Free Steroids Research Articles

Development of Methods to Quantify Free and Conjugated Steroids in Fatty Matrices by HPLC-MS/MS.

Steroids are called the “fingerprint” of oils, fats, and their derivatives. Different classes of steroids may be present in these matrices. Most of the methods developed to analyze these constituents involve the determination of free steroid content, although their conjugated forms are extremely important in determining the total composition. Thus, this article demonstrates that the coupling of sequential mass spectrometry and high-performance liquid chromatography obtained high sensitivity and high specificity of mass resolution to identify and quantify the main classes of steroids. Four methods were developed to quantify steroids free, esterified, glucosides, and acylated glucosides by internal standardization using betulin. The main validation parameters were tested and demonstrated good correlation results for the methods. The content of free steroids was the majority in all samples, whereas the content of glucoside steroids was the least abundant. The contents of free steroids quantified in the degummed soybean oil were significantly reduced in relation to the refined oil. A small amount of esterified steroids was superior in refined soybean oil than in degummed oil. Comparing the steroid content between degummed oil and biodiesel, we found that the concentration of free and esterified steroids decreases in the conversion to biodiesel, whereas the concentration of glucoside steroids increases slightly.

Development of a thin-film solid-phase microextraction (TF-SPME) method coupled to liquid chromatography and tandem mass spectrometry for high-throughput determination of steroid hormones in white sucker fish plasma.

Steroid hormones (SH) play a number of important physiological roles in vertebrates including fish. Changes in SH concentration significantly affect reproduction, differentiation, development, or metabolism. The objective of this study was to develop an in vitro high-throughput thin-film solid-phase microextraction (TF-SPME)-liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for targeted analysis of endogenous SH (cortisol, testosterone, progesterone, estrone (E1), 17β-estradiol (E2), and 17α-ethinylestradiol (EE2)) in wild white sucker fish plasma where the concentrations of the analytes are substantially low. A simple TF-SPME method enabled the simultaneous determination of free and total SH concentrations. The use of biocompatible coating allowed direct extraction of these hormones from complex biological samples without prior preparation. The carryover was less than 3%, thereby ensuring reusability of the devices and reproducibility. The results showed that TF-SPME was suitable for the analysis of compounds in the polarity range between 1.28 and 4.31 such as SH at different physicochemical properties. The proposed method was validated according to bioanalytical method validation guidelines. The limit of detection (LOD) and limit of quantification(LOQ) for cortisol, testosterone, progesterone, E1, E2, and EE2 were from 0.006 to 0.150ng/mL and from 0.020 to 0.500ng/mL, respectively. The recovery for the method was about 85%, and the accuracy and precision of the method for cortisol, testosterone, and progesterone were ≤ 6.0% and ≤ 11.2%, respectively, whereas those for E1, E2, and EE2 were ≤ 15.0% and ≤ 10.2%, respectively. On the basis of this study, TF-SPME demonstrated several important advantages such as simplicity, sensitivity, and robustness under laboratory conditions. Graphical abstract.

Phytochemical Analysis, Antioxidant Activity, Antimicrobial Activity, and Cytotoxicity of Chaptalia nutans Leaves.

Objective This work was to evaluate the chemical constitution of the hydromethanolic (30/70 methanol-water) macerating extract obtained from the leaves of C. nutans, as well as to study the antioxidant, antimicrobial, cytotoxic, and genotoxic activity of the species. Materials and methods. Phytochemical screening, antioxidant activity (total phenolic, total flavonoid, condensed tannins content, DPPH radical, and FRAP), antibacterial activity (P. aeruginosa, B. cereus, E. epidermidis, E. coli, S. aureus, E. faecalis, P. mirabilis, Candida glabrata (clinical isolate), Candida tropicalis (clinical isolate), C. krusei (clinical isolate), and C. albicans (clinical isolate)), and oxidative stress parameters (TBARS, carbonyl protein, and DCFH) were analyzed according to the literature. Toxicity of C. nutans was evaluated using an alternative method, D. melanogaster, as well as a locomotor assay. Results The phytochemical screening test of methanolic leaves extract revealed the presence of alkaloids, coumarins, quaternary bases, phenolics, flavonoids, tannins, and free steroids. A quantitative phytochemical study indicated the total phenol (30.17 ± 1.44 mg/g), flavonoid (21.64 ± 0.66 mg/g), and condensed tannins (9.58 ± 0.99 mg/g). DPPH (345.41 ± 5.35 μg/mL) and FRAP (379.98 ± 39.25 μM FeSO4/mg sample) show to extract of C. nutans leaves an intermediate value, indicating moderate antioxidant activity of the extract. Antibacterial results revealed only a positive result (antimicrobial activity) for the hexane fraction which significantly inhibited the microorganisms E. epidermidis, C. tropicalis, C. glabrata, and C. krusei at a concentration of 1000 μg/mL. TBARS, carbonyl protein, and DCFH demonstrate that the extract has the ability to protect the cell from protein and lipid damage, as well as the inhibition of oxygen-derived radicals at the three concentrations tested: 0.1, 1, and 10 mg/mL. Regarding the toxicity of C. nutans extract against D. melanogaster, it was found that until the concentration of 15 mg/mL, the extract showed no toxicity and that the LC50 obtained was 24 mg/mL. Results show that the C. nutans extract leaves used to prevent PQ damage were effective in reducing flies' mortality and improving locomotor capacity. Conclusion Our studies demonstrated for the first time that C. nutans crude leaf extract has high antioxidant capacity both in vitro and in vivo through different analysis techniques. These results make it possible to infer future applications in the pharmacological area, evidenced by the low toxicity observed in D. melanogatser, as well as the ability to neutralize different sources of RONS.

Role of preservative free steroids in treatment of inflammatory punctal stenosis guided by Anterior Segment Optical Coherence Tomography

Abstract Background Acquired inflammatory punctual stenosis is a disease commonly presented with epiphora and most common cause is long term treatment with preservative containing topical eye drops. The inflammation causes a decrease in the clearance of the medication and preservative, and their continued use will result in more inflammation and stenosis, and therefore a longer clearance time resulting in a higher concentration and longer transit time, leading to a vicious cycle. Objective This study was conducted to compare between the effectiveness of topical preservative free steroids eye drops and preservative free artificial tears in treatment of punctal edema in Egypt. Methods This prospective comparative study comprised 40 eyes with punctal edema. Group A: 20 eyes Patients in this group received preservative free hydrocortisone 2.5% eye drops 4 times daily for 1 week with tapering over 3 weeks and followed up after 1 month. And after 3 months. Group B: 20 eyes Patients in this group received one of the preservative free artificial tears available in market 4 times/day for 1 month. All subjects were subjected to history taking, slit lamp examination, Flourescene dye disappearance test for diagnosis and follow up once/month for 3 months and measuring the punctual diameter pretreatment, after 1 month and after 3 months. From treatment. Results Comparing the two groups, the study found that there was no statistically significant difference found between the two studied groups regarding punctual diameter pretreatment, at 1 month and at 3 months and a highly statistically significant symptomatic improvement after 3 months in preservative free steroids group more than the preservative free artificial tears group. Conclusion The present study compared the effectiveness of topical preservative free steroid eye drops and preservative free artificial tears as regards increase in punctual diameter and symptomatic improvement and found a statistically significant increase in symptomatic improvement in preservative free steroid group more than the preservative free artificial tears group as after 3 months and statistically significant increase in punctual diameters in both groups in comparison to control group.

Design and synthesis of 3-benzylaminocoumarin-7-O-sulfamate derivatives as steroid sulfatase inhibitors.

Steroid sulfatase (STS) is a sulfatase enzyme that catalyzes the conversion of sulfated steroid precursors to free steroid. The inhibition of STS could abate estrogenic steroids that stimulate the proliferation and development of breast cancer, and therefore STS is a potential target for adjuvant endocrine therapy. In this study, a series of 3-benzylaminocoumarin-7-O-sulfamate derivatives targeting STS were designed and synthesized. Structure-relationship activities (SAR) analysis revealed that attachment of a benzylamino group at the 3-position of coumarin improved inhibitory activity. Compound 3j was found to have the highest inhibition activity against human placenta isolated STS (IC500.13μM) and MCF-7 cell lines (IC50 1.35µM). Kinetic studies found compound 3j to be an irreversible inhibitor of STS, with KI and kinact value of 86.9nM and 158.7min-1, respectively.

P697 Efficacy and safety of ustekinumab in Crohn’s disease: A real-world study from Australia

Abstract Background Ustekinumab (UST), a human anti-IL12/23p40 monoclonal antibody, was approved in Australia for the treatment of adults with moderate to severe Crohn’s disease (CD) in 2017. The aim of this retrospective single centre study was to study the efficacy and safety of UST in CD in a real world cohort. Methods Patients with CD who began UST therapy between June 2017 and July 2019 were included. UST induction was given as an infusion (6 mg/kg) at week 0 followed by 90 mg subcutaneous injection (SC) at week 8 and 90 mg SC every 8 weeks as maintenance. Primary endpoint (PE) was steroid free clinical remission or steroid free clinical response at week 24. Secondary endpoints (SE) were: endoscopic response or remission, radiological response or remission, biochemical response (CRP < 5 mg/L or Calprotectin <150 μg/g), clinical response or remission at week 52. Results Seventy-six patients with CD were included in the study. 64.5% failed ≥1 anti-TNF and 13.1% failed anti-TNF and vedolizumab; 26(34.2%) patients were biologic-naïve. Median follow-up was 61 weeks. Ten patients (13.1%) discontinued UST, and the median time to discontinuation was 48 weeks; eight patients due to loss of response, one patient due to paradoxical worsening of arthralgia, one patient chose to stop treatment. Six patients underwent surgery whilst on UST. At week 12, 48 (63.1%) of patients achieved steroid free clinical remission or response [19/26 (73%) Anti-TNF naive and 29/49 (59%) anti-TNF exposed]; of these patients, 16/19 (84%) in the anti-TNF naive group and 19/29(65.5%) in the anti-TNF exposed group achieved PE. Forty-seven (61.8%) patients achieved PE. At 52 weeks, 68.2% (30/44), 67.5% (27/40), 56.1% (23/41), 69% (29/42) achieved endoscopic, radiological, biochemical and clinical endpoints respectively. Achieving steroid free clinical response or remission at week 12 was associated with achieving PE (79% vs. 42.8%, OR 3.6, p 0.01) and clinical SE (83.3 vs. 58.3%, p 0.001). Patients with B2/3 vs. B1(54% vs. 82%, p 0.09), ≥2 biological failure (72% vs. 33%, p 0.09), CRP < 6 baseline (55 % vs. 67.5%, p 0.3) were less likely to attain PE. Conclusion In a real-world cohort, UST appears efficacious and safe in medium and long term, with modest clinical, biochemical, radiological and endoscopic outcomes. Patients who achieve steroid-free clinical remission or response at 12 weeks are more likely to be in clinical remission or response at 24 and 52 weeks.

The effect of preservative free hydrocortisone 0.335% eye drops on tear film thickness in patients with moderate to severe dry eye disease

AbstractPurposeDry Eye Disease (DED) has been found to be associated with reduced pre‐corneal tear film thickness (TFT). Previous studies have shown that topical lubricants have the potential to increase TFT in patients with DED. In the present study, we investigated whether anti‐inflammatory therapy with topical low dose preservative free (PF) soft steroid, hydrocortisone 0.335% eye drops also has an effect on TFT in patients with moderate to severe DED.MethodsA total of 60 patients with moderate to severe DED were included. Patients were randomized to receive either hydrocortisone 0.335% for 12 days 4 times daily followed by 2 days twice daily instillation (intense treatment group) or 8 days 3 times daily followed by 3 days twice daily treatment (recommended treatment group). TFT was assessed using a custom‐built ultra‐high resolution optical coherence tomography system at baseline, at day 14 and day 28. Measurement of intraocular pressure (IOP) and standard clinical tests to assess signs and symptoms of DED, such as the Ocular Surface Disease Index (OSDI), were performed.ResultsFour weeks after treatment start, a significant increase in TFT was observed in both groups (5.3 ± 8.9%, p = 0.03 vs. baseline in the intense and 5.6 ± 13.1%, p = 0.04 vs. baseline in the recommended treatment group) with no difference between the two groups (p = 0.94). IOP did not change at the end of the treatment period in neither of the two treatment groups (p = 0.38, from 14.1 ± 1.8 to 13.9 ± 2.0 mmHg in the intense and from 12.8 ± 1.9 to 13.3 ± 2.1 mmHg in the standard treatment group). OSDI significantly decreased from baseline during the study period in both study groups (p < 0.001, by 19.5 ± 39.1% and 24.1 ± 33.9%, respectively) after PF hydrocortisone treatment.ConclusionsThe present data show that low dose hydrocortisone PF eye drops have the potential to restore TFT in patients with moderate to severe DED. This indicates that this anti‐inflammatory therapy option acts directly in the disease process.

7-keto-DHEAmetabolism in humans. Pitfalls in interpreting the analytical results in the antidoping field.

7-keto-DHEA (3β-hydroxy-androst-5-ene-7,17-dione) is included in section S1 of the World Antidoping Agency (WADA) List of Prohibited Substances. The detection of its misuse in sports needs special attention, since it is naturally present in urine samples. The main goal of this study is to investigate the in vivo metabolism of 7-keto-DHEA after a single administration to healthy volunteers and to better describe the relationship between arimistane (androst-5-ene-7,17-dione) and 7-keto-DHEA after the application of the common routine procedures to detect anabolic steroids in WADA accredited antidoping laboratories. Free, glucuro-, and sulpho-conjugated steroids extracted from urine samples obtained before and after the administration of 7-keto-DHEA were analyzed by different gas chromatographic (GC)-mass spectrometric (MS) techniques. Gas chromatography coupled to tandem MS to study the effect on the endogenous steroid profile, coupled to isotope ratio mass spectrometry (IRMS) to investigate the potential formation of androgens derived from DHEA and coupled to high resolution accurate mass spectrometry (HRMS) to investigate new diagnostic metabolites. The analysis by IRMS confirmed that there is no formation of DHEA from 7-keto-DHEA. Ten proposed metabolites, not previously reported, were described. These include reduced and hydroxylated structures that are not considered part of the steroid profile in antidoping analyses. They showed considerable responses in all fractions analyzed. Some deoxidation reactions (including arimistane formation) were found and most probably can be linked to the sample preparation or instrumental analysis. This is important when interpreting the results after the application of procedures to detect steroids in urine currently used in antidoping laboratories. 7-keto-DHEA metabolism in humans for antidoping purposes was studied and unexpected results were found. This could lead to a misinterpretation of the data, depending on the procedure applied and the analytical instrumentation used.

Rapid and structure-specific cellular uptake of selected steroids.

Steroid hormones and their respective nuclear receptors are essential mediators in numerous physiologic and pathophysiologic processes, ranging from regulation of metabolism, immune function, and reproductive processes to the development of hormone-dependent cancers such as those of the breast and prostate. Because steroids must enter cells before activating nuclear receptors, understanding the mechanisms by which cellular uptake occurs is critical, yet a clear understanding of these mechanisms has been elusive. It is generally assumed that diffusion-driven uptake is similar across various steroids whereas an elevated cellular concentration is thought to reflect active uptake, but these assumptions have not been directly tested. Here we show that intact cells rapidly accumulate free steroids to markedly elevated concentrations. This effect varies widely depending on steroid structure; more lipophilic steroids reach more elevated concentrations. Strong preferences exist for 3β-OH, Δ5-steroids vs. 3-keto, Δ4-structural features and for progestogens vs. androgens. Surprisingly, steroid-structure-specific preferences do not require cell viability, implying a passive mechanism, and occur across cells derived from multiple tissue types. Physiologic relevance is suggested by structure-specific preferences in human prostate tissue compared with serum. On the other hand, the presence of serum proteins in vitro blocks much, but not all, of the passive accumulation, while still permitting a substantial amount of active accumulation for certain steroids. Our findings suggest that both passive and active uptake mechanisms make important contributions to the cellular steroid uptake process. The role of passive, lipophilicity-driven accumulation has previously been largely unappreciated, and its existence provides important context to studies on steroid transport and action both in vitro and in vivo.

CUSSONIA SPICATA THUNB. IN TROPICAL AFRICA: PHYTOCHEMISTRY, PHARMACOLOGY, AND MEDICINAL POTENTIAL

Cussonia spicata is an evergreen tree widely used as herbal medicine throughout its distributional range in tropical Africa. The current study is aimed at providing a critical review of the phytochemistry, pharmacology, and evaluation of the medicinal potential of C. spicata. Documented information on the phytochemistry, pharmacology, and medicinal applications of C. spicata was collected from several online sources which included BMC, Scopus, SciFinder, Google Scholar, Science Direct, Elsevier, PubMed, and Web of Science. Additional information on the phytochemistry, pharmacology, and medicinal applications of C. spicata was gathered from pre-electronic sources such as book chapters, books, journal articles, and scientific publications sourced from the University library. This study showed that the bark, flowers, flower stalks, fruits, leaves, roots, root bark, and stems of C. spicata are used as antifebrile and emetic and herbal medicine for fever, nausea, vomiting, gonorrhea, venereal diseases, malaria, and mental illness. Phytochemical compounds identified from the leaves, root bark, stems, and stem bark of C. spicata include alkaloids, anthocyanins, anthracene glycosides, botulin, condensed tannins, free gallic acid, gallotannins, iridoids, pentacyclic triterpenoids, saponins, steroids, tannins, flavonoids, phenolics, triterpenoids, and volatile oils. Pharmacological research revealed that C. spicata crude extracts and compounds have acetylcholinesterase, antibacterial, antiviral, anti-inflammatory, antileishmanial, antiplasmodial, antiprotozoan, antioxidant, larvicidal, molluscicidal, spermicidal, and cytotoxicity activities. Future research should focus on evaluating the phytochemical, pharmacological, and toxicological properties of C. spicata crude extracts as well as compounds isolated from the species.

The study of photosynthesis pigments of canna hybrida herb of some varieties

Plants of Cannaceae family is a popular decorative and leafy plant with bright flowers and large leaves. According to the literature they contain amino acids, free carboxylic acids, flavonoids, anthocyanins, terpene and steroid compounds, cardioglycosides and alkaloids. They exhibit a wide range of biological activity, including antioxidant, antimicrobial, anti-inflammatory, cardioprotective, hemostatic, antihypertensive and anti-ischemic activity.The aim of the research was to study the qualitative composition and to determine the quantitative content of chlorophylls and carotenoids in herb of the Canna hybrida Suevia, Liberty, Livadia, Artek and Dark Night varieties.Materials and methods. The qualitative composition of photosynthetic pigments of herb of the Canna hybrida varieties Suevia, Liberty, Livadia, Artek and Dark Night differed not significantly by thin-layer chromatography method. In the analyzed objects the content of chlorophyll a, chlorophyll b, carotenoids and lycopene was determined by absorption spectrophotometry method.Results. At least 10 compounds appeared on the chromatograms of the studied samples, at least 4 of which were attributed to carotenoids and not less than 6 – to chlorophylls. It was established that the content of the total of chlorophylls in herb of the Canna hybrida Liberty, Artek, and Dark Night varieties exceeded the content of the carotenoids by approximately 5 times, in herb of the Canna hybrida Suevia and Livadia variety – almost 8 times. Approximately 65 % of the total content of chlorophyll was chlorophyll a, 35 % was chlorophyll b, which was characteristic of herb of the Canna hybrida Suevia, Liberty, Artek and Dark Night varieties. In herb of the Canna hybrida Livadia variety a fundamentally opposite situation was observed. The total content of chlorophyll b was in 2.3 times higher than the content of chlorophyll a.The highest content of chlorophyll a (1661.76 ± 41.54 mg/kg), chlorophyll b (943.79 ± 23.60 mg/kg) and lycopene (62.37 ± 1.56 mg/kg) was observe in herb of the Canna hybrida Suevia variety. The equally high content of carotenoids – 373.75 ± 9.34 mg/kg, 381.84 ± 9.55 mg/kg and 386.05 ± 9.65 mg/kg, was watched in herb of Canna hybrida Suyeviya, Liberty and Artek varieties respectively.Conclusions. The obtained results will be used in the development of quality control methods for herb of Canna hybrida and medicinal plant products on their basis.

Measurement of serum steroid profiles by HPLC-tandem mass spectrometry

Accurate investigations of adrenal hormone levels play a vital role in pediatric endocrinology for the detection of steroid-related disorders. In this study, we developed and validated a simultaneous assay for eight serum steroids, i.e., DHEA, androstenedione, testosterone, progesterone, 17‑hydroxyprogesterone, DHEA‑sulfate, pregnenolone‑sulfate and cholesterol-sulfate, using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Serum samples were prepared by liquid-liquid extraction with methyl t‑butyl ether. Quantitation by LC-MS/MS was performed in multiple reaction monitoring mode with an electrospray ionization source. The run time was 10 min. Analytical performance was evaluated, including imprecision, linearity, ion suppression, carry over and detection capabilities. Serum specimens from 59 children and 120 adults were analyzed to compare the distribution of steroid levels between the groups. All hormones were effectively extracted and separated using our method. The method was essentially free from potential interference and ion suppression. Within-run and between-run imprecision values were <20%. The lower limits of quantification varied from 0.025 to 10 ng/mL. The results were generally good and correlated with those obtained using immunoassay techniques. We developed the HPLC-MS/MS method for the simultaneous measurement of free and sulfated steroid hormones. The performance of the developed method was generally acceptable. Thus, this method may provide a novel approach to steroid profiling in children of age before adrenarche.

Analysis of steroids in urine by gas chromatography-capillary photoionization-tandem mass spectrometry

A new heated capillary photoionization (CPI) ion source design was developed to photoionize analytes inside a transfer capillary between a gas chromatograph (GC) and a mass spectrometer (MS). The CPI setup included a wide, oval-shaped vacuum-ultraviolet (VUV) transparent magnesium fluoride (MgF2) window to maximize photoionization efficiency and thus sensitivity. The source contained a nitrogen housing around the ionization chamber inlet to avoid undesirable hydrolysis and oxidation reactions with ambient air and to maximize the proportion of formed molecular radical cations of analytes. The feasibility of the ion source was studied by analyzing 18 endogenous steroids in urine as their trimethylsilyl (TMS) derivatives with gas chromatography-tandem mass spectrometry (GC–MS/MS). The method was validated and applied to human urine samples. To our best knowledge, this is the first time that a capillary photoionization ion source has been applied for quantitative analysis of biological samples. The GC-CPI-MS/MS method showed good chromatographic resolution (peak half-widths between 3.1 to 5.3 s), acceptable linearity (coefficient of determination between 0.981 to 0.996), and repeatability (relative standard deviation (RSD%) between 5 to 18%). Limits of detection (LOD) were between 2 to 100 pg mL−1 and limits of quantitation (LOQ) were between 0.05 to 2 ng mL−1. In total, 15 steroids were quantified either as a free steroid or glucuronide conjugate from the urine of volunteers. The new CPI source design showed excellent sensitivity for analysis of steroids in complex biological samples.

Phytotoxic potential of Senna occidentalis (L.) Link extracts on seed germination and oxidative stress of Ipê seedlings.

Senna occidentalis is an invasive plant producing a series of allelochemicals that might inhibit the development of other plants. The objective of this study was to assess the phytotoxic effect of S.occidentalis extracts on the germination, development and antioxidant defence of the native species Tabebuia chrysotricha, T.pentaphylla, T.roseoalba and Handroanthus impetiginosus (Ipê species). We evaluated the effects of chemicals extracted from S.occidentalis on the germination rate, germination speed index (GSI) and biometric parameters of the test species under controlled conditions. The effect of the extracts on the pigment content, amount of H2 O2 and malondialdehyde (MDA), and the activity of the antioxidant enzymes in roots and leaves were also tested. Alkaloids, coumarins, phenols, saponins, free steroids and condensed tannins were present in all extracts of S.occidentalis, while catechins were present only in leaf and stem extracts. Stem and root extracts caused a growth reduction in all Ipê species and total inhibition of seed germination in T.chrysotricha and T.roseoalba. All target species showed an increase in H2 O2 and MDA in radicles and leaves. Oxidative stress contributed strongly to the morphological changes, such as seed blackening, thinning and darkening of radicle tips and reduction of biomass allocation in all Ipê species. Although there was activation of antioxidant defence mechanisms, such as an increase in activity of ascorbate peroxidase (APX) and peroxidase (POD) enzymes, the joint action of the allelochemicals caused phytotoxicity, leading to cell dysfunction in all Ipê species.

Effects of hormonal contraceptive phase and progestin generation on stress-induced cortisol and progesterone release

The stress response differs between women using hormonal contraception and naturally cycling women. Yet, despite ample evidence showing that the stress response differs across the menstrual cycle in naturally cycling women, limited work has investigated whether the stress response differs across the hormonal contraceptive cycle, during which synthetic hormones are taken most of the month but not all of it. To induce a stress response, women using hormonal contraception completed the cold pressor test during either the active phase, when hormones are present, or during the inactive phase, when hormones are not present. Saliva was collected and assayed for free cortisol and progesterone levels prior to stress onset, immediately after stress termination, and 15-min post stress onset. Free cortisol and progesterone increased to a similar degree across both hormonal contraceptive phases in response to the cold pressor test. Post-hoc investigation indicates that the progestin “generation” (classification of synthetic progestins based on the compounds they are derived from) can differentially affect the free steroid response to cold pressor test stress, with the largest effects observed in women using formulations containing second-generation progestins. These findings indicate that progestin generation, particularly second-generation progestins, may have a more impactful influence on the stress response than hormonal contraceptive cycle phase. Potential mechanisms driving this effect and need for additional research are discussed.

GC-MS analysis of steroids and triterpenoids occurring in leaves and tubers of Tamus edulis Lowe

Tamus edulis Lowe is an endemic perennial plant belonging to Dioscoreaceae family. The plant has long climbing stems, ovate leaves, flowers in spikes, fleshy red berries and long tuberous roots. Young shoots and tuberous roots of T. edulis were used traditionally for nourishment and as a herbal medicine. Leaves and roots analyzed in the present study were collected in the northwest of Madeira island. The GC–MS analysis allowed to detect several steroids in free forms in diethyl ether extracts; and diosgenin with its isomer, yamogenin, in hydrolyzates from methanolic extracts.The obtained results reveal that Tamus edulis has some features common with other Dioscorea species, e.g., the presence of steroidal saponins with diosgenin and yamogenin as aglycones, or the phytosterol composition with predominating sitosterol. However, some other traits, like the relatively high content of free steroids (more than 1 mg/g d.w.) and their profile rich in cholesterol derivatives, can distinguish Tamus edulis from other Dioscorea species studied previously for their steroid and triterpenoid profile.

Salivary alpha-amylase and hormones levels of young adults with different body composition.

The aim of this study was to assess the differences in salivary biomarker levels of young adults classified according to body fat accumulation. One hundred and thirty-four volunteers were evaluated (mean age 21 ± 2 years). Body composition was calculated through skinfold thickness: supra-iliac, biceps, and triceps, sub-scapular. Body fat percentage (BF%) was used to classify subjects according to fat tissue accumulation: normal-weight (n = 37, 19 females, 18 males), overweight (n = 42, 30 females, 12 males), and obese (n = 55, 42 females, 13 males). Saliva samples were collected 30 min after awakening to determine salivary levels of 17-beta-estradiol. For salivary cortisol and alpha-amylase activity (sAA) three samples were obtained, just after awakening, 30 min after awakening and at bedtime. Oral contraceptive intake was considered for the female group. The results showed that overweight and obese females using oral contraceptive presented lower levels of 17-β-estradiol than normal-weight females. In overweight and obese males, sAA levels were higher 30 min post-awakening when compared with the normal-weight group. The comparison of sAA levels within time showed no difference for males; obese females showed significant higher values at bedtime than 30 min post-awakening. The salivary cortisol concentration showed higher values at morning decreasing significantly at bedtime for all groups. Concluding, differences in 17-β-estradiol and sAA levels were found in females and males, respectively, according to body fat accumulation, showing the usefulness of salivary biomarkers in the study of systemic conditions. PRACTICAL APPLICATIONS: Saliva is an advantageous biological fluid in innovative methods for diagnosis. Besides being a noninvasive method, salivary steroid measurements have the potential to provide a convenient assessment of serum free steroid concentrations.

Phytochemical Characterization and Effect of Cagaita Leaf Extracts on Aspergillus Sp.

ABSTRACT Fungi of the genus Aspergillus may promote damage to the physiological quality of forest seeds, and plant extracts have been used to control microflora associated with these seeds. Thus, the aim of the present work was to perform the phytochemical characterization of ethanolic and aqueous extracts obtained from Eugenia dysenterica leaves (cagaita), as well as to evaluate the effect of these extracts on the development of Aspergillus sp. isolated from Hymenaea stigonocarpa seeds (jatoba-do-cerrado). For this, qualitative phytochemical analysis of extracts was carried out and their effect on Aspergillus sp mycelial growth, sporulation and spore germination was evaluated. As a result, phenols, hydrolysable tannins, flavonols, flavanones, xanthones, flavones, free steroids and saponins were found in both extracts, which could be used in the search for new antifungal compounds in the treatment of stored forest seeds, since extracts have secondary compounds that allow inhibiting Aspergillus sp mycelial growth.

Type 2 Diabetes and Cancer: The Nitric Oxide Connection

Type 2 diabetes (T2DM) is now considered an independent risk factor for the development of some types of cancers, including liver, pancreas, endometrium, colon, rectum, breast, and bladder cancers. Several underlying mechanisms have been proposed that connect T2DM and cancer: hyperglycemia, hyperinsulinemia, increased levels of free steroid and peptide hormones, oxidative stress, and proinflammatory cytokines. Because nitric oxide (NO), a multifunctional gaseous signaling molecule, plays a critical role in carcinogenesis and tumor growth progression, undesirable changes of the NO system in T2DM may be an important missing link between these two pathogenic conditions. Increased inducible NO synthase (iNOS) activity is seen in T2DM (due to inflammatory cytokines, mitochondrial dysfunction, hyperglycemia and hypoxia), where detrimental amounts of NO can result in the initial carcinogenic transformation of normal cells and progression to malignancy. The uncoupling of endothelial NO synthase (eNOS) and increased generation of free radicals in hyperglycemic conditions can also lead to the formation of highly reactive nitrogen species such as peroxynitrite, which can lead to DNA damage, carcinogenic mutations, and activation of critical pathways involved in cell proliferation and apoptosis. In this review, we focused on the hypothesis that the imbalanced NO metabolism in pathologic conditions such as T2DM may contribute to the development of cancer. Overall, current evidence strongly suggests that an imbalanced NO metabolism in T2DM may contribute to the development of cancer.

Phytochemical and phytotoxic investigation of the aqueous leaf extract of Eugenia dysenterica DC. under laboratory conditions

The objective of this study was to carry out a phytochemical characterization of the aqueous leaf extract of Eugenia dysenterica DC., as well as to evaluate its phytotoxic potential on the germination and growth of Lactuca sativa L. and Zea mays L. under laboratory conditions. Concentrations of 250, 500, 1000, 2000 and 4000 mg/L of the extract in addition to the control were used for the bioassays. Under laboratory conditions this extract inhibited average growth of the radicle/primary root and hypocotyl/coleoptile leaves of lettuce and maize, with proportionality between the effect and the concentration being observed. The presence of phenols, hydrolysable tannins, flavonols, flavanones, flavanonols, xanthones, flavones, free steroids and saponins were identifiedin the aqueous extract. The aqueous extract of the plant species under study has a phytotoxic effect, and this effect is due to the presence of phenolic compounds, tannins, flavonoids and saponins.