Plants are important sources of biologically active natural products which differ widely in terms of structure and bio- logical properties. Some of the isolated plant constituents such as flavonoids, tannins, alkaloids, terpenes and others are responsible for many biological activities, such as analgesic activity, antiinflammatory activity, antiviral activity, antispas- modic activity, antiallergenic activity, and others.19) We can attribute this to the molecular diversity of these products. Many human physiological activities such as stimulation of phagocytic cells, host-mediated tumor activity, and a wide range of anti-infective actions have also been assigned to dif- ferent compounds.7) One of the biological activities of plant compounds that has attracted great interest is the capacity to stimulate macrophages. Macrophages are known to play an important role in host defense mechanisms. Being the first cells to par- ticipate in the immunological response, macrophages can be activated by a variety of stimuli and their principal functions include the phagocytosis of foreign particles, showing the presence of antigens, and the production of cytokines and of the intermediate compounds of oxygen (H2O2) and nitrogen (NO).4) Recent studies have also suggested that H2O2 plays an important role in the functions of macrophages.3,11,16,17) Currently, there is a strong tendency to study natural com- pounds that may be involved in the modulation of the im- munological system. A variety of materials derived from plants pertaining to the different classes of active agents have been reported to stimulate the immune system, with macrophage stimulation occurring in many cases.1,2,4,7,9—15,18) A simple, rapid and inexpensive method to measure the hydrogen peroxide (H2O2) produced by cells in in vitro cul- ture is based on the horseradish peroxidase (HRPO)-medi- ated oxidation of phenol red by H2O2 which results in the formation of a compound demonstrating increased ab- sorbance at 620 nm. The products of the oxidative burst are used to kill phago- cytosed pathogens and for the extracellular destruction of other cells. In addition to H2O2 and O2 2, two other highly re- active oxygen derivatives have been implicated in the killing process, i.e., hydroxyl radicals ( ·OH) and singlet oxygen (1O2). This coordinated sequence of biochemical reactions is initiated by an increase in oxygen uptake followed by the one-electron reduction of oxygen to superoxide anion (O2 2) using NADPH or NADH as the electron donor and catalyzed by an NAD(P)H oxidase. O2 2 is subsequently converted to hy- drogen peroxide (H2O2). 15,16) While the relative importance of O2 2, ·OH and 1O2 in oxy- gen-dependent killing is still debated, the role of H2O2 in both intra- and extracellular toxicity is well-established. The objective of the present study was to investigate the effect of 27 natural products on mouse peritoneal macro- phage functions by the liberation of H2O2. MATERIAL AND METHODS Animals Six-week old male Swiss mice weighing 18 to 25 g were supplied by the Animal House of the Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista- UNESP, Araraquara-São Paulo-Brasil. The animals were maintained in a polycarbonate box, with water and food available ad libitum. Plant Materials Natural products with varying chemical structures were isolated from various plants (see structures, Fig. 1). The purification procedures were performed by the Laboratório de Fitoquímica, Departamento de Química Orgânica, Universidade Estadual Paulista “Júlio de Mesquita Filho”, Araraquara, São Paulo, Brasil, where they are de- posited. Samples were checked for purity by TLC, GC-FID February 2001 Notes Biol. Pharm. Bull. 24(2) 201—204 (2001) 201 ∗ To whom correspondence should be addressed. e-mail: moreirar@fcfar.unesp.br © 2001 Pharmaceutical Society of Japan Release of Intermediate Reactive Hydrogen Peroxide by Macrophage Cells Activated by Natural Products Raquel Regina Duarte MOREIRA,*,a Iracilda Zeppone CARLOS,b and Wagner VILEGAS c Departamento de Princípios Ativos Naturais e Toxicologia Brasil,a Departamento de Análises Clínicas,b Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista “Júlio de Mesquita Filho,” CP 502, CEP 14801–902, Araraquara, São Paulo, Brasil, Departamento de Química Orgânica,c Instituto de Química, Universidade Estadual Paulista, Araraquara, São Paulo, Brasil. Received June 2, 2000; accepted October 3, 2000 By determining the hydrogen peroxide (H2O2) released in cultures of peritoneal macrophage cells from Swiss mice, we evaluated the action of 27 vegetable compounds (pristimerin, tingenone, jatrophone, palustric acid, lupeol, cladrastin, ocoteine, boldine, tomatine, yohimbine, reserpine, escopoletin, esculine, plumericin, dios- genin, deoxyschizandrin, p-arbutin, mangiferin, and others) using a 2 mg/ml solution of each compound (100 mmg/well). Macrophages are cells responsible for the development of the immunological response reaction, lib- erating more than one hundred compounds into the extracellular environment. Among these are the various cy- tokines and the intermediate compounds of nitrogen (NO) and oxygen (H2O2). This coordinated sequence of bio- chemical reactions is known as the “oxidative burst.” When we compared the results with those obtained with zy- mosan (an important stimulator of H2O2) we observed that the compounds showing the highest activity were sub- stances 2 (tingenone), 16 (reserpine) and 20. Other substances such as compounds 1, 4, 5, 6, 8, 12, 13, 14, 15, 17, 19, 23, 24, 26, and 27 also showed a certain activity, but with less intensity than the aforementioned ones. Com- pounds 3, 7, 9, 10, 11, 18, 21, 22 and 25 presented no activity. These results suggest that natural products (mainly tingenone and reserpine and others) with different chemical structures are strong immunological modulators. However, further tests are needed to determine the ‘oxidative burst’ in future studies. Key words natural product; macrophage; hydrogen peroxide and/or HPLC-UV methods. Purity was 95% or higher. Macrophage Cells and Determination of Hydrogen Peroxide (H2O2) Using the in vitro culture method and nat- ural products, we determined the reactive oxygen com- pounds.15,16) The method consists of the determination of the liberation of hydrogen peroxide (H2O2) in the culture of peri- toneal macrophages from Swiss mice. Peritoneal thioglyco- late-elicited macrophages from naive mice were obtained as reported previously.3) The peritoneal cavity was washed with 10 ml of cold phosphate buffered solution (PBS), the result- ing suspension was pelleted at 4 °C by centrifugation for 5 min at ca. 300 g, and the supernatant was removed. The 202 Vol. 24, No. 2 Fig. 1. Structures of Natural Products 1, pristimerin; 2, tingenone; 3, jatrophone; 5, palustric acid; 6, lupeol; 11, cladrastin; 12, ocoteine; 13, boldine; 14, tomatine; 15, yohimbine; 16, reserpine; 17, escopoletin; 18, esculine; 19, plumericin; 23, diosgenin; 24, deoxyschizandrin; 25, p-arbutin; 26, mangiferin. cells were resuspended at a concentration of 23106 cells/ml in a solution of phenol red containing 140 mM NaCl, 10 mM potassium phosphate, pH 7.0, 5.5 mM dextrose, 0.56 mM phe- nol red, and type II horseradish peroxidase, 0.01 mg/ml, (Sigma). Aliquots of 0.1 ml were transferred to flat-bottomed 96-well culture plates (Corning). A 50 m l volume of the nat- ural products solution (2 mg/ml, Sigma) or 50 m l of dimethyl sulfoxide (DMSO) was added. We used control without cells (culture media1DMSO); control with cells (culture media1 cells1DMSO); natural products dissolved in DMSO1cul- ture media1cells and positive control (Zymosan1culture media1cells). The samples were incubated for one hour at 37 °C in a humid atmosphere and the reaction was stopped by the addition of 10 m l of 4 N NaOH. Experiments were run in quadruplicate. Absorbance was determined with an auto- matic ELISA photometer with a 620 nm filter. The results were expressed as nanomoles of H2O2/23105 peritoneal cells, from a standard curve established in each test consist- ing of known molar concentrations of H2O2 in buffered phe- nol red. Statistical Analysis Data are expressed as mean6stan- dard deviation, and the Student’s t-test was used to determine the significance of the differences between the control and experimental groups. RESULTS AND DISCUSSION Many of our present medicines are directly or indirectly derived from higher plants. While several classic plant drugs have lost much ground to synthetic competitors, others have gained a new investigational or therapeutical status in recent years. Clinical plant-based research has made particularly re- warding progress in the important fields of therapy, such as yohimbine in male sexual dysfunction, reserpine as an anti- hypertensive agent, and boldine for digestive dysfunction.19) Diterpenoids isolated of plants from the genus Sideritis (Lamiaceae) have been used for their antiinflamatory and gastroprotective properties.5) Reactive oxygen metabolites (H2O2) have been suggested as potentially important signaling molecules in both intra- and intercellular reactions in a number of different cell types. It has been well documented that reactive species such as hy- drogen peroxide (H2O2) are produced by activated inflamma- tory cells.18) Figure 2 shows the concentrations of the control group DMSO, of zymosan and of 27 natural compounds that pre- sented a stimulatory effect detected by the determination of reactive compounds of oxygen (H2O2). The present study indicates that some natural products possess stronger activity than zymosan. The data presented in Fig. 2 summarize the immune response expressed as nmol after 1 h of incubation of several natural products. The com- pounds with a higher modulatory activity on the immune system were 2 and 20, which were able to release 385.81 and 285.16 nmol of H2O2, respectively, while zymosan (a power- ful immunostimulator) was able to release 275.16 nmol. Compound 16 also released a significative amount of H2O2 (198.91 nmol) when compared to zymosan. Comparison among compounds 8, 9 and 10 showed a marked decrease in the immune activity when the COOH February 2001 203 Fig. 2. Effects of Natural Products on Liberation on Hydrogen Peroxide Production by Mice Peritoneal Macrophages in Vitro The macrophages were cultured for 1 h in the presence of Control DMSO (C), Zymosan (250 mg/well) (Z) and isolated compounds (100 mg/well). Each bar represents the mean6S.D. of four animals. Representative results of one experiment repeated four times are given. Significantly different from control group, p,0.05. group is methylated. On the other hand, the introduction of a COOH group in the E-ring of 1 contributes to a decrease in the strong activity of 2. The high activity of 20 was also strongly reduced by re- moving the methylenedioxide group and by introducing the isoprene unit into the A-ring, as shown in compounds 21 and 22. Alteration in the chemical structure of the A and B rings of the aporfinic alkaloids (12, 13) also led to a significant de- crease in their activity. The activity of 12 was about five times higher than that of 13. Although the activities of compounds 5, 13, 15, 17, 19, 27 were about ten times lower than that of zymosan, they were still higher than that of the control. Comparison between compounds 23 and 14 showed that the sugar unit, the absence of the double bond and the nitro- genated E-ring of 14 did not significantly affect their activi- ties, since compounds 23 and 24 presented a statistically identical activity. Though the two coumarins (17, 18) did not present signifi- cant activity, in this case methylation of OH-6 led to a slightly higher activity of 17. Compounds 3, 7, 9, 10, 11, 18, 21, 22, and 25 did not pre- sent any activity. This note is the first communication of the activity of these purified components on macrophage cells. Many immunomodulating agents such polysaccharides (e.g., zymosan), protein-bound lipopolysaccharides (LPS) and proteins (e.g., concanavalin A, phytohemaglutinin) have been of interest in clinical research6,14) which have been re- ported to act primarily on cellular rather than humoral im- mune responses to restore the immunocompetency of im- paired hosts without hyperstimulating the normals. They augment macrophage chemotaxis, phagocytosis and promote interaction with other immunoregulatory lymphoid cells.8) On the basis of the results obtained, we may conclude that natural products might contribute to the induction of im- munostimulatory effects. However, further tests involving the ‘oxidative burst’ (O2 2), cytokines and NO production are needed. Therefore their mechanistic pharmacology is likely to be complex. Further phytochemical and pharmacological inves- tigations of these compounds is desirable too. Acknowledgements The authors are grateful to Marisa Campos Polesi Placeres, the technician of the Laboratório de Imunologia Clínica da Faculdade de Ciéncias Farmacêuticas of UNESP, Araraquara, São Paulo, Brazil. REFERENCES 1) Abdul K. M., Ramchender R. P., Immunopharmacol., 30, 231—236 (1995). 2) Barsucov A., Vetoshkin A., Vorontov E., Dubichev A., Planta Med., 56, 696— 697 (1990). 3) Carlos I. Z., Zini M. M. C., Sgarbi D. B. G., Angluster J., Alviano C. S., Silva C. L., Mycopathologia, 127, 189—194 (1994). 4) Hase K., Basnet P., Kadota S., Namba T., Planta Med., 63, 216—219 (1997). 5) Heras B., Hoult J. R. S., Planta Med., 60, 501—506 (1994). 6) Jain D., Kaur K., Sundaravadivel B., Salunke D. M., J. Biol. Chem., 26, 16098—16102 (2000). 7) Janeway C. J., “Immunobiology: The Immune System in Health and Disease,” ed. by Travers P., Garland Publishing Inc., New York, 1997. 8) Kapil A., Sharma S., J. Ethnopharmacol., 58, 89—95 (1997). 9) Kim H. M., Lee S. O., Lee E. H., Immunopharmacol., 21, 295—306 (1999). 10) Kreher B., Neszmélyi A., Jurcic K., Wagner H., Planta Med., 56, 572—573 (1990). 11) Marques M. F., Araujo A. R., Bolzani V. A. R., Young M. C. M., Plac- eres M. C. P., “Jornada Farmacêutica da UNESP,” ANAIS (abstract 65) (1999). 12) Mathew S., Kuttan G., Fitoterapia, 70, 35—43 (1999). 13) Nores M. M., Correges M. C., Benencia F., Coulombié F. C., J. Ethnopharmacol., 55, 99—106 (1997). 14) Nose M., Terawaki K., Oguri K., Ogihara Y., Yoshimatsu K., Shimo- mura K., Biol. Pharm. Bull., 21, 1110—1112 (1998). 15) Pick E., Keisari Y., J. Immunol. Methods, 38, 161—170 (1980) 16) Pick E., Mizel D., J. Immunol. Methods, 46, 211—226 (1981) . 17) Puri A., Saxena K. C., Srivastava V., Tandon J. S., J. Ethnopharmacol., 42, 31— 37 (1994). 18) Rochele L. G., Fischer B. M., Adler K. B., Free Radical Biology Medi- cine, 24, 863—868 (1998). 19) Simões C. M. O., Schenkel E. P., Gosamann G., Palazzo de Mello J. C., Mentz L. A., Ros Petrovick P., “Farmacognosia: Da Planta ao Medicamento,” ed. by U.F.R.G.S., Porto Alegre, 1999. 204 Vol. 24, No. 2