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In vitro study of the antioxidant and immunomodulatory activity of aqueous infusion of Bidens pilosa
Journal of Ethnopharmacology 93 (2004) 319–323
In vitro study of the antioxidant and immunomodulatory activity of aqueous infusion of Bidens pilosa夽 Celia Abajo a , Mar´ıa Ángeles Boffill b , Jaime del Campo a , Mar´ıa Alexandra Méndez a , Yisel González b , Montserrat Mitjans a , Mar´ıa Pilar Vinardell a,∗ a
Department of Fisiologia-Divisió, IV, Facultat de Farmàcia, Av. Joan XXIII s/n, E-08028 Barcelona, Spain b Unidad de Toxicolog´ıa, Villa Clara, Cuba Received 15 October 2003; received in revised form 5 March 2004; accepted 31 March 2004 Available online 28 May 2004
Abstract Bidens pilosa is an annual plant from tropical America with anti-inflammatory properties in hepatitis, laryngitis, headache and digestive disorders, among others. Its wide pharmacological applications can be attributed to its chemical composition, with inhibitory effects on pathogenic microorganisms and flavonoids, which show strong antioxidant capacities. We investigated the antioxidant activity of an aqueous infusion of Bidens pilosa by studying its protective effect on the hemolysis induced by an initiator of radicals such as 2,2 -azobis(2-amidinopropane) dihydrochloride (AAPH). The immunomodulatory activity of the infusion was tested using whole blood cells. Cytokine production increased in whole blood stimulated or not by lipopolysaccharides (LPSs). The infusion is also characterized by its capacity to protect erythrocytes from the phototoxic effect of chlorpromazine, which allows its use as a potential photoprotector. Finally, it did not show ocular irritation, as demonstrated by the effect on hemoglobin denaturation. This study supports the health benefits of the ingestion of the infusion. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Antioxidant; Free radicals; Bidens pilosa; Photoprotection; Cytokine
1. Introduction Bidens alba L. var. radiata Schultz-Bip, family Asteraceae, synonymous Bidens pilosa, is an annual plant from tropical America, 30–100 cm in height with yellow flowers. It is widely applied as an anti-inflammatory agent in hepatitis, laryngitis, headache and digestive disorders, among others. In Cuba, it is widely used in infusions for several disorders (Lastra et al., 2001). Its high pharmacological applications can be attributed to the chemical compounds, especially polyacetylenes, which inhibit pathogenic microorganisms and flavonoids, which are active anti-inflammatory agents (Pereira et al., 1999). Phytochemical studies of Bidens Abbreviations: AAPH, 2,2 -azobis-(2-amidinopropane) dihydrochloride; IC50 , inhibitory concentration 50; IL-1, interleukin-1; LPS, lipopolysaccharide; ROS, reactive oxygen species; TMB, 3,3 ,5,5 -tetramethylbenzidine; TNF␣, tumor necrosis factor ␣ 夽 A part of this paper was presented as a poster in the V Symposium of Oxidative Stress, La Havana, Cuba, 2003. ∗ Corresponding author. Tel.: +34 934024505; fax: +34 934035901. E-mail address: [email protected] (M. Pilar Vinardell).
pilosa have revealed the presence of alkaloids, saponins, flavonoids, polyacetylenes and triterpens (Hoffmann and Hölzl, 1988), acyl chalcones and glucosides and 1-phenyl hepta 1,3,5-triyne (Zollo et al., 1995). Bidens pilosa is used in traditional medicine to treat malaria (Brandäo et al., 1997) and shows high antibacterial activity (Rabe and van Staden, 1997). Increasing evidence suggests that oxidative damage to cell components has a relevant pathophysiological role in several types of human diseases (Ames et al., 1993). Reactive oxygen species (ROS) have been reported to damage erythrocytes in patients with blood pathologies (Vives-Corrons et al., 1995; Rice-Evans et al., 1986). Erythrocytes are highly susceptible to oxidative damage owing to the high polyunsaturated fatty acid content of their membranes and the high cellular concentrations of oxygen and hemoglobin, a powerful potential promoter of oxidative processes (Clemens et al., 1987). These are the most common sensitive configurations damaged by the action of oxygen free radical species, which may exert their damaging action by membrane lipid oxidation, leading to the lysis of red blood cells. Lipid peroxidation is one of the consequences
0378-8741/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2004.03.050
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of oxidative damage and has been suggested as a general mechanism of cell injury and death (i.e. hemolysis). In intact isolated erythrocytes, 2,2 -azobis-(2-amidinopropane) dihydrochloride (AAPH) induces the extensive oxidation of membrane components and promotes structural alterations that lead to the rupture of the erythrocyte membrane and to hemoglobin leakage (Sato et al., 1995). In this paper, we have studied the antioxidant and immunomodulatory activity of an infusion of Bidens pilosa, the most common preparation of this plant, with wide applications in popular medicine in Cuba and other countries around the world.
2. Material and methods 2.1. Plant material Aerial parts of fresh Bidens pilosa were collected from the Medicinal Plants Farm of Santa Clara, Villa Clara, Cuba, in June 2002. Plant material was authenticated by J. Matos, a specialist of the Flora and Fauna Entity. A voucher specimen is kept in the Herbarium of Villa Clara Medical University. This plant was dried at 50 ◦ C for 3 days. 2.2. Preparation of infusion An aqueous infusion of 8 g of the dried plant was prepared by addition of 250 ml of water. After 2 min of ebullition and 10 min at room temperature, it was filtered and prepared for use. 2.3. Chemicals The following reagents were obtained from Sigma Chemical Co. (St. Louis, MO): sodium chloride, sodium phosphate dibasic, chlorpromazine, 2,2 -azobis(2-amidinopropane) dihydrochloride, lipopolysaccharide (LPS) from Escherichia coli, LPS from Salmonella abortus equi. 2.4. Antioxidant effect Blood samples were obtained from healthy donors by venipuncture (Blood Bank, Hospital Clinic, Barcelona, Spain). Citrated blood was centrifuged at 1000 × g for 10 min and washed three times with phosphate-buffered saline (PBS). Supernatant and buffy coat were carefully removed by aspiration after each wash. Washed erythrocytes were finally suspended in buffered saline. We determined the hemolysis of erythrocytes mediated by AAPH (a peroxyl radical initiator) using a modification of a method described elsewhere (Miki et al., 1987). Twenty-five microlitre aliquots of erythrocyte suspension were incubated in the presence of 100 mM AAPH to induce hemolysis at 37 ◦ C for 2.5 h. The inhibitory concentration 50 (IC50 ) of the
hemolysis induced by AAPH was determined. Hemolysis was monitored by spectrophotometry at 540 nm. 2.5. Photoprotective activity The method used was an adaptation of the method described by Pape et al. (2001). Erythrocyte suspension was incubated for 30 min with the infusion equivalent to 2 mg/ml. Thereafter, the erythrocyte suspension was added to a 24-well plate with various concentrations of chlorpromazine, a photohemolytic compound. The cells were exposed to UVA (1000 W/cm2 ) and UVB (220 W/cm2 ) delivered through an OSRAM lamp for 120 min. The erythrocyte suspension was then centrifuged and hemolysis was monitored in the supernatant. 2.6. Cytokine production by whole blood cells stimulated by LPS We studied the effect of the extracts on the production of tumor necrosis factor ␣ (TNF␣) and interleukin-1  (IL-1) by whole blood cells stimulated with lipopolysaccharide from Escherichia coli and Salmonella abortus equi. Hundred microlitre of endotoxin solution (10 g/ml) was added to 200 l of human blood, 100 l of the infusion and 800 l of sterile saline solution (Sigma) in sterile polypropylene tubes. The assay mixture was incubated at 37 ◦ C for 18 h in a thermoblock. Cell-free supernatants obtained by centrifugation at 3000 × g for 1 min were stored at −80 ◦ C until cytokine measurement. The viability of the cells after exposure to the extracts was evaluated by the trypan blue exclusion test (Mascotti et al., 2000). Cytokines were determined in 96-well plates (Diaclone Research, France). Samples or standard were added at 100 l/well, and 50 l/well biotylinated anti-TNF␣ or anti-IL-1 was added to all wells and incubated for 3 h at room temperature. The plate was then washed three times, after which 100 l of streptavidin–HRP was added to all wells. The plate was incubated for 30 min at room temperature and washed three times. The chromogen 3,3 ,5,5 -tetramethylbenzidine (TMB) solution was added at 100 l/well and the plate was incubated at room temperature in the dark for 15 min. The chromogenic reaction was stopped by the addition of 100 l of 0.2 M H2 SO4 per well. The optical densities (OD) were read on a plate reader (Bio-Rad) at 450 nm. A linear standard curve was generated by plotting the average absorbance on the vertical axis versus the corresponding TNF␣ or IL-1 standard concentration on the horizontal axis. The amount of TNF␣ and IL-1 in each sample was determined by extrapolating OD values to TNF␣ or IL-1 concentrations in the standard curves. Human blood without stimulation was used as a negative control and the level of cytokines was used as a blank to calculate cytokine production.
C. Abajo et al. / Journal of Ethnopharmacology 93 (2004) 319–323
2.7. Study of potential eye irritant action The method is based on the hemoglobin denaturation induced by irritant products (Hayashi et al., 1995; Vinardell et al., 1999) and was modified using hemoglobin from human erythrocytes (Mitjans et al., 2003). Hemoglobin was exposed to various concentration of the infusion in 96-well plates and its absorbance was recorded. Increasing concentrations of irritant products progressively reduce hemoglobin absorbance. Non-irritant products do not alter hemoglobin absorbance. 2.8. Statistical analysis The differences between control and treatment tests were analyzed using the Student’s t-test. They were considered significant at P < 0.05 (represented by an asterisk). All experiments were repeated at least three times. Data are expressed as means ± S.E.M.
3. Results and discussion
% Hemolysis Inhibition
In this study, we first tested the efficacy of the aqueous infusion of Bidens pilosa as an inhibitor of AAPH-induced erythrocyte hemolysis. AAPH, a water-soluble free radical generator, was used to stimulate the in vivo conditions of oxidative stress and peroxyl radicals were generated by thermal decomposition of an azo compound in oxygen. The advantages of this method are that the AAPH decomposes thermally to generate radicals without biotransformation or enzymes and the rate of radical generation is easily controlled by adjusting the concentration of initiator (Ko et al., 1997). The antioxidant effect of the infusion of Bidens pilosa on the hemolysis induced by AAPH is shown in Fig. 1, which represents the percentage of hemolysis inhibition at several concentrations. The amount of Bidens pilosa infusion that halved the hemolysis induced by AAPH was 6 l, which corresponds to an IC50 of 1.19 mg of dry weight per millilitre of infusion. Thus, the oxidative hemolysis of erythrocytes
321
induced by AAPH was suppressed by an aqueous infusion of Bidens pilosa, which is a very active antioxidant and exerts its protective effect at low amounts. The activity of the infusion of Bidens pilosa may be attributed to the compounds found by HPLC (data not shown). We detected that the infusion contains gallic acid and polymeric polyphenolic material. A small part of this material is of proanthocyanidin type, since it was depolymerised by thiolysis. The rest are hydrolysable tannins, as ascertained by their elution time in HPLC and their UV absorption at 280 and 320 nm. The antioxidant activity of polyphenolic material has been demonstrated in biological models and extensively reviewed (Rice-Evans et al., 1996). The photoprotective effect of Bidens pilosa has been described after incubation of erythrocytes treated with the infusion in presence of chlorpromazine, a phototoxic substance (Torinuki and Tagami, 1986; Eberlein-Konig et al., 1997). Fig. 2 shows the photohemolysis induced by chlorpromazine in erythrocytes pre-incubated or not with the infusion of Bidens pilosa. The hemolysis induced by chlorpromazine in erythrocytes incubated with the infusion and in non-incubated erythrocytes differs significantly. The photohemolysis induced by chlorpromazine revealed a HC50 of 65.44 mg/ml. This value increases in blood cells incubated in presence of the infusion, with a HC50 of 112.10 mg/ml, which represents about 40% of the photoprotection induced by the infusion. It is known that the LPS from Escherichia coli induces the production of cytokines by human whole blood, which allows the study in vitro of the immunomodulatory action of several compounds (Hermann et al., 2003). The aqueous infusion of Bidens pilosa showed an increase in cytokine production after stimulation with LPS from Escherichia coli and Salmonella abortus equi. Fig. 3 shows the effect of the infusion of Bidens pilosa on the production of TNF␣ by
120 100 80 60 40 20 0 0
1
10
Concentration (mg/mL)
Fig. 1. Antioxidant action of infusion of Bidens pilosa as determined by the inhibition of hemolysis in human whole blood induced by AAPH (Mean ± S.E.M.).
Fig. 2. Photohemolysis induced by chlorpromazine in presence and absence of the infusion of Bidens pilosa (2 mg/ml). The symbol ( ) indicates significant differences.
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Fig. 3. Individual results of TNF␣ production by human whole blood exposed to LPS from Salmonella abortus equi and the effect of the incubation with 100 l of the aqueous infusion of Bidens pilosa. The symbol ( ) indicates significant differences.
Fig. 5. Effect of the aqueous infusion of Bidens pilosa (100 l) on TNF␣ and IL-1 production by human whole blood without previous stimulation. The symbol ( ) indicates significant differences.
Fig. 4. Effect of the aqueous infusion of Bidens pilosa (100 l) on the TNF␣ and IL-1 production by human whole blood stimulated by LPS from Escherichia coli. The symbol ( ) indicates significant differences.
whole blood stimulated by LPS in the presence of Bidens pilosa significantly increases. To test whether the effect of Bidens pilosa depends on the presence of LPS, we incubated whole blood with only the infusion of Bidens pilosa and determined cytokine production. The levels of cytokine in whole blood from healthy donors were very low and were used as blank. Fig. 5 shows the effect of Bidens pilosa on the production of TNF␣ and IL-1 in non-stimulated whole blood. TNF and IL-1 production was doubled, which may be due to the action of Bidens pilosa alone. IL-1 and TNF␣ are currently regarded as central regulatory cytokines in the induction of macrophage antimicrobial activities (Wang et al., 1994). The antimicrobial activity of the infusion may increase the amount of cytokine (Rabe and van Staden, 1997) in a similar way to other plants (Gomez-Flores et al., 2000; Rimbach et al., 2000). Fig. 6 shows the hemoglobin denaturation induced by Bidens pilosa. Increased amounts of the infusion did not enhance the denaturation of hemoglobin, which points to the non-irritant action of the infusion, thus allowing its use in ophthalmic preparations (Mitjans et al., 2003).
whole blood stimulated by LPS from Salmonella abortus equi in various subjects. The addition of Bidens pilosa to whole blood significantly increases (P < 0.01) TNF␣ production (between four and eight times, depending on the subject). Fig. 4 shows the production of TNF␣ and IL-1 by whole blood stimulated by LPS from Escherichia coli and the effect of Bidens pilosa. TNF␣ and IL-1 production in
Hemoglobin absorbance
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2
1.5
1
0.5
0 0
10
20
30
40
50
60
70
80
Bidens pilosa aqueous infusion (µL)
Fig. 6. Potential ocular irritation as determined by the measurement of human hemoglobin absorbance after incubation with various amounts of the aqueous infusion of Bidens pilosa (error bars smaller than symbols).
C. Abajo et al. / Journal of Ethnopharmacology 93 (2004) 319–323
In this paper, we have demonstrated in vitro some properties of the aqueous infusion of Bidens pilosa that are in accordance with the general use of the infusion in traditional medicine. However, its mechanism of action associated with boosting the immune response remains to be elucidated. The aqueous infusion of Bidens pilosa was found to enhance cytokine production by whole blood. Monokines such as interleukin-1 and TNF␣ are essential mediators of host inflammatory responses to natural immunity. These results shed some light on the mechanism of action of Bidens pilosa. The regulation of immune parameters induced by the infusion of Bidens pilosa may be clinically relevant in numerous disease processes like chronic viral infection, tuberculosis, AIDS and cancer. In summary, although the data presented in this paper yielded an incomplete picture of the effects of the infusion of Bidens pilosa on the immune system, we have demonstrated that the infusion enhances cytokine production by whole blood. Further studies with animal models are necessary to clarify how and to what extent this activation occurs in vivo.
Acknowledgements We thank Robin Rycroft for language assistance and Ariadna Selga for HPLC analysis. This study was supported by the project 501 from the Fundació Bosch i Gimpera of the Universitat de Barcelona.
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Hermann, C., von Aulock, S., Graf, K., Hartung, T., 2003. A model of human whole blood lymphokine release for in vitro and ex vivo use. Journal of Immunological Methods 275, 69–79. Hoffmann, B., Hölzl, J., 1988. New chalcones from Bidens pilosa. Planta Medica 54, 52–54. Ko, F.N., Hsiao, G., Kuo, Y.H., 1997. Protection of oxidative hemolysis by demethyldiioseugenol in normal and beta-thalassemic red blood cells. Free Radical Biology and Medicine 22, 215–222. Lastra, H., Valdés, I., Ponce de León, R.H., 2001. Bidens pilosa Linné. Revista Cubana de Planta Medica 1, 232–233. Mascotti, K., Mc Cullough, J., Burger, S.R., 2000. HPC viability measurement: trypan blue versus acridine orange and propidiym iodide. Transfusion 40, 693–696. Miki, M., Ramai, H., Mino, M., Yamamoto, Y., Niki, E., 1987. Free radical chain oxidation of rat red blood cells by molecular oxygen and its inhibition by ␣-toxopherol. Archives in Biochemistry and Biophysics 258, 373–380. Mitjans, M., Abajo, C., del Campo, J., Vinardell, M.P., 2003. Estudio comparativo de dos métodos alternativos para evaluar la irritación ocular utilizando hemoglobina. Revista de Toxicologia 22, 131. Pape, W.J., Maurer, T., Pfannenbecker, U., Steling, W., 2001. The red bood cell phototoxicity test (photohaemolysis and haemoglobin oxidation): EU/COLIPA validation programme on phototoxicity (phase II). Atla-Alternatives to Laboratory Animals 29, 145–162. Pereira, R.L., Ibrahin, T., Lucchetti L da Silva, A.J., Goncalves de Morales, V.L., 1999. Immunosuppressive and anti-inflammatory effects of methanolic extract and the polyacetylene isolated from Bidens pilosa L. Immunopharmacology 43, 31–37. Rabe, R., van Staden, J., 1997. Antibacterial activity of South African plants used for medicinal purposes. Journal of Ethnopharmacology 56, 81–87. Rice-Evans, C., Omorphos, S.C., Baysal, E., 1986. Sickle cell membranes and oxidative damage. Biochemical Journal 237, 265–269. Rice-Evans, C., Miller, N.J., Paganga, G., 1996. Structure-antioxidant activity relationship of flavonoids and phenolic acids. Free Radical Biology and Medicine 20, 933–956. Rimbach, G., Guo, Q., Moini, H., Saliou, C., Takayama, K., Virgili, F., Packer, L., 2000. Nitric oxide synthesis and TNF-␣ secretion in RAW 2647 macrophages. Mode of action of a fermented papaya preparation. Life Sciences 67, 679–694. Sato, Y., Kamo, S., Rakahasi, T., Suzuki, Y., 1995. Mechanism of free radical-induced hemolysis of human erythrocytes: hemolysis by a water-soluble radical initiator. Biochemistry 34, 8940–8949. Torinuki, W., Tagami, H., 1986. Role of complement in chlorpromazineinduced phototoxicity. Journal of Investigative Dermatology 86, 142– 144. Vinardell, M.P., Gonzalez, S., Infante, M.R., 1999. Adaptation of hemoglobin denaturation for assessment of ocular irritation of surfactants and manufactured products. Journal of Toxicology – Cutaneous and Ocular Toxicology 18, 375–384. Vives-Corrons, J.L., Miguel-Garc´ıa, A., Pujades, M.A., Miguel-Sosa, A., Cambiazzo, S., Linares, M., Dibarrarts, M.T., Calvo, M.A., 1995. Increased susceptibility of microcytic red blood cells to in vitro oxidative stress. European Journal of Haematology 55, 327–331. Wang, Y., Huang, D.S., Watson, R.R., 1994. In vivo and in vitro cocaine modulation on production of cytokines in C57BL/6 mice. Life Sciences 54, 401–411. Zollo, A., Kuiate, J.R., Menut, C., Lamaty, G., Bessiere, J.M., Chalcat, J.C., Garry, R.P., 1995. Aromatic plants of tropical central Africa. XX: the occurrence of 1-phenyl-1,3,5-triyne in the essential oil of Bidens pilosa L. from Cameroon. Flavour and Fragrance Journal 10, 97–100.
In vitro study of the antioxidant and immunomodulatory activity of aqueous infusion of Bidens pilosa夽 Celia Abajo a , Mar´ıa Ángeles Boffill b , Jaime del Campo a , Mar´ıa Alexandra Méndez a , Yisel González b , Montserrat Mitjans a , Mar´ıa Pilar Vinardell a,∗ a
Department of Fisiologia-Divisió, IV, Facultat de Farmàcia, Av. Joan XXIII s/n, E-08028 Barcelona, Spain b Unidad de Toxicolog´ıa, Villa Clara, Cuba Received 15 October 2003; received in revised form 5 March 2004; accepted 31 March 2004 Available online 28 May 2004
Abstract Bidens pilosa is an annual plant from tropical America with anti-inflammatory properties in hepatitis, laryngitis, headache and digestive disorders, among others. Its wide pharmacological applications can be attributed to its chemical composition, with inhibitory effects on pathogenic microorganisms and flavonoids, which show strong antioxidant capacities. We investigated the antioxidant activity of an aqueous infusion of Bidens pilosa by studying its protective effect on the hemolysis induced by an initiator of radicals such as 2,2 -azobis(2-amidinopropane) dihydrochloride (AAPH). The immunomodulatory activity of the infusion was tested using whole blood cells. Cytokine production increased in whole blood stimulated or not by lipopolysaccharides (LPSs). The infusion is also characterized by its capacity to protect erythrocytes from the phototoxic effect of chlorpromazine, which allows its use as a potential photoprotector. Finally, it did not show ocular irritation, as demonstrated by the effect on hemoglobin denaturation. This study supports the health benefits of the ingestion of the infusion. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Antioxidant; Free radicals; Bidens pilosa; Photoprotection; Cytokine
1. Introduction Bidens alba L. var. radiata Schultz-Bip, family Asteraceae, synonymous Bidens pilosa, is an annual plant from tropical America, 30–100 cm in height with yellow flowers. It is widely applied as an anti-inflammatory agent in hepatitis, laryngitis, headache and digestive disorders, among others. In Cuba, it is widely used in infusions for several disorders (Lastra et al., 2001). Its high pharmacological applications can be attributed to the chemical compounds, especially polyacetylenes, which inhibit pathogenic microorganisms and flavonoids, which are active anti-inflammatory agents (Pereira et al., 1999). Phytochemical studies of Bidens Abbreviations: AAPH, 2,2 -azobis-(2-amidinopropane) dihydrochloride; IC50 , inhibitory concentration 50; IL-1, interleukin-1; LPS, lipopolysaccharide; ROS, reactive oxygen species; TMB, 3,3 ,5,5 -tetramethylbenzidine; TNF␣, tumor necrosis factor ␣ 夽 A part of this paper was presented as a poster in the V Symposium of Oxidative Stress, La Havana, Cuba, 2003. ∗ Corresponding author. Tel.: +34 934024505; fax: +34 934035901. E-mail address: [email protected] (M. Pilar Vinardell).
pilosa have revealed the presence of alkaloids, saponins, flavonoids, polyacetylenes and triterpens (Hoffmann and Hölzl, 1988), acyl chalcones and glucosides and 1-phenyl hepta 1,3,5-triyne (Zollo et al., 1995). Bidens pilosa is used in traditional medicine to treat malaria (Brandäo et al., 1997) and shows high antibacterial activity (Rabe and van Staden, 1997). Increasing evidence suggests that oxidative damage to cell components has a relevant pathophysiological role in several types of human diseases (Ames et al., 1993). Reactive oxygen species (ROS) have been reported to damage erythrocytes in patients with blood pathologies (Vives-Corrons et al., 1995; Rice-Evans et al., 1986). Erythrocytes are highly susceptible to oxidative damage owing to the high polyunsaturated fatty acid content of their membranes and the high cellular concentrations of oxygen and hemoglobin, a powerful potential promoter of oxidative processes (Clemens et al., 1987). These are the most common sensitive configurations damaged by the action of oxygen free radical species, which may exert their damaging action by membrane lipid oxidation, leading to the lysis of red blood cells. Lipid peroxidation is one of the consequences
0378-8741/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2004.03.050
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of oxidative damage and has been suggested as a general mechanism of cell injury and death (i.e. hemolysis). In intact isolated erythrocytes, 2,2 -azobis-(2-amidinopropane) dihydrochloride (AAPH) induces the extensive oxidation of membrane components and promotes structural alterations that lead to the rupture of the erythrocyte membrane and to hemoglobin leakage (Sato et al., 1995). In this paper, we have studied the antioxidant and immunomodulatory activity of an infusion of Bidens pilosa, the most common preparation of this plant, with wide applications in popular medicine in Cuba and other countries around the world.
2. Material and methods 2.1. Plant material Aerial parts of fresh Bidens pilosa were collected from the Medicinal Plants Farm of Santa Clara, Villa Clara, Cuba, in June 2002. Plant material was authenticated by J. Matos, a specialist of the Flora and Fauna Entity. A voucher specimen is kept in the Herbarium of Villa Clara Medical University. This plant was dried at 50 ◦ C for 3 days. 2.2. Preparation of infusion An aqueous infusion of 8 g of the dried plant was prepared by addition of 250 ml of water. After 2 min of ebullition and 10 min at room temperature, it was filtered and prepared for use. 2.3. Chemicals The following reagents were obtained from Sigma Chemical Co. (St. Louis, MO): sodium chloride, sodium phosphate dibasic, chlorpromazine, 2,2 -azobis(2-amidinopropane) dihydrochloride, lipopolysaccharide (LPS) from Escherichia coli, LPS from Salmonella abortus equi. 2.4. Antioxidant effect Blood samples were obtained from healthy donors by venipuncture (Blood Bank, Hospital Clinic, Barcelona, Spain). Citrated blood was centrifuged at 1000 × g for 10 min and washed three times with phosphate-buffered saline (PBS). Supernatant and buffy coat were carefully removed by aspiration after each wash. Washed erythrocytes were finally suspended in buffered saline. We determined the hemolysis of erythrocytes mediated by AAPH (a peroxyl radical initiator) using a modification of a method described elsewhere (Miki et al., 1987). Twenty-five microlitre aliquots of erythrocyte suspension were incubated in the presence of 100 mM AAPH to induce hemolysis at 37 ◦ C for 2.5 h. The inhibitory concentration 50 (IC50 ) of the
hemolysis induced by AAPH was determined. Hemolysis was monitored by spectrophotometry at 540 nm. 2.5. Photoprotective activity The method used was an adaptation of the method described by Pape et al. (2001). Erythrocyte suspension was incubated for 30 min with the infusion equivalent to 2 mg/ml. Thereafter, the erythrocyte suspension was added to a 24-well plate with various concentrations of chlorpromazine, a photohemolytic compound. The cells were exposed to UVA (1000 W/cm2 ) and UVB (220 W/cm2 ) delivered through an OSRAM lamp for 120 min. The erythrocyte suspension was then centrifuged and hemolysis was monitored in the supernatant. 2.6. Cytokine production by whole blood cells stimulated by LPS We studied the effect of the extracts on the production of tumor necrosis factor ␣ (TNF␣) and interleukin-1  (IL-1) by whole blood cells stimulated with lipopolysaccharide from Escherichia coli and Salmonella abortus equi. Hundred microlitre of endotoxin solution (10 g/ml) was added to 200 l of human blood, 100 l of the infusion and 800 l of sterile saline solution (Sigma) in sterile polypropylene tubes. The assay mixture was incubated at 37 ◦ C for 18 h in a thermoblock. Cell-free supernatants obtained by centrifugation at 3000 × g for 1 min were stored at −80 ◦ C until cytokine measurement. The viability of the cells after exposure to the extracts was evaluated by the trypan blue exclusion test (Mascotti et al., 2000). Cytokines were determined in 96-well plates (Diaclone Research, France). Samples or standard were added at 100 l/well, and 50 l/well biotylinated anti-TNF␣ or anti-IL-1 was added to all wells and incubated for 3 h at room temperature. The plate was then washed three times, after which 100 l of streptavidin–HRP was added to all wells. The plate was incubated for 30 min at room temperature and washed three times. The chromogen 3,3 ,5,5 -tetramethylbenzidine (TMB) solution was added at 100 l/well and the plate was incubated at room temperature in the dark for 15 min. The chromogenic reaction was stopped by the addition of 100 l of 0.2 M H2 SO4 per well. The optical densities (OD) were read on a plate reader (Bio-Rad) at 450 nm. A linear standard curve was generated by plotting the average absorbance on the vertical axis versus the corresponding TNF␣ or IL-1 standard concentration on the horizontal axis. The amount of TNF␣ and IL-1 in each sample was determined by extrapolating OD values to TNF␣ or IL-1 concentrations in the standard curves. Human blood without stimulation was used as a negative control and the level of cytokines was used as a blank to calculate cytokine production.
C. Abajo et al. / Journal of Ethnopharmacology 93 (2004) 319–323
2.7. Study of potential eye irritant action The method is based on the hemoglobin denaturation induced by irritant products (Hayashi et al., 1995; Vinardell et al., 1999) and was modified using hemoglobin from human erythrocytes (Mitjans et al., 2003). Hemoglobin was exposed to various concentration of the infusion in 96-well plates and its absorbance was recorded. Increasing concentrations of irritant products progressively reduce hemoglobin absorbance. Non-irritant products do not alter hemoglobin absorbance. 2.8. Statistical analysis The differences between control and treatment tests were analyzed using the Student’s t-test. They were considered significant at P < 0.05 (represented by an asterisk). All experiments were repeated at least three times. Data are expressed as means ± S.E.M.
3. Results and discussion
% Hemolysis Inhibition
In this study, we first tested the efficacy of the aqueous infusion of Bidens pilosa as an inhibitor of AAPH-induced erythrocyte hemolysis. AAPH, a water-soluble free radical generator, was used to stimulate the in vivo conditions of oxidative stress and peroxyl radicals were generated by thermal decomposition of an azo compound in oxygen. The advantages of this method are that the AAPH decomposes thermally to generate radicals without biotransformation or enzymes and the rate of radical generation is easily controlled by adjusting the concentration of initiator (Ko et al., 1997). The antioxidant effect of the infusion of Bidens pilosa on the hemolysis induced by AAPH is shown in Fig. 1, which represents the percentage of hemolysis inhibition at several concentrations. The amount of Bidens pilosa infusion that halved the hemolysis induced by AAPH was 6 l, which corresponds to an IC50 of 1.19 mg of dry weight per millilitre of infusion. Thus, the oxidative hemolysis of erythrocytes
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induced by AAPH was suppressed by an aqueous infusion of Bidens pilosa, which is a very active antioxidant and exerts its protective effect at low amounts. The activity of the infusion of Bidens pilosa may be attributed to the compounds found by HPLC (data not shown). We detected that the infusion contains gallic acid and polymeric polyphenolic material. A small part of this material is of proanthocyanidin type, since it was depolymerised by thiolysis. The rest are hydrolysable tannins, as ascertained by their elution time in HPLC and their UV absorption at 280 and 320 nm. The antioxidant activity of polyphenolic material has been demonstrated in biological models and extensively reviewed (Rice-Evans et al., 1996). The photoprotective effect of Bidens pilosa has been described after incubation of erythrocytes treated with the infusion in presence of chlorpromazine, a phototoxic substance (Torinuki and Tagami, 1986; Eberlein-Konig et al., 1997). Fig. 2 shows the photohemolysis induced by chlorpromazine in erythrocytes pre-incubated or not with the infusion of Bidens pilosa. The hemolysis induced by chlorpromazine in erythrocytes incubated with the infusion and in non-incubated erythrocytes differs significantly. The photohemolysis induced by chlorpromazine revealed a HC50 of 65.44 mg/ml. This value increases in blood cells incubated in presence of the infusion, with a HC50 of 112.10 mg/ml, which represents about 40% of the photoprotection induced by the infusion. It is known that the LPS from Escherichia coli induces the production of cytokines by human whole blood, which allows the study in vitro of the immunomodulatory action of several compounds (Hermann et al., 2003). The aqueous infusion of Bidens pilosa showed an increase in cytokine production after stimulation with LPS from Escherichia coli and Salmonella abortus equi. Fig. 3 shows the effect of the infusion of Bidens pilosa on the production of TNF␣ by
120 100 80 60 40 20 0 0
1
10
Concentration (mg/mL)
Fig. 1. Antioxidant action of infusion of Bidens pilosa as determined by the inhibition of hemolysis in human whole blood induced by AAPH (Mean ± S.E.M.).
Fig. 2. Photohemolysis induced by chlorpromazine in presence and absence of the infusion of Bidens pilosa (2 mg/ml). The symbol ( ) indicates significant differences.
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Fig. 3. Individual results of TNF␣ production by human whole blood exposed to LPS from Salmonella abortus equi and the effect of the incubation with 100 l of the aqueous infusion of Bidens pilosa. The symbol ( ) indicates significant differences.
Fig. 5. Effect of the aqueous infusion of Bidens pilosa (100 l) on TNF␣ and IL-1 production by human whole blood without previous stimulation. The symbol ( ) indicates significant differences.
Fig. 4. Effect of the aqueous infusion of Bidens pilosa (100 l) on the TNF␣ and IL-1 production by human whole blood stimulated by LPS from Escherichia coli. The symbol ( ) indicates significant differences.
whole blood stimulated by LPS in the presence of Bidens pilosa significantly increases. To test whether the effect of Bidens pilosa depends on the presence of LPS, we incubated whole blood with only the infusion of Bidens pilosa and determined cytokine production. The levels of cytokine in whole blood from healthy donors were very low and were used as blank. Fig. 5 shows the effect of Bidens pilosa on the production of TNF␣ and IL-1 in non-stimulated whole blood. TNF and IL-1 production was doubled, which may be due to the action of Bidens pilosa alone. IL-1 and TNF␣ are currently regarded as central regulatory cytokines in the induction of macrophage antimicrobial activities (Wang et al., 1994). The antimicrobial activity of the infusion may increase the amount of cytokine (Rabe and van Staden, 1997) in a similar way to other plants (Gomez-Flores et al., 2000; Rimbach et al., 2000). Fig. 6 shows the hemoglobin denaturation induced by Bidens pilosa. Increased amounts of the infusion did not enhance the denaturation of hemoglobin, which points to the non-irritant action of the infusion, thus allowing its use in ophthalmic preparations (Mitjans et al., 2003).
whole blood stimulated by LPS from Salmonella abortus equi in various subjects. The addition of Bidens pilosa to whole blood significantly increases (P < 0.01) TNF␣ production (between four and eight times, depending on the subject). Fig. 4 shows the production of TNF␣ and IL-1 by whole blood stimulated by LPS from Escherichia coli and the effect of Bidens pilosa. TNF␣ and IL-1 production in
Hemoglobin absorbance
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Bidens pilosa aqueous infusion (µL)
Fig. 6. Potential ocular irritation as determined by the measurement of human hemoglobin absorbance after incubation with various amounts of the aqueous infusion of Bidens pilosa (error bars smaller than symbols).
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In this paper, we have demonstrated in vitro some properties of the aqueous infusion of Bidens pilosa that are in accordance with the general use of the infusion in traditional medicine. However, its mechanism of action associated with boosting the immune response remains to be elucidated. The aqueous infusion of Bidens pilosa was found to enhance cytokine production by whole blood. Monokines such as interleukin-1 and TNF␣ are essential mediators of host inflammatory responses to natural immunity. These results shed some light on the mechanism of action of Bidens pilosa. The regulation of immune parameters induced by the infusion of Bidens pilosa may be clinically relevant in numerous disease processes like chronic viral infection, tuberculosis, AIDS and cancer. In summary, although the data presented in this paper yielded an incomplete picture of the effects of the infusion of Bidens pilosa on the immune system, we have demonstrated that the infusion enhances cytokine production by whole blood. Further studies with animal models are necessary to clarify how and to what extent this activation occurs in vivo.
Acknowledgements We thank Robin Rycroft for language assistance and Ariadna Selga for HPLC analysis. This study was supported by the project 501 from the Fundació Bosch i Gimpera of the Universitat de Barcelona.
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