Furthermore, in vitro experiments showed that flavonoid aglycones displayed better anticancer potency than their homologous glycosides. On the other hand, there are reports showing that glycosylation enhances the bioactivity of flavonoids, which may be attributed to the facilitated transmembrane delivery driven by glycoside binding to glucose transporters on the cell membrane. Zou et al. proved that these two inhibitors of glucose transporters could inhibit the absorption of cyanidin-3-O-glucoside. Manzano et al. reported that peonidin-3-O-glucose inhibits the activities of the glucose transporters which could further affect glucose absorption and transport by cells. Alzaid et al. demonstrated that anthocyanin extracts from berries significantly reduced SGLT1 and GLUT2 expression by cells. Despite the aforementioned efforts, the effects of glycosylation on the functions of anthocyanins, especially their antitumour function, remain indistinct. The purpose of this research was to explore the glycosylation of anthocyanin on tumour cell inhibition and the related mechanism. We compared the tumour inhibitory efficiency of glycosylated anthocyanins on mouse colon cancer cells with that of the anthocyanidin aglycone and found that glycosylation significantly increased the cytotoxicity of anthocyanins to tumour cells. Then, blueberry production we demonstrated that the inhibitory pathway of anthocyanin was highly related to the energy metabolism of tumour cells, in which the glycosides of anthocyanins exerted a decisive influence.

For the first time, to our knowledge, we proposed a potential mechanism by which glycosides of anthocyanins enhance tumour cell inhibition through energy metabolism and glucose transport.Mitochondria are responsible for supporting life under aerobic conditions and are thus considered the source of signals initiating apoptotic cell death. During apoptosis, the decrease in MMP serves as a landmark event in early apoptosis. To further explore the mechanism of apoptosis, we detected decreases in MMP by FACS. When the cell membrane potential is reduced, the transformation of JC-1 is easily detected by the change fromred fluorescence to green fluorescence. Thus, the ratio of mitochondrial depolarisation was determined by the relative ratio of red-green fluorescence. The mitochondrial membrane potential is relatively higher in control group of MC38 cells stained with JC-1 , while the MMP of the positive control group dropped rapidly . The aggregated JC-1 within normal mitochondria was dissociated into monomeric form after treatment with BAE of different ANC concentrations for 24 h, indicating a decrease in MMP and mitochondrial damage. Low concentrations of ANC resulted in a distinguishable ratio of cells with low mitochondrial membrane potential, although it had little effect on cell viability. According to the results shown in Fig. 1, ANC shows selectivity between tumour cells and normal cells at concentrations below 500 μM. Here, we investigated the mitochondrial membrane potential of L929 cells at an ANC concentration of 500 μM. As shown in Fig. 3g, only 9.9 ± 0.20% of the cells had a low membrane potential, which was consistent with the survival rate.

The selectivity between tumour cells and normal cells might be attributed to the fact that the energy metabolism of tumour cells is more vigorous than that of normal cells.The reduction in mitochondrial membrane potential after apoptosis induces changes in membrane permeability. With the increase in membrane permeability, some apoptosis-inducing factors including cytochrome c are released from the mitochondrial matrix into thecytoplasm. There are key regulators of caspases in mitochondria, which are major factors in many apoptotic processes. The leakage of cytochrome c indicates the disassembly of the apoptosome, which is based on the activation of downstream caspases. As activation of the caspase cascade could lead to a series of events during cell apoptosis, it plays a crucial role in a variety of apoptotic pathways. The caspase protease family consist of initiative group and executive group during apoptotic process. The initiation of mitochondria-mediated apoptotic pathway by caspase-9 resulting in executing apoptosis by caspase-3. Hence, we detected the caspase-3 and caspase-9 activity using Caspase Activity Assay Kits to further explore the mechanism of apoptosis. Figure 4a reveals the changes of caspase-3 activity in MC38 cells after exposing to BAE with different ANC concentrations for 48 h. The activity of caspase-3 increased to 132.5 ± 2.3%, 155.1 ± 3.6%, 169.4 ± 2.3% and 764.5 ± 3.0% for ANC doses of 100, 200, 500, and 1000 μM compared with the control, respectively. Irreversible morphological changes of cells occurred when the activity of caspase-3 accumulated to a certain threshold. In addition, caspase-3 is the junction between the mitochondrial pathway and the death receptor pathway. This fact explains why the activity of caspase-3 changes in accordance with early cell apoptosis . Figure 4b presents the changes of caspase-9 activity after 48 h of treatment with ANC concentrations of 100, 200, 500, and 1000 μM.

The caspase-9 activity had negligible changes when the ANC concentration was below 500 μM, but a significant increase was found when the ANC concentration was 500 μM . As caspase-9 is an important initiator of apoptosis, its enzyme activity coincides with late apoptosis . The changes in caspase-3/9 activity correlate with early cell apoptosis , indicating that cell apoptosis proceeds through the mitochondrial route.The mitochondrial damage often occurs simultaneously with an increase in excessive reactive oxygen species on account of blockade of electron transport in the oxidation respiratory chain. Here, we detected the content of intracellular ROS. Figure 5d showed the ROS level in MC38 cells decreased with increasing ANC concentration, suggesting the strong antioxidant capacity of ANC. This result suggests that electron transport in the oxidation respiratory chain was not blocked and mitochondrial damage was not caused by the increase in ROS. The lack of oxidative phosphorylation substrates could also cause mitochondrial damage. To further explore the effects of ANC on mitochondrial, we assessed indicators of mitochondrial activity, such as the levels of cellular NADH. NAD is a coenzyme that exists in all cells. It includes NAD+ and NADH . NADH is obtained by reduction of the glycolate dinucleotide, which is produced in the cycle of glycofermentation and cellular respiration. This molecule is also a marker in the mitochondrial energy production chain. NADH produced in the mitochondria can be directly used for ATP synthesis. After treatment with ANC for 2 h, changes in the NADH and ATP contents in the cells were detected . After the 2 h treatment, there was little difference in the cell number; thus, the discrepancy in energy metabolism was caused only by the concentration of ANC. The results showed significant declines in NADH after treatment with ANC . When the concentration of ANC increased to 1000 μM, NADH was only 18.6 ± 0.1% of that in normal cells. The ratio of NAD+ / NADH increased with increasing concentrations of ANC . NAD+ is mainly distributed in the cytoplasm, while NADH is mainly located in mitochondria. Thus, the concentration of NAD+ changed little, but the reduction of NADH would lead to a higher ratio of NAD+ /NADH. The lower ratio of NAD+ /NADH could provide the driving force for the oxidation respiratory chain. Therefore, the decrease in this ratio may inhibit cell proliferation. The ATP content in the cells was determined as a function of ANC loading. As shown in Fig. 5c, when the ANC concentration was below 500 μM, few changes occurred in ATP, which remained 76.7 ± 0.2% of that of the control group after treatment for 2 h at 500 μM. When the ANC concentration was up to 1000 μM, a sharp decline in ATP was observed, with 16.5 ± 0.1% of the control group. These findings provide direct evidence that ANC could damage mitochondria by reducing oxidative phosphorylation agents.Multi-trophic interactions play important roles in shaping complex food webs throughout ecosystems. Many of these interactions occur around plants, which are at the base of food webs as members of the first trophic level. Plants are attacked by numerous herbivorous insects and defend themselves through mechanisms such as toxin production and the release of chemical cues from their roots and shoots that may recruit natural enemies of these insects . In response, blueberry in container herbivorous insects have evolved mechanisms to tolerate plant toxins and, in some cases, even to sequester them for use against their natural enemies. These mechanisms include, but are not limited to, metabolic detoxification, target site insensitivity via amino acid substitutions in the molecular targets of toxins, and/or clearing of toxins by ABC transporters and organic anion transport polypeptides expressed in the digestive system, the blood-brain barrier, and the Malpighian tubules .

When an herbivorous species evolves sequestration of toxins from its host plants, this has the potential to drive evolutionary changes across multiple trophic levels: while natural enemies do not feed directly on the producers of the toxins , they nevertheless encounter them upon attacking toxin-sequestering herbivorous hosts or prey. As an illustration of this concept, previous studies have shown that nematodes in regions with the western corn rootworm, which sequesters toxic benzoxazinoids from maize, have evolved behavioral and metabolic resistance to these toxins in their host . Toxins provide self-defense across a vast variety of organisms. Among distinct classes of toxins, cardiac glycosides represent a prevalent group produced by a diverse array of species, including plants such as milkweeds, foxglove, and oleander; insects such as certain chrysoline beetles and fireflies; and amphibians such as the cane toad . Several other species, while not capable of synthesizing CGs themselves, have evolved mechanisms that allow CG resistance, facilitating CG sequestration. Examples of species that sequester CGs for self-defense are the monarch butterfly , milkweed bugs, various clades of milkweed and dogbane beetles, the tiger keelback snake, and the African crested rat . CGs are typically produced as cocktails of compounds with varying hydrophobicities and all inhibit the sodium pump , hindering its function and disrupting ion flow across membranes of cells in the nervous system and other tissues . Na+ /K+ -ATPases are highly conserved among animal species, making their interactions with CGs a powerful model system for studying processes central to coevolution and chemical ecology, including in the context of multitrophic interactions . Milkweeds are common in North and Central America and serve as an important food source for a variety of specialized herbivorous insect species. Previous studies have shown that several of these specialists, including the monarch butterfly, milkweed and dogbane beetles, and the large milkweed bug , can sequester CGs in different compartments of their bodies for protection against natural enemies and that this is facilitated by TSI . TSI evolved in herbivores from at least six orders of insects and parallel TSI-conferring substitutions were found in the first extracellular loop of the Na+ /K+ -ATPase at sites 111, 119, and 122 . While monarch caterpillars and beetle grubs contain high CG levels in their hemolymph, milkweed bugs form storage compartments in which, aided by transporters, they can store high concentrations of hydrophilic CGs . Milkweed bugs further store hydrophobic CGs throughout the fat body . Like CG-sequestering insect herbivores, their predators and parasites must also confront high levels of the toxins. Nematodes of the genus Steinernema are entomopathogenic nematodes that parasitize insects. They can be found in soil as infective juveniles or within their insect hosts where they undergo further development . Milkweed and dogbane beetle larvae are present in the soil as root feeders . Furthermore, monarch caterpillars and milkweed bugs regularly visit the soil as well, for example when disturbed . When herbivorous insects move in or on soil, their risk of becoming infected by EPNs increases markedly. The species S. carpocapsae is known to occur in soil around milkweed plants and to naturally infect insects that feed on either below ground or above ground plant parts . While this species is a generalist, its presence near milkweed plants prompts the question of whether it can overcome the toxicity of CGs ingested by milkweed-feeding herbivores. This is particularly poignant in light of our recent finding that S. carpocapsae, but not any of its known congeners, evolved a substitution in the first extracellular loop of its Na+ /K+ – ATPase . This substitution was shown to have a large effect on TSI to CGs in insects and evolved in many insect herbivores specialized on milkweeds, suggesting a possibility of parallel evolution across trophic levels . Our research addresses the following questions: Does S. carpocapsae infect CGcarrying insects with more success than other Steinernemaspecies that lack TSI? I.e., does CG sequestration by herbivorous insects influence EPN infection?