Background
Mammary cancer is a type cancer that arises from the mammary glands. In humans, it is commonly known as breast cancer due to the anatomical location at the breast. In animals, it is still known as mammary cancer. Globally, breast cancer is the most frequent cancer amongst women as it accounts for high mortality rate especially in non-developing countries due to late diagnosis and population increase. Interestingly, breast cancer also can develop in males, though relatively very rare (less than 1%) [
1] and the pathophysiology remains uncertain [
2,
3].
Since 2003, there have been several new updates of breast cancer classifications. However, until now, the fundamentals of breast cancer classification are still based on pathology and molecular biology [
4,
5]. Evidently, pathological classification is based on characteristics seen under light microscopy of biopsy specimens. In 2003, the report from World Health Organization (WHO) stated that there are 20 major tumour types [
6]. The debate on the classes of mammary cancers is still on [
6]. However, a majority of the accepted positions are that most mammary cancers are derived from epithelium lining ducts and lobules [
5]. Thus, these mammary cancers are pathological classified as ductal or lobular carcinoma [
6]. Besides, as part of pathological classification, the presence of pathological grades such as the presence of acinar, glandular and pleomorphic in mammary cancers morphology also would be able to determine the prognosis of the patient [
7,
8]. Conversely, the biological classification is mostly based on endocrinology gene expression, which are oestrogen receptor (ER) positive/negative, progesterone receptor (PR)-positive/negative, human epidermal growth factor (HER2)-positive or HER2-negative type of mammary cancer [
5,
6]. However, there is still space to study the molecular biology classification of mammary cancer due to some overlapping amongst immunohistochemistry surrogate and many molecular classes and subtypes [
4]. However, there are two types of breast cancer cells that have gained interest amongst researchers. They are the oestrogen receptor (ER) positive breast cancer, MCF-7 [
9] and oestrogen receptor (ER) negative breast cancer, MDA-MB-231 [
10].
Since ancient immemorial times, medicines from herbal and natural products were widely used in every culture throughout the world. Medicinal plants generally known as herbs played significant roles in the development of drugs and the outcomes have been promising. Therefore, in the view of exploration for an alternative medicine, particularly breast cancer studies, local Asian plant named,
Ardisia crispa (Thunb.) A. DC plant was selected due to evidence that the plant exhibits anti-inflammatory activity that can be relevant to anti-breast cancer. More so, anti-inflammation is often associated with inhibition of angiogenesis [
11], which co-jointly regulate the activation of cell chemotaxis, migration, and proliferation, and thus has the potential of suppressing tumour growth and metastases. Hence, the inhibition of angiogenesis is one of the most promising strategies in the development of novel anti-cancer therapies, and in the treatment of other human diseases associated with angiogenesis.
Phytochemical analysis from the leaves extract of
A. crispa showed the presence of many phytochemicals compound such as flavonoids, phenolics, saponins, tannins, terpenoids, and steroids [
12]. In a previous study by [
11], it was revealed that the root of
A. crispa contains various phytochemical compound such as phenolic, flavonoid and saponin when hydroethanolic is used as a solvent system. This plant extract also showed several biological activities such as anti-inflammatory and anti-hyperalgesic [
11,
13], antipyrexic [
14] and antiulcer [
15]. Besides that,
A. crispa have been reported to possess cytotoxic effect against human liver cancer (HepG2), skin cancer cells [
16,
17] and mouse mammary cancer (4 T1) [
12]. It is believed that the plant has anti-inflammatory properties by inhibiting angiogenesis process. It is also proposed that anti-inflammatory mechanism could partly involve in the anticancer activity. Therefore, plants which exert anti-inflammatory activity will usually exert anticancer activity.
Oxidative stress is one of the pathways of carcinogenesis and the phenomenon is often associated with inflammatory cells. The connection between antioxidative and anticancer activities has been widely subjected to extant empirical endeavours. Ardisia crispa is an evergreen flowering plant that belongs to the family Myrsinaceae. It is widely distributed over Asian regions including Malaysia. Local Malaysian people know this plant as ‘pokok mata ayam’ or ‘pokok mata pelanduk’. Traditionally, the root extract of A. crispa is believed to be useful in the treatment of several human ailments such as liver cancer, swelling, rheumatism, cough, fever, diarrhoea, broken bones, women dysmenorrhoeal, respiratory tract infections, and traumatic injuries.
Thus far, no empirical submission has been reported on the cytotoxic and antioxidative properties of any Ardisia species against breast cancer cell including Ardisia crispa. This study was conducted with the intention of discovering the true potential of the local herbs for anti-breast cancer activity which could perhaps reduce the side effects of current treatment. It could also be used synergistically with the available one to subsequently improve their pharmacological and toxicological effect and prognosis of the treatment. Furthermore, complementary methods such as using herbs or vitamins to treat cancer or relieve side effects of cancer is not something new.
Discussion
The cytotoxic effect of HEAC and its partitions were assessed based on the minimum concentration of extract that giving at least 50% of the cancer cell survivability (IC
50). The four categories of extracts which are; very active (IC
50 ≤ 20 μg/mL), moderately active (IC
50 > 20–100 μg/mL), weakly active (IC
50 > 100–1000 μg/mL) and inactive (IC
50 > 1000 μg/mL), [
23,
29]. For pure compound or drug, IC
50 value less than 4 μg/mL is considered potent [
30,
31].
Cytotoxic analysis revealed that, HEAC and EAEAC possessed moderate cytotoxic effect against MCF-7 with IC
50 57.35 ± 19.33 μg/mL and 54.98 ± 14.10 μg/mL, respectively. HEAC and EAEAC showed weak cytotoxic effect (IC
50 > 100–1000 μg/mL) on MDA-MB-231. For AQEAC, the IC
50 value against MCF-7 was more than 1000 μg/mL and MDA-MB-231 was 347.44 ± 98.78 μg/mL indicating that AQEAC has poor cytotoxic effect against breast cancer. In this study, it showed the response of breast cancer cell lines toward
A. crispa extract and its partitions was variable. It is in agreement with previous studies that the response towards each breast cancer was difference depending on the classification and degree of malignancy of cancer cells [
32]. Moreover, differences in cell line, plant extract, solvent used, and plant source also contribute to the difference cytotoxic effect possessed by the plant [
33]. However, from the results, it might suggest that oestrogen receptor (ER) positive breast cancer susceptible to hydromethanolic and ethyl acetate extracts of
A. crispa.
Overall, A. crispa plant had high TP, TFC values and antioxidant capacity. However, amongst extracts, EAEAC had the highest level of TPC, TFC and antioxidative activities followed by HEAC and AQEAC. HEAC and EAEAC revealed more total phenolic and flavonoid contents as compared to aqueous extract (AQEAC). This result explained the weak cytotoxicity effect exhibited by AQEAC as the phytochemical compounds contribute significantly in the ethnopharmacological medicinal values of the plants.
It is also revealed from the analysis conducted that EAEAC possessed the highest scavenging activities as high antioxidative activities were contributed by the highest level of TPC and TFC values of the extarct. Ethyl acetate might be the good solvent for extracting phytochemical compounds with antioxidant properties. This is similar to previous studies conducted by [
34], that TPC of ethyl acetate (EA) extract of
Alpinia mutica (1.55 ± 0.16 mg GAE/g extract) was the highest when compared to its crude hydromethanolic, hexane and aqueous extract. The results from the same study also revealed that EA of
Alpinia mutica possessed the highest antioxidant capacity (EC
50: 0.125 ± 0.04 μg/mL) which correlates with its TPC finding. TPC was considered high when TPC level higher than 10 mg GAE/g extract [
28]. The level of TFC and TPC are the same in descending order; EAEAC > HEAC > AQEAC because flavonoid is a subgroup of phenolics compound.
For antioxidant capacity determination,
A. crispa possess high antioxidant capacity which is good to scavenge free radical in the body. The benchmark for the plant was considered high TPC and TFC values 10 mg GAE/g extract and 10 mg RE/ g extract, respectively, while for DPPH and ABTS scavenging assays were EC
50 < 10 mg/mL [
26,
35]. EAEAC contains highest TPC and TFC followed with HEAC and AQEAC. The findings of DPPH and ABTS scavenging assays also showed that
Ardisia crispa leaves extract possess high antioxidant capacity and the results were in agreement with TPC and TFC findings. EAEAC has the highest scavenging activity followed by HEAC and AQEAC. These results in agreement with many previous studies who found that the level of TPC and TFC in extract play a major role for antioxidant capacity [
36‐
39]. The difference of antioxidant capacities among HEAC, EAEAC and AQEAC was due to the solvent used during partitioning. The change in location of hydroxyl group attached to aromatic ring in phenolics compound and benzene ring of flavonoids specifically affect the antioxidant properties [
40,
41].
Notwithstanding that the EAEAC exhibit the highest antioxidative activity, it is not a potent cytotoxic plant. This is because, not necessarily phenolic and flavonoid compounds are responsible for anticancer properties. The phytochemical compound of plants are of multicomponent mixture. Other phytochemical constituents such as saponins also exhibit anticancer properties [
42,
43]. Besides, phenolic has broad secondary plant metabolites and many subgroup/class. Moreover, it can presence with combination of other compounds such as terpenoids, saponins, glycosides, chlorophyl, lipid, protein, polysaccharides and cyanides [
44]. For example, terpenoids and saponins are phytochemical compounds that have anticancer effect [
45‐
47]. Hence, it is plausible that during partitioning of HEAC with ethyl acetate and aqueous solvent, the chemical characteristic or solubility of some phytochemical composition was altered, thus making it toxic or less potent. Since, phytochemical compound is a complex multicomponent form, separation of the compounds possibly alter the action of mixture compound, resulting in the loss of synergestic effect of the extract. Higher phenolic and flavonoid compounds in EAEAC as compared to HEAC and AQEAC, could be due to some phenolic subgroup/subclass reacts strongly with the Folin–Ciocalteu reagent [
48,
49]. Therefore, the use of solvent partitioning method might contribute to structural alteration effect of the phytochemical compounds and affect the therapeutic value. Based on reference value and other studies, HEAC is considered among extracts that have good scavenging activitiy. Futhermore, HEAC is in the same category of cytotoxic effect as EAEAC against MCF-7 and MDA-MB-231. The only criteria that need to be considered to choose among HEAC and EAEAC is the safetly level. Extracts with good scavenging activities might be beneficial in complimentary with chemotherapeutic drugs in order to minimizing the side effects from the chemotherapeutic agent. In addition, it can perhaps prevent the carcinogenesis pathway.