Study On The Synthesis Of 8-alkyl-berberine Homologues And Their Pharmacological Activities

The isoquinoline alkaloids berberine, is an important active component of Rhizoma Coptidis, and used in the treatment of bacterial gastrointestinal diseases such as diarrhea. Modern scientific research indicated that berberine has not only antimicrobial activity in vitro and in vivo, but also other important pharmacological activities such as lipid-modulating, antihyperglycemic, antitumor, antihypertension and antiarrhythmia. To increase the pharmaceutica activity of berbeine 8-alkyl-berberine homologues with seriate different lipophilic property were synthesized in our laboratory by introducing different length alkyl at C8 position, some pharmacological effects of 8-alkyl-berberine homologues were studied including the antimicrobial activity, lipid-modulating effect, antihyperglycemic effect and toxicity. The methods and results are as follows:1. The synthesis and molecular structure identification of 8-alkyl-berberine homologuesThe synthesis approaches to 8-alkyl-dihydroberberine were carried out in three solvent, aether, dioxane and tetrahydrofuran. The result showed that the highest recovery of the compound had been achieved when synthesized in tetrahydrofuran. Compared with aether, the synthesized yield of 8-alkyl-dihydroberberine enhanced 4 times. The structure of 8-alkyl-berberine homologues synthesized was affirmed with element analysis, Ultraviolet spectrum, Infrared spectrum, ¹H NMR and ¹³CNMR.2. The antimicrobial activity of 8-alkyl-berberine homologues in vitroThe sensitivity of 18 microorganism come from G⁺ bacteria, G^- bacteria and fungus to 8-alkyl-berberine homologues respectively were evaluated by inhibition zone method and MICs of 8-alkyl-berberine homologues against 6 sensitive microorganisms were determined by turbidimetric method respectively.The result showed that G⁺ bacteria were more sensitive than G^- bacteria and fungus. The antimicrobial activity increased as the length of aliphatic chain elongated and then decreased gradually when the alkyl chain exceeded 8 carbon atoms. 8-octyl-berberine showed the highest antimicrobial activity among all compounds.3. The antimicrobial mechanism of 8-alkyl-berberine homologues in vitroWith rabbit red-blood cell and its cell membrane, B. subtilis (G⁺) and E. coli (G^- ) as experimental material, the hemolysis effect on rabbit red-blood cell, disturbing effect on cell membrane stability and fluorescence quenching effect of 8-alkyl-berberine homologues on membrane protein or bacterial protein were determined by fluorescence spectrum analytical method.Results showed that antimicrobial activity of the compound was stronger, the effect including hemolysis effect, disturbing effect on cell membrane stability and fluorescence quenching effect on membrane protein were stronger. 8-hexyl-berberine or 8-octyl-berberine showed highest disturbing effect on cell membrane stability and membrane protein of rabbit red-blood cell membrane, whole cell membrane stability and bacterial protein of B. subtilis (G⁺) and E. coli (G^-). The experimental data were compared by quantitative analysis method between B. subtilis (G⁺) and E. coli (G^- ), result showed that the disturbing ability to cell membrane stability of B. subtilis (G⁺) is far more than E. coli (G^-), but fluorescence quenching ability to whole cells of B. subtilis (G⁺) and E. coli (G^- ) is nearly same while same compound.4. The lipid-modulating effect of 8-alkyl-berberine homologues in vivoWith experimental hyperlipidemia rats as animal model, the Lipid-modulating effects of 8-alkyl-berberine homologues are studied.Result showed that the lipophilic properties of 8-alkyl-berberine homologues are beneficial to exerting Lipid-modulating effect in vivo. The long chain homologue 8-dodecyl-berberine and 8-cetyl-berberine showed better Lipid-modulating effect than berberine and other 8-alkyl-berberine homologues. For example, the serum TC value of 8-dodecyl-berberine and 8-cetyl-berberine group decreased respectively by 83% and 97% compared with berberine group. Dose-effect experiment showed that the decreasing of serum TC, serum TG and AI, and increasing of serum HDL-C of 8-dodecyl-berberine and 8-cetyl-berberine in experimental hyperlipidemia rats are dose dependent.5. Preliminary study on toxicity of 8-alkyl-berberine homologues in vivoWith kuming mouse or SD rats as animal model, the LD₅₀ of 8-alkyl-berberine homologues was determined according to modified karber's method, and the liver and kidney toxicity of interesting molecule 8-octyl-berberine, 8-dodecyl-berberine and 8-cetyl-berberine were observed at dosage of 125mg/kg.bw for 6 weeks.Result showed that the toxicity of 8-alkyl berberine derivatives was stronger than that of berberine. However, by elongating the aliphatic chain, toxicity decreased gradually. 8-ethyl-berberine showed highest toxicity. 8-octyl-berberine, 8-dodecyl-berberine and 8-cetyl-berberine didn't showed obvious liver and kidney toxicity on the experimental condition of dosage and time.