Polyphenolic Profiles and Cytotoxic Effect of Iraqi Morus alba leaves Ethyl Acetate Extract

Morus alba, member of the Moraceae family, is a perennial tree utilized in folk medicine, preparing the modern drug, and considered the main food for silkworms. However, data on chemical content in the leaves is still limited; the main objective of this study is to detect the presence and determine the concentration of different polyphenolic constituents in the leaves of the Morus alba plant by reverse phase-high performance liquid chromatography (RP-HPLC) and evaluate the cytotoxic effect of ethyl acetate extract of this plant on human breast cancer (AMJ-13) cell line. Phytochemical analysis of the Morus alba leaves ethyl acetate extract led to identifying and quantification of six polyphenolic constituents designated as phenolic acids (caffeic, chlorogenic, and p-coumaric acid), catechins (epicatechin) and flavonoids (luteolin and apigenin) in which p-coumaric acid exhibited the highest concentration follow by luteolin (identified for the first time), chlorogenic acid, caffeic acid, apigenin and epicatechin as the least. According to estimates, the ethyl acetate extract with a high concentration of polyphenolic constituents gave the best findings as cytotoxic against breast cancer AMJ-13 cell line with an IC50 value of 129.5 µg/ml.

Morus alba L. (of family Moraceae) is a deciduous, monoecious tree of moderate size with 1.8 meters in width and 3 meters in height 1 . It is native to Japan, China, and India and is sometimes distributed in other places like North America, Europe, and Africa. It is commonly named white mulberry. Morus alba is distributed where silkworms are elevated throughout the world. Its leaf is the main food behoof for silkworms 2 . The leaves of the mulberry are used as powdered juice and tea in Japan. White mulberry is utilized in various parts of the world as a vegetable and is also grown for fruit production in European countries 1 . The root bark, fruit, and leaf of white mulberry have a prolonged history in conventional medicine of Chinese. Its root bark is an expectorant, anti-inflammatory, antitussive, and diuretic agent. The mulberry fruit act as an analgesic and tonic agent. The mulberry leaves can be used for antitussive, pyrolysis treatment, blood cooling, and improvement of eye problems 3 . As it is rich in minerals and contains metabolizable energy, protein, and small antinutritional agents such as tannic acid, mulberry leaves have been considered an alternative source of protein for poultry production. White mulberry is an excellent source of â-carotene, ascorbic acid, and antioxidant compounds like rutin 4 . Also, this plant contains several active phytochemical compounds such as flavonoids, phytosterols, tannins, sitosterols, triterpenes, saponins, benzofuran derivatives, anthocyanins, anthraquinones glycosides, oleanolic acid, and others 5 . In addition to the nutritive value, the leaves of the mulberry are safe and normal medicinal agents reported to have antimicrobial, antidiabetic, antimutagenic, anticancer, antioxidant, anxiolytic, antistress, anthelmintic, immunomodulatory, nephroprotective, hypocholesterolemic, h e p a t o p r o t e c t i v e 6 -8 , h y p o u r i c e m i c 9 , neuroprotective 10 , anti-inflammatory 11 , and cardioprotective actions 12 . To best knowledge, few reports have been published on Morus alba, so it sparked our interest in conducting scientific research on the polyphenolic composition and cytotoxic activity of Iraqi Morus alba ethyl acetate leaves extract.

Plant material
Morus alba leaves (Family Moraceae) were cultivated and assembled in Baghdad, Iraq. The plant leaves were washed with tap water (H 2 O), dehydrated in shadow (at room temperature), and then ground as powder by mortar and pestle.

Preparation of Morus alba extract
Extraction was made with some modifications as process mentioned by Iswantini et al. (14) . Powder leaves of Morus alba (25 g) were macerated with 250 ml of n-hexane for one day and then filtrated. The plant materials left were extracted with 250 ml of aqueous ethanol (ethanol-water 85:15 v/v) by soxhlet apparatus for 12 hrs. The crude extract was filtered and reduced in volumes under vacuum to obtain a dry residue. The dried extract was hanging in the water and fractionated with ethyl acetate (100 ml x 2), as shown in figure 2. The ethyl acetate extract was dried under vacuum to give 0.408 gm, and the percentage yield was 1.632% and then stored for further examination.

Qualitative and Quantitative determination of phenolic compounds (flavonoid, phenolic acid, and catechin) by rP-hPlC
RP-HPLC technique is used in the identification, and quantitative estimation of phenolic acids (caffeic, chlorogenic, and p-coumaric acid), catechins (epicatechin), and flavonoids (luteolin and apigenin) in the Morus alba ethyl acetate extract of leaf part, in which using a mobile phase (Isocratic) consist of acetonitrile: water (1:1). The C18 Column (250 mm x 4.6 mm, with particle size 5 ìm) was utilized as stationary phase. The 20 ìL injection volume and 1.0 ml/min flow rate were used, and the injection concentration was 1 mg /ml. The UV detection was performed at 265 nm (15) .

Cytotoxic evaluation of the ethyl acetate extract of Morus alba
To evaluate the cytotoxic effect of Morus alba ethyl acetate extract of leaf part, breast cancer cell (AMJ13) was taken from an Iraqi patient with breast cancer and maintained in RPMI-1640, supplemented with penicillin (100 unit/mL), streptomycin (100 µg/mL), and fetal bovine (10%). The cells were passaged using Trypsin-EDTA, incubated at 37 °C, and reseed twice per 7 days at 50% confluence 16 .
On 96-well plates, the MTT test was performed, and at 1×10 4 cells per well, the cell line was seeded 17 . Cells were treated with the serial concentrations of ethyl acetate fraction (tested compound) when a confluent monolayer was done (after 24 hrs.). After treatment with 72 hrs, the viability of the cell was determined through the elimination of the intermedia, a solution of MTT (28 µL) was inserted, and the cells were incubated for about 1.5 hrs. at 37 °C. After the solution of MTT was eliminated, the residual crystal was dissolved in the wells through an addendum of Dimethyl Sulphoxide (130 µL), then incubated for about 15 min (at 37 °C) with stirring 18 . The test was done in triplicate, and the absorbance  was computed at 492 nm (wavelength of the test) on a microplate reader. The cell growth inhibitory rate (the cytotoxicity percentage) was determined according to the next equation 19 : % Cell viability = (absorbance of treated cell / Absorbance of non-treated cell) X 100 % Cytotoxicity = 100 -cell viability statistical analysis Data obtained statistically utilizing the unpaired T-test were analyzed with Graph Pad Prism 6. For the triplicate measurements, values were given as mean ± SD (20) .

Qualitative and Quantitative determination of phenolic compounds by rP-hPlC
The qualitative estimation of phenolic acids, flavonoids, and catechins in the Morus alba ethyl acetate extract by RP-HPLC was made      The results show that the concentration of p-coumaric acid was higher than the concentration of other constituents in the sample (ethyl acetate extract), and the amounts of phenolic acids are higher than that of flavonoids in Morus alba leaves, as shown in table (1).

Cytotoxic assay
The cytotoxic assay was done to investigate the cytotoxicity of Morus alba leaves ethyl acetate extract on the human tumor cell line, breast ductal carcinoma AMJ-13 cells, by the MTT test.
The Morus alba leaves ethyl acetate extract revealed a cytotoxic effect verse the AMJ-13 cell line with a maximum cytotoxic activity at 1000 µg/mL and a minimum cytotoxic activity at 31.2 µg/mL as shown in figure 16 and table 2. This effect depends on the concentration.

ConClusion
This study showed that the phytochemical found in leaves extract of Iraqi Morus alba could be considered an important source of medicine and vital for good health. In this study, six polyphenolic compounds have been identified, some of them for the first time, in the leaves extract of Morus alba by RP-HPLC. The result shows that the RP-HPLC method can be adopted for qualitative and quantitative determinations of phenolic acids, flavonoids, and catechins in Morus alba dried leaves extract. In addition, the dried leaves ethyl acetate extract of this plant was active against breast cancer AMJ13 cell line with IC50 value of 129.5 ìg/ml, and the good cytotoxic property of white mulberry is mainly related to the existence of polyphenolic compounds and other related compounds. aCknowledgMent I am beholden to the college of the pharmacy / University of Baghdad to award the facilities and the opportunity to do my work.

Conflict of Interest
There is no conflict of interst.