Dong Woo Kim

Determination and validation of tetrodotoxin in human whole blood using hydrophilic interaction liquid chromatography–tandem mass spectroscopy and its application

A sensitive analytical method was developed for the quantitative determination of tetrodotoxin (TTX), a powerful sodium channel blocker, in human postmortem whole blood. The sample mixture was cleaned up using cation exchange SPE catridge after protein precipitation by methanol and then separated on a PC-HILIC (phosphorylcholine hydrophilic interaction liquid chromatography) column (150 mm × 2.0mm i.d., 5 μm) using a isocratic elution of 1% acetic acid and acetonitrile. The identification of TTX was performed on tandem mass spectrometry with electrospray ionization interface in positive ion mode. The retention time of voglibose (internal standard) and TTX was 5.1 and 6.0 min, respectively. TTX and internal standard (voglibose) were monitored and quantitated using the ion transitions: the respective precursor to product ion combinations, m/z 320/302 for TTX and m/z 268/92 for voglibose in the multiple reaction monitoring (MRM) mode. The recovery of TTX and voglibose was 61.4% and 62.8%, respectively and the good accuracy (97.7-103.9%), linearity (2-1200 ng/mL) and reproducibility were shown in this method. The limit of detection and limit of quantification were 0.32 ng/mL and 1.08 ng/mL, respectively. This method was applied in the case of three fishermen who were poisoned (including one death) by unknown fish on their boat in October 2010. In this case, the levels of TTX were 27.2, 30.0 and 29.7 ng/mL in heart blood, peripheral blood and serum of a victim, were 3.1 and 12.1 ng/mL in peripheral blood and 3.9 and 12.8 ng/mL in serum of two survivors, respectively.

Rhododendric acid A, a new ursane-type PTP1B inhibitor from the endangered plant Rhododendron brachycarpum G. Don

In spite of the critical role of the natural products in drug discovery, surprising little attention has been placed on endangered and rare plant species that could play a pivotal role in pharmaceutical and fiber development. Protein tyrosine phosphatase-1B (PTP1B), which blocks insulin signaling, has been gaining interest to be a promising therapeutic target for the treatment of type 2 diabetes mellitus. Bioassay-guided fractionation on the leaves of Rhododendron brachycarpum G. Don (Ericaceae) yielded seven PTP1B inhibitory triterpenoids, including a new triterpene, rhododendric acid A (1). Their PTP1B inhibitory potency and their lipophilicity were investigated to provide a feasible scaffold that may overcome the innate limitation of the previously reported PTP1B inhibitors.

Bioassay-guided isolation of fatty acid synthase inhibitory diterpenoids from the roots of Salvia miltiorrhiza Bunge

Fatty acid synthase (FAS) is considered as a novel drug target for the development of anticancer and anti-obesity agents. Bioassay-guided fractionation of a n-hexane-soluble extract prepared from the roots of Salvia miltiorrhiza Bunge (Labiatae), using an in vitro enzyme assay, led to the isolation of five abietane diterpenoids: 15,16-dihydrotanshinone I (1), cryptotanshinone (2), tanshinone I (3), tanshinone IIA (4), and dansenspiroketallactone (5). Compounds 1–5 were tested for their in vitro FAS inhibitory activity and, except for compound 5 (IC50 > 100 μM), compounds 1–4 inhibited the enzyme activity with IC50 values ranging from 12.0 to 30.3 μM. Our findings may be partially related to the anticancer activity of abietane diterpenoids from the plant, suggesting a further study on the anticancer potential of tanshinone derivatives.

Extract of Allium tuberosum Rottler ex Spreng Promoted the Hair Growth through Regulating the Expression of IGF-1

Allium tuberosum Rottler ex Spreng (ATRES) has been used as a traditional medicine for the treatment of abdominal pain, diarrhea, and asthma. In this study, we investigated the hair growth promoting activities of ATRES on telogenic C57BL6/N mice. Hair growth was significantly increased in the dorsal skin of ethanol extract of ATRES treated mouse group compared with the control mouse group. To enrich the hair promoting activity, an ethanol-insoluble fraction was further extracted in sequence with n-hexane, dichloromethane, ethyl acetate, n-butanol, and distilled water. Interestingly, we found that extraction with n-butanol is most efficient in producing the hair promoting activity. In addition, the soluble fraction of the n-butanol extract was further separated by silica gel chromatography and thin layer chromatography (TLC) resulting in isolating four single fractions which have hair growth regeneration potential. Furthermore, administration of ATRES extracts to dorsal skin area increased the number of hair follicles compared with control mouse group. Interestingly, administration of ATRES extract stimulated the expression of insulin-like growth factor-1 (IGF-1) but not of keratin growth factor (KGF) or vascular endothelial growth factor (VEGF). Taken together, these results suggest that ATRES possesses strong hair growth promoting potential which controls the expression of IGF-1.

Development of a liquid chromatography-tandem mass spectrometry method for the determination of Grayanotoxins in rat blood and its application to toxicokinetic study.

A sensitive and specific high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the determination of Grayanotoxin I (GTX I) and Grayanotoxin III (GTX III) in rat whole blood. Grayanotoxins (GTXs) and clindamycin as internal standard (IS) were extracted from rat blood via solid-phase extraction using PEP solid-phase extraction cartridges. Chromatographic separation of the analytes was achieved on a Kinetex C18 (100 × 2.1 mm, 2.6 µm) reversed-phase column using a gradient elution with the mobile phase of 1% acetic acid in water and methanol at a flow rate of 0.2 mL/min. Electrospray ionization mass spectrometry was operated in the positive ion mode with multiple reaction monitoring. The calibration curves obtained were linear over the concentration range of 1-100 ng/mL with a lower limit of quantification of 1 ng/mL for GTXs. The relative standard deviation of intra-day and inter-day precision was below 6.8% and accuracy ranged from 94.8 to 106.6%. The analytes were stable in the stability studies. The validated method was successfully applied to the quantification and toxicokinetic study of GTXs in rats for the first time after oral administration of 11.52 mg/kg mad honey and 0.35 mg/kg GTX III, respectively.