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 24 h of incubation. However, a lack of FCS caused a total loss of HYP-DCHA after less than 24 h incubation time. Supplying HYP-DCHA loaded on ANP in an FCS-free medium resulted in a recovery of about 60% after 24 h 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.

由于天然Hyperforin分子的化学稳定性有限，近年来，一种更稳定形式的Hyperforin，即Hyperforin二环己基铵盐（HYP-DCHA）已用于离体和体外实验，但在典型情况下其实际稳定性细胞培养条件从未被研究过。

在这种贡献下，在典型的细胞培养条件下检查了HYP-DCHA的稳定性。对于通过HPLC分析确定的HYP-DCHA的进一步稳定化，研究了添加和不添加胎牛血清（FCS）的不同细胞培养基。此外，检查了白蛋白纳米颗粒作为HYP-DCHA的稳定载体系统。在这种情况下，检查了HYP-DCHA和白蛋白纳米颗粒（ANP）之间的相互作用，涉及了HYP的大小和载量。在HaCaT单层细胞（人角质形成细胞系）上体外研究了HYP-DCHA在细胞培养基中提供或在ANP上装载对细胞活力和细胞毒性的影响。

孵育24小时后，完全回收了含有FCS的培养基中提供的HYP-DCHA。但是，缺乏FCS会导致在不到24小时的孵育时间后HYP-DCHA完全丧失。孵育24小时后，在无FCS的培养基中提供加载在ANP上的HYP-DCHA，可使回收率提高约60％。与FCS一起在培养基中提供的HYP-DCHA表现出HaCaT细胞活力的缓慢剂量依赖性降低，而没有任何细胞毒性作用（抗增殖作用）。缺乏FCS的HYP-DCHA治疗导致存活率显着更快下降，这主要归因于细胞毒性。后者对于装载HYP-DHCA的ANP是正确的，尽管存在FCS，但仍观察到细胞毒性增加。

结果表明，在细胞培养条件下，广泛使用的HYP-DCHA的稳定性相当有限。特别是缺乏FCS会导致HYP-DCHA降解和/或氧化，可能会导致细胞毒性增加。相比之下，FCS补充剂在细胞培养条件下可以相当稳定地稳定HYP-DCHA，而白蛋白纳米颗粒可以起到相同的稳定作用，尽管对细胞本身的细胞毒性作用增加了。