Limonoids and alkaloids from Tetradium austrosinense (Hand.-Mazz.) T.G.Hartley.

A pair of new enantiomeric indolopyridoquinazoline-type alkaloids, (+)-1,7S,8R- and (-)-1,7R,8S-trihydroxyrutaecarpine (3a and 3b), and a new limonoid-tyrosamine hybrid, austrosinin (8), along with six known alkaloids and limonoids, were isolated from the stems with leaves of Tetradium austrosinense. Their structures were elucidated on the basis of analysis of MS, NMR, ECD and time-dependent density functional theory-based electronic circular dichroism (TDDFT-ECD) calculations, as well as proposed biosynthetic pathway. An anti-inflammatory bioassay in vitro showed 8 had significant immunosuppressive effect against the production of pro-inflammatory cytokine TNF-α in lipopolysaccharide (LPS)-stimulated RAW264.7 cells.

B-seco Limonoids with anti-inflammatory activity from Tetradium fraxinifolium (Hook.) T.G.Hartley.

Fraxinifolines A-F (1-6), six new B-seco limonoids, together with four known A,D-di-seco ones, were isolated from the twigs with leaves of Tetradium fraxinifolium. Their structures with absolute configurations were elucidated on the basis of analysis of MS, NMR, single-crystal X-ray diffraction and biogenetic pathway. An anti-inflammatory bioassay in vitro showed limonoids 1-3 had significant immunosuppressive effect against the production of pro-inflammatory cytokines (IL-1ß and/or TNF-α) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells.

Tribulus terrestris L. extract ameliorates atherosclerosis by inhibition of vascular smooth muscle cell proliferation in ApoE-/- mice and A7r5 cells via suppression of Akt/MEK/ERK signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Atherosclerosis (AS) is one of major threatens of death worldwide, and vascular smooth muscle cell (VSMC) proliferation is an important characteristic in the progression of AS. Tribulus terrestris L. is a well-known Chinese Materia Medica for treating skin pruritus, vertigo and cardiovascular diseases in traditional Chinese medicine. However, its anti-AS activity and inhibition effect on VSMC proliferation are not fully elucidated. AIMS: We hypothesize that the furostanol saponins enriched extract (FSEE) of T. terrestris L. presents anti-AS effect by inhibition of VSMC proliferation. The molecular action mechanism underlying the anti-VSMC proliferation effect of FSEE is also investigated. MATERIALS AND METHODS: Apolipoprotein-E deficient (ApoE-/-) mice and rat thoracic smooth muscle cell A7r5 were employed as the in vivo and in vitro models respectively to evaluate the anti- AS and VSMC proliferation effects of FSEE. In ApoE-/- mice, the amounts of total cholesterol, triglyceride, low density lipoprotein and high density lipoprotein in serum were measured by commercially available kits. The size of atherosclerotic plaque was observed by hematoxylin & eosin staining. The protein expressions of α-smooth muscle actin (α-SMA) and osteopontin (OPN) in the plaque were examined by immunohistochemistry. In A7r5 cells, the cell viability and proliferation were tested by MTT and Real Time Cell Analysis assays. The cell migration was evaluated by wound healing assay. Propidium iodide staining followed by flow cytometry was used to analyze the cell cycle progression. The expression of intracellular total and phosphorylated proteins including protein kinase B (Akt) and mitogen-activated protein kinases (MAPKs), such as mitogen-activated extracellular signal-regulated kinase (MEK), extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), were detected by western blotting analysis. RESULTS: FSEE significantly reduced the area of atherosclerotic plaque in high-fat diet-fed ApoE-/- mice. And FSEE increased the protein expression level of α-SMA and decreased the level of OPN in atherosclerotic plaque, which revealed the inhibition of VSMC phenotype switching and proliferation. In A7r5 cells, FSEE suppressed fetal bovine serum (FBS) or oxidized low density lipoprotein (oxLDL)-triggered VSMC proliferation and migration in a concentration dependent manner. FSEE protected against the elevation of cell numbers in S phase induced by FBS or oxLDL and the reduction of cell numbers in G0/G1 phase induced by oxLDL. Moreover, the phosphorylation of Akt and MAPKs including MEK, ERK and JNK could be facilitated by FBS or oxLDL, while co-treatment of FSEE attenuated the phosphorylation of Akt induced by oxLDL as well as the phosphorylation of MEK and ERK induced by FBS. In addition, (25R)-terrestrinin B (JL-6), which was the main ingredient of FSEE, and its potential active pharmaceutical ingredients tigogenin (Tigo) and hecogenin (Heco) also significantly attenuated FBS or oxLDL-induced VSMC proliferation in A7r5 cells. CONCLUSION: FSEE presents potent anti- AS and VSMC proliferation activities and the underlying mechanism is likely to the suppression of Akt/MEK/ERK signaling. The active components of FSEE are JL-6 and its potential active pharmaceutical ingredients Tigo and Heco. So, FSEE and its active compounds may be potential therapeutic drug candidates for AS.

Vascular Protective Effect and Its Possible Mechanism of Action on Selected Active Phytocompounds: A Review.

Vascular endothelial dysfunction is characterized by an imbalance of vasodilation and vasoconstriction, deficiency of nitric oxide (NO) bioavailability and elevated reactive oxygen species (ROS), and proinflammatory factors. This dysfunction is a key to the early pathological development of major cardiovascular diseases including hypertension, atherosclerosis, and diabetes. Therefore, modulation of the vascular endothelium is considered an important therapeutic strategy to maintain the health of the cardiovascular system. Epidemiological studies have shown that regular consumption of medicinal plants, fruits, and vegetables promotes vascular health, lowering the risk of cardiovascular diseases. This is mainly attributed to the phytochemical compounds contained in these resources. Various databases, including Google Scholar, MEDLINE, PubMed, and the Directory of Open Access Journals, were searched to identify studies demonstrating the vascular protective effects of phytochemical compounds. The literature had revealed abundant data on phytochemical compounds protecting and improving the vascular system. Of the numerous compounds reported, curcumin, resveratrol, cyanidin-3-glucoside, berberine, epigallocatechin-3-gallate, and quercetin are discussed in this review to provide recent information on their vascular protective mechanisms in vivo and in vitro. Phytochemical compounds are promising therapeutic agents for vascular dysfunction due to their antioxidative mechanisms. However, future human studies will be necessary to confirm the clinical effects of these vascular protective mechanisms.

Vibrational Spectroscopy-Based Chemometrics Analysis of Clinacanthus nutans Extracts after Postharvest Processing and Extract Effects on Cardiac C-Kit Cells.

Chemical constituents in plants can be greatly affected by postharvest processing, and it is important to identify the factors that lead to significant changes in chemistry and bioactivity. In this study, attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy was used to analyze extracts of Clinacanthus nutan (C. nutans) leaves generated using different parameters (solvent polarities, solid-liquid ratios, ultrasonic durations, and cycles of extraction). In addition, the effects of these extracts on the viability of cardiac c-kit cells (CCs) were tested. The IR spectra were processed using SIMCA-P software. PCA results of all tested parameter sets were within acceptable values. Solvent polarity was identified as the most influential factor to observe the differences in chemical profile and activities of C. nutans extracts. Ideal extraction conditions were identified, for two sample groups (G1 and G2), as they showed optimal total phenolic content (TPC) yield of 44.66 ± 0.83 mg GAE/g dw and 45.99 ± 0.29 mg GAE/g dw and CC viability of 171.81 ± 4.06% and 147.53 ± 6.80%, respectively. Validation tools such as CV-ANOVA (p < 0.05) and permutation (R 2 and Q 2 plots were well intercepted to each other) have further affirmed the significance and reliability of the partial least square (PLS) model of solvent polarity employed in extraction. Hence, these approaches help optimize postharvest processes that encourage positive TPC and CCs results in C. nutans extracts.

Monoterpene indole alkaloids from Melodinus cochinchinensis (Lour.) Merr.

Melodicochinines A - D (1-4), four new monoterpene indole alkaloids (MIAs), along with 21 known ones, were isolated from the stems and twigs of Melodinus cochinchinensis. Their structures were elucidated on the basis of extensive spectroscopic analysis. A ubiquitin-rhodamine 110 assay showed that 11-methyloxytabersonine had potential inhibitory effect against ubiquitin-specific protease 7 (USP7).

Extracellular matrix from decellularized mesenchymal stem cells improves cardiac gene expressions and oxidative resistance in cardiac C-kit cells.

OBJECTIVE: Myocardial infarction remains the number one killer disease worldwide. Cellular therapy using cardiac c-kit cells (CCs) are capable of regenerating injured heart. Previous studies showed mesenchymal stem cell-derived (MSC) extracellular matrices can provide structural support and are capable of regulating stem cell functions and differentiation. This study aimed to evaluate the effects of human MSC-derived matrices for CC growth and differentiation. METHODS: Human Wharton's Jelly-derived MSCs were cultured in ascorbic acid supplemented medium for 14 days prior to decellularisation using two methods. 1% SDS/Triton X-100 (ST) or 20 mM ammonia/Triton X-100 (AT). CCs isolated from 4-week-old C57/BL6N mice were cultured on the decellularised MSC matrices, and induced to differentiate into cardiomyocytes in cardiogenic medium for 21 days. Cardiac differentiation was assessed by immunocytochemistry and qPCR. All data were analysed using ANOVA. RESULTS: In vitro decellularisation using ST method caused matrix delamination from the wells. In contrast, decellularisation using AT improved the matrix retention up to 30% (p < 0.05). This effect was further enhanced when MSCs were cultured in cardiogenic medium, with a matrix retention rate up to 90%. CCs cultured on cardiogenic MSC matrix (ECMcardio), however, did not significantly improve its proliferation after 3 days (p < 0.05), but the viability of CCs was augmented to 67.2 ± 0.7% after 24-h exposure to H2O2 stress as compared to 42.9 ± 0.5% in control CCs (p < 0.05). Furthermore, CCs cultured on cardiogenic MSC matrices showed 1.7-fold up-regulation in cardiac troponin I (cTnI) gene expression after 21 days (p < 0.05). CONCLUSION: Highest matrix retention can be obtained by decellularization using Ammonia/Triton-100 in 2-D culture. ECMcardio could rescue CCs from exogenous hydrogen peroxide and further upregulated the cardiac gene expressions, offering an alternate in vitro priming strategy to precondition CCs which could potentially enhance its survival and function after in vivo transplantation.

Guided evaluation and standardisation of mesenchymal stem cell culture conditions to generate conditioned medium favourable to cardiac c-kit cell growth.

Mesenchymal stem cells (MSCs) are known to secrete cardioprotective paracrine factors that can potentially activate endogenous cardiac c-kit cells (CCs). This study aims to optimise MSC growth conditions and medium formulation for generating the conditioned medium (CdM) to facilitate CC growth and expansion in vitro. The quality of MSC-CdM after optimisation of seeding density during MSC stabilisation and medium formulation used during MSC stimulation including glucose, ascorbic acid, serum and oxygen levels and the effects of treatment concentration and repeated CdM harvesting were assessed based on CC viability in vitro under growth factor- and serum-deprived condition. Our data showed that functional CdM can be produced from MSCs with a density of 20,000 cells/cm2, which were stimulated using high glucose (25 mM), ascorbic acid supplemented, serum-free medium under normoxic condition. The generated CdM, when applied to growth factor- and serum-deprived medium at 1:1 ratio, improved CC viability, migration and proliferation in vitro. Such an effect could further be augmented by generating CdM concentrates without compromising CC gene and protein expressions, while retaining its capability to undergo differentiation to form endothelial, smooth muscle and cardiomyocytes. Nevertheless, CdM could not be repeatedly harvested from the same MSC culture, as the protein content and its effect on CC viability deteriorated after the first harvest. In conclusion, this study provides a proof-of-concept strategy to standardise the production of CdM from MSCs based on rapid, stepwise assessment of CC viability, thus enabling production of CdM favourable to CC growth for in vitro or clinical applications.

Water extract of Clinacanthus nutans leaves exhibits in vitro, ex vivo and in vivo anti-angiogenic activities in endothelial cell via suppression of cell proliferation.

BACKGROUND: Clinacanthus nutans (Burm. f.) Lindau. has traditionally been using in South East Asia countries to manage cancer. However, scientific evidence is generally lacking to support this traditional claim. This study aims to investigate the in vitro, ex-vivo and in vivo effects of C. nutans extracts on angiogenesis. METHODS: C. nutans leaves was extracted with 50-100% ethanol or deionised water at 1% (w/v). Human umbilical veins endothelial cell (HUVEC) proliferation was examined using MTT assay. The in vitro anti-angiogenic effects of C. nutans were assessed using wound scratch, tube formation and transwell migration assays. The VEGF levels secreted by human oral squamous cell carcinoma (HSC-4) cell and HUVEC permeability were also measured. Besides, the rat aortic ring and chick embryo chorioallantoic membrane (CAM) assays, representing ex vivo and in vivo models, respectively, were performed. RESULTS: The MTT assay revealed that water extract of C. nutans leaves exhibited the highest activity, compared to the ethanol extracts. Therefore, the water extract was chosen for subsequent experiments. C. nutans leaf extract significantly suppressed endothelial cell proliferation and migration in both absence and presence of VEGF. However, the water extract failed to suppress HUVEC transmigration, differentiation and permeability. C. nutans water extract also did not suppress HSC-4 cell-induced VEGF production. Importantly, C. nutans water extract significantly abolished the sprouting of vessels in aortic rings as well as in chick embryo CAM. CONCLUSION: In conclusion, these findings reveal potential anti-angiogenic effects of C. nutans, providing new evidence for its potential application as an anti-angiogenic agent.

Dose-Dependent Increase in Unconjugated Cinnamic Acid Concentration in Plasma Following Acute Consumption of Polyphenol Rich Curry in the Polyspice Study.

Spices that are rich in polyphenols are metabolized to a convergent group of phenolic/aromatic acids. We conducted a dose-exposure nutrikinetic study to investigate associations between mixed spices intake and plasma concentrations of selected, unconjugated phenolic/aromatic acids. In a randomized crossover study, 17 Chinese males consumed a curry meal containing 0 g, 6 g, and 12 g of mixed spices. Postprandial blood was drawn up to 7 h at regular intervals and plasma phenolic/aromatic acids were quantified via liquid chromatography tandem mass spectrometry (LC-MS/MS). Cinnamic acid (CNA, p < 0.0001) and phenylacetic acid (PAA, p < 0.0005) concentrations were significantly increased with mixed spices consumption, although none of the other measured phenolic/aromatic acids differ significantly between treatments. CNA displayed a high dose-exposure association (R² > 0.8, p < 0.0001). The adjusted mean area under the plasma concentration-time curve until 7 h (AUC0⁻7 h) for CNA during the 3 increasing doses were 8.4 ± 3.4, 376.1 ± 104.7 and 875.7 ± 291.9 nM.h respectively. Plasma CNA concentration may be used as a biomarker of spice intake.

Clinacanthus nutans Extracts Are Antioxidant with Antiproliferative Effect on Cultured Human Cancer Cell Lines.

Clinacanthus nutans Lindau leaves (CN) have been used in traditional medicine but the therapeutic potential has not been explored for cancer prevention and treatment. Current study aimed to evaluate the antioxidant and antiproliferative effects of CN, extracted in chloroform, methanol, and water, on cancer cell lines. Antioxidant properties of CN were evaluated using DPPH, galvinoxyl, nitric oxide, and hydrogen peroxide based radical scavenging assays, whereas the tumoricidal effect was tested on HepG2, IMR32, NCL-H23, SNU-1, Hela, LS-174T, K562, Raji, and IMR32 cancer cells using MTT assay. Our data showed that CN in chloroform extract was a good antioxidant against DPPH and galvinoxyl radicals, but less effective in negating nitric oxide and hydrogen peroxide radicals. Chloroform extract exerted the highest antiproliferative effect on K-562 (91.28 ± 0.03%) and Raji cell lines (88.97 ± 1.07%) at 100 µ g/ml and the other five cancer cell lines in a concentration-dependent manner, but not on IMR-32 cells. Fourteen known compounds were identified in chloroform extract, which was analysed by gas chromatography-mass spectra analysis. In conclusion, CN extracts possess antioxidant and antiproliferative properties against cultured cancer cell lines, suggesting an alternate adjunctive regimen for cancer prevention or treatment.

The resolution of aglinin A epimers and their NMR assignments.

Aglinin A (1) is a mixture of C(24)-epimeric 20S,24-epoxy-24,25-dihydroxy-3,4-secodammar-4(28)-en-3-oic acid and present in plants of the family Meliaceae. The two epimers of 1 were resolved through an acetonide reaction, and the absolute configurations of two derivatives were deduced by the analysis of their (13)C NMR differences induced by γ-gauche or steric effect. Based on it, the (13)C NMR assignment of 24R-1 and 24S-1 was also established.

Arylnaphthalene lignan glycosides and other constituents from Phyllanthus reticulatus.

Two new arylnaphthalene lignan glycosides, named reticulatusides A (1) and B (2), together with eight known compounds were isolated from the 95% EtOH extract of the whole plant of Phyllanthus reticulatus. The structures of the new compounds were elucidated by spectroscopic methods.

Minor dammarane saponins from the hongshen extract of Shenmai injection.

A new dammarane-type triterpenoid saponin, (20R)-ginsenoside ST(2) (1), along with five known saponins was isolated from the hongshen extract of Shenmai injection. The structure of 1 was elucidated to be (20R)-dammar-23(E)-ene-3ß,6α,12ß,20,25-pentol 6-O-ß-D-glucopyranoside by means of spectroscopic methods.

Lycopodium alkaloids from Huperzia serrata.

A new lycopodane-type Lycopodium alkaloid, 6α-hydroxy-5,15-oxide-lycopodane (1), and seven known alkaloids were isolated from the whole plants of Huperzia serrata. Their structures were elucidated by means of spectroscopic methods. 12-Deoxyhuperzine O (2) was reported as a naturally occurring alkaloid for the first time, and showed an antagonist effect on the N-methyl-d-aspartate receptor with an IC(50) value of 0.92 µM.

[Identification of main related substances in potassium sodium dehydroandrographolide succinate].

To identify the structure of three related substances in potassium sodium dehydroandrographolide succinate (PSDS), an HPLC preparation method was used to separate the impurities. These main impurities were identified using LC-ESI/TOFMS, LC-ESI/MSn, NMR, UV and IR. One of the main impurities was a hydrolyzed and oxidized product of PSDS, which has not been reported previouely. The other two impurities were hydrolyzed products of PSDS after losing different succinic acids. The results indicate that PSDS can be easily hydrolyzed and oxidized. It should be stored at cool and dry places.

Two new N-oxide Lycopodium alkaloids from Huperzia serrata.

Two new Lycopodium alkaloids, N-oxidehuperzine E (1) and N-oxidehuperzine F (2), along with two known alkaloids, huperzines E (3) and F (4), were isolated from Huperzia serrata (Thunb.) Trev. Their structures were elucidated by spectroscopic and chemical transformations.

Two new isomeric alpha-tetralones from Pyrola calliantha.

Callianthones A ( 1) and B ( 2), a pair of new isomeric alpha-tetralones, together with a known alpha-tetralone ( 3) and four known flavonoids ( 4 - 7) were isolated from the 50 % EtOH extract of Pyrola calliantha. The structures and absolute configurations of the two new isomers were established to be (2 S,4 R) - and (2 S,4 S)-2,4-dihydroxy-2,7-dimethyl-3,4-dihydronaphthalen-1(2 H)-one ( 1 and 2, respectively) on the basis of spectral analysis, including 2 D NMR, model studies, and CD spectra.

Halophilols A and B, two new stilbenes from Iris halophila.

A new monomeric stilbene, halophilol A (1), and a new tetrastilbene, halophilol B (2), along with three known oligostilbenes were isolated from the seeds of Iris halophila. Their structures were established on the basis of the spectral data. The oligostilbene skeleton is encountered for the first time in the Iridaceae family. Bioactivity tests showed that 1 had moderate cytotoxicity against KB and HMEC cell lines (IC50 = 17.28 microM, 22.47 microM respectively), while 2 was inactive.