Anti-SARS-CoV-2 activity of various PET-bottled Japanese green teas and tea compounds in vitro.

The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a serious threat to global public health. The emergence of SARS-CoV-2 variants is a significant concern regarding the continued effectiveness of vaccines and antiviral therapeutics. Thus, natural products such as foods, drinks, and other compounds should be investigated for their potential to treat COVID-19. Here, we examined the in vitro antiviral activity against SARS-CoV-2 of various polyethylene terephthalate (PET)-bottled green Japanese teas and tea compounds. Six types of PET-bottled green tea were shown to inhibit SARS-CoV-2 at half-maximal inhibitory concentrations (IC50) of 121- to 323-fold dilution. Our study revealed for the first time that a variety of PET-bottled Japanese green tea drinks inhibit SARS-CoV-2 infection in a dilution-dependent manner. The tea compounds epigallocatechin gallate (EGCG) and epicatechin gallate showed virucidal activity against SARS-CoV-2, with IC50 values of 6.5 and 12.5 µM, respectively. The investigated teas and tea compounds inactivated SARS-CoV-2 in a dose-dependent manner, as demonstrated by the viral RNA levels and infectious titers. Furthermore, the green teas and EGCG showed significant inhibition at the entry and post-entry stages of the viral life cycle and inhibited the activity of the SARS-CoV-2 3CL-protease. These findings indicate that green tea drinks and tea compounds are potentially useful in prophylaxis and COVID-19 treatment.

Anti-malarial activity of traditional Kampo medicine Coptis rhizome extract and its major active compounds.

BACKGROUND: Herbal medicine has been a rich source of new drugs exemplified by quinine and artemisinin. In this study, a variety of Japanese traditional herbal medicine ('Kampo') were examined for their potential anti-malarial activities. METHODS: A comprehensive screening methods were designed to identify novel anti-malarial drugs from a library of Kampo herbal extracts (n = 120) and related compounds (n = 96). The anti-malarial activity was initially evaluated in vitro against chloroquine/mefloquine-sensitive (3D7) and-resistant (Dd2) strains of Plasmodium falciparum. The cytotoxicity was also evaluated using primary adult mouse brain cells. After being selected through the first in vitro assay, positive extracts and compounds were examined for possible in vivo anti-malarial activity. RESULTS: Out of 120 herbal extracts, Coptis rhizome showed the highest anti-malarial activity (IC50 1.9 µg/mL of 3D7 and 4.85 µg/mL of Dd2) with a high selectivity index (SI) > 263 (3D7) and > 103 (Dd2). Three major chlorinated compounds (coptisine, berberine, and palmatine) related to Coptis rhizome also showed anti-malarial activities with IC50 1.1, 2.6, and 6.0 µM (against 3D7) and 3.1, 6.3, and 11.8 µM (against Dd2), respectively. Among them, coptisine chloride exhibited the highest anti-malarial activity (IC50 1.1 µM against 3D7 and 3.1 µM against Dd2) with SI of 37.8 and 13.2, respectively. Finally, the herbal extract of Coptis rhizome and its major active compound coptisine chloride exhibited significant anti-malarial activity in mice infected with Plasmodium yoelii 17X strain with respect to its activity on parasite suppression consistently from day 3 to day 7 post-challenge. The effect ranged from 50.38 to 72.13% (P < 0.05) for Coptis rhizome and from 81 to 89% (P < 0.01) for coptisine chloride. CONCLUSION: Coptis rhizome and its major active compound coptisine chloride showed promising anti-malarial activity against chloroquine-sensitive (3D7) and -resistant (Dd2) strains in vitro as well as in vivo mouse malaria model. Thus, Kampo herbal medicine is a potential natural resource for novel anti-malarial agents.

Kamiuntanto increases prefrontal extracellular serotonin levels and ameliorates depression-like behaviors in mice.

Kamiuntanto (KUT; Jia Wei Wen Dan Tang in Pinyin) is a traditional Japanese Kampo medicine that is used to treat psychological dysfunction. However, the mechanisms of action of KUT are not understood. To investigate the mechanisms underlying the ameliorative properties of KUT, the effects of KUT on abnormal behaviors of isolation-reared mice and on the prefrontal monoaminergic system were examined. KUT (1000 mg/kg) reversed encounter-induced hyperactivity and increased immobility in the forced swim test in isolation-reared mice, as also found for an antidepressant, fluoxetine (30 mg/kg). In vivo microdialysis showed that KUT (1000 mg/kg) transiently increased the level of extracellular serotonin (5-HT) in the prefrontal cortex. In contrast, an incomplete KUT formula excluding Bambusae Caulis (BC), a component herb of KUT, did not reverse abnormal behaviors of isolation-reared mice or increase prefrontal extracellular 5-HT. Furthermore, the antidepressant-like effect of KUT in the forced swim test was prevented by pretreatment with GR127935, a 5-HT1B antagonist. These findings suggest that KUT may ameliorate depressive symptoms via 5-HTergic systems, and that BC plays an important role in the antidepressant-like effects of KUT.

Identification of BMI1 Promoter Inhibitors from Beaumontia murtonii and Eugenia operculata.

B-Cell-specific Moloney murine leukemia virus insertion region 1 (BMI1) is a core component of the polycomb repressive complex 1 (PRC1). Abnormal expression of BMI1 is associated with a number of human malignances and cancer stem cells (CSCs), which cause chemotherapy resistance. Therefore, small molecules that inhibit BMI1 expression are potential candidates for cancer therapy. In this study, a cell-based reporter gene assay was developed that allowed BMI1 promoter activity to be measured in 293T human embryonic kidney cells based on luciferase expression levels. Using this screening assay, the methanol-soluble extracts of Beaumontia murtonii and Eugenia operculata were selected as leads. Bioassay-guided fractionation of the extracts led to the isolation of three known cardenolides (1-3) and one new compound (4) from B. murtonii and two known triterpenoids (5 and 6) and one new compound (7) from E. operculata. These seven compounds inhibited BMI1 promoter activity (IC50 range 0.093-23.0 µM), and the most active compound, wallichoside (1), was further evaluated. Western blot analysis revealed that wallichoside (1) decreases BMI1 protein levels in HCT116 human colon carcinoma cells, and flow cytometry analysis showed that it significantly reduced levels of the CSC biomarker epithelial cell adhesion molecule. Wallichoside (1) also inhibited sphere formation of Huh7 human hepatocellular carcinoma cells, indicating that it diminished the self-renewal capability of CSCs.

Prophylactic Administration of Aucubin Inhibits Paclitaxel-Induced Mechanical Allodynia via the Inhibition of Endoplasmic Reticulum Stress in Peripheral Schwann Cells.

Paclitaxel is a chemotherapeutic agent that causes peripheral neuropathy as its major dose-limiting side effect. However, the peripheral neuropathy is difficult to manage. A study we recently conducted showed that repetitive administration of aucubin as a prophylactic inhibits paclitaxel-induced mechanical allodynia. However, the mechanisms underlying the anti-allodynic activity of aucubin, which is a major component of Plantaginis Semen, was unclear. In addition to mechanical allodynia, aucubin inhibited spontaneous and mechanical stimuli-induced firing in spinal dorsal horn neurons; however, catalpol, a metabolite of aucubin, did not show these effects. Furthermore, paclitaxel induced the expression of CCAAT/enhancer-binding protein homologous protein, a marker of endoplasmic reticulum (ER) stress, in the sciatic nerve and a Schwann cell line (LY-PPB6 cells); however, this effect was inhibited by aucubin. These results suggest that aucubin inhibits paclitaxel-induced mechanical allodynia through the inhibition of ER stress in peripheral Schwann cells.

Cerasoidine, a Bis-aporphine Alkaloid Isolated from Polyalthia cerasoides during Screening for Wnt Signal Inhibitors.

A new bis-aporphine alkaloid, cerasoidine (1), was isolated from the root extract of Polyalthia cerasoides together with the known bis-aporphine bidebiline E (2) during screening for compounds with Wnt signal inhibitory activities. The structure of cerasoidine (1) was established by X-ray analysis and shown by chiral HPLC analyses and electronic circular dichroism to be a 57:43 mixture of R(-)- and S(+)-atropisomers. Bidebiline E (2) exhibited inhibition of transcriptional activity of TCF/ß-catenin with an IC50 value of 20.2 µM and was also found to inhibit Wnt signaling by decreasing nuclear ß-catenin.

Coronaridine, an iboga type alkaloid from Tabernaemontana divaricata, inhibits the Wnt signaling pathway by decreasing ß-catenin mRNA expression.

Four alkaloids, voacangine (1), isovoacangine (2), coronaridine (3), and coronaridine hydroxyindolenine (4), were isolated from the MeOH extract of Tabernaemontana divaricata aerial parts by activity-guided fractionation for Wnt signal inhibitory activity. Compounds 1-4 exhibited TCF/ß-catenin inhibitory activities with IC50 values of 11.5, 6.0, 5.8, and 7.3 µM, respectively. Of these, coronaridine (3) decreased ß-catenin levels in SW480 colon cancer cells, while this decrease in ß-catenin was not suppressed by a co-treatment with 3 and MG132, a proteasome inhibitor. These results suggested that the decrease observed in ß-catenin levels by coronaridine (3) did not depend on a proteasomal degradation process. On the other hand, the treatment of SW480 cells with coronaridine (3) caused a decrease in ß-catenin mRNA levels. Thus, coronaridine (3) may inhibit the Wnt signaling pathway by decreasing the mRNA expression of ß-catenin.

Boehmenan, a lignan from Hibiscus ficulneus, showed Wnt signal inhibitory activity.

The Wnt signal pathway modulates numerous biological processes, and its aberrant activation is related to various diseases. Therefore, inhibition of the Wnt signal may provide an effective (or efficient) strategy for these diseases. Cell-based luciferase assay targeting the Wnt signal (TOP assay) revealed that Hibiscus ficulneus extract inhibited the Wnt signal. The activity-guided isolation of the MeOH extract of H. ficulneus stems yielded four known (1-4) lignans along with myriceric acid (5). Compounds 1-4 potently inhibited the Wnt signal with TOPflash IC50 values of 1.0, 4.5, 6.3, and 1.9 µM, respectively. Compound 1 exhibited cytotoxicity against both Wnt-dependent (HCT116) and Wnt-independent (RKO) cells. Western blot analysis showed that 1 decreased the expression of full, cytosolic and nuclear ß-catenin along with c-myc in STF/293 cells. Our results suggested that 1 may have inhibited the Wnt signal by decreasing ß-catenin levels.

Sesquiterpenes with TRAIL-resistance overcoming activity from Xanthium strumarium.

The ability of TRAIL to selectively induce apoptosis in cancer cells while sparing normal cells makes it an attractive target for the development of new cancer therapy. In search of bioactive natural products for overcoming TRAIL-resistance from natural resources, we previously reported a number of active compounds. In our screening program on natural resources targeting overcoming TRAIL-resistance, activity-guided fractionations of the extract of Xanthium strumarium led to the isolation of five sesquiterpene compounds (1-5). 11α,13-dihydroxanthinin (2) and 11α,13-dihydroxanthuminol (3) were first isolated from natural resources and xanthinosin (1), desacetylxanthanol (4), and lasidiol p-methoxybenzoate (5) were known compounds. All compounds (1-5) showed potent TRAIL-resistance overcoming activity at 8, 20, 20, 16, and 16 µM, respectively, in TRAIL-resistant AGS cells. Compounds 1 and 5 enhanced the levels of apoptosis inducing proteins DR4, DR5, p53, CHOP, Bax, cleaved caspase-3, cleaved caspase-8, and cleaved caspase-9 and also decreased the levels of cell survival protein Bcl-2 in TRAIL-resistant AGS cells in a dose-dependent manner. Compound 1 also enhanced the levels of DR4 and DR5 proteins in a time-dependent manner. Thus, compounds 1 and 5 were found to induce both extrinsic and intrinsic apoptotic cell death. Compound 1 also exhibit TRAIL-resistance overcoming activity in DLD1, DU145, HeLa, and MCF7 cells but did not decrease viability in non-cancer HEK293 cells up to 8 µM.

Prenylated flavonoids and resveratrol derivatives isolated from Artocarpus communis with the ability to overcome TRAIL resistance.

In a screening program on natural products that can abrogate tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) resistance, four new prenylated flavonoid and resveratrol derivatives (1-4) were isolated from Artocarpus communis, together with eight known prenylflavonoids (5-12). The structures of 1-4 were elucidated spectroscopically. Pannokin D [corrected] (1) (2 µM) and artonin E (5) (3 µM) potently exhibited the ability to overcome TRAIL resistance. Artonin E (5) induced caspase-dependent apoptosis in combination with TRAIL, increased caspase 3/7 activity, and enhanced the protein levels of p53 and DR5. Moreover, this substance decreased cell viability in combination with TRAIL and enhanced the protein levels of DR5, and these effects were mediated by increases in the production of ROS (reactive oxygen species). Thus, artonin E (5) was found to induce extrinsic apoptotic cell death by the ROS- and p53-mediated up-regulation of DR5 expression in AGS cells.

9-Hydroxycanthin-6-one, a ß-Carboline Alkaloid from Eurycoma longifolia, Is the First Wnt Signal Inhibitor through Activation of Glycogen Synthase Kinase 3ß without Depending on Casein Kinase 1α.

Wnt signaling regulates various processes such as cell proliferation, differentiation, and embryo development. However, numerous diseases have been attributed to the aberrant transduction of Wnt signaling. We screened a plant extract library targeting TCF/ß-catenin transcriptional modulating activity with a cell-based luciferase assay. Activity-guided fractionation of the MeOH extract of the E. longifolia root led to the isolation of 9-hydroxycanthin-6-one (1). Compound 1 exhibited TCF/ß-catenin inhibitory activity. Compound 1 decreased the expression of Wnt signal target genes, mitf and zic2a, in zebrafish embryos. Treatment of SW480 cells with 1 decreased ß-catenin and increased phosphorylated ß-catenin (Ser 33, 37, Tyr 41) protein levels. The degradation of ß-catenin by 1 was suppressed by GSK3ß-siRNA, while compound 1 decreased ß-catenin even in the presence of CK1α-siRNA. These results suggest that 1 inhibits Wnt signaling through the activation of GSK3ß independent of CK1α.

Scopadulciol, Isolated from Scoparia dulcis, Induces ß-Catenin Degradation and Overcomes Tumor Necrosis Factor-Related Apoptosis Ligand Resistance in AGS Human Gastric Adenocarcinoma Cells.

Scopadulciol (1), a scopadulan-type diterpenoid, was isolated from Scoparia dulcis along with three other compounds (2-4) by an activity-guided approach using the TCF reporter (TOP) luciferase-based assay system. A fluorometric microculture cytotoxicity assay (FMCA) revealed that compound 1 was cytotoxic to AGS human gastric adenocarcinoma cells. The treatment of AGS cells with 1 decreased ß-catenin levels and also inhibited its nuclear localization. The pretreatment of AGS cells with a proteasome inhibitor, either MG132 or epoxomicin, protected against the degradation of ß-catenin induced by 1. The 1-induced degradation of ß-catenin was also abrogated in the presence of pifithrin-α, an inhibitor of p53 transcriptional activity. Compound 1 inhibited TOP activity in AGS cells and downregulated the protein levels of cyclin D1, c-myc, and survivin. Compound 1 also sensitized AGS cells to tumor necrosis factor-related apoptosis ligand (TRAIL)-induced apoptosis by increasing the levels of the death receptors, DR4 and DR5, and decreasing the level of the antiapoptotic protein Bcl-2. Collectively, our results demonstrated that 1 induced the p53- and proteasome-dependent degradation of ß-catenin, which resulted in the inhibition of TCF/ß-catenin transcription in AGS cells. Furthermore, 1 enhanced apoptosis in TRAIL-resistant AGS when combined with TRAIL.

Calotropin: a cardenolide from calotropis gigantea that inhibits Wnt signaling by increasing casein kinase 1α in colon cancer cells.

Wnt signaling plays key roles in embryonic development and various human diseases. Activity-guided testing to isolate Wnt signaling inhibitors from the methanol extract of Calotropis gigantea (Asclepiadaceae) exudutes identified six Wnt inhibitory cardenolides (1-6), of which 1, 3, 5, and 6 exhibited potent TCF/ß-catenin inhibitory activities (IC50 0.7-3.6 nM). Calotropin (1) inhibited Wnt signaling by decreasing both nuclear and cytosolic ß-catenin in a dose-dependent manner, and promoted degradation of ß-catenin by increasing the phosphorylation of ß-catenin at Ser45 through casein kinase 1α (CK1α). Moreover, 1 significantly increased CK1α protein and mRNA levels. The results suggest that 1 inhibits the Wnt signaling pathway by increasing CK1α protein levels. To the best of our knowledge, calotropin is the first small molecule to increase CK1α levels.

Ricinine: a pyridone alkaloid from Ricinus communis that activates the Wnt signaling pathway through casein kinase 1α.

Wnt signaling plays important roles in proliferation, differentiation, development of cells, and various diseases. Activity-guided fractionation of the MeOH extract of the Ricinus communis stem led to the isolation of four compounds (1-4). The TCF/ß-catenin transcription activities of 1 and 3 were 2.2 and 2.5 fold higher at 20 and 30µM, respectively. Cells treated with ricinine (1) had higher ß-catenin and lower of p-ß-catenin (ser 33, 37, 45, Thr 41) protein levels, whereas glycogen synthase kinase 3ß (GSK3ß) and casein kinase 1α (CK1α) protein levels remained unchanged. Cells treated with pyrvinium, an activator of CK1α, had lower ß-catenin levels. However, the combined treatment of pyrvinium and 1 led to higher ß-catenin levels than those in cells treated with pyrvinium alone, which suggested that 1 inhibited CK1α activity. Furthermore, 1 increased ß-catenin protein levels in zebrafish embryos. These results indicated that 1 activated the Wnt signaling pathway by inhibiting CK1α.

Chromomycins A2 and A3 from marine actinomycetes with TRAIL resistance-overcoming and Wnt signal inhibitory activities.

A biological screening study of an actinomycetes strain assembly was conducted using a cell-based cytotoxicity assay. The CKK1019 strain was isolated from a sea sand sample. Cytotoxicity-guided fractionation of the CKK1019 strain culture broth, which exhibited cytotoxicity, led to the isolation of chromomycins A2 (1) and A3 (2). 1 and 2 showed potent cytotoxicity against the human gastric adenocarcinoma (AGS) cell line (IC50 1; 1.7 and 2; 22.1 nM), as well as strong inhibitory effects against TCF/ß-catenin transcription (IC50 1; 1.8 and 2; 15.9 nM). 2 showed the ability to overcome tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) resistance. To the best of our knowledge, the effects of chromomycins A2 (1) and A3 (2) on TRAIL resistance-overcoming activity, and on the Wnt signaling pathway, have not been reported previously. Thus, 1 and 2 warrant potential drug lead studies in relation to TRAIL-resistant and Wnt signal-related diseases and offer potentially useful chemical probes for investigating TRAIL resistance and the Wnt signaling pathway.

Metabolomics analysis of peony root using NMR spectroscopy and impact of the preprocessing method for NMR data in multivariate analysis.

Peony root is an important herbal drug used as an antispasmodic analgesic. To evaluate peony roots with different botanical origins, producing areas, and post-harvest processing, 1H NMR-based metabolomics analysis was employed. Five types of monoterpenoids, including albiflorin (4), paeoniflorin (6), and sulfonated paeoniflorin (25), and six other compounds, including 1,2,3,4,6-penta-O-galloyl-ß-D-glucose (18), benzoic acid (21), gallic acid (22), and sucrose (26) were detected in the extracts of peony root samples. Among them, compounds 4, 6, 18, and total monoterpenoids including 21 were quantified by quantitative 1H NMR (qHNMR). Compound 25 was detected in 1H NMR spectra of sulfur-fumigated white peony root (WPR) extracts indicating that 1H NMR was a fast and effective method for identifying sulfur-fumigated WPR. The content of 26, the main factor affecting extract yield, increased significantly in peony root after low-temperature storage for one month, whereas that in WPR did not increase due to the boiling treatment after harvesting. We investigated the impact of preprocessing methods to such analysis for NMR data from commercial samples, resulting that the data matrix transformed from qHNMR spectra and normalized to internal standard were optimum for multivariate analysis. The multivariate analysis demonstrated that among commercial samples derived from P. lactiflora, peony root samples in Japanese market (PR) had high contents of 18 and 22, and red peony root (RPR) samples had high content of monoterpenoids represented by 6; and among RPR samples, those derived from P. veitchii showed higher contents of 18 and 22 than those from P. lactiflora. The 1H NMR-based metabolomics method coupled with qHNMR was useful for evaluation of peony root and would be applicable for other crude drugs.

Quality assessment of Rheum species cultivated in Japan by focusing on M2 polarization of microglia.

In traditional Japanese medicine, Rhei Rhizoma is used as a purgative, blood stasis-resolving and antipsychotic drug. The latter two properties are possibly related to anti-inflammatory effects. Microglia regulate inflammation in the central nervous system. M1 microglia induce inflammation, while M2 microglia inhibit inflammation and show neurotrophic effects. This study investigated the effects from water extracts of roots of cultivated Rheum species in Nagano Prefecture, Japan (strain C, a related strain to a Japanese cultivar, 'Shinshu-Daio'; and strain 29, a Chinese strain) and 3 kinds of Rhei Rhizoma available in the Japanese market, and also examined their constituents on the polarization of cultured microglia. All extracts significantly decreased M1 microglia, and strains C and 29 significantly increased M2 microglia. Furthermore, the extracts of both strains significantly increased the M2/M1 ratio. Among the constituents of Rhei Rhizoma, ( +)-catechin (2), resveratrol 4'-O-ß-D-(6″-O-galloyl) glucopyranoside (5), isolindleyin (8), and physcion (15) significantly increased the M2/M1 ratio. The contents of the constituents in water extract of each strain were quantified using HPLC. The extracts of strains C and 29 contained relatively large amounts of 2 and 5; and 2, 8, and 15, respectively. This study showed the water extracts of roots of cultivated Rheum strains in Japan had the effects of M2 polarization of microglia, suggesting that these strains become the candidate to develop anti-inflammatory Rhei Rhizoma. Moreover, the suitable chemical composition to possess anti-inflammatory activity in the brain was clarified for the future development of new type of Rhei Rhizoma.

The extract based on the Kampo formula daikenchuto (Da Jian Zhong Tang) induces Bdnf expression and has neurotrophic effects in cultured cortical neurons.

Reductions in brain-derived neurotrophic factor (BDNF) expression levels have been reported in the brains of patients with neurological disorders such as Alzheimer's disease. Therefore, upregulating BDNF and preventing its decline in the diseased brain could help ameliorate neurological dysfunctions. Accordingly, we sought to discover agents that increase Bdnf expression in neurons. Here, we screened a library of 42 Kampo extracts to identify those with the ability to induce Bdnf expression in cultured cortical neurons. Among the active extracts identified in the screen, we focused on the extract based on the Kampo formula daikenchuto. The extract of daikenchuto in the library used in this study was prepared using the mixture of Zingiberis Rhizoma Processum (ZIN), Zanthoxyli Piperiti Pericarpium (ZAN), and Ginseng Radix (GIN) without Koi. In this study, we defined DKT as the mixture of ZIN, ZAN, and GIN without Koi (DKT extract means the extract prepared from the mixture of ZIN, ZAN, and GIN without Koi). DKT extract significantly increased endogenous Bdnf expression by mediated, at least in part, via Ca2+ signaling involving L-type voltage-dependent Ca2+ channels in cultured cortical neurons. Furthermore, DKT extract significantly improved the survival of cultured cortical neurons and increased neurite complexity in immature neurons. Taken together, our findings suggest that DKT extract induces Bdnf expression and has a neurotrophic effect in neurons. Because BDNF inducers are expected to have therapeutic potential for neurological disorders, re-positioning of Kampo formulations such as daikenchuto may lead to clinical application in diseases associated with reduced BDNF in the brain.

Anti-Trypanosoma cruzi activity of Coptis rhizome extract and its constituents.

BACKGROUND: Current therapeutic agents, including nifurtimox and benznidazole, are not sufficiently effective in the chronic phase of Trypanosoma cruzi infection and are accompanied by various side effects. In this study, 120 kinds of extracts from medicinal herbs used for Kampo formulations and 94 kinds of compounds isolated from medicinal herbs for Kampo formulations were screened for anti-T. cruzi activity in vitro and in vivo. METHODS: As an experimental method, a recombinant protozoan cloned strain expressing luciferase, namely Luc2-Tulahuen, was used in the experiments. The in vitro anti-T. cruzi activity on epimastigote, trypomastigote, and amastigote forms was assessed by measuring luminescence intensity after treatment with the Kampo extracts or compounds. In addition, the cytotoxicity of compounds was tested using mouse and human feeder cell lines. The in vivo anti-T. cruzi activity was measured by a murine acute infection model using intraperitoneal injection of trypomastigotes followed by live bioluminescence imaging. RESULTS: As a result, three protoberberine-type alkaloids, namely coptisine chloride, dehydrocorydaline nitrate, and palmatine chloride, showed strong anti-T. cruzi activities with low cytotoxicity. The IC50 values of these compounds differed depending on the side chain, and the most effective compound, coptisine chloride, showed a significant effect in the acute infection model. CONCLUSIONS: For these reasons, coptisine chloride is a hit compound that can be a potential candidate for anti-Chagas disease drugs. In addition, it was expected that there would be room for further improvement by modifying the side chains of the basic skeleton.

Metabolite profiling of Drynariae Rhizoma using 1H NMR and HPLC coupled with multivariate statistical analysis.

Drynariae Rhizoma has been used to treat bone diseases and kidney deficiency in traditional medicine. Recently its aqueous extract was reported to enhance memory function. Although the Japanese standards for non-Pharmacopoeial crude drugs 2022 prescribed Drynaria roosii as the botanical origin, some counterfeits and both raw and stir-fired crude drugs are available in markets. To distinguish Drynariae Rhizoma derived from D. roosii appropriately from others and verify the validity of uses of stir-fried ones, 1H NMR-based metabolite profiling coupled with HPLC were performed. Raw samples derived from D. roosii contained naringin (1), neoeriocitrin (2), 5,7-dihydroxychromone-7-O-neohesperidoside (3), caffeic acid 4-O-ß-D-glucoside (4), protocatechuic acid (5), trans-p-coumaric acid 4-O-ß-D-glucoside (6), and kaempferol 3-O-α-L-rhamnoside 7-O-ß-D-glucoside (8). Stir-fried samples were characterized by presence of 5-hydroxymethyl-2-furaldehyde (13), and were divided into two types; one possessing similar composition to raw samples (Type I) and another without above components except 5 (Type II). Quantitative analyses using qHNMR and HPLC, followed by principal component analysis demonstrated that the raw samples had higher contents of 1 (0.93-9.86 mg/g), 2 (0.74-7.59 mg/g), 3 (0.05-2.48 mg/g), 4 (0.27-2.51 mg/g), 6 (0.14-1.26 mg/g), and 8 (0.04-0.52 mg/g), and Type II had a higher content of 5 (0.84-1.32 mg/g). The counterfeit samples derived from Araiostegia divaricata var. formosana were characterized by higher content of ( -)-epicatechin 3-O-ß-D-allopyranoside (10) (1.44-11.49 mg/g) without 1 and 2. These results suggested that Drynariae Rhizoma samples derived from other botanical origins and Type II stir-fried samples cannot substitute for D. roosii rhizome.