Chromatography Conditions Development by Design of Experiments for the Chemotype Differentiation of Four Bauhinia Species

Abstract

The extensive use of medicinal herbs to traditionally treat disease persists for generations, and scientific evidence on plant-derived extracts has indicated their numerous biological activities. The Bauhinia, popular known as cow's paw ("pata de vaca"), with more than 60 native species, are extensively used in Brazilian popular medicine for the control of diabetes. Therefore, in 2009, B. forficata, B. variegata and/or B. affinis were included in the Brazilian National List of Medicinal Plants of Interest to SUS (RENISUS - Brazil). In this context, this work reports the results of the chemical differentiation of B. forficata, B. variegata, B. longifolia, and B. affinis using liquid chromatography coupled to high-resolution mass spectrometry and unsupervised chemometric tools. Chromatographic conditions were optimized by using the design of experiments (DoE) and chromatographic knowledge. Furthermore, the chemical profile of the studied species was analyzed by principal component analysis (PCA) and hierarchical cluster analysis that differentiated the four species of Bauhinia, and 55 compounds were also inferred by MS2 experiments, some of them for the first time in B. affinis. In this manner, this work provides important information that could be used in quality control, development of new pharmaceuticals, and food products based on Bauhinia leaves, as well as to explain ethnomedicinal properties, pharmacological and toxicological actions.

Keywords: Bauhinia; HRMS; chemotype differentiation; dereplication; design of experiments; hierarchical cluster analysis.

FIGURE 1

Contour plots obtained in DoE—Step 1 of the experimental design.

FIGURE 2

Contour plot obtained in DoE—Step 2. [* The values in the contour chart refer to the number of chromatographic bands. ** Fixed condition: Kinetex^® Biphenyl column, pH 4.2 (formic acid 200 μM), temperature 40°C, and flow rate 0.5 ml/min X axis (time—minutes) and Y axis organic modifier.

FIGURE 3

Final chromatogram after DoE—Step 2 of leaf ethanolic extract Bauhinia forficata (A) base ion chromatogram in the negative ionization mode (-All/MS), and (B) by UV at 254 nm. Chromatographic conditions: Kinetex^® Biphenyl column, flow rate 0.7 ml/min, temperature 50°C. Gradient: 5%–70% B.

FIGURE 4

Survey view (SV) and base peak chromatograms (BPC) in negative ionization mode (-All/MS) for B. forficata (1a and 1b), B. variegata (2a and 2b), B. affinis (3a and 3b), and B. longifolia (4a and 4b).

FIGURE 5

Hierarchical clustering analysis of the 4 studied Bauhinia species.

FIGURE 6

Principal component analysis (PCA) of B. forficata, B. variegata, B. longifolia, and B. affinis. Upper: PC1 vs. PC2 (Score graphs). Bottom: PC2 vs. PC3 (loading graphs).