Evaluation of novel cosmetic shampoo formulations against Malassezia species: Preliminary results of anti-dandruff shampoo formulations.

OBJECTIVES: Malassezia species are common, clinically relevant, and lipid-dependent yeasts of humans. They are also the leading causes of the dandruff problem of humans, and the azoles are used primarily in their topical and systemic treatment. Resistance to azoles is an emerging problem among Malassezia sp., which indicates the need of new drug assessments that will be effective against dandruff and limit the use of azoles and other agents in treatment. Among them, the efficacy of various combinations of piroctone olamine and climbazole against Malassezia sp. is highly important. Here, we assessed the efficacies of various piroctone olamine and climbazole formulations against Malassezia sp. in comparison with ketoconazole. METHODS: A total of nine formulations were included in the study, where each formulation was prepared from different concentrations of piroctone olamine and climbazole and both. All formulations contained the same ingredients as water, surfactants, hair conditioning agents, and preservatives. Malassezia furfur CBS1878, Malassezia globosa CBS7874, and Malassezia sympodialis CBS9570 were tested for antifungal susceptibility of each formulation by agar diffusion method. Sizes of the inhibition zones were compared with standard medical shampoo containing 2% ketoconazole, and the data were analyzed by Dunnett's multiple-comparison test. RESULTS: For all Malassezia sp. strains, climbazole 0.5% and piroctone olamine/climbazole (0.1%/0.1% and 0.1%/0.5%) combinations were found to have the same effect as the medical shampoo containing 2% ketoconazole. Piroctone olamine/climbazole 1.0%/0.1% formulation showed the same efficacy as 2% ketoconazole on M. furfur and M. sympodialis, while 0.1%/0.5% formulation to only M. furfur. For M. globosa, none of the formulations tested were as effective as ketoconazole. CONCLUSION: The species distribution of Malassezia sp. varies depending on the anatomical location on the host. According to the results of this study, climbazole and piroctone olamine combinations seem to be promising options against the dandruff problem with their high antifungal/anti dandruff efficacy.

The Effects of Helichrysum italicum Extract on the Extracellular Matrix of the Skin.

OBJECTIVE: An in-vitro study was performed to investigate the molecular basis of the wound healing and skin protective features of Helichrysum italicum (HI), a medicinal plant from the Mediterranean basin. MATERIALS AND METHODS: A dermal fibroblast cell line culture was treated with HI hydro-alcoholic extract to detect the gene expression levels of three selected primers: FGF-2, HAS-2 and MMP-9. Cell proliferation assay was performed using a XTT reagent. RNA isolations were carried out from both the extract treated study cell group and the control cell group using a TRI reagent. GAPDH was used as the reference gene. Gene expressions were determined by real time RT-qPCR. The results were represented as 'Target/GAPDH Fold Change'. Statistical evaluation was performed by Student's t test. RESULTS: HI extract caused statistically significant upregulation of FGF-2 (P=0.0473) and HAS-2 (P=0.0335) gene expressions compared to the untreated control cells. The treatment ended with 1.74 and 3.10 fold changes for FGF-2 and HAS-2, respectively. CONCLUSION: In general, it may be considered that HI has certain anabolic effects on the extracellular matrix of the skin because of the significant increases it causes in FGF-2 and HAS-2. Therefore, it may have a promising future in anti-aging studies and cosmetic dermatology. The results obtained in this study may also partially explain the molecular basis of the health benefits of HI on skin, including improvement in wound healing, and protection against the detrimental effects of ultraviolet exposure.

Effectiveness of topical Nigella sativa for vitiligo treatment.

Vitiligo is a pigment disease characterized by the disruption of melanocyte structure and function. Its etiology is unknown; however, genetic predisposition, biochemical factors, and neural mechanisms are thought to be effective. Although many agents are being used for its treatment, generally there is no absolute cure. The aim of the present study is to evaluate the effectiveness of topical Nigella sativa seed oil on vitiligo patients. Thirty-three vitiligo patients were included in the study. Totally 47 areas were evaluated in all patients. Cream containing N sativa seed oil was topically applied to hands, face, and genital region two times a day for 6 months. Statistically significant repigmentation was detected in hands, face, and genital region, the three treatment areas, and the p values are found .005, .001, and .004, respectively. N sativa can be used as an adjuvant therapy that can contribute to the treatment especially in sensitive skin areas like genital region.

Efficacy of two plant extracts against acne vulgaris: Initial results of microbiological tests and cell culture studies.

BACKGROUND: Acne vulgaris is a common skin disease characterized by increased sebum production, inflammation, and colonization of Propionibacterium acnes (P. acnes) on pilosebaceous follicles. AIMS: To determine the efficacy of two different plant extracts against P. acnes and to analyze the gene expression levels of IL-1α, SRD5A1, and TNFα in HaCaT cells treated with these plant extracts. METHODS: Anti-acne extract 1 (AE1) consisted of Juglans regia (walnut husk), Myrtus communis (myrtle leaves), Matricaria chamomilla (chamomilla flowers), Urtica dioica (stinging nettle leaves), and Rosa damascena (rose flowers). Anti-acne extract 2 (AE2) contained Brassica oleracea var. botrytis (broccoli) and B. oleracea var. italica (cauliflower). The antimicrobial activities of the extracts were tested on two different P. acnes strains: the reference strain of P. acnes (ATCC 51277) and the clinical isolate from a patient. The minimum inhibitory concentration (MIC) of the extracts was determined using the broth dilution method. Human keratinocyte cells were used for in vitro tests. Gene expression analyses were performed with RT-qPCR. RESULTS: The MIC values of the extracts were below 1/2048 µg/mL. In the gene expression analysis, AE1 increased the expression level of TNFα (1.1719, P < 0.0001), suppressed the expression level of IL-1α, SRD5A1 (0.0588, P = 0.0231; 0.3081, P = 0.0351), respectively. AE2 suppressed gene expression level of IL-1α, SRD5A1, TNFα (0.3815, P = 0.0254; 0.3418, P = 0.0271; 0.1997, P = 0.0623). CONCLUSIONS: Both herbal extracts demonstrated strong antibacterial and anti-inflammatory activity in this preliminary trial. In conclusion, the topical application of these botanical extracts can be good candidates for local acne treatment.

Proprietary Herbal Extract Downregulates the Gene Expression of IL-1α in HaCaT Cells: Possible Implications Against Nonscarring Alopecia.

BACKGROUND: Currently while, topical minoxidil and oral finasteride are the only medications approved in androgenetic alopecia (AGA), the cause oriented treatment and immunsupressive treatment are being performed in telogen effluvium (TE) and alopecia areata (AA) respectively. Considering the inflammatory factors in the pathogenesis of these three nonscarring alopecia forms, we have formulated a mixture for topical usage composed of six different herbal extracts (HE) which have already known antiinflammatory and antioxidant features. MATERIALS AND METHODS: In addition to performing the phytochemical analysis of HE, we detected the gene expression level of IL-1α, the crucial hair loss mediator, for the putative efficacy in nonscarring alopecia. Cell proliferation assay was performed by XTT reagent. After determination of non-cytotoxic concentration, HaCaT cells were treated with HE. RNA isolations were carried out from both non-treated and treated cell groups by using TRI-reagent. Gene expressions of IL-1α and as control GAPDH were determined by RT-qPCR analysis. RESULTS: Results were represented as "IL-1α/GAPDH Fold Change". HE solution caused statistically significant downregulation of IL-1α gene expressions (p<0.0001), compared to untreated control cells. HE treatment ended up with 0.1900 fold change for IL-1α. CONCLUSION: IL-1α is a direct growth inhibitory agent in hair follicles and an important actor in the pathogenesis of AGA , TE, and AA. Considering together the vitamins, flavonoids, and trace elements identified in the phytochemical analyses and downregulation of IL-1α in HaCaT cells, our HE may be an auxiliary agent in the therapy of these three nonscarring alopecia forms.

Minoxidil Downregulates Interleukin-1 Alpha Gene Expression in HaCaT Cells.

INTRODUCTION: Minoxidil has been used topically to stimulate hair growth for male androgenetic alopecia (AGA) for more than 3 decades. It is currently being used for female AGA and alopecia areata (AA) as well. Although much time has passed since its first use, our understanding of its mechanism of action is highly limited. Therefore, we examined the inflammatory properties of AGA and AA, two entities in which minoxidil is being used as a therapeutic agent. We investigated the in vitro expression levels of cytokine interleukin-1 alpha (IL-1α), a potent inhibitor of hair growth, in minoxidil-treated human keratinocyte (HaCaT) cells to determine whether this molecule exerts anti-inflammatory effects. MATERIALS AND METHODS: Cellular proliferation was examined using the Cell Proliferation Kit II (XTT) reagent. After determining a noncytotoxic concentration, HaCaT cells were treated with minoxidil. RNA was isolated from both untreated and treated cells with TRI Reagent®. Expression of the IL-1α gene was determined by reverse transcription quantitative polymerase chain reaction analysis and is reported relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which served as a control. RESULTS: Results are presented as IL-1α/GAPDH fold change. Minoxidil treatment downregulated IL-1α expression by 0.3433-fold compared with untreated cells (P = 0.001). CONCLUSION: This anti-inflammatory effect of minoxidil, as evidenced by significant downregulation of IL-1α gene expression in HaCaT cells, may represent one of its mechanisms of action in alopecia.

In vitro evaluation of natural thermal mineral waters in human keratinocyte cells: a preliminary study.

We aimed to test the anti-inflammatory and angiogenic properties of two different thermal waters at the cellular level in human keratinocyte cells in the present study. Two different thermal waters, thermo-mineral BJ1 (Bursa, Turkey) and oligomineral BG (Bolu, Turkey), were tested in human keratinocyte (HaCaT) cell line. HaCaT cells were incubated for 3 days with thermal waters; RNA isolation was carried out in the treated and untreated cells. The gene expressions of TNFα, IL-1α, and VEGF were measured using the RT-qPCR. The tested thermal waters significantly decreased the expression of IL-1α (BJ1 93% p = 0.0024 and BG 38% p = 0.0303). BJ1 and BG thermal waters downregulated the expression of TNFα (59% p = 0.0001 and 23% p = 0.0238 respectively). Furthermore, BJ1 and BG significantly downregulated the gene expression of VEGF (98% p = 0.0430 and 15% p = 0.0120). The observed decrease in the gene expression of TNFα and IL1α could be interpreted as an anti-inflammatory effect of mineral waters on HaCaT cells. Moreover, the suppressed VEGF expression might be an indicator of the antiangiogenic effect on human keratinocytes. Therefore, we hypothesized that depending on their specific chemical composition such as silica (128 mg/L) in BJ1 and hydrogen sulfide (1.2 mg/L) in BG, thermal waters suppress pro-inflammatory cytokines and angiogenic growth factor. These preliminary findings might give insight on the underlying mechanisms of the therapeutic benefits observed in some skin diseases such as rosacea and psoriasis.

A proprietary herbal extract against hair loss in androgenetic alopecia and telogen effluvium: a placebo-controlled, single-blind, clinical-instrumental study.

INTRODUCTION: Currently there are only a limited number of drugs available for treatment of androgenetic alopecia and telogen effluvium. However, certain plants and their standardized extracts may provide some clinical benefits against hair loss. We formulated a herbal shampoo and a solution to evaluate their efficacy, safety, and synergy in hair loss. METHODS: We conducted a randomized, placebo-controlled, single-blind, clinical and instrumental study for 6 months on 120 subjects with androgenetic alopecia and telogen effluvium, confirmed by pull test and phototricogram. Each subject was examined monthly. At the end of the study, a self-assessment test was carried out. RESULTS: Herbal formulations were found to be more effective in preventing and reducing hair loss than placebo at every assessment point. Anagen/telogen ratios improved significantly in the study group. In addition, concomitant use of the shampoo and solution were found to be more effective than single product use. CONCLUSION: We interpret this eutrophic effect for scalp hair as the final outcome of the entire content of our herbal formula, which has antiandrogenic, anti-inflammatory, antioxidative, angiogenic, and hair-stimulating features. In combination, these features help prevent hair from falling out and reducing hair loss.

Effect of Ficus carica leaf extract on the gene expression of selected factors in HaCaT cells.

BACKGROUND: Ficus carica Linn. (Fc), common fig, has been traditionally used for many metabolic, cardiovasculary, respiratory, gastrointestinal, and skin disorders. Several studies were performed showing its anti-inflammatory, anti-angiogenic, anticancerogenic, and tissue-protective effects. In all of those studies, the positive effects of Fc were concluded as the result of its antioxidant and anti-inflammatory features due to the polyphenols it contains. AIMS: To study the phenolic compounds of Fc extract and to investigate the molecular basis of anti-inflammatory, anti-angiogenic, antimitotic, and anti-androgenic effects of Fc leaf extract in vitro. MATERIALS AND METHODS: The gene expression levels of vascular endothelial growth factor (VEGF), tumor necrosis factor-alpha (TNF-a), interleukin 1-alpha (IL-1a), and 5 alpha-reductase type II (SRD5A2) were tested in human keratinocyte cells (HaCaT) by RT-qPCR. RESULTS: The gene expression analysis showed that the plant extract caused statistically significant downregulation of VEGF, TNF-a, IL-1a, and SRD5A2 compared to the untreated cells. DISCUSSION: These preliminary results of this in vitro study may partially explain the clinical success of Fc in the traditional medicine. CONCLUSION: Topical Fc leaf extract may be beneficial for some inflammatory disorders and androgen-dependent disorders of the skin such as androgenetic alopecia.

Minoxidil Acts as an Antiandrogen: A Study of 5α-reductase Type 2 Gene Expression in a Human Keratinocyte Cell Line.

Although more than three decades have passed since the first use of minoxidil in androgenetic alopecia (AGA), its mechanisms of action have still not been comprehensively understood. 5α-reductase (5α-R) has an active role as the predominant enzyme in both AGA and female pattern hair loss (FPHL), which are also the main therapeutic indications of topical minoxidil. But there is insufficient literature data regarding the interaction of minoxidil and the enzyme 5α-R. Herein, we studied the in vitro expression levels of 5α-R type 2 (5α-R2) in a minoxidil-treated human keratinocyte cell line (HaCaT) in order to elucidate the relation of these two parameters. Cell proliferation assay was performed by a XTT reagent (a yellow tetrazolium salt). After determination of non-cytotoxic concentration, HaCaT cells were treated with minoxidil. Ribonucleic acid (RNA) isolations were carried out from both non-treated and treated cell groups using a TRI reagent (an RNA, DNA, and protein isolation reagent). Gene expressions of 5α-R2 as study material and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the control were determined by real time-quantitative polymerase chain reaction (RT-qPCR) analysis. Results were represented as 5α-R2 / GAPDH fold change. Minoxidil treatment resulted in a 0.22 fold change for 5α-R2 (p < 0.0001). This antiandrogenic effect of minoxidil, shown by significant downregulation of 5α-R2 gene expression in HaCaT cells, may be one of its mechanisms of action in alopecia.