Natural products have many healing effects on the skin with minimal or no adverse effects. In this study, we analyzed the regenerative properties of a waste product (hydrolate) derived from Helichrysum italicum (HH) on scratch-tested skin cell populations seeded on a fluidic culture system. Helichrysum italicum has always been recognized in the traditional medicine of Mediterranean countries for its wide pharmacological activities. We recreated skin physiology with a bioreactor that mimics skin stem cell (SSCs) and fibroblast (HFF1) communication as in vivo skin layers. Dynamic culture models represent an essential instrument for recreating and preserving the complex multicellular organization and interactions of the cellular microenvironment. Both cell types were exposed to two different concentrations of HH after the scratch assay and were compared to untreated control cells. Collagen is the constituent of many wound care products that act directly on the damaged wound environment. We analyzed the role played by HH in stimulating collagen production during tissue repair, both in static and dynamic culture conditions, by a confocal microscopic analysis. In addition, we performed a gene expression analysis that revealed the activation of a molecular program of stemness in treated skin stem cells. Altogether, our results indicate a future translational application of this natural extract to support skin regeneration and define a new protocol to recreate a dynamic process of healing.

OBJECTIVE: An in-vitro study was performed to investigate the molecular basis of the wound healing and skin protective features of Helichrysum italicum (HI), a medicinal plant from the Mediterranean basin.
METHODS: A dermal fibroblast cell line culture was treated with HI hydro-alcoholic extract to detect the gene expression levels of three selected primers: FGF-2, HAS-2 and MMP-9. Cell proliferation assay was performed using a XTT reagent. RNA isolations were carried out from both the extract treated study cell group and the control cell group using a TRI reagent. GAPDH was used as the reference gene. Gene expressions were determined by real time RT-qPCR. The results were represented as \'Target/GAPDH Fold Change\'. Statistical evaluation was performed by Student\'s t test.
RESULTS: HI extract caused statistically significant upregulation of FGF-2 (P=0.0473) and HAS-2 (P=0.0335) gene expressions compared to the untreated control cells. The treatment ended with 1.74 and 3.10 fold changes for FGF-2 and HAS-2, respectively.
CONCLUSIONS: In general, it may be considered that HI has certain anabolic effects on the extracellular matrix of the skin because of the significant increases it causes in FGF-2 and HAS-2. Therefore, it may have a promising future in anti-aging studies and cosmetic dermatology. The results obtained in this study may also partially explain the molecular basis of the health benefits of HI on skin, including improvement in wound healing, and protection against the detrimental effects of ultraviolet exposure.

Wound healing is a dynamic process involving different cell types with distinct roles according to the stages of healing. Fibroblasts and stem cells actively participate in tissue regeneration. A proper stimulation could contribute to enhance wound healing process-es. Helichrysum italicum (H. italicum) is a medical plant well described for its pharmacological, antimicrobial, and anti-inflammatory activities. Aim of the present work was to examine the effect of the hydrolat derivate from H. italicum on stem cells isolated from skin and fibroblasts in vitro in presence or absence of tissue damage. The viability and proliferation of all cell types cultured in dif-ferent conditions were analyzed by MTT and BrdU assays. Cell proliferation after wound was analyzed with scratch test. Also, the expression of the main genes involved in tissue repair was evaluated by RT-qPCR analysis. Here we describe the capability of hy-drolat of H. italicum to promote tissue regeneration after scratch test both in stem cells and in fibroblasts. Moreover, the gene ex-pression analysis revealed that, hydrolat of H. italicum is also able to enhance stemness related. In conclusion our results are en-couraging, highlighting novel regenerative properties of hydrolat of H. italicum and paving the way for future application of this wasting product in accelerating wound healing.

The aim of this study is to investigate how climate change influences the distribution of economically and environmentally important species of P. abyssinica and H. citrispinum in Ethiopia. The species distribution modeling intends to forecast species\' ecological niche ranges and habitat suitability by employing a variety of environmental parameters as predictors, which is vital for conservation planning and restoration success. Six representative concentration pathways (RCP 2.6, 4.5, and 8.5 for the years 2050 and 2070) with the same resolution of 2.5 min that shows the emission scenarios were used for the prediction. To predict the current and future distributions of H. citrispinum and P. abyssinica 56 and 45 occurrence records from National Herbarium, Addis Ababa University, GBIF, and available literatures were used respectively.
The MaxEnt model predicted habitat suitability for H. citrispinum species with an Area Under Curve (AUC) value of 0.961 ± 0.027, and 0.809 ± 0.045 for P. abyssinica, indicating excellent discriminatory ability or accuracy under the current climate scenario. The Future distribution of suitable habitat for both H. citrispinum and P. abyssinica plant species was accurately predicted with AUC values of 0.960 ± 0.017 and 0.780 ± 0.35, respectively under future climatic scenarios. The jackknife test result indicates that environmental variables such as topographic position index (92.5%), precipitation of the driest quarter (3%) and precipitation in the coldest quarter (1.8%) are associated with the distributions of H. citrispinum, while topographic position index (36.6%), precipitation of driest quarter (21.4%), precipitation of warmest quarter (16.2%) and precipitation seasonality (13.9%) were found to be limiting environmental variables for P. abyssinica under current and future climatic conditions in Ethiopia. The prediction map and interception calculation for both present and projected (in the 2050s and again in the 2070s) climate change scenarios indicate significant habitat loss, decreased, and fragmentation under all RCPs (2.6, 4.5, and 8.5) scenarios for P. abyssinica while habitat gain, and increasing for H. citrispinum in Ethiopia.
Topographic position index (TPI) is the most impactful predictor variable on the distribution of the two species. Consequently, potentially habitable areas (with diverse aspects and slopes) are increasing for H. citrispinum while decreasing for P. abyssinica.

Helichrysum italicum has piqued the interest of many researchers in recent years, mostly for its essential oil, but increasingly for its polyphenolic content as well. In the current study, we examine the polyphenolic composition of H. italicum grown in Bulgaria. The polyphenolic complex was fractionated with solvents of various polarities, including hexane, chloroform, ethyl acetate, and butanol, in order to assess the biological impact of the components. HPLC-PDA and UHPLC-MS/MS were used to examine all fractions. The green coffee fingerprint profile was employed as a \"surrogate standard\" in the polyphenolic components detection approach. From the UHPLC-MS/MS analysis, we identified 60 components of the polyphenolic complex such as quercetin 3-O-glucuronide, quercetin acetyl-glycoside, isorhamnetin acetyl-glycoside, isorhamnetin caffeoyl-glycoside, quercetin caffeoyl-malonyl-glycoside, isorhamnetin coumaroyl-glycoside, coumaroyl-caffeoylquinic acid, and diCQA-acetyl-derivative were first reported in the composition of H. italicum. The biological activity of the fractions was evaluated in vitro and in silico, which included the fight against oxidative stress (hydrogen peroxide scavenging activity (HPSA), hydroxyl radical scavenging activity (HRSA), metal-chelating activity (MChA)) and nitrosative (nitric oxide scavenging activity) (NOSA)), in vitro anti-inflammatory, and anti-arthritic activity. Results are presented as IC50 ± SD μg/mL. The analysis showed that the EtOAc fraction was characterized by highest HPSA (57.12 ± 1.14 μg/mL), HRSA (92.23 ± 1.10 μg/mL), MChA (5.60 ± 0.17 μg/mL), and NOSA (89.81 ± 2.09 μg/mL), while the hexane and chloroform fractions showed significantly higher in vitro anti-inflammatory activity (30.48 ± 2.33 μg/mL, 62.50 ± 1.69 μg/mL) compared to the standard ibuprofen. All three fractions showed potential anti-arthritic activity (102.93 ± 8.62 μg/mL, 108.92 ± 4.42 μg/mL, 84.19 ± 3.89 μg/mL).

Helichrysum stoechas is a singular halophyte that has been shown to have anti-inflammatory, antioxidant, and allelopathic properties. In the work presented herein, we have characterized its inflorescences hydromethanolic extract and assessed its antifungal activity for the pre- and postharvest management of tomato crop diseases. Gas chromatography-mass spectrometry characterization of the extract showed that 4-ethenyl-1,3-benzenediol, 2,3-dihydro-benzofuran, quinic acid, 3,5-dihydroxy-6,7,8-trimethoxy-2-phenyl-4H-1-benzopyran-4-one, 1,6-anhydro-β-D-glucopyranose, catechol, scopoletin, and maltol were the main constituents. The co-occurrence of pyranones, benzenediols, and quinic acids as phytoconstituents of H. stoechas extract resulted in promising in vitro minimum inhibitory concentrations of 500, 375, 500, 187.5, 187.5, and 375 μg·mL-1 against mycelia of Alternaria alternata, Colletotrichum coccodes, Fusarium oxysporum f. sp. lycopersici, Rhizoctonia solani, Sclerotinia sclerotiorum, and Verticillium dahliae, respectively. Further, to assess the potential of H. stoechas inflorescence extract for postharvest tomato crop protection, ex situ tests were conducted against C. coccodes, obtaining high protection at a dose of 750 μg·mL-1. Taking into consideration that the demonstrated activity is among the highest reported to date for plant extracts and comparable to that of the synthetic fungicides tested as positive controls, H. stoechas inflorescence extract may be put forward as a promising biorational and may deserve further testing in field-scale studies.

Helichrysum italicum (Roth) G. Don., immortelle, is a plant species used in ethnomedicine and the food industry as a spice added to food, beverages, and bakery products. It has been shown to possess various biological activities, such as antioxidant and antibacterial activity, making it useful as a natural preservative. We investigated the phytochemical profile and biological activity of H. italicum essential oils from wild-grown plant material collected from natural habitats in the Republic of Croatia and Bosnia and Herzegovina. Using high-resolution scanning electron microscopy (SEM), a visual investigation of plant organs (stem, leaf, and flower) was performed, confirming the presence of essential oil reservoirs on the surface of all examined plant organs. Essential oils were isolated by hydrodistillation in the Clevenger apparatus. The chemical composition of the essential oils was determined using the GC-MS analytical technique. Cytotoxic activity tests were performed in vitro on three cell lines: skin (fibroblast), lung, and breast cancer. Using statistical tools, the synergistic and selective effects of H. italicum essential oil on healthy and tumor cells were correlated to chemical composition and cytotoxic activity. The synergistic and antagonistic effects of H. italicum essential oil\'s individual components were simulated by testing pure compounds and their mixture of cytotoxic activity on fibroblasts and breast cancer cells. The results confirm that essential oil\'s biological activity is much greater than the sum of the effects of its components. The present data are novel contributions to the body of knowledge on the biological activity of this species used in the food industry.

Silver nanoparticles (AgNPs), which have recently gained attention due to their antimicrobial activity, can also be produced by green synthesis. The aims of this study were to (i) characterise green synthesized AgNPs using microwave-assisted aqueous extracts of Galium aparine (G-AgNPs) and Helichrysum arenarium (H-AgNPs) and (ii) investigate the combined antimicrobial effects of the G- and H-AgNPs in different ratios. Nanoparticle formation and reactions were determined with UV-Vis spectroscopy. The G-AgNPs were 52.0±10.9 nm in size, with a 0.285±0.034 polydispersity index (PDI), and a -17.9±0.9 mV zeta potential. For H-AgNPs these characteristics were 23.9±1.0 nm, 0.280±0.032, and -21.3±2.7 mV, respectively. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) confirmed that the particles were monodisperse and spherical. The Fourier transform-infrared spectroscopy (FT-IR) results showed the presence of reducing agents that stabilised the AgNPs. Three different nanoformulations (NF-1, NF-2, and NF-3) were prepared by combining these two synthesised nanoparticles in different ratios and their antimicrobial activity was tested against E. coli, S. aureus, C. albicans, and A. flavus. Our study is the first to show that combining AgNPs from two different biological sources can produce effective nanoformulations with improved antibacterial activity against E. coli and S. aureus. These nanoformulations showed lower minimum inhibitory concentrations (31.25 µg/mL against E. coli with all NFs; 62.5 µg/mL for NF-1 and 125 µg/mL for NF-2/3 against S. aureus) than G-AgNPs (62.5 µg/mL for E. coli) or H-AgNPs (125 µg/mL for S. aureus) alone. Their high combined inhibitory effect against E. coli (NF-1-3) was synergistic and against S. aureus (NF-2 and NF-3) potentially additive. Considering such promising results, we believe our study provides some direction for new research and strategies in antimicrobial therapeutics.
Srebrne se nanočestice (AgNP), koje su već neko vrijeme u središtu pažnje zbog svojih antimikrobnih svojstava, mogu proizvoditi i zelenom sintezom. Cilj je ovog istraživanja bio (i) opisati (karakterizirati) zelenu sintezu različitih AgNP-a pomoću vodenih ekstrakata čekinjaste broćike (Galium aparine) (G-AgNPs) i pješčarskoga smilja (Helichrysum arenarium) (H-AgNPs), dobivenih metodom mikrovalne ekstrakcije, te (ii) utvrditi antimikrobno djelovanje kombinacije tih dvaju nanosustava u različitim omjerima. Oblikovanje nanočestica i kemijske reakcije utvrđene su pomoću UV-Vis spektroskopije. Veličina G-AgNP-a bila je 52,0±10,9 nm, njihov polidisperzivni indeks (PDI) 0,285±0,034, a zeta potencijal -17,9±0,9 mV. Osobine H-AgNP-a bile su sljedeće: veličina 23,9±1,0 nm, PDI 0,280±0,032, a zeta potencijal -21,3±2,7 mV. Mikroskopijom atomskih sila (engl. atomic force microscopy, krat. AFM) i pretražnom elektronskom mikroskopijom (engl. scanning electron microscopy, krat. SEM) potvrđeno je da su čestice monodisperzivne i sferične. Rezultati infracrvene spektroskopije s Fourierovom transformacijom (engl. Fourier transform-infrared spectroscopy, krat. FT-IR) potvrdili su prisutnost reduktivnih agenasa koji su stabilizirali srebrne nanočestice. Zatim su pripremljene tri formulacije nanočestica (NF-1, NF-2 i NF-3) kombinacijom sintetiziranih nanočestica u različitim omjerima, a njihova antimikrobna djelotvornost testirana je na mikroorganizmima E. coli, S. aureus, C. albicans i A. flavus. Naše je istraživanje prvo koje dokazuje da kombinacija srebrnih nanočestica dobivenih iz dvaju bioloških izvora može biti djelotvorna te da ima poboljšano antibakterijsko djelovanje protiv E. coli i S. aureus u odnosu na zasebne nanosustave. Minimalna inhibicijska koncentracija kombinacija iznosila je 31,25 µg/mL za E. coli u svim nanoformulacijama te 62,5 µg/mL za S. aureus s NF-1, odnosno 125 µg/mL s NF-2 i NF-3, a minimalne inhibicijske koncentracije G-AgNP-a odnosno H-AgNP-a zasebno su iznosile 62,5 µg/mL za E. coli (G-AgNP), odnosno 125 µg/mL za S. aureus (H-AgNP). To kombinirano antibakterijsko djelovanje protiv E. coli bilo je sinergijsko, a protiv S. aureus naizgled aditivno. S obzirom na ovako obećavajuće rezultate, smatramo da naše istraživanje daje smjer za razvoj novih strategija u antibakterijskom liječenju.

The Afromontane and Afroalpine areas constitute some of the main biodiversity hotspots of Africa. They are particularly rich in plant endemics, but the biogeographic origins and evolutionary processes leading to this outstanding diversity are poorly understood. We performed phylogenomic and biogeographic analyses of one of the most species-rich plant genera in these mountains, Helichrysum (Compositae-Gnaphalieae). Most previous studies have focused on Afroalpine elements of Eurasian origin, and the southern African origin of Helichrysum provides an interesting counterexample. We obtained a comprehensive nuclear dataset from 304 species (≈50% of the genus) using target-enrichment with the Compositae1061 probe set. Summary-coalescent and concatenation approaches combined with paralog recovery yielded congruent, well-resolved phylogenies. Ancestral range estimations revealed that Helichrysum originated in arid southern Africa, whereas the southern African grasslands were the source of most lineages that dispersed within and outside Africa. Colonization of the tropical Afromontane and Afroalpine areas occurred repeatedly throughout the Miocene-Pliocene. This timing coincides with mountain uplift and the onset of glacial cycles, which together may have facilitated both speciation and intermountain gene flow, contributing to the evolution of the Afroalpine flora.

The phytochemical investigation of the MeOH and CH2Cl2-MeOH (1:1) extracts from the flowers and twigs of Helichrysumfoetidum (L.) Moench (Asteraceae), which showed antileishmanial and antiplasmodial activities during the preliminary screening, led to the isolation of four undescribed compounds, including two ent-beyer-15-ene-type diterpenoids, foetidumins A (1) and B (2), one flavonoid, foetidumin C (3) and one chalcopyrone, foetidumin D (4). Additionally, fourteen known compounds comprising, two ent-beyer-15-ene-type diterpenoids (5-6), six flavonoids (7-12), two steroids (13-14), three triterpenoids (15-17), and one glyceryl monostearate (18) were also isolated. The chemical structures of foetidumins A-D were fully elucidated by analyses of their spectroscopic data. The structure and the stereochemistry of foetidumin A (1) were confirmed by SC-XRD analyses. Among the tested compounds, foetidumin C (3), erythroxylol A (6), and kaempferol (7) displayed the highest antileishmanial potency with IC50 values of 13.0, 11.8, and 11.1 μM, respectively. Foetidumin C (3) had no cytotoxicity toward Vero cells with the selectivity index > 3.59. Meanwhile, extracts of flowers and twigs had higher activity against Plasmodium falciparum chloroquine-sensitive (Pf3D7) strain with IC50 values of 3.66 and 10.52 μg/mL, respectively.