Development of a mitochondria-based centrifugal ultrafiltration/liquid chromatography/mass spectrometry method for screening mitochondria-targeted bioactive constituents from complex matrixes: Herbal medicines as a case study
Introduction
Mitochondria are the powerhouses of cells and serve as the primary energy source. Mitochondria also have many other important roles, including adjustment of apoptotic cell death, reactive oxygen species generation, maintenance of calcium homeostasis and regulation of lipid metabolism [1]. Accordingly, mitochondrial dysfunction leads to a number of human diseases, including cancer, neurodegenerative diseases, ischemia–reperfusion injury, diabetes and obesity [2]. Mutations to mitochondrial DNA can also cause a number of human disorders [2]. Thus, strategies to prevent mitochondrial damage or to manipulate mitochondrial functions in a clinically useful manner may provide effective therapies for a variety of human diseases [2]. A large number of therapeutically applied substances have been specifically designed to affect mitochondrial functions to exert their therapeutic effects as antitumor agents, neuroprotective agents, immunosuppressants, antiviral drugs, potassium channel openers, sulfonylureas and anesthetics [3]. Therefore, mitochondria have become a prominent intracellular pharmacological target for new drug development [4], [5], [6], [7].
Herbal medicines (HMs) are increasingly of interest as sources of bioactive compounds for the discovery of promising new drugs [8]. A rapidly expanding body of literature suggests that many HMs affect mitochondrial function to exert their therapeutic effects, including anticancer [9], antiaging [10], anti-diabetes [11], anti-obesity [12], neuroprotection [13], cardioprotection [14] and hepaticprotection [15]. The bioactive constituents in HMs that interact with mitochondria have not been identified, severely limiting an understanding of their mechanisms and hindering drug development from medicinal herbs. Hence, screening of the bioactive constituents in HMs that act on mitochondria is urgently required.
Because of the large number of substances in HMs, bioactive constituent screening and analysis are extremely difficult, even for well-documented formulations for specific diseases. The conventional approach for screening mitochondria-targeted bioactive constituents includes extraction and isolation of the HM constituents and pharmacological tests of the purified compounds [16]. This method is inefficient for the direct screening of bioactive compounds from complex agents in addition to being labor-intensive, expensive, and time- and sample-consuming. High-throughput screening [17], [18] and high-content screening [19] methods have also been used to efficiently identify mitochondria-targeted compounds. However, these methods are designed to screen pure compounds and are not suitable for the direct detection of multiple bioactive compounds from complex matrixes. Consequently, further development of screening approaches to efficiently detect mitochondria-targeted bioactive constituents from natural complex matrixes such as HMs is required.
Centrifugal ultrafiltration (CU) employs centrifugal force and a semi-permeable membrane to retain suspended solids and high-molecular-weight solutes while liquid and low-molecular-weight solutes are allowed to permeate the membrane, depending on the nominal molecular weight cut-off of the membrane. Thus, due to its simple operation, high speed and high dependability, CU has become a useful technique for screening bioactive compounds from complex objects bound to biomacromolecules such as bovine serum albumin [16], Plasmodium falciparum thioredoxin reductase [20], α-glucosidase [21], quinone reductase-2 [22], DNA [23], retinoid X receptors [24], β-amyloid protein [25] and liposomes [26]. Liquid chromatography/mass spectrometry (LC/MS) has been widely applied for the simultaneous separation and identification of active constituents in complex mixtures [20], [21], [22], [23]. The combination of LC/MS with CU permits the efficient screening and identification of active constituents in HMs. However, no screening method has been reported for the direct identification of mitochondria-targeted constituents from complex matrixes.
This study was initially designed to develop a rapid and efficient screening method coupling CU with LC/MS to discover potential mitochondria-targeted bioactive constituents from HMs used to treat mitochondrial disorders, exemplified by two Chinese traditional medicines, Polygoni Cuspidati Rhizoma et Radix (PCRR) and Scutellariae Radix (SR). Using this new method, called the screening method for mitochondria-targeted bioactive constituents (SM-MBC), fractions containing bioactive constituents that specifically bind the mitochondria were isolated by the CU technique. The ultrafiltrates were then collected, evaporated and injected into the LC/MS system for separation and identification. A pharmacological verification trial confirmed that SM-MBC is an effective protocol for the efficient screening of potential bioactive constituents that bind mitochondria from complex mixtures (Fig. 1). SM-MBC may be useful for an in-depth comprehension of drug mechanism of action and drug discovery using medicinal herbs as sources of lead compounds.
Section snippets
Chemicals and materials
Nifedipine (NP, lot no. 100338-201103), captopril (CP, lot no. 100318-200602), baicalin (lot no. 110715-200514), baicalein (lot no. 11595-201306) and wogonin (lot no. 11514-200403) were obtained from the National Institute for Food and Drug Control (Beijing, China). Thiabendazole (TZ, lot no. 5838), rotenone (RN, lot no. 031201AG-AC), trimetazidine (TD, lot no. JLISA-BE), amiodarone (AR, lot no. 77290) and rhodamine 123 (Rh123, lot no. LK80N08) were purchased from J&K Scientific Ltd. (Beijing,
Preparation of intact and pure myocardial mitochondria
The successful development of a mitochondria-based screening method depends on a good supply of intact and pure mitochondria. It is therefore advisable to employ an optimized protocol for mitochondria preparation based on organelle release followed by differential and Nycodenz density gradient centrifugation (optimization of the mitochondrial preparation procedure is shown in the Supplementary Material). The structural integrity, biological functions and purity of the prepared mitochondrial
Conclusion
We developed an efficient SM-MBC for the systematic screening of mitochondria-targeted bioactive constituents from complex matrices, such as PCRR and SR. This method exhibited excellent recognition, separation and identification capabilities that were validated using positive and negative controls and possessed advantages such as efficient use of labor, a simple procedure, and low time and sample requirements. We successfully detected and identified 19 bioactive constituents from PCRR and SR
Acknowledgment
Financial support from the Specialized Research Fund for the Doctoral Program of Higher Education (Grant 20130001110057), the National Natural Science Foundation of China (Grant 81373921) and the National Science and Technology Major Project for Major New Drugs Innovation and Development of China (Grant 2013ZX09508104) is gratefully acknowledged.
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