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 evaluated 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 insulin-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.
Hair Growth Hair Loss Human Follicle Dermal Papilla Cells Caffeine Dihydrotestosterone IGF-1 VEGF Reactive Oxygen Species1. 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 immensely shapes the external appearance and contributes decisively to self-esteem. As a consequence, hair growth disorders and hair loss can significantly affect the well-being and quality of life of those affected and contributes to negative self-esteem as well as an increased prevalence of psychosocial problems [1] [2] .
Hair consists essentially of keratin and is embedded with its root in the scalp. The hair root is anchored in the epidermis and enclosed by the hair follicle. This follicle consists of an epithelial and a dermal (mesenchymal) component, whose interactions play an essential role regarding the regulation of hair follicle growth [3] . On average, the scalp hair grows 0.3 - 0.5 mm per day, which is about 15 cm in a year [4] . The resulting hair length is essentially determined by the growth rate and the duration of sustained growth. Hair growth is subject to a fixed cycle consisting of several phases of varying periods. This cycle is divided into the anagen, catagen, and telogen phases [5] . In contrast to many animals, the hair cycles of the individual hair follicles run asynchronously in humans [6] . The anagen phase is the growth phase where a new hair root is formed and hair production begins. This phase is characterized by increased cell proliferation and lasts for 2 - 8 years, depending on age, sex, and growth site [7] [8] . It is followed by the catagen phase, which lasts several weeks and is defined by an apoptosis-controlled regression of the cells and reduced cell proliferation. That leads to controlled degeneration of the hair follicle, causing it to tighten, and the connection between the hair and the hair root is lost [9] . The hair then enters a resting phase (telogen) lasting 2 - 4 months, during which the loose hair is detached from the scalp by mechanical influences or pushed out of the follicle by the new, regrowing hair [9] . Due to the cyclic growth phases, no hair grows indefinitely and remains in the anagen phase. Instead, it may enter the catagen stage according to a fixed rhythm controlled by numerous complex physiological and pathological factors.
The dermal papilla at the base of the hair follicle is crucial for hair follicle morphogenesis and cycling [10] . Dermal papilla cells release growth factors that induce epithelial cells to proliferate, leading to hair shaft growth and acceleration of hair regeneration [11] . They secrete numerous growth factors such as insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF), which are necessary for maintaining the anagen phase, cell proliferation, and migration [12] [13] [14] .
Since the hair continuously enters the telogen phase, a person loses about 70 - 100 hairs per day. This natural hair loss is usually not noticeable on the head, as the new hair grows back at the same time. In case of damage to the hair root or follicle in the growth phase, nutrients deficiency, mechanical stress, hormonal imbalances, or if many hairs enter the telogen phase at once, it leads to increased hair loss (alopecia) and thinning of the hair [15] [16] . As both men and women are affected by increased hair loss and thinning hair, especially with age, there is an urgent need to develop new approaches and products that support hair growth, strengthen hair and prevent hair loss [17] [18] [19] .
Olive mill wastewater (OMWW) is a phenol-rich by-product of olive oil production 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.
2. Materials and Methods2.1. 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 (0.2 µm). Final dilutions were prepared with cell growth medium. Besides, the effects of caffeine (Caesar & Loretz GmbH, Hilden, Germany) and dihydrotestosterone (Sigma-Aldrich, St. Louis, MO, USA) on cells were also investigated. For this purpose, a caffeine stock solution with a concentration of 10 mg/mL was prepared in phosphate-buffered saline (PBS) and filtered (0.2 µm). The final dilution of 0.25 mM caffeine was prepared in cell growth medium. For dihydrotestosterone (DHT), a 10 mM stock solution was prepared in ethanol and then diluted to 0.1 mM in cell growth medium.
2.2. Cultivation of Human Follicle Dermal Papilla Cells
Human follicle dermal papilla cells (HFDPC) obtained from PromoCell (Heidelberg, Germany) were cultured at 37˚C and 5% CO2 in follicle dermal papilla cell growth medium (PromoCell, Heidelberg, Germany) supplemented with 100 units/mL penicillin and 100 µg/mL streptomycin (Capricorn Scientific, Ebsdorfergrund, Germany).
2.3. 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% CO2. Then the cells were washed with PBS and treated with the samples for 24 h (100 µL) and 48 h (200 µL). After sample incubation, 0.25 mg/mL MTT in cell growth medium was added, and the plate was incubated at 37˚C for 4 hours. Subsequently, the MTT solution was removed, and the cells were lysed with dimethyl sulfoxide (Merck KgaA, Darmstadt, Germany) while shaking at 300 rpm for 1 hour. Finally, the absorbance was measured at 570 nm and 650 nm (reference wavelength) using SynergyTM HTX multi-mode microplate reader with the software Gen5TM 2.07 (BioTek Instruments Inc., Winooski, VT, USA). Results are shown in relation to the untreated control (incubated with cell culture medium or incubated with 1% ethanol in medium), and dimethyl sulfoxide served as blank value. All experiments were performed three times with at least four replicates. HFDPCs from passages 3 to 6 were used. Statistical analyses were performed on results as described in Section 2.6 Statistical Analysis.
2.4. 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% CO2. Then the cells were washed with PBS and treated with the samples for 24 h (0.5 mL) and 48 h (1 mL). Cell-free supernatants were collected, and the concentrations of Insulin-like growth factor 1 (IGF-1) and Vascular Endothelial Growth Factor (VEGF) were determined using commercial ELISA kits using the manufacture’s protocols without adaptions (R&D Systems Inc. Minneapolis, MN, USA). The absorbance was measured at 450 nm and 570 nm (reference wavelength) using SynergyTM HTX multi-mode microplate reader with the software Gen5TM 2.07 (BioTek Instruments Inc., Winooski, USA). Results are shown in relation to the untreated control. All experiments were performed three times with at least two replicates.
2.5. Reactive Oxygen Species Assay in Vitro
10,000 HFDPCs (passage 5 to 7) per well, in growth medium without phenol red (PromoCell, Heidelberg, Germany), were seeded in a black 96-well plate with a clear bottom (Corning Life Science, Tewksbury, MA, USA) and incubated overnight at 37˚C and 5% CO2. Cells were then washed twice with PBS, and 100 µL of the samples containing 50 µM 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA, Sigma-Aldrich, St. Louis, MO, USA) were added. That was followed by incubation at 37˚C for 30 min before the cells were washed again, and 100 µL of 100 µM H2O2 (Sigma-Aldrich, St. Louis, MO, USA) was applied. After incubation at 37˚C and 5% CO2 for 45 min, a final washing step with PBS was carried out before measuring the fluorescence intensity (FLU, ex./em. 485/528 nm) using the microplate reader (100 µL of phosphate buffer, bottom read). The DCFH radical scavenging activity was calculated based on Equation (1). All experiments were performed three times with at least four replicates.
DCHFradicalscavengingactivity ( % ) = 100 × FLU control − FLU sample FLU control (1)
2.6. 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 using GraphPad Prism® v8.3.0 (538) (San Diego, CA, USA).
3. Results3.1. Influence of OMWW Extract on the Viability of HFDPCs
First, the influence of the OMWW extract in different dilutions, 0.25 mM caffeine, and 0.1 mM dihydrotestosterone, on HFDPC viability was examined via MTT assay over 24 h and 48 h to identify which dilutions are suitable for the following in vitro experiments and to determine whether the samples affect cell proliferation. After a 24 h-incubation with OMWW 1:100, the HFDPC showed viability of 6.3% ± 1.56%, and after 48 h 3.14% ± 0.05% (Figure 1). In addition, a decrease in cell viability was observed after incubation with OMWW 1:200 (24 h 16.80% ± 8.01%, 48 h 2.35% ± 0.18%) and OMWW 1:250 (24 h 68.22% ± 11.80%, 48 h 3.82% ± 1.41%). Cells treated with OMWW 1:500 showed a significant increase in viability of 124.90% ± 2.71% compared to the untreated control after 24 h. This effect was not visible with 48 h incubation (108.58% ± 7.69%). Treatment of cells with 0.25 mM caffeine for 24 h also resulted in a slightly increased cell viability of 110.21% ± 4.90%. This effect was not visible at 48 h incubation. The remaining samples (OMWW 1:750 and 0.1 mM DHT) showed no significant effect on cell viability (Figure 1). Based on these results, OMWW 1:500, OMWW 1:750, 0.25 mM caffeine, and 0.1 mM DHT samples were chosen for further in vitro studies.
3.2. 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 investigated in this work. While incubation of the cells with both OMWW extract dilutions had no significant effect on the secretion of IGF-1 after 24 h, incubation for 48 h resulted in a significant increase of IGF-1 in the supernatant for cells treated with OMWW 1:500 (346.67% ± 55.72%) and OMWW 1:750 (290.39% ± 50.33%) (Figure 2). Similar observations were made during incubation with caffeine. While treatment of the cells for 24 h resulted in a slight increase in IGF-1 (149.68% ± 20.88%), a 48-hour incubation caused a significant increase of IGF-1 (535.84% ± 117.55%). The supernatant of dihydrotestosterone-treated HFDPC contained 146.59% ± 9.46% (24 h) respectively and 203.44% ± 79.57% (48 h) IGF-1 in relation to the untreated control.
The secretion of the growth factor VEGF was not significantly affected by the samples after 24 h of incubation (Figure 3). Moreover, treatment with OMWW 1:500, OMWW 1:750, and caffeine for 48 h resulted in a slight decrease in VEGF release to 69.45% ± 2.47%, 78.90% ± 2.33%, and 81.37% ± 2.48% respectively.
3.3. OMWW Extract Reduces the Production of Reactive Oxygen Species in vitro
To evaluate the antioxidant potential of the samples on human follicle dermal papilla cells, the formation of reactive oxygen species (ROS) was triggered by exposure of the cells to H2O2. HFDPC were incubated with the samples for 30 min before exposure to H2O2 to investigate the ability of the samples to reduce the formation of ROS. Due to studies by Rossi et al. [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 1:250, 1:500 and 1:750 lead to a reduction of free radicals by 60.76% ± 5.29%, 47.40% ± 6.20% and 24.10% ± 2.01% respectively. While caffeine showed no significant DCFH radical scavenging activity (−15.30% ± 10.51%), incubation with DHT increased ROS production by 72.36% ± 15.83%.
4. Discussion
In this work, the potential of the phenol-rich olive mill wastewater extract was investigated, concerning the support of hair growth and the possible reduction as well as prevention of hair loss in vitro. Primary human follicle dermal papilla cells were used as a screening model for the experiments. These are specialized mesenchymal cells that play a major role in hair morphogenesis and regeneration by
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 investigated growth factor secreted by HFDPCs in vitro and in vivo is the vascular endothelial growth factor (VEGF), which stimulates vasculogenesis and angiogenesis [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 H2O2-induced 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 ROS-induced hair loss through its antioxidant properties.
5. Conclusion
This work demonstrates the potential of polyphenol-rich OMWW extract for use in hair care. Due to the positive influence of the substance on cell proliferation and release of the growth factor IGF-1 of the hair follicle dermal papilla cells, OMWW could contribute to improved and prolonged hair growth in vivo. 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.
Acknowledgements
The authors would like to acknowledge the financial support by Gianni Lo Franco, Antonio Lo Franco and Bandino Lo Franco (Fattoria la Vialla, Italy; fattoria@lavialla.it) and the samples provided. We thank Prof. Dr. Adriana Albini, Department of Medicine and Surgery, University Milano-Bicocca, Milan, Italy and Scientific and Technology Pole, IRCCS MultiMedica, Milan, Italy for her scientific support of the project. We are grateful to Dres. Massimo and Daniele Pizzichini (Genelab srl, ENEA) for the phenol rich purified extract (OMWW extract, A009).
Conflicts of Interest
The authors declare no conflicts of interest.
Cite this paper
Sittek, L.-M., Schmidts, T.M. and Schlupp, P. (2021) Polyphenol-Rich Olive Mill Wastewater Extract and Its Potential Use in Hair Care Products. Journal of Cosmetics, Dermatological Sciences and Applications, 11, 356-370. https://doi.org/10.4236/jcdsa.2021.114029
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