Kyung-Mi Joo

Pharmacokinetic study of ginsenoside Re with pure ginsenoside Re and ginseng berry extracts in mouse using ultra performance liquid chromatography/mass spectrometric method

Ginsenoside Re is the major ginsenoside in ginseng berry(GB) extract and its pharmacokinetics were studied following the intravenous and oral administration of pure Re or ginseng berry extract in mouse with doses of 10 and 50 mg/kg using ultra performance liquid chromatography mass spectrometric (UPLC/MS) method which can simultaneously determine ginsenoside Re, Rg1 and Rh1 in mouse serum. The serum samples were pretreated by protein precipitation and chromatographic separation was performed on AQUITY UPLC BEH C(18) column using gradient elution with the mobile phase of 5 mM ammonium formate and acetonitrile. Analytes and digoxin (I.S.) were analyzed and identified using an electrospray negative ionization mass spectrometry in the selected ion monitoring mode with the linear concentration range of 5.0-5000 ng/mL and lower limits of detection (LLOD) under 2.5 ng/mL. Ginsenoside Re was rapidly cleared from the body with a short half-life (0.2+/-0.03 h for male and 0.5+/-0.08 h for female mice after i.v.) and oral absorption was generally poor (F% 0.19-0.28). Notably, GB extract showed a superior oral absorption of ginsenoside Re (F% 0.33-0.75) at equivalent ginsenoside Re dose to pure ginsenoside Re, indicating that GB extract might be a good form for ginsenoside Re intake.

Determination of spatial distribution of melamine-cyanuric acid crystals in rat kidney tissue by histology and imaging matrix-assisted laser desorption/ionization quadrupole time-of-flight mass spectrometry.

After the outbreak of acute renal failure associated with melamine-contaminated pet food, many attempts have been made to uncover the mechanism underlying the renal toxicity caused by melamine and melamine-related compounds. Using rat models, we investigated the renal crystal formation following the ingestion of a melamine-cyanuric acid mixture (M+CA, 1:1) to gain insight into the M+CA-induced renal toxicity. M+CA did not induce toxicity in precision-cut kidney slices, suggesting that M+CA does not have a direct nephrotoxicity. On the contrary, oral administration of M+CA for 3 days induced nephrotoxicity as determined by increased serum blood urea nitrogen and creatinine, reduced creatinine clearance, and enlarged kidneys in the animals treated with 50 mg/kg M+CA (melamine, 25 mg/kg, and cyanuric acid, 25 mg/kg; 2 of 10 animals) and 100 mg/kg M+CA (9 of 9 animals). While urine crystals were found in all animals treated with M+CA (25-100 mg/kg), histological examination revealed that renal crystals could be observed only in the kidneys of animals showing signs of nephrotoxicity. Remarkably, at 50 mg/kg M+CA, crystals were observed mainly in the medulla region of the kidney, while at 100 mg/kg, crystals were disseminated throughout the cortex and medulla regions. To further investigate the crystal formation by M+CA, matrix-assisted laser desorption/ionization quadrupole time-of-flight (MALDI-Q-TOF) imaging mass spectrometry detecting melamine distribution through monitoring the product ion (m/z 85, M + H) from melamine (m/z 127, M + H) was developed to directly obtain the image of melamine distribution in the kidney. The distribution image of melamine in kidney tissue confirmed that dense points of melamine were located only in the medulla region at 50 mg/kg M+CA, while at 100 mg/kg, they were disseminated widely from the cortex to medulla. These results demonstrated that M+CA ingestion could lead to crystal formation in kidney tubules along the osmotic gradient and that renal crystal formation is closely linked with M+CA-induced nephrotoxicity.

Procoagulant and prothrombotic activation of human erythrocytes by phosphatidic acid

Increased phosphatidic acid (PA) and phospholipase D (PLD) activity are frequently observed in various disease states including cancers, diabetes, sepsis, and thrombosis. Previously, PA has been regarded as just a precursor for lysophosphatidic acid (LPA) and diacylglycerol (DAG). However, increasing evidence has suggested independent biological activities of PA itself. In the present study, we demonstrated that PA can enhance thrombogenic activities in human erythrocytes through phosphatidylserine (PS) exposure in a Ca(2+)-dependent manner. In freshly isolated human erythrocytes, treatment of PA or PLD induced PS exposure. PA-induced PS exposure was not attenuated by inhibitors of phospholipase A(2) or phosphatidate phosphatase, which converts PA to LPA or DAG. An intracellular Ca(2+) increase and the resultant activation of Ca(2+)-dependent PKC-alpha appeared to underlie the PA-induced PS exposure through the activation of scramblase. A marginal decrease in flippase activity was also noted, contributing further to the maintenance of exposed PS on the outer membrane. PA-treated erythrocytes showed strong thrombogenic activities, as demonstrated by increased thrombin generation, endothelial cell adhesion, and erythrocyte aggregation. Importantly, these procoagulant activations by PA were confirmed in a rat in vivo venous thrombosis model, where PA significantly enhanced thrombus formation. In conclusion, these results suggest that PA can induce thrombogenic activities in erythrocytes through PS exposure, which can increase thrombus formation and ultimately contribute to the development of cardiovascular diseases.

Simultaneous determination of two Amadori compounds in Korean red ginseng (Panax ginseng) extracts and rat plasma by high-performance anion-exchange chromatography with pulsed amperometric detection.

A new simple, rapid and sensitive high-performance anion-exchange chromatography method with pulsed amperometric detection (HPAEC-PAD) was developed and validated for the simultaneous determination of two Amadori compounds, arginyl-fructose and arginyl-fructosyl-glucose in Korean red ginseng (Panax ginseng) extracts, rat plasma. Separation of the two target analytes was efficiently undertaken on CarboPac PA1 anion-exchange column with isocratic elution (400 mM sodium hydroxide and deionized water (90:10, v/v)) at flow rate 0.7 mL/min within 15 min of single chromatographic run. Under optimized conditions, the detection limits (signal-to-noise ratio equal to 3) were 20 and 25 ng/mL for arginyl-fructose and arginyl-fructosyl-glucose, respectively. Calibration curves of peak area for the two analytes were linear over three orders of magnitude with a correlation coefficients greater than 0.999. The accuracy of the method was tested by recovery measurement of the spiked samples which yielded good results of 94.15-102.62%. This method was successfully applied to the quantification of arginyl-fructose and arginyl-fructosyl-glucose in herbal extracts and in the plasma samples from rat.

Influence of N-glycan processing disruption on tyrosinase and melanin synthesis in HM3KO melanoma cells.

Abstract:  Tyrosinase, a type I membrane glycoprotein, is synthesized and glycosylated in the endoplasmic reticulum (ER) and Golgi. The enzyme is subsequently transported to melanosomes where it participates in melanogenesis. Previous studies showed that the disruption of early ER N‐glycan processing by deoxynojirimycin (DNJ), an inhibitor of α‐glucosidase, suppresses tyrosinase enzymatic activity and melanogenesis. However, the disruption of late glycan processing, mainly performed by ER and Golgi α‐1,2‐mannosidases, on tyrosinase enzymatic activity and melanogenesis remains to be investigated. Following treatment of HM3KO human melanoma cells with deoxymannojirimycin (DMJ), an inhibitor of α‐1,2‐mannosidase, transport of tyrosinase to the melanosome, enzymatic activity, and melanogenesis were reduced in a dose‐dependent manner. However, DMJ did not directly inhibit tyrosinase enzymatic activity and expression. Interestingly, an extract of Streptomyces subrutilus culture medium (ESSCM) containing DMJ and DNJ as the main components inhibited glycosylation and transport of tyrosinase to the melanosome as well as melanin synthesis, but with no negative effects on cell viability. These inhibitory effects of ESSCM were stronger than those of DMJ or DNJ alone. Tyrosinase glycosylation and melanogenesis in HM3KO melanoma cells were more effectively inhibited by DMJ and DNJ combined than DMJ or DNJ alone. Accordingly, we propose that ESSCM is a potential candidate for treating undesirable hyperpigmentation conditions, such as melasma, postinflammatory melanoderma, and solar lentigo.

Relationship of ceramide–, and free fatty acid–cholesterol ratios in the stratum corneum with skin barrier function of normal, atopic dermatitis lesional and non-lesional skins

One of the main causes for atopic dermatitis (AD) is skin barrier dysfunction which can be represented as increased trans-epidermal water loss (TEWL). Skin barrier dysfunction is induced by the disruption of stratum corneum (SC), a dense protein–lipid matrix which functions as a barrier to exogenous irritants and allergens. Recently a relationship between altered SC lipid profiles and impaired skin barrier function has been described [1,2]. SC lipids are composed of ceramides (CER), free fatty acids (FFA) and cholesterol (CHOL)[3]. Ishikawa et al. [4] reported that 9 out of 11 CER classes found in human SC, exhibit statistically significant correlations with TEWL in the aspect of contents and average total carbon numbers. van Smeden et al. [5] further demonstrated the reduction of carbon-chain length in FFAs and CERs in AD lesion, enlightening the roles of respective FFA and CER species in skin barrier function. Of note, these studies employed total protein content as normalization criteria to reduce sampling errors during SC tapestripping, and large inter-individual variations in the amount of SC lipids. However, synthetic pathway and distribution of protein are completely different from lipids, and inflammation and immune reaction in AD may alter the protein expression profiles substantially. Moreover, physical properties of protein are distinct from lipids, and accordingly separate steps of sample extraction are necessary, further reducing its value as normalization criteria for lipid quantitation. Here, we introduced CHOL, another SC lipid class as normalization criteria to investigate the profiles of CER and FFA species in AD non-lesional, lesional and normal skins. The protocol was approved by the institutional review board of Ajou University Medical Center. SC samples were collected from lesional and non-lesional skins of Korean AD patients (10 patients, 7 males and 3 females, SCORAD 25.8 5.8, TEWL 11.7 3.6 and 29.7 6.3 for non-lesional and lesional area, respectively, Fig. 1A and B) and healthy volunteers (4 males and 6 females for normal, TEWL 12.3 4.5). With authentic standards (Sigma–Aldrich, St. Louis, MO, USA, Avanti Polar Lipids, Alabaster, AL and Evonik), CER-AP (a-hydroxy fatty acid conjugated to phytosphingosine), CER-NP (nonhydroxy fatty acid conjugated to phytosphingosine), CER-NDS (nonhydroxy fatty acid conjugated to dihydrosphingosine), CER-NS (nonhydroxy fatty acid conjugated

Rapid, simultaneous and nanomolar determination of pyroglutamic acid and cis-/trans-urocanic acid in human stratum corneum by hydrophilic interaction liquid chromatography (HILIC)-electrospray ionization tandem mass spectrometry.

A rapid, sensitive and specific hydrophilic interaction liquid chromatography coupled to tandem mass spectrometric (HILIC-MS/MS) method for the simultaneous determination of pyroglutamic acid, cis- and trans-urocanic acid in human skin stratum corneum (SC) were developed and validated. This method was carried out without derivatization or addition of ion-pair additives in mobile phase. The analytes were extracted by PBS buffer solution and analyzed using an electrospray positive ionization mass spectrometry in the multiple reaction monitoring (MRM) mode. Chromatographic separation was performed on an AQUITY UPLC amide column using gradient elution with the mobile phase of water and acetonitrile. The standard curves were linear over the concentration range of 1.0-250 ng/mL with a correlation coefficient higher than 0.999 with an LLOQ of 0.5 ng/mL. The lower limits of detection (LLOD) of these analytes were lower than 0.2 ng/mL. The intra- and inter-day precisions were measured to be below 7.7% and accuracies were within the range of 94.3-102.6%. The validated method was successfully applied to determine the level of pyroglutamic acid and cis-/trans-urocanic acid in the SC samples from forearm and forehead region of 19 human volunteers.

Enzymatic Hydrolysis of Green Tea Seed Extract and Its Activity on 5α-Reductase Inhibition

Two kaempferol glycosides were isolated from green tea seed extract (GTSE). After conducting a structure analysis, these two compounds were identified as kaempferol-3-O-[2-O-β-D-galactopyranosyl-6-O-α-L-rhamnopyranosyl]-β-D-glucopyranoside (compound 1) and kaempferol-3-O-[2-O-β-D-xylopyranosyl-6-O-α-L-rhanmopyranosyl]-β-D-glucopyranoside (compound 2). These two compounds were hydrolysed by o-glycolytic enzymes for the production of kaempferol. After performing several reactions, we found the optimum enzyme combination, a reaction with β-galactosidase and hesperidinase. Finally, we produced kaempferol of above 95% purity. The 5α-reductase inhibition activities of GTSE hydrolysate (GTSE-H) containing kaempferol were evaluated by the contact cell-based metabolic method using a stable HEK 293 cell line. GTSE-H showed a good inhibition effect on HEK 293 cell lines both type 1 and type 2 on 5α-reductase. Especially, GTSE-H inhibited type 2 with kaempferol content dependency. The results indicate that the inhibition activity of hydrolysate on 5α-reductase type 2 increases in accordance with kaempferol content.

Determination of hexavalent chromium in cosmetic products by ion chromatography and postcolumn derivatization.

Chromium hydroxide green [Cr2O(OH)4] and chromium oxide green (Cr2O3) are colouring agents for use in cosmetic products. These colourants may contain chromium (VI), which cause skin allergies through percutaneous adsorption on the skin. Eye shadow is a representative cosmetic product in which significant colourants are used. We analysed the chromium (VI) in the eye shadows by ion chromatography and post column derivatization. We optimize conditions of chromium (VI) analysis in eye shadows. During the pretreatment procedure, there are no exchange of chromium (III) to chromium (VI). This method has a limit of quantification for chromium (VI) of 1.0 μg l−1, recovery rate of 100 ± 3% and analysis time less than 10 min. This result is 300 times more sensitive than the high‐performance liquid chromatography method. We applied the optimized method to analyse 22 eye shadows and 6 colouring agents. 2 out of 22 of the products contained more than 5 mg l−1. In our previous work, 5 mg l−1 of Cr represented a threshold level. There was much more Cr(VI) in the colouring agents. The Cr(VI) in one of the colouring agents was 97.6 mg l−1.

Determination of N-nitrosodiethanolamine, NDELA in cosmetic ingredients and products by mixed mode solid phase extraction and UPLC-tandem mass spectrometry with porous graphitic carbon column through systemic sample pre-cleanup procedure.

A rapid, sensitive, accurate and specific ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) method for the detection of N-nitrosodiethanolamine (NDELA), a highly toxic contaminant in cosmetic raw materials and products was developed and validated. Systematized sample preparation steps were developed according to product types. Various SPE cartridges and columns were examined to establish the condition of SPE and chromatographic separation for NDELA. Sample cleanup steps consisting of solvent and liquid-liquid extraction tailored to the various sample matrix types were established prior to mixed mode SPE (Bond Elut AccuCAT). Chromatographic separation was achieved within 7 min on a porous graphitic carbon (PGC) column using a gradient elution with the mobile phase of 1mM ammonium acetate containing 0.1% acetic acid and methanol. NDELA was monitored using an electrospray positive ionization mass spectrometry in the multiple reaction monitoring (MRM) mode (m/z 134.9>103.7(quantifier) and 73.7(qualifier ion)) with d8-NDELA (m/z 143.1>111.0) as internal standard. The standard curves were linear over the concentration range of 1-100 ng/mL with a correlation coefficient higher than 0.99. The limit of detection (LOD) and the limit of quantification (LOQ) was 10 and 20 μg/kg, respectively (0.5 and 1 ng/mL in standard solution). The intra- and inter-day precisions were estimated to be below 11.1% and accuracies were within the range of 90.8-115.8%. The validated method was successfully applied to the analysis of real samples including raw materials, skin care, make-up, shampoos and hair products.