Development of organic three-phase laminar flow microfluidic chip for extraction of ginsenosides from Panax ginseng.

BACKGROUND: Herbal extracts contain multiple active constituents, so the sample preparation based on the liquid-liquid extraction (LLE) is demanding, especially when a study subsequent to extraction is needed. Since the laminar flow occurring in microchannels can be formed between two miscible organic phases, a new method of extracting polar compounds from the crude extract of Panax ginseng Meyer in aqueous ethanol by pure n-butanol in the three-phase laminar flow microfluidic chip was established. METHODS: A new chip consisting of long microchannels with a guide structure was employed to improve the extraction efficiency caused by the low diffusion ability of saponins. The method was evaluated by using the extraction yields and purities of ginsenosides Rg1, Re and Rb1 as the indicators, and extraction conditions such as flow rate, temperature and other governing factors were optimized. RESULTS: Using the new chip method, the extraction efficiencies of ginsenoside Rg1, Re and Rb1 were 63.1%, 69.5% and 71.6%, respectively, which are higher than the 26% achieved in a previous report. The extraction yields of 1.53, 0.51, 0.90 mg/g were also higher than those obtained previously by the successive laminar flow microchip method. CONCLUSION: The proposed new microfluidic chip method has simplified the sample pretreatment steps to improve the yield of ginsenoside extraction from ginseng samples.

Nucleic acid amplification test (NAAT) conducted in a microfluidic chip to differentiate between various ginseng species.

Panax ginseng and Panax quinquefolius have different medicinal properties and market values; however, they can be difficult to distinguish from one another based on physical appearances alone. Therefore, a molecular test that can be performed in commercial settings is needed to overcome this difficulty. A locus that contains a single nucleotide polymorphism (SNP) site to differentiate between P. ginseng and P. quinquefolius has been selected. An isothermal nucleic acid amplification test (NAAT) has been developed for use in a microfluidic chip; this NAAT method, which is based on lesion-induced DNA amplification (LIDA), amplifies the extracted plant genomic samples and enhances the detection of specific SNPs. This NAAT method was used to authenticate five ginseng root samples which indicated that two of the five samples appear to be mislabeled. These authentication results were consistent with those obtained from next generation sequencing (NGS) although this molecular test is more affordable and faster than NGS.

The genetic authentication of Panax ginseng and Panax quinquefolius based on using single nucleotide polymorphism (SNP) conducted in a nucleic acid test chip.

Panax ginseng and Panax quinquefolius, which are commonly called Chinese ginseng and American ginseng respectively, have different medicinal properties and market values; however, these samples can be difficult to differentiate from one another based on physical appearances of the samples especially when they are in powdery or granular forms. A molecular technique is thus needed to overcome this difficulty; this technique is based on the nucleic acid test (NAT) conducted on the microfluidic chip surface. Three single nucleotide polymorphism (SNP) sites (i.e. N1, N2, N3) on the Panax genome that differ between P. ginseng (G) and P. quinquefolius (Q) have been selected to design probes for the NAT. Primers were designed to amplify the antisense strands by asymmetric PCR. We have developed three different NAT methodologies involving surface immobilization and subsequent (stop flow or dynamic) hybridization of probes (i.e. N1G, N1Q, N2G, N2Q, N3Q) to the antisense strands. These NAT methods consist of two steps, namely immobilization and hybridization, and each method is distinguished by what is immobilized on the microfluidic chip surface in the first step (i.e. probe, target or capture strand). These three NATs developed are called probe-target method 1, target-probe method 2 and three-strand complex method 3. Out of the three methods, it was found that the capture strand-target-probe method 3 provided the best differentiation of the ginseng species, in which a 3' NH2 capture strand is first immobilized and the antisense PCR strand is then bound, while N2G and N3Q probes are used for detection of P. ginseng (G) and P. quinquefolius (Q) respectively.

Screening of high-efficiency and low-toxicity antitumor active components in Macleaya cordata seeds based on the competitive effect of drugs on double targets by a new laminar flow chip.

It is urgent to obtain targeted drugs that selectively bind to pathological targets rather than physiological targets in the early stage of drug screening. G-Quadruplex has become one of the important targets in the development of anti-tumor drugs. However, drugs that target quadruplexes may also bind to dsDNA, which may lead to adverse reactions. In this study, a new three-phase laminar flow chip was constructed to enable the multi-components of a traditional Chinese medicine extract to dynamically and competitively bind with G-quadruplex DNA (on target) and double-stranded DNA (off target), so as to select high-efficiency and low-toxicity anti-tumor drugs. The results showed that there were five compounds in the extracts of Macleaya cordata seeds that exhibited obvious differences in binding to the two targets. Furthermore, the binding constants and modes of four identified alkaloids as they bound to two DNA targets were verified by fluorescence spectra and molecular docking methods. The toxicity to HepG2 and LO2 cells from the four alkaloids was also compared. The results showed that sanguinarine and chelerythrine could be used as candidate drugs with stronger binding to HT24 than DNA26. The chip can also be used for other types of double-target screening of other traditional Chinese medicine extracts or compound libraries.

Microchip electrophoretic separation and fluorescence detection of chelerythrine and sanguinarine in medicinal plants.

A new method has been developed for separation of chelerythrine and sanguinarine in medicinal plants used in traditional Chinese medicine (TCM). The separation is achieved by microchip electrophoresis (CE) using laser-induced fluorescence detection. The CE separation is achieved by using a hydro-organic medium as the electrolyte buffer. The experimental results are consistent with the prediction by theory in terms of resolution and migration speed because of the low Joule heat generated in microchip CE. In addition, formamide was found to have a potential for separation of molecules with similar chemical structures. Based on these findings, a run buffer containing 50% formamide was used to separate chelerythrine (CHE) and sanguinarine (SAN). The influencing factors, such as solvent of run buffer, pH of buffer, separation distance, and separation voltage, were optimized. Baseline separation of chelerythrine and sanguinarine was achieved within 120 s under an electrical voltage of 1.8 kV. Good linearity was observed in the concentration range of 0.15-550 µg mL(-1) (r=0.9993) for CHE and in the range of 0.3-600 µg mL(-1) (r=0.9998) for SAN. A low limit of detection (LOD) was achieved because of the high sensitivity achieved by laser-induced fluorescence detection (i.e. 5.0 ng mL(-1) and 2.0 ng mL(-1) for CHE and SAN, respectively). The contents of CHE are found to be 641.8±7.5 and 134.0±2.3 mg/kg in extracts of Macleaya cordata and Chelidonium majus, respectively, with good recovery of above 99%. The corresponding values for SAN found in these Chinese herbal extracts are 681.8±7.9 mg/kg and 890.5±8.9 mg/kg, respectively.

Real-time detection of the early event of cytotoxicity of herbal ingredients on single leukemia cells studied in a microfluidic biochip.

A microfluidic approach has been developed for the real-time detection of drug effects, based on the quantitative measurement of calibrated cytosolic calcium ([Ca(2+)](i)) on single cancer cells. This microfluidic method is rapid by detecting the early event of cytotoxicity of drug candidates on cancer cells, without waiting for a couple of days needed for cell seeding and drug treatment by conventional assays. The miniaturized biochip consists of a V-shaped structure for the single-cell selection and retention. Various test reagents such as the chemotherapy drug (daunorubicin), an ionophore (ionomycin), and herbal ingredients from licorice (isoliquiritigenin or IQ) were investigated for their abilities to stimulate sustained cellular [Ca(2+)](i) elevations. The microfluidic results obtained in hours have been confirmed by conventional cytotoxicity assays which take days to complete. Moreover, any color or chemical interference problems found in the conventional assays of herbal compounds could be resolved. Using the microfluidic approach, IQ (50 microM) has been found to cause a sustained [Ca(2+)](i) elevation and cytotoxic effects on leukemia cells. The microfluidic single-cell analysis not only reduces reagent cost, and demands less cells, but also reveals some phenomena due to cellular heterogeneity that cannot be observed in bulk analysis.

Artesunate derived from traditional Chinese medicine induces DNA damage and repair.

Artesunate is a semisynthetic derivative from artemisinin, a natural product from the Chinese herb Artemisia annua L. It exerts antimalarial activity, and, additionally, artemisinin and its derivatives are active against cancer cells. The active moiety is an endoperoxide bridge. Its cleavage leads to the formation of reactive oxygen species and carbon-centered radicals. These highly reactive molecules target several proteins in Plasmodia, which is thought to result in killing of the microorganism. DNA damage induced by artemisinins has not yet been described. Here, we show that artesunate induces apoptosis and necrosis. It also induces DNA breakage in a dose-dependent manner as shown by single-cell gel electrophoresis. This genotoxic effect was confirmed by measuring the level of gamma-H2AX, which is considered to be an indication of DNA double-strand breaks (DSB). Polymerase beta-deficient cells were more sensitive than the wild-type to artesunate, indicating that the drug induces DNA damage that is repaired by base excision repair. irs1 and VC8 cells defective in homologous recombination (HR) due to inactivation of XRCC2 and BRCA2, respectively, were more sensitive to artesunate than the corresponding wild-type. This was also true for XR-V15B cells defective in nonhomologous end-joining (NHEJ) due to inactivation of Ku80. The data indicate that DSBs induced by artesunate are repaired by the HR and NHEJ pathways. They suggest that DNA damage induced by artesunate contributes to its therapeutic effect against cancer cells.

Same-single-cell analysis for the study of drug efflux modulation of multidrug resistant cells using a microfluidic chip.

Since multidrug resistance (MDR) is a major cause of failure in cancer chemotherapy, we report a microfluidic approach combined with the same-single-cell analysis to investigate the modulation of MDR, manifested as the inhibition of drug efflux. A microfluidic chip that was capable of selecting and retaining a single multidrug-resistant cancer cell was used to investigate drug efflux inhibition in leukemia cell lines. Three advantages of the microfluidic-based same-single-cell analysis (dubbed as SASCA) method have been revealed. First, it readily detects the modulation of drug efflux of anticancer compounds (e.g., daunorubicin) by MDR modulators (e.g., verapamil) among cellular variations. Second, SASCA is able to compare the different cellular abilities in response to drug efflux modulation based on the drug transport kinetics of single cells. Third, SASCA requires only a small number of cells, which may be beneficial for investigating drug resistance in minor cell subpopulations (e.g., cancer "stem" cells).

From traditional Chinese medicine to rational cancer therapy.

Many natural products and derivatives thereof belong to the standard repertoire of cancer chemotherapy. Examples are Vinca alkaloids, taxanes and camptothecins. In recent years, the potential of natural products from plants, notably from medicinal plants used in traditional Chinese medicine (TCM), has been recognized by the scientific community in the Western world. To provide an example of the most recent developments in this field, we have selected several compounds, namely artesunate, homoharringtonine, arsenic trioxide and cantharidin, that are found in natural TCM products and that have the potential for use in cancer therapy. Controlled clinical studies have shown that homoharringtonine and arsenic trioxide can exert profound activity against leukaemia. Increased knowledge of the molecular mechanisms of TCM-derived drugs and recent developments in their applications demonstrate that the combination of TCM with modern cutting-edge technologies provides an attractive strategy for the development of novel and improved cancer therapeutics.

Chemical analysis of raw, dry-roasted, and honey-roasted licorice by capillary electrophoresis.

In herbal medicine, licorice is usually processed using a roasting procedure which might modify the chemical compositions in licorice. To test this hypothesis, licorice root samples were roasted under various conditions (with or without honey) and subsequently extracted by refluxing with 95% ethanol. The analysis of chemical compositions of licorice root extracts was achieved by capillary electrophoresis. The running buffer has been optimized to be 50 mM sodium tetraborate (pH 9.01) containing 5 mM beta-cyclodextrin. Thermal decomposition of glycyrrhizin, which was a major ingredient in licorice, was first studied in detail, indicating the conversion of glycyrrhizin to glycyrrhetinic acid. The licorice extracts were then analyzed to indicate the above thermal conversion did occur in the licorice samples. This finding may shed some light on understanding the differences in the therapeutic values of raw versus roasted licorice in herbal medicine.