SAMPLE PREPARATION

Solvent: MeOH
Instructions:
- Extraction time: 30 min
- Extraction method: Ultrasound
- Material/solvent ratio: 1:30 w/v
- Temperature of extraction: 30oC
- Filter extracts

Figure 1. Fruits Extracts.

1. DETERMINATION OF ANTIOXIDANT CAPACITY, ANTHOCININS AND

FLAVONOIDS

1.1 Total phenolic content (TPC)

The TPC of samples was determined by the Folin Ciocalteu’s colorimetric method

described by Aguiar et al. [1]. Briefly, 50 µL of the sample extracts were mixed with 3

mL of distilled water and 250 µL of Folin Ciocalteau reagent. After 5 min, 750 μL of

20% (w/v) Na2CO3 were added and the resulting mixture was vortexed for 2 min and

incubated at room temperature in the dark for 30 min. The absorbance of the solution

was measured at 750 nm using a UV-Vis Spectrophotometer (Perkin Elmer Lambda 25,

ILC-Instrumentos de Laboratório e Científicos, Lda., Portugal). Gallic acid (25–400

mg/L) was used as standard to prepare a calibration curve (Figure 2), from which TPC
was determined in terms of mg of gallic acid equivalent in one gram of fresh mass (mg

GAE/g)

Figure 2. Calibration curve of Gallic acid

1.2 Total Flavonoid Content (TFC)

The TFC of samples was determined using the aluminum chloride colorimetric assay

described by Aguiar et al. [1] , with some modifications. Briefly, an aliquot of 1 mL of

sample was added to a 10-mL volumetric flask containing 4 mL of distilled water and

300 μL of 5% NaNO2. The mixture was allowed to stand for 5 min, and then 300 µL of

10% AlCl3 were added. After 1 min, 2 mL of NaOH (1M) were added to the mixture

and the total volume was increased to 10 mL with distilled water. The resulting mixture

was shaken vigorously for 2 min and the absorbance was measured at 415 nm. The

calibration curve was constructed using quercetin standard solution at different

concentration levels (10 to 500 mg/L) (figure 3), and the TFC of samples was expressed

as mg of quercetin equivalent per gram of fresh mass (mg QE/g).

 Figure 3. Calibration curve of quercetin

1.3 Total Anthocyanin Content (TAC)

The TAC of samples extracts was determined by the pH differential method proposed

by Giusti and Wrolstad [2]. Each sample extract was diluted separately at a 1:10 ratio

with 0.025 M potassium chloride buffer (pH = 1) and 0.4 M sodium acetate buffer (pH

= 4.5). After an equilibration period of 15 min at room temperature, the absorbance of

each solution was measured at 520 and 700 nm. The TAC was expressed as cyanidin-3-

glucoside equivalents per gram of fresh mass (mg C3GE/g) using the following

equation:

TAC (mg C3GE/g) = (A × MW × DF × V × 1000)/(ε × l × m),

where A is the absorbance calculated as A = (A 520 – A700)pH 1 – (A520 – A700)pH 4.5, MW is

the molecular weight of cyanidin-3-glucoside (449.2), DF is the dilution factor, V is the

volume of extract (L), 1000 is the conversion factor from gram to milligram, ε is the

molar absorptivity (26,900 L/mol.cm), l is the cell path length (1 cm), and m is the

sample mass (g).

2.4 Determination of Antioxidant Capacity in vitro

2.41 DPPH method

Briefly, 150 µL of extract was mixed with 2850 μL methanolic solution of DPPH (60

µM). The mixture was shaken vigorously and allowed to stand for 30 min in the dark at

room temperature. Afterward, the absorbance values of these solutions were recorded

spectrophotometrically at 515 nm using a control containing the same concentration of

DPPH radicals. The radical scavenging activity (RSA) was determined as a percentage

of DPPH· discoloration using the following formula:

RSA (%) = [(Acontrol − Asample)/Acontrol] × 100

Where Acontrol is the absorbance of DPPH radical in methanol and A sample is the

absorbance of DPPH radical solution mixed with sample extract. A calibration curve

was obtained using Trolox standard solution at different concentrations (10-1200 μM).

The results obtained were also expressed as µM Trolox equivalent per gram of fresh

mass (µM TE/g). All samples were carried out in triplicate

2.4.2 ABTS method

The ABTS radical cation (ABTS·+) was prepared by mixing a 7.4 mM ABTS solution

with a 2.6 mM potassium persulfate solution at a ratio of 1:1 (v/v) and stored in the dark

at room temperature for 12 h. Before use, this solution was diluted with methanol to

obtain an absorbance of 0.700 ± 0.020 at 734 nm. Then 2850 μL of the ABTS·+ solution

were mixed with 150 µL of fruit and vegetable extracts and allowed to react for 30 min

in dark at room temperature. Finally, the absorbance was taken at 734 nm. The ABTS ·+

scavenging capacity of the extract was determined using the following formula:

ABTS·+ scavenging effect (%) = [(Acontrol − Asample)/Acontrol] × 100

where Acontrol is the absorbance of ABTS·+ solution in methanol and Asample is the

absorbance of ABTS·+ solution mixed with sample extract. The calibration curve

between % ABTS·+ scavenging capacity and known solutions of Trolox (10-400 μM)

was then established and the results were expressed as µM Trolox equivalent per gram

of fresh mass (µM TE/g).

2.4.3 Reducing power assay

1 mL of different concentrations of sample extract (0.05-50 mM) was to 2.5 mL of

0.2M sodium phosphate butter (pH 6.6) and 2.5 mL of 1% potassium ferricyanide

solution. The reaction mixture was vortexed well and then incubated at 50ºC for 20 min

using a water bath. At the end of the incubation, 2.5 mL of 10% trichloroacetic acid was

added to the mixture and centrifuged at 3000 rpm for 10 min. the supernatant (2.5 mL)

was mixed with 2.5 mL of deionized water and 0.5 mL of 0.1% ferric chloride. The

colored solution was read at 700 nm against the blank with reference to standard using

UV spectrophotometer.

2.5 Results

Assay Arctic Lingonberry Cloudberry Stoneberry Straw

bramble
Total phenolic content 449.76±5.28 7419.71±26.51 309.27±12.04 246.57±28.78 374.69±
(mg GAE/100 g)
Total flavonoids (mg 47.27±0.90 5675.23±89 45.40±0.94 4.39±0.47 143.95
QE/100 g)
Total anthocyanins ND 113.90±0.70 ND ND ND
(mg/100 g)
Reducing power (mg 105.32±1.26 1550.02±23.52 158.21±1.05 151.40±0.99 65.53±
GAE/100 g)
DPPH (mg TE/100 g) 55.74±1.27 836.80±3.53 121±2.79 97.32±1.10 96.14±

ABTS (mg TE/100 g) 697±17.61 1317.07±2.14 1138.03±13.21 684.81±6.57 1238.24

Table 1: Total phenolic content and antioxidant activity of methanolic plant extracts by
multiple assays (average of triplicates).
According to the obtained results, the lingonberry extract has the highest content in all

evaluated tests. Figure 4 shows the comparison of bioactive compounds and their

antioxidant activity in studied methanolic plant extracts. It is important to note that all

tests were done in triplicate.

Figure 4. Bioactive compounds and antioxidant activity.

3. DETERMINATION OF CYTOTOXICITY EFFECT OF PLANT EXTRACT

To perform the cytotoxic assay, the methanolic extracts were evaporated under a
nitrogen stream to dryness (figure 5).

Figure 5. Drying of extracts by nitrogen stream

The cytotoxic effect of plant extract was determined with the following concentrations:

200, 300, 400, 500 and 600 µg/mL. The sample's mother solution was made with

DMSO, since when using water to dissolve the dry extract, it is left with small particles

in suspension.

3.1 Measurement of inhibition activity towards MCF-7 and PC-9 cell proliferation

The MCF-7 cells were grown in 90 % RPMI 1640 supplemented with 10 % of FBS, 1%

Antibiotic-Antimycotic solution, 1% MEM Non-Essential Amino Acids solution, 1 mM

sodium pyruvate and 10 μg/mL human insulin. The PC-9 cells were grown in 90 %

RPMI 1640 supplemented with 10 % of FBS, 1% Antibiotic-Antimycotic. Cells were

maintained in 25 cm petri dish monolayer incubated in a humidified atmosphere

containing 5% CO2 and 95% air at 37 °C. Afterward, the cells were harvested at

70−80% confluence, using 0.25 % trypsin-EDTA solution for used for all cytotoxicity

assays.

The cytotoxicity was determined by MTT assay, the cells (2 × 104 / well) were seeded

in a 96 well microplate and cultured in the presence of 200 at 600 μg/mL, of the

methanolic extracts, and then were incubated for 48 h. The cells trated with 100 μL

MTT reagents (5 mg/mL) and incubated for 3 h at 37 ºC to obtain purple-coloured

formazan. The colour was dissolved in 100 μL of DMSO and estimated by measuring

the absorbance at 570 nm in an ELISA microplate reader. All values were calculated as

a percent of the unviable cell number compared to the control from three independent

experiments performed in triplicate.

3.2 Results

The results show that the extracts had better cytotoxic power in cancerogenic cells in

breast cancer. In table 2 samples the percentage of toxicity of the samples in those of the

cell lines studied.

Table 2. % Cytoxicity of samples extract in MCF-7 and PC-9 cell lines

Artic
Concentration Bramble Lingonberry Cloudberry Stoneberry Strawberry
(µg/mL)
MCF-7 cells
200 70.78 ± 5.5 81.01 ± 8.50 87.98 ± 6.35 95.12 ± 4.43 84.90 ± 3.69
300 67.85 ± 3.6 76.20 ± 3.91 88.59 ± 8.26 91.17 ± 7.93 69.98 ± 4.98
400 60.77 ± 6.51 60.52 ± 1.88 55.84 ± 2.90 66.67 ± 2.65 63.09 ± 2.74
500 58.43 ± 9.46 58.79 ± 12.23 45.56 ± 8.41 63.24 ± 5.64 65.05 ± 5.15
600 54.12 ± 6.20 32.33 ± 3.69 47.18 ± 7.71 60.78 ± 2.33 59.17 ± 1.82
  PC-9 cells
200 86.53 ± 4.33 86.73 ± 4.95 74.84 ± 12.92 86.67 ± 3.1 63.69 ± 6.07
300 81.51 ± 2.19 68.19 ± 12.13 75.86 ± 6.53 86.85 ± 6.4 60.41 ± 7.56
400 91.36 ± 6.62 52.95 ± 8.11 69.19 ± 6.01 87.51 ± 0.9 54.45 ± 6.32
500 90.54 ± 1.34 54.37 ± 6.26 63.46 ± 7.35 90.37 ± 4.4 57.63 ± 7.02
600 88.69 ± 5.66 35.90 ± 3.99 60.62 ± 8.30 92.58 ± 6 54.40 ± 4.24

Figure 6. Cytotoxicity of MCF-7 and PC-9 cells by different samples extract.

For the lingonberry and cloudberry samples IC50 was calculated using Software

OriginPro 9. 497.55 µg/mL was the IC50 concentration obtained for lingonberry sample in

MFC-7 cells, while 513.42 µg/mL was the IC50 concentration obtained for cloudberry

sample in the same line cell. In PC-9 cell line, IC50 was 466.64 µg/mL for the lingonberry

extract.

In relation to the remaining samples, IC50 was not possible to determine within the

working range. Based on the literature [3,4] the antiproliferative activities of the extracts

are generally categorized into four groups: ≤20 µg/mL, active; >20–100 µg/mL,

moderately active; >100–1000 µg/mL, weakly active; and >1000 µg/mL, inactive.

References

1. Aguiar, J.; Gonçalves, J.L.; Alves, V.L.; Câmara, J.S. Chemical Fingerprint of Free

Polyphenols and Antioxidant Activity in Dietary Fruits and Vegetables Using a Non-

Targeted Approach Based on QuEChERS Ultrasound-Assisted Extraction Combined

with UHPLC-PDA. Antioxidants 2020, 9, 305.

2. Giusti, M.M.; Wrolstad, R.E. Characterization and Measurement of Anthocyanins by

UV-Visible Spectroscopy. In Current Protocols in Food Analytical Chemistry; John

Wiley & Sons, Inc.: New Jersey, 2001; p. F1.2.1-F1.2.3.

3. Baharum, Z.; Akim, A.M.; Hin Taufiq-Yap, Y.; Hamid, R.A.; Kasran, R. In Vitro

Antioxidant and Antiproliferative Activities of Methanolic Plant Part Extracts of

Theobroma cacao. Molecules 2014, 19, 18317–18331.

4. Nordin, M.L.; Abdul Kadir, A.; Zakaria, Z.A.; Abdullah, R.; Abdullah, M.N.H. In vitro

investigation of cytotoxic and antioxidative activities of Ardisia crispa against breast

cancer cell lines, MCF-7 and MDA-MB-231. BMC Complement. Altern. Med. 2018, 18,

87.