Stachys byzantina K. Koch in the Treatment of Skin Inflammation: A Comprehensive Evaluation of Its Therapeutic Properties

Abstract

Stachys byzantina is a plant widely cultivated for food and medicinal purposes. Stachys species have been reported as anti-inflammatory, antibacterial, anxiolytic, and antinephritic agents. This study aimed to evaluate the anti-inflammatory potential of the ethanolic extract (EE) from the aerial parts of S. byzantina and its most promising fraction in models of acute and chronic inflammation, including a psoriasis-like mouse model. The EE was fractionated into hexane (HF), dichloromethane (DF), ethyl acetate (AF), and hydroalcoholic (HD) fractions. Screening for anti-inflammatory activity based on nitric oxide inhibition (IC₅₀ μg/mL: HF 24.29 ± 5.87, EE 176.45 ± 18.65), hydroxyl radical scavenging (HF 3.89 ± 0.61, EE 6.38 ± 2.25), β-carotene/linoleic acid assay (HF 10.13 ± 3.81, EE 25.64 ± 2.12), and ORAC identified HF as the most active fraction. Topical application of HF effectively reduced croton oil- and phenol-induced ear edema in mice, with no statistical difference to the reference drugs. A formulation containing HF showed significant activity in the imiquimod-induced psoriasis model, reducing pro-inflammatory cytokines and nitric oxide production in macrophages, with no cytotoxicity to skin cells. Phytochemical analysis of HF revealed the presence of terpenes, steroids (491.68 ± 4.75 mg/g), phenols (34.30 ± 4.96 mg/g), flavonoids (151.77 ± 6.66 mg/g), and α-tocopherol, which was identified and quantified by HPLC-UV analysis (10.56 ± 0.97 mg/g of HF). These findings highlight the therapeutic potential of S. byzantina for skin inflammation, particularly contact dermatitis and psoriasis, encouraging further studies, including in human volunteers.

Figure 1

HF and EE kinetics in ORAC assay. Two-way ANOVA followed by Bonferroni test. The values obtained represent the mean ± s.e.m. of R.F.U. (Relative Fluorescence Unit). The data were obtained by six readings, each one being carried out within 10 min. Equal letters indicate no statistical difference (p < 0.05).

Figure 2

Evaluation of HF cytotoxicity in J774A.1, L929 and HaCaT cells. The concentrations used varied between 12.5 and 100 μg/mL. DMEM culture medium was used as the negative control. Viability above 70% characterizes a nontoxic sample.

Figure 3

Determination of IL-6, IL12, and TNF-α cytokines in J774A.1 cell line stimulated with E. coli LPS. HF concentrations range from 125 to 15.6 μg/mL. ANOVA followed by the Dunnet test. Significant values: ****p < 0.0001 and ***p < 0.001.

Figure 4

Determination of ^•NO radical concentration in J774A.1 cell line stimulated with LPS 3 from E. coli and IFN-γ. HF concentrations ranged from 125 to 15.6 μg/mL. ANOVA 4 followed by the Dunnet test. Significant values: ****p < 0.0001.

Figure 5

Effect of HF on the inflammatory stimulus induced by croton oil and phenol, in addition to representative photographs of the clinical appearance of mice ears (n = 7 and 8 animals, respectively). Negative control (vehicle - acetone), dexamethasone (Dexa) 0.1 mg/20 μL and HF at 0.1; 0.5 and 1.0 mg/20 μL were topically administered immediately after topical application of 2.5% croton oil and phenol 10% (v/v). Values in each column represent the mean ± s.e.m. of the weight difference between ear fragments (mg). ANOVA, followed by the Newman-Keuls test. Equal letters indicate no statistical difference (p < 0.05).

Figure 6

Effect of HF on the weight difference between ear fragments in the imiquimod-induced ear edema test (n = 8 animals). Negative control (vehicle - formulation), clobetasol 0.5 mg/g, HF 6 and 12% were topically applied for 10 days to investigate the antipsoriatic activity. Values in each column represent the mean ± s.e.m. of weight (mg) and thickness (μm) of the right ear edema fragments for each group. ANOVA, followed by the Newman-Keuls test. Means represented by equal letters indicate no statistical difference (p < 0.05).

Figure 7

Representative photomicrographs (100× magnification) of the ear tissues on the last day of the imiquimod-induced ear edema test. The variation in edema thickness between groups is shown by the arrow. Means for each group were as follows: (a) vehicle (477.68 μm), (b) clobetasol (251.45 μm), (c) HF 6% (352.84 μm), (d) HF 12% (299.03 μm), and (e) left ear (186.17 μm).

Figure 8

Representative photomicrographs (400× magnification) of the ear tissue on the last day of the imiquimod-induced ear edema test. The presence of nuclei in the corneal layer indicates parakeratosis (in brackets). Leukocyte infiltrate is indicated by arrows. Vasodilation was highlighted by a circle. Increased epidermal thickness (hyperkeratosis), typical of psoriasis lesions is shown in braces. (a) vehicle, (b) clobetasol, (c) HF 6%, (d) HF 12%, and (e) left ear.

Figure 9

Chromatogram and UV spectra obtained by HPLC-UV analysis. The identification of α-tocopherol was carried out by comparing the UV spectra and retention time, and by coelution. Chromatographic conditions: Methanol and UHQ water (98:2, v/v) as mobile phase, at a flow rate of 1.3 mL/min. α-tocopherol standard at 25 μg/mL and PHEX at 1000 μg/mL were diluted in the mobile phase. The Injection volume was 20 μL, and the temperature was 25 °C. Agilent Eclipse Plus C18 column was used. UV detection was performed at 205 nm.