Anti-oxidative and anti-neuroinflammatory effects of ethyl acetate extract fractions of Suaeda asparagoides MIq

Purpose: To evaluate the in vitro antioxidant and anti-neuroinflammatory effects of Suaeda asparagoides ethylacetate extract (SAE) in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. Methods: The antioxidative activity of SAE was evaluated by measuring 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging activity spectrometrometrically. Cell viability was evaluated by 3-(4, 5dimethylthiazol-2-yl)-2, 5- diphenyl-tetrazolium bromide (MTT) assay, while LPS-stimulated BV-2 microglia were used to study the expression and production of inflammatory mediators, including nitric oxide (NO), inducible NO synthase (iNOS) and tumor necrosis alpha (TNF-α). Results: Pretreatment with SAE prior to LPS treatment significantly inhibited excessive production of NO (p < 0.001 at 20, 40, 80 and 100 μg/mL) in a dose-dependent manner, and was associated with down-regulation of expression of inducible nitric oxide synthase (iNOS). SAE also suppressed the LPSinduced increase in TNF-α level (p < 0.01at concentrations of 40 and 80 μg/mL) in BV-2 cells. Furthermore, DPPH-generated free radicals were inhibited by SAE in a concentration-dependent manner. Conclusion: These results indicate that SAE possesses strong anti-oxidant properties, and inhibits excessive production of pro-inflammatory mediators, including NO, iNOS and TNF-α, in LPS-stimulated BV-2 cells

Protective effect of (-)-epigallocatechin gallate on ultraviolet b-induced skin damage in hairless mice

Purpose: To investigate the protective effect of green tea (-)-epigallocatechin gallate (EGCg) on ultraviolet B (UV-B)-induced skin damages in hairless mice in order to develop a natural sunscreen compound for use in skin care products and cosmetics. Methods: EGCg was dissolved in acetone at concentrations of 1.0, 10.0 and 50.0 mg/mL, and topically applied to the skin of hairless mice at doses of 0.2 mL/cm2, with acetone as control. The mice were then irradiatd m2 UV-B for 30 min daily. EGCg treatment and UV-B irradiation were carried out daily for 28 consecutive days. The mice were then sacrificed and their dorsal skin examined by transmission electron microscopy (TEM) on the 28th day. Results: UV-B irradiation induced severe macroscopic skin damage including chapping, cracking and abnormal desquamation in the treated hairless mice. EGCg showed dose-dependent protective effects against UV-B induced damage on the skin. Treatments with 10.0 and 50.0 mg/mL EGCg alleviated UVB-induced skin damage by suppressing both keratinocyte apoptosis and mitochondrial dysfunction, along with inhibiting the production of melanin pigment. Conclusion: Topical application of green tea EGCg shows dose-dependent protective effect against UV-B-induced damage on hairless mouse skin. Thus, the plant compound can potentially be used as an alternative agent for photoprotection against UV-B exposure.

Analgesic synergism of gabapentin and carbamazepine in rat model of diabetic neuropathic pain

Purpose: To evaluate synergy in the analgesic effects of a combination therapy of carbamazepine (CBZ) and gabapentin (GBP) in diabetic neuropathic pain. Methods: Neuropathic pain was produced in rats by a single intraperitoneal injection of streptozotocin (STZ) at 60 mg/kg. CBZ, GBP, and their combination were orally administered at varying doses (GBP 30 - 180 mg/kg; CBZ 20 - 40 mg/kg) comparable to their therapeutic doses in humans. Nociceptive responses in the diabetic rats were assessed using hot plate test. Results: Hot plate latency significantly increased with oral administration of GBP at a dose of 180 mg/kg when compared with control group (p < 0.05), while at a dose of 90 mg/kg, the increase was not significant. Oral administration of CBZ at doses of 20 and 40 mg/kg did not produce any significant impact on hot plate latency. However, a combination of GBP at 90 mg/kg and CBZ at 20 mg/kg produced significant increase in latency, compared with control group and other groups (p < 0.05), except the group that received 180 mg/kg GBP. The combination of low dose GBP 30 mg/kg and carbamazepine 30 mg/kg had no significant effect on latency (p > 0.05). Conclusion: The results obtained in this study provide useful information on the combination therapy of GBP and CBZ, which may be applied in the treatment of pain in diabetic neuropathy.

Effect of inulin supplementation in male mice fed with high fat diet on biochemical profile and α-amylase gene expression

Purpose: To evaluate the preventive and therapeutic effects of inulin supplementation in Naval Medical Research Institute (NMRI) male mice fed with high fat diet. Methods: NMRI male mice (n = 36) were divided into three groups. Control (C1), obese (O1) and experimental mice (E1) were fed during 8 weeks as follows: C1 with normal rodent pellet, O1 with high fat diet, and E1 with high fat diet plus 20 % inulin. C2, O2, and E2 were fed as follows: C2 with normal rodent pellets for 12 weeks; O2 with high fat diet during 8 weeks and switched to normal rodent pellet during next 4 weeks; and E2 with high fat diet over a period of 8 weeks and switched to normal rodent pellet plus 20 % inulin for 4 weeks. Body weight, serum glucose, triglycerides, total cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL), and hepatic α-amylase gene expression were measured. Results: Groups receiving high fat diet showed higher weight (30.71 ± 0.66 g in O2, p < 0.001), nonfasting blood glucose levels (257.69 ± 5.10 mg/dl in O2, p < 0.001), TG (282.15 ± 1.83 mg/dl in O2, (p < 0.001)), and cholesterol levels (335.72 ± 2.23 mg/dl in O2, (p < 0.001)), compared with control. In C2 group, mean body weight was 25.71 ± 0.54 g, non-fasting blood level 161.54 ± 4.48 mg/dl, TG level 214.29 ± 5.54 mg/dl, and cholesterol level 164.29 ±4.57 mg/dl. Compared to obese group, mice receiving inulin showed lower blood glucose levels (223.10 ± 8.7 mg/dl in E2, p < 0.001), body weight (27.86 ± 0.57 g in E2, p < 0.001), TG (232.14 ± 4.02 mg/dl in E2, p < 0.001) and cholesterol (249.97 ± 2.28 in E2, p < 0.001). A slight decrease in hepatic α-amylase gene expression was observed only in E1. Conclusion: Besides its sweetening properties, inulin may also find use as a potential anti-obesity compound.

Anti-cancer and antioxidant properties of phenolics isolated from Toona sinensis A Juss acetone leaf extract

Purpose: To investigate the antioxidant and anticancer activities of phenolics from the leaf extract of Toona sinensis (TS). Methods: Acetone leaf extract of TS was screened for total phenolic and flavanoid contents, and the flanonoids were subjected to high performance liquid chromatographic (HPLC) analysis. Antioxidant properties were assessed via oxygen radical absorbance capacity (ORAC), peroxyl radical scavenging capacity (PSC) and cellular antioxidant activity (CAA), while anti-proliferative activity ins HepG2 cell line was assessed using methylene blue assay. Results: The extract contained 36.02 ± 0.24 mg of gallic acid equiv/g dry weight (DW) and 20.24 ± 1.73 mg of catechin equiv/g DW of total phenolic and total flavonoid, respectively. The levels of rutin and quercitrin were 0.51 and 19.55 mg/g, respectively. Epicatechin, gallic acid, quercitin, isoquercetin were not detected. The extract showed significant antioxidant potential and high anti-proliferation capacity with low cytotoxicity against HepG2 cell in vitro. The underlying mechanism of anti-proliferative effect was induction of apoptosis. Conclusion: TS leaf extract possesses significant in vitro antioxidant properties and anti-proliferative effect against HepG2 cells, which make it a potential anticancer drug source.

In vitro antioxidative activity of moss extract, and effect of moss on serum lipid level of mice fed with high-fat diet

Purpose: To evaluate the potential of active compounds derived from moss in the prevention and treatment of various diseases. Methods: Three species of moss were extracted with deionized water at 95 °C, and with 70.5 % ethanol at 85 °C. Analysis of total phenolic contents (TPC) of the extracts were performed by FolinCiocalteu (FC) method. The antioxidant activity of the extracts were determined using three methods, namely, by 2,2\'-azino-bis(3-ethylbenzothiazoline-6-sulphonic) acid (ABTS), 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP). In vivo effects were evaluated in mice fed high fat diet (HFD) supplemented with 20 % ground moss. Cholesterol levels in HFD were evaluated by ophthalaldehyde method. Serum triglyceride levels were measured using triglyceride (TG) kit, while blood insulin level and leptin concentration were measured by enzyme-linked immunosorbent assay (ELISA) kit. Results: The moss extracts exhibited antioxidative effects, as evidenced of . TPC of 47.20 ± 11.20 to 119.87 ± 11.51 mg GAE/mg, respectively. ABTS scavenging activity was 1078.11 ± 18.95 to 2587.33 ± 46.19 μmol Trolox/mg, DPPH scavenging activity of were 42.11 ± 8.22 to 298.78 ± 20.02 μmol Trolox/mg, and FRAP value of 393.19 ± 24.64 to 1070.14 ± 17.92 μmol Trolox/mg, respectively. Mice fed with 20 % ground moss did not show any significant effect (p < 0.05) on visceral weight and blood lipid levels of HFD, while leptin concentrations reduced significantly to 4.74 ± 0.00 and 0.20 ± 0.00 ng/dL) relative to HFD alone (26.72 ± 6.53 ng/dL). Conclusion: Moss can potentially be used as an antioxidative ingredient, for the improvement of overall human health, suggesting that important medical benefits associated with moss consumption. However, further investigations are required to ascertain this.

Dose-dependent protection of reseveratrol against spinal cord ischemic-reperfusion injury in rats

Purpose: To examine the protective effects of resveratrol (RESV) against spinal cord ischemic reperfusion (SCIR) injury. Methods: Forty-eight male rats were divided into six groups: sham-operated (control-I), SCIR-treated (SCIR-II), rats receiving 20 mg/kg of RESV with SCIR (RESV 20+SCIR-III), rats receiving 40 mg/kg of RESV with SCIR (RESV 40+SCIR-IV), rats receiving 60 mg/kg of RESV with SCIR (RESV 60+SCIR-V), and rats receiving 50 mg/kg of methylprednisolone (MP) with SCIR (MP + SCIR-VI), for 7 days prior to IR (pre-treatment) and 7 days after IR (post-treatment). Results: The levels of oxidative markers (TBARS, MPO) and inflammatory markers (IL-1β, IL-6, TNF-α, and NF-p65) were concomitantly suppressed in RESV-treated rats, which showed improved locomotor function. A pronounced increase in the activities of antioxidant enzymes (SOD, CAT and GSH) was noted in the RESV group compared with the MP and SCIR groups. RESV and MP supplementation increased neuronal count with decreased nuclear degeneration. RESV (40 mg) exhibited greater protective effect than 20 mg and 60 mg of RESV and 50 mg of MP. Conclusion: The results show the neurotherapeutic potential of RESV (40 mg) to attenuate oxidative stress and the inflammatory response to SCIR injury.

Isolation and identification of two galangin metabolites from rat urine and determination of their in vitro hypolipidemic activity

Purpose: To investigate the lipid-lowering activity of two metabolites of galangin, namely, galangin-3-Oβ-D-glucuronic acid (GG-1) and galangin-7-O-β-D-glucuronic acid (GG-2). Methods: Female Sprague-Dawley rats were orally administered with galangin. The two metabolites of galangin were isolated from urine sample and purified using Sephadex LH-20 and semi-preparative high performance liquid chromatography (HPLC). The structures of the metabolites were identified by analyzing spectroscopic data. Hypolipidemic activity was evaluated in HepG2 cells. The down- or upregulation of lipogenic genes was detected using real-time quantitative polymerase chain reaction (qPCR). Results: Both metabolites of galangin showed hypolipidemic activity. These activities are closely associated with the down-regulation of lipogenic genes such as SREBP-1a, SREBP-1c, and SREBP-2 transcription factors, and the downstream genes such as FAS, ACC, and HMGR were revealed by realtime qPCR data. Conclusion: The results show that both metabolites possess better lipid-lowering activities than galangin. These hypolipidemic activities are closely associated with inhibiting key genes or proteins that regulated the biosynthesis of both cholesterol and triglycerides.

Antitumor effect and mechanism of action of polysaccharides extracted from Polygonum perfoliatum L whole plant in human lung carcinoma A549 cell line

Purpose: To optimize the extraction conditions of polysaccharides from Polygonum perfoliatum L. (PSDP) and to evaluate their anti-tumor activities on A549 cell line. Methods: Extraction of PSDP was optimized using Box-Behnken design (BBD). Three factors of response surface methodology (RSM) including extraction time, ratio of water to raw material and number of extractions were employed to optimize the yield of PSDP. The cytotoxic effect of PSDP on human lung carcinoma A549 cell line was evaluated in vivo, while its effects on expressions of caspase3, caspase-9, Bcl-2 and Bax were determined by western blot assay. Result: BBD was significant and applicable to PSDP extraction. Based on the contour plots, response surface plots and variance analysis, it predicted that the optimum conditions for PSDP extraction were: 1.58 h (extraction time); 30.18 mL/g (ratio of water to raw material); and 2.02 (number of extractions). PSDP had significant inhibitory effect on the growth of A549 cells in a concentration- and timedependent manner (p < 0.05). After treatment with PSDP, caspase-3, caspase-9 and Bax were significantly up-regulated (p < 0.05), whereas Bcl-2 was down-regulated, all concentration-dependently. Conclusion: RSM analysis is an appropriate method to optimize PSDP extraction. The results also indicate that PSDP has significant anti-tumor effect against A549 cells, most likely via inducing mitochondria-mediated apoptosis.

Anticonvulsant and sedative effect of Fufang Changniu pills and probable mechanism of action in mice

Purpose: To investigate the anticonvulsant and sedative effects of Fufang Changniu Pills (FCP) and its probable mechanism of action in mice. Methods: The water decoction of FCP was prepared and the main constituents were determined by high performance liquid chromatography (HPLC). The anticonvulsant activities of FCP were evaluated by maximal electroshock (MES) and pentylenetetrazole (PTZ)-induced seizures in mice. Pentobarbital sodium-induced sleeping time and locomotor activity measurements were performed to evaluate the sedative effects of FCP in mice. Finally, PTZ-induced chronic seizures were established, and expressions of gamma-aminobutyric acid A receptor (GABA-A) and glutamic acid decarboxylase 65 (GAD65) in the brains of the mice were assayed by western blot in order to explore the probable mechanisms of action of the drug. Results: Gallic acid, liquiritin, cinnamyl alcohol, cinnamic acid and glycyrrhizic acid were detected in FCP decoction. FCP (50, 100 and 200 mg/kg) showed significant anticonvulsant and sedative effects on epileptic mice induced by MES (p < 0.05) and PTZ (p < 0.05). Moreover, pentobarbital sodium-induced sleeping time and locomotor activity tests showed that FCP possesses sedative effect (p < 0.05). Western blot data indicate that FCP significantly up-regulated GABA-A and GAD 65 in the brains of chronic epileptic rats (p < 0.05). Conclusion: FCP has significant anticonvulsant and sedative effects, and the mechanism of its action may be related to the up-regulation of GABA-A and GAD 65 in mice brain.

Bioprospecting for culturable actinobacteria with antimicrobial properties isolated from rivers in Colombian Orinoquia

Purpose: To Isolate and characterize Actinobacteria with antimicrobial activity from Guaviare River (Colombia). Methods: Water and sediment samples were collected from Guaviare River. Direct plating, heat and CaCO3 methods were used to isolate Actinobacteria. Six bacterial strains were tested using T-Streak method: Escherichia coli ATCC 23724, Staphylococus aureus ATCC 25923, Acinetobacter baumannii ATCC 19606, Bacillus subtilis ATCC 21556, Klebsiella pneumoniae ATCC 700603, Chromobacterium violaceum ATCC 31532. Strains of Fusarium sp. H24, Trichoderma harzianum H5 and Colletotrichum gloeosporioides were tested using Kirby-Bauer method. Isolates with high antimicrobial activity were selected for further taxonomic identification. Results: A total of 374 actinobacteria isolates were obtained. Seven isolates exhibited high antimicrobial activity (p < 0.05) and were confirmed as members of Streptomycetaceae family. Of these, three isolates showed differential phenotypic and genotypic profiles, indicating that they may represent new species. Conclusions: To date, this is the first study of this type in Colombian Orinoquia and indicates that this promising source of Actinobacteria from aquatic sediments with the ability to produce antimicrobial secondary metabolites.

Synthesis and antibacterial study of some s-substituted aliphatic analogues of 2-mercapto-5-(1-(4-toluenesulfonyl) piperidin-4-yl)-1,3,4-oxadiazole

Purpose: To synthesize a series of analogues of 1,3,4-oxadiazole and to evaluate their antibacterial activity. Methods: Ethyl piperidin-4-carboxylate (1) was mixed with 4-toluenesulfonyl chloride (2) in benignant conditions to yield ethyl 1-(4-toluenesulfonyl)piperidin-4-carboxylate (3) and then 1-(4- toluenesulfonyl)piperidin-4-carbohydrazide (4). Intermolecular cyclization of 4 into 2-mercapto-5-(1-(4- toluenesulfonyl) piperidin-4-yl)-1,3,4-oxadiazole (5) was obtained on reflux with CS2 in the presence of KOH. Molecule 5 was stirred with alkyl halides, 6a-i, in DMF in the presence of LiH to synthesize the final compounds, 7a-i. The structures of these molecules were elucidated by Fourier transform infra-red (FTIR) spectroscopy, proton nuclear magnetic resonance (1H-NMR) and electron impact mass spectrometry (EI-MS). Antibacterial activity was evaluated against five bacterial strains, namely, Salmonella typhi, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis, with ciprofloxacin used as standard antibacterial agent. Results: Out of nine synthesized derivatives, compound 7a was the most active against three bacterial strains, S. typhi, E. coli and P. aeruginosa, with minimum inhibitory concentration (MIC) of 9.11 ± 0.40, 9.89 ± 0.45 and 9.14 ± 0.72 μM, respectively, compared with 7.45 ± 0.58, 7.16 ± 0.58 and 7.14 ± 0.18 μM, respectively, for the reference standard (ciprofloxacin). Similarly, compounds 7a - 7c showed relatively good antibacterial activity against B. subtilis strain while compound 7e - 7g revealed good results against S. typhi bacterial strain. Conclusion: The results indicate that S-substituted derivatives of the parent compound are more effective antibacterial agents than the parent compound, even with minor differences in substituents

Determination of acrylamide levels in selected commercial and traditional foods in Syria

Purpose: To determine acrylamide (AA) levels in different brands of commercial and traditional foodstuffs available in Syria by ultra-performance liquid chromatography-mass spectrometery (UPLCMS). Methods: A total of 63 samples were analyzed. Food samples were defatted by hexane and then extracted with methanol 98 % in a vortex mixer. Thereafter, Carrez I and Carrez II were added to precipitate proteins from the co-extractives and then centrifuged to obtain a clear aqueous extract that was evaporated to dryness. The extract was dissolved in 1 mL of water, eluted through a preconditioned Oasis HLB cartridge and then filtered. The filtrate was analyzed by UPLC-MS/MS to determine AA content. Results: Among the commercial foods tested, the highest acrylamide quantity was found in potato products (396 ± 3.59 – 1844 ± 5.29 μg kg−1) and the lowest in corn products (183 ± 2.64 – 366 ± 4.58 μg kg−1). This was followed by biscuits (57 ± 2.64 – 1433 ± 2.51 μg kg−1), breakfast cereals (121 ± 8.73 – 245 ± 3.60 μg kg−1), bread (119 ± 1.73 – 263 ± 3.60 μg kg−1) and then coffee (113 ± 2.64 - 64 ± 3.05 μg kg−1). Regarding traditional foods, the highest level of AA was found in AL- Mshabak (481 ± 2.08 μg kg−1) and AL-Awamat (421 ± 2.64 μg kg−1) followed by AL-Namora (282 ± 4.35 μg kg−1) and AL-Kenafa (242 ± 2.64 μg kg−1). It was also observed that the lowest amount of AA was in fried bread (230 μg kg−1), AL-Fatayer (192 ± 3.51 μg kg−1) and AL-Baqlawa (172 ± 4.35 μg kg−1) while Eid Aqras (130 ± 4.58 μg kg−1) and AL-Brazeq (167 ± 3.78 μg kg−1) contained the least amount of AA. Conclusion: The results indicate that the highest levels of AA are found in the most commonly consumed foods. There was significant difference (p < 0.05) in AA levels among different food items and within different brands of the same product.

Liquid chromatographic-mass spectrometric method for determination of drug content uniformity of two commonly used dermatology medications in a split-tablet dosage form

Purpose: To develop and validate a simple, efficient and reliable Liquid chromatographic-mass spectrometric (LC-MS/MS) method for the quantitative determination of two dermatological drugs, Lamisil® (terbinafine) and Proscar® (finasteride), in split tablet dosage form. Methods: Thirty tablets each of the 2 studied medications were randomly selected. Tablets were weighed and divided into 3 groups. Ten tablets of each drug were kept intact, another group of 10 tablets were manually split into halves using a tablet cutter and weighed with an analytical balance; a third group were split into quarters and weighed. All intact and split tablets were individually dissolved in a water: methanol mixture (4:1), sonicated, filtered and further diluted with mobile phase. Optimal chromatographic separation and mass spectrometric detection were achieved using an Agilent 1200 HPLC system coupled with an Agilent 6410 triple quadrupole mass spectrometer. Analytes were eluted through an Agilent eclipse plus C8 analytical column (150 mm × 4.6 mm, 5 μm) with a mobile phase composed of solvent A (water) containing 0.1% formic acid and 5mM ammonium formate pH 7.5, and solvent B (acetonitrile mixed with water in a ratio A:B 55:45) at a flow rate of 0.8 mL min-1 with a total run time of 12 min. Mass spectrometric detection was carried out using positive ionization mode with analyte quantitation monitored by multiple reaction monitoring (MRM) mode. Results: The proposed analytical method proved to be specific, robust and adequately sensitive. The results showed a good linear fit over the concentration range of 20 - 100 ng mL-1 for both analytes, with a correlation coefficient (r2) ≥ 0.999 and 0.998 for finasteride and terbinafine, respectively. Following tablet splitting, the drug content of the split tablets fell outside of the proxy USP specification for at least 14 halves (70 %) and 34 quarters (85 %) of FIN, as well as 16 halves (80 %) and 37 quarters (92.5 %) of TBN. Mean weight loss, after splitting, was 0.58 and 2.22 % for FIN half- and quarter tablets, respectively, and 3.96 and 4.09 % for TBN half- and quarter tablets,respectively. Conclusion: The proposed LC-MS/MS method has successfully been used to provide precise drug content uniformity of split tablets of FIN and TBN. Unequal distribution of the drug on the split tablets is indicated by the high standard deviation beyond the accepted value. Hence, it is recommended not to split non-scored tablets especially, for those medications with significant toxicity

Determination of borneol and other chemical compounds of essential oil of Dryobalanops aromatica exudate from Malaysia

Purpose: To determine borneol and other chemical compounds of essential oil derived from the exudate of Dryobalanops aromatica aromatica in Malaysia. Methods: Exudate was collected from D. aromatica and subjected to fractional distillation to obtain essential oil. Gas chromatography-mass spectrometry (GC-MS) was performed to characterize the composition of the isolated essential oil. Results: Essential oil (7.58 %) was obtained with the highest yield (3.24 %) in the first 2 h of fractional distillation. Thirty compounds which accounted for 97.56 % of total essential oil composition were identified by GC-MS, and they include sesquiterpenes (46.87 %), monoterpenes (31.05 %), oxygenated monoterpenes (16.76 %) and oxygenated sesquiterpenes (2.13 %). Borneol (0.74 %) was also detected in the essential oil. Conclusion: Borneol and other terpenoid compounds are present in the essential oil of the exudate of D. aromatica