Investigation On The Bioactive Essence Screening And Quality Evaluation For Radix Isatidis Based On Biothermodynamics

Bioactive essence is the most important base for modernation of Traditional Chinese Medicine (TCM), especially for TCM quality control and evaluation while it is also the difficult and hot spot in TCM research. However, due to the complexity of TCM and the restriction of technology, together with the limit of novel ideas, there is no breakthrough in researches on screening of TCM bioactive essence based on analytical models of that so called "three chemical and four pharmacological levels". It is difficult to control quality and characteristics of TCM by the currently used models and methods on TCM quality control guided by the theory of "compound only", let alone to reflect the efficiency and safety of TCM.Therefore, in this paper, doublely guided by theories of Traditional Chinese Medicine and Thermodynamics, Banlangen, with the features of confirmed clinical therapeutic effect but undefined bioactive essence used as experimental model, was observed on the field of metabolism of microorganisms and immunocytes which has been called biothermoactivity, along with the regular pharmacological experiments and the correlative analysis with chemical componets. Based on the results of experiments mentioned above, the bioactive essence of Banlangen with the effects of antibacteria, antivirus and immunoactivity was elucidated preliminarily. Meanwhile, the method of Banlangen bioactive screening and the model of Banlangen quality control based on biological value (biothermodynamics) were also established preliminarily. The researches and results were shown as follow:1. Sample collection and preparation together with chemical analysisAll of Banlangen herbs used in this project were standard ones. The traditional procedure of water boiling being used as main preparation technology, the samples of Banlangen at different levels were obtained as follow: water extraction (G.E.), total organic acid (T.O.A), total polysaccharides (T.P.S) and different kinds of polar extractants. The chemical fingerprint spectrum was established and the content of T.O.A and T.P.S were assayed, together with the content and average molecular weight of polysaccharides (P.S) was calculated. The chemical differences and characteristics among different levels of essences were elucidated preliminarily to provide chemical data for correlative analysis between bioactive essence and bioactivity with establishment of bioactive essence screening and quality evaluation.2. Experiments on biothermoactivity of Banlangen based on biothermodynamic expressionFrom three aspects such as antibactria, antivirus and immunoenhancement, the pharmacological effects and differences among varied extracts from Banlangen were observed systematically by the method of biothermodynamics. The technological parameters of biothermodynamic method were optimized and confirmed. Methodlogical results showed that this method appeared good precision, reproducibility and stability. Also, the thermodynamic differences in the effects of G.E., polar extractants, T.O.A, T.P.S on Escherichia coli, Staphylococcus aureus, mumps virus and lymphocytes were also observed. The results showed that there is appearant dependent relationship between concentration of sample and the perameter K which expressed the growth ratio. Other thermodynamic perameters such as the peak heat rating Pm, output of heat Q reflected characteristics of Banlangen on organismic model and the correlative analysis demonstrated that the results were highly coincident with that of regular pharmacological experiments.3. Regular pharmacological demonstration on bioactivities of different extracts from BanlangenThe reliability of thermodynamic methods was demonstrated by regular pharmacology. The results showed that the pharmacological effects on antibacteria, antivirus and immunoenhancement of different polar extracts such as T.O.A, T.P.S and different chemical parts from Banlangen were highly coincident with biothermodynamics. (antibacterial activity, P<0.01, antiviral activity, P<0.05, immuno enhancement activity P<0.01). The sequence of the polar extracts on antibacteria and antivirua was: nBuOH E.> EtOAc E.> CHCl₃ E.> G. E.> P.E and EtOAc E.> CHCl3 E.> nBuOH E.> G. E> P.E. E.>R. E. respectively. The water extract (G.E.) and residue of extraction (R.E.) could significantly enhance the multiplication of splenic lymphocytes in mice with fine activity of R.E. All of the extracts including nBuOH E.,EtOAc E.,CHCl₃ E.,P.E. E. could promote the multiplication of lymphocytes although they were at low multiplicative ratio compared with G.E. or R.E.4. The preliminary establishment on bioactivity screening method of Bailangen based on biothermoactivity and regular pharmacology (from effect to effect)The correlative analysis showed that the results from thermodynamics were identical to that from pharmacology, demonstrating that it is objective, accurate and feasible to use biothermodynamics as bioactivity screening system from the viewpoint of energy metabolism. By using this system, the high throughput screening of TCM bioactivities could be obtained, reflecting the intergrity and systematics of TCM. The preliminary results showed that technology of TCM biothermoactive detection based on thermodynamic expression had advantages of real time, on line, sensitive, high performance and well suitable. To some extent, bioactive equations expressed the characteristics on intergrity and nonlinearity of TCM. Combined with biothermoactive information provided by biothermodynamics, it could be used as a kind of fine model and method on TCM bioactive essence screening.5. The preliminary investigation on bioactive essence of Banlangen based on chemical components, biothermoactivity and regular pharmacology (essence-effect-effect)By investigations on different polar extracts, varied chemical parts of Banlangen on biothermoactivity, pharmacological activity and chemical material essence, along with the statistical methods such as canonical correlation analysis and multiple regression analysis to correlatively analyse the relationship among essence, biothermoactivity and pharmacology, the final effects of the extracts were elucidated. Meanwhile, T.O.A was regarded as antibacterial material essence, T.P.S as immuno enhancement material essence. Besides, the chemical composition of T.P.S was investigated and the fingerprint spectrum of T.O.A was established and the active compounds were identified as benzenecarboxylic acid and orthohydroxybenzoic acid. The preliminary results showed that the bioactive information provided by thermodynamic method could be a fine model on TCM bioactive essence screening.6. The preliminary formulation on method and index of Banlangen quality evaluation based on biological value (thermodynamics) The traditional recognition on quality of Banlangen being used as references, 10 batches of the normal herbs of Banlangen were investigated and the mathematical model on quality evaluation of Banlangen was built. The model could give an accurate judgement of Banlangen quality with only 1.39% possibility of wrong judgement which was far low from 50% possibility of wrong judgement of chemical fingerfrint in quality evaluation only by content determination. In addition, the normal wave scope was preliminarily determined on normal herbs of Banlangen biothermodynamic expression. The scope for reference on Banlangen acting on Escherichia coli was K₁ 0.01—0.04 min^-1,K₂ 0.004～0.007 min^-1, P¹_m 0.4～2mW,P²_m 1.2～2.8 mW,T¹_m 220～260 min,T²_m 700～850 min Q 30—130 J, on mumps virus was K 0.005—0.018 min^-1,P_m 140～260μW,Q 35～55mJ and on lymphocytes K 0.01—0.04mm^-1, P_m 80～180μW,Q 30⁶0mJ.