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Paeoniae Radix, the dried root of Paeonia lactiflora, is one of the most important ingredients in Kampo medicine. It is known that Paeoniae Radix is derived from various P. lactiflora cultivars, including medicinal and horticultural cultivars, and that cultivar identification by DNA analysis has been unsuccessful. We attempted to develop sequence characterized amplified region (SCAR) markers as useful DNA markers for the identification and herbal medicine authentication of two cultivars developed in Japan, 'Bonten' and 'Kitasaisho,' which are two superior medicinal strains of P. lactiflora. Sequence-related amplified polymorphism (SRAP) analysis was conducted on fourteen P. lactiflora cultivars, and polymorphic fragments specific to 'Bonten' or 'Kitasaisho' were detected. Then, SCAR markers for 'Bonten' and 'Kitasaisho' were developed from the sequence information of these polymorphic fragments. Thirty cultivars of P. lactiflora and five herbal medicine samples were used to validate the specificity of the developed SCAR markers. As a result, we confirmed that our SCAR markers can identify 'Bonten' or 'Kitasaisho' from the plant samples and the herbal medicine samples. Thus, we have successfully designed two highly specific DNA markers and established an easy, rapid, and cost-efficient method to identify specific cultivars of P. lactiflora. Our SCAR markers are expected to contribute to the maintenance of P. lactiflora cultivars such as 'Bonten' as superior medicinal strains, the development of more elite cultivars in the future, and the deterrence of outflow of original cultivars to foreign countries.

We attempted to conduct an intraspecific analysis of 30 peony cultivars in Japan and to authenticate five herbal medicine samples derived from Paeoniae Radix by polymorphism analysis of the TEOSINTE BRANCHED1, CYCLOIDEA, and PCF (TCP) gene region. We focused on cultivar-dependent differences in leaf margin undulation and analyzed the sequence of the related TCP gene region. As a result, we found that the nucleotide sequences of 29 cultivars of Paeonia lactiflora except 'America' exhibit interspecific variations compared with the nucleotide sequences of Paeonia suffruticosa and Paeonia tenuifolia. Therefore, in the dendrogram constructed on the basis of the sequence similarity in the TCP gene region, the 29 cultivars of P. lactiflora were separated from P. suffruticosa, P. tenuifolia, and 'America', and clustered into three subgroups. There were 16 variations containing heterogenous DNA sequences within P. lactiflora species, and two characteristic variations in subgroup I. Some P. lactiflora cultivars showed the same nucleotide sequence in the TCP gene region, whereas the five herbal medicine samples showed different sequences, although all of them could be authenticated. The results suggest that Paeoniae Radix in the Japanese crude drug market can be authenticated by analysis of the TCP gene region., (© 2021. The Japanese Society of Pharmacognosy.)

The three Forsythia species, F. suspensa, F. viridissima and F. koreana, have been used as herbal medicines in China, Japan and Korea for centuries and they are known to be rich sources of numerous pharmaceutical metabolites, forsythin, forsythoside A, arctigenin, rutin and other phenolic compounds. In this study, de novo transcriptome sequencing and assembly was performed on these species. Using leaf and flower tissues of F. suspensa, F. viridissima and F. koreana, 1.28-2.45-Gbp sequences of Illumina based pair-end reads were obtained and assembled into 81,913, 88,491 and 69,458 unigenes, respectively. Classification of the annotated unigenes in gene ontology terms and KEGG pathways was used to compare the transcriptome of three Forsythia species. The expression analysis of orthologous genes across all three species showed the expression in leaf tissues being highly correlated. The candidate genes presumably involved in the biosynthetic pathway of lignans and phenylethanoid glycosides were screened as co-expressed genes. They express highly in the leaves of F. viridissima and F. koreana. Furthermore, the three unigenes annotated as acyltransferase were predicted to be associated with the biosynthesis of acteoside and forsythoside A from the expression pattern and phylogenetic analysis. This study is the first report on comparative transcriptome analyses of medicinally important Forsythia genus and will serve as an important resource to facilitate further studies on biosynthesis and regulation of therapeutic compounds in Forsythia species.

The root of Angelica acutiloba Kitagawa is an important crude drug in Kampo medicines (traditional Japanese medicine). Chemical evaluation of crude drugs is crucial to ensuring the safety and efficacy of herbal medicine; however, there is currently no chemical standard for the A. acutiloba crude drug in Japanese pharmacopoeia. (E)-ferulic acid (FA) is an important active ingredient of Angelica spp., including A. sinensis (Oliv.) Diels, and has been suggested as a marker for quality evaluation of those crude drugs. However, it has been controversial whether FA is a reliable marker constituent of A. acutiloba. To achieve effective extraction of FA from A. acutiloba, we compared three different extraction methods: alkaline hydrolysis, ethanol extraction, and hexane extraction. FA levels in these extracts were assessed using high performance liquid chromatography (HPLC), and alkaline hydrolysis was found to be the most effective. Furthermore, in the hydrolysate, FA was distinctly identified by thin layer chromatography (TLC) analysis. These results provide useful information for the quality control of the A. acutiloba crude drug.

Background: Hepatitis C virus (HCV) is a major cause of liver disease and a potential cause of substantial morbidity and mortality worldwide. The overall prevalence of HCV infection is 2%, representing 120 million people worldwide. Current standard treatment using pegylated interferon and ribavirin is effective in only 50% of the patients infected with HCV genotype 1, and is associated with significant side effects. Therefore, it is still of importance to develop new drugs for treatment of HCV. Antiviral substances obtained from natural products, including medicinal plants, are potentially good targets to study. In this study, we evaluated Indonesian medicinal plants for their anti-HCV activities., Methods: Ethanol extracts of 21 samples derived from 17 species of medicinal plants explored in the East Java region were tested. Anti-HCV activities were determined by a cell culture method using Huh7.5 cells and HCV strains of 9 different genotypes (1a to 7a, 1b and 2b)., Results: Four of the 21 samples tested showed antiviral activities against HCV: Toona sureni leaves (TSL) with 50% inhibitory concentrations (IC50) of 13.9 and 2.0 μg/ml against the HCV J6/JFH1-P47 and -P1 strains, respectively, Melicope latifolia leaves (MLL) with IC50 of 3.5 and 2.1 μg/ml, respectively, Melanolepis multiglandulosa stem (MMS) with IC50 of 17.1 and 6.2 μg/ml, respectively, and Ficus fistulosa leaves (FFL) with IC50 of 15.0 and 5.7 μg/ml, respectively. Time-of-addition experiments revealed that TSL and MLL inhibited both at the entry and post-entry steps while MMS and FFL principally at the entry step. TSL and MLL inhibited all of 11 HCV strains of all the genotypes tested to the same extent. On the other hand, FFL showed significantly weaker inhibitory activities against the HCV genotype 1a strain, and MMS against the HCV strains of genotypes 2b and 7a to a lesser extent, compared to the other HCV genotypes., Conclusions: Ethanol extracts of TSL, MLL, MMS and FFL showed antiviral activities against all the HCV genotypes tested with the exception that some genotype(s) showed significant resistance to FFL and to MMS to a lesser extent. These plant extracts may be good candidates for the development of anti-HCV drugs.

A new bisindole alkaloid, bisnicalaterine A (1), consisting of two vobasine-type skeletons, and 3-epivobasinol (2) and 3-O-methylepivobasinol (3), with vobasine-type skeletons, were isolated from the leaves of Hunteria zeylanica, and their structures were elucidated on the basis of spectroscopic data and chemical correlation. Bisnicalaterine A showed moderate cytotoxicity against various human cancer cell lines.

Two new acylated triterpenoid saponins were isolated from the branches of Schima noronhae by bioassay-guided purification. Their chemical structures were established on the basis of spectroscopic analysis and chemical means as 3-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-galactopyranosyl-(1-->3)-[beta-D-glucopyranosyl-(1-->2)]-beta-D-glucuronopyranosyl 22-O-angeloyl-A1-barrigenol (1) and 3-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-galactopyranosyl-(1-->3)-[beta-D-glucopyranosyl-(1-->2)]-beta-D-glucuronopyranosyl 22-O-angeloylerythrodiol (2). Compounds 1 and 2 showed cell growth inhibitory activity against both HeLa and DLD1 cells at a concentration of less than 10 microM.

A new furostan-type saponin (1) was isolated from the methanolic extract of Brunfelsia grandiflora leaves, together with four known compounds. The chemical structure of 1 was determined by spectroscopic analysis and chemical reaction to be 26-O-beta-D-glucopyranosyl 22alpha-methoxyfurost-3beta,26-diol 3-O-beta-D-xylopyranosyl(1-->3)-{beta-D-glucopyranosyl(1-->2)}-beta-D-glucopyranosyl(1-->4)-beta-D-glucopyranoside. Compound 1 showed potent leishmanicidal activity in vitro against Leishmania major.

In the screening of biologically active constituents from woody plants, the methanol extract of leaves of Chamaecyparis obtusa showed potent neurite outgrowth-promoting activity in neuronal PC12 cells. The ethyl acetate-soluble fraction of the methanol extract showed potent activity and was separated by means of various chromatographic methods to give the two new compounds 1 and 2, as well as 11 known lignan and sesquiterpene derivatives. The structures of the new compounds were determined to be 9-O-acetyldihydrosesamin (1) and 9-O-(11-hydroxyeudesman-4-yl)dihydrosesamin (2), respectively, in NMR studies including 2D-NMR experiments. Of the 13 compounds, the known compound hinokinin (5) and the new compound 2 showed potent neurite outgrowth-promoting activity in PC 12 cells.

The cytotoxicity against A172 human malignant glioma cells was examined for 14 alkaloids from the roots of Aconitum yesoense var. macroyesoense and of Aconitum japonicum and from the seeds of Delphinium elatum as well as for 25 semisynthetic derivatives. The major alkaloid constituents of A. yesoense var. macroyesoense, kobusine (2) and pseudokobusine (3), a minor alkaloid constituent of A. japonicum, aljesaconitine A (5), and six alkaloid derivatives, N-deethyldelcosine (10), N-deethyldelsoline (11), 12-benzoylluciculine (18), 12-anisoylluciculine (19), 6,11-dibenzoylpseudokobusine (28), and 6-veratroylpseudokobusine (29), had only very weak activity. Four acylated alkaloid derivatives, 12-acetylluciculine (23), 11-veratroylpseudokobusine (30), 11-(m-trifluoromethylbenzoyl)pseudokobusine (32), and 11-(m-trifluoromethylbenzoyl)kobusine (39), exhibited more potent activity, while pseudokobusine 11-cinnamoate (31), 11-anisoate (33), and 11-p-nitrobenzoate (34) were found to be the most potent cytotoxic agents.

The new Daphniphyllum alkaloids, pordamacrines A (1) and B (2), have been isolated from the leaves of Daphniphyllum macropodum, and their structures were elucidated on the basis of interpretation of spectroscopic data and by the single-crystal X-ray diffraction analysis of 2. Pordamacrines A (1) and B (2) exhibited moderate vasorelaxant effects on the rat aorta.

Ishigami et al. (Ishigami et al., 1998) reported that squalene monohydroperoxide (SQOOH) induced skin damage in hairless mice. Kohno and Takahashi (Kohno and Takahashi, 1993) reported that SQOOH induced cytotoxicity against Chinese hamster lung fibroblasts. We have already evaluated the efficacy of extracts obtained from Brazilian herbal medicines in protecting the normal human epidermis keratinocytes [NHEK(B)] against the cytotoxicity caused by SQOOH. The EtOAc extract was separated by silica-gel column chromatography into eight fractions. Fractions (Fr) 1,3 and 5 significantly protected rat basophilic leukemia (RBL-2H3) cells from the release of beta-hexosaminidase due to SQOOH. Additionally, Fr5-1 was most effective in a Gunze three-dimensional cultured human skin model (Vitrolife-skin) against the cytotoxicity due to SQOOH and the release of interleukin (IL)-2 and IL-4. The mixture of cinchonains Ia and Ib and the mixture of cinchonains IIa and IIb were isolated from Fr3 and Fr5-1, respectively. The results suggest that the addition of SQOOH caused the reduction in cell viability and the release of beta-hexosaminidase and cytokines as chemical mediators. The extract of Catuaba (Anemopaegma mirandum) prevented these toxic effects with the main active agents suggested to be cinchonains IIa and IIb.

From the Brazilian medicinal plant Carucaá (Cordia multispicata), oleanane- and ursane-type triterpenoids were previously reported as anti-androgenic constituents of the plant. In this study, purification of the polar elements of the EtOAc-soluble fraction of the plant revealed nine novel dammarane-type triterpenes, named cordianols A-I (1-9) along with the known compound cordialin A (10). The structures of these new compounds were elucidated by means of spectral methods including HRFABMS, (1)H NMR, (13)C NMR, and 2D NMR (HMQC, HMBC, NOESY). Absolute configuration at C-23 of compound 7 was determined by an excitone chirality method. Some of these new compounds revealed a hemiketal structure on the A ring and a hydroxylated or epoxidated 20(22)-(E)-ene side chain and showed weak anti-androgenic activity.

From the methanol extract of Smilax bracteata rhizomes, six new phenolic compounds, (2S,3S)-5-O-beta-D-glucopyranosyloxy-6-methyl-3'-methoxy-3,7,3'-trihydroxyflavan (1), (2S,3S)-5-O-beta-D-glucopyranosyloxy-6-methyl-4'-methoxy-3,7,4'-trihydroxyflavan (2), 3beta-(3',5'-dihydroxyphenyl)-2alpha-(4' '-hydroxyphenyl)dihydrobenzofuran-5-carbaldehyde (3), (1-p-O-coumaroyl-6-O-feruroyl)-beta-D-fructofuranosyl-alpha-D-glucopyranoside (4), (1-p-O-coumaroyl-3,6-di-O-feruroyl)-beta-D-fructofuranosyl-alpha-D-glucopyranoside (5), and (6-O-feruroyl)-beta-D-fructofuranosyl-(6-O-acetyl)-alpha-D-glucopyranoside (6) were isolated together with five known compounds. Their structures were established by spectral data interpretation.

Compounds 1-6 were isolated from the AcOEt soluble fraction of leaves of the Brazilian medicinal plant, Cordia multispicata, and their structures were elucidated to be 3beta,25-epoxy-21beta-acetoxy-3alpha,22beta-dihydroxyurs-12-en-28-al (1), 3beta,25-epoxy-28-acetoxy-3alpha,21beta,22beta-trihydroxyurs-12-ene (2), 21beta-acetoxy-22beta-hydroxy-3-oxours-12-en-28-al (3), 28-acetoxy-6beta, 21beta,22beta-trihydroxy-3-oxours-12-ene (4), 21beta,22beta-dihydroxy-3-oxours-1 2-en-28-al (5) and 3beta,21beta,22beta-trihydroxyurs-I2-en-28-al (6), respectively, by means of spectral data, especially two dimensional NMR techniques. Triterpenes having the hemiketal structure at the A-ring, an acyloxy group at C-22 and/or ketone at C-3 showed potent anti-androgenic activity.

The structure of a new type of sesterterpenoid, designated as alborosin, isolated from Gentianella alborosea, has been deduced from a spectroscopic investigation.

A novel sesterterpenoid designated as nitiol (1), possessing enhancement activity of IL-2 gene expression in a human T cell line, was isolated from the Peruvian folk medicine "Hercampuri" (Gentianella nitida). The structure was elucidated by extensive spectroscopic investigation.

The role of the substituents at C3 and C8 of jesaconitine (1) on jesaconitine-induced analgesia and toxicity was examined. 3-O-Acetyljesaconitine (2), 3-O-anisoyljesaconitine (3), and 3-deoxyjesaconitine (6) showed dose-dependent analgesic action, and the potency of their compound-induced analgesia and toxicity was lower than those of 1. The most remarkable difference was found in the toxicity. The results indicate that the C3 hydroxy function of 1 participate in the induction of toxicity rather than of analgesia. 8-O-Linoleoyl-14-anisoylaconine (5), 8-O-methyl-14-anisoylaconine (7), 8-O-ethyl-14-anisoylaconine (8), 14-anisoylaconine (4) and 8-deoxy-14-anisoylaconine (9) showed lower activities than jesaconitine-induced analgesia and toxicity. The analgesic activity of 7 was almost the same as that of 8, but the toxicity of 7 was lower than that of 8. The analgesic activity of 9 was lower than that of 4, but the toxicities of both derivatives were not apparent. These facts indicate that the C8 function of 1 is important to the induction of analgesia and toxicity, and also that this function participates differently in the induction of the analgesia and toxicity. Subsequently, it was suggested that substituents at C3 and C8 of 1 played important roles of the induction of the analgesia and toxicity, and that the modes of this participation were not the same in analgesia and toxicity.