Oxidative stress can be generated in the connective tissue of the skin during its UV radiation, inflammatory processes like wound healing, and in skin aging [1–3]. The most promising treatments of these pathological changes include herbal extracts, vitamins, and antioxidant food supplements, which have been reported widely to scavenge free radicals from skin cells [1, 4, 17, 18].
It has been shown that hydrogen peroxide, like other reactive oxygen species, plays a substantial role in the metabolism of the main component of ECM, collagen [14, 19, 20]. We also found that hydrogen peroxide used at concentration of 0.3 mM caused a decrease in collagen biosynthesis in human skin fibroblasts by 54 %. Pretreatment of cells with anethole at a low concentration of 0.5 µM completely prevented this alteration. These changes at the protein level were correlated with alterations in the mRNA expression of type I collagen, and is in agreement with other studies [14, 20]. Anethole at the concentration of 0.5 µM totally abrogated the H2O2-induced alteration of COL1A1 gene.
Extracellular collagen plays an important role in the maintenance of the structural integrity of ECM, and its level is determined by the balance between synthesis and degradation . MMPs, which are zinc-dependent endopeptidases, degrade components of ECM and, therefore, play an important role in physiologic and pathological remodeling . MMP-2 (gelatinase A) and MMP-9 (gelatinase B) are key enzymes in the degradation of ECM collagen and are regulated through activation of latent proenzymes (pro-MMPs).
In our study we have shown that H2O2 exhibited a stimulating effect on the activity of both MMP-2 forms (72 and 66 kDa) (33 and 73 %, respectively) and that 0.5 µM of anethole completely protected against these changes. These results suggest that the effect of hydrogen peroxide was mediated by the induction of MMP-2 synthesis and activation at the translational and post-translational level. There is evidence that H2O2 is involved in the induction of MMP-2 at the mRNA level [14, 15]. Furthermore, the authors reported that H2O2 not only directly activates MMPs, but also causes a decrease in the expression of their inhibitors, such as TIMP2. The significant negative correlations between collagen content and MMP-2 activity, which have been found in our study, suggest that the increase in enzyme activity can contribute to the decrease in collagen synthesized in H2O2-treated cells. MMP-2 is known to digest native type I collagen and generate the 3/4- and 1/4-fragments characteristic of vertebrate collagenases .
Similarly, both forms of MMP-9 (95 and 88 kDa) were significantly influenced by hydrogen peroxide, much more than MMP-2, since a 3- and 3.6-fold increase in their activity, respectively, was observed. Anethole at all concentrations used (0,5, 1, and 10 µM) significantly inhibited the increase in the intensity of both bonds corresponding to the molecular masses of MMP-9, with the most efficient suppression demonstrated at 0.5 µM. Furthermore, anethole at concentration of 10 µM inhibited the activity of both 95 and 88 kDa MMP-9 by 30 and 38 %, respectively, in comparison to the untreated control. It has been reported that anethole used at a higher concentrations of 50 and 100 µM inhibited the activity of both MMP-2 and MMP-9 in HT 1080 cells suggesting its antimetastatic activity . However, in contrast to MMP-2, no significant correlations between collagen content in the medium and MMP-9 activity were detected.
We also examined the effect of anethole on H2O2-induced cytotoxicity and apoptosis of skin fibroblasts. Treatment of cells with 0.3 mM H2O2 significantly decreased viability of cells compared with the control cultures, confirming previous data of its toxic effect on fibroblasts [15, 25, 26]. Anethole significantly suppresses the H2O2-induced cytotoxicity at a concentration of 1 μM. We did not observe a more efficient prevention at higher concentrations. Several authors have reported that hydrogen peroxide can induce apoptosis in fibroblast cell cultures [16, 17, 26]. In our study, after a 24 h treatment of cells with 300 µM of H2O2, the percent of apoptotic cells increased 4-times and apoptosis was significantly attenuated by anethole at concentrations of 0.5 and 1 µM.
It is well known that a decrease in the content of dermal collagen, a major ECM protein, results in the loss of tensile strength and elasticity of skin, increases its fragility, and impaired wound healing, all of which are characteristic of aged skin. Skin aging has general relevance for many degenerative connective tissue diseases such as osteoarthritis, osteoporosis, and arteriosclerosis [1–3]. Therefore, agents with the ability to scavenge ROS, elevate ECM collagen levels or inhibit major collagen-degrading enzymes, would be useful in the development of effective agents in pharmacotherapy of various connective tissue diseases.
Components of essential oils containing a phenol group in their structure have considerable antioxidant properties. It has been shown in experiments that the volatile fractions of Thymus sp. and Eugenia sp. and their main components thymol and eugenol are strong antioxidants with activity comparable to BHT and even higher .
Phenylpropanoids, to which anethole belongs, work either by direct scavenging of reactive oxygen species or by acting as chain-breaking peroxyl radical scavengers . Anethole might chelate the zinc ion present in the catalytic site of MMPs and directly inhibit their activities, as well as exerting its antioxidant effects and inhibit their activity by attenuating oxidative stress. This is because oxidative stress activates nuclear factor Kappa B (NF-κB), an oxidant sensitive transcriptional factor, which plays a crucial role in the expression of MMP-2 as well as type I collagen [24, 29]. It has been determined that anethole at a concentration of 1 mM completely blocks NF-κB activation induced by TNF, phorbol ester, ceramide, or okadaic acid and partially by H2O2 . In contrast, in our study, anethole was effective at a concentration lower than 1 µM. Anethole and its sulfated analogs have also been shown to increase the level of cellular glutathione (GSH) which, as an endogenous antioxidant, plays a key role in the protection against ROS damage .
It is worth mentioning that the majority of recently introduced antioxidants is hydrophilic, which inhibits membrane passage and their antioxidant applications. Essential oils can be absorbed through the skin. This process is not active but occurs by simple diffusion, therefore, volatile components must be in contact with the skin for a certain period of time. They also reach the bloodstream, and, for example, anethole is expelled with the air breathed out after about 20–40 min. Anethole is a small, lipophilic molecule which permeates through cell membranes. It is worth noticing that (E)-anethole is non-toxic, non-irritant, and non sensitizing. It has no genotoxic activity and is not significantly carcinogenic .
In conclusion, the results of the present study indicate that anethole exhibits protective properties against hydrogen peroxide-induced toxicity and collagen metabolism changes in human skin fibroblast cultures, which can suggest its therapeutic properties in oxidative stress-related skin diseases. However, further study is needed to elucidate the exact mechanism of this action.