Fluorescence Spectroscopic Analysis on the Synergistic Mechanism of Diazepam and Ethanol†

doi:10.7503/cjcu20170710

Abstract

Abstract:

The synergistic mechanism of diazepam and ethanol was investigated based on fluorescence spectroscopic method by simulating the physiological environment in vitro and the in vivo experiments in mice. The experimental results revealed that the presence of ethanol in the blood can reduce the equilibrium binding constant of diazepam-HSA complex in the early metabolism of diazepam, which resulted in the increase of the free diazepam proportion, accelerating the metabolic rate of diazepam, and shorten the half-life and storage time of diazepam in the blood, thereby enhancing its bioactive efficacy.

Key words: Diazepam, Alcohol dependence, Fluorescence, Human serum albumin, Spectral analysis

CLC Number:

O657

TrendMD:

CUI Shengfeng, WAN Jingwei, ZHOU Chenghe. Fluorescence Spectroscopic Analysis on the Synergistic Mechanism of Diazepam and Ethanol^†[J]. Chem. J. Chinese Universities, 2018, 39(6): 1178.

Figures/Tables 10

Fig.1 UV-Vis absorption spectra of HSA in the presence of various concentrations of diazepam(298 K, pH=7.4) c(HSA)=1.5×10-5 mol/L; a. diazepam only; concentrations of diazepam are 0—1.6×10-5 mol/L for curves b—j at increment of 0.2. The inset corresponds to the absorbance at 278 nm with different concentrations of diazepam.

Fig.2 Emission spectra of HSA in the presence of diazepam(T=298 K)c(HSA)=1.5×10-5 mol/L; concentrations of diazepam are 0—1.4×105 mol/L for curves a—h at increment of 0.2; i. diazepam only.

Fig.3 Stern-Volmer plots of diazepam-HSA system

Table 1 Stern-Volmer constants of the diazepam-HSA system*

T/K	10⁴ K_SV/(L·mol^-1)	10^-12 K_q/(L·mol^-1·s^-1)	R	SD
298	4.32	6.75	0.998	0.015
303	4.03	6.30	0.997	0.017
310	3.62	5.66	0.999	0.004

Fig.4 Modified Stern-Volmer plots of diazepam-HSA system

Table 2 Modified Stern-Volmer constants of diazepam-HSA system(pH=7.4)

T/K	10⁴ K_a/(L·mol^-1)	R	SD
298	8.68	0.999	0.06
303	6.74	0.999	0.08
310	4.42	0.999	0.06

Fig.5 Scatchard plots of diazepam-HSA system

Table 3 Binding constants and sites of diazepam-HSA system(pH=7.4)

T/K	10⁴K_b/(L·mol^-1)	n	SD	R
298	8.21	1.08	0.003	0.997
303	7.08	1.14	0.001	0.997
310	4.47	1.12	0.001	0.995

Table 4 Effect of ethanol on the Kb value of diazepam-HSA system*

Concentration of ethanol/(mg·mL^-1)	10⁴K_b/(L·mol^-1)	K_b/K₀(%)	R	SD
0.2	3.25	39.6	0.992	0.005
0.4	1.30	15.8	0.991	0.002
0.6	2.88	35.1	0.993	0.004
0.8	6.37	77.6	0.997	0.006
1.0	3.82	46.5	0.993	0.005
1.2	2.81	34.2	0.993	0.004
1.4	4.62	56.3	0.992	0.007
1.6	3.78	46.0	0.994	0.005
1.8	4.36	53.1	0.991	0.006

Table 5 Concentrations of diazepam and nordiazepam in the heart blood of mice

Concentration of ethanol/ (mg·kg^-1)	Concentration of diazepam/(μg·mL^-1)			Concentration of nordiazepam/(μg·mL^-1)
Concentration of ethanol/ (mg·kg^-1)	20 min	40 min	60 min	20 min	40 min	60 min
0	1.245	0.873	1.096	4.345	6.486	7.135
600	0.773	0.606	0.801	3.752	6.738	11.365
1200	0.783	0.736	0.752	3.124	6.906	12.653
1800	0.521	0.504	0.691	3.426	7.094	12.974

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