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Canscora decussata (Roxb.) Schult (Gentianaceae) inhibits LPS-induced expression of ICAM-1 and E-selectin on endothelial cells and carageenan-induced paw-edema in rats
Journal of Ethnopharmacology 89 (2003) 211–216
Canscora decussata (Roxb.) Schult (Gentianaceae) inhibits LPS-induced expression of ICAM-1 and E-selectin on endothelial cells and carageenan-induced paw-edema in rats Babita Madan, B.C. Mandal, Sarvesh Kumar, B. Ghosh∗ Molecular Immunology and Immunogenetics Laboratory, Institute of Genomics and Integrative Biology, University of Delhi Campus (North), Mall Road, Delhi 110007, India Received 11 March 2003; received in revised form 10 July 2003; accepted 25 July 2003
Abstract Inflammation is characterized pathologically by an increased supply of blood to the affected area, vasodilation, and infiltration of phagocytic, monocytic, and polymorphonuclear cells into the site of inflammation. The infiltration of leukocytes is in part regulated by the increased expression of cell adhesion molecules viz. ICAM-1 and E-selectin on endothelial cells. We investigated the effect of ethanolic extract prepared from plant Canscora decussata on expression of ICAM-1 and E-selectin on endothelial cells. Our results demonstrated that treatment of human endothelial cells with the extract down-regulated the LPS-induced expression of ICAM-1 and E-selectin. To study further the anti-inflammatory activity of the extract, we evaluated its effect in a rat model of carageenan-induced paw-edema. Our results showed that the extract was able to reduce the edema formation in a dose-dependent manner. These results, therefore, indicate that Canscora decussata extract could be useful towards developing effective anti-inflammatory agent(s) in the future. © 2003 Published by Elsevier Ireland Ltd. Keywords: Canscora decussata; Paw-edema; Carageenan; Anti-inflammatory; ICAM-1; E-selectin; Endothelial cells
1. Introduction Inflammation normally a localized defensive process is caused by soluble antigen, live organisms, and chemical or mechanical stress upon tissue, which serves to destroy and/or dilute the injurious materials, and remove the injured tissues. It is characterized pathologically by an increased supply of blood to the affected area, increased capillary permeability caused by retraction of the endothelial cells and infiltration of phagocytic, monocytic and polymorphonuclear cells into the site of tissue insult. The accumulation and subsequent activation of leukocytes is one of the central events in the pathogenesis of all forms of inflammation. Leukocytes migrate to and accumulate at the site of inflammation by locally produced chemoattractants, and are then activated to secrete granular contents and to release active
Abbreviations: ICAM-1, intercellular adhesion molecule-1; LPS, lipopolysaccharide; TNF-␣, tumor necrosis factor-␣; IL-1, interleukin1; CdEE, Canscora decussata ethnolic extract ∗ Corresponding author. Tel.: +91-11-2766-7602; fax: +91-11-2766-7471. E-mail address: [email protected] (B. Ghosh). 0378-8741/$ – see front matter © 2003 Published by Elsevier Ireland Ltd. doi:10.1016/S0378-8741(03)00281-2
oxygen metabolites during chemotaxis and phagocytosis. These active metabolites cause damage to the underlying tissue leading to inflammation. The migration of the leukocytes to the site of inflammation is regulated in part by the expression of cell adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and E-selectin (Springer, 1994). These cell adhesion molecules are induced on endothelial cells by various pro-inflammatory cytokines like IL-1 and TNF-␣ and also by bacterial LPS (reviewed by Mantovani et al., 1997). It is well established that in various inflammatory diseases, the expression of these proteins is upregulated on endothelial cells (Calderon and Lockey, 1992; Gorski, 1994). Inhibition of these molecules using specific monoclonal antibodies (mAbs) has been found to be beneficial for controlling various inflammatory diseases (Gorski, 1994; Weiser et al., 1997). Canscora decussata (Gentianaceae), popularly known as shankhpushpi is a herb and is used as a laxative and a tonic. Several medicinal properties have been attributed to it in the traditional medicine in India. The crude dried powder of the herb and its ethanolic extract has shown anti-convulsant activity (Dikshit et al., 1972). The ethanolic
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extract of the herb lowers blood pressure in albino rats and also shows stimulant action on the smooth muscle of intestine, uterus, and bronchus. It also has spermicidal activity in rats (Ramachandran, 1992). Also the purified xanthones from Canscora decussata have shown inhibitory activity against Mycobacterium tuberculosis H37 RV (Ghosal et al., 1978). No studies have, however, been undertaken to explore the anti-inflammatory activity of this plant. In the present study, we investigated the ability of extract prepared from the plant Canscora decussata on expression of ICAM-1 and E-selectin on endothelial cells and on inflammatory response in rat model of carageenan-induced paw-edema. Our results show that an extract of Canscora decussata inhibits the expression of ICAM-1 and E-selectin on endothelial cells and is also effective in vivo as it reduces the edema formation in rats in carageenan-induced paw-edema assay.
2. Methodology 2.1. Materials TNF-␣, anti-ICAM-1, and anti-E-selectin antibodies were purchased from Pharmingen, CA. M199, l-glutamine, penicillin, streptomycin, amphotericin, endothelial cell growth factor, trypsin, Pucks saline, HEPES, o-phenylenediamine dihydrochloride and anti-mouse IgG-HRP were purchased from Sigma Chemical Co., USA. Carrageenan was purchased from S.D. Fine Chemical Ltd., India. 2.2. Preparation of Canscora decussata extract Dried powdered rhizomes of Canscora decussata (100 mg) was macerated in 4 ml of ethanol:water (1:1) overnight at 25 ◦ C under shaking and the percolate was collected. Maceration and percolate collection was continued for 2 days. The percolate was pooled and vacuum dried. The dried material (40 mg) was resuspended in 400 l 50% ethanol before use and is designated CdEE. 2.3. Cells and cell culture Primary endothelial cells were isolated from umbilical cord by mild trypsinization (Gupta and Ghosh, 1999). The endothelial cells obtained were maintained in M199 medium supplemented with 20% heat inactivated fetal calf serum, 2 mM l-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, 0.25 g/ml amphotericin, endothelial cell growth factor (50 g/ml) and heparin (5 U/ml). The cells were subcultured by dislodging with 0.125% trypsin–0.01 M EDTA solution in Pucks saline and HEPES buffer. The cells were used between passages three to four. The viability of cells was determined by trypan blue exclusion test and the pu-
rity of endothelial cells were determined by E-selectin expression. 2.4. Modified ELISA for measurement of ICAM-1 and E-selectin For measuring the expression of ICAM-1 on surface of endothelial cells cell-ELISA was used as described before (Madan et al., 2001). HUVECs, plated to confluence in 96-well plates were incubated with or without varying dilutions of CdEE for 1 h followed by treatment with LPS (1 g/ml) for 16 h for ICAM-1 or for 4 h for E-selectin expression. Following incubation, the cells were fixed with 1.0% glutaraldehyde, non-specific binding was blocked using non-fat dry milk (3.0% in PBS) and cells were incubated overnight at 4 ◦ C with antibodies to ICAM-1 or E-selectin or control IgG Ab (0.25 g/ml). This was followed by washing cells with PBS and incubation with peroxidase-conjugated goat anti-mouse secondary Ab (1 g/ml). The cells were again washed with PBS and exposed to the peroxidase substrate (o-phenylenediamine dihydrochloride 40 mg/100 ml in citrate phosphate buffer, pH 4.5). Colour development reaction was stopped by the addition of 2N sulfuric acid. Absorbance was determined at 490 nm by an automated microplate reader (Spectramax 190, Molecular Devices, USA). 2.5. Carageenan-induced paw-edema in rats Anti-inflammatory activity of Canscora decussata extract on carageenan-induced paw-edema in rats was determined by a mercury plethysmometer designed in the laboratory following the method of Sanches et al. (1998). For experiments male Sprague–Dawley rats weighing 125–160 g were taken. Animals were allowed free access to food and water throughout the experiment. Experimental protocol was approved by the institutional ethical committee. Edema was induced by subcutaneous injection of carageenan (0.1 ml of 1% solution w/v in 0.9% saline) into subplantar region of the left hind paw. The volume of each paw upto tibio-tarsal articulation was measured before the injection of carageenan, and 1.30, 3.00, and 4.30 h after the injection of carageenan. Rats were divided into five groups of four each: Group A, rats were injected with carageenan; Group B, rats were administered with phenylbutazone (50 mg/kg) intraperitoneally 1 h prior to carageenan injection; Group C, D, and E, rats were administered with 40, 55, and 70 mg/kg CdEE, respectively, intraperitoneally 1 h prior to carageenan injection. The inflammatory response is presented as increase in paw volume and inhibition percentage of edema according to the equation: %I =
VC − VT × 100 VC
where VC is arithmetic mean of the increase in paw volume in the control group. VT is arithmetic mean of the increase in paw volume in the treated group.
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Fig. 1. Concentration-dependent inhibition of LPS-induced ICAM-1 expression by CdEE: endothelial cells grown to confluence in 96-well plates were incubated without or with indicated dilutions of CdEE for 1 h prior to induction without or with LPS (1 g/ml) for 16 h. Following this, ICAM-1 level on the cells was measured by ELISA as described in Section 2. The data presented are representative of three independent experiments. Values shown are mean ± S.D. of quadruplicate wells.
3. Results 3.1. CdEE is non-toxic to endothelial cells To determine any possible toxic effect of CdEE on endothelial cells, the cells grown to confluence in 96-well plates were incubated with varying concentrations of CdEE
for 24 h. Viability of the cells was determined by trypan blue exclusion test and their morphology was observed under microscope. It was observed that the time of incubation (upto 24 h) and the concentrations of the extracts used in subsequent experiments did not affect the viability or morphology of the endothelial cells (data not shown).
Fig. 2. Concentration-dependent inhibition of LPS-induced E-selectin expression by CdEE: endothelial cells grown to confluence in 96-well plates were incubated without or with indicated dilutions of CdEE for 1 h prior to induction without or with LPS (1 g/ml) for 4 h. Following this, E-selectin level on the cells was measured by ELISA as described in Section 2. The data presented are representative of three independent experiments. Values shown are mean ± S.D. of quadruplicate wells.
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3.2. CdEE inhibits ICAM-1 expression on endothelial cells in a concentration-dependent manner To determine the effect of CdEE on the expression of ICAM-1 on endothelial cells, the cells were incubated with or without CdEE at the indicated concentrations for 1 h prior to treatment with LPS (1 g/ml) for 16 h. ICAM-1 was ex-
pressed at low levels on unstimulated endothelial cells and its expression was induced over fivefolds by LPS stimulation (Fig. 1). Interestingly, CdEE had no effect on the constitutively expressed levels of ICAM-1, whereas it led to a significant reduction (approximately 70%) in the LPS-induced ICAM-1 expression in a concentration-dependent manner (Fig. 1).
Fig. 3. (A) Effect of Canscora decussata extract on carageenan-induced paw-edema in rats: the rats in all the five groups were treated as described in Section 2. The increase in paw volume was measured using a plethysmometer before the injection of carageenan (0.1 ml of 1% solution), and 1.30, 3.00 and 4.30 h after the injection of carageenan. Control rats (filled square), phenylbutazone treated and carageenan injected rats (open circle), Canscora decussata (40 mg/kg) treated and carageenan injected rats (filled triangle), Canscora decussata (55 mg/kg) treated and carageenan injected rats (open triangle) and Canscora decussata (70 mg/kg) treated and carageenan injected rats (filled diamond). (B) The rats in all the five groups were treated and increase in paw volume was measured as described in (A). The percentage inhibition of paw-edema was calculated as detailed in Section 2. Experiments were repeated six times, data are average of three independent experiments.
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3.3. CdEE inhibits E-selectin expression on endothelial cells in a concentration-dependent manner To determine the effect of CdEE on the expression of E-selectin, the cells were incubated with or without CdEE at the indicated concentrations for 1 h prior to treatment with LPS (1 g/ml) for 4 h. As shown in Fig. 2, E-selectin was expressed at very low levels on unstimulated endothelial cells and its expression was induced over sevenfolds by LPS stimulation. CdEE had no effect on the constitutively expressed levels of E-selectin, whereas it led to a significant reduction in the LPS-induced E-selectin expression in a concentration-dependent manner (upto a maximum of 75%). 3.4. CdEE prevents paw-edema in rats To determine the effect of CdEE in an in vivo inflammatory response, we develop a rat model of carageenan-induced paw-edema as described in Section 2. In control animals, the subplantar injection with carageenan (0.1 ml of 1% solution) induced local edema in paw of rats. There was an increase in the paw volume progressively with time as measured using plethesmometer. As shown in Fig. 3A maximal increase in the paw volume of rats was observed between 3 and 5 h after carageenan injection. After 5 h there was a decrease in the edema (data not shown). Intraperitoneal injection of rats with CdEE (40–70 mg/kg) 1 h prior to injecting carageenan led to a decrease in the carageenan-induced paw-edema. As shown in Fig. 3A and B, CdEE inhibited paw-edema in a dose-dependent manner, inhibiting edema by 21.8, 39.0, and 59.0%, respectively at a dose of 40, 55, and 70 mg/kg at 4.30 h after carageenan injection. The maximal inhibition was observed at a dose of 70 mg/kg. In control experiments, the treatment with solvent (50% ethanol) had no effect on carageenan-induced paw-edema in rats and also had no effect on LPS-induced expression of ICAM-1 or E-selectin on endothelial cells (data not shown). The inhibition of paw-edema at this dose was comparable to that observed with phenylbutazone (55.0%). It is noticed that CdEE at the dose of 70 mg/kg inhibited the paw-edema in a time-independent manner (Fig. 3B). In contrast, at lower doses the inhibition was decreased by 4 h, probably because the effective molecule(s) in the extract are metabolized with time (Fig. 3A).
4. Discussion and conclusions Our results for the first time demonstrate that CdEE can be used for controlling cell trafficking by inhibiting the expression of ICAM-1 and E-selectin. CdEE effectively inhibits LPS-induced expression of ICAM-1 and E-selectin in a dose-dependent manner and it is also effective in vivo as it decreases the edema in rat paw induced with carageenan. It is interesting to note that our earlier experiments with an
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aqueous extract of Canscora decussata, in contrast to the ethanolic extract, induced the expression of cell adhesion molecules in human umbilical vein endothelial cells (Madan and Ghosh, 2002). Extracts from various plant materials have been reported to be effective in preventing carageenan-induced paw-edema. For example, methanol, chloroform and ethanol extracts of plant Angelica pubescens are effective in inhibiting carageenan-induced paw-edema by 65.0% at a dose of 100 mg/kg (Chen et al., 1995). The dichloromethane extract of Diospyros leucomelas reduced the paw-edema by 46.3% at a dose of 100 mg/kg (Recio et al., 1995). The alcoholic and ether extracts of Curcuma longa are also effective in inhibiting carageenan-induced paw-edema by 50% at a doses of 300 and 40 mg/kg (Yenganarayanan et al., 1976). Our results demonstrated that Canscora decussata extract is effective at a dose range of 70 mg/kg body weight and about 60% inhibition in paw-edema is observed at this dose. This shows that Canscora decussata extract is more effective in preventing paw-edema as compared to Diospyros leucomelas and Curcuma longa at a lower dose compared to these extracts. Earlier phytochemical studies indicated the presence of various xanthones in Canscora decussata extract (Ghosal et al., 1978). It would, therefore, be interesting to investigate whether the inhibition of carageenan-induced paw-edema by is due to any of these xanthones. In various vascular and inflammatory diseases, the adhesive property of the vasculature is primarily altered due to the upregulation of expression of cell adhesion molecules. The upregulated expression of cell adhesion molecules leads to the infiltration of leukocytes from the blood vessels to the underlying tissues and their accumulation leads to inflammation. As CdEE inhibits the expression of ICAM-1 and E-selectin on endothelial cells, this explains the mechanism underlying the ability of this extract to prevent edema formation in rat paw. The inhibition of cell adhesion molecules using various approaches, such as mAbs specific to cell adhesion molecules, peptides derived from adhesion molecules and small molecules, such as glucocorticoids, curcumin, serine proteinase and proteosome inhibitors has been used successfully in vitro and in various animal models of inflammatory diseases. Our present results indicate that an ethanolic extract prepared from Canscora decussata, exhibits considerable anti-inflammatory activity as demonstrated by its in vitro ability to down-regualte the expression of ICAM-1 and E-selectin and in an in vivo paw-edema model of rat. Thus, the extract offers a novel therapeutic target for controlling various pathological conditions associated with upregulation of endothelial leukocyte adhesion molecules and could be employed in conditions where down-regulation of cell adhesion molecules is required. Canscora decussata extract, therefore, could be used in the future to identify and characterize lead molecules for developing anti-inflammatory drugs.
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Acknowledgements We thank Dr. Asgar Ali, Hamdard University, Delhi for his help for designing the mercury plethysmometer. Council of Scientific and Industrial Research, India supported this work.
References Calderon, E., Lockey, R.F., 1992. A possible role for adhesion molecules in asthma. The Journal of Allergy and Clinical Immunology 90, 852– 865. Chen, Y.F., Tsai, H.Y., Wu, T.S., 1995. Anti-inflammatory and analgesic activities from roots of Angelica pubescens. Planta Medica 61, 2–8. Dikshit, S.K., Tewari, P.V., Dixit, S.P., 1972. Anticonvulsant activity of Canscora decussata Roem. & Sch. Indian Journal of Physiology and Pharmacology 16, 81–83. Ghosal, S., Biswas, K., Chaudhuri, R.K., 1978. Chemical constituents of Gentianaceae XXIV: anti-mycobacterium tuberculosis activity of naturally occurring xanthones and synthetic analogs. Journal of Pharmaceutical Science 67, 721–722. Gorski, A., 1994. The role of cell adhesion molecules in immunopathology. Immunology Today 15, 251–255. Gupta, B., Ghosh, B., 1999. Curcuma longa inhibits TNF-a induced expression of adhesion molecules on human umbilical vein endothelial cells. International Journal of Immunopharmacology 21, 745–757.
Madan, B., Gade, W.N., Ghosh, B., 2001. Curcuma longa activates NF-B and promotes adhesion of neutrophils to human umbilical vein endothelial cells. Journal of Ethnopharmacology 75, 25–33. Madan, B., Ghosh, B., 2002. Canscora decussata promotes adhesion of neutrophils to human umbilical vein endothelial cells. Journal of Ethnopharmacology 79, 229–235. Mantovani, A., Bussolino, F., Introna, M., 1997. Cytokine regulation of endothelial cell function: from molecular level to bedside. Immunology Today 18, 231–240. Ramachandran, K. (1992). Wealth of India (Raw Materials), vol. 3. Publications and Information Directorate, Council of Scientific and Industrial Research, Delhi, p. 205. Recio, M.C., Giner, R.M., Manez, S., Gueho, J., Julien, H.R., Hostettmann, K., Rios, J.L., 1995. Investigations on the steroidal anti-inflammatory activity of triterpenoids from Diospyros leucomelas. Planta Medica 61, 9–12. Sanches, S., Bartrones, R., Rodriguez, L., Gonzalez, P., Planas, M.E., 1998. Protective effect of nifedipine against carageenan-induced inflammation. Pharmacology 56, 131–136. Springer, T.A., 1994. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76, 301– 314. Weiser, M.R., Gibbs, S.A.L., Hechtman, H.B., 1997. Strategies to inhibit cellular adhesion molecules. In: Paul, L.C., Issekutz, T.B. (Eds.), Adhesion Molecules in Health and Disease. Marcel Dekker, New York, p. 55. Yenganarayanan, R., Saraf, A.P., Balwani, J.H., 1976. Comparison of anti-inflammatory activity of various extracts of Curcuma longa (Linn). Indian Journal of Medical Research 64, 601–608.
Canscora decussata (Roxb.) Schult (Gentianaceae) inhibits LPS-induced expression of ICAM-1 and E-selectin on endothelial cells and carageenan-induced paw-edema in rats Babita Madan, B.C. Mandal, Sarvesh Kumar, B. Ghosh∗ Molecular Immunology and Immunogenetics Laboratory, Institute of Genomics and Integrative Biology, University of Delhi Campus (North), Mall Road, Delhi 110007, India Received 11 March 2003; received in revised form 10 July 2003; accepted 25 July 2003
Abstract Inflammation is characterized pathologically by an increased supply of blood to the affected area, vasodilation, and infiltration of phagocytic, monocytic, and polymorphonuclear cells into the site of inflammation. The infiltration of leukocytes is in part regulated by the increased expression of cell adhesion molecules viz. ICAM-1 and E-selectin on endothelial cells. We investigated the effect of ethanolic extract prepared from plant Canscora decussata on expression of ICAM-1 and E-selectin on endothelial cells. Our results demonstrated that treatment of human endothelial cells with the extract down-regulated the LPS-induced expression of ICAM-1 and E-selectin. To study further the anti-inflammatory activity of the extract, we evaluated its effect in a rat model of carageenan-induced paw-edema. Our results showed that the extract was able to reduce the edema formation in a dose-dependent manner. These results, therefore, indicate that Canscora decussata extract could be useful towards developing effective anti-inflammatory agent(s) in the future. © 2003 Published by Elsevier Ireland Ltd. Keywords: Canscora decussata; Paw-edema; Carageenan; Anti-inflammatory; ICAM-1; E-selectin; Endothelial cells
1. Introduction Inflammation normally a localized defensive process is caused by soluble antigen, live organisms, and chemical or mechanical stress upon tissue, which serves to destroy and/or dilute the injurious materials, and remove the injured tissues. It is characterized pathologically by an increased supply of blood to the affected area, increased capillary permeability caused by retraction of the endothelial cells and infiltration of phagocytic, monocytic and polymorphonuclear cells into the site of tissue insult. The accumulation and subsequent activation of leukocytes is one of the central events in the pathogenesis of all forms of inflammation. Leukocytes migrate to and accumulate at the site of inflammation by locally produced chemoattractants, and are then activated to secrete granular contents and to release active
Abbreviations: ICAM-1, intercellular adhesion molecule-1; LPS, lipopolysaccharide; TNF-␣, tumor necrosis factor-␣; IL-1, interleukin1; CdEE, Canscora decussata ethnolic extract ∗ Corresponding author. Tel.: +91-11-2766-7602; fax: +91-11-2766-7471. E-mail address: [email protected] (B. Ghosh). 0378-8741/$ – see front matter © 2003 Published by Elsevier Ireland Ltd. doi:10.1016/S0378-8741(03)00281-2
oxygen metabolites during chemotaxis and phagocytosis. These active metabolites cause damage to the underlying tissue leading to inflammation. The migration of the leukocytes to the site of inflammation is regulated in part by the expression of cell adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and E-selectin (Springer, 1994). These cell adhesion molecules are induced on endothelial cells by various pro-inflammatory cytokines like IL-1 and TNF-␣ and also by bacterial LPS (reviewed by Mantovani et al., 1997). It is well established that in various inflammatory diseases, the expression of these proteins is upregulated on endothelial cells (Calderon and Lockey, 1992; Gorski, 1994). Inhibition of these molecules using specific monoclonal antibodies (mAbs) has been found to be beneficial for controlling various inflammatory diseases (Gorski, 1994; Weiser et al., 1997). Canscora decussata (Gentianaceae), popularly known as shankhpushpi is a herb and is used as a laxative and a tonic. Several medicinal properties have been attributed to it in the traditional medicine in India. The crude dried powder of the herb and its ethanolic extract has shown anti-convulsant activity (Dikshit et al., 1972). The ethanolic
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extract of the herb lowers blood pressure in albino rats and also shows stimulant action on the smooth muscle of intestine, uterus, and bronchus. It also has spermicidal activity in rats (Ramachandran, 1992). Also the purified xanthones from Canscora decussata have shown inhibitory activity against Mycobacterium tuberculosis H37 RV (Ghosal et al., 1978). No studies have, however, been undertaken to explore the anti-inflammatory activity of this plant. In the present study, we investigated the ability of extract prepared from the plant Canscora decussata on expression of ICAM-1 and E-selectin on endothelial cells and on inflammatory response in rat model of carageenan-induced paw-edema. Our results show that an extract of Canscora decussata inhibits the expression of ICAM-1 and E-selectin on endothelial cells and is also effective in vivo as it reduces the edema formation in rats in carageenan-induced paw-edema assay.
2. Methodology 2.1. Materials TNF-␣, anti-ICAM-1, and anti-E-selectin antibodies were purchased from Pharmingen, CA. M199, l-glutamine, penicillin, streptomycin, amphotericin, endothelial cell growth factor, trypsin, Pucks saline, HEPES, o-phenylenediamine dihydrochloride and anti-mouse IgG-HRP were purchased from Sigma Chemical Co., USA. Carrageenan was purchased from S.D. Fine Chemical Ltd., India. 2.2. Preparation of Canscora decussata extract Dried powdered rhizomes of Canscora decussata (100 mg) was macerated in 4 ml of ethanol:water (1:1) overnight at 25 ◦ C under shaking and the percolate was collected. Maceration and percolate collection was continued for 2 days. The percolate was pooled and vacuum dried. The dried material (40 mg) was resuspended in 400 l 50% ethanol before use and is designated CdEE. 2.3. Cells and cell culture Primary endothelial cells were isolated from umbilical cord by mild trypsinization (Gupta and Ghosh, 1999). The endothelial cells obtained were maintained in M199 medium supplemented with 20% heat inactivated fetal calf serum, 2 mM l-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, 0.25 g/ml amphotericin, endothelial cell growth factor (50 g/ml) and heparin (5 U/ml). The cells were subcultured by dislodging with 0.125% trypsin–0.01 M EDTA solution in Pucks saline and HEPES buffer. The cells were used between passages three to four. The viability of cells was determined by trypan blue exclusion test and the pu-
rity of endothelial cells were determined by E-selectin expression. 2.4. Modified ELISA for measurement of ICAM-1 and E-selectin For measuring the expression of ICAM-1 on surface of endothelial cells cell-ELISA was used as described before (Madan et al., 2001). HUVECs, plated to confluence in 96-well plates were incubated with or without varying dilutions of CdEE for 1 h followed by treatment with LPS (1 g/ml) for 16 h for ICAM-1 or for 4 h for E-selectin expression. Following incubation, the cells were fixed with 1.0% glutaraldehyde, non-specific binding was blocked using non-fat dry milk (3.0% in PBS) and cells were incubated overnight at 4 ◦ C with antibodies to ICAM-1 or E-selectin or control IgG Ab (0.25 g/ml). This was followed by washing cells with PBS and incubation with peroxidase-conjugated goat anti-mouse secondary Ab (1 g/ml). The cells were again washed with PBS and exposed to the peroxidase substrate (o-phenylenediamine dihydrochloride 40 mg/100 ml in citrate phosphate buffer, pH 4.5). Colour development reaction was stopped by the addition of 2N sulfuric acid. Absorbance was determined at 490 nm by an automated microplate reader (Spectramax 190, Molecular Devices, USA). 2.5. Carageenan-induced paw-edema in rats Anti-inflammatory activity of Canscora decussata extract on carageenan-induced paw-edema in rats was determined by a mercury plethysmometer designed in the laboratory following the method of Sanches et al. (1998). For experiments male Sprague–Dawley rats weighing 125–160 g were taken. Animals were allowed free access to food and water throughout the experiment. Experimental protocol was approved by the institutional ethical committee. Edema was induced by subcutaneous injection of carageenan (0.1 ml of 1% solution w/v in 0.9% saline) into subplantar region of the left hind paw. The volume of each paw upto tibio-tarsal articulation was measured before the injection of carageenan, and 1.30, 3.00, and 4.30 h after the injection of carageenan. Rats were divided into five groups of four each: Group A, rats were injected with carageenan; Group B, rats were administered with phenylbutazone (50 mg/kg) intraperitoneally 1 h prior to carageenan injection; Group C, D, and E, rats were administered with 40, 55, and 70 mg/kg CdEE, respectively, intraperitoneally 1 h prior to carageenan injection. The inflammatory response is presented as increase in paw volume and inhibition percentage of edema according to the equation: %I =
VC − VT × 100 VC
where VC is arithmetic mean of the increase in paw volume in the control group. VT is arithmetic mean of the increase in paw volume in the treated group.
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Fig. 1. Concentration-dependent inhibition of LPS-induced ICAM-1 expression by CdEE: endothelial cells grown to confluence in 96-well plates were incubated without or with indicated dilutions of CdEE for 1 h prior to induction without or with LPS (1 g/ml) for 16 h. Following this, ICAM-1 level on the cells was measured by ELISA as described in Section 2. The data presented are representative of three independent experiments. Values shown are mean ± S.D. of quadruplicate wells.
3. Results 3.1. CdEE is non-toxic to endothelial cells To determine any possible toxic effect of CdEE on endothelial cells, the cells grown to confluence in 96-well plates were incubated with varying concentrations of CdEE
for 24 h. Viability of the cells was determined by trypan blue exclusion test and their morphology was observed under microscope. It was observed that the time of incubation (upto 24 h) and the concentrations of the extracts used in subsequent experiments did not affect the viability or morphology of the endothelial cells (data not shown).
Fig. 2. Concentration-dependent inhibition of LPS-induced E-selectin expression by CdEE: endothelial cells grown to confluence in 96-well plates were incubated without or with indicated dilutions of CdEE for 1 h prior to induction without or with LPS (1 g/ml) for 4 h. Following this, E-selectin level on the cells was measured by ELISA as described in Section 2. The data presented are representative of three independent experiments. Values shown are mean ± S.D. of quadruplicate wells.
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3.2. CdEE inhibits ICAM-1 expression on endothelial cells in a concentration-dependent manner To determine the effect of CdEE on the expression of ICAM-1 on endothelial cells, the cells were incubated with or without CdEE at the indicated concentrations for 1 h prior to treatment with LPS (1 g/ml) for 16 h. ICAM-1 was ex-
pressed at low levels on unstimulated endothelial cells and its expression was induced over fivefolds by LPS stimulation (Fig. 1). Interestingly, CdEE had no effect on the constitutively expressed levels of ICAM-1, whereas it led to a significant reduction (approximately 70%) in the LPS-induced ICAM-1 expression in a concentration-dependent manner (Fig. 1).
Fig. 3. (A) Effect of Canscora decussata extract on carageenan-induced paw-edema in rats: the rats in all the five groups were treated as described in Section 2. The increase in paw volume was measured using a plethysmometer before the injection of carageenan (0.1 ml of 1% solution), and 1.30, 3.00 and 4.30 h after the injection of carageenan. Control rats (filled square), phenylbutazone treated and carageenan injected rats (open circle), Canscora decussata (40 mg/kg) treated and carageenan injected rats (filled triangle), Canscora decussata (55 mg/kg) treated and carageenan injected rats (open triangle) and Canscora decussata (70 mg/kg) treated and carageenan injected rats (filled diamond). (B) The rats in all the five groups were treated and increase in paw volume was measured as described in (A). The percentage inhibition of paw-edema was calculated as detailed in Section 2. Experiments were repeated six times, data are average of three independent experiments.
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3.3. CdEE inhibits E-selectin expression on endothelial cells in a concentration-dependent manner To determine the effect of CdEE on the expression of E-selectin, the cells were incubated with or without CdEE at the indicated concentrations for 1 h prior to treatment with LPS (1 g/ml) for 4 h. As shown in Fig. 2, E-selectin was expressed at very low levels on unstimulated endothelial cells and its expression was induced over sevenfolds by LPS stimulation. CdEE had no effect on the constitutively expressed levels of E-selectin, whereas it led to a significant reduction in the LPS-induced E-selectin expression in a concentration-dependent manner (upto a maximum of 75%). 3.4. CdEE prevents paw-edema in rats To determine the effect of CdEE in an in vivo inflammatory response, we develop a rat model of carageenan-induced paw-edema as described in Section 2. In control animals, the subplantar injection with carageenan (0.1 ml of 1% solution) induced local edema in paw of rats. There was an increase in the paw volume progressively with time as measured using plethesmometer. As shown in Fig. 3A maximal increase in the paw volume of rats was observed between 3 and 5 h after carageenan injection. After 5 h there was a decrease in the edema (data not shown). Intraperitoneal injection of rats with CdEE (40–70 mg/kg) 1 h prior to injecting carageenan led to a decrease in the carageenan-induced paw-edema. As shown in Fig. 3A and B, CdEE inhibited paw-edema in a dose-dependent manner, inhibiting edema by 21.8, 39.0, and 59.0%, respectively at a dose of 40, 55, and 70 mg/kg at 4.30 h after carageenan injection. The maximal inhibition was observed at a dose of 70 mg/kg. In control experiments, the treatment with solvent (50% ethanol) had no effect on carageenan-induced paw-edema in rats and also had no effect on LPS-induced expression of ICAM-1 or E-selectin on endothelial cells (data not shown). The inhibition of paw-edema at this dose was comparable to that observed with phenylbutazone (55.0%). It is noticed that CdEE at the dose of 70 mg/kg inhibited the paw-edema in a time-independent manner (Fig. 3B). In contrast, at lower doses the inhibition was decreased by 4 h, probably because the effective molecule(s) in the extract are metabolized with time (Fig. 3A).
4. Discussion and conclusions Our results for the first time demonstrate that CdEE can be used for controlling cell trafficking by inhibiting the expression of ICAM-1 and E-selectin. CdEE effectively inhibits LPS-induced expression of ICAM-1 and E-selectin in a dose-dependent manner and it is also effective in vivo as it decreases the edema in rat paw induced with carageenan. It is interesting to note that our earlier experiments with an
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aqueous extract of Canscora decussata, in contrast to the ethanolic extract, induced the expression of cell adhesion molecules in human umbilical vein endothelial cells (Madan and Ghosh, 2002). Extracts from various plant materials have been reported to be effective in preventing carageenan-induced paw-edema. For example, methanol, chloroform and ethanol extracts of plant Angelica pubescens are effective in inhibiting carageenan-induced paw-edema by 65.0% at a dose of 100 mg/kg (Chen et al., 1995). The dichloromethane extract of Diospyros leucomelas reduced the paw-edema by 46.3% at a dose of 100 mg/kg (Recio et al., 1995). The alcoholic and ether extracts of Curcuma longa are also effective in inhibiting carageenan-induced paw-edema by 50% at a doses of 300 and 40 mg/kg (Yenganarayanan et al., 1976). Our results demonstrated that Canscora decussata extract is effective at a dose range of 70 mg/kg body weight and about 60% inhibition in paw-edema is observed at this dose. This shows that Canscora decussata extract is more effective in preventing paw-edema as compared to Diospyros leucomelas and Curcuma longa at a lower dose compared to these extracts. Earlier phytochemical studies indicated the presence of various xanthones in Canscora decussata extract (Ghosal et al., 1978). It would, therefore, be interesting to investigate whether the inhibition of carageenan-induced paw-edema by is due to any of these xanthones. In various vascular and inflammatory diseases, the adhesive property of the vasculature is primarily altered due to the upregulation of expression of cell adhesion molecules. The upregulated expression of cell adhesion molecules leads to the infiltration of leukocytes from the blood vessels to the underlying tissues and their accumulation leads to inflammation. As CdEE inhibits the expression of ICAM-1 and E-selectin on endothelial cells, this explains the mechanism underlying the ability of this extract to prevent edema formation in rat paw. The inhibition of cell adhesion molecules using various approaches, such as mAbs specific to cell adhesion molecules, peptides derived from adhesion molecules and small molecules, such as glucocorticoids, curcumin, serine proteinase and proteosome inhibitors has been used successfully in vitro and in various animal models of inflammatory diseases. Our present results indicate that an ethanolic extract prepared from Canscora decussata, exhibits considerable anti-inflammatory activity as demonstrated by its in vitro ability to down-regualte the expression of ICAM-1 and E-selectin and in an in vivo paw-edema model of rat. Thus, the extract offers a novel therapeutic target for controlling various pathological conditions associated with upregulation of endothelial leukocyte adhesion molecules and could be employed in conditions where down-regulation of cell adhesion molecules is required. Canscora decussata extract, therefore, could be used in the future to identify and characterize lead molecules for developing anti-inflammatory drugs.
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Acknowledgements We thank Dr. Asgar Ali, Hamdard University, Delhi for his help for designing the mercury plethysmometer. Council of Scientific and Industrial Research, India supported this work.
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