Insights into tyrosinase inhibition by compounds isolated from Greyia radlkoferi Szyszyl using biological activity, molecular docking and gene expression analysis.

Greyia radlkoferi ethanol extract and its five compounds were tested for their inhibitory activity against the mushroom tyrosinase enzyme and melanin production on melanocytes. The crude extract showed significant tyrosinase inhibition with IC50 of 17.96µg/ml. This is the first report of the isolation of these 5 compounds from Greyia radlkoferi. 2',4',6'-Trihydroxydihydrochalcone showed the highest tyrosinase inhibition at 17.70µg/ml (68.48µM), with low toxicity when compared with crude extract. This compound is therefore, a key component in the crude extract, which is responsible for tyrosinase inhibitory activity. The RT-qPCR indicated that the mechanism of action is most likely post transcriptional. Further, the molecular docking study showed that tyrosinase inhibitory activity depends on interaction of the compound with Cu2+ ions at the active site. This is the first report of the tyrosinase inhibitory activity of the G. radlkoferi extract and molecular insights on interaction of its compounds with Cu2+ ions as the driving factor for tyrosinase inhibition. These results suggest that the extract of G. radlkoferi and the compound 2',4',6'-trihydroxydihydrochalcone have great potential to be further developed as pharmaceutical or cosmetic agents for use against dermatological disorders associated with melanin.

Evaluation of Wound Healing and Antibacterial Potential of Greyia radlkoferi Szyszyl. Ethanolic Leaf Extract.

Angiogenesis is an essential mechanism in both physiological and pathological functions, such as wound healing and cancer metastasis. Several growth factors mediate angiogenesis, including vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF). This study evaluated the potential wound healing activity of Greyia radlkoferi Szyszyl (GR) and its effect on growth factors regulating angiogenesis. The ethanolic leaf extract of GR was evaluated for antibacterial activity against wound associated bacteria; Staphylococcus aureus and Pseudomonas aeruginosa. It exhibited antibacterial activity against two strains of S. aureus (ATCC 25293 and ATCC 6538) displaying a minimum inhibitory concentration (MIC) at 250 and 500 µg/ml, respectively. The antioxidant activity of the extract was investigated for nitric oxide (NO) scavenging activity and showed a fifty percent inhibitory concentration (IC50) of 1266.5 ± 243.95 µg/ml. The extract was further investigated to determine its effect on the proliferation and modulation of growth factors secreted by human keratinocytes (HaCaT). Its effect on wound closure was evaluated using the scratch assay, where non-toxic concentrations were tested, as determined by the antiproliferative assay against HaCat cells (IC50 > 400 µg/ml). Results showed that the extract significantly inhibited wound closure, with a percentage closure of 60.15 ± 1.41% (p < 0.05) and 49.52 ± 1.43% (p < 0.01) at a concentration of 50 and 100 µg/ml, respectively, when compared to the 0.25% Dimethyl sulfoxide vehicle control (65.86 ± 1.12%). Quantification of secreted growth factors from cell-free supernatant, collected from the scratch assay, revealed that the extract significantly decreased the concentration of platelet-derived growth factor (PDGF-AA) at both 50 (p < 0.05) and 100 µg/ml (p < 0.001) (443.08 ± 77.36 and 178.98 ± 36.60 pg/ml) when compared to the 0.25% DMSO vehicle control (538.33 ± 12.64 pg/ml). Therefore, whilst the extract showed antibacterial activity against wound associated bacteria, it did not induce wound healing but rather showed a significant inhibition of wound closure, which was confirmed by the inhibition of PDGF-AA, a major growth factor involved in angiogenesis. Therefore, the GR extract, should be considered for further investigation of anti-angiogenic and anti-metastatic properties against cancer cells.

The Activity of Gold Nanoparticles Synthesized Using Helichrysum odoratissimum Against Cutibacterium acnes Biofilms.

The human skin is home to millions of bacteria, fungi, and viruses which form part of a unique microbiome. Commensal microbes, including Cutibacterium acnes can occasionally become opportunistic resulting in the onset of dermatological diseases such as acne. Acne is defined as a chronic inflammatory disorder based on its ability to persist for long periods throughout an individual's life. The synthesis of gold nanoparticles (AuNPs) was performed using the bottom-up approach by reduction of a gold salt (HAuCl4.3H2O) by the methanol extract (HO-MeOH) and aqueous decoction prepared from the dried aerial parts of Helichrysum odoratissimum (HO-Powder). The HO-MeOH and HO-Powder AuNPs were prepared as unstabilised (-GA) or stabilized (+GA) by the omission or addition of Gum Arabic (GA) as the capping agent. The characterization of the AuNPs was performed using Transmission Electron Microscopy (TEM), dynamic light scattering (DLS), Ultraviolet-Visual spectroscopy (UV-Vis), Thermogravimetric Analysis (TGA), X-Ray Diffraction (XRD) and Zeta-potential. The MBIC50 values for HO-MeOH - GA and HO-MeOH + GA were 1.79 ± 0.78% v/v and 0.22 ± 0.16% v/v, respectively. The HO-Powder AuNPs showed potent inhibition of C. acnes cell adhesion to the 96-well plates. The HO-MeOH - GA and HO-Powder + GA exhibited IC50 of 22.01 ± 6.13% v/v and 11.78 ± 1.78% v/v, respectively. The activity of the AuNPs validated the anti-adhesion activity of the methanol extract in the crude form. The study emphasizes the selectivity of H. odoratissimum AuNPs for the prevention of C. acnes cell adhesion and not antimicrobial activity, which may prevent the emergence of resistant strains of C. acnes through reduced bactericidal or bacteriostatic activity, while targeting mechanisms of pathogenesis.

Exploring the Anti-Acne Potential of Impepho [Helichrysum odoratissimum (L.) Sweet] to Combat Cutibacterium acnes Virulence.

The Gram-positive bacterium Cutibacterium acnes (previously Propionibacterium acnes), plays an important role in the pathogenesis and progression of the dermatological skin disorder acne vulgaris. The methanolic extract of Helichrysum odoratissimum (L.) Sweet (HO-MeOH) was investigated for its ability to target bacterial growth and pathogenic virulence factors associated with acne progression. The gas chromatography-mass spectrometry (GC-MS) analysis of HO-MeOH identified α-humulene (3.94%), α-curcumene (3.74%), and caryophyllene (8.12%) as major constituents, which correlated with previous reports of other Helichrysum species. The HO-MeOH extract exhibited potent antimicrobial activity against C. acnes (ATCC 6919) with a minimum inhibitory concentration (MIC) of 7.81 µg/ml. It enhanced the antimicrobial activity of benzoyl peroxide (BPO). The extract showed high specificity against C. acnes cell aggregation at sub-inhibitory concentrations, preventing biofilm formation. Mature C. acnes biofilms were disrupted at a sub-inhibitory concentration of 3.91 µg/ml. At 100 µg/ml, HO-MeOH reduced interleukin-1α (IL-1α) cytokine levels in C. acnes-induced human keratinocytes (HaCaT) by 11.08%, highlighting its potential as a comedolytic agent for the treatment of comedonal acne. The extract exhibited a 50% inhibitory concentration (IC50) of 157.50 µg/ml against lipase enzyme activity, an enzyme responsible for sebum degradation, ultimately causing inflammation. The extract's anti-inflammatory activity was tested against various targets associated with inflammatory activation by the bacterium. The extract inhibited pro-inflammatory cytokine levels of IL-8 by 48.31% when compared to C. acnes-induced HaCaT cells at 7.81 µg/ml. It exhibited cyclooxygenase-II (COX-II) enzyme inhibition with an IC50 of 22.87 µg/ml. Intracellular nitric oxide (NO) was inhibited by 40.39% at 7.81 µg/ml when compared with NO production in lipopolysaccharide (LPS)-induced RAW264.7 cells. The intracellular NO inhibition was potentially due to the 2.14 fold reduction of inducible nitric oxide synthase (iNOS) gene expression. The HO-MeOH extract exhibited an IC50 of 145.45 µg/ml against virulent hyaluronidase enzyme activity, which is responsible for hyaluronan degradation and scar formation. This study provides scientific validation for the traditional use of H. odoratissimum as an ointment for pimples, not only due to its ability to control C. acnes proliferation but also due to its inhibitory activity on various targets associated with bacterial virulence leading to acne progression.

Sideritis Perfoliata (Subsp. Perfoliata) Nutritive Value and Its Potential Medicinal Properties.

Sideritis perfoliata L. subsp. perfoliata is an endemic species of the Eastern Mediterranean region with several uses in traditional medicine. The present study aims to explore the unknown properties of S. perfoliata investigating the nutritional content as well as the antioxidant, anticancer, antituberculosis, antiwrinkle, anti-acne, hyper/hypo-pigmentation and antibacterial activities. Mineral content, nutritional value, the composition and antioxidant properties of the essential oil, the antityrosinase, the antibacterial activity and anti-elastase potential of the extract, were evaluated. The antiproliferative activity of S. perfoliata against cervical cancer (HeLa), human melanoma (UCT-Mel-1), human hepatocellular carcinoma (HepG2) and human epidermoid carcinoma (A431) was investigated. Cytotoxic effects on normal human keratinocyte (HaCat) and kidney epithelial (Vero) cell lines were also determined. Sideritis perfoliata exhibited high nutritional value of proteins and minerals (K, P, Mg, Fe, Zn, Cu). The most abundant components of the essential oil were found to be α-pinene, ß-phelladrene, valeranone, ß-pinene and sabinene. The ethanolic extract of S. perfoliata displayed moderate antioxidant potential and antibacterial activity against Prevotella intermedia. Noteworthy elastase and moderate anticancer potential against the human liver cancer cell line (HepG2) was observed with IC50 values of 57.18 ± 3.22 µg/mL and 64.27 ± 2.04 µg/mL respectively. The noteworthy in vitro activity of S. perfoliata could be due to the presence of flavonoids and phenols in the leaves, having high nutritional value. Sideritis perfoliata could potentially be useful to reduce the appearance of wrinkles and for the treatment of liver cancer. The moderate antibacterial, antioxidant and elastase activity of the plant can be linked to the traditional use of S. perfoliata for the treatment of wounds and inflammation.

Viability Reagent, PrestoBlue, in Comparison with Other Available Reagents, Utilized in Cytotoxicity and Antimicrobial Assays.

This study compared different commercially available viability reagents. The growth indicator reagents include p-iodonitrotetrazolium violet (INT), PrestoBlue, and Alamar Blue which were used for antimicrobial analysis against Streptococcus mutans, Prevotella intermedia, Propionibacterium acnes, and Mycobacterium tuberculosis. PrestoBlue and Alamar Blue are resazurin based reagents that resulted in a quick and easily distinguishable colour change that allowed for visual readings. INT and Sodium 3'-[1-(phenyl amino-carbonyl)-3,4-tetrazolium]-bis-[4-methoxy-6-nitro] benzene sulfonic acid hydrate (XTT) are tetrazolium based reagents which are converted to a formazan dye in the presence of metabolically active mitochondria enzyme. For cell viability analysis, reagents XTT and PrestoBlue were compared. PrestoBlue was able to clearly indicate the minimum inhibitory concentration (MIC) of various positive drug controls on various microbial strains. PrestoBlue was also a good indicator of the 50% inhibitory concentration (IC50) of positive drug controls on various cell lines.