Jintana Sattayasai

Analgesic, antipyretic, anti-inflammatory and toxic effects of andrographolide derivatives in experimental animals

Andrographolide (1) and 14-deoxy-11,12-didehydroandrographolide (2) are active constituents of Andrographis paniculata (Burm. f.), family Acanthaceae. A. paniculata extracts are reported to have antiviral, antipyretic, immunostimulant and anticancer activities. In this study, 1 and its 14-acetyl- (4) and 3,19-isopropylidenyl- (3) derivatives, as well as 2 and its 3,19-dipalmitoyl-derivative (5), were intraperitoneally tested for their analgesic, antipyretic, anti-inflammatory and acute toxicity effects in animal models. Analgesic effects were tested in mice using hot plate and writhing tests to distinguish the central and peripheral effects, respectively. The results showed that, at 4 mg/kg, all tested substances have significant analgesic effects, and the highest potency was seen with 3, 4 and 5. Increasing the dose of 3 and 5 to 8 mg/kg did not increase the analgesic effect. In the writhing test, 3 and 5, but not 1, showed significant results. In a baker’s yeast-induced fever model, 3 and 5 significantly reduced rats’ rectal temperature (p < 0.05). In a carrageenan-induced inflammation model, 1, 3 and 5 significantly reduced rats’ paw volume. Doses of 3 and 5 up to 100 mg/kg did not show any serious toxic effects. From this study, 3 and 5 are the most interesting derivatives, showing much greater potency than their parent compounds. These could be further developed as analgesic, antipyretic and anti-inflammatory agents, without any serious toxicity.

Protective Effects of Mangosteen Extract on H2O2-Induced Cytotoxicity in SK-N-SH Cells and Scopolamine-Induced Memory Impairment in Mice

Mangosteen extracts (ME) contain high levels of polyphenolic compounds and antioxidant activity. Protective effects of ME against β-amyloid peptide (Aβ), induced cytotoxicity have been reported. Here, we further studied the protective effects of ME against oxidative stress induced by hydrogen peroxide (H2O2) and polychlorinated biphenyls (PCBs), and demonstrated the protection against memory impairment in mice. The cytoprotective effects of ME were measured as cell viability and the reduction in ROS activity. In SK-N-SH cell cultures, 200 μg/ml ME could partially antagonize the effects of 150 or 300 µM H2O2 on cell viability, ROS level and caspase-3 activity. At 200, 400 or 800 µg/ml, ME reduced AChE activity of SK-N-SH cells to about 60% of the control. In vivo study, Morris water maze and passive avoidance tests were used to assess the memory of the animals. ME, especially at 100 mg/kg body weight, could improve the animal’s memory and also antagonize the effect of scopolamine on memory. The increase in ROS level and caspase-3 activity in the brain of scopolamine-treated mice were antagonized by the ME treatment. The study demonstrated cytoprotective effects of ME against H2O2 and PCB-52 toxicity and having AChE inhibitory effect in cell culture. ME treatment in mice could attenuate scopolamine-induced memory deficit and oxidative stress in brain.

Effects of andrographolide on sexual functions, vascular reactivity and serum testosterone level in rodents

In this study, effects of andrographolide from Andrographis paniculata on sexual functions, vascular reactivity and serum testosterone level in experimental animals were observed. The suspension of andrographolide in 5% DMSO was administered orally at the dose of 50mg/kg to male ICR mice. The female mice involved in mating were made receptive by hormonal treatment. The mating behaviors, mounting latency and mounting frequency, were determined and compared with the standard reference drug sildenafil citrate. Administration of andrographolide significantly decreased the mounting latency at 120 and 180 min and increased mounting frequency at 180 min after treatment. In endothelium-intact rat aortic strips, norepineprine-induced contraction was reduced by preincubation with andrographolide. Administration of 50mg/kg andrographolide orally to male mice once daily for 2, 4, 6 or 8 weeks had no significant effects on sperm morphology and motility. Interestingly, at week 4, serum testosterone level in mice treated with andrographolide was significantly increased when compared to the control. Thus, the effects of andrographolide on vascular response to norepinephrine and testosterone level observed in this study might be contributed to the sexual enhancing properties observed.

Biphasic effects of Morus alba leaves green tea extract on mice in chronic forced swimming model

In this study, the effects of an aqueous extract of Morus alba leaves green tea (ME) on mouse behaviors (depression, anxiety, climbing activity and thermal response), muscle coordination and muscle strength were studied. Male IRC mice received a single intraperitoneal injection of either the ME, desipramine or diazepam. Thirty minutes after injection, the mice were tested in all experimental models. A significant antidepressant‐like effect could be detected in the animals receiving either 100 or 200 mg/kg ME. The effect of 200 mg/kg ME in decreasing the immobility time was comparable to 10 mg/kg desipramine. With higher dose (1000 mg/kg), a significant increase in immobility time could be observed. In the elevated plus maze, no increase in time in the open arm could be observed in mice treated with ME at either 100 or 200 mg/kg. However, high doses of ME (500 or 1000 mg/kg) decreased both time in the open arm and the number of entries in the maze. No change in thermal response could be seen in mice treated with ME at doses up to 500 mg/kg, however, at 1000 mg/kg, the response time to heat was increased significantly. The ME at either 500 or 1000 mg/kg also decreased muscle coordination, strength and climbing activity significantly when compared with the control. This study suggests that ME possesses an antidepressant‐ without an anxiolytic‐like effect, however, at high doses, the extract might show the sedative effect and alter other functions such as muscle strength, animal activity in the maze and pain response. Copyright

Antipyretic, analgesic and anti-oxidative activities of Aquilaria crassna leaves extract in rodents

In Thailand, the leaves of Aquilaria crassna have been used traditionally for the treatments of various disorders, but without any scientific analysis. In this study, the antipyretic, analgesic, anti-inflammatory and anti-oxidative properties of A. crassna leaves extract were investigated at a wide dose range in rodents. Experimental animals were treated orally with an aqueous extract of Aquilaria crassna leaves (ACE). They were tested for antipyretic (Bakers yeast-induced fever in rats), analgesic (hot plate test in mice) and anti-inflammatory (carrageenan-induced paw edema in rats) activities. An anti-oxidative effect of ACE was evaluated by using the DPPH anti-oxidant assay. The results showed that, after 5 hours of yeast injection, 400 and 800 mg/kg ACE significantly reduced the rectal temperature of rats. Mice were found significantly less sensitive to heat at an oral dose of 800 mg/kg ACE, after 60 and 90 min. No anti-inflammatory activity of ACE at an 800 mg/kg dose could be observed in the rat paw assay. An anti-oxidative activity of ACE was observed with an IC 50value of 47.18 μg/ ml. No behavioral or movement change could be observed in mice after oral administration of ACE (800 or 8,000 mg/kg) for seven consecutive days. Interestingly, from the second day of treatment, animals had a significant lower body weight at the 8,000 mg/kg dose of ACE compared to the control. No toxicity was identified and the results of this study state clearly that Aquilaria crassna leaves extracts possess antipyretic, analgesic and anti-oxidative properties without anti-inflammatory activity.

Synthesis and evaluation of N1-substituted-3-propyl-1,4-benzodiazepine-2-ones as cholecystokinin (CCK2) receptor ligands

A novel synthetic approach towards N1‐alkylated 3‐propyl‐1,4‐benzodiazepineswas developed in five synthetic steps from 2‐amino‐4‐chlorobenzophenone, in which the N‐oxide 4 served as a key intermediate. The structure‐activity relationship optimization of this 3‐propyl‐1,4‐benzodiazepine template was carried out on the N1‐position by selective alkylation reactions and resulted in a ligand with an improved affinity on the cholecystokinin (CCK2) receptor. The N‐allyl‐3‐propyl‐benzodiazepine 6d displayed an affinity towards the CCK2 (CCK‐B) receptor of 170 nM in a radiolabelled receptor‐binding assay. The anxiolytic activity of this allyl‐3‐propyl‐1,4‐benzodiazepine 6d was subsequently determined in in‐vivo psychotropic assays. This novel lig and had ED50 values of 4.7 and 5.2 mg kg−1 in the black and white box test and the x‐maze, respectively, and no significant sedation/muscle relaxation was observed.

Dose-dependent effects of glutamate in pyridoxine-induced neuropathy

In order to explore the effects of glutamate in a pyridoxine megadose-induced neuropathy, rats were received glutamate either 0.5 or 1 g/kg/day orally with or without pyridoxine 0.8 g/kg/day intraperitoneally for 14 days. The animals motor coordination, the muscle power and the thermal threshold were observed daily. The nerve conduction velocity was measured at day 0 and day 15 of the treatment. Glutamate either 0.5 or 1 g/kg/day appeared to have no effect on motor coordination, the nerve conduction velocity and the muscle power score compared with control. However, the thermal response latency was significantly decreased (from day 9) in animals treated with 1 g/kg/day glutamate. In pyridoxine-induced neuropathy rats, glutamate 0.5 g/kg/day significantly decreased the effects of pyridoxine on the sciatic nerve conduction velocity, the muscle power score and the motor coordination. Interestingly, glutamate at a dose of 1 g/kg/day worsened the neurotoxic effects cause by pyridoxine.

In‐vitro and in‐vivo antivenin activity of 2‐[2‐(5,5,8a‐trimethyl‐2‐methylene‐decahydro‐naphthalen‐1‐yl)‐ethylidene]‐succinaldehyde against Ophiophagus hannah venom

Objectives Curcuma zedoaroides A. Chaveerach & T. Tanee, locally known as Wan‐Paya‐Ngoo‐Tua‐Mia, is commonly used in the North‐Eastern part of Thailand as a ‘snakebite antidote’. The aim of this study was to isolate the active compound from the rhizome of C. zedoaroides, to determine its structure and to assess its antagonistic activity in vitro and in vivo against King cobra venom.

A Biotin-Coupled Bifunctional Enzyme Exhibiting Both Glutamine Synthetase Activity and Glutamate Decarboxylase Activity

Purpose: To purify and study native form and enzymatic activity of the 42 kDa biotin-coupled protein (p42), which is related to glutamate action in chick retina. Methods: p42 was purified using molecular filtration in the presence of 0.7 M sodium chloride. Purity and identification of p42 were studied by SDS-PAGE, 2D-PAGE, LC-MS/MS, and MALDI-TOF MS. The native form of p42 was investigated using native-PAGE and Ferguson plot. Biotin-coupled property was examined by Western blot analysis. Enzymatic actions of p42 were studied using glutamate as substrate in the presence or absence of glutamine. Results: p42 was successfully purified from chick retinal protein solution using the molecular filtration. Western blot analysis with avidin showed that p42 was a biotin-coupled protein. Using SDS-PAGE, 2D-PAGE, LC-MS/MS, and MALDI-TOF MS, purified p42 was identified as a glutamine synthetase with four isoforms. Native-PAGE, followed by Ferguson plot analysis, showed two molecular forms of p42 corresponding to homotetramers and homooctamers. Enzymatic reaction followed by paper chromatography showed that p42 catalyzed the synthesis of glutamine from glutamate in the presence of ammonium ion, ATP, and magnesium ion. At prolonged reaction time, γ-aminobutyric acid (GABA) was also formed. With glutamate and glutamine present at equal concentrations in the reaction mixture, GABA could be rapidly detected, but GABA could not be detected when glutamate concentration was more than four-fold that of glutamine. The results indicated that p42 also had glutamate decarboxylase activity. Both enzymatic activities were inhibited by avidin. High concentrations of Mn2+ inhibited synthetase activity of p42 but not decarboxylase activity. Conclusion: p42 was purified from chick retinal protein solution using molecular filtration in the presence of sodium chloride. The protein was a biotin-coupled bifunctional enzyme that contained glutamine synthetase activity and glutamate decarboxylase activity. Biotin was possibly involved in these activities. Mn2+ showed different effects on the two activities.

In vivo evaluation of substituted 3-amino-1,4-benzodiazepines as anti-depressant, anxiolytic and anti-nociceptive agents.

Oxazepam (CAS 604-75-1) 4a served as building block in the synthesis of substituted 3-amino-1,4-benzodiazepines, which were subsequently tested in various CNS animal models. The hydroxy group of oxazepam was either activated as a chloride (Method A) or as a phosphor-oxy derivative (Method B) giving the desired 3-amino-1,4-benzodiapines 6a-6r in high yields with primary and secondary amines in a typical nucleophilic substitution reaction. Eighteen 3-substituted 1,4-benzodiazepines were prepared and served as new chemical entities and for lead structure discovery. The mixed cholecystokinin (CCK) antagonist 6e showed anxiolytic and antidepressant effects from 10 microg/kg in mice in the elevated x-maze test and the forced swimming test. The CCK1 antagonist 6 g has shown antidepressant effects from the same dose, but lacked anxiolytic properties. Both compounds potentiated at a dose of 0.5 mg/kg morphine antinociception with a maximum possible effect (MPE) about 35%. By assessing initially the MPE of antinocipection for the 18 newly synthesised benzodiazepines in the tail-flick test, 4 other benzodiazepines were found active. In further in vive evaluation the cyclohexyl derivative 6 i displayed anxiolytic, antidepressant and antinociceptive properties as single agent at a dose of 5 mg/kg without toxicity. The benzodiazepines 6i and 6p, which initially showed a higher MPE in terms of morphine potentiation (43/44%) showed analgesic effects as single agents, without having anxiolytic or antidepressant properties. The amino-piperidinyl derivative 6p displayed a similar dose-response relationship to morphine, but was 3 times more potent.