Phylogenetic relationship of Glycyrrhiza lepidota, American licorice, in genus Glycyrrhiza based on rbcL sequences and chemical constituents

Two known saponins, licorice-saponin H2 and macedonoside A, were isolated from the stolons of Glycyrrhiza lepidota (American licorice) as major saponins. Since licorice-saponin H2 and macedonoside A are minor saponins isolated from the three glycyrrhizin-producing species (i.e. G. glabra, G. uralensis, G. inflata) and the three macedonoside C-producing species (i.e. G. macedonica, G. echinata, G. pallidiflora), respectively, the present study suggests that G. lepidota is an intermediate of both glycyrrhizin-producing and macedonoside C-producing species. The phylogenetic tree constructed from the nucleotide sequences of ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit gene (rbcL) of these seven Glycyrrhiza plants indicated that G. lepidota was separated from the other six Glycyrrhiza species, and this phylogenetic relationship was in accordance with their saponin compositions.     

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.     

Field survey of Glycyrrhiza plants in Central Asia (1). Characterization of G. uralensis,G. glabra and the putative intermediate collected in Kazakhstan

The characteristics of Glycyrrhiza plants from 12 collection sites in southeastern Kazakhstan were investigated. G. uralensis was observed at 9 of the sites from Almaty to Shu, and G. glabra was observed at 8 sites. At 4 sites near Shu, and 1 site near Almaty, G. glabra and G. uralensis grew together forming a mixed population, and intermediate-type plants between them were also observed at 3 sites. Although two nucleotide substitutions of the chloroplast rbcL gene were observed between G. uralensis and G. glabra, rbcL sequences of the intermediate-types were divided into G. uralensis-type (G-A type) and G. glabra-type (A-T type). HPLC analysis of the roots indicated that species-specific flavonoids, glabridin and glycycoumarin, were detected in the roots of G. glabra and G. uralensis, respectively, but neither flavonoid was detected in underground parts of the intermediate-types. HPLC analysis of their leaves indicated a significant difference among G. uralensis, G. glabra and the intermediate-type plants. Both G. glabra-specific and G. uralensis-specific compounds were detected in the leaves of the intermediate-type, thus suggesting that the intermediate plants are hybrids of G. glabra and G. uralensis.     

Phenolic constituents of licorice. IV. Correlation of phenolic constituents and licorice specimens from various sources, and inhibitory effects of licorice extracts on xanthine oxidase and monoamine oxidase

The roots and/or rhizomes of Glychyrrhiza uralensis, G. glabra and G. inflata, and commercial licorice specimens from various regions or countries were analyzed by high-performance liquid chromatography (HPLC), and classified into three types based on their phenolic constituents. i) Type A: The roots and rhizomes of G. uralensis, commercial licorice specimens from northwestern region of China (Seihoku-kanzo) and from northeastern region of China (Tohoku-kanzo) in Japanese markets, and also several licorice specimens from Chinese markets. They contain licopyranocoumarin (6), glycycoumarin (7) and/or licocoumarone (8), which were not found in G. glabra and G. inflata. ii) Type B: The root and rhizome of G. glabra, and the licorice specimens imported from the Soviet Union and Afghanistan. They contain glabridin (9) and glabrene (10), which were not found in the samples of the other two Glycyrrhiza species. A root sample of Glycyrrhiza species from Turkey also contains 9 and 10. iii) Type C: The root sample of G. inflata. They contain licochalcones A (11) and B (12), which were not found in the samples of the other two Glycyrrhiza species. Commercial licorice specimens obtained in Japan, which were imported from Sinkiang of China (Shinkyo-kanzo), and some licorice specimens obtained from Chinese markets, have also been found to contain 11 and 12. The phenolics 6-12, characteristic constituents of types A, B or C, were not found in a specimen of cortex-free licorice from a Japanese market (kawasari-kanzo). Extracts of some licorice specimens of types A and B, and all of the licorice specimens of type C inhibited 40-56% of the xanthine oxidase activity at the concentration of 30 micrograms/ml. Extracts of some licorice specimens of types A and B also showed inhibitory effects on monoamine oxidase (44-64% inhibition, at the concentration of 30 micrograms/ml), which were slightly weaker than that of harmane hydrochloride.     

Analysis and comparison of Radix Glycyrrhizae (licorice) from Europe and China by capillary-zone electrophoresis (CZE)

A simple capillary-zone electrophoresis (CZE) method for the analysis of plant specimens, Glycyrrhiza glabra L., G. uralensisFisch. and G. inflata Bat. (Leguminosae) as well as commercial licorices from Europe and China was developed. Contents of glycyrrhizin (GL), glycyrrhetic acid (GA), glabridin (GLAB), liquiritin (LQ) and licochalcone A (LC(A)) in ethanolic extracts were investigated. Optimum separation was achieved with sodium tetraborate buffer (pH 9.22; 70 mM); voltage, 25 kV. Recovery rate for GL was found to be 101.90+/-2.54%. Adequate correlation was observed between GL contents measured by CZE and HPLC (r=0.977). Advantages over conventional HPLC analysis of Glycyrrhiza species are short analysis time (<15 min), simple running buffer preparation and the none-use of organic solvents. Using the present CZE method, it was demonstrated that (1) G. glabra was distinguished from G. uralensis especially by phenolic compounds GLAB (G. glabra: 0.19+/-0.11%; n=53) and LQ (G. uralensis, 1.34+/-0.34%, n=10); (2) on average, GL contents were higher in Chinese commercial licorices; (3) relatively high LC(A) contents were especially detected in a Chinese commercial licorice (origin estimated as G. inflata); (4) Glycyrrhiza species were also distinguished by applying PCA on the basis of CZE peak area data of GL, GLAB, GA, LQ and LC(A); and (5) liquiritin apioside was found in all samples.     

Comparative study of Glycyrrhiza glabra L. and Glycyrrhiza echinata L. of different origin. Medicinal plant polysaccharides III

Comparative study was carried out among the polysaccharides of Chinese, Lithuanian and Hungarian origin Glycyrrhiza glabra L. as well as the Hungarian origin Glycyrrhiza echinata L. Monosaccharide composition, measured as alditol acetates, of Chinese and Hungarian G. glabra was very similar, but the Lithuanian G. glabra and the G. echinata were quite different. The monosaccharides of polysaccharides isolated from root and stem of G. echinata show significant differences (Table II.). All investigated samples contain glucuronic acid, the G. echinata contains also galacturonic acid (Table II.). Some fractions with higher uronic acid content and the acidic polysaccharides isolated from these fractions were studied and their monosaccharide composition was determined (Table III. and IV.).     

Pharmaceutical botanical studies on some Glycyrrhiza species

Some Glycyrrhiza species grown in several domestic research gardens of medicinal plants were collected by the Osaka University of Pharmaceutical Sciences and were cultivated to compare their morphological properties. HPLC profile analysis was performed and index compounds of MeOH extracts of aerial parts and EtOAc extracts of subterranean parts were determined. Glycyrrhizin contents and growth rates of the underground parts of some types of Glycyrrhiza uralensis and Glycyrrhiza glabra were compared and four excellent types were selected as candidates for cultivation. One of them was due to Kanzo-Yashiki (Enzan, Yamanashi prefecture), where G. uralensis was cultivated in the Edo period. Alkaloidal constituents of G. uralensis and G. glabra were also investigated and anabasine (an insecticide) and a new tricyclic alkaloid were obtained.     

药用甘草离体快速繁殖体系的建立

筛选合适的甘草快速繁殖技术体系,为甘草的大规模生产生物活性物质、基因工程和人工制种奠定基础。以乌拉尔甘草、黄甘草和光果甘草为供试材料,以其子叶茎段和侧芽茎段作外植体,对影响甘草快速繁殖形成和发生的因素进行研究。结果显示乌拉尔甘草诱导的丛生苗数较多,是3个供试材料中最好的;甘草适宜的外植体是子叶茎段。利用甘草子叶茎段和侧芽茎段作外植体进行快速繁殖,具有生长周期短,再生频率高,适于规模化工厂生产。     

Comparative analysis of ten strains of Glycyrrhiza uralensis cultivated in Japan

Comparative analysis of 10 strains of Glycyrrhiza uralensis cultivated in Kyoto, Japan, was undertaken to characterize their variations. Based on the chemical characteristics of their leaves and underground parts, the 10 strains were divided into two chemotypes, the China type and Kazakhstan type. The contents of licoleafol in the leaves of the China type (0-0.03% of dry weight) were lower than those of the Kazakhstan type (0.05-1.16% of dry weight). In addition, a China type-specific unidentified compound was also detected in the leaves of China-type plants. Glycyrrhizin contents in the underground parts of the China type (2.08-5.12% of dry weight) were relatively higher than those of the Kazakhstan type (0.75-2.55% of dry weight). Contents of glycycoumarin, a species-specific flavonoid of G. uralensis, in the underground parts of China-type plants (0.07-0.28% of dry weight) were higher than those of Kazakhstan-type plants (0.01-0.08% of dry weight). These 10 strains were also divided into two genotypes, the GA type and AT type, based on their chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit gene (rbcL) sequences, although there was no correlation between the chemotype and the rbcL genotype.     

Metabolic profiling of roots of liquorice (Glycyrrhiza glabra) from different geographical areas by ESI/MS/MS and determination of major metabolites by LC-ESI/MS and LC-ESI/MS/MS

Liquid chromatography electrospray mass spectrometry (LC-ESI/MS) has been applied to the full characterization of saponins and phenolics in hydroalcoholic extracts of roots of liquorice (Glycyrrhiza glabra). Relative quantitative analyses of the samples with respect to the phenolic constituents and to a group of saponins related to glycyrrhizic acid were performed using LC-ESI/MS. For the saponin constituents, full scan LC-MS/MS fragmentation of the protonated (positive ion mode) or deprotonated (negative ion mode) molecular species generated diagnostic fragment ions that provided information concerning the triterpene skeleton and the number and nature of the substituents. On the basis of the specific fragmentation of glycyrrhizic acid, an LC-MS/MS method was developed in order to quantify the analyte in the liquorice root samples. Chinese G. glabra roots contained the highest levels of glycyrrhizic acid, followed by those from Italy (Calabria).     

Pharmaceutical evaluation of cultivated Glycyrrhiza uralensis roots in comparison of their antispasmodic activity and glycycoumarin contents with those of licorice

In China, the collection of wild Glycyrrhiza uralensis, one of the raw materials of Chinese licorice, has been restricted to prevent desertification. To compensate for the reduced supply of wild Glycyrrhiza plants, cultivation programs of G. uralensis have been initiated in eastern Inner Mongolia. The goal of the present study was to compare the chemical and pharmacological properties of cultivated G. uralensis roots to those of licorice prepared from wild Glycyrrhiza plants. The antispasmodic effect of boiled water extract of 4-year-old cultivated G. uralensis roots and licorice on carbachol-induced contraction in mice jejunum was similar (ED(50): 134+/-21 microg/ml vs. 134+/-16 microg/ml). In addition, glycycoumarin content, which is an antispasmodic and species-specific ingredient of G. uralensis, was similar when comparing the boiled water extracts of 4-year-old cultivated roots and licorice (0.10+/-0.02% vs. 0.10+/-0.06%). These data suggest that cultivated G. uralensis roots may be an adequate replacement for the generation of licorice in the context of the restriction of wild Glycyrrhiza plant collection.     

光果甘草三萜皂苷类化学成分研究

采用聚酰胺—大孔树脂柱色谱、ODS中压柱色谱以及半制备液相色谱分离技术,对光果甘草(Glycyrrhiza glabra L.)水提取物中三萜皂苷类成分进行分离纯化,共获得10个三萜皂苷类化合物,依据理化性质及NMR、MS波谱数据鉴定化合物结构,分别鉴定为3β-O-[β-D-glucuronpyranosyl-(1→2)-β-D-glucuronpyranosyl]-30β-O-β-Dglucuronpyranosyl-oleanane-11-oxo-12(13)-ene (1)、3β-O-[β-D-glucuronpyranosyl-(1→2)-β-D-glucuronpyranosyl]-30β-O-α-L-rhamnopyranosyl-oleanane-11-oxo-12(13)-en-22β,30-diol (2)、uralsaponin C (3)、licorice-saponin A3 (4)、licoricesaponin P2 (5)、22β-acetoxyl-glycyrrhizin (6)、macedonoside A (7)、29-hydroxyl-glycyr...     

甘草鲨烯合成酶基因的分离及植物表达载体的构建

采用RT-PCR技术从乌拉尔甘草(Glycyrrhiza uralensis)中克隆甘草鲨烯合成酶(Squalene Syn-thase,SS)基因,并通过酶切连接的方法构建相关植物表达载体。结果表明,两个SS基因编码区长分别为1242bp、1239bp,分别编码412、413个氨基酸残基的多肽,与Hayashi等报道的光果甘草两个SS基因(GgSQS1和GgSQS2)同源性高达98%,分别命名为GuSQS1和GuSQS2(GenBank登录号分别为:AM182329,AM182330);植物表达载体构建的鉴定结果表明,已将GuSQS1和GuSQS2序列分别正向插入到双T-DNA表达载体中的CaMV 35S启动子和NOS终止子之间,重组质粒分别命名为130/35S-GuSQS1和130/35S-GuSQS2,为今后的次生代谢基因工程研究奠定基础。     

甘草挥发性成分GC-MS分析

目的对甘草挥发性成分进行研究。方法用水蒸汽蒸馏法提取甘草挥发性成分并采用气相色谱-质谱技术对其进行分析分离、鉴定。结果初步分离出40余个峰,鉴定出32种成分,用峰面积归一化法确定其相对含量。在被测物质中,醇、酚、酯类化合物有2,6-双(1,1-二甲基乙基)-4-甲基苯酚(20.16%)、邻苯二甲酸二甲酯(6.82%)等8种;酸类化合物有正十六酸(8.33%)、正十八酸(4.68%)等8种;烃类化合物有正十九烷(4.71%)、正十八炕(4.16%)、正十七烷(4.02%)等14种;还有2个酮类化合物。结论通过对甘草挥发性成分的研究,为甘草资源的进一步开发利用提供实验依据。     

后莫紫檀素、美迪紫檀素对人肝癌细胞抑制作用的研究

观察刺果甘草活性成分后莫紫檀素 (homopterocarpin)、美迪紫檀素 (medicarpin)的体外抗癌活性 ,通过人肝癌细胞 (Hep 2 )进行体外实验 ,结果表明它们均有抑制和杀灭人肝癌细胞的作用 ,并且美迪紫檀素的作用强于后莫紫檀素。此 2个单体化合物是刺果甘草的活性成分     

Variation of glycyrrhizin and liquiritin contents within a population of 5-year-old licorice (Glycyrrhiza uralensis) plants cultivated under the same conditions

Cultivated licorice plants (Glycyrrhiza uralensis FISCH.) contain smaller amounts of the triterpene saponin glycyrrhizin than wild licorice plants. To resolve this problem and to breed strains with high-glycyrrhizin content we determined the glycyrrhizin content of 100 samples of G. uralensis that were propagated from seed and grown under the same conditions in the field for 5 years. There was a 10.2-fold variation in glycyrrhizin content among these plants, ranging from 0.46 to 4.67% (average 2.11±0.90%). There was also a wide variation in liquiritin content, ranging from 0.11 to 2.65% (average 1.00±0.49%). The glycyrrhizin content was positively correlated with that of liquiritin in the taproots (r(2)=0.5525). Our results indicate that there are various genetic strains for glycyrrhizin and liquiritin synthesis within a population of plants propagated from seed. The selected high-glycyrrhizin and liquiritin strains will be useful for licorice production and studies on biosynthetic analysis of glycyrrhizin and liquiritin.     

Obtaining Glycyrrhizic Acid and Its Practically Useful Salts from a Commercial Licorice Root Extract

We describe an optimized method for obtaining glycyrrhizic acid (GA, 90.5 ± 1.5%), which is the main triterpene glycoside of licorice root (Glycyrrhiza glabra L., Gl. uralensis Fisher) extract, and its trisodium and monoammonium salts from a commercial licorice root extract containing 20.0 ± 1.5% GA.__________Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 39, No. 2, pp. 30 – 33, February, 2005.     

Identification of Fritillaria pallidiflora using diagnostic PCR and PCR-RFLP based on nuclear ribosomal DNA internal transcribed spacer sequences

Fritillaria pallidiflora Schrenk (Liliaceae) is a commonly used antitussive herb. There are 9 species of Fritillaria recorded as herbal drugs in the Chinese Pharmacopoeia. The other species are often marketed as F. pallidiflora, and thus, the therapeutic effects of F. pallidiflora are not achieved. Methods to distinguish F. pallidiflora from the 8 other species of Fritillaria are limited by the current morphological and chemical methods. In this study, we report two molecular authentication methods based on the sequences of nuclear ribosomal DNA internal transcribed spacer (nrDNA ITS) regions. For diagnostic PCR, we designed a pair of species-specific primers to authenticate F. pallidiflora. The PCR program consisted of only two steps for every repeated cycle. For PCR-RFLP, we identified a distinctive site which can be recognized by the restriction endonuclease Eco81I in the nrDNA ITS1 region of F. pallidiflora. PCR-RFLP analysis was established to differentiate F. pallidiflora from the other species of Fritillaria. These methods provide effective and accurate identification of F. pallidiflora.     

Effect of hot water extract of a glycyrrhizin-deficient strain of Glycyrrhiza uralensis on contact hypersensitivity in mice

To evaluate the medicinal properties of a glycyrrhizin (GL)-deficient strain of Glycyrrhiza uralensis, we investigated the anti-allergic effect of the hot water extract obtained from its roots on contact hypersensitivity in mice, and compared it with that of the hot water extract of a commercial crude drug, Glycyrrhiza Radix. The hot water root extract of the GL-deficient strain contained glucoglycyrrhizin (GGL) and rhaoglucoglycyrrhizin (RGL) instead of GL, and it showed anti-allergic activity against contact hypersensitivity in a fashion similar to that of the crude drug extract. We further confirmed the presence of glycyrrhetinic acid (GA), a major metabolite of GL, in mice serum after oral administration of the hot water root extract of a GL-deficient strain. We demonstrated that GGL underwent hydrolysis by intestinal microflora of mice to form GA. These results suggest that a GL-deficient strain of G. uralensis is a useful medicinal resource since the glycosides of GA work in a fashion similar to that of GL when orally administered.