Comparison of Metabolomics Approaches for Evaluating the Variability of Complex Botanical Preparations: Green Tea (<i>Camellia sinensis</i>) as a Case Study

A challenge that must be addressed when conducting studies with complex natural products is how to evaluate their complexity and variability. Traditional methods of quantifying a single or a small range of metabolites may not capture the full chemical complexity of multiple samples. Different metabolomics approaches were evaluated to discern how they facilitated comparison of the chemical composition of commercial green tea [<i>Camellia sinensis</i> (L.) Kuntze] products, with the goal of capturing the variability of commercially used products and selecting representative products for in vitro or clinical evaluation. Three metabolomic-related methodsuntargeted ultraperformance liquid chromatography–mass spectrometry (UPLC-MS), targeted UPLC-MS, and untargeted, quantitative ¹HNMRwere employed to characterize 34 commercially available green tea samples. Of these methods, untargeted UPLC-MS was most effective at discriminating between green tea, green tea supplement, and non-green-tea products. A method using reproduced correlation coefficients calculated from principal component analysis models was developed to quantitatively compare differences among samples. The obtained results demonstrated the utility of metabolomics employing UPLC-MS data for evaluating similarities and differences between complex botanical products

Plasma concentration-time profiles following oral administration of the last (14^th) daily dose of DB844.

<p>Symbols and error bars represent geometric means and SEs, respectively, for DB844 (△) and DB820 (○). The monkeys (n = 7) were treated with DB844 at 6 mg/kg×14 days, from 28–41 days post infection. The insert graph shows the extended profile up to 28 days post the last daily dose of DB844.</p

HPLC/UV chromatograms and concentration-time profiles of DB844/metabolites following incubation of DB844 with male vervet monkey liver microsomes.

<p>A: HPLC/UV chromatograms; B: Concentration-time profiles of DB844 and metabolites. Incubation mixtures (1 ml at pH7.4, in triplicate) contained 10 µM DB844 and 0.2 mg/ml monkey liver microsomes. Aliquots were taken at 0.2, 5, 15, 30, and 120 min and evaluated for concentrations of DB844 and six metabolites (M1A, M1B, M2A, M2B, M3, and DB820). Metabolites M4A and M4B were not quantified due to lack of synthetic standards.</p

Parasitaemia pattern in monkeys infected with <i>T.b. rhodesiense</i> KETRI 2537 and subsequently treated with DB844.

<p>Symbols and error bars represent means and SEs, respectively, of 7 animals; monkeys were treated with DB844 at 6 mg/kg×14 days, from 28–41 days post infection; Log parasitaemia values were determined by microscopic examination of wet smears of blood using the matching method of Herbert and Lumsden, 1976 <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001734#pntd.0001734-Ndungu1" target="_blank">[23]</a>.</p

Treatment outcome in monkeys treated with DB844 while in second stage <i>T. b. rhodesiense</i> infection.

<p>Key: ID = identity in the laboratory; PP = pre-patent period; DPI = days post infection; CSF = cerebrospinal fluid; Tryps = trypanosomes; WC = white cells; EoT = end of treatment; ToC = test of cure; Neg = Negative; Pos = positive; WD = withdrawn from the experiment after 10^th drug dose due to toxicity; Numbers in square brackets = maximum number of white cell counts observed during any of the four weekly samplings between 0–27 DPI; I: DB844 5 mg/kg×10 days per os; 28–37 DPI; II: DB844 6 mg/kg×14 days per os; 28–41 DPI.</p

Transient infection and DB844 induced changes in clinical chemistry indicators of liver and kidney function.

<p>Symbols represent mean ± SE (n = 7) of aspartate aminotransferase (AST, ▪), alanine aminotransferase (ALT, □), total bilirubin (•), direct bilirubin (0), blood urea nitrogen (BUN, ▴) and albumin (◊); monkeys were treated with DB844 at 6 mg/kg×14 days, from 28–41 days post infection.</p

Changes in red cell distribution width in monkeys following infection and subsequent treatment with DB844.

<p>Symbols and error bars represent means and SEs, respectively, of seven monkeys that were treated with DB844 at 6 mg/kg×14 days, from 28–41 days post infection.</p

Chemical structure of DB844 and DB820.

<p>Chemical structure of DB844 and DB820.</p

Haematologic effects of <i>T. b. rhodesiense</i> KETRI2537 infection and treatment with DB844 in vervet monkeys.

<p>Key: RBC = red blood cells; WBC = White blood cells; g/dl = grams/decilitre; fl = femtolitres; p-values<0.05 indicate values that were significantly different from pre-infection baseline (day 0) values (Repeated measures Anova with Fishers PLSD post hoc test); Monkey were treated with DB844 at 6 mg/kg×14 days, from 28–41 days post infection.</p

Parasitaemia progression in vervet monkeys following infection.

<p>Monkeys were infected with <i>T. b. rhodesiense</i> KETRI 2537 on Day 0. Symbols represent means and standard error of the mean (n = 16).</p