Polyphenol-Rich Olive Mill Wastewater Extract and Its Potential Use in Hair Care Products

In this work, the influence of phenol-enriched olive mill wastewater (OMWW) extract on hair growth was investigated in vitro on human follicle dermal papilla cells. OMWW has already shown great potential for use in skincare products, and its high polyphenol content is predestined to have a positive effect on hair growth. The studies included caffeine, a positive modulator of hair growth, and dihydrotestosterone, which causes hair loss in vivo, as controls. The impact of the investigated compounds on hair growth was eva-luated by studies on cell viability and proliferation, the release of growth factors (insulin-like growth factor-1 and vascular endothelial growth factor), and the reduction of reactive oxygen species formation. OMWW showed a positive influence on the proliferation of the human follicle dermal papilla cells. Moreover, the extract leads to a significantly increased secretion of insu-lin-like growth factor-1, and a considerable reduction in reactive oxygen species formation was observed. Overall, our results show that the investigated phenol-enriched OMWW extract is a promising ingredient for hair care to improve hair growth, prevent hair loss due to oxidative stress and maintain a healthy scalp.


Introduction
Besides its protective function against heat, UV radiation, and exposure to cold, the human scalp hair also has an important psychological role. The hair im-duction with great potential for use in skincare to improve skin health and protection and with beneficial effects on skin aging [20]. Since studies of polyphenols from green tea and Annurca Apples showed a beneficial effect on hair growth, the influence of OMWW extract on human follicle dermal papilla cells (HFDPC) and their secretion of growth factors (IGF-1 and VEGF) was investigated in this work [21] [22]. In addition, the protective function of the extract regarding the formation of reactive oxygen species (ROS) in the cells was examined.

Olive Mill Wastewater Extract and Sample Preparation
The olive mill wastewater used for this study was provided by Agriturismo La Vialla (Castiglion Fibocchi (Arezzo), Italy). The phenol-enriched purified extract (OMWW extract, quantitative composition [20]) was obtained from Massimo and Daniele Pizzichini according to the Patent formulation (Patent 8815815) [23]. Before use, the OMWW extract was sonicated for 10 min, centrifuged at 500 x g for 20 s, and filtrated (0.

Cell Viability Assay
The effect of the samples on the viability of the HFDPCs was determined by MTT assay in accordance with Mosmann [24]. HFDPCs were seeded in 96-well plates (2500 cells per well) and incubated overnight at 37˚C and 5% CO 2

ELISA of Growth Factors IGF-1 and VEGF
HFDPCs (passages 5 to 8) were cultured in 48-well plates (2500 cells per well) in 1 mL growth medium for four days at 37˚C and 5% CO 2 . Then the cells were

Statistical Analysis
Data are given as arithmetic mean values ± standard deviation. To verify statistically significant differences between the mean values of different groups, a two-way ANOVA, followed by a Dunnett test, was carried out. Thus, p ≤ 0.05 is considered to indicate a significant difference. Statistical analysis was performed

OMWW Extract Increases the Release of the Growth Factor IGF-1 from HFDPCs
Since various studies show that growth factors and cytokines released by dermal papilla cells are essential for the division, proliferation, and differentiation of cells in the hair follicle and thus for hair growth, the influence of OMWW extract release of the growth factors IGF-1 and VEGF from HFDPC was investi-      [23] and Schlupp et al. [20], the OMWW extract was used diluted 1:500, 1:750, and additionally, the dilution 1:250 was tested. The ROS production was significantly reduced by the OMWW dilutions in a concentration-dependent manner ( Figure 4). Thus, OMWW

Discussion
In this work, the potential of the phenol-rich olive mill wastewater extract was  influencing follicle development and growth through the supply of multipotent stem cells, nutrients and growth factors [25] [26]. Due to their essential role in hair growth, HFDPC has been used in numerous studies as an in vitro screening model for the evaluation of various hair growth modulators [27]. To validate the experiments and to include suitable positive and negative reference substances, caffeine and dihydrotestosterone (DHT) were also tested. DHT is an activated form of testosterone resulting from systemic and local metabolism in the scalp, which leads to androgenic alopecia in men by shortening the hair follicle growth phase [28]. It is a known inhibitor of human hair follicle proliferation and induces apoptosis in HFDPC [29]. Our applied concentration of 0.1 mM DHT is based on studies by Lee et al. [30] and Shin et al. [31], who investigated the effect of DHT on human dermal papilla cells. Caffeine was chosen as a reference for this work because it is commonly used in hair care products and is claimed to stimulate hair growth and prevent hair loss. Fischer et al. showed that caffeine leads to a significant stimulation of hair follicle growth, reduces the negative influence of testosterone [32], and increases IGF-1 protein expression in male and female hair follicles in vitro [33]. In addition, the substance decreases the activity of the enzyme 5-reductase, which is responsible for the conversion of testosterone into dihydrotestosterone [34]. Following the studies of Fischer et al., we used caffeine with a concentration of 0.25 mM (equivalent to 0.005%). The cell viability studies (Figure 1) indicate that caffeine has a slightly proliferative effect at 24 h incubation time. Zhuang et al. [35] also demonstrated that caffeine has a proliferative effect on HFDPC. In our experiments, OMWW at a dilution of 1:500 also showed a positive influence on cell proliferation that was slightly stronger than the proliferative effect of caffeine. Since proliferation is one of the most tested markers of HFDPC activity and the cells are critical for hair growth in the anagen phase of the hair cycle [36], the results imply that OMWW extract may positively influence hair growth by increasing the proliferation of follicle dermal papilla cells. Treatment of HFDPC with other polyphenol-rich extracts like Ecklonia cava and Annurca apple extracts led to increased cell proliferation as well [22] [37]. In addition, Schlupp et al. [20] showed that OMWW extract was also beneficial for skin cell viability. In contrast to the study by Kiesewetter et al. [38], no inhibitory effect of DHT on cell proliferation could be detected in our studies, whereby a higher concentration (0.1 mM) was used in our experiments.
Considering that growth factors released by dermal papilla cells such as insulin-like growth factor-1 (IGF-1), fibroblast growth factor-5, vascular endothelial growth factor (VEGF), and platelet-derived growth factor have already been identified as essential mediators of hair growth by controlling processes such as division, proliferation, and differentiation of cells in the hair follicle, the influence of OMWW extract release of the growth factors IGF-1 and VEGF from HFDPC was investigated in this work [39] [40] [41]. Numerous studies, such as those by Rajendran et al. [12], showed that IGF-1 is produced and secreted by dermal papilla cells in vitro, which was confirmed by our experiments ( Figure   2). A significantly increased secretion could be detected after 48 hours of treatment with OMWW 1:500, OMWW 1:750, and caffeine. IGF-1 is an insulin-like essential growth factor in many biological systems [42]. Regarding hair growth, IGF-1 plays a crucial role in regulating hair follicle development, maintaining the anagen phase, and is considered one of the key mediators of hair growth [14] [43] [44]. An in vivo study by Zhao et al. [45] demonstrated by systemic and local injection of IGF-1 that the growth factor is essential for the maintenance of hair growth and prevents hair follicles from prematurely entering the catagen phase. The study by Shin et al. showed that increased IGF-1 secretion in vitro in HFDPC also has positive effects on hair growth ex vivo [37]. Thus, treatment with the phenolic extract of Ecklonia cava resulted not only in an increased secretion of IGF-1 in vitro but also in an elongation of human hair shaft length ex vivo. Regarding DHT, no significant effect on the release of IGF-1 was observed ( Figure 2). Nevertheless, an in vivo study by Zhao et al. [46] showed that DHT administration in mice resulted in decreased IGF-1 expression, associated with a reduction in proliferating cells in hair follicles and inhibition of hair regrowth. Since our data show that DHT has no direct effect on the secretion of IGF-1 in HFDPC in vitro, the association of the parameters DHT, IGF-1 and hair loss in vivo seem to be more complex. Further investigations are required in vitro to determine the exact mechanism of action and connection. Another frequently giogenesis [47]. Concerning hair growth, several studies have shown that VEGF can promote hair growth, increase follicle size and hair thickness [41] [48] [49]. Incubation of HFDPCs with the samples for 24 hours did not result in any significant change regarding VEGF release (Figure 3). After 48 h, OMWW 1:500, OMWW 1:750, and caffeine lead to a significant reduction of VEGF secretion, whereas DHT does not influence the growth factor. Investigations by Kim et al. [50] indicated that a 48 h incubation of dermal papilla cells with 20 ppm caffeine leads to a significant increase of VEGF. Despite the higher applied concentration of caffeine, we could not confirm this in our experiments. In general, it should be noted that the study of DHT in our experiments did not show a significant effect on the release of growth factors in vitro, implying that the relationship of DHT and hair loss in vivo must have a much more complex mechanism. The entire process of hair growth is very intricate and not yet fully understood. It is known that the growth factors studied have a positive influence on hair growth, but the detailed mechanisms of action have not yet been unraveled. Therefore, more in vitro data need to be collected on this topic. Overall, based on the existing literature and our in vitro studies, it can be suggested that both OMWW extract and caffeine may have a positive effect on the anagen phase and thus on hair growth due to their positive impact on the release of the growth factor IGF-1.
Oxidative stress is associated with inflammatory processes, UV radiation, and cellular aging. Increased oxidative stress contributes to damage of cellular DNA, proteins, and lipids, which may lead to cell cycle arrest, cell senescence, and cell death [51]. This stress can also have a major impact on hair growth and balding.
For example, Upton et al. [52] showed that oxidative stress promotes the secretion of TGF-β1 in dermal papilla cells, an inhibitor of hair growth, and suppresses the release of the important growth factor IGF-1. In our study, the H 2 O 2induced ROS production was significantly reduced in a concentration-dependent manner by OMWW extract (Figure 4). This antioxidant potential and concentration dependence were also demonstrated in the studies by Schlupp et al. in dermal cells [20] and Rossi et al. in endothelial cells [23]. That shows the clear advantage of OMWW extract over caffeine in terms of the potential use in hair care products. Although caffeine showed similar positive effects as OMWW in the other experiments, caffeine has no beneficial effect on ROS formation. Dihydrotestosterone led to a significant increase in ROS production. That is consistent with literature data showing that DHT increases intracellular ROS levels and thereby induces cell death and senescence [30] [53]. Other antioxidants, such as ascorbic acid, were shown to stimulate the growth of HFDPCs and promote hair shaft elongation in vitro and in vivo [54]. Thus, OMWW can protect the essential hair follicle cells from oxidative stress or DHT and reduce ROSinduced hair loss through its antioxidant properties.

Conclusion
This work demonstrates the potential of polyphenol-rich OMWW extract for In addition, its antioxidant capabilities could prevent oxidative stress and the formation of free radicals on the scalp and in the hair follicles. Consequently, OMWW would contribute to the maintenance of a healthy scalp and possibly prevent hair loss due to oxidative stress.