**1. Introduction**

Breast cancer is a major public health problem, representing the highest incidence rate of death for women [1,2]. The interest in products of natural origin from plants with different pharmacological activities that can be used in chemotherapy is increasingly broad. Many natural compounds have shown anticancer activities [3]. The most famous antineoplastic drug, paclitaxel, used for the treatment of breast cancer, was isolated from the bark of *Taxus brevifolia* Nutt. [4]. Furthermore, among the natural products, there are aloe-emodin, which is isolated from the root of *Rheum palmatum* L. and the leaves of aloe vera. Numerous in vitro studies have demonstrated that aloe-emodin is able to reduce the viability and proliferation of different human cancer cell lines, inducing apoptotic cell death and inhibiting adhesion and the migration process [5,6]. Another compound is curcumin, the active ingredient of turmeric, which is a polyphenolic compound with a broad range of medicinal properties, such as antibreast-cancer activity. Curcumin is able to enhance the effects of chemotherapeutic agents such as paclitaxel [3]. A natural stilbene and nonflavonoid polyphenol, resveratrol is present in grapes, peanuts and red wine. This natural product possesses anti-inflammatory, antioxidant, cardioprotective and anticancer properties [7]. Moreover, a synergistic effect has been reported by the combination of genistein

**Citation:** Colone, M.; Maggi, F.; Rakotosaona, R.; Stringaro, A. *Vepris macrophylla* Essential Oil Produces Notable Antiproliferative Activity and Morphological Alterations in Human Breast Adenocarcinoma Cells. *Appl. Sci.* **2021**, *11*, 4369. https:// doi.org/10.3390/app11104369

Academic Editor: Hari Prasad Devkota

Received: 2 April 2021 Accepted: 7 May 2021 Published: 12 May 2021

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and doxorubicin [8]. Additionally, rosemary (*Rosmarinus officinalis* L.) extract can increase the activity of the anti-breast-cancer agents tamoxifen, trastuzumab, and paclitaxel [9]. Currently, one of the most interesting approaches in the fight against this tumor comprises new therapeutic strategies using natural products in combination with synthetic drugs in order to increase the therapeutic index of the drug and reduce the many undesirable side effects caused by the doses used in typical chemotherapy protocols. After chemotherapy or radiotherapy, a number of adverse effects in patients occur [10]. A natural flavonoid, quercetin has anti-inflammatory and antioxidative effects. After high dose chemotherapy, quercetin, administered in capsules of 250 mg (twice daily for four weeks), can reduce oral mucositis events in patients with blood malignancy [11]. A combination of curcumin and α-tocopherol regulates rat liver enzymes via inhibition of oxidative stress, revealing protection against cisplatin-induced hepatotoxicity [12]. Naringenin, a natural flavanone isolated from *Thymus vulgaris* L., has growth inhibitory and chemosensitization effects on human breast and colorectal cancer [13]. Naringenin can reduce the nephrotoxicity induced by daunorubicin treatment in rats [14]. Several drugs derived from natural products have already received clinical approval, and many are currently undergoing in clinical trials. To date, the Food and Drug Administration (FDA) has approved the administration of resveratrol and quercetin [15].

Essential oils (EOs) are products obtained from vegetable raw material [16]. They are complex, multicomponent systems composed of volatile small molecular weight components, mainly terpenes and non-terpene components. EOs have been used for a long time by various traditional medicine systems as antiseptic agents [17]. Nowadays, scientific evidence is available showing that certain EOs have antimicrobial, antimycotic, antiviral, antioxidant, immunomodulant and anticancer properties [18,19]. Some of these characteristics are related to their functions in plants. *Vepris macrophylla* (Baker) I. Verd. (Rutaceae) is a tree endemic to Madagascar, where it is known by many names, such as itampody, ampodiberavina, mampodifotsy, mampody (evoking its euphoric properties) (Figure 1) [20]. Indeed, the roots of *V. macrophylla* are traditionally used to manufacture euphoric alcoholic beverages. In Malagasy ethnomedicine, each part of the plant has a specific use. The root bark, grated and macerated in water, is administered as a drink to treat nervous depression or apathetic states. Fruits are used to prepare steam baths during convalescence periods following infectious diseases. The leaves are used to regulate the coronary blood flow. [21–25]. The EO obtained from *V. macrophylla* leaves showed antimicrobial activities for the treatment of infectious diseases [26]. Furthermore, it was effective against phytopathogenic fungi such as *Phytophthora cryptogea* and *Fusarium avenaceum* [27].

In our previous study, the *V. macrophylla* EO showed notable effects on human tumor cells, notably breast adenocarcinoma and colon carcinoma cell lines, highlighting its cytotoxicity [26]. These results pushed us to further explore the effects of this EO on tumor cells. Thus, in the present study, we evaluated the antiproliferative activity of *V. macrophylla* EO, at different times and concentrations, on human breast cell line SKBR3.

Furthermore, the ultrastructural morphological alterations of SKBR3 cells were evaluated by fluorescence and scanning electron microscopy analyses.

**Figure 1.** Vepris macrophylla tree.

### **2. Materials and Methods**

### *2.1. Plant Material and Essential Oil (EO) Extraction and Composition*

The EO was obtained by hydrodistillation of *V. macrophylla* leaves, collected in the east coast of Madagascar (Sahamamy/Analalava), using a portable alembic as reported in Maggi et al. [26]. Following GC and GC-MS analysis the following major components were detected in the EO chemical profile: geranial (33.2%), neral (23.1%), citronellol (14.5%), and myrcene (8.3%).

### *2.2. Cell Culture*

SKBR3 cell line from human breast cancer was obtained from American Type Culture Collection (ATCC, Rockville, MD, USA) and was grown in Dulbecco's Modified Eagle's Medium (DMEM) medium with 10% fetal bovine serum (HyClone™ FBS (U.S.A. origin), Characterized), 1% nonessential amino acids, 1% L-glutamine, 100 IU per mL penicillin, 100 IU per mL streptomycin, in a humidified atmosphere at 37 ◦C with 5% CO2.

### *2.3. Cell Viability Assay*

MTT assay was utilized to evaluate cell viability. Briefly, cells were seeded for 24 h in a 96-well plate (NunclNunclonTM, NuncGmbH & Co., Wiesbaden, Germany) with a density of 1.2 × 10<sup>4</sup> cells/well. Then were treated with *V. macrophylla* EO at concentration of 1.25; 2.5; 5 and 10 μg/mL for 24, 48 and 72 h. After the incubation period, 0.5 mg/mL of MTT (Sigma, Deisenhofen, Germany) was added to each well for 2 h at 37 ◦C and the cells were dissolved with 200 μL/well of dimethylsulfoxide (Merck, Darmstadt, Germany). The absorbance of formazan was read at 570 nm on a scanning microtiter spectrophotometer plate reader. The results were calculated as the percentage of viability in relation to the untreated cells standardized to 100%. They are the mean ± SD of three separate experiments done in triplicate.

### *2.4. Immunfluorescence Microscopy*

SKBR3 cells were grown for 24 h on 12 mm diameter coverslips and treated with *V. macrophylla* EO for an incubation time of 24 and 48 h. Cells were then fixed with 4% paraformaldehyde for 30 min and were permeabilized with 0.5% Triton X-100 (Sigma Chemicals Co., St. Louis, MO, USA) for 5 min. For actin detection, cells were stained with FITC-phalloidin (Sigma) at room temperature for 30 min. For nuclei detection, cells were stained with Hoechst 33258 (Sigma-Aldrich, St. Louis, MO, USA; #861405) at 37 ◦C for 15 min. After the washing with PBS coverslips were mounted with glycerol-phosphate and images were acquired with a Nikon Microphot fluorescence microscope (Nikon Instruments, Melville, NY, USA) equipped with a Zeiss CCD camera (Carl Zeiss, Oberkochen, Germany).

### *2.5. Scanning Electron Microscopy (SEM)*

SEM analysis allowed the study of cell surface modifications induced by *V. macrophylla* EO. Samples were grown for 24 h on glass coverslips and treated for 24, 48 and 72 h with *V. macrophylla* EO with a concentration of 0.01; 0.1; 1.25 and 2.5 μg/mL. Then cells were fixed in 2.5% glutaraldehyde in 0.2 M Na-cacodylate buffer for 2 h and postfixed with 1% (*w/w*) OsO4 for 1 h. Subsequently, cells were dehydrated using an ethanol gradient. After the passage in 100% ethanol, the samples were submitted to drying with CO2 (Critical point dryer CPD 030, Bal-Tec AG, Lichtenstein) and gold coated by sputtering (SCD 050 Blazers device, Bal-Tec). Samples were observed with a scanning electron microscope FE-SEM Quanta Inspect F (FEI—Thermo Fisher Scientific; Eindhoven—The Netherlands).

### *2.6. Statistical Analysis*

All data were repeated in at least three different experiments, and results are expressed as the mean ± standard deviation. Statistical differences were determined using a one-way ANOVA test and values with *p* < 0.05 being considered significant.
