Nutritional, phytochemical, and in vitro anticancer potential of sugar apple (Annona squamosa) fruits

In plants, Fruits and their wastes are the main sources of bioactive compounds. Currently, Annona fruits have attracted the attention of people interested in health-promoting foods due to their phytochemical content that their activities were not studied before. This study aimed to explore the potential antioxidant, antimicrobial, and in vitro anticancer activity of two cultivars Annona squamosa (Annona b. and Annona h.) seed, peel, and pulp. We also meausred phenolic, flavonoid, sulfated polysaccharide, tannins, and triterpenoids. Polyphenol identification was determined using RP-HPLC. Results of the antioxidant activity revealed that the highest activity was observed for Annona h. seed extract using DPPH and ABTS assays with IC50 6.07 ± 0.50 and 9.58 ± 0.53 µg/ml, respectively. The antimicrobial activity against various pathogenic strains revealed that the peel extracts of both Annona b. and Annona h. exhibited the best antimicrobial activity. We also assessed the IC50 values for anticancer activity in all six Annona b. and Annona h samples against four cancer cell lines colon (Caco-2), prostate (PC3), liver (HepG-2), and breast (MCF-7) using MTT assay. Annona b. and Annona h seed extracts had the lowest IC50 values for four cancer cell lines with 7.31 ± 0.03 and 15.99 ± 1.25 for PC-3 and MCF-7, respectively. Both seed extracts, Annona b. and Annona h., showed significantly down-regulated mRNA expression of Bcl-2 and up-regulated p53 in all treated cell lines. Apoptosis was evaluated using nuclear staining, flow cytometric analysis, and immunohistochemistry of the proliferation marker (Ki-67). Additional studies are required to characterize the bioactive compounds responsible for the observed activities of Annona seed and determine its mechanism as an anticancer drug.


Results and discussion
Proximate composition analysis. The proximate compositions of different parts of Annona spp. were presented in Table 1. The obtained results showed that the moisture content of the pulp of the two species (Annona b. and Annona h.) varied from 81.66 ± 1.35 to 83.40 ± 0.61%, respectively. The highest protein content was recorded in Annona b. peels with a concentration of 3.30 ± 0.16%. The fat content ranged from 0.96 ± 0.23 to 29.21 ± 1.12% for Annona b. pulp and Annona b. seeds, respectively. The carbohydrate content ranged from 93.77 ± 1.78 to 94.36 ± 1.26%; the crude fiber content ranged from 66.64 ± 3.92 to 94.36 ± 1.26% for Annona b. pulp and Annona b. pulp, respectively. Finally, the ash content varied from 2.12 ± 0.19 to 3.14 ± 0.28%. Diversity in the gross chemical composition could be attributed to several factors, such as cultivar, ripening stage of fruit, climatic conditions, fertilization and irrigation system, and harvesting time 22 . It has been reported that the protein content of Annona muricata (L.) peels and seeds were 1.56 ± 0.01 and 2.73 ± 0.10%, respectively 23,24 , which is less than current findings in peels and much more in seeds in both species. At the same time, other studies showed that the protein content of Annona crassiflora pulp was 1.38 ± 0.01% 25 , which was less than the results of the present investigation (2.133 ± 0.33%). Annona b. and Annona h. contained all essential minerals and, Annona b. samples had the highest mineral concentrations, as shown in Table 1. On the other hand, Annona b. pulp showed the highest concentration of all measured minerals except potassium, which found to be high in Annona Table 1. Proximate chemical composition of different parts for Annona b. and Annona h. Means ± SD (standard deviations) in the same row followed by the same letters are not significantly different (P < 0.05). Each data point is the average of three replications.  27,28 . Utilization of bioactive compounds such as natural phenolics with antioxidant activity plays a crucial role in the food industry for the formulation of health-promoting foods. The findings of the present work are comparable with those described in the literature 8,28 . It is worth mentioning that the matrices studied in the current work were approximately 1.5-fold richer than those described in the previous studies in terms of total phenolic content. Notably, this is the first study exploring the concentrations of tannins, triterpenoids, and sulfated polysaccharides in Annona h. and Annona b., as these aspects have never been reported previously.

Phenolic compound profile (RP-HPLC). Phenolic compounds of the seed extracts of both Annona b.
and Annona h. are shown in Fig. 1B. Results revealed that the main differences between the two extracts are based on the presence or absence of vanillin, p-coumaric acid, ferulic acid, rutin, ellagic acid, and benzoic acid and that the relative amounts of all the compounds are present. The most abundant compounds in the Annona b. seed extract were gallic acid, p-hydroxybenzoic acid, syringic acid, ferulic acid, ellagic acid, benzoic acid, o-coumaric acid, and salicylic acid. These compounds are by far the most abundant compounds, not only in the seed of Annona b. but also in the seed of Annona h. except for ferulic acid, ellagic acid, and benzoic acid. Cinnamic acid was detected in low quantity in Annona b. seed extract and was not found in Annona b. seed extract. Moreover, cinnamic acid derivatives (p-Coumaric acid, ferulic acid, and o-coumaric acid) were detected only in Annona b. seed extract at concentrations of 1.96, 5.08, and 49.02 mg/100 g, respectively. Janicke et al. 29 reported that dietary fiber is a rich source of hydroxycinnamic acids, ferulic acid, and p-coumaric acid, all of which may contribute to its protective effect against colon cancer. Besides Pei et al. 30 confirmed the antioxidant, antiinflammatory, anti-mutagenic, anti-ulcer, and anticancer activities of p-coumaric acid. Also, many studies have shown that o-coumaric acid has antioxidant, and antitumor biological activity 31,32 . Antioxidant activity. Figure 2 shows the IC 50 values of free radical scavenging activity of the extracts toward DPPH and ABTS. Concerning DPPH, the IC 50 values of ascorbic acid and the seed extracts of Annona b. and Annona h. were 6.39 ± 0.04, 7.88 ± 0.28, and 6.07 ± 0.50 µg/ml, respectively, and the peel extract of Annona b. was 61.78 ± 3.16 µg/ml. This finding indicates that the seed extracts exhibited the highest antioxidant capacity; this may be attributed to the greater accumulation of polyphenols and flavonoids in the seed than in other parts of the plant. Regarding ABTS, IC 50 values were arranged in ascending order as follows; Annona b. seed, Annona h. peel, and Annona b. pulp, followed by Annona h. pulp, then Annona b. peel. The seed parts of both species exhibited the highest scavenging activity against both DPPH and ABTS free radicals (Fig. 2). The results revealed the potency of the Annona extracts as an excellent antioxidant. It is well known that antioxidants play a crucial role in the prevention of cancer and other oxidative stress related diseases 33 . Albuquerque et al. 34 reported the antioxidant activity of Annona cherimola Mill. pulp, peel, and seed, had IC 50 values of 452 ± 0.01, 330 ± 0.00, and 319 ± 0.08 µg/ml, respectively. These results were relatively high compared to our results for Annona squamosal, indicating that Annona h. and Annona b have high antioxidant activity compared to other reported Annona species. Numerous studies revealed the antioxidant activity for phenolic compounds and their ability to scavenge free radicals that disrupt biological molecules such as DNA, proteins, and lipids 35 . Antimicrobial activity. The results of the antimicrobial assay of the peel, seed, and pulp extracts of the two Annona spp. against six pathogenic microorganisms are shown in Table 2. E. coli, B. subtilis, C. albicans, K. pneumoniae, S. senftenberg, and S. aureus were used to test pathogenic microorganisms due to the prevalence of these microbes as foodborne pathogens. The peel extract of Annona h. exhibited excellent activities against K. pneumoniae ATCC 12296 and S. senftenberg ATCC 8400, with inhibition zones of 14.70 ± 1.07 mm and 16.10 ± 1.25 mm, respectively. This study is the first investigation to survey the antimicrobial activity of the peel, seed, and pulp extracts of Annona b. and Annona h., while many investigations have evaluated the antimicrobial activity of Annona Linn. leaf extracts 36,37 . The highest antimicrobial activity of Annona b. seed extract was observed against   [39][40][41] . Overall, the peel and seed extracts exhibited better antimicrobial activities than the pulp extracts, which might be due to the presence of active ingredients at higher concentrations in the seed and peel than in the pulp. Annona extracts (peel and seed) with high antimicrobial activity could be used to develop some functional food products to increase their shelf-life and boost the overall health benefits.  Table 3). The most active extract (seed) of both Annona species caused a collapse in spindle shape with cellular shrinking in the treated human cancer cell lines. This obvious morphological alteration was more severe in the seed extract of Annona b. than that of Annona h. (Fig. 3A). These morphological signs of cell death were detected by fluorescence after dual staining of the treated cancer cells with ethidium bromide and acridine orange (EB/AO) as shown in Fig. 3B. Nuclei of viable, healthy cells can only be stained with AO, and they appeared with green fluorescence, but early   (Fig. 3B). This result was confirmed by quantitative detection of the apoptosis percentage as a response to Annona b. seed and Annona h. seed extract treatment on HepG-2, Caco-2, and MCF-7 cell lines using annexin V/propidium iodide (PI)-flow cytometric analysis (Fig. 4A,B). Results revealed a significant elevation in total apoptosis percentages (> 48%) in the Annona b. seed extract-treated Caco-2, HepG-2, MCF-7, and PC-3 cells in comparison with those treated by Annona h. seed extract (< 39%). The most affected cell lines were PC-3 (51.27 ± 0.1%) and HepG-2 (50.14 ± 0.32%), followed by MCF-7 (49.30 ± 0.07%) and Caco-2 (48.81 ± 0.16%) after Annona b. seed extract treatment. On the other hand, the most affected cell lines were MCF-7 (38.99 ± 0.37%) and Caco-2 (33.05 ± 0.3%) followed by HepG-2 (32.67 ± 0.33%) then PC-3 (29.43 ± 0.035%) after Annona h. seed extract treatment. Natural products are still important sources to discover new anticancer drugs 42 . The National Cancer Institute has collected about 35,000 plant samples from 20 countries and has screened around 114,000 extracts for anticancer activity 43 . Aidy et al. 44 reported the anticancer potential activity of Annona muricata against breast, prostate, pancreatic, liver, lung, and colon cancers. Also, the anticancer properties of graviola (Annona muricata) were reported by Islam et al. 45 . Another report mentioned the anticancer potential of Annona muricata L. leaf extract 46 . Annona b. and Annona h. seed extracts and its polyphenols may be potentiating their anticancer activity. Polyphenols from diet have previously been documented to interfere many of biochemical pathways involved in cancer progression 47 . Furthermore, polyphenols may act as immune system booster, as well as help in living cells protection from ROS damage.

In vitro cytotoxicity and anticancer activity of different Annona h. and Annona b. parts.
Many clinical trials of polyphenols treatments confirming the protective mechanism of them due to variations in dose, timing, and other conflicting factors 48 . Thus, this in vitro study aimed to evaluate the anticancer potential of two Egyptian Annona species fruits and their byproducts as a first step for use in medical applications.  Fig. 5A. Furthermore, the seed extract of Annona b. enhanced the expression of p53 by more than fourfold, which was higher than in Annona h-treated cancer cells (2.15 to 3.10-fold) (Fig. 5B). Bcl-2 itself is an anti-apoptotic gene that prevents the initiation steps of apoptosis and programmed cell death 49 . These results suggested that Annona b. seed extract had a higher potency against apoptosis induction than Annona h. seed extract in all tested cancer cell lines. Wild type p53 is an important regulatory protein in the induction of apoptosis following DNA damage induced by anticancer drugs 50 . This tumor suppressor protein leads to the arrest of growth of viable cells in the G1 phase or apoptosis 51 . Further in vitro studies are required to determine the detailed anticancer mechanism of Annona b. seed extract. Figure 5C shows the immunohistochemistry for the proliferation marker (Ki-67) of HepG-2 cell line after treatment for 48 h with Annona b. and Annona h. seed extracts. These extracts were able to reduce Ki-67 level as indicated by increased purple color-stained cells (particularly in Annona b-treated HepG-2 cells) in comparison with brown color-stained untreated control cells (Fig. 5C-I). Figure 5C-II reveals that the Ki 67 level (2.51 ± 0.39%) was lower with Annona b. seed extract treatment than with Annona h. seed extract treatment HepG-2 cells (21.06 ± 1.93%). The potent inhibitory effect of seed extract of Annona b. on Ki-67 may attribute to their stronger apoptotic activity (Figs. 3B and 4) that was confirmed by higher percentage of the annexinstained HepG2 population (50.14%) and reddish fluorescence-stained nuclei compared to 32.67% apoptosis and yellowish-orange stained nuclei in the case of Annona h. Additionally, the apoptosis-dependent anticancer effect of Annona b. seed was validated by up-regulation of p53 expression by sevenfold while it was twofold in the case of Annona h. seed-treated HepG2 cell line (Fig. 5B).  Chemical analysis. Crude protein content determination, in dried powder form, was conducted using the Kjeldahl procedure according to Nelson and Sommers 52 . The crude fat and fiber contents were estimated in accordance with Folch et al. 53 , and Prosky et al. 54 , respectively. The mineral content, including Na, K, Ca, Mg, Cu, Mn, Zn, and Fe, was measured by microwave plasma atomic emission spectrometry (MP-AES) (Agilent 4100 MP-AES, USA), according to AOAC 55 . Phosphorus content was determined spectrophotometrically 56 . The mineral content was expressed as mg/kg of dry weight (dw). All measurements were carried out in triplicate.

Ki-67 expression in HepG2 cells after the treatment with the most powerful anticancer Annona extract.
Phytochemical content. The total phenolic content was determined using Folin-Ciocalteu reagent, as described by Dewanto et al. 57 . The total flavonoid content of the extracts was quantified by a colorimetric method described by Sakanaka et al. 58 . Sulfated polysaccharides were measured by the toluidine blue assay as described by Albano and Mourao 59 . The triterpenoid content was assessed colorimetrically via reaction of the triterpenoids with vanillin using ursolic acid as a standard 60 . The tannin content was determined colorimetrically following the method described by Price et al. 61 . All measurements were carried out in triplicate.

HPLC analysis for phenolics. Quantitative analyses of the phenolic content of the water extracts of the
Annona h. and Annona b. seed were carried out by following the procedure described by Tomaino et al. 62 using an RP-HPLC system (Agilent1260; Santa Clara, CA, USA) at 284 nm wavelengths with a C18 column (aKinetex 5lJm EVO C18 106 mm × 4.6 mm, Phenomenex, USA) maintained at 35 °C. The values for the main phenolic compounds were expressed in mg/100 g.  www.nature.com/scientificreports/ 700 nm. The reducing power was expressed as ascorbic acid equivalents (ASE)/mg. ASE is the reducing power of 1 mg of sample, which is equivalent to the reducing power of 1 nmol of ascorbic acid. The DPPH assay was carried out according to the method described by Shimada et al. 64 , and the absorbance of the solution at 517 nm was noted. For the ABTS radical scavenging assay, the method described by Re et al. 65 was adopted, and the absorbance was measured at 734 nm after 7 min. All experiments were carried out in triplicate.   In vitro detection of apoptosis-dependent anticancer effect using fluorescence microscope. Cancer cells were seeded in six well plates and incubated for cell attachment in 5% CO 2

Immunohistochemical detection of proliferation marker (Ki-67).
After trypsinization, the untreated and treated HepG-2 cells were centrifuged and washed with PBS buffer and then 10% formalin in PBS was added to cell pellets. The fixed cell specimens were dehydrated in ascending grades of alcohol and immersed in xylene for one hour (three times), followed by impregnation in melted paraffin to form solid paraffin blocks. Next, a rotator microtome was used to cut each block into 3-5 μm thick sections transferred onto positively charged slides. Slides were dried at 60-70 °C for 1-2 h then dewaxed by immersion in xylene three times for 5 min and rehydrated in descending grades of ethanol. After that, slides were incubated in 3% H 2 O 2 for 10 min, then washed in PBS buffer twice for 3 min and placed in 10 mM citrate buffer (pH 6), followed by heating at 60 °C for 10-20 min. After cooling and washing in PBS, the slides were separately soaked overnight in primary antibody (anti-Ki-67). Slides were washed in PBS, covered with biotinylated goat anti-polyvalent secondary antibody for 10 min, and then streptavidin peroxidase was added. After 10 min, the substrate of the secondary antibody (3,3′-diaminobenzidine) was added followed by washing in PBS and placed in a hematoxylin bath for 1-4 min. This was followed with a PBS wash (1 min) and then water (3 min). The percentage of immunostained cells was evaluated by imaging analysis software cellSens (CellSens Dimension 1.12) using a phase-contrast microscope (Olympus, Japan) 69 .
Statistical analysis. Data were expressed as mean ± standard error of the mean by the multiple comparison one-way analysis of variance (ANOVA) and Tukey's test using the SPSS16 software program with probability (p)-values < 0.05 considered statistically significant.

Conclusion
Nutritional composition analysis of two Annona species peels, seeds, and pulp containing relatively high contents from protein, fat, fibers, carbohydrates, and good mineral quantity was done. The high potassium and calcium levels mean the species parts may be used for food supplementation, to increase the quality of food. Based on the current data the significantly high levels of phenolic compounds in Annona seeds, exhibited very high antioxidant potential compared to the peels and pulp. The polyphenol compounds in Annona seeds might induce effective bactericidal action against foodborne pathogens. The polyphenol rich extracts, probably o-Coumaric acid (49.02 mg/100 g), may be responsible for their apoptosis-dependent anticancer behavior against (Caco-2), prostate (PC3), liver (HepG-2), and breast (MCF-7) cancer cell lines. This is the first report on the possible molecular mechanism of action Annona b. seed extract has on cancer cell proliferation. Additional investigations will be required to characterize the bioactive compounds that may be responsible for the observed activities of the Annona seed to determine the potential mechanisms that make it an anticancer drug. Furthermore, Annona seed extract could be used in other food application and the manufacturing of nutraceuticals. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.