Due to the limited chemical stability of the natural hyperforin molecule, a more stable form of hyperforin, i.e., the hyperforin dicyclohexylammonium salt (HYP-DCHA) has been used for ex vivo and in vitro experiments in recent years, but its actual stability under typical cell culture conditions has never been studied before. In this contribution the stability of HYP-DCHA was examined under typical cell culture conditions. Different cell culture media with and without fetal calf serum (FCS) supplementation were studied with regard to further stabilization of HYP-DCHA determined with HPLC analysis. Furthermore, albumin nanoparticles were examined as a stabilizing carrier system for HYP-DCHA. In this context, the interaction between HYP-DCHA and albumin nanoparticles (ANP) was examined with regard to size and loading with HYP . The effects of HYP-DCHA either supplied in cell culture medium or loaded on ANP on viability and cytotoxicity were studied in vitro on HaCaT monolayers (human keratinocyte cell line). HYP-DCHA supplied in FCS-containing medium was recovered completely after 24h of incubation. However, a lack of FCS caused a total loss of HYP-DCHA after less than 24h incubation time. Supplying HYP-DCHA loaded on ANP in an FCS-free medium resulted in a recovery of about 60% after 24h incubation. HYP-DCHA supplied in medium along with FCS showed a slow dose-dependent decrease in viability of HaCaT cells without any cytotoxic effects (antiproliferative effect). Treatment with HYP-DCHA with a lack of FCS resulted in a significantly faster decrease in viability which was mainly due to cytotoxicity. The latter was true for HYP-DHCA-loaded ANP where increased cytotoxicity was observed despite the presence of FCS. The results show that the stability of the widely used HYP-DCHA is rather limited under cell culture conditions. Especially a lack of FCS leads to degradation and/or oxidation of HYP-DCHA probably causing an increased cytotoxicity. In contrast, FCS supplementation fairly stabilizes HYP-DCHA under cell culture conditions while albumin nanoparticles may serve the same stabilization purpose despite increasing cytotoxic effects onto the cells themselves.

Benzoic acid-derived compounds, such as polyprenylated benzophenones and xanthones, attract the interest of scientists due to challenging chemical structures and diverse biological activities. The genus Hypericum is of high medicinal value, as exemplified by H. perforatum. It is rich in benzophenone and xanthone derivatives, the biosynthesis of which requires the catalytic activity of benzoate-coenzyme A (benzoate-CoA) ligase (BZL), which activates benzoic acid to benzoyl-CoA. Despite remarkable research so far done on benzoic acid biosynthesis in planta, all previous structural studies of BZL genes and proteins are exclusively related to benzoate-degrading microorganisms. Here, a transcript for a plant acyl-activating enzyme (AAE) was cloned from xanthone-producing Hypericum calycinum cell cultures using transcriptomic resources. An increase in the HcAAE1 transcript level preceded xanthone accumulation after elicitor treatment, as previously observed with other pathway-related genes. Subcellular localization of reporter fusions revealed the dual localization of HcAAE1 to cytosol and peroxisomes owing to a type 2 peroxisomal targeting signal. This result suggests the generation of benzoyl-CoA in Hypericum by the CoA-dependent non-β-oxidative route. A luciferase-based substrate specificity assay and the kinetic characterization indicated that HcAAE1 exhibits promiscuous substrate preference, with benzoic acid being the sole aromatic substrate accepted. Unlike 4-coumarate-CoA ligase and cinnamate-CoA ligase enzymes, HcAAE1 did not accept 4-coumaric and cinnamic acids, respectively. The substrate preference was corroborated by in silico modeling, which indicated valid docking of both benzoic acid and its adenosine monophosphate intermediate in the HcAAE1/BZL active site cavity.

Benzoic acid is a building block of a multitude of well-known plant natural products, such as paclitaxel and cocaine. Its simple chemical structure contrasts with its complex biosynthesis. Hypericum species are rich in polyprenylated benzoic acid-derived xanthones, which have received attention due to their biological impact on human health. The upstream biosynthetic sequence leading to xanthones is still incomplete. To supply benzoic acid  for  xanthone biosynthesis, Hypericum calycinum cell  cultures use  the  CoA-dependent non-β-oxidative pathway, which starts with peroxisomal cinnamate CoA-ligase (HcCNL). Here, we use the xanthone-producing cell cultures to identify the transcript for benzaldehyde dehydrogenase (HcBD), a pivotal player in the non- β-oxidative pathways. In addition to benzaldehyde, the enzyme efficiently catalyzes the oxidation of trans-cin-namaldehyde in vitro. The enzymatic activity is strictly dependent on the presence of NAD+as co-factor. HcBD is localized to the cytosol upon ectopic expression of reporter fusion constructs. HcBD oxidizes benzaldehyde, which moves across the peroxisome membrane, to form benzoic acid. Increases in the HcCNL and HcBD tran-script levels precede the elicitor-induced xanthone accumulation. The current work addresses a crucial step in the yet incompletely understood CoA-dependent non-β-oxidative route of benzoic acid biosynthesis. Addressing this step may offer a new biotechnological tool to enhance product formation in biofactories.