Pharmaceutical Biology

Published by Informa Healthcare
Online ISSN: 1744-5116
Print ISSN: 1388-0209
Context: The widely used antineoplastic drug cyclophosphamide causes pulmonary toxicity by inducing oxidative stress. Selenium, a dietary micronutrient, has been found to protect various organs from oxidative injuries. Objective: This study was designed to investigate the protective efficacy of an organoselenium compound 2-(5-selenocyanato-pentyl)-benzo[de]isoquinoline 1,3-dione against cyclophosphamide-induced pulmonary toxicity in Swiss albino mice. Materials and methods: Cyclophosphamide (25 mg/kg b.w.) was administered intraperitoneally for 10 d and the organoselenium compound (3 mg/kg b.w.) was given by oral gavage in concomitant and pretreatment schedules. Various biochemical parameters related to oxidative stress and antioxidant enzymes along with histology of lungs were evaluated to assess the effect of the test compound. Results: The oral LD50 of the test compound was more than 1000 mg/kg b.w. in Swiss albino mice. The test compound substantially ameliorated cyclophosphamide-induced pulmonary injury by reducing the levels of reactive oxygen species, reactive nitrogen species, and lipid peroxidation, respectively, by 14.88, 18.54, and 21.10% in concomitant treatment schedule and by 23.89, 35.73, and 30.76% in the pretreatment schedule as well as by restoring the level of reduced glutathione and activities of glutathione-S-transferase, superoxide dismutase, catalase, and glutathione peroxidase, respectively, by 36.88, 42.43, 38.0, 35.0, and 34.06% in the concomitant treatment schedule and by 66.02, 59.29, 57.23, 71.59, and 57.22% in the pretreatment schedule. The test compound also attenuated cyclophosphamide-induced histological alterations of lung tissue. Discussion and conclusion: The test compound emerged as an efficient antioxidant protecting lungs tissue from cyclophosphamide-induced injury.
Chemical composition of the essential oil of S. lavandulifolia.
(A) Effect of monoterpenes on cell viability. U373-MG cells were treated with a-pinene and 1,8-cineole (a range of concentrations from 10 to 400 lM) for 24 h. Cell viability was measured using MTT assay. Experiments were performed in triplicate and results are expressed as mean ± S.D. *p50.05 versus control. (B) Cytoprotective effect of monoterpenes. U373-MG cells were pretreated with a-pinene and 1,8-cineole (a range of concentrations from 10 to 100 lM) for 24 h, previous to the treatment with H 2 O 2 (1 mM) for 30 min. Cell viability was measured using the MTT assay. Experiments were performed in triplicate and results are expressed as mean ± S.D. *p50.05 versus control, # p50.05 versus H 2 O 2 -treated cells. (C) Effect of monoterpenes on cell morphology. U373-MG cells were pretreated with a-pinene and 1,8-cineole (10 and 25 lM) for 24 h, previous to the treatment with H 2 O 2 (1 mM) for 30 min. Representative images are shown.  
Effect of monoterpenes on intracellular ROS production. U373-MG cells were pretreated with a-pinene and 1,8-cineole (10 and 25 lM) for 24 h, previous to the treatment with H 2 O 2 (1 mM) for 30 min. The intracellular ROS production was measured using the dichlorofluorescein assay. Data are expressed as percentage of ROS production, mean ± SD, #p50.05, versus H 2 O 2 -treated cells.  
(A) Effect of monoterpenes on protein expression of antioxidant enzymes. U373-MG cells were pretreated with a-pinene and 1,8-cineole (10 and 25 lM) for 24 h, previous to the treatment with H 2 O 2 (1 mM) for 30 min. The levels of protein expression of CAT, SOD, GR, GPx, and HO-1 were measured by Western blots. (B) Effect of monoterpenes on Nrf2 protein expression. U373-MG cells were pretreated with a-pinene and 1,8-cineole (10 and 25 lM) for 24 h, previous to the treatment with H 2 O 2 (1 mM) for 30 min. Nuclear and cytoplasmic Nrf2 protein expression were measured by Western blots. Data are expressed as mean ± SD. Results are representative of three independent experiments. *p50.05 versus control, # p50.05 versus H 2 O 2 .  
Context: Salvia lavandulifolia has been employed in folk medicine for the treatment of memory and dementia problems. This specie contains numerous bioactive terpenes which may contribute to its effectiveness. Objective: To analyze the composition of essential oil of S. lavandulifolia and to investigate the potential in vitro cytoprotective and antioxidant activities of its major compounds, α-pinene and 1,8-cineole, against H2O2-induced oxidative stress in the U373-MG cell line. Materials and methods: Chemical composition was analyzed by gas chromatography; antioxidant capacity was measured using the ORAC assay, and cytoprotective activity was evaluated using the MTT assay (for cell viability) (range of concentrations: 10-400 μM), DCFH-DA assay (for intracellular ROS generation), thiobarbituric acid reactive substances (TBARS) method (for lipid peroxidation), and spectrofometric techniques and Western blot (for enzymatic activity and protein expression, respectively) at 10 and 25 µM. Results: α-Pinene (18.39%) and 1,8-cineole (19.57%) were identified as major compounds in S. lavandulifolia essential oil. Pretreatments with these monoterpenes protected U373-MG cells against H2O2-induced oxidative injury by attenuating the loss of cell viability (IC50 : 79.70 µM to α-pinene and 66.23 µM to 1,8-cineole) and cell morphology, inhibiting ROS production (the most active compound was 1,8-cineole by decreasing the ROS production over 30-45% at 10 and 25 μM) and lipid peroxidation and increasing the endogenous antioxidant status (glutathione levels and CAT, SOD, GR, GPx, and HO-1 activity and protein expression). Conclusions: These findings demonstrate for the first time the effects of the monoterpenes 1,8-cineole and α-pinene identified in S. lavandulifolia essential oil as regulators of cellular redox balance in astrocytes.
Extracts of Artemisia annua (L.) (Asteraceae) and artemisinins are used for treatment of malaria, parasitic infections and have potent anticancer properties in cell lines. Eucalyptus oil and 1,8-cineole have antimicrobial, immune-stimulatory, anti-inflammatory, antioxidant, analgesic, and spasmolytic effects. Codling moth, Cydia pomonella, (L.) (Tortricidae), is a major cosmopolitan pest of the apple, potentially causing damage translating to 40 billion US dollars per year, globally. Currently used control measures are either hazardous to agricultural workers and harmful to environment, or ineffective. The potential of plant-derived semiochemicals for codling moth control is heavily understudied. This study evaluated the potential of A. annua extracts, and two chemicals that this plant contains, artemisinin and 1,8-cineole, for preventing apple feeding and infestation by neonate Cydia pomonella larvae. We studied effects of A. annua extracts, artemisinin and 1,8-cineole on apple infestation by neonate codling moth larvae using fruit choice assay in laboratory experiments. Preference of fruit treated with test solutions versus fruit treated with solvent was recorded and analyzed. Crude A. annua extracts prevented fruit feeding at 1, 3, and 10 mg/ml. Artemisinin had feeding deterrent effects at 10 and 30 mg/ml, and 1,8-cineole at 100 and 300 mg/ml. A. annua contains chemicals that prevent apple infestation by codling moth neonates. Artemisinin and 1,8-cineole are among them, but there are other, polar constituents of A. annua, which have similar effects. There is a potential of using our findings in codling moth control and production of codling moth-resistant apples.
Chemical structure of 1,8-cineole which is the principal constituent of the many medicinal plant essential oils.
Effect of 1,8-cineole on normal gastrointestinal transit in rat.
Antidiarrheal activity of 1,8-cineole on the diarrhea parameters that induced by castor oil in rats.
Context: 1,8-Cineole, a terpene, characterized as a major constituent occurring in the essential oils of several aromatic plants. It is widely used in pharmaceutical industry, as a food additive and for culinary purposes. Objective: This study investigates the inhibitory effect of 1,8-cineole on transit time and diarrhea in animal models. Materials and methods: Acute toxicity and lethality of 1-8-cineole was determined by Lork's guidelines. The antidiarrheal effect of 1,8-cineole was investigated by determining the intestinal transit and enterpooling in rats. In all experiments, different doses of 1,8-cineole (20-120 mg/kg), atropine, and loperamide were administered orally. Results: The LD50 of 1,8-cineole for oral administration was estimated to be 1280 mg/kg. 1,8-Cineole (20-120 mg/kg) did not show a significant decrease in small intestine transit (p > 0.05); however, the highest dose displayed a significant decrease in comparison with atropine (p < 0.05). This substance decreased the peristaltic index value to 68 ± 0.36% at a dose of 120 mg/kg compared with the control group (85.22 ± 4.31%) in the castor oil transit test. 1,8-Cineole significantly delayed the onset of diarrhea to -142.33 ± 6.08 min at 120 mg/kg, while the time was 103.66 ± 20.73 min for the control and >240 min for the loperamide. Moreover, 1,8-cineole significantly decreased intestinal fluid accumulation (p < 0.05). Conclusions: This study demonstrated antispasmodic and antisecretory activities of 1,8-cineole and rationalized the traditional use of the plant containing various levels of this terpene in the treatment of gastrointestinal complains such as diarrhea.
Context: Endophytes colonizing medicinal plants are diverse, constituting a rich bioresource for novel natural products. Objective: Myrothecium sp. isolate M1-CA-102 was the most promising among the 16 Myrothecium isolates screened. The bioactive potential of the crude extract from the Calophyllum apetalum Willd. endophytic Myrothecium sp. (Alb. & Schwein.) Ditmar (Incertae sedis) isolate M1-CA-102 and its thin layer chromatography (TLC) fractions were screened based on antioxidant, anti-inflammatory, antimicrobial activities, and cytotoxicity. Materials and methods: The antioxidant activity was measured by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) radical scavenging capacities. Further, 15-lipoxygenase (15-LOX) and human cyclooxygenase-2 (COX-2) inhibition were assessed at different concentrations (25, 50, and 100 μg/mL for the crude extract, 5, 25, and 50 μg/mL for the TLC fractions). DNA-nicking assay as an indicator of the capacity of extracts to scavenge hydroxyl radical was recorded at a concentration of 50 μg/mL. Cell cytotoxicity was recorded by colorimetric 3-(4,5-dimethylthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Antibacterial (Bacillus subtilis) and anti-Candida (Candida albicans) assays were performed by the microdilution method. Results: The DPPH and ABTS IC50 values of M1-CA-102 extract were 10 and 6 μg/mL compared with 6.1 and 7.03 μg/mL for the positive control quercetin. The cytotoxicity IC50 value of M1-CA-102 extract was 37 μg/mL, while the M-I TLC fraction was 21 μg/mL. The M1-CA-102 extract gave an IC50 value of 58 and 8 μg/mL for 15-LOX and COX-2, respectively. The MIC values for antimicrobial activity for M1-CA-102 extract ranged from 35 to 54 μg/mL, while for the TLC fractions, it ranged from 91 to 515 μg/mL. Conclusion: The results indicate that Myrothecium M1-CA-102 isolated from C. apetalum is a potential source of natural metabolites of pharmaceutical importance.
Sinomenium acutum (Thumb.) Rehd. et Wils. (Menispermaceae, SA) has been used as a traditional Chinese medicine in the treatment of various diseases for hundreds of years; it possesses favorable effects against autoimmune diseases, especially rheumatoid arthritis (RA). A great number of investigations have been done on SA in the last decade, but they are usually scattered across various publications. The purpose of this article is to summarize and review the published scientific information about the chemical constituents, pharmacological effects, pharmacokinetics, and clinic applications of this plant since 2000. The information for 89 cases included in this review was compiled. The SA contains alkaloids, sterols, phospholipids, and some other components. A great deal of pharmacological and clinic research has been done on sinomenine, a main compound from SA, which mainly focuses on the immune system, cardiovascular system, and nervous system. Previous studies strongly support its potential as an effective adaptogenic herbal remedy. There is no doubt that SA is being widely used now and will have extraordinary potential for the future.
DDB analogs and their EBV-EA inhibition ability. 
Syntheses of DDB analogs. 
Dimethyl dicarboxylate biphenyl (DDB) is a clinically used hepatoprotectant and has also been found to have chemopreventive activity. Sixteen novel analogs (5-20) were designed, synthesized, and evaluated for their cancer preventive activity. The 2,2'-bismethyl ester (5-18) and ether (19, 20) DDB analogs were synthesized by insertion of various linear alkyl, short fatty acid, polar, and aromatic groups. All synthesized analogs were evaluated in an in vitro short-term 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced Epstein Barr virus early antigen (EBA-EA) activation assay. Three of the most potent compounds were also tested for inhibitory effects on skin tumor promotion in an in vivo two-stage mouse-skin carcinogenesis test using 7,12-dimethylbenz[a]anthracene (DMBA) as an initiator and TPA as a promoter. Compound 19 with bisprenyl ethers had the most significant cancer preventive activity (100% inhibition of activation at 1 × 10(3) mol ratio/TPA, 78.4%, 49.7%, and 10.9% inhibition at 5 × 10(2), 1 × 10(2), 1 × 10 mol ratio/TPA, respectively) in vitro. Compound 19 also exhibited a remarkable inhibitory effect on skin tumor promotion in the in vivo two-stage mouse-skin carcinogenesis test. Thus, DDB analog 19 could be a valuable candidate as a cancer preventive agent or as a lead for the development of new antitumor promoter drugs.
Effect of the methanol extract of I. frutescens on plasma lipid levels of high-fat diet induced hyperlipidemic rats.
Effect of I. frutescens fractions on plasma lipid levels in triton WR-1339-induced hyperlipidemic mice.
Effect of fraction 3 of Ichnocarpus frutescens and orlistat on fat excretion in feces of mice fed with high-fat diet.
Ichnocarpus frutescens (L.) R.Br. (Apocynaceae) is used to treat diabetes and hyperlipidemia in folk medicine. Objective: The crude methanol extract and fractions of I. frutescens were investigated for antihyperlipidemic effect. Fresh leaves of I. frutescens were extracted with methanol and fractionated with hexane, benzene, ethyl acetate, acetone, and methanol. The active acetone fraction was subfractionated, which resulted in active fraction 3. The antihyperlipidemic effects of the methanol extract and fractions of I. frutescens were studied in triton WR-1339-induced and high-fat diet (HFD) obese animals. Further, lipid absorption and excretion were studied. The methanol extract significantly reduced total cholesterol (TC) by 29.63% and triglyceride (Tg) by 51.10% at 400 mg/kg in triton WR-1339-induced animals and significantly reduced TC (27.81%) and Tg (37.03%) at 400 mg/kg in HFD animals. Fraction 3 showed significant reduction in TC (25.03%) and Tg (58.05%) at 200 mg/kg. Feeding of HFD consisting 3% of fraction 3 increased feces weight and Tg level in mice. Fraction 3, showed significant decrease in plasma Tg level at the second hour, after oral administration of the lipid emulsion to rats. The observed properties apparently validate the folk medicinal use of this plant in amelioration of hyperlipidemia.
Act1 mRNA expressed in synovioblast cells and SW982 cells. (A) Gel image of RT-PCR product for Act1 mRNA. Lines 1, 2: Act1 mRNA (415 bp) in primary cultured RA fibroblast-like synovial cells; line 3: Act1 mRNA in SW982 cells; lines 4–6: b -actin mRNA (220 bp) in SW982 cells; M: Trans DNA Marker I. (B) Representative Western blot bands of Act1 and b -actin are shown. Line 1: SW982; lines 2–3: primary cultured RA fibroblast-like synovial cells. 
Act1 mRNA levels are induced by different concentrations and different incubation times of IL-17 in SW982 cells. (A) Gel image of RT-PCR product for Act1 mRNA 4 h after stimulation by IL-17. Lines 1-6: Act1 mRNA in 100, 50, 20, 10, 5, 0 ng/ml IL-17; lines 1 0-6 0 : the corresponding b-actin mRNA. (B) The quantitive analysis for Act1 mRNA in SW982 cells. The Act1 mRNA level was increased with the elevated concentration of IL-17. The data are expressed as mean AE SEM (n ¼ 9 per group). **p50.01, ***p50.001, compared with 0 ng/ml IL-17. (C) Gel image of RT-PCR product for Act1 mRNA after stimulation by 20 ng/ml IL-17. Lines 1-6: Act1 mRNA in 0 min, 15 min, 30 min, 1 h, 2 h, 4 h after induction by IL-17; lines 1 0-6 0-: the corresponding b-actin mRNA. (D) The quantitative analysis for Act1 mRNA in SW982 cells. The Act1 mRNA level was increased with the stimulation time of IL-17. The data are expressed as mean AE SEM (n ¼ 9 per group). *p50.05, **p50.01, ***p50.001, compared with IL-17 at 0 min.
Traf3ip2 siRNA significantly decreased Act1 protein expression level in SW982 cells. (A) Representative Western blot bands of Act1 are shown. Lines 1-2: control-siRNA; line 3: Traf3ip2-siRNA. (B) The DY-547-labeled SW982 cells after transfection with Traf3ip2 gene for 48 h. Left: common microscopy; right: fluorescence microscopy (Â400).
IL-6 and IL-8 were induced in SW982 cells with various treatments. IL-17 (20 ng/ml) significantly increased IL-6 and IL-8 in SW982 cells, while transfection of Traf3ip2 siRNA markedly decreased IL-6 and IL-8 level compared to transfection controlsiRNA group. The data are expressed as mean AE SEM (n ¼ 9 per group). **p50.01 compared with the control group; ##p50.01 compared with the control-siRNA group.
Context: Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammation in the synovial membrane of affected joints. It has been shown that several kinds of cytokine were increased in synovial fluid, while the underlying mechanism remains poorly understood. Objectives: NF-κB activator 1 (Act1) is a recently identified protein binding to the IκB kinase complex. Our study aimed to investigate the expression of Act1 induced by cytokine IL-17 stimulation in SW982 cells. Materials and methods: The human synovial sarcoma cell line SW982 and primary cultured RA fibroblast-like synovial cells were used. RT-PCR and Western blot assays were selected to investigate the genetic and protein expression of Act1. Additionally, four independent Act1 small interfering RNA (siRNA) oligonucleotides were designed and obtained according to the GenBank cDNA, the sequence of Act1 (Traf3ip2). Finally, enzyme-linked immunosorbent assay (ELISA) double antibody sandwich was used to assay supernatant IL-6 and IL-8 concentrations. Results: The Act1 mRNA expression level increased significantly after stimulation with IL-17 (5-100 ng/ml) in SW982 cells. Additionally, the level of Act1 mRNA expression correlated positively with the concentration of IL-17 (p < 0.01). IL-17 induced IL-6 and IL-8 in SW982 cells was in a concentration- and time-dependent way. Furthermore, ELISA assay revealed that IL-17 (20 ng/ml) significantly increased IL-6 (1927.4 ± 288.77 versus 786.5 ± 172.42 ng/ml, p < 0.01) and IL-8 levels (984.8 ± 95.09 ng/ml versus 307.1 ± 90.83 ng/ml, p < 0.01) compared with control group after stimulation for 24 h. However, transfection of Traf3ip2 siRNA markedly decreased IL-6 (995.9 ± 115.30 ng/ml versus 1816.1 ± 273.27 ng/ml, p < 0.01) and IL-8 levels (575.6 ± 65.96 ng/ml versus 929.4 ± 124.39 ng/ml, p < 0.01) compared to transfection negative control. These findings suggested that IL-6 and IL-8 level induced by IL-17 in SW982 cells could be reversed by down-regulation of Act1 expression level with Traf3ip2 siRNA. Conclusion: Our results suggested that Act1 might play a key role in the pathophysiology and the treatment of RA.
Syzygium cumini (L.) Skeels (Myrtaceae), commonly known as jamun, is an Indian plant, traditionally well known for its medicinal properties including antidiabetic activity. To isolate the antidiabetic compounds from Syzygium cumini seeds and evaluate their activity using aldose reductase (AR) and protein-tyrosine phosphatase 1B (PTP1B) inhibition assays. The dried seeds were extracted with methanol and partitioned with ethyl acetate, butanol, and water. The extracts were screened for antidiabetic activity at a concentration of 100 µg/mL using in vitro AR and PTP 1B inhibition assays. The highly enriched fractions obtained from broad ethyl acetate fraction yielded maslinic acid (1), 5-(hydroxymethyl) furfural (2), gallic acid (3), valoneic acid dilactone (4), rubuphenol (5), and ellagic acid (6). Structures were elucidated by (1)H-NMR and (13)C-NMR. The initial ethyl acetate fraction showed AR inhibitory activity with the IC50 value of 2.50 μg/mL and PTP1B enzyme inhibition with the IC50 value of 26.36 μg/mL. Compounds 3, 4, 5, and 6 were found to inhibit AR with IC50 values of 0.77, 0.075, 0.165, and 0.12 μg/mL while the compounds 4, 5, and 6 inhibited PTP1B with IC50 values of 9.37, 28.14, and 25.96 μg/mL, respectively. The results of this study demonstrate that the isolated constituents show promising in vitro antidiabetic activity and, therefore, can be candidates for in vivo biological screening using relevant models to ascertain their antidiabetic activity.
Context: Tamarindus indica L. (Leguminosae) is widely used as a traditional medicine for the management of diabetes mellitus (DM) in India, in addition to its anti-inflammatory activity. The present study has been designed to understand the correlation involved between antidiabetic and anti-inflammatory action of aqueous seed extract of T. indica (TSE) in diabetic rats. Objective: In view of the fact that fatty acid synthesis and insulin release from islets of pancreas are regulated by sterol regulatory element-binding proteins (SREBP-1c) and cytosolic calcium, respectively, the objectives of present study were to determine the influence of TSE on SREBP-1c mRNA and to investigate the intracellular islets calcium [Ca²⁺](I) involvement and β-cell mass preservation in insulin secretagogue action of TSE. Materials and methods: The effect of 4 weeks oral treatment (120 and 240 mg/kg) of high-performance liquid chromatography (HPLC) standardized TSE was studied in streptozotocin (STZ)-induced diabetic male Wistar rats. Reverse transcription-PCR (RT-PCR) and a spectrofluorometer were used for mRNA concentration and islets [Ca²⁺](I) determination, respectively. The TUNEL assay was followed to study the pancreatic apoptosis. Results: TSE (120 and 240 mg/kg) showed positive correlation with [Ca²⁺](I) and insulin release. The anti-inflammatory action of TSE was significant on nitric oxide (NO) and tumor necrosis factor-α (TNF-α) in addition to a favorable effect on β-cell neogenesis and improved mRNA concentration of SREBP-1c. Discussion and conclusion: The results suggest that anti-inflammatory action of Tamarind seeds on β-cell cells of islets and cytokines contribute toward its antidiabetic activity by way of complex mechanisms of [Ca²⁺](I) handling and through SREBP-1c gene in liver.
Abstract Context: Formononetin, an isoflavone, can inhibit the proliferation of cancer cells, including those of the prostate. However, its antitumor mechanism remains unclear. Aim: To investigate whether the insulin-like growth factor 1 (IGF-1)/insulin-like growth factor 1 receptor (IGF-1 R) signaling pathway mediates the formononetin antitumor effect on prostate cancer cells. Materials and methods: The viability of PC-3 cells was measured by MTT assay 48 h after formononetin treatment (25, 50 and 100 μM). Formononetin-induced cell apoptosis was measured by Hoechst 33258 staining and flow cytometry. Expression of Bax mRNA was detected by real-time PCR, and the expression levels of Bax and IGF-1 R proteins were detected by western blots. Results: At concentrations >12.5 μM, formononetin significantly inhibited the proliferation of human prostate cancer cells. Formononetin increased Bax mRNA and protein expression levels and decreased the expression levels of pIGF-1 R protein in a dose-dependent manner. Conclusion: High concentrations of formononetin-induced apoptosis in androgen-independent prostate cancer cells through inhibition of the IGF-1/IGF-1 R pathway.
Tephrosia sinapou ethyl acetate extract inhibited carrageenin- induced mechanical and thermal hyperalgesia in an opioid receptor- dependent manner. Mice were treated with naloxone (0.1–1 mg/kg, s.c., 1 h) before T. sinapou (100 mg/kg, i.p.), morphine (5 mg/kg, i.p.) or vehicle. After additional 30 min, mice received carrageenin injection (100 m g/paw). The intensity of mechanical (Panel A) and thermal (Panel B) hyperalgesia were measured 3 (open bars) and 5 (closed bars) h after carrageenin injection by the electronic pressure-meter and hot-plate test. Results are presented as means Æ SEM of experiments performed with five mice per group and are representative of two separated experiments. * p 5 0.05 compared to the carrageenin positive control vehicle group, 
Tephrosia sinapou ethyl acetate extract inhibited neutrophil recruitment (myeloperoxidase activity) by an opioid-dependent mechanism. Panel A: mice were treated with vehicle or T. sinapou (100 mg/ kg, i.p.) 30 min before carrageenin (100 mg/paw) stimulus. Panel B: mice were treated with naloxone (0.1-1 mg/kg, s.c., 1 h) before T. sinapou (100 mg/kg, i.p.), morphine (5 mg/kg, i.p.) or vehicle. After additional 30 min, mice received carrageenin injection (100 mg/paw). The neutrophil recruitment was evaluated by the myeloperoxidase activity at 1, 3 and 5 h (Panel A) and at 5 h (Panel B) after carrageenin injection using a kinetic colorimetric assay. Results are presented as means AE SEM of experiments performed with five mice per group and are representative of two separated experiments. *p50.05 compared to the carrageenin positive control vehicle group, #p50.05 compared to the naloxone negative control group (one-way ANOVA followed by Tukey's test).
Tephrosia sinapou ethyl acetate extract inhibited carrageenin- induced TNF a production in the paw skin by an opioid-dependent mechanism. Panel A: mice were treated with vehicle or T. sinapou (100 mg/kg, i.p.) 30 min before carrageenin (100 m g/paw) stimulus. Panel B: mice were treated with naloxone (1 mg/kg, s.c., 1 h) before T. sinapou (100 mg/kg, i.p.), morphine (5 mg/kg, i.p.) or vehicle treatment. After additional 30 min, mice received carrageenin (100 m g/paw) stimulus. Paw skin samples were collected 1, 3 and 5 h (Panel A) and at 3 h (Panel B) after carrageenin injection, and TNF a levels were determined by ELISA. Results are presented as means Æ SEM of experiments performed with five mice per group and are representative of two separated experiments. * p 5 0.05 compared to carrageenin þ vehicle group, # p 5 0.05 compared to the naloxone negative control group (one-way ANOVA followed by Tukey’s test). 
Tephrosia sinapou ethyl acetate extract inhibited carrageenininduced IL-1b production by an opioid-dependent mechanism. Panel A: mice were treated with vehicle or T. sinapou (100 mg/kg, i.p.) 30 min before carrageenin (100 mg/paw) stimulus. Panel B: mice were treated with naloxone (1 mg/kg, s.c., 1 h) before T. sinapou (100 mg/kg, i.p.), morphine (5 mg/kg, i.p.) or vehicle. After additional 30 min, mice received carrageenin (100 mg/paw) stimulus. Paw skin samples were collected 1, 3 and 5 h (Panel A) and at 3 h (Panel B) after carrageenin injection, and IL-1b levels were determined by ELISA. Results are presented as means AE SEM of experiments performed with five mice per group and are representative of two separated experiments. *p50.05 compared to carrageenin þ vehicle group, #p50.05 compared to the naloxone negative control group (one-way ANOVA followed by Tukey's test).
Tephrosia sinapou ethyl acetate extract inhibited acetic acidinduced writhing response in mice by an opioid-dependent mechanism. Panel A: mice were treated i.p. with T. sinapou ethyl acetate extract (1-10 mg/kg, 30 min), morphine (5 mg/kg, i.p., 30 min) or vehicle before i.p. stimulus with acetic acid (0.8% diluted in saline). Panel B: mice were treated with T. sinapou (10 mg/kg, i.p., 30 min) or vehicle and another group was treated with naloxone (1 mg/kg, s.c., 1 h) before T. sinapou (10 mg/kg, i.p., 30 min) before i.p. stimulus with acetic acid (0.8% diluted in saline). The writhing score was evaluated during 20 min after stimulus injection. Results are presented as means AE SEM of experiments performed with five mice per group and are representative of two separated experiments. *p50.05 compared to the vehicle group, #p50.05 compared to the vehicle group, the dose of 1 mg/kg (Panel A) and compared to the naloxone negative control group (Panel B; one-way ANOVA followed by Tukey's test).
Unlabelled: CONTEXT. Tephrosia toxicaria is currently known as Tephrosia sinapou (Buc'hoz) A. Chev. (Fabaceae) and is a source of compounds such as flavonoids that inhibit inflammatory pain. Objective: To investigate the analgesic effect and mechanisms of the ethyl acetate extract of T. sinapou in inflammatory pain in mice. Materials and methods: Behavioral responses were evaluated using mechanical (1-24 h) and thermal hyperalgesia (0.5-5 h), writhing response (20 min) and rota-rod (1-5 h) tests. Neutrophil recruitment (myeloperoxidase activity), cytokines (tumor necrosis factor [TNF]α and interleukin [IL]-1β), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) serum levels were determined by colorimetric assays. Pharmacological treatments were opioid receptor antagonist (naloxone, 0.1-1 mg/kg) and control opioid (morphine, 5 mg/kg). Inflammatory stimuli were carrageenin (100 µg/paw), complete Freund's adjuvant (CFA, 10 µl/paw), prostaglandin E2 (PGE2, 100 ng/paw) and acetic acid (0.8%). Results: The intraperitoneal pre-treatment with extract inhibited in a dose-dependent (30-300 mg/kg) dependent manner the mechanical hyperalgesia induced by carrageenin (up to 93% inhibition). The post-treatment (100 mg/kg) inhibited CFA-induced hyperalgesia (up to 63% inhibition). Naloxone (1 mg/kg) prevented the inhibitory effect of the extract over carrageenin-induced mechanical (100%) and thermal (100%) hyperalgesia, neutrophil recruitment (52%) and TNFα (63%) and IL-1β (98%) production, thermal threshold in naïve mice (99%), PGE2-induced mechanical hyperalgesia (88%) and acetic acid-induced writhing response (49%). There was no significant alteration in the rota-rod test, and AST and ALT serum levels by extract treatment. Discussion and conclusion. Tephrosia sinapou ethyl acetate extract reduces inflammatory pain by activating an opioid receptor-dependent mechanism.
The roots of Phytolacca americana L. (Phytolaccaceae) may be toxic. Despite heated controversy over the toxic compounds of P. americana, especially esculentosides, relevant studies remain scarce. The objective of this study is to screen the toxic fractions and compounds of P. americana, to determine the controlling indices, and to provide evidence for unraveling the mechanism. Petroleum ether (PE), CH2Cl2, n-BuOH, and water fractions were isolated from 70% ethanol extract of P. americana. The n-BuOH fraction was dissolved in 50% ethanol and precipitated by adding ethyl ether. The resultant supernatants and precipitates were referred to as SUPs and SEDs fractions, respectively. SUPs fraction was separated by column chromatography into four main stimulating esculentosides that were identified by HR-ESI/MS and NMR as EsA, EsB, EsC, and EsF. The irritating effects of esculentosides on rabbit conjunctivae (500 μg/eye) was observed by pathological examination and those on macrophages (5, 25, 50 and 100 μg/mL) were evaluated by detecting changes of NO, TNF-α, and IL-1β levels. n-BuOH, SUP fractions, and EsC induced severe conjunctival edema. The four esculentosides induced dose-dependent releases of proinflammatory mediators NO, TNF-α, and IL-1β from macrophages, and releasing amounts peaked after 2 h of treatment. EsC and EsF induced macrophages to release mediators most significantly. EsC (50 μg/mL) functioned more effectively than EsF did, and similarly n-BuOH and SUPs fractions functioned more effectively than the esculentoside mixture. Thus, the four esculentosides exerted proinflammatory effects synergistically. All extracted esculentosides, especially EsC, induced inflammatory stimulation. Phytolacca americana-induced irritation of the gastrointestinal tract may be associated with esculentosides such as EsC.
Context: Previous in vitro studies have demonstrated that emodin (1,3,8-trihydroxy-6-methyl-anthraquinone), an anthraquinone derivative from the rhizome of Rheum palmatum L., can inhibit the activation of P2X₇ receptors (P2X₇R) as a potential antagonist. However, the effects of emodin on P2X₇R-related inflammatory processes remain unclear. Objective: This study aimed to investigate the effects of emodin on different inflammation responses of macrophages induced by ATP, the natural ligand of P2X₇R. Materials and methods: Rat peritoneal macrophages were treated with millimolar ATP and emodin (0.1, 0.3, 1, 3, 10 µM) or brilliant blue G (BBG, 0.1, 1, 10 µM). Cytosolic Ca²⁺ concentration ([Ca²⁺]c) was detected by fluorescent Ca²⁺ imaging. Interleukin-1β (IL-1β) release was measured by rat IL-1β ELISA kits. Reactive oxygen species (ROS) generation was examined by dihydroethidium (DHE) fluorescent staining. Phagocytic activity was tested by neutral red uptake assay. Results: We found that the [Ca²⁺](c) increase evoked by ATP (5 mM) was inhibited by emodin, in a dose-dependent manner with IC₅₀ of 0.5 μM. Furthermore, emodin reduced the IL-1β release induced by ATP (2 mM) in lipopolysaccharide (LPS)-activated macrophages, with an IC₅₀ of 1.6 μM. Emodin also strongly suppressed the ROS production and phagocytosis attenuation triggered by ATP (1 mM), with IC₅₀ values of 1 μM and 0.7 μM, respectively. Besides, BBG, a specific antagonist of P2X₇R, exhibited similar suppressive effects on these inflammation responses. Conclusion: These results showed the inhibitory effects of emodin on ATP-induced [Ca²⁺](c) increase, IL-1β release, ROS production and phagocytosis attenuation in rat peritoneal macrophages, by inhibiting the activation of P2X₇R.
Cynanchum taiwanianum T. Yamaza (Asclepiadaceae) is a medicinal herb used in folk medicine for the treatment of several inflammation-related diseases such as hepatitis and dermatitis in Taiwan. In the present study, we investigated the anti-inflammatory effect of C. taiwanianum T. Yamaza rhizome aqueous extract (CTAE). The present study investigated the anti-inflammatory effect of CTAE using IL-1β-induced NRK-52E cells. Production of NO and PGE(2) by ELISA, the mRNA and protein expression of iNOS and COX-2, phosphorylation of IκBα, and activation of NF-κB by RT-PCR and western blotting were determined. The CTAE significantly (P < 0.05) inhibited NO and PGE(2) production (decreased by 46.1% and 51%, respectively), and also significantly (P < 0.05) attenuated protein and mRNA expression of iNOS and COX-2 (decreased by 90% and 55% for iNOS and by 72% and 74%% for COX-2, respectively) in IL-1β-induced NRK-52E cells, in a dose-dependent manner, without obvious cytotoxic effects. Furthermore, the CTAE suppressed the NF-κB nuclear translocation, in terms of inhibition of IκBα phosphorylation. Our results provided evidence for its folkloric uses and suggest that the anti-inflammatory activities of CTAE may result from the inhibition of inflammatory mediators, such as NO and PGE(2), and an upstream suppression of a NF-κB-dependent mechanism, might be involved.
2,7-Dihydroxy-3-methylanthraquinone (DDMN) is reported to have a remarkable anticancer activity against gastric cancer SGC-7901 cells. The objective of this study is to study the anticancer effect and mechanism of DDMN on SGC-7901 cells. The MTT assay was used to determine the effect of DDMN on cell viability of SGC-7901 cells, and the cytotoxic effect was evaluated by the IC50 value. After treatment with different doses of DDMN (10, 20, and 40 μM) for 48 h, flow cytometry was used to investigate the apoptosis of SGC-7901 cells induced by DDMN. Further, western blotting was performed to study anticancer mechanism by assaying apoptosis-related proteins containing Mcl-1, Bcl-xl, Bcl-2, Bax, Bak, Bad, cytochrome c, caspase-3, and caspase-9. Finally, xenograft assay was used to further evaluate the effect of DDMN on SGC-7901 cells by determining body weight of nude mice, tumor volumes, and apoptosis-related proteins. These results suggest that DDMN can significantly inhibit (IC50 value = 20.92 μM) the proliferation of SGC-7901 cells and induce apoptosis of SGC-7901 cells demonstrated by flow cytometry analysis. Additionally, the results of western blotting indicated that DDMN can suppress the expression of anti-apoptotic proteins Bcl-xl and Bcl-2, increase the expression of pro-apoptotic proteins Bax, Bad (40 μM), caspase-3 and caspase-9, and evidently promote the release of cytochrome c from the mitochondria to the cytoplasm. The xenograft assay further confirmed that DDMN had significant anticancer effects on SGC-7901 cells. DDMN had significant anticancer effect on SGC-7901 cells in vitro and in vivo related to mitochondria-mediated apoptosis.
Liver disease is a serious ailment and the scenario is worsened by the lack of precise therapeutic regimens. Currently available therapies for liver ailments are not apposite and systemic toxicity inhibits their long term use. Medicinal plants have been traditionally used for treating liver diseases since centuries as the toxicity factor appears to be on the lower side. Several phytochemials have been identified which have significant hepatoprotective activity with minimal systemic adverse effects which could limit their long term use. The scenario calls for extensive investigations which can lead to development of lead molecules for hepatoprotective molecules of future. This review deals with the biological activity, mode of action and toxicity and forthcoming application of some of these leads. These generally have strong antioxidative potential and cause induction of antioxidant enzymes like superoxide dismutase, reduced glutathione and catalase. Additional mechanisms of hepatoprotection include stimulation of heme oxygenase-1 activity, inhibition of nitric oxide production, hepatocyte apoptosis and nuclear factor-κB activation. Out of the several leads obtained from plant sources as potential hepatoprotective agents, silymarin, andrographolide, neoandrographolide, curcumin, picroside, kutkoside, phyllanthin, hypophyllanthin, and glycyrrhizin have been established as potent hepatoprotective agents. The hepatoprotective potential of several herbal medicines has been clinically evaluated. Significant efficacy has been seen with silymarin, glycyrrhizin and Liv-52 in treatment of hepatitis, alcoholic liver disease and liver cirrhosis.
The bacterium Pseudomonas syringae pv. syringae (Pss) is a pathogen of many plant species and causes, for example, brown spot disease in bean plants (Phaseolus vulgaris). Pss excretes the syringolins, natural product molecules that act as a virulence factors and inhibit the proteasome of the host plants. Proteasome inhibitors belong to an important class of anticancer agents and bortezomib (Velcade(®)) has been Food and Drug Administration-approved for the treatment of multiple myeloma (MM) and mantle cell lymphoma. Syringolins represent a new class of proteasome inhibitors and the present work was undertaken to design a potent syringolin-inspired analogue (TIR-203) for anticancer drug development. TIR-203 was tested against human MM and neuroblastoma (NB) cells. Cancer cells were treated with TIR-203 at various concentrations (0-10 µM) and the cell viability was measured using the MTS assay. To determine the effects on proteasomal activities, the cell culture-based proteasome inhibition assay was used. Syringolin A (SylA) and bortezomib were included as controls. TIR-203 inhibited the cell proliferation of MM and NB cells in a dose-dependent manner at significantly lower concentrations than SylA. In MM cells, TIR-203 effectively inhibited the chymotrypsin-like (CT-L), caspase-like (C-L), and trypsin-like (T-L) activities of the proteasome. In NB cells, TIR-203 inhibited the CT-L and C-L activities, but not the T-L activity. Discussion and conclusions: The newly designed proteasome inhibitor TIR-203 is more potent than the natural product SylA and strongly inhibits the cell viability and proteasomal activity of MM and NB cells.
Effects of TPL on the growth of K562 cells and K562/A02 cells. The cells were treated with various concentrations of TPL for 72 h, respectively. Viable cells were evaluated by MTT assay.
The effect of TPL on miR-21 and PTEN expression in K562/A02 cells. A: the miR-21 levels in K562 and K562/A02 cells; B: PTEN protein expression in K562 and K562/A02 cells.
The effects of miR-21 on adriamycin cytotoxicity in K562 and K562/A02 cells. A: miR-21 levels in K562 cells; B: miR-21 levels in K562/A02 cells; C: cell viability of K562 cells; D: cell viability of K562/A02 cells.
The effects of miR-21 on PTEN protein level in K562 and K562/A02 cells. A,B: PTEN protein levels in K562 cells; C,D: PTEN protein levels in K562/A02 cells.
The effects of miR-21 on PTEN promoter activity in K562 cells. miR-21 markedly decreased the activity of the PTEN-3'UTR reporter.
Context: Multidrug-resistance is a serious obstacle encountered in leukemia treatment. Recent studies have shown microRNA-21 (miR-21) is overexpressed in several types of cancer and contributes to tumor resistance to chemotherapy. In our previous studies, we found triptolide (TPL) could enhance adriamycin-induced cytotoxicity and apoptosis in K562/A02 cells. Objective: In the present study, we investigated the mechanism of TPL on the sensitivity of K562/A02 cells to adriamycin. Materials and methods: Cell viability was assessed by methyl thiazolyl tetrazolium (MTT) assay. Expression of mature miR-21 was determined by SYBER green PCR. The miR-21 mimics and inhibitors were chemically synthesized and transfected into K562 cells or K562/A02 cells. PTEN protein levels was determined by western blots. PTEN promoter activity was measured by luciferase assays. Results: TPL (5 nmol/L) increased the sensitivity of K562/A02 to adriamycin. When adriamycin was combined with 5 nmol/L TPL, the mean apoptotic population of K562/A02 cells was increased from 4.3 to 18.5%, respectively. K562/A02 cells showed a significant reduction in miR-21 and phosphatase and tensin homolog deleted on chromosome ten (PTEN) expressions after TPL treatment. K562/A02 cells that were transfected with the miR-21 inhibitor had a significantly higher PTEN protein level than the control. K562 cells that were pre-treated with PTEN siRNA had increased survival rate compared to the control group. Discussion and conclusion: Our findings indicated that triptolide modulates the sensitivity of K562/A02 cells to adriamycin by regulating miR-21 expression. Triptolide inhibited miR-21 expression and enhanced PTEN levels in K562/A02 cells.
Ginkgo biloba L. (Ginkgoaceae) leaves have been used as an herbal medicine that has a complex range of biological activities. However, when we consider that biological activity of plant extracts is highly variable according to the source, location, and harvest season, technology to obtain the natural products with homogeneity is extremely important. We established the technology to obtain the cambial meristematic cells (CMCs) of Ginkgo biloba, which were expanded in vitro with homogeneity through a suspension culture and then determined the anti-inflammatory activity of fractionated samples prepared from the ethanol extract of CMCs. We determined the anti-inflammatory activity of samples using lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. Especially, influence of sample treatment on the expression of various indicators, such as nitric oxide (NO), inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, mitogen-activated protein (MAP) kinases, transcription factor, and cytokines, involved in inflammatory activity was assessed. A fractionated sample demonstrated 53.4% inhibition of LPS-induced NO production from the cells. Additionally, when fractionated samples were treated, iNOS and COX-2 expressions were almost completely suppressed. Fractionated samples also inhibited the phosphorylation of LPS-induced extracellular signal-regulated (ERK) and p38 MAP kinases more than 60%. IκB phosphorylation and subsequent nuclear factor (NF)-κB activation were also suppressed by fractionated samples. The expression of pro-inflammatory cytokines, IL-6 and tumor necrosis factor (TNF)-α, was significantly inhibited by the sample treatment. Fractionated samples from the ethanol extract of Ginkgo biloba CMCs could potentially be the source of a powerful anti-inflammatory substance.
Juncus effusus L. var. decipiens BUCHEN. f. leschenaultii GAY has been used in traditional medicine for the treatment of anxiety and insomnia. The objective of this study was to evaluate the effects of ethanol extract from the pith of Juncus effusus (JEE) on anti-inflammatory activities in RAW 264.7 cells. The production of inflammatory mediators and the underlying mechanisms using 3.1, 6.3, and 12.5 μg/mL concentrations of JEE were investigated. In addition, the topical anti-inflammatory effects of JEE (0.5, 1, and 2 mg/mL) on 12-O-tetradecanoylphorobol-13 acetate (TPA)-induced ear edema and oral administration of JEE (50, 100, and 200 mg/kg) on carrageenan-induced paw-edema were studied in mice. JEE reduced the release of nitric oxide (NO, IC50 value = 1.98 μg/mL), prostaglandin E2 (IC50 value = 5.5 μg/mL), and pro-inflammatory cytokines, IL-1β (IC50 value = 4.74 μg/mL) and IL-6 (IC50 value = 20.48 μg/mL). JEE also suppressed the protein expression of inducible NO synthase and cyclooxygenase-2 in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Mechanism studies showed attenuation of LPS-induced activation of NF-κB by JEE via abrogation of IκBα degradation and a subsequent decrease in nuclear p65 level. Phosphorylation of all three MAP kinases (ERK, JNK, and p38) in LPS-stimulated RAW 264.7 cells was also suppressed in a dose-dependent manner. In acute inflammation models of mice, topical application (1 and 2 mg) and oral administration (50, 100, and 200 mg/kg) of JEE ameliorated TPA-induced ear edema and carrageenan-induced paw edema, respectively, in dose-dependent manners. These results indicate that JEE exhibited anti-inflammatory activities by suppressing the production of inflammatory mediators in LPS-stimulated RAW 264.7 cells and by attenuating edema in mice.
Sedative effect in the exploratory cylinder test of Ternstroemia sylvatica extracts after intraperitoneal administration in mice. 
Context: Fruits of Ternstroemia sylvatica Schltdl. and Cham. (Theaceae) are used in Mexican traditional medicine to alleviate anxiety, sleep disorders and seizures; however, the active principles have not been identified. Objective: To identify the neuroactive principles of T. sylvatica fruits using neuropharmacological tests on mice. Materials and methods: The methanol and aqueous extracts of pericarp or seeds of T. sylvatica fruits were intraperitoneally administered (1-562 mg/kg, single doses) to mice. The exploratory cylinder, hole board, open field, Rota-rod and sodium pentobarbital-induced hypnosis tests were used to evaluate the CNS depressant effect after 30 min single administration of extracts. From aqueous seeds extract, triterpene glycoside 28-O-[β-l-6-rhamnopyranosyl]-R1-barrigenol was isolated an active compound. Results: Crude extracts of T. sylvatica fruits, separated from seed and pericarp, showed sedative effect in mice. The aqueous (ED50 = 4.9 ± 0.8 mg/kg) seed extracts is the most active among them. This extract also decrease locomotor activity and disrupt motor coordination of mice. This extract was also the most toxic extract (LD50 = 5.0 ± 1.4 mg/kg; i.p.). The triterpene glycoside 28-O-[β-l-6-rhamnopyranosyl]-R1-barrigenol was identified in this extract as one of the active sedative compounds (ED50 = 0.12 ± 0.01 mg/kg) also with toxic effect (LD50 = 1.11 ± 0.23 mg/kg). Conclusion: The results suggest that T. sylvatica fruits has toxic activity rather than CNS depressant activity in mice and that this effect might be related to the presence of 28-O-[β-l-6-rhamnopyranosyl]-R1-barrigenol, one of the active principles of T. sylvatica fruits with sedative and toxic effect.
Context: 3,4-Dihydroxyacetophenone (DHAP) has been reported to possess cardiovascular pharmacological effects. Objective: This study was designed to determine whether DHAP could improve endothelial function in obese rats. Materials and methods: Wistar rats were randomly divided into control, obesity, and DHAP groups and fed a normal, high-fat, and high-fat plus DHAP (10 mg kg⁻¹ d⁻¹) diet, respectively, for 8 weeks. Endothelial-dependent vasodilatation was assessed. Endothelial nitric oxide synthase (eNOS) activity and nitric oxide (NO) production in endothelial cells were determined. Nuclear transcription factor kappa B (NF-κB) expression and superoxide production in aorta were evaluated. Results: DHAP treatment significantly decreased plasma triglycerides (0.94 ± 0.31 mmol/l versus 1.36 ± 0.29 mmol/l, p < 0.05) and free fatty acids (0.53 ± 0.15 mmol/l versus 0.99 ± 0.24 mmol/l, p < 0.05), reduced serum tumor necrosis factor α (35.56 ± 9.28 pg/ml versus 68.3 ± 10.24 pg/ml, p < 0.05) and malondialdehyde (2.94 ± 0.58 pg/ml versus 6.45 ± 0.70 pg/ml, p < 0.05), and increased serum adiponectin levels (164.5 ± 34.5 μg/l versus 84.5 ± 20.4 μg/l, p < 0.05). DHAP enhanced endothelial-dependent vasodilatation and improved endothelial function in obese rats (p < 0.05). eNOS activity and NO production in endothelial cells significantly decreased and NF-κB activation and superoxide production in aorta significantly increased in obese rats compared with the control group (p < 0.05). However, DHAP treatment significantly up-regulated the eNOS–NO pathway and decreased NF-κB activation and superoxide production (p < 0.05). Conclusion: DHAP improved endothelial function in obese rats. This beneficial effect may be associated with up-regulation of the eNOS–NO pathway by improving lipid metabolism and reducing oxidative stress and inflammation activity.
The diseases of plants and humans due to pathogenic fungi are increasing. Among the substances used to combat fungi, the azoles are of primary interest, both in agricultural field both in health. To avoid fungal resistance phenomena, the synthesis and tests of new derivatives are necessary. This article discusses the synthesis and the antifungal activity of pyrazolo[3,4-c]isothiazole and isothiazolo[4,3-d]isoxazole derivatives against three fungi that are pathogenic only for plants and two fungi that are opportunistic in humans and plants. The compounds were prepared starting from 2-cyano-3-ethoxy-2-butenethioamide. The antifungal activity of the compounds was determined by measuring the inhibition of growth of the fungi tested at 20, 50, and 100 µg/mL in comparison with the controls. Results demonstrated that several compounds were able to control the mycelial growth of the tested fungi, even if they showed different sensitivity to the different azole-derivatives. In general Magnaporthe grisea (T.T. Hebert) Yaegashi & Udagawa was the most sensitive fungus, being blocked almost entirely by 4-chloro derivative even at 20 µg/mL, a concentration at which the reference commercial compound tricyclazole was nearly ineffective. These findings demonstrate that the pyrazolo[3,4-c]isothiazole derivatives have a wide spectrum of activity on phytopathogenic and opportunistic fungi. In particular the 4-chloro derivative seems to have a great potential as new product to combat M. grisea in the agricultural field.
Context: Resveratrol (3,5,4'-trihydroxystilbene) is a phytoalexin synthesized by plants, most notably grapes, against microbial invasion or ultraviolet stimulation, and is known to exert antioxidant, anticancer, and antiobesity effects. Objective: This study was conducted to find resveratrol derivatives with higher anti-obesity activity compared to resveratrol and to verify their mechanism of action. Materials and methods: The inhibitory effect of resveratrol and its derivatives on adipocyte differentiation in 3T3-L1 cells was studied using Oil Red O staining, and the effects on the intracellular expression of fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) were measured via Western blot analysis. Results: A derivative of resveratrol, 4-[2-(3,5-dimethoxyphenyl)vinyl]pyridine (DPVP), exerted inhibitory effects against 3T3-L1 adipocyte differentiation (IC(50) = 13.5 µM) and FAS expression. Notably, it displayed higher activity at concentrations lower than 25 µM compared to resveratrol. Discussion and conclusion: DPVP is considered to have greater potential as an anti-obesity substance, as it exhibits excellent activity at low concentrations compared to resveratrol.
Presence/absence tests for alkaloids of 31 medicinal vascular plant species from 31 genera and 26 families of eastern Nicaragua provided a baseline for bioactivity tests. Objective: To determine the bioactivity and cytoxicity of aqueous extracts of widely used medicinal species in eastern Nicaragua. Ethnomedicinal applications were obtained from interviews of traditional healers. We used Dragendorff's reagent to test alkaloids and brine shrimp for cytotoxicity of aqueous extracts. Twenty-nine of the 31 species tested positive for alkaloids. The median lethal concentration that kills 50% of the larvae within 24 h of contact with the extract (LC(50) was less than 1000 µg/mL for 4 (13%) species (the usual cytotoxic category), 1001-5000 µg/mL for 23 (74%) species, and between 5001-7500 µg/mL for the remaining 4 (13%) species. Twenty-five of the ethnomedicines contain alkaloids but are not cytotoxic. In contrast to first suppositions, we suggest that this is a good and desirable, and perhaps expected, outcome. Medicinal plants that are cytotoxic may obviously control or kill bacteria or other pathogens, but may also negatively affect the patient; some high alkaloid levels have been associated with carcinogens. Thus, perhaps the majority of effective medicinals should be expected to be noncytotoxic. We suggest that this is a new paradigm for consideration of the overall value and effectiveness of medicinals. Of course, medicinals also can be effective in numerous ways (e.g., organ stimulation or other physiological functions) other than simply as antimicrobials or antipathogens.
Effect of partially purified protein against CCl 4 -induced histological liver injury: (A) normal control rats showing hepatic cells with well-preserved cytoplasm (H), well brought out central vein (CV) and prominent nucleus and nucleolus; (B) liver section of CCl 4 -treated rats showing massive fatty changes (F), necrosis (NC), and the loss of cellular boundaries; (C) liver section of rats treated with CCl 4 and protein 100 mg/kg bw, retaining normal hepatic architecture with fewer areas of fatty change, and necrosis. (See colour version of this figure online at www.informahealthcare. com/phb).  
It has been found that many proteins from silkworm (Bombyx mori L.) fecal matter have been active against human immunodeficiency virus, Sendai virus, herpes simplex virus type-1, and nuclear polyhedrosis virus. A partially purified 35 kDa protein from silkworm was screened for its hepatoprotective activity, and in vitro antioxidant, and antiviral properties against camelpox and goatpox viruses. The study investigated the efficiency of the partially purified 35 kDa protein from silk worm fecal matter against CCl₄-induced liver damage measured in terms of enzyme levels such as aspartate aminotransferase (AST), alanine amino transferase(ALT), alkaline phosphatase (ALP) and total bilirubin, which maintain liver integrity. In vitro antioxidant potential of this protein was determined based on its ability to scavenge 2, 2-diphenylpicrylhydrazyl (DPPH) and superoxide anions scavenging activity. Further, in vitro cytotoxic effect on Vero cells and antiviral activity against goatpox and camelpox viruses were also studied. The protein had significant hepatoprotection against CCl₄-induced liver damage and scavenging of DPPH radical and superoxide anion activity. However, the protein did not inhibit the multiplication of either virus tested at its maximum non-toxic concentration (MNTC) in vitro. The partially purified 35 kDa protein from silk worm Bombyx mori L fecal matter possessed protective effect against CCl₄-induced oxidative stress in rat model. The protein was found to be ineffective against camelpox and goatpox viruses at its MNTC in vitro.
Context: In the course of searching potential antitumor agents from a library of chalcones synthesized under microwave irradiations, the brine shrimp lethality (BSL) assay and a 3D structure-activity relationship (3DQSAR) studies were followed by the antitumor evaluation of most potent analogues. Objective: The objective of the current study was to effectively use the BSL assay for the identification of potential cytotoxic analogues from a set of compounds. Methods: We applied the comparative molecular field analysis (CoMFA) and devised 3DQSAR on 33 synthesized chalcones leading to prediction of five related compounds with improved activity. The scope of BSL assay for the prediction of antitumor potency was tested through the in vitro antitumor studies against six human tumor cell-lines, docking studies and the tubulin-polymerization assay. Results: The newly designed compounds 34-38 displayed very promising cytotoxic potency. From our results, the BSL toxicity, antitumor efficacy and docking outcomes could be easily co-related. Conclusion: The study draws a very good relationship between a simple, inexpensive, and bench-top BSL assay and the antitumor potential of the cytotoxic compounds. Devising the CoMFA analysis helped in designing chalcones with improved cytotoxic potential as displayed through their BSL and cytotoxic activity against human tumor cell lines. The studies are noteworthy as such comprehensive studies were never performed before on the BSL assay. The present studies widen the scope of the BSL model that may prove quite helpful as a preliminary screen in the antitumor drug designing and synthesis expeditions.
Context: Raspberry ketone (RK) is a natural phenolic compound of red raspberry. The dietary intake of RK has been reported to exert anti-obese actions and alter the lipid metabolism in vivo and human studies. Objective: To elucidate a possible mechanism for anti-obese actions of RK, the effects of RK on the adipogenic and lipogenic gene expression in 3T3-L1 adipocytes were investigated. Materials and methods: 3T3-L1 maturing pre-adipocytes were treated from day 2 to day 8 of differentiation and mature adipocytes for 24 h on day 12 with 1, 10, 20, and 50 μM of RK. Triacylglycerols were assessed by spectrophotometry and gene expression by quantitative real-time polymerase chain reaction (qRT-PCR). Results: Treatment of adipocytes with RK suppressed adipocyte differentiation and fat accumulation in a concentration-dependent manner. RK suppressed the expression of major genes involved in the adipogenesis pathway including peroxisome proliferator-activated receptor-γ (PPARγ) and CCAAT enhancer binding protein-α (C/EBPα), which led to further down-regulation of adipocyte fatty acid-binding protein-2 (aP2). In addition, treatment with 10 μM of RK also reduced mRNA levels of lipogenic genes such as acetyl-CoA carboxylase-1 (ACC1), fatty acid synthase (FASN), and stearoyl-CoA desaturase-1 (SCD1). In mature adipocytes, RK increased the transcriptional activities of genes involved in lipolysis and the oxidative pathways including adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL), and carnitine palmitoyl transferase-1B (CPT1B). Discussion and conclusion: These findings suggest that RK holds great promise for an herbal medicine with the biological activities altering the lipid metabolism in 3T3-L1 adipocytes.
Growing adipose tissue is thought to require adipogenesis, angiogenesis, and extracellular matrix (ECM) remodeling. Close examination of developing adipose tissue microvasculature reveals that angiogenesis often precedes adipogenesis. Since our previous study demonstrated that Ob-X, the anti-angiogenic herbal composition composed of Melissa officinalis L. (Labiatae), Morus alba L. (Moraceae), and Artemisia capillaris Thunb. (Compositae), reduced adipose tissue mass in obese mice, we hypothesized that adipogenesis can be inhibited by Ob-X. To investigate the effects of the anti-angiogenic herbal extracts Ob-X on adipogenesis in 3T3-L1 adipocytes. After differentiated 3T3-L1 adipocytes were treated with Ob-X, we studied the effects of Ob-X on triglyceride accumulation and expression of genes involved in adipogenesis, angiogenesis, and ECM remodeling. Treatment of cells with Ob-X inhibited lipid accumulation and adipocyte-specific gene expression caused by troglitazone or monocyte differentiation-inducing (MDI) mix. Ob-X reduced mRNA levels of angiogenic factors (vascular endothelial growth factor-A, -B, -C, -D, and fibroblast growth factor-2) and matrix metalloproteinases (MMPs; MMP-2 and MMP-9), whereas it increased mRNA levels of angiogenic inhibitors [(thrombospondin-1, tissue inhibitor of metalloproteinase-1 (TIMP-1), and TIMP-2)] in differentiated cells. MMP-2 and MMP-9 activities were also decreased in Ob-X-treated cells. These results suggest that the anti-angiogenic herbal composition Ob-X inhibits differentiation of preadipocytes into adipocytes. These events may be mediated by changes in the expression of genes involved in lipogenesis, angiogenesis, and the MMP system. Thus, by reducing adipogenesis, anti-angiogenic Ob-X provides a possible therapeutic approach for the prevention and treatment of human obesity and its related disorders.
Context: Obesity is associated with a number of diseases with metabolic abnormalities such as type 2 diabetes (T2D). Medicinal plants have been widely used for the treatment of obesity and related complications. Objective: In this study, we investigated the antidiabetic properties of the extract of twigs of Cinnamomum cassia Blume (Lauraceae) (Cinnamomi Ramulus; CR) in 3T3-L1 murine preadipocytes. Materials and methods: 3T3-L1 cells were differentiated into adipocytes for 3 d in insulin-conditioned medium and then treated with CR extract at concentrations of 100 and 500 μg/mL for 6 d. Adipocyte differentiation was measured by Oil Red O staining, and the expression of master transcription factors, peroxisome proliferator-activated receptor-gamma (PPARγ), CCAAT/enhancer binding protein-alpha (C/EBPα), and sterol regulatory element binding protein-1c (SREBP-1c), and lipid metabolism factors were investigated by reverse transcription-polymerase chain reaction (RT-PCR). The activation of the AMP-activated protein kinase (AMPK)/insulin signaling pathway was assessed by western blot analysis. Results: CR extract significantly reduced lipid accumulation and down-regulated the expression of PPARγ, C/EBPα, and SREBP-1c in 3T3-L1 adipocytes. CR extract also suppressed the expression of fatty acid synthase (FAS), acyl-CoA synthase, and perilipin. Moreover, CR extract markedly up-regulated the phosphorylation of AMPK and acetyl-CoA carboxylase (ACC). In addition, CR extract effectively increased the expression levels of glucose transporter-4 (GLUT-4), phosphatidylinositol 3-kinase (PI3K), and insulin receptor substrate-1 (IRS-1) in 3T3-L1 adipocytes. Discussion and conclusion: These results suggest that CR extract may have therapeutic potential as a natural agent for the improvement of T2D via regulation of the insulin-dependent signaling pathway.
Context: Linum usitatissimum L. (Linaceae), commonly known as flaxseed, is a good source of dietary fiber and lignans. Earlier we reported cardioprotective, antihyperlipidemic, and in vitro antioxidant activity of flax lignan concentrate (FLC) obtained from flaxseed. Objectives: To isolate secoisolariciresinol diglucoside (SDG) from FLC and to evaluate the antihyperlipidemic activity of SDG in poloxamer-407 (P-407)-induced hyperlipidaemic mice. Material and methods: FLC was subjected to column chromatography and further subjected to preparative HPTLC to isolate SDG. The chemical structure of the isolated compound was elucidated by UV, IR, (1)H NMR, (13)C NMR, DEPT, COSY, HSQC, HMBC, ROESY, MS, and specific optical rotation was recorded. Further, we have investigated the antihyperlipidaemic effect of SDG (20 mg/kg) in P-407-induced hyperlipidaemic rats. Hyperlipidaemia was induced by intraperitoneal administration of P-407 (30% w/v). Serum lipid parameters such as total cholesterol (TC), triglycerides (TG), and high-density lipoprotein cholesterol (HDL-C) levels were measured. Results and discussion: The structure and stereochemistry of the isolated compound were confirmed on the basis of 1D and 2D spectral data and characterized as SDG. Finally, isolated pure SDG was screened using a P-407-induced mice model for its antihyperlipidemic action using serum lipid parameters. The isolated SDG (20 mg/kg) significantly reduced serum cholesterol, triglyceride (p < 0.001), very low-density lipoprotein (p < 0.05), and non-significantly increased HDL-C. Conclusion: Finally, it was concluded unequivocally that SDG showed antihyperlipidaemic effects in P-407-induced hyperlipidaemic mice. Isolated pure SDG confirms that SDG is beneficial in the prevention of experimental hyperlipidemia in laboratory animals.
Decoctions of Baliospermum montanum Müll. Arg. (Euphorbiaceae) leaves are reported to be useful in the treatment of asthma and other respiratory complications in the Ayurvedic system. Objective: To evaluate the mast cell stabilization and antihistaminic activities of the chloroform (BMLC) and ethanol (BMLE) extracts of the leaves of Baliospermum montanum. The stabilization potential was studied on mouse peritoneal mast cells and the antihistaminic activity was carried out by determining the mortality rate of mice treated with toxicant (compound 48/80) and the effect on elevation of histamine release upon degranulation. The increased number of intact mast cells (43.640 ± 1.7% and 61.57 ± 1.79% at 200 and 400 mg/ kg, respectively) suggested that the BMLC stabilized the mast cell degranulation and showed decreased elevation of histamine. BMLC extract was found to be most effective against degranulation and release of histamine from mast cells. Identifying the lead from this plant will be a definite target for treating allergic diseases.
The incidence and mortality of thrombotic disorders are rapidly increasing throughout the world. Therefore, attempts have been made to develop new anticoagulant and antithrombotic drugs. Our previous studies showed that a novel protein, named Fu-P, had fibrinogenolytic activity and much higher fibrinolytic activity on the fibrin plate than urokinase in vitro. The antithrombotic activities of Fu-P in vivo are investigated here for the first time. Antithrombotic activity of Fu-P was studied in a rat model of artery-vein bypass thrombosis. The anticoagulant activity of Fu-P was measured by clotting assay of activated partial thrombinplastin time and prothrombin time (PT). The effects of Fu-P on the factor Xa and thrombin were assayed using the chromogenic substrate S-2765 and S-2238. Results: Intravenous injection of Fu-P produced a 58.4% inhibition ratio of thrombus formation at 0.1 mg/kg body weight, while heparin produced 42.5% inhibition ratio of thrombus formation at 0.6 mg/kg body weight. Fu-P significantly prolonged fibrinogen clotting time, activated partial thrombinplastin time and thrombin time, which also prolonged PT. The inhibition assay of the coagulant factors using chromogenic substrates S-2238 and S-2765 showed that Fu-P was not the inhibitor of the thrombin and Xa. These findings demonstrated that the novel fibrinolytic enzyme (Fu-P) might also be used as a natural agent for thrombolytic therapy or thrombosis prevention.
In addition to bergapten ( 1 ), xanthotoxin ( 2 ), isopimpinellin ( 3 ), imperatorin ( 4 ), and pimpinellin ( 5 ), for the first time 5,7-disubstituted simple coumarins, limettin (5,7-dimethoxycoumarin) ( 6 ) and a new compound 5-methoxy-7-(3,3-dimethylalliloxy)-coumarin ( 7 ), have been isolated from the fruits of Heracleum mantegazzianum Somm. et Lev. The new compound 7 is a derivative of anisocoumarin B. The structures of 6 and 7 were determined on the basis of 1D and 2D NMR spectra.
Effect of various concentrations of PEGylated G-CSF on proliferative responses of the NFS-60 cells as measured by the MTS/WST-8 assays (the error bars in the chart represent standard error of mean for four replicate wells). The NFS-60 cells were seeded at 7 Â 10 5 cells/ml concentration and stimulated with various concentrations of PEGylated G-CSF. The MTS/WST-8 assays were performed after 48 h of incubation with PEGylated G-CSF. Note: DOD ¼ OD stimulated À OD unstimulated .  
Effect of cell concentrations on proliferative responses of the NFS-60 cell line as measured by the MTS/WST-8 assays (the error bars in the chart represent standard error of mean for four replicate wells). Various cell concentrations of the NFS-60 cell line were seeded and stimulated with 800 IU/ml of PEGylated G-CSF. The MTS/WST-8 assays were performed after 48 h of stimulation with PEGylated G-CSF. Note: DOD ¼ OD stimulated À OD unstimulated .  
Kinetics of the NFS-60 cell proliferation upon stimulation with PEGylated G-CSF as measured by the MTS/WST-8 assays (the error bars in the chart represent standard error of mean for six replicate wells and star denotes statistical significance at p50.05). The NFS-60 cells were seeded at 7 Â 10 5 cells/ml concentration and stimulated with 800 IU/ml of PEGylated G-CSF. The MTS/WST-8 assay was performed after 24, 48, 72, and 96 h of stimulation with PEGylated G-CSF. The dark and light colored lines represent results from the MTS and the WST-8 (CCK kit) assays, respectively.  
Context: Granulocyte colony stimulating factor (G-CSF) has been commonly used to treat neutropenia caused by chemotherapy, radiotherapy, and organ transplants. Improved in vitro efficacy of G-CSF has already been observed by conjugating it to polyethylene glycol (PEG). Objective: The in vivo bioassay using tetrazolium dye with the NFS-60 cell line has been recommended for G-CSF but no such monographs are available for PEGylated G-CSF in pharmacopeias. In the present study, the assay recommended for G-CSF was evaluated for its suitability to PEGylated G-CSF. Materials and methods: The generally used MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]-based assay was compared with a bioassay employing a water-soluble tetrazolium dye, WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium], using NFS-60 cells at a concentration of 7 × 10(5) cells/ml against 800 IU/ml of PEGylated G-CSF at 24, 48, 72, and 72 h time points to determine the efficacy of PEGylated G-CSF. Further, the optimized WST-8 dye-based assay was used to test the potency of various commercially available PEGylated G-CSF preparations. Results: The results demonstrated enhanced sensitivity of the WST-8-based assay over the conventional MTS-based assay for determining the potency of PEGylated G-CSF using the NFS-60 cell line. Conclusion: Our study demonstrates the potential application of WST-8-based bioassays for other biotherapeutic proteins of human and veterinary interest.
Millettia griffoniana Baill. (Fabaceae), which contains isoflavonoids like griffonianone C (Griff C), is commonly used in the folk medicine in Cameroon to treat various ailments. Possible health benefits of Griff C which include alleviation of menopausal symptoms, limitation of bone resorption, and lowering of the risks of cancer and cardiovascular diseases attracted our interest. The effects of Griff C on the regulation of the expression of proliferation markers such as proliferating cell nuclear antigen (PCNA), cyclin D1 (CD1) and Ki-67 are investigated here. Its role in apoptosis or cell survival, through the phosphatidylinositol 3 kinase-Akt (PI3K-Akt) signaling pathway is further studied. Semiquantitative real-time PCR was performed to analyze the effects of Griff C on gene expression in MCF-7 cells. Western blot analysis was used to assess the role of Griff C on the expression of phosphorylated Akt in MCF-7 cells. Results: Griff C induced a 4.84-fold increase in the expression of Ki-67 mRNA at the concentration of 10(-8) M and a 3.90-fold increase of CD1 mRNA at 10(-7) M. Griff C slightly increased the phosphorylation of Akt at its serine 473 residue. Akt phosphorylation was inhibited by the PI3K inhibitor, LY294002, but not by the specific estrogen receptor antagonist, fulvestrant. These findings suggest that Griff C can modulate proliferation of MCF-7 cells. Our results also suggest that Griff C can affect the PI3K-related signaling pathway. Thus, Griff C may exert part of its low proliferative and antiapoptotic effects by a nongenomic mode of action.
Daucus carota L. ssp. carota (Apiacea) is widely distributed throughout the world and has many uses in traditional medicine. The present study investigates the chemopreventive effects of oil extract of D. carota umbels on 7,12-dimethyl benz(a)anthracene (DMBA)-induced skin cancer in mice. D. carota oil extract (DCOE) was prepared by extracting the dried umbels with 50:50 acetone:methanol. Skin papilloma were initiated by DMBA and promoted by 12-O-tetradecanoyl phorobol-13-acetate (TPA). The extract was administered to animals via gavage (0.02 mL of 100% oil), intraperitoneal (0.3 mL of 2% oil), and topical (0.2 mL of 5, 50, and 100% oil) routes for 20 weeks. Tumor appearance, incidence, yield, and volume were compared with those of a non-treated control group. Topical 100% treatment delayed tumor appearance, and inhibited tumor incidence and yield by 40 and 89%, respectively. Topical 50% treatment inhibited tumor incidence and yield by 30 and 83%, respectively, whereas the 5% treatment inhibited tumor yield by 36%. Tumor volume was decreased by 99, 91, and 70% following topical treatments with 100, 50, and 5% oil, respectively. Intraperitoneal treatment inhibited tumor yield by 43%, and decreased tumor volume by 85%, whereas gavage treatment showed minimal effects on both. Intraperitoneal and topical treatment decreased infiltration and hyperplasia with an increase in the level of hyperkeratosis. These findings demonstrate that DCOE has remarkable antitumor activity against DMBA-induced skin cancer compared with non-treated animals paving the ground for further investigations.
Context: Corchorus olitorius L. (Malvaceae) has industrial importance in world jute production and is a widely cultivated and consumed crop in Cyprus and in some Arabic countries. Objective: The present study investigated cytotoxic and genotoxic effects of leaf extracts (LE) and seed extracts (SE) of the C. olitorius on the multiple myeloma-derived ARH-77 cells. The extracts were also evaluated for their total phenol content (TPC) and free radical scavenging activity (FRSA). Materials and methods: C. olitorius was collected from Nicosia, Cyprus. TPC and FRSA were measured by Folin-Ciocalteu and DPPH free radical methods, respectively. Cytotoxicity was evaluated by the MTT assay (4-2048 µg/mL range), and DNA damage (at IC50 and ½IC50) was measured by the comet assay. Results and discussion: The LE had significantly higher total phenol (78 mg GAE/g extract) than the SE (2 mg GAE/g extract) with significantly higher FRSA (IC50 LE: 23 µg/mL and IC50 SE: 10 401 µg/mL). Both LE and SE exerted cytotoxic effects on cells after 48 h. The IC50 of SE (17 µg/mL) was lower than LE (151 µg/mL), which demonstrates its higher cytotoxicity on cells. The extracts were applied at 150 and 75 µg/mL for LE and at 17 and 8.5 µg/mL for SE, and the results of the comet assay revealed that the extracts induced genotoxic damage on ARH-77 cells. In both 48 h leaf and seed extract treatments, genotoxic damage significantly increased with increasing concentrations at relevant cytotoxic concentrations. Conclusion: To our knowledge, this is the first report demonstrating the high cytotoxic potential of C. olitorius SE and the genotoxic potential of LE and SE.
Anti-nociceptive effect of magnesium sulfate (MS 1, 5, 15, 30, and 45 mg/kg, s.c.) (A) and MK-801 (0.005, 0.01, and 0.03 mg/kg, s.c.) (B) in acetic acid-induced writhing test. Each bar represents the mean ± SEM for a group of 6-8 rats, analyzed by the Kruskal-Wallis test, followed by the Mann-Whitney U-test. (A) **p50.01 versus the saline group, +p50.05, ++p50.01 versus the MS 15 group, ##p50.01 versus the MS 1 group. (B) **p50.01 versus the saline group, ++p50.01 versus the MK-801 0.03 group. s.c., subcutaneous; i.p., intraperitoneal.
Effect of L-NAME (10 mg/kg, i.p.) on the antinociceptive effect of magnesium sulfate (MS 15 mg/kg, s.c.) (A) or MK-801 (0.01 mg/kg, s.c.) (B) in acetic acid-induced writhing test. Each bar represents the mean ± SEM for a group of 6-8 rats, analyzed by the Kruskal-Wallis test, followed by the Mann-Whitney U-test. (A) **p50.01 versus the saline group. (B) *p50.05 versus the saline group, ++p50.01 versus the MK-801 0.01 group. s.c., subcutaneous; i.p., intraperitoneal. 
Effect of methylene blue (MB 0.5 mg/kg, s.c.) on the antinociceptive effect of magnesium sulfate (MS 15 mg/kg, s.c.) (A) or MK-801 (0.01 mg/kg, s.c.) (B) in acetic acid-induced writhing test. Each bar represents the mean ± SEM for a group of 6-8 rats, analyzed by the Kruskal-Wallis test, followed by the Mann-Whitney U-test. (A) *p50.05, **p50.01 versus the saline group. (B) **p50.01 versus the saline group, +p50.05 versus the MK-801 0.01 group. s.c., subcutaneous; i.p., intraperitoneal. 
Effect of glybenclamide (Gly 3 mg/kg, s.c.) on the antinociceptive effect of magnesium sulfate (MS 15 mg/kg, s.c.) (A) or MK801 (0.01 mg/kg, s.c.) (B) in acetic acid-induced writhing test. Each bar represents the mean ± SEM for a group of 6-8 rats, analyzed by the Kruskal-Wallis test, followed by the Mann-Whitney U-test. (A) *p50.05, **p50.01 versus the saline group. (B) *p50.05 versus the saline group, ++p50.01 versus the MK-801 0.01 group. s.c., subcutaneous; i.p., intraperitoneal.
Effects of MK-801 (0.03 mg/kg, s.c.), MS (15 mg/kg, s.c.), and saline on motor performance assessed using the rotarod test. Each bar represents the mean time (in seconds) the rats remained on rotarod ± SEM for a group of six rats, analyzed by the Kruskal-Wallis test, followed by the Mann-Whitney U-test. Statistical significance was not found between any group. s.c., subcutaneous.
Magnesium and MK-801 (dizocilpine), antagonists of N-methyl-d-aspartate receptors, are involved in the processing of pain. This study determines whether magnesium sulfate (MS) and MK-801 affects visceral inflammatory pain and determines a possible mechanism of action. Analgesic activity was assessed using the acetic acid-induced writhing test in rats. MS (1-45 mg/kg) or MK-801 (0.005-0.03 mg/kg) was administrated subcutaneously (s.c.). To assess possible mechanisms of action, we examined the effects of l-NAME (10 mg/kg, intraperitoneal), methylene blue (0.5 mg/kg, s.c.), and glibenclamide (3 mg/kg, s.c.) on the effect of MS or MK-801. MS and MK-801 showed biphasic and linear dose-response pattern, respectively. MS reduces the number of writhing on the dose of 1, 5, and 15 mg/kg by 60, 50, and 78%, respectively, while it has no effects on the doses of 30 and 45 mg/kg. MK-801 (0.005-0.03 mg/kg) showed decrease in the number of writhing by 33-79%. The mean effective doses of MS and MK-801 were 6.6 (first phase) and 0.009 mg/kg, respectively. Both drugs did not impair the rotarod performance. l-NAME, methylene blue, and glybenclamide reduced the effect of MK-801 by 100, 43, and 64%, respectively, but not the effect of MS. The results suggest that MS and MK-801 may be useful analgesics in the management of visceral inflammatory pain, at doses that do not induce motor impairment. The modulation of NO/cGMP/K+ATP pathway plays an important role in the antinociceptive mechanism of MK-801, but does not contribute to the antinociceptive effect of MS.
We recently reported that F2, an oligomer procyanidin fraction isolated from grape seeds, triggered an original form of cell death in U-87 human glioblastoma cells with a phenotype resembling morphological characteristics of paraptosis. However, the specific death mode induced by F2 and the mechanism of its action have not been assessed so far. In the present work, we therefore further investigated the death mode of human glioblastoma cells induced by F2 and gained insight into the nature of the signaling pathways activated by F2 in glioblastoma cells. Cell viability assay using MTT, (AO/EB) double staining, Western blot analysis, and Ca2+ assay using fura-2. Morphology studies revealed extensive cytoplasmic vacuolization in dying cells and no apoptotic body formation, membrane bleb formation, or nuclear fragmentation, though some was accompanied by MAPK activation and new protein synthesis, and was independent of caspase activation. Moreover, we demonstrated the involvement of calcium mobilization in F2-induced U-87 cell signaling. Altogether we showed that F2 induced a kind of cell death resembling paraptosis in U-87 cells. The current report complements previous studies on the characterization of F2-induced U-87 cell death, enhances our understanding of the action mechanism of F2 on glioma, and helps in the development of novel antitumor therapeutics.
The production of antimicrobial compounds by macrofungi is not unexpected because they have to compete with other organisms for survival in their natural hostile environment. Previous studies have indicated that macrofungi contain secondary metabolites with a range of pharmacological activities including antimicrobial agents. To investigate macrofungi for antimicrobial activity due to the increasing need for new antimicrobials as a result of resistance in the bacterial community to existing treatments. Forty-seven different specimens of macrofungi were collected across Queensland, Australia. Freeze-dried fruiting bodies were sequentially extracted with three solvents: water, ethanol, and hexane. These extracts were tested against representative Gram+ve, Staphylococcus aureus and Gram-ve, Escherichia coli bacteria. Overall water and ethanol extracts were more effective against S. aureus than E. coli, whereas a small number of hexane extracts showed better results for their antimicrobial potential against E. coli at higher concentrations only. Encouraging results were found for a number of macrofungi in the genera Agaricus (Agaricaceae), Amanita (Amanitaceae), Boletus (Boletaceae), Cantharellus (Cantharellaceae), Fomitopsis (Fomitopsidaceae), Hohenbuehelia (Pleurotaceae), Lentinus (Polyporaceae), Ramaria (Gomphaceae), and Strobilomyces (Boletaceae) showing good growth inhibition of the pathogens tested. The present study establishes the antimicrobial potential of a sample of Australian macrofungi that can serve as potential candidates for the development of new antibiotics.
Abstract Context: Estrogens in non-small-cell lung cancer (NSCLC) are important, and their interaction with epidermal growth factor receptor (EGFR) might be crucial. Objective: This study investigates the effect of exemestane, an aromatase inhibitor, and erlotinib, an EGFR inhibitor, on human NSCLC cell lines; H23, H358 and A549. Materials and methods: A cell proliferation assay was used for measuring cell number, apoptosis assay for detecting apoptosis and necrosis and immunoblotting for beclin-1 and Bcl-2 proteins detection. An immunofluorescence assay was used for EGFR localization. A migration assay and zymography were used for cell motility and metalloproteinases (MMPs) expression, respectively. Results: Exemestane, erlotinib or their combination decreased cell proliferation and increased apoptosis. Exemestane's half maximal inhibitory concentration (IC50) was 50 μM for H23 and H358 cells and 20 μM for A549. The IC50 of erlotinib was 25 μM for all cell lines. Apoptosis increase induced by exemestane was 58.0 (H23), 186.3 (H358) and 34.7% (A549) and by erlotinib was 16.7 (H23), 65.3 (H358) and 66.3% (A549). A synergy effect was observed only in H23 cells. Noteworthy, the combination of exemestane and erlotinib decreased beclin-1 protein levels (32.3 ± 19.2%), an indicator of autophagy, in H23 cells. The combination of exemestane and erlotinib partially reversed the EGFR translocation to mitochondria and decreased MMP levels and migration. Discussion and conclusions: The benefit from a dual targeting of aromatase and EGFR seems to be regulated by NSCLC cell content. The diverse responses of cells to agents might be influenced by the dominance of certain molecular pathways.
Context: Mere15 is a novel antitumor polypeptide purified from Meretrix meretrix Linn. (Veneridae). Previous studies have shown that the polypeptide induced cell death via intrinsic mitochondrial pathway. Objective: In the present study, the effects of Mere15 on cell adhesion, migration, invasion, as well as secretion and expression of matrix metalloproteinases (MMPs) were studied in human lung adenocarcinoma A549 cells. Materials and methods: The effect of Mere15 on cell adhesion, migration and invasion were studied by cell adhesion and transwell assay. The expression of MMPs was determined by gelatin zymography and RT-PCR analysis. Results: The ability of cell adhesion was decreased by 86.74% at the concentration of 12.0 μg/mL of Mere15. And the migration and invasion of A549 cells were decreased with the inhibition ratio of, 69.22 and 53.84% when treated with 15.0 μg/mL of Mere15. Further study also revealed that treatment of the cancer cells with Mere15 (15.0 μg/mL), the secretion of MMP-2 and MMP-9 were down-regulated with the inhibition ratio of 72.00 and 93.24% and the inhibition rate of mRNA expression of MMP-2 and MMP-9 was 57.54 and 91.22%, respectively. Discussion and conclusion: The study demonstrates that Mere15 inhibits tumor growth via both pro-apoptotic and antimetastasis pathways, and the polypeptide has potential to be developed as a multi-target therapeutic agent for the treatment of human lung cancer.
Context: Curcumin exhibits growth-suppressive activity against a variety of cancer cells, but low bioavailability restricts its application in chemotherapeutic trials. Nowadays, a growing number of curcumin derivatives or analogs are known, hoping to replace curcumin and circumvent this problem. Hydrazinobenzoylcurcumin (HBC) has been synthesized and identified as a potent inhibitor of cell proliferation in previous reports. Objective: This study presents a novel mechanism of cell autophagy induced by HBC in the human non-small lung epithelial carcinoma (A549) cells. Materials and methods: Cells were cultured and treated with HBC at different concentrations (10-80 μM) and at different time periods (1-24 h). Microscopic analysis was used to detect the morphology changes and autophagolysosomes of A549 cells. An acridine orange staining assay was conducted to evaluate the autophagolysosomes and autophagic vacuoles was analyzed by monodansylcadaverine (MDC) and GFP-LC3 transfection analysis. Western blotting was used to assess the conversion of microtubule-associated protein light chain 3 (LC3). Results: HBC could induce A549 cells autophagolysosomes formation in a dose and time-dependent manner and the inhibitory rate of HBC (80 μM) on the viability of A549 cells reached 76.68 ± 5.81% after 24 h of treatment. Autophagic vacuoles increased in a concentration-dependent manner in HBC-treated cell. Furthermore, conversion of LC3-I to LC3-II, accumulation of GFP-tagged LC3 positive intracellular vacuoles and increased fusion of autophagosomes with lysosomes suggested the occurrence of autophagy. Conclusion: Our data indicate that HBC induced A549 cell autophagy, which is a novel cell death mechanism induced by curcumin derivatives.
Context: The in vitro and in vivo antitumor activities of ardisiphenol D, a natural product isolated from the roots of Ardisa brevicaulis Diels (Myrsinaceae), have been studied. Objective: Previously, we have isolated and identified some chemical constituents from this plant. Furthermore, these compounds showed significant inhibition of the proliferation of human pancreatic PANC-1, human lung A549, human gastrointestinal carcinoma SGC 7901, human breast MCF-7, and human prostate PC-3 cancer cells. In the present paper, a major resorcinol derivative called ardisiphenol D was further studied for its antitumor mechanism. Materials and methods: MTT assay was used to detect the proliferation of A549 cancer cells. Apoptosis induced by ardisiphenol D was observed by Hoechst 33258 fluorescence staining. Caspase-3 enzyme activity was measured by a commercial caspase-3 enzyme activity detection kit. Protein expression of bax, bcl-2, and caspase-3 was tested by Western blots. In vivo antitumor activity of ardisiphenol D was evaluated by determination of A549 tumor growth in nude mice. Results: Ardisiphenol D significantly inhibited the proliferation of A549 cells with an IC50 of 0.997 μM with a 48 h treatment. Hoechst 33258 fluorescence staining results indicated the apoptosis of A549 cells induced by 3.125 μM of ardisiphenol D. About 0.39 and 0.78 μM of ardisiphenol D also potently increased the caspase-3 enzyme activity in 24 h. Furthermore, 0.39-3.125 μM of ardisiphenol D induced the activation of caspase-3 protein and the up-regulation of the ratio of bax/bcl-2 protein expression in A549 cells. After i.p. injection, ardisiphenol D (5 mg/kg) also strongly suppressed the A549 tumor growth in nude mice. Discussion and conclusion: Ardisiphenol D induced apoptosis of A549 cells via activation of caspase-3 and up-regulation of the ratio of bax/bcl-2 protein expression. Ardisiphenol D also strongly suppressed the A549 tumor growth in nude mice and exerted antitumor activity in vivo.
Context: A new caffeic acid derivative, named (7'Z)-3-O-(3, 4-dihydroxyphenylethenyl)-caffeic acid (CADP), extracted from Abacopteris penangiana (Hook.) Ching. Objective: To elucidate the neuroprotective effect of CADP against H₂O₂-induced cytotoxicity in PC12 cells and D-galactose (D-gal)-induced neurotoxicity in mice brain. Materials and methods: CADP was isolated from the methanol extract of the rhizomes of A. penangiana. In vitro, the protective effect of CADP (0.1-10 μM) against H₂O₂-induced oxidative damage on PC12 cells was investigated by a MTT assay. In vivo, behavioral tests and antioxidant enzymes measurements were performed to investigate the protective effect of intraperitoneal (i.p.) injection of CADP (5 or 10 mg/kg/day) for 2 weeks on D-gal-induced neurotoxicity in mice. Results: The results showed that CADP significantly attenuated cell toxicity in a dose-dependent manner, and the EC₅₀ value of CADP was 0.83 ± 0.02 μM. In vivo, it was found that CADP significantly improved the behavioral performance of D-gal-treated mice in both Morris water maze (MWM) test and step-down avoidance test. As compared with model group, CADP (5, 10 mg/kg/day) attenuated the decrease in superoxide dismutase (SOD) activities by 40.5 and 75.4%, respectively; attenuated the decrease in glutathione peroxidase (GPx) activities by 53.8 and 73.2%, respectively; attenuated the decrease in catalase activities by 12.0 and 53.3%, respectively; reduced the increased levels of malondialdehyde (MDA) by 38.6 and 79.9%, respectively. Discussion and conclusion: The results suggested that CADP has significant neuroprotective effects which can be attributed to inhibiting the generation of free radical and enhancing the activity of endogenous antioxidant enzymes.
Top-cited authors
Kundan Bora
Hong Zhang
  • Shanghai University of Traditional Chinese Medicine
Ilkay Erdogan Orhan
  • Gazi University, Faculty of Pharmacy
Subhash Laxmanrao Bodhankar
  • Bharati Vidyapeeth's College of Pharmacy
Berrin Ozçeli̇k
  • Gazi University