Evaluation of antioxidant and anticancer effects of Piper betle L (Piperaceae) leaf extract on MCF-7 cells, and preparation of transdermal patches of the extract

Purpose: To determine the antioxidant and anticancer effects of Piper betle (P. betle) leaf extract on human breast cancer MCF-7 cells, and to develop transdermal patches containing the extract. Methods: The leaf extract of P. betle was prepared by maceration method, and its antioxidant activity was evaluated using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. Cytotoxicity and suppression of cell migration (indices of anticancer activity) were also assessed in MCF-7 cells by sulforhodamine B (SRB) and wound healing assays, respectively. Transdermal patches were developed using the casting method, and the resultant patches were evaluated with regard to their physical appearance and mechanical properties before and after a stability test. Results: The extract exhibited antioxidant activity with half-maximal inhibitory concentration (IC50) of 30.0 ± 0.1 μg/mL. It also showed cytotoxicity with an IC50 of 114.3 ± 14.9 μg/mL, and significantly suppressed the migration of MCF-7 cells at a dose of 25 μg/mL. Based on desirable characteristics, patch base formulations containing 4.2 % pectin, 0.4 % hydroxyl propyl methylcellulose (HPMC), 0.4 % polyvinyl pyrrolidine K-90 (PVP-K90) and 3 % propylene glycol (PG) were selected for incorporation into the extract. Conclusion: Leaf extract of P. betle exhibits potential anti-breast cancer properties. A transdermal patch containing 0.03 % of the extract can be successfully developed for treatment of breast cancer.


INTRODUCTION
Breast cancer is one of the deadliest diseases, and the most common cancer in women worldwide. Currently, treatment failures in breast cancer are associated with drug resistance and drug toxicity [1]. Thus, there is need for development of newer and less toxic chemotherapeutic agents. Recently, many researchers have focused attention on medicinal plant extracts rich in flavonoids and phenolic compounds as potential sources of alternative chemotherapeutic agents. This is due to reports from many studies showing that these compounds exert anticancer effects [2,3]. Moreover, positive correlations have been established between the antioxidant activities of plant extracts and their proliferative inhibition effects [4].
Piper betle L (Piperaceae) are generally named as betel leaf. It is one of many medicinal plants that have become popular in cancer studies [5]. Studies have shown that leaf extract of P. betle possess phenolic compounds such as hydroxychavicol, chavibetol and eugenol [5,6]. In addition, it has been noted that leaf extract of P. betle exhibits antioxidant activity, as revealed through DPPH, hydroxyl, nitric oxide and superoxide anion radical scavenging activities, as well as ferric reducing antioxidant power (FRAP) assay. Moreover, the anti-proliferative effect of P. betle leaf extract has been documented against B lymphocyte cell line (Raji cells) [7], KERATIN-forming tumor cell line (KB cells) [8], and MCF-7 cells [9]. These findings indicate the potential of the extract for use in the breast cancer treatment. However, not much is known about the effect of the leaf extract of P. betle on MCF-7 cell migration. Cancer metastasis is a crucial step in the prognosis of cancer patients [10]. The wound healing assay is usually used to determine migration of cancer cells. The inhibitory potential of P. betle leaf extract on the migration of MCF-7 cells can be demonstrated though its suppression of relative closure of scratch wound in the wound healing assay. If the extract suppresses healing of the scratch would, it may be reasonably concluded that it reduces cancer cell metastasis, implying that its application may result in good prognosis of cancer patients. Then, the extract could be considered as exerting good chemo-preventive effect.
Transdermal patches are dosage forms formulated for effective delivery of active principles/compounds at controlled rates, and within predictable times [11,12]. Transdermal administration is one of the promising alternative routes for delivery of anticancer drugs due to its safety, ease of administration, reduced frequency of dosing, by passing of hepatic first-pass effect, and reduce incidence of adverse side effects, when compared with the oral route [11].
This present study was examined the determination of the antioxidant and anticancer activities of leaf extract of P. betle on human breast cancer (MCF-7) cells, and development of transdermal patches of the extract.

EXPERIMENTAL Preparation of P. betle leaf extract
The P. betle leaves were collected from the botanical garden of School of Pharmaceutical Sciences, University of Phayao, from October to December, 2018. The plant sample was identified by Dr. Prachaya Srisanga, Herbarium Curator at The Botanical Garden Organization, Queen Sirikit Botanic Garden (QBG). A voucher specimen was deposited at the QBG Herbarium, Chiang Mai (QBG voucher number 110895). The fresh leaves were washed, chopped into small pieces and air-dried. They were further dried at 50 o C using a hot-air oven for 48 h. The dried leaves were ground into powder, and were extracted with 95 % ethanol for 72 h. Following filtration, the extract was concentrated using a rotary evaporator (Heidolph, Germany) at controlled temperature of 50 o C. The percentage yield of the extract was determined using Eq 1.
where WCE is dried weight of the crude extract, and WLP is dried weight of the P. betle leaf powder.

Phenolic content
In the evaluation of total phenolic content (TPC) of the extract, 40 µL of the extract (200 µg/mL) was mixed with 80 µL of Folin-Ciocalteu reagent for 5 min. Thereafter, 7 % sodium carbonate (Na 2 Co 3 ) was added to the mixture, and the reaction mixture was incubated for 30 min. The solution absorbance was determined at 750 nm using a microplate reader (Synergy H1, Biotek Instruments, Friedrichshall, Germany). The TPC was determined using a gallic acid standard curve prepared using gallic acid with a serial concentrations of 20 -100 µg/mL. The TPC was presented as gallic acid equivalent (GAE) per gram of crude extract.

Flavonoid content
In the evaluation of total flavonoid content (TFC) of the extract, 100 µL of 2 % aluminum chloride (AlCl 3 ) solution was added to the sample solution (100 µg/mL, 100 µL). After incubation for 10 min, the absorbance intensity was measured at 415 nm. The TFC was calculated from the standard curve of rutin (20 -100 µg/mL) and presented as rutin equivalent (RE) per g of the crude extract.

Antioxidant activity
In the evaluation of antioxidant activity, the extract solution was prepared by dissolving the extract in 95 % ethanol to obtain concentrations of 0.05 -2.0 mg/mL. Then, 100 µL of each extract solution was separately mixed with 100 µL of 5.0 mM DPPH in a 96-well plate. Following incubation at room temperature for 20 min, absorbance of the control (without the test sample, A blank ) and that of the sample (A sample ) were determined at 540 nm. Gallic acid served as a positive control. The DPPH radical scavenging ability (D) was determined using Eq 2.

Cell culture and cytotoxicity assay
In the determination of cytotoxic activity of P. betle extract, the human breast cancer cell, MCF-7, (ATCC #HTB-22, Manassas, USA) was cultured in Dulbecco's Modified Eagle Medium (DMEM) supplementing of 10 % fetal bovine serum (FBS), 100 µg/mL streptomycin and 100 U/mL penicillin. The SRB method was used to determine the cytotoxic activity as previously described [12,13]. Cells were plated, exposed to the medium containing P. betle extract (0 -250 µg/mL) for 48 h and then cells were stained with 0.4 % SRB for 30 min at room temperature. After incubation time, cells were discarded SRB dye, washed several times to remove excess SRB dye, and solubilized in Tris base buffer (10 mM, pH 7.4). The absorbance were measured at 540 nm using a microplate reader (Opsys MR™, Dynex Technologies, USA).

Suppression of cell migration
In the determination of anti-migratory effect of the extract, the cells were plated onto 24-wells culture plate for 24 h at 37 °C. Then, cell was created a straight wound by scratching the cell with a sterile pipette tip, and the cells were separately incubated with various doses of the extract (0 -100 µg/mL) for 48 h. Thereafter, the area of the uncovered region of the wound was measured using an inverted microscope (TS100, Nikon, Japan) at a magnification of x10 [14,15]. The percentage of relative closure of the scratch was calculated from the area data.

Formulation of transdermal patch base
The transdermal patch base was formulated using a film casting method. Three types of polymers i.e. pectin, HPMC and PVP-K 90 were used as film-formers, while propylene glycol (PG) or PEG-400 act as plasticizer, and paraben served as a preservative. Based on the best physical appearance of the preliminary trial batch (data not shown), the three types of polymers were combined at various ratios to generate a fixed polymer amount at 5 % (w/w) in each patch formulation. The amount of plasticizer used was varied from 1 to 5% (w/w). The compositions and formulation code of the preliminary trial batches are shown in Table 1.
The polymers were completely dissolved in distilled water. Then, the plasticizer and preservative were added under continuous stirring for 60 min. The polymer solutions were left at room temperature overnight to remove air bubbles. Then, 50 g of the polymer solution was poured on a petri dish and dried at 40 o C using a hot-air oven (Memmert, Schwabach, Germany). The dried patches were removed from the petri dish, wrapped in aluminum foil, and preserved in a desiccator prior to use.

Physical appearance
The patches were visually observed for color, homogeneity and flexibility.

Mechanical properties
The tensile strength of each patch was evaluated by a texture analyzer (TA.XT. plus, Stable Micro System, Surrey, UK). The patch was cut into shape of rectangular of 7 cm in length, and 1 cm in width. The patch was fixed between two cell grips of the instrument, with one grip fixed, and the other movable. A force was gradually applied to pull the patch until it was torn at the center. The tensile strength was calculated from Eq 3.

Preparation and characterization of patches containing P. betle leaf extract
Patch base formulations with good physical appearance and mechanical properties were selected for incorporation of the extract. The extract was dissolved in the blended polymer solution before mixing with other ingredients. The patches containing the extract were evaluated for their physical appearance and mechanical properties as described above. Weight variation, uniformity of thickness, and water uptake of the P. betle leaf extract containing patches were determined as indicated.

Weight variation
A set of three patches from each patch base, and patches containing the extract formulation with a diameter of 1 cm 2 were weighed on a digital balance (New Classic MF, ML802, Mettler Toledo, Switzerland) and the mean values were calculated.

Thickness uniformity
The thicknesses of the patches were measured using a vernier caliper (Macoh, Thailand) at five different places, and the mean values were determined.

Water uptake
Patches of each formulation were cut into 1 x 1 cm squares. The squares were accurately weighed and soaked in distilled water (2 mL) at 37 o C for 5 min. The patches were removed from the water and hung in the air to remove excess free water on their surfaces. They were then weighed again, and water uptake (W) was calculated as shown in Eq 5.

Stability studies
Stability studies on the prepared patches were carried out using heat/cool cycling test. In heat/cool cycling test, the patches were packed into aluminum foil and kept at 4 °C for 24 h, followed by kept at 60 °C for 24 h. This process was repeated in five cycles. The physical appearance, tensile strength, elongation, weight variation, thickness uniformity and water uptake of the prepared patch formulations were evaluated when first prepared, and after heat/cool cycling test.

Statistical analysis
The data values are shown in term of means ± standard deviation (SD). Statistical data analysis was done by a one-way analysis of variance (ANOVA) (Sigma Stat software version 3.5). Set value of p < 0.05 was considered statistical significance.

Physical appearance, TPC, TFC and antioxidant activity of extract
The leaf extract of P. betle was obtained as a dark-green paste at a percentage yield of approximately 1.4 % (w/w). The crude extract had TPC of 626.4 ± 60.5 mg GAE/g, and TFC of 138.5 ± 20.2 mg RE/g. It exhibited antioxidant activity with IC 50 of 30.0 ± 0.1 µg/mL, relative to that of gallic acid (IC 50 of 8.2 ± 0.5 µg/mL).

Cytotoxic effect of P. betle extract on breast cancer cells
The extract exerted cytotoxic activity on MCF-7 cells. The MCF-7 cell viability was decreased when increasing dose of the extract and it decreased the MCF-7 cell viability with an IC 50 of 114.3  14.9 µg/mL ( Figure 1). In addition, it showed a significant impact at a concentration of 50 µg/mL.

Anti-migratory effect of extract of P. betle on breast cancer cells
The results showed that the P. betle extract suppressed migration of the cancer cells in a concentration-dependent manner, with a significant effect at a concentration of 25 µg/mL (Figure 2 a). At this concentration, the extract suppressed the migration of breast cancer cells by approximately 30 %, relative to the control group (Figure 2 b).

Characteristics of patch base formulations
Ten patch base formulations were given product codes F-1 to F-10 ( Table 1). All formulations were yellowish, homogeneous, flexible and smooth films (data not shown). There were no problems removing the prepared patch base formulations from the petri dishes after ovendrying. The tensile strength and percentage elongation indicated the strength and elasticity of the patches, respectively [16]. The mechanical properties of the patch bases are shown in Figure 3. All patches of the prepared formulations exhibited percentage elongation at break values in the range of 29.88 -72.88 %, and they showed tensile strength values in the range of 0.35 -3.49 N/mm 2 . In general, preferable patches for transdermal application should be flexible enough to follow the movements of the skin without breaking and the films should be strong enough to prevent abrasion of the films during contact with clothing [17]. Patch base formulation F-3 was selected for use in the preparation of transdermal patch containing the extract because it exhibited a high percentage elongation at break of 76.13 ± 12.68 % with a tensile strength of 1.19 ± 0.31 N/mm 2 ( Figure 3). The extract was added to the patch formulation at the level of 0.03 % (w/w).

Characteristics of patch base and patch containing leaf extract of P. betle
The patch base was light yellow in color, while the patch containing the extract appeared dark green in color due to the color of the extract (Figure 4). The mechanical properties, weight, thickness and water uptake of the patches are shown in Table 2. Compared with the transdermal patch base, the tensile strength of patch containing the extract was increased, whereas the elongation was decreased.

Stability
The results of stability test showed that the weight, thickness and percentage elongation of the patch base were decreased, whereas percentage water uptake and the tensile strength of the patch were increased. Similar results were obtained with the patch containing the extract ( Table 2).

DISCUSSION
Nowadays, there are attempts to discover potent anticancer agents from herbal medicine, especially anti-cancer agents with low toxicity. A few studies have reported that medicinal plants with high antioxidant activities suppress the proliferation of cancer cells [2,3]. The present study was assessed the antioxidant and anticancer effects of P. betle leaf extract on the human breast cancer cells, MCF-7. The results indicated that the P. betle leaf extract of had high antioxidant activity, most likely due to the presence of phenolic and flavonoid compounds in the extract. In addition, it has been reported that phenolic compounds and flavonoids have both chemotherapeutic and chemo-preventive effects [2][3][4]. The results revealed that the leaf extract of P. betle exerted dose-dependent anticancer activity against breast cancer cells. These findings are in agreement with previous reports [9]. Moreover, the extract inhibited metastasis of MCF-7 cells. Thus, the leaf extract of P. betle inhibits cancer metastasis and exerts chemo-preventive effects.
Reactive oxygen species (ROS) have been related with cancer pathogenesis. The anticancer activity of the P. betle extract may be due to its high antioxidant activity. In a study, it was revealed that hydroxychavicol, a crucial phenolic in P. betle leaf extract, was metabolized to an electrophile which conjugated with reduced glutathione; this increases the ROS sensitivity of cancer cells, resulting in enhanced apoptosis [18]. The cytotoxic and anti-migratory effects of the extract on MCF-7 cells suggest that local treatment i.e. application of the extract on the breast skin might produce effective outcomes. Consequently, the P. betle leaf extract was converted into transdermal patches, based on the finding that it was preserved in the patch formulation, and it exerted prolonged anticancer effect after the patch was applied on the skin.
To develop transdermal patch formulations, pectin was selected as a major component of the polymer blend due its good gelling property, high stability, biocompatibility and affordability [19,20]. The tensile strength of the prepared patch base decreased with increasing plasticizer concentration, due to the fact that plasticizers reduced the intermolecular forces between the chains of adjacent macromolecules [21]. Stability tests revealed that the prepared patch was stable throughout the study period. However, it is important to investigate the long-term stability and release profiles of the prepared patches in further studies.

CONCLUSION
The results obtained in present study indicate that the leaf extract of P. betle exerts antioxidant activity, also inhibits the viability and migration of MCF-7 cells. Thus, the extract has promising potential for development into an anticancer agent for breast cancer. This study is the first to develop P. betle patch against breast cancer. A transdermal patch containing the extract was successfully developed. The developed patch has potential uses for local treatment of breast cancer.

Acknowledgement
This study was financially supported by School of Pharmaceutical Sciences, University of Phayao, Thailand and Thailand Research Fund (Grant no. MRG6080071). The authors would also like to express their appreciation to Mr Kamchai Saepang for technical assistance with texture analysis.