tap water ad libitum. The animal quarters were maintained at 21"-24 "C, and 40%40% humidity with a 12 h lightdark cycle. Preparation of plant extracts. Fresh green leaves of

Cedrela tubifora Bert. collected during late spring, Author to whom correspondence should be addressed. CCC 0951-418X/96/010037-05 0 1996 by John Wiley & Sons, Ltd.

were identified and deposited at the Department of Botany, Science School, University of Buenos Aires. The plant material was washed with distilled water and blended with 10 mM potassium phosphate buffer, pH 7.2 containing 0.35 M KCI (1 g plant materiaVmL). The sap obtained was filtered through cheesecloth and then clarified by centrifugation at 10 OOO x g for 30 min. The final extract had a concentration of 41 mg of dried plant materiaVmL as determined by lyophilization. Haemolytic assay for the study of the classical and alternative pathway of mouse complement. The inhibition of the clas-

sical (CP) and alternative (AP) pathways activities of mouse complement by C. tubiflora extracts was determined in sera of BALBk mice using the method of Van Dijk et al. (1980). Mice were anaesthetized with ether and bled from the retroorbital venous plexus by means of capillary tubes and serum was separated by centrifugation. Serum pooled from at least four animals was tested for complement activities. The serum concentration giving rise to 50% haemolysis of the target cells (1AP,,, for alternative pathway or 1 CP,, unit for classical pathway) was calculated by means of the Von Krogh equation (Mayer, 1961). The haemolytic activity of mouse serum was expressed in AP,, or CPmunitdml. For determining the anticomplementary activity of C. tubiflora extracts, undiluted sera were mixed with different concentrations of the extracts (0.25-10 mg/ mL) or heparin (Abbott) (0.2-10mg/mL), used as a positive control, and incubated for 30 min at 37 "C. The dilutions were made in Verona1 saline buffer (VSB) containing 5mM veronal and 1 5 0 m ~ N a C at l pH7.4 supplemented with 0.5 mM Mg2+ and 0.15 mM Ca" (CP) or 8 mM ethylene glycol-bis(2-aminoethyl) tetraacetic acid (AP). The residual haemolytic complement activity was determined by a method using sensitized (CP) or normal (AP) rabbit erythrocytes as target cells (Van Dijk etal., 1980; Rademaker et al., 1981; Klerx et Accepted (revised) 16 January 1995

38

F. BHNENCIA E T A L .

al., 1983, 1985a, 1985b). The AP and CP activities in the presence of the extracts were read from the Von Krogh plots. Thus, the activities were obtained as APSo or CPm unitdml and transformed to percentages of inhibition with respect to the activity of serum tested in the absence of the extracts. A water lysed control (100% lysis) and a buffer control (OYo lysis) were used. Isolation and culture of murine peritoneal macrophages. Mice

were killed by cervical dislocation. Resident peritoneal cells were obtained by washing the peritoneal cavity with MEM (Gibco), containing 2% heat inactivated calf serum and 5 U/mL heparin (Abbott). Cell viability, determined by the trypan blue exclusion method, was always greater than 95% and the proportion of macrophages in the peritoneal exudate was over 90% as determined by acridine orange staining (Goldstein and Blomgren, 1973). Cell suspensions containing 3 x lo6 macrophages were plated onto glass coverslips in Leighton tubes. The cultures were incubated with MEM supplemented with 5% heat inactivated calf serum and 50 pg/mL of gentamycin (control) or different dilutions of the plant extract prepared in the same medium. After 24 h incubation at 37 "C the phagocytic capability and respiratory burst activity of these cells was measured. Isolation and culture of polymorphonuclear (PMN) leukocytes. PMN leukocytes were obtained using the method

of Badwey et al. (1983). Cells were allowed to attach to glass coverslips for 1h at 37 "C and then the cultures were washed in order to remove non-adherent cells. The percentage of PMN in the adherent population was over 90% as determined by differential counting on Giemsa stained cells. Then, cells were incubated for 45 min with MEM alone (control) or different dilutions of plant extract in the same medium. Subsequently, phagocytic activity and respiratory burst of these cells were evaluated. Phagocytic assay. The phagocytic activity of treated or control cells was tested using sheep erythrocytes (SE) sensitized with a subagglutinating dilution of goat antiSE serum in the case of macrophages or zymosan particles (Sigma Chemical Co., MO) in the case of PMN. After treatment with C. tubiflora extracts, cell cultures were challenged with a suspension of the appropriate opsonized particles. Two hundred cells were scored in each coverslip and the percentage of phagocytic cells was recorded. Nonspecific ingestion was discarded considering as positive only those cells ingesting more than four particles (Losche et al., 1988). Cultures treated in a similar way with different concentrations of 2-deoxyglucose (2DG) (Sigma Chemical Co., MO) were used as positive controls.

black by precipitated formazan, the oxygen dependent reduction product of NBT (Wilkinson, 1981). At least 200 cells were scored for each experiment.

RESULTS

Anticomplementary effect of C. tubifloru extracts The activation of murine classical (CP) and alternative (AP) complement pathways were studied with a method that uses sensitized (CP) or normal (AP) rabbit erythrocytes as target cells. As shown in Fig. 1, preincubation of mouse serum with different concentrations of the extract caused a dose dependent reduction in the haemolytic activities in both pathways. The effect was more pronounced in the CP where a significant reduction was observed at an extract concentration of 0.5 mg/ mL and 5 mg/mL caused a total inhibition of the haemolytic activity. Although the extract was also capable of inhibiting the AP, 100% inhibition was not reached even at the higher extract concentration tested (10 mg/ mL). Preincubation of the target cells with the extract did not prevent complement mediated lysis (data not shown) discounting a protective effect of C. tubiflora extracts on the erythrocyte membrane. As a positive control on both CP and AP pathways, heparin, a sulphated polysaccharide with known anticomplementary activity (Klerx et al., 1985a), was used. Although this compound was also capable of inhibiting the serum haemolytic activity on both CP and AP complement pathways, this effect was less pronounced than that exerted by C. tubiflora extract (Fig. 1). Heat inactivated serum of BALB/c mice showed no haemolytic activity. % Inhibition of hemolytic activity

7

loo[

0

2

4

e

8

10

12

Concentration (mg/mL) Qualitative nitroblue tetrazolium (NBT) reduction assay.

After treatment with C. tubijlora extracts, PMN or macrophage cultures were incubated for 30 min at 37 "C in a 5% C 0 2 atmosphere with a suspension of 2 x 106 opsonised zymosan particledml in MEM containing 0.5mg/mL of NBT (Sigma Chemical Co, MO.) as described by Losche et al. (1988). Cells were scored as positive when ingested particles were stained blue-

Figure 1. Anticomplementary effect of C. tubiflora extracts. Mouse serum was incubated for 30 min (alternative pathway) or 60 rnin (classical pathway) with different concentrations of C. tubiflora extract or heparin (as positive control) before the addition of sensitized (CP) or normal rabbit erythrocytes (AP). After treatment, remaining haemolytic capacity was titrated and expressed as percentage of inhibition respect to the activity of untreated sera. W C. tubiflora CP, t heparin CP, +C. tubiflora AP, 0 heparin AP.

IMMUNOMODULATORY ACTIVITIES OF CEDRELA TUBIFLORA AQUEOUS EXTRACTS

39

Effect of C . tubifloru extract treatment on the phagocytic capability of peritoneal macrophages As shown in Table 1, treatment of macrophages for 24 h caused a significant inhibition in phagocytic capability at nearly all the extract concentrations tested ( 0 . 2 5 4 mg/mL). The antiphagocytic activity resulted not only in a significant reduction in the number of cells which had incorporated opsonized erythrocytes, but also in the number of erythrocytes ingested per single cell (Fig. 2). According to the trypan blue exclusion method, no significant differences in the total number of viable cells were observed between treated (98.00+4.30%) and control (96.00% f5.20%) cultures, after an incubation of 24 h in the presence of the maximum extract concentration tested (4 mg/mL). 2-Deoxyglucose is a compound capable of inhibiting phagocytosis (Michl et al., 1976) and was used as a positive control in our experiments. Cell cultures treated in parallel with different concentrations of 2DG showed a dose dependent inhibition of their phagocytic activity. C. tubifrora extracts appeared to be considerably more antiphagocytic than 2DG since 50% inhibition of phagocytic activity was obtained at an extract concentration of 0.77 mg/mL, whereas 5.5 mg/mL of 2DG was needed for a similar effect (data not shown). Effect of C. tubijiora extracts on the oxidative metabolism of macrophages Phagocytosis of particles is usually accompanied by an oxidative burst. To test the effect of the extracts on the generation of oxygen radicals, the NBT assay was used. Table 1 shows that treatment produced a significant decrease in NBT reduction by macrophages at all the extract concentrations tested (0.25-4.00 mg/mL). At 2 mg/mL the phagocytic capability of these cells was completely reduced but 33% of them were still NBT positive. Taking into account the fact that treatment not only reduced the number of phagocytic cells but also the number of particles ingested per single cell, this result could be explained by the intracellular reduction of formazan salts in treated cells ingesting less than four particles (scored as non-phagocytic cells). Table 1. Effect of C. tubifora extracts on phagocytic capability and oxidative metabolism of macrophages Extract concentration

lmg/mLl

Phagocytosis

(%I

0.12 0.25 0.50

91.00k3.90 80.00k3.4OC 65.00f3.00' 37.00+2.30'

2.00 4.00

O.OOb O.OOb

1 .oo

NET positive cells'

(%I ND

81.00f3.20' 60.00k4.50' 39.00+ 6.40" 33.00k 1.90c ND

92.00f4.70 The phagocytic capability and oxidative metabolism of the cells were tested after 24 h of incubation with different concentraControl

96.00k4.2

tions of C. tubiflora extracts (treated cells) or with medium alone (control). Values are expressed as meankSD of three independent measurements. 'Percentage of cells containing intracellular precipitated formazan, the oxygen dependent reduction product of NBT. Statistical differences vs control: bp