Magister Pharmaceuticae - MPharm / Due to the contribution of hypertension to various cardiovascular diseases, many studies are currently focused on identifying efficient bioactive compounds with antihypertensive activity and thus reducing the levels of cardiovascular disease. ACE inhibitors are an important component of the therapeutic regimen for treating hypertension, but due to the increase in the prevalence of side effects of synthetic compounds, alternative and complementary medicines which may consist of pure bioactive compound or a combination of various compounds from natural sources are gaining importance in overcoming hypertension. Hypericum perforatum has been studied for various activities including anti-bacterial, anti-depressant, anti-oxidant properties, but studies on its cardiovascular effects specifically ACE inhibitory activity have not yet been explored. In this study, ACEI assay-guided fractionation of the ethanol extract of Hypericum perforatum was carried out other to isolate a compound with ACE inhibition. A compound – (3-hydroxy 4, 4 dimethyl-4-butyrolactone) was isolated from an active fraction of the plant extract and was tested for ACE inhibition and its chemical structure elucidated using 1HNMR and C13NMR spectrometry and further characterized using mass spectrometry and FTIR.

Abstract Hypericum perforatum L. (St. John’s wort) is a medicinal plant widely utilized for the treatment of depression. The antidepressant activity is mainly attributed to the phenolic compounds hypericins and hyperforins, which also have a wide range of other pharmacologically interesting properties. The biosynthetic routes leading to hypericins and hyperforins are poorly understood, although a polyketide pathway including type III polyketide synthases (PKSs) has been suggested to be involved. Furthermore, a gene called hyp-1 is assumed to attend to the final stages of the hypericin biosynthesis. In the present work, the biosynthesis of hypericins and hyperforins in H. perforatum was further studied by focusing on the elucidation of the precursors and genes involved. The incorporation of isotopically labelled branched-chain amino acids into hyperforins was investigated as well as the possibilities to enhance the production of hyperforins in H. perforatum in vitro cultures by feeding them with amino acid precursors. Furthermore, two novel cDNAs encoding for type III PKSs were isolated from H. perforatum. The functions of these new genes, designated HpPKS1 and HpPKS2, as well as the role of hyp-1 were elucidated by comparing their expression with the levels of hypericins and hyperforins in H. perforatum tissues. The enzymatic activity of the recombinant HpPKS2 protein was also analyzed. To study Hyp-1 at a protein level, a protein extraction method was optimized for tissues of Hypericum species. The results show the incorporation of valine and isoleucine into the acyl side chain of hyperforin and adhyperforin, respectively. Through the biotransformation of the amino acid precursors, it is possible to enhance the levels of adhyperforin, but not hyperforin, in H. perforatum shoot cultures, which demonstrates the tight regulation of the hyperforin biosynthesis. A correlation between HpPKS1 expression and hyperforins was detected in H. perforatum tissues. The localization of HpPKS2 mRNA in dark glands in which hypericins accumulate as well as the octaketide synthase activity of the recombinant HpPKS2 suggest that HpPKS2 is associated with possible co-operating tailoring enzymes in the biosynthesis of hypericins. The presence of both hyp-1 mRNA and Hyp-1 protein in distinct places compared with hypericins in H. perforatum tissues does not support the idea that Hyp-1 would be involved in the biosynthesis of hypericins in dark glands, although mobility of the Hyp-1 protein was shown to be possible. The present thesis extends knowledge about the biosynthesis of hypericins and hyperforins in H. perforatum by providing new candidate genes for their biosynthesis and by identifying precursors for hyperforins. Moreover, new information was obtained about the role of hyp-1 in H. perforatum.