Phytochemical Investigation of Corchorus olitorius L . Leaves Cultivated in Iraq and it ’ s In Vitro Antiviral Activity

The aim of our study was to investigate the antiviral activity of the Corchorus olitorius family Tiliaceae cultivated in Iraq against measles virus, and to demonstrate an overview about chemical constituents and pharmacological activity of Corchorus olitorius L. About150 gm Leaves of Corchorus. olitorius were defatted by maceration in hexane for 24 hrs. The defatted plant materials were subjected for extraction after filtration using Soxhlet apparatus, with aqueous methanol 85% as a solvent extraction for 24 hours, the extract was filtered, and the solvent was evaporated under reduced pressure using a rotary evaporator to get a dry extract of about 12 gm. About 4 gm from the residue was suspended in 100ml water, about 3-4ml of 5% sodium hydroxide was added to obtain a basic solution having PH 10 and partitioned with ethyl acetate (3x100ml), the aqueous layer collected and evaporated to dryness. MTT-cell viability assay (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyl tetrazolium bromide) was conducted on 96-well plates (Falcon), Vero cells were seeded at 1× 10 cells/well to obtain a multiplicity of infection (MOI 10), and at 5 × 10 cells/well to obtain a multiplicity of infection (MOI 5). Different statistical result revealed a significant antiviral activity of the aqueous layer of Corchorus olitorius leaves against measles virus. The preliminary phytochemical tests showed the presence of phenols and flavonoids in the aqueous layer of Corchorus olitorius leaves. The antiviral activity of Corchorus olitorius leaves is mainly due to the phenolics and flavonoids that detected in the aqueous layer.


Introduction
Therapeutic plants have been used since ancient times for the treatment of a variety of diseases (1).
Current estimates of the number of types of flowering vegetation range between 200 000 and 250 000 in some 300 families and 10 500 genera, and despite a rapidly expanding books and researches on phytochemistry, only a tiny percentage of the entire varieties have been examined chemically (2) .
Almost all organisms need to convert and interconvert a great number of organic and natural substances to permit live, grow, and reproduce.In contrast to these |major metabolic pathways (primary metabolite), which synthesize, degrade, and generally interconvert compounds commonly encountered in all organisms, there also exists an area of metabolism concerned with substances that have a very limited spread in nature.Such compounds, called "secondary metabolites", which gives almost all the pharmacologically active natural products (3) .
The genus Corchorus (jute) which belongs to the family of Tiliaceae, involves about 50-60 species allocated in the tropics, subtropics and warm temperate areas of the world (4) .Corchorus species is one of the main genera containing cardiac glycosides, especially in the seeds (2) .Corchorus olitorius are tall, annual herbs, reaching a height of two to four meters.The plant could be unbranched, or with only a few side divisions.The leaves alternately distributed, simple, lanceolate, finely serrated or lobed margin.The flowers are small (2-3 cm in diameter) and yellow, it has 5 petals.
The fruit of the plant contains many seeds inside in the form of a capsule, Corchorus olitorius is an important green leafy vegetable in many areas including Egypt, Southern Asia, Japan, India, China, Lebanon, Palestine, Syria, Jordan, Tunisia, and Nigeria, it is a cultivated plant here in Iraq.It has diverse common names bush okra, nalta jute, jute mallow and Jew's mallow, ewedu, melokhyia and moroheiya (4,5) .

Traditional use
Different parts of Corchorus.olitorius have been utilized to relieve pain, aches, chronic cystitis, dysentery, enteritis and pectoral pain (7) , the leaves have been utilized in case of gonorrhea, chronic cystitis, fever, and tumors (8)   ,the seeds were utilized as demulcent, diuretic, purgative, also used in chronic cystitis, in cases of cardiac diseases like heart failure due to its content of cardenolides cardiac glycosides (5,9) .It is highly consumed in Japan as "healthy vegetable" due to its high content of carotenoids, vitamin C, B1, B2 and E, many minerals and bioactive compounds (5,10) .
Measles is one of the most common communicable diseases around the world and may cause serious complications and sometimes death, about 350 children loss his life every day around the world (11) .
Corchorus olitorius is one of the plants that's used in folk medicine for the treatment of measles (12) .

Collection of plant materials
Corchorus olitorius leaves were harvested from a farm in al-Utafiyah district in Baghdad City, during July 2017.The plant was identified and authenticated by Prof. Dr. Sukaena Abass / Department of Biology / College of Sciences/ University of Baghdad.Leaves were washed thoroughly, dried under shade, and grinded in a mechanical grinder to a fine powder.

Extraction
About 150 gm of shade-dried pulverized leaves were defatted by maceration with hexane for 24 hours then filtered and allowed to dry at room temperature.The defatted plant materials were extracted using Soxhlet apparatus in which the powder packed in the thimbles and extracted with 1.75L of aqueous methanol 85% as a solvent extraction for 24 hours.The extract was filtered, and the solvent was evaporated under reduced pressure using a rotary evaporator to get a dry extract of about 12 gm (34) .About 4 gm from the residue was suspended in 100ml water, about 3-4ml of 5% sodium hydroxide was added to obtain a basic solution having pH 10. and partitioned with ethyl acetate (3x100ml), the aqueous layer collected and evaporated to dryness (35) . (34)est for flavonoids 0.1 g of plant extract (aqueous layer) was dissolved in 20 ml of 80% ethanol and filtered.The filtrate was used for the following tests: (a) 3 ml of the filtrate was mixed with 4 ml of 1% aluminum chloride in methanol in a test tube and the color was observed.Formation of yellow color indicated the presence of flavonoids.(b) 3 ml of the filtrate was mixed with 4 ml of 1% potassium hydroxide in a test tube and the color was observed.A dark yellow color indicated the presence of flavonoids.

Test for phenols
0.25 g of each plant extract (aqueous layer) was dissolved in 10 ml of distilled water and filtered.1% aqueous ferric chloride (FeCl3) solution was added to the filtrate.The appearance of intense green, blue or black color indicates the presence of phenols compounds in the test samples.

Evaluation of the antiviral activity of the aqueous layer Experimental design
The dried extract (about 0.9gm) obtained from the aqueous layer was used in this test and symbolled as (sample tested compound).

Cell lines and culture
The Vero (transformed monkey kidney) cell lines were obtained from the Iraq biotech Cell Bank Unit and maintained in RPMI-1640 supplemented with 10% Fetal bovine, 100 units/mL penicillin, and 100 µg/mL streptomycin.Cells were passaged 37 times including the passage number from the reference, using Trypsin-EDTA reseeded at 50% confluence twice a week and incubated at 37 °C (36)   .

Virus cells
The Schwartz Edmonston attenuated measles vaccine strain was obtained from the Iraq biotech Cell Bank Unit. (37)o determine the cell cytotoxic effect of the measles virus, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) cell viability assay was conducted as on 96-well plates (Falcon), Vero cells were seeded at 1× 10 4 cells/well to obtain a multiplicity of infection (MOI 10), and at 5 × 10 3 cells/well to obtain multiplicity of infection (MOI 5).After 24hrs.or a confluent monolayer is achieved.Cells were treated with Cells were treated with different concentrations of the tested compounds (100 μg/ml, 50 μg/ml, 25μg/ml, 12.5μg/ml and 6.25 μg/ml; first: virus alone, virus with tested sample and second: the tested sample alone.The procedure was performed by adding the (virus and or tested sample) at first for 2 hrs.at room temperature to allow virus attachment and penetration.After that, cells were washed with (phosphate buffered saline) PBS and serial dilution of the tested sample was added to the non-infected and infected cells.Cell viability was measured after 72 hrs. of infection by removing the medium, adding 28 µl of 2 mg/ml solution of MTT (Sigma Aldrich-co) and incubating for 3 hrs.at 37°C.After removing the MTT solution by pouring, the crystals remaining in the wells were solubilized by the addition of 130 µl of DMSO (Dimethyl Sulphoxide) (BDH, England) followed by 37°C incubation for 15 min with shaking.The absorbency was determined by a microplate reader (Gennex Lab.USA) at 492 nm (test wavelength); the assay was performed in triplicate (36) .Endpoint parameter that is calculated for each individual cell line is the percentage of cytotoxicity, it was calculated by the following equations:

𝑽𝒊𝒂𝒃𝒊𝒍𝒊𝒕𝒚 𝒑𝒆𝒓𝒄𝒆𝒏𝒕𝒂𝒈𝒆 = (𝑨 − 𝑩)/𝑨 × 𝟏𝟎𝟎
Where A is the mean optical density of untreated wells (control) and B is the optical density of treated (tested) wells.

Control test
In order to investigate the cytotoxicity effect of the tested sample itself; the Vero cells are treated with different concentrations from the aqueous layer.

The Phytochemical composition
Preliminary examination of the aqueous fraction results is shown in Table 1.

Table 1. Preliminary examination of the aqueous fraction for phenols and flavonoids. phytochemicals
Aqueous layer flavonoids + phenols +

The Anti-Cytotoxic or antiviral effect of the plant extract
The results demonstrate the significant anticytotoxic effect of different concentrations from aqueous layer against the toxicity effect of measles virus on Vero cells using both the obtained multiplicity of infection (MOI of 5 and 10).The obtained result against the cytotoxic effect of measles virus is shown in figures (1, 2) and tables (2, 3).The results obtained from the control test are demonstrated in figure (3) and table (4).

Discussion
In the present study, maceration of plant material in hexane for overnight was used as defatting method.Next day the solvent removed by filtration and the defatted plant was dried then extracted with alcohol (85% ethanol) to get the crude extract.fractionation of the crude extract is recommended for the separation other, it depends on several factors like differences in the polarity and solubility.polyphenols like (flavonoids and phenolic acids) have acidic properties (38) , adding base like 5%NaOH will produce water-soluble salts, fractionating the aqueous phase with ethyl acetate will make the lipophilic and nonpolar compounds to stay in the Ethyl acetate phase leaving the water-soluble salts (flavonoids and phenolic compounds) in the aqueous phase (35) .MTT is a water-soluble yellow dye that is absorbed by living cells and reduced by the action of mitochondrial dehydrogenases.The reduction product is a water-insoluble blue formazan, that must thenbe dissolved for a colorimetric way of measuring by using different types of solvents like DMSO.Metabolically inactive cells do not produce significant amounts of formazan Conversely which can be detected by the microplate reader, the quantity of formazan produced per cell in a given time depends on the metabolic activity of the cells, so Formazan production is directly proportionate to the living cell number.The MTT (Tetrazolium (MTT) Assay for Cellular Viability and Activity) procedure assesses the activity and number of living cells at the end of an experiment (37) .The results demonstrate the significant anticytotoxic effect of different concentrations from aqueous layer against the toxicity effect of measles virus on Vero cells using both the obtained multiplicity of infection (MOI of 5 and 10).
The obtained result clarifies a linear relationship between the concentration of the tested sample and the degree of cytoprotecting effect (antiviral effect) against the cytotoxic effect of measles virus.The result obtained from the control test shows no cytotoxic effect against Vero cells by the tested sample, since even in high concentration which is 100μg/mL only five percent cells were killed, while only 1 percent cell killed at 12.5 μg/mL.this result augments the effect of the tested sample as antiviral (anticytotoxic) against measles virus.Natural flavonoid compounds with antiviral activity were identified in 1940, and many reviews were available about their antiviral activity especially against human immunodeficiency virus (HIV).several types of research demonstrate the mechanism of action through inhibition of various enzymes involved in the life cycle of viruses such as HIV-1 inverse transcriptase, DNA polymerases, and HIV-1 enzyme proteinase (39)   .
In general, the presence of two vicinal hydroxyl groups on an aromatic ring is the most important structural requirement for inhibition of integrase protein (HIV -IN) leading to inactivate viral integration and replication which is the situation with most flavonoids.the dicaffeoylquinic and Lchicoric acid derivatives have been proved to show antiviral activity although it doesn't have such adjacent hydroxyl group (40) .It may exert its action by inhibiting the viruscell fusions in the early stage of the replication cycle and by inhibiting the cell-cell fusions at the end stage of the replication cycle (41) .

Figure1.
Figure1.Cytoprotecting chart; increasing the concentration of the tested sample (aqueous layer) reduce the cytotoxicity of measles virus on Vero cells using MOI of 10.

Figure 3 .
Figure 3.Control test.cytotoxicity chart using a different concentration of the tested sample (aqueous layer) against Vero cells.