Within the 212 compounds that were isolated from different parts of the plant, acetogenins (AGEs) were reported to be the major phytochemicals, followed by alkaloids and phenols (45). These secondary metabolites were first isolated from multiple parts of plants belonging to the Annonaceae family in 1982 by Tempesta, and acetogenins were further determined to have toxicity towards the P-388 lymphocytic leukaemia in mice (46). Since then, more than 120 AGEs have been identified from ethanol, methanol or organic-based extracts from different parts of the plant, ranging from the leaves and the seeds (47) to even the fruit’s outer skin.
AGEs were later determined to be metabolites that can be distinguished by the presence of a methyl-substituted α,β-unsaturated γ-lactone (48). Since their discovery, more than 500 AGEs have been discovered from different parts of the plant (1). These metabolites were later classified into several groups on the basis of the availability of tetrahydrofuran (THF) and hydroxyl groups, as well as on factors such as the terminal γ-lactone ring and the characteristics aliphatic chain substituents (49). On the basis of these characteristics, AGEs can be divided into 10 different types, which include 1) linear AGEs (AGEs without the THF rings), 2) epoxy-AGEs (without THF rings), 3) AGEs with mono-THF α,α′-dihydroxylated γ-lactone, 4) AGEs with a mono-THF α-hydroxylated γ-lactone, 5) AGEs with mono-THF and several lactone moieties, 6) AGEs with a neighbouring bis-THF α,α′-dihydroxylated γ-lactone, 7) AGEs with a neighbouring bis-THF α-hydroxylated γ-lactone, 8) AGEs with a non-adjacent bis-THF γ-lactone, 9) AGEs with a saturated lactone bis-THF and 10) miscellaneous AGEs (50).
Multiple forms of AGEs have been studied for their mechanisms that can be used against particular targets, such as insects and tumour cells. Acetogenins have been shown to be very effective against insects and can be used as insecticides and insect repellent. Multiple parts of the plant, including the roots, leaves, unripe fruits and seeds, have been shown to have insecticide and insect repellent properties (51), (52), (53). Against the larva of the moth Plutella xylostella, a pest of cabbage, 5 mg/mL ethanolic extracts of A. muricata leaves were proven to be effective in killing 100% of the larvae tested. Further tests using lower concentrations of the ethanolic extracts even showed an ability to significantly reduce the survival of the larvae population (54). Crude extracts from the plant were also shown to be effective against Aedes aegypti, in combination with silver nanoparticles, which makes A. muricata extracts a good candidate to be used to control the spread of dengue fever (55). A similar anti-parasitic effect was also observed when methanolic extracts of the seed were used against Entamoeba histoltica, Molinema dessetae and Artemia salina, which was later deduced to be due to the presence of acetogenins (56). The role of acetogenins in inhibiting the growth of insect larvae was suggested to be due to the THF ring inhibition of the mitrochondrial complex I through NADH oxidase inhibition (52).
The capacity of acetogenins to inhibit NADH oxidase was also shown to be important for their anti-tumour function. The inhibition of NADH function is evident, as reported by Morré et al. (1995). The report suggested that exposure to an acetogenin called bullatacin specifically inhibited NADH oxidase enzyme functions isolated from Hela cells as HL-60 cancer cells (57).
Acetogenins were also shown to be capable of blocking ATP production in mitochondria. This mechanism of action was shown to be effective against cancer cells that produce higher amounts of ATP in comparison to normal cells, thus limiting the ability of cancer cells to grow (58). Interestingly, AGE toxicity was observed in the cancer cells, with a minimal negative impact on the normal cells. Studies using AA mimetics have suggested that acetogenin analogues synthesised in vitro have shown toxicity towards the HCT-8 and HT29 cell lines, with negative toxicity towards the normal human cell line HELF (59). Similarly, a study using A. muricata leaf extracts also showed toxicity towards cancer cell lines MCF-7, MDA-MB-231 and 4 T1, but less toxicity towards the normal breast cell, MCF10A (60), which shows that these AGEs specifically target cancer cells, not normal cells. These observations thus suggest the potential use of AGEs as treatment options against cancer.
Annona muricata and Its Biological Properties against Cancer
A dysfunctional apoptotic pathway is one of the main contributors to carcinogenesis. The inability of cells to execute apoptosis to remove cancer cells was observed in multiple cancer types, including breast, pancreatic, ovarian and colorectal cancers (61–65). This occurrence creates imbalances between cell proliferation and cell death and can be caused by disruptions in the normal functions of the apoptotic pathway. Similar to other types of cancer, failure to carry out apoptosis in breast cancer cells might be attributed to the disruptions within the apoptotic pathway. Defects within the intrinsic pathway, for example, have been associated with the progression of breast cancer. Defects in the regulation of cytochrome release (66), apoptosome formation (67) and caspase activation (68) have all been shown to be present within breast cancer cells.
The capability of inducing cancer cell cytotoxicity has been one of the main reasons behind the increased interest among scientists in the benefits of CAM, especially plant-based CAMs. A. muricata leaves, among other CAMs, are good candidates to treat cancer caused by viruses. Extracts from A. muricata leaves were shown to have the capacity to induce apoptosis in Hela cells, suggesting that the extracts have the potential to be used as a treatment against virus-induced cancer cells (69). Such a potential was also evident in the prevention of skin papillomagenesis in laboratory mice. In this study, ethanolic extracts of A. muricatawere shown to inhibit tumour growth in a two-stage skin papillomagenesis model, as evidenced by the presence of only slight hyperplasia in mice groups treated with A. muricata extracts in comparison to an untreated mice group (70).
In terms of specific effects against breast cancer, several studies have indicated the potential use of this plant in potential therapeutic treatments in patients. Extracts from A. muricata were shown to inhibit the proliferation of breast cancer cells by inducing cytotoxic activity in lung cancer cell lines (71). Gomes further reported that A. muricata extracts have the most cytotoxic effect when compared to extracts from Lantana camara, Handroanthus impetiginosus and Mentzella aspera. A separate study by Rachman showed that the ethanol extracts of leaves of the soursop plant extracted into ethanol induce cytotoxic activity within the breast cancer cell line MCF7 (72). Another study by Gavamukulya also showed that a similar ethanol extract of the soursop plant leaves was found to be highly cytotoxic in vitro against the two human breast cancer cell lines MDA and SKBR3 (31).
The cytotoxic effect of the plant’s extracts were also proposed by multiple in vitro studies using cultured liver cancer cells, suggesting that they can potentially be used as a treatment option against liver cancer. The growth and viability of the liver cancer cell HepG2 was shown to be inhibited following incubation with an ethanol extract of A. muricata. The cytotoxic effect observed in the HepG2 cell line was suggested to be a result of induction of the apoptosis pathway through the production of reactive oxygen species (ROS) (73). In a separate study using a similar cell line, Liu (74) also demonstrated the capability of A. muricata extracts to induce apoptosis. The study showed that the treatment of HepG2 resulted in the up regulation of heat shock protein 70 (HSP70), glucose-regulated protein 94 (GRP94) and protein disulphide isomerase 5 (PDI-related protein 5). A bioinformatic analysis of the up-regulation of these proteins suggested that the treatment of HepG2 cells with A. muricata extracts can trigger the apoptotic pathway by means of endoplasmic reticulum (ER) stress (74).
Graviola leaf extract (GLE), flavonoid-enriched extract and acetogenin-enriched extract (AEF) administered in vivo and in vitro were also shown to negatively affect the proliferation of prostate cancer. A study conducted by Yang (75) suggested that GLE, FEF and AEF all showed the capacity to down-regulate prostate cancer, with GLE being the most efficient at doing so. This study not only showed the efficacy of A. muricata extracts at inhibiting prostate cancer but also the importance of using whole-leaf extracts to achieve the highest inhibitor efficacy in combating cancer (75).
A. muricata-induced cytotoxicity in cancer cells has also encouraged scientists to further examine the molecular pathways that lead to such observations. According to a study conducted by Torres (49), the activation of extracellular signal-regulated kinases (ERK) and the phosphatidylinositol 3′kinases (PI3K/Akt) pathways play a crucial role in the proliferation and survival of pancreatic cancer cell, and the inhibition of these pathways leads to the inhibition of pancreatic cell growth. A similar study also revealed that the treatment of pancreatic cancer cells with A. muricata extracts resulted in a decrease in the activation of both ERK and Akt pathways in pancreatic cancer cells. Thus, the inhibition of these pathways is in agreement with the decreased viability of pancreatic cancer cells treated with the plant extract (76). Besides that, A. muricata was also shown to inhibit metastasis. A study performed on pancreatic cancer cells by Torres showed that the migratory capacity of pancreatic cancer cells was reduced after treatment with a graviola extract, as evaluated by a transwell assay, suggesting that the natural extract reduces the motility of pancreatic cancer cells. The motility and migration of cancer cells is associated with the arrangements of the cortical actin and microtubules network. Additionally, cellular ATP depletion has been associated with a reorganisation of the actin cytoskeleton and a suspension of the dynamics of microtubules, which is known to induce mitotic arrest. Thus, graviola extracts cause a disruption of the cortical actin network that can inhibit the motility of cancer cells (76).
Moghadamtousi showed that A. muricata induces apoptosis in lung cancer cells (77). This finding was confirmed by high-content screening (HCS) multiple cytotoxicity analyses that examined the characteristics of apoptosis before and after treatment with A. muricata extracts, including nuclear condensation, mitochondrial membrane potential (MMP), cytochrome c leakage and perturbation in membrane symmetry. The analyses showed that A549 cells treated with A. muricata extracts experience inhibition in growth capability as well as up-regulation of the apoptosis pathway.
Cancer is a disease caused by cell cycle dysfunction. The ability to block the cell cycle progression in cancer cells can effectively elevate the anti-cancer potential of natural products (78). A. muricata extracts, for example, were shown to have the potential to induce G1 cell cycle arrest (28). The study also showed that treatments of HCT116 and HT-29 cells resulted in the up-regulation of the apoptotic pathway, as suggested by an increase in the production of ROS, an increase in detectable cytochrome c, and an increase in initiator and executioner caspases in both of the tested cell lines. Furthermore, an increase in the levels of Bax protein was also observed by flow cytometry, which further suggested the activation of the apoptotic pathway (79).
Annona muricata Extracts in Conjunction with Conventional Treatment
Despite the various benefits associated with the use of A. muricata extracts in inhibiting cancer cell lines, their usage alongside chemotherapy and radiation therapy has not been explored in such experiments and is in need of immediate attention. However, one cell culture-based study revealed a promising outlook on the potential use of these plant extracts alongside current radiotherapy and chemotherapy methods. The study, conducted at Sebelas Maret University in Indonesia, revealed a synergistic interaction between A. muricataLinn leaves extracts with doxorubicin in reducing the development of Hela cells. In the study, 38.5 μg/ml polyketide derivatives isolated from the plant were shown to have a synergistic effect with every concentration of doxorubicin used during the treatment of Hela cells (80).