Evaluations of saponin properties of HPLC analysis of Platycodon grandiflorum A.DC

Platycodon root, one of the most important Chinese herbal medicines, has been used as an antiphlogistic, antitusivie, and expectorant agent since ancient times. In the Japanese Pharmacopoeia XIV this is listed as the root of Platycodon grandiflorum A. De Candolle (Campanulaceae) and called KIKYOU (Platycodi Radix) in Japanese. HPLC analysis showed that commercial samples of P. Radix all contained platycodins, and a total of 12 peaks were identified by co-HPLC analysis with authentic samples isolated earlier from this laboratory. The peak purity and identity were checked with a photodiode array detector. The contents of the major saponins, platycodins A, C, and D, were determined and the peak-area ratios of platycodins A, C, and D, were shown to be correlated with their sources of origin. Fourteen commercial samples of Platycodon root, the origin of which was Platycodon grandiflorum, were collected from China (5 samples), Korea (5 samples), and Japan (4 samples). The commercial samples from China, Korea, and Japan each gave a distinct HPLC pattern with peak-area ratio of platycodins A, C, and D, of 1:2:3 and 2:8:1, respectively. HPLC analysis showed that those on the Japanese market were either imported from China or Korea based upon their HPLC patterns.     

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.     

一测多评法同时测定桔梗中3种桔梗皂苷的含量

目的 探索一测多评法应用于同时测定桔梗中3种桔梗皂苷含量的可行性。方法 采用HPLC-ELSD为检测手段,考察桔梗提取物在不同色谱系统和不同色谱柱上的色谱行为,确定3种待测成分的色谱峰定位方法。以桔梗皂苷D为内标,建立其与桔梗皂苷D₃、桔梗皂苷E的相对校正因子;通过外标法测定桔梗皂苷D的含量,并由建立的相对校正因子计算出其他2种桔梗皂苷的含量;将计算的结果与外标法测定结果相比较,以验证“一测多评”法在桔梗质量评价中的可行性。结果 方法重现性和耐用性良好,用于10批桔梗药材的含量测定,计算结果与外标法测定结果无显著差异。结论 一测多评法可用于同时测定桔梗中多种皂苷的含量。     

Genetic and chemical comparison of Boi (<Emphasis Type="Italic">Sinomeni Caulis</Emphasis><Emphasis Type="Italic">et Rhizoma</Emphasis>) and Seifuto (<Emphasis Type="Italic">Caulis Sinomenii</Emphasis>)

Boi and its original plant Sinomenium acutum from Japan were compared with Seifuto and its botanical origins from China in terms of their internal transcribed spacer (ITS) sequences and major chemical components. Boi, Seifuto, and their botanical origins overall showed seven variable sites in the ITS sequence and six genotypes. Japanese S. acutum and Boi had one nucleotide variation at position 593 to show two genotypes (J1 and J2) and their heterozygote (J3). Seifuto samples and their botanical origins, S. acutum and S. acutum var. cinereum from China, showed three genotypes (C1, C2, and C3), which did not agree with the botanical classification, indicating that they cannot be distinguished according to their ITS sequences. All Seifuto samples from Henan market showed the same ITS genotype (C1). The Japanese and Chinese genotypes differed in the nucleotide position 424, which can be used to distinguish the country of origin of these materials. In the HPLC analysis of six major components, sinomenine (1), magnoflorine (2), menisperine (3), 6-O-methyllaudanosoline glucoside (4), liriodendrin (5), and menisdaurin (6), all were detected in Boi, whereas five (all except for menisdaurin) were detected in Seifuto. The main component in the rhizome of Seifuto was sinomenine, whereas magnoflorine was the main component in the rhizome and the climbing stem of Boi. The content of sinomenine in Seifuto was almost twice that in Boi. Although the individual content of alkaloids 1–4 differed between Boi and Seifuto, the total contents of these alkaloids were comparable between them both in the climbing stem and rhizome.     

中、日、韩三国当归中阿魏酸、藁本内酯及总多糖的含量比较

目的:探讨中、日、韩三国当归的内在质量。方法:利用高效液相色谱法和比色法,测定产于中国、日本和韩国的3种当归中阿魏酸、藁本内酯和总多糖的含量。结果:中国产当归中阿魏酸、藁本内酯和总多糖的含量明显高于日、韩当归。结论:从阿魏酸、藁本内酯和总多糖的含量看,中国产当归质量较好。     

桔梗生长年限和采收期与质量的相关性研究

目的确定桔梗最佳生长年限与最佳采收期。方法蒽酮比色法测定多糖含量;HPLC测定桔梗皂苷D含量;改进的总苷称重法测定总皂苷含量。结果以两年生,9月下旬至11月上旬采收的桔梗质量最佳。结论三种指标具有代表性,能够较全面反映药材质量。     

A comparative study on commercial samples of the roots of Paeonia vitchii and P. lactiflora

A total of 37 commercial samples of paeoniae radix (Paeonia lactiflora Pall. = P. albiflora Pall, and P. vitchii Lynch), were collected from Taiwan's herbal market. The contents of eight constituents (gallic acid, oxypaeoniflorin, albiflorin, paeoniflorin, benzoic acid, pentagalloylglucose, paeonol, and benzyoylalbiflorin) in these samples were determined by high-performance liquid chromatography. It was found that constituent contents in P. lactiflora samples were generally higher than in P. vitchii samples except for the albiflorin/oxypaeoniflorin ratio and paeonol content. The major compounds of P. lactiflora were paeoniflorin, albiflorin, and pentagalloylglucose, while those in P. vitchii were paeoniflorin, pentagalloylglucose, and oxypaeoniflorin. The peak-area ratio of albiflorin/oxypaeoniflorin was higher than unity in P. lactiflora samples but lower than unity in P. vitchii, and the ratio can be used to differentiate the origin of Paeonia species clearly. In addition, the former had higher contents in the core wood but the latter in the cortex.     

Population differences in major functional polymorphisms of pharmacokinetics/pharmacodynamics-related genes in Eastern Asians and Europeans: implications in the clinical trials for novel drug development

Drug lag, recently discussed extensively in Japan, can be divided into two phases: clinical development time and application review time. The former factor is still an important problem that might be improved by promoting multi-regional clinical trials and considering the results from other similar populations with Japanese, such as Koreans and Chinese. In this review, we compare the allelic or genotype frequencies of 30 relatively common functional alleles mainly between Eastern Asians and Europeans as well as among 3 major populations in Eastern Asian countries, Japan, Korea, and China, in 12 pharmacokinetics (PK)/pharmacodynamics (PD)-related genes; CYP2C9 (*2 and *3), CYP2C19 (*2, *3 and *17), 13 CYP2D6 haplotypes including *4, *5 and *10, CYP3A5 (*3), UGT1A1 (*28 and *6), NAT2 (*5, *6 and *7), GSTM1 and GSTT1 null genotypes, SLCO1B1 521T>C, ABCG2 421C>A, and HLA-A*31:01 and HLA-B*58:01. In this review, differences in allele frequencies (AFs) or genotype frequencies (GFs) less than 0.1 (in the cases of highest AF (GF) ≥0.1) or less than 0.05 (in the cases of lowest AF (GF) <0.1) were regarded as similar. Between Eastern Asians and Europeans, AFs (or GFs) are regarded as being different for many alleles such as CYP2C9 (*2), CYP2C19 (*2, *3 and *17), CYP2D6 (*4 and *10), CYP3A5 (*3), UGT1A1 (*28 and *6), NAT2 (*5*7), GSTT1 null and ABCG2 421C>A. Among the 3 Eastern Asian populations, however, only AFs of CYP2C19*3, CYP2D6*10, HLA-A*31:01 and HLA-B*58:01 are regarded as dissimilar. For CYP2C19*3, the total functional impact on CYP2C19 could be small if the frequencies of the two null alleles CYP2C19*2 and *3 are combined. Regarding CYP2D6*10, frequency difference over 0.1 is observed only between Japanese and Chinese (0.147). Although environmental factors should be considered for PK/PD differences, we could propose that among Japan, Korea, and China, genetic differences are very small for the analyzed common PK-related gene polymorphisms. On the other hand, AFs of the two HLA alleles important for cutaneous adverse drug reactions are diverse even among Eastern Asians and thus should be taken into account.     

In vitro inhibitory effect of triterpenoidal saponins from platycodi radix on pancreatic lipase

In the process of investigating anti-obesity effect of Platycodi Radix, we found that aqueous extract of Platycodi Radix might inhibit intestinal absorption of dietary fat by inhibiting pancreatic lipase (PL) activity. In order to clarify the anti-obesity mechanism of Platycodi Radix, activity-guided isolation was performed to find active components. The total saponin fraction of Platycodi Radix appeared to have a potent inhibitory activity against the hydrolysis of triolein emulsified with phosphatidycholine by pancreatic lipase in vitro. Based on these results, further purification of active components yielded 10 known triterpenoidal saponins, among these compounds, platycodin A, C, D, and deapioplatycodin D exhibited significant inhibitory effects on PL at the concentration of 500 microg/mL with 3.3, 5.2, 34.8, and 11.67% pancreatic lipase activity vs control, respectively. Platycodin D was found to inhibit the PL activity in a dose-dependent manner. Therefore, the anti-obesity effect of Platycodi Radix might be due to the inhibition of pancreatic lipase by its saponins.     

Simultaneous determination of seven saponins in the roots of <Emphasis Type="Italic">Codonopsis lanceolata</Emphasis> by liquid chromatography–mass spectrometry

We developed a rapid and simple analytical method for the simultaneous determination of seven 3,28-bidesmosidic triterpenoid saponins in the roots of Codonopsis lanceolata. The saponins are lancemaside A, lancemaside B, lancemaside C, lancemaside E, lancemaside G, foetidissimoside A, and aster saponin Hb. Root samples were extracted with 50% methanol and prepared for analysis. Saponins were detected by reversed-phase high-performance liquid chromatography with electrospray ionization mass spectrometry, and ginsenoside Rb₁ was used as an internal standard. The overall recoveries of all saponins were 92–116%, and the relative standard deviation values of intra- and inter-day precision were lower than 3.7 and 7.7%, respectively. Eight root samples collected from Korea and Japan were analyzed using the developed method. Lancemaside A was the most abundant saponin in the root samples from Korea, ranging from 2.65 to 3.64 mg/g dry root. However, the maximum content of lancemaside A among Japanese samples was 0.101 mg/g dry root.     

Herbological studies on Chinese crude drug Ma-huang. Part 1-On the botanical origin of Ma-huang in ancient China and the origin of Japanese Ma-huang

The botanical origin of a Chinese crude drug Ma-huang in ancient China and the origin of Japanese Ma-huang were herbologically studied. The results showed that the plants of Ephedra sinica Stapf, E. intermedia Schrenk & C. A. Meyer and E. equisetina Bunge were used as Ma-huang in China, and the first species was considered to be of high quality. The characters of Mao-zhou Ma-huang and Tong-zhou Ma-huang printed in Tu-jing-ben-cao, published in the Song Dynasty in China, were identified as E. likiangensis Florin and E. intermedia, respectively, and both species were recognized as excellent Ma-huang in the Ming Dynasty. The word origin of Katsune-kusa, the Japanese name for Ma-huang in the Heian Era, was etymologically considered as meaning the plant having reddish brown roots. In Japan, the plant of Equisetum ramosissimum Desf. var.japonicum Milde, of the family Equisetaceae, was substituted for Ma-huang in the middle of Edo Era, and it was designated that this action was based on the confusion of Ephedra plants and Equisetum plants those days in China.     

Simultaneous analysis of six major compounds in Osterici Radix and Notopterygii Rhizoma et Radix by HPLC and discrimination of their origins from chemical fingerprint analysis

Osterici Radix, the dried roots of Ostericum koreanum Maximowicz, has long been used to treat cold, headache and arthralgia in Chinese medicine. Notopterygii Rhizoma et Radix, the dried rhizomes and roots of Notopterygium incisum Ting et H.T. Chang or Notopterygium forbesii Boiss (family: Umbelliferae), are well-known traditional Chinese medicines. These all herbal medicines were named kangwhoal in China, Japan and Korea. However no discrimination methods for the origin and no simultaneous analysis methods for bioactive components are currently available. We developed simultaneous analysis of six biomarkers including nodakenin (1), oxypeucedanin (2), bisabolangelone (3), notopterol (4), imperatorin (5), and isoimperatorin (6) using HPLC method. HPLC chromatographic separation was conducted with C18 column and mobile phase of acetonitrile (A) — water (B) (gradient, (A) 35%–65% (30 min)). The analytical condition was validated with specificity, selectivity, linearity (correlation coefficients of all bio marker were more than 0.999.), recovery (94.9–106.8%), precision and accuracy (intra and inter RSD < 2.99%). In addition, principal component analysis and cluster analysis was performed on analytical data of 38 commercial kangwhoal samples from different places. We confirmed to classify kangwhoal origins and this study was applied to quality evaluation and chemotaxonomy of medicinal herbs.     

Quantitative analysis of betaine in Lycii Fructus by HILIC-ELSD

A rapid and simple high-performance liquid chromatography (HPLC) method with evaporative light scattering detection (ELSD) was developed for the determination of betaine from Lycii Fructus. Betaine was separated with an Atlantis hydrophilic interaction liquid chromatography silica column (4.6 × 150 mm, 5 μm, 100 Å) by isocratic elution using 30 mM ammonium acetate buffer and acetonitrile (20:80, v/v %) as the mobile phase. The flow rate was 1.0 mL/min, and the temperature for the spray chamber and drift tube was set at 30 and 50 °C, respectively. The method was fully validated with respect to linearity, precision, accuracy, stability and robustness. The HPLC/ELSD method was applied successfully to the quantification of betaine in the extract of Lycii Fructus. The HPLC analytical method for pattern recognition analysis was validated by repeated analysis of twenty-six L. barbarum L. from China (BC01–BC26), 3 L. barbarum L. (BJ27–BJ29) from Japan, 12 L. chinense Miller from China (CC30–CC41) and 51 L. chinense Miller samples (CK42–CK92) from Korea. The results indicate that the established HPLC/ELSD method is suitable for quality evaluation of Lycii Fructus.     

Blood cadmium levels in the populations of Masan, Korea, and Miyagi, Japan: an inter-regional comparison

Blood samples were collected from factory workers (with no occupational exposure to metals including cadmium) in Masan (167 subjects), Korea, in 1986, and Miyagi prefecture (270 subjects) Japan, in 1987. The samples were analyzed for cadmium in a single laboratory by flameless atomic absorption spectrophotometry under strict quality control. The effect of aging was detected in Masan and possibly also in Miyagi. The effect of smoking was evident among male smokers in Miyagi. Comparison with findings in a sister study on blood cadmium levels in China (taking the effects of smoking and aging into account) showed regional differences: the levels were lower in China, higher in Korea and probably highest in Japan. The smoking-associated increase in blood cadmium level per cigarette was 2-3 times higher in China than in Japan. The blood cadmium levels in the study regions appeared to be associated with the cadmium content of local rice as well as with local rice-eating habits.     

Comparison of urinary bromide levels among people in East Asia,and the effects of dietary intakes of cereals and marine products

Groups of people with no occupational exposure to Br-containing chemicals (29-54 year old, mostly women) in Japan, China and Korea, respectively, offered spot urine samples. Those in China and Korea offered 24 h duplicates of foods of the day. Urine samples were analyzed for Br by ECD-gas chromatography (ECD-GC) after derivatization to methyl bromide, and the Br-U was adjusted for a specific gravity of 1.016. Food intake data were based on national statistics and supplemented by the food duplicate data. Mean Br-U was 5.4 and 6.5 mg/l for Japanese men and women, respectively. Mean levels were in a range of 1.8-2.8 mg/l for four groups of Chinese, and 8-12 mg/l for the four groups of women in Korea. Br-U levels among Korean women were close levels reported for occupational exposure to 1- or 2-bromopropane, or methyl bromide. Regression analyses showed that Br-U levels were influenced by the intake of marine products (such as sea algae, sea fish and shellfish) and fruits, and inversely relate to intakes of cereals and potato.     

High performance liquid chromatographic fingerprint evaluation of the quinolizidine alkaloids from commercial Radix Sophorae Flavescentis

Fifteen commercial samples of Radix Sophorae Flavescentis were collected from different parts of China. HPLC analysis showed that the commercial samples all contained quinolizidine alkaloids and a total of nine chromatographic peaks were identified by referring to standard compounds, HPLC/MS analysis and comparison of the physicochemical data of the isolated peak with the literature. The contents of the major alkaloids were determined and the ratio of the major alkaloids contents was shown to be correlated with their source of origin. The commercial samples from China gave a distinct HPLC pattern showing the main alkaloids contents. The sample preparations were researched and the extraction conditions of the alkaloids were optimized. Reproducible HPLC fingerprints can be obtained for the quinolizidine alkaloids under the well-controlled extraction conditions. The HPLC fingerprint analysis method is suitable for the quality control of the Radix Sophorae Flavescentis and the standardization of phytomedicines.     

Molecular identification of "Chuanxiong" by nucleotide sequence and multiplex single base extension analysis on chloroplast trnK gene

Chloroplast trnK gene sequences of Cnidium officinale and Ligusticum chuanxiong were determined to establish an effective method for identifying Japanese Senkyu and Chinese Chuanxiong, the two which have the same drug name in Chinese characters, similar external feature, but different botanical origins. Three sites of nucleotide differences were found between these 2 species at positions 767,924 and 964 from upstream in trnK gene sequence, allowing molecular identification of the two plants and crude drugs. Further, three kinds of specific primers of 14 mer, 23 mer and 30 mer long were designed to detect these 3 sites of marker nucleotides. By using multiplex single base extension (MSBE) analysis with the 3 specific primers, C. officinale and L. chuanxiong could be distinguished clearly by the electrophoretograms, where 3 peaks with different color of ddTMP, ddCMP and ddTMP were observed in case of C. officinale and those of ddGMP, ddAMP and ddGMP in L. chuanxiong. Moreover, trnK gene sequence of "Dongxiong," a kind of Chuanxiong cultivated in Northeast China, suggested that its botanical origin was C. officinale.     

基于经皮给药研究制川乌与白芍配伍对大鼠皮肤及肝微粒体中CYP450活性的影响

目的：研究经皮给予制川乌-白芍药对后,配伍药对大鼠皮肤、肝脏药物代谢酶的影响,以探讨制川乌-白芍药对的配伍机制。方法：大鼠连续经皮给药制川乌、白芍、制川乌与白芍配伍凝胶剂14 d后,采用探针底物与皮肤、肝微粒体体外孵育,通过高效液相色谱法（HPLC）检测皮肤微粒体中1种探针底物及肝微粒体中4种探针底物的浓度,计算代谢速率,考察各给药组大鼠皮肤、肝微粒体CYP450酶活性的变化。结果：与空白组比较,白芍、制川乌对大鼠皮肤微粒体中的CYP3A4酶具有明显的抑制作用（P<0.05）;与白芍单用组比较,制川乌与白芍配伍给药使大鼠皮肤微粒体中的CYP3A4酶活性显著下降（P<0.05）;与制川乌组比较,制川乌与白芍配伍给药使大鼠皮肤中微粒体的CYP3A4酶的活性显著增加（P<0.05）。与空白组比较,白芍、制川乌对大鼠肝微粒体中的CYP3A4、CYP1A2、CYP2E1、CYP2D6酶都具有明显的抑制作用（P<0.05）;与制川乌组、白芍组比较,制川乌与白芍配伍使大鼠肝微粒体中CYP3A4、CYP1A2、CYP2E1、CYP2D6酶活性均明显增加（P<0.05）。结论：制川乌、白芍均为CYP3A4、CYP1A2、CYP2E1、CYP2D6药酶的抑制剂,两药配伍可引起大鼠皮肤、肝微粒体中药酶活性的改变而致活性成分的代谢速度改变,这可能是制川乌与白芍配伍发挥"增效减毒"的作用机制之一。     

Norms and alcohol consumption: Japanese in Japan, Hawaii and California.

The study compared age/sex norms and alcohol consumption of a sample of Japanese from Japan and Japanese Americans in Hawaii and California. Age norms included acceptable drinking levels for the following age groups: 16, 21, 40 and 60, both male and female. There were significant differences in norms across the sites with the Japanese in Japan holding the most tolerant views of heavier drinking for men, followed by California and Hawaii. The Japanese-American samples were more tolerant of female drinking than were the Japanese. There was a high degree of similarity between Hawaii and California, whereas Japanese respondents in Japan differed in their responses. A correlation between drinking and norms by site indicated a high degree of congruence between drinking and more tolerant norms for drinking in all of the samples: Those who drank the most also had the most permissive norms toward drinking. The differences in norms between Japanese in Japan and Japanese Americans were explained in terms of enculturation and acculturation.     

Molecular analysis of medicinally-used Chinese and Japanese Curcuma based on 18S rRNA gene and trnK gene sequences.

Curcuma drugs have been used discriminatingly for invigorating blood circulation, promoting digestion, and as a cholagogic in China. However, there is confusion about the drug's botanical origins and clinical uses because of morphological similarity of Curcuma plants and drugs. In order to develop an ultimate identification, molecular analysis based on 18S rRNA gene and trnK gene sequences were performed on 6 Curcuma species used medicinally in China and Japan. The 18S rRNA gene sequences were found to be of 1810 bps in length. In comparison with the common sequence of C. longa, C. phaeocaulis, C. wenyujin and C. aromatica, that of C. kwangsiensis had one base substitution, and the same base difference was observed between the Chinese and the Japanese populations of C. zedoaria. The trnK gene sequences were found to span 2698-2705 bps. There were base substitutions, small deletions or insertions at some sites between the trnK coding region and matK region among each species. Based on the base substitutions, C. zedoaria and C. kwangsiensis specimens were divided into two groups, respectively. An identical sequence was detected in C. phaeocaulis and in the Chinese population of C. zedoaria, as well as in the Japanese population of C. zedoaria and in one group of C. kwangsiensis with a purple-colored band in leaves. New taxonomic information to be used for authenticating Curcuma drugs was obtained.