| Ginkgo Biloba flavonoids are main pharmaceutical constituents of Ginkgo Biloba Extract (GBE),exhibiting widespread pharmacological active effect. They not only can be used for prevention and cure cardiovascular disease ,but also can serve as an inhibitor to plate-activating factor(PAF). Thus, Ginkgo Biloba Extract and related preparation attract a lot of attention. It has been reported that flavonoids play an important role in the treatment of trachea disease by influence on epinephrine of the body, so it is highly necessary to determine epinephrine of the body sensitively.Protein serves as a transport carrier for drugs , and the binding of drugs with protein has a great influence not only on the distribution, metabolism and excretion of drugs in the body but also on their bioactive. Thus, the study of the binding characteristics of flavonoids and epinephrine to protein and their sensitive determination methods can provide scientific basis for the drugs being taken rationally.This thesis studied the interaction mechanism of both flavonoids and epinephrine with bovine serum albmin(BSA) using the techniques including fluorescence, resonance Light Scattering(RLS), CD spectra and absorption spectrscopies.Chapter 1: We summarize (1) development of extraction and determination methods of GBE, (2)the methods used to study the interaction of drug with protein, (3) determination methods for epinephrine and (4) development of rare earth ion co-luminescence effect and rare earth ion complex as fluorescence probe of protein(BSA). 87 references are cited here.Chapter 2: A sensitive method for determination contents of total flavone of Ginkgo Biloba Extract was established. The experiments indicate that the fluorescence of Ginkgo Biloba Extract(GBE) is greatly enhanced by forming complex with Al3+. Based on this, a fluorimetric method for determination of total flavonoids in GBE is proposed by taking quercetin as standard sample . Under optimum conditions, a linear relationship has been obtained between the fluorescence intensity and the concentration of quercetin in the range of 0.00075 ～ 0.5 0μg/ml, and the detection limit is 0.451 ng/ml(S/N=3). The method is applied for the determination of total flavonoids in GBE and recovery test using standard addition method, and the results obtained are satisfactory.Chapter 3: The fluorescence quenching reactions of quercetin with bovine serum albmin(BSA) in solution were studied. The quenching mechanism of BSA by quercetin was interpreted using the Stern-Volmer equation. The binding constant K values and the number of binding sites(f) at different temperatures were calculated, respectively. In addition , the thermodynamic function enthalpy(AH),entropy(AS) and free energy(AG) for the reaction were also calculated according to Van't Hoff equation, on which the pattern of interaction force was discussed. Furthermore, the interaction distance between the quercetin and the residues of BSA was calculated using Forster energy transfer theory, and it was found that the conformation of the BSA was changed as addition of quercetin according to the synchronous fluorescence, UN-Vis absorption and circular dichroism(CD) spectra.The reaction of Ginkgo Biloba Extract(GBE) with bovine serum albumin was investigated by the fluorescence quenching teccnology. The binding constant K values and the number of binding sites (f) at different temperatures were calculated, respectively. In addition, the thermodynamic parameters for the reaction were also calculated, on which the interaction force of the drug with serum albumin were indicated. The synchronal fluorescence spectra of tryptophane and tyrosine were also used to study the changes of the conformation of bovine serum albumin .Chapter 4: A sensitive fiuorimetric method for the determination of epinephrine is described in this paper. The method is based on the reaction of epinephrine with formaldehyde (HCHO) in an acid medium and potassium hexacyanoferrate (III) (K3[Fe(CN)6]) in borax buffer (pH=9.5). Experiments indicate that the reaction product can emit strong fluorescence. Ascorbic acid is used to consume excess potassium hexacyanoferrate and stabilize the fluorescent product. Under optimum conditions, a linear relationship had been obtained between the fluorescence intensity and the concentration of epinephrine in the range of 1.4×10-9～2.1×10-6mol/l, and the detection limit is 2.4×10-10mol/l (4.3×10-11g/ml, S/N=3). The method is applied for the determination of E in actual sample, and the result obtained is satisfactory. The mechanism of the reaction was also discused.Chapter 5: A new fluorimetric method for the determination of epinephrine was established by rare earth ion co-luminescence effect of Tb-Gd-E-Tris system .Under optimum conditions, a linear relationship has been obtained between the the fluorescence intensity and the concentration of epinephrine in the range of 1.8×10-8 2.5×10-6mol/l, and the detection limit is 4.5×10-9mol/l (S/N=3). The method was applied for the determination of epinephrine in actual sample and recovery test in urine by standard addition method, and the results obtained are satisfactory. The mechanism of the co-luminescence in the system was also discused.Chapter 6: A new sensitive fluorimetric method for the determination of protein(BSA) was established using Tb(III)-epinephrine complex as probe. The ascorbic acid( AA) and the sodium dodecylsulfate(SDS) were added for stablizing and enhanceing the fluorescence of this system, respectively. Under optimum conditions, a linear relationship has been obtained between the the fluorescence intensity and the concentration of protein in the range of 5.2×10-9-1.3×10-5g/ml, and the detection limit is 1.3ng/ml (S/N=3). The method was applied for the determination of protein in actual sample by standard addition method, and the results obtained are satisfactory. The mechanism of the interaction between Tb(III)-epinephrine complex and bovine serum albmin(BSA) was was studied.The chief charteristics of this thsis are as follows:(1) Quercetin was served as standard substance to establish a sensitive method for determination contents of total flavone of Ginkgo Biloba Extract, which is a sensitive,simple and rapid method.(2) Several methods including fluorescence, UN-Vis absorption and circular dichroism(CD)spctra were used to study the interaction of both -Ginkgo Biloba Extract(GBE) and quercetin with bovine serum albmin(BSA), respectively, and their binding constant K values, the number of binding sites(f), the thermodynamic parameters and the interaction distance were calculated. In addition, the interaction force of the drugs with serum albumin was indicated. (3) Using formaldehyde(HCHO), potassium hexacyanoferrate(III) (K3[Fe(CN)6]) and ascorbic acid(AA) as condensing , oxidizing and stabilizing agents, respectively, the most sensitive fluorimetric method for the determination of epinephrine was proposed so far.(4) According to the rare earth ion co-luminescence effect of Tb-Gd-E-Tris system found, a sensitive fluorimetric method for the determination of epinephrine was established(5) It is found that protein can obviously enhance the fluorescence of the Tb3+-E system in presence of both ascorbic acid and sodium dodecylsulfate. According to this, a new sensitive fluorimetric method for the determination of protein was established. The interaction mechanism between protein and Tb(III)-epinephrine complex was studied by resonance light scattering(RLS), CD spectra, fluorescence polarization and fluorescence lifetime. |
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