الفهرس 
REVIEW OF LITERATUREOnly 14 pages are availabe for public view
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Abstract
COVID-19 is a respiratory disease caused by SARS-CoV-2, it characterized by fever, coughing, shortness of breath, and/or chest pain. Between the four genera of the Coronaviruses (CoVs); alpha, Beta, Gamma & Delta, two beta-CoVs and two alpha-CoVs have been linked to the common cold and self-limiting respiratory infections in humans, two extremely pathogenic human beta-CoVs; SARS-CoV in 2003, MERS-CoV in 2012 and SARS-CoV-2 in 2019 have recently been added to this list.
Although there are vaccines and efforts are being made in the vaccine development against viruses, the need for alternative solutions was urgent, due to the RNA viruses’ proclivity for mutational alterations that render the approved vaccines no longer effective and the majority of clinically used drugs being used to relieve COVID-19 symptoms rather than their side effects.
Plants are the most abundant source of both traditional and modern medicine, as well as, food for humans and animals. Countless beneficial harvests obtained from plants, whether directly or indirectly, attest to their significance for both humans and other living organisms.
According to the WHO, 80% of the world’s population use herbal medicines for some aspect of primary healthcare; hence, the exploration of medicinal plants’ therapeutic potential to treat human and animals is made possible by scientific study of these plants.
This study took this trend of using medicinal plants in treating viral diseases, and the used plants were Cuphea ignea and Moringa oleifera.
At first, both plant leaves powders were extracted by 70% ethanol and the solutions were evaporated using the rotary device at 45°C.
The dry extract residues were dissolved in 10% DMSO and were diluted, then they were tested for their cytotoxicity on Vero-E6 and MDCK cells via crystal violet assay, in which cell-extract dilution mixtures were co-incubated, fixed, stained, and the optical density were measured through the plate reader at λmax = 570, using the Anthos Zenyth plate reader (Anthos Labtec Instruments, Heerhugowaard, Netherlands), and the results were drawn by GraphPad Prism 5.01 using nonlinear regression, and CC50 was calculated.
The results of this test revealed that:
Cuphea ignea crude extract was safe on both cell types with CC50 of 16.3 g and over 100 mg/ml, and Moringa oleifera crude extract also was detected as a secure compound with CC50 of 73.3 mg and over 100 mg/ml, on Vero-E6 and MDCK, respectively.
After that the antiviral activity of each extract was assessed against SARS-CoV-2 and H1N1, the assay was performed using crystal violet method as the cytotoxicity test, except the incubation of the cells and the extract dilutions with the virus, finally, IC50 was calculated by plotting log inhibitor vs. normalized response.
It was found that Cuphea ignea crude extract was active against both viruses with IC50 of 58.7 µg/ml and 43.6 µg/ml, and Moringa oleifera extract was defined also as an active compound with IC50 of 16.12 µg/ml and 23.3 µg/ml against SARS-CoV-2 and H1N1, respectively.
from CC50 and IC50 of both plant crude extracts, selectivity index (SI) was calculated, which defines the availability of the tested compound for further investigations as its value ≥ 10.
The results showed both plants as a promising antiviral agent against both viruses with large SI values reached over 1000 for both plants against H1N1, and over 278 for Cuphea ignea, and over 1000 for Moringa oleifera against SARS-CoV-2.
According to SI values of both plants, they needed further studies, hence, the crude plants extract undergone successive fractionation using the following solvents; petroleum ether, methylene chloride and butanol, respectively. The collected liquid fractions and the remaining solution were dried via rotary evaporation giving the dry residues of the fractions of the following weights; 0.07 g, 0.08 g, 6 g, and 6 g for Cuphea ignea, and 0.1345 g, 0.413 g, 7.5 g, and 8 g for Moringa oleifera.
The fractions of both plants were tested for cytotoxicity and antiviral activities using crystal violet assay as applied on the crude plants, and the results showed the following: The fractionation process for both plants increased the activity of the fractions than the crude, and the most active and safest fraction of Cuphea ignea and Moringa oleifera against SARS-CoV-2 was petroleum ether with IC50 of 13.9 µg/ml and 8.8 µg/ml alike IC50 of the crude of 58.7 µg/ml and 16.12 µg/ml, respectively, while against H1N1 were methylene chloride of 6.0 µg/ml and butanol of 6.3 µg/ml for Moringa oleifera alike its crude of 23.3 µg/ml, while for Cuphea ignea was residual aqueous fraction of 6.3 µg/ml and methylene chloride of 6.5 µg/ml, while the crude was 43.62 µg/ml .
After the confirmation of the activity against both viruses, both plant extracts were studied for their mechanism of action against both viruses, whether directly on virus (virucidal) or indirectly by hindering cell receptors (adsorption) or interfering with the replication cycle.
Cuphea ignea crude ethanolic extract exerted inhibitory effects against both viruses with virucidal action over 95%, with interference in adsorption over 90% & 65 %, and in replication steps over 70% & 25%, against SARS-CoV-2 and H1N1, respectively.
While Moringa oleifera crude extract, above 85% was the virucidal action against both viruses and for replication the inhibition % were 74% and 21%, and for adsorption was 75% and 48%, against SARS-CoV-2 and H1N1, respectively.
Due to the promising activity of both plants’ fractions, investigations being done on them to identify the phytoconstituents that may be the reason for their different therapeutic activities.
For the analysis of gaseous and heat-sensitive phytochemicals presented in each plant fraction, GC-MS was performed, while HPLC for the liquid content.
GC-MS analysis identified many phytochemicals (terpenes, fatty acids, phenolic acids, and amino acids, and so on) in plants fractions, which had many therapeutic properties as antioxidants and antivirals against many viruses such as: 9,12,15-Octadecatrienoic acid, ethyl ester (Z,Z,Z), and its methyl ester, 9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z), Hexadecanoic acid and its methyl and ethyl ester, phytol, quinic acid, Cyclopentasiloxane, decamethyl, Oleamide, beta.-Sitosterol, vitamin E, loliolide , and 3-(Prop-2-enoyloxy), and so on .
HPLC analysis was performed for the phenolic content of the fractions by using different standards as catechin, quercetin, kaempferol, Rutin, chlorogenic acid, and gallic acid in concentration of 1 mg/mL, and the results revealed the presence of Rutin and chlorogenic acid in butanol and the residual aqueous fractions of Moringa oleifera and absence of all standard compounds in Cuphea ignea, this may be due to the difference in plant cultivation method, time, place, and the harvest time, and other factors.
The quantitative HPLC analysis also revealed that Moringa oleifera cultivated in Egypt can be considered as a good source of flavonoids and phenolic acids such as, Rutin and chlorogenic acids, in which they presented in both fractions in high amounts reached in butanol fraction to 153.17±0.21 mg/g extract and 12.69±0.20 mg/g extract, for Rutin and chlorogenic acid, respectively.
Presence of both compounds in high amounts in butanol and the residual aqueous fractions may be the reason for the promising action of both fractions.
Rutin and chlorogenic acid were tested for their cytotoxicity and antiviral activity against SARS-CoV-2 and the results revealed the anti-SARS activity of both compound with IC50 of 14.26 µg/ml and 11.5 µg/ml, while SI value of 12.7 µg/ml and 4 µg/ml, respectively.
The significant safety observed on MDCK and Vero-E6 Cells indicated the ability of ethanolic extract of both plants and their fractions to maintain cell viability upon co-incubation, subsequently, they need further studies to be formulated in suitable pharmaceutical form or involved in antiviral products.
In conclusion this research contributes to the study of Cuphea ignea and Moringa oleifera and their fractions as antiviral agents against two RNA viruses showing strong virucidal action against them and was focused on the profiling of the phytochemicals responsible for the activity, using several analyses like GC-MS and HPLC which helped in these health benefits.