A multiple biomarker assay for quality assessment of botanical drugs using a versatile microfluidic chip

Abstract

Quality control is critical for ensuring the safety and effectiveness of drugs. Current quality control method for botanical drugs is mainly based on chemical testing. However, chemical testing alone may not be sufficient as it may not capture all constituents of botanical drugs. Therefore, it is necessary to establish a bioassay correlating with the drug’s known mechanism of action to ensure its potency and activity. Herein we developed a multiple biomarker assay to assess the quality of botanicals using microfluidics, where enzyme inhibition was employed to indicate the drug’s activity and thereby evaluate biological consistency. This approach was exemplified on QiShenYiQi Pills using thrombin and angiotensin converting enzyme as “quality biomarkers”. Our results demonstrated that there existed variations in potency across different batches of the intermediates and preparations. Compared with chromatographic fingerprinting, the bioassay provided better discrimination ability for some abnormal samples. Moreover, the chip could function as “affinity chromatography” to identify bioactive phytochemicals bound to the enzymes. This work proposed a multiple-biomarker strategy for quality assessment of botanical drugs, while demonstrating for the first time the feasibility of microfluidics in this field.

Mass Spectrometry-Sensitive Probes Coupled with Direct Analysis in Real Time for Simultaneous Sensing of Chemical and Biological Properties of Botanical Drugs

“…As a result, four ions, namely 20 (rosmarinic acid), 14 (protocatechualdehyde), 5 (danshensu), and 33 (salvianolic acid B), were identified as key variables positively correlated with ACE inhibition, and ion 20 (rosmarinic acid), 30 (rosmarinic acid + danshensu), and 3 (caffeic acid) were found to be highly associated with thrombin inhibition. The result corresponded well with some bioactivity studies ,,, while highlighting that protocatechualdehyde could be investigated as a potential anti-ACE agent. However, validation studies are necessary to confirm the screening result by using purified compounds.…”

“…Interestingly, a weak correlation is observed ( r = 0.39, p < 0.05) when plotting thrombin inhibition against ACE inhibition (Figure B). It could be attributed to the fact that some active constituents in DSI, such as danshensu (ion 5), rosmarinic acid (ion 20), and salvianolic acid B (ion 33) exhibit inhibitory activities against both the enzymes. ,,− Therefore, variations in the contents of these constituents may result in similar changes in ACE and thrombin inhibitions. Another interesting finding was that sample 26 showed no obvious difference in activity compared with other samples, although a large difference was observed in their MS fingerprints.…”

“…As a result, there has been increasing interest in developing biological assays that correlate with the drug’s known or intended mechanism of action to complement the chemical tests. This trend has also led to the emergence of different bioassays for the assessment of botanical drug activity using animals, microorganisms, cells, and enzymes. − Of the bioassays that have been designed, optical schemes such as colorimetry and fluorescence represent the most widely used mechanisms. However, these optical sensing methods suffer from background interference or autofluorescence from constituents contained in botanical drugs, , compromising the detection sensitivity and dynamic range.…”

“…This trend has also led to the emergence of different bioassays for the assessment of botanical drug activity using animals, microorganisms, cells, and enzymes. − Of the bioassays that have been designed, optical schemes such as colorimetry and fluorescence represent the most widely used mechanisms. However, these optical sensing methods suffer from background interference or autofluorescence from constituents contained in botanical drugs, , compromising the detection sensitivity and dynamic range. Moreover, few of the bioassays meet the assay requirements which include multiplexing, sensitivity, and speed.…”

“…To evaluate this platform for practical application we tested Danshen ( Salvia miltiorrhiza ) Injection (DSI), a Chinese medicine widely prescribed for cardiovascular and cerebrovascular diseases. Previous studies have demonstrated the drug exerts its therapeutic effects through inhibition of several critical enzymes, including thrombin and angiotensin converting enzyme (ACE). − Therefore, the two target enzymes are selected as quality biomarkers to develop the dual-enzyme assay. The testing result demonstrates this platform can detect the chemical constituents and bioactivities in a sensitive, reliable, and efficient manner, which highlights its great potential in quality assessment of botanical drugs.…”