Bioactive potential of tropical highland apple ( Malus domestica cv. Anna) crude extract: opportunities for food waste revalorization

Background: The third most produced fruit crop in the world, apples ( Malus domestica Borkh) are one of the most accessible and widely consumed fruits, with known benefits for human health. Although they are a temperate crop, apple cultivation has been successfully implemented in lower latitudes. Tropical highland Costa Rican apples (cv. Anna) have shown high total polyphenol content, and cytotoxic effects against human cancer cell lines. However, most reports originate from purified fractions, obtained using methods which are not easily translatable for commer‑ cial applications. In this study, we prepared a polyphenol‑rich bioactive extract from Costa Rican Anna apples, using food‑grade solvents and simple techniques, aiming towards easy translation of the extraction protocols to small local producers. Results: The whole apple crude extract (AE) was investigated for its total polyphenol content, general phytochemical profile, cytotoxicity against human breast and lung cancer cells, and regenerative potential in murine skin fibroblast monolayers. The AE showed total polyphenol amounts comparable to that obtained using more refined extractions in previous studies, and exhibited cytotoxic activity against human breast (MCF7) and lung (NCI‑H460) cancer cells, and inhibition of cell proliferation in the scratch‑wound‑healing assay. Conclusions: Food‑grade simple protocols were successful for obtaining a polyphenol‑rich bioactive extract from Costa Rican Anna apples. The easy‑to‑implement extraction protocols and biochemical tests could provide a source of bioactive phytochemicals to be used in circular production systems.


Background
Apple fruits (Malus domestica Borkh, Rosaceae) are worldwide accessible and low-cost fruits with high nutritional value, diverse bioactive phytochemical content, and several health-promoting properties confirmed in clinical trials, including antioxidant, anti-inflammatory, cardioprotective and neuroprotective effects, gastrointestinal benefits, and modulatory activities in the glucose and lipid metabolism [1,2].The health benefits of dietary apple intake have been attributed, at least partially, to their antioxidant polyphenol content [3].Antioxidant plant polyphenols have been shown to reduce the risk of developing chronic diseases associated with the accumulation of age-related oxidative damage [4][5][6][7][8].The phytochemical composition and bioactive activities of apples have been described elsewhere [2,3,[9][10][11][12].
After bananas and watermelons, apples were listed as the third most produced fruit crop in the world in 2020, reaching over 86 million tons, with China as the main producer (46.9%), followed by the United States (5.4%), Turkey (5.0%),Poland (4.1%), and India (3.2%) [13].Although they are traditionally a temperate crop, apples have been increasingly adapted to cultivation at low latitudes, with higher temperatures and longer photoperiods [14].Tropical highland conditions may also provide high solar irradiation, which is known to promote flavonoid accumulation and diversity in plants [15].Accordingly, high levels of UV irradiation have been shown to increase anthocyanin and flavonol content in fruit peel in apples [16].
One of the most prominent apple varieties cultivated in tropical high-altitude settings is M. domestica cultivar Anna (Golden Delicious x Red Hadassiya), a low chilling requiring variety created in Israel in 1963 [14,17].Anna apples cultivated in Costa Rica highlands have shown higher and more diverse total polyphenol content in peel extract than other cultivars from temperate regions, as well as cytotoxic effects against gastric (AGS) and colon (SW-620) human carcinoma cells [18,19].
In this study, we prepared a whole fruit crude extract from Anna apples cultivated in the same Costa Rican highland region, using food-grade solvents and simple techniques, aiming towards easy translation of the extraction protocols to small local producers.The whole apple extract was evaluated for total polyphenol content, general phytochemical profile, cytotoxic activity against human breast (MCF7) and human lung (NCI-H460) cancer cells, and regenerative potential in NIH-3T3 murine skin fibroblast monolayers.

Apple extract (AE) preparation
Apple fruits (Malus domestica cv.Anna) were purchased in a ripe state (Fig. 1) from local producers located in San Marcos, Tarrazú, San José, Costa Rica (permit R-CM-ITCR-002-2021-OT). Whole fruits (without seeds) were rinsed in water, sliced, freeze-dried (Bench Top FDB-8602, OPERON), grinded (1 mm, MF-10 basic IKA, WERKE), and preserved at − 20 °C until extraction.The powdered material was extracted (1:10, plant material to solvent) by three consecutive water-ethanol macerations at room temperature, followed by ethanol elimination in a vacuum evaporator (40 °C, R-300, BUCHI).The identity of all fruits collected was authenticated by Botanist M.Sc.Elizabeth Arnáez-Serrano, Biology Department, Costa Rica Institute of Technology and were previously confirmed with the support of the Costa Rican National Herbarium [19].
High-performance Thin Layer Chromatography (HPTLC) was performed using the freeze-dried AE powder dissolved in methanol (1 mg mL −1 ).The method was developed using a CAMAG HPTLC system (CAMAG, Muttenz, Switzerland).Sample application (10 µL) was done on a 20 × 10 cm HPTLC glass plate coated with Silica gel F 254 (Merck, Darmstadt, Germany).Samples were applied to the HPTLC plate with the aid of the CAMAG Automatic Sample Applicator 4 (ATS4) as 8 mm bands.The first application position (x axis) was 20 mm and the sample bands were applied at a distance of 8 mm from the base of the HPTLC plate (application position y).The distance between tracks was 11 mm.Chromatography development was done in a CAMAG Automatic Developing Chamber 2 (ADC 2) with the following settings: 30 s pre-drying, 20 min saturation with filter paper, humidity control with MgCl 2 (10 min, 33% relative humidity), Locally grown fruits may be recognized by the green-yellow spots on their peel, and their tangy acidic flavor.B. Topographic map of the Costa Rican region where the cultivars are grown, which locates at about 1500 m.a.s.l.Geographically, it is located between coordinates 83°58′16" to 84°9′28" west longitude, and 9°26′46" to 9°43′7" north latitude [20] 70 mm migration distance (solvent front position), 5 min drying time, 10 ml of mobile phase for HPTLC development and 25 mL of mobile phase for HPTLC chamber saturation.The mobile phase was comprised of ethyl acetate: water: formic acid (100: 30: 20).The chromatograms were exposed to UV light (254 nm and 366 nm) and white light.The derivatization of the chromatograms with the natural product reagent (2-aminoethyl diphenyl borinate), anisaldehyde reagent, phosphomolybdic acid, fast blue salt B and the Dragendorff reagent were performed with the CAMAG Derivatizer.If the derivatization process required heating, The CAMAG TLC Plate Heater III was used.All reagents were purchased from SIGMA; prepared and applied according to the CAMAG Derivatizer instructions.Pre-and post-derivatization, images were taken, processed, and documented with the CAMAG TLC Visualizer.All HPTLC steps were programmed and monitored with the CAMAG VisionCATS software (version 2.5).Image comparison views were created with this software as well.
Total phenolic content (TPC) in the apple extract (AE) was determined using a modified Folin-Ciocalteu spectrophotometric method as described by Rojas-Garbanzo et al. [23].Briefly, AE were incubated with Folin-Ciocalteu reagent and sodium carbonate solution (75 g/L) at 50 °C for 15 min.The absorbance was measured at λ = 620 nm (FLUOstar OPTIMA, BMG LABTECH).TPC was calculated by comparing against an external calibration curve of gallic acid (10-80 mg GAE L −1 , r 2 = 0.9909).Results were expressed as µg of gallic acid equivalents (GAE) per gram of AE.TPC are shown as the mean ± SD (n = 3).

Cytotoxicity by MTT assay
Cells lines NIH-3T3, NCI-H460, and MCF7 were seeded onto 96-well plates (1 × 10 5 cells cm −2 ) and treated for 24 h with increasing concentrations of the apple extract (AE) diluted in the respective culture medium.Cell viability was measured by the MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assay as described by Calvo-Castro et al. [24].The formazan crystals were dissolved in ethanol 95%, and the absorbance was measured at 570 nm (FLUOstar OPTIMA, BMG LABTECH).Cell viability was normalized as relative percentages in comparison to untreated controls.The half-maximal inhibitory concentration (IC50) for each extract in each cell line was determined from dose-response linear dispersion curves.Data is shown as the mean ± SD (n = 3).Normality was tested with the Kolmogorov-Smirnov test and D'Agostino-Pearson test.Differences between AE treatments in the three cell lines were analyzed by two-way ANOVA followed by a Bonferroni post-test.All statistical analyses were performed using the software package Graph-Pad Prism (version 8.01; GraphPad Software, USA).

Scratch wound healing assay
NIH-3T3 fibroblasts were seeded on 24-well plates (1 × 10 5 cells cm −2 ).Confluent monolayers were scraped using a standard 1 mL micropipette tip, leaving an empty and homogeneous cross-shaped area [25], followed by treatment for 72 h with non-cytotoxic AE concentrations (38 and 58 µg GAE mL −1 ).The lesion was photographed by light microscopy every 24 h, and percentage wound area over time was calculated using Image J (https:// www.imagej.nih.gov/ ij/).Data is shown as the mean ± SD (n = 3).

Phytochemical analysis of the apple extract (AE)
The absence of bands in the HPTLC when exposed to UV light at 366 nm (Fig. 2, lane 1) and the few bands in the phosphomolybdic acid derivatization at visible light (Fig. 2, lane 5) suggest low compound diversity in the sample.Nonetheless, polyphenol and flavonoid content in the AE was confirmed by positive Folin-Ciocalteu and Shinoda tests, respectively (Table 1).Moreover, the HPTLC derivatized with the natural product reagent (Fig. 2, lane 2), presented two intense dark blue bands (at Rfs of 0.08 and 0.15) while exposed to UV light at 366 nm, common for phenolic compound.These bands were derivatized with phosphomolybdic acid and returned a blue color at visible light (Fig. 2, lane 5).This indicates that such substances are probably glycones.Together, the results for these bands suggest the presence of anthocyanins.
The extract was negative for containing alkaloids, saponins, steroids and terpenoids (Table 1).The absence of red-blue bands after treatment with anisaldehyde at visible light (Fig. 2, lane 3) and at 366 nm (Fig. 2, lane 4) confirms the lack of triterpenes and steroids in the sample.The lack of bands after staining with the Dragendorff reagent at visible light (Fig. 2, lane 7) confirms the absence of alkaloids in the sample.The lack of bands with the Fast Blue B Salt derivatization at visible light (Fig. 2, lane 6) could indicate the absence of aflatoxins in the extract.

Cytotoxicity of the apple extract (AE)
The effect of the AE on the viability of human cancer cells MCF7 (breast cancer) and NCI-H460 (lung cancer), as well as NIH-3T3 murine skin fibroblasts, was tested (MTT assay) after treatment for 24 h.The AE was most effective against MCF7 cells, requiring a half-maximal inhibitory concentration (IC50) 6.5 times and 3.9 times lower than in the H460 and 3T3 cell lines, respectively (Fig. 3).

Scratch wound healing assay
The effect of AE wound healing was tested by monitoring the regeneration of a simulated lesion on a monolayer of murine skin fibroblasts (NIH-3T3) (Fig. 4).While the untreated control regained full confluence (98.71 ± 2.24%) after 48 h, treatment with AE (in concentrations below the respective IC50) resulted in delayed wound regeneration, showing a prolonged effect Fig. 2 High-performance Thin Layer Chromatography (HPTLC) of whole apple (Malus domestica cv.Anna) freeze-dried AE powder (1 mg mL −1 ) (Image Comparison View, CAMAG).The HPTLCs were developed with UV light at 366 nm (1); or derivatized and developed with the natural products reagent and at 366 nm (2); with anisaldehyde at visible light (3); with anisaldehyde at 366 nm (4); with phosphomolybdic acid at visible light ( 5), with Fast Blue B Salt staining at visible light (6); and with Dragendorff reagent at visible light (7) Table 1 General physicochemical composition of the whole apple (Malus domestica cv.Anna) extract (AE) *Analyzed by food official methods of analysis of the association of official analytical chemists (AOAC) at Centro de Investigación y de Servicios Químicos y Microbiológicos (CEQUIATEC), Costa Rica.

Parameter
Apple extract (AE) even after 72 h at the highest concentration, with only 20.72 ± 11.97% lesion restoration.

Discussion
In this study, food-grade tropical highland whole apple (Malus domestica var.Anna) crude extract (AE) obtained by ethanol extraction showed slightly higher total polyphenol content (767.40 ± 65.83 mg GAE g −1 ) than previous studies using apples from the same source (up to 619.6 ± 19.5 mg GAE g −1 , methanol-based extraction) [18,19].The discrepancy is probably due to different extraction and analytical techniques, and natural fruit variation caused by seasonal cultivation, ripeness, and storage conditions.Nonetheless, the presence of phenolic compounds in the AE was confirmed by HPTLC, Folin-Ciocalteu and Shinoda tests (Table 1, Fig. 2).Moreover, the phytochemical analysis also showed no evidence of alkaloids, saponins, steroids, terpenoids and aflatoxins in the AE (Table 1, Fig. 2), supporting the potential use of whole apple crude extract for human intake in future applications.
Comparably, Tow et al. [26] showed that considerable amounts of polyphenols (up to 539.84 ± 8.90 mg polyphenols GAE g −1 , extracted with 50% ethanol) with cytotoxic activities (against cervical HeLa, liver Hep-G2, and colon HT-29 human cancer cells) can be recovered from frozen apple industrial byproducts.Considering that over 20 million tons of apple residues from agroindustry are produced per year globally [27], food-grade simple protocols as described in this investigation could be implemented for obtaining relevant amounts of total polyphenols from apple sources, including underutilized waste material, which could be converted into high-value recycled products with nutritional, medicinal, and cosmetic applications [27][28][29][30].Although apple production in tropical highland regions is not as extensive as in temperate latitudes [13], circular economy strategies should be encouraged for the upcoming challenges on food security and human health in the near future.
On the other hand, non-cytotoxic doses of the AE did not improve the regeneration of a simulated lesion on a monolayer of murine skin fibroblasts (NIH-3T3); on the contrary, the AE seemed to delay or inhibit the regeneration process.The scratch-adjacent cells treated with AE remained attached to the flask, but with reduced migration into the simulated lesion in comparison to the control, and in an apparent dose-dependent manner.Given that we only monitored the surface area covered with cells over 72 h, future experiments should quantify cell amounts, doubling times and viability, over an extended period of time, and after removing the AE treatment and reverting to control conditions.Even though this observation speaks against possible regenerative applications, and while the effect might only be cytostatic, the prolonged inhibition of cell migration or proliferation at relatively low concentrations supports the chemopreventive potential of the AE.Future studies should also explore the impact of gastrointestinal digestion of the extracts on the biological effects reported here, and consider other forms of administration.

Conclusions
In this study, we verified that a food-grade whole fruit crude extract from tropical highland Costa Rican Anna apples showed similar total polyphenol content than previously reported in more refined extractions [18,19], and exhibited cytotoxic activity against human breast (MCF7) and lung (NCI-H460) cancer cells.Overall, the simple food-grade extraction protocol and easy-to-implement low-cost phytochemical tests could be put into practice by small producers for obtaining relevant amounts of bioactive polyphenols from apple materials, aiming towards their conversion into added-value products in circular production systems.