Biocatalysis of Platycoside E and Platycodin D3 Using Fungal Extracellular ß-Glucosidase Responsible for Rapid Platycodin D Production.

Platycodi radix (i.e., Platycodon grandiflorum root) products (e.g., tea, cosmetics, and herbal supplements) are popular in East Asian nutraceutical markets due to their reported health benefits and positive consumer perceptions. Platycosides are the key drivers of Platycodi radixes' biofunctional effects; their nutraceutical and pharmaceutical activities are primarily related to the number and varieties of sugar side-chains. Among the various platycosides, platycodin D is a major saponin that demonstrates various nutraceutical activities. Therefore, the development of a novel technology to increase the total platycodin D content in Platycodi radix extract is important, not only for consumers' health benefits but also producers' commercial applications and manufacturing cost reduction. It has been reported that hydrolysis of platycoside sugar moieties significantly modifies the compound's biofunctionality. Platycodi radix extract naturally contains two major platycodin D precursors (platycoside E and platycodin D3) which can be enzymatically converted to platycodin D via ß-d-glucosidase hydrolysis. Despite evidence that platycodin D precursors can be changed to platycodin D in the Platycodi radix plant, there is little research on increasing platycodin D concentrations during processing. In this work, platycodin D levels in Platycodi radix extracts were significantly increased via extracellular Aspergillus usamii ß-d-glucosidase (n = 3, p < 0.001). To increase the extracellular ß-d-glucosidase activity, A. usamii was cultivated in a culture media containing cellobiose as its major carbon source. The optimal pH and temperature of the fungal ß-d-glucosidase were 6.0 and 40.0 °C, respectively. Extracellular A. usamii ß-d-glucosidase successfully converted more than 99.9% (w/v, n = 3, p < 0.001) of platycoside E and platycodin D3 into platycodin D within 2 h under optimal conditions. The maximum level of platycodin D was 0.4 mM. Following the biotransformation process, the platycodin D was recovered using preparatory High Performance Liquid Chromatography (HPLC) and applied to in vitro assays to evaluate its quality. Platycodin D separated from the Platycodi radix immediately following the bioconversion process showed significant anti-inflammatory effects from the Lipopolysaccharide (LPS)-induced macrophage inflammatory responses with decreased nitrite and IL-6 production (n = 3, p < 0.001). Taken together, these results provide evidence that biocatalysis of Platycodi radix extracts with A. usamii may be used as an efficient method of platycodin D-enriched extract production and novel Platycodi radix products may thereby be created.

Rapid cell-deformability sensing system based on slit-flow laser diffractometry with decreasing pressure differential.

A slit-flow apparatus with a laser-diffraction method has been developed with significant advances in ektacytometry design, operation and data analysis. In the slit-flow ektacytometry, the deformation of red blood cells subjected to continuously decreasing shear stress in slit-flow can be quickly measured with adopting a laser-diffraction technique. Both the laser-diffraction image and pressure were measured with respect to time, which enable to determine the elongation index (EI) and the shear stress. The range of shear stress is 0-35 Pa and the measuring time is < 2 min. The EI is determined from an isointensity curve in the diffraction pattern using an ellipse-fitting program. The present study proposed the deformability index (DI) as a new measure of the RBC deformability, which is defined as an integral area under the EI curve between 0 and tau10 (tau(w) = 0-10). The key advantage of this design is the incorporation of a disposable element that holds the blood sample, which enables the present system to be easily used in a clinical setting.

Slit-flow ektacytometry: laser diffraction in a slit rheometer.

BACKGROUND: Deformability of red blood cells (RBCs) is a determinant of blood flow resistance as RBCs pass through small capillaries of the microcirculation. Available techniques for measuring RBC deformability often require a washing process after each measurement, which is not optimal for day-to-day clinical use. METHODS: A laser diffraction technique has been combined with slit-flow rheometry, which shows significant advances in ektacytometric design, operation, and data analysis. The essential features of this design are its simplicity (ease of operation and no moving parts) and a disposable element that is in contact with the blood sample. RESULTS: With slit ektacytometry, the deformation of RBCs subjected to continuously decreasing shear stress in a slit flow can be quickly measured with extremely small quantities of blood. The measurements with the slit ektacytometer were compared with those of LORCA and a strong correlation was apparent. The deformability of the hardened RBCs was markedly lower than that of the normal RBCs. In addition, the young cells showed higher values of the elongation index than did the old cells. CONCLUSIONS: The newly developed slit ektacytometer can measure RBC deformability with ease and accuracy. In addition, the slit ektacytometer can be easily used in a clinical setting owing to the incorporation of a disposable element that holds the blood sample.

Measurement of blood viscosity using a pressure-scanning capillary viscometer.

A newly designed pressure-scanning capillary viscometer is extended to measure the viscosity of whole blood over a range of shear rates without the use of anticoagulants in a clinical setting. In the present study, a single measurement of pressure variation with time replaces the flow rate and pressure drop measurements that are usually required for the operation of a capillary tube viscometer. Using a pressure transducer and capillary, we measured the variation of pressure flowing through capillary tube with respect to time, p(t), from which viscosity and the shear rate were mathematically calculated. For water and anticoagulant-added bloods, there was an excellent agreement found between the results from the pressure scanning capillary viscometer and those from a commercially available rotating viscometer. Also, the pressure-scanning capillary viscometer measured the viscosity of whole blood without heparin or EDTA. This new method overcomes the drawbacks of conventional viscometers in the measurement of whole blood viscosity. First, the pressure-scanning capillary viscometer can accurately and consistently measure the whole blood viscosity over a range of shear rates in less than 2 min without any anticoagulants. Second, this design provides simplicity (i.e., ease of operation, no moving parts, and disposable) and low cost.

Blood flow resistance with vibration and its effect on blood cell migration.

The present study investigated the effect of transverse vibration on the hemo-rheological characteristics of blood flow using a newly designed pressure-scanning capillary viscometer. As a transverse vibration was applied, aggregated blood cells become disaggregated. Frequency of vibration was found to be the main parameter causing hemo-rheological changes. For RBC suspension in a non-aggregating medium (Dextran 40), increasing frequency of vibration caused decreased flow resistance. Meanwhile, flow resistance for whole blood increased with frequency of vibration. These seemingly contradictory results could be interpreted without conflict when a comprehensive mechanism of cell migration under vibration is elucidated. The present study confirmed that vibration diminishes RBC aggregation, which triggers two different cell migration mechanisms and subsequently resulted in either increasing or decreasing the flow resistance.