Raw material and chemicals

Jackfruit fruits were provided by Frutos Tropicales de la Bahia, S.P.R. of R.L. in Compostela, Nayarit, Mexico, at the physiological maturity stage. The fruits were kept at room temperature (28 ± 3 °C, 70-75 % RH) for 5-6 days until ripe. The chemicals n-hexane (65 % purity), methanol (99.80 % purity), and glacial acetic acid (99.7 % purity) were purchased from Jalmek (San Nicolás de los Garza, Nuevo León, Mexico). HPLC-grade acetonitrile was obtained from TEVIA (Fairfield, Ohio, USA). Authentical standards of Table 1 were purchased from Sigma-Aldrich (St. Louis, MO, USA) and BioCrick (Chengdu, China).

Ultrasound-assisted extraction of HVBC

A sample of 50 g of jackfruit pulp lyophilized at -50 °C and 0.12 mbar (Labconco FreeZone Freeze-Dry, model 4.5, Kansas, MO, USA) was mixed with n-hexane in a 1:10 ratio (g of sample:mL of solvent). The mixture was placed in an ultrasonic bath (CD-4820, Guangdong, China) for 30 min at 42 kHz. Subsequently, the mixture was filtered and concentrated in a rotary evaporator (IKA, RV10, USA) under reduced pressure (-90 kPa) at 200 rpm and 40 °C, until solvent-free extract. Partition of the extract was carried out by dissolving it in a methanol-hexane mixture (2:3 v/v). The immiscible phases were separated in a separatory funnel for 3 h at 4 °C. The methanolic partition was concentrated under reduced pressure and further dried with a gentle N₂ stream at 500 mg, and finally was stored in an amber vial at -20 °C until analysis (^{Ruiz-Montañez et al., 2015}).

HPLC-MS analysis of HVBC

The identification of HVBC was performed by using high-performance liquid chromatography coupled to mass spectrometry with a 1260 Infinity HPLC system (Agilent, Santa Clara, USA) equipped with a quaternary pump coupled to a 6120 quadrupole mass detector (Agilent, Santa Clara, USA). A Poroshell 120 EC-C18 column (2.7 μm, 4.6 × 50 mm) thermostated at 25 °C was employed for separation. A sample of 5 mg was diluted in 5 mL of methanol and filtered, and 5 μL were injected and eluted at a flow rate of 0.1 mL/min. A solution of acetonitrile (solvent A) and acidified water with 0.2 % glacial acetic acid (solvent B) was used as mobile phase in a gradient of 90/10 (vA/vB). The mass spectra were performed with N₂ as a gas pressure nebulizer (2 psi), the dry gas flow rate was 9 L/min, and the solvation temperature of 300 °C. The capillary voltage was adjusted to 3000 V in negative scanning mode. For identification, the injection of authentical standards was made under the same chromatographic conditions, for tentative identification, the molecular mass acquired was compared with reports in the bibliography; as well as a comparison of the mass spectral obtained was made with NIST and mass-spectra MassBank databases. The samples were analyzed in triplicate.

Identification of HVBC in the methanolic extract of jackfruit

The HVBC in the methanolic fraction of jackfruit extract were identified and those metabolites reported with anticancer activity were chosen to develop postulates about their possible mechanisms of action against cancer cells. Different compounds were identified, such as cudraflavone C, artocarpin, apigenin, moracin C and β-carotene (Table 1).

Compounds previously reported in jackfruit: ^{bFang et al. (2008}), ^{cZheng et al. (2014}), ^{dYao et al. (2016}), ^{eSun et al. (2017}), and ^{fRanasinghe et al. (2019}).

Postulates of the mechanisms for inhibition of carcinogenesis by HVBC present in jackfruit extract

According to the HVBC identified and the reported studies on the mechanisms for inhibition in different phases of the cancer process, the following postulates are proposed.

Apigenin has antimutagenic and antiproliferative properties against cancer cells

Apigenin is a dietary flavonoid present in several plants. Important anti-inflammatory, antioxidant, antibacterial, and antiviral activities have been attributed to this compound. Currently, apigenin has been extensively investigated for its anticancer activity and low toxicity. In vitro and in vivo studies have shown its capacity of suppressing human cancers by triggering apoptosis and autophagy. Mechanisms involve cell cycle arrest, suppressing cell migration and invasion, and promoting an immune response (^{Yan et al., 2017}). In this sense, ^{Shukla et al. (2014}) reported that apigenin treatment of androgen-resistant human prostate cancer cell lines PC-3 and DU145 resulted in apoptosis and reduced the cell viability caused by a decrease in Bcl-2 and Bcl-xL. Additionally, an increase in Bax protein concentration, accompanied by dose-dependent suppression of XIAP proteins, c-IAP1, c-IAP2, and survivin, was observed.

Regarding autophagy, studies carried out by ^{Tong et al. (2012}) reported that apigenin-triggered autophagy induces the activation of AMPK-activated protein kinase and the mTOR pathway in human keratinocytes. In this circumstance, autophagy plays a cytoprotective role in apigenin-induced cytotoxicity in cancer cells.

Other immune responses have been reported, studies with human and mouse mammary carcinoma cells developed by ^{Coombs et al. (2016}) demonstrated that apigenin could target STAT1, causing inhibition of PD-L1 expression induced by IFN-γ. Meanwhile, apigenin treatment induced the proliferation of Jurkat T cells PD1-expressing and interleukin-2 synthesis when co-cultured with MDA-MB-468 cells. According to a previous study conducted by ^{Kuo et al., (1992}) apigenin is capable of reversing mutations in Salmonella T98 strains and hamster ovary cells. This is due to a possible induction of a cellular defense system, through metabolizing enzymes such as glutathione-S-transferase (GST) or excision repair enzymes. ^{Babcook & Gupta (2012}) suggest that apigenin acts to downregulate insulin-like growth factor 1 (IGF-1R), which is the predominant receptor in mitogenesis, transformation, and protection from apoptosis. Apigenin instead allows IGF-1R to form a protein complex with IGFBP-3, which regulates cell proliferation through competitive inhibition (^{Burger et al., 2005}) (Figure 1a).

Artocarpin is capable of generating cytotoxicity in cancer cells

^{Tsai et al. (2017}) reported that artocarpin has a cytotoxic effect on cancer cells by inducing the production of reactive oxygen species. Mainly hydrogen peroxide (H₂O₂) is formed during the generation of the superoxide anion radical (O₂ ^-), which allows the formation of the OH radical. ^{Zhou et al. (2006}) informed that H₂O₂ induces apoptosis in MCF-7 breast cancer cells mediated by the phosphorylation of MARK, ERK, p28, and JNK proteins. The H₂O₂ promotes the expression of the MKP-1 protein. MKP-1 is an early stress response protein that inhibits the activation of apoptosis by p38 and JNK pathways. In addition, they allow the activation of the P53 protein, being the main responsible for triggering apoptosis through the caspase pathway (Figure 1b). In another study, the mechanism of inhibition of HDGF (a nuclear protein with mitogenic activity) was determined by small interfering RNA (^{Shik et al., 2013}). These authors propose that the increase in ROS contributes to apoptosis by improving the activation of p53 and PUMA expression.

Figure 1 Possible mechanisms of action for the carcinogenesis inhibition of a) apigenin, b) artocarpin, c) cudraflavone C, d) moracin C and e) β-carotene. 

Cudraflavone C selectively induces apoptosis in cancer cells by inhibiting the PI3K-AKT pathway

^{Soo et al. (2017}) found that cudraflavone C could inhibit the PI3K-AKT pathway. This pathway modulates the regulation of cell survival, cell cycle progression, and cell growth. The abnormal activation of the PI3K pathway causes the alteration of the mechanisms that control cell growth and survival. This behavior favors competitive growth, metastatic capacity, and often greater resistance to drug treatments (^{Carnero et al., 2008}). Besides, cudraflavone C could cause apoptosis in the PI3K-AKT pathway through the activation of NFκB and induce a signaling cascade that ends in caspase 3 (Figure 1c).

Moracin C counteracts inflammatory activity in the cancer process through the inhibition of the NF-κB and MAPK pathways

According to ^{Yao et al. (2016}), this flavonoid can inhibit the NF-κB and MAPK pathways, being the two main mechanisms to produce inflammatory cytokines initiated by lipopolysaccharides (LPS), such as IL-1β, IL-6, and TNF-α encoding cytokines, as well as inflammation-associated enzymes including COX-2 and iNOS (Figure 1d).