The effects of 20-hydroxyecdysone on nuclear shape, heterochromatin quantity and gray-level co-occurrence matrix texture analysis of adrenal zona fasciculata cells in an obese gerbil (Gerbillus tarabuli) model for metabolic syndrome: a correlational study.

The aim of this study was to reveal the effects of obesity and phytotherapy with 20-hydroxyecdysone (20E) on the nuclei of adrenal zona fasciculata (ZF) in the gerbil Gerbillus tarabuli by analyzing nuclear shape and gray-level co-occurrence matrix (GLCM) texture characteristics and by quantifying heterochromatin. Twelve gerbils were divided into three groups: control (C), HC and HC-20E (animals receiving a high-calorie-diet without or with a supplement of 20E, respectively). The adrenals were removed and fixed for histological and statistical analysis. Principal component analysis showed a positive correlation of area, perimeter and textural correlation in C. Nevertheless, a negative correlation was recorded for contrast and entropy. The obesity caused a disorder in nuclear texture; negative correlation was noted with heterochromatin fraction, which may be related to increased ZF activity. However, administration of 20E seems to improve the nuclear state by preserving circularity, uniformity and homogeneity of nuclei as well as the proportion of heterochromatin, which could be a sign of a downregulation of cell activity.Our results suggest that new techniques of image processing could contribute to the understanding of nuclear changes associated with obesity and its possible therapy in this gerbil model for metabolic syndrome.

Anti-Adipogenic Activity of Rhaponticum carthamoides and Its Secondary Metabolites.

Besides their common use as an adaptogen, Rhaponticum carthamoides (Willd.) Iljin. rhizome and its root extract (RCE) are also reported to beneficially affect lipid metabolism. The main characteristic secondary metabolites of RCE are phytoecdysteroids. In order to determine an RCE's phytoecdysteroid profile, a novel, sensitive, and robust high-performance thin-layer chromatography (HPTLC) method was developed and validated. Moreover, a comparative analysis was conducted to investigate the effects of RCE and its secondary metabolites on adipogenesis and adipolysis. The evaluation of the anti-adipogenic and lipolytic effects was performed using human Simpson-Golabi-Behmel syndrome cells, where lipid staining and measurement of released glycerol and free fatty acids were employed. The HPTLC method confirmed the presence of 20-hydroxyecdysone (20E), ponasterone A (PA), and turkesterone (TU) in RCE. The observed results revealed that RCE, 20E, and TU significantly reduced lipid accumulation in human adipocytes, demonstrating their anti-adipogenic activity. Moreover, RCE and 20E were found to effectively stimulate basal lipolysis. However, no significant effects were observed with PA and TU applications. Based on our findings, RCE and 20E affect both lipogenesis and lipolysis, while TU only restrains adipogenesis. These results are fundamental for further investigations.

Sustainable Production of Ajuga Bioactive Metabolites Using Cell Culture Technologies: A Review.

The genus Ajuga (Lamiaceae) is rich in medicinally important species with biological activities ranging from anti-inflammatory, antitumor, neuroprotective, and antidiabetic to antibacterial, antiviral, cytotoxic, and insecticidal effects. Every species contains a unique and complex mixture of bioactive metabolites-phytoecdysteroids (PEs), iridoid glycosides, withanolides, neo-clerodane terpenoids, flavonoids, phenolics, and other chemicals with high therapeutic potential. Phytoecdysteroids, the main compounds of interest, are natural anabolic and adaptogenic agents that are widely used as components of dietary supplements. Wild plants remain the main source of Ajuga bioactive metabolites, particularly PEs, which leads to frequent overexploitation of their natural resources. Cell culture biotechnologies offer a sustainable approach to the production of vegetative biomass and individual phytochemicals specific for Ajuga genus. Cell cultures developed from eight Ajuga taxa were capable of producing PEs, a variety of phenolics and flavonoids, anthocyanins, volatile compounds, phenyletanoid glycosides, iridoids, and fatty acids, and demonstrated antioxidant, antimicrobial, and anti-inflammatory activities. The most abundant PEs in the cell cultures was 20-hydroxyecdysone, followed by turkesterone and cyasterone. The PE content in the cell cultures was comparable or higher than in wild or greenhouse plants, in vitro-grown shoots, and root cultures. Elicitation with methyl jasmonate (50-125 µM) or mevalonate and induced mutagenesis were the most effective strategies that stimulated cell culture biosynthetic capacity. This review summarizes the current progress in cell culture application for the production of pharmacologically important Ajuga metabolites, discusses various approaches to improve the compound yield, and highlights the potential directions for future interventions.

Comparative Transcriptomic Analysis of Genes in the 20-Hydroxyecdysone Biosynthesis in the Fern Microsorum scolopendria towards Challenges with Foliar Application of Chitosan.

Microsorum scolopendria is an important medicinal plant that belongs to the Polypodiaceae family. In this study, we analyzed the effects of foliar spraying of chitosan on growth promotion and 20-hydroxyecdysone (20E) production in M. scolopendria. Treatment with chitosan at a concentration of 50 mg/L in both young and mature sterile fronds induced the highest increase in the amount of accumulated 20E. Using RNA sequencing, we identified 3552 differentially expressed genes (DEGs) in response to chitosan treatment. The identified DEGs were associated with 236 metabolic pathways. We identified several DEGs involved in the terpenoid and steroid biosynthetic pathways that might be associated with secondary metabolite 20E biosynthesis. Eight upregulated genes involved in cholesterol and phytosterol biosynthetic pathway, five upregulated genes related to the methylerythritol 4-phosphate (MEP) and mevalonate (MVA) pathways, and several DEGs that are members of cytochrome P450s and ABC transporters were identified. Quantitative real-time RT-PCR confirmed the results of RNA-sequencing. Taken together, we showed that chitosan treatment increased plant dry weight and 20E accumulation in M. scolopendria. RNA-sequencing and DEG analyses revealed key enzymes that might be related to the production of the secondary metabolite 20E in M. scolopendria.

Effect of Ecdysterone on the Hepatic Transcriptome and Lipid Metabolism in Lean and Obese Zucker Rats.

Conflicting reports exist with regard to the effect of ecdysterone, the predominating representative of steroid hormones in insects and plants, on hepatic and plasma lipid concentrations in different rodent models of obesity, fatty liver, and diabetes, indicating that the effect is dependent on the rodent model used. Here, the hypothesis was tested for the first time that ecdysterone causes lipid-lowering effects in genetically obese Zucker rats. To test this hypothesis, two groups of male obese Zucker rats (n = 8) were fed a nutrient-adequate diet supplemented without or with 0.5 g ecdysterone per kg diet. To study further if ecdysterone is capable of alleviating the strong lipid-synthetic activity in the liver of obese Zucker rats, the study included also two groups of male lean Zucker rats (n = 8) which also received either the ecdysterone-supplemented or the non-supplemented diet. While hepatic and plasma concentrations of triglycerides and cholesterol were markedly higher in the obese compared to the lean rats (p < 0.05), hepatic and plasma triglyceride and cholesterol concentrations did not differ between rats of the same genotype fed the diets without or with ecdysterone. In conclusion, the present study clearly shows that ecdysterone supplementation does not exhibit lipid-lowering actions in the liver and plasma of lean and obese Zucker rats.

Analytical strategy for the detection of ecdysterone and its metabolites in vivo in uPA(+/+)-SCID mice with humanized liver, human urine samples, and estimation of prevalence of its use in anti-doping samples.

Ecdysteroids are of interest as potential sport performance enhancers, due to their anabolic effects. The current study aimed to analyze levels of the most abundant ecdysteroid, ecdysterone (20-hydroxyecdysone, 20-OHE) in easily available dietary supplements, and, outline an analytical strategy for its detection, and that, of its metabolites, (1) following administration of pure 20-OHE to uPA(+/+)-SCID mice with humanized liver, (2) in a human volunteer after ingestion of two supplements, one with a relatively low, and the other a high, concentration of 20-OHE, and, (3) to estimate the prevalence of use of 20-OHE in elite athletes (n = 1000). Of the 16 supplements tested, only five showed detectable levels of 20-OHE, with concentrations ranging from undetectable up to 2.3 mg per capsule. Urine of uPA(+/+)-SCID urine showed the presence of 20-OHE and its metabolite, 14 deoxy ecdysterone, within 24 hours (hr) of ingestion. In humans, both the parent and the metabolite were detectable within 2 to 5 hr of ingestion, with the metabolite being detectable for longer than the parent. After ingestion of a low dose supplement, the parent and metabolite were detectable for 70 and 48 hr, while following the higher dose it was 96 and 48 hr, respectively. Analysis of urines from athletes (n = 1000) confirmed four positives for 20-OHE, suggesting a prevalence of use of 0.4%. Prevalence of its use by elite athletes was relatively low, however, this needs to be confirmed in other populations, and with other related ecdysteroids.

Two Ecdysteroids Isolated from Micropropagated Lychnis flos-cuculi and the Biological Activity of Plant Material.

Genetically uniform plant material, derived from Lychnis flos-cuculi propagated in vitro, was used for the isolation of 20-hydroxyecdysone and polypodine B and subjected to an evaluation of the antifungal and antiamoebic activity. The activity of 80% aqueous methanolic extracts, their fractions, and isolated ecdysteroids were studied against pathogenic Acanthamoeba castellani. Additionally, a Microtox® acute toxicity assay was performed. It was found that an 80% methanolic fraction of root extract exerts the most potent amoebicidal activity at IC50 of 0.06 mg/mL at the 3rd day of treatment. Both ecdysteroids show comparable activity at IC50 of 0.07 mg/mL. The acute toxicity of 80% fractions at similar concentrations is significantly higher than that of 40% fractions. Crude extracts exhibited moderate antifungal activity, with a minimum inhibitory concentration (MIC) within the range of 1.25-2.5 mg/mL. To the best of our knowledge, the present report is the first to show the biological activity of L. flos-cuculi in terms of the antifungal and antiamoebic activities and acute toxicity. It is also the first isolation of the main ecdysteroids from L. flos-cuculi micropropagated, ecdysteroid-rich plant material.

Antihyperglycaemic, haemorheological and antioxidant activities of Lychnis chalcedonica L. extract in a streptozotocin-induced rat model of diabetes mellitus.

Background New therapeutic strategies, such as the use of agents to correct rheological disorders, are needed for the prevention and treatment of angiopathy in diabetic patients. The aim of this work was to study the antihyperglycaemic, haemorheologic and antioxidant activities of an extract from the flowering plant Lychnis chalcedonica L. (ELC) and 20-hydroxyecdysone using the streptozotocin-induced model of diabetic rats. Methods The streptozotocin-induced model of diabetes was produced using streptozotocin at a dose of 50 mg/kg (ip). Animals from the experimental groups were treated with ELC (150 mg/kg) or 20-hydroxyecdysone (1.1 mg/kg) intragastrically in 1% aqueous starch mucilage daily, for 14 days; rats of control groups received an equal volume of starch mucilage. The following parameters were measured: glucose concentration (GC) in blood, whole blood viscosity (WBV), conjugated dienes in RBC membranes. Macro- and microrheological indicators (viz. plasma viscosity, haematocrit, RBC aggregation (T1/2) and the RBC elongation index (EI)) were additionally measured in rats that received ELC, and in the control group. Results After treatment with ELC, the GC in rats was 19% lower than that in the control group (14.7 ± 0.9 mM compared to 18.2 ± 1.1 mM). Rats with streptozotocin-induced diabetes have hyperviscosity syndrome, which is characterized by increased WBV, increased RBC aggregation and decreased deformability. ELC treatment reduced WBV at shear rates of 10-90 s-1 by 5-8%, and T1/2 and EI in the experimental group were 31% and 5-10% higher compared to the control group. 20-Hydroxyecdysone decreased WBV at shear rates of 10-90 s-1 by 3-11%. Finally, ELC and 20-hydroxyecdysone lowered the content of conjugated dienes by 27% and by 26% compared to the control groups. Conclusion In the streptozotocin-induced diabetic rat model, ELC showed measurable antihyperglycaemic activity; ELC and 20-hydroxyecdysone demonstrated similar haemorheological, and antioxidant activities.

Andean Prumnopitys Andina (Podocarpacae) Fruit Extracts: Characterization of Secondary Metabolites and Potential Cytoprotective Effect.

The fruits from the Chilean Podocarpaceae Prumnopitys andina have been consumed since pre-Hispanic times. Little is known about the composition and biological properties of this fruit. The aim of this work was to identify the secondary metabolites of the edible part of P. andina fruits and to assess their antioxidant activity by means of chemical and cell-based assays. Methanol extracts from P. andina fruits were fractionated on a XAD7 resin and the main compounds were isolated by chromatographic means. Antioxidant activity was determined by means of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), ferric reducing power (FRAP), trolox equivalent antioxidant capacity (TEAC) and oxygen radical absorbance capacity (ORAC) assays. The cytoprotective activity of the extract against oxidative and dicarbonyl stress was evaluated in human gastric epithelial cells (AGS). The total intracellular antioxidant activity (TAA) of the extract was determined in AGS cells. The inhibition of meat lipoperoxidation was evaluated under simulated gastric digestion conditions. Rutin, caffeic acid ß-glucoside and 20-hydroxyecdysone were identified as major components of the fruit extract. Additional compounds were identified by high-performance liquid chromatography diode-array detector mass spectrometry (HPLC-DAD-MSn) and/or co-injection with standards. Extracts showed dose-dependent cytoprotective effects against oxidative and dicarbonyl-induced damage in AGS cells. The TAA increased with the pre-incubation of AGS cells with the extract. This is the first report on the composition and biological activity of this Andean fruit.

Combined Use of Deep Eutectic Solvents, Macroporous Resins, and Preparative Liquid Chromatography for the Isolation and Purification of Flavonoids and 20-Hydroxyecdysone from Chenopodium quinoa Willd.

: Deep eutectic solvents (DESs) were used in combination with macroporous resins to isolate and purify flavonoids and 20-hydroxyecdysone from Chenopodium quinoa Willd by preparative high-performance liquid chromatography (HPLC). The extraction performances of six DESs and the adsorption/desorption performances of five resins (AB-8, D101, HPD 400, HPD 600, and NKA-9) were investigated using the total flavonoid and 20-hydroxyecdysone extraction yields as the evaluation criteria, and the best-performing DES (choline chloride/urea, DES-6) and macroporous resin (D101) were further employed for phytochemical extraction and DES removal, respectively. The purified extract was subjected to preparative HPLC, and the five collected fractions were purified in a successive round of preparative HPLC to isolate three flavonoids and 20-hydroxyecdysone, which were identified by spectroscopic techniques. The use of a DES in this study significantly facilitated the preparative-scale isolation and purification of polar phytochemicals from complex plant systems.

Poststerone increases muscle fibre size partly similar to its metabolically parent compound, 20-hydroxyecdysone.

In mice, poststerone is a major in vivo metabolite of the worldwide popular anabolic food supplement 20-hydroxyecdysone (20E). Here we present the first study on this ecdysteroid in view of the in vivo anabolic effect of its parent compound, 20E in mammals. We have monitored muscle fibre type cross sectional areas (CSA) of developing rats after treatment with poststerone as we did in a previous study with 20E. The muscle mass and fibre CSAs of soleus and EDL were increased by poststerone in a muscle specific manner as by 20E but there were some differences. Notably, the CSAs of type I and type IIa fibres in the soleus were less elevated by poststerone than by 20E. However poststerone increased the CSA of each four fibre types (I, IIa, IIx, IIb) in the EDL more effectively than 20E did. Poststerone, like 20E, also increased the number of myonuclei in the EDL of both hind limbs. Overall, this shows for the first time that poststerone having steroid nucleus and no side chain of 20E has a partly overlapping effect with that of 20E.

Crustacean cardioactive peptide (CCAP) of the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae): Molecular characterization, distribution and its potential roles in larva-pupa ecdysis.

Insects must undergo ecdysis for successful development and growth, and the crustacean cardioactive peptide (CCAP) is one of the most important hormone in this process. Here we reported a cDNA encoding for the CCAP precursor cloned from the oriental fruit fly, Bactrocera dorsalis, a most destructive insect pest of agriculture. The CCAP mature peptide (PFCNAFTGC-NH2) of B. dorsalis was generated by post-translational processing and found to be highly comparable with other insects. RT-qPCR showed that mRNA of CCAP in B. dorsalis (BdCCAP) was predominantly expressed in the central nervous system (CNS) and midgut of 3rd-instar larvae. By using immunohistochemical analysis, we also localized the endocrine cells that produce CCAP in the CNS, ring gland and midgut of 3rd-instar larvae of B. dorsalis. The synthetic CCAP mature peptide could induce the expression of mRNA of adipokinetic hormone (AKH), the metabolic neuropeptides in insects. The expression of BdCCAP mRNA in the CNS, but not in the midgut, could be upregulated in the response to the challenge of insect molting hormone, 20-hydroxyecdysone.

Transcriptome analysis of abscisic acid induced 20E regulation in suspension Ajuga lobata cells.

Ajuga lobata D. Don is a medicinal plant rich in 20-hydroxyecdysone (20E), alkaloids, and other active substances. In this study, the cell suspension was incubated for 7 days, followed by the analysis on the effects of abscisic acid (ABA) on the regulation of 20E synthesis. Then A. lobata suspension cells treated with 0.15 mg/l ABA were used as material, with the Illumina technology applied for transcriptome sequencing. Digital analysis on the gene expression profile was carried out on ABA treated and control samples, respectively. Finally, transcriptomics was applied to assess the molecular response of A. lobata induced by ABA through applying transcriptomics by evaluating differentially expressed genes. The results suggested that ABA promoted 20E accumulation, while longer processing time caused cell browning. A total of 154 genes were significantly regulated after ABA treatment, with 99 up-regulated and 55 down-regulated, respectively. In addition to 20E-related pathways, the genes belonged to the ko00900 (terpenoid backbone biosynthesis) pathway (six differentially expressed genes [DEGs]), ko00100 (steroid biosynthesis) pathway (four DEGs), and ko00140 (steroid hormone biosynthesis) pathway (six DEGs). Providing a better understanding of the 20E biosynthetic pathway and its regulation, in particular in plants, this study is necessary.

Larvicidal efficacy of Adiantobischrysene from Adiantum latifolium against Oryctes rhinoceros through disrupting metamorphosis and impeding microbial mediated digestion.

BACKGROUND: Oryctes rhinoceros Linn. (Coleoptera: Scarabaeidae) is a serious pest of coconuts and other palms. Symbiotic gut bacteria play significant roles in the digestion of cellulosic materials as well as in some other physiological processes essential for the existence of O. rhinoceros larvae. The study was undertaken to isolate a compound with antibacterial and larvicidal activities from the leaves of Adiantum latifolium Lam. following a bioassay-guided method. RESULTS: Methanol extract (ME) of dry leaf powder of A. latifolium showed larvicidal activity against third-instar O. rhinoceros (LD50 , 5018 mg/kg) with antibacterial activity on its gut microbiota. An in vitro study showed the bacteria Bacillus cereus, Micrococcus lylae, Stenotrophomonas maltophilia, Kocuria rosea, Burkholderia mallei, Staphylococcus epidermidis, S. arlettae and Corynebacterium afermentans identified from the larval gut were sensitive to ME. Bioactivity-guided isolation of the compound by liquid-liquid extraction and column chromatography resulted in Adiantobischrysene which showed antibacterial and larvicidal activity (LD50 , 8.4 mg/kg) and led to weight loss and precocious metamorphosis in larvae. An enzyme immunoassay showed a large peak in 20-hydroxyecdysone that commits larvae to precocious metamorphosis. CONCLUSION: This study demonstrated that the antibacterial and metamorphosis disrupting activity of Adiantobischrysene make it a natural pesticidal compound against O. rhinoceros. © 2018 Society of Chemical Industry.

Quantification Analysis and In Vitro Anti-Inflammatory Effects of 20-Hydroxyecdysone, Momordin Ic, and Oleanolic Acid from the Fructus of Kochia scoparia.

BACKGROUND: The fructus of Kochia scoparia Schrader (Chenopodiaceae) is a traditional herbal medicine that has been used for treating gonorrhea and dermatitis. OBJECTIVE: We investigated the anti-inflammatory activities of three marker compounds, including 20-hydroxyecdysone, momordin Ic, and oleanolic acid, from the fructus of K. scoparia. MATERIALS AND METHODS: The simultaneous analysis of three components was performed using high-performance liquid chromatography and high-performance thin-layer chromatography. We evaluated the anti-inflammatory effects of the nine marker compounds by determining their anti-inflammatory activities in the murine macrophage cell line RAW 264.7. RESULTS: Among three marker compounds, momordin Ic, but not 20-hydroxyecdysone and oleanolic acid, had inhibitory effects on the production of inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in LPS-treated RAW264.7 macrophages. The effects of three marker compounds on prostaglandin E2(PGE2) were also evaluated. All three compounds significantly reduced PGE2 production in LPS-treated cells. CONCLUSIONS: We suggest that momordin Ic is the most potent phytochemical of the fructus of K. scoparia as an anti-inflammatory agent. SUMMARY: Simultaneous analysis of three phenylpropanoids in the Kochia scoparia was established using HPLC-PDA systemThe momordin Ic had inhibitory effects on production of inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in LPS-treated RAW264.7 macrophagesThe momordin Ic, 20-hydroxyecdysone, and oleanolic acid significantly reduced PGE2 production in LPS-treated cells. Abbreviations used: HPLC: High-performance liquid chromatography; TNF-α: Tumor necrosis factor alpha; IL-6: Interleukin-6; PGE2: Pro-inflammatory mediator prostaglandin E2; LPS: Lipopolysaccharide.

Voltage-sensitive potassium channels expressed after 20-Hydroxyecdysone treatment of a mosquito cell line.

The goal of this research was to express receptors and ion channels in hormone-treated insect cell lines. Treatment of Anopheles gambiae Sua1B cells with 20-hydroxyecdysone showed an inhibition of cell growth over a time course of three days, with no change in cellular morphology. The effect of 20-hydroxyecdysone was enhanced in the presence of the potassium channel blocker 4-aminopyridine, but not tetraethylammonium. Concentration-response curves of 4-aminopyridine in the presence of 42 µM (1 mg/ml) 20-hydroxyecdysone showed similar IC50 values (6-10 µM) across 3 day exposures. Whole cell patch clamp confirmed the expression of delayed-rectifier (Kv2) potassium channels in hormone-supplemented Sua1B cells, whereas untreated Sua1B cells showed no evidence of Kv2 expression. The hormone-induced expression of Kv2 channels occurred in as little as 4 h after treatment, but were not observed after 24 h of exposure to 20-hydroxyecdysone, suggesting they played a role in cell death. The expressed channels had current-voltage relationships diagnostic for the Kv2 subtype, and were inhibited with an IC50 = 13 mM of tetraethylammonium. Overall, these parameters were similar to Anopheles gambiae Kv2 potassium channels expressed in HEK-293 cells. The induced presence of ion channels (and possibly receptors) in these cells has potential utility for high throughput screening and basic neuroscience research.

The phytoecdysteroid ß-ecdysone is genotoxic in Rodent Bone Marrow Micronuclei and Allium cepa L. Assays.

ETHNAOPHARMACOLOGIAL RELEVANCE: In South America, the ß-ecdysone ecdysteroid has been found in species of the genus Pfaffia Mart. Due to the similar morphology of its roots to the Panax ginseng C. A. Mey. (Korean ginseng), some species of this genus has been known as Brazilian ginseng and have been used as tonic and aphrodisiac, as well as for the treatment of diabetes and rheumatism. AIM OF THE STUDY: Here we report a cytogenotoxic evaluation of ß-ecdysone (a natural ecdysteroid found in plants) in Rodent Bone Marrow Micronuclei and Allium cepa Assays. MATERIALS AND METHODS: Three ß-ecdysone (pure) concentrations (based in human therapeutic dosage) were used in the Micronucleus Assay. The animals were treated during two consecutive days. Micronucleated cells were counted in 2000 polychromatic erythrocytes per animal. For A. cepa L. Assay, one ß-ecdysone concentration was analyzed. The onions bulbs were exposed for 24h. RESULTS: The Micronucleus Assay showed genotoxic effects for all treatments, expressed by an increase of micronucleated cells. In A. cepa L. Assay, cell abnormalities associated to the malfunction/non-formation of mitotic spindle (aneugenic effect) and chromosomal bridges (clastogenic effect) were observed. CONCLUSIONS: The results indicate a cytogenotoxic activity of ß-ecdysone. Therefore, the popular use of Pfaffia and others species containing ß-ecdysone should be considered with caution.

Sida tuberculata (Malvaceae): a study based on development of extractive system and in silico and in vitro properties

Sida tuberculata (Malvaceae) is a medicinal plant traditionally used in Brazil as an antimicrobial and anti-inflammatory agent. Here, we aimed to investigate the different extractive techniques on phytochemical parameters, as well as to evaluate the toxicity and antioxidant capacity of S. tuberculata extracts using in silico and in vitro models. Therefore, in order to determine the dry residue content and the main compound 20-hydroxyecdysone (20E) concentration, extracts from leaves and roots were prepared testing ethanol and water in different proportions. Extracts were then assessed by Artemia salina lethality test, and toxicity prediction of 20E was estimated. Antioxidant activity was performed by DPPH and ABTS radical scavenger assays, ferric reducing power assay, nitrogen derivative scavenger, deoxyribose degradation, and TBARS assays. HPLC evaluation detected 20E as main compound in leaves and roots. Percolation method showed the highest concentrations of 20E (0.134 and 0.096 mg/mL of extract for leaves and roots, respectively). All crude extracts presented low toxic potential on A. salina (LD50 >1000 µg/mL). The computational evaluation of 20E showed a low toxicity prediction. For in vitro antioxidant tests, hydroethanolic extracts of leaves were most effective compared to roots. In addition, hydroethanolic extracts presented a higher IC50 antioxidant than aqueous extracts. TBARS formation was prevented by leaves hydroethanolic extract from 0.015 and 0.03 mg/mL and for roots from 0.03 and 0.3 mg/mL on egg yolk and rat tissue, respectively (P<0.05). These findings suggest that S. tuberculata extracts are a considerable source of ecdysteroids and possesses a significant antioxidant property with low toxic potential.

ROLE OF SERUM AND ION CHANNEL BLOCK ON GROWTH AND HORMONALLY-INDUCED DIFFERENTIATION OF Spodoptera frugiperda (Sf21) INSECT CELLS.

A neuronal morphological phenotype can be induced in cultured Spodoptera frugiperda insect cells (Sf21) by supplementing serum-containing media with 20-hydroxyecdysone (20-HE) and/or insulin. In this study, the primary objectives were to determine any role of ion channels in mediating the morphological change in cells treated with 20-HE and insulin, and whether serum was required to observe this effect. Results showed serum-free media also induced growth of processes in Sf21 cells, but at a lower percentage than that found previously in cells bathed in serum-containing media. Veratridine, a sodium channel activator, increased cell survival when applied in combination with 20-HE to Sf21 cells, and the effect was blocked by tetrodotoxin (1 µM) a known sodium channel blocker. Cobalt, a calcium channel blocker, showed significant inhibition of cell process growth when applied in combination with both 20-HE and 20-HE plus veratridine. Cobalt also showed significant inhibition of cell process growth when applied in combination with insulin. Thus, some type of sodium channel, as well as a mechanism for transmembrane calcium ion movement, are apparently expressed in Sf21 cells and are involved in the differentiation process. These cell lines may be used in a wide variety of endeavors, including the screening of insecticides, as well as foster basic studies of neurodevelopment and ecdysone action.

New septanoside and 20-hydroxyecdysone septanoside derivative from Atriplex portulacoides roots with preliminary biological activities.

The phytochemical investigation of a Tunisian plant Atriplex portulacoides (Chenopodiaceae) led to the isolation of two new compounds designated as portulasoid (2) and septanoecdysone (3) along with the known 20-hydroxyecdysone (20HE) (1). Their chemical structures were elucidated on the basis of extensive spectroscopic methods including ES-HRMS, 1D and 2D-NMR. The isolated compounds were finally tested for their antioxidant activity by using DPPH, ABTS(+), Fe(3+) and catalase assays and also for their antibacterial and anticholinesterase activities.