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.

Superior Efficacy of Lipid Emulsion Infusion Over Serum Alkalinization in Reversing Amitriptyline-Induced Cardiotoxicity in Guinea Pig.

BACKGROUND: Tricyclic antidepressants (TCAs) are a major cause of fatal drug poisoning due to their cardiotoxicity. Alkalinization by sodium bicarbonate (NaHCO3) administration, the first-line therapy for TCA-induced cardiotoxicity, can occasionally yield insufficient efficacy in severe cases. Because most TCAs are highly lipophilic, lipid emulsion may be more effective than alkalinization. However, it remains to be determined whether lipid emulsion is more beneficial than alkalinization in reversing amitriptyline-induced cardiotoxicity. METHODS: Hemodynamic variables were recorded from in vivo guinea pig models and Langendorff-perfused hearts. Whole-cell patch-clamp experiments were conducted on enzymatically isolated ventricular cardiomyocytes to record fast sodium currents (INa). Lipid solutions were prepared using 20% Intralipid. The pH of the alkaline solution was set at 7.55. We assessed the effect of lipid emulsion on reversing amitriptyline-induced cardiotoxicity, in vivo and in vitro, compared to alkalinization. The data were evaluated by Student t test, 1-way repeated-measures analysis of variance, or analysis of covariance (covariate = amitriptyline concentration); we considered data statistically significant when P < .05. RESULTS: In the in vivo model, intervention with lipids significantly reversed the amitriptyline-induced depression of mean arterial pressure and prolongation of QRS duration on electrocardiogram more than alkalinization (mean arterial pressure, mean difference [95% confidence interval]: 19.0 mm Hg [8.5-29.4]; QRS duration, mean difference [95% confidence interval] -12.0 milliseconds [-16.1 to -7.8]). In the Langendorff experiments, perfusion with 1% and 2% lipid solutions demonstrated significant recovery in left ventricular developed pressure (LVdevP), maximum change rate of increase of LVdevP (dP/dtmax) and rate-pressure product compared with alkaline solution (LVdevP [mm Hg], alkaline 57 ± 35, 1% lipid 94 ± 12, 2% lipid 110 ± 14; dP/dtmax [mm Hg/s], alkaline 748 ± 441, 1% lipid 1502 ± 334, 2% lipid 1753 ± 389; rate-pressure product [mm Hg·beats·minute], alkaline 11,214 ± 8272, 1% lipid 19,025 ± 8427, 2% lipid 25,261 ± 4803 with analysis of covariance). Furthermore, lipid solutions (0.5%-4%) resulted in greater recovery of hemodynamic parameters at 3 µM amitriptyline. Amitriptyline inhibited INa in a dose-dependent manner: the half-maximal inhibitory concentration (IC50) was 0.39 µM. The IC50 increased to 0.75 µM in the alkaline solution, 3.2 µM in 1% lipid solution, and 6.1 µM in 2% lipid solution. Furthermore, the lipid solution attenuated the use-dependent block of sodium channels by amitriptyline more than alkaline solution. On 30 consecutive pulses at 1 Hz, the current decreased to 50.1 ± 2.1, 60.3 ± 1.9, and 90.4% ± 1.8% in standard, alkaline, and 1% lipid solution, respectively. Even 0.5% lipid solution showed greater effects than the alkaline solution in all experiments. CONCLUSIONS: Lipid emulsion significantly suppressed amitriptyline-induced INa, inhibition, which was likely related to the marked improvement in hemodynamic status observed in vivo and in isolated perfused hearts. These results suggest the superiority of lipid emulsion as the first-line therapy for TCA-induced cardiotoxicity compared to alkalinization therapy.

The effect of lipid emulsion on intracellular bupivacaine as a mechanism of lipid resuscitation: an electrophysiological study using voltage-gated proton channels.

BACKGROUND: Lipid resuscitation has become a standard treatment for local anesthetic (LA) systemic toxicity, but its mechanisms remain to be fully elucidated. Although the partitioning effect is one of the proposed mechanisms, it is difficult to evaluate its impact independently from several other mechanisms or to examine the intracellular concentration of a LA, which is primarily responsible for LA systemic toxicity. We recently reported that LAs as weak bases reduced voltage-gated proton currents by increasing intracellular pH, which could be estimated from the reversal potentials of the channels (Vrev). Using this characteristic, we examined the partitioning effect in detail and showed its impact on lipid resuscitation. METHODS: A whole-cell voltage clamp technique was used to record proton channel currents in a rat microglial cell line (GMI-R1). We used Intralipid® 20% as lipid emulsion. The effects of lipid emulsion on the intracellular concentrations of LAs were evaluated by measuring the current amplitude and the Vrev. The intracellular concentrations of LAs were calculated by the Henderson-Hasselbalch equation, using estimated intracellular pH. To confirm the importance of partitioning, we separated lipid by centrifugation. Data are means ± SD unless otherwise stated. RESULTS: Bupivacaine (1 mM) decreased proton currents to 43% ± 10% of the control and shifted the Vrev to positive voltages (from -88.0 ± 4.1 to -76.0 ± 5.5 mV, n = 5 each, P = 0.02). An addition of the lipid emulsion recovered the currents to 79% ± 2% of the control and returned the Vrev toward the control value (to -86.0 ± 7.1 mV, n = 5, P = 0.03). Both recoveries of the current and Vrev in the centrifuged aqueous extract were almost the same as in the 4% lipid solution (-85.6 ± 4.9 mV, n = 5, P = 0.9, 95% confidence interval for difference = -9.3 to 8.6). When 1 mM bupivacaine was applied extracellularly, the intracellular concentration of the charged form of bupivacaine was estimated to reach about 18.1 ± 3.9 mM but decreased to 5.4 ± 1.8 mM by the 4% lipid solution. CONCLUSIONS: Here we quantitatively evaluated for the first time the partitioning effect of lipid emulsion therapy on the intracellular concentration of bupivacaine in real-time settings by analyzing behaviors of voltage-gated proton channels. Our results suggested that lipid emulsion markedly reduced the intracellular concentration of bupivacaine, which was mostly due to the partitioning effect. This could contribute to our understanding of the mechanisms underlying lipid resuscitation, especially the importance of the partitioning effect.