Nitric Oxide-Scavenging Properties of Some Chalcone Derivatives

NITRIC OXIDE: Biology and Chemistry Vol. 6, No. 2, pp. 242–246 (2002) doi:10.1006/niox.2001.0396, available online at http://www.idealibrary.com on

Nitric Oxide-Scavenging Properties of Some Chalcone Derivatives Felipe Herencia,* M. Pilar Lo´pez-Garcı´a,† Amalia Ubeda,* and M. Luisa Ferra´ndiz* ,1 *Department of Pharmacology, and †Department of Biochemistry and Molecular Biology, University of Valencia, 46100 Burjassot, Spain Received January 17, 2001, and in revised form July 13, 2001; published online October 22, 2001

The implication of NO in many inflammatory diseases has been well documented. We have previously reported that some chalcone derivatives can control the iNOS pathway in inflammatory processes. In the present study, we have assessed the NO-scavenging capacity of three chalcone derivatives (CH8, CH11, and CH12) in a competitive assay with HbO 2, a wellknown physiologically relevant NO scavenger. Our data identify these chalcones as new NO scavengers. The estimated second-order rate constants (k s) for the reaction of the three derivatives with NO is in the same range as the value obtained for HbO 2, with CH11 exerting the greatest effect. These results suggest an additional action of these compounds on NO regulation. © 2001 Elsevier Science (USA) Key Words: chalcone derivatives; nitric oxide scavengers; nitric oxide; oxyhemoglobin; spermineNONOate.

Free radicals are involved in a variety of physiological and pathological processes (1). Thus, reactive nitrogen intermediates such as nitric oxide (NO) 2 play a central role in a number of pathologies including inflammatory and immune diseases (2). NO is an important physiological regulator of functions such as vasodilatation and neurotransmission. Nevertheless, under pathological conditions, high concentrations of NO can be either beneficial (e.g., when antibacterial, antiparasitic, or antiviral effects are required) or detrimental, if they lead to inflammatory reactions and injury for host tissues. Such detrimental effects can be mediated 1

To whom correspondence should be addressed at Departamento de Farmacologia, Facultad de Farmacia, Av. Vicent Andres Estelles s/n, 46100 Burjasot, Valencia, Spain. Fax: 34-963864292. E-mail: [email protected]. 2 Abbreviations used: NO, nitric oxide; iNOS, inducible nitric oxide synthase; HbO 2, oxyhemoglobin; DAN, 2,3-diaminonaphthalene; SPNO, spermine-NONOate; metHb, methemoglobin; TRI, 2,3naphthotriazole. 242

by reaction of persistent high amounts of NO with concomitantly produced superoxide anions, generating highly toxic species, such as peroxynitrite and hydroxyl radicals (3, 4). Thus, inhibition of NO synthesis or NO scavenging seems to be an attractive therapeutic approach in some diseases. Chalcone skeleton has been considered the biological precursor of flavonoids. This family of natural products is formed by a huge number of compounds possessing a wide array of biological effects (5, 6). Because of their chemical heterogenity, no general molecular mechanism underlying all their activities can be expected. However, part of the therapeutic effect of flavonoids has been ascribed to their free radical scavenging properties (7). The NO-scavenging capacity of different flavonoids has been described (8) but little is known of chalcone effects on this radical. Recently, we have reported that chalcone derivatives CH8, CH11, and CH12 inhibit nitrite production (the stable metabolite of NO) and can reduce the expression of inducible nitric oxide synthase (iNOS) in mouse peritoneal macrophages (9, 10). We have also shown that CH11 is a superoxide scavenger (10). The present study was designed to determine if NO scavenging could participate in the inhibitory effects of these chalcone derivatives (Table 1) on the NO pathway. MATERIALS AND METHODS Chemicals Chalcone derivatives were prepared according to procedures previously described by Herencia et al. (10). Stock solutions (10 mM) were prepared in methanol and stored at ⫺20°C until used. Oxyhemoglobin (HbO 2, human Ao, ferrous) and 2,3-diaminonaphthalene (DAN) was obtained from Sigma (St. Louis, MO). HbO 2 stock solutions (25 mg/ml) were prepared in 0.1 M Hepes buffer, pH 7.4, aliquoted, and kept at ⫺80°C until use. The actual HbO 2 concentration in the assays 1089-8603/01 $35.00 © 2001 Elsevier Science (USA) All rights reserved.

CHALCONE AS NO-SCAVENGER

was always verified spectrophotometrically. SpermineNONOate (SPNO) was purchased from Cayman Chemicals (Ann. Arbor, MI) and prepared as 10 mM stock aliquots in NaOH 0.01 M. NO-Scavenging Assay The potential NO-scavenging properties of chalcone derivatives were examined using a competitive “quenching” method, as recommended for conditions under which the product derived from the reaction with NO is unkown (11). An in vitro kinetic competition assay using oxyhemoglobin as detector molecule (8) was designed and here adapted to the particular characteristics of the tested compounds. The assay is based on the sensitive detection of the inhibitory effect of chalcones on the very rapid NO-induced HbO 2 oxidation to methemoglobin (metHb) and nitrate. NO was generated in situ using the spontaneous decomposition of the SPNO adduct at pH 7.4. Under these conditions, SPNO decomposes to yield 2 mol NO/mol adduct at known and constant rates, estimated in 2.4 and 0.9 nmol NO/min for SPNO concentrations of 100 and 50 ␮M, respectively (12). The incubations were performed in 0.1 M potassium phosphate/1 mM EDTA buffer, pH 7.4, in 1-ml cuvettes at 25°C. Briefly, solutions of HbO 2 (concentration ranging from 6 to100 ␮M) were exposed to the NO donor, in the simultaneous presence of varying concentrations of the different chalcones. HbO 2 oxidation was checked spectrophotometrically (13) following the decrease in the absorbance at 576 nm during a 5-min assay period. Rates of metHb formation were corrected from the spontaneous HbO 2 oxidation (always less than 8% of the overall rate). Appropriate controls were carried out to verify (i) that in the absence of SPNO, chalcones or solvent (⬍0.1%, v/v, methanol) alone are not able to oxidate HbO 2, which excludes the generation of H 2O 2 or superoxide; (ii) that the final reaction product (metHb) is stable and not recycled back to HbO 2 upon exposure to chalcones; and (iii) the linearity of NO flux during the whole assay period. Estimation of the second-order rate constants of chalcone derivatives was performed as previously described (8, 11). The reported k s value for the HbO 2 reaction with NO, 3 ⫻ 10 7 M ⫺1 s ⫺1 (14), was taken for calculation. The validity of the results obtained in the kinetic competition assay with HbO 2 was further confirmed using DAN as an alternative detector molecule, as previously described (11, 15). Briefly, this assay is based on the nitrosation of DAN (from SPNO-

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generated NO flux) to form a fluorescent stable triazole complex (2,3-naphthotriazole, TRI). A 200 ␮M DAN concentration was chosen in order to provide that over 90% of the NO generated from 50 ␮M SPNO is scavenged in the absence of chalcones. Incubations were carried out for 20 min at 37°C, in the absence or presence of chalcones. Supernatants were then made 10 mM in NaOH, and TRI formation was fluorimetrically determined. At least three separate experiments were performed, and the data are expressed as means ⫾ SD. RESULTS Oxidation of HbO 2 to metHb is a well-characterized specific assay for the determination of NO. In the kinetic competition method, scavengers of NO compete with HbO 2 for the radical and thus decrease the rate of HbO 2 oxidation. This depends on the reaction rate of the scavenger with NO as compared to that of HbO 2. The competitive assay of NO-induced HbO 2 oxidation was here properly adapted to evaluate the potential NO-scavenging ability of three chalcone derivatives. The HbO 2 spectrum is shown in Fig. 1A (insert). Due to the UV-VIS spectral characteristics of the tested compounds (with maximal absorbance wavelengths between 280 and 425 nm, Table 1), metHb formation was necessarily monitored following the decrease of the HbO 2 peak at 576 nm. As shown in Fig. 1A, upon exposure to SPNO, HbO 2 oxidation proceeded linearly for at least 10 min, thus reflecting a constant NO flux. It was also verified that the rate of metHb formation varied linearly with SPNO concentration (Fig. 1B). A fixed concentration of 100 ␮M SPNO (which provides a mean rate of 1.58 ⫾ 0.35 nmol metHb/min, n ⫽ 6) was selected as optimal for further experiments. The estimated rates of metHb formation from 100 ␮M SPNO in control assays (with no chalcone added) did not vary for HbO 2 initial concentrations ranging from 6 to 100 ␮M (not shown). A high initial HbO 2 concentration (60 ␮M) was chosen to maximally increase the sensitivity of the competition assay, and to ensure that the detector molecule was nonlimiting for the whole incubation period. The three chalcone derivatives studied efficiently competed with HbO 2 for NO in a concentrationdependent manner. As illustrated in Fig. 2A for CH11, this was reflected by a significant decrease on the rate of metHb formation. Results also showed that the three compounds behaved as potent and efficient scavengers since NO-induced HbO 2 oxidation could be completely blocked (⬎90% inhibition of metHb formation was

© 2001 Elsevier Science (USA). All rights reserved.

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HERENCIA ET AL.

FIG. 1. Oxyhemoglobin oxidation by NO generated from SPNO. (A) HbO 2 (40 ␮M) was exposed to 100 ␮M SPNO. MetHb formation was followed by the decrease in absorbance at 576 nm every minute for 10 min (insert: HbO 2 absorption spectrum). (B) Variation of the rate of metHb formation as a function of SPNO concentration in the assay.

achieved with chalcone concentrations in the same range as that of HbO 2 in the assay). Logarithmic transformation of HbO 2 oxidation rates as a function of chalcone concentration (Fig. 2B) allowed the calculation of IC 50 values. A mean of 47.1 ⫾ 8.9, 24.4 ⫾ 8.1, and 46.8 ⫾ 5.3 ␮M was obtained for CH8, CH11, and CH12, respectively. For evaluation of the biological relevance of chalcone reactivity toward NO, it is also important to consider the reaction rate. The estimated second-order rate constants for the NO scavenging reaction of chalcones are shown in Table 1.

The feasibility of NO scavenging by chalcones was confirmed using DAN as detector molecule. As illustrated in Fig. 3 for CH11 and CH12, the tested compounds were able to efficiently reduce the amount of TRI generated from the DAN-NO reaction which confirms that competition with DAN indeed occurs. DISCUSSION An interesting potential therapeutic target to control undesirable effects of NO overproduction could be the

TABLE 1

Effects of Chalcone Derivatives on NO Scavenging ␭ max (nm)

k s (⫻10 7 M ⫺1 s ⫺1)

n

CH 8

280–322

4.0 ⫾ 0.8

3

CH 11

425

8.1 ⫾ 2.2

3

CH 12

339

4.0 ⫾ 0.8

3

Compound

Chemical structure

© 2001 Elsevier Science (USA). All rights reserved.

CHALCONE AS NO-SCAVENGER

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FIG. 2. Inhibitory effects of chalcone derivatives on HbO 2 oxidation by NO. (A) HbO 2 (60 ␮M) was exposed or not (autooxidation) to 100 ␮M SPNO, in the presence of variable concentrations of CH11. (B) Rate of metHb oxidation by NO as a function of chalcone concentration in the assay. This figure shows a representative experiment out of three.

scavenging of this radical in certain pathophysiological states such as septic shock. For example, hemoglobin itself has been shown to react with NO under physiological conditions, and may exert beneficial effects in endotoxic shock (16). Some natural compounds such as flavonoids present in Ginkgo biloba extract Egb 761 have been described as NO scavengers in different experimental assays (17). We have previously shown that some chalcone derivatives can control the NO pathway and exert mod-

FIG. 3. 2,3-Naphthotriazole (TRI) formation in the absence and presence of chalcones. 200 ␮M DAN was exposed to 50 ␮M SPNO at 37°C, in the presence of variable concentrations of CH11 and CH12, as indicated. Following the incubation period, TRI formation was monitored fluorimetrically.

ulatory effects in inflammatory processes (9). In the present study, the results indicate that the three chalcone derivatives tested (CH8, CH11, and CH12) are excellent NO scavengers. The potency of these compounds was in the same range as hemoglobin, a wellknown, physiologically relevant, endogenous NO scavenger. CH8 and CH12 showed equal potency, whereas the k s value determined for CH11 indicated twofold more NO-scavenging capacity, suggesting that this property expands the role of this chalcone as antioxidant agent. CH11 was previously found to exert potent superoxide-scavenging effects in vitro and in vivo, cytoprotective actions in oxidative stress, downregulation of iNOS expression, and anti-inflammatory effects (18). The ability of our compounds to decrease the massive amounts of NO produced in inflammatory processes can be related to the inhibition of iNOS expression and also to the NO-scavenging property demonstrated in this study. In addition, the activity of CH11 on superoxide and NO may allow the use of this chalcone in situations with excessive generation of these radicals, including ischemia-reperfusion injury and endotoxic shock. To date, no general mechanism for the otherwise known free radical-scavenging properties of chalcones and/or flavonoids has been described. It has been reported by Bors et al. (19) that the 2,3-double bond in conjugation with a 4-oxo function, which is responsible for electron delocalization from the B ring, is important for radical scavenging and antioxidative potential of

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flavonoids (19). Besides in the case of CH11, the pair of electrons of the dimethylamine can be delocalized in the aromatic ring reaching the 2,3-double bond in conjugation with the 1-oxo function and could react with NO. Although NO overproduction can be controlled by NOS inhibitors, they affect frequently constitutive isoforms at doses normally used, which leads to hypertension and a decreased local blood flow (20). There is also evidence that chronic inhibition of NOS can result in an increased iNOS expression followed by overproduction of NO upon removal of the inhibitor (21). In conclusion, NO scavenging is an alternative to NOS inhibition and may be an interesting approach to reduce toxic levels of NO without totally eliminating low levels which may be beneficial. ACKNOWLEDGMENTS We thank Jose´ N. Domı´nguez, Jaime E. Charris, and Gricela M. Lobo (Laboratory of Organic Synthesis, Faculty of Pharmacy, Central University of Venezuela, Caracas) for kindly providing us chalcone derivatives. F. Herencia was the recipient of a Carmen and Severo Ochoa grant from Ayuntamiento de Valencia. This work was supported by Grant SAF97-0249 from CICYT.

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