基于分子热力学特征探讨黄连解毒汤水煎自沉淀形成机制

Formation mechanism of precipitation in Huanglian Jiedu Decoction based on molecular thermodynamic characteristics

目的全面解析黄连解毒汤(HJD)水煎自沉淀来源,并从分子热力学角度探讨HJD组方配伍中药间相互作用热力学参数及结合能力强弱,以期揭示中药复方水煎自沉淀形成机制。方法通过拆方研究,锁定产生水煎自沉淀的主要组方配伍;通过等温滴定量热技术(isothermal titration calorimetry,ITC)分别测定不同配伍中药相互作用过程中的能量和热力学参数变化,包括结合常数(Ka)、解离常数(Kd)、结合计量比(n)、焓变(ΔH)、熵变(ΔS)、吉布斯自由能变(ΔG),进而阐明不同组方配伍中药间结合能力的差异。结果拆方研究结果表明,能够产生明显自沉淀现象的配伍有黄芩-黄连、黄芩-黄柏,其中黄芩-黄连生成沉淀最明显,黄芩-黄柏次之;而黄芩-栀子、黄连-黄柏、黄柏-栀子、黄连-栀子配伍则未发现明显自沉淀生成。应用ITC分别考察黄芩-黄连、黄芩-黄柏、黄芩-栀子3种配伍关系,结果显示黄芩-栀子相互作用过程中能量变化显著低于黄芩-黄连和黄芩-黄柏相互作用的能量变化,3组的ΔH<0,-TΔS>0,ΔG<0,|ΔH|>|-TΔS|,均为焓驱动的自发反应过程;黄芩水煎液滴定黄连、黄柏、栀子水煎液的Ka依次为1.283×10~4、4.680×10~3、1.973×10~3,数值逐渐减小,反之K_d依次增大(7.794×10^(-5)、2.137×10^(-4)、5.068×10^(-4))。结论 HJD水煎自沉淀形成过程中,存在焓驱动非共价键结合的化学反应,而非简单的物理凝聚吸附沉淀;并且黄芩-黄连成分间结合力大于黄芩-黄柏成分间结合力,而黄芩-栀子间的相互作用极弱。

Objective To explore all sources of precipitation, parameters of interaction and binding abilities of different Chinese materia medica（CMM） combinations during the formation process of the precipitation in Huanglian Jiedu Decoction（HJD） using the thermodynamic method. The study was aim to reveal the mechanism of the formation of precipitation in CMM decoction. Methods First, the possible CMM combinations which had obvious precipitation phenomenon when combined were determined by the research on decomposed recipes. Then isothermal titration calorimetry（ITC） was used to determine the binding heat and thermodynamic parameters of binding reactions, thermodynamic parameters contents binding constant（Ka）, dissociation constant（Kd）, estimate of stoichiometric ratio（n）, enthalpy change（ΔH）, entropy change（ΔS）, Gibbs free energy change（ΔG）, these data were used to explain the differences in binding abilities between different CMM combinations. Results The original decomposed recipes experiment indicated the CMM combinations that could produce obvious precipitate when they were mixed were Scutellariae Radix-Coptidis Rhizoma and Scutellariae Radix-Phellodendri Chinensis Cortex. Precipitation amount of Scutellariae Radix-Coptidis Rhizomawas the most. There was no precipitate in Scutellariae Radix-Gardeniae Fructus, Coptidis Rhizoma-Phellodendri Chinensis Cortex, Coptidis Rhizoma-Gardeniae Fructus or Phellodendri Chinensis Cortex-Gardeniae Fructus. In ITC, the thermodynamic parameters of the three experience groups were the same, ΔH 0,-TΔS 0, ΔG 0, ｜ΔH｜ ｜-TΔS｜, indicating the three reactions were all enthalpy-driving reactions. The Ka of Scutellariae Radix decoction titrated Coptidis Rhizoma decoction was 1.283 × 10~4, Scutellariae Radix decoction titrated Phellodendri Chinensis Cortex deccotion was 4.680 × 10~3, Scutellariae Radix decoction titrated Gardeniae Fructus decoction was 1.973 × 10~3. The value decreased gradually. The binding heat of Scutellariae Radix and Gardeniae Fructus was much smaller than the binding heats of Scutellariae Radix and Coptidis Rhizoma, Scutellariae Radix and Phellodendri Chinensis Cortex. Conclusion During the formation process of the precipitation in HJD, the main source of acid-base complex-precipitation were Scutellariae Radix-Coptidis Rhizoma and Scutellariae Radix-Phellodendri Chinensis Cortex; Analysis of molecular thermodynamic indicates the binding energy of Scutellariae Radix and Coptidis Rhizoma was larger than that of Scutellariae Radix and Phellodendri Chinensis Cortex, compared with them, the binding energy of Scutellariae Radix and Gardeniae Fructus is tiny.