[Development of Functional Compounds Using Chemically Unstable Compounds Obtained from Medicinal Plants].

Sulfur- or nitrogen-containing compounds from medicinal plants exhibit various biological activities such as anticancer potential. Developing efficient strategies to isolate or synthesize these compounds or their derivatives is a remarkable achievement. We have isolated several sulfur-containing compounds such as tetrahydro-2H-difuro[3,2-b:2',3'-c]furan-5(5aH)-one derivatives from Allium plants. We have devised a unique approach for the rapid preparation of thiopyranones using the regioselective sequential double Diels-Alder reaction; we used a naturally-occurring chemically-unstable intermediate such as thioacrolein, which is produced from allicin, a major component in garlic. The cytotoxicity of the synthetic thiopyranones against cancer stem cells (CSCs) was equal to or higher than that of (Z)-ajoene, the reference compound.

Cytotoxic activities of alkaloid constituents from the climbing stems and rhizomes of Sinomenium acutum against cancer stem cells.

From the methanolic extract of the climbing stems and rhizomes of Sinomenium acutum, two new aporphine analogues, acutumalkaloids I and II, were isolated together with fifteen known compounds including lysicamine. The chemical structures of the isolated new compounds were elucidated based on chemical/physicochemical evidence such as NMR and MS spectra. For acutumalkaloids I and II, the absolute configurations were established by comparison of experimental and predicted electronic circular dichroism (ECD) data. We compared anti-proliferative activities of isolated compounds with reported naturally occurring Wnt/ß-catenin pathway inhibitor, nuciferine. Among the isolated compounds, we found lysicamine have anti-proliferative activity against both of HT-29 human colon cancer cell line and its cancer stem cells (CSCs). The IC50 values of lysicamine against non-CSCs and its CSCs were lower than that of nuciferine. In addition, the results of western blotting analysis suggested that lysicamine inhibited the expression of Wnt/ß-catenin pathway target protein such as survivin. These results suggested that lysicamine show cytotoxic activity via inhibition of Wnt/ß-catenin pathway.

New biofunctional effects of oleanane-type triterpene saponins.

In the current review, we describe the novel biofunctional effects of oleanane-type triterpene saponins, including elatosides, momordins, senegasaponins, camelliasaponins, and escins, obtained from Aralia elata (bark, root cortex, young shoot), Kochia scoparia (fruit), Polygala senega var. latifolia (roots), Camellia japonica (seeds), and Aesculus hippocastanum (seeds), considering the following biofunctional activities: (1) inhibitory effects on elevated levels of blood alcohol and glucose in alcohol and glucose-loaded rats, respectively, (2) inhibitory effects on gastric emptying in rats and mice, (3) accelerative effects on gastrointestinal transit in mice, and (4) protective effects against gastric mucosal lesions in rats. In addition, we describe (5) suppressive effects of the extract and chakasaponins from Camellia sinensis (flower buds) on obesity based on inhibition of food intake in mice. The active saponins were classified into the following three types: (1) olean-12-en-28-oic acid 3-O-monodesmoside, (2) olean-12-ene 3,28-O-acylated bisdesmoside, and (3) acylated polyhydroxyolean-12-ene 3-O-monodesmoside. Furthermore, common modes of action, such as involvements of capsaicin-sensitive nerves, endogenous NO and PGs, and possibly sympathetic nerves, as well as common structural requirements, were observed. Based on our findings, a common mechanism of action might mediate the pharmacological effects of active saponins. It should be noted that the gastrointestinal tract is an important action site of saponins, and the role of the saponins in the gastrointestinal tract should be carefully considered.

Quantitative analysis of taxiphyllin, a cyanogenic glycoside, in the leaves of Hydrangea macrophylla var. thunbergii.

The dried and fermented leaves of Hydrangea macrophylla var. thunbergii are currently used as crude drugs (Sweet Hydrangea Leaf) with a sweet taste for patients with diabetes. In recent years, cases of food poisoning with symptoms of vomiting etc. have been reported after drinking a decoction of this crude drug. Cyanogenic glycosides have been suggested as potential causative agents. However, cyanogenic glycosides from H. macrophylla var. thunbergii was ambiguous. In the present study, we found that the leaves contained the cyanogenic glycoside taxiphillin (1). Next, the content of 1 in leaves of different sizes, colors, parts, and growth periods was quantified. In addition, we prepared the leaves of plants grown in five types of soils with different pH values (pH 5.0-7.5). The content of 1 in the leaves of the plants grown in these soils was quantified. The content of 1 varied greatly, with more than a three-fold difference, depending on when the leaves were collected from the plants. Furthermore, we compared the content of 1 in the crude drug obtained under different processing conditions for H. macrophylla var. thunbergii. The results showed that 1 was mostly hydrolyzed during plant processing. It has been suggested that cyanogenic glycosides are not the causative constituents of food poisoning.

Analysis of Active Compounds Using Target Protein Cofilin-Cucurbitacins in Cytotoxic Plant Bryonia cretica.

We examined a two-step target protein binding strategy that uses cofilin as the target protein to analyze the active constituents in Bryonia cretica. In the first step, we prepared the target protein, and used it to analyze the compounds binding to it in the second step. We used the methanolic extract of B. cretica as a library of possible active compounds. We conducted LC-MS analysis using information from our previous study. The peaks in the HPLC profile were identified as cucurbitacin D, isocucurbitacin D, and cucurbitacin I. As far as we know, there is no known study of the activity of isocucurbitacin D in this research field. Therefore, we examined the effects of isocucurbitacin D on cell proliferation and cofilin protein in human fibrosarcoma cell line HT1080 to confirm the effectiveness of this strategy. The cytotoxicity assay, the fibrous/globular actin ratio assay, and the immunoblotting analysis revealed that isocucurbitacin D showed a cytotoxic effect with disruption of target protein cofilin. The target protein binding strategy is a direct and straightforward method for finding new drug seeds from crude sources, such as natural plant extracts.

Construction of sulfur-containing compounds with anti-cancer stem cell activity using thioacrolein derived from garlic based on nature-inspired scaffolds.

Sulfur-containing compounds, such as cyclic compounds with a vinyl sulfane structure, exhibit a wide range of biological activities including anticancer activity. Therefore, the development of efficient strategies to synthesize such compounds is a remarkable achievement. We have developed a unique approach for the rapid and modular preparation of nature-inspired cyclic and acyclic sulfur-containing compounds using thioacrolein, a naturally occurring chemically unstable intermediate. We constructed thiopyranone derivatives through the regioselective sequential double Diels-Alder reaction of thioacrolein produced by allicin, a major component in garlic, and two molecules of silyl enol ether as the diene partner. The cytotoxicity toward cancer stem cells of the thiopyranones was equal to or higher than that of (Z)-ajoene (positive control) derived from garlic, and the thiopyranones had higher chemical stability than (Z)-ajoene.

Inhibitory effects of cynaropicrin and related sesquiterpene lactones from leaves of artichoke (Cynara scolymus L.) on induction of iNOS in RAW264.7 cells and its high-affinity proteins.

The methanolic extract of the leaves of artichoke (Cynara scolymus L.) was found to inhibit nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Among the constituents of the extract, six sesquiterpene lactones (cynaropicrin, grosheimin, 11ß,13-dihydrocynaropicrin, 3ß-hydroxy-8α-[(S)-3-hydroxy-2-methylpropionyloxy]guaia-4(15),10(14),11(13)-trien-1α,5α,6ßH-12,6-olide, 3ß-hydroxy-8α-[2-methoxymethyl-2-propenoyloxy]guaia-4(15),10(14),11(13)-trien-1α,5α,6ßH-12,6-olide, and deacylcynaropicrin) inhibited NO production and/or inducible nitric oxide synthase (iNOS) induction. The acyl group having an α,ß-unsaturated carbonyl group at the 8-position and the α-methylene-γ-butyrolactone moiety were important for the strong inhibitory activity. Our results suggested that these sesquiterpene lactones inhibited the LPS-induced iNOS expression via the suppression of the JAK-STAT signaling pathway in addition to the κNF-κB signaling pathway. With regard to the target molecules of the sesquiterpene lactones, high-affinity proteins of cynaropicrin were purified from the cell extract. ATP/ADP translocase 2 and tubulin were identified and suggested to be involved in the cytotoxic effects of cynaropicrin, although the target molecules for the inhibition of iNOS expression were not clarified.

One-pot enantioselective synthesis of (S)-spirobrassinin and non-natural (S)-methylspirobrassinin from amino acids using a turnip enzyme.

The enantioselective synthesis of (S)-(-)-spirobrassinin, which features a unique sulfur-containing spirooxindole skeleton, was achieved by focusing on the phytoalexin generation in Brassicaceae plants. Specifically, (S)-(-)-spirobrassinin was obtained in a one-pot fashion from L-tryptophan through a reaction involving S-spirocyclization with various turnip enzymes and constituents, i.e., using the turnip as a reaction reagent, catalyst, and reaction vessel. Surprisingly, this strategy also enabled the one-pot enantioselective synthesis of the novel non-natural spirooxindole (S)-(-)-5-methylspirobrassinin from 5-methyl-DL-tryptophan.

Correction to: Inhibition of melanin production by anthracenone dimer glycosides isolated from Cassia auriculate seeds.

The article Inhibition of melanin production by anthracenone dimer glycosides isolated from Cassia auriculata seeds, written by Weicheng Wang, Yi Zhang, Souichi Nakashima, Seikou Nakamura, Tao Wang, Masayuki Yoshikawa and Hisashi Matsuda.

Effects of Sanoshashinto on left ventricular hypertrophy and gut microbiota in spontaneously hypertensive rats.

In our previous study, we found that the methanolic extract of Sanoshashinto () (SHXXTM) exhibited significant vasorelaxant effects in vitro and antihypertensive effects in vivo, and baicalin and berberine were the main antihypertensive constituents in SHXXTM. We also speculated that the baicalin-berberine (BB) combination produced vasorelaxant effects by activating the NO/cGMP pathway, and the BKCa channel and the DAG/PKC/CPI-17 pathway were involved. In this study, we examined the vasorelaxant effects using helical strips of rat aorta pretreated with different activators or inhibitors. The results suggested that the KATP channel and the voltage-dependent Ca2+ channel (VDCC) were also involved in the vasorelaxant effects. Furthermore, we found that SHXXTM and the BB combination reduced left ventricular hypertrophy and altered gut microbiota. Together, the results indicated that Sanoshashinto might have comprehensive effects on ameliorating hypertension.

Accelerative effects of carbazole-type alkaloids from Murraya koenigii on neurite outgrowth and their derivative's in vivo study for spatial memory.

Murraya koenigii is a medicinal plant that contains several carbazole-type alkaloids as its characteristic constituents. Blood-brain barrier permeable constituents of M. koenigii accelerated neurite outgrowth in PC-12 cells. Nine compounds were isolated from M. koenigii and their effects on neurite outgrowth were examined. Murrayamine-E (8) at 10 µM showed significant effect. Focusing on the carbazole skeleton, we synthesized derivatives to attenuate cytotoxicity. 9-Benzyl-9H-carbazol-4-ol (15) exhibited strong neurite outgrowth accelerative effect. In addition, the novel object recognition test and the Morris water maze test were performed to evaluate memory improvement of 15 in APdE9 mice. Compound 15 tended to improve spatial memory in the Morris water maze test. These results suggest that carbazole derivative 15 would be a seed compound for Alzheimer's disease drug.

Antihypertensive constituents in Sanoshashinto.

It has been reported that Sanoshashinto (SanHuangXieXinTang, ), which is composed of Rhei Rhizoma, Scutellariae Radix, and Coptidis Rhizoma, exhibits vasorelaxant effects in vitro and lowers blood pressure of patients. Based on this discovery, in this study, a mixture containing those three materials and combinations of them were extracted with methanol, and the extracts were fractionated into different parts. Effects of all extracts and fractions on high concentration of potassium chloride (High K+)- or noradrenaline (NA)-induced contractions of isolated rat aortic rings or helical strips were examined. Qualitative and quantitative HPLC analyses of the extracts and the fractions revealed that the contents of baicalin and berberine in Sanoshashinto methanol extract (SHXXTM) were higher than those of the other constituents. All pharmacological and HPLC data were analyzed by principal component analysis (PCA) software and the results indicated that baicalin, berberine, palmatine, baicalein, and wogonoside contributed significantly to the pharmacological activity. Furthermore, spontaneously hypertensive rats (SHRs) that were orally given SHXXTM or a baicalin-berberine combination showed significantly reduced increase in the rate of systolic blood pressure (SBP) compared to the control group. These findings suggested that Sanoshashinto has significant vasorelaxant effects in vitro and antihypertensive effects in vivo, and baicalin and berberine, which were the principal constituents of Scutellariae Radix and Coptidis Rhizoma, were the main antihypertensive constituents in Sanoshashinto. It was speculated that baicalin and berberine produced vasorelaxant effects by activating the NO/cGMP pathway and that the BKCa channel and the DAG/PKC/CPI-17 pathway were also involved.

Anti-inflammatory effect of isopimarane diterpenoids from Kaempferia galanga.

Two new isopimarane diterpenes, 1α-hydroxy-14α-methoxyisopimara-8(9),15-diene (7) and 1α,14α-dihydroxyisopimara-8(9),15-diene (9) and eight known isopimarane diterpenes including (-)-sandaracopimaradiene (1), 6ß-acetoxysandaracopimaradiene-9α-ol (2), sandaracopimaradiene-7ß,9α-diol (3), sandaracopimaradiene-1α,9α-diol (4), 6ß-acetoxysandaracopimaradiene-9α-ol-1-one (5), 6ß-acetoxysandaracopimaradiene-1α,9α-diol (6), 6ß,14α-dihydroxyisopimara-8(9),15-diene (8), and 6ß,14ß-dihydroxyisopimara-8(9),15-diene (10) were isolated from hexane fraction of Kaempferia galanga ethanol extract. Compounds 5, 6, 8, and 9 exerted the good anti-inflammatory effect on lipopolysaccharide-stimulated nitric oxide production from RAW264.7 cells with IC50 of 11.2, 7.7, 14.3, and 12.1 µM, respectively. These four compounds inhibited nitric oxide synthase (iNOS) mRNA expression. Compounds 5 and 6 also suppressed cyclooxygenase 2 (COX-2) mRNA expression; in addition, compound 6 had mild inhibitory effect on TNF-α mRNA. Among these compounds, 5 dramatically inhibited iNOS and COX-2 mRNA expression. The influential structures were proposed to be oxygen substitute at C-1, C-6, and α-OH at C-14.

BBB-permeable aporphine-type alkaloids in Nelumbo nucifera flowers with accelerative effects on neurite outgrowth in PC-12 cells.

Blood-brain barrier (BBB)-permeable components in the methanolic extract of Nelumbo nucifera flowers showed accelerative effects on neurite outgrowth in PC-12 cells. Among the constituents isolated from N. nucifera flowers in our previous study, aporphine-type alkaloids, lirinidine, asimilobine, N-methylasimilobine, and pronuciferine, showed accelerative effects. Lirinidine, N-methylasimilobine, and an alkaloid-rich diethyl ether fraction at low concentrations increased the expression of mRNAs coding for TrkA, Vav3, and Rac1. In addition, good permeability of asimilobine and N-methylasimilobine was confirmed using an in vitro BBB model. Asimilobine and N-methylasimilobine are considered to be suitable as seed compounds of drugs for Alzheimer's disease, because of their activity and BBB permeability.

6-Hydroxythiobinupharidine Inhibits Migration of LM8 Osteosarcoma Cells by Decreasing Expression of LIM Domain Kinase 1.

BACKGROUND/AIM: Osteosarcoma is the most malignant type of bone tumor. Patients with osteosarcoma metastases have a poorer prognosis than those without metastases. Thus, the prognosis of osteosarcoma patients with metastases must be improved. MATERIALS AND METHODS: The present study investigated the inhibitory effects of 6-hydroxythiobinupharidine isolated from Nuphar pumilum on migration of LM8 murine osteosarcoma cells by a migration assay and also examined the expression of proteins related to actin dynamics by western blot. The present study also developed an automatic cell counting system using machine learning to count migrated cells by Fiji and Trainable Weka Segmentation. RESULTS: 6-Hydroxythiobinupharidine inhibited migration of LM8 osteosarcoma cells in a dose-dependent manner, and decreased protein expression of Lin11, Isl-1, and Mec-3 domain kinase 1 (LIMK1) and the levels of phosphorylated Cofilin. CONCLUSION: 6-Hydroxythiobinupharidine suppressed migration of LM8 osteosarcoma cells by decreasing expression of LIMK1. 6-Hydroxythiobinupharidine could be potentially used as an anti-metastatic compound.

Inhibition of melanin production by anthracenone dimer glycosides isolated from Cassia auriculata seeds.

The methanol extract of Cassia auriculata seeds was found to inhibit melanogenesis in B16 melanoma 4A5 cells under conditions of theophylline stimulation. Two new phlegmacin-type anthracenone dimer glycosides, auriculataosides A and B, were isolated from the active methanol fraction, and their inhibitory effects were observed in the concentration range of 0.03 to 0.3 µM. Inhibition of microphthalmia-associated transcription factor, tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2 protein expression was observed, suggesting that the inhibition of these factors is part of the mechanism of action underlying melanogenesis inhibition.

Anti-proliferative effect of auriculataoside A on B16 melanoma 4A5 cells by suppression of Cdc42-Rac1-RhoA signaling protein levels.

Auriculataoside A, an anthracenone dimer glycoside isolated from Cassia auriculata seed, shows anti-proliferative effects on cell line B16 melanoma 4A5 cells with an IC50 value of 0.82 µM. However, it shows no such effect on normal human dermal fibroblast (HDF) cells. To evaluate the mode of action underlying the anti-proliferative effect of auriculataoside A on cells, we examined changes in whole protein expression after treatment with auriculataoside A and found that the expression Cdc42, RhoA, and Rac1, which are Rho family GTPases, was reduced. Auriculataoside A also arrested the cell cycle at G1 phase. These results suggest that the suppression of the above proteins induced G1 arrest. In addition, auriculataoside A also suppressed the expression of ß-catenin and c-Myc proteins. This action of auriculataoside A could be one of the mechanisms underlying its selective anti-proliferative effect on B16 melanoma cells.

Cyclic sulfur-containing compounds from Allium fistulosum 'Kujou'.

Sulfur-containing compounds, allicin and ajoene, etc., were isolated from Allium species. In a recent study, some sulfur-containing cyclic compounds were isolated from A. sativum, A. cepa, and A. fistulosum. Four new compounds with multiple rings with methyl disulfide or propyl disulfide at the side chain of the 7-position, kujounins A3 (1), B1 (2), B2 (3) and B3 (4), and two new thiolane type compounds with methoxy and methyl sulfoxide moiety at the 2- and 5-positions, and allium sulfoxides A2 (5) and A3 (6), were isolated from the acetone extract of the fresh white parts of Allium fistulosum 'Kujou' with three known compounds, kujounin A1 (7) and A2 (8), and allium sulfoxide A1 (9). The chemical structures of the new compounds were elucidated on the basis of physicochemical evidence. The kujounins had a rare molecular skeleton, which was tetrahydro-2H-difuro[3,2-b:2',3'-c]furan-5(5aH)-one.