Hiroaki Takemoto

Evaluation of volatile components from spikenard : valerena-4,7(11)-diene is a highly active sedative compound

Valerena-4,7(11)-diene and β-maaliene were isolated from spikenard for the first time, and the effects of inhaling these compounds were investigated. Both compounds reduced the locomotor activity of mice dose-dependently, even at a low dose. Valerena-4,7(11)-diene had a particularly profound effect, with the strongest sedative activity observed at a dose of 0.06%. Caffeine-treated mice that showed an area under the curve (AUC) for locomotor activity that was double that of controls were calmed to normal levels by administration of valerena-4,7(11)-diene. The continuous sleep time of pentobarbital-treated mice was prolonged by about 2.7 times with valerena-4,7(11)-diene, an effect similar to that of chlorpromazine administered orally.

Induction of the UDP-Glucuronosyltransferase 1A1 during the Perinatal Period Can Cause Neurodevelopmental Toxicity.

Anticonvulsants can increase the risk of developing neurotoxicity in infants; however, the underlying mechanism has not been elucidated to date. Thyroxine [3,5,3′,5′-l-tetraiodothyronine (T4)] plays crucial roles in the development of the central nervous system. In this study, we hypothesized that induction of UDP-glucuronosyltransferase 1A1 (UGT1A1)—an enzyme involved in the metabolism of T4—by anticonvulsants would reduce serum T4 levels and cause neurodevelopmental toxicity. Exposure of mice to phenytoin during both the prenatal and postnatal periods significantly induced UGT1A1 and decreased serum T4 levels on postnatal day 14. In the phenytoin-treated mice, the mRNA levels of synaptophysin and synapsin I in the hippocampus were lower than those in the control mice. The thickness of the external granule cell layer was greater in phenytoin-treated mice, indicating that induction of UGT1A1 during the perinatal period caused neurodevelopmental disorders. Exposure to phenytoin during only the postnatal period also caused these neurodevelopmental disorders. A T4 replacement attenuated the increase in thickness of the external granule cell layer, indicating that the reduced T4 was specifically associated with the phenytoin-induced neurodevelopmental disorder. In addition, these neurodevelopmental disorders were also found in the carbamazepine- and pregnenolone-16-α-carbonitrile–treated mice. Our study is the first to indicate that UGT1A1 can control neurodevelopment by regulating serum T4 levels.

Theaflavin Synthesized in a Selective, Domino-Type, One-Pot Enzymatic Biotransformation Method with Camellia sinensis Cell Culture Inhibits Weight Gain and Fat Accumulation to High-Fat Diet-Induced Obese Mice

The polyphenolic compound theaflavin, which is the main red pigment present in black tea, is reported to elicit various physiological effects. Because of the extremely low concentration of theaflavin present in black tea, its extraction from black tea leaves in quantities sufficient for use in medical studies has been difficult. We have developed a simple, inexpensive, selective, domino-type, one-pot enzymatic biotransformation method for the synthesis of theaflavin that is suitable for use in medical studies. Subsequent administration of this synthetic theaflavin to high-fat diet-induced obese mice inhibited both body weight gain and visceral fat accumulation, with no significant difference in the amount of faeces between the experimental and control mice.

Inhalation Administration of the Sesquiterpenoid Aristolen-1(10)-en-9-ol from Nardostachys chinensis Has a Sedative Effect via the GABAergic System

Spikenard, the dried roots of Nardostachys chinensis, contains sesquiterpenoids and is widely used as an herbal tranquilizer. We previously demonstrated that spikenard vapor showed a sedative effect when administered by inhalation, and we identified hydrocarbon sesquiterpenoids as active components. Here we investigated the other components that contribute to the effects of spikenard. Six oxygenated sesquiterpenoids, including aristolane- and guaiane-types, were isolated from an acetone extract of spikenard. We evaluated the sedative activities of these oxygenated compounds using an inhalation administration method in a caffeine-treated excitatory mouse model. We identified aristolen-1(10)-en-9-ol and patchouli alcohol as highly effective sedative components. These compounds inhibited locomotion in mice by approximately 60% at a dose of 300 µg/cage. In addition, aristolen-1(10)-en-9-ol prolonged pentobarbital-induced sleep to the same extent as 1 mg/kg diazepam. This effect completely disappeared with the administration of the GABAA-benzodiazepine receptor antagonist flumazenil (3 mg/kg), suggesting that the sedative effect of aristolen-1(10)-en-9-ol is expressed via the GABAergic system. Furthermore, differently from diazepam, inhalation of aristolen-1(10)-en-9-ol for 1 h did not affect the motor coordination in the rota-rod test. In the present study, we identified active components and provided evidence supporting the traditional sedative use of spikenard. Our research suggests that aristolen-1(10)-en-9-ol may be an effective aromatherapy, providing mild sedation.

Cytotoxic activity of two natural sesquiterpene lactones, isobutyroylplenolin and arnicolide D, on human colon cancer cell line HT-29

In this study, we found that two sesquiterpene lactones, isobutyroylplenolin and arnicolide D, from Centipeda minima L. (Compositae) exerted stronger cytotoxic activity than cisplatin on the human colon carcinoma HT-29 cell line. Furthermore, the cytotoxicity of these two compounds on normal cells was weaker than that of cisplatin. Treatment with isobutyroylplenolin and arnicolide D increased the levels of intracellular reactive oxygen species and decreased the levels of nuclear factor-κB protein, resulting in cell cycle arrest in G1 phase and apoptosis. We also discuss the difference in structure and activity between these two compounds.

Synthesis of Theaflavins and Their Functions

Numerous epidemiological and interventional clinical studies have consistently reported that black tea is good for human health. The polyphenolic compound, theaflavin, and its galloyl esters (theaflavins) are the primary red pigments in black tea that possess several health benefits, including fat-reducing and glucose-lowering capabilities and lifestyle-related disease prevention related to anti-obesity, anticancer, anti-atherosclerotic, anti-inflammatory, antiviral, antibacterial, anti-osteoporotic, and anti-dental caries properties. These compounds are produced by key enzymes, such as polyphenol oxidase and peroxidase, from parent green tea catechins present in fresh green tea leaves during the production of black tea leaves or the fermentation of green tea. However, theaflavins are only present in low concentrations in black tea; thus, their extraction from black tea leaves at sufficient levels for use in medical studies has been difficult. To circumvent this issue, different procedures for the synthesis of theaflavins using chemical oxidizing reagents or enzymes have been studied; however, low yields have limited their utility. Recently, however, several biosynthetic methods have been developed for the mass production of theaflavins. Using these methods, the physiological functions of theaflavins in lifestyle-related diseases in mice and humans have also been studied. In this review, we present the synthesis of theaflavins and their health benefits.

Two flavone C -glycosides as quality control markers for the manufacturing process of ephedrine alkaloids-free Ephedra Herb extract (EFE) as a crude drug preparation

As part of our continuing study of ephedrine alkaloids-free Ephedra Herb extract (EFE) in pursuit of its approval as a crude drug preparation, we identified two quantitative markers for the quality control of the manufacturing process of EFE and sought to establish cost-effective and simple methods for quantitative analyses. We analysed Ephedra Herb extracts grown in different habitats and collection years by liquid chromatography/high-resolution mass spectrometry (LC/HRMS) and detected two notable peaks common to each extract. These peaks were identified as vicenin-2 (1) and isovitexin 2″-O-rhamnoside (2). Quantitative analyses using the isocratic condition of LC/MS showed that the content percentages of 1 and 2 in EFE were 0.140–0.146% and 0.350–0.411%, respectively. We concluded that 1 and 2 were adequate quality control markers for quantitative analysis of EFE. Furthermore, we quantitatively analysed apigenin (3), an aglycon common to 1 and 2, and found that the conversion factors of 1 to 3 and 2 to 3 were 1.3 and 1.5, respectively. Therefore, we concluded that 3 was a secondary standard for quantifying the contents of 1 and 2 in EFE. A series of results obtained from this study will be valuable for the quality control of EFE.

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 β1 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.

A simple, enzymatic biotransformation method using fresh green tea leaves efficiently generates theaflavin-containing fermentation water that has potent physiological functions in mice and humans

The polyphenolic compound theaflavin, the main red pigment in black tea, possesses many beneficial properties, such as fat-reducing and glucose-lowering capabilities. To produce theaflavin-containing fermentation water on a large scale, we have developed a simple, inexpensive, and selective enzymatic biotransformation method to obtain sufficient levels from fresh green tea leaves. Subsequent administration of theaflavin-containing fermentation water to obese mice on a high-fat diet inhibited body weight gain, decreased casual blood glucose and fasting blood glucose levels, and lowered mesenteric and total fat composition. To note, there were no significant differences observed in food consumption between the experimental and control (water without theaflavin) mice groups. Next, we investigated the effect of this water on blood glucose levels in healthy humans and found that it significantly inhibited blood glucose levels. Thus, we showed that theaflavin-containing fermentation water can be efficiently generated from fresh green tea leaves and demonstrated its significantly potent effects in vivo.

Effects of L-citrulline diet on stress-induced cold hypersensitivity in mice.

Background: L-citrulline is an amino acid discovered in watermelon (Citrullus lanatus, Cucurbitaceae) and is a known component of the nitric oxide (NO) cycle that plays an important role in adjusting blood circulation and supplying NO and a key component of the endothelium-derived relaxing factor. Objective: The objective of this study is to evaluate the effect of L-citrulline on a newly established stress-induced cold hypersensitivity mouse model. Materials and Methods: When normal mice were forced to swim in water at 25°C for 15 min, their core body temperature dropped to 28.9°C, and then quickly recovered to normal temperature after the mice were transferred to a dry cage at room temperature (25°C). A 1-h immobilization before swimming caused the core body temperature to drop to ca. 24.1°C (4.8°C lower than normal mice), and the speed of core body temperature recovery dropped to 57% of the normal control. We considered this delay in recovery from hypothermia to be a sign of stress-induced cold hypersensitivity. Similar cold hypersensitivity was induced by administration of 50 mM L-NG-nitroarginine methyl ester, a NO synthesis inhibitor. Results: In this study, we showed that recovery speed from the stress-induced hypothermia remarkably improved in mice fed a 1% L-citrulline-containing diet for 20 days. Furthermore, the nonfasting blood level of L-arginine and L-citrulline increased significantly in the L-citrulline diet group, and higher serum nitrogen oxide levels were observed during recovery from the cold. Conclusions: These results suggested that oral L-citrulline supplementation strengthens vascular endothelium function and attenuates stress-induced cold hypersensitivity by improving blood circulation.