## 4 Discussion  
It has been demonstrated that chemotherapy alone reduced the ovarian reserve in the growing follicles by activating the apoptosis in the granulosa cells [25; 26; 27; 28]. Besides, oxidative and nitrosative stress are known to damage cellular macromolecules during follicle growth leading to follicular atresia and other damages in the intraocular microenvironment, developing the so called premature ovarian insufficiency [29; 30]. Granulosa cells are easily affected by ROS, mostly by H\({}_{2}\)O\({}_{2}\)[27]. Therefore, in this paper we provided new insights regarding the oxidative stress effects after cyclophosphamide treatments and _P. sempervirens_ therapy of preserving the ovarian physiological function. In accordance with our previous work [15], _P. sempervirens_ hidroalcoholic extract has showed a strong antioxidant effect in vitro against DPPH and ABTS radicals, as well as in the in vitro cell migration assay. In addition, the same extract was investigated in an in vivo turpentine oil-induced inflammation model, where three different dilutions of the _P. sempervirens_ extract have decreased the oxidative and nitrosative stress markers, such as TOS, OSI, MDA, and NO; meanwhile TAR, SH, CAT, and superoxide dismutase enzyme (SOD) were significantly enhanced in a dose-dependent manner [15]. Here we showed that 200 mg/kg CPA in a unique injection disturbed the homeostasis leading to oxidative and nitrosative stress, as seen in Figure 1, in TOS, OSI, and MDA levels. While it is assumed that CPA metabolic byproducts, such as phosphoramide mustard, create such oxidative injuries in the ovarian reserve, antioxidants are more than required to defend cells and allow them to function in that environment. As such, employing _P. sempervirens_ as an adjuvant pretherapy, did manage to counteract the excess of ROS/RNS by decreasing values of TOS, OSI, and MDA to near-normal or even higher. The same was observed for NO and 3 NT. The dose-dependent effect was not clearly noticed, but an interesting observation in TOS and OSI levels was that P2 dose induced a greater inhibition as compared with P1 and P3 doses, meaning that _P. sempervirens_ chemical composition has a high concentration of polyphenols, which can easily become pro-oxidants above a certain dose [15]. It is known and well investigated that polyphenols, mostly flavonoids, can manifest a pro-oxidant reactivity under certain conditions, leading to undesirable effects, many of them related to oxidative stress [31]. Other studies have shown that in vivo dietary polyphenols can interact with transition metal ions in the gastrointestinal tract and since these ions are often unabsorbed, pro-oxidant effects could most likely occur, and among these changes, plasma ROS markers are most prominent to reflect them [32]. With respect to antioxidants, these not only serve as ROS or RNS neutralizers, but other biological effects have been demonstrated over the years, including the inhibition of cyclooxygenase, lipoxygenase activity, metaloproteases, and also regulating many signaling pathways (MAPK, P13K, NF-kB) [33; 34; 35]. The antioxidant defense markers investigated in our in vivo experimental model (TAC, CAT, GPx and SH), revealed a contradictory situation. As seen in the results section, TAC is not significantly influenced by CPA, meanwhile the P2 and P3 doses of _P. sempervirens_ extract managed to significantly enhance the TAC levels as compared with CPA group. On the other hand, the endogenous antioxidant enzymes, GPx and CAT, behaved opposite to each other, after the injection with 200 mg/kg BW, namely CAT significantly decreased, while GPx significantly increased. This is an unusual situation, if compared with previous studies, where endogenous antioxidant enzymes are strongly depleted in various in vivo models of oxidative injuries [36; 37; 38; 39; 40]. It is intriguing that GPx reacted in such a significant up-rising way, while the literature has confirmed so many times that this enzyme behaves similarly to its counterparts during stress.