Skin aging is caused by an intrinsic or natural mechanism affecting the skin and other body organs related to hormonal changes occurring with age, and by extrinsic mechanisms associated with ultraviolet radiation exposure, which causes free radical species generation [1
]. Reactive oxygen species (ROS) are responsible for inducing the activator protein-1 (AP-1), a transcription factor that promotes collagen and elastin breakdown by up regulation of the matrix metalloproteinases (MMPs) [2
]. This environmental stress generated from ROS and reactive nitrogen species (RNS), combined with increased secretion of inflammatory mediators and enzyme expression (e.g., collagenase and elastase), causes inflammation and decreases the tensile strength and elasticity of the skin [3
]. Also in the antimicrobial domain, the incidence of skin and soft tissue infections is growing as a consequence of increasing bacteria resistance to drugs in a wide clinical spectrum [4
Natural ingredients for skin care are becoming popular due to their protective and defensive role against generation of free radicals and reduced production of oxidative enzymes, such as inducible nitric oxide synthase (iNOS) associated with inflammation and inflammatory diseases; collagenase and elastase that usually cause degradation of the extracellular matrix of the skin; and tyrosinase, the enzyme that catalyzes the most important step in melanin biosynthesis [5
]. Thus, an increasing interest in finding natural sources able to inhibit these enzymes, and using them as potential cosmetic ingredients in the design of creams or lotions for topical application is an interesting topic [6
Cosmeceutical formulations containing bioactive ingredients—such as phytonutrients, microbial metabolites, dairy products, minerals, and animal proteins—displaying medical-drug like benefits able to improve the structure, function, and appearance of the skin, are becoming more and more appellative solutions [7
Particularly, mushrooms, which are recognized as nutritionally important foods and reported as having important medicinal benefits, are slowly finding their way into the cosmetic industry in cosmeceutical applications, either as creams, lotions, or ointments [7
]. Agaricus bisporus
(J.E.Lange) Imbach, Lentinula edodes
(Berk.) Pegler, and Pleurotus
species are the most cultivated mushrooms worldwide, with a commercial production projected to increase significantly in the coming years [8
]. A. bisporus
has been known to show medicinal potential related with antitumor, antimicrobial, immunomodulatory, anti-inflammatory, and antioxidant properties [9
]. L. edodes,
popularly known as “shiitake”, has been reported to halve the proliferation of tumor cells and to strengthen the immune system [10
]. This mushroom has several bioactive compounds such as l
-ergothioneine and lentinan known to exert strong antioxidant [11
] and anti-inflammatory [13
] activities, respectively. Pleurotus ostreatus
(Jacq. ex Fr.) P. Kumm., has also attracted attention due to its content in polysaccharides, proteins, organic acids, vitamins, and minerals [14
], being reported as having anti-inflammatory [15
], antioxidant [16
], immunomodulatory, antimicrobial, and antitumor effects [17
]. All these aforementioned medicinal properties of mushroom extracts and related bioactive constituents, reinforce the interest in these natural matrices for cosmetic applications.
Some mushroom-based cosmetics can be found in the market; examples include Aveeno Positively Ageless
, which contains shitake (L. edodes
) extract (www.aveeno.com
) used to improve skin’s health; Sekkisei Cream
containing Cordyceps sinensis
(Berk.) Sacc., extract (www.coracosmetics.com
), used to strengthen skin’s natural defense system and to suppress melanin production, thereby correcting hyperpigmentation; Embellir Refresh Massage
that contains Ganoderma lucidum
(Curtis) P. Karst extract (www.menard-cosmetic.com
) used as a cleansing and massage cream and also as an efficient anti-aging skin care product.
The present study aimed to evaluate the cosmeceutical potential of A. bisporus, L. edodes, and P. ostreatus ethanolic extracts through the assessment of their anti-inflammatory, anti-bacterial, antioxidant, and anti-tyrosinase activities, and further incorporation in a cosmetic base cream. The bioactive properties of the final formulations were confirmed and related with the chemical ingredients present in the extracts, namely with phenolic acids and ergosterol.
3. Materials and Methods
3.1. Standards and Reagents
Acetonitrile 99.9% was of high-performance liquid chromatography (HPLC) grade from Lab-Scan (Lisbon, Portugal), methanol was of analytical grade and supplied by Pronalab (Lisbon, Portugal). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was obtained from Alfa Aesar (Ward Hill, MA, USA). Dulbecco’s modified Eagle’s minimum essential medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, Griess reagent system (Promega), DMSO, sulphorodamine B (SRB), lipopolysaccharide (LPS), 3,4-dihydroxy-l-phenylalanine (l-DOPA), and mushroom tyrosinase enzyme were obtained from Sigma-Aldrich Co. (Saint Louis, MO, USA). The culture media Muller Hinton broth (MHB) and Tryptic Soy Broth (TSB) were obtained from Biomerieux (Marcy l’Etoile, France). Blood agar with 7% sheep blood and Mac Conkey agar plates were purchased from Biomerieux Marcy l’Etoile, France). The dye p-iodonitrotetrazolium chloride (INT) was purchased from Sigma-Aldrich (Spruce Street; St. Louis, MO, USA) and was used as microbial growth indicator.
3.2. Mushroom Species and Preparation of the Ethanolic Extracts
Fresh samples of Agaricus bisporus (J.E.Lange) Imbach, Lentinula edodes (Berk.) Pegler, and Pleurotus ostreatus (Jacq. ex Fr.) P. Kumm) were purchased from a local supermarket in the Northeast of Portugal. For each mushroom species, the fruiting bodies were dried at 30 °C in an oven, reduced to a fine powder (20 mesh) and the treated portions mixed together to obtain homogeneous samples for further use.
To obtain the extracts, 3.0 g of the dried powder was mixed with 150 mL of ethanol and extracted in a Soxhlet apparatus for 4 h (12 cycles). Finally, the solvent was evaporated under reduced pressure (rotary evaporator Büchi R-210, Flawil, Switzerland) to obtain the dried ethanolic extracts [35
3.3. Chemical Characterization of the Extracts
3.3.1. Analysis of Phenolic Acids
Dried extracts were re-dissolved in a water/ethanol mixture (80:20, v
) at a concentration of 20 mg/mL and filtered through a 0.22 μm nylon disposable filter. The Shimadzu 20A series ultra-fast liquid chromatograph (UFLC, Shimadzu Corporation, Kyoto, Japan), operating at 0.5 mL/min and equipped with a Waters Spherisorb S3 ODS2 C18 column (3 μm, 150 × 4.6 mm) thermostatted at 35 °C, was used for phenolic acids identification and quantification. A binary solvent mixture consisting of 0.1% formic acid in water (A) and acetonitrile (B) was used. The detection was performed using a photodiode array detector (PDA) at 280 nm as the preferred wavelength [27
]. The phenolic acids were quantified using calibration curves obtained from commercial standards, namely: protocatechuic acid (y
= 0.999), p
-hydroxybenzoic acid (y
= 0.999), p
-coumaric acid (y
= 0.998), and cinnamic acid (y
= 0.998). The results were expressed as μg per g of extract.
3.3.2. Analysis of Ergosterol
Each dried mushroom extract was re-dissolved in methanol at a final concentration of 10 mg/mL and filtered through a 0.22 μm nylon disposable filter. The ergosterol identification and quantification was performed in HPLC-UV equipment, an integrated system composed by a pump (Knauer, Smartline system 1000, Berlin, Germany), a degasser system (Smartline manager 5000), an auto-sampler (AS-2057 Jasco, Easton, MD, USA), and a UV detector (Knauer Smartline 2500) according to a methodology developed in-house [19
]. Chromatographic separation was achieved with an Inertsil 100A ODS-3 reversed-phase column (5 μm, 4.6 × 150 mm, BGB Analytik AG, Boeckten, Switzerland) operating at 35 °C (7971R Grace oven). The mobile phase was acetonitrile/methanol (70:30, v
) and detection was performed at 280 nm. Ergosterol was quantified using a calibration curve obtained from a commercial standard (y
+ 0.20476; R2
= 0.99624). The results were expressed as mg per g of extract.
3.4. Evaluation of the Anti-Inflammatory Activity
Anti-inflammatory activity was evaluated following the procedure reported by Taofiq et al. [15
]. Briefly, the RAW 264.7 macrophage cell line is cultured in DMEM medium and supplemented with 10% heat-inactivated fetal bovine serum, glutamine, and antibiotics at 37 °C under 5% CO2
, in humidified air. For each experiment, cells were detached with a cell scraper to achieve a cell density of 5 × 105
cells/mL. Trypan blue dye exclusion test was used to estimate the proportion of dead cells that were found to be less than 5%. Cells were seeded in 96-well plates at 150,000 cells/well and allowed to stay overnight for proper attachment to the plate. Then, cells were treated with extract solutions (concentration of 400–50 μg/mL, for each extract) for 1h. Dexamethasone (50 μM) was used as a positive control. Then, macrophage cells with the extract were stimulated with LPS (1 μg/mL) for a period of 18 h. The effect of all the tested samples in the absence of LPS was also evaluated, in order to observe if they induced changes in nitric oxide (NO) basal levels. In negative controls, no LPS was added. The extracts and LPS were dissolved in supplemented DMEM.
Each ethanolic extract was dissolved in 50% DMSO at 8 mg/mL and submitted to successive dilutions (400 to 50 μg/mL). Griess reagent System kit which contains sulphanilamide, N-(1-napthyl) ethylenediamine hydrochloride (NED), and nitrite solutions was used to quantify nitric oxide. A reference curve of nitrite (sodium nitrite 100 μM to 1.6 μM; y = 0.0065x + 0.1309; R2 = 0.9988) was prepared in a 96-well plate. The cell culture supernatant (100 μL) was transferred to the plate and mixed with sulphanilamide and NED solutions, 5–10 min each, at room temperature. The produced nitric oxide was determined by measuring the absorbance at 540 nm (microplate reader ELX800 Biotek, Bio-Tek Instruments, Inc.; Winooski, VT, USA), and compared with the standard calibration curve. EC50 values were calculated from the graph reporting the percentage inhibition of NO production versus extract concentration.
3.5. Evaluation of the Anti-Tyrosinase Activity
Each ethanolic extract was dissolved in 50% DMSO at 10 mg/mL and submitted to successive dilutions (10 to 0.625 mg/mL). Tyrosinase inhibition assay was performed using L-DOPA as substrate according to the procedure described by Yoon et al. [36
] with slight modifications. The assays were carried out in a 96-well microplate with each well containing 40 μL of the sample, 80 μL of phosphate buffer (0.1 M, pH 6.8), 40 μL of tyrosinase enzyme (60 units/mL), and 40 μL of l
-DOPA (3.5 mM). The mixture was incubated for 10 min at 37 °C, and the absorbance measured at 475 nm using a microplate reader (ELX800 Bio-Tek Instruments, Inc.). l
-ascorbic acid was used as the positive control and the results were compared with a control comprising 50% DMSO in place of the sample. The percentage of tyrosinase inhibition was calculated as follows: [(Acontrol
] × 100. EC50
values were calculated from the calibration curve tyrosinase inhibition percentage versus extract concentration.
3.6. Evaluation of the Antioxidant Activity
3.6.1. Reducing Power
Each ethanolic extract was dissolved in ethanol at 50 mg/mL and submitted to successive dilution (50 mg/mL to 1.56 mg/mL). The extract solutions with the different concentrations (0.5 mL) were mixed with sodium phosphate buffer (200 mmol/L, pH 6.6, 0.5 mL) and potassium ferricyanide (1% w
, 0.5 mL). The mixture was incubated at 50 °C for 20 min, followed by addition of trichloroacetic acid (10% w
, 0.5 mL). Then, 0.8 mL of the mixture was transferred to a 48-well plate, followed by addition of deionized water (0.8 mL), ferric chloride (0.1% w
, 0.16 mL) and the absorbance measured at 690 nm in the Microplate Reader mentioned above [37
]. The sample concentration providing 0.5 of absorbance (EC50
values) was calculated from the calibration curve absorbance (690 nm) versus extract concentrations. Trolox was used as positive control.
3.6.2. DPPH Scavenging Activity
The prepared ethanolic extracts were dissolved in ethanol at 50 mg/mL and successive dilutions (50 mg/mL to 1.56 mg/mL) were made. For the reaction mixture, 30 μL of each extract solution and 270 μL of methanol containing DPPH radicals (6 × 10−5
mol/L) were added. The mixture was allowed to stand in the darkness for 1 h. The absorbance was further measured at 515 nm in the Microplate Reader mentioned above [37
]. The radical scavenging activity (RSA) was calculated using the equation: %RSA = [(ADPPH
] × 100, where AS
is the absorbance of the sample solution with the sample and ADPPH
is the absorbance of the DPPH solution. The concentrations responsible for 50% of RSA (EC50
values) were calculated from the graphs of RSA percentages versus extract concentrations. Trolox was used as positive control.
3.7. Evaluation of the Antibacterial Activity
3.7.1. Bacteria Strains
As multiresistant bacteria are more problematic due to their resistance to a wide range of antibiotics, clinical isolates were used instead of ATCC bacteria. Therefore, bacteria strains correspond to clinical isolates obtained from patients hospitalized in various departments at the Hospital Center of Trás-os-Montes and Alto Douro (Vila Real, Portugal). Two Gram-negative bacteria (Escherichia coli (isolated from urine), and Pseudomonas aeruginosa (isolated from expectoration)) and three Gram-positive bacteria (Enterococcus faecalis (isolated from urine), methicillin-sensitive Staphylococcus aureus (MSSA) (isolated from wound exudate), and methicillin-resistant Staphylococcus aureus (MRSA) (isolated from expectoration)) were tested. All the tested bacteria were placed to grow in the appropriate fresh medium, 24 h, and kept in the oven at 37 °C before analysis in order to maintain the exponential growth phase.
3.7.2. Characterization of the Antibiotic Susceptibility of the Bacteria Strains
Microorganism identification and susceptibility tests were performed using MicroScan panels (MicroScan®
; Siemens Medical Solutions Diagnostics, West Sacramento, CA, USA) by microdilution plate method. The interpretation criteria were based on Interpretive Breakpoints as indicated in Clinical and Laboratory Standards Institute (CLSI) [38
]; Committee of L’Antibiogramme de la Société Française de Microbiologie (CA-SFM) [39
]; and in the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [40
3.7.3. Determination of the Minimal Inhibitory Concentration (MIC)
Each ethanolic extract was dissolved in 50% DMSO at 220 mg/mL. The determination of the minimal inhibitory concentration (MIC) was performed by the microdilution method and the rapid p
-iodonitrotetrazolium chloride (INT) colorimetric assay according to [41
] with some modifications. Initially, 50 μL of the extract solution in 50% DMSO at 220 mg/mL was added to 450 μL of medium TSB or MHB, according to the bacteria requirements, making a solution of 22 mg/mL at 5% DMSO. Then, 190 μL of this extract solution was added to each well of the 96-well microplate and therefore submitted to successive dilutions over the wells containing 90 μL of MHB or TSB media, starting by taking 100 μL of the higher concentration to the right below one and thus repeating this procedure sequentially. Afterwards, 10 μL of inoculum (1.5 × 108
CFU/mL) was added to all the microplate wells thus achieving a concentration range of 20 to 0.156 mg/mL. Three negative controls were prepared (one with MHB/TSB 5% DMSO, another one with the extract, and the third one with medium and antibiotic). For the Gram negative bacteria, colistin was used at a concentration based on the obtained MIC (Table S1, Supplementary Materials
); for the Gram positive bacteria, vancomycin was used (Table S2, Supplementary Materials
). A positive control was also performed with MHB/TSB medium at 5% DMSO and the inoculum. The plates were then incubated at 37 °C, for 24 h, in an oven (Jouan, Berlin, Germany). The MIC of the samples was determined after adding INT (0.2 mg/mL, 40 μL) and after incubation at 37 °C for 30 min. Viable microorganisms reduced the yellow dye to a pink color. MIC was defined as the lowest extract concentration that prevented this change, thus showing complete inhibition of bacterial growth.
3.8. Development of the Cosmeceutical Formulations Based on Mushroom Extracts
The cosmetic cream used in this study is a commercial base cream purchased from Fagron Iberica S.A.U. (Barcelona, Spain). It is described as a bright, white to yellowish color; free from fragrances, colorants, parabens, mineral oils, sodium lauryl sulphate (SLS), propylene glycol, and ethoxylates; and composed of purified water, O/W emulsifiers, emollient, lubricant, pro-liposome, vitamin E, chelating agents, silicone, and preservatives. According to the supplied technical data, all ingredients are recognized as safe by the US Food and Drug Administration (FDA), Regulation (EC) No 1907/2006—REACH and the National Health Surveillance Agency (ANVISA) of Brazil. Considering the MIC values for the antibacterial activity, the highest ones were observed for A. bisporus and P. ostreatus, (~10 mg/mL). For L. edodes, the highest EC50 value was found for the antioxidant activity (~25 mg/mL). In order to cover all the bioactivities and considering the dilutions necessary to carry out the assays, the base creams were incorporated with 10 × MIC value for A. bisporus and P. ostreatus and 10 × EC25 value for L. edodes, which correspond to a scale of 100 mg of extract per gram of base cream. The base-cream and the extract were carefully mixed to guarantee sample homogeneity and analyzed immediately after incorporation to study the ability of the extracts to maintain their bioactivities in the cosmetic base cream.
The final cream formulations were extracted with methanol for 30 min, filtered, dried in a rotary evaporator, and re-dissolved in a water:methanol mixture (80:20, v/v) at 20 mg/mL for phenolic acids analysis; in methanol at 10 mg/mL for ergosterol analysis; in 50% DMSO at 200 mg/mL for anti-inflammatory activity evaluation; in 50% DMSO at 200 mg/mL for anti-tyrosinase activity evaluation; in 100% DMSO at 200 mg/mL for antioxidant activity evaluation; and in 5% DMSO at 220 mg/mL for antibacterial activity evaluation.
3.9. Statistical Analysis
For all the experiments, three independent samples were prepared and all the assays were carried out in triplicate. The results are expressed as mean values ± standard deviation (SD). The differences between the samples were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s honestly significant difference post hoc test with α = 0.05, coupled with Welch’s statistic. This treatment was carried out using IBM SPSS Statistics for Windows version 23.0 program (IBM Corp., Armonk, NY, USA).