94 LONGEVITY
The solution was then subjected to ultrasonic treatment for 15 minutes with agitation, followed by filtration through a 0.2 µm PTFE filter.
Ex vivo efficacy tests Skin explants were collected with informed consent from a 31-year-old female Caucasian donor (phototype II/III) during abdominal surgery. Post-surgery, the explants were maintained in OxiProteomics® medium at 37°C in a humidified atmosphere containing 5% CO2
Cannabis Sativa Hairy Roots (CSHR)
Grinding These explants were then divided into
nine experimental groups, with three explants per group (Table 1). The culture medium was refreshed every 24 hours. Explant sections, each 5 µm thick, were
prepared using a cryostat (Leica). The sections were fixed in a solution of 95% ethanol and 5% acetic acid. In situ protein carbonylation was detected using a fluorescent probe (excitation at 647 nm/emission at 650 nm), specifically designed to bind to carbonyl groups.4 DAPI (4’,6-diamidino-2-phenylindole) was used to label cell nuclei. For immunodetection, the skin sections
were fixed in a 4% paraformaldehyde solution, followed by permeabilization with 0.1% Triton for ten minutes. Non-specific binding sites were blocked using a 3% BSA (Bovine Serum Albumin) solution in PBS (pH 7.4). The sections were then incubated with
primary antibodies for biomarker detection in a 3% BSA/PBS solution. After washing to remove excess primary antibody, the sections were incubated for one hour with a fluorophore- conjugated secondary antibody. Cell nuclei were labelled with DAPI, and excess antibody and DAPI were removed through a series of PBS washes. Fluorescent images were captured using
an epi-fluorescent microscope (EVOS M5000 Imaging System; 40x objective), maintaining consistent acquisition times and resolutions across all samples. The raw images, including the full range of fluorescence signal intensities (16-bit .TIFF format), were analysed using ImageJ software. The fluorescence intensity of targeted
biomarkers was quantified by integrating the specific fluorescence signal above the threshold, normalized to the evaluated area. Carbonylation levels were quantified by integrating the fluorescence signal across different skin compartments (dermis, epidermis, and stratum corneum) and the entire skin sample.
Treatment(s)
Water + Ethanol
Microwaves* and mixing
Filtration Concentration Drying CSHR dry extract
Figure 2: Extraction procedure to obtain Cannabis sativa hairy root (CSHR) extract *Microwave- assisted extraction was compared to a classical extraction without microwaves
The number of P16INK4A positive cells was
quantified by counting labelled cells and normalizing to the length of the evaluated skin sections. Sirtuin-1 levels were quantified using nuclei as the region of interest (ROI). Protein concentration was accurately
measured using the Bradford Protein Assay Dye Reagent (Bio-Rad™), following the manufacturer’s instructions. Proteins were separated by high-resolution electrophoresis (SDS-PAGE, 4-20% gradient; Thermo Scientific™) and transferred to a 0.2 µm nitrocellulose membrane (Bio-Rad 1620233). To confirm successful protein transfer, the
membrane was incubated in ponceau red solution (Thermo Scientific) for five to ten minutes, followed by multiple washes with Milli-Q water. The membrane was then washed
TABLE 1: EXPERIMENTAL GROUPS FOR THE EX VIVO TESTS Description
Control 0.003% CSHR extract
0.001% CSHR extract 1% Resveratrol
Stress (UVA)
0.001% CSHR extract + Stress 1% Resveratrol + Stress
Not treated No stress
Topically treated with products (30 uL/cm2 No Stress
Not treated UV-A irradiation (λ=365 nm; J/cm2)
0.003% CSHR extract + Stress Topically treated with products (30 uL/cm2 UV-A irradiation (λ=365 nm; J/cm2
) PERSONAL CARE November 2025 ) for 24h ) for 24h Sampling
25h after the treatment
in Tris-buffered saline (TBS, pH 7.6) with 0.1% Tween (TBS-T) and incubated for 30 minutes in TBS with 3% BSA (TBS-BSA) for blocking. Biomarker quantification was normalized
relative to the control group (set at 100%) to obtain mean values and standard deviations. Statistical analyses were performed using GraphPad software (La Jolla, California, USA) with one-way ANOVA and Dunnett’s post-hoc test for multiple comparisons, or an unpaired t-test with Welch’s correction against the control or stress group (95% confidence interval). Efficacy percentages for experimental
groups were calculated using the control group as the maximum efficiency reference (100%) and the UV-A stress group as the minimum efficiency reference (0%).
Efficacy % (group X)
=
Biomarker level (Stress) - Biomarker level (group X)
Biomarker level (Stress) - Biomarker level (Control)
* 100 An induction value (%) was obtained for
the experimental groups using the following formula:
Induction % (group X)
3h and 24h after the stress
=
Biomarker level (group X) Biomarker level (Control)
- 1 * 100
Results and discussion Hairy roots biomass cultivation and extraction process Microwave-assisted extraction resulted in
www.personalcaremagazine.com Solid waste
Water + Ethanol
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76 |
Page 77 |
Page 78 |
Page 79 |
Page 80 |
Page 81 |
Page 82 |
Page 83 |
Page 84 |
Page 85 |
Page 86 |
Page 87 |
Page 88 |
Page 89 |
Page 90 |
Page 91 |
Page 92 |
Page 93 |
Page 94 |
Page 95 |
Page 96 |
Page 97 |
Page 98 |
Page 99 |
Page 100 |
Page 101 |
Page 102 |
Page 103 |
Page 104 |
Page 105 |
Page 106 |
Page 107
