Kazufumi Toume

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-β-d-glucoside (4), protocatechuic acid (5), trans-p-coumaric acid 4-O-β-d-glucoside (6), and kaempferol 3-O-α-l-rhamnoside 7-O-β-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-β-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.

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-β-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.

Peony root is an important herbal drug used as an antispasmodic analgesic. To evaluate peony roots with different botanical origin, 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-β-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.

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.

Essential oils (EOs) comprised of various bioactive compounds have been widely detected in the Curcuma species. Due to the widespread distribution and misidentification of Curcuma species and differences in processing methods, inconsistent reports on major compounds in rhizomes of the same species from different geographical regions are not uncommon. This inconsistency leads to confusion and inaccuracy in compound detection of each species and also hinders comparative study based on EO compositions. The present study aimed to characterize EO compositions of 12 Curcuma species, as well as to detect the compositional variation among different species, and between the plant specimens and their related genetically validated crude drug samples using headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry. The plant specimens of the same species showed similar EO patterns, regardless of introducing from different geographical sources. Based on the similarity of EO compositions, all the specimens and samples were separated into eight main groups: C. longa; C. phaeocaulis, C. aeruginosa and C. zedoaria; C. zanthorrhiza; C. aromatica and C. wenyujin; C. kwangsiensis; C. amada and C. mangga; C. petiolata; C. comosa. From EOs of all the specimens and samples, 54 major compounds were identified, and the eight groups were chemically characterized. Most of the major compounds detected in plant specimens were also observed in crude drug samples, although a few compounds converted or degraded due to processing procedures or over time. Orthogonal partial least squares-discriminant analysis allowed the marker compounds to discriminate each group or each species to be identified.

Fibroblast growth factor 21 (FGF21), which is mainly synthesized and secreted by the liver, plays a crucial role in systemic glucose and lipid metabolism, ameliorating metabolic diseases. In this study, we screened the WAKANYAKU library derived from medicinal herbs to identify compounds that can activate Fgf21 expression in mouse hepatocyte AML12 cells. We identified Scutellaria baicalensis root extract and one of its components, wogonin, as an activator of Fgf21 expression. Wogonin also enhanced the expression of activating transcription factor 4 (ATF4) by a mechanism other than ER stress. Knockdown of ATF4 by siRNA suppressed wogonin-induced Fgf21 expression, highlighting its essential role in wogonin’s mode of action. Thus, our results indicate that wogonin would be a strong candidate for a therapeutic to improve metabolic diseases by enhancing hepatic FGF21 production.

Investigation of the non-polar (rich in chlorophyll) and polar fractions of the ethyl acetate extract of Gymnosporia senegalensis (Lam.) Loes. leaves led to the isolation of nine compounds, including one new megastigmane derivative, (3 R*,5 S*,6 R*,7 E,9ξ)-7-megastigmene-3,6,9-triol-3- O-β-D-(6′- O-galloyl)glucopyranoside (1), four known galloylated derivatives (2, 3, 8, 9), three known chlorophyll derivatives (4–6), and one known fatty acid (7). The structures of the isolates were determined by means of spectroscopic and spectrometric data, as well as by comparison with literature values. The extracts and isolated compounds were tested for their antibacterial, antifungal, and antioxidant activities. The isolates showed weak to high antimicrobial activities with phaeophytin A (4) being the most active one whereas phaeophorbide-a (5) and Chlorine e₆ trimethyl ester (6) exhibited the highest antioxidant activities.

Aim: Massa Medicata Fementata(MMF) is a crude drug used in East Asia to treat anorexia and dyspepsia. It is prepared from wheat and several herbs through microbial fermentation usingAspergillussp. andRhizopussp. There is great difference in the quality of commercial MMF, and the microbes of MMF are suggested to affect its quality. We investigated the effects of microbial fermentation on the quality of MMF.Methods: Raw materials of MMF were mixed according to the ratio listed in the National Standard for Chinese Patent Drugs, and MMF was prepared using pure cultures ofAspergillus oryzaeorRhizopus oryzae. Digestive enzyme activities (α‐amylase, protease, and lipase) and volatile compounds were measured using an analytical kit and GC–MS, respectively.Results: Enzyme activity increased in MMF. MMF prepared withA.oryzae(MMF‐A) showed higher α‐amylase and lipase activities than that prepared withR.oryzae(MMF‐R). Protease activity was marginally higher in MMF‐R than in MMF‐A. GC–MS analysis revealed that terpenoids decreased with fermentation; however, 2,3‐butanediol, acetoin, and guaiacol were detected in MMF only. C8 compounds such as 1‐octen‐3‐ol were higher in MMF‐A than MMF‐R; however, aromatic compounds such as 4‐vinylguaiacol and pyrazines were higher in MMF‐R than MMF‐A.Conclusion: Microbial fermentation contributes to increased enzyme activity and changes in MMF volatiles. These properties of MMF were considerably affected by the microbes used, and it is proposed in this study that it is important to have microbial control in the production of commercial MMF.

Three phenylpropanoid-conjugated iridoid glucosides, acetylgaertneric acid (1), acetyldehydrogaertneroside (2), and dehydrogaertneric acid (10), together with nine known related iridoid glucosides (3–9, 11, and 12), two coumaroyl alkaloids, one benzenoid, and three flavonoid glucosides were isolated from leaves of Morinda morindoides (Rubiaceae). Structures of these isolated compounds were determined using spectroscopic analysis. Compounds 1–18 and previously isolated compounds (19–29) were evaluated for anti-trypanosomal activity against Trypanosoma cruzi Tulahuen strain (trypomastigote and amastigote) together with cytotoxicity against host cells, new-born mouse heart cells. Among them, molucidin (21) and prismatomerin (22) exhibited good anti-trypanosomal activity (IC₅₀ of 4.67 and 5.70 µM, respectively), together with cytotoxicity (CC₅₀ of 2.76 and 3.22 μM, respectively). Compounds 1–18 did not show anti-malarial activity against a chloroquine/mefloquine-sensitive strain of Plasmodium falciparum.

Two phenylpropanoid-conjugated iridoids, deglucosyl gaertneroside (1) and morindoidin (2), were isolated from the leaves of Morinda morindoides (Rubiaceae) by activity-guided fractionation using an anti-malarial activity assay. The known related iridoids molucidin (3) and prismatomerin (4), two lignans, abscisic acid, two megastigmanes, and two flavonol glycosides were also identified. The structures of isolated compounds were elucidated using spectroscopic analysis. The isolated compounds were evaluated for anti-malarial activity against the chloroquine/mefloquine-sensitive strains of Plasmodium falciparum together with cytotoxicity against adult mouse brain cells. Potent anti-malarial activity of 3 and 4 (IC₅₀ of 0.96 and 0.80 μM, CC₅₀ of 1.02 and 0.88 μM, and SI of 1.06 and 1.10, respectively) was shown, while new iridoids 1 and 2 and pinoresinol (5) displayed moderate activity (IC₅₀ of 40.9, 20.6, and 24.2 μM) without cytotoxicity (CC₅₀ > 50 μM). These results indicate that 1–5 may be promising lead compounds for anti-malarial drugs. In addition, our results imply the necessity of the quality control of the extract of M. morindoides leaves based on the contents of 1–5 in terms of the safety and efficacy.