Multistep Approach Points to Compounds Responsible for the Biological Activity and Safety of Hydrolates from Nine Lamiaceae Medicinal Plants on Human Skin Fibroblasts

Abstract

As byproducts of essential oil distillation, hydrolates are used in natural cosmetics/biomedicine due to their beneficial skin effects. However, data on their safety with relevant biological targets, such as human skin cells, are scarce. Therefore, we have tested nine hydrolates from the Lamiaceae family with skin fibroblasts that are responsible for extracellular collagenous matrix builds. Thyme, oregano, and winter savoury hydrolates showed several times higher total phenolics, which correlated strongly with their radical scavenging and antioxidative capacity; there was no correlation between their viability profiles and the reducing sugar levels. No proteins/peptides were detected. All hydrolates appeared safe for prolonged skin exposure except for 10-fold diluted lavender, which showed cytotoxicity (~20%), as well as rosemary and lavandin (~10%) using viability, DNA synthesis, and cell count testing. Clary sage, oregano, lemon balm, and thyme hydrolates (10-fold diluted) increased fibroblast viability and/or proliferation by 10-30% compared with the control, while their viability remained unaffected by Mentha and winter savoury. In line with the STITCH database, increased viability could be attributed to thymol presence in oregano and thyme hydrolates in lemon balm, which is most likely attributable to neral and geranial. The proliferative effect of clary sage could be supported by alpha-terpineol, not linalool. The major volatile organic compounds (VOCs) associated with cytotoxic effects on fibroblasts were borneol, 1,8-cineole, and terpinene-4-ol. Further research with pure compounds is warranted to confirm the roles of VOCs in the observed effects that are relevant to cosmetic and wound healing aspects.

Keywords: STITCH database; anti-proliferative effects; hydrolates; natural cosmetics; proliferative effects; skin fibroblast viability; volatile organic compounds.

Figure 1

Electrophoretically resolved sodium dodecyl sulphate polyacrylamide gel (16%) with 1 mL of hydrolates concentrated on SpeedVac (room temperature under vacuum) until a 10 μL volume, mixed with 10 μL of 2 × Laemmli buffer in denaturing conditions and stained with CBB 250-R. M. piperita (MP), R. officinalis (RO), L. officinalis (LO), T. vulgaris (TV), S. sclarea (SS), S. montana ssp. variegata (SM), L. intermedia (LI), O. vulgare (OV), and M. officinalis (MO). M—protein weight markers in kilodaltons (kDa). Each marker band in the M lane represents 1 μg of protein.

Figure 2

Viability assessments and cell counts of primary human skin fibroblasts treated with nine plant hydrolates. (A) MTT assay with three different hydrolates concentrations in a primary cell culture medium. (B) DNA synthesis assessment by BrdU testing with the highest concentration point (10%) applied in the MTT test. Results are expressed as optical density values and above or under them as a percentage in relation to the control (average ± SD). (C) The number of cells before and after the treatment was obtained by crystal violet and trypan blue staining and counting. A two-way ordinary ANOVA test was applied in (A) and one-way ANOVA with Dunnett’s multiple comparison tests were applied in (B,C) to show significant differences among different hydrolates treatments compared with the control. All tests were conducted with three biological batches, each in triplicates. *** denotes a difference at p < 0.0005; ** denotes a difference at p < 0.005; * denotes a difference at p < 0.05. M. piperita (MP), R. officinalis (RO), L. officinalis (LO), T. vulgaris (TV), S. sclarea (SS), S. montana ssp. variegata (SM), L. intermedia (LI), O. vulgare (OV), and M. officinalis (MO).

Figure 3

PCA ordination of variables based on component correlations. TPC—total phenolic content; TRS—total reducing sugars; DPPH—2,2-diphenyl-1-picrylhydrazyl assay; ABTS—2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic Acid) assay. Red arrows denote viability testing by the MTT assay with different hydrolates concentrations. The red and black ellipses and the green distorted rectangles represent a separation of groups of hydrolates.

Figure 4

(A) Overview of the structural formulas of volatile organic compounds (VOCs) with abundance ≥ 10%, divided into three groups based on their exclusive presence in hydrolates that either promote or suppress fibroblast viability, including those with no or contradictory effects. (B) The abundance of up to the top five volatile compounds per hydrolate that each contribute at least a 10% share. Colours: grey shade—contradictory or no effects on the proliferation; * dark rose—significantly cytotoxic; pale rose—significantly cytotoxic for at least one of the three viability tests employed. MolView, a web application and intuitive, open-source program, was used to draw the chemical structural formulas (https://molview.org accessed on the 3 April 2023).

Figure 5

The confidence view of the protein networking of carvacrol (representative of the major VOCs from hydrolates that exert no net effect), thymol (representative of the major VOCs from hydrolates that exert a positive viability effect) and camphor (representative of the major VOCs from hydrolates that exert a negative viability effect), performed using the STITCH database http://stitch.embl.de/ accessed on the 9 April 2023. Only first-line protein targets of chemical compounds are shown with the host species of Homo sapiens. Thicker lines represent more robust associations. Green lines show chemical–protein interactions; protein–protein interactions are in grey, small and large node sizes differentiate between proteins with and without 3D structures, and coloured and white circles denote first- and second-line protein targets, respectively. The codes represent gene name abbreviations that code the designated proteins.