Cornus Mas L. Extract As a Multifunctional Material for Manufacturing Cosmetic Emulsions

Chinese Journal of

Natural Chinese Journal of Natural Medicines 2018, 16(4): 02840292 Medicines

doi: 10.3724/SP.J.1009.2018.00284

Cornus mas L. extract as a multifunctional material for manufacturing cosmetic emulsions

Nizioł-Łukaszewska Z1, Wasilewski T2, Bujak T1, Gaweł-Bęben K1, Osika P1*, Czerwonka D2

1 The University of Information Technology and Management in Rzeszow, Department of Cosmetology, Tyczyn 36020, Poland; 2 The University of Technology and Humanities in Radom, Department of Chemistry, Radom 26600, Poland Available online 20 Apr., 2018

[ABSTRACT] Dogwood fruits are a valuable source of active ingredients, such as phenolic compounds, vitamin C, iridoids, flavonoids and anthocyanins. Plant extracts and substances derived from latin Cornus mas L. exhibit not only strong antibacterial but also antioxidant and tonicity properties, effectively preventing the development of inflammation in living organisms. In the present study, we attempted to obtain the innovative, multi-functional plant extract from the fruit of dogwood (Cornus mas L.). During the extraction process a mixture of water, glycerol, and vegetable oil were used as an extractant. The usage of such mixtures of solvents enabled us to extract a variety of active substances, soluble in both water and oils. The obtained extracts were analyzed for their physicochemical and biochemical properties, in order to apply the extract in a body nutrient lotion. The results clearly showed that such extract could be an innovative and multi-functional raw material used in cosmetics industry. [KEY WORDS] Cornus mas; Extract; Antioxidant activity; Emulsions; Formulation/stability [CLC Number] R944 [Document code] A [Article ID] 2095-6975(2018)04-0284-09

crease the amount of waste (packaging) generated in manu- Introduction facturing plants. Manufacturers look for multifunctional cos- From the physicochemical point of view, skin care cosmetic ingredients primarily in materials of natural origin, e.g., metics are available mainly in the form of water in oil (W/O) plant extracts. Their complex chemical compositions make it or oil in water (O/W) emulsions. They contain approximately possible to obtain cosmetics which deliver multi-faceted ac- 70%−80% of water, 10%−30% of hydrophobic substances tivity (e.g. moisturizing, soothing, and nourishing effects) and a range of additives such as emulsifiers, nutrients and using just one ingredient. Moreover, through an appropriate moisturizing agents, preservatives, and fragrances. Cosmetic selection of extraction solvents, formulators are able to obtain emulsions for facial care exhibit a high viscosity (creams). In specific groups of chemical substances, e.g. moisturizing or contrast, body care emulsions are less viscous and, as a result, soothing agents, and antioxidants [1-2]. cosmetics (balms and lotions) flow easily inside their pack- The present study attempted to obtain an innovative mul- aging. This property allows for their problem-free dispensa- tifunctional extract of dogwood (Cornus mas) berries. Multi- [1] tion from the packaging and application to the skin . ple studies have shown that extracts and plant substances Recent years cosmetic manufacturers have searched for derived from Cornus mas demonstrate potent antioxidant, multifunctional materials in order to minimize costs and ob- antibacterial, toning and astringent effects, and effectively tain products with a wide spectrum of effects. With such solu- prevent inflammations in living organisms [3-6]. In addition, tions, it is possible to reduce the number of materials that their health-promoting properties are largely attributable to need to be purchased for the process, and consequently, de- phenolic compounds, vitamin C, iridoids, flavonoids and, in particular, anthocyanins [7-9]. The amount of these compounds  [Received on] 19-May-2017 in dogwood berries considerably exceed the content of an- [Research funding] This work was financially supported by The thocyanins found in other fruit [10-11]. The main anthocyanins University of Information Technology and Management in Rzeszow include delphinidin-3-galactoside, cyanidin 3-galactoside and as part of the project “Plant extracts as an innovative, multi-functional [12] cosmetic raw materials”. pelargonidin-3-galactoside. According to reference , the [*Corresponding author] E-mail: [email protected] most abundant anthocyanin is cyanidin 3-galactoside. Dog- These authors have no conflict of interest to declare. wood berries contain also a significant amount of phenolic

– 284 – Nizioł-Łukaszewska Z, et al. / Chin J Nat Med, 2018, 16(4): 284292 acids such as elagic acid, p-cumaroylhhexoside, and chloro- and after 6 min in the dark, 1 200 µL of sodium carbonate genic acid and bioactive compounds from the family of fla- (7.5%) was added. The samples were incubated in the dark vonoles: quercitine-3-O-glucuronide, kaempferol-3-O-gala- for 2 h and theabsorbance at 740 nm was then measured (He- ctoside, which are able to modify the activity of some en- lios Aquamate by Thermo Scientific). zymes. Iridoids such as loganic acid or cornuside, hydrolyz- The total phenolic concentration was calculated from a −1 able tannins, and elagic and gallic acid derivatives are the Gallic acid (GA) calibration curve (10−100 mg·L ). The data main anti-inflammatory and antimicrobial agents identified in were expressed as mg of Gallic acid equivalents (GA) in 1 L dogwood fruit. The research also demonstrated that fresh of extract, averaged from 3 measurements. dogwood berries are rich in vitamin C. The vitamin C content Determination of flavonoid content is twice as high as in oranges [13] and seven times higher than The total flavonoid content was determined spectropho- [18-19] in blueberries [14]. Active substances contained in dogwood tometrically by the procedure reported with modifica- berries have a surprisingly beneficial effect on human skin and tion using aluminum nitrate nanohydrate. To prepare a sample, can successfully replace synthetic substances [15]. Research 600 µL of an appropriate extract or solvent was mixed with conducted by Sultana and Lee [16] has revealed that extracts 2 400 µL of reaction mixture (80% C2H5OH, 10% Al(NO3)3 × −1 derived from dogwood berries possess a powerful antioxidant 9 H2O, 1 mol·L C2H3KO2). After 40 min of incubation at room temperature the absorbance was measured at 740 nm activity and inhibit the enzyme elastase which is responsible using an Helios Aquamate by Thermo Scientific spectropho- for the degradation of elastin. The inhibition of elastin degra- tometer. dation also contributes to delaying the ageing processes. Con- The flavonoid content was calculated from a quercetin sequently, dogwood extracts can be used as an innovative ingre- hydrate calibration curve (10−100 mg·L−1) and expressed as dient of cosmetic products for face and body care as well. mg of quercetin equivalents (QU) in 1 L of extract, averaged The study reported here made an attempt to determine from 3 measurements. what type of dogwood extract allowed obtaining a cosmetic DPPH• radical scavenging activity assay with a broad spectrum of effects. The extraction process was The DPPH• radical scavenging activity by dogwood fruit conducted using a mixture of water, glycerin, and a plant oil extracts was performed as previously reported [20]. 1 mL of as an extraction solvent. This mixture of solvents makes it extract or appropriate solvent was mixed with 1 mL of possible to obtain a number of active substances, both water- 25 mmol·L−1 DPPH• solution in 96% ethanol. After 40 min of and fat-soluble, from dogwood berries. After completing the incubation at room temperature, the absorbance of the sample extraction process the mixture was separated, yielding wa- was measured at λ = 515 nm using AquaMate spectropho- ter-glycerin and oil extracts. The extracts were subsequently tometer (Thermo Scientific). 96% of ethanol was used as a subjected to an analysis of physicochemical and biochemical blank sample. All the samples were analyzed in triplicate. The properties. Next, the extract was utilized into a formulation of percentage of DPPH• scavenging was calculated for each a nourishing body balm. The effects of the extract on the sample based on the following equation: properties of the finished cosmetic was also determined. • % of DPPH scavenging = [1 – (As/Ac)] × 100

Material and Methods where As is absorbance of the sample and Ac is absorbance of the control sample (DPPH• solution). Extract derivation method ABTS•+ radical scavenging activity assay Following the removal of seeds, the plant material, The scavenging of ABTS•+ free radical was evaluated Cornus mas Bolestraszycki, was dried at 40 oC until constant using a modified protocol [21-22]. The scavenging reaction is weight was achieved. A total of 5 g of dogwood was reduced based on discoloration of the green ABTS radical cation in size and agitated with 100 g of water, glycerin, and sun- (ABTS•+). To prepare the ABTS•+ solution, 19.5 mg of ABTS flower oil mixed at the ratio of 25 : 25 : 50, W/W. The extrac- and 3.3 mg of potassium persulfate were mixed with 7 mL of tion process was conducted for 24 h without any access of light, phosphate buffer pH = 7.4 and dissolved for 16 h in darkness. and the mixtures were stirred at 300 r·min−1 with mechanical stirrer (ChemLand OS20-PRO, screw propeller stirrer shaft). The solution was diluted to reach the absorbance at λ = 414 Next, the extracts were decanted and filtered under re- nm around 1.0. 20 µL of dogwood extract or appropriate sol- •+ duced pressure through filter paper. Consequently, the ob- vent was mixed with 980 µL diluted ABTS solution and in- •+ tained extracts were separated to water, glycerin, and oil phases cubated for 10 min. The decrease in ABTS absorbance was in a separatory funnel for 24 h. The extracts were stored in measured at λ = 414 nm using AquaMate spectrophotometer dark glass bottles at 4 °C until use. (Thermo Scientific), alongside with distilled water as a blank Determination of total phenolic content control. All the samples were analyzed in triplicate. The per- The determination of total phenolic content in extracts centage of ABTS•+ scavenging calculations were based on the was performed spectrophotometrically by the Folin-Ciocalteu following equation: [17] •+ method . Briefly, 300 µL of analyzed extracts or solvents % of ABTS scavenging = [(1 – (As/Ac)] × 100 were mixed with 1 500 µL of 1 : 10 Folin-Ciocalteu reagent where As is absorbance of the sample and Ac is absorbance of

– 285 – Nizioł-Łukaszewska Z, et al. / Chin J Nat Med, 2018, 16(4): 284292 the control sample (ABTS•+ solution). Oil fractions were diluted in DPBS containing 1% (W/V) of Determnation of irritation potential of Cornus mas fractions lecithin (Ecospa) as an emulsifier. Following treatment the in vitro cell culture cells were washed with 150 µL of DPBS and incubated with 100

Normal human skin fibroblast cell line BJ CRL-2522 was µL of acidified ethanol (50% C2H5OH, 1% CH3COOH, 49% purchased from the American Type Culture Collection (ATCC, H2O) for 5 min at room temperature, on a rotating platform. LGC Standards). The cells were maintained in Eagle’s Mini- The absorbance was measured at λ = 540 nm using Filtes- mum Essential Medium (EMEM, Sigma-Aldrich) supple- Max F5 Multi-Mode microplate reader (Molecular Devices). mented with 10% foetal bovine serum (FBS, EURx) at 37 °C The mean optical density of the control cells was set to in a humidified incubator with 5% CO2. 100% viability and used to calculate the percentage of vi- Neutral Red Release Irritation Assay able cells following extract treatment. The experiment was 1 × 104 of BJ CRL-2522 cells were plated per well onto performed twice with 5 replicates for each of the experi- 96-well plates and grown for 24 h in EMEM supplemented mental conditions. Significant differences between obtained with 10% of FBS. The cells were incubated with 100 µL of values were analyzed using One-way ANOVA and Tukey’s EMEM containing 1% of FBS and 33 µg·mL−1 of neutral red test, with significance level of 95%. (Sigma-Aldrich) for 3 h. The cells were then washed once Preparation of prototypes of nourishing body balms with 150 µL of DPBS and treated with 100 µL of diluted A prototype of a nourishing body balm was designed and dogwood extract fractions at the concentrations (V/V) of 50%, formulated with a composition containing water-glycerin and 25% and 10%, respectively, for 15 min. The control cells oil extracts of dogwood. The product was then compared to were treated with equal volume of the used diluent. Wa- an equivalent formulation which, instead of extracts, con- ter-glycerin fractions of Cornus mas extract were diluted in tained a mixture of water and glycerin, as well as sunflower Dulbecco’s Phosphate Buffered Saline (DPBS, Sigma-Aldrich). oil. The balm formulations are presented in Table 1.

Table 1 Formulation tested in the present study Body care emulsion with Cornus Mas extract Standard body care emulsion Phase Ingredient [wt, %] [wt, %] Oil Cornus mas extract 10 - A Heliantus Annuus seed oil - 10 Sorbitan monolaurate 1.5 1.5 Glycerin-water Cornus mas extract 10 - Glycerin - 5 B Water 25 30 Polyglyceryl-4 laurate/succinate and Aqua 2.5 2.5 Xanthan Gum 0.8 0.8 C Water 49.2 49.2 D Preservative 1.0 1.0

The products compositions were selected in a way to en- (using an IKA homogenizer). As soon as the products were sure that the cosmetics can be classified as eco-friendly (nat- prepared, they were further tested. ural, organic). Those eco-friendly cosmetics may contain Evaluation of balm stability many components, such as Natragem E 145 (Polyglyceryl-4 Emulsion stability was evaluated using two types of tests. Laurate/Succinate, primary emulsifier, Croda), Span 20 (Sor- The first examination (centrifugation test) involved placing a bitan monolaurate, co-emulsifier, Croda), Xanthan Gum (vis- product sample (approx. 3 g in weight) in a test tube, followed cosity modifier, Standard Lublin, Poland), mixture Potassium by centrifugation at 3 000 r·min−1 for 30 min. After the test, Sorbate and Sodium Benzoate (Euxyl K712, Schulke&Myer), the products were evaluated visually. vegetable glycerin (Standard Lublin, Poland), sunflower oil The second examination (turbidimetric test) was per- (Gustav Hees) and double distilled water. formed using a Turbiscan Lab analyzer (Formulaction SA, The preparation procedure was as follows: different France). This method involves measuring the relationship phases (A, B, and C) were weighed and mixed (at room tem- between the amount of light emitted by the source and the perature) in separate beakers. Phase B was then combined amount of light reaching the detector after going through a with phase A, and the two phases were mixed for 1 min. Then, cell (cuvette) with the test sample, and the amount of reflected phases C and D were added, and all the ingredients were light reaching the back-scattering detector. The product sam- mixed for another minute. After that, the emulsions were ho- ples were scanned directly after emulsion preparation in a mogenized at the rotational speed of 4 000 r·min−1 for 2 min daily cycle for 14 days. The test samples were stored at 40 oC

– 286 – Nizioł-Łukaszewska Z, et al. / Chin J Nat Med, 2018, 16(4): 284292 in order to accelerate processes that destabilize the structure corded and analyzed using EZ-Yield Software. of the emulsion. Patch test Emulsion stability can be evaluated and compared by The study involved 12 healthy people (7 women and means of the Turbiscan Stability Index (TSI). The parameter 5 men) aged 12−48 years. None of the study participants were takes into account all individual measurements performed taking any medication for a minimum of two weeks before during experiments, and the TSI value is determined by aver- the epidermal patch test. Information about the health of the aging them. Additionally, TSI values are calculated with the respondents was obtained in an interview before the tests. The formula: study was performed by person qualified in conducting the

n 2 epidermal patch test. For the embodiment of the epidermal  ()xxiBS TSI  i0 patch test, there were used IQ Ultra Diagnostics company n 1 Chemotechniqe chambers. According to the manufacturer’s Where instructions each of the extracts was applied in an amount of xi isaverage backscattering for each minute of measurement; 30 µL in each chamber. xBS, is average xi; and n os number of scans. Epidermal patch tests were made in accordance with the Evaluation of color guidelines of the International Contact Dermatitis Research The determination of colorimetric parameters, which re- Group. Two identical patches with extracts were applied on lates to the color of the formulations, were determined using a skin on the back of respondents. The first patch was removed Konica Minolta CR-400 colorimeter. The measurement was after 24 h, while the second one (in case of no allergic reac- performed in the C.I.E. system based on determining three tion) was kept for additional 96 h from the first test. Inter- * * trichromatic components: L, a , and b . Every color deter- pretation of the results was carried out according with the mined in the C.I.E. space was defined by the following three international recording system epidermal patch test results. components: The scale for the interpretation of patch test results is as L, indicates lightness (intensity of color brightness); follows: * a , designates a value between red and green; IR, Irritant reaction; +++, Extreme positive reaction; * b , designates a value between yellow and blue. ++, Strong positive reaction; +, Weak positive reaction; The mean color parameters determined during measure- and ?+, Doubtful reaction. ments were used for calculating a change in color saturation Error Analysis * * (ΔC ), color tone (Δh ) and total color difference (ΔE) ac- The points in the charts represent mean values from a se- cording to the formulas below: ries of three or five independent measurements. The t distri- * * 2 * 2 * 2 * 2 ΔCx = [(ax ) + (bx ) ] × 1/2 − [(ab ) + (bb ) ] × 1/2 bution was used to calculate confidence limits for the mean * * * 2 * * 2 * * 2 ΔEx = [(Lx − Lb ) + (ax − ab ) + (bx − bb ) ] × 1/2 values. Confidence intervals, which constitute a measuring * * 2 * * 2 * 2 Δhx = [(ΔEx ) − (Lx − Lb ) + (ΔCx ) ] × 1/2 error, were determined for the confidence level of 0.90. Error where x is tested formulation and b is base formulation. values are presented in the figures. The results were processed using the following classification Statistical analysis of absolute color differences (ΔE*): 0–1, unidentifiable (deviation invisible); 1–2, slight deviation identifiable by a per- Results and disscusion son experienced in distinguishing nuances of color; 2–3.5, Flavonoid and total phenolic contents moderate deviation identifiable even by an ordinary person; Natural polyphenols are the wide group of secondary 3.5–5, marked deviation; and over 5, large color deviation. plant metabolites. Despite the fact that they are produced in Rheological properties of balm prototypes relatively small amounts, they play important role in plant The values of the dynamic viscosity coefficient (η) for metabolism. They are responsible for UV radiation or pest the products were determined by means of a Brookfield vis- protection [23]. One of the most significant groups of poly- cometer called HADV-III Ultra type, with the measurement phenols is flavonoids such as flavonoles, flavones, izofla- range of 100 to 13 000 000 mPa·s. The measurements were vones, and antocyanidines. conducted at 20 oC and spindle rotation speeds of 1, 10, 50, From the chemical point of view, polyphenols are sub- and 100 r·min−1. The measurements were performed in tripli- stances that consist of at least two hydroxyl groups bonded cate and the final results were their arithmetic average. with six-membered aromatic ring. Polyphenols characterized The flow limits of the test products were determined with by higher molecular masses contain more than one ring, a Brookfield RV DV III+ viscometer equipped with a set of where the second ring is often heterocyclic and contains oxy- vane spindles. The measurements were recorded during each gen atom. Flavonoids, cumarynes, and tanins are the exam- second of the test progressing at constant rotational speeds of ples of this type of polyphenols. Important polyphenolic the spindle: 0.1, 1, and 5 r·min−1. The flow limit corresponds molecules with a medium mass are anthocyanins-natural col- to the maximum value of shear stress which, when exceeded, orants. Plants synthesis also represents high mass polypheno- induces product fluidization. The measurements were re- lic molecules such as lignin.

– 287 – Nizioł-Łukaszewska Z, et al. / Chin J Nat Med, 2018, 16(4): 284292

The chemical structure of polyphenols determines their 1 000 g in water-glycerin phase (WG + O + D) and 5.01 mg/ high reducing potential, which makes them effective antioxi- 1 000 g in oil phase (O + WG + D) of extract, respectively. [24] dant agents with a high radical scavenging activity . While Our results showed that total polyphenolic content in wa- multiple health benefits are recorded after supplementing diet ter-glycerin extract from dogwood fruit are over two fold with polyphenols, the profound studies over influence of higher than their content in water extract from dogwood these molecules on skin condition are also conducted. leaves about 264.9 mg/1 000 g of extract [27]. Further research has proven that preparations rich in polyphenols benefits skin after direct application in several ways. Not only they are the effective antioxidants, but also inhibit several skin enzymes (elastase, collagenase and hya- luronidase), which decompose structural skin proteins. Poly- phenols have antiphlogistic, antibacterial and anti-acne properties and act as low (2−12 SPF) or medium (12−30 SPF) UV filters [25]. Plant material with relative high polyphenolic content is the fruit of cornelian cherry. It is estimated on average level of 370 mg·g−1 of fresh weight but may differ significantly depending on cultivar or year of harvest [26]. The aim of the present study was to estimate total phenolic and flavonoid content in two-phase cornelian cherry extracts (Figs. 1 and 2). Extract with a potential cosmetic application was prepared by the extraction of dried Cornus mas Fig. 2 Analysis of flavonoids extracts from Cornus mas L. fruits. fruit with a two-phase solvent mixture: sunflower oil (O) and The graphs show mean values ± SD, n =3 the mixture of water and glycerin (WG). To estimate the absolute level of active compounds total phenolic and flavonoid The quantity of polyphenols estimated in raw sunflower content were determined for both raw solvents (WG, O), wa- oil was similar to data obtained by other authors, depending ter-glycerin (WG + O) and oil (O + WG) phases after extrac- on the degree of oil manufacturing process it was assessed on tion without dogwood fruit and phases after extraction with the level between 4.9 mg/1 000 g and 12.0 mg/1 000 g [28-29]. plant material: water-glycerin (WG + O + D) and oil extract What is more, the obtained data indicated that both pol- (O + WG + D). yphenols and flavonoid contained in raw sunflower oil (O) may partially migrate to water-glycerin phase (WG) during extraction. The quantity of polyphenols initially assessed in raw sunflower oil on the level of 9.59 mg/1 000 g was low- ered after extraction with water-glycerin phase to 4.78 mg/1 000 g of extract, which was over a half less than initial value. Also, the oil phase of extract with plant material was characterized by lower level, relative to rough sunflower oil, of phenolic content and was equal 5.01 mg/1 000 g. The fact of migration of polyphenols may be connected with the polar character of these residues and their good water solubility. Dogwood berries are a valuable source of multiple active ingredients. The antioxidant activity of dogwood extracts is primarily attributable to the presence of phenolic compounds, flavonoids, anthocyanins, and vitamin C. Factors affecting the antioxidant activity of phenolic compounds include the num- Fig. 1 Analysis of total phenols extracts from Cornus mas L. ber and positions of hydroxyl groups in the molecular struc- fruits. The graphs show mean values ± SD, n = 3 ture, and solubility [30]. The level of phenolic compounds has been shown to correlate very well with their antioxidant ac- Total polyphenolic content was assessed on the basis of tivity [31]. The content of antioxidant compounds is influenced Gallic acid (GA) calibration curve (10−100 mg/1 000 g) and significantly by cultivation conditions and plant variety. Cul- expressed in milligram equivalents of GA per 1 000 g of ex- tivated varieties of dogwood have a lower antioxidant activity tract (mg (GA)/1 000 g). than wild forms [32]. It has also been shown that natural poly- Both phases of the extract contained significant amounts phenols found in dogwood berries exhibit varying degrees of of polyphenolic compounds. The phases contained 540.03 mg/ antioxidant activity depending on the extraction solvent used [33].

– 288 – Nizioł-Łukaszewska Z, et al. / Chin J Nat Med, 2018, 16(4): 284292

Matthäus et al. [34] have demonstrated that the higher the po- 0.31 mg·mL−1 (ABTS•+). larity of the solvent used in the extraction process is, the In contrast, studies conducted by Gülçin et al. [27] have weaker the antioxidant effect we receive. proven that antioxidant activity of aqueous extracts derived Antioxidant activity from dogwood leaves is increased significantly only in lower The antioxidant activity of the subjected extracts was as- levels of extract concentrations (to 20 µg·mL−1). In range sessed by DPPH• and ABTS•+. The antioxidant activity was 20−60 µg·mL−1 of extract concentration, the antioxidant abil- determined for six samples (WG + O + D, WG + O, WG, O + ity remains at the constant level of about 25%. WG + D, O + WG, O). All the extracts assessed in the present High ability of DPPH and ABTS scavenging was also study demonstrated a free radical scavenging ability corre- observed for the O + WG + D extract. It might be associated lated with extract concentration (Figs. 3 and 4). with the content of nonpolar ingredients, which may enhance antioxidant abilities, and one of them is vitamin E, in which sunflower oil is rich. The analysis of commercial extracts available on Euro- pean market has shown that the vast majority of the offered products are water-glycerin and water-glycol extracts. New method of extraction developed in the present study allowed to extract both polar and nonpolar active compounds from plant material and, as a final result, to obtain innovative cosmetic ingredient. Active compounds extracted by the oil phase would easily penetrate epidermal barrier [1-2] due to their lipophilic character. It would result in higher nutritional value for this type extract. Irritation potential of Cornus mas extracts in vitro Due to the potential application of oil and water-glycerin fractions of Cornus mas extract in cosmetic formulations their Fig. 3 Analysis of antioxidant properties of extracts from irritation potential was analyzed in vitro, using normal human Cornus mas L. fruits skin fibroblasts. The analysis was based on the Neutral Red Release Assay, where the cells are pre-loaded with neutral red dye and treated for a short time period with the potentially irritating agent. Irritation of the cells results in the release of [35] neutral red and decrease in A540 absorbance in the cell lysate . Unfortunately, the current literature lacks the data describing the influence of Cornus mas fruit extracts on the viability of skin fibroblasts. Surprisingly, several studies showed significant cytotoxicity of extracts from fruits of Cornus mas and Cornus officinalis against human cancer cell lines, including leukemia, ovary, prostate, lung, breast, bone and liver cancers [36-38]. On the other hand, extracts from various Cornus species, as well as compounds extracted from this extract, show significant neuroprotective activity, protecting neuronal cells from oxidative stress and apoptosis [39-41]. Our in vitro studies showed that water-glycerin fraction Fig. 4 Analysis of antioxidant properties of extracts from of Cornus mas extract could cause irritation of human skin Cornus mas L. fruits fibroblasts (Fig. 5A). Treatment of the cells with the highest examined concentration (50%, V/V) reduced their viability up Extract WG + O + D showed the strongest antioxidant to about 25%. However, irritation potential of this fraction properties in both DPPH and ABTS tests. Our results indi- was comparable with irritation caused by the extractant con- cated that the strong antioxidant activity of the extract may be trols, suggeting that the irritation of fibroblasts was caused by connected to high amount of water-soluble compounds, in- the solvent rather than by bioactive compounds extracted cluding not only polyphenols, such as flavonoids and antho- from Cornus mas fruits. Treatment of skin fibroblasts with oil cyanins, but also vitamin C. It was also possible that phenolic fractions in concentrations of 50%, 25% and 10% (V/V) did compounds present in sunflower oil may partially migrate to not affect cellular viability (Fig. 5B). To summarize, the re- the water-glycerin phase. The largest increase in antioxidant sults obtained from the in vitro experiments suggested that oil activity for WG + O + D extract have been observed at a fractions of Cornus mas extracts used in cosmetic formula- concentration of 0.04 to 0.16 mg·mL−1 (DPPH•) and 0.08 to tions would not cause skin irritation.

– 289 – Nizioł-Łukaszewska Z, et al. / Chin J Nat Med, 2018, 16(4): 284292

Fig. 5 Irritation potential of water : glycerin (A) and oil (B) fractions of Cornus mas extract against normal human skin fibroblasts (ATCC-2522) following 15 minutes treatment. The graphs show mean values ± SD, n = 6

Patch test (in vivo) dogwood extract contributed to the formation of stable dis- No allergic or irritation reactions were observed during persed systems. The effect might be produced by amino acids, patch testing of extracts in human subjects. proteins and other macromolecular substances extracted from Properties of body balm with Cornus mas extracts dogwood berries. The impact of dogwood extract on the color The balms formulated according to Table 1 were charac- of the balms was also assessed. The results are listed in Table 2. terized by a relatively high stability. Following the centrifuga- Table 2 Colorimetric parameters used in the present study tion tests, the products revealed no visual signs of instability. and test results More detailed findings were obtained using a Turbiscan ana- Sample lyzer. The results are shown in Fig. 6. Parameter Standard body care Body care emulsion with emulsion Cornus Mas extract L* 77.62 76.54 a* −0.52 0.94 b* 77.62 76.84 ΔC* - 0.10 Δh* - 0.80 ΔE* - 1.90

Both the product enriched with dogwood extract and the color of the base product was white, which is typically associated with emulsions. In the dogwood-containing product, a slight shift in color parameters was noted from green (−a) towards red (+a): Δa* was 1.46. For yellow (+b), a mild decrease in color saturation was observed: Δb* was around 0.78. Fig. 6 A change of the Turbiscan Stability Index (TSI) for the The difference in color between individual products was base emulsion and emulsion witch Cornus extract. The graphs slight, however the total change in color ΔE* reached 1.9. A show mean values ± SD, n = 5 deviation of this magnitude is easily identifiable by a person experienced in distinguishing nuances of color. The color of Determining the TSI value is helpful for designing emul- dogwood extract is largely attributed to anthocyanins, par- sion compositions, which has been used for evaluating the ticularly mono- and diglycosides of pelargonidin, cyanidin [42] stability of terpene-containing emulsions . In the present and delphinidin [12]. Differences between rheological parame- study, tests carried out for the balm prototypes found that the ters of the balms obtained were evaluated on the basis of vis- addition of extracts contributed only slightly to an improve- cosity (Fig. 7) and flow limit tests (Fig. 8). ment in system stability. Differences were noticeable six days According to the results from the present study, neither after product preparation. The TSI value recorded for the viscosity nor flow limit measurements revealed significant emulsion containing dogwood extracts was 2.3, and for the differences between the balms. Slightly higher values, typi- base emulsion it was 2.6. As the time progressed, the differ- cally not exceeding 10%, were noted for the emulsion based ence increased. After 14 days, the TSI values were 3.8 and 5.3, on dogwood extract. The results were definitely favorable respectively. The data showed that substances contained in from the viewpoint of product application.

– 290 – Nizioł-Łukaszewska Z, et al. / Chin J Nat Med, 2018, 16(4): 284292

References

[1] Handbook of Cosmetic Science and Technology [M]. Marcel Dekker Inc, 2001. [2] Multifunctional Cosmetics [M]. Marcel Dekker Inc, 2001. [3] Wang W, Sun F, An Y, et al. Morroniside protects human neuroblastoma SH-SY5Y cells against hydrogen perox- ide-induced cytotoxicity [J]. Eur J Pharmacol, 2009, 613(1-3): 19-23. [4] Seeram NP, Nair MG. Inhibition of lipid peroxidation and structure-activity-related studies of the dietary constituents anthocyanins, anthocyanidins, and catechins [J]. J Agric Food Chem, 2002, 50(9): 5308-5312. [5] Demir F, Kalyoncu IH. Some nutritional, pomological and physical properties of cornelian cherry (Cornus mas L.) [J]. J Fig. 7 The dependence of the dynamic viscosity of the emul- Food Eng, 2006, 60: 335-341. sions as a function of spindle rotation speed. The graphs show [6] Kyriakopoulos AM, Dinda B. Cornus mas (Linnaeus) novel mean values ± SD, n =5 devised medicinal preparations: bactericidal effect against staphylococcus aureus and pseudomonas aeruginosa [J]. Mol- ecules, 2015, 20(6): 11202-11218. [7] Tural S, Koca I. Physicochemical and antioxidant properties of cornelian cherry fruits (Cornus mas L.) grown in Turkey [J] Sci Hortic, 2008, 116: 362-366. [8] Klimenko SW. The cornelian cherry (Cornus mas L.) collection, preservation and utilization of genetic resources [J]. J Fruit Ornam Plant Res, 2004, 12: 93-98. [9] Pawlowska AM, Camangi F, Braca A. Quali-quantitative analysis of flavonoids of Cornus mas L. (Cornaceae) fruits [J]. Food Chem, 2010, 119: 1257-1261. [10] Gunduz K, Saracoglu O, Özgen M, et al. Antioxidant, physical and chemical characteristics of cornelian cherry fruits (Cornus mas L.) at different stages of ripenees [J]. Acta Sci Pol Horto- rum Cultus, 2013, 12(4): 59-66. [11] Yilmaz KU, Ercisli S, Zengin Y, et al. Preliminary characteri-

sation of cornelian cherry (Cornus mas L.) genotypes for their Fig. 8 The values of the yield stress for prototypes of cosmetic physico-chemical properties [J]. Food Chem, 2008, 114: emulsions. The graphs show mean values ± SD, n = 5 408-412. [12] Vareed SK, Reddy MK, Schutzki RE, et al. Anthocyanins in Cornus alternifolia, Cornus controversa, Cornus kousa and Conclusions Cornus florida fruits with health benefits [J]. Life Sci, 2006, In the present study, we demonstrated the properties and 78(7): 777-84. [13] Seeram NP, Schutzki R, Chandra A, et al. Characterization, possibilities for the application of a hydrophilic/hydrophobic quantification and bioactivities of anthocyanins in Cornus spe- dogwood extract in body care balms. A single-stage extraction cies [J]. J Agric Food Chem, 2002, 50(9): 2519-2523. process using both hydrophilic and hydrophobic solvents [14] Pantelidis GE, Vasilakakis M, Manganaris GA, et al. Antioxi- make it possible to extract a broad range of active substances dant capacity, phenol, anthocyanin and ascorbic acid contents from dogwood berries, both soluble in water and non-polar in raspberries, blackberries, red currants, gooseberries and substances. It was demonstrated that the hydrophobic and Cornelian cherries [J]. Food Chem, 2007, 102: 777-783. hydrophilic phases of the extract were associated with a rapid [15] Polinicencu C, Popescu H, Nistor C. Vegetal extracts for cos- increase in free radical neutralizing ability. Furthermore, both metic use: 1. Extracts from fruits of Cornus mas. Preparation phases were found to be characterized by a high content of and characterization [J]. Cluj Med, 1980, 53: 160-163. phenolic compounds and polyphenols. Dogwood extracts [16] Sultana N, Ho Lee N. Antielastase and free radical scavenging added to emulsion formulations played a vital role in increas- activities of compounds from the stems of Cornus kousa [J]. Phytother Res, 2007, 21(12): 1171-1176. ing emulsion stability and demonstrated a favorable effect on [17] Singleton VL, Orthofer R, Lamuela-Raventos RM. Analysis of the rheological properties of the product. The results pointed total phenols and other oxidation substrates and antioxidants by clearly to benefits associated with the addition of extracts of means of Folin-Ciocalteu reagent [J]. Meth Enzymol, 1999, 299: this type to the formulations of body care cosmetics. Finally, a 152-178. dogwood extract may be an innovative and thus multifunc- [18] Woisky R, Salatino A. Analysis of propolis: some parameters tional material for the cosmetic industry. and procedures for chemical quality control [J]. J Apic Res,

– 291 – Nizioł-Łukaszewska Z, et al. / Chin J Nat Med, 2018, 16(4): 284292

1998, 37: 99-105. vitro antioxidant potentials of three edible fruits: cornelian [19] Matejic JS, Dzamic A, Randjelovic V, et al. Total phenolic cherry, Japanese persimmon and cherry laurel [J]. Food Chem content, flavonoid concentration, antioxidant and antimicrobial Toxic, 2012, 50(9): 3329-3335. activity of methanol extracts from three Seseli taxa L. [J]. Cent [33] Matthäus B. Antioxidant activity of extracts obtained from Eur J Biol, 2012, 7(6): 1116-1122. residues of different oilseeds [J]. J Agric Food Chem, 2001, [20] Brand-Williamis W, Cuvelier M, Berset C. Use of a free radical 50(12): 3444-3452. method to evaluate antioxidant activity [J]. LWT Food Sci [34] Sielicka M, Samotyja U. Solvent influence on antioxidant Technol, 1995, 28: 25-30. activity assay of selected cold-pressed plant oils [J]. PhD Inter [21] Re R, Pellegrini N, Protegente A, et al. Antioxidant activity J, 2011, 2011: 67-74. applying and improved ABTS radical cation decolorization as- [35] Zanatta CF, Ugartondo V, Mitjans M. Low cytotoxicity of say [J]. Free Radic Biol Med, 1999, 26(9-10): 1231-1237. creams and lotions formulated with Buriti oil (Mauritia flexu- [22] Bartosz G. Reactive oxygen species: destroyers or messengers [J]. osa) assessed by the neutral red release test [J]. Food Chem Biochem Pharm, 2009, 77(8): 1303-1315. Toxicol, 2008, 46(8): 2776-2781. [23] Phenolic compounds biochemistry [M]. Springer, 2006. [36] Yousefi B, Abasi M, Abbasi MM, et al. Anti-proliferative [24] Medvidovic-Kosanovic M, Seruga M, Jakobek L, et al. properties of Cornus mass fruit in different human cancer cells Eletrochemical and antioxidant properties of (+)-catechin, [J]. Asian Pac J Cancer Prev, 2015, 16(14): 5727-5731. quercetin and rutin [J]. Croat Chem Acta, 2014, 83: 197-207. [37] Liao CL, Lin JH, Lien JC, et al. The crude extract of Corni [25] Zillich OV, Schweiggert-Weisz U, Eisner P, et al. Polyphenols Fructus inhibits the migration and invasion of U-2 OS human as active ingredients for cosmetic products [J]. Int J Cosmet Sci, osteosarcoma cells through the inhibition of matrix metallo- 2015, 37(5): 455-464. proteinase-2/-9 by MAPK signalling [J]. Environ Toxicol, 2015, [26] Związki aktywne owoców derenia [M]. Uniwersytet Przyrod- 30(1): 53-63. niczy we Wrocławiu, 2012. [38] Savikin K, Zdunic G, Jankovic T, et al. In vitro cytotoxic and [27] Gülçin I, Beydemir S, Küfrevioğlu Öİ. Evaluation of antioxi- antioxidative activity of Cornus mas and Cotinus coggygria [J]. dant activity of Cornelian Cherry (Cornus Mas L.) [J]. Acta Nat Prod Res, 2009, 23(18): 1731-1739. Aliment, 2005, 34(2): 193-202. [39] Wang H, Nair MG, Strasburg GM, et al. Antioxidant and an- [28] Chandran J, Soban Kumar DR, Reshma MV, et al. Compara- ti-inflammatory activities of anthocyanins and their aglycon, tive study on the total phenolic content and radical scavenging cyanidin, from tart cherries [J]. J Nat Prod, 1999, 62(2): 294-296. activity of common edible vegetable oils [J]. J Food Biochem, [40] Jeong EJ, Kim TB, Yang H, et al. Neuroprotective iridoid 2014, 38: 38-48. glycosides from Cornus officinalis fruits against glutamate-in- [29] Siger A, Nogala-Kalucka M, Lampart-Szczapa E. The content duced toxicity in HT22 hippocampal cells [J]. Phytomedicine, and antioxidant activity of phenolic compounds in cold-pressed 2012, 19(3-4): 317-321. plant oils [J]. J Food Lipids, 2008, 15: 137-149. [41] Hong SY, Jeong WS, Jun M. Protective effects of the key [30] Lim SH, Choi SH, Oh YI, et al. Anti-oxidative effects of compounds isolated from Corni fructus against β-amyloid- in- ﬂavonoids enriched Corni fructus extract and the mechanism [J]. duced neurotoxicity in PC12 cells [J]. Molecules, 2012, 17(9): Afr J Pharm Pharmacol, 2011, 5: 506-514. 10831-10845. [31] Moyer RA, Hummer KE, Finn CE, et al. Antocyanins, pheno- [42] Pérez-Mosqueda LM, Trujillo-Cayado LA, Carrillo F, et al. lics, and antioxidant capacity in diverse small fruits: vaccinium, Formulation and optimization by experimental design of rubus, and ribes [J]. J Agric Food Chem, 2002, 50(3): 519-525. eco-friendlyemulsions based on d-limonene [J]. Colloid Surf B [32] Celep E, Aydın A, Yesilada E. A comparative study on the in Biointerfaces, 2015, 128: 127-131.

Cite this article as: Nizioł-Łukaszewska Z, Wasilewski T, Bujak T, Gaweł-Bęben K, Osika P, Czerwonka D. Cornus mas L. extract as a multifunctional material for manufacturing cosmetic emulsions [J]. Chin J Nat Med, 2018, 16(4): 284-292.

– 292 –