Involvement of cardiac glycosides targeting Na/K-ATPase in their inhibitory effects on c-Myc expression via its transcription, translation and proteasomal degradation.

Cardiac glycosides (CGs) have been used for decades to treat heart failure and arrhythmic diseases. Recent non-clinical and epidemiological findings have suggested that CGs exhibit anti-tumor activities. Therefore, CGs may be repositioned as drugs for the treatment of cancer. A detailed understanding of the anti-cancer mechanisms of CGs is essential for their application to the treatment of targetable cancer types. To elucidate the factors associated with the anti-tumor effects of CGs, we performed transcriptome profiling on human multiple myeloma AMO1 cells treated with periplocin, one of the CGs. Periplocin significantly down-regulated the transcription of MYC (c-Myc), a well-established oncogene. Periplocin also suppressed c-Myc expression at the protein levels. This repression of c-Myc was also observed in several cell lines. To identify target proteins for the inhibition of c-Myc, we generated CG-resistant (C9) cells using a sustained treatment with digoxin. We confirmed that C9 cells acquired resistance to the inhibition of c-Myc expression and cell proliferation by CGs. Moreover, the sequencing of genomic DNA in C9 cells revealed the mutation of D128N in α1-Na/K-ATPase, indicating the target protein. These results suggest that CGs suppress c-Myc expression in cancer cells via α1-Na/K-ATPase, which provides further support for the anti-tumor activities of CGs.

Corrigendum: Identification of an alternative glycyrrhizin metabolite causing liquorice-induced pseudohyperaldosteronism and the development of ELISA system to detect the predictive biomarker.

[This corrects the article DOI: 10.3389/fphar.2021.688508.].

Suppressive effects of processed aconite root on dexamethasone-induced muscle ring finger protein-1 expression and its active ingredients.

Processed aconite root (PA), the tuberous root of Aconitum carmichaelii prepared by autoclaving, is a crude drug used in Japanese traditional Kampo medicine and traditional Chinese medicine for the symptoms of kidney deficiency, that is related to the muscle atrophy in modern medicine. The objective of the present study is to evaluate the effectiveness of PA on muscle atrophy and to find its active ingredients using dexamethasone-induced muscle ring finger protein-1 (MuRF1) mRNA expression in murine myoblast C2C12 cells. Dexamethasone-induced MuRF1 expression was significantly suppressed by methanol-soluble part of boiling water extract of PA in a concentration-dependent manner with its IC50 value of 1.5 mg/ml. By the activity-guided fractionations of PA extract using the partition between organic solvents and its aqueous solution, the activity of PA did not transfer into the fraction containing aconitine-type diterpenoid alkaloids but into BuOH layer. Then, we found higenamine and salsolinol as the active ingredients in PA. Higenamine and salsolinol significantly suppressed dexamethasone-induced MuRF1 expression, and their IC50 values were 0.49 and 50 µM, respectively. The contents of higenamine and salsolinol in the decoctions of commercially available fourteen PA products are 0.12 and 14 µg/ml as the average values, and varied with the coefficient of variation (CV) values of 97 and 63%, respectively. Higenamine also significantly suppressed dexamethasone-induced mRNA expressions of muscle atrophy F-box protein (MAFbx)/atrogin1, casitas B-lineage lymphoma-b (Cbl-b), troponin, branched-chain amino acid aminotransferase 2 (BCAT2), and Bcl-2 binding and pro-apoptotic protein3 (Bnip3). Although the quality control of PA is regulated by the contents of diterpene alkaloids, salsolinol and higenamine can be used as the marker compounds to certificate the pharmacological activities of PA.

Identification of an Alternative Glycyrrhizin Metabolite Causing Liquorice-Induced Pseudohyperaldosteronism and the Development of ELISA System to Detect the Predictive Biomarker.

Liquorice is usually used as crude drug in traditional Japanese Kampo medicine and traditional Chinese medicine. Liquorice-containing glycyrrhizin (GL) can cause pseudohyperaldosteronism as a side effect. Previously, we identified 18ß-glycyrrhetyl-3-O-sulfate (3) as a GL metabolite in Eisai hyperbilirubinuria rats (EHBRs) with the dysfunction of multidrug resistance-related protein (Mrp2). We speculated that 3 was associated with the onset of liquorice-induced pseudohyperaldosteronism, because it was mainly detected in serum of patients with suspected to have this condition. However, it is predicted that other metabolites might exist in the urine of EHBRs orally treated with glycyrrhetinic acid (GA). We explored other metabolites in the urine of EHBRs, and investigated the pharmacokinetic profiles of the new metabolite in EHBRs and normal Sprague-Dawley rats. We further analyzed the serum concentrations of the new metabolite in the patients of pseudohyperaldosteronism. Finally, we developed the analyzing method of these metabolites as a preventive biomarker for the onset of pseudohyperaldosteronism using an enzyme-linked immunosorbent assay (ELISA). We isolated a new GL metabolite, 18ß-glycyrrhetyl-3-O-sulfate-30-O-glucuronide (4). Compound 4 significantly inhibited rat type-2 11ß-hydroxysteroid dehydrogenase (11ß-HSD2) and was a substrate of both organic anion transporter (OAT) 1 and OAT3. Compound 4 was also detected in the serum of patients with suspected pseudohyperaldosteronism at an approximately 10-fold lower concentrations than 3, and these concentrations were positively correlated. Compound 4 showed a lower serum concentration and weaker inhibitory titer on 11ß-HSD2 than 3. We developed an enzyme-linked immunosorbent assay system using an anti-18ß-glycyrrhetyl-3-O-glucuronide (3MGA) monoclonal antibody to measure the serum concentration of 3 to facilitate the measurement of biomarkers to predict the onset of pseudohyperaldosteronism. Although we found 4 as the secondary candidate causative agent, 3 could be the main potent preventive biomarker of liquorice-induced pseudohyperaldosteronism. Compound 3 was detected in serum at a higher concentration than GA and 4, implying that 3 may be a pharmacologically active ingredient mediating not only the development of pseudohyperaldosteronism but anti-inflammatory effects in humans administered GL or other liquorice-containing preparations.

Binding activity of Valeriana fauriei root extract on GABAA receptor flunitrazepam sites and distribution of its active ingredients in the brain of mice - A comparison with that of V. officinalis root.

ETHNOPHARMACOLOGICAL RELEVANCE: Valeriana fauriei root (VF) is a crude drug registered in the Japanese Pharmacopeia 17th Edition and a known substitute for V. officinalis (VO). Although VO has been pharmacologically evaluated for its sedative effects and mechanism of action, data regarding VF remain scarce. AIM OF THE STUDY: We compared the binding affinity of VF and VO extracts, as well as examined the active ingredients in the VF extract, on flunitrazepam sites of γ-aminobutyric acid receptor type A (GABAA receptor). Furthermore, we confirmed whether these active ingredients were distributed in the brain of mice orally administered the VF extract. MATERIALS AND METHODS: We prepared the assay system to evaluate the binding activity of flunitrazepam sites of GABAA receptor using a 96-well plate and assessed the activities of VF and VO extracts. We then analyzed their constituents using HPLC with principal component analysis (PCA) and evaluated active ingredients correlated with their activities. The distribution of active ingredients in the plasma and brain of mice orally administered the VF extract prepared with different emulsifiers were analyzed by LC-MS/MS. RESULTS: The ethanol extract of VF exhibited significantly higher activity on flunitrazepam sites of GABAA receptor than VO. For the VF extract, kessyl glycol diacetate (KGD) was markedly associated with the binding activities; however, active ingredients included KGD, kessyl glycol 8-acetate (KG8), α-kessyl acetate (α-KA), and coniferyl isovalerate (CI). For VO, valerenic acid and five other compounds were associated with the binding affinity on flunitrazepam sites of GABAA receptor. On emulsifying the VF extract with a fat-soluble glycerin fatty acid ester, the plasma and brain distributions of KGD tended to be higher, those of KG8 were significantly more than 10-times higher, and those of α-KA was lower than those of the VF extract emulsified with water-soluble gum arabic, after oral administration in mice. CONCLUSIONS: Based on the binding activity on flunitrazepam sites of GABAA receptor and brain distribution, KGD, KG8, and α-KA can be considered active ingredients of VF. The addition of a fat-soluble emulsifier promoted the absorption of KGD, the main active ingredient, and KGD was metabolized to KG8 in the body. The present results suggest a possible mechanism underlying the sedative effect for VF, and these three compounds can be used as marker compounds to evaluate the quality of VF products.

Periplocin and cardiac glycosides suppress the unfolded protein response.

The unfolded protein response (UPR) controls protein homeostasis through transcriptional and translational regulation. However, dysregulated UPR signaling has been associated with the pathogenesis of many human diseases. Therefore, the compounds modulating UPR may provide molecular insights for these pathologies in the context of UPR. Here, we screened small-molecule compounds that suppress UPR, using a library of Myanmar wild plant extracts. The screening system to track X-box binding protein 1 (XBP1) splicing activity revealed that the ethanol extract of the Periploca calophylla stem inhibited the inositol-requiring enzyme 1 (IRE1)-XBP1 pathway. We isolated and identified periplocin as a potent inhibitor of the IRE1-XBP1 axis. Periplocin also suppressed other UPR axes, protein kinase R-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6). Examining the structure-activity relationship of periplocin revealed that cardiac glycosides also inhibited UPR. Moreover, periplocin suppressed the constitutive activation of XBP1 and exerted cytotoxic effects in the human multiple myeloma cell lines, AMO1 and RPMI8226. These results reveal a novel suppressive effect of periplocin or the other cardiac glycosides on UPR regulation, suggesting that these compounds will contribute to our understanding of the pathological or physiological importance of UPR.

Isolation and characterization of a novel glucosyltransferase involved in production of emodin-6-O-glucoside and rhaponticin in Rheum palmatum.

Anthraquinones are widely distributed in various organisms and known as bioactive ingredients. Some of the anthraquinones accumulate as glycosides in higher plants. Plant secondary product glycosyltransferases (PSPGs) are the well-characterized enzymes producing plant secondary metabolite glycosides. However, PSPGs involved in the formation of anthraquinone glycosides remains unclear. The rhizome of Rheum palmatum contains anthraquinones as laxative agents, some of which are accumulated as glucosides. We isolated a glucosyltransferase, R. palmatum UDP-glycosyltransferase (RpUGT) 1 from the rhizome of R. palmatum, and characterized functionally. RpUGT1 glucosylated emodin yielding emodin-6-O-glucoside, and it also glucosylated rhapontigenin, a compound belonging to stilbenes, yielding rhaponticin. The expression patterns of RpUGT1 and the accumulation of the metabolites revealed that RpUGT1 contributes to the production of these glucosides in R. palmatum. These results may provide important information for the substrate recognition of the PSPGs for anthraquinones and stilbenes.

Honey isomaltose contributes to the induction of granulocyte-colony stimulating factor (G-CSF) secretion in the intestinal epithelial cells following honey heating.

We have previously discovered that heated honey but not unheated honey could induce the secretion of granulocyte-colony stimulating factor (G-CSF) in the MCE301 intestinal epithelial cells. The objective of this study was to identify compounds in honey that could contribute to this activity. We bought several kinds of commercial honey samples derived from different flowers, as well as corn syrup samples, in the markets of China and Japan, and heated them at 180 °C for 30 min. MCE301 cells were treated with the medium containing the samples, and G-CSF levels in the medium were measured by ELISA. By comparing their activities and sugar contents, we discovered that isomaltose was primarily implicated. The optimum heating conditions for isomaltose were at 180 °C for 60 min or at 200 °C for 15-30 min, and these time- and temperature-dependencies were similar to those of honey in our previous study. When heated isomaltose was partitioned by dialysis, the active ingredients were transferred into a high-molecular-weight fraction. By size-exclusion HPLC analysis, the average molecular weight of heated isomaltose was 790 kDa. When heated isomaltose was hydrolyzed by acids, glucose was subsequently produced. Maltose, sucrose, turanose, and trehalose did not exhibited any activity when heated at 180 °C for 60 min, indicating that the glucose groups with α(1 → 6)-binding in the isomaltose molecule play important roles in its activity when oxidatively polymerized by heat. The stimulating activity of heated isomaltose was inhibited by toll-like receptor 4 (TLR4) inhibitor, suggesting that heated isomaltose activates TLR4 to induce G-CSF. Since G-CSF is clinically used for cancer patients to accelerate their recovery from neutropenia following chemotherapy or accompanied with aplastic anemia, these findings indicate that honey which contains high level of isomaltose could improve immunosuppressive conditions when honey is heated, and that heated isomaltose might be of potential therapeutic use in patients with compromised immunity caused by chemotherapeutic agents.

Decaturenol A and known oxalicine related meroterpenoids isolated from Penicillium decaturense RO050, and their new biological activities.

Decaturenol A (1), a new oxalicine related meroterpenoid, has been isolated from Penicillium decaturense RO050 along with seven known compounds (2-8). The structure of 1 was elucidated by spectroscopic data. The effects of isolated compounds (1-8) on endoplasmic reticulum (ER) stress-induced cell death in HT22 hippocampal nerve cells and on the interleukin 10 (IL-10)-induced expression of CD163, a M2 phenotype marker, in human monocyte-derived macrophages were evaluated. While decaturenol A (1) exhibited a protective effect on ER stress-induced cell death in HT22 cells at 10 µM, on the other hand oxalicine A (7) showed cytotoxic activity (IC50 = 5.9 µM). Additionally, decaturenol A (1), decaturins D (2), E (3), and B (4) inhibited the IL-10-induced expression of CD163 each at a concentration of 20 µg/mL.

Neoline, an active ingredient of the processed aconite root in Goshajinkigan formulation, targets Nav1.7 to ameliorate mechanical hyperalgesia in diabetic mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Goshajinkigan (GJG), a traditional Japanese Kampo formula, has been shown to exhibit several pharmacological actions, including antinociceptive effects. Processed aconite root (PA), which is considered to be an active ingredient of GJG, has also been demonstrated to have an ameliorative effect on pain, such as diabetic peripheral neuropathic pain. We recently identified neoline as the active ingredient of both GJG and PA that is responsible for its effects against oxaliplatin-induced neuropathic pain in mice. AIM OF THE STUDY: In the present study, we investigated whether GJG, PA, and neoline could inhibit Nav1.7 voltage-gated sodium channel (VGSC) current and whether neoline could ameliorate mechanical hyperalgesia in diabetic mice. MATERIALS AND METHODS: To assess the electrophysiological properties of GJG extract formulation, powdered PA, and neoline on Nav1.7 VGSCs, whole-cell patch clamp recording was performed using human HEK293 cells expressing Nav1.7 VGSCs. In addition, the ameliorative effects of neoline on diabetic peripheral neuropathic pain were evaluated using the von Frey test in streptozotocin (STZ)-induced diabetic model mice. RESULTS: GJG extract formulation significantly inhibited Nav1.7 VGSC peak current. Powdered PA also inhibited Nav1.7 VGSC peak current. Like GJG and PA, neoline could inhibit Nav1.7 VGSC current. When diabetic mice were treated with neoline by intraperitoneal acute administration, the mechanical threshold was increased in diabetic mice, but not in non-diabetic mice, in a behavioral study. CONCLUSION: These results suggest that neoline might be a novel active ingredient of GJG and PA that is one of responsible ingredients for ameliorating mechanical hyperalgesia in diabetes via the inhibition of Nav1.7 VGSC current at least.

Mariannamides A and B, new cyclic octapeptides isolated from Mariannaea elegans NBRC102301.

Two new cyclic octapeptides, mariannamides A (1) and B (2), have been isolated from Mariannaea elegans NBRC102301, a Pinus densiflora-derived filamentous fungus. Their structures were elucidated to be cyclo-(l-Leu1-l-Pro1-l-Pro2-l-Leu2-l-Ile1-l-Pro3-l-Val1-l-Ile2) and cyclo-(l-Leu1-l-Pro1-l-Pro2-l-Leu2-l-Ile1-l-Pro3-l-Val1-l-Val2) based on spectroscopic data and Marfey's method. Mariannamide A (1) promoted mRNA expression of sirtuin 1 (SIRT1) in C2C12 cells, a mouse skeletal muscle myoblast cell line, and showed the antimicrobial activity against Escherichia coli and Cryptococcus neoformans.

Identification of glycyrrhizin metabolites in humans and of a potential biomarker of liquorice-induced pseudoaldosteronism: a multi-centre cross-sectional study.

Liquorice [main ingredient, glycyrrhizin (GL)] is widely used as a food sweetener and herbal medicine. Occasionally, liquorice consumption causes pseudoaldosteronism as a side effect which causes oedema, hypokalaemia, and hypertension due to hyperactivity of mineral corticoid receptor. We aimed to detect GL metabolites in human blood and urine samples and to determine the pathological relationship between GL metabolites and pseudoaldosteronism. For this multi-centre, retrospective, cross-sectional study, we recruited patients who had visited Center for Kampo Medicine in Keio University Hospital, Department of Japanese Oriental (Kampo) Medicine in Chiba University Hospital, Clinic of Japanese Oriental (Kampo) Medicine in Kanazawa University Hospital, and Department of Oriental Medicine in Kameda Medical Center from November 2011 to July 2018. We collected laboratory data including concentration of serum potassium, plasma activity of renin and aldosterone, and residual blood and/or urine samples of participants who had experienced symptoms/signs of pseudoaldosteronism in the form of increase in blood pressure and occurrence or aggregation of oedema while taking liquorice-containing herbal preparations, and measured GL metabolites using a highly selective liquid chromatography tandem mass spectrometer system. We registered 97 participants (mean age 60 ± 15 years; male:female 14:83). 18ß-glycyrrhetinic acid (GA) was detected in 67 serum samples (median 122 nM, range 5 nM-1.8 µM) and 18ß-glycyrrhetyl-3-O-sulfate (compound 3) in 68 samples (median 239 nM, range 2 nM-4.2 µM). 3-Monoglucuronyl 18ß-glycyrrhetinic acid, 22α-hydroxy-18ß-glycyrrhetyl-3-O-sulfate-30-glucuronide, 22α-hydroxy-18ß-glycyrrhetyl-3-O-sulfate, and GL itself were not or rarely detected. We could not find any correlation between blood pressure or peripheral oedema and serum concentration of GL metabolites. Sulfotransferase 2A1 catalysed the metabolic reaction of GA to compound 3, a major GL metabolite in human blood. High serum concentration of compound 3 was related to lower renin, aldosterone, and potassium levels, suggesting a pathological relationship between compound 3 and liquorice-induced pseudoaldosteronism. This is the first study to identify the association between a novel metabolite, compound 3, and the incidence of pseudoaldosteronism, highlighting it as a promising biomarker.

Neoline is the active ingredient of processed aconite root against murine peripheral neuropathic pain model, and its pharmacokinetics in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Processed aconite root (PA), the root of Aconitum carmichaeli (Ranunculaceae), is a crude drug used in traditional Chinese or Japanese kampo medicine to treat pain associated with coldness. In our previous study, PA and its active ingredient, neoline, alleviated oxaliplatin-induced peripheral neuropathy in mice. AIM OF THE STUDY: The present study investigated the effects of PA on a murine peripheral neuropathy model induced by intraperitoneal injection of paclitaxel and partial ligation of the sciatic nerve (Seltzer model), and identified its active ingredients. MATERIALS AND METHODS: PA powder (1 g/kg/day) was orally administered, and either neoline or benzoylmesaconine (10 mg/kg/day) was subcutaneously injected into the murine model. Mechanical hyperalgesia was evaluated via the von Frey filament method. PA extract was orally administered to rats; blood samples were chronologically collected, and the plasma concentrations of Aconitum alkaloids were measured. The contents of Aconitum alkaloids in commercial PA products were also measured. RESULTS: PA extract and neoline significantly attenuated the mechanical hyperalgesia induced by either paclitaxel or partial ligation of the sciatic nerve in mice. In the plasma samples of rats treated with PA extract, higher concentrations of benzoylmesaconine and neoline were apparent among Aconitum alkaloids. The contents of benzoylmesaconine and neoline varied among PA products with different processing procedures. Subcutaneous injection of benzoylmesaconine did not attenuate the hyperalgesia induced by each paclitaxel, partial ligation of the sciatic nerve, or oxaliplatin in mice. CONCLUSIONS: The present results indicate that PA and its active ingredient, neoline, are promising agents for the alleviation of neuropathic pain. Neoline can be used as a marker compound to determine the quality of the PA products for the treatment of neuropathic pain.

Cytotoxic phenylethanoid glycosides from Digitalis davisiana Heywood: Evaluation of structure activity relationships and chemotaxonomical significance of isolated compounds.

The phytochemical studies on the aerial parts of Digitalis davisiana Heywood led to the isolation of three undescribed phenylethanoid glycosides named as digidavisoside A (5), digidavisoside B (7), and davisoside (8), along with 9 known compounds, ferruginoside B (1), isolugrandoside (2), lugrandoside (3), maxoside (4), 3″″-O-methylmaxoside (6), trans-lamiuside E (9), digiciliside B (10), p-hydroxyacetophenone (11), and chrysoeriol (12). For the first time compound 11 was reported for Digitalis genus. The chemotaxonomical significance of these compounds in Plantaginaceae family was evaluated and 3'-O-glucosyl substituted phenylethanoid glycosides 4-8 and 10 were found to be chemotaxonomically important for the family. Cytotoxic activity of the aqueous fraction of the methanolic extract was also tested against HEp-2 (human larynx epidermoid carcinoma) and HepG2 (human hepatocellular carcinoma) cancer cell lines. The aqueous fraction showed stronger cytotoxicity on HEp-2 cells than on HepG2. Therefore, the cytotoxic activity of 1-4, 6, 7 and 9 were tested against HEp-2 and L929 (mouse fibroblast cell) cell lines. Other isolated compounds could not be tested due to their insufficient amount. The results were evaluated in the point of structure-activity relationships. IC50 values against HEp-2 cells were established in a range of 71.9-220 µM. Maxoside (4), isolugrandoside (2) and lugrandoside (3) showed higher cytotoxicity against HEp-2 cell line than other isolated compounds.

18ß-glycyrrhetyl-3-O-sulfate would be a causative agent of licorice-induced pseudoaldosteronism.

Licorice-induced pseudoaldosteronism is a common adverse effect in traditional Japanese Kampo medicine, and 3-monoglucuronyl glycyrrhetinic acid (3MGA) was considered as a causative agent of it. Previously, we found 22α-hydroxy-18ß-glycyrrhetyl-3-O-sulfate-30-glucuronide (1), one of the metabolites of glycyrrhizin (GL) in the urine of Eisai hyperbilirubinuria rats (EHBRs) treated with glycyrrhetinic acid (GA), and suggested that it is also a possible causative agent of pseudoaldosteronism. The discovery of 1 also suggested that there might be other metabolites of GA as causal candidates. In this study, we found 22α-hydroxy-18ß-glycyrrhetyl-3-O-sulfate (2) and 18ß-glycyrrhetyl-3-O-sulfate (3) in EHBRs' urine. 2 and 3 more strongly inhibited rat type 2 11ß-hydroxysteroid dehydrogenase than 1 did in vitro. When EHBRs were orally treated with GA, GA and 1-3 in plasma and 1-3 in urine were detected; the levels of 3MGA were quite low. 2 and 3 were shown to be the substrates of organic anion transporter (OAT) 1 and OAT3. In the plasma of a patient suffering from pseudoaldosteronism with rhabdomyolysis due to licorice, we found 8.6 µM of 3, 1.3 µM of GA, and 87 nM of 2, but 1, GL, and 3MGA were not detected. These findings suggest that 18ß-glycyrrhetyl-3-O-sulfate (3) is an alternative causative agent of pseudoaldosteronism, rather than 3MGA and 1.

B-ring-homo-tonghaosu, isolated from Chrysanthemum morifolium capitulum, acts as a peroxisome proliferator-activated receptor-γ agonist.

The capitula of Chrysanthemum morifolium and C. indicum are used to prepare Chrysanthemi Flos in traditional Japanese Kampo medicine. In our previous study, we reported on the agonistic effect of methanol extract of C. indicum capitulum on peroxisome proliferator-activated receptor (PPAR)-γ. We further isolated (E)-tonghaosu from C. indicum capitulum as one of the active ingredients. In the present study, we aimed to evaluate the PPAR-γ agonistic activity of a methanol extract of C. morifolium capitulum (MCM) in which (E)-tonghaosu could not be detected. MCM exhibited PPAR-γ agonistic activity in a concentration-dependent manner, and at a dose of 100 µg/ml, it showed similar activity to pioglitazone (30 µM), a standard PPAR-γ agonist. Through activity-guided fractionation, we isolated two geometric isomers, (E)- (1) and (Z)-B-ring-homo-tonghaosu (2), as the active ingredients of MCM. Both compounds exerted concentration-dependent PPAR-γ agonistic effects, and 1 had higher activity than 2. At 1.4 µM, 1 had similar activity to pioglitazone (30 µM), which was achieved by 2 at a concentration of 140 µM. Thus, 1 has the potential to become a lead compound for the drug discovery of PPAR-γ agonists. We compared the activities and the contents of (E)-, (Z)-tonghaosu, 1, and 2 among 13 commercial samples of Chrysanthemi Flos, including those derived from both C. morifolium and C. indicum. Their PPAR-γ agonistic activities were not related to the contents of these compounds. 1 and 2 were detected in the samples derived from both species but (E)- and (Z)-tonghaosu were not detected in the samples derived from C. morifolium; hence (E)- and (Z)-tonghaosu can serve as marker compounds to identify the capitula of C. indicum in Chrysanthemi Flos samples.

The immunostimulatory effects and chemical characteristics of heated honey.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Chinese medicine (TCM), honey has been used as an additive in the heat-processing of herbal medicines to enhance their immunostimulatory activities. AIM OF THE STUDY: We investigated the immunostimulatory activity of heated honey in vitro and in vivo. MATERIALS AND METHODS: For the in vitro study, we compared the differences among the inducible effects of honey subjected to various heating conditions on granulocyte colony-stimulating factor (G-CSF) secretion from the cultured enterocytes and investigated the active ingredient. For the in vivo study, we conducted a survival test of mice infected by Streptococcus pyogenes with and without oral administration of heated honey. RESULTS: We found that heating the honey induced the appearance of G-CSF secretions from the cultured enterocytes, and that this appearance depended on the heating temperature and time. No G-CSF secretions appeared when honey was not heated. Mice infected with Streptococcus pyogenes that were fed heated honey revealed prolonged survival. The active ingredient in heated honey was a high-molecular compound with about 730 kDa. When this compound was hydrolyzed, galactose, glucose, rhamnose, α-ribofuranose ß-ribofuranose 1,5':1',5-dianhydride, and 5-hydroxymethylfurfural were generated. CONCLUSIONS: Heated honey reveals immunostimulatory activity both in vitro and in vivo. These results support the scientific evidences of the TCM theory.

Isolation of a novel glycyrrhizin metabolite as a causal candidate compound for pseudoaldosteronism.

Pseudoaldosteronism is a common adverse effect associated with traditional Japanese Kampo medicines. The pathogenesis is mainly caused by 3-monoglucuronyl glycyrrhetinic acid (3MGA), one of the metabolites of glycyrrhizin (GL) contained in licorice. We developed an anti-3MGA monoclonal antibody (MAb) and an ELISA system to easily detect 3MGA in the plasma and urine of the patients. However, we found that some metabolites of GL cross-reacted with this MAb. Mrp2-deficient Eisai Hyperbilirubinemia rats (EHBRs) were administered glycyrrhetinic acid (GA), and we isolated 22α-hydroxy-18ß-glycyrrhetyl-3-O-sulfate-30-glucuronide (1) from the pooled urine with the guidance of positive immunostaining of eastern blot as the new metabolite of GL. The IC50 of 1 for type 2 11ß-hydroxysteroid dehydrogenase (11ß-HSD2) was 2.0 µM. Similar plasma concentrations of 1 and GA were observed 12 h after oral administration of GA to EHBR. Compound 1 was eliminated via urine, whereas GA was not. In Sprague-Dawley (SD) rats orally treated with GA, compound 1 was absent from both the plasma and the urine. Compound 1 was actively transported into cells via OAT1 and OAT3, whereas GA was not. Compound 1, when produced in Mrp2-deficiency, represents a potential causative agent of pseudoaldosteronism, and might be used as a biomarker to prevent the adverse effect.

A glucosyltransferase specific for 4-hydroxy-2,5-dimethyl-3(2H)-furanone in strawberry.

4-Hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) is a key aroma compound in Fragaria × ananassa (strawberry). A considerable amount of HDMF is converted into HDMF ß-D-glucoside and accumulated in mature strawberry fruits. Here we isolated a novel UDP-glucose: HDMF glucosyltransferase, UGT85K16 from Fragaria × ananassa. UGT85K16 preferentially glucosylated the hydroxyl group of HDMF and its structural analogs. Although UGT85K16 also catalyzed the glucosylation of vanillin, its affinity and efficiency toward HDMF was higher. The expression of UGT85K16 mRNA correlated with the accumulation of HDMF and its glucoside in Fragaria × ananassa plants. These results suggest that UGT85K16 might be UDP-glucose: HDMF glucosyltransferase in strawberries. ABBREVIATIONS: DMMF: 2,5-dimethyl-4-methoxy-3(2H)-furanone; EHMF: 2(5)-ethyl-4-hydroxy-5(2)-methyl-3(2H)-furanone; GBV: glycosidically bound volatile; HDMF: 4-hydroxy-2,5-dimethyl-3(2H)-furanone; HMF: 4-hydroxy-5-methyl-3(2H)-furanone; HMMF: 4-hydroxy-5-methyl-2-methylene-3(2H)-furanone; PSPG: Plant secondary product glycosyltransferase; RT-PCR: reverse transcription-PCR; OMT: O-methyltransferase; UGT: UDP-glycosyltransferase.