Antiproliferative Activities of Diterpenes from Leaves of Isodon trichocarpus against Cancer Stem Cells.

Two new diterpenes named trichoterpene I (1) and trichoterpene II (2) were isolated from the extract from the leaves of Isodon trichocarpus together with 19 known diterpenes. Their chemical structures were elucidated on the basis of chemical and physicochemical properties. Among them, oridonin (3), effusanin A (4), and lasiokaurin (9) with the α,ß-unsaturated carbonyl moiety showed antiproliferative activities against breast cancer MDA-MB-231 and human astrocytoma U-251 MG cells [i.e., non-cancer stem cells (non-CSCs)] and their cancer stem cells (CSCs) isolated by sphere formation. In particular, compound 4 (IC50 = 0.51 µM) showed a higher antiproliferative activity against MDA-MB-231 CSCs than against MDA-MB-231 non-CSCs. The antiproliferative activity toward CSCs of compound 4 was equal to adriamycin (positive control, IC50 = 0.60 µM).

Plantainoside B in Bacopa monniera Binds to Aß Aggregates Attenuating Neuronal Damage and Memory Deficits Induced by Aß.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by dementia. The most characteristic pathological changes in AD brain include extracellular amyloid-ß (Aß) accumulation and neuronal loss. Particularly, cholinergic neurons in the nucleus basalis of Meynert are some of the first neuronal groups to degenerate; accumulating evidence suggests that Aß oligomers are the primary form of neurotoxicity. Bacopa monniera is a traditional Indian memory enhancer whose extract has shown neuroprotective and Aß-reducing effects. In this study, we explored the low molecular weight compounds from B. monniera extracts with an affinity to Aß aggregates, including its oligomers, using Aß oligomer-conjugated beads and identified plantainoside B. Plantainoside B exhibited evident neuroprotective effects by preventing Aß attachment on the cell surface of human induced pluripotent stem cell (hiPSC)-derived cholinergic neurons. Moreover, it attenuated memory impairment in mice that received intrahippocampal Aß injections. Furthermore, radioisotope experiments revealed that plantainoside B has affinity to Aß aggregates including its oligomers and brain tissue from a mouse model of Aß pathology. In addition, plantainoside B could delay the Aß aggregation rate. Accordingly, plantainoside B may exert neuroprotective effects by binding to Aß oligomers, thus interrupting the binding of Aß oligomers to the cell surface. This suggests its potential application as a theranostics in AD, simultaneously diagnostic and therapeutic drugs.

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.

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.

Asymmetric Nitrogen-Containing Dimer from Aerial Parts of Mercurialis leiocarpa and Its Synthesis by Mimicking Generation Process through Radical Intermediates.

An asymmetric nitrogen-containing dimer, leiocarpanine A, was isolated from the aerial part of Mercurialis leiocarpa as a new compound. The new generation process of leiocarpanine A was estimated and a concise synthesis of leiocarpanine A could be detailed based on mimicking the generation process through the radical intermediates. In general, a lot of reaction step and organic reagents are required for the synthesis of asymmetric nitrogen-containing dimers. However, our new synthesis method enables a concise synthesis of asymmetric nitrogen-containing dimers through radical intermediates by only liquid-separation. This synthetic method provides a rapid and concise pathway to construct a library of nitrogen-containing dimers that might be useful for drug discovery. In addition, it is useful to elucidate the generation process of leiocarpanine A.

Structures of Cyclic Organosulfur Compounds From Garlic (Allium sativum L.) Leaves.

Five new cyclic organosulfur compounds, foliogarlic disulfanes A1 (1), A2 (2), and A3 (3) and foliogarlic trisulfane A1 (4) and A2 (5), were isolated from the leaves of Allium sativum (garlic). The chemical structures of these compounds were elucidated on the basis of physicochemical evidence including Nuclear Magnetic Resonance (NMR) and Mass Spectrometry (MS). Compounds 1-5 were obtained as complex compounds with disulfane or trisulfane and tetrahydro-2H-difuro[3,2-b:2',3'-c]furan-5(5aH)-one. In addition, the hypothetical biosynthetic pathways of these compounds were suggested.

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.

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.

Biofunctional Effects of Thiohemiaminal-Type Dimeric Sesquiterpene Alkaloids from Nuphar Plants.

Dimeric sesquiterpene thioalkaloids from the rhizomes of Nuphar pumilum exhibited immunosuppressive effects using a sheep erythrocyte plaque forming cell (PFC) assay, as well as an anti-metastasis effect, and rapid apoptosis-inducing effects in tumor cell lines. In particular, dimeric sesquiterpene thioalkaloids with a hydroxy group (6-hydroxythiobinupharidine, 6,6'-dihydroxythiobinupharidine, 6-hydroxythionuphlutine B) showed substantial effects, whereas dimeric sesquiterpene thioalkaloids lacking the hydroxy group (thiobinupharidine, thionuphlutine B, 6'-hydroxythionuphlutine B, neothiobinupharidine, thionuphlutine B ß-sulfoxide, neothiobinupharidine ß-sulfoxide) and monomeric sesquiterpene alkaloids (nupharidine, 7-epideoxynupharidine, nupharolutine) showed weak activity. In this review, we summarize our studies of the biofunctional effects of these alkaloids.

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.

Cytotoxicity of sesquiterpene alkaloids from Nuphar plants toward sensitive and drug-resistant cell lines.

Multi-drug resistance (MDR) is a critical problem in cancer chemotherapy. MDR causes the overexpression of ATP-binding cassette (ABC) transporters and mutations in tumor suppressor genes and oncogenes. To tackle this issue, in this study, we focused on Nuphar plants, which have been traditionally used as food. Sesquiterpene alkaloids (1-3) were isolated from N. japonicum and dimeric sesquiterpene thioalkaloids (4-10) were isolated from N. pumilum. P-glycoprotein-overexpressing CEM/ADR5000 cells were cross-resistant to 6,6'-dihydroxythiobinupharidine (10). Using in silico molecular docking, we calculated the binding energies and simulated the interactions of these compounds with the corresponding amino acid residues at the binding site of P-gp. In addition, we investigated the cytotoxicity of these compounds towards cell lines overexpressing other ABC transporters (BCRP, ABCB5), cell lines with a knocked out tumor suppressor gene TP53 or cell lines overexpressing a deletion-activated EGFR oncogene. These cell lines were sensitive or only minimally cross-resistant to these compounds compared with their corresponding wild-type cell lines.

Comparison of lawsone contents among Lawsonia inermis plant parts and neurite outgrowth accelerators from branches.

Lawsonia inermis L. (Lythraceae) is cultivated in many countries, including Japan, China, India and Egypt. Its leaves are well known as hair dye and exhibit antibacterial, anticancer, antifungal and anticonvulsant activities. However, there are few reports on its branches. 2-Hydroxy-1,4-naphthoquinone (lawsone) is a characteristic compound in its leaves. However, it remains to be clarified whether lawsone is present in other plant parts or not. In this study, we measured lawsone contents in the extracts of L. inermis flowers, leaves and branches by HPLC with tandem mass spectrometry. The extracts of L. inermis flowers, leaves and branches contained 116.9, 486.2 and 5.4 µg/g lawsone, respectively. Lawsone content was much lower in branches than the other plant parts. Next, in order to identify the biological constituents in the branches, we isolated nine known compounds and examined their effects on neurite outgrowth in PC12 cells. Among the constituents isolated, 2-[4-(3-hydroxypropyl)-2-methoxyphenoxy]-propane-1,3-diol (1) and quercetin 7-O-ß-D-glucopyranoside (8) showed accelerative effects on neurite outgrowth in PC12 cells.

New diterpenes from Nigella damascena seeds and their antiviral activities against herpes simplex virus type-1.

Nigella species are rich source of dolabellane diterpenes. During our study of Nigella species, new dolabellane diterpenes, damasterpenes V-VIII were isolated. The structural determination of new compounds damasterpenes V-VIII is described with consideration of their absolute configurations. The antiviral activities against herpes simplex virus type-1 of the isolated compounds and their derivatives are also evaluated. Damasterpene V (inhibition 35.0%) and 2-phenylacetyl 13-benzoyl damasterpenol (32.0%) showed significant antiviral activity at 10 µM.