38 ANTI-POLLUTION
has generated a large amount of inedible by-products, including fruit and vegetable peels, seeds and leaves. From these wastes, it is possible to obtain bioactive compounds and active ingredients of interest for the wellness industry, strongly contributing to the paradigm shift towards a circular economy. The strawberry active is 100% upcycled: it is made up from fruit waste of large-scale distribution and it is completely plant-based. Its good composition is a key requirement since consumers are increasingly sensitive and informed, and critically check the INCI list of the cosmetic products they are going to choose.
The seek safe beauty products, with few
ingredients and low environmental impact following the trend of clean beauty, at the basis of which there is the philosophy of ‘less is more’. Clean beauty cosmetics are characterized
by a short INCI list. The inclusion of multifunctional ingredients in the formula is the smartest way to achieve it. The strawberry active is mostly fruit juice and imparts an intense natural colour and fragrance to the cosmetics in which it is incorporated. In this sense, it is a perfect ingredient that
answers to the needs of both formulator and consumer: colorant, perfume and active in a single INCI name. Its activity has, in fact, been successfully tested in vitro for its antioxidant and revitalising action.
Antioxidant efficacy The aim of this study was to evaluate the antioxidant effect of the strawberry active in vitro on the human keratinocyte cell line HaCaT. The evaluation was carried out on cells exposed to 0,5% v/v of the test item for 24 hours and subsequently menadione was added as an oxidizing stimulus.4 The antioxidant activity was quantified as
a reduction in ROS induced by exposure to menadione, by means of the dichlorofluorescein assay.
Test system HaCaT cells HaCaT cells are spontaneously transformed and immortalized aneuploid keratinocytes derived from adult human skin. The cell line was routinely maintained in the laboratory in DMEM medium supplemented with glutamine, antibiotics and fetal bovine serum (FBS).
Dichlorofluorescin diacetate The 2’,7’ – dichlorofluorescin diacetate (DCFDA) quantitatively measures the reactive oxygen species in live cells. This fluorogenic dye interacts with the hydroxyl, peroxyl and other ROS species within the cell. Once penetrated, DCFDA is
deacetylated by cellular esterases forming a non-fluorescent compound which is then oxidized by ROS present in the cell generating the fluorescent compound 2’,7’ – dichlorofluorescein (DCF). The DCF fluorescence is then measured in fluorescence spectroscopy with excitation at 485 nm and emission 535 nm.
PERSONAL CARE November 2023
TABLE 1: EFFECTS OF MENADIONE ON HACAT CELLS. RESULTS ARE EXPRESSED AS RELATIVE FLUORESCENCE UNIT (RFU)
MENADIONE (µM) 0
20 50
100
6000 5000 4000 3000 2000
MEAN 3486 4298 4775 5349
STD. DEVIATION 56.569
208.597 70.491
205.061
STATISTICAL SIGNIFICANCE (P VALUE) -
0,0029 <0.0001 <0.0001
PERCENTAGE INCREASE (%) -
+23.31 +36.98 +53.44
*** *** **
0
20 Menadione (µM) Figure 1: Menadione-induced oxidative stress in HaCaT cells without the strawberry active
Test method Cells from routine culture were seeded in 96-well black plates and allowed to adhere overnight. The next day, the cells were exposed to 0.5% v/v of the strawberry active in complete medium. After 24 hours of exposure to the strawberry
active, the medium of each well was replaced with serum-free buffer and subsequently dissolved DCFDA was added to the buffer. Forty-five minutes later, the buffer with the probe was aspirated and replaced with buffer containing 10% FBS and menadione at different concentrations for one hour. At the end of the menadione exposure, the
fluorescence was measured by a microplate reader. To analyze the test results, an unpaired t-test was applied to compare the mean fluorescence of two experimental groups.
Results Development of oxidative stress model HaCaT cells were exposed for one hour to three concentrations of menadione: 20, 50 and 100 μM. Menadione is capable of inducing oxidative stress in HaCaT cells in a concentration dependent manner. The induction compared to the untreated
control is statistically significant in the treatment at 20 µM (**) and highly relevant at 50 and 100 µM (***) with an increase in oxidative stress of approximately 24, 37 and 53% respectively. The results of exposure to menadione are shown in Table 1 and Figure 1.
Antioxidant efficacy model Pretreating cells with the strawberry active, menadione-induced oxidative stress is partially reduced. The results of exposure to menadione after pretreatment with the strawberry active
are shown in Table 2 and Figure 2. The pretreatment with the strawberry active
at 0.5% v/v (Table 2) reduces menadione- induced oxidative stress and the reduction is statistically significant for all three menadione concentrations tested. In fact, the reduction in ROS levels averages
around 17% compared to the levels induced by menadione alone and the maximum efficacy is manifested against the stress induced by menadione at a concentration of 20 μM. The study can be considered valid because
no bacterial contamination was found in the treatment chambers and, at the same time, increased concentration-dependent fluorescence was recorded in the cells treated with menadione used as an oxidative treatment (Figure 1). Regarding the antioxidant efficacy, a positive
effect was found in cells pretreated with the strawberry active at a concentration of 0.5% v/v (Figure 2), with a reduction in ROS levels by an average of 17% compared to those induced by menadione alone.
Revitalising effects The purpose of this study is to evaluate the revitalising effect of the strawberry active on the HaCaT human keratinocytes in vitro. The evaluation was done on cells exposed to dilution of the active or glycerin in serum-free medium.
The study was conducted in the absence
of serum in order to minimize the promotion of cell proliferation and viability induced by factors external to the test items. In fact, fetal bovine serum contains growth factors and other proteins that support cell proliferation in such quantities as to overcome the potential promoting effect of the test item.
www.personalcaremagazine.com
50
100
Fluorescence (RFU)
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