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Anti-inflammatory activities of Trichilia glabra aqueous leaf extract
Journal of Ethnopharmacology 71 (2000) 293 – 300 www.elsevier.com/locate/jethpharm
Anti-inflammatory activities of Trichilia glabra aqueous leaf extract Fabia´n Benencia *, Marı´a Cecilia Courre`ges, Fe´lix Carlos Coulombie´ Laboratory of Virology, Faculty of Science, Uni6ersity of Buenos Aires, Pabello´n II, Piso 4, Ciudad Uni6ersitaria, (1428) Nun˜ez, Buenos Aires Argentina Received 3 May 1999; received in revised form 7 February 2000; accepted 14 February 2000
Abstract Trichilia glabra L. aqueous leaf extract exerted a significant antiinflammatory effect ‘in vivo’ in the zymosan-induced inflammation model. The extract impaired the ‘in vitro’ activities of polymorphonuclear leukocytes and complement, components of mouse immune system closely related to the inflammatory response induced by zymosan. In particular, a significant reduction in the phagocytic capability and respiratory burst response of mouse polymorphonuclear leukocytes together with an inhibition in the hemolytic activity of mouse complement was observed. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Anti-inflammatory activity; Trichilia glabra; Aqueous extract; Zymosan-induced inflammation complement; Neutrophils
1. Introduction Plants belonging to Meliaceae family have been widely used in ethnomedicine. Antiviral, antihelmintic, anti-inflammatory and antirheumatic activities have been reported (Bhakuni et al., 1969; Fujiwara et al., 1982; Patel, 1986; Andrei et al., 1990; Bray et al., 1990; Coulombie´ et al., 1992). The anti-inflammatory and antirheumatic properties of some members of this family, such as Azedirachta indica A. Juss, Mefia azedarach L. and Cedrela tubifibra Bert. have been explained by their action on the immune response (Labadie et al., 1989; Courre`ges et al., 1994). In previous * Corresponding author. Fax: +54-1-7820458. E-mail address: [email protected] (F. Benencia)
reports we observed that aqueous leaf extracts of Melia azedarach L, Cedrela tubifibra Bert., Cedrela lilloi L. and Trichilia elegans L. exerted inhibitory activities on both murine complement activation and phagocytosis mediated by mouse peritoneal exudate cells and PMN leukocytes (Benencia et al., 1996; Nores et al., 1997). A significant diminution in the delayed-type hypersensitivity response of mice have been reported for Cedrela lilloi L., Trichifia elegans L. and Melia azedarach L. (Nores et al., 1997; Courre`ges et al., 1998). Besides, we have found that Cedrela tubffiora Bert. and Trichilia glabra L. extracts also interfered on some mechanisms related with the human immune function such as: the hemolytic activity of human complement, Tlymphocytes proliferation, phagocytic capability
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and oxidative metabolism of peripheral blood monocytes and polymorphonuclear leukocytes (Benencia et al., 1995; Benencia and Coulombie´, 1998). In this report we describe the effect of aqueous leaf extract of the Meliaceae tree Trichilia glabra L. on the zymosan-induced immediate inflammation in the mouse model, and the in vitro phagocytic activity and burst respiratory response of polymorphonuclear ieukocytes and the activation of the complement system, elements of the immune response strongly related to inflammatory processes induced by zymosan.
2. Materials and methods
2.1. Animals Inbred BALB/c mice, 8 – 10 weeks old, reared in our own animal house, were used in all experiments. Mice were housed 5 per cage with sterile wood chip bedding and provided with chow pellets and tap water ad libitum. The animals quarters were maintained at 21 – 240°C, and 40 – 60% humidity with a 12 h light-dark cycle.
2.2. Preparation of plant extract T. glabra L. fresh green leaves were collected in Buenos Aires city in November 1997 and identified at the Department of Botany, Faculty of Agronomy and Veterinary, University of Buenos Aires where a voucher specimen is deposit (Argentina BAA 2722). 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 materiallmi). The sap obtained was filtered through cheesecloth and then darified by centrifugation at 10.000 x g for 30 min. The final extract had a concentration of 40 mg of dried plant material/ml as determined by lyophilization.
2.3. Hemolytic assay for the study of the alternati6e pathway of mouse complement The inhibition of the alternative pathway (AP)
activity of mouse complement by T. glabra L. extract was determined in sera of BALB/c mice by using the method of Van Dijk et al. 1980. Mice were anesthetized with ether and bled from the retro-orbital venous plexus by means of capillary tubes and serum was separated by centrifugation. Serum pooled from at least ten animals were tested for complement activities. The serum concentration giving rise to 50% haemolysis of the target cells (1 AP50 unit) was calculated by means of the Von Krogh equation (Mayer et al., 1961). So the hemolytic activity of mouse serum is expressed in AP50 units/ml. For determining the anticomplementary activity of T. glabra extract, undiluted sera were mixed with different concentrations of the extract (0.25–10 mg/ml) and incubated for 30 min at 370C. The dilutions were made in Veronal saline buffer (VSB) containing 5mM veronal and 150 mM NaCI at pH 7.4 supplemented with 8 mM ethyleneglycol-bis (2–aminoethyl) tetraacetic acid. The residual hemolytic complement activity was determined by a method using normal rabbit erythrocytes as target cells (Van Dijk et al., 1980; Rademaker et al., 1981; Klerx et al., 1983, 1985a,b). The AP activity in the presence of the extract was read from the Von Krogh plots. Thus, the activity was obtained as AP50 units/mi and transformed to percentages of inhibition with respect to the activity of serum tested in absence of the extracts. A water lysed control (100% lysis) and a buffer control (0% lysis) were used. Heparin was used as a positive control
2.4. 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 coverAps for 1 h 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 at 37°C with minimum essential medium (MEM) (GIBCO) alone (control) or different con-
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centrations of plant extract in the same medium. Subsequently, phagocytic activity and respiratory burst of these cells were evaluated.
control= [average thickness (in mm) of the footpad of treated animals/average thickness (in mm) of the footpad of control animals]× 100.
2.5. Phagocytic assay
2.8. Statistics
The phagocytic activity of treated or control cells was tested by zymosan particles (Sigma Chemical Co., MO). After treatment with T. glabra L. extract, cell cultures were challenged with a suspension of zymosan containing 2 x 106 opsonised particles/ml particles. Two hundred cells were scored in each coverslip and the percentage of phagocytic cells was recorded. Unspecific ingestion was discarded considering positive only those cells that have ingested more than 4 particles (Losche et al., 1988).
The statistical significance of the data was determined by Student’s t-test. A P value lesser than 0.05 was taken as significant.
2.6. Qualitati6e nitroblue tetrazolium (NBT) reduction assay After treatment with T. glabra extract, PMN cultures were incubated for 30 min at 370°C in a 5% CO2 atmosphere with a suspension of 2 x 106 opsonised zymosan particles/ml or 0.5 MM phorbol 12-myristate 13-acetate (Sigma Chemical Co., Mo.) (PMA) in MEM containing 0.5 mg/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-black by precipitated formazan, the oxygen dependent reduction product of NBT (Wilkinson, 1981). At least 200 cells were scored for each experiment.
2.7. Zymosan-induced inflammation The effect of T. glabra L. extract on zymosaninduced inflammation was studied in groups of 10 mice. Inflammation was induced by injecting 300 mg of zymosan suspended in 25 ml of sterilized phosphate buffered saline (PBS) in the left hind foot pad. Different concentrations (1, 2 and 4 mg/ml) of T. glabra extract in PBS (final volume: 0.2 ml) were injected intravenously 6 and 3 hours before zymosan injection. Control mice were injected with P13S. Foot-pad swelling was measured 6 hs after induction with a precision caliper. Data were registered as: % of inhibition with respect to
3. Results
3.1. Anti-inflammatory acti6ity of T. glabra L. extract in the zymosan-induced immediate inflammation model In order to investigate the in vivo effect of the extract we used the zymosan-induced immediate inflammation model. Herewith, a significant inflammation in the paw is observed 3 h after zymosan inoculation. Animals were intraperitoneally treated with different concentrations (14 mg/ml) of T glabra extract 6 and 3 h previous to zymosan administration. As shown in Fig. 1, treatment with the extract significantly reduced the zymosan-induced oedema. These effect was dose dependent, obtaining nearly a 50% reduction in inflammation with the highest extract concentration tested (4 mg/ml).
3.2. Anticomplementary effect of T. glabra L. extract The activation of murine alternative complement pathway was studied with a method that uses normal rabbit erythrocytes as target cells. As shown in Fig. 2, preincubation of mouse serum with different concentrations of the extract caused a dose dependent reduction in the hemolytic activity of the serum, being 5.1 mg/ml the extract concentration capable to inhibit this activity to 50% (IC50%). As a positive control heparin, a sulphated polysaccharide with known anticomplementary activity (Klerx et al., 1985a), was used. Although this compound was also capable of inhibiting the serum hemolytic activity this effect was less pronounced than that exerted by T.
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glabra L. extract (Fig. 2). Preincubation of the target cells with the extract did not prevent complement mediated lysis (data not shown) discounting a protective effect of T. glabra extract on the erythrocyte membrane. Heat inactivated serum of Balbic mice showed no hemolytic activity.
3.3. Effect of T. glabra extract on PMN leukocytes acti6ities PMN cultures were incubated for 45 min with different concentrations of extract (0.12 – 2 mg/
ml). The effect of treatment on the phagocytic capability and the NBT reduction capacity of murine PMN leukocytes is shown in Table 1. A significant decrease in both the phagocytic capability and the respiratory burst response of these cells was observed with the higher extract concentrations tested (0.5, 1 and 2 mg/ml). Similar results were obtained when a post-receptor stimulus (PMA) was employed (Table 1). No significant differences in the total number of viable cells between control (92.009 4.50%) and T. glabra L. extract treated cultures (89.009 5.30%) were observed according to the trypan blue exclusion method.
Fig. 1. Effect of T. glabra aqueous extract on acute foot-pad swelling in mice induced by zymosan. Groups of 10 animals were inoculated endovenously with different concentrations of the extract (1, 2 and 4 mg/ml) in saline (final volume: 0.1 ml) or equal volume of saline (control). These doses were administrated 6 and 3 h previous to zymosan inoculation in the right foot-pad (0 time). Six hours later food pad thickness was measured in treated and control animals.* P B 0.05.
F. Benencia et al. / Journal of Ethnopharmacology 71 (2000) 293–300
Fig. 2. Anticomplementary effect of T glabra extract. Mouse serum was incubated for 30 min with different concentrations of T. glabra extract (initial extract concentration: 40 mg of dried plant materiallmi as determined by lyophilization) or heparin in VS13 before the addition of normal rabbit erythrocytes. After treatment, remaining hemolytic capacity was titrated and expressed as percentage of inhibition respect to the activity of untreated sera.
4. Discussion The present study shows that Trichilia glabra L. aqueous leaf extract exert in vivo inhibitory activities in the zymosan-induced immediate inflammation. In this case the inflammatory response is mediated by PMN and complement (Schalkwijk, et al., 1985). First, the activation of the alternative complement pathway by zymosan generates vasodilatation and swelling while producing chemotactic factors that recruits PMN to the affected zone. These cells liberate reactive oxygen compounds generating tissular damage thus increasing inflammation. T. glabra L. extract significantly diminished in a dose dependent way the induced paw oedema in treated mice. These results agree with those observed by other authors who worked on Meliaceae plants used in folk medicine for the
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treatment of rheumatic and inflammatory diseases. Particularly, Cedrela lilloi L., Trichilia elegans L. and Melia azedarach L. extracts were able to diminish in a dose dependent way the inflammation caused during delayed-type hypersensitivity responses (Nores et al., 1997; Courre`ges et al., 1998). According to the in vivo results we decide to investigate the in vitro effect of the extract over murine complement and PMN. First, the extract was capable to diminish in a dose dependent way the in vitro hemolytic activity of the alternative complement pathway. No attempts were done in order to investigate the effect on the classical pathway since, in the in vivo model, only the alternative pathway is responsible for the observed inflammation. Similar anticomplementary activities were previously obtained with aqueous extracts of other Meliaceae plants such as Azadirachta indica (Van der Nat et al., 1987), Mumromia pumila (Labadie et al., 1989), Wia azederach L., Cedrela tubitiora Bert., C. filloi L. and T. elegans L. (Benencia et al., 1994; Nores et al., 1997). The anticomplementary activities observed with T. glabra L. extracts on AP pathway can be attributed either to complement inhibition or to complement consumption. Further studies will be needed to comprise the mechanism(s) involved in such anticomplementary behaviour of T. glabra L. extract. Phytochemical studies were carried out in our laboratory to establish the chemical. nature of the components responsible for the anticomplementary activity reported here. The fractionation procedure was followed by testing the biological activity of each fraction. T. glabra L. leaves were extracted sequentially in a Soxhlet with chloroform, methanol and water. The aqueous extract was poured into ethanol obtaining a precipitate which was dissolved in water and dialysed obtaining a crude polysaccharide fraction. After applying this fraction to DEAE Sephadex A-25 resin, the supernatant and washings obtained were combined to constitute the non-bound fraction (NB) solution. Elution of the bound material was performed in batch with NaCI solutions of increasing concentrations (0.1, 0.5 and 1.0 M in Tris-HCL buffer). The NB solution, selected due to its higher anticomple-
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mentary activity, was dialysed, concentrated and applied to a Sepharose CL-213 column. According to the carbohydrate elution pattern six peaks were obtained: N131-6. Only NB4 showed anticomplementary activity. The estimated M, of the polysaccharide active fraction was 3,16 x 1C D. Polysaccharides with anticomplementary activity were also found in A. indica and M. pumila (Labadie et al., 1989). On the other hand, T. glabra L. extract significantly reduced the in vitro activity of the other component of the immediate inflammation. Both the phagocytic activity and burst oxidative response of PMN leukocytes was impaired by extract treatment. It is noteworthy to comment that in other members of the same family such as Azadirachta indica A. Juss, aqueous extracts were capable of inhibiting luminol-enhaced chemiluminiscence of human PMN leukocytes (Van der Nat et al., 1987) while Melia azedarach L. extracts inhibited the phagocytic capability and luminolenhaced cherniluminiscence of murine peritoneal exudate cells (Courre`ges et al., 1994). Inhibition of monocyte-macrophage phagocytosis have also been reported for Cedrela tubiflora Bert., C. lilloi L., T. glabra L. and T. elegans L. leaf aqueous extracts (Benencia et al., 1994; Nores et al., 1997; Benencia and Coulombie´, 1998). Chemical studies
were performed in order to isolate the antiphagocytic and burst response inhibiting principle. We determined that It can be recovered from dried leaves by hot extraction with organic solvents. Plant material was dessicated and sucessively extracted in a Soxhlet apparatus with chloroform and methanol. The organic extract was concentrated at reduced pressure and then applied to a Silica Gel 60 column which was sucessively eluted with two volumes of different proportions of a chloroform: methanol mixture (95:5, 90:10 and 80:20). Each fraction was evaporated and resuspended in distilled water in order to determine their antiphagocytic activity. A single active fraction was eluted with the 95:5 mixture. This fraction was evaporated at reduced pressure, resuspended in methanol and applied to a LH-20 column which was eluted with methanol. A peak with antiphagocytic activity was obtained. This fraction was subjected to silica gel preparative thin layer chromatography (TLC) using a 95:5 chloroform: methanol mixture as eluant. Seven fractions with different migration patterns were obtained. One of them with an Rf value 0. 12 proved to be bioactive. This fraction was chromatographed in a TLC system using a mixture of ethyl-acetate: chloroform (11) as solvent, and the activity was eluted as a single band in this system.
Table 1 Effect of T. glabra extract on the phagocytic capability and the oxidative metabolism of polymorphonuclear leukocytesa Extract conc. (mg/ml)
2.00 1.00 0.50 0.25 0.12 0.00
% of phagocytic cellsb
18.4 92.1* 21.3 95.3* 23.9 91.2* 87.1 93.8(n.s.) 91.2 96.4 (n.s.) 94.4 92.7
% NBT positive cellsc Zymosan
PMA
14.7 96.2 17.5 9 6.7 20.8 94.1 80.5 98.5 (n.s.) 84.296.7 (n.s.) 89.4 93.8
31.6 9 8.3* 35.8 97.4* 54.7 9 3.6* 78.896.2 (n.s.) 82.295.1 (n. s.) 84.5 9 3.6
a The phagocytic capability and oxidative metabolism of the PMN cells were tested after 45 min of incubation with different concentrations of T.glabra extract (treated cells) or with medium alone (control). Values are expressed as mean 9SD of three independent measurements b % Phagocytic cells = (No of positive cells/ No of total cells)×100, positive cells were those having at least 4 internalized zymosan particles. c % NBT positive cells = (No of positive cells/ No of total cells)×100, where positive cells were those containing intracellular precipitated formazan. Cells were stimulated with zymosan or PMA. * Statistical differences vs. control: PB0.01
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An absorption spectrum of this fraction revealed a single absorption peak at 220 rim, thus indicating a peptidic nature of the active principle. This also agrees with what is observed in A. indica, where the immunosuppressive activity over phagocytic cells is exerted by small organic compounds (Van der Nat et al., 1992). Taking into account all these data, it is possible that the in vivo anti-inflammatory effect may be the result of the action of different compounds present in the extract affecting different components of the immune system. References Andrei, G.M., Coulombie´, F.C., Courre`ges, M.C., de Torres, R.A., Coto, C.E., 1990. Meliacine, an antiviral compound from Melia azedarach L. inhibits interferon production. Journal of Interferon Research 10, 469–475. Badwey, J.A., Robinson, J.M., Lazdens, J.K., Briggs, R.T., Karnowsky, M.J., Karnowsky, M.L., 1983. Comparative biochemical and cytochemical studies on superoxide and peroxide in mouse macrophages. Journal of Cell Physiology 115, 208 – 216. Benencia, F., Courre`ges, M.C., Coulombie´, F.C., 1994. Effect of Melia azedarach L. leaf extracts on human complement and polymorphonuclear leukocytes. Journal of Ethnopharmacology 41, 53 – 57. Benencia, F., Courre`ges, M.C., Nores, M.M., Coulombie´, F.C., 1995. Immunomodulatory activities of Cedrela tubiflora leaf aqueous extracts. Journal of Ethnopharmacology 49, 133 – 139. Benencia, F., Courre`ges, M.C., Coulombie´, F.C., 1996. In vitro activities of Cedrela tubiflora aqueous leaf extracts on murine macrophages, polymorphonuclear leukocytes and complement. Phytotherapy Research 10, 37–41. Benencia, F., Coulombie´, F.C., 1998. Immunomodulatory activities of Trichilia glabra leaf aqueous extracts. Phytotherapy Research 1213, 167–171. Bhakuni, D.S, Dhar, M.L., Dhawan, B.N., 1969. Screening of Indian plants for biological activity. Part 11. Indian Journal of Experimental Biology 7, 250–262. Bray, D.H., Warhurst, D.C., Connolly, J.D., O’Neill, M.J.L., 1990. Plants as sources of antimalarial drugs. Part 7. Activity of some species of Meliaceae plants and their constituent limonoids. Phytotherapy Research 4, 29–35. Coulombie´, F.C., Andrei, G.M., Laguens, R.P., de Torres, R.A., Coto, C.E., 1992. Partially purified leaf extracts of Melia azedarach L. inhibit Tacaribe virus growth in neonatal mice. Phytotherapy Research 6, 15–19. Courre`ges, M.C., Benencia, F., Coto, C.E, Massouh, E.J, Coulombie´, F.C., 1994. In vitro antiphagocytic effect of Melia azedarach L. leaf extracts on mouse peritone & exudate cells. Journal of Ethnopharmacology 43, 135–140.
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Courre`ges, M.C., Benencia, F., Coulombie´, F.C., Coto, C.E., 1998. Immunomodulatory activity of Melia azedarach L. Aqueous leaf extracts on murine lymphocytes. Phytomedicine 5 (1), 47 – 53. Fujiwara, T., Takeda, T., Ogihara, Y., Shimizu, M., Nomura, T., 1982. Studies on the structure of polysaccharides from the bark of Melia azedarach. Chemical and Pharmaceutical Bulletin 30, 4025 – 4030. Klerx, J.P.A.M., Beukelman, C.J., Van Dijk, H., Willers, J.M.N., 1983. Microassay for colorimetric estimation of complement activity in guinea pig, human and mouse serum. Journal of lmmunological Methods 63, 215 – 220. Klerx, J.P.A.M., Van Dijk, H., Van der Waden, W.J., Willers, J.M.N., 1985a. Analytic study of the differential anticomplementary effects of dextran sulphate and heparin in the assay for the mouse alternative pathway. International Archives of Allergy and Applied Immunology 78, 182 – 189. Klerx, J.P.A.M., Van Oosterhout, A.J.M., Van Dijk, H., Nouwenberg, E.A., Willers, J.M.N., 1985b. Anti-complementary amines are immunological adjuvants in mice. Immunology Letters 10, 281 – 286. Labadie, R.P., Van der Nat, J.M., Simmons, J.M., Kroes, B.H., Kosasi, S., Van Der Berg, A.J.J., ’t Hart, L.A., Van der Sluis, W.G., Abeyesera, A., Bamunarachchi, A., De Silva, K.T.D., 1989. An ethnopharmacognostic approach to the search for immunomodulators of plant origin. Planta Medica 55, 339 – 348. Losche, W., Michel, E., Hepinstall, S., Groenewegen, W.A., Pescarmona, G.P., Thielmann, K., 1988. Inhibition of the behavior of human polymorphonuclear leukocytes by an extract of Chrysanthemum parthenium. Planta Medica 54, 381 – 384. Mayer, M.M. and Kabat, E.A. (Eds.), 1961. Experimental Immunochemistry. Springfield IL, p. 133. Nores, M.M., Courre`ges, M.C., Benencia, F., Coulombie´, F.C., 1997. Immunomodulatory activity of Cedrele lilloi and Trichilia elegans aqueous leaf extracts. Journal of Ethnopharmacology 55, 99 – 106. Patel, N.G., 1986. Indians traditional medicine: Ayurveda. In: Steiner, R.P. (Ed.), Folk medicine: the art and science. American Chemical Society Publications, Washington DC, p. 157. Rademaker, P.M., Van Dijk, H., Willers, J.M.N., 1981. Involvement of membrane-associated sialic acid in the resistance of sheep erythrocytes to lysis by mouse complement. Immunology Letters 3, 221 – 225. Schalkwijk, J., van den Berg, W.B., van de Putte, L.B.A., Joosten, L.A.B., van der Sluis, M., 1985. Effects of experimental joint inflammation on bone marrow and periarticular bone. A study of two types of arthritis, using a variable degrees of inflammation. British Journal of Experimental Pathology 66, 435 – 443. Van der Nat, J.W., Klerx, J.P.A.M., Van Dijk, H., De Silva, K.T.D., Labadie, R.P., 1987. Immunomodulatory activity of an aqueous extract of Azadirachta indica stem bark. Journal of Ethnopharmacology 19, 125 – 131.
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F. Benencia et al. / Journal of Ethnopharmacology 71 (2000) 293–300 Van Dijk, H., Rademaker, P.M., Willers, J.M.N., 1980. Estimation of classical pathway of mouse complement activity by use of sensitized rabbit erythrocytes. Journal of Immunological Methods 39, 257 – 268. Wilkinson, P.C., 1981. Neutrophil function test. In: Thompson, R.A. (Ed.), Techniques in Classical Immunology. Blackwell Scientific Publications, USA, p. 283.
Van der Nat, J.W., Van der Sluis, L.A., ’t Hart, L.A., Van Dijk, H., de Silva, K.T.D., Labadie, R.P., 1992. Activity guided isolation and identification of Azadirachta indica bark extract constituents which specifically inhibit chemiluminiscence production by activated human polymorphonuclear leukocytes. Planta Medica 57, 65– 68.
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Anti-inflammatory activities of Trichilia glabra aqueous leaf extract Fabia´n Benencia *, Marı´a Cecilia Courre`ges, Fe´lix Carlos Coulombie´ Laboratory of Virology, Faculty of Science, Uni6ersity of Buenos Aires, Pabello´n II, Piso 4, Ciudad Uni6ersitaria, (1428) Nun˜ez, Buenos Aires Argentina Received 3 May 1999; received in revised form 7 February 2000; accepted 14 February 2000
Abstract Trichilia glabra L. aqueous leaf extract exerted a significant antiinflammatory effect ‘in vivo’ in the zymosan-induced inflammation model. The extract impaired the ‘in vitro’ activities of polymorphonuclear leukocytes and complement, components of mouse immune system closely related to the inflammatory response induced by zymosan. In particular, a significant reduction in the phagocytic capability and respiratory burst response of mouse polymorphonuclear leukocytes together with an inhibition in the hemolytic activity of mouse complement was observed. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Anti-inflammatory activity; Trichilia glabra; Aqueous extract; Zymosan-induced inflammation complement; Neutrophils
1. Introduction Plants belonging to Meliaceae family have been widely used in ethnomedicine. Antiviral, antihelmintic, anti-inflammatory and antirheumatic activities have been reported (Bhakuni et al., 1969; Fujiwara et al., 1982; Patel, 1986; Andrei et al., 1990; Bray et al., 1990; Coulombie´ et al., 1992). The anti-inflammatory and antirheumatic properties of some members of this family, such as Azedirachta indica A. Juss, Mefia azedarach L. and Cedrela tubifibra Bert. have been explained by their action on the immune response (Labadie et al., 1989; Courre`ges et al., 1994). In previous * Corresponding author. Fax: +54-1-7820458. E-mail address: [email protected] (F. Benencia)
reports we observed that aqueous leaf extracts of Melia azedarach L, Cedrela tubifibra Bert., Cedrela lilloi L. and Trichilia elegans L. exerted inhibitory activities on both murine complement activation and phagocytosis mediated by mouse peritoneal exudate cells and PMN leukocytes (Benencia et al., 1996; Nores et al., 1997). A significant diminution in the delayed-type hypersensitivity response of mice have been reported for Cedrela lilloi L., Trichifia elegans L. and Melia azedarach L. (Nores et al., 1997; Courre`ges et al., 1998). Besides, we have found that Cedrela tubffiora Bert. and Trichilia glabra L. extracts also interfered on some mechanisms related with the human immune function such as: the hemolytic activity of human complement, Tlymphocytes proliferation, phagocytic capability
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and oxidative metabolism of peripheral blood monocytes and polymorphonuclear leukocytes (Benencia et al., 1995; Benencia and Coulombie´, 1998). In this report we describe the effect of aqueous leaf extract of the Meliaceae tree Trichilia glabra L. on the zymosan-induced immediate inflammation in the mouse model, and the in vitro phagocytic activity and burst respiratory response of polymorphonuclear ieukocytes and the activation of the complement system, elements of the immune response strongly related to inflammatory processes induced by zymosan.
2. Materials and methods
2.1. Animals Inbred BALB/c mice, 8 – 10 weeks old, reared in our own animal house, were used in all experiments. Mice were housed 5 per cage with sterile wood chip bedding and provided with chow pellets and tap water ad libitum. The animals quarters were maintained at 21 – 240°C, and 40 – 60% humidity with a 12 h light-dark cycle.
2.2. Preparation of plant extract T. glabra L. fresh green leaves were collected in Buenos Aires city in November 1997 and identified at the Department of Botany, Faculty of Agronomy and Veterinary, University of Buenos Aires where a voucher specimen is deposit (Argentina BAA 2722). 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 materiallmi). The sap obtained was filtered through cheesecloth and then darified by centrifugation at 10.000 x g for 30 min. The final extract had a concentration of 40 mg of dried plant material/ml as determined by lyophilization.
2.3. Hemolytic assay for the study of the alternati6e pathway of mouse complement The inhibition of the alternative pathway (AP)
activity of mouse complement by T. glabra L. extract was determined in sera of BALB/c mice by using the method of Van Dijk et al. 1980. Mice were anesthetized with ether and bled from the retro-orbital venous plexus by means of capillary tubes and serum was separated by centrifugation. Serum pooled from at least ten animals were tested for complement activities. The serum concentration giving rise to 50% haemolysis of the target cells (1 AP50 unit) was calculated by means of the Von Krogh equation (Mayer et al., 1961). So the hemolytic activity of mouse serum is expressed in AP50 units/ml. For determining the anticomplementary activity of T. glabra extract, undiluted sera were mixed with different concentrations of the extract (0.25–10 mg/ml) and incubated for 30 min at 370C. The dilutions were made in Veronal saline buffer (VSB) containing 5mM veronal and 150 mM NaCI at pH 7.4 supplemented with 8 mM ethyleneglycol-bis (2–aminoethyl) tetraacetic acid. The residual hemolytic complement activity was determined by a method using normal rabbit erythrocytes as target cells (Van Dijk et al., 1980; Rademaker et al., 1981; Klerx et al., 1983, 1985a,b). The AP activity in the presence of the extract was read from the Von Krogh plots. Thus, the activity was obtained as AP50 units/mi and transformed to percentages of inhibition with respect to the activity of serum tested in absence of the extracts. A water lysed control (100% lysis) and a buffer control (0% lysis) were used. Heparin was used as a positive control
2.4. 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 coverAps for 1 h 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 at 37°C with minimum essential medium (MEM) (GIBCO) alone (control) or different con-
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centrations of plant extract in the same medium. Subsequently, phagocytic activity and respiratory burst of these cells were evaluated.
control= [average thickness (in mm) of the footpad of treated animals/average thickness (in mm) of the footpad of control animals]× 100.
2.5. Phagocytic assay
2.8. Statistics
The phagocytic activity of treated or control cells was tested by zymosan particles (Sigma Chemical Co., MO). After treatment with T. glabra L. extract, cell cultures were challenged with a suspension of zymosan containing 2 x 106 opsonised particles/ml particles. Two hundred cells were scored in each coverslip and the percentage of phagocytic cells was recorded. Unspecific ingestion was discarded considering positive only those cells that have ingested more than 4 particles (Losche et al., 1988).
The statistical significance of the data was determined by Student’s t-test. A P value lesser than 0.05 was taken as significant.
2.6. Qualitati6e nitroblue tetrazolium (NBT) reduction assay After treatment with T. glabra extract, PMN cultures were incubated for 30 min at 370°C in a 5% CO2 atmosphere with a suspension of 2 x 106 opsonised zymosan particles/ml or 0.5 MM phorbol 12-myristate 13-acetate (Sigma Chemical Co., Mo.) (PMA) in MEM containing 0.5 mg/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-black by precipitated formazan, the oxygen dependent reduction product of NBT (Wilkinson, 1981). At least 200 cells were scored for each experiment.
2.7. Zymosan-induced inflammation The effect of T. glabra L. extract on zymosaninduced inflammation was studied in groups of 10 mice. Inflammation was induced by injecting 300 mg of zymosan suspended in 25 ml of sterilized phosphate buffered saline (PBS) in the left hind foot pad. Different concentrations (1, 2 and 4 mg/ml) of T. glabra extract in PBS (final volume: 0.2 ml) were injected intravenously 6 and 3 hours before zymosan injection. Control mice were injected with P13S. Foot-pad swelling was measured 6 hs after induction with a precision caliper. Data were registered as: % of inhibition with respect to
3. Results
3.1. Anti-inflammatory acti6ity of T. glabra L. extract in the zymosan-induced immediate inflammation model In order to investigate the in vivo effect of the extract we used the zymosan-induced immediate inflammation model. Herewith, a significant inflammation in the paw is observed 3 h after zymosan inoculation. Animals were intraperitoneally treated with different concentrations (14 mg/ml) of T glabra extract 6 and 3 h previous to zymosan administration. As shown in Fig. 1, treatment with the extract significantly reduced the zymosan-induced oedema. These effect was dose dependent, obtaining nearly a 50% reduction in inflammation with the highest extract concentration tested (4 mg/ml).
3.2. Anticomplementary effect of T. glabra L. extract The activation of murine alternative complement pathway was studied with a method that uses normal rabbit erythrocytes as target cells. As shown in Fig. 2, preincubation of mouse serum with different concentrations of the extract caused a dose dependent reduction in the hemolytic activity of the serum, being 5.1 mg/ml the extract concentration capable to inhibit this activity to 50% (IC50%). As a positive control heparin, a sulphated polysaccharide with known anticomplementary activity (Klerx et al., 1985a), was used. Although this compound was also capable of inhibiting the serum hemolytic activity this effect was less pronounced than that exerted by T.
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glabra L. extract (Fig. 2). Preincubation of the target cells with the extract did not prevent complement mediated lysis (data not shown) discounting a protective effect of T. glabra extract on the erythrocyte membrane. Heat inactivated serum of Balbic mice showed no hemolytic activity.
3.3. Effect of T. glabra extract on PMN leukocytes acti6ities PMN cultures were incubated for 45 min with different concentrations of extract (0.12 – 2 mg/
ml). The effect of treatment on the phagocytic capability and the NBT reduction capacity of murine PMN leukocytes is shown in Table 1. A significant decrease in both the phagocytic capability and the respiratory burst response of these cells was observed with the higher extract concentrations tested (0.5, 1 and 2 mg/ml). Similar results were obtained when a post-receptor stimulus (PMA) was employed (Table 1). No significant differences in the total number of viable cells between control (92.009 4.50%) and T. glabra L. extract treated cultures (89.009 5.30%) were observed according to the trypan blue exclusion method.
Fig. 1. Effect of T. glabra aqueous extract on acute foot-pad swelling in mice induced by zymosan. Groups of 10 animals were inoculated endovenously with different concentrations of the extract (1, 2 and 4 mg/ml) in saline (final volume: 0.1 ml) or equal volume of saline (control). These doses were administrated 6 and 3 h previous to zymosan inoculation in the right foot-pad (0 time). Six hours later food pad thickness was measured in treated and control animals.* P B 0.05.
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Fig. 2. Anticomplementary effect of T glabra extract. Mouse serum was incubated for 30 min with different concentrations of T. glabra extract (initial extract concentration: 40 mg of dried plant materiallmi as determined by lyophilization) or heparin in VS13 before the addition of normal rabbit erythrocytes. After treatment, remaining hemolytic capacity was titrated and expressed as percentage of inhibition respect to the activity of untreated sera.
4. Discussion The present study shows that Trichilia glabra L. aqueous leaf extract exert in vivo inhibitory activities in the zymosan-induced immediate inflammation. In this case the inflammatory response is mediated by PMN and complement (Schalkwijk, et al., 1985). First, the activation of the alternative complement pathway by zymosan generates vasodilatation and swelling while producing chemotactic factors that recruits PMN to the affected zone. These cells liberate reactive oxygen compounds generating tissular damage thus increasing inflammation. T. glabra L. extract significantly diminished in a dose dependent way the induced paw oedema in treated mice. These results agree with those observed by other authors who worked on Meliaceae plants used in folk medicine for the
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treatment of rheumatic and inflammatory diseases. Particularly, Cedrela lilloi L., Trichilia elegans L. and Melia azedarach L. extracts were able to diminish in a dose dependent way the inflammation caused during delayed-type hypersensitivity responses (Nores et al., 1997; Courre`ges et al., 1998). According to the in vivo results we decide to investigate the in vitro effect of the extract over murine complement and PMN. First, the extract was capable to diminish in a dose dependent way the in vitro hemolytic activity of the alternative complement pathway. No attempts were done in order to investigate the effect on the classical pathway since, in the in vivo model, only the alternative pathway is responsible for the observed inflammation. Similar anticomplementary activities were previously obtained with aqueous extracts of other Meliaceae plants such as Azadirachta indica (Van der Nat et al., 1987), Mumromia pumila (Labadie et al., 1989), Wia azederach L., Cedrela tubitiora Bert., C. filloi L. and T. elegans L. (Benencia et al., 1994; Nores et al., 1997). The anticomplementary activities observed with T. glabra L. extracts on AP pathway can be attributed either to complement inhibition or to complement consumption. Further studies will be needed to comprise the mechanism(s) involved in such anticomplementary behaviour of T. glabra L. extract. Phytochemical studies were carried out in our laboratory to establish the chemical. nature of the components responsible for the anticomplementary activity reported here. The fractionation procedure was followed by testing the biological activity of each fraction. T. glabra L. leaves were extracted sequentially in a Soxhlet with chloroform, methanol and water. The aqueous extract was poured into ethanol obtaining a precipitate which was dissolved in water and dialysed obtaining a crude polysaccharide fraction. After applying this fraction to DEAE Sephadex A-25 resin, the supernatant and washings obtained were combined to constitute the non-bound fraction (NB) solution. Elution of the bound material was performed in batch with NaCI solutions of increasing concentrations (0.1, 0.5 and 1.0 M in Tris-HCL buffer). The NB solution, selected due to its higher anticomple-
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mentary activity, was dialysed, concentrated and applied to a Sepharose CL-213 column. According to the carbohydrate elution pattern six peaks were obtained: N131-6. Only NB4 showed anticomplementary activity. The estimated M, of the polysaccharide active fraction was 3,16 x 1C D. Polysaccharides with anticomplementary activity were also found in A. indica and M. pumila (Labadie et al., 1989). On the other hand, T. glabra L. extract significantly reduced the in vitro activity of the other component of the immediate inflammation. Both the phagocytic activity and burst oxidative response of PMN leukocytes was impaired by extract treatment. It is noteworthy to comment that in other members of the same family such as Azadirachta indica A. Juss, aqueous extracts were capable of inhibiting luminol-enhaced chemiluminiscence of human PMN leukocytes (Van der Nat et al., 1987) while Melia azedarach L. extracts inhibited the phagocytic capability and luminolenhaced cherniluminiscence of murine peritoneal exudate cells (Courre`ges et al., 1994). Inhibition of monocyte-macrophage phagocytosis have also been reported for Cedrela tubiflora Bert., C. lilloi L., T. glabra L. and T. elegans L. leaf aqueous extracts (Benencia et al., 1994; Nores et al., 1997; Benencia and Coulombie´, 1998). Chemical studies
were performed in order to isolate the antiphagocytic and burst response inhibiting principle. We determined that It can be recovered from dried leaves by hot extraction with organic solvents. Plant material was dessicated and sucessively extracted in a Soxhlet apparatus with chloroform and methanol. The organic extract was concentrated at reduced pressure and then applied to a Silica Gel 60 column which was sucessively eluted with two volumes of different proportions of a chloroform: methanol mixture (95:5, 90:10 and 80:20). Each fraction was evaporated and resuspended in distilled water in order to determine their antiphagocytic activity. A single active fraction was eluted with the 95:5 mixture. This fraction was evaporated at reduced pressure, resuspended in methanol and applied to a LH-20 column which was eluted with methanol. A peak with antiphagocytic activity was obtained. This fraction was subjected to silica gel preparative thin layer chromatography (TLC) using a 95:5 chloroform: methanol mixture as eluant. Seven fractions with different migration patterns were obtained. One of them with an Rf value 0. 12 proved to be bioactive. This fraction was chromatographed in a TLC system using a mixture of ethyl-acetate: chloroform (11) as solvent, and the activity was eluted as a single band in this system.
Table 1 Effect of T. glabra extract on the phagocytic capability and the oxidative metabolism of polymorphonuclear leukocytesa Extract conc. (mg/ml)
2.00 1.00 0.50 0.25 0.12 0.00
% of phagocytic cellsb
18.4 92.1* 21.3 95.3* 23.9 91.2* 87.1 93.8(n.s.) 91.2 96.4 (n.s.) 94.4 92.7
% NBT positive cellsc Zymosan
PMA
14.7 96.2 17.5 9 6.7 20.8 94.1 80.5 98.5 (n.s.) 84.296.7 (n.s.) 89.4 93.8
31.6 9 8.3* 35.8 97.4* 54.7 9 3.6* 78.896.2 (n.s.) 82.295.1 (n. s.) 84.5 9 3.6
a The phagocytic capability and oxidative metabolism of the PMN cells were tested after 45 min of incubation with different concentrations of T.glabra extract (treated cells) or with medium alone (control). Values are expressed as mean 9SD of three independent measurements b % Phagocytic cells = (No of positive cells/ No of total cells)×100, positive cells were those having at least 4 internalized zymosan particles. c % NBT positive cells = (No of positive cells/ No of total cells)×100, where positive cells were those containing intracellular precipitated formazan. Cells were stimulated with zymosan or PMA. * Statistical differences vs. control: PB0.01
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An absorption spectrum of this fraction revealed a single absorption peak at 220 rim, thus indicating a peptidic nature of the active principle. This also agrees with what is observed in A. indica, where the immunosuppressive activity over phagocytic cells is exerted by small organic compounds (Van der Nat et al., 1992). Taking into account all these data, it is possible that the in vivo anti-inflammatory effect may be the result of the action of different compounds present in the extract affecting different components of the immune system. References Andrei, G.M., Coulombie´, F.C., Courre`ges, M.C., de Torres, R.A., Coto, C.E., 1990. Meliacine, an antiviral compound from Melia azedarach L. inhibits interferon production. Journal of Interferon Research 10, 469–475. Badwey, J.A., Robinson, J.M., Lazdens, J.K., Briggs, R.T., Karnowsky, M.J., Karnowsky, M.L., 1983. Comparative biochemical and cytochemical studies on superoxide and peroxide in mouse macrophages. Journal of Cell Physiology 115, 208 – 216. Benencia, F., Courre`ges, M.C., Coulombie´, F.C., 1994. Effect of Melia azedarach L. leaf extracts on human complement and polymorphonuclear leukocytes. Journal of Ethnopharmacology 41, 53 – 57. Benencia, F., Courre`ges, M.C., Nores, M.M., Coulombie´, F.C., 1995. Immunomodulatory activities of Cedrela tubiflora leaf aqueous extracts. Journal of Ethnopharmacology 49, 133 – 139. Benencia, F., Courre`ges, M.C., Coulombie´, F.C., 1996. In vitro activities of Cedrela tubiflora aqueous leaf extracts on murine macrophages, polymorphonuclear leukocytes and complement. Phytotherapy Research 10, 37–41. Benencia, F., Coulombie´, F.C., 1998. Immunomodulatory activities of Trichilia glabra leaf aqueous extracts. Phytotherapy Research 1213, 167–171. Bhakuni, D.S, Dhar, M.L., Dhawan, B.N., 1969. Screening of Indian plants for biological activity. Part 11. Indian Journal of Experimental Biology 7, 250–262. Bray, D.H., Warhurst, D.C., Connolly, J.D., O’Neill, M.J.L., 1990. Plants as sources of antimalarial drugs. Part 7. Activity of some species of Meliaceae plants and their constituent limonoids. Phytotherapy Research 4, 29–35. Coulombie´, F.C., Andrei, G.M., Laguens, R.P., de Torres, R.A., Coto, C.E., 1992. Partially purified leaf extracts of Melia azedarach L. inhibit Tacaribe virus growth in neonatal mice. Phytotherapy Research 6, 15–19. Courre`ges, M.C., Benencia, F., Coto, C.E, Massouh, E.J, Coulombie´, F.C., 1994. In vitro antiphagocytic effect of Melia azedarach L. leaf extracts on mouse peritone & exudate cells. Journal of Ethnopharmacology 43, 135–140.
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