The Study Of Allicin And Its Interaction With Protein In Blood And The Intestinal Absorption Characteristics In Rat

Objective:A method used to extract and purify allicin from fresh garlic, and its results were compared with the results of the way that using alliin and enzyme to synthesis allicin.Study the methodology validation of method which is used to contain the allicin from European pharmacopoeia(EP8.0).Determination the plasma protein binding of allicin with rats and human.Study the intestine absorption characteristics of allicin in different intestinal segments in rats.Methods:1.Take 2 kg fresh garlic peeled and add 95% ethanol to precipitate protein after homogenating, then get the relatively high concentrations of allicin through a series of concentration extraction purification. Using chromatography in the European pharmacopoeia method to determin the content of allicin in allicin injection,and make the complete methodology verified.2.Established the determination method of allicin in biological samples by using 1% methanol and formic acid as aqueous solution gradient elution,and determined the Human and Rat Plasma Protein Binding of Allicin with Ethylparaben as Substitute Reference Standard by HPLC.3.Using fluorescence spectroscopy and synchronous fluorescence spectrometry with 280 nm as the excitation wavelength to scan the action of fluorescence spectra after allicin acting on Bovine Hemoglobin(Bovine Hemoglobin, BHB), Bovine Serum Albumin(Bovine Serum Albumin,BSA)and Human Serum Albumin(Human Serum Albumin,HSA),and set the wavelength difference respectively Δλ=15 and 60 nm to scansynchronous fluorescence spectra, and calculation the function parameters.4.Use ussing chamber to detect the absorption characteristics of allicin in rat duodenum, jejunum,ileum and colon.Results:1.The concentration of allicin extracted from fresh garlic was up to 8.27 mg·ml-1;the results of methodology validation of the method which was used to measure the content of allicin fall out to well.2.In this experiment,we measured that 1 mg hydroxyl ethyl benzene was equivalent to 10.8 mg allicin under the condition of chromatography.With different concentration of allicin including 75,150 and 300 μg·ml-1,the protein binding rates of allicin in rat and human were 23.2%,19.8%,28.1% and 15.5%,25.8%,40.0%.23.2%,19.8%,28.1%;15.5%,25.8%,40.0%,and the statistical inspection results showed that the protein conjugation rate of allicin in the rat and human plasma had significant difference(p<0.05).3.Allicin and alliin were not directly acted on the BHB while the hydrogen sulfide can act on BHB.The Ksv between hydrogen sulfide and BHB at 298 and 310 K were 1.00×104 L·mol-1(R=0.9969) and 1.94×104 L·mol-1(R=0.9979 respectively,the maximum dynamic fluorescence quenching rate constant Kq at 298 and 310 K were1.00×1012and1.94×1012 L·(mol·s)-1respectively;All of allicin,alliin and hydrogen sulfide can interact with BSA and HSA.4.The Papp of allicin in four intestinal segments of rats were all greater than 1×10-6 cm·s-1,and the Papp was not affected by drug concentration,the absorption rate constant of allicin in the intestine increaseed with the concentration of allicin.Conclusion:1.The allicin can be directly extracted from fresh garlic isolated to get high concentration of allicin and purified by molecular distillation way subsequent further.Direct extraction method can obviously improve the concentration of a solution ofallicin by comparing with the way that using alliin and enzyme to synthesis.The way to measure the content of allicin in EP8.0 measurement method can be used in the lab.2.The protein binding of allicin in human plasma was greater than in the rat plasma,allicin was under proof combination drugs with protein, the plasma protein binding of allicin had obvious differences between different species.3.Allicin and its precursor alliin did not interact with BHB directly;part of allicin entered into red blood cells to converted to hydrogen sulfide and another combined with plasma proteins once added into blood,after interaction with hemoglobin.4.Allicin could be absorbed in different intestines of rats and the ileum was the main absorption site.Speculating the absorption rate of allicin was between 20-70 %.