Toxicological assessment of the Achyrocline satureioides aqueous extract in the Caenorhabditis elegans alternative model.

Achyrocline satureioides, popularly called "marcela" in Brazil, is used in traditional medicine in South America. A. satureioides, inflorescences are used for many conditions, including to minimize the Sars-Cov-2 symptoms. Therefore, the aim of this study was to determine the toxicity profile of A. satureioides aqueous extract (ASAE), using the Caenorhabditis elegans (C. elegans) alternative model. Survival, reproduction, development, and transgenerational assays were performed. The effects of ASAE were investigated under conditions of thermal stress and presence of oxidant hydrogen peroxide (H2O2). In addition, C. elegans strains containing high antioxidant enzyme levels and elevated lineages of daf-16, skn-1 and daf-2 regulatory pathways were examined. The ASAE LC50 value was found to be 77.3 ± 4 mg/ml. The concentration of ASAE 10 mg/ml (frequently used in humans) did not exhibit a significant reduction in worm survival at either the L1 or L4 stage, after 24 or 72 hr treatment. ASAE did not markedly alter the body area. In N2 strain, ASAE (10 or 25 mg/ml) reversed the damage initiated by H2O2. In addition, ASAE protected the damage produced by H2O2 in strains containing significant levels of sod-3, gst-4 and ctl - 1,2,3, suggesting modulation in these antioxidant systems by this plant extract. ASAE exposure activated daf-16 and skn-1 stress response transcriptional pathways independently of daf-2, even under extreme stress. Data suggest that ASAE, at the concentrations tested in C. elegans, exhibits a reliable toxicity profile, which may contribute to consideration for safe use in humans.

Pomolic acid and its glucopyranose ester promote apoptosis through autophagy in HT-29 colon cancer cells.

BACKGROUND: Colon cancer remains a leading cause of death globally. Pomolic acid (PA) can be separated from the ethyl acetate fraction of achyrocline satureioides. AIM: To determine the effects of PA and its glucopyranose ester, pomolic acid-28-O-ß-D-glucopyranosyl ester (PAO), on colon cancer HT-29 cells. METHODS: 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay was used to measure cell viability. Apoptosis was detected via hoechst 33342 staining. PI single staining was identified by flow cytometry to determine the cycle and scratch assay was used to observe the migration of HT-29 cells. The levels of mRNA and proteins were evaluated by q polymerase chain reaction and western blotting, respectively. RESULTS: PA and PAO considerably inhibited the growth of the HT-29 cell line in a time and dose-dependent manner. After the administration of PA and PAO for 24 and 48 h, cell apoptosis was significantly promoted and HT-29 cells were arrested in the G0/G1 stage. The Bax/Bcl2 ratio was also increased, which activated cysteinyl aspartate specific proteinase 3, leading to apoptosis; it also increased the expression of light chain 3 II/I and Beclin1, which activated autophagy and caused cell death. This in turn increased the expression of p62 to promote cell apoptosis, inhibiting the levels of signal transducer and activator of transcription 3 (STAT3) and p-STAT3, suppressing the level of Bcl2, and promoting cell. CONCLUSION: Both PA and PAO provide novel therapeutic strategies for treating colorectal cancer.

Achyrocline satureioides Hydroalcoholic Extract as a Hypoallergenic Antimicrobial Substitute of Natural Origin for Commonly Used Preservatives in Cosmetic Emulsions.

Achyrocline satureioides is a South American herb used in traditional medicine to treat a wide range of ailments. The healing and antimicrobial effects of this plant have already been covered by many studies, which have confirmed its beneficial effects on human health. In this study, the antimicrobial effect of A. satureioides hydroalcoholic extract against Escherichia coli ATCC10536, Staphylococcus aureus ATCC25923, Staphylococcus epidermidis ATCC12228 and Lactobacillus acidophilus INCQS00076 was determined. The cytotoxicity of the extract was tested on human HaCaT keratinocytes showing very favourable effects on the proliferation and renewal of keratinocytes. According to the results of the HPLC and GC-MS analyses, the lyophilized extract contained only a minimal amount of fragrance allergens. The extract was then used in two cosmetic formulations, and one of them showed a significant synergistic interaction with other cosmetic components. We suggest the use of A.satureioides hydroalcoholic extract as a suitable antimicrobial component of natural origin for cosmetic preparations as a substitute for commonly used preservatives that can cause skin irritation and as a material with its own biological activity.

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.

Effect of Hydrogel Containing Achyrocline satureioides (Asteraceae) Extract-Loaded Nanoemulsions on Wound Healing Activity.

Achyrocline satureioides (Lam.) DC extract-loaded nanoemulsions have demonstrated potential for wound healing, with promising effects on keratinocyte proliferation. We carried out the first in vivo investigation of the wound healing activity of a hydrogel containing A. satureioides extract-loaded nanoemulsions. We prepared hydrogels by adding the gelling agent (Carbopol® Ultrez) to extract-loaded nanoemulsions (~250 nm in diameter) obtained by spontaneous emulsification. The final flavonoid content in formulation was close to 1 mg/mL, as estimated by ultra-fast liquid chromatography. Permeation/retention studies using porcine ear skin showed that flavonoids reached deeper layers of pig ear skin when it was damaged (up to 3.2 µg/cm² in the dermis), but did not reach the Franz-type diffusion cell receptor fluid. For healing activity, we performed a dorsal wound model using Wistar rats, evaluating the lesion size, anti-inflammatory markers, oxidative damage, and histology. We found that extract-loaded formulations promoted wound healing by increasing angiogenesis by ~20%, reducing inflammation (tumor necrosis factor α) by ~35%, decreasing lipid damage, and improving the re-epithelialization process in lesions. In addition, there was an increase in the number of blood vessels and hair follicles for wounds treated with the formulation compared with the controls. Our findings indicate that the proposed formulation could be promising in the search for better quality healing and tissue reconstruction.

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.

The challenge of flavonoid/cyclodextrin complexation in a complex matrix of the quercetin, luteolin, and 3-O-methylquercetin.

The complexation of herbal constituents with cyclodextrin has been a useful tool to improve their aqueous solubility. However, the simultaneous complexation of these compounds still lacks detailed studies. The present study investigated the multicomplexation of quercetin (QCT), luteolin (LUT), and 3-O-methylquercetin (3OMQ) with (2-hydroxypropyl)-ß-cyclodextrin (HPßCD), when they are simultaneously contained in a flavonoid-enriched fraction (FEF) of Achyrocline satureioides. The phase-solubility diagram revealed a linear correlation between the flavonoids solubility and the HPßCD concentration, demonstrating the formation of complexes with a 1:1 stoichiometric ratio, which was confirmed by ESI-MS. Negative ΔG0 values indicated that complexation was spontaneous. Flavonoids/HPßCD interactions were evidenced by FT-IR, DSC, SEM, and 1D and 2D NMR. The last one showed the formation of inclusion complexes by insertion of the B-ring of the flavonoids into the cavity of HPßCD. Unexpectedly, the FEF/HPßCD complex showed a radical scavenger potential lower than the FEF. The HPLC analysis revealed that the complex contained different flavonoid ratio than the fraction. Thus, the antioxidant capacity of the samples was demonstrated to be related to the ratio among the flavonoids, rather than to the total flavonoids. These new findings are very useful for developing herbal cyclodextrin-based products from A. satureioides or other herbal products.

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 (Asteraceae) Extract-Loaded Nanoemulsions as a Promising Topical Wound Healing Delivery System: In Vitro Assessments in Human Keratinocytes (HaCaT) and HET-CAM Irritant Potential.

Achyrocline satureioides (Lam.) DC Asteraceae extracts (ASEs) have been investigated for the treatment of various skin disorders. This study reports the effects of ASE-loaded nanoemulsions (NEASE) on the cellular viability, death by necrosis, and migration of immortalized human keratinocytes (HaCaT cell line), as well as the irritant potential through the hen's egg chorioallantoic membrane test (HET-CAM). NEASE exhibited a polydispersity index above 0.12, with a droplet size of 300 nm, ζ-potential of -40 mV, and content of flavonoids close to 1 mg/mL. No cytotoxicity of the ASE was observed on HaCaT by MTT assay (up to 10 µg/mL). A significant increase of HaCaT viability was observed to NEASE (up to 5 µg/mL of flavonoids), compared to treatment with the ASE. The necrosis death evaluation demonstrated that only NEASE did not lead to cell death at all the tested concentrations. The scratch assay demonstrated that NEASE was able to increase the cell migration at low flavonoid concentrations. Finally, the HET-CAM test proved the non-irritative potential of NEASE. Overall, the results indicate the potential of the proposed formulations for topical use in wound healing, in view of their promising effects on proliferation and migration in keratinocytes, combined with an indication of the absence of cytotoxicity and non-irritating potential.

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 satureioides compounds, achyrobichalcone and 3-O-methylquercetin, induce mitochondrial dysfunction and apoptosis in human breast cancer cell lines.

Natural products are a valuable source of new molecules and are important for drug discovery. Many chemotherapeutics currently in clinical use were originated from natural sources and are effective cytotoxic agents. In this study, we investigated the cytotoxic and pro-apoptotic effects of achyrobichalcone (ACB) and 3-O-methylquercetin (3OMQ), two novel compounds isolated from the Achyrocline satureioides plant. Because extracts from this plant have been shown to have anticancer activity in vitro, we evaluated ACB and 3OMQ using a human breast cancer cell line, MDA-MB-231, and a nontumorigenic human breast epithelial cell line, MCF-12A. We found that ACB demonstrates cytotoxic effects on MDA-MB-231 cells, but not MCF-12A cells. 3OMQ also demonstrated cytotoxic effects on MDA-MB-231 cells, but with lower selectivity compared to treated MCF-12A cells. Cell death by both compounds was associated with caspase-9 and caspase-3/7 activation. Using high-resolution respirometry, we found that ACB and 3OMQ were able to cause acute mitochondrial dysfunction in MDA-MB-231-treated cells. These results suggest that apoptosis in MDA-MB-231 cells is induced through the activation of the mitochondrial-dependent pathway. Collectively, these findings suggest that ACB is a strong candidate for further anticancer in vivo tests.

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.

Purification, structural elucidation, antioxidant capacity and neuroprotective potential of the main polyphenolic compounds contained in Achyrocline satureioides (Lam) D.C. (Compositae).

Achyrocline satureioides (Lam) D.C (Compositae) is a native medicinal plant of South America traditionally utilized for its anti-inflammatory, sedative and anti-atherosclerotic properties among others. Neuroprotective effects have been reported in vivo and could be associated to its elevated content of flavonoid aglycones. In the present study we performed the isolation and structure elucidation of the major individual flavonoids of A. satureioides along with the in vitro characterization of their individual antioxidant and neuroprotective properties in order to see their putative relevance for treating neurodegeneration. Exact mass, HPLC-MS/MS and 1H NMR identified dicaffeoyl quinic acid isomers, quercetin, luteolin, isoquercitrin, and 3-O-methylquercetin as the mayor polyphenols. Flavonoids intrinsic redox properties were evaluated in the presence of the endogenous antioxidants GSH and Ascorbate. Density Functional Theory (DFT) molecular modeling and electron density studies showed a theoretical basis for their different redox properties. Finally, in vitro neuroprotective effect of each isolated flavonoid was evaluated against hydrogen peroxide-induced toxicity in a primary neuronal culture paradigm. Our results showed that quercetin was more efficacious than luteolin and isoquercitrin, while 3-O-methylquercetin was unable to afford neuroprotection significantly. This was in accordance with the susceptibility of each flavonoid to be oxidized and to react with GSH. Overall our results shed light on chemical and molecular mechanisms underlying bioactive actions of A. satureioides main flavonoids that could contribute to its neuroprotective effects and support the positive association between the consumption of A. satureioides as a natural dietary source of polyphenols, and beneficial health effect.

Chemical constituents from Achyrocline satureioides (I) / 中草药

Objective To study the chemical constituents from Achyrocline satureioides. Methods Chemical constituents from A. satureioides were separated and purified by a variety of chromatographic techniques, and their structures were identified by various spectroscopic methods. Results Twenty-one compounds were isolated from ethyl acetate fraction of the plant, which were α-amyrin (1), ergosta-4,6,8,22-tetraene-3-one (2), (R)-24-ethylcholest-4-en-3,6-dione (3), clovandiol (4), caryolane-1,9β-diol (5), lepidissipyrone (6), 5,7-dihydroxy-3,6-dimethoxyflavone (7), quercetin-7-O-β-D-glucoside (8), pinocembrin (9), (2R,3R)-taxifolin (10), quercetin-4’-O-β-D-glucoside (11), helichrysetin (12), 3-[5,7-dihydroxy-2,2-dimethyl-8-(2-(S)-methyl-butanoyl)-2H-chromen-6- yl-methyl]-6-ethyl-4-hydroxy-5-methyl-pyran-2-one (13), quercetin (14), protocatechuic acid (15), (+)-(3R)-3-hydroxyl-4,4- dimethyl-4-butyrolactone (16), (4S,5R)-5-(4’-methyl-3’-pentenyl)-4-hydroxy-5-methyldihydrofuran-2-one (17), genkwanin (18), quercetin-3-methyl ether (19), galangin (20), and neosakuranin (21). Conclusion Compounds 1, 4, 5, 16, 17, and 21 are obtained from the genus for the first time, and compounds 1-12, 16-18, and 21 are obtained from the plant for the first time.

Endophytic Fungus Isolated From Achyrocline satureioides Exhibits Selective Antiglioma Activity-The Role of Sch-642305.

Glioblastoma is the most devastating primary brain tumor. Current treatment is palliative, making necessary the development of new therapeutic strategies to offer alternatives to patients. Therefore, endophytes represent an interesting source of natural metabolites with anticancer potential. These microorganisms reside in tissues of living plants and act to improve their growth. Evidence revealed that several medicinal plants are colonized by endophytic fungi producer of antitumor metabolites. Achyrocline satureioides is a Brazilian medicinal plant characterized by its properties against gastrointestinal disturbances, anticancer and antioxidant effects. However, there are no reports describing the endophytic composition of A. satureioides. The present study proposes the isolation of endophytic fungus from A. satureioides, extract preparation, phytochemical characterization and evaluation of its antiglioma potential. Our data showed that crude extracts of endophyte decreased glioma viability with IC50 values of 1.60-1.63 µg/mL to eDCM (dichloromethane extract) and 37.30-55.12 µg/mL to eEtAc (ethyl acetate extract), respectively. Crude extracts induced cell death by apoptosis with modulation of redox status. In order to bioprospect anticancer metabolites, endophytic fungus extracts were subjected to guided fractionation and purification yielded five fractions of each extract. Six of ten fractions showed selective antiproliferative activity against glioma cells, with IC50 values ranged from 0.95 to 131.3 µg/mL. F3DCM (from eDCM) and F3EtAc (from eEtAc) fractions promoted C6 glioma toxicity with IC50 of 1.0 and 27.05 µg/mL, respectively. F3EtAc fraction induced late apoptosis and arrest in G2/M stage, while F3DCM promoted apoptosis with arrest in Sub-G1 phase. Moreover, F3DCM increased antioxidant defense and decreased ROS production. Additionally, F3DCM showed no cytotoxic activity against astrocytes, revealing selective effect. Based on promising potential of F3DCM, we identified the production of Sch-642305, a lactone, which showed antiproliferative properties with IC50 values of 1.1 and 7.6 µg/mL to C6 and U138MG gliomas, respectively. Sch-642305 promoted arrest on cell cycle in G2/M inducing apoptosis. Furthermore, this lactone decreased glioma cell migration and modulated redox status, increasing superoxide dismutase and catalase activities and enhancing sulfhydryl content, consequently suppressing reactive species of oxygen generation. Taken together, these results indicate that metabolites produced by endophytic fungus isolated from A. satureioides have therapeutic potential as antiglioma agent.

Anticancer activity of flavonoids isolated from Achyrocline satureioides in gliomas cell lines.

Achyrocline satureioides, popularly known as "marcela", is a medicinal plant found in South America. This plant is rich in flavonoids, which have been reported to exert numerous biological activities. The aim of this study was to purify, identify and evaluate the mechanisms underlining anticancer activity of A. satureioides flavonoids in glioma cell lines (U87, U251 and C6) as well as their comparative toxicity in normal brain cells (primary astrocytes, neurons and organotypic hippocampal cultures). The main flavonoids present in A. satureioides are luteolin, quercetin, 3-O-methyl-quercetin and achyrobichalcone, the later a very unique metabolite present in this plant. Isolated flavonoids as well as A. satureioides extracts reduced proliferation and clonogenic survival, and induced apoptosis of glioma cell lines. In addition, A. satureioides flavonoids potentiated the cytotoxic effect and apoptosis induction by the glioma chemotherapeutic temozolomide (TMZ). Importantly, A. satureioides flavonoids were less cytotoxic to astrocytes, neuron:astrocytes co-cultures and hippocampal cultures if compared to gliomas. Investigation of 10 cancer-related pathways showed a reduced activation of MYC and the Map kinases ERK and JNK by A. satureioides flavonoid-enriched extract, an effect not observed when individual flavonoids were evaluated. Altogether, the herein presented results show that A. satureioides extract possesses a combination of flavonoids, some unique for this plant, which have synergistic anticancer activity and potential for further studies in vivo.

Ultrasonic assisted extraction to obtain bioactive, antioxidant and antimicrobial compounds from marcela / Extração assistida por ultrassom para obtenção de compostos bioativos, antioxidantes e antimicrobianos de marcela

ABSTRACT: The aim of this study was to evaluate the effect of extraction conditions on bioactive compounds, as well as on antioxidant activity, and the antimicrobial activity of the extracts with the highest antioxidant characteristics. The extracts were obtained by conventional method and ultrasound-assisted extraction at various temperatures (20°C, 40°C and 60°C). Total phenolics, total flavonoids, antimicrobial activity and antioxidant activity were quantified by the methods of DPPH, FRAP, and ORAC, respectively. The conventional extraction method and ultrasound method influenced the phenolic content at all the tested temperatures. Flavonoids were not influenced by extraction methods. The antioxidant activity (DPPH) was highest in the ultrasonic method at temperatures of 40°C and 60°C; however, in the case of the FRAP method the best results were for the conventional extraction method. The conventional and ultrasonic methods did not influence the IC50 at temperatures of 20°C and 40°C, but using ORAC the antioxidant activity was influenced by the methods at all temperatures. The extract obtained at 60°C by the ultrasound method had high antimicrobial action in relation to the strains of Salmonella sp., Escherichia coli, and Staphylococcus aureus. Extraction ultrasonic-assisted can be considered adequate to obtain extracts of marcela, which are rich in bioactive compounds with high antioxidant activity.

RESUMO: O objetivo deste trabalho foi avaliar o efeito das condições de extração sobre os compostos bioativos, bem como sobre a atividade antioxidante e atividade antimicrobiana do extrato com maior característica antioxidante. Os extratos foram obtidos pelo método convencional e extração assistida por ultrassom, variando a temperatura (20oC, 40oC e 60oC). Foram quantificados os fenólicos totais, flavonóides totais, a atividade antimicrobiana, e atividade antioxidante pelos métodos DPPH, FRAP, e ORAC. Os métodos de extração convencional e ultrassom influenciaram no teor de fenólicos em todas as temperaturas testadas. Os flavonóides não sofreram influência dos métodos de extração. A atividade antioxidante (DPPH) foi superior no método ultrassom nas temperaturas de 40°C e 60°C, entretanto pelo método FRAP os melhores resultados foram na extração convencional. Os métodos convencional e ultrassom não influenciaram no IC50 nas temperaturas de 20oC e 40oC, mas a atividade antioxidante pelo método ORAC sofreu influencia dos métodos em todas as temperaturas. O extrato obtido a 60°C pelo método ultrassom possui elevada ação antimicrobiana frente a cepas de Salmonella sp., Escherichia coli, e Staphylococcus aureus. A extração assistida por ultrassom pode ser considerada adequada para obtenção de extratos de marcela, que são ricos em compostos bioativos com alta atividade antioxidante.

Supplementation with Achyrocline satureioides Inflorescence Extracts to Pregnant and Breastfeeding Rats Induces Tissue-Specific Changes in Enzymatic Activity and Lower Neonatal Survival.

Achyrocline satureioides (AS, family Asteraceae) is a plant widely used in traditional medicine for stomach, digestive, and gastrointestinal disorders during pregnancy. Studies regarding the indiscriminate use of plant infusions during pregnancy are limited. Recent reports have shown that chronic flavonoid supplementation induces toxicity in vivo and raises the mortality rates of healthy subjects. Therefore, we investigated whether supplementation of pregnant and lactating Wistar rats with two AS inflorescence extracts, consisting of an aqueous (AQ) extract similar to a tea (47 mg·kg-1·day) and a hydroethanolic (HA) extract (35 mg·kg-1·day-1) with a higher flavonoid content, could induce redox-related side effects. Total reactive antioxidant potential (TRAP), thiobarbituric reactive species (TBARS), and total reduced thiol (SH) content were evaluated. Superoxide dismutase (SOD) and catalase (CAT) activities were additionally quantified. Our data suggest that both AQ and HA of AS inflorescence extracts may induce symptoms of toxicity in concentrations of (47 mg·kg-1·day) and (35 mg·kg-1·day-1), respectively, in mothers regarding the delivery index and further decrease of neonatal survival. Of note, significant tissue-specific changes in maternal (liver, kidney, heart, and hippocampus) and pups (liver and kidney) biochemical oxidative parameters were observed. Our findings provide evidence that may support the need to control supplementation with the AQ of AS inflorescence extracts during gestation due to potential toxicity in vivo, which might be related, at least in part, to changes in tissue-specific redox homeostasis and enzymatic activity.