4.13.1. Lethal Dose 50% (LD50) Test

LD<sup>50</sup> is the amount of the substance (usually per body weight) required to kill 50% of the test population within a specific time. In order to observe the overall effect of our compound on a living subject, 18 male Balb/C mice were divided into three groups of animals in each group. Every group was treated with one concentration of F13b1/PV-EA, 1 time every week until 2 weeks, for a total of two times. Three doses (12.5, 25 and 50 µg/mL) of pure compound was dissolved in 10% tween 20 and given orally by gavage to the mice. For purpose of calculating the consumable dose (dosage compound for each mouse) we used the formula below:

(Concentration of the compound) × (mouse body weight) × 6 (the rate of metabolism mice to the human) = consumable dose

Animals were monitored for 30 min, 2, 4, 8, 24 and 48 h for up to two weeks after the dosing. Any signs to the animals involving toxicity and/or mortality and behavior changes were observed keenly and recorded throughout the experimental period for 14 days.

#### 4.13.2. In Vivo Anti-Tumor Assessment

To construct an allograft breast carcinoma model, Tubo cancer cell lines were grown and harvested under appropriate conditions. Tubo cells are a cloned cell line established in vitro from a BALB-neuT mouse mammary carcinoma. 1.5 <sup>×</sup> <sup>10</sup><sup>6</sup> cells were suspended in 0.2 mL PBS and were injected this subcutaneously into the right flank of BALB/c mice (*n* = 6). After the tumor inoculation (approximately 7 days later) the animals were randomly divided into four groups of six mice:

Group 1: Negative control (just received 10% tween 20 orally) once daily for 2 weeks.

Group 2: Standard drug control group as a positive control (Tubo induced + tamoxifen 10 mg/kg dissolved in 10% tween 20 via oral administration) once daily for 2 weeks.

Group 3: Experimental group A (Tubo induced + low dose of compound dissolved in 10% Tween 20 via oral administration) once daily for 2 weeks.

Group 4: Experimental group B (Tubo induced + high dose of compound dissolved in 10% Tween 20 via oral administration) once daily for 2 weeks.

For calculating the consumable dose of F13b1/PV-EA in the experimental groups, we used two concentrations (12.5 and 25 µg/mL) based on the LD<sup>50</sup> result. The mice were euthanized at the end of 14 days treatment period and the tumors were then excised and weighted (Figure 20). The tumor volumes were measured according to the formula below:

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

where A: length, B: width *Molecules* **2020**, *25*, x 19 of 21

**Figure 20.** In vivo anti-tumor assessment in mouse. (**A**) breast cancer injection; (**B**) oral gavage treatment; (**C**, **D**) tumor isolation and (**E**) tumor mass. **Figure 20.** In vivo anti-tumor assessment in mouse. (**A**) breast cancer injection; (**B**) oral gavage treatment; (**C**,**D**) tumor isolation and (**E**) tumor mass.

4.13.3. Histological Analysis and Scoring Method H & E staining was performed in order to analyze the histopathological samples.

#### After separating tumor from the body, it was instantly was fixed in 10% formalin overnight, 4.13.3. Histological Analysis and Scoring Method

*4.14. Statistical Analysis*

**5. Conclusions**

embedded in paraffin, cut into 4 µm sections and stained with hematoxylin-eosin (H&E). Tissue scoring was conducted according to Elston and Ellis method [25], with some slight modifications. Randomly five different sections from each H & E slide view at 100× magnification and mean score is calculated from these five sections and the scoring is referred to in Table 4. Only structures which could not be misinterpreted as anything except mitotic figures were taken into account. For apoptosis only hyperchromatin or pyknotic nuclei with hollow signs were counted: After separating tumor from the body, it was instantly was fixed in 10% formalin overnight, embedded in paraffin, cut into 4 µm sections and stained with hematoxylin-eosin (H & E). Tissue scoring was conducted according to Elston and Ellis method [25], with some slight modifications. Randomly five different sections from each H & E slide view at 100× magnification and mean score is calculated from these five sections and the scoring is referred to in Table 4. Only structures which

> Mitotic index = The numbers of mitotic cells/the number of total cells Apoptotic index = The numbers of apoptotic cells/the number of total cells

**Apoptotic Count Score Mitotic Count Score** 0–4 1 <25 1 5–10 2 25–50 2 11–15 3 51–100 3 >15 4 >100 4 Adapted from Elston and Ellis [25], with some slight modifications.

For statistical assessment t-test was applied for non-dose responses. Dose response experiments were studied using the Omnibus test followed by Rodger's method. All values are declared as mean

To conclude, the results from the present study provide an understanding of the cytotoxic effect of *Pistacia vera* red hull. Cytotoxicity studies of *Pistacia vera* red hull ethyl acetate (PV-EA) extract on different cancer cell lines and subsequent chemical purification of bioactive PV-EA have led to the isolation of gallic acid and quercetin. Purified compound (F13b1/PV-EA) was shown to possess cytotoxic effects against MCF-7 cells. This finding may have an impact on the future of cancer

± S.D. Probability values \* *p* < 0.05 was considered as statistically significant.

could not be misinterpreted as anything except mitotic figures were taken into account. For apoptosis only hyperchromatin or pyknotic nuclei with hollow signs were counted:

Mitotic index = The numbers of mitotic cells/the number of total cells

Apoptotic index = The numbers of apoptotic cells/the number of total cells


**Table 4.** Scoring method for apoptosis and mitotic indexes.

Adapted from Elston and Ellis [25], with some slight modifications.

#### *4.14. Statistical Analysis*

For statistical assessment *t*-test was applied for non-dose responses. Dose response experiments were studied using the Omnibus test followed by Rodger's method. All values are declared as mean ± S.D. Probability values \* *p* < 0.05 was considered as statistically significant.
