This study was conducted to evaluate the phytochemical contents and antimalarial properties of the oils extracted from the leaves of Malaysian Plectranthus amboinicus in mice infected with Plasmodium berghei. The essential oils were extracted and prepared by using steam distillation technique and subjected to phytochemical screening by using GC-MS. Antimalarial activity of different extract doses of the essential oil was tested in vivo in ICR mice infected with Plasmodium berghei (PZZ1/100) during early, established and residual infections. In all, 5 compounds made up 88.34% of total oil and the major chemical compounds were carvacrol (85.14%), thymoquinone (1.65%), terpinen-4-ol (0.70%), octenol (0.62%) and thymol (0.23%). Antimalarial assay showed this essential oil as a potential prophylactic agent with the percentage chemosuppression of 45.23%, 18.28%, 45.38% and 58.26% while treated with 50, 200, 400 and 1000 µL/kg respectively of essential oil and curative agent with percentage of chemo suppression of 54.10%, 47.35%, 56.75% and 65.38% while treated with the above dose of essential oil. Statistically no reduction of parasitemia was calculated for suppressive test. The extract has prophylactic and curative effects on P.berghei in mice
The quality of RNA is crucial when performing microarray experiments. This is particularly important when dealing with preimplantation embryos, from which a minimum yield of RNA of good quality can be produced. We report the optimization of several RNA extraction methods applied to preimplantation embryos at different stages of development. The quality of the samples was confirmed using a microarray and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis. A total of 30 cultured two-cell stage embryos of ICR mice were pooled at the 8-cell, morula, and blastocyst stages. The embryos were divided into two groups comprising DNase-treated and non-DNase-treated RNA samples. Total RNA was extracted using a Pico Pure RNA Isolation Kit following the manufacturer protocol, with some modifications. Lysed samples were bound to a silica-based filter, treated with deoxyribonuclease I (DNase I), and washed several times before elution. RNA concentration and integrity were evaluated using an Agilent 2100 Bioanalyzer and an RNA 6000 Pico Assay kit. Although concentrations of non-DNase-treated RNAs were higher than DNase-treated RNA, DNase-treated RNA gave a higher RNA integrity number compared with non-DNase-treated RNA. Inclusion of DNase treatment in the RNA extraction procedure gave the best quality RNA samples from preimplantation embryos, as validated by microarray and RT-qPCR quality control.
Malaria, caused by the Plasmodium parasite is still a health problem worldwide due to resistance of the pathogen to current anti-malarials. The search for new anti-malarial agents has become more crucial with the emergence of chloroquine-resistant Plasmodium falciparum strains. Protein kinases such as mitogen-activated protein kinase (MAPK), MAPK kinase, cyclin-dependent kinase (CDK) and glycogen synthase kinase- 3(GSK-3) of parasitic protozoa are potential drug targets. GSK-3 is an enzyme that plays a vital role in multiple cellular processes, and has been linked to pathogenesis of several diseases such as type II diabetes and Alzheimer's disease. In the present study, the antiplasmodial property of LiCl, a known GSK-3 inhibitor, was evaluated in vivo for its antimalarial effect against mice infected with Plasmodium berghei. Infected ICR mice were intraperitoneally administered with LiCl for four consecutive days before (prophylactic test) and after (suppressive test) inoculation of P. berghei-parasitised erythrocytes. Results from the suppressive test (post-infection LiCl treatment) showed inhibition of erythrocytic parasitemia development by 62.06%, 85.67% and 85.18% as compared to nontreated controls for the 100 mg/kg, 300 mg/kg and 600 mg/kg dosages respectively. Both 300 mg/kg and 600 mg/kg LiCl showed similar significant (P<0.05) suppressive values to that obtained with chloroquine-treated mice (86% suppression). The prophylactic test indicated a significantly (P<0.05) high protective effect on mice pre-treated with LiCl with suppression levels relatively comparable to chloroquine (84.07% and 86.26% suppression for the 300 mg/kg and 600 mg/kg LiCl dosages respectively versus 92.86% suppression by chloroquine). In both the suppressive and prophylactic tests, LiCl-treated animals survived longer than their non-treated counterparts. Mortality of the non-treated mice was 100% within 6 to 7 days of parasite inoculation whereas mice administered with LiCl survived beyond 9 days. Healthy non-infected mice administered with 600 mg/ kg LiCl for four consecutive days also showed decreased mortality compared to animals receiving lower doses of LiCl; three of the seven mice intraperitoneally injected with the former dose of LiCl did not survive more than 24 h after administration of LiCl whereas animals given the lower LiCl doses survived beyond four days of LiCl administration. To date, no direct evidence of anti-malarial activity in vivo or in vitro has been reported for LiCl. Evidence of anti-plasmodial activity of lithium in a mouse infection model is presented in this study.
This study was conducted to examine the potential for implantation and sustainable fetal development of mouse embryos cultured from the pronuclear to blastocyst stage. Pronuclear embryos from ICR mice (Harlan Sprague-Dawley) were cultured in Sydney IVF sequential media (Cook) to the blastocyst stage in medium only or co-cultured with autologous cumulus cells. We also experimented with co-culture in 100 µL drops. Drop co-culture produced blastocyst formation rates with a mean of 47.0%, which was significantly higher (P < 0.05) compared to embryos cultured in identical culture conditions except without cumulus cells at 27.3%. Blastocysts obtained in vitro in Cook medium only and co-cultured in Cook medium with cumulus cells were transferred to pseudopregnant females of ICR strain. The day of blastocyst transfer into surrogate females was designated as post-transfer of blastocyst day 1 (PT 1). The implantation and fetal development was compared to embryo transfer of in vivo derived blastocysts, which served as controls. There were no statistical differences for implantation and fetal development rates for blastocysts cultured in vitro in either Cook medium only or co-culture in Cook medium with cumulus cells compared to in vivo-derived blastocysts. The advantage of the co-culture system is in generating more blastocysts available for transfer.
Actinodaphne sesquipedalis Hook. F. Var. Glabra (Kochummen), also known as "Medang payung" by the Malay people, belongs to the Lauraceae family. In this study, methanol leaf extract ofA. sesquipedaliswas investigated for their acute toxicity and gastroprotective effects to reduce ulcers in rat stomachs induced by ethanol. The rats were assigned to one of five groups: normal group (group 1), ulcer group (group 2), control positive drug group (group 3) and two experimental groups treated with 150 mg/kg (group 4) and 300 mg/kg (group 5) of leaf extract. The rats were sacrificed an hour after pretreatment with extracts, and their stomach homogenates and tissues were collected for further evaluation. Macroscopic and histological analyses showed that gastric ulcers in rats pretreated with the extract were significantly reduced to an extent that it allowed leukocytes penetration of the gastric walls compared with the ulcer group. In addition, an ulcer inhibition rate of >70% was detected in rats treated with both doses ofA. sesquipedalisextract, showing a notable protection of gastric layer. Severe destruction of gastric mucosa was prevented with a high production of mucus and pH gastric contents in both omeprazole-treated and extract-treated groups. Meanwhile, an increase in glycoprotein uptake was observed in pretreated rats through accumulation of magenta color in Periodic Acid Schiff staining assay. Analysis of gastric homogenate from pretreated rats showed a reduction of malondialdehyde and elevation of nitric oxide, glutathione, prostaglandin E2, superoxide dismutase and protein concentration levels in comparison with group 2. Suppression of apoptosis in gastric tissues by upregulation of Hsp70 protein and downregulation of Bax protein was also observed in rats pretreated with extract. Consistent results of a reduction of gastric ulcer and the protection of gastric wall were obtained for rats pretreated withA. sesquipedalisextract, which showed its prominent gastroprotective potential in rats' stomach against ethanol-induced ulcer.
Acmella uliginosa (Sw.) Cass. is a medicinal herbaceous plant that is commonly used by the Malay community in Malaysia to relieve pain often associated with mouth ulcers, toothache, sore throat, and stomach ache.
A novel synthetic compound from the 2-benzoyl-6-benzylidenecyclohexanone analogue, namely 2-benzoyl-6-(3-bromo-4-hydroxybenzylidene)cyclohexen-1-ol (BBHC), showed pronounced nitric oxide inhibition in IFN-γ/LPS-induced RAW 264.7 cells. Based on this previous finding, our present study aimed to investigate the antinociceptive effects of BBHC via chemical and thermal stimuli in vivo. The investigation of the antinociceptive activity of BBHC (0.1, 0.3, 1.0 and 3.0 mg/kg, i.p.) was initiated with 3 preliminary screening tests, then BBHC was subjected to investigate its possible involvement with excitatory neurotransmitters and opioid receptors. The potential acute toxicity of BBHC administration was also studied. Administration of BBHC significantly inhibited acetic acid-induced abdominal constrictions, formalin-induced paw licking activity and developed notable increment in the latency time. BBHC's ability to suppress capsaicin- and glutamate-induced paw licking activities, as well as to antagonise the effect of naloxone, had indicated the possible involvement of its antinociception with TRPV1, glutamate and opioid receptors, respectively. The antinociceptive activities of BBHC was not related to any sedative action and no evidence of acute toxic effect was detected. The present study showed that BBHC possessed significant peripheral and central antinociceptive activities via chemical- and thermal-induced nociceptive murine models without any locomotor alteration and acute toxicity.
The immunoregulatory properties of mesenchymal stem cells (MSC) have been demonstrated on a wide range of cells. Here, we describe the modulatory effects of mouse bone marrow-derived MSC on BV2 microglia proliferation rate, nitric oxide (NO) production and CD40 expression. Mouse bone marrow MSC were co-cultured with BV2 cells at various seeding density ratios and activated with lipopolysaccharide (LPS). We show that MSC exert an anti-proliferative effect on microglia and are potent producers of NO when stimulated by soluble factors released by LPS-activated BV2. MSC suppressed proliferation of both untreated and LPS-treated microglia in a dose-dependent manner, significantly reducing BV2 proliferation at seeding density ratios of 1:0.2 and 1:0.1 (p
The objective of this study is to synthesize and evaluate acute toxicity of the bacterial cellulose (BC)/acrylamide (Am) hydrogels as noncytotoxic and biocompatible oral drug delivery vehicles. A novel series of solubilized BC/Am hydrogels were synthesized using a microwave irradiation method. The hydrogels were characterized by Fourier transform infrared spectroscopy (FTIR), swelling ratio, porosity, drug release, and in vitro and in vivo biocompatibility experiments. FTIR spectra revealed that the BC crystallinity and gel fraction decreased as the NaOH concentration increased from 2% to 10% w/v, whereas the optical transparency, pH sensitivity, and porosity were enhanced with increasing alkali concentration. Theophylline was used as a model drug for drug loading and release studies. The percentage of drug released was higher at pH 7.4 compared to pH 1.5. In vitro cytotoxicity and hemolytic tests indicated that the BC/Am hydrogel is noncytotoxic and hemocompatible. Results of acute oral toxicity tests on ICR mice suggested that the hydrogels are nontoxic up to 2000 mg/kg when administered orally, as no toxic response or histopathological changes were observed in comparison to control mice. The results of this study demonstrated that the pH-sensitive smart hydrogel makes it a possible safe carrier for oral drug delivery.
In an earlier report, we demonstrated an antipsychotic-like activity of a methanolic extract of Morinda citrifolia Linn fruit in mouse models and postulated the contribution of its bioactive principles, scopoletin and rutin. Moreover, the antidopaminergic activities of scopoletin and rutin were reported in isolated vas deferens preparations. In the present study, scopoletin and rutin were assessed for antipsychotic-like activity using apomorphine-induced climbing behavior and methamphetamine-induced stereotypy in mice. The results of this study revealed that scopoletin and rutin (0.05, 0.1, 0.5, and 1 mg/kg, p.o.) had a "U-shaped" dose-dependent effect on climbing and stereotyped behaviors induced by apomorphine and methamphetamine, respectively, in mice. A significant reduction in climbing and stereotyped behaviors caused by scopoletin and rutin was observed only at a dose 0.1 mg/kg. This study suggests that scopoletin and rutin can alleviate positive symptoms of schizophrenia only at a specific dose. Further studies evaluating the effects of scopoletin and rutin on animal models for negative symptoms of schizophrenia are required for a novel drug discovery in the treatment of neuropsychiatric diseases.
In earlier ex vivo studies, we reported the biphasic effect of a methanolic extract of unripe Morinda citrifolia fruit (MMC) on dopamine-induced contractility in isolated rat vas deferens preparations. The present in vivo study was designed and undertaken to further explore our earlier ex vivo findings. This study examined the effect of the ethyl acetate fraction of a methanolic extract of unripe Morinda citrifolia Linn. fruit (EA-MMC; 5-100 mg/kg, p.o.) on the dopaminergic system using mouse models of apomorphine-induced climbing time and climbing behavior, methamphetamine-induced stereotypy (sniffing, biting, gnawing, and licking) and haloperidol-induced catalepsy using the bar test. Acute treatment with EA-MMC at a low dose (25 mg/kg, p.o.) significantly attenuated the apomorphine-induced climbing time and climbing behavior in mice. Similarly, EA-MMC (5 and 10 mg/kg, p.o.) significantly inhibited methamphetamine-induced stereotyped behavior in mice. These results demonstrated that the antidopaminergic effect of EA-MMC was observed at relatively lower doses (<25 mg/kg, p.o.). On the other hand, EA-MMC showed dopaminergic agonistic activity at a high dose (3,000 mg/kg, p.o.), which was evident from alleviation of haloperidol (a dopamine D2 blocker)-induced catalepsy in mice. Therefore, it is concluded that EA-MMC might possess a biphasic effect on the dopaminergic system, i.e., an antagonistic effect at lower doses (<25 mg/kg, p.o.) and an agonistic effect at higher doses (>1,000 mg/kg, p.o.). However, further receptor-ligand binding assays are necessary to confirm the biphasic effects of M. citrifolia fruit on the dopaminergic system.
Morinda citrifolia L. commonly known as noni or Indian mulberry belongs to the family Rubiaceae. Noni fruit juice has recently become a very popular remedy for the treatment of several diseases, including psychiatric disorders. This study aimed to investigate the anticraving effect of Tahitian Noni® Juice (TNJ) against ethanol seeking behavior in ICR male mice using the conditioned place preference (CPP) test. The CPP procedure consisted of four phases: preconditioning, conditioning, extinction, and reinstatement. During conditioning, intraperitoneal (i.p.) injections of ethanol (2 g/kg body weight (bw)) and normal saline (10 ml/kg bw) were given on alternate days for 12 days. Then, the animals were subjected to extinction trials for the next 12 days to weaken CPP. Finally, CPP was reinstated in the extinguished animals by a single low-dose priming injection of ethanol (0.4 g/kg bw, i.p.). The effect of TNJ (as a source of drinking water) on different phases of ethanol CPP in mice was studied. TNJ-treated mice showed a significant reduction in ethanol seeking behavior in the CPP test. The reference drug, acamprosate (ACAM) also showed a similar effect in the CPP test. The outcome of this study suggests that TNJ is effective in attenuating ethanol craving in mice and could be utilized for the treatment of alcohol dependence. Further clinical studies in this direction are warranted to support the present preclinical findings.
The first objective of the present study was to determine the appropriate dose of methamphetamine (Meth) to induce a successful conditioned place preference (CPP) in mice. The next objective was to examine the effect of a methanolic extract of M. citrifolia unripe fruit (MMC) against Meth-induced CPP in mice. In answering to the first objective, following the preconditioning test, an intraperitoneal injection of a fixed dose of Meth (0.5 or 1 or 2 mg/kg, i.p.) or saline (10 ml/kg, i.p.) was given on alternate days during the 10 days conditioning period followed by a postconditioning test conducted in Meth-free state. The first experiment revealed that 0.5 mg/kg of Meth could be an appropriate fixed low dose to induce CPP in mice. Meanwhile, in other experiments, the effect of MMC and bupropion (BUPR) against the expression, extinction, and reinstatement of Meth (0.5 mg/kg)-induced CPP in mice, respectively, was investigated. In a separate set of studies on each phase, an oral administration of MMC (1, 3 and 5 g/kg, p.o.) or BUPR (20 mg/kg, p.o.) was given 60 min prior to CPP postconditioning testing or extinction testing or reinstatement testing in mice. Extinction trials were conducted in Meth-free state to weaken CPP over the next 5 days. Reinstatement test was conducted by a single low dose priming injection of Meth (0.1 mg/kg, i.p.). The present study, however, failed to establish a successful extinction and reinstatement of Meth-CPP in mice. Further studies using other doses of Meth are warranted for a successful establishment of all phases of Meth CPP in mice. This study also demonstrates that MMC (3 and 5 g/kg, p.o.) and BUPR (20 mg/kg, p.o.) could attenuate the expression of Meth-induced CPP in mice.
Mouse macrophages pre-labeled with [3H]arachidonic acid (20:4) were shown to release metabolites generated by the lipoxygenase and cyclo-oxygenase pathways following in vitro addition of heat-killed Salmonella typhi. These metabolites were maximally released after 60-90 min of incubation and consisted of prostaglandins (85%), leukotriene C (6%), di-HETEs, leukotrienes D and E (4%), mono-HETEs (2%) and other metabolites (3%). Of the metabolites generated by the cyclo-oxygenase pathway (prostaglandins), 6-keto PGF1 alpha and PGE2 were generated at a ratio of 1.2 to 1. The significance and importance of these results are discussed.
This study investigated the antinociceptive effects of zerumbone in chemical behavioural models of nociception in mice. Zerumbone given through intraperitoneal route (i.p.) produced dose-related antinociception when assessed on acetic acid-induced abdominal writhing test in mice. In addition, the i.p. administration of zerumbone exhibited significant inhibition of the neurogenic pain induced by intraplantar (i.pl.) injection of capsaicin and bradykinin. Likewise, zerumbone given by i.p. route reduced the nociception produced by i.pl. injection of glutamate and phorbol myristate acetate (PMA). The antinociception caused by zerumbone in the acetic acid test was significantly attenuated by i.p. pre-treatment of mice with l-arginine (nitric oxide precursor) and glibenclamide (ATP-sensitive K(+) channel inhibitor). However, the antinociception of zerumbone was enhanced by methylene blue (non-specific gyanylyl cyclase inhibitor). Together, these results indicate that zerumbone produces pronounced antinociception against chemical models of nociception in mice. It also strongly suggests that the l-arginine-nitric oxide-cGMP-PKC-K(+) ATP channel pathways, the TRPV1 and kinin B2 receptors play an important role in the zerumbone-induced antinociception.
There are no definite reports regarding the effects of chronic fluoxetine on animal models of epilepsy. Since chronically administered fluoxetine, in comparison to acutely administered fluoxetine has different effects on CNS, the present study was undertaken to investigate the effect of acute and chronic fluoxetine pretreatment, on a median anticonvulsant dose (ED50) of phenytoin in male ICR albino mice. Additionally, the effects of fluoxetine pretreatment on median convulsive current (CC50) in the presence and absence of phenytoin were investigated and results were compared. The maximal electroshock seizure (MES) test was used to estimate the ED50of phenytoin. The electroshock threshold test was used to estimate CC50. ED50and CC50values were calculated by probit analysis. The effects of the chronic and acute fluoxetine groups on the ED50of phenytoin were significantly different (P<0.05), and on CC50this difference was not statistically significant. Chronic fluoxetine insignificantly increased the ED50of phenytoin and decreased the CC50while acute fluoxetine decreased the ED50of phenytoin and increased the CC50. Our results indicate that chronic fluoxetine does not have an antiepileptic property and it may have dubious proconvulsant properties, contrary to acute fluoxetine.
Ardisia crispa (Family: Myrsinaceae) has been used as a traditional medicine for various ailments. Previous studies showed that Ardisia crispa possesses antimetastatic and anti-inflammatory properties. Nevertheless, research done on the plant is still limited. Therefore, the present study was designed to evaluate the suppression effect of Ardisia crispa root hexane (ACRH) extract on 7, 12-dimethylbenz (α) anthracene (DMBA)-induced mice skin tumor promotion in ICR mice with topical application twice weekly for 10 weeks. Results showed significant difference between treatment groups (mice treated with 30 mg/kg, 100 mg/kg and 300 mg/kg of ACRH extract; denoted as group I, II and III respectively) for tumor incidence and tumor burden (P<0.05). Significant reduction in tumor incidence (20%), tumor burden (1.5 ± 0.50), tumor volume (2.49 ± 1.70) and delayed latency period of tumor formation was observed in group I (30 mg/kg) in comparison to carcinogen control. This study indicates that ACRH extract could be a promising skin tumor promotion suppressing agent at a lower dosage (30 mg/kg). Further studies are required to elucidate the underlying mechanism(s) leading to this effect.
Chikungunya virus (CHIKV) outbreaks have led to a serious economic burden, as the available treatment strategies can only alleviate disease symptoms, and no effective therapeutics or vaccines are currently available for human use. Here, we report the use of a new cost-effective approach involving production of a recombinant antiviral peptide-fusion protein that is scalable for the treatment of CHIKV infection. A peptide-fusion recombinant protein LATA-PAP1-THAN that was generated by joining Latarcin (LATA) peptide with the N-terminus of the PAP1 antiviral protein, and the Thanatin (THAN) peptide to the C-terminus, was produced in Escherichia coli as inclusion bodies. The antiviral LATA-PAP1-THAN protein showed 89.0% reduction of viral plaque formation compared with PAP1 (46.0%), LATA (67.0%) or THAN (79.3%) peptides alone. The LATA-PAP1-THAN protein reduced the viral RNA load that was 0.89-fold compared with the untreated control cells. We also showed that PAP1 resulted in 0.44-fold reduction, and THAN and LATA resulting in 0.78-fold and 0.73-fold reductions, respectively. The LATA-PAP1-THAN protein inhibited CHIKV replication in the Vero cells at an EC50 of 11.2μg/ml, which is approximately half of the EC50 of PAP1 (23.7μg/ml) and protected the CHIKV-infected mice at the dose of 0.75mg/ml. We concluded that production of antiviral peptide-fusion protein in E. coli as inclusion bodies could accentuate antiviral activities, enhance cellular internalisation, and could reduce product toxicity to host cells and is scalable to epidemic response quantities.
Lack of vaccine and effective antiviral drugs against chikungunya virus (CHIKV) outbreaks have led to significant impact on health care in the developing world. Here, we evaluated the antiviral effects of tetracycline (TETRA) derivatives and other common antiviral agents against CHIKV. Our results showed that within the TETRA derivatives group, Doxycycline (DOXY) exhibited the highest inhibitory effect against CHIKV replication in Vero cells. On the other hand, in the antiviral group Ribavirin (RIBA) showed higher inhibitory effects against CHIKV replication compared to Aciclovir (ACIC). Interestingly, RIBA inhibitory effects were also higher than all but DOXY within the TETRA derivatives group. Docking studies of DOXY to viral cysteine protease and E2 envelope protein showed non-competitive interaction with docking energy of -6.6±0.1 and -6.4±0.1 kcal/mol respectively. The 50% effective concentration (EC50) of DOXY and RIBA was determined to be 10.95±2.12 μM and 15.51±1.62 μM respectively, while DOXY+RIBA (1:1 combination) showed an EC50 of 4.52±1.42 μM. When compared, DOXY showed higher inhibition of viral infectivity and entry than RIBA. In contrast however, RIBA showed higher inhibition against viral replication in target cells compared to DOXY. Assays using mice as animal models revealed that DOXY+RIBA effectively inhibited CHIKV replication and attenuated its infectivity in vivo. Further experimental and clinical studies are warranted to investigate their potential application for clinical intervention of CHIKV disease.