Bioactive Bacterial Nanocellulose Membranes Enriched With Eucalyptus Globulus Labill. Leaves Aqueous Extract For Anti-Aging Skin Care Applications

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Abstract: Bacterial nanocellulose (BNC) membranes, with remarkable physical and mechanical properties, emerged as a versatile biopolymeric carrier of bioactive compounds for skin care applications. In this study, BNCmembranes were loaded with glycerol (as plasticizer and humectant agent) and different doses(1-3 ug cm-2)of an aqueous extract obtained from the hydro-distillation of Eucalyptus globulus Labill. leaves (HDE), for application as sheet facial masks. All membranes are resistant and highly malleable in dry and wet states, with similar or even better mechanical properties than those of a commercial BNC mask. Moreover, the HDE was found to confer a dose-dependent antioxidant activity to pure BNC. Additionally, upon 3months of storage at 22-25°C and 52% relative humidity (RH) or at 40C and 75% RH, it was confirmed that the antioxidant activity and the macroscopic aspect of the membrane with 2 ug cm-2 of HDE were maintained. Membranes were also shown to be non-cytotoxic toward HaCaT and NIH/3T3 cells, and the membrane with 2ug cm-2 of HDE caused a significant reduction in the senescence-associated β-galactosidase activity in NIH/3T3 cells. These findings suggest the suitability and potential of the obtained membranes as bioactive facial masks for anti-aging applications.

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Keywords: bacterial nanocellulose; Eucalyptus globulus Labill. leaves; aqueous extract; antioxidant activity; sheet facial masks; anti-aging; skin care applications

1. Introduction

The world population is considerably aged due to the decline in birth rate and the improvement in survival, and consequent increase in the average life expectancy, associated with the medical and technological developments achieved over the last decades [1,2]. The global population aged 60 or above is expected to reach about 2.1 billion by 2050, representing an increase of approximately twofold from 2017, and the number of people aged 80 or over is expected to nearly triple in the same period 3]. However, despite improved longevity, the aging of the skin is an inevitable process, and therefore, growing demand for anti-aging skin care products is expected.

Skin aging is a complex biological process combining endogenous and exogenous mechanisms [4]. Endogenous aging is mainly determined by genetic factors and hormonal changes that occur with the normal aging process [4]. On the other hand, exogenous aging is mediated by external factors (e.g., overexposure to ultraviolet radiation (photoaging), gravity, smoking, pollution, and poor nutrition)[5]. In both aging processes, but particularly the exogenous one, it is well accepted that the increased oxidative stress, induced by reactive oxygen species (ROS) and resulting from the unbalance between ROS production and antioxidant defense, plays a crucial role [6-8]. With age, there is also a decrease in the ability of human skin cells to repair damaged DNA, which contributes to skin aging as well [8]. Cistanche Extract Anti Radiation Thus, the aging process causes several biochemical alterations in skin composition, with effects on its structure and function [9], resulting in visible signs like wrinkling, dryness, loss of elasticity, thinning, rough texture, or irregular pigmentation [7,10]. Hence, with the growing pursuit for health and well-being, in which skin health and aesthetics are also included, the development of cosmetics incorporating bioactive compounds to prevent or attenuate skin aging and its external signs, thus improving skin appearance, has been at the forefront of research and innovation in the cosmetic industry over the last decades. In this context, and among cosmetics for topical treatment, beauty masks, and particularly sheet facial masks, are a growing market, with an expected compound annual growth rate(CAGR) of 8.1% from 2019 to 2027 [11]. This type of mask is especially attractive to modern consumers, largely due to its simple and easy application, fast use, and effectiveness]12. Several materials can be used as the support matrix of these sheet masks, namely synthetic polymers (e.g., poly(vinyl alcohol) and silicone, fabrics (e.g., non-woven and cotton), hydrogels(e.g., collagen- and silk-based hydrogels)or cellulose nanofibers, such as bacterial nanocellulose(BNC)[13]. With the increasing demand for naturally derived skin care products, BNC has gained relevance and now stands as a superior bio-based material, already explored, and commercialized by some of the top cosmetics companies (e.g., Lancome, Elizabeth Arden, and DHC)[14] that are following the trend of green-oriented consumers' demand.

Bacterial nanocellulose is an extracellular polysaccharide produced by several non-pathogenic bacteria from different genera (e.g., Komagataeibacter (formerly Gluconacetobacter), Agrobacterium, Rhizobium, Escherichia, and Aerobacter), through fermentation in the presence of oxygen and carbon sources(e.g., glucose)[1516]. BNC is synthesized in the air-culture medium interface as an ultrafine 3D nanofiber network, showing high water content(>90%), high hydrophilicity, and a nanoporous structure [17]. When produced in static culture, BNC is obtained as a gelatinous hydrogel-like membrane with variable thickness and the shape of the culture vessel (viz. in situ moldability), which is an advantage when a predefined shape is desired [18]. The set of unique properties of BNC also embraces high crystallinity and purity, remarkable mechanical strength, and thermal stability [19]. Besides being biodegradable, BNC has also demonstrated good skin tolerance and biocompatibility in several in vivo studies [20-22], making it an ideal material for skin care products. In this regard, and because of its porous nanostructure, the applicability and effectiveness of BNC as a carrier for the delivery of skin active compounds have been the focus of several works, for instance, for the delivery of caffeine [23], silk-sericin [24], retinol [25], or rutin [26]. For further reading, a recent review provides a comprehensive overview of the versatility and applicability of BNC in green cosmetics, including as a carrier of skin active substances [27].

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Natural bioactive compounds are widely used in anti-aging skin care cosmetics [28]. cistanche herba Plant extracts, in particular, are a rich source of bioactive compounds (e.g., polyphenols, terpenoids, vitamins, among others) with potential multiple actions on the skin, with antioxidant and antimicrobial activities of tyrosinase inhibition effects being the main benefits reported so far [29]. Therefore, given the current trend for natural products in cosmetics, plant extracts appear among the most promising ingredients for the development of skincare products. Moreover, the incorporation of natural extracts into BNC membranes has also been investigated for cosmetic purposes. Examples are the works reporting the incorporation of propolis extract, known to have antiseptic and astringent activities[13,30], extracts from oat and rosemary with moisturizing effects [13], or the cosmetic formulation composed of Adansonia digitata fruit, Hibiscus sabdariffa flower, Coffea arabica seedcake, Kigelia Africana fruit, and Acacia and Crocus chrysanthus bulb extracts, with anti-aging properties, namely wrinkle-smoothing [21]. Recently, a review made a critical insight into the importance of associating BNC with plant phenolics to prevent UV-induced skin damage [31].

Eucalyptus globulus Labill. the evergreen tree is a well-known source of bioactive compounds, namely phenolic compounds such as phenolic acids, flavonoids, or hydrolyzable tannins [32-36]. The antioxidant and antimicrobial activities of E.globulus extracts containing these compounds have been demonstrated in several studies [34,35]. Concerning application in the cosmetic field, a recent study demonstrated the protective effect, both in vitro (in human dermal fibroblasts) and in vivo (hairless mice), of a 50% ethanolic extract from dried commercial E.globulus biomass against UV-induced photoaging, with promising results regarding the prevention of wrinkle formation and skin dryness [37]. However, to the best of our knowledge, E.globulus extracts have never been explored, in combination with a polymeric substrate, to produce bioactive skin masks for anti-aging skin care.

With this perspective, the purpose of the present work was to produce BNC mem-branes loaded with an aqueous extract derived from the hydro-distillation (hydro-distillation extract, HDE) of E.globulus leaves. Hydro-distillation is a method commonly used by the industry for the extraction of essential oils [38]. In addition, glycerol (G), a widely used humectant ingredient in cosmetics [39], was also incorporated into the BNC membranes (7.5 mg cm-2) to increase their flexibility and, at the same time, their conformability to the skin. Thus, the goal of this work was to obtain an entirely bio-based material with functional properties for potential application as an anti-aging sheet facial mask. BNC membranes loaded with different doses of the hydro-distillation extract were characterized in terms of their morphology, mechanical properties, thermal stability, and moisture-uptake capacity. Moreover, the chemical antioxidant activity of the different membranes was also evaluated, as well as their in vitro cytotoxicity (in human keratinocytes and mouse fibroblasts cell lines). Additionally, the antioxidant activity stability after 3 months of storage, in the dark, at 22-25°Cand 52% relative humidity (RH) or at 40°C and 75% RH of the membrane with 2 ug cm-2 of HDE was assessed. Finally, the in vitro anti-senescence activity of the BNC membrane with 2 μg cm-2 of HDE was further investigated.

2. Materials and Methods

2.1. Chemicals and Materials

Citric acid (≥99.5%), dimethyl sulfoxide (DMSO) (≥99.0%), disodium hydrogen phosphate(≥99.0%), glucose(≥99.5%), glycerol(≥99.5%), magnesium nitrate hexahydrate (≥99.0%), potassium chloride(≥99.0%), potassium dihydrogen phosphate (≥99.0%), potassium sulphate (>99.0%), sodium bicarbonate (>99.5%), sodium chloride (>99.0%),and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)(≥97.5%)were purchased from Sigma-Aldrich(Lisbon, Portugal). All other chemicals were of laboratory grade. cistanche penis growth Calcium chloride, Dulbecco's Modified Eagle's Medium (DMEM), etoposide, Folin-Ciocalteu's phenol reagent, magnesium chloride, sodium pyruvate, trypsin-EDTA solution, N-(2-hydroxyethyl)piperazine-N'-(2-ethane sulfonic acid),4-(2-hydroxyethyl piperazine-1-ethane sulfonic acid (HEPES), and 2,2-diphenyl-1-picrylhydrazyl(DPPH) were supplied by Sigma-Aldrich (Lisbon, Portugal). Peptone and yeast extract were acquired from Himedia Laboratories GmbH (Einhausen, Germany). Fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Gibco (Carlsbad, CA, USA).

Samples from a BNC commercial sheet facial mask were used in the mechanical and moisture uptake assays, for comparison purposes. The BNC commercial sheet facial mask (Superstart Probiotic Boost Skin Renewal Biocellulose Mask, Elizabeth Arden, New York, NY, USA)is described as being derived from natural coconut water and embedded in a cosmetic formulation with skin renewal properties containing, among other ingredients, probiotics(Lactococcus ferment lysate, Lactobacillus), plant extracts(Althea rose flower extract, Polymnia sonchifolia root juice, Crithmum maritimum extract), caffein, and humectants, such as glycerol, hyaluronic acid, or polyethylene glycol(PEG)-450. Samples of the BNC-commercial sheet were dried in a ventilated oven at 358C before use. cistanche salsa benefits Fresh E.globulus Labill. leaves were collected from adult trees from an industrial plantation of The Navigator Company near Aveiro at Sever do Vouga (Bracal)(Aveiro, Portugal) and submitted to a hydro-distillation process until complete extraction of the essential oil fraction(2-3 h) using a modified Clevenger-type apparatus. In the end, an aqueous extract, the HDE, was recovered and lyophilized, stored, and protected from light in a desiccator, at room temperature, until use [40].

2.2. Total Phenolic Content of HDE

Total phenolic content (TPC)of HDE was determined using the Folin-Ciocalteu method, as described elsewhere [41], with some modifications. Briefly, in a 96-well plate, 150 μL of Folin-Ciocalteu reagent previously diluted（1∶10， o/ø） with water and 120 L of aqueous sodium carbonate solution （75 g L-）were added to 30 μL of an aqueous HDE solution in concentrations ranging from 1.95 ugs to 500 μg mL-1 of extract. The reaction mixtures were incubated in the dark at room temperature for 60 min. After the incubation period, the absorbance was measured at 760 nm against a blank (water instead of extract) in a Thermo Scientific MultiskanTM FC microplate reader (Thermo Fisher Scientific Inc., Waltham, MA, USA). TPC was calculated as gallic acid equivalents(GAE) from the calibration curve of gallic acid standard solutions (5.13-205.0 ug mL-1) and expressed as mg of GAE of lyophilized extract. The assay was performed five times, and each sample was analyzed in triplicate.

2.3. Production of the BNC Membranes

BNC membranes were produced in our laboratory using Hestrin-Schramm (HS)liquid culture medium (20g L-1glucose,5g L-1 peptone,5g L-l yeast extract,2.7g L-1 disodium hydrogen phosphate, 1.15 g L-Citric acid, pH5) inoculated with the acetic acid bacteria Gluconacetobacter sacchari under static culture conditions [42]. After 4-6 days of culture at 30°C, the BNC membranes were harvested and treated twice with 0.5 M NaOH at 80°C for 30 min. Afterward, membranes were washed several times with distilled water to eliminate the remaining medium components and bacterial cells. Finally, BNC membranes were whitened with a 1% sodium hypochlorite aqueous solution followed by repeated washes with distilled water until neutral pH was reached. Purified membranes were stored in ultrapure water at +4°C until use.

2.4. Preparation of the BNC-G-HDE Membranes

Wet BNCmembranes （diameter∶ ca.7.0±0.5 cm; thickness∶ 7000± 1000 μm）were loaded with different doses of HDE (expressed as mass of HDE per surface area of the membrane) and glycerol (7.5 mg cm-2)(Table 1), using the impregnation method. Succinctly, wet BNC membranes were weighted (about 180 mg of dry BNC) and drained by hand-pressing with laboratory-grade absorbent paper until water content decreased to nearly 40%(estimated by weight loss). Then, membranes were soaked in 5 mL of an aqueous solution containing the respective doses of both HDE and glycerol and maintained at room temperature until complete absorption of the HDE-G solution. Finally, BNC membranes were dried (in Petri dishes) at 35 °C in a ventilated oven (Venticell Eco line, MMM group, Planegg, Germany) for at least 20 h. Dried membranes were stored and protected from the light in a desiccator at room temperature until use. For comparison purposes, pure BNC membranes (without the extract and glycerol) were dried as previously described, and membranes loaded with glycerol (BNC-G) were prepared following the same procedure.

2.5. Characterization of Membranes

2.5.1.Thickness

The thickness of dry membranes was measured in five random sites using a hand-held digital micrometer（Mitutoyo Corporation， Tokyo， Japan） with an accuracy of 1 μm

2.5.2. Morphology

The surface and cross-section (cryo-fractured) morphologies of the membranes were analyzed by scanning electron microscopy(SEM). Micrographs were obtained using a high-voltage microscope (HR-FESEMSU 70 Hitachi, Tokyo, Japan) operated at 4.0 kV. Prior to image acquisition, samples were placed on steel support and coated with carbon.

2.5.3. Mechanical Performance

The mechanical performance of membranes in dry and wet (80% moisture) states were evaluated by tensile tests. The moisture content of wet membranes was defined according to the moisture content determined in the commercial BNC. Tensile tests were performed on a uniaxial Instron 5564 testing machine (Instron Corporation, Norwood, MA, USA) in the traction mode at a crosshead velocity of 10 mm min-I using a 500 N static load cell. All measurements were conducted in at least five replicates using rectangular test specimens （5×1 cm²） and a gauge length of 30 mm. Stress （MPa）and strain （%） curves were plotted， and Young's modulus, tensile strength, and the elongation at break were determined using Instron BlueHill 3 software. In these assays, specimens of a commercial BNC facial mask were also tested in the same conditions, for comparison.

2.5.4. Thermal Stability

Thermogravimetric analysis (TGA) was carried out with a SETSYS Setaram TGA analyzer (SITARAM Instrumentation, Lyon, France) equipped with a platinum cell. The samples were heated from room temperature to 800C at a constant rate of 10 °C min-1 under a nitrogen atmosphere.

2.5.5. Moisture Uptake Capacity

The moisture uptake capacity of all membranes was evaluated by placing dried specimens (2 ×2 cm-)of each membrane in a desiccator, at room temperature, with relative humidity at ca. 52% using a saturated magnesium nitrate aqueous solution (52.89 ± 0.22% at 25°C)[43]. The specimens were taken from the desiccator and weighed after 0.5 h,1 h,2.5 h, 24 h, and 48 h. All membranes were tested in triplicate. Moisture uptake was calculated as follows:

where Whois the specimen's initial weight, and Wwis the weight at each time point.

2.6. In Chermico Antioxidant Activity

The antioxidant activity of the HDE-loaded membranes was estimated using the DPPH free radical scavenging method, following as previously reported procedure [44], with some modifications. The reaction is based on the decrease in the absorbance of the DPPH solution resulting from the radical scavenging by the antioxidant compounds, with the consequent change in color of the DPPH solution from purple to pale yellow [45]. Briefly, specimens （2 ×5 cm²）of each membrane were added to3.75 mL of ethanol（EtOH） and phosphate-buffered saline （PBS）solution（60∶40; pH5.5）. Then， 250 μL of DPPH（1 mM）solution in ethanol was added, and the resulting mixtures were incubated in the dark with gentle mixing (100rpm) at 22°C for 0.5 h,1 h, and 2.5h, and then the absorbance at 517nm was read against the blank (EtOH: PBS1× pH5.5)using a MultiskanTM FC microplate reader (Thermo Scientific, Waltham, Massachusets, EUA). For comparison, reaction mixtures containing the HDE diluted in EtOH: PBS1×pH5.5 at a final concentration equivalent to the maximum quantity of HDE that could be released from each membrane (HDE 1:2.5 μg mL-1; HDE1.5∶3.75 μg mL-1; HDE 2∶5.0 μg mL-1; HDE 3∶7.5 μg mL-）were also included in the assay. A control consisting of EtOH: PBS 1× pH5.5 with DPPH and without membrane was used. Three independent assays were carried out for each sample. The DPPH radical scavenging activity was calculated from the absorbance of each sample (A sample)with respect to the control DPPH absorbance (ApppH) as follows:

2.7. Evaluation of the Stability of the BNC-G-HDE2 Membrane under Storage

Based on the"Guidelines on stability testing for cosmetic products"[46], the storage stability of BNC-G-HDE2 was evaluated. For this， samples（14 cm²） of BNC-G（as control）and BNC-G-HDE2 membranes were placed in glass vials and stored for 3 months in the dark at the expected normal storage condition, at room temperature(22-25°C) and 52% RH (a condition I, using a magnesium nitrate saturated solution [43]) and at the accelerated condition of 40℃C and 75% RH (a condition I, using a sodium chloride saturated solution [43]). The relative humidity was periodically monitored using a thermohydrometer to ensure constant humidity during the entire storage period. All samples were analyzed before and after 1,2, and 3 months of storage regarding their antioxidant activity(as described in Section 2.6). The macroscopic aspect of membranes was also recorded. For each condition, all samples were analyzed in triplicate.

2.8. In Vitro Biological Assays

2.8.1. Cell Culture

Human keratinocytes(HaCaT, from CLS, Cell Lines Service, Eppelheim, Germany)and mouse fibroblasts(NIH/3T3, ATCC CRL-1658, Manassas, VA, USA) cell lines were cultured using Dulbecco's Modified Eagle's Medium (DMEM), at 37°C, in a humidified5%CO2-95% air atmosphere. The medium was supplemented with 10%(o/v) heat-inactivated fetal bovine serum（FBS）， 1%（o/ø）pen/strep，3.7 g L-Sodium bicarbonate， and 1 mM sodium pyruvate. Cells were detached with trypsin-EDTA solution 1×.

2.8.2. Cytotoxicity Assay

The cytotoxicity of the membranes was evaluated using an MTT reduction assay. Samples （2.5 cm²） of the membranes （BNC-G， BNC-G-HDE1， BNC-G-HDE1.5， BNC-G-HDE2， BNC-G-HDE3)were sterilized twice by UV radiation on each side during 20 min and further incubated, for 24 h, in 2.5 mL of complete DMEM medium at 37°C in a humidified 5%CO,-95% air atmosphere, to prepare each membrane extract.

Meanwhile,2×10 and 1×104 cells/well were seeded in 96-well plates for HaCaT and NIH/3T3 cells, respectively. After 24 h, the same volume of the membrane extract was used to replace the culture medium, and cells were further incubated for 24 h at 37°C. As a control, cells were treated in the same way as described for samples but exposed only to DMEM medium. After 24 h, the medium was removed, and the cytotoxicity was determined as previously described [47]. Briefly, a fresh solution of MTT(0.5 mg L-1)prepared in Krebs medium (pH 7.4) was added and incubated at 37°C for 2h (HaCaT cells) or 4h (NIH/3T3 cells). After that, the MTT solution was replaced by DMSO and incubated for l0 min, with shaking, to completely dissolve the formazan crystals. After incubation, the absorbance was measured at 570 nm in a spectrophotometer (SLT spectra I). The results of 3 independent experiments, with 3 replicates each, were expressed as a percentage (%) of the absorbance value obtained in control and graphically presented as % of MTT reduction.

2.8.3. Anti-Senescence Activity

The anti-senescence activity of the BNC-G-HDE2 membrane was evaluated using the β-galactosidase(β-gal) staining assay. The BNC-G membrane was used as a control. To prepare the membrane extracts, sterilized samples (10 cm-)were incubated in 10 mL of complete DMEM medium as previously described for the cytotoxicity assay.

NIH/3T3 cells were seeded in 12-well plates at a density of 2.5× 104 cells/well and allowed to stabilize for 24 h. Subsequently， 12.5 μM etoposide was used to induce cellular senescence in NIH/3T3 for 24 h. Etoposide-stimulated cells were then treated with the BNC-G-HDE2 membrane extract for another 24 h. cistanche tubulosa dosage reddit As a control, non-treated cells were incubated with DMEM medium or with extracts of BNC-G and BNC-G-HDE membranes. After incubation, the culture medium was removed, and cells were washed with PBS and marked with β-gal solution prepared as described by the manufacturer(Cell Signaling Technology, Danvers, MA, USA). The analysis of the positive cells for senescence was performed at 20×magnification using a widefield microscope(Carl Zeiss, Oberkochen, Germany). At least 3 independent experiments were performed in replicate, and the percentage of β-gal-positive cells was determined using four microscopic images.

2.9. Statistical Analysis

In the treatment of the results from the characterization, antioxidant activity, and storage stability test of the membranes, a one-way analysis of variance(ANOVA) followed by Tukey's test was used to assess the level of significance. The results are expressed as the mean ±standard deviation of the mean. Regarding in vitro assays, the normality of the data distribution was assessed by the D'Agostino-Pearson and Shapiro-Wilk normality tests. Statistical comparisons between groups were performed by ANOVA followed by Dunnett's post-hoc test or unpaired Students' t-test. The results are presented as the mean ± standard error of the mean of the indicated number of experiments.

Significance was accepted at values of p<0.05. All statistical calculations were performed using GraphPad Prism software(8.0.2, GraphPad Software Inc., San Diego, CA, USA).

This article is extracted from Materials 2022, 15, 1982. https://doi.org/10.3390/ma15051982 https://www.mdpi.com/journal/materials