Electrochemical evidence reveals the effects of L-cysteine and surfactant on the stability of artemisinin (qinghaosu)
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《中华医药杂志》英文版
【Abstract】 Artemisnin (qinghaosu,QHS) is a natural drug that has potent antimalarial activity. It is generally believed that the antimalarial activity of QHS stems from the iron or heme-mediated cleavage of its unique endoperoxy bond to produce carbon-centered radicals that kill parasites. However, the low aqueous solubility of QHS limits its bioavailability. In this study, we employed electrochemistry approach to examine the effects on QHS stability from L-cysteine (L-Cys), an endogenous low-molecular-weight thiol, and surfactants that may be used for QHS formulation to increase solubility. We found that L-Cys can directly interact with QHS without iron mediation to form a binary adduct, stabilizing QHS as presented by the negative shift of the reduction potential from-0.64V to-1.03V. Cationic surfactant benzalkonium bromide (DBDAB) can also join the interaction with QHS-L-Cys to form a ternary adduct, impairing the effect of L-Cys on QHS stability. When the concentrations of DBDAB and L-Cys reached a 2∶1 stoichiometric ratio, DBDAB completely saturated L-Cys by interactions at both sides of L-Cys, freed QHS and thus fully inhibited the effect of L-Cys on QHS. These findings are important information for understanding the antimalarial mechanism and efficacy of QHS and for assaying pharmaceutical application in formulating QHS.
【Key words】 artemisinin;L-Cysteine;surfactants;stability
INTRODUCTION
Artemisinin (Qinghaosu, QHS), isolated from a Chinese herb Qinghao, is a natural 1,2,4-trioxane featuring a unique endoperoxide bond (Fig.1). QHS and its derivatives are the most promising drugs to counter malaria[1,2]. Recent works indicated that these natural and synthetic compounds also exhibit broad anti-cancer bioactivities[3-6]. It is commonly accepted that the QHS antimalarial activity stems from the parasiticidal attack of the carbon radicals or intermediates generated from a cascade of chemical reactions triggered by the intraparasitic iron or heme that catalyzes the cleavage of the endoperoxide bridge in the trioxane[1,2,7]. The facts that antioxidants such as ascorbate and glutathione can inhibit the antimalarial activity[8,9] whereas free radical-generating compounds enhance the activity[8,10] support the idea of the oxidative free radicals as the active species.
L-cysteine (L-Cys) is one of the endogenous low-molecular-weight thiols in physiological systems and a component for glutathione synthesis. L-Cys concentrations in cells and plasma are respectively 0.15~0.25 mmol/L (in rat liver)[11] and 1025 μM [12], and the concentration of free L-Cys increases in the infected erythrocytes probably due to the catabolism of hemoglobin[13]. These amounts of L-Cys are enough to influence the QHS activity by acting as an antioxidant. Research has shown that, mediated by traces of iron, L-Cys can react with QHS to produce a new species that incorporates the sulfur moiety of cysteine[7].
Due to the poor solubility of QHS in both water and oil, the QHS bioavailability in pharmaceutical preparations is low[14]. To increase the solubility of QHS in aqueous environments, one alternative is to use surfactants to aggregate or coat the drug. This method has also been broadly employed in other drug preparations[13,16]. However, whether the surfactants could affect the stability and efficacy of QHS? This question has not been addressed so far. In this study, we used cyclic voltammetry (CV) to examine the electrochemical behaviors of QHS in a binary system with L-Cys and a ternary system with both L-Cys and cationic surfactant benzalkonium bromide (DBDAB) (Fig. 1). (We demonstrated that L-Cys can increase the stability of QHS by directly interacting with QHS without iron mediation and DBDAB can limit the effect of L-Cys on QHS in solution. These findings may be informative for understanding the mode of action of QHS and for QHS pharmaceutical application.)
MATERIALS AND METHODS
QHS was provided kindly from Tropical Medicine Institute, Guangzhou University of Traditional Chinese Medicine, China. QHS was dissolved in absolute ethanol as stock solution. L-Cysteine (L-Cys) was purchased from Sigma. Dodecyl-benzyl-dimethyl-ammoniun-bromide (DBDAB) was purchased from Dongkang Pharmaceutical Company Ltd (Guangzhou, China). Lauryl sodium sulfate (SDS) was purchased from the Chemical Reagent Factory of Guangzhou (China). Supporting electrolyte for cyclic voltammetry was Britton-Robinson buffer solution at pH 7.2.
Electrochemical measurements were performed at a CHI660A electrochemistry work station (CH Instruments Co., USA). The electrochemical cell consisted of a three electrode system where a silver electrode (Ф3 mm) was used as the working electrode, a platinum wire as a counter electrode and a saturated calomel electrode (SCE) as the reference electrode.
Britton-Robinson buffer (pH 7.2) solution containing 20% (v/v) ethanol was used for cyclic voltammetry measurement. The voltammetric experiments were performed in the potential range of 0.00 ~-1.30 V (vs SCE) with a scan rate of 100 mV/s in the Britton-Robinson buffer after deaeration for 10 min.
RESULTS AND DISCUSSION
As shown in Figure 2, in the Britton-Robinson buffer of pH 7.2, QHS (1.0 mM) displayed a reduction peak at-0.64V (Curve A) in CV scanning, reflecting that QHS was reduced on the electrode surface. The peak current of the reduction peak decreased when L-Cys was added into the buffer solution (Curve B), indicating the occurrence of QHS-L-Cys interaction. When L-Cys concentration increased to 0.04mM, the QHS reduction peak disappeared, accompanied with the appearance of a new reduction peak at-1.03 V (Curve C). The peak current at-1.03 V linearly increased upon increasing amounts of either L-Cys or QHS being added (Fig. 3), suggesting the formation of a binary QHS-L-Cys complex or adduct resulted from the direct interaction between QHS and L-Cys. It must be pointed out that, given the fact that the concentration of QHS was in heavy excess over L-Cys in the solution, the formation of the QHS-L-Cys species may not be in 1∶1 stoichiometric ratio. The appearance of-1.03 V signal only indicated that the small amount of the QHS-L-Cys complex formed can diffuse to the electrode surface and be sensitively detected although [L-Cys] << [QHS]. Moreover, based on Gibbs free energy calculation (Table 1), the formation of the QHS-L-Cys complex increased the activation energy and thus the stability of QHS, leading to the shift of the reduction peak negatively from-0.64V to-1.03V.
Note:△G =-nF △E, where n is the number (2) of reduction electrons, F is Faraday constant, and △E is the difference of potential
Figure 1 Structural formulae of arte misinin (QHS), DBDBA and L-cysteine
Iron or heme catalyzed QHS reaction to form toxic free radicals that kill malaria parasites is a commonly accepted mechanism for QHS antimalarial activity[1,2,17]. Evidence has also been obtained for non-heme iron mediated interaction between QHS and L-Cys, which was regarded as another possible pathway for the cleavage of the endoperoxide bridge[7].
The current electrochemical results clearly demonstrated that without iron mediation QHS and L-Cys can directly interact to form an adduct, resulting in an enhanced stability of QHS. Considering the fact that there are chemically sufficient amounts of L-Cys in human body, the direct interaction between QHS and L-Cys can be regarded as a major side reaction in parallel to the ironorheme-mediated reactions. If this is a case, the side reaction could play a role in the QHS degradation and anti-parasitic efficacy.
To study the influence of pH on the reduction of QHS in the presence of L-Cys, CV scanning was performed for the binary system at different pH values andthe electrochemical behaviors were summarized in Table 2. In acidic condition, only the reduction peak of QHS was observed. With pH value increasing from pH 1.1 to 5.5, the QHS peak descended and the reduction potential of QHS shifted gradually from-0.41V to-0.54V. The reduction peak then disappeared, with the peak at-1.03V developing as the pH reaching 6.2. In alkaline condition, the peak current of QHS reduction at-1.03V increased with the enhancing pH value, and decreased significantly when pH>12 and then disappeared completely as pH>13.
A: QHS (1.0mM); B: A + L-Cys (0.01mM);
C: A + L-Cys (0.04mM).
Figure 2 Cyclic voltammograms of QHS in the presence of L-Cys in Britton-Robinson buffer (pH 7.2) containing 20% ethanol (v/v) at silver electrode
These electrochemical behaviors are closely related to the electrostatic property of L-Cys. L-Cys has an isoelectric point at pH 5.02 and the apparent ionization constants of L-Cys are 1.71 and 8.33 respectively. Under the extreme acidic condition pH = 1.5, L-Cys exists mostly as a neutral compound in the solution and thus the binary system displayed only the reduction peak of QHS with high potentials. With pH increasing, L-Cys was gradually ionized as a zwitterion to interact with QHS, leading to the decrease of QHS on the electrode surface and thus the decrease of the peak currents. When pH reached 6.2, the deprotonated L-Cys increased to a concentration for the formation of the QHS-L-Cys species which can be detected as the CV signal at-1.03V. The peak currents of-1.03V increased with the detection of more QHS-L-Cys complex formed in neutral to alkaline conditions. Based on the fact that QHS can form a C-S covalent bond with L-Cys in the presence of iron[7], the binary adduct of QHS-L-Cys may also form through the thiol group. The electrode reaction can be depicted as Scheme 1A. Under strong alkaline conditions (pH>12), the lactone bond of QHS was easy to be hydrolyzed and L-Cys was apt to form cystine, the binary adduct may decompose and the reduction peak of the adduct thus disappeared. These observations indicate that neutral to alkaline pH in the range of 7.2~11.2 are the optimal condition for the formation of the binary adduct of QHS and L-Cys.
In order to detect the effect of surfactants, anionic surfactant SDS and cationic surfactant DBDAB were respectively added into the binary system containing QHS and L-Cys. While there was nothing happened with the SDS addition, the addition of DBDAB into the binary system caused a positive shift of the reduction peak from-1.03 V to-0.93 V (Fig. 4, Curve B → Curve C). This peak at-0.93 V was not observed in the control binary systems containing either QHS + DBDAB (data no shown) or L-Cys + DBDAB (Fig. 5, Curve A), suggesting that this new reduction peak is for the specific ternary interaction, perhaps the formation of a ternary adduct, in the system. In view of the free energy difference due to the potential shift from-1.03V to-0.93V (Table 1), DBDBA, in other words, decreased the effect of L-Cys on the stability of QHS by interacting with L-Cys preferably in the carboxyl side (Scheme 1B). Again, the excess amount of QHS does not support the 1∶1∶1 stoichiometry of the ternary complex in solution. The observed signal at-0.93V only indicates that the Ag electrode can sensitively detect the ternary complex even in a small amount.
To verify whether the three components in the ternary system were involved in the interaction, the concentration of DBDAB, QHS or L-Cys was respectively increased while the other two components remained unchanged. As Curves C,D,E shown in Figure 4,when the concentration of DBDAB in the ternary system was gradually raised, the increase of the peak current at-0.93V was accompanied with the growth of the reduction peak of QHS at-0.64V. When the concentration of DBDAB reached 2 folds of L-Cys concentration, the peak at-0.93V disappeared and the peak at-0.64V fully developed, entirely resembling the CV curve of QHS only (Fig. 4, Curves A & E). This suggests that DBDAB completely saturated L-Cys by interactions at both sides of carboxyl and mercapto groups of L-Cys and thus the Ag electrode detected free QHS only (Scheme 1C). Obviously, cationic surfactant DBDAB took part in the interaction in the ternary system containing QHS and L-Cys by impairing and then breaking down the QHS-L-Cys interaction.
On the other hand, Figure 5 shows the representative electrochemical spectra for the influence of QHS concentration on the formation of the ternary adduct. Double amounts of L-Cys and DBDAB were used in this case for better observation of the change of the peak current. The CV spectra clearly demonstrated that (omit)
A:[QHS] = 1.0mmol/L; B:[L-Cys] = 0.04mmol/L
Figure 3 Influence of L-Cys and QHS concen
trations on peak current at-1.03 V
system in Britton-Robinson buffer (pH 7.2) containing 20% ethanol (v/v). A: QHS (1 mM);B: QHS (1 mM) + L-Cys (0.04 mM); C: B + DBDAB (5μM); D: B + DBDAB (0.02 mM);E: B + DBDAB (0.08 mM)
Figure 4 Cyclic voltammograms of the ternary
A: L-Cys (0.08 mM) + DBDAB (0.01 mM);B: A + QHS (0.4 mM); C: A + QHS (0.8 mM);D: A + QHS (1.2 mM).Figure 5 Influence of QHS concentration on the peak current of the ternary adduct
the reduction peak at-0.93 V developed upon QHS being added into the solution containing L-Cys and DBDAB ([L-Cys]∶[DBDAB] =1∶1,mole ratio) and the peak current increased with the concentration increase of QHS. Similar CV changes were detected with increasing concentrations of L-Cys.
Based on the electrochemical behaviors described above, an electrode mechanism can be proposed to interpret the interaction on the electrode surface (Scheme 1). At neutral pH 7.2, L-Cys exists as a zwitterion to interact with QHS through-SH group which although remains no deprotonation (Scheme 1A). Cationic surfactant DBDAB may electrostatically interact with L-Cys (firstly with the carboxyl group) on the electrode surface to form ternary adducts (Scheme 1B). This explains the observation that anionic surfactant SDS had no interaction in the system. The electrostatic interaction between DBDAB and the carboxyl group of L-Cys weakened the interaction between QHS and the mercapto group of L-Cys, the reduction peak therefore shifted positively by 0.1 V from-1.03 V for the binary adduct to-0.93 V for the ternary adduct. With the concentration of DBDAB rising, cationic DBDAB may replace QHS to interact with L-Cys at the mercapto group side (Scheme 1C), and free QHS was released from the weakened interaction with L-Cys, leading to the development of the reduction peak of QHS at-0.64 V. When the concentration of DBDAB reached the double amount of L-Cys, DBDAB completely replaced QHS to saturate L-Cys in 2:1 stoichiometry, we thus observed that the peak for the ternary adduct disappeared and the peak for free QHS fully developed.
Conclusions
Surfactants have been widely utilized in the formulation of drugs to increase solubility[18-20]. Surfactant aggregation with QHS has also been studied to enhance the poor solubility of QHS and increase its bioavalability[13]. However, the introduction of surfactants may also create other side effects[19]. Our current observation clearly demonstrated that electrochemical measurement can be used to sensitively monitor the formation of the ternary complex of cationic surfactant DBDAB with QHS and endogenous L-Cys under a physiological relevant condition. The CV scanning further revealed that high amount of the cationic surfactant can block the interaction between QHS and L-Cys and thus inhibit the effect of L-Cys on QHS stability. These interesting findings provide important information for better understanding QHS efficacy and effecting factors and for global consideration in drug formulation of QHS in terms of increasing solubility.
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【Acknowledgements and funding】 This work was partially supported by the Guangdong Province and Guangzhou Natural Science Foundation of China (No.021190, 2003Z3D2041). We would like to thank Professors Y.T. Zhu and M.Y. Zhang from Tropical Medicine Institute, Guangzhou University of Traditional Chinese Medicine, China, for kindly providing Qinghaosu sample and for their helpful suggestions
1 Department of Chemistry, Jinan University, Guangzhou,Guangdong Province 510632, China
2 Institute of Life and Health Engineering, Jinan University, Guangzhou,Guangdong Province 510632, China
* Correspondence to Professor Qing-Yu He, Institute of Life and Health Engineering, Jinan University, Guangzhou,Guangdong Province 510632, China,
Tel. & Fax: 86-20-85227039,E-mail: qing-yu.he@163.com
(Editor Guo Hui-ling)ARTICLES(YANG Pei-hui,SU Zhang-yi,)
【Key words】 artemisinin;L-Cysteine;surfactants;stability
INTRODUCTION
Artemisinin (Qinghaosu, QHS), isolated from a Chinese herb Qinghao, is a natural 1,2,4-trioxane featuring a unique endoperoxide bond (Fig.1). QHS and its derivatives are the most promising drugs to counter malaria[1,2]. Recent works indicated that these natural and synthetic compounds also exhibit broad anti-cancer bioactivities[3-6]. It is commonly accepted that the QHS antimalarial activity stems from the parasiticidal attack of the carbon radicals or intermediates generated from a cascade of chemical reactions triggered by the intraparasitic iron or heme that catalyzes the cleavage of the endoperoxide bridge in the trioxane[1,2,7]. The facts that antioxidants such as ascorbate and glutathione can inhibit the antimalarial activity[8,9] whereas free radical-generating compounds enhance the activity[8,10] support the idea of the oxidative free radicals as the active species.
L-cysteine (L-Cys) is one of the endogenous low-molecular-weight thiols in physiological systems and a component for glutathione synthesis. L-Cys concentrations in cells and plasma are respectively 0.15~0.25 mmol/L (in rat liver)[11] and 1025 μM [12], and the concentration of free L-Cys increases in the infected erythrocytes probably due to the catabolism of hemoglobin[13]. These amounts of L-Cys are enough to influence the QHS activity by acting as an antioxidant. Research has shown that, mediated by traces of iron, L-Cys can react with QHS to produce a new species that incorporates the sulfur moiety of cysteine[7].
Due to the poor solubility of QHS in both water and oil, the QHS bioavailability in pharmaceutical preparations is low[14]. To increase the solubility of QHS in aqueous environments, one alternative is to use surfactants to aggregate or coat the drug. This method has also been broadly employed in other drug preparations[13,16]. However, whether the surfactants could affect the stability and efficacy of QHS? This question has not been addressed so far. In this study, we used cyclic voltammetry (CV) to examine the electrochemical behaviors of QHS in a binary system with L-Cys and a ternary system with both L-Cys and cationic surfactant benzalkonium bromide (DBDAB) (Fig. 1). (We demonstrated that L-Cys can increase the stability of QHS by directly interacting with QHS without iron mediation and DBDAB can limit the effect of L-Cys on QHS in solution. These findings may be informative for understanding the mode of action of QHS and for QHS pharmaceutical application.)
MATERIALS AND METHODS
QHS was provided kindly from Tropical Medicine Institute, Guangzhou University of Traditional Chinese Medicine, China. QHS was dissolved in absolute ethanol as stock solution. L-Cysteine (L-Cys) was purchased from Sigma. Dodecyl-benzyl-dimethyl-ammoniun-bromide (DBDAB) was purchased from Dongkang Pharmaceutical Company Ltd (Guangzhou, China). Lauryl sodium sulfate (SDS) was purchased from the Chemical Reagent Factory of Guangzhou (China). Supporting electrolyte for cyclic voltammetry was Britton-Robinson buffer solution at pH 7.2.
Electrochemical measurements were performed at a CHI660A electrochemistry work station (CH Instruments Co., USA). The electrochemical cell consisted of a three electrode system where a silver electrode (Ф3 mm) was used as the working electrode, a platinum wire as a counter electrode and a saturated calomel electrode (SCE) as the reference electrode.
Britton-Robinson buffer (pH 7.2) solution containing 20% (v/v) ethanol was used for cyclic voltammetry measurement. The voltammetric experiments were performed in the potential range of 0.00 ~-1.30 V (vs SCE) with a scan rate of 100 mV/s in the Britton-Robinson buffer after deaeration for 10 min.
RESULTS AND DISCUSSION
As shown in Figure 2, in the Britton-Robinson buffer of pH 7.2, QHS (1.0 mM) displayed a reduction peak at-0.64V (Curve A) in CV scanning, reflecting that QHS was reduced on the electrode surface. The peak current of the reduction peak decreased when L-Cys was added into the buffer solution (Curve B), indicating the occurrence of QHS-L-Cys interaction. When L-Cys concentration increased to 0.04mM, the QHS reduction peak disappeared, accompanied with the appearance of a new reduction peak at-1.03 V (Curve C). The peak current at-1.03 V linearly increased upon increasing amounts of either L-Cys or QHS being added (Fig. 3), suggesting the formation of a binary QHS-L-Cys complex or adduct resulted from the direct interaction between QHS and L-Cys. It must be pointed out that, given the fact that the concentration of QHS was in heavy excess over L-Cys in the solution, the formation of the QHS-L-Cys species may not be in 1∶1 stoichiometric ratio. The appearance of-1.03 V signal only indicated that the small amount of the QHS-L-Cys complex formed can diffuse to the electrode surface and be sensitively detected although [L-Cys] << [QHS]. Moreover, based on Gibbs free energy calculation (Table 1), the formation of the QHS-L-Cys complex increased the activation energy and thus the stability of QHS, leading to the shift of the reduction peak negatively from-0.64V to-1.03V.
Note:△G =-nF △E, where n is the number (2) of reduction electrons, F is Faraday constant, and △E is the difference of potential
Figure 1 Structural formulae of arte misinin (QHS), DBDBA and L-cysteine
Iron or heme catalyzed QHS reaction to form toxic free radicals that kill malaria parasites is a commonly accepted mechanism for QHS antimalarial activity[1,2,17]. Evidence has also been obtained for non-heme iron mediated interaction between QHS and L-Cys, which was regarded as another possible pathway for the cleavage of the endoperoxide bridge[7].
The current electrochemical results clearly demonstrated that without iron mediation QHS and L-Cys can directly interact to form an adduct, resulting in an enhanced stability of QHS. Considering the fact that there are chemically sufficient amounts of L-Cys in human body, the direct interaction between QHS and L-Cys can be regarded as a major side reaction in parallel to the ironorheme-mediated reactions. If this is a case, the side reaction could play a role in the QHS degradation and anti-parasitic efficacy.
To study the influence of pH on the reduction of QHS in the presence of L-Cys, CV scanning was performed for the binary system at different pH values andthe electrochemical behaviors were summarized in Table 2. In acidic condition, only the reduction peak of QHS was observed. With pH value increasing from pH 1.1 to 5.5, the QHS peak descended and the reduction potential of QHS shifted gradually from-0.41V to-0.54V. The reduction peak then disappeared, with the peak at-1.03V developing as the pH reaching 6.2. In alkaline condition, the peak current of QHS reduction at-1.03V increased with the enhancing pH value, and decreased significantly when pH>12 and then disappeared completely as pH>13.
A: QHS (1.0mM); B: A + L-Cys (0.01mM);
C: A + L-Cys (0.04mM).
Figure 2 Cyclic voltammograms of QHS in the presence of L-Cys in Britton-Robinson buffer (pH 7.2) containing 20% ethanol (v/v) at silver electrode
These electrochemical behaviors are closely related to the electrostatic property of L-Cys. L-Cys has an isoelectric point at pH 5.02 and the apparent ionization constants of L-Cys are 1.71 and 8.33 respectively. Under the extreme acidic condition pH = 1.5, L-Cys exists mostly as a neutral compound in the solution and thus the binary system displayed only the reduction peak of QHS with high potentials. With pH increasing, L-Cys was gradually ionized as a zwitterion to interact with QHS, leading to the decrease of QHS on the electrode surface and thus the decrease of the peak currents. When pH reached 6.2, the deprotonated L-Cys increased to a concentration for the formation of the QHS-L-Cys species which can be detected as the CV signal at-1.03V. The peak currents of-1.03V increased with the detection of more QHS-L-Cys complex formed in neutral to alkaline conditions. Based on the fact that QHS can form a C-S covalent bond with L-Cys in the presence of iron[7], the binary adduct of QHS-L-Cys may also form through the thiol group. The electrode reaction can be depicted as Scheme 1A. Under strong alkaline conditions (pH>12), the lactone bond of QHS was easy to be hydrolyzed and L-Cys was apt to form cystine, the binary adduct may decompose and the reduction peak of the adduct thus disappeared. These observations indicate that neutral to alkaline pH in the range of 7.2~11.2 are the optimal condition for the formation of the binary adduct of QHS and L-Cys.
In order to detect the effect of surfactants, anionic surfactant SDS and cationic surfactant DBDAB were respectively added into the binary system containing QHS and L-Cys. While there was nothing happened with the SDS addition, the addition of DBDAB into the binary system caused a positive shift of the reduction peak from-1.03 V to-0.93 V (Fig. 4, Curve B → Curve C). This peak at-0.93 V was not observed in the control binary systems containing either QHS + DBDAB (data no shown) or L-Cys + DBDAB (Fig. 5, Curve A), suggesting that this new reduction peak is for the specific ternary interaction, perhaps the formation of a ternary adduct, in the system. In view of the free energy difference due to the potential shift from-1.03V to-0.93V (Table 1), DBDBA, in other words, decreased the effect of L-Cys on the stability of QHS by interacting with L-Cys preferably in the carboxyl side (Scheme 1B). Again, the excess amount of QHS does not support the 1∶1∶1 stoichiometry of the ternary complex in solution. The observed signal at-0.93V only indicates that the Ag electrode can sensitively detect the ternary complex even in a small amount.
To verify whether the three components in the ternary system were involved in the interaction, the concentration of DBDAB, QHS or L-Cys was respectively increased while the other two components remained unchanged. As Curves C,D,E shown in Figure 4,when the concentration of DBDAB in the ternary system was gradually raised, the increase of the peak current at-0.93V was accompanied with the growth of the reduction peak of QHS at-0.64V. When the concentration of DBDAB reached 2 folds of L-Cys concentration, the peak at-0.93V disappeared and the peak at-0.64V fully developed, entirely resembling the CV curve of QHS only (Fig. 4, Curves A & E). This suggests that DBDAB completely saturated L-Cys by interactions at both sides of carboxyl and mercapto groups of L-Cys and thus the Ag electrode detected free QHS only (Scheme 1C). Obviously, cationic surfactant DBDAB took part in the interaction in the ternary system containing QHS and L-Cys by impairing and then breaking down the QHS-L-Cys interaction.
On the other hand, Figure 5 shows the representative electrochemical spectra for the influence of QHS concentration on the formation of the ternary adduct. Double amounts of L-Cys and DBDAB were used in this case for better observation of the change of the peak current. The CV spectra clearly demonstrated that (omit)
A:[QHS] = 1.0mmol/L; B:[L-Cys] = 0.04mmol/L
Figure 3 Influence of L-Cys and QHS concen
trations on peak current at-1.03 V
system in Britton-Robinson buffer (pH 7.2) containing 20% ethanol (v/v). A: QHS (1 mM);B: QHS (1 mM) + L-Cys (0.04 mM); C: B + DBDAB (5μM); D: B + DBDAB (0.02 mM);E: B + DBDAB (0.08 mM)
Figure 4 Cyclic voltammograms of the ternary
A: L-Cys (0.08 mM) + DBDAB (0.01 mM);B: A + QHS (0.4 mM); C: A + QHS (0.8 mM);D: A + QHS (1.2 mM).Figure 5 Influence of QHS concentration on the peak current of the ternary adduct
the reduction peak at-0.93 V developed upon QHS being added into the solution containing L-Cys and DBDAB ([L-Cys]∶[DBDAB] =1∶1,mole ratio) and the peak current increased with the concentration increase of QHS. Similar CV changes were detected with increasing concentrations of L-Cys.
Based on the electrochemical behaviors described above, an electrode mechanism can be proposed to interpret the interaction on the electrode surface (Scheme 1). At neutral pH 7.2, L-Cys exists as a zwitterion to interact with QHS through-SH group which although remains no deprotonation (Scheme 1A). Cationic surfactant DBDAB may electrostatically interact with L-Cys (firstly with the carboxyl group) on the electrode surface to form ternary adducts (Scheme 1B). This explains the observation that anionic surfactant SDS had no interaction in the system. The electrostatic interaction between DBDAB and the carboxyl group of L-Cys weakened the interaction between QHS and the mercapto group of L-Cys, the reduction peak therefore shifted positively by 0.1 V from-1.03 V for the binary adduct to-0.93 V for the ternary adduct. With the concentration of DBDAB rising, cationic DBDAB may replace QHS to interact with L-Cys at the mercapto group side (Scheme 1C), and free QHS was released from the weakened interaction with L-Cys, leading to the development of the reduction peak of QHS at-0.64 V. When the concentration of DBDAB reached the double amount of L-Cys, DBDAB completely replaced QHS to saturate L-Cys in 2:1 stoichiometry, we thus observed that the peak for the ternary adduct disappeared and the peak for free QHS fully developed.
Conclusions
Surfactants have been widely utilized in the formulation of drugs to increase solubility[18-20]. Surfactant aggregation with QHS has also been studied to enhance the poor solubility of QHS and increase its bioavalability[13]. However, the introduction of surfactants may also create other side effects[19]. Our current observation clearly demonstrated that electrochemical measurement can be used to sensitively monitor the formation of the ternary complex of cationic surfactant DBDAB with QHS and endogenous L-Cys under a physiological relevant condition. The CV scanning further revealed that high amount of the cationic surfactant can block the interaction between QHS and L-Cys and thus inhibit the effect of L-Cys on QHS stability. These interesting findings provide important information for better understanding QHS efficacy and effecting factors and for global consideration in drug formulation of QHS in terms of increasing solubility.
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【Acknowledgements and funding】 This work was partially supported by the Guangdong Province and Guangzhou Natural Science Foundation of China (No.021190, 2003Z3D2041). We would like to thank Professors Y.T. Zhu and M.Y. Zhang from Tropical Medicine Institute, Guangzhou University of Traditional Chinese Medicine, China, for kindly providing Qinghaosu sample and for their helpful suggestions
1 Department of Chemistry, Jinan University, Guangzhou,Guangdong Province 510632, China
2 Institute of Life and Health Engineering, Jinan University, Guangzhou,Guangdong Province 510632, China
* Correspondence to Professor Qing-Yu He, Institute of Life and Health Engineering, Jinan University, Guangzhou,Guangdong Province 510632, China,
Tel. & Fax: 86-20-85227039,E-mail: qing-yu.he@163.com
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