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Development of an at-line coupling of LC-QTOF-ESI-MS/MS to steroid 5-alpha reductase inhibition assay, a fast bioactive targeting and guided purification of natural complex sample, Impatiens balsamina Linn

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Abstract

This study provides a rapid and accurate method for screening steroid 5-alpha reductase (S5αR) inhibitors in Impatiens balsamina Linn (IB). using at-line LC-QTOF-ESI-MS/MS coupling S5αR inhibitory assay. IB (Balsami- naceae) is an annual herbaceous plant cultivated in tropical and subtropical regions. It has been used in tradi- tional Chinese and Thai medicine for treatment of hair loss and various skin conditions, potentially through anti- androgenic mechanisms. A combined approach of S5αR inhibitory assay and LC-QTOF-ESI-MS/MS was devel- oped to rapidly screen for target biomarkers and guide their isolation using preparative HPLC. The toxicity of both the extract and isolated biomarkers was evaluated on skin cells, keratinocytes, and fibroblasts. Eight bioactive compounds were identified as two naphthoquinone, two fatty acid derivatives, three nitrogenous compounds and one aromatic derivative. The most potent bioactive markers, identified as 2-methoxy-1,4-naph- thoquinone (2MN) and impateinol, were targeted and isolated using preparative HPLC, yielding 5.0 % and 3.5 %, respectively. These compounds exhibited S5αR inhibitory activity higher than that of finasteride drug by 10 and 2 times, respectively. Both the isolated biomarkers and the extract demonstrated a broad therapeutic index. The developed method in this study proved to be both rapid and accurate, making it suitable for screening and targeting S5αR inhibitors in herbal plants or complex matrix samples. It facilitated the fast-guided isolation of bioactive compounds, highlighting its potential for future applications in drug discovery research.

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5α-reductase inhibitors (5ARI) include finasteride and dutasteride, and are commonly prescribed in the treatment of benign prostatic hyperplasia and androgenic alopecia. 5ARIs are associated with several known adverse effects (AEs), with varying reported prevalence rates. The aim was to review and summarize findings from published literature detailing AEs associated with 5ARI use. A secondary aim was to review potential mechanisms of action, which may account for these observed and reported AEs. A PubMed search was conducted on articles published from 1992 to 2012, which reported AEs with 5ARIs. Priority was given to randomized, placebo-controlled trials. Studies investigating potential mechanisms of action for 5ARIs were included for review. AE data reported from available trials were summarized and reviewed. Reported AEs with 5ARIs include sexual dysfunction, infertility, mood disorders, gynecomastia, high-grade prostate cancer, breast cancer, and cardiovascular morbidity/risk factors, although their true association, prevalence, causality, and clinical significance remain unclear. A pooled summary of all randomized, placebo-controlled trials evaluating 5ARIs (N = 62,827) revealed slightly increased rates over placebo for decreased libido (1.5%), erectile dysfunction (ED) (1.6%), ejaculatory dysfunction (EjD) (3.4%), and gynecomastia (1.3%). The limited data available on the impact of 5ARIs on mood disorders demonstrate statistically significant (although clinically minimal) differences in rates of depression and/or anxiety. Similarly, there are limited reports of reversible, diminished fertility among susceptible individuals. Post-marketing surveillance reports have questioned the actual prevalence of AEs associated with 5ARI use and suggest the possibility of persistent symptoms after drug discontinuation. Well-designed studies evaluating these reports are needed. 5ARIs are associated with slightly increased rates of decreased libido, ED, EjD, gynecomastia, depression, and/or anxiety. Further studies directed at identifying prevalence rates and persistence of symptoms beyond drug discontinuation are required to assess causality. Trost L, Saitz TR, and Hellstrom WJG. Side effects of 5-alpha reductase inhibitors: A comprehensive review. Sex Med Rev 2013;1:24–41.
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Unlabelled: The antioxidant and antimicrobial activities as well as the quantity of phenolic substances of Impatiens balsamina L. stem extracts obtained with various solvent were determined in this study. All of the extracts possessed moderate antioxidant potential in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and reducing power assays. Antimicrobial activity was estimated using the cylinder-plate and agar dilution methods against four bacterial and six fungal strains. The extracts showed good antimicrobial activity especially antifungal activity against all of the tested microorganisms. The total phenolic and flavonoid contents ranged from 2.88 to 13.63 mg gallic acid equivalents/g dried extract and 0.98 to 7.87 mg quercetin equivalents/g dried extract, respectively. The results presented here indicate that the I. balsamina stem extracts have antioxidant and antimicrobial properties and are therefore a potential source of antioxidant and antimicrobial agents for the food and pharmaceutical industries. Practical application: Our work indicates that the I. balsamina stem may be a good candidate as natural antioxidant and antimicrobial agents. It can be applied in food industry for preservation.
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Lawsone (1), lawsone methyl ether (2), and methylene-3,3'-bilawsone (3) are the main naphthoquinones in the leaf extracts of Impatiens balsamina L. (Balsaminaceae). Antimicrobial activities of these three naphthoquinones against dermatophyte fungi, yeast, aerobic bacteria and facultative anaerobic and anaerobic bacteria were evaluated by determination of minimal inhibitory concentrations (MICs) and minimal bactericidal or fungicidal concentrations (MBCs or MFCs) using a modified agar dilution method. Compound 2 showed the highest antimicrobial activity. It showed antifungal activity against dermatophyte fungi and Candida albicans with the MICs and MFCs in the ranges of 3.9-23.4 and 7.8-23.4 µg mL(-1), respectively, and also had some antibacterial activity against aerobic, facultative anaerobic and anaerobic bacteria with MICs in the range of 23.4-93.8, 31.2-62.5 and 125 µg mL(-1), respectively. Compound 1 showed only moderate antimicrobial activity against dermatophytes (MICs and MFCs in the ranges of 62.5-250 and 125-250 µg mL(-1), respectively), but had low potency against aerobic bacteria, and was not active against C. albicans and facultative anaerobic bacteria. In contrast, 3 showed significant antimicrobial activity only against Staphylococus epidermidis and Bacillus subtilis (MIC and MBC of 46.9 and 93.8 µg mL(-1), respectively).
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Triterpenoid saponins, impatienosides A-G, together with 12 known saponins, were isolated from the whole plants of Impatiens siculifer. Their structures were established on the basis of extensive 1D and 2D NMR and MS analyses coupled with chemical degradation. Cytotoxic activities of the isolated saponins were evaluated against three human cancer cell lines: human myeloid leukemia HL-60 cells, human stomach KATO-III adenocarcinoma, and human lung A549 adenocarcinoma.
Article
Human or rat microsomal 5 alpha-reductase activity, as measured by enzymic conversion of testosterone into 5 alpha-dihydrotestosterone or by binding of a competitive inhibitor, [3H]17 beta-NN-diethulcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one ([3H]4-MA) to the reductase, is inhibited by low concentrations (less than 10 microM) of certain polyunsaturated fatty acids. The relative inhibitory potencies of unsaturated fatty acids are, in decreasing order: gamma-linolenic acid greater than cis-4,7,10,13,16,19-docosahexaenoic acid = cis-6,9,12,15-octatetraenoic acid = arachidonic acid = alpha-linolenic acid greater than linoleic acid greater than palmitoleic acid greater than oleic acid greater than myristoleic acid. Other unsaturated fatty acids such as undecylenic acid, erucic acid and nervonic acid, are inactive. The methyl esters and alcohol analogues of these compounds, glycerols, phospholipids, saturated fatty acids, retinoids and carotenes were inactive even at 0.2 mM. The results of the binding assay and the enzymic assay correlated well except for elaidic acid and linolelaidic acid, the trans isomers of oleic acid and linoleic acid respectively, which were much less active than their cis isomers in the binding assay but were as potent in the enzymic assay. gamma-Linolenic acid had no effect on the activities of two other rat liver microsomal enzymes: NADH:menadione reductase and glucuronosyl transferase. gamma-Linolenic acid, the most potent inhibitor tested, decreased the Vmax. and increased Km values of substrates, NADPH and testosterone, and promoted dissociation of [3H]4-MA from the microsomal reductase. gamma-Linolenic acid, but not the corresponding saturated fatty acid (stearic acid), inhibited the 5 alpha-reductase activity, but not the 17 beta-dehydrogenase activity, of human prostate cancer cells in culture. These results suggest that unsaturated fatty acids may play an important role in regulating androgen action in target cells.
Article
Dinaphthofuran-7,12-dione derivatives named balsaminones A (1) and B (2) were isolated from the pericarp of Impatiens balsamina L. together with the known compound 2-methoxy-1,4-naphthoquinone (3). Their structures were elucidated by spectral techniques. These compounds have significant antipruritic activity.
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The 35% EtOH extract of aerial parts of Impatiens balsamina L. has been investigated for activity against testosterone 5alpha-reductase. Activity-guided fractionation led to the identification of the bisnaphthquione derivative named impatienol (1), 3-hydroxy-2-¿[3-hydroxy-1,4-dioxo (2-naphthyl)] ethyl¿ naphthalene-1, 4-dione, which exhibited significant testosterone 5alpha-reductase inhibitory activity. This 5alpha-reductase inhibitory compound has been previously synthesized, but this is the first report of its isolation from a natural source.
Article
In different cell systems, the lipido-sterolic extract of Serenoa repens (LSESr, Permixon inhibits both type 1 and type 2 5alpha-reductase activity (5alphaR1 and 5alphaR2). LSESr is mainly constituted of fatty acids (90+/-5%) essentially as free fatty acids (80%). Among these free fatty acids, the main components are oleic and lauric acids which represent 65% and linoleic and myristic acids 15%. To evaluate the inhibitory effect of the different components of LSESr on 5alphaR1 or 5alphaR2 activity, the corresponding type 1 and type 2 human genes have been cloned and expressed in the baculovirus-directed insect cell expression system Sf9. The cells were incubated at pH 5.5 (5alphaR2) and pH 7.4 (5alphaR1) with 1 or 3nM testosterone in presence or absence of various concentrations of LSESr or of its different components. Dihydrotestosterone formation was measured with an automatic system combining HPLC and an on-line radiodetector. The inhibition of 5alphaR1 and 5alphaR2 activity was only observed with free fatty acids: esterified fatty acids, alcohols as well as sterols assayed were inactive. A specificity of the fatty acids in 5alphaR1 or 5alphaR2 inhibition has been found. Long unsaturated chains (oleic and linolenic) were active (IC(50)=4+/-2 and 13+/-3 microg/ml, respectively) on 5alphaR1 but to a much lesser extent (IC(50)>100 and 35+/-21 microg/ml, respectively) on 5alphaR2. Palmitic and stearic acids were inactive on the two isoforms. Lauric acid was active on 5alphaR1 (IC(50)=17+/-3 microg/ml) and 5alphaR2 (IC(50)=19+/-9 microg/ml). The inhibitory activity of myristic acid was evaluated on 5alphaR2 only and found active on this isoform (IC(50)=4+/-2 microg/ml). The dual inhibitory activity of LSESr on 5alpha-reductase type 1 and type 2 can be attributed to its high content in free fatty acids.
Article
Liquid chromatography (LC) is currently considered as the gold standard in pharmaceutical analysis. Today, there is an increasing need for fast and ultra-fast methods with good efficiency and resolution for achieving separations in a few minutes or even seconds. A previous article (i.e. method transfer for fast LC in pharmaceutical analysis. Part I: isocratic separation) described a simple methodology for performing a successful method transfer from conventional LC to fast and ultra-fast LC in isocratic mode. However, for performing complex separations, the gradient mode is often preferred. Thus, this article reports transfer rules for chromatographic separations in gradient mode. The methodology was applied for the impurity profiling of pharmaceutical compounds, following two strategies. A first approach, using short columns (20-50mm) packed with 3.5microm particles and optimized HPLC instrumentation (with reduced extra-column and dwell volumes), was applied for the separation of a pharmaceutical drug and eight related impurities. Special attention was paid to the dwell (gradient delay) volume, which causes the most detrimental effect for transferring a gradient method. Therefore, the dwell volume was simultaneously decreased with the column dead volume. Under optimal conditions, it was possible to reduce the analysis time by a factor of 10, with an acceptable loss in resolution since the column length reduction is less critical in gradient than isocratic mode. The second tested approach was Ultra Performance Liquid Chromatography (UPLC), where sub-2microm particles were used simultaneously with very high pressures (up to 1000bar). A complex pharmaceutical mixture containing 12 compounds was separated in only 1.5min allowing a reduction of the analysis time by a factor of 15 in comparison to a conventional method, with similar peak capacity.