Introduction
Phenolic compounds present in Achyrocline satureioides are known to have therapeutic benefits like antioxidant, anti‐inflammatory, and antitumour properties. The main polyphenols present in the plant are quercetin (QCT), luteolin (LUT), 3‐O‐methylquercetin (3OMQ), and achyrobichalcone (ACB). However, the effective isolation and purification of these compounds from A. satureioides inflorescences are not an easy task.
Objective
To develop an efficient high‐performance counter‐current chromatography (HPCCC) method for quick separation and purification of naturally occurring phenolic compounds from the extract of A. satureioides.
Methodology
A two‐step HPCCC semi‐preparative isolation method was developed using a solvent system composed of n‐hexane/ethyl acetate/methanol/water (0.8:1.0:0.8:1.0) and dichloromethane/methanol/water (3.5:3.5:2.5).
Results
The HPCCC method was used to obtain two fractions. The first fraction (F1) contained high levels of ACB, among other constituents, while the second fraction (F2) contained mostly QCT, LUT, and 3OMQ. Besides the high ACB content, F1 contained three other flavonoid‐aglycones (kaempferol, 97.3%; isokaempferide, 92.4%; and 3,3′‐di‐O‐methylquercetin, 95.2%) identified by an ultra‐performance liquid chromatography system coupled to a quadrupole time‐of‐flight with high‐definition mass spectrometry (UPLC‐QTOF/HDMS) and nuclear magnetic resonance (NMR) analysis. Purity levels of ACB, 3OMQ, QCT, and LUT were 98.0, 97.0, 97.5, and 90.2%, respectively.
Conclusion
This is the first time that high purity ACB and six other flavonoids were obtained from A. satureioides inflorescences by HPCCC. These excellent results reveal the potential and versatility of HPCCC as a technique to produce different types of products from this plant species on a semi‐preparative scale: enriched fractions, new metabolites, or high purity compounds.
The biological properties of Achyrocline satureioides have been mostly ascribed to its major flavonoids quercetin (QCT), luteolin (LUT), and 3-O-methylquercetin (3OMQ). The present study aimed to optimize the extraction by dynamic maceration of the major phenolic compounds in order to obtain in a subsequent step a flavonoid-enriched fraction (FEF) using high performance countercurrent chromatography (HPCCC). A 3-level Box-Behnken design (BBD) was applied to maximize the extraction of the substances, using the plant : solvent ratio (X1
), extraction time (X2
), and ethanol concentration (X3
) as factors. One-step HPCCC semipreparative separation with a solvent system composed of hexane : ethyl acetate : methanol : water (0.9 : 0.9 : 0.8 : 1.0, v/v) was employed to obtain the FEF. The second-order polynomial model was able to fit the experimental data adequately. The linear and quadratic terms of X3
were the most significant factors that affected all the responses. The positive linear term of X3
indicated a substantial increase in extraction yield, while the negative quadratic term showed a nonlinear tendency. Linear terms of X1
suggested a tendency to solvent saturation, except for QCT. The terms of X2
did not affect the responses substantially. The HPCCC method was found to be efficient and rapid for separating the FEF with 71% (w/w) flavonoid content. Overall, the developed extraction procedure coupled with HPCCC proved to be efficient for obtaining an enriched fraction with a very high content of flavonoids from A. satureioides.
Natural products are a valuable source of new molecules and are important for drug discovery. Many chemotherapeutics currently in clinical use were originated from natural sources and are effective cytotoxic agents. In this study, we investigated the cytotoxic and pro‐apoptotic effects of achyrobichalcone (ACB) and 3‐O‐methylquercetin (3OMQ), two novel compounds isolated from the Achyrocline satureioides plant. Because extracts from this plant have been shown to have anticancer activity in vitro, we evaluated ACB and 3OMQ using a human breast cancer cell line, MDA‐MB‐231, and a nontumorigenic human breast epithelial cell line, MCF‐12A. We found that ACB demonstrates cytotoxic effects on MDA‐MB‐231 cells, but not MCF‐12A cells. 3OMQ also demonstrated cytotoxic effects on MDA‐MB‐231 cells, but with lower selectivity compared to treated MCF‐12A cells. Cell death by both compounds was associated with caspase‐9 and caspase‐3/7 activation. Using high‐resolution respirometry, we found that ACB and 3OMQ were able to cause acute mitochondrial dysfunction in MDA‐MB‐231‐treated cells. These results suggest that apoptosis in MDA‐MB‐231 cells is induced through the activation of the mitochondrial‐dependent pathway. Collectively, these findings suggest that ACB is a strong candidate for further anticancer in vivo tests.
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