**In Vitro and In Vivo Anticancer Activity of the Most Cytotoxic Fraction of Pistachio Hull Extract in Breast Cancer**

#### **Maryam Seifaddinipour <sup>1</sup> , Reyhaneh Farghadani <sup>2</sup> , Farideh Namvar 3,\*, Jamaludin Bin Mohamad 1,\* and Nur Airina Muhamad 1,\***


Academic Editors: Severina Pacifico and Simona Piccolella Received: 17 January 2020; Accepted: 11 March 2020; Published: 13 April 2020

**Abstract:** Pistacia (*Pistacia vera*) hulls (PV) is a health product that has been determined to contain bioactive phytochemicals which have fundamental importance for biomedical use. In this study, PV ethyl acetate extraction (PV-EA) fractions were evaluated with the use of an MTT assay to find the most cytotoxic fraction, which was found to be F13b1/PV-EA. After that, HPTLC was used for identify the most active compounds. The antioxidant activity was analyzed with DPPH and ABTS tests. Apoptosis induction in MCF-7 cells by F13b1/PV-EA was validated via flow cytometry analysis and a distinctive nuclear staining method. The representation of genes like *Caspase 3*, *Caspase 8*, *Bax*, *Bcl-2*, *CAT* and *SOD* was assessed via a reverse transcription (RT\_PCR) method. Inhabitation of Tubo breast cancer cell development was examined in the BALB-neuT mouse with histopathology observations. The most abundant active components available in our extract were gallic acid and the flavonoid quercetin. The F13b1/PV-EA has antiradical activity evidence by its inhibition of ABTS and DPPH free radicals. F13b1/PV-EA displayed against MCF-7 a suppressive effect with an IC<sup>50</sup> value of 15.2 ± 1.35 µg/mL. Also, the expression of *CAT*, *SOD*, *Caspase 3*, *Caspase 8* and *Bax* increased and the expression of *Bcl-2* decreased. F13b1/PV-EA dose-dependently inhibited tumor development in cancer-induced mice. Thus, this finding introduces F13b1/PV-EA as an effectual apoptosis and antitumor active agent against breast cancer.

**Keywords:** pistacia (*Pistacia vera*) hulls; breast cancer; anticancer

### **1. Introduction**

The pistachio hull refers to the epicarp which has a reddish/yellow color during development and when it ripens, it is a rosy and light yellow [1,2]. Usually, collected pistachio nuts are encased in this shell which is removed by a dehulling process. During the pistachio dehulling process many types of by-products are generated that are currently considered as an agriculture waste and to a lesser extent, are used as fodder by local livestock farmers. Hulls may also be used as an herbal medicine for stomach pains and the prevention of diarrhea and to improve hemorrhoids. Pistachio hull has caught the attention of researchers in recent years due to its natural phenolic and antioxidant compounds. Recent literature has proven that pistachio hull extracts have antioxidant, antimicrobial and antimutagenicity activities. Several reports have validated and established the pharmacological activities and medicinal properties of pistachio hull [1,3,4]. In a report by Tomaino the antioxidant

activity of the polyphenols extracts from natural shelled pistachios (NP) was determined. In the rats treated with NP a remarkable decrease was observed for CAR-induced histological paw damage, nitrotyrosine formation and neutrophil infiltration. These results demonstrated that the polyphenols display antioxidant properties in lower doses [1].

In Goli [4] report pistachio hulls were extracted with three different solvents (water, methanol and ethyl acetate) and its total phenol content were determined using the Folin–Ciocalteu method. Additionally, the effect of water and methanolic extracts on the stability of soybean oil that was heated to 60 ◦C was ascertained. The pistachio hull extract (PHE) slowed down the process of oil deterioration at 60 ◦C with a concentration-dependent increase between 0.02–0.06%. The 0.06% PHE showed a similar activity pattern to butylhydroxyanisole (BHA) and butylated hydroxytoluene (BHT) of. Thus, pistachio hulls, which at the moment are mainly considered as agricultural waste, contain antioxidants that may be compatible for adding them to food products [4].

It should be noted that the content of antioxidant compounds could vary depending on the extraction procedures adopted. Indeed, for pistachio it has been demonstrated by Garavand et al. [5], who studied and measured the phytochemical substances and radical scavenging activity of pistachio hull extracts, obtained using diverse solvents (water, ethanol, and butanol). Their results showed that ultrasound-assisted aqueous extraction of the hull using ultrasound power (35 kHz) was more effective in increasing the phytochemicals content than a sonochemical ultrasonication method (130 kHz). The amount of vanillic acid, *p*-coumaric acid, naringenin, and catechin in the ultrasound-assisted extracts increased as demonstrated by high-performance liquid chromatography-mass spectrometry. The content of phenolics and antioxidant properties of the aqueous extract decreased remarkably after post-extraction sonication. Contrariwise, the amount of phenolics and flavonoids improved with microwave-assisted extraction in a power-dependent trend [5]. Grace et al. [6] described the presence of anacardic acids, fatty acids, carotenoids, tocopherols and phytosterols as the main components in pistachio hulls. Quercetin-3-*O*-glucoside together with smaller concentrations of quercetin, myricetin and luteolin flavonoids were found in a polar (P) extract. Gallotannins and other phenolic compounds esterified with a gallic acid moiety were characterized in the P extract. Release of nitric oxide (NO) and reactive oxygen species (ROS) were inhibited by the P extract in lipopolysaccharide-stimulated RAW 264.7 macrophage cells. In addition, in the macrophages the non-mitochondrial oxidative burst associated with inflammatory response were reduced by the P extract [6].

Bulló, et al. [7] also investigated the anti-inflammatory properties of polyphenol extracts from natural raw shelled pistachios (NP). For the determination of the amount of protection offered by NP against lipopolysaccharide (LPS)-induced inflammation, the monocyte/macrophage cell line J774 was utilized. The in vitro study illustrated that pre-treatment with NP decreased the TNF-α and IL-1β production and degradation of IκB-α, although not significantly. These results show that, at lower doses, the polyphenols present in pistachios possess anti-inflammatory properties [7].

The hulls of pistachio have been shown to have in vitro antioxidant and in vivo photoprotective effects [2], and also exhibit antimicrobial and antimutagenicity [8] as well as enzyme inhibitory and also possess radical scavenging activities [4].

Some phytochemical assessments have revealed the presence of wide ranging levels of phenolic and flavonoids compounds such as gallic acid, catechin, cyanidin-3-*O*-galactoside, eriodictyol-7-*O*-glucoside and epicatechin in the skin of pistachio, which is even 10 times richer than the seeds [9].

In our previous study, we have demonstrated a promising cytotoxic effect and anti-angiogenesis potential of the ethyl acetate extract from pistachio (*Pistacia vera*) hulls (PV-EA) against MCF-7 breast cancer cells [10]. Therefore, in the current study, we have taken this research a step further and investigated the anticancer activity of the most cytotoxic fraction of PV-EA through the utilization of in vitro and in vivo models of breast cancer.

#### **2. Results** *Molecules* **2020**, *25*, x 3 of 21 *Molecules* **2020**, *25*, x 3 of 21

#### *2.1. Separation of the Bioactive Compound 2.1. Separation of the Bioactive Compound 2.1. Separation of the Bioactive Compound*

Dried hulls of *Pistacia vera* were extracted with ethyl acetate. The ethyl acetate extract (16 g) was fractionated in three steps by column chromatography on silica gel 60, which yielded some fractions in each step. After doing an MTT assay and choosing the most cytotoxic fraction in every step, we continued with the next step until the isolation and purification of final fraction (F13b1/PV-EA) that was about 10 mg with an IC<sup>50</sup> 15.2 µg/mL (Figure 1). Preparative HPTLC using 100% methanol as the mobile phase and silica gel as the stationary phase with 10 concentrations or tracks was done. The image and spectrum of spots were scanned at two wavelengths (254 and 320 nm). All spectra were the same and in an identical region (Figures 2–4). Chemical profiling of F13b1/PV-EA was investigated by the use of HPTLC again. After comparing retention times, first with blended standards (gallic acid, cyanidin and the flavonoid quercetin and a second time only with gallic acid and the purified compound from pistachio it was found that gallic acid and quercetin were present in the F13b1/PV-EA fraction (Figure 5). Dried hulls of *Pistacia vera* were extracted with ethyl acetate. The ethyl acetate extract (16 g) was fractionated in three steps by column chromatography on silica gel 60, which yielded some fractions in each step. After doing an MTT assay and choosing the most cytotoxic fraction in every step, we continued with the next step until the isolation and purification of final fraction (F13b1/PV-EA) that was about 10 mg with an IC<sup>50</sup> 15.2 µg/mL (Figure 1). Preparative HPTLC using 100% methanol as the mobile phase and silica gel as the stationary phase with 10 concentrations or tracks was done. The image and spectrum of spots were scanned at two wavelengths (254 and 320 nm). All spectra were the same and in an identical region (Figures 2,3 and 4). Chemical profiling of F13b1/PV-EA was investigated by the use of HPTLC again. After comparing retention times, first with blended standards (gallic acid, cyanidin and the flavonoid quercetin and a second time only with gallic acid and the purified compound from pistachio it was found that gallic acid and quercetin were present in the F13b1/PV-EA fraction (Figure 5). Dried hulls of *Pistacia vera* were extracted with ethyl acetate. The ethyl acetate extract (16 g) was fractionated in three steps by column chromatography on silica gel 60, which yielded some fractions in each step. After doing an MTT assay and choosing the most cytotoxic fraction in every step, we continued with the next step until the isolation and purification of final fraction (F13b1/PV-EA) that was about 10 mg with an IC<sup>50</sup> 15.2 µg/mL (Figure 1). Preparative HPTLC using 100% methanol as the mobile phase and silica gel as the stationary phase with 10 concentrations or tracks was done. The image and spectrum of spots were scanned at two wavelengths (254 and 320 nm). All spectra were the same and in an identical region (Figures 2,3 and 4). Chemical profiling of F13b1/PV-EA was investigated by the use of HPTLC again. After comparing retention times, first with blended standards (gallic acid, cyanidin and the flavonoid quercetin and a second time only with gallic acid and the purified compound from pistachio it was found that gallic acid and quercetin were present in the F13b1/PV-EA fraction (Figure 5).

**Figure 1.** Flow chart from the three steps of bioassay guided fractionation of F13b/PVLH-EAE. **Figure 1.** Flow chart from the three steps of bioassay guided fractionation of F13b/PVLH-EAE. **Figure 1.** Flow chart from the three steps of bioassay guided fractionation of F13b/PVLH-EAE.

**Figure 2.** The image and spectrum of all 10 HPTLC spots scanned at 254 nm wavelength on 2 sides **A** and **B**. **Figure 2.** The image and spectrum of all 10 HPTLC spots scanned at 254 nm wavelength on 2 sides **A** and **B**. **Figure 2.** The image and spectrum of all 10 HPTLC spots scanned at 254 nm wavelength on 2 sides (**A**) and (**B**).

*Molecules* **2020**, *25*, x 4 of 21

*Molecules* **2020**, *25*, x 4 of 21

*Molecules* **2020**, *25*, x 4 of 21

**Figure 3.** The image and spectrum of all 10 HPTLC spots scanned at 320 nm wavelength on 2 sides **A** and **B**. **Figure 3.** The image and spectrum of all 10 HPTLC spots scanned at 320 nm wavelength on 2 sides (**A**) and (**B**). **Figure 3.** The image and spectrum of all 10 HPTLC spots scanned at 320 nm wavelength on 2 sides **A** and **B**. **Figure 3.** The image and spectrum of all 10 HPTLC spots scanned at 320 nm wavelength on 2 sides and **B**.

**Figure 4.** The image and spectrum of all 10 spots scanned at **A**) 254 nm and **B**) 320 nm wavelengths. **Figure 4. Figure 4.** The image and spectrum of all 10 spots scanned at ( The image and spectrum of all 10 spots scanned at **A A** ) 254 nm and ( ) 254 nm and **B B** ) ) 320 nm wavelengths. 320 nm wavelengths. **Figure 4.** image and spectrum of all 10 spots scanned at ) nm and **B**) 320 nm wavelengths.

**Figure 5.** *Cont.*

*Molecules* **2020**, *25*, x 5 of 21

**Figure 5.** HPTLC analysis. **A**) Injection of blended standard and, **B**) Injection of gallic acid standard, **C**) injection of F13b1/PV-EA to the column. **Figure 5.** HPTLC analysis. (**A**) Injection of blended standard and, (**B**) Injection of gallic acid standard, (**C**) injection of F13b1/PV-EA to the column.

#### *2.2. Cytotoxic effect of F13b1/PV-EA toward MCF-7 Cells 2.2. Cytotoxic e*ff*ect of F13b1*/*PV-EA toward MCF-7 Cells*

An evaluation of the cytotoxic properties of F13b1/PV-EA in the MCF-7 cell line was performed using the prescribed MTT assay. An evaluation of the cytotoxic properties of F13b1/PV-EA in the MCF-7 cell line was performed using the prescribed MTT assay.

\*\*\* \*\*\* \*\*\* \*\*\* \*\*\* \*\*\* \*\*\* **60 80 100 120 % Viability of MCF7 cells** Different concentrations ranging from 7.8 to 250 µg/mL of the compound were used and the amount of formazan formed was specified and detected after 24, 48 and 72 h of incubation. Figure 6 display that F13b1/PV-EA resulted in dose-dependent and time-dependent decline in cell viability with increasing concentration and treatment period. The results suggest that cell growth was prevented when the cells were incubated in the presence of the compound.

\*\*\*

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#### **40** *2.3. Apoptotic Morphological Variations*

were done in triplicate.

**Figure 6.** Shows the growth inhibition effects of F13b1/PV-EA on MCF-7 cells noted at different **Control 7.8 15.6 31.2 62.5 125 250 24h 100 87.4 84.9667 51.7033 35.0533 28.84 16.63 48h 100 69.7667 54.17 33.9167 12.4933 10.8933 7.0933 72h 100 66.5667 51.4 29.1767 4.4333 2.4733 2.02** \*\*\* \*\*\* \*\*\* \*\*\* \*\*\* \*\*\* **0 20 Concentrations (µg/ml)** Figure 7 shows the results acquired after performing the AO/PI tests. From the data, it can be seen that the compound has dose-dependent effects on cell viability and induces apoptotic morphological variations in treated cells. The results show reduced viability as more apoptotic cells (red in color) were seen at all three concentrations of treatment. In addition, Hoechst 33342 staining (Figure 8), also revealed that the F13b1/PV-EA stimulates apoptotic morphological variations. The cells underwent amazing nuclear changes when treated. However, in the untreated group, the cells were uniformly stained by the fluorescence Hoechst dye indicating the nuclei of the cells were virgin. However, with increasing concentration level of the compound, there was an increase of intensity captured on fluorescence signals and luminous points where the cells expressed apoptotic morphological variations.

intervals (24, 48 and 72 h) and concentrations. (\*\*\* *p* value < 0.001). All of the in vitro experiments

using the prescribed MTT assay.

**C**) injection of F13b1/PV-EA to the column.

*2.2. Cytotoxic effect of F13b1/PV-EA toward MCF-7 Cells*

**Figure 5.** HPTLC analysis. **A**) Injection of blended standard and, **B**) Injection of gallic acid standard,

**Figure 6.** Shows the growth inhibition effects of F13b1/PV-EA on MCF-7 cells noted at different intervals (24, 48 and 72 h) and concentrations. (\*\*\* *p* value < 0.001). All of the in vitro experiments were done in triplicate. **Figure 6.** Shows the growth inhibition effects of F13b1/PV-EA on MCF-7 cells noted at different intervals (24, 48 and 72 h) and concentrations. (\*\*\* *p* value < 0.001). All of the in vitro experiments were done in triplicate. However, with increasing concentration level of the compound, there was an increase of intensity captured on fluorescence signals and luminous points where the cells expressed apoptotic morphological variations.captured on fluorescence signals and luminous points where the cells expressed apoptotic morphological variations.

However, with increasing concentration level of the compound, there was an increase of intensity

**Figure 7.** Fluorescent images of MCF-7 cells dyed by AO/PI. Untreated cells (×200) and treated with three concentration of F13b1/PV-EA for 48 h (×200). **Figure 7.** Fluorescent images of MCF-7 cells dyed by AO/PI. Untreated cells (×200) and treated with three concentration of F13b1/PV-EA for 48 h (×200). **Figure 7.** Fluorescent images of MCF-7 cells dyed by AO/PI. Untreated cells (×200) and treated with three concentration of F13b1/PV-EA for 48 h (×200).

**Figure 8.** Fluorescent images of Hoechst 33,342 stained MCF-7 cells. Untreated cells (×200) and cells **Figure 8.** Fluorescent images of Hoechst 33,342 stained MCF-7 cells. Untreated cells (×200) and cells treated with 8, 16 and 32 µg/mL of F13b1/PV-EA for 48 h (×200). **Figure 8.** Fluorescent images of Hoechst 33342 stained MCF-7 cells. Untreated cells (×200) and cells treated with 8, 16 and 32 µg/mL of F13b1/PV-EA for 48 h (×200).

treated with 8, 16 and 32 µg/mL of F13b1/PV-EA for 48 h (×200).

#### *2.4. Flow Cytometer Analysis 2.4. Flow Cytometer Analysis*

By utilizing PI staining, we tried to establish whether MCF-7 cells treated with F13b1/PV-EA underwent apoptosis accompanied by alteration in the cell cycle, and the distribution index was also noted. This was in tandem with growth in the Sub-G1 population with increasing concentrations as shown in Figure 9. As depicted in the mentioned figure, high concentration treatment with the compound (32 µg/mL) led to a growth in the percentage of Sub-G1 phase up to 62.1% ± 0.41 when compared to the control cells which were at 2.8% ± 0.86%, thus indicating a change in arrested cells towards a Sub-G1 population which is known as apoptotic cells. The population of cells that possesses sub-diploid DNA content is a clear indication of DNA fragmentation happening at the time of apoptosis. By utilizing PI staining, we tried to establish whether MCF-7 cells treated with F13b1/PV-EA underwent apoptosis accompanied by alteration in the cell cycle, and the distribution index was also noted. This was in tandem with growth in the Sub-G1 population with increasing concentrations as shown in Figure 9. As depicted in the mentioned figure, high concentration treatment with the compound (32 µg/mL) led to a growth in the percentage of Sub-G1 phase up to 62.1% ± 0.41 when compared to the control cells which were at 2.8% ± 0.86%, thus indicating a change in arrested cells towards a Sub-G1 population which is known as apoptotic cells. The population of cells that possesses sub-diploid DNA content is a clear indication of DNA fragmentation happening at the time of apoptosis. *Molecules* **2020**, *25*, x 7 of 21 *2.4. Flow Cytometer Analysis* By utilizing PI staining, we tried to establish whether MCF-7 cells treated with F13b1/PV-EAunderwent apoptosis accompanied by alteration in the cell cycle, and the distribution index was also noted. This was in tandem with growth in the Sub-G1 population with increasing concentrations as

*Molecules* **2020**, *25*, x 7 of 21

**Figure 9.** MCF-7 cell cycle analysis of untreated cells (×200) and cells treated with 8, 16 and 32 µg/mL of F13b1/PV-EA for 48-h interval (×200). **Figure 9.** MCF-7 cell cycle analysis of untreated cells (×200) and cells treated with 8, 16 and 32 µg/mL of F13b1/PV-EA for 48-h interval (×200).

#### *2.5. RT- PCR Evaluation 2.5. RT-PCR Evaluation*

The link of some genes containing *Caspase 3, Caspase 8*, *Bax*, *Bcl-2*, *CAT* and *SOD* with apoptosis induced by F13b1/PV-EA were observed using RT-PCR. The link of some genes containing *Caspase 3*, *Caspase 8*, *Bax*, *Bcl-2*, *CAT* and *SOD* with apoptosis induced by F13b1/PV-EA were observed using RT-PCR.

\*\*\* \*\*\* **5 6 7** As explained in Figures 10 and 11, *Caspase 3*, *Caspase 8*, *CAT*, *Bax* and *SOD* gene expressions increased, respectively, when compared to the control (gene expression in cancer cells without any treatment). Further investigation revealed that the compound treatment lowered the expression level of *Bcl-2* over time. These results show that F13b1/PV-EA could stimulate apoptosis by shifting the regulation of apoptotic genes exclusively through the up-regulation of *Bax* and down-regulation of *BCL-2*. **Figure 9.** MCF-7 cell cycle analysis of untreated cells (×200) and cells treated with 8, 16 and 32 µg/mL of F13b1/PV-EA for 48-h interval (×200). *2.5. RT- PCR Evaluation* The link of some genes containing *Caspase 3, Caspase 8*, *Bax*, *Bcl-2*, *CAT* and *SOD* with apoptosis induced by F13b1/PV-EA were observed using RT-PCR.

**Figure 10.** The *Bax*, *Bcl-2*, *Caspase 3* and *Caspase 8* genes expression of MCF-7 cells treated with 15 µg/mL of F13b1/PV-EA for 24 h. (\* *p* < 0.05, \*\*\* *p* < 0.001). **Figure 10.** The *Bax*, *Bcl-2*, *Caspase 3* and *Caspase 8* genes expression of MCF-7 cells treated with 15 µg/mL of F13b1/PV-EA for 24 h. (\* *p* < 0.05, \*\*\* *p* < 0.001).

**8**

**10**

**12**

**14**

*Molecules* **2020**, *25*, x 8 of 21

\*\*\*

*Molecules* **2020**, *25*, x 8 of 21

**Figure 11.** The *CAT* and *SOD* genes expression of MCF-7 cells treated with 15 µg/mL of F13b1/PV-EA for 24 h. (\*\*\* *p* value < 0.001). **Figure 11.** The *CAT* and *SOD* genes expression of MCF-7 cells treated with 15 µg/mL of F13b1/PV-EA for 24 h. (\*\*\* *p* value < 0.001). regulation of apoptotic genes exclusively through the up-regulation of *Bax* and down-regulation of *BCL-2*.

#### As explained in Figures 10 and 11, *Caspase 3, Caspase 8*, *CAT*, *Bax* and *SOD* gene expressions *2.6. Analysis of Radical Scavenging E*ff*ect 2.6. Analysis of Radical Scavenging Effect*

increased, respectively, when compared to the control (gene expression in cancer cells without any treatment). Further investigation revealed that the compound treatment lowered the expression level of *Bcl-2* over time. These results show that F13b1/PV-EA could stimulate apoptosis by shifting the regulation of apoptotic genes exclusively through the up-regulation of *Bax* and down-regulation of *BCL-2*. *2.6. Analysis of Radical Scavenging Effect* Examination to evaluate the antioxidant activity of F13b1/PV-EA, ABTS and DPPH free radical scavenging activity was performed. Figure 12 shows that F13b1/PV-EA demonstrated antiradical activity by inhibiting ABTS radical with IC<sup>50</sup> values less than 125 µg/mL. F13b1/PV-EA displayed a dose-dependent activity and the ABTS scavenging effect has measured at 63% at a concentration of 125 µg/mL. In addition, pure compound displayed a dose-dependent activity and the DPPH scavenging effect was 38.8% at a concentration of 1000 µM. F13b1/PV-EA thus displayed a moderate inhibitory effect on DPPH free radicals (Figure 13). Examination to evaluate the antioxidant activity of F13b1/PV-EA, ABTS and DPPH free radical scavenging activity was performed. Figure 12 shows that F13b1/PV-EA demonstrated antiradical activity by inhibiting ABTS radical with IC<sup>50</sup> values less than 125 µg/mL. F13b1/PV-EA displayed a dose-dependent activity and the ABTS scavenging effect has measured at 63% at a concentration of 125 µg/mL. In addition, pure compound displayed a dose-dependent activity and the DPPH scavenging effect was 38.8% at a concentration of 1000 µM. F13b1/PV-EA thus displayed a moderate inhibitory effect on DPPH free radicals (Figure 13).

**40**

**125 250 500 1000 0 20 Figure 12.** Inhibition activity of F13b1/PV-EA and comparison of a BHA group with treated samples. Data are expressed as mean ± standard division. **Figure 12.** Inhibition activity of F13b1/PV-EA and comparison of a BHA group with treated samples. Data are expressed as mean ± standard division.

**BHA 80.5333 84.6333 89.8 96.1333 Sample 63.9333 94.0667 95.8167 99.2667**

**Concentrations (µg/ml)**

### *2.7. Animal Study*
