Chemical Composition and DNA Damage Protection Activity of Black Mulberry

(Morus nigra L.) Fruit extract in Aqueous, Methanolic and Ethanolic Solutions

Research Plan

Proponent/s: Espera, J., Magbanua, F., Peralta, Z.

A. Problems Being Addressed

Deoxyribonucleic acid (DNA) controls genetic characteristics of living organisms

consequently DNA is an important molecule to be studied in the welfare of human race

(Liu, 2017). Therefore DNA damages and its important segment could occur at

endogenous level as well as external factors, thus posting threat to cellular levels (John,

1987; Lu et.al, 2015). And if DNA is damaged and not repaired, it will lead to mutation

and possible diseases such as skin cancer due to ultraviolet radiation (Clancy, 2008).

There are various types of damage to DNA, this includes oxidative damage,

hydrolytic damage and others. This damages could result in the development of a variety

of cancers including colon, breast, and prostate (O’Driscoll, 2012). Treatments for this

diseases are costly and beyond the reach of general public (Hai Tao, 2017). There are

also some methods for DNA repair like nucleotide excision repair. The history of the

discovery of NER, its association with genetic disorders, mechanistic features, and

relationship with other cellular pathways has been extensively reviewed in 2005 in

several articles in DNA Repair and Mutagenesis (Friedberg et al. 2005).

  Morus alba L. (M. alba), one of the most valuable plants rich in natural

ingredients, have demonstrated that extracts, fractions and major constituents from M.

alba exhibit numerous pharmacological activities such as antioxidant, anti-inflammatory,

anticancer, antimicrobial, antifungal, skin-whitening, antidiabetic, anti-hyperlipidemic,

anti-atherosclerotic, anti-obesity, cardioprotective, cognitive enhancing,

hepatoprotective, anti-platelet, anxiolytic, anti-asthmatic, anthelmintic, antidepressant,

and immunomodulatory activities (Lim & Choi, 2019).

B. Objectives

 Generally, this study aims to screen the fruit extract in three different solutions

(aqueous, methanolic, and ethanolic) of Black mulberry for its protective effects

on DNA against the mutagenic and toxic effects of UV and H2O2.

 Specifically, the study aims to determine the phytochemical compounds present

in fruit extract and in the Positive control (Ascorbic acid), determine if there is a

significant difference in the protective effects of the fruit extract of Morus nigra

treated in three different solutions: aqueous solution, methanolic solution, and

ethanolic solution by measuring the dna damage using PCR assay.

C. Statement of Hypothesis

Based on the statement of the problem, the researchers assumed that there is no

significant difference between the varying ratios of Black Mulberry extract in aqueous,

methanolic and ethanolic solutions in terms of protective effects in DNA damage.

D. Procedures

Figure 1: Flowchart of Procedure

Research Design

A pretest posttest design is an experiment where measurements are taken both before and

after a treatment. The design means that you are able to see the effects of some type of treatment

on a group.

R experimental group 01 x 03

R control group 02 x 04

Figure 2: Schematic Diagram of the Research Design

Plant Material and Plant Grinding

M. nigra will be collected at Gen luna St, Salvacion Subdv., Brgy 6, Victorias City .The

fruit will be carefully washed using tap water to remove the dust and will be dried. The dried

fruit will be milled individually using a small electric miller. The powdered fruit will be

transferred to a glass covered cans and will be placed in the refrigerator before the extraction

process.

Plant Extraction

The aqueous extract of the fruit will be made in distilled water. The mixture will be taken

into 300 ml sterile conical flasks, plugged with sterile cotton and will be kept in Shaking

Incubator with the 200 rpm for 24 hrs. (Niamah, et. al., 2015). The Soxhlet method will be used

for the ethanolic extract of the plant (Redfern, et.al, 2014).The methanolic extract will be made

from plant succus that will be prepared from 100g of plant leaves to be evaporated to dryness
and dissolved in 100 mL of 100% methanol (MeOH) overnight (Larson, et.al , 2016).

α-Diphenyl-β- Picrylhydrazyl (DPPH) Free Radical Scavenging Activity

The DPPH assay is used to predict antioxidant activities by mechanism in which

antioxidants act to inhibit lipid oxidation, so scavenging of DPPH radical and therefore

determinate free radical scavenging capacity. The method is widely used due to relatively short

time required for the analysis. The DPPH scavenging potential of M. nigra will be measured

according the method described by Brand Williams with slight modifications. Different

concentrations of M. nigra will be prepared in methanol and used. 500µL of each concentration

of M. nigra will be added to 500µL of 0.1M DPPH. Color blank to negate the color of plant

extracts were prepared as above by replacing methanol with DPPH. Positive control will be

prepared by adding 500µL of DPPH to 500µL of methanol. Ascorbic acid will be used as

standard (same concentrations as plant extract) and prepared in the similar manner as the test. All

tubes is to be incubated in dark at room temperature for 30 min (Dhawal, et. al., 2018).

Reducing Power

The reducing power will be determined according to the method of Oyaizu (Berk, et.al.

2011). Each extract (0.2 to 1.0 mg/ml) in water (2.5 ml) will be mixed with 2.5 ml of 200 mm

sodium phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricynide and the mixture will

be incubated at 50°C for 20 min. Then, 2.5 ml of 10% trichloroacetic acid will be added, and the

mixture is to be centrifuged at 200 for 10 min. The upper layer (2.5 ml) will be mixed with 2.5

ml of deionized water and 0.5 ml of 0.1% ferric chloride.

Chelating Effects on Ferrous Ions

The chelating effect is going to be determined according to the method of Dinis et al.

Briefly, 1 ml (2 mg/ml) of the extracts in water will be added to 1 ml of water and a solution of 2

mm FeCl2 (0.05 ml). The reaction initiated by the addition of 5 mm ferrozine (0.2 ml). Then, the

mixture is going to be shaken vigorously and left at room temperature for 10 min. Absorbance of

the solution will be measured spectrophotometrically at 562 nm (Berk, et.al, 2011).

Assay for Total Phenolics

Total phenolic constituent of the water extracts will be determined by employing the

methods given in the literature involving Folin–Ciocalteu reagent and Gallic acid as standard. 1

ml of extract solution containing 2000 μg extract is going to be added to a volumetric flask. 45

ml distilled water and 1 ml Folin–Ciocalteu reagent is going to be added and flask was shaken

vigorously. After 3 min, a 3 ml of Na2CO3 (2%) solution will be added and the mixture will be

allowed to stand for 2 h by intermittent shaking (Berk, et.al, 2011).

Assay for Total Flavonoids

 Total flavonoid content is going to be determined using the Dowd method as adapted by

Arvouet-Grand et al. Briefly, 1 ml of 2% aluminium trichloride (AlCl3) in methanol will be

mixed with the same volume of the water extracts (2000 μg). Absorption readings at 415 nm is

going to be taken after 10 min against a blank sample consisting of a 1 ml extract solution with 1

ml methanol without AlCl3 (Berk, et.al., 2011).

DNA Damage Protection Potential

DNA damage protection activities of the extracts will be evaluated on pBR322 plasmid

DNA (vivantis). Plasmid DNA oxidized with H2O2 + UV treatment in the presence of extracts

and checked on 1% agarose gels according to Russo et al. [40] after some modifications. In brief,

the experiments will be performed in a volume of 10 μl in a microfuge tube containing 3 μl

pBR322 plasmid DNA (172 ng/μl), 1 μl of 30% H2O2, and 5 μl of extract in the concentrations

of 5, 10, 15 and 20 mg/ml, respectively. The reactions will be initiated by UV irradiation and

continued for 5 min on the surface of a UV transilluminator (DNR-IS) with an intensity of 8000

μW/cm2 at 302 nm at room temperature. After irradiation, the reaction mixture (10 μl) along

with gel loading dye (6×) is going to be loaded on a 1% agarose gel for electrophoresis.

Untreated pBR322 plasmid DNA was used as a control in each run of gel electrophoresis along

with partially treated plasmid, i.e. only UV or only H2O2 treatment. Gels will be stained with

EtBr and photographed with the Gel documentation system (Berk, et.al, 2011).

Data Gathering
 In gathering the data, the researchers will use PCR assay to measure its protective effects.

A convenient way to determine the presence of lesions in DNA is by PCR. This scheme has been

used in different experiments to monitor DNA repair of UV or chemically produced DNA

lesions (Anachkova & Russev, 2009). The lower the rate is, solution is effective.

Data Analysis

After the tests, the results will be gathered and analysed. The researchers will use

Analysis of Variance (ANOVA) as an analysis tool. ANOVA is used to determine the influence

that independent variables have on the dependent variable in a regression study (Kenton, 2019).
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