Studies are on-going to search for natural-based anti-proliferative and chemopreventive agents which can act as alternatives to the chemically-synthesised drugs and which are potentially less toxic and contain less side effects. In this study, we tested the antioxidant abilities and cytotoxic effects of the extracts of P. betle on the breast cancer cells, MCF-7.
The antioxidant activities of P. betle have been reported in numerous studies but mostly concentrated on the aqueous or polar extracts. However, variation in antioxidant activities can still occur depending on varieties, location and growth conditions of the plant, hence data on antioxidant activities are still relevant and important
[23, 24]. In this study, we used solvents of varying polarities to separate antioxidants of low, medium and high polarity, using water, methanol, ethyl acetate and hexane, to provide a better insight into the antioxidative properties of this plant.
Overall, in the assessment of the antioxidant capacities of the plant extracts, the ethyl acetate extract showed the highest ferric reducing and radical scavenging activities against DPPH, superoxide anion and nitric oxide radicals. However, the ethyl acetate extract was not as potent as the aqueous extract in scavenging the hydroxyl radicals, implying selective scavenging effect of antioxidants in the former. Ethyl acetate is the most optimal solvent for extraction of antioxidants in P. betle, implying that the antioxidants in P. betle are mainly of medium polarity. In contrast, the antioxidant activities of the aqueous, methanol and hexane extracts were many folds lower than the ethyl acetate extract, implying minimal contribution of these extracts towards protection against oxidative damage. Many studies have reported positive correlation between phenolic compounds in plants and their antioxidant activities, showing the importance of phenolic compounds as antioxidants
[25, 26]. Our correlation analyses also support this observation. Indeed, the ethyl acetate extract of P. betle had the highest phenolic content, implying that the antioxidant activities may have been contributed by the phenolics. We identified the presence of catechin, morin and quercetin through HPLC analyses. These three compounds are well-known antioxidants and could have contributed to the observed antioxidant activities
. Previous studies have identified several phenolic compounds in the leaves of P. betle including β-sitosterol, dotriacontanoic acid, tritriacontane, stearic acid, hydroxychavicol, chevibetol and allylpyrocatechol, together with their glucosides
[28, 29]. Many of these compounds including chevibetol, hydroxychavicol and allylpyrocatechol have antioxidant activities
Studies comparing the anti-proliferative effect of extracts of P. betle leaves on breast cancer cells are lacking. In this study, amongst the four solvent extracts, the ethyl acetate extract had the most potent anti-proliferative effect on breast cancer cells and this was observed to be more potent than Scutellaria baicalensis and Patrinia scabiosaefolia, popular Chinese medicinal herbs that are traditionally used for treating cancer. Furthermore, the ethyl acetate extract also contained the highest phenolic content and antioxidant activities which could contribute towards the protective effects. A study in China reported a positive correlation between the antioxidant activities of several Chinese medicinal herbs and their anti-cancer effects on MCF-7 cells
. Hydroxychavicol, a component of P. betle leaf showed anti-proliferative effect towards oral carcinoma cell line
 and may have the same anti-proliferative effect against MCF-7 cells. Toxicology studies in rats showed no signs of toxicity and hepatotoxicity of P. betle up to a concentration of 1.5 g/kg body weight, implying its safety against normal cells and its specificity in targetting cancer cells
Epidemiology and clinical studies have revealed the involvement of reactive oxygen species (ROS) in carcinogenesis
[34, 35]. Tumor cells have increased production of ROS, causing oxidative stress and disturbing the redox state, leading to DNA damage, mutations and altered gene expression which contributes to carcinogenesis. At the same time, cancer cells have reduced capacity to remove ROS due to altered antioxidant defense systems. However, ROS also play important roles in inducing apoptosis, implying an anti-cancer effect. Hence finding the right balance between ROS and antioxidant defense levels in cancer cells is important to ensure that cancer progression can be inhibited while at the same time maintaining apoptosis.
For this reason, we investigated the effect of the ethyl acetate extract on antioxidant enzyme levels in MCF-7-treated cells to ascertain the possible protective effects of these enzymes against oxidative damage. SOD catalyses the dismutation of superoxide anion into water and H2O2 whereas CAT and GPx protect against oxidative damage by converting H2O2 into water. Accumulation of H2O2 can lead to production of the highly reactive hydroxyl radicals, causing DNA damage.
CAT activation is postulated to exert control on breast cancer progression
. Indeed, activities of CAT have been reported to be lowered in breast cancer patients
[37, 38]. Acatalasemic and hypocatalasemic mice, which have drastically decreased CAT levels in the blood and tissues, were more susceptible to mammary carcinoma than their wild type counterparts
. A recent study reported that CAT overexpression in MCF-7 cells led to less proliferation and migration of the cancer cells
. The plant extract used in this study could have increased CAT activities possibly by directly inducing increased expression of CAT, thereby inhibiting proliferation of the cancer cells.
SOD activities are low in many cancers implying reduced protection against ROS
[41, 42]. Low levels of manganese superoxide dismutase (MnSOD) in nonaggressive breast cancer cells caused accumulation of superoxide anion which acted as second messengers, promoting cancer cell proliferation
. In contrast, over-expression of SOD in cancer cell lines, including MCF-7, inhibited tumor growth, possibly acting as tumor-supressor proteins
. One of the mechanisms for this is through the influence of MnSOD on transcription factor activity. Over-expression of MnSOD in MCF-7 cells have been reported to reduce transcriptional activity of the transcription factors AP-1 and NF-κB and decreased expression of interleukin IL-1 and IL-6
, contributing towards tumor supression. MnSOD can also supress tumors by altering ROS levels in cancer cells and it was reported that H2O2 production by MnSOD contributed to the tumor-supressing properties
. The increased SOD activities in our study imply the ability of the plant extract to remove superoxide anions and possibly inhibit tumor growth. Furthermore, the plant extract may also directly scavenge superoxide anion since the in vitro analysis showed its potent superoxide anion scavenging activity.
There are five different forms of GPx in humans, however, GPx1 and 4 are more relevant to breast cancer. Generally, all GPx could inhibit initiation and metastastis although this may differ according to the types and stages of the cancers
. GPx1 is postulated to prevent initiation of cancer through ROS-mediated DNA damage whereas GPx4 could inhibit growth of established tumors
. Our study did not show significant changes in GPx activities throughout the incubation times indicating the plant extract has little or no effect on this enzyme.
Antioxidant-rich plants such as raspberry, blueberry and soybeans could inhibit the growth of several cancer cell lines, including breast cancer
. Phenolic compounds such as epigallocatechin gallate, catechin, genistein and quercetin suppressed growth of breast cancer cells
[1, 2] implying the importance of antioxidants towards the anti-proliferative effects of cells. Anti-cancer agents with antioxidant activities may exert their beneficial effects by balancing levels of ROS so as not to cause further proliferation of cancer cells while still allowing apoptosis to occur. In addition to the radical scavenging properties of the ethyl acetate extract observed in this study, another study had also reported the ability of extracts of P. betle to scavenge ROS including H2O2, superoxide radicals and hydroxyl radicals
 and this effect was attributed to hydroxychavicol, a major phenolic present in the plant
. Increased activities of the antioxidant enzymes in this study implied the ability of the extracts of P. betle to remove ROS and protect against oxidative damage while at the same time inhibiting cell proliferation. Studies have indicated that in addition to influencing antioxidant enzymes, antioxidants may inhibit carcinogenesis through other non-antioxidant action such as by modulating signaling pathways involved in cellular functions such as proliferation, cell growth and differentiation, by influencing activities of cancer-related enzymes such as cyclooxygenase-2 and phase I or II metabolizing enzymes or by inducing cell cycle arrest