Health benefits and apples: animal and in vitro studies

Antioxidant activity

Apples, and especially apple peels, have been found to have a potent antioxidant activity and can greatly inhibit the growth of liver cancer and colon cancer cells [31,32]. The total antioxidant activity of apples with the peel was approximately 83 μmol vitamin C equivalents, which means that the antioxidant activity of 100 g apples (about one serving of apple) is equivalent to about 1500 mg of vitamin C. However, the amount of vitamin C in 100 g of apples is only about 5.7 mg [32]. Vitamin C is a powerful antioxidant, but this research shows that nearly all of the antioxidant activity from apples comes from a variety of other compounds. Vitamin C in apples contributed less than 0.4% of total antioxidant activity.

Antiproliferative activity

Apples have been shown to have potent antiproliferative activity in several studies. When Caco-2 colon cancer cells were treated with apple extracts, cell proliferation was inhibited in a dose-dependent manner reaching a maximum inhibition of 43% at a dose of 50 mg/mL. The same trend was seen in Hep G2 liver cancer cells with maximal inhibition reaching 57% at a dose of 50 mg/mL [32]. Eberhardt et al. [32] proposed that it is the unique combination of phytochemicals in the apples that are responsible for inhibiting the growth of tumor cells. Apples had the third highest antiproliferative activity when compared to eleven other commonly consumed fruits [17].Different varieties of apples had different effects on liver cancer cell proliferation [33]. At a dose of 50 mg/mL, Fuji apple extracts inhibited Hep G2 cell proliferation by 39% and Red Delicious extracts inhibited cell proliferation by 57%. Northern Spy apples had no effect on cell proliferation [33]. Apples without peels were significantly less effective in inhibiting Hep G2 cell proliferation when compared to apples with the peel, suggesting that apple peels possess significant antiproliferative activity. Wolfe et al. [31] demonstrated that apple peels alone inhibited Hep G2 cell proliferation significantly more than whole apples. For example, apple peels from Idared apples had an EC50 of 13.6 mg/mL whereas the whole apple had an EC50 of 125.1 mg/mL. The EC50 refers to the dose of the apple that is required to inhibit cell proliferation by 50%.There has been some concern that apple antioxidants do not directly inhibit tumor cell proliferation, but instead they indirectly inhibit cell proliferation by generating H2O2 in reaction with the cell culture media [34]. However, more recently it has been reported that apple extracts did not generate H2O2 formation in WME, DMEM, or DMEM/Ham F12 media, and H2O2 addition to culture medium did not inhibit Hep G2 cell proliferation or Caco-2 colon cancer cell proliferation [35]. Additionally, the addition of catalase did not block the antiproliferative activity of apple extracts.

Inhibition of lipid oxidation

Addition of apple phenolics to human serum decreased diphenylhexatriene-labeled phosphatidylcholine (DPHPC) oxidation in a dose dependent manner [36]. DPHPC is incorporated into low-density lipoprotein (LDL), high-density lipoprotein and very low-density lipoprotein (VLDL) fractions and is an indicator of oxidation. Apple ingestion led to a decrease in DPHPC oxidation, reflecting the apples antioxidant activity in vivo [36]. The protective effects of apples on LDL oxidation reached its peak at three hours following apple consumption and returned to baseline levels by 24 hours [36]. Diphenylhexatriene labeled propionic acid (DPHPA) binds to serum albumin and is a good measure of oxidation within the aqueous phase of human serum. Mayer et al. (2001) also found that consumption of apples also led to a decrease in albumin DPHPA oxidation, reaching peak activity at 3 hours.Although apple juice typically contains less phenolics than whole apples, it is still a widely consumed source of dietary antioxidants. Pearson et al [37] examined the effects of six commercial apple juices and Red Delicious apples (whole apples, peels alone, and flesh alone) on human LDL oxidation in vitro. LDL oxidation was measured using headspace analysis of hexanal produced from copper-induced lipid oxidation in vitro. The dose of the apple juices and whole apple, apple peel and apple flesh, were standardized for gallic acid equivalents, and each LDL solution was treated with 5 μM gallic acid equivalents for each apple sample. LDL oxidation inhibition varied greatly between brands of fruit juice, ranging from 9 to 34% inhibition and whole apples inhibited LDL oxidation by 34%. Apple peels inhibited LDL oxidation by 34%, while the flesh alone showed significantly less inhibition (21%) [37].

Rats fed apple juice also had a decreased level of malondialdehyde (MDA), a marker of lipid peroxidation [38]. Quercetin, a major flavonoid in apples, had no effect on lipid oxidation when ingested by rats, suggesting that quercetin alone is not responsible for the apple's ability to inhibit lipid oxidation [38]. Other antioxidants and the interaction between the different apple antioxidants, including quercetin, may contribute to the antioxidant activity of apples. The effect of apple juice on lipid oxidation has also been examined in vivo in human subjects. In a study involving four women and one man, ingestion of high levels of a 1:1 mixture of apple juice and black currant juice increased the antioxidant status of the blood and decreased lipid oxidation [39]. Glutathione peroxidase also increased in humans consuming apple juice. Plasma MDA decreased over the seven-day intervention period when the subjects ingested the highest dose of the apple juice and black currant mixture (1500 mL). Despite the antioxidant effect on lipoproteins, apple juice intake had a pro-oxidant effect on plasma proteins in both humans and rats [38,39].