Improved quantitative analysis of tenuifolin using hydrolytic continuous-flow system to build prediction models for its content based on near-infrared spectroscopy.

This study used two types of analyses and statistical calculations on powdered samples of Polygala root (PR) and Senega root (SR): (1) determination of saponin content by an independently developed quantitative analysis of tenuifolin content using a flow reactor, and (2) near-infrared spectroscopy (NIR) using crude drug powders as direct samples for metabolic profiling. Furthermore, a prediction model for tenuifolin content was developed and validated using multivariate analysis based on the results of (1) and (2). The goal of this study was to develop a rapid analytical method utilizing the saponin content and explore the possibility of quality control through a wide-area survey of crude drugs using NIR spectroscopy. Consequently, various parameters and appropriate wavelengths were examined in the regression analysis, and a model with a reasonable contribution rate and prediction accuracy was successfully developed. In this case, the wavenumber contributing to the model was consistent with that of tenuifolin, confirming that this model was based on saponin content. In this series of analyses, we have succeeded in developing a model that can quickly estimate saponin content without post-processing and have demonstrated a brief way to perform quality control of crude drugs in the clinical field and on the market.

Metabolite profiling of Drynariae Rhizoma using 1H NMR and HPLC coupled with multivariate statistical analysis.

Drynariae Rhizoma has been used to treat bone diseases and kidney deficiency in traditional medicine. Recently its aqueous extract was reported to enhance memory function. Although the Japanese standards for non-Pharmacopoeial crude drugs 2022 prescribed Drynaria roosii as the botanical origin, some counterfeits and both raw and stir-fired crude drugs are available in markets. To distinguish Drynariae Rhizoma derived from D. roosii appropriately from others and verify the validity of uses of stir-fried ones, 1H NMR-based metabolite profiling coupled with HPLC were performed. Raw samples derived from D. roosii contained naringin (1), neoeriocitrin (2), 5,7-dihydroxychromone-7-O-neohesperidoside (3), caffeic acid 4-O-ß-D-glucoside (4), protocatechuic acid (5), trans-p-coumaric acid 4-O-ß-D-glucoside (6), and kaempferol 3-O-α-L-rhamnoside 7-O-ß-D-glucoside (8). Stir-fried samples were characterized by presence of 5-hydroxymethyl-2-furaldehyde (13), and were divided into two types; one possessing similar composition to raw samples (Type I) and another without above components except 5 (Type II). Quantitative analyses using qHNMR and HPLC, followed by principal component analysis demonstrated that the raw samples had higher contents of 1 (0.93-9.86 mg/g), 2 (0.74-7.59 mg/g), 3 (0.05-2.48 mg/g), 4 (0.27-2.51 mg/g), 6 (0.14-1.26 mg/g), and 8 (0.04-0.52 mg/g), and Type II had a higher content of 5 (0.84-1.32 mg/g). The counterfeit samples derived from Araiostegia divaricata var. formosana were characterized by higher content of ( -)-epicatechin 3-O-ß-D-allopyranoside (10) (1.44-11.49 mg/g) without 1 and 2. These results suggested that Drynariae Rhizoma samples derived from other botanical origins and Type II stir-fried samples cannot substitute for D. roosii rhizome.

Genetic and Chemical Diversity of Commercial Japanese Valerian.

In order to investigate the relationship between the chemical composition of essential oils and haplotypes of the psbA-trnH intergenic spacer region of chloroplast DNA (psbA-trnH) in Valerianae Fauriei Radix (Japanese Valerian; JV), we analyzed the DNA sequence and GC-MS metabolome of JV from Japanese markets and of herbal specimens from related species. DNA analysis revealed that JV products from Japan consisted of three haplotypes, namely AH-1, -2 and -5 reported in our previous study. The GC-MS metabolome revealed five chemotypes (J1, J2, C, K and O), of which J1, J2 and C were detected in the JV products from Japan. Chemotypes J1 and J2, with kessyl glycol diacetate (KGD) as the main volatile component, were found in the products of Japanese origin whereas chemotype C, with 1-O-acetyl-2,10-bisaboladiene-1,6-diol (ABD), was found in the products of Chinese and Korean origin. The haplotypes were correlated with the chemotypes: haplotype AH-1 for chemotype J1, AH-2 for chemotype J2 and AH-5 for chemotype C, suggesting that the chemical diversity of JV is not attributed to the environmental factors rather to the genetic factors. Since KGD and ABD were reported to have sedative effects and nerve growth factor (NGF)-potentiating effects, respectively, understanding the chemotypes and selecting an appropriate one would be important for the application of JV. The psbA-trnH haplotypes could be useful DNA markers for the quality control and standardization of JV.

Comparison of various commercially available cinnamon barks using NMR metabolomics and the quantification of coumarin by quantitative NMR methods.

Cinnamon bark is an important spice worldwide. In this study, the chemical diversity of various commercially available cinnamon barks that differed in their production areas and utility applications (culinary spice or medicines) were investigated by the use of 1H NMR metabolomics. Our results indicated that principle component analysis (PCA) and hierarchical cluster analysis (HCA) of the 1H NMR spectra of the cinnamon bark methanolic extracts including the deduction of their species by nucleotide sequence analysis enabled differentiation of the cinnamon barks according to their species, production areas and utility applications. The constituents of Vietnam cinnamon were found to differ significantly from the other samples investigated based on PCA score plots and HCA constellation dendrograms. Coumarin was found to be a key compound for the discrimination of Vietnamese cinnamon by multivariate analysis of the 1H NMR spectral data and direct comparison of the 1H NMR spectra. In addition, coumarin was quantified using quantitative NMR methods. As a result, coumarin was contained in Vietnamese cinnamon at a higher level compared to other cinnamons. This study indicated that 1H NMR metabolomics could deduce spices, utility, and producing area of commercially available cinnamon barks. Furthermore, combining quantitative 1H NMR methods with 1H NMR metabolomics enable quantification of coumarin in cinnamon bark on a single measurement.

Characterization of metabolites in Saposhnikovia divaricata root from Mongolia.

Saposhnikoviae Radix (SR), derived from the dried root and rhizome of Saposhnikovia divaricata, is a popular crude drug used in traditional Chinese and Japanese medicine. To evaluate the metabolites of S. divaricata roots from Mongolia and to investigate their geographical variation, we developed the HPLC method, determined the contents of 9 chromones and 4 coumarins, and conducted multivariate statistical analysis. All Mongolian specimens contained prim-O-glucosylcimifugin (1) and 4'-O-ß-D-glucosyl-5-O-methylvisamminol (3), and their total amount (5.04-25.06 mg/g) exceeded the criterion assigned in the Chinese Pharmacopoeia. Moreover, the content of 1 (3.98-20.79 mg/g) was significantly higher in the Mongolian specimens than in Chinese SR samples. The specimens from Norovlin showed the highest contents of 1 and 3. The total levels of dihydropyranochromones were higher in the specimens from Bayan-Uul. The orthogonal partial least squares-discriminant analysis revealed that the Mongolian specimens tended to be separated into three groups based on growing regions, in which several chromones contributed to each distribution. Furthermore, 1H NMR analysis revealed that Mongolian specimens had less amount of sucrose and a substantial amount of polyacetylenes. Thus, in this study, the chemical characteristics of Mongolian S. divaricata specimens were clarified and it was found that the specimens from the northeast part of Mongolia, including Norovlin, had the superior properties due to higher amounts of major chromones.

Microbial Community Structure and Chemical Constituents in Shinkiku, a Fermented Crude Drug Used in Kampo Medicine.

Shinkiku (Massa Medicata Fermentata) is a traditional crude drug used to treat anorexia and dyspepsia of elder patients in east Asia. Shinkiku is generally prepared by the microbial fermentation of wheat and herbs. Shinkiku is also used in Japanese Kampo medicine as a component of (Hangebyakujutsutemmato). However, the quality of shinkiku varies by manufacture because there are no reference standards to control the quality of medicinal shinkiku. Thus, we aim to characterize the quality of various commercially available shinkiku by chemical and microbial analysis. We collected 13 shinkiku products manufactured in China and Korea and investigated the microbial structure and chemical constituents. Amplicon sequence analysis revealed that Aspergillus sp. was common microorganism in shinkiku products. Digestive enzymes (α-amylase, protease, and lipase), organic acids (ferulic acid, citric acid, lactic acid, and acetic acid), and 39 volatile compounds were commonly found in shinkiku products. Although there were some commonalities in shinkiku products, microbial and chemical characteristic considerably differed as per the manufacturer. Aspergillus sp. was predominant in Korean products, and Korean products showed higher enzyme activities than Chinese products. Meanwhile, Bacillus sp. was commonly detected in Chinese shinkiku, and ferulic acid was higher in Chinese products. Principal component analysis based on the GC-MS peak area of the volatiles also clearly distinguished shinkiku products manufactured in China from those in Korea. Chinese products contained higher amounts of benzaldehyde and anethole than Korean ones. Korean products were further separated into two groups: one with relatively higher linalool and terpinen-4-ol and another with higher hexanoic acid and 1-octen-3-ol. Thus, our study revealed the commonality and diversity of commercial shinkiku products, in which the commonalities can possibly be the reference standard for quality control of shinkiku, and the diversity suggested the importance of microbial management to stabilize the quality of shinkiku.

Verification of a thrombus induction method at the target point inside the blood pump using a fibrinogen coating for a thrombus detection study.

Although the magnetically levitated centrifugal blood pump (mag-lev pump) is considered superior to other pumps in antithrombogenicity, thrombotic complications are still reported. Research into thrombus detection inside a mag-lev pump is very important for solving this problem. Our research group has already proposed a method to detect a thrombus inside a mag-lev pump in real time without an additional sensor, which is named the impeller vibration method. To efficiently advance our research with reproducibility, a preconditioning method to induce thrombus inside the pump was thought to be necessary. Therefore, this study aimed to develop a preconditioning method that induces thrombus formation. To verify this method, in vitro experiments for thrombus detection were performed. A mag-lev pump developed at Tokyo Institute of Technology was used. A fibrinogen solution was coated on the inner surfaces of the bottom housing to induce thrombus formation at the target point inside the pump. The thrombus is detected by utilizing the phenomenon that the phase difference between the impeller displacement and input current to the magnetic bearing increases when a thrombus is formed inside a pump. Five hundred mL of porcine blood anticoagulated with heparin sodium was circulated in the mock circuit, and protamine sulfate was administered. Flow rate (1 L/min), impeller vibrational frequency (70 Hz), and vibrational amplitude (30 µm) were set to constant. The experiment was terminated when the phase difference increased by over 2° from the minimum value. The experiments were performed in fibrinogen-coated (group F, n = 5) and non-coated pumps (group N, n = 5). In group F, thrombus formation was observed at the fibrinogen-coated point of the housing. In contrast, a relatively small thrombus was observed in varying locations such as the housing or the impeller in group N. Thrombus formation time (the time from when the phase difference takes the minimum value to when the experiment is terminated) was different between the two groups. The mean time was significantly shorter in group F (44 ± 29 minutes) than in group N (143 ± 38 minutes; p = 0.0019). Therefore, a preconditioning method that induced thrombus formation at the target point inside a blood pump was successfully developed.

Identifying the compounds that can distinguish between Saposhnikovia root and its substitute, Peucedanum ledebourielloides root, using LC-HR/MS metabolomics.

Previously, we established a 1H NMR metabolomics method using reversed-phase solid-phase extraction column (RP-SPEC), and succeeded in distinguishing wild from cultivated samples of Saposhnikoviae radix (SR), and between SR and its substitute, Peucedanum ledebourielloides root (PR). Herein, we performed LC-HR/MS metabolomics using fractions obtained via RP-SPEC to identify characteristic components of SR and PR. One and three characteristic components were respectively found for SR and PR; these components were isolated with their m/z values and retention times as a guide. The characteristic component of SR was identified as 4'-O-ß-D-glucosyl-5-O-methylvisamminol (1), an indicator component used to identify SR in the Japanese Pharmacopoeia. In contrast, the characteristic components of PR were identified as xanthalin (2), 4'-O-ß-D-apiosyl (1 → 6)-ß-D-glucosyl-5-O-methylvisamminol (3), and 3'-O-ß-D-apiosyl (1 → 6)-ß-D-glucosylhamaudol (4) based on spectroscopic data such as 1D- and 2D-NMR, MS, and specific optical rotation. Among them, 4 is a novel compound. For the correlation between the NMR metabolomics results in the present and our previous report, only 1 and 2 were found to correlate with the chemical shifts, and the other compounds had no correlation. As the chemical shifts for compounds 1, 3, and 4 were similar to each other, especially for the aglycone moiety, they could not be distinguished because of the sensitivity and resolution of 1H NMR. Accordingly, combining NMR and LC/MS metabolomics with their different advantages is considered useful for metabolomics of natural products. The series of methods used in our reports could aid in quality evaluations of natural products and surveying of marker components.

Quality Evaluation and Characterization of Fractions with Biological Activity from Ephedra Herb Extract and Ephedrine Alkaloids-Free Ephedra Herb Extract.

Previously, we reported that the c-Met inhibitory effect of Ephedra Herb extract (EHE) is derived from ingredients besides ephedrine alkaloids. Moreover, analgesic and anti-influenza activities of EHE and ephedrine alkaloids-free Ephedra Herb extract (EFE) have been reported recently. In this study, we examined the fractions containing c-Met kinase inhibitory activity from EHE and the fractions with analgesic and anti-influenza activities from EFE, and elucidated the structural characteristics of the active fractions. Significant c-Met kinase activity was observed in 30, 40, and 50% methanol (MeOH) eluate fractions obtained from water extract of EHE using Diaion HP-20 column chromatography. Similarly, 20 and 40% MeOH, and MeOH eluate fractions obtained from water extract of EFE were found to display analgesic and anti-influenza activities. Reversed phase-HPLC analysis of the active fractions commonly showed broad peaks characteristic of high-molecular mass condensed tannin. The active fractions were analyzed using 13C-NMR and decomposition reactions; the deduced structures of active components were high-molecular mass condensed tannins, which were mainly procyanidin B-type and partly procyanidin A-type, including pyrogallol- and catechol-type flavan 3-ols as extension and terminal units. HPLC and gel permeation chromatography (GPC) analyses estimated that the ratio of pyrogallol- and catechol-type was approximately 9 : 2, and the weight-average molecular weight based on the polystyrene standard was >45000. Furthermore, GPC-based analysis was proposed as the quality evaluation method for high-molecular mass condensed tannin in EHE and EFE.

Detection of thrombosis in a magnetically levitated blood pump by vibrational excitation of the impeller.

The use of contactless support technology for the impeller has led to an increase in the durability of ventricular assist devices (VADs), and these have been in clinical use worldwide. However, pump thrombosis and stroke are still issues to be solved. We have developed a method for detecting the thrombosis in a magnetically levitated blood pump without the need for additional sensors or other equipment. In the proposed method, a sinusoidal current is applied to the electromagnets used for the magnetic bearing, resulting in vibration of the impeller. The phase difference between the current and displacement of the impeller increases with pump thrombosis. First, we describe the principle by which the pump thrombosis is detected. Pump thrombosis reduces the narrowest fluid gap in the pump and this gives rise to a change in the phase difference. Second, we report on experiments in which we changed the narrowest fluid gap using oriented polypropylene tape and showed that decreasing the narrowest fluid gap resulted in an increase in phase difference. For these experiments, the measurements were repeated three times for each condition. Third, we examine the relationship between the pump thrombosis and the phase difference evaluated by observations of the underside of the impeller when operating the pump with porcine blood. Since light was unable to penetrate the blood layer, the erythrocytes were removed for this observation. Only one observation was made. The results showed the phase difference rapidly increased at the same moment when the pump thrombosis was observed. This implies the proposed method has the potential to detect the early stages of pump thrombosis. Finally, in vitro experiments to detect thrombosis when using whole porcine blood in the pump were conducted. The experiment was carried out five times. To intentionally form a thrombus inside the pump, the activated clotting time was controlled to be less than 200 s. In every case, the phase difference increased by more than one degree after tens of minutes. Then, the pump was disassembled and a small amount of pump thrombosis was observed. We conclude that real-time diagnosis of pump thrombosis may be realized by measuring the phase difference without the need for additional sensors.

1H NMR-based metabolomic analysis coupled with reversed-phase solid-phase extraction for sample preparation of Saposhnikovia roots and related crude drugs.

1H NMR-based metabolomics has been applied in research on food, herbal medicine, and natural products. Although excellent results were reported, samples were directly extracted with a deuterated solvent (e.g., methanol-d4 or D2O) in most reports. As primary metabolites account for most of the results, data for secondary metabolites are partially reflected. Consequently, secondary metabolites tend to be excluded from factor loading analysis, serving as a significant unfavorable feature of 1H NMR-based metabolomics when investigating biologically active or functional components in natural products and health foods. Reversed-phase solid-phase extraction column (RP-SPEC) was applied for sample preparation in 1H NMR-based metabolomics to overcome this feature. The methanol extract from Saposhnikoviae radix (SR), an important crude drug, was fractionated with RP-SPEC into 5% methanol-eluting fractions, and the remaining fraction was collected. Each fraction was subjected to 1H NMR-based metabolomics and compared to results from conventional 1H NMR-based metabolomics. Based on principal component analysis (PCA) and partial least squares projections to latent structures discriminant analysis (PLS-DA), the 5% methanol fraction and conventional method reflected the amount of saccharides such as sucrose on the PC1/PLS1 axes, and wild and cultivated samples were discriminated along those axes. The remaining fraction clearly distinguished SR from Peucedanum ledebourielloides root. The compounds responsible for this discrimination were deemed falcarindiol derivatives and other unidentified secondary metabolites from the s-plot on PLS-DA. The secondary metabolites from original plants were, therefore, presumed to be concentrated in the remaining fraction by RP-SPEC treatment and strongly reflected the species differences. The developed series is considered effective to perform quality evaluation of crude drugs and natural products.

Metabolomic profiling of Saposhnikoviae Radix from Mongolia by LC-IT-TOF-MS/MS and multivariate statistical analysis.

Saposhnikoviae Radix (SR) is a commonly used crude drug that is obtained from the root and rhizome of Saposhnikovia divaricata which is distributed throughout China, Korea, Mongolia, and Russia. To evaluate the quality of Mongolian S. divaricata, metabolomic profiling of 43 plant specimens from different regions of Mongolia, as well as 8 SR samples and 2 plant specimens from China, were conducted by liquid chromatography-ion-trap-time-of-flight-mass spectrometer (LC-IT-TOF-MS). LC-MS profiles of the specimens showed uniformity and 30 compounds were tentatively identified, including 13 chromones and 17 coumarins. Among them, 16 compounds were isolated and unambiguously verified by comparing them with the spectroscopic data of standard compounds. Orthogonal partial least squares-discriminant analysis (OPLS-DA) based on LC-MS data from 7 Mongolian specimens and 8 Chinese SR samples as well as 2 plant specimens revealed that these 2 groups were clearly distinguishable and that Mongolian specimens were characterized by an abundance of prim-O-glucosylcimifugin (1). Moreover, the OPLS-DA of the Mongolian specimens showed that they can be discriminated by their growing regions based on the content of 8 chromones. The total content of dihydrofurochromones 1-3 was relatively higher in the specimens from Khalkhgol in the far eastern part of Mongolia, while contents of 10, 11, 15, and 16 were higher in those from Holonbuir in the eastern part. Based on this research, the roots of S. divaricata from Mongolia have potential as a new resource of SR in Kampo medicine.

Analgesic Effects of Ephedra Herb Extract, Ephedrine Alkaloids-Free Ephedra Herb Extract, Ephedrine, and Pseudoephedrine on Formalin-Induced Pain.

The analgesic effect of Ephedra Herb (EH) is believed to be derived from the anti-inflammatory action of pseudoephedrine (Pse). We recently reported that ephedrine alkaloids-free EH extract (EFE) attenuates formalin-induced pain to the same level as that achieved by EH extract (EHE), which suggests that the analgesic effect of EH may not be due to ephedrine alkaloids (EAs). To examine the contribution of EAs to the analgesic effect of EH, mice were injected with formalin to induce a biphasic pain reaction (first phase, 0-5 min; second phase, 10-45 min) at various time points after oral administration of the following test drugs: ephedrine (Eph), Pse, "authentic" EHE from Tsumura & Co. (EHE-Ts), EFE, and EHE that was used as the source of EFE (EHE-To). Biphasic pain was suppressed at 30 min after administration of Eph, EHE-Ts, and EHE-To. At 6 h after administration of EFE, EHE-To, and Pse-and at 4 to 6 h after administration of EHE-Ts-only second-phase pain was suppressed; however, the effect of Pse at 6 h was not significant. These results suggested that EHE has a biphasic analgesic effect against biphasic formalin-induced pain: in the first phase of analgesia (30 min after administration), biphasic pain is suppressed by Eph; in the second phase of analgesia (4-6 h after administration), second-phase pain is alleviated by constituents other than EAs, although Pse may partially contribute to the relief of second-phase pain.

High-Resolution Liquid Chromatography-Mass Spectrometry-Based Metabolomic Discrimination of Citrus-Type Crude Drugs and Comparison with Nuclear Magnetic Resonance Spectroscopy-Based Metabolomics.

Five Citrus-type crude drugs (40 samples) were classified using liquid chromatography-mass spectrometry (LC-MS)-based metabolomics. The following six flavonoid derivatives were identified as contributors from the loading plots of multivariate analysis: naringin (1), neohesperidin (2), neoeriocitrin (3), narirutin (9), hesperidin (10), and 3,5,6,7,8,3',4'-heptamethoxyflavone (12). Three coumarin derivatives, namely, meranzin (6), meranzin hydrate (7), and meranzin glucoside (8), were also identified as contributors. Furthermore, compared with our previous studies on proton (1H) and 13C NMR spectroscopy-based metabolomics, the present study revealed that the Citrus-type crude drugs were distinguished with the same pattern; however, the contributors differed between the 1H and 13C NMR spectroscopy-based metabolomics. The high dynamic range of NMR spectroscopy provided broad coverage of the metabolomes including the primary and secondary metabolites. However, LC-MS appeared to be superior in detecting secondary metabolites with high sensitivity, some of which occurred in quantities that were undetectable using NMR spectroscopy.

Measuring real-time blood viscosity with a ventricular assist device.

Ventricular assist devices assist in blood circulation and form a crucial component of artificial hearts. While it is important to measure parameters such as the flow rate, pressure head and viscosity of the blood, implanting additional devices to do such measurements is inadvisable. To this end, we demonstrate the adaptation of a ventricular assist device for the purpose of measuring blood viscosity. Such an approach eliminates the need for additional dedicated viscometers in artificial hearts. In the proposed method, the blood viscosity is measured by applying radial vibrational excitation to the impeller in a ventricular assist device using its magnetic levitation system. During the measurement, blood is exposed to a combination of a low shear rate (≈100/s) generated by the radial vibration of the impeller and a high shear rate (>10,000/s) generated by the impeller's rotation. The apparent viscosity of blood depends on the shear rate, so we determined which shear rate was the dominant one in the proposed method. The measurement results showed that the viscosity measured by the proposed method was in good agreement with the reference viscosity measured with a high shear rate. The mean absolute deviation in the measurements using the proposed method and those obtained using a concentric cylindrical viscometer at a high shear rate was 0.12 mPa s for four samples of porcine blood, with viscosities ranging from 2.32 to 2.75 mPa s.

Development of an Intelligent Ventricular Assist Device with a Function of Sensorless Thrombus Detection.

Thrombus is one of the major problems in ventricular assist devices (VADs). However, method for detecting thrombus in early stage has not been established yet. In this study, we propose an intelligent function that the VAD itself can detect thrombus automatically and alert it to medical staffs. In the proposed method, thrombus formation inside a blood pump is detected by monitoring blood viscosity. This viscosity measurement is performed by using magnetic levitation system for the impeller. Hence, it can be implemented without any additional sensors or mechanisms in principle. For verification of the method, at first, we visualized inside of the pump during thrombus formation with measuring blood viscosity by using erythrocytes removed porcine blood. The result showed that the viscosity of the blood increased as blood coagulation progressed. Then, we conducted in vitro principle verification experiments with three different whole porcine blood. In all experiments, the measured blood viscosity increased and small thrombus was observed inside the pump. From these results, we confirmed that the proposed method has a possibility to detect and predict the thrombus in early stage.

13C-NMR-based metabolic fingerprinting of Citrus-type crude drugs.

Five Citrus-type crude drugs (40 samples) were classified using 13C-NMR spectra-based metabolomics. The following eight metabolites were identified from the loading plots of multivariate analysis of the 13C-NMR spectra; naringin, neohesperidin, narirutin, synephrine, sucrose, α-glucose, ß-glucose, and limonene. 13C-NMR spectra-based metabolic fingerprinting is a promising strategy for classifying crude drugs.

Mechanism for Decreased Gene Expression of ß4-Galactosyltransferase 5 upon Differentiation of 3T3-L1 Mouse Preadipocytes to Adipocytes.

Upon differentiation of cells, remarkable changes in the structures of glycans linked to lipids on cell surface have been observed. Lactosylceramide (Lac-Cer) serves as a common precursor for a series of glycosphingolipids with diverse structures. In the present study, we examined the underlying mechanism for the biosynthesis of Lac-Cer upon differentiation of 3T3-L1 mouse preadipocytes to adipocytes. TLC analysis showed that the amounts of Lac-Cer decrease in 3T3-L1 adipocytes compared to 3T3-L1 preadipocytes. In accordance with this change, the gene expression level of ß4-galactosyltransferase (ß4GalT) 5, which was identified as Lac-Cer synthase, decreased drastically upon differentiation of 3T3-L1 preadipocytes. The analysis of the transcriptional mechanism of the ß4GalT5 gene demonstrated that the core promoter region is identified between nucleotides -299 and -1 relative to the translational start site. During adipocyte differentiation, the expression levels and promoter activities of the ß4GalT5 gene decreased dramatically. Since the Specificity protein 1 (Sp1)-binding sites in the promoter region were critical for the promoter activity, it is suggested that Sp1 plays an important role for the expression of the ß4GalT5 gene in 3T3-L1 cells. The gene and protein expression of Sp1 decreased significantly upon differentiation of 3T3-L1 preadipocytes. Taken together, the present study suggest that the expression of the ß4GalT5 gene decreases through reduced expression of the Sp1 gene and protein upon differentiation of 3T3-L1 peradipocytes to adipocytes, which may lead to the decreased amounts of Lac-Cer in 3T3-L1 adipocytes.

Ephedrine Alkaloids-Free Ephedra Herb Extract, EFE, Has No Adverse Effects Such as Excitation, Insomnia, and Arrhythmias.

Ephedrine alkaloids-free Ephedra Herb extract (EFE) has been developed to eliminate the adverse effects caused by ephedrine alkaloid-induced sympathetic hyperactivation. Previously, we reported that EFE possesses analgesic, anti-influenza, and cancer metastatic inhibitory effects at comparable levels to that of Ephedra Herb extract (EHE). However, it has not yet been demonstrated that EFE is free from the known side effects of EHE, such as excitation, insomnia, and arrhythmias. In this study, the incidence of these adverse effects was compared between mice administered EHE and those administered EFE. Increased locomotor activity in an open-field test, reduced immobility times in a forced swim test, and reduced sleep times in a pentobarbital-induced sleep test were observed in EHE-treated mice, when compared to the corresponding values in vehicle-treated mice. In contrast, EFE had no obvious effects in these tests. In electrocardiograms, atrial fibrillation (i.e., irregular heart rhythm, absence of P waves, and appearance of f waves) was observed in the EHE-treated mice. It was suggested that this atrial fibrillation was induced by stimulation of adrenaline ß1 receptors, but not by hypokalemia. However, EFE did not affect cardiac electrophysiology. These results suggest that the abovementioned side effects are caused by ephedrine alkaloids in EHE, and that EFE is free from these adverse effects, such as excitation, insomnia, and arrhythmias. Thus, EFE is a promising new botanical drug with few adverse effects.