Development of a validated HPTLC-bioautographic method for evaluation of aromatase inhibitory activity of plant extracts and their constituents.

INTRODUCTION: Aromatase is a CYP450 enzyme that catalyses the conversion of androgens into oestrogens, where the decrease in the production of oestrogens aided by aromatase inhibitors is considered a target in post-menopausal breast cancer therapy. TLC-bioautography is a technique employed for combining chromatographic separations on TLC plates with bioassays. This is the first report to evaluate aromatase inhibitory activity using this technique. OBJECTIVES: The aim of this study is to develop and validate a new TLC-bioautographic method for determination of aromatase inhibitory activity in 14 plant extracts. Two quantitation methods, the peak area method and reciprocal iso-inhibition volume (RIV) method, were compared and investigated to attain reliable results. Factors affecting the enzymatic reaction (temperature, pH, enzyme and substrate concentrations … etc.) were also investigated to attain the optimum parameters. METHODOLOGY: TLC assisted by digital image processing was implemented for quantitative estimation of the aromatase inhibition of 14 plant extracts using chrysin as positive control. The fluorometric substrate dibenzyl fluorescein (DBF) was utilised for the assay, where inhibitory compounds were visualised as dark spots against a blue fluorescent background. Two software programs, Sorbfil® videodensitometer (in the peak area method) and ImageJ® (in the RIV method), were thoroughly validated using the International Council on Harmonisation (ICH) guideline and used for quantitation. RESULTS: The RIV method showed superiority over the peak area method in the quantitation results of the tracks with non-homogenous background with %RSD values of 0.98 and 1.49 compared with 2.86 and 3.58, respectively. Further, the methods allow the comparison of the activity of different unknown inhibitory compounds without the need for a reference or a positive control. CONCLUSION: Using the TLC-bioautographic method by image processing combined with the RIV quantitation method, simultaneous separation and quantitation of aromatase inhibitory components could be applied to estimate the relative activity of various plant extracts.

Chemometric evaluation of alfalfa sprouting impact on its metabolic profile using HPTLC fingerprint-efficacy relationship analysis modelled with partial least squares regression.

Sprouting is a commonly applied food processing practice specially in Western countries. Tracking the impact of sprouting of Medicago sativa L. (alfalfa) seeds on their phytochemical composition and curative efficacy was implemented in the current study. Sprouting of alfalfa seeds under controlled conditions for eleven days was performed in a biochemical incubator and three samples were randomly taken each day. A total of thirty-six samples (three ungerminated seeds and thirty-three sprouts samples) were collected, extracted and their cytotoxic, antioxidant and antimicrobial activities against five pathogenic microbial strains were measured. Samples were subjected to High performance thin layer chromatography (HPTLC) as a pattern-oriented strategy for metabolite fingerprinting to discover the fluctuations occurring during the sprouting process accompanied by multivariate chemometric analysis. Unsupervised pattern recognition was carried out using Principal Component Analysis (PCA) after extracting the chromatographic fingerprints from HPTLC chromatograms using ImageJ® software. PCA- loading plots demonstrated that luteolin-7-O-glucoside, ferulic acid and P-coumaric acid were the metabolically significant markers. Thus, simultaneous quantification of these crucial three markers in different aged alfalfa seeds/ sprouts extracts was performed using a newly developed and validated HPTLC-image analysis method. The results of the biological activities together with the quantitative data were further subjected to a Partial Least Squares Regression (PLSR) model for implementing HPTLC fingerprint-efficacy relationship analysis. The results obtained from metabolic pool profiling revealed that sprouting can cause remarkable changes in the phytochemical, nutritional and efficacy characteristics of alfalfa seeds.

Integrated in silico-in vitro strategy for screening of some traditional Egyptian plants for human aromatase inhibitors.

ETHNOPHARMACOLOGICAL RELEVANCE: Aromatase enzyme (CYP19) is widely known as a critical target protein for treating hormone-dependent breast cancer. Natural products from traditional medicinal plants continue to be an active source of aromatase inhibitors. Meanwhile, high cost of experimental work and low hit rate associated with HTS have stimulated the implementation of in-silico virtual screening to resolve these pitfalls, where coupling of both classical wet lab procedure and VS may offer a more deepened access to bioactive compounds with less work and time waste. AIM OF THE STUDY: In this work, a sequential structure-based and ligand-based virtual screening strategy was utilized for investigating an in-house database of 1720 phytochemical constituents of 29 medicinal plants and natural products used in traditional Egyptian medicine to search for compounds with the potential to be used as inhibitors of the human aromatase enzyme. MATERIALS AND METHODS: The suggested strategy included using Glide docking with its feature 'extra precision (XP)' for carrying out structure-based virtual screening (SBVS) where the resulting hits were further promoted to ligand-based virtual screening (LBVS) through the development of two pharmacophore and QSAR models; one for steroidal and the other for non-steroidal aromatase inhibitors. RESULTS: The combined results revealed that Artemisia annua, Zingiber officinale, Cicer arietinum, Annona muricata and Vitex agnus castus were the top scoring plants in terms of in-silico activity scores, respectively. The hydro-alcoholic extracts and different solvent fractions of the top scoring plants were subsequently tested experimentally for their aromatase inhibitory activity, by the aid of in-vitro fluorometric assay. The rank ordering of the activities for the plants agreed with the ordering predicted on the basis of SBVS and LBVS workflow implemented. CONCLUSION: The suggested strategy provides a reliable means of prospecting in-silico screening of natural products databases in the search for new dug leads as aromatase inhibitors. The hits so obtained can then be subjected to further phytochemical studies, to isolate and identify suitable compounds for further in-vitro testing.

Cornigerin, a new sesqui-lignan from the hepatoprotective fractions of Cynara cornigera L.

The ethanol extract of Cynara cornigera L. was fractionated and the fractions were subjected to hepatoprotective assays using Wistar albino rats at a dose of 500 and 250mg/kg. The liver injury was induced in rats using carbon tetrachloride. Biochemical parameters such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and total bilirubin were estimated as reflections of the liver condition, with silymarin as a positive control. Phytochemical investigation and chromatographic separation of the hepatoprotective fractions led to the isolation of a new sesqui-lignan namely cornigerin (1), along with eight known compounds: apigenin (2), luteolin (3), ß-sitosterol glycoside (4), apigenin 7-O-ß-D-glucopyranoside (5), luteolin-7-O-ß-D-glucopyranoside (6), apigenin-7-O-rutinoside (7), cynarin 1,5-di-O-caffeoylquinic acid (8), and apigenin-7-O-ß-D-glucuronide (9). This is the first report for the isolation of 8 and 9 from this plant.