Quinazolinone-Peptido-Nitrophenyl-Derivatives as Potential Inhibitors of SARS-CoV-2 Main Protease.

The severe acute respiratory syndrome coronavirus 2 main protease (SARS-CoV-2-Mpro) plays an essential role in viral replication, transcription, maturation, and entry into host cells. Furthermore, its cleavage specificity for viruses, but not humans, makes it a promising drug target for the treatment of coronavirus disease 2019 (COVID-19). In this study, a fragment-based strategy including potential antiviral quinazolinone moiety and glutamine- or glutamate-derived peptidomimetic backbone and positioned nitro functional groups was used to synthesize putative Mpro inhibitors. Two compounds, G1 and G4, exhibited anti-Mpro enzymatic activity in a dose-dependent manner, with the calculated IC50 values of 22.47 ± 8.93 µM and 24.04 ± 0.67 µM, respectively. The bio-layer interferometer measured real-time binding. The dissociation kinetics of G1/Mpro and G4/Mpro also showed similar equilibrium dissociation constants (KD) of 2.60 × 10-5 M and 2.55 × 10-5 M, respectively, but exhibited distinct association/dissociation curves. Molecular docking of the two compounds revealed a similar binding cavity to the well-known Mpro inhibitor GC376, supporting a structure-function relationship. These findings may open a new avenue for developing new scaffolds for Mpro inhibition and advance anti-coronavirus drug research.

GSK-3 inhibitors enhance TRAIL-mediated apoptosis in human gastric adenocarcinoma cells.

Resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis has been reported in some cancer cells, including AGS human gastric adenocarcinoma cells. Reducing this resistance might shed light on the treatment of human gastric adenocarcinoma. In this study, we examined whether glycogen synthase kinase-3 (GSK-3) inhibitors can restore TRAIL responsiveness in gastric adenocarcinoma cells. The effect of two GSK-3 inhibitors, SB-415286, and LiCl, on apoptosis signaling of TRAIL in human gastric adenocarcinoma cell lines and primary gastric epithelial cells was analyzed. Both inhibitors can sensitize gastric adenocarcinoma cells, but not primary gastric epithelial cells, to TRAIL-induced apoptosis by increasing caspase-8 activity and its downstream signal transmission. Adding p53 siRNA can downregulate GSK-3 inhibitor-related sensitization to TRAIL-induced apoptosis and caspase-3 activity. GSK-3 inhibitors strongly activate the phosphorylation of JNK. Inhibition of JNK leads to earlier and more intense apoptosis, showing that the activation of JNK may provide anti-apoptotic equilibrium of pro-apoptotic cells. Our observations indicate that GSK-3 inhibitors can sentize AGS gastric adenocarcinoma cells to TRAIL-induced apoptosis. Therefore, in certain types of gastric adenocarcinoma, GSK-3 inhibitor might enhance the antitumor activity of TRAIL and mightbe a promising candidate for the treatment of certain types of gastric adenocarcinoma.

Drynaria fortunei Promoted Angiogenesis Associated With Modified MMP-2/TIMP-2 Balance and Activation of VEGF Ligand/Receptors Expression.

Background and Purpose: Drynaria fortunei J. Sm (D. fortunei), known as Gu-Sui-Bu, is used in traditional Chinese medicine to treat common injuries, including bone fractures and bruising. The specific functional mechanisms of the angiogenic and endothelial cell migration properties of D. fortunei are currently unclear. Thus, the purpose of this study is to validate the potential angiogenic and cellular migration properties and related mechanisms by D. fortunei both in vivo and in vitro. Experimental Approach: The present study investigates, both in vivo and in vitro, the wound healing effects of D. fortunei as associated with angiogenesis, specifically by the modulation of matrix metalloproteinases (MMPs) and upregulation of vascular endothelial growth factor (VEGF) ligand/receptors. In order to determine the potential angiogenic effects of D. fortunei, in vivo neovascularization of chick chorioallantoic membranes (CAMs) assay, and directed in vivo angiogenesis assay (DIVVA) were performed, while in vitro scratch wound healing, migration, and matrix-induced tube formation assays were performed by using human umbilical vascular endothelial cells (HUVECs). Furthermore, we used qPCR to analyze the gene expressions and Western blot to observe protein expressions of MMP-2, MMP-14, TIMP-2, RECK, and VEGF/VEGFRs. Results: This study identified five major compounds from the water extract of D. fortunei: protocatechuic acid, caffeic acid 4-O-ß-D-glucopyranoside, 5,7-dihydroxychromone-7-O-rutinoside, neoeriocitrin, and naringin. D. fortunei was confirmed to activate in vivo angiogenesis by CAM and DIVVA assays. D. fortunei further exhibited in vitro angiogenic effects associated with cell migration, as demonstrated by the tube formation assay, transwell migration assay, and scratch wound healing assay. The extracellular MMP-2 activity was found to be dose-dependently augmented both in vitro and in vivo by D. fortunei. The mRNA and protein expressions of MMP-2, and MMP-14 were increased; while the tissue inhibitor metalloproteinase-2 (TIMP-2), and reversion-inducing cysteine-rich protein with kazal motifs (RECK) were both decreased. Furthermore, D. fortunei activated the gene and protein expressions of VEGF-A, -B, and VEGFR-2, -3. Conclusion: D. fortunei increased MMP-2 activity, thereby stimulating angiogenesis and cell migration, both in vivo and in vitro, as a result of MMP-2 and TIMP-2 balance modulation and the activation of VEGF/VEGFRs expression.

The characteristics and prescription patterns of Chinese herbal medicine in clinical practice for the treatment of anemia.

OBJECTIVE: Chinese herbal medicine (CHM) is frequently applied to patients to improve the symptoms and signs associated with anemia. The aim of this study is to use the claims data from the National Health Insurance Research Database (NHIRD) in Taiwan to analyze CHM prescription patterns and to identify the frequency and combinations of CHM commonly used to treat anemia. MATERIALS AND METHODS: A total of 41,028 patients were diagnosed with anemia in Taiwan within the defined study period. After randomly equal matching for age and sex, data from 7682 patients characterized as CHM users and non-users were analyzed. Network analyses of the 30 most frequently applied herbs and formulas were used to indicate CHM combinations in patients with anemia. RESULTS: Those patients with anemia who were older, office workers, and lived in central areas of Taiwan had higher tendencies toward CHM usage. Based on considerations of comorbidities, anemia patients associated with chronic kidney diseases, diabetes mellitus, and hypertensive diseases preferred Western medical management and demonstrated a lesser likelihood of combining treatment with CHM; by contrast, those with coronary artery disease demonstrated a higher tendency for CHM use. Notably, Astragalus membranaceus (AM) and Gui-Pi-Tang (GPT) were the most commonly prescribed CHM single herb and formula, respectively. The core prescription pattern consisted of AM, Salvia miltiorrhiza (SM), Angelica sinensis (AS), GPT, and Si-Wu-Tang (SWT), as indicated by the associations and frequency of CHM utilization by traditional Chinese medicine (TCM) physicians. CONCLUSION: This study demonstrates that CHM may be applied as an integral element of treatment for patients with anemia. It also provides insight regarding individual therapy and common clinical practices of TCM physicians in the treatment of anemia. Further research is required to explore potential interactions and possible mechanisms at play with CHM management of anemia.

Radix Paeoniae Rubra stimulates osteoclast differentiation by activation of the NF-κB and mitogen-activated protein kinase pathways.

BACKGROUND: Radix Paeoniae Rubra (RPR), a traditional Chinese herb, has anti-inflammatory and immuno-regulatory properties. This study explored the effects of RPR on stimulation of osteoclast differentiation in RAW264.7 cells and peripheral blood mononuclear cells (PBMC)s. METHODS: The mature osteoclasts were measured by bone resorption assays and TRAP staining. JNK, ERK, p38 and NF-κB inhibitors were used applied in order to verify their contribution in RPR-induced osteoclast differentiation. The NF-κB and MAPK pathways were evaluated by western blotting, RT-PCR and luciferase assay. RESULTS: RPR induced osteoclast differentiation in a dose-dependent manner and induced the resorption activity of osteoclasts differentiation of RAW264.7 cells and PBMCs. Western blotting showed that RPR treatment induced phosphorylation of JNK, ERK, and p38 in RAW 264.7 cells. Treatment of JNK, ERK, and p38 MAP kinase inhibitors verified the contribution of JNK, ERK and p38. RPR treatment induced c-Fos and NFATc1 protein expression; NF-κB inhibitor treatment and luciferase assay verified the contribution of the NF-κB pathway. CONCLUSIONS: This study demonstrated the interesting effect, in which RPR stimulated osteoclast differentiation in murine RAW264.7 cells and human monocytes.

Hepatoprotective effect of the ethanol extract of Polygonum orientale on carbon tetrachloride-induced acute liver injury in mice.

Polygonum orientale L. (Polygonaceae) fruits have various medicinal uses, but their hepatoprotective effects have not yet been studied. This study investigated the hepatoprotective activity of the ethanolic extract of P. orientale (POE) fruits against carbon tetrachloride (CCl4)-induced acute liver injury (ALI). Mice were pretreated with POE (0.1, 0.5, and 1.0 g/kg) or silymarin (0.2 g/kg) for 5 consecutive days and administered a dose of 0.175% CCl4 (ip) on the 5th day to induce ALI. Blood and liver samples were collected to measure antioxidative activity and cytokines. The bioactive components of POE were identified through high-performance liquid chromatography (HPLC). Acute toxicity testing indicated that the LD50 of POE exceeded 10 g/kg in mice. Mice pretreated with POE (0.5, 1.0 g/kg) experienced a significant reduction in their serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), and alkaline phosphatase (ALP) levels and reduction in the extent of liver lesions. POE reduced the malondialdehyde (MDA), nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) levels, and increased the activity of superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GRd) in liver. HPLC revealed peaks at 11.28, 19.55, and 39.40 min for protocatechuic acid, taxifolin, and quercetin, respectively. In summary, the hepatoprotective effect of POE against CCl4-induced ALI was seemingly associated with its antioxidant and anti-proinflammatory activities.

Hirsutane-Type Sesquiterpenes with Inhibitory Activity of Microglial Nitric Oxide Production from the Red Alga-Derived Fungus Chondrostereum sp. NTOU4196.

The marine red alga Pterocladiella capillacea is an economic alga for the food industry in Taiwan, and its associated highly diversified fungi have not been investigated meticulously thus far. The EtOAc extract of the fermented broth of Chondrostereum sp. NTOU4196, a fungal strain isolated from P. capillacea, was found to exhibit significant nitric oxide (NO) production inhibitory activity in lipopolysaccharide-activated murine RAW 264.7 cells at a concentration of 100 µg/mL in the preliminary screening. Therefore, separation of the active principles from the fermented broths was performed, and that has led to the isolation of eight new 5,5,5-tricyclic hirsutane-type sesquiterpenes, namely, chondroterpenes A-H (1-8), together with seven known analogues. They were identified by analyses of spectroscopic data and comparison with literature values. Among the new isolates, chondroterpene A (1) exhibited more significant NO production inhibitory activity in murine BV-2 microglial cells, and of all the isolated compounds, hirsutanol A (9) exerted limited cytotoxic effects and the most potent inhibitory activity on NO production.

Development of a new type of multifunctional fucoidan-based nanoparticles for anticancer drug delivery.

Fucoidan, a sulfated marine polysaccharide, has many potential biological functions, including anticancer activity. Recently, fucoidan has been reported to target P-selectin expressed on metastatic cancer cells. Increasing research attention has been devoted to the developments of fucoidan-based nanomedicine. However, the application of traditional chitosan/fucoidan nanoparticles in anticancer drug delivery may be limited due to the deprotonation of chitosan at a pH greater than 6.5. In this study, a mutli-stimuli-responsive nanoparticle self-assembled by fucoidan and a cationic polypeptide (protamine) was developed, and their pH-/enzyme-responsive properties were characterized by circular dichroism (CD) spectroscopy, dynamic light scattering (DLS), and zeta potential analysis. Enzymatic digestion and acidic intracellular microenvironment (pH 4.5-5.5) in cancer cells triggered the release of an anticancer drug (doxorubicin) from the nanoparticles. The protamine/fucoidan complex nanoparticles with P-selectin mediated endocytosis, charge conversion and stimuli-tunable release properties showed an improved inhibitory effect against a metastatic breast cancer cell line (MDA-MB-231).

Genistein suppresses leptin-induced proliferation and migration of vascular smooth muscle cells and neointima formation.

Obesity is a strong risk factor for the development of cardiovascular diseases and is associated with a marked increase in circulating leptin concentration. Leptin is a peptide hormone mainly produced by adipose tissue and is regulated by energy level, hormones and various inflammatory mediators. Genistein is an isoflavone that exhibits diverse health-promoting effects. Here, we investigated whether genistein suppressed the atherogenic effect induced by leptin. The A10 cells were treated with leptin and/or genistein, and then the cell proliferation and migration were analysed. The reactive oxygen species (ROS) and proteins levels were also measured, such as p44/42MAPK, cell cycle-related protein (cyclin D1 and p21) and matrix metalloproteinase-2 (MMP-2). Immunohistochemistry and morphometric analysis were used for the neointima formation in a rat carotid artery injury model. Genistein (5 µM) significantly inhibited both the proliferation and migration of leptin (10 ng/ml)-stimulated A10 cells. In accordance with these finding, genistein decreased the leptin-stimulated ROS production and phosphorylation of the p44/42MAPK signal transduction pathway. Meanwhile, genistein reversed the leptin-induced expression of cyclin D1, and cyclin-dependent kinase inhibitor, p21. Genistein attenuated leptin-induced A10 cell migration by inhibiting MMP-2 activity. Furthermore, the leptin (0.25 mg/kg)-augmented neointima formation in a rat carotid artery injury model was attenuated in the genistein (5 mg/kg body weight)-treated group when compared with the balloon injury plus leptin group. Genistein was capable of suppressing the atherogenic effects of leptin in vitro and in vivo, and may be a promising candidate drug in the clinical setting.

Nanoparticle Targeting CD44-Positive Cancer Cells for Site-Specific Drug Delivery in Prostate Cancer Therapy.

Prostate cancer is one of the leading causes of cancer death in adult men and is a multistage disease with therapeutic challenges of local recurrent advanced tumors and distant metastatic disease. CD44 is a multifunctional and multistructural cell surface glycoprotein that is involved in cell-cell interactions, cell proliferation, and cell migration. In the study, we produced negatively charged and biocompatible hyaluronic acid-based nanoparticles as a therapeutic system for targeting CD44-positive cancer cells. Subsequently, we confirmed the delivery of bioactive epigallocatechin-3-gallate and site-specific inhibition of prostate tumor growth. In this study, hyaluronic acid-based nanoparticles successfully encapsulated epigallocatechin-3-gallate and were efficiently internalized into cancer cells via CD44 ligand receptor recognition, induced cell cycle arrest at G2/M phase, and inhibited prostate cancer cell growth. Furthermore, in vivo assays indicated that these nanoparticles specifically bind CD44 receptors and increase apoptosis of cancer cells, leading to significant decreases in prostate tumor activity and tumor tissue inflammation.

Piceatannol Exerts Anti-Obesity Effects in C57BL/6 Mice through Modulating Adipogenic Proteins and Gut Microbiota.

Obesity is a global health concern. Piceatannol (Pic), an analog of resveratrol (Res), has many reported biological activities. In this study, we investigated the anti-obesity effect of Pic in a high-fat diet (HFD)-induced obese animal model. The results showed that Pic significantly reduced mouse body weight in a dose-dependent manner without affecting food intake. Serum total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL) levels, and blood glucose (GLU) were significantly lowered in Pic-treated groups. Pic significantly decreased the weight of liver, spleen, perigonadal and retroperitoneal fat compared with the HFD group. Pic significantly reduced the adipocyte cell size of perigonadal fat and decreased the weight of liver. Pic-treated mice showed higher phosphorylated adenosine 5'-monophosphate-activated protein kinase (pAMPK) and phosphorylated acetyl-CoA carboxylase (pACC) protein levels and decreased protein levels of CCAAT/enhancer-binding protein C/EBPα, peroxisome proliferator-activated receptor PPARγ and fatty acid synthase (FAS), resulting in decreased lipid accumulation in adipocytes and the liver. Pic altered the composition of the gut microbiota by increasing Firmicutes and Lactobacillus and decreasing Bacteroidetes compared with the HFD group. Collectively, these results suggest that Pic may be a candidate for obesity treatment.

Berberine-loaded targeted nanoparticles as specific Helicobacter pylori eradication therapy: in vitro and in vivo study.

AIM: The aim of this work was to develop fucose-conjugated nanoparticles and control the release of berberine, and demonstrate that these particles come into contact with Helicobacter pylori and enhance the suppressive effect of berberine on H. pylori growth. MATERIALS & METHODS: Fucose-chitosan/heparin nanoparticle-encapsulated berberine was prepared and delivery efficiency was monitored by confocal laser scanning microscopy. Anti-H. pylori activities were investigated by determining the calculated bacterial colonies and immunohistochemistry staining analysis. RESULTS: Analysis of a simulated gastrointestinal medium indicated that the proposed drug carrier effectively controls the release of berberine, which interacts specifically at the site of H. pylori infection, and significantly increases berberine's suppressive effect on H. pylori growth. In an in vivo study, the berberine-loaded fucose-conjugated nanoparticles exhibited an H. pylori clearance effect. CONCLUSION: These findings indicate that berberine-loaded fucose-conjugated nanoparticles exert an H. pylori clearance effect and effectively reduce gastric inflammation in an H. pylori-infected animal study.

Antiviral activity of chemical compound isolated from Artemisia morrisonensis against hepatitis B virus in vitro.

The compound p-hydroxyacetophenone (PHAP) isolated from Artemisia morrisonensis was found to have potential anti-HBV effects in HepG2 2.2.15 cells. We clarified its antiviral mode further and HBV-transfected Huh7 cells were used as the platform. During viral gene expression, treatment with PHAP had no apparent effects on the viral precore/pregenomic RNA. However, the 2.4-kb preS RNA of viral surface gene increased significantly relative to the 2.1-kb S RNA with PHAP. Promoter activity analysis demonstrated that PHAP had a potent effect on augmenting the viral preS promoter activity. The subsequent increase in the large surface protein and induce endoplasmic reticular (ER) stress has been reported previously. Interestingly, PHAP specifically reduced ER stress related GRP78 RNA/protein levels, but not those of GRP94, in treated Huh7 cells while PHAP also led to the significant intracellular accumulation of virus. Moreover, treatment with the ER chaperone inducer thapsigargin relieved the inhibitory effect of PHAP based on the supernatant HBV DNA levels of HBV-expressed cells. In conclusion, this study suggests that the mechanism of HBV inhibition by PHAP might involve the regulation of viral surface gene expression and block virion secretion by interference with the ER stress signaling pathway.

Quantitative analysis of fructo-oligosaccharides in Gynura divaricata subsp. formosana by high performance anion exchange chromatography-pulsed amperometric detection.

High performance anion exchange chromatography-pulsed amperometric detection was employed in this study to conduct quantitative analysis of the inulin-related fructo-oligosaccharides present in Gynura divaricata subsp. formosana. Result obtained for the 1-kestose (GF2), nystose (GF3) and 1F-beta-fructofuranosyl-nystose (GF4) contents were 146.60 +/- 0.04, 24.70 +/- 0.75 and 16.60 +/- 0.91 microg/g, fresh weight, and 68.90 +/- 0.02, 7.60 +/- 1.34 and 149.30 +/- 0.06 microg/g (mean +/- RSD), dry weight, respectively. Using this method, the limit of quantitation was 20 microg/mL and the linear detectability between 0-250 microg/mL. The developed method provides a practical analysis for these low caloric value, prebiotic and non-digestible carbohydrates in the genus Gynura.

Antihepatitis B virus constituents of Solanum erianthum.

Eleven compounds were isolated from the methanolic extract of the leaves of Solanum erianthum D. Don, including five alpha-linolenic acid analogs, alpha-linolenic acid (1), 13S-hydroxy-9(Z),11(E)-octadecadienoic acid (2), 9S-hydroxy-10(E),12(Z), 15(Z)-octadectrienoic acid (3), 9(Z),11(E)-octadecadienoic acid (4), and octadecanoic acid (5); two benzofuran-type lactones, loliolide (6) and dihydroactinidiolide (7); two steroidal alkaloids, solasonine (8) and solamargine (9); a flavonol glycoside, camelliaside C (10); and a flavone, 5-methoxy-(3,4"-dihydro-3",4"-diacetoxy)-2",2'-dimethylpyrano-(7,8:5",6")-flavone (11). Among these isolated compounds, 9 showed the most potent activity against HBsAg, with an IC50 of 1.57 microM, followed by 8 (IC50 is 5.89 microM). In the testing against HBeAg, 11 was the only active compound with an IC50 of 36.11 microM. Compound 9 also revealed strong inhibition of DNA replication towards HBV and its IC50 was 2.17 microM. However, alpha-linolenic acid (1) showed a prominent selected index (SI), both in anti-HBsAg and inhibition of DNA replication with SI values of 7.75 and 7.18, respectively. This is the first report that unsaturated fatty acid 1, steroidal alkaloid glycoside 9 and flavone 11, all showed excellent activity against HBV. These results provide lead candidates in the development of anti-HBV drugs from natural sources.

Hypoglycemic constituents of Gynura divaricata subsp. formosana.

Gynura divaricata Kitam. subsp. formosana is a folk medicine used as a hypoglycemic agent for diabetes patients in Taiwan. Guided by the hexose transport assay, the hypoglycemic constituents of the aerial part of this plant were disclosed through chromatographic methods. They are fructooligosaccharides, including beta-D-fructofuranose, sucrose, 1-kestose, nystose, and 1(F)-beta-fructofuranosylnystose. The hexose transport assay indicated that nystose was the most potent among these compounds, showing a 46.7% difference from pinitol in the stimulation index at a concentration of 0.5 mg/mL.

Analgesic and Anti-Inflammatory Activities of the Ethanolic Extract of Artemisia morrisonensis Hayata in Mice.

The aim of this study was to investigate the possible analgesic and anti-inflammatory mechanisms of the ethanolic extract of A. morrisonensis Hayata (AM(EtOH)). Two models were employed for evaluation of the analgesic effects: acetic acid-induced writhing response and formalin-induced paw licking. The results demonstrated that AM(EtOH) decreased writhing response for both the acetic acid assay and the licking time in the formalin test. The anti-inflammatory effect was evaluated by paw edema of mice induced by λ-carrageenan. AM(EtOH) significantly decreased induced paw edema three to four hours after λ-carrageenan injection. Additionally, the results indicated that the anti-inflammatory mechanism of AM(EtOH) may be due to the declined levels of nitric oxide (NO) and malondialdehyde (MDA) in the edematous paw. Furthermore, AM(EtOH) decreased the tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) levels, leading to the reduction of prostaglandins and subsequently alleviated edema. Isolation and purification of the AM(EtOH) extract determined p-hydroxyacetophenone to be a major component at 130 mg/g of extract. No mortality was observed in the acute toxicity test given at the dose of 10 g/kg. This study demonstrated the possible mechanisms for the analgesic and anti-inflammatory effects of AM(EtOH) for mice and provided evidence for the ethnobotanical uses of A. morrisonensis in treating inflammatory diseases.

Oleanane-type triterpenoids from the leaves and twigs of Fatsia polycarpa.

Seven new oleanane-type triterpenoids (1-7), named fatsicarpains A-G, and the known compounds 3α-hydroxyolean-11,13(18)-dien-28-oic acid (8) and 3α-hydroxyolean-11-en-28,13ß-olide (9) were isolated from the leaves and twigs of Fatsia polycarpa on the basis of bioassay-guided fractionation. The structures of compounds 1-7 were elucidated through spectroscopic analyses and single-crystal X-ray crystallography of 1, 8, and 9. Cytotoxicity against HepG2 2.2.15 and AGS cells and antihepatitis B virus (HBV) and antibacterial activities of 1-9 were also evaluated in vitro.

Influence of gallate esterification on the activity of procyanidin B2 in androgen-dependent human prostate carcinoma LNCaP cells.

PURPOSE: Present study assessed the influence of gallate esterification on the anti-cancer activity of procyanidin B2 (B2) in androgen-dependent human prostate carcinoma LNCaP cells employing B2-3,3'-di-O-gallate (B2-G(2)), two mono-gallate esters B2-3-O-gallate (B2-3G) and B2-3'-O-gallate (B2-3'G) and the parent compound B2, all isolated from grape seed extract (GSE). MATERIALS AND METHODS: Study compounds were isolated from GSE by several chromatographic steps and structures determined by a combination of enzymatic hydrolysis, mass spectrometry and comparisons with standards. Cells, treated with these compounds, were assessed for viability and apoptosis and examined by western blotting. RESULTS: Gallate esters B2-G(2), B2-3G and B2-3'G significantly decreased LNCaP cell viability; however, B2 and gallic acid were ineffective. Furthermore, only B2-G(2) also significantly decreased cell growth. Decreases in cell viability were largely due to apoptosis induction with B2-G(2) and B2-3'G exhibiting comparable effects, whereas B2-3G was less effective. In mechanistic studies, B2-G(2) and B2-3'G treatments caused caspases-9 and -3 and PARP cleavage, and down-regulated Bcl-2, Bcl-Xl and androgen receptor levels. CONCLUSION: Together, our findings demonstrate anti-PCA efficacy of B2-G(2) and suggest that a gallate ester moiety at 3' position of procyanidin B2 contributes more extensively toward the biological activity of the di-gallate ester than esterification of position 3.

Hydrophobic metabolites from Rhododendron formosanum and their allelopathic activities.

Eighteen components were isolated from the methanolic extract of leaves of Rhododendron formosanum Hemsl. (Ericaceae), an endemic species in Taiwan, and evaluated for their allelopathic properties. Of the isolated compounds, 3beta-friedelinol, 5,6beta-epoxy-5beta-stigmastan-3beta-ol, 5,6alpha-epoxy-5alpha-stigmastan-3beta-ol, lupeol and ursolic acid revealed inhibitory effects at 10(-4) M or above, whereas alpha-tocopherol, friedelin and beta-amyrin acetate exhibited stimulatory effects on the radicle growth of the test plants at the same concentration or above. However, squalene and alpha-amyrin showed either a stimulatory or inhibitory effect. The remaining four components, coumarin, beta-sitosterol, adian-5-en-3beta-ol and 3beta-acetoxyurs-12-en-28-al, were not significantly effective in the present bioassay, but have been reported as allelopathic agents elsewhere.