Some Physical And Chemical Properties Of The Bioactive Components Responsible For Antinociceptive Activity Of Haruan (Channa Striatus) Extracts

Haruan (Channa striatus) fillet and mucus extracts have been proven to exhibit a dosedependent antinociceptive activity and enhanced morphine effect. This current study is to establish some of the physical and chemical properties of the bioactive components responsible for haruan antinociceptive activity. Five types of solvents (water, methanol, ethanol, chloroform and chloroform:methanol) were used to extract the respective components from freeze-dried haruan fillet. Various salts (NaCI, K Cl, CaCh, MgCh and their combinations (MS» were used to study the involvement of ionic components in mediating the antinociceptive activity of haruan extract. The aqueous portion obtained after a chloroform:methanol extraction of fresh haruan fillet was filtered using three types of Millipore filters (30,000, 10,000 and 5,000 Nominal Molecular Weight Limit (NMWL), respectively. Lastly, the aqueous portion, filtered using the 5,000 NMWL Millipore filters, was purified using the Hig

Possible Antinociceptive Mechanism and Site of Activity of Haruan (Channa Striatus) Crude Aqueous Extract in Mice

The present study was carried out to determine the possible mechanism of antinociception and site of activity of the crude aqueous extract of Haruan (Channa striatus) (ASH) in mice using the abdominal constriction test. The ASH, obtained after chloroform:methanol (CM) (2:l; vlv) extraction (24 hrs) of the fresh Haruan fillet, was evaporated to remove the methanol residue and used throughout the study. The first study was carried out to ascertain the dry weight and antinociceptive profile of ASH. The second study was carried out to determine the amino acids and fatty acids compositions, as well as the polypeptide profile of ASH. The third study was carried out to determine the actual onset and offset of ASH activity after its subcutaneous (SC) or intraperitoneal (IP) administration at four different sets of time (0, 5, 30 and 60 min). The fourth and fifth studies were carried out to determine the involvement of opioid and non-opioid receptors, respectively, in the ASH antinociceptive activity. All of the antagonists of opioidergic, muscarinic, nicotinic, a- and P-adrenergic, dopaminergic, serotonergic and y-aminobutyric acid (GABA) receptors were administered (SC) 10 min prior to ASH (SC) administration. The sixth study was carried out to determine the role of L argininelnitric oxide/cyclic 3'5'-guanosine monophosphate (L-arginine/NO/cGMP) pathway in the ASH antinociceptive activity. The precursor (L-arginine) and inhibitor @IG-nitro-L-arginine methyl esters (L-NAME)) for NO, as well as the inhibitor for cGMP (methylene blue (MB), were administered (SC) 5 min before ASH administration (SC). In all of the above-mentioned studies that involved the use of antinociceptive test, the 0.6% acetic acid-induced abdominal constriction test in mice was used as an assay to evaluate the ASH antinociceptive activity. All data obtained were analysed using the One-way Analysis of Variance (ANOVA) followed by the Tukey test with P<0.05 as the limit of significance. From the data obtained, the ASH, which exhibited significant (P<0.05) and concentration/dosage-dependent antinociceptive activity, yielded 1.89g/10.0ml of white coloured powder after subjection to the freeze-drying process. The ASH was also found to contain all the important amino acids with major amino acids found are glycine (35.77% -+ 0.58), alanine (10.19% + 1.27), lysine (9.44 + 0.56), aspartic acid (8.53 -+ 1.15) and proline (6.86% -+ 0.78). Furthermore, the ASH was also found to contain high composition of palmitic acid (C16:O) (35.93% * 0.63), oleic acid (C18: 1) (22.96% A 0.40), stearic acid (C18:O) (15.31% * 0.33), linoleic acid (C18:2) (1 1.45% * 0.3 1) and arachidonic acid (C20:4) (7.44% rt 0.83). The ASH was also found to produce at least four major fractions (at the retention times of 8.919, 9.841, 10.263 and 10.744), when subjected to the high performance liquid chromatography (HPLC) process, that are believed to be polypeptides. The onset time and the offset time of the ASH antinociceptive activity, which are concentration-dependent and concentration independent, occurred between 0 to 5 min, and 60 min after its SC administration. Interestingly, changing the route of administration from SC to IP caused significant (Pc0.05) increase in the ASH antinociceptive activity with the concentration-independent onset time of activity observed immediately after the ASH administration with no apparent offset time. The activity was found to reach the maximum effect 30 min after the ASH administration regardless of the route of administration used. Pretreatment with naloxone at all dosages did not cause any significant changes in the ASH antinociceptive activity indicating that the activity did not involve an opioid receptor mechanism, and thus confirmed the report made by Dambisya et al. (1999). Re-treatment with various types of non-opioid receptor antagonists demonstrated the involvement of at least four =of receptors (muscarinic, GABA*, a-adrenergie d s e r o t o ~ g k ) in the mechanism of ASH antinociceptive activity. Pre-treatment with atropine and bicuculine almost completely blocked (P<0.05), while pre-treatment with phenoxybenzamine and methysergide significantly (Pc0.05) reduced half of the ASH activity. The role of Larginine/ NO/cGMP pathway in ASH antinociceptive activity was also observed after pretreatment of the ASH with L-arginine, L-NAME or MB, but not with D-arginine. Pretreatment with L-arginine was found to significantly (PC0.05) reduce the ASH antinociceptive activity, whereas pretreatment with L-NAME or MB were found to enhance (Pc0.05) the activity. Based on the finding, low concentration of NO, limited by the presence of higher concentration of ASH, and inhibition of cGMP system play important role in ASH antinociceptive activity. However, the actual mechanism underlying this phenomenon is yet to be fully understood.As a conclusion, we suggest that the ASH-produced antinociceptive activity could be due to the presence of various types of amino acids and fatty acids, as well as four major fractions, and involved activation of at least four types of the non-opioid receptors (namely the muscarinic, GABA*, a-adrenergic and serotonergic) and the Larginine/ NO/cGMP pathway

Anticarcinogenic activity of Labisia pumila against 7, 12- dimethylbenz (a) anthracene (DMBA)/croton oil-induced mouse skin carcinogenesis.

Labisia pumila or locally known as Kacip Fatimah, is one of the most popular medicinal herb in Malaysia. Anticarcinogenic activity of this medicinal herb however, has not been reported until today. In this paper, the in vivo anticarcinogenic activity of L. pumilaethanol extract on two-stage mouse skin carcinogenesis model is reported. In the present study, we investigated whether L. pumila ethanol extract have an effect on tumor growth in vivo. Therefore, varying doses (25, 50 and 100 mg/kg bwt) of L. pumilaethanol extract were tested on 7, 12-dimethylbenz(a)anthracene (DMBA)/croton oil-induced mouse skin carcinogenesis. At the end of the experiment of 20 weeks, animals in carcinogen control group developed a mean number of 5.70 ± 1.3 skin tumors per tumor-bearing mouse and on the 16th week prompted a tumor incidence of 100%. Animals that have been treated with 25, 50 and 100 mg/kg bwt of L. pumila extract topically for 30 min developed a mean number of 3.60 ± 1.1, 3.20 ± 0.8 and 2.40 ± 0.7 skin tumors per tumor-bearing mouse with tumor incidence of 90, 60 and 50%, respectively. The tumor volume per tumor-bearing mice of carcinogen control animals was 121.03 ± 3.46 mm3, which was significantly (p<0.05) reduced to 92.27 ± 2.68, 69.24 ± 3.93 and 54.24 ± 4.38 mm3 for the animals treated with 25, 50 and 100 mg/kg bwt of L. pumila extract, respectively. In terms of tumor incidence and tumor burden, the highest dose (100 mg/kg bwt) of L. pumila ethanol extract was almost equipotent with curcumin (10 mg/kg bwt). The extract of L. pumila not only decreased the tumor incidence, tumor burden and tumor volume in DMBA/croton oil-induced mice but also delayed the skin tumor growth as compared to carcinogen control group. Further histopathological examination revealed that tumors from animals that have been treated with L. pumila showed intact basement membrane as compared to the tumors from the untreated animals. This finding suggested that L. pumila extract was able to suppress the progression of benign tumors to malignant stage in DMBA/croton oil-induced mice. Further studies should be carried out in order to identify the active compound responsible for the anticarcinogenic activities and the mechanism of action of L. pumilaat the molecular level

Antiproliferative and Proapoptotic Effects of Labisia pumila Ethanol Extract and Its Active Fraction in Human Melanoma HM3KO Cells

The present study was to determine the anticancer potential of Labisia pumila in in vitro models. Results from the study revealed that ethanol extract of L. pumila was more cytotoxic against HM3KO cells while having reduced effects on nonmalignant cells as compared to aqueous and hexane extracts. Thus, ethanol extract was selected to be further separated by using the bioassay-guided fractionation method to give an active fraction, SF2Lp. Results obtained from the flow cytometry analysis showed that SF2Lp was able to arrest the HM3KO cell cycle at the G1 phase, while morphological findings from AO-EB nuclear staining assays along with the Apoptotic Index confirmed the induction of apoptosis by SF2Lp in HM3KO cells. Results from the mechanistic study further revealed that SF2Lp treatment was able to concurrently increase the expression level of p53 and pro-apoptotic protein Bax and also reduce the expression level of anti-apoptotic protein BCl-2 in HM3KO cells, directly contributing to the increase in Bax/Bcl-2 ratio. These findings, therefore, suggested that L. pumila was able to inhibit HM3KO cell growth possibly by arresting the cell cycle at G1 phase and inducing apoptosis in HM3KO cells via the up- and down-regulation of Bax/Bcl-2 protein, mediated through a p53-dependent pathway

Antioxidant and anti-proliferative activities of Roselle juice on Caov-3, MCF-7, MDA-MB-231 and HeLa cancer cell lines.

Roselle (Hibiscus sabdariffa Linn) extract has been scientifically proven to possess high antioxidant activity, anti-proliferation and anti-carcinogenic properties. This study was conducted to evaluate the antioxidative capacity of commercialized Roselle juice (RJ) at three storage periods and its anti-proliferative effect on breast (MCF-7 and MDA-MB-231), ovarian (Caov-3) and cervical (HeLa) cancer cell lines. The antioxidant activity of 1 week (WRJ), 1 month (MRJ) and 1 year (YRJ) juice samples each at 0.001 to 10% concentration range were determined through 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay with L-ascorbic acid as positive control. EC50 values of WRJ, MRJ, and YRJ were found to be 3.733±0.247, 3.717±0.637 and 3.383±0.711%, respectively. These values were compared to 0.217±0.616% for positive control. The difference in antioxidant activity between different storage periods of RJ was not significant (p>0.05) but all samples exhibited increasing activity with increasing concentrations. RJ at the same concentrations were tested using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay on the four cell lines to obtain the percentage viability of the cells. The cells were incubated for 72 h after inoculation with RJ and the control group was without treatment. The IC50 was found to be highest for Caov-3 cells (2.267±1.193%) whereas MCF-7 cells exhibited the lowest (0.432±0.278%) IC50 value after treatment with MRJ. All determinations were analyzed using ANOVA through SPSS with p0.05). The study showed that commercialized Roselle juice has strong antioxidant capacity and anti-proliferative activity on the four cancer cell lines despite different storage periods. However, further study should be conducted to establish its anti-cancer mechanisms

Single nucleotide polymorphism (SNPS) analysis of Mu-opioid receptors (OPRM1) using denaturing high performance liquid chromatography (DHPLC) among the intravenous drug users

Objectives: The genetic polymorphisms of OPRM1 among the intravenous drug users (IVDUs) and healthy controls were investigated and the risk of addiction in relation to OPRM1 was predicted. Methods: PCR-denaturing high performance liquid chromatography (DHPLC) method was developed to investigate SNPs in the coding regions of OPRM1 in 93 IVDUs and 100 healthy controls. Subjects were confirmed to be drug addicts and their personality was studied using validated Tridimensional Personality Questionnaires (TPQ). Results: Based on the results obtained, seven SNPs were detected; two of them were previously associated with addiction. Homozygous OPRM1:c.118GG and heterozygous OPRM1:c.118AG variants were found to have higher frequencies among the IVDUs and healthy controls. In addition, carriers of OPRM1:c.118G allele scored higher for novelty seeking (NS) and harm avoidance (HA) with more explorative, neurotic and uninhibited personalities. We identified a new variant of OPRM1:c.77C>G which is located at the N-terminus of the G-coupled protein receptor and possibly decreases the binding affinity of its ligands among the IVDUs. Conclusion: In conclusion, DHPLC allows the detection of new and existing variants of OPRM1. Genotyping of OPRM1:c.118A>G and assessment of personalities using TPQ provide valuable tools for determination of addiction risk

Effects of Cytochrome P450 Inhibitors on Itraconazole and Fluconazole Induced Cytotoxicity in Hepatocytes

Itraconazole and fluconazole have been reported to induce hepatotoxicity in patients. The present study was designed to investigate the role of cytochrome P450 inhibitors, SKF 525A, and curcumin pretreatment on the cytotoxicity of antifungal drugs fluconazole and itraconazole. For 3 consecutive days, female rats were administered daily SKF 525A or curcumin (5 and 25 mg/kg). Control rats received an equivalent amount of dosed vehicle. The animals were anaesthetized 24 hours after receiving the last dose for liver perfusion. Hepatocytes were then exposed to various concentrations of antifungal drugs. In vitro incubation of hepatocytes with itraconazole revealed significantly lower viability when compared to fluconazole as assessed by lactate dehydrogenase, aspartate aminotransferase and alanine aminotransferase activities. The cytotoxicity of itraconazole was enhanced when incubated with hepatocytes pretreated with SKF 525A. SKF 525A had no effects on the cytotoxicity of fluconazole. Curcumin failed to either increase or decrease the cytotoxicity of both antifungal drugs. ATP levels also showed significant decrease in both itraconazole and fluconazole incubated hepatocytes. However, SKF 525A pretreated hepatocytes had significantly lower ATP levels after itraconazole incubations. Collectively, these results confirm the involvement of cytochrome P450 in the cytoprotection in itraconazole induced hepatocyte toxicity. Differences of the effects of SKF 525A on the cytotoxicity induced by itraconazole and fluconazole may be due to the differences on the metabolism of each antifungal drug in vivo

In vitro characterization and in vivo performance of mefenamic acid-sodium diethyldithiocarbamate based liposomes

Mefenamic acid (MFA) is a hydrophobic drug with low dissolution rate. This study aimed to develop stable and reproducible aqueous formulations of MFA using liposomes as drug carriers. The drug entrapment, particles size and drug release profiles, and stability and reproducibility of the liposomes were determined. In addition, the maximum tolerated dose (MTD) was determined in rats via the oral and intraperitoneal routes of administration. Also, the anti-inflammatory efficacy of these liposomes was evaluated using carrageenan-induced paw edema model in rats. MFA-DDC based liposomes demonstrated a drug entrapment efficacy of 93.6%, particles size of 170.9 nm, and polydispersity index of 0.24 which were not statistically affected when stored in room and refrigerated temperatures for at least 4 weeks. The MTD of the intraperitoneally administrated MFA-loaded liposomes was 20 mg MFA/kg, whereas for those of oral administrations, it was up to 80 mg MFA/kg. Intraperitoneal dose (80 mg MFA/kg) of MFA-DDC liposomes induced extrapyramidal symptoms associated with significant elevation in serum potassium and muscle enzymes. Moreover, significant inhibition of paw edema was demonstrated by the oral and intraperitoneal routes. These findings suggest that MFA-DDC based liposomes are an effective formulation of MFA and recommend the use of bioequivalence assessments with commercial formulations

Comparison of conjugated linoleic acid and other fatty acid content of milk fat of mafriwal and jersey cows.

Special attention has been given to the milk Fatty Acids (FA) that have a beneficial effect for human health such as mono and poly unsaturated fatty acids in particularly the Conjugated Linoleic Acids (CLA). This study was undertaken to investigate the milk fat contents of CLA variables (CLA and CLA-desaturase index) and other FA composition of Mafriwal and Jersey cows under same feeding system. In addition, the relationship between these two CLA variables with milk production and milk fat percent was determined. All the cows were grazed on pasture and given 5.5 kg of concentrate per head daily. Milk FA composition was determined using gas chromatography after extraction of milk fat using modified Folch's method. The results showed a significant variation (p<0.05) in the FA contents of the two breeds. The cis-9, trans-II CLA and CLA-desaturase index in milk fat of Mafriwal were significantly higher (p<0.05) than that of Jersey cows. Mafriwal cows produced significantly (p<0.05) higher concentrations of C18:0, C18:1cis-9, C18:3 and C20:1 than that of Jersey, while Jersey cows produced significantly (p<0.05) higher concentrations of C12:0 and C14:0 than Mafriwal cows. Additionally, significant positive correlations were observed between CLA variables and milk production. This study indicates that the breed of cows has an effect on CLA and other FA composition of milk fat and Mafriwal cows produced significantly higher percentages of CLA than Jersey cows which would provide better benefits for human health. Furthermore, the milk fat content of CLA and CLA-desaturase index were positively related to the milk production

Comparative ultrastructural hepatic alterations induced by free and liposome-encapsulated mefenamic acid

Mefenamic acid (MFA) is used as an anti-inflammatory, antinociceptive, and antipyretic agent for treatment of a wide range of pathological disorders. While the uncertainty of its safety and the poor oral bioavailability constitute the major limiting factors of its medical use, considerable efforts including liposomal encapsulation are needed to achieve maximum therapeutic advantages. The current work was conducted to investigate the ultrastructural alterations in the liver induced by free MFA and its liposomal preparation. Female Sprague-Dawley rats were treated with daily oral doses of either free MFA or MFA entrapped in Tween 80 inoculated liposomes at the concentration of 80 mg/kg for 28 days. Ultrathin sections were prepared from biopsies taken from the liver of each member of all animals under study and subjected to examination by transmission electron microscopy. The liver of rats that were exposed to liposomal MFA showed more ultrastructural alterations than the rats treated with the free drug. While both groups of rats demonstrated sinusoidal dilatation, Kupffer cell hyperplasia, mitochondrial damage, and nuclear alterations, rats treated with liposome-encapsulated MFA induced an increase in the multiple lysosomes formation, hepatocytic steatosis, and apoptotic activity than free MFA-treated rats. The ultrastructural findings of the present study indicate that the use of liposomal MFA induces more hepatic damage than the use of free MFA