Achyrocline satureioides infusion, popularly prepared and consumed, has an in vitro protective effect on human neural cells exposed to rotenone.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional harvest of Achyrocline satureioides (AS) occurs at dawn on Good Friday in some South American countries. Inflorescences are traditionally used as infusions for several disorders, including neuropsychiatric disorders. Pillows and cushions are popularly filled with AS to attenuate the symptoms of depression, anxiety, and sleep disturbances. However, evidence for the potential beneficial effects of AS on human neural cells remains unclear. AIM OF THE STUDY: An in vitro model of SH-SY5Y human neural cells was applied to evaluate the effect of AS infusion, prepared as commonly used, on cells exposed to rotenone and to investigate its potential for neuropsychiatric disorders. MATERIALS AND METHODS: A hot aqueous extract was obtained from a traditionally prepared AS inflorescence infusion and chemically characterized by high-resolution mass spectrometry and spectrophotometric quantification of total polyphenols, tannins, and flavonoids. The SH-SY5Y cell cultures were treated with AS extract at concentrations of 1, 3, 5, 10, 50, 100, and 300 µL/mL to determine the potential cyto- and genotoxic effects of AS on neural cells using MTT, Neutral Red, and GEMO assays. Apoptosis modulation was assessed using flow cytometry and apoptosis-modulating genes were evaluated by qRT-PCR. The protective effect of AS on the neurotoxicity triggered by rotenone exposure (30 nM) was determined by analyzing cellular viability and oxidative markers such as lipid peroxidation and protein carbonylation, and DNA damage was assessed by micronucleus assay. RESULTS: The AS extract, as traditionally prepared, had estimated concentrations of 409.973 ± 31.107 µg/mL, 0.1041 ± 0.0246 mg GAE/mL, and 63.309 ± 3.178 mg QE/mL of total tannins, total polyphenols, and flavonoids, respectively. At concentrations of 30 and 100 µl/mL, AS decreased apoptotic events, whereas the highest concentration (300 µl/mL) increased apoptosis compared to that in the control (p < 0.05). In cells exposed to rotenone, AS treatment induced cell proliferation, reduced DNA damage (as evaluated by micronuclei), and reduced lipid and protein oxidation. CONCLUSIONS: The data indicate the non-cytotoxic and beneficial effects of AS extract on human neural cells by reducing cellular mortality and oxidative stress in neural cells triggered by rotenone exposure.

Achyrocline B (3,5 dihydroxy-6,7,8-trimethoxyflavone) synergizes with 5-fluorouracil allowing for dose reduction and reduced off-target toxicity in the treatment of colonic and pancreatic cancers.

Surgically unresectable colorectal and pancreatic carcinomas have a high rate of mortality as current therapeutic options are limited. One common chemotherapeutic used to broadly treat both cancers is 5-flurouracil (5-Fu); however, treatment serves only to slow progression of the disease and comes with many side effects due to 5-Fu's intrinsic toxicity. Thus, strategies to decrease the dose of 5-Fu utilized therapeutically as well as reduce 5-Fu's off-target toxicity are paramount. Using cell models of colorectal and pancreatic cancers, we show that cotreatment with Achyrocline B (3,5 dihydroxy-6,7,8-trimethoxyflavone, AcB), a natural flavone from Achyrocline bogotensis, allows for four-fold reduction in 5-Fu dosage without loss of efficacy. We further show that the action of AcB is due to continued cell cycle progression despite 5-Fu pressure to synchronize at the G1/S threshold. In addition to AcB's effect on cancer cells, we found that AcB can directly reduce toxicity of 5-Fu in cells mimicking non-cancerous tissues. These in vitro results are then supported by xenograft modeling. AcB was shown to increase apoptosis in tumors leading to degeneration of the outer tumoral boundary. Furthermore, in 5-Fu treated animals it was found that AcB provided protection to the intestinal tract as indicated by preserved histological and immunohistochemical features. These results show promise for a new adjuvant therapy for colorectal and pancreatic carcinomas that not only reduces tumor progression, but more importantly has the potential to improve patient quality of life.

Achyrocline satureioides as an adjuvant therapy for the management of mild viral respiratory infections in the context of COVID-19: Preliminary results of a randomized, placebo-controlled, and open-label clinical trial.

We evaluated the impact of an Achyrocline satureioides inflorescence infusion on the clinical outcomes of viral respiratory infections, including those caused by SARS-CoV-2, in a monocentric, randomized, open-label, placebo-controlled clinical trial. Patients with symptoms of viral respiratory infection, including suspected cases of COVID-19, were included and assigned to receive either A. satureioides (n = 57) or Malus domestica (n = 67) infusions twice a day for 14 days. All participants were included before the RT-PCR results, performed using a nasopharyngeal swab. The patients were further divided into subgroups according to real-time polymerase chain reaction results: SARS-CoV-2-positive and SARS-CoV-2-negative subgroups for statistical analyses. We assessed clinical outcomes, such as the latency to resolution of cough, dyspnea, fever, sore throat, chest pain, smell and taste dysfunctions, diarrhea, nausea, abdominal pain, and loss of appetite; hospitalization; and mortality with questionnaires and medical records. The subjects that received early A. satureioides infusion showed a significant reduction in the average number of days with respiratory and neurological symptoms compared with the control group (M. domestica infusion). We conclude that A. satureioides is a safe agent and, in combination with standard care, improves viral respiratory infection symptoms, especially those related to COVID-19.

Achyrocline satureioides review: From the pharmacochemical diversity to the technological development of products.

Achyrocline satureioides (Lam.) DC (Asteraceae) is a native species of the southeastern subtropical and temperate region of South America, popularly known as "marcela" or "macela". This species is recognized, in traditional medicine, by diverse biological activities such as digestive, antispasmodic, anti-inflammatory, antiviral, sedative, hepatoprotective, among others. Some of these activities have been related to the presence of phenolic compounds, including flavonoids, phenolic acids, terpenoids in the essential oils, coumarins and phloroglucinol derivatives reported to the species. The approaches on the technological development of phytopharmaceutical products of this species provided relevant advances in the optimization of the extraction and product obtention, especially spray-dried powders, hydrogels, ointments, granules, films, nanoemulsions and nanocapsules. The most relevant biological activities described for the extracts or derivative products from A. satureioides were antioxidant, neuroprotective, antidiabetic, antiobesity, antimicrobial, anticancer effects, and obstructive sleep apnea syndrome. The scientific and technological findings reported for the species, in conjunction with its traditional use and cultivation, reveal the high potential of the species for diverse industrial applications.

Hexanic extract of Achyrocline satureioides: antimicrobial activity and in vitro inhibitory effect on mechanisms related to the pathogenicity of Paenibacillus larvae.

INTRODUCTION: Paenibacillus larvae is a spore-forming bacillus, the most important bacterial pathogen of honeybee larvae and the causative agent of American foulbrood (AFB). Control measures are limited and represent a challenge for both beekeepers and researchers. For this reason, many studies focus on the search for alternative treatments based on natural products. AIM: The objective of this study was to determine the antimicrobial activity of the hexanic extract (HE) of Achyrocline satureioides on P. larvae and the inhibitory activity on some mechanisms related to pathogenicity. MATERIAL AND METHODS: The Minimum Inhibitory Concentration (MIC) of the HE was determined by the broth microdilution technique and the Minimum Bactericidal Concentration (MBC) by the microdrop technique. Swimming and swarming motility was evaluated in plates with 0.3 and 0.5% agar, respectively. Biofilm formation was evaluated and quantified by the Congo red and crystal violet method. The protease activity was evaluated by the qualitative technique on skim milk agar plates. RESULTS: It was determined that the MIC of the HE on four strains of P. larvae ranged between 0.3 and 9.37 µg/ml and the MBC between 1.17 and 150 µg/ml. On the other hand, sub-inhibitory concentrations of the HE were able to decrease swimming motility, biofilm formation and the proteases production of P. larvae.

Endophytic Fungus of Achyrocline satureioides: Molecular Identification, Chemical Characterization, and Cytotoxic Evaluation of its Metabolites in Human Melanoma cell line.

Endophytic fungi are important sources of anticancer compounds. An endophytic fungus was isolated from the medicinal plant Achyrocline satureioides, and molecularly identified as Biscogniauxia sp. (family Xylariaceae) based on partial nucleotide sequences of the internal transcribed spacer genomic region (GenBank Accession No. ON257911). The chemical characterization and cytotoxic properties of secondary metabolites produced by Biscogniauxia sp. were evaluated in a human melanoma cell line (A375). The fungus was grown in potato-dextrose liquid medium for 25 days, and the extracted compounds were subjected to solid-phase fractionation to obtain the purified FDCM fraction, for which the metabolites were elucidated via ultra-performance chromatography coupled to a mass spectrometer. In the present study, 17 secondary metabolites of Biscogniauxia sp., including nine polyketide derivatives, five terpenoids, and three isocoumarins, were putatively identified. This is the first study to report of the ability of Biscogniauxia sp. in the production of isocoumarin orthosporin; the terpenoids nigriterpene A and 10-xylariterpenoid; the polyketide derivatives daldinin C, 7'dechloro-5'-hydroxygriseofulvin, daldinone D, Sch-642305, curtachalasin A, cytochalasin E, epoxycytochalasins Z8, Z8 isomer, and Z17. Furthermore, this study has reported the biosynthesis of Sch-642305 by a Xylariaceae fungus for the first time. FDCM significantly reduced the viability and proliferation of human melanoma cells at half-maximal inhibitory concentrations ​​of 10.34 and 6.89 µg/mL, respectively, and induced late apoptosis/necrosis and cell cycle arrest in G2/M phase after 72 h of treatment. Given its ability to produce unique metabolites with promising cytotoxic effects, Biscogniauxia sp. of A. satureioides may be a reservoir of compounds with important therapeutic applications.

In vitro evaluation of nanocomposites of linseed mucilage and k-carrageenan loaded with Achyrocline satureioides nanoemulsion: a gradual-release candidate of antimicrobials for the treatment of bovine mastitis.

This research paper presents the development and evaluation of pioneering nanocomposites (NCs) based on the combination of k-carrageenan and linseed mucilage. When loaded with macela extract nanoemulsion they present an innovative approach for the sustained release of antimicrobial herbal constituents, specifically tailored for bovine mastitis treatment. The NCs, encompassing various ratios of k-carrageenan and linseed mucilage polymers (8:2, 7:3, and 5:5 w/w) with 1.25 mg of macela extract/g of gel, underwent in vitro assessment, emphasizing viscosity, degradation speed, release of herbal actives from macela nanoemulsion and antimicrobial activity. The NCs exhibited thermoreversible characteristics, transitioning from liquid at 60°C to a gel at 25°C. NCs allowed a gradual release of phenolic compounds, reaching approximately 80% of total phenolics release (w/v) within 72 h. NCs inhibited the growth of MRSA (ATCC 33592) until 8 h of incubation. No toxic effect in vitro of NCs was found on MAC-T cells. Thus, the developed materials are relevant for the treatment of bovine mastitis, especially in the dry period, and the data support future evaluations in vivo.

In Vitro Evaluation of Anti-Rotaviral Activity and Intestinal Toxicity of a Phytotherapeutic Prototype of Achyrocline bogotensis (Kunth) DC.

Viruses represent the primary etiologic agents (70-80%) of acute diarrheal disease (ADD), and rotavirus (RV) is the most relevant one. Currently, four rotavirus vaccines are available. However, these vaccines do not protect against emerging viral strains or are not available in low-income countries. To date, there are no approved drugs available against rotavirus infection. In this study, we evaluated the in vitro anti-rotaviral activity and intestinal toxicity of a phytotherapeutic prototype obtained from Achyrocline bogotensis (Kunth) DC. (PPAb); medicinal plant that contains compounds that inhibit the rotavirus replication cycle. Virucidal and viral yield reduction effects exerted by the PPAb were evaluated by immunocytochemistry and flow cytometry. Furthermore, the toxic impact of the PPAb was evaluated in polarized human intestinal epithelial C2BBe1 cells in terms of cytotoxicity, loss of cytoplasmic membrane asymmetry, and DNA fragmentation by MTT and fluorometry. PPAb concentrations under 0.49 mg/mL exerted significant virucidal and viral yield reduction activities, and concentrations under 16 mg/mL neither reduced cell viability, produced DNA fragmentation, nor compromised the C2BBe1cell membrane stability after 24-h incubation. Based on these results, the evaluated phytotherapeutic prototype of Achyrocline bogotensis might be considered as a promising alternative to treat ADD caused by rotavirus.

The association of extracts of Achyrocline satureioides and the fungus Beauveria bassiana against the tick Rhipicephalus microplus.

The tick Rhipicephalus microplus (Canestrini) is a major economic hurdle to the global livestock industry. The incorrect and indiscriminate control carried out by synthetic chemical compounds has contributed to the emergence of parasite resistance present today to many products available in the veterinary market. Effective, economically viable and potentially safe alternatives need to be investigated, including herbal medicine and biological control by entomopathogenic fungi. In this study, extracts from Achyrocline satureioides (Lam.) DC. obtained by extraction with solvents of different polarities associated and not associated with the entomopathogenic fungus Beauveria bassiana (Balsamo-Crivelli) Vuillemin were evaluated on different stages of the life cycle of R. microplus. The activity of plant extracts, fungus and their associations in ticks was analyzed in vitro through the adult immersion test, larval packet test and larval immersion test. The highest efficacy on engorged females (43.3%) was reached by the hexanic partition (ASh) at 5 mg/mL + fungus, reducing larval hatchability. ASh associated with B. bassiana also caused high mortality of larvae, reaching LC50 of 6.23 and 2.65 mg/mL in the packet and immersion tests, respectively. The results obtained showed that extracts, when used in combination with the fungus, have their action enhanced, reaching 100% mortality of larvae. Among the evaluated methodologies, it was observed that the larval immersion test allows better assessment, due to the time of contact with the larvae. The results obtained with A. satureioides associated with the fungus are considered promising and open new perspectives for future product development.

Acaricidal and anthelmintic action of ethanolic extract and essential oil of Achyrocline satureioides.

Although Brazilian livestock is considered one of the most productive in the world, the losses caused by parasites still greatly limit these activities and its incorrect control through traditional synthetic chemicals has caused problems such as parasitic resistance. The present study evaluated the anthelmintic and acaricidal action of ethanolic extract (EE) and essential oil (OE) obtained from Achyrocline satureioides inflorescences against an endo and an ectoparasite of great importance in sheep and cattle. The action against Haemonchus contortus was evaluated by the Larval Development Test (LDT) and the Egg Hatching Test (EHT), while the acaricidal potential against Rhipicephalus (Boophilus) microplus was evaluated by the Adult Immersion Test (AIT) and Larval Package Test (LPT). Chemical analyzes were performed by liquid chromatography coupled to mass of spectrometry (LC-MS) and gas chromatography coupled to mass spectrometry (GC-MS) in order to evaluate the chemical composition of EE and EO, respectively. Luteolin (m/z 285), quercetin (m/z 301) and methyl quercetin (m/z 315) were the major compounds detected in the A. satureioides EE, while 41 compounds were identified in EO. Among them, the major compounds were caryophyllene oxide (15.4%), limonene (10.9%), trans-calamenene + Î´-cadinene (8.0%), (E)-caryophyllene (5.5%), spathulenol (5.1%) and α-pinene (4.5%). Against H. contortus, by EHT, the EE and EO showed LC50 of 83.14 mg mL-1 and 10.42 mg mL-1, respectively. In LDT, LC50 were 0.16 mg mL-1 and 0.42 mg mL-1, respectively by EE and EO. In the AIT assay for R. (B.) microplus tick LC50 obtained for EE and EO were 76.43 mg mL-1 and 114.35 mg mL-1, respectively. EE at 100 mg mL-1 completely inhibited egg laying, while the EO at 50 mg mL-1 completely inhibited larval hatching. No significant larvicidal effect was observed in LPT for both extracts. The EE and EO presented a high efficacy at low concentrations in the inhibition of egg hatching and larval development of H. contortus. Although both extracts did not show consistent larvicidal action, the results were significant in inhibiting the oviposition of R. (B.) microplus engorged females.

Fractionation of hexane extracts from Achyrocline satureioides and their biological activities against Paenibacillus larvae.

Previous studies carried out in our laboratory described the antimicrobial activity of the whole hexanic extract (HE) of Achyrocline satureioides (Lam.) DC against Paenibacillus larvae, the causal agent of American Foulbrood (AFB) a disease of the honey bee larvae. In this study, the HE was partitioned into five main fractions by chromatographic techniques leading to the isolation of four known compounds: two prenylated phloroglucinol α-pyrones (1 and 3), 5,7-dihydroxy-3,8-dimethoxyflavone (gnaphaliin A) (2), and 23-methyl-6-O-demethylauricepyrone (4). Isolated compounds were further analyzed towards structural elucidation using 1H RMN and 13C RMN spectroscopic techniques. For the first time, the antimicrobial activity of the isolated compounds was evaluated against P. larvae strains by broth microdilution method and compared with that of the whole HE. Compounds 1-4 displayed minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values ranging between 0.07 and 62.5 µg/mL and 0.26 and 12.5 µg/mL, respectively. The lowest MIC and MBC values were obtained with compounds 3 and 4, respectively. The antimicrobial activity of each single compound and the combination of them showed that the presence of all compounds is needed for the antimicrobial efficacy of whole HE.

Achyrocline satureioides (Lam) DC extracts acting as enzyme modulators: digestion, inflammation, and hemostasis / Extratos de Achyrocline satureioides (Lam) DC atuando como moduladores enzimáticos: digestão, inflamação e hemostasia

Achyrocline satureioides is popularly known for its richness in phenolic compounds and medicinal properties (anti-inflammatory, analgesic, and hepatoprotective). The present study aimed at broadening the knowledge about the pharmacological potential exerted by the aqueous and ethanolic extracts of A. satureioides. These extracts were characterized by HPLC and tested for their modulatory action on phospholipases A2 and proteases of snake venoms. In addition, they were tested on the activities of digestive enzymes. Snake venoms were used as tools since they have enzymes with high functional and structural homology to human enzymes. The results demonstrate that the extracts of A. satureioides act as enzymatic inhibitors or potentiators, interfering in processes related to the hemostasis, such as coagulation and thrombus dissolution. In addition, the anti-genotoxic activity and inhibitions exerted on digestive enzymes suggests their potential use in the prevention and/or treatment of several pathologies. New studies could provide information on how the compounds present in the extracts and the different enzymes interact.

A Achyrocline satureioides é popularmente conhecida por sua riqueza em compostos fenólicos e por suas propriedades medicinais (anti-inflamatória, analgésica e hepatoprotetora). No presente estudo, com o objetivo de ampliar o conhecimento sobre o potencial farmacológico exercido por esses extratos, os extratos aquoso e etanólico de A. satureioides foram caracterizados por HPLC e testados quanto à sua ação modulatória sobre as fosfolipases A2 e proteases de peçonhas de serpentes. Além disso, também foram testados em atividades de enzimas digestivas. As peçonhas de serpentes foram usadas como ferramentas por apresentarem enzimas com alta homologia funcional e estrutural às humanas. Os resultados demonstram que os extratos de A. satureioides atuam como inibidores ou potencializadores enzimáticos, interferindo em processos relacionados à hemostasia, como coagulação e dissolução do trombo. Além do mais, destacam seu potencial antigenotóxico e as inibições exercidas sobre as enzimas digestivas direcionando seu potencial de uso na prevenção e/ou tratamento de diversas patologias. Novos estudos poderão fornecer informações sobre os mecanismos de interação entre os compostos presentes nos extratos e as diferentes enzimas.

Antimycobacterial activity of Achyrocline flaccida (Asteraceae) aqueous extract from Southern Brazil.

Achyrocline flaccida aqueous extract was obtained by macerating wildflowers. The phytochemical profile present in the A. flaccida aqueous extract was elucidated by HPLC-ESI-MS/MS. Toxicity was evaluated in vitro by comet assay in peripheral blood mononuclear cells (PBMCs) and in vivo using Caenorhabditis elegans as a model. The antioxidant activity was also evaluated, and antimycobacterial activity was assessed by the broth microdilution method. The compounds present in the aqueous extract mainly belonged to the flavonoid class (89%). The concentrations that showed protective effects in C. elegans against oxidative stress and antimycobacterial activity had no toxic effects. The antimycobacterial activity test demonstrated that the concentration of 1,560 µg mL-1 inhibited the growth and eradication of the mycobacterial tested strains. Based on our findings, the A. flaccida aqueous extract presents a viable potential in developing new phytotherapeutic drugs against mycobacteria of clinical relevance.

Achyrophenols A-F: Polycyclic Polyphenol Lactone Skeletons and a Nor-Ursane-Type Triterpenoid from Achyrocline Satureioides.

Two novel derivatives of 2-methyl-1-(2,3,4,6-tetrahydroxyphenyl)propan-1-one and (S)-2-methyl-1-(2,3,4,6-tetrahydroxyphenyl)butan-1-one (1 and 2), four novel six-membered lactone phenols (3-6), and a nor-ursane type triterpenoid (7) named Achroacid, were isolated from the aerial part of Achyrocline satureioides. The absolute configurations of 1-7 are presented by spectroscopic data and X-ray crystallographic analysis. A DP4plus evaluation was applied to determine the final stereochemistry for 1 and 2. The biosynthesis pathway of 1 and 2 was proposed. 1 has potential on anti-Gram-negative bacteria. Both 1 and 2 exhibited a significant impact on anti-H1299 cells. Compounds 3-7 showed moderate cancer cell lethality and significant anti-inflammatory activities.

3-O-Methylquercetin from Achyrocline satureioides-cytotoxic activity against A375-derived human melanoma cell lines and its incorporation into cyclodextrins-hydrogels for topical administration.

3-O-Methylquercetin (3OMQ), a natural 3-O-methylflavonoid, was isolated from Achyrocline satureioides and purified using the high-performance counter current chromatography (HPCCC) on a semi-preparative scale. High-purity 3OMQ (98%) was obtained with excellent recovery (81.8% (w/w)) and good yield (190 mg/100 g of plant). Isolated 3OMQ was evaluated against the A375 human amelanotic melanoma cancer cell line and A375-derived with different degrees of aggressiveness (A375-A7, A375-G10, and A375-PCDNA3). The results showed that 3OMQ reduced the cell viability of all strains, demonstrating time- and dose-dependent responses. 3OMQ was used to obtain hydrogels for the topical treatment of melanoma. Thus, 3OMQ was incorporated into hypromellose hydrogels with/without different cyclodextrins (CDs). The 3OMQ formulations showed permeation/retention in all skin layers, namely stratum corneum, epidermis, and dermis. A significant amount of 3OMQ was found in the replication site of the melanoma cells (epidermis and dermis). Altogether, these results demonstrate that 3OMQ can be isolated from Achyrocline satureioides by HPCCC on a semi-preparative scale and exhibit cytotoxic activity against melanoma cells. Its incorporation into an HPMC hydrogel containing HP-ß-CD yielded a formulation with excellent technological and biopharmaceutical characteristics for evaluating the topical management of melanoma.

Achyrocline flaccida essential oil from Brazil: phytochemical composition, genotoxicity, protective effects on Caenorhabditis elegans, and antimycobacterial activity.

Achyrocline flaccida (Weinm) DC. essential oil was obtained by hydrodistillation of wild flowers from southern Brazil. We explored, for the first time, the phytochemical composition, toxicity, resistance to oxidative stress in Caenorhabditis elegans, and antimycobacterial activities of A. flaccida essential oil. Twenty-four compounds were identified by GC-FID-MS and the major constituents identified were α-pinene (41.10%) and caryophyllene (30.52%). The essential oil showed no signs of genotoxicity in the comet assay and presented relevant antioxidant capacity since it prevented ROS production in the C. elegans model. Furthermore, the minimal inhibitory concentrations (MIC) results showed that M. abscessus, M. massiliense, and M. fortuitum had their growth inhibited by A. flaccida essential oil. Therefore, the essential oil of this plant is a promising alternative in the search for new compounds capable of decreasing oxidative stress and treating mycobacteriosis.

Topical hydrogel containing Achyrocline satureioides oily extract (free and nanocapsule) has anti-inflammatory effects and thereby minimizes irritant contact dermatitis.

Inflammatory dermatoses are prevalent worldwide, with impacts on the quality of life of patients and their families. The aim of this study was to determine the anti-inflammatory effects of Achyrocline satureioides oily extracts and nanocapsules on the skin using a mouse model of irritant contact dermatitis induced by croton oil, and a skin inflammation model induced by ultraviolet B (UVB) radiation. The mice were treated with 15 mg/ear oily extract (HG-OLAS) or nanocapsules (HG-NCAS) of A. satureioides incorporated into Carbopol® 940 hydrogels. We found that HG-OLAS and HG-NCAS formulations reduced ear edema in croton oil-induced lesions with maximum inhibitions of 54±7% and 74±3%, respectively. HG-OLAS and HG-NCAS formulations decreased ear edema induced by UVB radiation (0.5 J/cm2), with maximum inhibitions of 68±6% and 76±2% compared to the UVB radiation group, respectively. HG-OLAS and HG-NCAS modulated myeloperoxidase (MPO) activity after croton oil induction. Furthermore, croton oil and UVB radiation for 6 and 24 h, respectively, stimulated polymorphonuclear cells infiltration. The topical treatments reduced inflammatory processes, as shown by histological analysis. Together, the data suggest that topical application of A. satureioides oily extracts and nanocapsules produced antiedematogenic and anti-inflammatory effects. They constitute a compelling alternative for treatment of skin injuries.

A stability-indicating ultra-fast liquid chromatography method for the assay of the main flavonoids of Achyrocline satureioides (Marcela) in porcine skin layers and nanoemulsions.

INTRODUCTION: Achyrocline satureioides (marcela or macela) is a plant widely used in folk medicine in South America. Recently, there has been increasing interest for the development of skin care products containing A. satureoides extracts, due to its well-documented antioxidant, antiherpetic, and wound healing properties. OBJECTIVES: The present study aimed to develop and validate a yet unexplored stability-indicating and robust ultra-fast liquid chromatography (UFLC) method for the simultaneous quantification of the main flavonoids of A. satureioides in extracts, nanoemulsions, and porcine skin layers. MATERIAL AND METHODS: The chromatographic separation of flavonoids quercetin, luteolin, and 3-O-methylquercetin was performed on a Luna C18 analytical column (100 mm × 2.0 mm i.d.; particle size 2.5 µm) using isocratic elution with methanol/phosphoric acid 1% (48:52 v/v) with a flow rate of 0.3 mL/min at 40°C. RESULTS: The method was found to be specific, linear (R > 0.998), precise, accurate, and robust for all flavonoids assayed in A. satureioides extract, nanoemulsions, and porcine ear skin. A low matrix effect was noted for all complex matrices. The stability-indicating UFLC method was evaluated by submitting isolated flavonoids, a mixture of standards, and A. satureioides extract to acidic, alkaline, oxidative, UV-A/UV-C light, and thermal stress conditions. No peaks were found co-eluting with the flavonoids of interest in all matrices. The robustness of the method was confirmed using Plackett-Burman experimental design. CONCLUSION: The short run time (8 min) and reliability of the method could be useful for the determination of A. satureioides flavonoids in topical product development since extracts of this medicinal plant have been used to treat various skin disorders.

Box-Behnken Design for Extraction Optimization Followed by High Performance Countercurrent Chromatography: Production of a Flavonoid-enriched Fraction from Achyrocline Satureioides.

The biological properties of Achyrocline satureioides have been mostly ascribed to its major flavonoids quercetin (QCT), luteolin (LUT), and 3-O-methylquercetin (3OMQ). The present study aimed to optimize the extraction by dynamic maceration of the major phenolic compounds in order to obtain in a subsequent step a flavonoid-enriched fraction (FEF) using high performance countercurrent chromatography (HPCCC). A 3-level Box-Behnken design (BBD) was applied to maximize the extraction of the substances, using the plant : solvent ratio (X1 ), extraction time (X2 ), and ethanol concentration (X3 ) as factors. One-step HPCCC semipreparative separation with a solvent system composed of hexane : ethyl acetate : methanol : water (0.9 : 0.9 : 0.8 : 1.0, v/v) was employed to obtain the FEF. The second-order polynomial model was able to fit the experimental data adequately. The linear and quadratic terms of X3 were the most significant factors that affected all the responses. The positive linear term of X3 indicated a substantial increase in extraction yield, while the negative quadratic term showed a nonlinear tendency. Linear terms of X1 suggested a tendency to solvent saturation, except for QCT. The terms of X2 did not affect the responses substantially. The HPCCC method was found to be efficient and rapid for separating the FEF with 71% (w/w) flavonoid content. Overall, the developed extraction procedure coupled with HPCCC proved to be efficient for obtaining an enriched fraction with a very high content of flavonoids from A. satureioides.