2.7.1. LD<sup>50</sup> Tests (Lethal Dose 50 Test)

**Figure 12.** Inhibition activity of F13b1/PV-EA and comparison of a BHA group with treated samples. Data are expressed as mean ± standard division. LD<sup>50</sup> is the amount of a material that results in loss of life of 50% (one half) of animals in an experiment. The LD<sup>50</sup> is one way to measure the short-term poisoning potential (acute toxicity) of a material. In this study three concentrations of F13b1/PV-EA were tested (12.5, 25 and 50 µg/mL) and in the concentration of 50 µg/mL, 50% of the mice died. This result indicated that a concentration of 50 µg/mL or higher was poisonous to mice and it was best for our main animal experiments to use *Molecules*  concentrations of less than 50 µg/mL, which meant only using 12.5 and 25 µg/mL. **2020**, *25*, x 9 of 21

**Figure 13.** Shows F13b1/PV-EA radical inhibition activity and comparison of BHA group with treated samples. Data are expressed as mean ± standard division. **Figure 13.** Shows F13b1/PV-EA radical inhibition activity and comparison of BHA group with treated samples. Data are expressed as mean ± standard division.

#### *2.7. Animal Study*  2.7.2. Average Tumor Volume

2.7.1. LD<sup>50</sup> Tests (Lethal Dose 50 Test) LD<sup>50</sup> is the amount of a material that results in loss of life of 50% (one half) of animals in an experiment. The LD<sup>50</sup> is one way to measure the short-term poisoning potential (acute toxicity) of a material. In this study three concentrations of F13b1/PV-EA were tested (12.5, 25 and 50 µg/mL) and To investigate the effects of F13b1/PV-EA in the inducement of apoptosis in Tubo breast cancer cells, mice were treated with two different concentrations of the compound (12.5 and 25 µg/mL), tamoxifen was used as a standard drug and one group without any treatment. At the end of the experiment, the mice were euthanized, tumors were excised from the mice and weighted. The tumor volumes (Table 1) were measured according to the formula below:

$$\text{tumor volume} = \mathbf{A} \times \mathbf{B}^2 \times 0.5$$

concentrations of less than 50 µg/mL, which meant only using 12.5 and 25 µg/mL. where A: length, B: width.



where A: length, B: width. Statistical analysis showed that the total tumor volume in all treatment groups was smaller than Statistical analysis showed that the total tumor volume in all treatment groups was smaller than that of the control group.

### that of the control group. 2.7.3. Histological Analysis

2.7.3. Histological Analysis

**Table 1.** The tumor volume of Tubo cancer cells in the four treatment groups. **Group Tumor Volumes** Negative control group 10.7 ± 1.2 cm<sup>3</sup> Positive control group 0.95 ± 0.7 cm<sup>3</sup> Experimental group A(12.5 µg/mL) 2.5 ± 0.8 cm<sup>3</sup> A histopathological examination by H & E staining was done to confirm the effect of treatment by isolated compounds present in *Pistacia vera* hull extract F13b1/PV-EA. Five different sections from each H & E slide were monitored at 100× magnification (Figures 14–17) and mean score was calculated from these five sections (Table 2). The scoring method is described in Table 4 in the Materials and Methods section.

A histopathological examination by H&E staining was done to confirm the effect of treatment by isolated compounds present in *Pistacia vera* hull extract F13b1/PV-EA. Five different sections from

Experimental group B (25 µg/mL) 0.8 ± 0.7 cm<sup>3</sup>

Materials and Methods section.

each H & E slide were monitored at 100× magnification (Figures 14–17) and mean score was calculated from these five sections (Table 2). The scoring method is described in Table 4 in the

> **Table 2.** Apoptotic index, and mitotic index in animal treated with F13b1/PV-EA. **Experimental Group Apoptotic Index Mitotic Index** Negative control 1 (2 ± 1) 1 (15 ± 3) Positive control 2 (9 ± 0.5) 1 (4 ± 0.8) Low-dose 2 (5 ± 0.1) 1 (9 ± 1) High-dose 3 (11 ± 0.9) 1 (5 ± 0.7)

**Figure 14.** Negative control group with a high density of mitotic cells with dense nuclei (100×). **Figure 14.** Negative control group with a high density of mitotic cells with dense nuclei (100×). *Molecules* **2020**, *25*, x 11 of 21

**Figure 15.** Standard drug control group with a high density of apoptotic cells with disintegrated nuclei in the cancer islands (100×). **Figure 15.** Standard drug control group with a high density of apoptotic cells with disintegrated nuclei in the cancer islands (100×).

**Figure 16.** Low dose treatment group with a high density of mitotic cells with dense nuclei (100×).

nuclei in the cancer islands (100×).

*Molecules*  **Figure 16. Figure 16. 2020**, *25*, x Low dose treatment group with a high density of mitotic cells with dense nuclei (100 Low dose treatment group with a high density of mitotic cells with dense nuclei (100×) <sup>12</sup>.×). of 21

**Figure 17.** High dose treatment group with a high density of apoptotic cells with disintegrated nuclei in the cancer islands (100×). **Figure 17.** High dose treatment group with a high density of apoptotic cells with disintegrated nuclei in the cancer islands (100×).


**3. Discussion Table 2.** Apoptotic index, and mitotic index in animal treated with F13b1/PV-EA.

confirmation using the HPTLC method. Chemical profiling of F13b1/PV-EA showed the presence of

Phytochemical investigations conducted previously on the hulls of ripe pistachio have led to the identification of structurally varied secondary metabolites [15]. Barreca, et al. [9] in their research identified 20 derivatives from extracts of hull of pistachio, the most plentiful being gallic acid, followed by 4-hydroxybenzoic acid, protocatechuic acid, naringin, eriodictyol-7-*O*-glucoside, isorhamnetin-7-*O*-glucoside, quercetin-3-*O*-rutinoside, isorhamnetin-3-*O*-glucoside and catechin. The key difference between the red and green hulls was the presence of anthocyanins in the green hulls. For the first time, differently galloylated hydrolysable tannins, anthocyanins, and minor anacardic acids were identified. Thus pistachio hulls have structurally varied and potentially

One of the phenolic compounds is gallic acid (GA), chemically known as 3,4,5 trihydroxybenzoic acid [16]. Gallic acid is structured in such a way that it has phenolic groups that are a source of activated hydrogen atoms so that generated radicals can be delocalized over the phenolic moieties [17]. Another polyphenolic flavonoid compound is quercetin (3,3′,4′,5,7 pentahydroxyflavone) that is ubiquitous in plants and foods of plant origin. The most notable property of quercetin is its ability to act as an antioxidant. Quercetin seems to be a strong flavonoid

to ascertain and determine the bioactive constituents that are present. Thus, the Isolation and identification of the bioactive compounds present were performed accordingly to help identify which

compound(s) play a role in the safe and effective use for therapeutic purposes.

gallic acid and quercetin.

bioactive phenolic compounds [9].

#### **3. Discussion**

One of the unique characteristics of cancer is the capability of malignant cells to elude apoptosis [11,12]. Therefore, an all-inclusive perception of the apoptotic signaling pathways that are involved is of crucial importance for the discovery and development of target selective therapeutics. Mouse models are useful tool for carcinogenic study. They will greatly enrich the understanding of pathogenesis and molecular mechanisms for cancer. [13,14]. According to our previous study [10], the molecular and cellular response exerted by the PV-EA on the MCF-7 is noteworthy, thus it is vital to ascertain and determine the bioactive constituents that are present. Thus, the Isolation and identification of the bioactive compounds present were performed accordingly to help identify which compound(s) play a role in the safe and effective use for therapeutic purposes.

In this study, we managed to purify 14 fractions from the ethyl acetate extract of this plant in three steps and they were characterized using diverse spectroscopic analyses with subsequent confirmation using the HPTLC method. Chemical profiling of F13b1/PV-EA showed the presence of gallic acid and quercetin.

Phytochemical investigations conducted previously on the hulls of ripe pistachio have led to the identification of structurally varied secondary metabolites [15]. Barreca, et al. [9] in their research identified 20 derivatives from extracts of hull of pistachio, the most plentiful being gallic acid, followed by 4-hydroxybenzoic acid, protocatechuic acid, naringin, eriodictyol-7-*O*-glucoside, isorhamnetin-7-*O*-glucoside, quercetin-3-*O*-rutinoside, isorhamnetin-3-*O*-glucoside and catechin. The key difference between the red and green hulls was the presence of anthocyanins in the green hulls. For the first time, differently galloylated hydrolysable tannins, anthocyanins, and minor anacardic acids were identified. Thus pistachio hulls have structurally varied and potentially bioactive phenolic compounds [9].

One of the phenolic compounds is gallic acid (GA), chemically known as 3,4,5-trihydroxybenzoic acid [16]. Gallic acid is structured in such a way that it has phenolic groups that are a source of activated hydrogen atoms so that generated radicals can be delocalized over the phenolic moieties [17]. Another polyphenolic flavonoid compound is quercetin (3,30 ,40 ,5,7-pentahydroxyflavone) that is ubiquitous in plants and foods of plant origin. The most notable property of quercetin is its ability to act as an antioxidant. Quercetin seems to be a strong flavonoid for defending the body against reactive oxygen species, which is very important in cancer therapy [15].

Young et al. [18] have examined polyphenols as potential inhibitors of UGDP-glucose dehydrogenase (UGDH) activity. Gallic acid and quercetin decreased the specific activity of UGDH and inhibited the proliferation of MCF-7 human breast cancer cells. Western blot analysis showed that gallic acid and quercetin did not affect UGDH protein expression, suggesting that UGDH activity is inhibited by polyphenols at the post-translational level. Kinetics studies using human UGDH revealed that gallic acid was a non-competitive inhibitor with respect to UDP-glucose and NAD+. In contrast, quercetin showed a competitive inhibition and a mixed-type inhibition with respect to UDP-glucoseand NAD+, respectively. These results indicate that gallic acid and quercetin are effective inhibitors of UGDH that exert strong antiproliferative activity in breast cancer cells.

By evaluating the cytotoxic properties of F13b1/PV-EA on MCF-7 cell line, it was observed that there was decreased cell viability in tandem with increasing concentration and time of treatment. Multiple papers about gallic acid and its pharmacological activities have been published. Gallic acid has shown some activities that include in the following: angiogenesis, repression of cell viability and reproduction in human glioma cells, prevention of the propagation of HeLa cervical cancer cells, inhibition of ribonucleotide reductase, induction of apoptosis in humoral cell lines, prevention of lymphocyte duplication and cyclooxygenases in human HL-60 promyelocytic leukemia cells, stimulation inactivating phosphorylation via ATM-Chk2 activation and anti-oxidant activity [11,18,19].

The effects of three different doses of F13b1/PV-EA on MCF-7 was evaluated through acridine orange/propidium iodide (AO/PI) staining and fluorescence microscopy. The tests confirmed that the compound has a dose-dependent effect on cell viability and induces apoptotic morphological changes in the treated cells. The results show reduced viability with the presence of higher levels of apoptotic cells for all three treatment concentrations.

To get a better understanding of the efficacy of the bioactive compound on the nucleus, treated cells were stained with Hoechst stain. The cells was seen to have gone through major nuclear changes upon treatment. In the control, the cells were uniformly stained via the fluorescent Hoechst stain, indicating the nuclei of the cells were intact. However, with increasing concentrations of the compound, there was a growth of severity seen captured on the fluorescence signals and luminous points where the cells exhibited apoptotic morphological changes.

By utilizing PI staining, it was established whether the MCF-7 cells treated with F13b1/PV-EA underwent apoptosis and if it was accompanied by notable alterations in the cell cycle, and the distribution index was also investigated via PI staining. This was accompanied by growth in the Sub-G1 population with increasing concentrations. This cells population possessed a sub-diploid DNA content which is indicative of DNA fragmentation occurring during apoptosis. F13b1/PV-EA was thus shown to be able to bring about apoptosis by changing the regulation of apoptotic genes, particularly through up-regulation of *Bax* and down-regulation of *BCL-2*.

*Caspase 3*, *Caspase 8*, *CAT*, *Bax* and *SOD* genes expression increased compared to control (gene expression in cancer cells without any treatment). Further investigations revealed that compound treatment eventually decreased the expression level of Bcl2.

The cytotoxicity and anti-cancer effects of hydro-alcoholic extracts of pistachio shell on HepG2 and L929 cells was elucidated by Harandi et al. [20]. Cell viability of HepG2 and L929 was decreased after 24 and 48 h of treatment with IC<sup>50</sup> 1500 and 1000 µg/mL for HepG2 and 2000 and 1500 µg/mL for L929. *Bax* and *P53* genes were shown to be up-regulated and *Bcl-2* gene was displayed to be down-regulated after treatment.

ROS are stabilized by reactions which in turn cause cellular damage and the formation of carcinogenic DNA adducts. The consumption of antioxidants has been shown to reduce the risks of getting cancer. Our compound displayed a dose dependent activity on ABTS and a slow inhibitory effect on DPPH free radicals.

Hashemi, et al. [21] investigated the antioxidant activity of a *Pistacia atlantica* extract. The antioxidant activity of the extract was 4.6 ± 0.66 µg/mL, while it was 25.41 ± 1.89 µg/mL for butylated hydroxytoluene (BHT). The total phenol, flavonoid and flavanol contents were 269 mg GAE/g, 40.7 mg RUT/g and 88.12 mg RUT/g, respectively. In recent times, identifying and an emphasis on chemical agents and natural products with the capability of preventing human cancer has been an important objective in preclinical cell culture and animal efficacy testing models. In clinical chemoprevention tests, according to the toxicity screening experiments, only the most active factors have potential as human chemopreventives.

Our animal experiment study results showed that the total tumor volume of all treatment groups were smaller than that those in the control group. According to several papers, quercetin has anti-proliferative effects to cancer, enhances the efficacy of chemotherapeutic factors, in vivo lymphocyte tyrosine kinase prevention and anti-tumor activity. LD<sup>50</sup> tests in rats showed that injection of boldine remarkable decreased breast cancer tumor size and at dose of 100 mg/kg body weight was well tolerated.

*Ferulago angulata* leaf hexane extract (FALHE) is capable of inducing apoptosis on MCF-7 cells. An in vivo study showed that FALHE reduce the tumor size from 2031 <sup>±</sup> 281 mm<sup>3</sup> to 432 <sup>±</sup> 201 mm<sup>3</sup> after treatment. Acute toxicity tests revealed an absence of toxic effects of the two compounds on mice [22].

This study explained the potential use of red *Pistacia vera* hull as chemopreventive drug, as it can be exploited as a new lead compound for prodrug therapy. Since MCF7 is an estrogen-receptor negative human breast cancer, it's good for potential future examination of our extract and its active compound in another estrogen receptor-responsive cell line like MDA-231 [23]. Also as extracts of plants may activate the immune system of the hosts and kill cancer cells, one can consider testing the concentrations

of various cytokines, tumor necrosis factor, etc. [24], before and after administration of PV. On the other hand, since 100% pure gallic acid and quercetin are commercially available, a combination of these two agents at the same concentration ratio as they exist in pistachio can be tested to examine the anti-tumor effect of PV extract. These results present an opening to new roads for discovery of anticancer drugs and treatment of cancer by promoting induced apoptosis.

#### **4. Materials and Methods**

#### *4.1. General Experimental Procedures*

Column chromatography (CC) was run on silica gel 60 column (Merck, Darmstadt, Germany). Thin layer chromatography (TLC) was performed on an aluminum supported silica gel 60 (Merck). The compound purity confirmation was confirmed on a HPTLC system (Gilson, Inc., Middleton, WI, USA) with a mobile phase of methanol (100%). Gas chromatography was performed on a Breeze2 system.

#### *4.2. Collection and Extraction of Plant*

The hulls of the *Pistacia vera* (PV) were procured from Kerman Province, Iran, and identified at the Herbarium in the Institute of Biological Science, University of Malaya, by Dr. Yong Kien Thai with voucher number KLU48697. The hull after drying and powdering was soaked in ethyl acetate. The extract was filtered from the residue by using filter paper and the residue was re-extracted with ethyl acetate solvent twice more. By using a rotary evaporator (R110 Rotavapor, Buchi Labortechnik AG, Flawil, Switzerland), the solvent was evaporated at a temperature of 40 ◦C, giving a dark brown crude extract and stored in 4 ◦C before further testing was done (Figures 18 and 19). *Molecules* **2020**, *25*, x 15 of 21

**Figure 18.** *Pistacia vera* tree, fruit and red hulls. Isolated fractions were monitored by TLC and finally seven appropriate fractions (F13a-F13g) were **Figure 18.** *Pistacia vera* tree, fruit and red hulls.

**Figure 19.** PV extraction method steps: deshelling, drying, maceration, evaporation and extraction.

14 fractions for choosing the most cytotoxic fraction, which was fraction number 13 (F13).

*Pistacia vera* ethyl acetate extract was chosen for next analysis and purification according to the data from our previous study. PV-EA (16 g) was subjected to column chromatography using a glass column (60 cm L 6 cm I.D) packed with Merck Kieselgel 60 stationary phase. Briefly, the silica gel was made into a slurry with solvent before it was packed into the column and it was allowed to equilibrate for at least one hour before use. The extract was then introduced on top of the silica surface. The column was generally eluted with combinations of solvents with a stepwise increase in the solvent polarities. In the first step hexane and ethyl acetate (70:30) was used as solvent. Isolated fractions were monitored by TLC and those samples displaying similar Rf values on the TLC were pooled to yield 14 fractions (designated F1-F14). The MTT cell viability assay was carried out on these

In the next step (step 2), fraction number 13 (F13) was subjected to glass column (60 cm L 6 cm I.D.) chromatography with combinations of ethyl acetate and dichloromethane of increasing polarity.

*4.3. Bioassay-Guided Fractionation of Pistacia vera Ethyl Acetate Extract*

**Figure 18.** *Pistacia vera* tree, fruit and red hulls.

**Figure 19.** PV extraction method steps: deshelling, drying, maceration, evaporation and extraction. **Figure 19.** PV extraction method steps: deshelling, drying, maceration, evaporation and extraction.

#### *4.3. Bioassay-Guided Fractionation of Pistacia vera Ethyl Acetate Extract 4.3. Bioassay-Guided Fractionation of Pistacia vera Ethyl Acetate Extract*

*Pistacia vera* ethyl acetate extract was chosen for next analysis and purification according to the data from our previous study. PV-EA (16 g) was subjected to column chromatography using a glass column (60 cm L 6 cm I.D) packed with Merck Kieselgel 60 stationary phase. Briefly, the silica gel was made into a slurry with solvent before it was packed into the column and it was allowed to equilibrate for at least one hour before use. The extract was then introduced on top of the silica surface. The column was generally eluted with combinations of solvents with a stepwise increase in the solvent polarities. In the first step hexane and ethyl acetate (70:30) was used as solvent. Isolated fractions were monitored by TLC and those samples displaying similar Rf values on the TLC were pooled to yield 14 fractions (designated F1-F14). The MTT cell viability assay was carried out on these 14 fractions for choosing the most cytotoxic fraction, which was fraction number 13 (F13). *Pistacia vera* ethyl acetate extract was chosen for next analysis and purification according to the data from our previous study. PV-EA (16 g) was subjected to column chromatography using a glass column (60 cm L × 6 cm I.D) packed with Merck Kieselgel 60 stationary phase. Briefly, the silica gel was made into a slurry with solvent before it was packed into the column and it was allowed to equilibrate for at least one hour before use. The extract was then introduced on top of the silica surface. The column was generally eluted with combinations of solvents with a stepwise increase in the solvent polarities. In the first step hexane and ethyl acetate (70:30) was used as solvent. Isolated fractions were monitored by TLC and those samples displaying similar Rf values on the TLC were pooled to yield 14 fractions (designated F1-F14). The MTT cell viability assay was carried out on these 14 fractions for choosing the most cytotoxic fraction, which was fraction number 13 (F13).

In the next step (step 2), fraction number 13 (F13) was subjected to glass column (60 cm L 6 cm I.D.) chromatography with combinations of ethyl acetate and dichloromethane of increasing polarity. Isolated fractions were monitored by TLC and finally seven appropriate fractions (F13a-F13g) were In the next step (step 2), fraction number 13 (F13) was subjected to glass column (60 cm L × 6 cm I.D.) chromatography with combinations of ethyl acetate and dichloromethane of increasing polarity. Isolated fractions were monitored by TLC and finally seven appropriate fractions (F13a-F13g) were combined and dried. After an MTT assay the most cytotoxic fraction was fraction number 2. In the last step of fractionation (step 3), fraction number 2 (F13b) was subjected to column chromatography with combinations of dichloromethane and methanol of increasing polarity. After TLC analysis on the isolated fractions, four appropriate fractions (F13b1-F13b4) were combined and dried for the next MTT assay. At the final stage, fraction number 1(F13b1) of about 10 mg was selected as the most effective fraction or pure compound.

#### *4.4. Cell Lines and Cell Culture*

The MCF-7 human breast adenocarcinoma cell line was procured via the American Type Culture Collection (ATCC, Manassas, VA, USA). Roswell Park Memorial Institute medium (RPMI-1640) supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin (Sigma-Aldrich, St. Louis, MO, USA) was used for the cultivation of MCF-7 and this were subsequently cultured in a humidified incubator using 5% CO2 at 37 ◦C.

#### *4.5. MTT Cell Proliferation Assay*

Briefly, 24 h prior to treatment, MCF-7 cells (5 <sup>×</sup> <sup>10</sup><sup>4</sup> cells/mL) were seeded in a 96-well plate. Dissolved compounds in RPMI were used in various concentrations (from 7.8 to 500 µg/mL). After 72 h in each well of plates was added 20 µL of MTT solution and then plates were incubated for further 4 h. In the next step, 150 µL of DMSO was put into each well and incubated for 10 min to solve the purple formazan crystals. The dose-response curves were mapped to obtain IC<sup>50</sup> values and identify the best active fractions or pure compound.

### *4.6. High-Performance Thin Layer Chromatography (HPTLC) Analysis*

High-performance thin-layer chromatography is an improved form of the normal thin-layer chromatography. Several augmentations can be made to the basic method of thin-layer chromatography to automate the different steps, increase the resolution achieved and allow more accurate quantitative measurements. In this method 10 tracks were applied on the TLC plate with silica gel 60 (for 10 × 10 cm) using micro syringe. The plates were saturated for 20 min in a twin trough glass chamber with the mobile phase of methanol (100%). The plates were placed in the mobile phase and dried. A densitometric scanning of plates were performed at 254 nm and 320 nm using a Camag TLC scanner III operated in reflectance–absorbance mode. To examine the chemical profiling of F13b1/PV-EA, analysis was carried out using a C<sup>18</sup> column on a Breeze2 system. First, 20 µL of blended standard (gallic acid, cyaniding and flavonoid quercetin) was injected to the column with water and acetonitrile solvent. Subsequently, 20 µL of standard of gallic acid and finally 20 µL of compound or F13b1/PV-EA was injected to the column. For comparing the three injections' properties and identification of the chemical profile of our compound, after each injection, the retention time (RT) or the amount of time that a compound spends on the column from injection to detection, was calculated.

### *4.7. Flow Cytometry Analysis*

In the next step, three different concentrations (8, 16 and 32 µg/mL) of F13b1/PV-EA was added for 2 days to MCF-7 cells that were seeded (5 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/well) in a 35 mm dish for 24 h. Then nuclear fractions from the cells were obtained according to the kit's propidium iodide staining protocol. The intensity of the fluorescence was detected using a FAC Scan flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed via Cell Quest software.

#### *4.8. Acridine Orange*/*Propidium Iodide Staining (AO*/*PI)*

After a 24-h incubation of the 1 <sup>×</sup> <sup>10</sup><sup>6</sup> cells/well of MCF-7 cells in a 6-well plate, the cells were treated with 8, 16 and 32 µg/mL of F13b1/PV-EA for 48 h. Then detached cells were dyed with AO/PI stain according to manufacturer's protocol and examined using fluorescence microscope.

#### *4.9. Hoechst 33342 Staining*

Using a functional vital dye the classical morphological criteria, the quantification and determination of cell death notation was carried out. The MCF-7 cells were treated using three different concentrations (8, 16 and 32 µg/mL) of F13b1/PV-EA for 48 h. Hoechst 33342, which is a specific stain used for AT-rich regions of double-stranded DNA was utilized. The cells were incubated for 15 min with Hoechst 33342 dye (5 µg/mL in PBS)) and subsequently visualized using a BHZ, RFCA microscope (Olympus, Tokyo, Japan) equipped with a fluorescent light source with an excitation wavelength of 330 nm and a barrier filter of 420 nm.

#### *4.10. Gene Expression Assay*

By employing the RT-PCR, the gene expression of *Bax*, *Bcl2*, *Caspase 3*, *Caspase 8*, *CAT* and *SOD* were analyzed. RNA was extracted from MCF-7 cells (3 <sup>×</sup> <sup>10</sup><sup>6</sup> cells/well) that were treated with 15 µg/mL of F13b1/PV-EA for 24 h, using the manufacturer's instructions for RNA extraction. The mRNA was transcribed in reverse to cDNA by adhering to the manufacturer's protocol using the Advantage RT-PCR kit. cDNA was amplified via a real time and sybr green kit. Table 3 shows the specific primers used for amplifying the cDNA.

#### *4.11. DPPH Radical Scavenging Assay*

The free radical scavenging activity of F13b1/PV-EA was assessed based on its effect trapping 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals. 0.1 mM methanolic DPPH solution was mixed with the varying concentrations of F13b1/PV-EA (125, 250, 500 and 1000 µg/mL), in an equal volume. After 30 min of incubation, the absorbance of the samples was read at 517 nm. In the control group water and BHA was used as a standard compound. The percentage of inhabitation of DPPH free radical was calculated according to the formula below:

Percentage of inhibition of DPPH free radical = Absorbance of control − Absorbance of sample/Absorbance of control × 100


**Table 3.** Primer sequence for amplifying cDNA.

#### *4.12. ABTS Radical Scavenging Assay*

In brief 1 mL of various concentrations of F13b1/PV-EA (125, 250, 500 and 1000 µg/mL), were mixed with 1 mL of ABTS·+ working solution. After incubation period of 1 h in room temperature in the dark, absorbance was read at 734 nm. To prepare the ABTS·+ stock solution, 7 mM of ABTS and 2.45 mM of potassium persulfate were mixed, incubated at room temperature for 12–16 h and finally ABTS·+ stock solution was diluted with distilled water to gain 0.70 ± 0.02. Percentage of inhibition of ABTS free radical was calculated according to formula in previous part.

#### *4.13. Experimental Animals*

The experiments on the animal were divided into 2 parts. First, the lethal dose 50% (LD50) test and after that in vivo anti-tumor assessment was carried out. For LD<sup>50</sup> testing and to determine the acute toxicity of the proposed compounds, 18 healthy male Balb/C mice (25 ± 5 g, five-weeks-old) were provided by the animal house of the University of Malaya Animal Experimental Unit (AEU), in clean, sterile and polyvinyl cages. The mice were maintained under standard conditions, temperature of 22–26 ◦C, 45–50% relative humidity with water, food and sterile diet under pathogen-free environment and maintained on a 12 h light/dark photo period. For the second part or in vivo anti-tumor assessment, 24 healthy female Balb/C mice were purchased from Pasteur Institute of Mashhad (Iran). The mice were maintained under conditions that were mentioned above. The animal studies were performed after approval of the protocol by the FOM Institutional Animal Care and Use Committee, University of Malaya (FOM, IACUC, ethic No.: 2016-190405/IBS/R/MS).
