Species identification of licorice using nrDNA and cpDNA genetic markers.

For the accurate identification of medicinal licorice species, nucleotide sequences of four types of DNA regions were researched for 205 specimens, including three species used as licorice: Glycyrrhiza uralensis, Glycyrrhiza glabra, and Glycyrrhiza inflata. The four DNA regions were the internal transcribed spacer (ITS) on nuclear ribosomal DNA, the rbcL gene, the matK gene, and the trnH-psbA intergenic region on chloroplast DNA (cpDNA). Ten genotypes were consequently recognized as combinations of the sequence data obtained from the four DNA regions. Species-specific genotypes were defined from the frequency of the appearance of species in each genotype and from the phylogenetic relationships of the 10 genotypes. This revealed the possibility of identifying licorice species based on the 10 genotypes. Next, comparison of species identifications by each DNA region suggested that efficient identification of licorice species is possible using the genetic information obtained from the ITS and trnH-psbA intergenic region. Additionally, concerning the phylogenetic relationships of the Glycyrrhiza species used as licorice, it is suggested from the genetic information of the four types of DNA regions that G. glabra is more closely related to G. inflata than to G. uralensis. In the G. uralensis examined, four genotypes were recognized as intra specific variations. The appearance frequency of each genotype in G. uralensis differed according to the area in China. G. uralensis may have expanded its distribution areas from western to eastern China because many licorices with the phylogenetic ancestral genotype were observed in western areas, while many with the derivative genotype were observed in eastern areas.

Constituent properties of licorices derived from Glycyrrhiza uralensis, G. glabra, or G. inflata identified by genetic information.

Constituent properties of licorices derived from Glycyrrhiza uralensis, G. glabra, and G. inflata are revealed by comparing 117 of licorice identified using four genetic markers; internal tracscribed spacer (ITS) on nuclear ribosomal DNA, rbcL gene, matK gene, and trnH-trnK1 intergenic region on chloroplast DNA. Regarding six main constituents of licorice; glycyrrhizin, liquiritin, liquiritin apioside, isoliquiritin, isoliquiritin apioside, and liquiritigenin, the constituent property of G. glabra resembles to that of G. inflata. On the other hand, the constituent property of G. uralensis is not similar to that of G. glabra or G. inflata and is characterized by a wide content variation of the six constituents compared to those of G. glabra and/or G. inflata. The mean contents of liquiritin, isoliquiritin, or liquilitigenin in G. uralensis are significantly higher than those of G. glabra or G. inflata. Therefore, the licorice species should be selected depending on these constituent properties for the traditional Chinese medicines or the Japanese Kampo medicines. Additionally, glycycoumarin, glabridin, and licochalcone A were reconfirmed as the species-specific typical constituents of G. uralensis, G. glabra, and G. inflata respectively. Therefore, it is resulted that the determination of the three species-specific constituents may be useful for the species identification of licorice. However, since 6% of licorice examined and hybrids were exceptions to the rule, their genetic information is necessary for the accurate species identification of licorice.

Reticulate evolution and phylogeography in Asarum sect. Asiasarum (Aristolochiaceae) documented in internal transcribed spacer sequences (ITS) of nuclear ribosomal DNA.

Phylogenetic analyses were performed for the taxonomically complicated group, Asarum sect. Asiasarum, using internal transcribed spacers (ITS) of nuclear ribosomal DNA. Direct sequences for 99 samples of a total of 14 taxa and geographic races and cloning analyses for 32 of these samples provided new insights that extensive reticulate evolution has occurred in this group. Eight taxa had slight or no polymorphism of the ITS sequences. On the other hand, the other five taxa had polymorphic ITS sequences composed of two ribotypes that were completely the same or almost the same as the sequences recognized in the taxa with only slight or no polymorphism, and were probably of diploid hybrid origin and to have retained their parental ribotypes. In terms of biogeographic implications, at least four interactions including migration, hybridization, and introgression, were presumed between the Japanese Archipelago and the continents, two times via a southern route, from the Korean Peninsula, and two times via a northern route, from Sakhalin or directly from the Eurasian continent.

Identification of medicinal Atractylodes based on ITS sequences of nrDNA.

Dried rhizomes of five species of Atractylodes (A. japonica, A. macrocephala, A. lancea, A. chinensis, and A. koreana), Compositae, have been used as crude drugs mainly for the treatment of stomach disorders and for their diuretic properties in Chinese and Japanese traditional medicines. The identification of the botanical origins of these crude drugs is generally difficult from their morphological and chemical features only. In this study, for identification with more reliable, nuclear ribosomal DNA (nrDNA), internal transcribed spacer (ITS) regions of five species of medicinal Atractylodes were sequenced. As a result, specific ITS genotypes were recognized by each species. The four species (A. japonica, A. macrocephala, A. lancea, and A. chinensis) prescribed in Chinese and Japanese Pharmacopoeias as botanical origins of crude Atractylodes drugs could be distinguished by their ITS sequences because they had difference genotypes on the ITS sequences. However, the genotype of A. koreana was the same as that of A. chinensis. Additionally, hybrids between A. lancea and A. chinensis were also recognized as nucleotide additives on their ITS sequences. In this study, several morphological characteristics were researched by their genotype, too. As this result, the hybrids recognized from the genetic analysis had intermediate morphological characteristics between A. lancea and A. chinensis. It was also recognized that A. lancea and A. chinensis except for their hybrids were significant differences. It is therefore suggested that ITS sequences of nrDNA would be useful for the identification of the crude drugs derived from Atractylodes species and their interspecific hybridizations.

Glycycoumarin from Glycyrrhizae Radix acts as a potent antispasmodic through inhibition of phosphodiesterase 3.

Glycyrrhizae Radix is used to treat abdominal pain as a component of Shakuyaku-kanzo-to, a traditional Chinese medicine formulation. We aim at clarifying the antispasmodic principles of Glycyrrhizae Radix, and consequently isolated glycycoumarin as a potent relaxant on the carbamylcholine (CCh)-induced contraction of mouse jejunum. In this paper we investigated the effects and the action mechanism of glycycoumarin on the contraction of mouse jejunum. Glycycoumarin inhibited the contraction induced by various types of stimulants, such as CCh, KCl, BaCl(2), and A23187 (calcium ionophore III) with IC(50) values of 2.93+/-0.94 micromol/l (1.08+/-0.35 microg/ml), 2.59+/-0.58 micromol/l (0.95+/-0.29 microg/ml), 4.09+/-1.82 micromol/l (1.51+/-0.67 microg/ml) and 7.39+/-5.19 micromol/l (2.72+/-1.91 microg/ml), respectively, with a potency similar to that of papaverine (a representative antispasmodic for smooth muscle). Furthermore, pretreatment with glycycoumarin enhanced the relaxation induced by forskolin on CCh-evoked contraction, similar to that by pretreatment with IBMX, a non-specific inhibitor of phosphodiesterases (PDEs). Pretreatment with glycycoumarin also enhanced the relaxation effect of rolipram, a specific inhibitor of PDE isozyme 4, as pretreatment with milrinone, a specific inhibitor of isozyme 3, did. Moreover, the effect of glycycoumarin was associated with dose-dependent accumulation of cAMP, but not cGMP, in mouse jejunum. These results indicate that glycycoumarin has an inhibitory effect on smooth muscle contraction induced by various types of stimulants through the inhibition of PDEs, especially isozyme 3, followed by the accumulation of intracellular cAMP.

The influence of the sennosides on absorption of glycyrrhetic acid in rats.

In the course of our clinical studies of Kampo medicine (traditional Japanese medicines), we observed the pharmacokinetic interactions between two herbs. When Onpito (TJ-8117, Kampo medicine) containing licorice and rhubarb was administered orally to human subjects, we observed that the AUC(0-lim) and Cmax of glycyrrhetic acid (GA) in plasma were lower than those treated with other Kampo medicines containing licorice. In this study, we demonstrate the pharmacokinetic interactions of GA derived from glycyrrhizinic acid (GL) in licorice and anthraquinones derived from rhubarb. To our knowledge, this is the first report to investigate the pharmacokinetic interactions between two herbs. When GL was orally co-administrated to rats with a non-effective dose of sennoside A having purgative activity, the AUC(0-lim) and Cmax of GA decreased. In addition, sennoside A did not affect the metabolism of GL by the intestinal bacteria in vitro. In the examination using an in situ loop of rat colon, the remaining ratio of GA rose drastically by the co-administration of sennoside A, sennidin A and rhein. Observed inhibition activity of these anthraquinones on GA absorption depended on the concentration of the components added. The maximum inhibition ratio was approximately 75% by rhein, 60% by sennoside A and 25% by sennidin A. We conclude that the decrease of the pharmacokinetic parameters of GA in human plasma observed in the clinical study of TJ-8117 is attributable to an interactive action of absorption from the intestinal tract by anthraquinones contained in or derived from rhubarb.

Pharmacokinetics of TJ-8117 (Onpi-to), a drug for renal failure (I): Plasma concentration, distribution and excretion of [3H]-(-)epicatechin 3-O-gallate in rats and dogs.

TJ-8117 (Onpi-to) is an herbal medicine extracted from a mixture of five crude medicinals (Rhei Rhizoma, Glycyrrhizae Radix, Ginseng Radix, Zingiberis Rhizoma and Aconiti Tuber), which has been developed as a drug for chronic renal failure. (-)Epicatechin 3-O-gallate (ECG), one of the active components of TJ-8117, was labeled with tritium and added to TJ-8117. Pharmacokinetics in plasma, tissue distribution and excretion of radioactivity were investigated following a single oral administration of TJ-8117 containing [3H]ECG ([3H]TJ-8117) in rats and dogs. 1. Following oral administration of [3H]TJ-8117, radioactivity exhibited linear pharmacokinetics in Cmax. Linearity of AUC(0-72 h) was lost at the highest dose of [3H]TJ-8117. Cmax and AUC(0-72 h) were higher in female rats than in male rats, a finding which suggested a sex difference in rats. Plasma levels of radioactivity displayed curves with one peak in dogs, which suggested a species difference between rats and dogs. 2. No accumulation was observed in any tissues in male rats. 3. Within 168 h after administration of [3H]TJ-8117 to male rats, 18.7%, 84.1% and 0.9% of the dose was excreted in urine, feces and expired air, respectively. Data from bile-duct cannulated rats indicated that at least 18.4% of the dose was absorbed.

Permeability of the flavonoids liquiritigenin and its glycosides in licorice roots and davidigenin, a hydrogenated metabolite of liquiritigenin, using human intestinal cell line Caco-2.

The present investigation focused on the transepithelial flux of liquiritigenin (LG), davidigenin (DG), liquiritin (LQ), and liquiritin apioside (LA) using the human colonic cell line Caco-2 as a model of human intestinal absorption. Apparent permeability coefficients (Papp) for the apical to basolateral flux of LG and DG were (16.0 +/- 0.727) x 10(-6) cm/s and (18.2 +/- 1.67) x 10(-6) cm/s, respectively. These Papp were higher than that of the transcellular transport marker propranolol (13.5 +/- 0.34) x 10(-6) cm/s (P < 0.01). Papp for the apical to basolateral flux of LQ and LA were (0.26 +/- 0.12) x 10(-6) cm/s and (0.075 +/- 0.005) x 10(-6) cm/s, respectively. These Papp were lower than that of the paracellular transport marker mannitol (0.64 +/- 0.04) x 10(-6) cm/s (LG, P < 0.01; LA, P < 0.001). These data suggested excellent absorption of LG and DG through the human intestinal epithelial cell line. On the contrary, poor absorption of LQ and LA was expected due to the little transepithelial flux of these compounds in the human colonic cell line Caco-2.

Pharmacokinetics of (-)-epicatechin-3-O-gallate, an active component of Onpi-to, in rats.

(-)-Epicatechin-3-O-gallate (ECG), a component of Rhei Rhizoma, is one of the active components of Onpi-to, a herbal medicine composed of five crude drugs (Rhei Rhizome, Glycyrrhizae Radix, Ginseng Radix, Zingiberis Rhizoma and Aconiti Tuber), which has been used in patients with chronic renal failure. Pharmacokinetics of ECG was investigated in male rats employing an HPLC-electrochemical detection method. 1. Following oral administration of ECG, ECG plasma levels revealed curves characterized by peaks at 0.065, 0.063 and 0.085 h corresponding to dosages of 12.5, 25.0 and 50.0 mg/kg at mean concentrations of 49.62, 212.89 and 464.04 ng/ml, respectively. Plasma levels subsequently declined bi-exponentially. ECG demonstrated nonlinear pharmacokinetics in terms of C(max) and AUC(0-inf). 2. The absolute bioavailability values (F) were 1.02, 1.47 and 3.30% at doses of 12.5, 25.0, and 50.0 mg/kg, respectively. 3. Following intravenous injection of ECG, plasma levels of ECG decreased with the gamma-elimination half-life (t(1/2gamma)) of 4.03 h. 4. Following oral administration of ECG, urinary levels of ECG were lower than the quantitation limit. Moreover, cumulative excretion of the metabolites, delta-(3,4-dihydroxyphenyl)-gamma-valerolactone and delta-(3-methoxy-4-hydroxyphenyl)-gamma-valerolactone, was 2.45 and 0.23% of dose, respectively, up to 30 h after dosing.

Pharmacokinetics of rhein from Onpi-to, an Oriental herbal medicine, in rats.

Onpi-to, an herbal medicine composed of five crude drugs (Rhei Rhizoma, Glycyrrhizae Radix, Ginseng Radix, Zingiberis Rhizoma and Aconiti Tuber), was administered orally to rats. Onpi-to includes 1.240% of total potential rhein derived from sennoside A, sennoside B, rhein 8-O-glucopyranoside and rhein. Plasma, urinary and biliary levels of rhein were determined by an HPLC-UV method. The plasma levels displayed curves characterized by maximum peaks at 8.3+/-5.2 min, 8.3+/-5.2 min and 20.0+/-21.9 min following dosages of 125, 250 and 500 mg/kg with mean concentrations of 1302.5+/-926.4, 2973.6+/-684.3 and 3118.8+/-1701.2 ng/ml, respectively, followed by a subsequent decline. Area under the concentration-time curve (AUC)(0-48 h) at doses of 125, 250 and 500 mg/kg were 752.3+/-321.5, 2443.3+/-554.4 and 4443.2+/-2641.3 ng.h/ml, respectively. In female rats, rhein plasma levels showed curves which had a maximum peak at 45.0+/-16.4 min after a dosage of 250 mg/kg with mean concentration of 3058.0+/-1533.7 ng/ml, followed by a subsequent decline. AUC(0-48 h) was 5537.7+/-1876.0 ng.h/ml. The cumulative urinary excretion of rhein and of conjugated rhein was 3.14+/-1.56% and 38.21+/-18.87% of dose, respectively, 48 h after dosing at 500 mg/kg of Onpi-to in male rats. The cumulative biliary excretion of rhein was 1.34+/-0.44% of dose 48 h after dosing at 500 mg/kg of Onpi-to in male rats.