Bioactive Compound Separation Method Tested on Herbal Extracts

Scientists from Northwest University and the Shaanxi Institute for Food and Drug Control, both in Xi’an, China, recently tested a new means of separating bioactive compounds from herbal extracts. Their findings were later published in the Journal of Chromatography A (1).

Medicinal herbs and tinctures alternative medicine. Selective focus. | Image Credit: © yanadjan - stock.adobe.com

A bioactive compound is a type of chemical found in plants and certain foods, which have been studied in preventing cancer, heart diseases, and other diseases (2). To successfully analyze bioactive compounds for discovering new drugs from herbal medicine, excellent pretreatments are critical. An herbal extract is a complex matrix that consists of various molecules with different physicochemical properties, where the analytes at trace level need enrichment for improving instrumental detectability. Further, the matrix effect of interferences can cause inaccurate measurement of the target compounds and even contamination of the analytical instruments. The matrix in natural products is not commonly known and exploring the effects of an interfering species prior to analyte determination can be time-consuming and costly. There are many challenges involved with separating and rapidly analyzing pharmacologically active small molecules that originate from herbal extracts.

Sorbent-based extraction methods for sample preparation continue to dominate in bioanalytical fields, since liquid-liquid extraction (LLE) typically suffers from disadvantages, such as poor extraction efficiency and consumption of large amounts of samples and toxic organic solvents. In recent years, special miniaturized extraction techniques, such as micro solid phase extraction (MSPE) and solid phase microextraction (SPME) have been developed. These have distinct advantages, such as high enrichment ability, high recovery, and low sample consumption. To advance the selective extraction process, numerous alternative methods have been proposed isolate bioactive compounds based on the high-affinity binding towards a certain target protein.

In this study, the scientists developed a rapid and highly selective method for separating bioactive compounds from herbal extracts using a protein affinity-selection spin column. The column was packed with new sorbent materials from integrating the recombinant β₂-adrenoceptor (β₂-AR) directly out of cell lysates onto the surface of microspheres. Protein affinity-selection spin column was placed in a centrifugal tube, where after the non-specific binders were released to the filtrate under the operational centrifugation, the specific binders on the spin column were cleaned with a washing solvent for liquid chromatography–mass spectrometry (LC-MS) analysis.

The known agonists of β₂-AR, which were retained and released on the protein affinity-selection spin column but not on the control column, successfully demonstrated the method, returning good recovery (79.4–95.7%) and high repeatability (RSD < 3.5%) values. Three ligands on the spin columns had their adsorption features best described with the Prism saturation binding model. With saturation binding, you vary the concentration of radioligand and measure binding at equilibrium, with the goal being to determine the K_d (ligand concentration that binds to half the receptor sites at equilibrium) and B_max (maximum number of binding sites) (3). The spin column’s high-affinity binding and large binding capacity, combined with the saturation binding model, lead to the spin column being a feasible means of effectively trapping trace analytes. The researchers determined that the spin column with the immobilized protein microspheres placed in the centrifugal filter device represents a promising tool, enabling the rapid and highly-selective extraction/isolation of the bioactive compounds in the herb medicines.

References

(1) Wang, T.; Wang, Y.; Zhao, S.; Han, Y.; Li, Q.; Liu, P.; Li, X.; Xiao, C. Target-Specific Affinity Separation of the Bioactive Compounds from Herbal Extract Using the Spin Column Packed with the Immobilized Protein Microspheres Prior to LC-MS Analysis. J. Chromatogr. A 2024, 1734, 465322. DOI: 10.1016/j.chroma.2024.465322

(2) Bioactive Compound. National Cancer Institute 2024. https://www.cancer.gov/publications/dictionaries/cancer-terms/def/bioactive-compound (accessed 2024-10-14)

(3) Key Concepts: Saturation Binding. GraphPad 2024. https://www.graphpad.com/guides/prism/latest/curve-fitting/reg_key_concepts_saturaton_binding.htm (accessed 2024-10-14)