الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
There are some issues with the available antiviral drugs such as drug resistance; also toxicity could be a problem. So the trend was to use the herbal medicine as anti-viral reduce the side effects of antiviral drugs.
Moringa is a plant rich in chemicals and elements such as protein, iron, calcium, ascorbic acid vitamin A and antioxidant compounds such as carotenoids, flavonoids, alkaloids, vitamin E and phenolic compounds, so they are useful in medical and nutrition terms, and have recently been used as an antiviral.
Coxsackie B virus (COX-BV) and hepatitis A virus (HAV) are the most important foodborne viruses that cause many human diseases. Including insulin-dependent diabetes, myocarditis, meningitis, hepatitis. One of the most important characteristics of these viruses is that the human faeces infected with the virus contains 107 - 1011 virus parts and that the pathogenic dose of the infection usually less than 100 viral parts and that these viruses have high stability in the acid and is resistant to heat, drought, radiation, detergent and environmental stresses. Although these viruses are non-enveloped therefore these viruses are difficult to resist.
The aim of this study is to reach some of the substances that have a positive activity of viruses to be used as a treatment for some food-borne viruses and which cause human pathogenic diseases such as viruses COX-BV and HAV.
A COX-BV was previously isolated and defined by Seif, et al., (2016), as for HAV was obtained from Holding Company for Biological Product and Vaccines (VACSERA). Propagation of COX-BV and HAV viruses were done in HEP-2 and Vero cells, respectively. These are done by infecting the cells with the viruses in the special flask and then were incubated in the co2 incubator at 37 °C. The cytopathic effects (CPE) of the viruses were monitored daily and the cells were frozen and that wed to obtain the virus. A centrifuge was then carried out to exclude cells and the concentration of viruses was determined using End Point Assay technique according to Reed and Muench formula (1938). The viruses were then saved at a temperature of - 70 ° C.
The Moringa oleifera Lam fresh leaves were collected from the farm and greenhouse of the Department of Agricultural Microbiology, Faculty of Agriculture, Ain Shams University during November and December 2016. Then they were washed with running tap water and were dried at 40°C for 4hr and then were grinded. Different phytochemicals were extracted from moringa leaves grinded by four different organic solvents; Chloroform (CL), ethyl acetate (E.A), Methanol 80% (M) and n-butanol (n.b). Where, 100 g of the grinded Moringa leaves were soaked in 1 liter of organic solvents for one week. Then, the extracts were filtered by Whatmann filter paper and the extracts were concentrated by Rotary Evaporator at 45°C. and kept at 4°C. The residual was resuspended in Dimethylsulphoxide (DMSO).
The total phenol content of the four extracts was estimated by the in terms of gallic acid equivalence by Folin-Ciocalteau reagent reagent using the spectrophotometer at a wavelength of 765 nm. The Folin-Ciocalteau assay relies on the transfer of electrons in alkaline medium from phenolic compounds to phosphomolybdic/ phosphotungstic acid complexes,The results indicated that the highest concentration of phenol was obtained from the E.A extract followed by the extract of n-b.
The effect of Moringa leaf extracts with four solvents on COX-BV and HAV viruses were studied in vitro and in vivo.
Cytotoxicity of the four extracts were evaluated on two types of cells; HEp-2 cell and Vero cell using [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] (MTT) assay. This test is used to assess cell survival, and from this test is determined the Toxic conc. and non-toxic conc. of extracts on cells as well as determining the inhibitory concentration of 50 percent of the cells (IC50). MTT technique is a colorimetric method, it is based on the reduction of MTT, and the soluble yellow tetrazolim pigment is water-soluble in living cell mitochondria to the water-soluble violet formasan crystals that are dissolved with the organic solvent dimethyl sulfoxide (DMSO) in the living cell cytoplasm. The amount of formazan produced is directly proportional to the number of living cells, then quantified at a wavelength of 570 nm. Two fold dilutions of the four extracts were added to the wells of cell culture plate and added the cells to it, and then Plates were incubated in 37 oC, then MTT solution were added to each well, the developed crystals were completely dissolved using Dimethylsulphoxide (DMSO) and the absorbance was read on a spectrophotometer at 570 nm. The results showed that, the 390 µg / ml concentration were considerably the toxic concentration of the four extracts on HEP-2 and Vero cell and 97.7µg/ml were considerably safe (Non-toxic concentration) on the same cells. The highest IC50 values for the four extracts on HEp-2 cells were for n.b extract and the lowest values for IC50 were for CL extract. For Vero cells, the results were the opposite. We also observed that the cell viability increases as the concentration of extracts decreases.
The effect of the nontoxic concentration of the four extracts were tested for the Inhibition of the activity of the COX-BV and the HAV by HEp-2 and Vero cells using end point assay technique. Three different ways were used (in order to study the mechanics of the effect of extracts on the inhibition of virus activity) compared to IFN-α2a as a positive control:
1- Pre-treatment: It means adding the nontoxic concentration of the extracts to the cells before infecting the cells with decimal dilutions of the virus in wells.
2- Co-treatment: It means adding the nontoxic concentration of the extracts to the cells and was mixed with decimal dilutions of the virus at the same time in the wells.
3- Post-treatment: It means adding the nontoxic concentration of the extracts to the cells after infecting the cells with decimal dilutions of the virus in the wells.
Wells are incubated at 37oC for 24 hours. At the end of each experiment, the wells were microscopically examined using the inverted microscope for detection of cellular changes / CPE and the scored CPE was recorded and the virus titer was calculated using Reed and Meunch (1938) equation, in comparison to that achieved under the effect of IFN-α2a. In order to find the best extract Able to make highest reduce of virus titre compared to IFN-α2a. It was found that the highest reduction of the COX-BV was detected in CL extract followed by E.A extract in pre-treatment compared to IFN-α2a, and the highest reduction of HAV was recorded in n.b extract in co-treatment compared with IFN-α2a.
Toxicity of Moringa leaves extracted by E.A and n.b were evaluated in vivo using male albino Wistar rat divided to 5 groups each one contained 5 animals, the E.A and n.b non-toxic concentration extracts 97.7 and 390 µg/ml toxic concentration were given oral gavage administered daily for 28 days.
1- The first group of rats was given doses of toxic conc. (390µg / ml) for the extract resulting from the extraction by E.A.
2- The second group of rats was given doses of non-toxic conc. (97.7µg/ml) for the extract resulting from the extraction by E.A.
3- The third group of mice rats given doses of toxic conc. (390g / ml 390) for the extract obtained by the extraction of n.b.
4- The fourth group of rats was given doses of non-toxic conc. (97.7µg/ml) of the extract obtained by the extraction of n.b
5- The fifth group of rats was given a salt solution (0.9% Nacl) as (negative control).
The animals were observed weekly for general appearance, behavior and signs of abnormalities. At the end of all weeks the animals were fasted overnight, blood samples were collected from eye vein for determining hematological ( White blood cells and lymphocytes ), serum biochemical values such as; glucose concentration, liver enzymes and renal function and histological studies in liver and kidney of rats (tissue were dyed with eosin and hematoxylin). The results showed that:
- Regarding the effect of toxic conc. (390g / ml 390) of E.A and n.b extracts on the activity and vitality of rats, it was found that, there was an increase in all the parameter that were measured compared to the negative control and showed the presence of apparent symptoms such as blood urine and the presence of ulcers around the mouth and nose area. As for the analysis of tissues, it was found that, there is a harmful effect on the liver and kidney tissues of mice.
- Regarding the effect of nontoxic concentration (97.7µg/ml) of E.A and n.b extracts, it was found that, all the parameter that were measured and mentioned Previously were normal compared to the negative control. As for the analysis of tissues, it was found that, there was no changed on the liver and kidney tissues of mice compared to negative control.
Effect of Nontoxic Concentration (97.7µg/ml) of E.A and n.b extracts were tested on the activity of COX-BV and HAV respectively, compared to IFNᾰ-2a were performed using male albino Wistar rats strain, where the rats were divided into eight groups each group contains five animals as follows: -
1- 1st group of rats was infected with COX-BV virus (103 TCID50/0.1 ml) and it was administered intra peritoneal as positive control.
2- 2nd group was negative control; which it treated with 0.9 % saline solution.
3- 3rd group was pre-treated with E.A extract daily via oral gavage for 72 hr, followed by infection with COX-BV as virus model (103 TCID50/0.1 ml) and it was administered intra peritoneal, then the E.A extract administration was continued daily via oral gavage for 35 days.
4- 4th group was injected with mixture of IFN–α 2a and COX-BV virus and it was administered intra peritoneal.
5- 5th group injected with IFN–α 2a in a single form and it was administered intra peritoneal.
6- 6th group was injected with HAV virus (103 TCID50/0.1 ml) and it was administered intra peritoneal as positive control.
7- 7th group was treated with n.b extract and HAV virus (103 TCID50/0.1 ml) mix and it was administered intra peritoneal, and then the n.b extract administration was continued daily via oral gavage for 35 days.
8- Finally, 8th group was treated with mixture of IFN–α 2a and HAV virus and it was administered intra peritoneal.
The animals were observed weekly for general appearance, behavior and signs of abnormalities. At the end of all the weeks the animals were fasted overnight and blood samples were collected from eye vein for determining hematological (WBCs and lymphocytes), serum biochemical values such as; glucose concentration, liver enzymes and renal function, cytokines values and virus antibodies (Ab), titre of viruses and MX gene values in intestinal of rats, and histological studies in intestinal of infected rats.
- For the effect of E.A extract on COX-BV activity, it was found that, there was an increase in the number of white blood cells and lymphocytes and high glucose levels. And the presence of symptoms of diarrhea in the feaces of the 1st group which was infected with the virus COX-BV only compared with 2nd group (negative control), the 3rd group and 4th group. As for the histopathological analysis of intestinal tissues, it was shown have a harmful effect on the intestinal tissues of the 1st group only compared with 2nd group (negative control), the 3rd group and 4th group.
- For the effect of n.b extract on HAV activity, it was found that, there was an increase in the number of white blood cells and lymphocytes in the 6th group (were infected with HAV only) compared to the 2nd group (negative control), 7th group and 8th group. As for the histopathological analysis of intestinal tissues, it was no shown have a harmful effect on the intestinal tissues of all groups (6th, 2nd and 7th group).
In addition to the parameters measured, other parameters were measured to Inference the effect of non-toxic concentration (97.7µg/ml) Moringa leaf extracts E.A and n.b on COX-BV and HAV viruses activity respectively. Viruses Titration were counted in the intestinal at the end of each week for a 5-week period on HEP-2 and Vero cells, respectively, using End Point assay, where The intestines were grinded in EMEM or 19.9 E and decimal dilutions of viruses were added to HEP-2 or Vero cells respectively in Wells of plates and then incubated at 37 °C. CPE was monitored by the inverted microscope, then the titer of viruses were calculated using Reed and Meunch (1938) equation. It was found that, the highest reduction in the number of viral molecules was found in the 3rd and 7th groups of rats (injected with (COX-BV or HAV respectively, and then given doses of non-toxic concentration (97.7µg/ml) of the E.A or n.b extracts, respectively), followed by the 4th and 8th groups of rats (That have been injected with mixed of COX-BV or HAV viruses and IFN-α2a) respectively followed by the 1st and 6th groups of rats injected with the virus (COX-BV or HAV) respectively, compared to 2nd group (negative control).
The level of antibodies (Ab) of the COX-BV and HAV were measured in the blood serum in all groups of rats at the end of each week for five weeks. where the level of antibodies in the blood is evidence of the presence of viral infection, which so infer from it, presence of the viral molecules in the body of mice, especially HAV viral because it does not multiply only in humans and monkeys. And the level of Ab is measured as it is an immunological significance that reflects the amount of Ab not binding with the virus, which gives an indication of the quantity of the virus found. The level of antibodies was measured using ELISA technique, where wells of microplate were covered with antigen of COX-BV or HAV viruses. The blood serum of rats was added until the antibodies were bound to the antigen in the microplate, then the enzyme conjugate was added, then the substrate was added. The optical density of each well was determined immediately, using the ELISA reader set to 450 nm. it was found that, The lowest values of Ab were detected in the 3rd and 7th groups of rats (injected with COX-BV or HAV ) respectively, and then given doses of non-toxic concentration (97.7µg/ml) of EA or n.b extracts, respectively), followed by the 4th and 8th groups of rats (That have been injected with mixed of COX-BV or HAV viruses and IFN-α2a) respectively followed by the 1st and 6th groups of rats injected with the virus COX-BV or HAV respectively, compared to 2nd group (negative control).
Some cytokines were estimated as immunological tests such as interleukin-6 (IL-6) and interferon gamma (IFN-γ) in the blood serum that were withdrawn from rats on the third, seventh and fifteenth day of viral injection using ELISA technique to infer the effect of Non-toxicity (97.7µg/ml) of Moringa leaf extracts derived from E.A and n.b on immune response of rats against infection with COX-BV and HAV virus in terms of the high or low concentration of IL-6 and IFN-γ, It was found that:
- For IL-6 concentration, the lowest concentration was found in the 4th and 8th groups of rats (That have been injected with mixed of COX-BV or HAV viruses and IFN-α2a) respectively, followed by the 1st and 6th groups of rats injected with the virus COX-BV or HAV respectively ,followed by the 3rd and 7th groups of rats (injected with COX-BV or HAV respectively, and then given doses of non-toxic concentration (97.7µg/ml) of EA or n.b extracts, respectively), compared to 2nd group (negative control).
- For IFN-γ concentration, the highest concentration was found in the 3rd and 7th groups of rats (injected with COX-BV or HAV respectively, and then given doses of non-toxic concentration (97.7µg/ml) of EA or n.b extracts) respectively, followed by the f4th and 8th groups of rats (That have been injected with mixed of COX-BV or HAV viruses and IFN-α2a) respectively, followed by the 1st and 6th groups of rats injected with the virus COX-BV or HAV respectively compared to the compared to 2nd group (negative control).
To supplement the immunological tests that have been assessed, the gene expression of the MX gene was evaluated in vitro (in Vero cell and HEp-2 cell) and in vivo (in the intestines of rats) were treated with non-toxic concentration of E.A and n.b Moringa leaf extracts (97.7 7µg / ml), IFN-α2a (5µg /ml) and untreated cells and rats (negative control). MX gene is estimated as an Indicates to IFN-α2a activity because it is a protein involved in the antiviral response and is one of the mechanisms of the effect of interferon on the inhibition of virus activity. In the cells, it is prevents replication of virus inside the cell as antiviral. Therefore, the gene expression of a MX gene was estimated In order to study mechanically the effect of non-toxic concentration (97.7µg/ml) of E.A and n.b Moringa leaf extracts on inhibition of virus activity. The gene expression of the MX gene was estimated using rt-RT-PCR. The results show that:
In vitro, the highest gene expression for the MX gene was found in HEp-2 cells treated with IFNᾰ-2a- , followed by HEp-2 cells treated with nontoxic conc. of E.A extract (97.7µg/ml), followed by Vero cells treated with IFNᾰ-2a, followed by Vero cells treated with nontoxic concentration of n.b extract (97.7µg/ml) compared to the control cells (cells untreated with IFN-α2a or nontoxic concentration (97.7µg/ml) of extracts).
- In vitro, the highest gene expression value of the MX gene was found in the group of rats treated with non-toxic concentration of E.A extract (97.7µg/ml), followed by rats treated with IFNᾰ-2a, followed by the group of rats treated with non-toxic concentration of n.b extract (97.7µg/ml), compared to the negative control group (rats untreated with extracts or IFN-α2a).
Following phenolic compounds, the total serum phenol content in the serum of rats treated and untreated with non-toxic concentration (97.7µg/ml) of Moringa leaf extracts derived from E.A and n.b were estimated by the Folin-Ciocalteau reagent using the spectrophotometer at a wavelength of 765 nm. The results showed that, the highest concentration of phenol was found in the serum rat treated with non-toxic concentration (97.7µg/ml) of the extract obtained by E.A, followed by the rats treated with non-toxic concentration (97.7µg/ml) of the extract obtained by n.b, compared to negative control group.
Conclusion
Phytochemicals were extracted from moringa leaves by four different solvents: CL, E.A, M and. n.b. Their effect on inhibition of COX-BV and HAV virus in vitro and in vivo were studied. It was found that, the best extract was E.A, It was recorded that it has the highest reduction of COX-BV virus titre in the pre-treatment (addition of the nontoxic concentration of the extracts before viral infection) and n.b, was recorded to have the highest reduction of HAV titre in the co-treatment (addition of the nontoxic concentration of the extracts mixed with viral infection) compared with IFN-2a.
Based on previous results, we can conclude that, Extracts obtained with E.A and n.b can be used as antivirals for COX-BV and HAV respectively.
Recommendation
It isrecommend using Moringa as a preventive do not a therapeutic.