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INTERNATIONAL JOURNAL OF SCIENTIFIC & TECHNOLOGY RESEARCH VOLUME 2, ISSUE 10, OCTOBER 2013 The Effect Of Combination Of Octadecanoic Acid, Methyl Ester And Ribavirin Against Measles Virus Reagan Entigu Ak Linton @ Jerah, Samuel Lihan, Ismail bin Ahmad Abstract: Ribavirin is a broad spectrum antiviral drug and has been used to treat various diseases. It has been used as a treatment for subacute
sclerosis panencephalitis (SSPE) caused by measles virus infection. However, there were several adverse effects when receiving ribavirin treatment.
Other than ribavirin and vaccine, there is no cure for the disease thus medicinal plants being studied for their potential active compounds to be used as
either mono or combined treatment with drugs. The objective of this study was to test antiviral activity of octadecanoic acid, methyl ester (OA), extracted
from Cymbopogon nardus (sweet lemon grass) against measles virus in both mono and combination with ribavirin. The cytotoxicity and antiviral activity
were tested at low concentration for the compound (25, 12.5 and 2.5 µg/ml) and ribavirin (0.1, 0.05 and 0.01 CC50). The cytotoxicity result showed that
the low concentrations of both compounds have low cytotoxicity on Vero cell although there was slight increment of toxicity when they were combined.
However, the combined treatment showed higher antiviral activity (p <0.05) compared to single treatment of both compounds (OA12.5 µg/ml +
RBV0.05CC50: 94.38 ± 1.5%, OA12.5 µg/ml: 67.09 ± 0.2%, RBV0.05CC50: 51.12 ± 2.1%). The result has also shown that decreasing of the
concentration of the combination could still maintained the antiviral activity comparable to single treatment and less cytotoxicity toward Vero cell. This
study has proven that OA can be combined with commercial drug such as ribavirin to produce higher antiviral activity at lower concentration for
combination of both compounds.

Index terms:
Antiviral, Cymbopogon nardus, cytotoxicity, measles, octadecanoic acid, methyl ester, ribavirin
1 Introduction

Although ribavirin showed antiviral activity against HCV, Stearic acid, methyl ester or stearate is a saturated 19 there was adverse effect to recipients. SARS patient that carbon-chained compound and it is also known as was treated with ribavirin developed hypoxemia, a state of octadecanoic acid methyl ester (OA). There are various low healthy hemoglobin to carry oxygen [9]. The advantage antiviral activities from fatty acids against viruses. Fatty acid of synergistic effect between drugs in combination therapy was able to inhibit the replication of HCV and synergistic is the additive effect or diminished resistance of viral effect with IFN-α was observed by Leu et al. [1]. Measles infection. However, multiple therapies may incur higher virus (MV) belongs to paramyxoviridae family along with the cost, increase in toxicity and could lead to treatment failure parainfluenza, mumps, Newcastle disease virus and compared to monotherapy using single drug [10]. several other viruses. The MV replicates first in the Combination of drug with herbs may increase or decrease respiratory tract and moved to lymphoid tissue for further the activity of either component [11] and the combination viral processes [2]. The MV causes high death rates could also cause various adverse effects such as bleeding annually and has claimed as much as 13 million lives of when patient mixed warfarin with Ginkgo biloba and mild children less than 6 years of age [3]. Although introduction serotonin syndrome when serotonin-reuptake inhibitors of vaccine has been for 50 years, the disease is still could mixed with Herpericum perforatum [12]. not be eradicated due to difficulty of vaccine distribution especially in rural or undeveloped areas such as Africa [4]. 2 Materials and Methods
Ribavirin is an antiviral drug used to treat respiratory syncytial virus, RSV and strains of influenza A and B [5]. It 2.1 Octadecanoic acid, methyl ester preparation
has also shown antiviral activity against RNA viruses such The octadecanoic acid, methyl ester (OA) was isolated from as influenza A and B, measles and parainfluenza [6]. The ribavirin has the ability to incorporate itself into viral RNA chromatography mass spectrometry, GCMS. It was and the reaction caused mutation that interferes with the prepared at different concentrations (25, 12.5 and 2.5 normal viral replication processes [7],[8]. µg/ml) by diluting in Dulbecco‘s Modified Eagle Medium, DMEM. The control drug, ribavirin was also prepared at 0.1, 2.2 Cell culture
The Vero cells were cultured and maintained in T-25 flask with HyClone DMEM/ low glucose 5% FBS (HyClone) added with penicillin streptomycin (AMRESCO tissue  Reagan Entigu Ak Linton @ Jerah, postgraduate culture grade). The Vero cells were subcultured once it student Virology laboratory, Department of Molecular reached 80-90% confluent. The Vero cells were also given geneticin to maintain the Signaling Lymphocyte Activating Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia. 2.3 Vaccine
Samuel Lihan, Senior Lecturer of Department of The vaccine used was the live attenuated Edmonston Molecular Biology, Faculty of Resource Science and Strain of the MMR vaccine (Serum Institute of India Ltd.). Technology, Universiti Malaysia Sarawak, 94300 Kota INTERNATIONAL JOURNAL OF SCIENTIFIC & TECHNOLOGY RESEARCH VOLUME 2, ISSUE 10, OCTOBER 2013 2.4 Antiviral assay
The Vero cells with the cell counts of 1.0 x 105 cells/ml
were plated onto the 96-well plate with DMEM 2% FBS and
incubated overnight until the cells are confluent. The
medium were discarded and the plates were washed with
DMEM twice and 10 µl of the measles vaccine was added
into the designated wells of the plate and incubated for 30
minutes to allow the adsorption of virus to the cells. Then,
100 µl of groups of ribavirin and octadecanoic acid, methyl
ester at different concentrations were added separately and
combined to each well and left in the 37 ºC 5% CO2
incubator for 48 hours.
2.5 Plate processing
After 48 hours, the medium were discarded and the cells Fig. 1: Cytotoxicity of octadecanoic acid, methyl ester
were fixed with 125 µl of cold TCA for each well which then (OA) and ribavirin (RBV) against Vero cell. The graph incubated for 1 hour at 40 ºC. The solution were removed showed the percentage of cell viability after different and washed 5 times with distilled water and left in 40 ºC for 1 hour. About 100 µl of eosin were added and left for 1 hour at room temperature. The plates were washed 5 times with 300 µl of acetic acid to wash the excess dye and left for 1 In the individual treatment, both compounds showed low day at room temperature. About 200 µl of 5mM NaOH were cytoxicity. The ribavirin had cytotoxicity ranging from 90.03 added and left for 20 minutes at room temperature. The ± 1.1% to 96.88 ± 0.3% cell viability while octadecanoic optical density, OD readings were taken with the Elisa acid, methyl ester had cytotoxicity ranging from 91.01 ± reader (Microplate reader Metertech Inc.) at 490nm 1.6% to 98.22 ± 1.7% cell viability. There was a slight increase in cytotoxicity when 0.05CC50 ribavirin combined with 12.5µg/ml octadecanoic acid, methyl ester compared 2.6 Statistical assay
to individual treatment (combined: 88.59 ± 1.5%, RBV Each treatment of fractions/subfractions was carried out in 0.05CC50: 92.11 ± 2.1%, OA 12.5 µg/ml: 96.53 ± 2.6%). 5 replicates. The data were analyzed with one-way ANOVA However the Vero cell viability increased slightly than test to compare between groups of treatment and Student individual treatment as the combined concentration became T-test between the highest activity of treated group with lower (combined: 98.60 ± 0.5%, RBV 0.01CC50: 96.88 ± control group. The criteria for statistical significance were
3 Results
The combination of octadecanoic acid, methyl ester (OA)
and ribavirin (RBV) would determine whether the
combination treatment could exhibit higher antiviral activity.
It was also vital that the combination used lower
cytotoxitcity and lower concentrations compared to
individual treatment. In this study, both compounds
(octadecanoic acid, methyl ester and ribavirin) were tested
for their cytotoxicity against Vero cell prior to antiviral assay
both individual and combined treatment (Fig. 1).
Fig. 2: Antiviral activity of octadecanoic acid, methyl
ester and ribavirin against measles disease virus. The graph was based on the viability of treated infected-Vero In this study, the inhibition of ribavirin ranged from 68.67 ± 0.4% to 40.59 ± 1.9% while the octadecanoic acid, methyl ester had inhibition activity ranging from 82.33 ± 1.0% to 58.79 ± 2.4% (Fig. 2). When OA combined with RBV at concentration of 12.5 µg/ml and 0.05CC50 respectively, there was an increase of inhibition activity observed. The individual treatment had lower activity (OA 12.5 µg/ml: 67.09 ± 0.2% and RBV 0.05CC50: 51.12 ± 2.1%) compared to combined treatment of the same concentration, 94.38 ± 1.5%. The combined treatment had higher (p <0.05) inhibition activity even when compared to individual treatments at double the concentrations (OA 25 µg/ml: INTERNATIONAL JOURNAL OF SCIENTIFIC & TECHNOLOGY RESEARCH VOLUME 2, ISSUE 10, OCTOBER 2013 82.33 ± 1.0% while RBV 0.1CC50: 68.67 ± 0.4%). By and cancer cells together with testing for other bioactivities alternating the concentration of ribavirin and octadecanoic such antimicrobial, antioxidant and antifungus. OA might acid, methyl ester, there was an influenced on the inhibition possess various bioactivities that are yet to be fully studied activity. When the OA concentration was decreased from and could also provide based-structure for other derivatives 12.5 to 2.5 µg/ml while maintaining RBV concentration at 0.05CC50, there was 16.06% decrease of activity. Whereas when the RBV concentration was decreased from 0.05 to Acknowledgement
0.01CC50 with fixed concentration of OA at 12.5 µg/ml, The research was funded under the grant E-science Vot no: 4 Discussions
In this study, the octadecanoic acid, methyl ester (OA) and G.Z. Leu, T.Y. Lin, and J.T.A. Hsu, ―Anti-HCV ribavirin (RBV) have shown their antiviral activity against activities of selective polyunsaturated fatty acids,‖ measles disease virus in both combined and separate treatment. Before the antiviral assay was conducted, it is vital to test the cytotoxicity of both compounds. Both compounds showed very low cytotoxicity against the Vero J.D. Kettering, ―Virology,‖ Essentials of Diagnostic cell line with more than 90% viability for all treatments. The Microbiology, L. A. Shimeld, ed., USA: Delmar cytotoxicity assay is important to determine the toxic level of tested compounds that causes cell death via necrosis or apoptosis [13]. The compound to be tested should be able WHO, ―Measles,‖ From World Health Organization to induce antiviral activity at low cytotoxicity toward cells because without carrying out cytotoxicity assay, the result of antiviral activity might not be important though it was tested at low concentration [14]. This would relate to the cytotoxicity of combined OA and RBV in this study that has Ministry of Health Malaysia, ―Measles prevention showed slight increment but able to exert high antiviral and control in Malaysia,‖ Handbook for healthcare activity. The concentration of RBV could be manipulated by lowering it further to decrease the cytotoxicity while maintaining concentration of OA to produce high antiviral e%20disease%20unit/Publications,IEC/MEASLES.
activity in combined treatment. Although this study used measles attenuated vaccine, it could still carry out viral processes that caused cell to lyse and cytophatic effect S.K. Trying, ―Antiviral Agents, Vaccines and such as multinucleated cells [2] as the effect could be Immunotherapies,‖ New York: Marcel Dekker, observed during the study. The antiviral results were based on the viability of treated (with OA and RBV) measles infected Vero cells in comparison to the control, measles R.T. D' Aquila, ―Antiviral treatment strategies,‖ infected Vero cells. The combination treatment of OA and Schaechter's Mechanisms of Microbial Disease, RBV in this study showed a promising enhanced antiviral 4th ed., M. Schaechter, N. C. Engleberg, V. J. activity of both compounds by inhibiting the viral processes DiRita, and T. Dermody, eds., Philadelphia: that leads to cell lysis. The combination of both compounds Lippincott Williams & Wilkins, pp. 435-444, 2007. showed higher antiviral activity compared to their separate treatment at double concentration. The control drug, J.D. Graci, and C.E. Cameron, ―Quasispecies, ribavirin has been known as a broad spectrum antiviral drug error catastrophe and the antiviral activity of ribavirin,‖ Virology (298), 175-180, 2002. dehydrogenase that reduces GTP hence restricting replication processes [6]. It has been used in combination N.M. Dixit, J.E. Layden-Almer, T.J. Layden, and with IFN-α for treatment against subacute sclerosis A.S. Perelson, ―Modelling how ribavirin improves panencephalitis (SSPE) virus infection in hamsters and interferon response rate in hepatitis C virus showed higher viral inhibition than individually while no infection,‖ Nature , 432 (7019), 922–924, 2004. significant toxicity increase when combined [15]. OA itself has been shown in a previous study by Reagan et al. [16] H. Chiou, C. Liu, M.J. Buttrey, H. Ku, H. Liu, H. and found to possessed antiviral activity against measles Kou, and Y. Lu, ―Adverse effects of ribavirin and disease. OA is classified as fatty acid methyl ester, FAMEs outcome in severe acute respiratory syndrome: and this group has been shown to have antimicrobial [17] experience in two medical centers,‖ CHEST and potential anticancer [18] properties. 5 Conclusion and recommendation
[10]. G.D. Schiff, W.L. Galanter, J. Duhig, M.J. The outcome of this preliminary study showed that Koronkowski, and A.E. Lodolce, ―A prescription for octadecanoic acid, methyl ester (OA) possessed important improving drug formulary decision making,‖ Public antiviral properties and it can be combined with drug such Library of Science Medicine, 9(5): e1001220, 2012. as ribavirin to exert higher activity. Further study is needed on cytotoxicity against different cells such as human normal INTERNATIONAL JOURNAL OF SCIENTIFIC & TECHNOLOGY RESEARCH VOLUME 2, ISSUE 10, OCTOBER 2013 MadhusudhanChetty, ―A review on herb-drug interactions,‖ Pharmaceutical Research and Development, 3(3), Article 17, 2013. [13]. M.B. Bolger, R. Fraczkiewicz, M. Entzeroth, and B. Steere, ―Concepts for in-vitro profiling: Drug activity, selectivity and liability,‖ Exploiting Chemical Diversity for Drug Discovery, P. A. Barlett, and M. Entzeroth, eds., The Royal Society of Chemistry, pp. 336-360, 2006. [14]. A.J. Vlientinck, T. De Bruyne, and D.A. Berghe, ―Plant substances as antiviral agents,‖ Current Organic Chemistry, 1 (4), 307-344, 1997. [15]. T. Takahashi, M. Hosoya, K. Kimura, K. Ohno, S. Mori, K. Takahashi, and S. Shigeta, ‗‘The cooperative effect of interferon-alpha and ribavirin on subacute sclerosing panencephalitis (SSPE) virus infections, in-vitro and in-vivo,‖ Antiviral Research, 37(1), 29–35, 1998. [16]. Reagan, E., Samuel, L. & Ahmad, I. B., ―Isolation of antiviral compound from Cymbopogon nardus methanolic fractions,‖ International Journal of Health and Pharmaceutical Sciences, 2(2), 1-7. [17]. M. Chandrasekaran, A. Senthilkumar, and V. Venkatesalu, ―Antibacterial and antifungal efficacy of fatty acid methyl esters from the leaves of Sesuvium portulacastrum L.,‖ European Review or Medical and Pharmacological Sciences, 15(7), 775–80, 2011. [18]. F.R. Yu, X.Z. Lian, H.Y. Guo, P.M. McGuire, R.D. Li, R. Wang, and F.H. Yu, ―Isolation and characterization of methyl esters and derivatives from Euphorbia kansui (Euphorbiaceae) and their inhibitory effects on the human SGC-7901 cells,‖ Journal of Pharmacy & Pharmaceutical Sciences: a publication Pharmaceutical Sciences, Société canadienne des Sciences Pharmaceutiques, 8(3), 528–35, 2005.


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