Cistus, Acacia, and Lemon verbena Valorization through Response Surface Methodology: Optimization Studies and Potential Application in the Pharmaceutical and Nutraceutical Industries.

Cistus ladanifer L., Acacia dealbata L., and Aloysia citrodora Paláu were subject to an optimization procedure for two extraction techniques (heat-assisted extraction (HAE) and ultrasound-assisted extraction (UAE)). The extracts were then analyzed by HPLC-DAD-ESI/MS for their phenolic profile (cistus-15 compounds, acacia-21 compounds, and lemon verbena-9 compounds). The response surface methodology was applied, considering four varying factors: ethanol percentage; extraction time; temperature/power; and S/L ratio, generating two responses (the major phenolic compound, or family of compounds, and the extraction yield). For cistus, both techniques optimized the extraction yield of punicalagins, with UAE proving to be the most efficient extraction method (3.22% ethanol, 22 min, 171 W, and 35 g/L). For acacia, HAE maximized the extraction of procyanidin (74% ethanol, 86 min, 24 °C, and 50 g/L), and UAE maximized the content of myricetin (65% ethanol, 8 min, 50 W, and 50 g/L). For lemon verbena, HAE favored the extraction of martynoside (13% ethanol, 96 min, 49 °C and 17 g/L) and forsythiaside UAE (94% ethanol, 25 min, 399 W, and 29 g/L). The optimal conditions for the extraction of compounds with high added value and potential for use in pharmaceuticals and nutraceuticals were defined.

Ungulates mitigate the effects of drought and shrub encroachment on the fire hazard of Mediterranean oak woodlands.

Climate change is increasing the frequency of droughts and the risk of severe wildfires, which can interact with shrub encroachment and browsing by wild ungulates. Wild ungulate populations are expanding due, among other factors, to favorable habitat changes resulting from land abandonment or land-use changes. Understanding how ungulate browsing interacts with drought to affect woody plant mortality, plant flammability, and fire hazard is especially relevant in the context of climate change and increasing frequency of wildfires. The aim of this study is to explore the combined effects of cumulative drought, shrub encroachment, and ungulate browsing on the fire hazard of Mediterranean oak woodlands in Portugal. In a long-term (18 years) ungulate fencing exclusion experiment that simulated land abandonment and management neglect, we investigated the population dynamics of the native shrub Cistus ladanifer, which naturally dominates the understory of woodlands and is browsed by ungulates, comparing areas with (no fencing) and without (fencing) wild ungulate browsing. We also modeled fire behavior in browsed and unbrowsed plots considering drought and nondrought scenarios. Specifically, we estimated C. ladanifer population density, biomass, and fuel load characteristics, which were used to model fire behavior in drought and nondrought scenarios. Overall, drought increased the proportion of dead C. ladanifer shrub individuals, which was higher in the browsed plots. Drought decreased the ratio of live to dead shrub plant material, increased total fuel loading, shrub stand flammability, and the modeled fire parameters, that is, rate of surface fire spread, fireline intensity, and flame length. However, total fuel load and fire hazard were lower in browsed than unbrowsed plots, both in drought and nondrought scenarios. Browsing also decreased the population density of living shrubs, halting shrub encroachment. Our study provides long-term experimental evidence showing the role of wild ungulates in mitigating drought effects on fire hazard in shrub-encroached Mediterranean oak woodlands. Our results also emphasize that the long-term effects of land abandonment can interact with climate change drivers, affecting wildfire hazard. This is particularly relevant given the increasing incidence of land abandonment.

Thymus mastichina (L.) L. and Cistus ladanifer L. for skin application: chemical characterization and in vitro bioactivity assessment.

ETHNOPHARMACOLOGICAL RELEVANCE: Thymus mastichina (L.) L. (TM) and Cistus ladanifer L. (CL) are two Portuguese autochthonous species with traditional skin application in folk medicine. TM is majorly known for its antiseptic and wound healing properties, as an external anti-inflammatory agent and for its application in folk cosmetics and hygiene products. Its use in acne vulgaris has also been reported. CL is traditionally used in remedies for wounds, ulcers and other skin ailments such as psoriasis and eczema. Its application has been found useful due to its anti-inflammatory, astringent, wound healing and antiseptic properties. AIM OF THE STUDY: With this work, we aimed to investigate relevant bioactivities related with the traditional application of TM and CL essential oils (EOs) and hydrolates (by-products of EO production) in skin ailments. Specifically their in vitro antioxidant, anti-inflammatory, cytotoxic, wound healing and antimicrobial properties were evaluated. The chemical composition of both EOs and respective hydrolates was also characterized. MATERIALS AND METHODS: Chemical characterization of EOs and hydrolates was performed by GC-FID and GC-MS. Cellular biocompatibility was evaluated using the MTT assay in macrophages (RAW 264.7) and fibroblasts (L929) cell lines. Anti-inflammatory activity was investigated by studying nitric oxide (NO) production by macrophages with Griess reagent. Wound healing potential was evaluated with the scratch-wound assay. The antioxidant potential was studied by the DPPH scavenging method. Antimicrobial activity was evaluated by broth microdilution assay against relevant microbial strains and skin pathogens, namely Staphylococcus aureus, Staphylococcus epidermidis, Cutibacterium acnes, Pseudomonas aeruginosa, Escherichia coli, Candida albicans and Aspergillus brasiliensis. RESULTS: The major compounds present in TM and CL EOs were 1,8-cineole and α-pinene, respectively. 1,8-cineole and E-pinocarveol were the major compounds in the correspondent hydrolates. CL EO presented the highest anti-inflammatory potential [EC50 = 0.002% (v/v)], still with significant cytotoxicity [IC50 = 0.012% (v/v)]. TM preparations presented anti-inflammatory potential, also presenting higher biocompatibility. The same profile was present on fibroblasts regarding biocompatibility of the tested preparations. CL EO and hydrolate increased fibroblasts' migration by 155.7% and 148.4%, respectively. TM hydrolate presented a milder activity than CL hydrolate, but wound healing potential was still present, increasing cell migration by 125.1%. All preparations presented poor antioxidant capacity. CL EO presented higher antimicrobial activity, with MICs ranging from 0.06% (v/v) to 2% (v/v), against different microorganisms. CONCLUSIONS: Anti-inflammatory and skin repairing potential were present for CL preparations. TM hydrolate presented an interesting biocompatible profile on both cell lines, also presenting anti-inflammatory potential. Furthermore, EOs from both species presented antimicrobial activity against a panel of different microorganisms. These in vitro bioactivities support some of their traditional skin applications, specifically regarding their antiseptic, wound healing and anti-inflammatory uses.

Phytochemical profile, in vivo anti-inflammatory and wound healing activities of the aqueous extract from aerial parts of Cistus ladanifer L.

Cistus ladanifer L. is a Mediterranean shrub, well recognized for its many pharmacological properties. The present study aimed to investigate the phytochemical profile, the in vivo anti-inflammatory and the wound healing activities of the aqueous extract from its aerial parts (AECL). The in vivo anti-inflammatory and the wound healing properties were evaluated by carrageenan-induced paw edema (CAECL) and burn wound in rats, respectively. The results showed that six compounds belonging to flavonoids and tannins were identified in the AECL extract. Moreover, the AECL administrated orally at a dose of 500 mg/kg significantly reduced edema with a percentage of 72 % in terms of inflammation inhibition, using indomethacin as reference drug. On the other hand, when administrated topically, both doses of CAECL cream (5 % and 10 %) manifested a significant anti-inflammatory action using diclofenac as reference drug with a percentage reduction of inflammation that reached 85 % (CAECL-10). As far as wound healing is concerned, the CAECL showed higher effect at a dose of 10 % (CAECL-10) producing a wound contraction of 96 %, similar to that of the reference drug. The findings of the present study indicate that the aqueous extract from aerial parts of Cistus ladanifer L. display strongly anti-inflammatory and wound healing effects to be advantageously used for medical purposes.

Influence of the Storage of Cistus ladanifer L. Bales from Mechanised Harvesting on the Essential Oil Yield and Qualitative Composition.

Cistus ladanifer is a Mediterranean native plant from which valuable products, such as essential oil, are obtained. Manual harvesting of the plants in wild shrublands is usual during short periods of time. Their mechanised harvesting could increase the volume of harvested plants and prevent fires, further storage of the plants collected being necessary. The objective of this work is to study the influence of the storage period of mechanically harvested bales on the essential oil yield and qualitative composition. The harvesting trials were carried out with an adapted commercial harvester baler and the storage of the bales was performed indoors during 1-7 days, 15-30 days and 100-120 days. Afterwards, the bales were crushed (30 mm) and distilled in a 30 litre stainless steel still with saturated steam (0.5 bar). The essential oil components were identified by GC-MS and quantified by GC-FID. The storage of mechanically harvested Cistus ladanifer does not decrease the oil yield of steam distillation on a pilot scale. However, it leads to differences in the quantitative composition of the essential oils, decreasing the total monoterpene compounds content and increasing that of oxygenated sesquiterpenes, especially when the biomass is stored for 100-120 days, without affecting its qualitative composition.

Fatty Acid Content and Composition of the Morphological Fractions of Cistus Ladanifer L. and Its Seasonal Variation.

Cistus ladanifer L. is a shrub from Cistaceae family, widespread in Mediterranean countries. Fatty acids (FA) have multiple roles in plants and are involved in adaption mechanisms to environmental conditions. This work evaluated the FA content and composition of each morphological fraction of C. ladanifer (leaves, stems, flower buds, flowers and seed heads) throughout a full year. Cistus ladanifer plants were collected in southern Portugal, during four consecutive seasons (18 plants/season), and the different morphological plant fractions (leaves, stems, flower buds, flowers and seed heads) were separated. Cistus ladanifer morphological fractions showed distinct FA compositions, being possible to discriminate three groups-the leaves that showed to be dominated by saturated FA (main 20:0) and contain branched-chain FA (iso-19:0 and iso-21:0); the stems that are composed mainly by SFA (main 22:0); and the reproductive organs that showed higher contents of polyunsaturated FA (PUFA) and the 16:0 as the main SFA. The FA composition of leaves changed over seasons, with replacement of the PUFA by monounsaturated FA and branched-chain FA during hot seasons. Regarding the other C. ladanifer morphological fractions, the FA composition was more stable over seasons, suggesting that leaves are more prone to adaptations to environmental changes.

Increasing the α-tocopherol content and lipid oxidative stability of meat through dietary Cistus ladanifer L. in lamb fed increasing levels of polyunsaturated fatty acid rich vegetable oils.

The aim of this study was to evaluate the effect of the dietary inclusion of Cistus ladanifer L. and vegetable oils on intramuscular fat composition, muscle antioxidant status and lipid oxidative stability, and explore the mechanism by which dietary Cistus ladanifer limits the lipid oxidation. Lambs were assigned 9 diets, corresponding to 3 levels of Cistus ladanifer (50, 100 and 200 g/kg DM) and 3 levels of oil (0, 40 and 80 g/kg DM). Oil supplementation increased highly peroxidizable fatty acid content and the peroxidizability index (P < .05). Muscle phenolic content, reducing ability and radical scavenging ability were not affected by Cistus ladanifer (P > .05). The highest level of Cistus ladanifer enhanced muscle resistance to lipid oxidation (P = .006) and increased α-tocopherol content (P = .005), reducing meat lipid oxidation over 7 days of storage (P < .001). Results showed that Cistus ladanifer has a protective effect against lipid oxidation through increasing α-tocopherol concentration in the muscle.

Inclusion of the aerial part and condensed tannin extract from Cistus ladanifer L. in lamb diets - Effects on growth performance, carcass and meat quality and fatty acid composition of intramuscular and subcutaneous fat.

Thirty-six lambs were used to evaluate the effect of three levels of Cistus ladanifer condensed tannins (CT; 0, 1.25 and 2.5%) and two ways of CT supply (C. ladanifer aerial parts and C. ladanifer CT extract) on lamb growth performance, carcass composition, meat quality and FA composition of intramuscular and subcutaneous fat. The basal diet was composed of dehydrated lucerne supplemented with 60 g/kg of soybean oil. The highest amount of CT (2.5%) had detrimental effects on growth performance, particularly when the aerial part of C. ladanifer was used. Conversely, 1.25% CT in diets did not affect growth performance and meat quality, and inclusion of C. ladanifer CT extract in diet at this level increased t11-18:1, but not c9,t11-18:2 content in fat. Feeding C. ladanifer CT extract might be a good approach for enhancing the nutritional value of ruminant fat, but further studies are needed to ensure upregulation of endogenous synthesis of c9,t11-18:2.

Pseudomonas edaphica sp. nov., isolated from rhizospheric soil of Cistus ladanifer L. in Spain.

During a study on biodiversity of bacteria inhabiting rhizospheric soil of rockrose (Cistus ladanifer L.), we isolated a strain coded RD25T in a soil from Northern Spain. The 16S rRNA gene sequence showed 99.5 % identity with respect to the closest related species Pseudomonas brenneri DSM15294T, and 99.4 % with respect to P. paralactis WS4672T. The following related Pseudomonas species showed 99.3 % or less identity, and therefore RD25T was classified within genus Pseudomonas. The phylogenetic analysis of 16S rRNA and the housekeeping genes rpoB, rpoD and gyrB suggested that this strain could be a novel species. The strain RD25T has several polar-subpolar flagella. It can grow at 36 °C, at 0-6 % NaCl concentration and a range of pH 5-9. Positive for arginine dihydrolase and urease production, and negative for reduction of nitrate. The strain is catalase and oxidase positive. Major fatty acids are C16 : 1 ω7c / C16 : 1 ω6c in summed feature 3, C16 : 0, and C18 : 1 ω7c / C18 : 1 ω6c in summed feature 8. The respiratory ubiquinone is Q9. The DNA G+C content was 59.9 mol%. The digital DNA-DNA hybridisation average values (dDDH) ranged between 30-61.2 % relatedness and the ANIb values ranged between 93.9-80.5 % with respect to the type strains of the closely related species. Therefore, the genotypic, genomic, phenotypic and chemotaxonomic data support the classification of strain RD25 as a novel species of genus Pseudomonas, for which the name P. edaphica sp. nov. is proposed. The type strain is RD25T (=LMG 30152T=CECT 9373T).

Quantitative Variation of Flavonoids and Diterpenes in Leaves and Stems of Cistus ladanifer L. at Different Ages.

The compounds derived from secondary metabolism in plants perform a variety of ecological functions, providing the plant with resistance to biotic and abiotic factors. The basal levels of these metabolites for each organ, tissue or cell type depend on the development stage of the plant and they may be modified as a response to biotic and/or abiotic stress. As a consequence, the resistance state of a plant may vary in space and time. The secondary metabolites of Cistus ladanifer have been quantified in leaves and stems throughout autumn, winter, spring and summer, and at different ages of the plant. This study shows that there are significant differences between young leaves, mature leaves and stems, and between individuals of different ages. Young leaves show significantly greater synthesis of flavonoids and diterpenes than mature leaves and stems, with a clear seasonal variation, and the differences between leaves at different growth stages and stems is maintained during the quantified seasons. With respect to age, specimens under one year of age secreted significantly lower amounts of compounds. The variation in the composition of secondary metabolites between different parts of the plant, the season and the variations in age may determine the interactions of Cistus ladanifer with the biotic and abiotic factors to which it is exposed.

Seasonal Variation of Cistus ladanifer L. Diterpenes.

The exudate of Cistus ladanifer L. consists mainly of two families of secondary metabolites: flavonoids and diterpenes. The amount of flavonoids present in the leaves has a marked seasonal variation, being maximum in summer and minimum in winter. In the present study, we demonstrate that the amount of diterpenes varies seasonally, but with a different pattern: maximum concentration in winter and minimum in spring-summer. The experiments under controlled conditions have shown that temperature influences diterpene production, and in particular, low temperatures. Given this pattern, the functions that these compounds perform in C. ladanifer are probably different.