[A new alkaloid from Ervatamia hainanensis].

In order to study the alkaloids from branches and leaves of Ervatamia hainanensis, silica gel, ODS, Sephadex LH-20 and HPLC chromatography were used to obtain six alkaloids from the branches and leaves of E. hainanensis with use of. Based on the physicochemical properties and spectral data, their structures were identified as 10-hydroxydemethylhirsuteine(1), 3R-hydroxycoronaridine(2), 3-(2-oxopropyl)coronaridine(3), pandine(4), 16-epi-vobasine(5), and 16-epi-vobasinic acid(6). Among them, compound 1 was a new monoterpenoid indole alkaloid, and compounds 5 and 6 were obtained from this plant for the first time.

1ß-OH-arenobufagin induces mitochondrial apoptosis in hepatocellular carcinoma through the suppression of mTOR signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Chansu, dried secretions from Bufonidae, has long been used for cancer treatment as a traditional Chinese medicine. In searching for effective anti-hepatoma agents from Chansu, our preliminary drug screening found that a bufadienolide, namely 1ß-hydroxyl-arenobufagin (1ß-OH-ABF), displays anti-hepatoma activities. However, the anti-hepatoma effects and molecular mechanisms of 1ß-OH-ABF have not been defined. AIM OF THE STUDY: To evaluate the anti-hepatoma activity of 1ß-OH-ABF against liver cancer Hep3B and HepG2 cells in vitro and in vivo, as well as explore the underlying mechanisms. MATERIALS AND METHODS: The anti-proliferative effects of 1ß-OH-ABF on liver cancer Hep3B, HepG2, HuH7, SK-HEP-1 and normal hepatocyte LO2 cells were examined by MTT assay and colony formation assay. Hoechst 33258 staining and Annexin V-FITC/PI staining assay were used to analyze apoptosis induced by 1ß-OH-ABF. The collapse of the mitochondrial membrane potential (ΔΨm) was detected by JC-1 staining assay. Western blotting was used to examine the expression levels of targeted proteins. The role of mTOR in 1ß-OH-ABF-induced apoptosis was investigated using small interfering RNA (siRNA) transfection. Zebrafish xenograft model was established to evaluate the anti-hepatoma effects of 1ß-OH-ABF in vivo. RESULTS: We found that 1ß-OH-ABF inhibits the proliferation of Hep3B, HepG2, HuH7, SK-HEP-1 cells but has little cytotoxicity towards LO2 cells. 1ß-OH-ABF induces mitochondria dysfunction and triggers mitochondria apoptotic pathway, which is accompanied by the loss of ΔΨm, upregulation and translocation of Bax, as well as cleavages of caspase-9, caspase-3 and PARP. Mechanistically, 1ß-OH-ABF markedly decreases the expression level of p-AKT/AKT and p-mTOR (Ser2248 and Ser2481)/mTOR in a time-dependent manner. Inhibition of mTOR by siRNA strengthens 1ß-OH-ABF-mediated apoptosis. Critically, 1ß-OH-ABF shows a marked in vivo anti-hepatoma effect on human Hep3B cell xenografts in zebrafish model. CONCLUSION: 1ß-OH-ABF induces mitochondrial apoptosis through the suppression of mTOR signaling in vitro and in vivo, indicating that 1ß-OH-ABF may serve as a potential agent for the treatment of liver cancer.

New cadinane sesquiterpenoids from Mikania micrantha.

Chemical investigation of the aerial parts of Mikania micrantha led to the isolation of eight sesquiterpenoids and ten diterpenoids, including five cadinane sesquiterpenoids (1-5), three bisabolene sesquiterpenoids (6 - 8), nine ent-kaurane diterpenoids (9-17), and an abietane diterpenoid (18). Among them, 1 - 3 are new and feature a rare lactone or furan ring derived from C-6 isopropyl group side chain. Compound 18 was isolated from genus Mikania for the first time, and was also the first example of abietane-type diterpenoids from this plant. Their structures were elucidated on the basis of extensive spectroscopic analyses (1D and 2D NMR, HRESIMS, and ECD). All compounds were examined for their inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophage cells, and compound 18 exhibited pronounced inhibition on NO production (IC50 = 11.04 µM), being comparable to the positive control, quercetin (IC50 = 11.15 µM).

Jatrogricaine A: a new diterpenoid with a 5/6/6/4 carbon ring system from the stems of Jatropha podagrica.

Jatrogricaine A (1), a new diterpenoid possessing a 5/6/6/4 carbon ring system, together with eight known diterpenoids (2-9) were isolated from the stems of Jatropha podagrica. Their structures were elucidated by extensive spectroscopic methods and the absolute configuration of 1 was determined by single crystal X-ray diffraction analysis. All compounds were evaluated for their anti-inflammatory activities in vitro, and compound 3 showed significant inhibitory effects against nitric oxide (NO) production in lipopolysaccharide (LPS)-induced RAW264.7 macrophage cells with an IC50 of 13.44 ± 0.28 µmol·L-1, being comparable to the positive control, quercetin (IC50 17.00 ± 2.10 µmol·L-1).

Anti-tumor effects and 3D-quantitative structure-activity relationship analysis of bufadienolides from toad venom.

Toad venom (venenum bufonis, also called Chan'su) has been widely used for centuries in China to treat different diseases, especially for cancer. Bufadienolides are mainly responsible for the anti-cancer effects of toad venom. However, systematic chemical composition and cytotoxicity as well as key pharmacophores of these bufadienolides from toad venom have not yet been defined clearly. To enrich the understanding of the diversity of bufadienolides and to find bufadienolides with better activities from toad venom. This study was carried out to isolate chemical constituents, research their anti-tumor effects and mechanisms by MTT assay, flow cytometry and Western blotting, and develop a CoMFA and CoMSIA quantitative structure-activity relationship (QSAR) model for illustrating the vital relationship between the chemical structures and cytotoxicities. Among 47 natural bufadienolides, most of bufadienolides (21 compounds isolated in this study and 26 compounds isolated previously) could significantly inhibit the proliferation of cancer cells, and compounds 1, 8, 12, 18 and 19 showed the most potent inhibitory activity against four types of human tumor cells. Compound 18 induced G2/M cell cycle arrest and apoptosis. Moreover, 3D contour maps generated from CoMFA and CoMSIA identified several pharmacophores of bufadienolides responsible for the anti-tumor activities. Our study might provide reliable information for future structure modification and rational drug design of bufadienolides with anticancer activities in medical chemistry.

Rearranged Phloroglucinol-Monoterpenoid Adducts from Callistemon rigidus.

Callisretones A (1) and B (2), two rearranged phloroglucinol-monoterpenoid adducts featuring an unprecedented isopropylcyclopenta[b]benzofuran backbone, together with their postulated biosynthetic precursors (3-9), were isolated from Callistemon rigidus. The previously assigned absolute configurations of viminalins H (7), L (8), and N (9) were revised and unequivocally established by X-ray diffraction data. A putative biosynthetic pathway toward callisretones A and B involving the rearrangement of the terpenoid motif is proposed. In addition, 1 and 2 showed inhibitory effects on nitric oxide production with IC50 values of 15.3 ± 1.0 and 17.7 ± 1.1 µM, respectively.

Two new diterpenoid lactones isolated from Andrographis paniculata.

In the present study, two new diterpenoid lactones, 3-deoxy-andrographoside (1) and 14-deoxy-15-methoxy-andrographolide (2), were isolated from the aerial parts of Andrographis paniculata. Their structures were elucidated by combination of NMR, MS, and chemical methods. The configurations of 1 and 2 were established based on the analysis of ROESY data and single crystal X-ray diffraction experiment.

A comparative analysis of chemical compositions in Camellia sinensis var. puanensis Kurihara, a novel Chinese tea, by HPLC and UFLC-Q-TOF-MS/MS.

Camellia sinensis var. puanensis Kurihara (Puan tea) is a kind of ancient tea plant newly found in Jiangxipo and the surrounding areas of Puan County (Guizhou, China). People there always believe that drinking Puan tea is beneficial to the promotion of health and prevention of diseases. However, detailed information on its compositions has not been reported. Therefore, in this study, the varieties and contents of purine alkaloids and polyphenols in Puan tea were identified and determined by HPLC and UFLC-Q-TOF-MS/MS. Our results showed that theacrine, but not caffeine, was the dominated purine alkaloid detected in Puan tea. Meanwhile, Puan tea contained B-type procyanidin dimer, trimer and dimer monogallate, which were not detected in Camellia sinensis, Camellia ptilophylla and Camellia assamica var. kucha. The obtained results could support the local uses of Puan tea in health and nutrition and contribute to the research of tea variety.

Isolation and identification of polyphenols from Marsilea quadrifolia with antioxidant properties in vitro and in vivo.

Marsilea quadrifolia is an edible aquatic medicinal plant used as a traditional health food in Asia. Four new polyphenols including kaempferol 3-O-(2″-O-E-caffeoyl)-ß-d-glucopyranoside (1), kaempferol 3-O-(3″-O-E-caffeoyl)-α-l-arabinopyranoside (3), 4-methy-3'-hydroxypsilotinin (4) and (±)-(E)-4b-methoxy-3b,5b-dihydroxyscirpusin A (18) together with 14 known ones (2, 5-17) were isolated from the ethanol extract of M. quadrifolia. Structures of the new compounds were elucidated by extensive spectroscopic analyses. In DPPH and oxygen radical absorbance capacity antioxidant assays, some compounds showed stronger antioxidant activities and quercetin (9) was the most potent antioxidant in both assays. In a restraint-induced oxidative stress model in mice, quercetin significantly attenuated the increase in plasma ALT and AST levels as well as liver MDA content of restrained mice. Liver SOD activity was also significantly increased by quercetin, indicating a significant in vivo antioxidant activity. As a rich source of polyphenols with strong antioxidant activities, M. quadrifolia may be developed to a product for relieving oxidative stress.

A New C23 Steroid from the Venom of Bufo bufo gargarizans.

A new C23 steroid, (3ß,5ß,14ß)-methyl (3-hydroxy-14,15-epoxy-20-oxo-21-norcholan-24-oate) (1), together with four known ones (2-5), were isolated from the venom of Bufo bufo gargarizans. Their structures were elucidated on the basis of extensive spectroscopic analysis. The cytotoxicity of these compounds was also evaluated against human hepatocarcinoma HepG2 cells. Compound 3 showed significant cytotoxicity with an IC50 value of 16.8 +/- 0.7 µM.

Isolation, chemotaxonomic significance and cytotoxic effects of quassinoids from Brucea javanica.

A new quassinoid, bruceene A (1) along with seventeen known quassinoids (2-18) was isolated from the fruits of Brucea javanica. The structure of 1 was elucidated by extensive spectroscopic methods, and was further confirmed by single-crystal X-ray diffraction analysis. Isolation of similar quassinoids 1-3 as those in genus Ailanthus from genus Brucea, indicated the close chemotaxonomic relationship between these two genera, which further supported the phylogenetic study by DNA analysis. Compounds 5, 7, 10 and 12 with a 3-hydroxy-3-en-2-one moiety showed potent inhibitory activities against the MCF-7 and MDA-MB-231 cells with IC50 values in the ranges 0.063-0.182 µM and 0.081-0.238 µM, respectively; while glycosidation at 3-OH significantly decreased the cytotoxicity. It was also found that the most potent compound 7 induced apoptosis in MCF-7 cells via the intrinsic mitochondrial apoptotic pathway.

Bufadienolides with cytotoxic activity from the skins of Bufo bufo gargarizans.

Twelve new bufadienolides (1-12), along with fourteen known analogues (13-26) were isolated from the skins of Bufo bufo gargarizans Cantor. Their chemical structures were elucidated on the basis of NMR, HRESIMS and X-ray diffraction analysis. Compound 1 was an unusual bufadienolide with 3,19-epoxy moiety and A/B trans ring junction. Compounds 2-4 were rare bufadienolides possessing 10-H or 10-carboxyl units. All the isolated compounds were tested for their cytotoxic effects on HepG2, A549 and HeLa cells. Six new compounds (2, 3, 5, 6, 10 and 12) displayed significant anti-proliferative activities with IC50 values ranging from 0.049 to 1.856 µM. Arenobufagin (24) exhibited the most potent cytotoxic activity with IC50 value 0.011 µM. In addition, the present data provided more insight into the structure-activity relationships of bufadienolides.

Structures and Chemotaxonomic Significance of Stemona Alkaloids from Stemona japonica.

A pair of new alkaloid stereo-isomers, stemocochinin (1) and isostemocochinin (2), was obtained from the roots of Stemona japonica Miq., along with seven known alkaloids, stemonamine (3), isostemonamine (4), maistemonine (5), isomaistemonine (6), croomine (7), stemonine (8), and protostemonine (9). The complete structure and stereochemistry of the pair of isomers were established by extensive analysis of the spectral data. Furthermore, our results indicated that S. japonica is chemically closer to S. sessilifolia than S. tuberosa, which are consistent with our previous DNA study on Stemona species.

[Bufadienolides from venom of Bufo bufo gargarizans].

Twelve compounds were isolated from the venom of Bufo bufo gargarizans. On the basis of their physical and chemical properties and spectral data, their structures were identified as resibufagenin (1), bufotalin (2), desacetylcinobufagin (3), 19-oxodesacetylcinobufotalin (4), cinobufotalin (5), 1beta-hydroxylbufalin (6), 12alpha-hydroxybufalin (7), bufotalinin (8), Hellebrigenin (9), telocinobufagin (10), hellebrigenol (11) and cinobufagin-3-hemisuberate methyl ester (12), respectively. Compounds 7 and 12 are new natural products.

In vivo angiogenesis screening and mechanism of action of novel tanshinone derivatives produced by one-pot combinatorial modification of natural tanshinone mixture from Salvia miltiorrhiza.

BACKGROUND: Natural products present in low quantity in herb medicines constitute an important source of chemical diversity. However, the isolation of sufficient amounts of these low abundant constituents for structural modification has been a challenge for several decades and subsequently halts research on the utilization of this important source of chemical entities for drug discovery and development. And, pro-angiogenic therapies are being explored as options to treat cardio-cerebral vascular diseases and wound healing recently. The present study investigates the pro-angiogenic potential of tanshinone derivatives produced by one-pot synthesis using zebrafish model. METHODOLOGY/PRINCIPAL FINDINGS: In order to address the difficulty of chemical modification of low abundant constituents in herb medicines, a novel one-pot combinatorial modification was used to diversify a partially purified tanshinone mixture from Salvia miltiorrhiza. This led to the isolation of ten new imidazole-tanshinones (Compounds 1-10) and one oxazole-tanshinone (Compound 11), the structures of which were characterized by spectroscopic methods in combination with single-crystal X-ray crystallographic analysis. The angiogenesis activities of the new tanshinone derivatives were determined in an experimental model of chemical-induced blood vessels damage in zebrafish. Of all the tested new derivatives, compound 10 exhibited the most potent vascular protective and restorative activity with an EC50 value of 0.026 µM. Moreover, the mechanism underlying the pro-angiogenesis effect of 10 probably involved the VEGF/FGF-Src-MAPK and PI3K-P38 signalling pathways by gene expression analysis and a blocking assay with pathways-specific kinase inhibitors. CONCLUSIONS/SIGNIFICANCE: Taken together, our study demonstrated the more distinctive pro-angiogenic properties of 10 than other tanshinones and revealed 10 has potential for development as a pro-angiogenic agent for diseases associated with insufficient angiogenesis. Our results highlighted the great potential of adopting a newly modified one-pot approach to enhance the chemical diversity and biological activities of constituents from natural products regardless of their abundances.

Hellebrigenin induces cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells through inhibition of Akt.

Hellebrigenin, one of bufadienolides belonging to cardioactive steroids, was found in skin secretions of toads and plants of Helleborus and Kalanchoe genera. In searching for natural constituents with anti-hepatoma activities, we found that hellebrigenin, isolated from traditional Chinese medicine Venenum Bufonis, potently reduced the viability and colony formation of human hepatocellular carcinoma cells HepG2, and went on to explore the underlying molecular mechanisms. Our results demonstrated that hellebrigenin triggered DNA damage through DNA double-stranded breaks and subsequently induced cell cycle G2/M arrest associated with up-regulation of p-ATM (Ser(1981)), p-Chk2 (Tyr(68)), p-CDK1 (Tyr(15)) and Cyclin B1, and down-regulation of p-CDC25C (Ser(216)). It was also found that hellebrigenin induced mitochondrial apoptosis, characterized by Bax translocation to mitochondria, disruption of mitochondrial membrane potential, release of cytochrome c into cytosol and sequential activation of caspases and PARP. In addition, Akt expression and phosphorylation were inhibited by hellebrigenin, whereas Akt silencing with siRNA significantly blocked cell cycle arrest but enhanced apoptosis induced by hellebrigenin. Activation of Akt by human insulin-like growth factor I (hIGF-I) could obviously attenuate hellebrigenin-induced cell death. In summary, our study is the first to report the efficacy of hellebrigenin against HepG2 and elucidated its molecular mechanisms including DNA damage, mitochondria collapse, cell cycle arrest and apoptosis, which will contribute to the development of hellebrigenin into a chemotherapeutic agent in the treatment of liver cancer.

Cyclodepsipeptides from the ascocarps and insect-body portions of fungus Cordyceps cicadae.

A new cyclodepsipeptide cordycecin A (1), together with four known ones beauvericin E (2), beauvericin J (3), beauvericin (4), and beauvericin A (5) was isolated from the ascocarps and insect-body portions of fungus Cordyceps cicadae. Their structures were identified by NMR and MS analyses. The absolute configuration of 1 was confirmed by crystal X-ray diffraction. Compounds 2-5 exhibited a significant inhibitory effect on HepG2 and HepG2/ADM cells with IC50 values ranging from 2.40±0.37 to 14.48±1.68 µM. Interestingly, compounds 3-5 showed cytotoxic activity against multiple drug resistant HepG2 cell line (HepG2/ADM) with IC50 value 25-fold more sensitive to doxorubicin.

Two new bufadienolides from the rhizomes of Helleborus thibetanus with inhibitory activities against prostate cancer cells.

Two new bufadienolide glycosides (1 and 2) with an A/B trans ring fusion together with nine known compounds (3-11) were isolated from the rhizomes of Helleborus thibetanus. The structures of new compounds were elucidated by extensive spectroscopic analyses in combination with single-crystal X-ray diffraction. The bufadienolides 1 and 3-6 exhibited potent cytotoxic activities against the prostate cancer cells.

New monoterpenoid alkaloids from the aerial parts of Uncaria hirsuta.

To investigate the chemical constituents of medicinal plant Uncaria hirsuta, three new monoterpenoid alkaloids, named hirsutanines A-C (1-3), were isolated. Their structures with absolute configurations were elucidated by means of NMR, X-ray diffraction and CD analysis. Compound 3 was the first dimeric monoterpenoid alkaloid obtained from genus Uncaria.