Identification Of Active Metabolites Research Articles

Antineoplastic Activity of Rhus trilobata Nutt. (Anacardiaceae) against Ovarian Cancer and Identification of Active Metabolites in This Pathology.

Rhus trilobata (RHTR) is a medicinal plant with cytotoxic activity in different cancer cell lines. However, the active compounds in this plant against ovarian cancer are unknown. In this study, we aimed to evaluate the antineoplastic activity of RHTR and identify its active metabolites against ovarian cancer. The aqueous extract (AE) and an active fraction (AF02) purified on C18-cartridges/ethyl acetate decreased the viability of SKOV-3 cells at 50 and 38 μg/mL, respectively, compared with CHO-K1 (>50 μg/mL) in MTT assays and generated changes in the cell morphology with apoptosis induction in Hemacolor® and TUNEL assays (p ≤ 0.05, ANOVA). The metabolite profile of AF02 showed a higher abundance of flavonoid and lipid compounds compared with AE by UPLC-MSE. Gallic acid and myricetin were the most active compounds in RHTR against SKOV-3 cells at 50 and 166 μg/mL, respectively (p ≤ 0.05, ANOVA). Antineoplastic studies in Nu/Nu female mice with subcutaneous SKOV-3 cells xenotransplant revealed that 200 mg/kg/i.p. of AE and AF02 inhibited ovarian tumor lesions from 37.6% to 49% after 28 days (p ≤ 0.05, ANOVA). In conclusion, RHTR has antineoplastic activity against ovarian cancer through a cytostatic effect related to gallic acid and myricetin. Therefore, RHTR could be a complementary treatment for this pathology.

Pharmacokinetics of noopept and its active metabolite cycloprolyl glycine in rats

The study of the pharmacokinetics of new drugs and the identification of active metabolites is a necessary step for effective and safe use in the clinical practice. It is especially important for peptide drugs due to their enzymatic instability, low bioavailability and poor permeability through the blood-brain barrier (BBB). The role of endogenous neuropeptides containing cyclic amino acids, proline, pyroglutamic acid, and glycine, in the regulation of memory processes is known as terminal peptide fragments. The development of nootropic drugs based on natural neuropeptides with high pharmacological activity and improved pharmacokinetic properties (enzymatic stability, high bioavailability, and good permeability through the BBB) is an important problem of modern neuropsychopharmacology. Developed drugs - representing short (di- and tri-) peptides appear to meet these requirements. In the Zakusov Research Institute of Pharmacology a nootropic agent noopept (N-phenylacetyl-prolyl-L-glycine ethyl ester), was developed and introduced into medical practice, studies of its pharmacokinetics in ratsrevealed that the noopept metabolite found in the rat plasma and brain, cyclo-prolyl-L-glycine (CPG), differed significantly in its pharmacokinetic parameters from noopept, but at the same time it had similar noopept multi-component spectrum of pharmacological action, namely the influence on higher integrative functions of memory.

Is the History Repeated? Can (2R,6R)-Hydroxynorketamine be Another Antidepressant?

Historically, identification of active metabolites has contributed to drug discovery for psychiatric disorders. It has led to the identification of new medications such as desipramine (a metabolite of imipramine) and paliperidone (a metabolite of risperidone). (R,S)-Ketamine, which has been regarded as the greatest breakthrough in depression research, is rapidly and stereoselectively metabolized into a variety of metabolites. Therefore, identification of an active substance after administration of (R,S)-ketamine is a critical issue, not only to delineate the underlying mechanisms but also to pave the way to develop a new antidepressant. Recently, one of the metabolites of (R,S)-ketamine, namely, (2R,6R)-hydroxynorketamine (HNK) was proposed as an active metabolite formed after administration of (R,S)-ketamine, and even as being essential for (R,S)-ketamine to exert its antidepressant effects. However, this is still controversial. Indeed, we demonstrated that the antidepressant effect of (2R,6R)-HNK is not as potent as that of its parent compounds ((R)-ketamine and (R,S)-ketamine), and that (2R,6R)-HNK is not essential for (R)-ketamine to exert its antidepressant effects. From the historical point of view, however, there is potential to discover new medications by further investigations of (2R,6R)-HNK. Therefore, more careful and thorough investigation of (2R,6R)-HNK is needed for the discovery of more efficacious and safer antidepressants.

Isolation and purification of (E)-3- (2, 3- dihydroxyphenyl) acrylic acid fromendophytic fungi Fusarium equsetiEF-32 and its anti-candidal and anticancer activities

The endophytic fungi Fusarium equseti was isolated from the healthy tissues of Terminalia pallida and the EtOAc extracts of the culture was subjected to primary antimicrobial screening against two fungal cultures, along with anticancer activity in breast cancer cell lines. As a result, potential extracts exhibiting anticancer and antimicrobial activity among them, were selected for further investigation and identification of active metabolites. Bioassay-guided fraction of the EtOAc extract of liquid culture were identified as (E)-3- (2,3- dihydroxy phenyl) acrylic acid by mass and spectral means (MS, IR NMR). In vitro anticancer and antimicrobial assay showed that compound was substantially active against Candida tropicalis and Candida albicans and breast cancer cell lines evident from MTT (3-(4, 5-Dimethylthiazol-2-yl)-2,5-DiphenyltetrazoliumBromide) assay.

Piper betle and its bioactive metabolite phytol mitigates quorum sensing mediated virulence factors and biofilm of nosocomial pathogen Serratia marcescens in vitro

Ethnopharmacological relevancePiper betle, a tropical creeper plant belongs to the family Piperaceae. The leaves of this plant have been well known for their therapeutic, religious and ceremonial value in South and Southeast Asia. It has also been reported to possess several biological activities including antimicrobial, antioxidant, antinociceptive, antidiabetic, insecticidal and gastroprotective activities and used as a common ingredient in indigenous medicines. In Indian system of ayurvedic medicine, P. betle has been well recognized for its antiseptic properties and is commonly applied on wounds and lesions for its healing effects. Aim of the studyTo evaluate the anti-quorum sensing (anti-QS) and antibiofilm efficacy of P. betle and its bioactive metabolite phytol against Serratia marcescens. Materials and methodsThe P. betle ethyl acetate extract (PBE) was evaluated for its anti-QS efficacy against S. marcescens by assessing the prodigiosin and lipase production at 400 and 500µgml−1 concentrations. In addition, the biofilm biomass quantification assay was performed to evaluate the antibiofilm activity of PBE against S. marcescens. Besides, the influence of PBE on bacterial biofilm formation was assessed through microscopic techniques. The biofilm related phenomenons like exopolysaccharides (EPS) production, hydrophobicity and swarming motility were also examined to support the antibiofilm activity of PBE. Transcriptional analysis of QS regulated genes in S. marcescens was also done. Characterization of PBE was done by separation through column chromatography and identification of active metabolites by gas chromatography -mass spectrometry. The major compounds of active fractions such as hexadecanoic acid, eugenol and phytol were assessed for their anti-QS activity against S. marcescens. Further, the in vitro bioassays such as protease, biofilm and HI quantification were also carried out to confirm the anti-QS and antibiofilm potential of phytol in PBE. ResultsPBE inhibits QS mediated prodigiosin pigment production in S. marcescens, which confirmed its anti-QS potential against S. marcescens. At 500µgml−1 concentration, PBE significantly inhibited the production of protease, lipase, biofilm and EPS to the level of 71%, 68%, 65% and 43% in S. marcescens, respectively. Further, their antibiofilm efficacy was confirmed through microscopic techniques. In addition, PBE effectively inhibited the hydrophobicity and swarming motility. Additionally, the results of qPCR analysis validated the downregulation of QS genes. Chromatographic techniques the presence of hexadecanoic acid, eugenol and phytol in PBE and the potential bioactive compound with anti-QS activity was identified as phytol. In vitro assays with phytol evidenced the potent inhibition of QS-controlled prodigiosin, protease, biofilm and hydrophobicity in S. marcescens, without exerting any deleterious effect on its growth. ConclusionThis study demonstrates the promising anti-QS and antibiofilm activities of PBE and its active metabolite phytol, and confirms the ethnopharmacological applications of these leaves against S. marcescens infections.

Stevia-derived compounds attenuate the toxic effects of ectopic lipid accumulation in the liver of obese mice: A transcriptomic and metabolomic study

There is a close interaction between Type 2 Diabetes, obesity and liver disease. We have studied the effects of the two most abundant Stevia-derived steviol glycosides, stevioside and rebaudioside A, and their aglycol derivative steviol on liver steatosis and the hepatic effects of lipotoxicity using a mouse model of obesity and insulin resistance. We treated ob/ob and LDLR-double deficient mice with stevioside (10 mg⋅kg−1⋅day-1 p.o., n = 8), rebaudioside A (12 mg⋅kg−1⋅day-1 p.o., n = 8), or steviol (5 mg⋅kg−1⋅day−1 p.o., n = 8). We determined their effects on liver steatosis and on the metabolic effects of lipotoxicity by histological analysis, and by combined gene-expression and metabolomic analyses. All compounds attenuated hepatic steatosis. This could be explained by improved glucose metabolism, fat catabolism, bile acid metabolism, and lipid storage and transport. We identified PPARs as important regulators and observed differences in effects on insulin resistance, inflammation and oxidative stress between Stevia-derived compounds. We conclude that Stevia-derived compounds reduce hepatic steatosis to a similar extent, despite differences in effects on glucose and lipid metabolism, and inflammation and oxidative stress. Thus our data show that liver toxicity can be reduced through several pathophysiological changes. Further identification of active metabolites and underlying mechanisms are warranted.

Glycosyl glycerides from hydroponic Panax ginseng inhibited NO production in lipopolysaccharide-stimulated RAW264.7 cells

BackgroundAlthough the aerial parts of hydroponic Panax ginseng are reported to contain higher contents of total ginsenosides than those of roots, the isolation and identification of active metabolites from the aerial parts of hydroponic P. ginseng have not been carried out so far. MethodsThe aerial parts of hydroponic P. ginseng were applied on repeated silica gel and octadecylsilane columns to yield four glycosyl glycerides (Compounds 1–4), which were identified based on nuclear magnetic resonance, infrared, fast atom bombardment mass spectrometry, and gas chromatography/mass spectrometry data. Compounds 1–4 were evaluated for inhibition activity on NO production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Results and conclusionThe glycosyl glycerides were identified to be (2S)-1-O-7(Z),10(Z),13(Z)-hexadecatrienoyl-3-O-β-d-galactopyranosyl-sn-glycerol (1), (2S)-1-O-linolenoyl-3-O-β-d-galactopyranosyl-sn-glycerol (2), (2S)-1-O-linolenoyl-2-O-linolenoyl-3-O-β-d-galactopyranosyl-sn-glycerol (3), and 2(S)-1-O-linoleoyl-2-O-linoleoyl-3-O-β-d-galactopyranosyl-sn-glycerol (4). Compounds 1 and 2 showed moderate inhibition activity on NO production in LPS-stimulated RAW264.7 cells [half maximal inhibitory concentration (IC50): 63.8 ± 6.4μM and 59.4 ± 6.8μM, respectively] without cytotoxicity at concentrations < 100μM, whereas Compounds 3 and 4 showed good inhibition effect (IC50: 7.7 ± 0.6μM and 8.0 ± 0.9μM, respectively) without cytotoxicity at concentrations < 20μM. All isolated compounds showed reduced messenger RNA (mRNA) expression of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α in LPS-induced macrophage cells with strong inhibition of mRNA activity observed for Compounds 3 and 4.

The Selenium Metabolite Methylselenol Regulates the Expression of Ligands That Trigger Immune Activation through the Lymphocyte Receptor NKG2D

For decades, selenium research has been focused on the identification of active metabolites, which are crucial for selenium chemoprevention of cancer. In this context, the metabolite methylselenol (CH3SeH) is known for its action to selectively kill transformed cells through mechanisms that include increased formation of reactive oxygen species, induction of DNA damage, triggering of apoptosis, and inhibition of angiogenesis. Here we reveal that CH3SeH modulates the cell surface expression of NKG2D ligands. The expression of NKG2D ligands is induced by stress-associated pathways that occur early during malignant transformation and enable the recognition and elimination of tumors by activating the lymphocyte receptor NKG2D. CH3SeH regulated NKG2D ligands both on the transcriptional and the posttranscriptional levels. CH3SeH induced the transcription of MHC class I polypeptide-related sequence MICA/B and ULBP2 mRNA. However, the induction of cell surface expression was restricted to the ligands MICA/B. Remarkably, our studies showed that CH3SeH inhibited ULBP2 surface transport through inhibition of the autophagic transport pathway. Finally, we identified extracellular calcium as being essential for CH3SeH regulation of NKG2D ligands. A balanced cell surface expression of NKG2D ligands is considered to be an innate barrier against tumor development. Therefore, our work indicates that the application of selenium compounds that are metabolized to CH3SeH could improve NKG2D-based immune therapy.

Abstract 3657: Identification of active metabolites of docosohexaenoic acid (DHA): A first step towards unraveling the fish oil/breast cancer conundrum.

Abstract The role of fish oil (FO) rich in omega-3 fatty acid (n-3FA) in breast cancer prevention remains controversial. Based on our data showing that a high n-3:n-6 ratio (≥10) is necessary to inhibit N-methyl-N-nitrosourea (MNU)-induced rat mammary carcinogenesis (Zhu Z, et al., Cancer Prev Res 4(10):1675, 2011), we hypothesize that this chemopreventive effect of FO may be due to one of its components (e.g., Docosohexaenoic acid, DHA, 22:6) and/or to the production of Active DHA Metabolites (ADM) which may be present in small and variable amounts in FO preparations used in different preclinical studies. To test this hypothesis, we used LC-TOF/MS followed by tandem mass spectroscopy to quantify ADM in the plasma and mammary tumors of rats fed a 20% CO diet and administered either saline or one of two doses of DHA (1.5 ml/kg and 3.0 ml/kg) by gavage twice a week for 8 weeks following ip MNU injection (50 mg/kg) at 21 days of age. The lower dose of DHA was also given in combination with Tamoxifen (Tam) (0.6 ppm) to test the hypothesis that n-3FA may inhibit the development of antiestrogen resistant tumors. We observed that both doses of DHA similarly inhibited mammary carcinogenesis and that the combination of Tam and DHA was more effective than each of the individual agents. DHA administration caused a significant dose-dependent increase in the plasma levels of several ADM including 4-OH-DHA, 7-OH-DHA, 14-OH-DHA, and 17-OH-DHA. The levels of these ADM were ∼10-fold higher in the tumors of the rats given DHA compared to control, irrespective of the dose of DHA administered. The most abundant ADM in the tumors was 14-OH-DHA followed in order by 17-OH-DHA, 4-OH-DHA, and 7-OH-DHA. In order to compare the biological activity of these ADM to that of the parent compound, we tested in vitro their ability to induce PPARγ activation using a PPARγ reporter assay since PPARγ activation is considered to be a potential mechanism by which n-3FA exert their antitumor action. We observed that every ADM was more effective than DHA by at least 2-fold in inducing PPARγ activity with the following order of magnitude of the effect, 7-OH-DHA &amp;gt; 14-OH-DHA &amp;gt; 4-OH-DHA &amp;gt; 17-OH-DHA. In summary, our studies identify for the first time ADM which may be responsible for the chemopreventive effect of FO. This finding may have major translational significance since compounds with well-defined chemical structure given at optimal concentrations are likely to be more effective chemopreventive agents than FO which contains an inconsistent mixture of FA with varying biological activity. In addition, since DHA inhibits carcinogenesis in Tam-treated rats, these ADM may complement the effect of Tam by inhibiting the development of hormone independent tumors. This work is supported by grant KG081632 from Susan G. Komen for the Cure. Citation Format: Andrea Manni, John P. Richie, Neil Trushin, Bogdan Prokopczyk, A Patterson, John P. Vanden Heuvel, Sharlene Washington, Haifang Xu, Cesar Aliaga, Arunangshu Das, Krishne Gowda, Shantu Amin, Jason Liao, Henry Thompson, Karam El-Bayoumy. Identification of active metabolites of docosohexaenoic acid (DHA): A first step towards unraveling the fish oil/breast cancer conundrum. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3657. doi:10.1158/1538-7445.AM2013-3657