Inhibitory effect of Ephedra herba on human norovirus infection in human intestinal organoids.

Human norovirus (HuNoV) is a major cause of acute gastroenteritis and foodborne diseases worldwide with public health concern, yet no antiviral therapies have been developed. In this study, we aimed to screen crude drugs, which are components of Japanese traditional medicine, ''Kampo'' to see their effects on HuNoV infection using a reproducible HuNoV cultivation system, stem-cell derived human intestinal organoids/enteroids (HIOs). Among the 22 crude drugs tested, Ephedra herba significantly inhibited HuNoV infection in HIOs. A time-of-drug addition experiment suggested that this crude drug more preferentially targets post-entry step than entry step for the inhibition. To our knowledge, this is the first anti-HuNoV inhibitor screen targeting crude drugs, and Ephedra herba was identified as a novel inhibitor candidate that merits further study.

Evaluation of rooting characteristics of Ephedra cuttings by anatomy and promising strain selection based on rooting characteristics and alkaloid content.

The differences in rooting characteristics of cuttings prepared from E. sinica strains were investigated and found that cuttings prepared from strains with high rooting characteristics showed approximately 90% of the cuttings were rooted, whereas cuttings prepared from low rooting characteristics did not root. To understand the reason for this substantial difference, the anatomy of nodes was examined and found that adventitious roots were generated from the cortex and parenchyma in pith. Calculations of the correlation coefficients between the rooting rate and the value of anatomy indicated that the rooting rate was positively correlated with the parenchyma in pith in the node. On the basis of the positive correlation, it is possible to estimate the rooting characteristics of new strains without having to prepare cuttings. Next, we conducted a screening for E. sinica strains on the basis of total alkaloids content [ephedrine (E) + pseudoephedrine (PE)] and selected strains having no less than 0.7% total alkaloids content as defined by the Japanese Pharmacopoeia 18th edition. Strains having characteristic E or PE content were uncovered: E-rich strains had 100% E content and PE-rich strains had 99% PE content. We were able to select E. sinica strains on the basis of two factors: high rooting rate of cuttings and high or characteristic alkaloid content. These strains are valuable for breeding.

Seasonal variation of alkaloids and polyphenol in Ephedra sinica cultivated in Japan and controlling factors.

We investigated the seasonal variation of alkaloids (ephedrine and pseudoephedrine), total polyphenol, and sugar contents in Ephedra sinica cultivated in Japan and elucidated the controlling factors for the variation. In 2018, alkaloids and polyphenol contents increased dramatically from May to July, decreased to their lowest in October, and slightly increased again in November. The reduction of alkaloids and polyphenol contents in the autumn may be affected by precipitation in summer. In 2020, alkaloids and polyphenol contents started to decrease in late July when rainfall was abundant from July to August. In contrast, sucrose and starch contents continued to increase until September and remained high until October. Vascular bundles and fiber developed, and herbal stem weight increased from August to October. Alkaloids and total polyphenol contents tended to increase in November. At the same time, starch and sucrose contents decreased dramatically, whereas glucose and fructose contents increased. Sugar content decreased from October and was lowest in November. The seasonal variation of alkaloids and total polyphenol contents exhibited a contrasting tendency to the seasonal variation of sugar content and tissue development. The seasonal variation of alkaloids and total polyphenol contents was caused by the seasonal variation of sugar content and tissue development. In addition, it is suggested that anatomy may be used for alkaloids content estimation in Ephedra plants.

Development of Highly Sensitive Method for Sugar Determination in Herbal Medicine; Application of Monosaccharides and Oligosaccharides in Japanese Angelica Root and Rehmannia Root.

We have developed a simple and accurate method for quantifying sugars in herbal medicines, which have hitherto been difficult to quantify. Using ultra performance liquid chromatography-quadrupole-time-of-flight (UPLC-Q-TOF)-MS and two types of columns with different chemical properties, we determined the optimum conditions for separating nine sugars (fructose, galactose, glucose, mannitol, sucrose, melibiose, raffinose, manninotriose, and stachyose) commonly found in herbal medicines. Separation was completed within 10 min when an apHera NH2 HPLC column was used, although galactose and glucose could not be separated. On the other hand, the nine sugars were completely separated within 16 min when a hydrophilic interaction chromatography (HILIC)pak VG-50 2D column was used. The calibration curves obtained using those two columns gave good linearity for the sugar standards, and the coefficient of determination was 0.995 or higher. Both columns showed excellent performance with short analysis time and high sensitivity. Using our developed method, we were able to quantify sugars in galactose-free herbal medicines within 10 min and in herbal medicines containing galactose within 16 min. We revealed that our method could be used for the analysis of sugars in Angelica acutiloba and Rehmannia glutinosa roots.

Study on Catalpol Content in Rehmannia glutinosa Root, an Important Ingredient in Kampo Prescriptions.

Rehmannia glutinosa is an important medicinal plant in Asia, and its roots are used as an ingredient in herbal medicine. However, the roots exhibit different medicinal effects depending on the processing conditions. Since the catalpol content differs greatly during the process, the catalpol content is an essential index for quality evaluation. R. glutinosa roots have various weights, diameters, and lengths, and there are differences between individuals and within an individual immediately after harvest. We found that, catalpol content in the roots tended to increase as root diameter increased. Furthermore, it has been reported that catalpol content decreased with drying, and our results also supported this phenomenon. To clarify the reason for the decrease in catalpol content, we investigated the effect of ß-glucosidase in R. glutinosa cells. An in situ assay for ß-glucosidase activity revealed that the activity in the tissue inside the cambium disappeared one month after drying under natural conditions, and the activity in the tissue outside the cambium completely disappeared after two months. Because catalpol content remained almost unchanged even after drying for two months, it was clarified that ß-glucosidase activity had minimal involvement in the decrease in catalpol content in R. glutinosa roots. Based on the above results, we proposed that slicing the roots and rapidly removing water by natural drying is best to obtain dry root with little loss of catalpol content.

Development of SCAR Markers to Identify Medicinal Cultivars of Paeonia lactiflora.

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.

Study on morphological and genetic diversity of Rehmannia glutinosa cultivated in Japan.

It is said that Rehmannia glutinosa is grouped into two types, Akaya and Kaikei, in Japan. However, previous reports of genetic analysis of R. glutinosa in commercial products suggest the existence of varieties other than these two, and therefore, it is inappropriate to simply classify them into these two varieties. In this study, we clarified the diversity of R. glutinosa cultivated in Japan on the basis of morphological observation and genetic analysis. We conducted principal component analysis (PCA) of R. glutinosa morphology, including leaf surface color, leaf undersurface anthocyanin coloration, root shape, and the ratio of string root. We also performed (1) sequence-related amplified polymorphism (SRAP) analysis and (2) polymorphism analysis of the TEOSINTE BRANCHED1, CYCLOIDEA, and PCF (TCP) gene region. We were able to separate Akaya type from Kaikei type, and to divide Kaikei type into three small groups. These two gene analysis methods were also useful in estimating the patrilineal and matrilineal strains of a hybrid origin. Our findings revealed that Akaya type and Kaikei type can be distinguished on the basis of morphological and genetic analyses, and that Kaikei type cultivated in Japan exhibited morphological and genetic diversity.

Polymorphism analysis of TCP gene region to intraspecific analysis of Paeonia lactiflora, and application for authentication of Paeoniae Radix.

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.

Ephedra Herb, Mao, Inhibits Antigen-Induced Mast Cell Degranulation by Induction of the Affinity Receptor for IgE Internalization.

PURPOSE: Ephedra herb (Mao) exerts potent anti-allergic effects. This study aimed to examine the underlying mechanisms of Mao on allergic inflammation using in vitro cultured mast cells (MCs) and an in vivo model of MC-dependent anaphylaxis. METHODS: Bone marrow-derived MCs (BMMCs) were presensitized with anti-2,4-dinitrophenol (DNP) immunoglobulin E (IgE) and challenged with antigens (Ag; DNP-human serum albumin). Degranulation responses and cell surface high-affinity receptor for IgE (FcεRI) expression were assessed with/without Mao treatment. Passive systemic anaphylaxis (PSA)-treated mice were administered Mao and the pathophysiological responses were evaluated. RESULTS: Mao inhibited Ag-induced BMMC degranulation, but not polyclonal activation with phorbol 12-myristate 13-acetate (PMA) and ionomycin, indicating that Mao inhibits IgE-dependent activation of BMMCs. Mao-treated BMMCs exhibited significant reductions in expression of surface IgE and its receptor FcεRI. Analysis of subcellular localization revealed that Mao induces FcεRI internalization in BMMCs without degranulation. In the PSA mouse model, Mao administration prevented antigen-induced hypothermia. Mao administration significantly reduced cell surface expression of IgE-bound FcεRI on peritoneal MCs. CONCLUSIONS: Mao induced FcεRI internalization in MCs, thereby inhibiting Ag-induced IgE-dependent degranulation. The inhibitory effects of Mao on MC degranulation may offer a novel therapeutic approach for allergic diseases.

Studies on cultivation of Angelica acutiloba using paper pot (Part 2): effect of difference in taproot base diameter of seedlings on growth after planting and quality of toki produced from paper pot seedlings.

By using Angelica acutiloba seedlings produced by short-period raising in paper pot (PP), we investigated the effect of taproot base diameter on the growth after planting and evaluated the quality of toki (, Angelicae Acutilobae Radix) produced from PP-grown seedlings (PT). In this study, PP-grown seedlings of seven classes which are sorted difference taproot base diameter (P1-P7) were transplanted. As a result, the survival rates were not less than 90% for P6 (taproot base diameter: 10.0-12.7 cm) and P7 (taproot base diameter: 12.8-17.3 cm), and the bolting rates were 20.5% and 24.1% for P6 and P7, respectively. The traditionally grown seedling (TS) showed the highest available harvest rate of 82.3%, followed by P6 (71.8%) and P7 (69.0%). P7 showed the highest fresh weight and dry weight per 1a (100 m2), followed by P6 and TS. However, PP-grown seedlings are prone to bolting, and it is expected the yield can be increased by preventing bolting. In addition, PT satisfied the regulations of JP17 in terms of, for example, dilute ethanol-soluble extract content, total ash content, and acid-insoluble ash content. It was also found that PT has a higher proportion of lateral roots with respect to the total weight than toki produced from traditionally grown seedlings (TT). The total (Z)-ligustilide contents were 0.07% in PT and 0.12% in TT. It was revealed that it is possible to produce toki that satisfies JP17 regulations; moreover, the period required for toki production was shortened by 6 months with PP-grown seedlings.

Quality evaluation of Angelicae acutilobae radix: individual differences and localization of (Z)-ligustilide in Angelica acutiloba root.

It has been difficult to evaluate the quality of Angelicae acutilobae Radix (Toki) because of large differences in the contents of its chemical constituents. In this research, we revealed individual differences and localization of (Z)-ligustilide in Toki cultivated and processed under the same conditions. Thirteen Toki samples (dry weight: 68.2 g-132.3 g) were divided and categorized into 13 parts and the (Z)-ligustilide content of each part was quantified. Total (Z)-ligustilide content ranged from 0.08% to 0.22% and the maximum difference between samples was approximately 2.8-fold. In addition, the localization of (Z)-ligustilide was examined. (Z)-Ligustilide content was the highest in lateral root at 0.19%, followed by main root at 0.13%, and the lowest in root head at 0.09%. Furthermore, the content tended to increase as the root became thinner. In particular, the difference in content between the inner side of upper root head (removed 5 mm from the epidermis 0.06%) and the 1.1-3.0 mm in diameter lateral root (0.24%) was largest at approximately 4.1-fold. We revealed that not only differences among individuals but also localization is a factor affecting the quality of Toki. In contrast, individuals with higher root part (main root + lateral root) weight ratio in whole root dry weight had higher (Z)-ligustilide content. The difference in (Z)-ligustilide content among individuals is due to the balance between root head part and root other than head part. It is possible to predict (Z)-ligustilide content from weight ratio of root part to whole root.

Evaluation of white Kwao Krua (Pueraria candollei Grah. ex Benth.) products sold in Thailand by molecular, chemical, and microscopic analyses.

The tuberous roots of Pueraria candollei Grah. ex Benth. (Fabaceae), commonly known as white Kwao Krua, are used to relieve menopausal symptoms in Thai traditional medicine because they contain phytoestrogens. Black and red Kwao Krua crude drugs exist as well, but they have different botanical origins and pharmacological activities. There is a high demand for white Kwao Krua products, but because of the limited availability of the plant material, it is suspected that the adulteration and misidentification of white Kwao Krua crude drugs and products occur. In this study, we authenticated white Kwao Krua products collected from Thai herbal markets by molecular, chemical, and microscopic analyses. The nucleotide sequences in the internal transcribed spacer (ITS) and trnH-psbA regions of 23 samples of authentic P. candollei were analyzed, and both regions were found to have intraspecific DNA polymorphisms. Based on the single nucleotide polymorphisms in the ITS1 region, species-specific primer sets of P. candollei were designed to authenticate white Kwao Krua and differentiate it from red and black Kwao Krua. Only the PCR products of KWP02 were not amplified by the primer sets. Isoflavonoid contents and microscopic features were used to support the results of molecular analysis to clarify the botanical origin of white Kwao Krua. Molecular, chemical and microscopic methods confirmed that all the Thai Kwao Krua products examined in this study contained authentic "white Kwao Krua" as claimed on their labels.

The combination of ITS2 and psbA-trnH region is powerful DNA barcode markers for authentication of medicinal Terminalia plants from Thailand.

The dried fruits of Terminalia plant (Combretaceae) called "Samo" have been used as herbal medicine in Thai traditional medicine. Four "Samo" crude drugs, namely, Samo thai, Samo thed, Samo dee-ngu, and Samo phiphek, are used as the main ingredients in Triphala and Trisamo recipes. Their commercial products are available in processed and powdered form, but are difficult to authenticate by conventional methods. In this study, we aimed to discriminate species of genus Terminalia for the identification of their crude drugs by a DNA barcoding technique. A total of 208 closely related nucleotide sequences were obtained from nine Terminalia species collected from Thailand and the DDBJ/EMBL/GenBank database. An effective DNA barcode marker was selected from six DNA loci (matK, rbcL, psbA-trnH, ITS, ITS1, and ITS2) and their two-locus combination. All sequences were analyzed by three major methods: (1) BLAST search; (2) the genetic divergence method using Kimura 2-parameter (K2P) distance matrices; and (3) tree topology analysis based on the neighbor-joining method. Comparison of the six candidate DNA loci indicated that ITS identified Terminalia with 100% accuracy at the species and genus levels in the BLAST1 method. ITS2 showed the highest K2P variability. The data from the single markers and the two-locus combinations revealed that only the two-locus combinations, namely, the combinations of rbcL, ITS, ITS1, and ITS2 with psbA-trnH, clearly discriminated all the species. From the results of DNA sequence analysis and the three methods, ITS2 is recommended for the identification of Terminalia species to supplement psbA-trnH.

Rapid identification of Aconitum plants based on loop-mediated isothermal amplification assay.

OBJECTIVE: Aconitum plants (Ranunculaceae) exhibit toxicity, and accidental ingestion of the plants has been reported in Japan. Identifying the cause of poisoning is important for emergency medical treatment, and a rapid and simple detection technique is required for the identification of poisoning cause. In the present study, we developed a rapid and simple method for detecting Aconitum plant DNA using a loop-mediated isothermal amplification (LAMP) assay. RESULTS: Specific LAMP primers for Aconitum plants were designed based on the trnL-trnF intergenic spacer region. Using the LAMP primers, the LAMP assay included an initiation reaction of 10 min followed by amplification for 20 min at the isothermal reaction temperature of 65 °C. The LAMP reaction was demonstrated to be specific and highly sensitive to Aconitum plants, given that the assay can be used for 1 pg of purified DNA. Using raw extracted DNA as template, the entire detection procedure from DNA extraction to final detection required only 30 min. Moreover, the protocol identified samples containing approximately 5 mg of Aconitum plants cooked and digested with artificial gastric juice. The currently proposed protocol exhibits good potential as a screening method of Aconitum plant poisoning for emergency medical care.

Morphological and genetic differences between Coptis japonica var. anemonifolia H. Ohba and Coptis japonica var. major Satake in Hokuriku area.

Coptis japonica is widely distributed in Japan, and its dried rhizome is a source of the domestic herbal medicine Coptidis Rhizoma ( Oren). There are three varieties of C. japonica, two of which, namely, C. japonica var. anemonifolia and C. japonica var. major, are important as sources of traditional medicines. Coptis japonica var. anemonifolia and C. japonica var. major are distinguishable on the basis of their ternate or biternate compound leaves, respectively. In the Hokuriku area, where both C. japonica var. anemonifolia and C. japonica var. major grow naturally, some individual plants cannot be identified unambiguously on the basis of leaf morphology because changes in leaf morphology may occur due to intra-variety variation or crossbreeding between the two varieties. In addition, genetic differences between the two varieties have remained unclear. In this study, we employed new genetic and morphological classification approaches to discriminate between the two varieties. Based on the single nucleotide polymorphisms of the tetrahydroberberine oxidase gene, we found four conserved SNPs between the two varieties and were able to classify C. japonica into two varieties and crossbreeds. Furthermore, we introduced a new leaf type index based on the overall degree of leaflet dissection calculated by surface area of a leaflet and length of leaflet margin and petiolule. Using our new index we were able to discriminate between the two varieties and their crossbreeds more accurately than is possible with the conventional discrimination method. Our genetic and morphological classification methods may be used as novel benchmarks to discriminate between the two varieties and their crossbreeds.

A Novel Clerodane Diterpene from Vitex cofassus.

New clerodane diterpene, 16-hydroxy-pentandralactone (1) and known diterpene acuminolide (2) were isolated from the methanol extract of Vitex cofassus leaves. The chemical structure and the absolute configuration of 1 were determined by MS, NMR and electron circular dichroism (ECD) experiments. The isolated compounds were evaluated for their antiproliferative activities against a panel of human tumor cell lines, including a multidrug-resistant (MDR) cell line. Both compounds showed potent antiproliferative activities against all the tested cell lines with IC50 values of 5.4-11.4 µM. Their effects on cell viability were also tested using vascular endothelial growth factor (VEGF)-stimulated human umbilical vein endothelial cells (HUVECs). Compound 1 inhibited VEGF-stimulated HUVEC proliferation in a dose-dependent manner. Based on these results, compound 1 could be a candidate for antitumor agent and inhibitor of angiogenesis.

Pharmacological evaluation for improvement of Kanazawa Sutra, medicinal thread for anal fistula.

Kanazawa Sutra (KanS) is a medicinal thread that is used for the treatment of anal fistula. It is used as a substitute for Kshara Sutra (KS) which is used in Ayurvedic medicine. KanS is composed of Ficus carica latex (FCL), Capsicum annuum tincture (CAT), Achyranthes fauriei Kshara (which is processed ash from the whole plant) and powdered Curcuma longa rhizome (CLR). In this study, we evaluated the ingredients of KanS by measuring nitric oxide (NO) production in murine macrophage-like cell line J774.1 as well as examining cytotoxicity to rat skeletal muscle myoblasts (L6) and L6 differentiation, with a view to improving its pharmacological effect. We focused on Mallotus japonicus bark (MJB), which is described in the Japanese Pharmacopeia and belongs to the Euphorbiaceae family. Its biological activities were evaluated in a similar manner to the evaluation of KanS ingredients. We found that MJB extracts showed similar biological activity to Euphorbia neriifolia latex (ENL), an ingredient of KS. We conclude that the NO inhibitory activity of KanS is mainly due to CLR, and its cytotoxicity to L6 and inhibitory activity on L6 differentiation are mainly due to CLR and FCL. As CAT has no characteristic activity, the biological activity and the anal fistula treatment ability of KanS would be improved by substituting MJB for CAT.

Molecular Analysis of Terminalia spp. Distributed in Thailand and Authentication of Crude Drugs from Terminalia Plants.

Terminalia, a large genus of Combretaceae, is distributed in Tropical Asia, Africa, and America. Some Terminalia plants are used in folk medicine because they possess powerful medicinal properties. Dried fruits of Terminalia bellirica and Terminalia chebula are used as the main ingredient in Triphala, a famous polyherbal formulation in Ayurvedic medicine and Thai folk medicine, because of their laxative, detoxifying, and rejuvenating effects. To clarify the phylogenetic relationships of medicinal Terminalia species (T. bellirica, T. chebula, and T. catappa) and authenticate their crude drugs, "Samo" and Triphala, nucleotide sequencing alignments in the internal transcribed spacer one-two (ITS 1-2) regions of Terminalia plants collected in Thailand were performed. The amplified fragments of Terminalia species were approximately 800 bp in length. To compare these sequences and DDBJ registered data, a molecular phylogenetic tree was constructed. Phylogenetic analysis clearly separated the sequences into two groups: Asian Terminalia and African Terminalia with some exceptions. In the analyzed sequences, the length of the ITS1-5.8S-ITS2 region was 674 bp in T. chebula, and 677 bp in T. bellirica and T. catappa. Eighty-one single nucleotide polymorphisms (SNPs) and nine insertion-deletions (indels) were observed, and the nucleotide sequences of this region showed species-specific sequences. Based on these differences, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and amplification refractory mutation system (ARMS) were applied to identify medicinal Terminalia species. Moreover, the ARMS method was chosen for fingerprinting analysis of Samo crude drugs and Triphala formulations because it was a fast, cost-effective, and reproducible approach.