Determination of Perilla frutescens compound stability

The assessment of cell proliferation after treatment revealed an immediate effect on the cells for all PF extracts, which is reflected by the drop of p-values (Appendix V, Figure 3). The most pronounced and long-lasting significant effe t as aptu ed ith . g V“ PF water extract (Appendix V, Figure 3 A). The stability of PF in cell culture medium was therefore exemplarily assessed by the detection of the molecular composition of VS PF water extract at time points 0, 1, 2, 6 and 24h in comparison to control samples of extract prepared in water (Appendix V, Table 2). A variety of different molecules could be found in the employed extract based on previous findings of Buchwald-Werner et al., who analyzed the same PF material [163]. In addition to this, the development of a chromatographic separation discussed in a previous publication, allowed the tentative identification of further compounds based on their logD vs. retention time behavior (Appendix IV, Figure 3). In the following the stability of those known and tentatively identified PF compounds, i.e. RA or apigenin and luteolin as well as their O- or C- glucuronide or glucoside conjugates (Appendix V, Table 2 A-C & Figure 29), was investigated in order to draw conclusions on their potential to affect the physiology of IPEC-J2 cells. In comparison to PF extract controls prepared in water, which showed a stable abundance of all compounds within 24 h, in cell culture medium most of the compounds were found with decreasing abundance over time (Appendix V, Table 2 & Figure 29).

83 Figure 29 Stability of VS PF water extract compounds in the absence of cells either solved in cell culture medium (dashed grey) or water (bold black) as control at time points 0, 1, 2, 6 and 24 h. For clarity reasons, only positive standard deviations are given, which were calculated out of n=3. Diglu = diglucuronide moiety.

Apigenin has been demonstrated to be readily oxidized to form a reactive phenoxyl radical, but only in the presence of H₂O₂ or xanthine oxidase [340-342]. In contrast to the here captured distinct decrease of apigenin abundance (Appendix V, Table 2 A & Figure 29), Long et al. detected only a slight degradation in pure cell culture medium within the same time period [285]. However, they measured the stability of individual substances as opposed to

0

84 the mixture of a plethora of different compounds contained in PF extract. Those may chemically react with one another or with cell culture medium components, thus forming undesired decomposition products like e.g. H₂O₂ [336, 343-345]. This effect might furthermore be enhanced due to the here conducted experimental procedure, which included several purification and dilution steps prior to LC-MS analysis. The required quantity of VS PF water extract solved in cell culture medium was therefore increased compared to cell proliferation and gene expression experiments. Consequently, the generation of distinct quantities of H₂O₂ can be assumed. In the presence of transition metal ions H2O2 would be converted to hydroxyl radical, thus enhancing oxidation processes and therewith the degradation of PF extract compounds. Those aspects likely contribute to the degradation of apigenin with the here applied experimental conditions (Appendix V, Table 2). In contrast to apigenin and luteolin -the latter also revealing a fast degradation- their conjugates were detected with enhanced stability, which is especially apparent for apigenin 7-O-glycosides and 7-O-(di)glucuronides (Appendix V, Table 2 A & Figure 29 B). This would imply the involvement of the C7-OH group in the instability of non-conjugated apigenin and luteolin in cell culture medium. Musialik et al. determined the highest OH-acidity for C7-OH in polar organic solvents. This in turn may result in a deprotonation of the group and the formation of a phenolate anion, which was detected to readily react with radicals [346].

However C-conjugated apigenin, in which C7-OH is not blocked, was still detected to be stable during the experimental time range, which might be attributed to steric effects. In contrast the here observed O-conjugated luteolins were found to degrade over time. The presence of the B-ring catechol moiety might therefore contribute to their susceptibility to degradation compared to O-conjugated apigenin, which merely contains one OH-group in its B-ring (Appendix V, Table 2 A & B). In fact, O-substitution with glycosides or glucuronides was reported to stabilize flavonoids by Zenkevich et al. and Shirai et al., since it blocks OH-groups involved in antioxidant as well as pro-oxidant activities [347, 348]. Nevertheless, further mechanisms seem to be crucial for the stability of conjugated flavonoids.

Comparable to findings of Long et al., RA is rapidly degraded. Of all the compounds observed, it was found to be the most prone to decomposition in cell culture medium (Figure 29, E). Two catechol groups within its structure most likely result in the generation of two quinone moieties [349]. RA was furthermore reported to produce H₂O₂ and superoxide

85 [285, 350] and an iron catalyzed oxidation of RA was captured by Fujimoto et al., however in EtOH solution [349]. Some studies discuss the issue of a heightened oxygen partial pressure present in in vitro experiments as one of the main problems causing polyphenol oxidation and generation of H₂O₂ [351, 352]. However conducted control measurements, in which PF extract was solved in water, but otherwise treated like PF extract samples in cell culture medium, revealed no decrease of compounds within 24 h (Figure 29, A-D, bold black line).

Although the presence of a non-physiological high oxygen concentration might contribute to the degradation of PF extract, it may only destabilize the compounds in combination with cell culture media. Components like metal catalyst are however more likely to impair PF extracts compound stability. In this regard, iron or copper ions, which are essential components of cell culture media, catalyze the generation of ROS via Haber-Weiss and Fenton reaction [334, 335, 353]. Jungbluth et al. proposed the degradation of flavonols in aqueous media in the context of a metal-catalyzed oxidation by Cu(II), Fe(II) or Fe(III) [354].

The possibility of flavonoid autooxidation was also described, whereupon phenoxyl radicals are generated [355, 356], which react with oxygen to form quinone products and superoxide in the presence of transition metals [334, 357]. In contrast flavonoids have also been observed to be able to chelate transition metals to form flavonoid-metal complexes at physiological pH, in this regard preventing metal catalyzed reactions [358-360]. The formation of those complexes is favored e.g. by a catechol moiety in the B-ring, as it is present in the structure of luteolin and rosmarinic acid [361, 362]. Metal-flavonoid complexes were even found to possess an enhanced superoxide scavenging activity compared to the parent flavonoid, in this manner e.g. exerting increased cytoprotective effects [363].

Beyond that, most wholesome properties ascribed to flavonoids are connected with their antioxidant activity. Their capability to capture ROS was found to be especially pronounced in the presence of some structural features, like a catechol moiety in the B-ring, a C3 and C5 hydroxyl substitution, a double bond between C2 and C3 and a C4 keto group [361, 364].

Luteolin and apigenin possess the latter three structural features, whereas only luteolin has a catechol moiety in the B-ring. However the processes causing a fast degradation of PF extract compounds in cell culture medium are apparently outbalancing their capability to scavenge ROS or chelate metal ions in the employed experimental setup. Consequently the

86 detected loss of flavonoid abundance would result in the generation of degradation products. In this regard Kern et al. detected the appearance of small phenolic acid degradation products like gallic acid after the rapid decrease of anthocyanidines in cell culture. Since gallic acid itself has been reported to inhibit cancer cell proliferation in vitro and in vivo, its generation will likely result in data misinterpretation [345, 365-367].

Oxidation mechanisms of flavonoids and the related generation of products were proposed by Zenkevich et al. and Jorgensen et al. by means of quercetin [348, 368]. Zhou et al.

tentatively identified further oxidation products of quercetin, which included taxifolin, low molecular weight compounds and dimers. They moreover proposed the respective degradation pathways [369]. Dimerization, hydroxylation and the increase of decomposition products were also found by Sang et al. [343, 344], Zenkevich et al. [348] and Ramesova et al. [351] for EGCG, quercetin and luteolin. Furthermore adjacent hydroxyl-groups, as present in catechol type flavonoids like luteolin, facilitate a fast degradation via the formation of potentially harmful quinones and semiquinones [285, 368, 370-372]. With regard to the described literature, MS data was assessed for newly generated compounds (Appendix V, Table 2). Although some m/z were found to increase over time, they couldn´t be assigned to oxidation products discussed in the studies above. However, polar degradation products of PF extract compounds may not be present within the sample anymore due to the SPE purification procedure employed in this study. Increasing abundances of m/z were however not limited to low molecular weight products, which are likely to occur after chemical decomposition reactions. Compounds with high m/z were also found to increase, which might be due to chemical polymerization of the PF extract compounds in cell culture medium (Appendix V, Table 2, D).

4.7. Conclusion - Effects of Perilla frutescens on cell proliferation and gene