Analysis of Phytonutrients, Anti-Mutagenic and Chemopreventive Effects of Tropical Fruit Extracts

Thailand is located in the tropics and a wide variety of fruits are grown commercially. However, studies regarding the phytonutrients, anti-mutagenic and chemopreventive effects of these fruits are limited. Thus, phytochemical profiles and inhibition of key enzymes involved in obesity and diabetes, together with anti-mutagenic and chemopreventive properties of eight tropical fruit extracts cultivated in Thailand, including Psidium guajava ‘Kimju’, Psidium guajava ‘Keenok’, Ananas comosus ‘Pattavia’, Ananas comosus ‘Phulae’, Durio zibethinus ‘Chanee’, Durio zibethinus ‘Monthong’, Carica papaya ‘Khaekdum’ and Mangifera indica ‘Namdokmai’ were investigated. Different cultivars were also compared. Results showed that M. indica ‘Namdokmai’ was the most antioxidant-rich extract containing abundant 4-hydroxybenzoic acid and its derivative, gallic acid, as the main phenolics. M. indica ‘Namdokmai’ also exhibited high inhibitory capacities (>60% inhibition under studied conditions) against lipase, α-amylase and α-glucosidase, key enzymes as drug targets for controlling obesity and type 2 diabetes. Interestingly, all fruit extracts suppressed food mutagen-induced DNA mutations assayed by the Ames test, especially M. indica ‘Namdokmai’ and C. papaya ‘Khaekdum’ (>50% inhibition at 200 µg/plate). The M. indica ‘Namdokmai’ was also the most potent extract for suppression of cancer promotion (>90% inhibition at 200 µg/mL) followed by P. guajava ‘Kimju’, P. guajava ‘Keenok’ and C. papaya ‘Khaekdum’. Results potentially indicated that fruit intake after overcooked meat consumption might supplement nutrients and fiber and also reduce DNA mutation sources.


Introduction
Non-communicable diseases (NCDs) including cancer, diabetes, hypertension and cardiovascular diseases are leading global causes of public healthcare. Worldwide, new cancer cases in 2020 numbered 19.3 million [1], which is increasing annually, suggesting a rise in cancer prevalence as an important issue facing healthcare systems. However, many studies have supported the beneficial effects of fruit and vegetable intake. The previous cohort studies had suggested that consumption of fruits and vegetables at approximately 5 serving/day could effectively lower risk of mortality [2]. Besides, a systematic review and dose-response meta-analysis from 95 studies revealed that fruit and vegetable consumption correlated with decreased risk of cardiovascular disease, cancers and mortality. The study also revealed that fruit and vegetable intake should exceed 600 g/person/day to reduce cancer risk [3]. Reduction of cancer risk through consumption of fruits and vegetables may result from their phytochemical contents [4]. Furthermore, meta-analysis also showed analyzer (Mettler-Toledo AG, Greifensee, Switzerland) and were below 10%. The powdery samples were kept at −20 • C until required for further analysis.
Extraction of the fruit samples was performed as previously described [19] with some modifications as follows. Powdered samples (100 g) were extracted using a solvent mixture comprising methanol, acetone and water in a ratio of 2:2:1 (400 mL) for 24 h. Filtrates were obtained using a Büchner funnel filtration set equipped with a Whatman no.54 filter paper and an aspirator pump. Solvent removal was conducted by a rotary vacuum evaporator (Büchi Corporation, New Castle, DE, USA) with water bath temperature set at 40 • C. Removal of water-soluble components from crude extracts was conducted using a solid phase extraction (SPE) Sep-Pak C18 cartridge (Waters Corporation, Milford, MA, USA). The cartridge (5 g) was rinsed with methanol followed by distilled water twice (50 mL each). Dried crude extracts were dissolved in distilled water (500 mL), assisted with ultrasonic agitation. Then the extract was pumped into a pre-rinsed cartridge and eluted with distilled water (100 mL) with a flow rate of 5 mL/min. The filtrate was then re-extracted by liquid-liquid extraction using ethyl acetate (25 mL) to collect the remaining water-insoluble components (fraction 1) and discard the remaining water-soluble compounds, which cannot be moved to ethyl acetate. Finally, the solid phase attached within the cartridge was eluted using a mixture of methanol and acetone (1:1) (100 mL). The obtained eluate (fraction 2) was combined with the ethyl acetate fraction (fraction 1), and all solvents were removed using the rotary vacuum evaporator. The dried extract was stored at −20 • C until required for use.
Total phenolic contents (TPCs) were determined as previously described [22,23] with some modifications as follows. The fruit extracts (25 µL) in 10% (v/v) dimethyl sulfoxide (DMSO) were mixed with 10% (v/v) Folin-Ciocalteu reagent (50 µL) and incubated for 5 min. To the mixture, 7.5% (w/v) saturated sodium bicarbonate (200 µL) was added and mixed well. The mixture was then incubated in dark at room temperature (25 • C) for 2 h. The TPCs were measured at 765 nm using a Synergy TM HT 96-well UV-visible microplate reader (BioTek Instruments, Inc., Winooski, VT, USA) with a Gen 5 data analysis software. Gallic acid in a range of 0-200 µg/mL was used as a standard, and the TPCs were expressed as mg gallic acid equivalents (GAE)/g extract.
Total flavonoid contents (TFCs) were analyzed according to a well-established protocol [22] with some modifications as follows. The fruit extracts in 10% (v/v) DMSO (165 µL) were mixed with 5% (w/v) sodium nitrite (9 µL). After 6 min of incubation, 10% (w/v) aluminum chloride hexahydrate (18 µL) was added, and incubated for another 5 min. To the mixture, 1 M sodium hydroxide (60 µL) was added, and the TFCs were measured at 510 nm using the 96-well UV-visible microplate reader. Quercetin at the concentration ranging 0-100 µg/mL was used as a standard, and the TFCs were expressed as mg quercetin equivalent (QE)/g extract.

Determination of Antioxidant Activities
The antioxidant activities were performed using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, ferric ion reducing antioxidant power (FRAP), and oxygen radical absorbance capacity (ORAC) assays as previously described [23]. The dried extracts were dissolved in 10% (v/v) DMSO before analyzing antioxidant activities. The DPPH radical scavenging activity was evaluated using the DPPH free radical solution, a Trolox standard in the concentrations of 0.01-0.64 mM, and a detection at 520 nm. The FRAP activity was determined using FRAP reagent, the Trolox standard of 7.81-250.00 µM, and a detection at 595 nm. Lastly, the ORAC activity was determined using a fluorescein reagent, the Trolox standard of 3.12-100.00 µM, and the detection at an excitation wavelength of 485 nm and emission wavelength of 528 nm. All assays were performed on the 96-well UV-visible microplate reader and expressed as µmol Trolox equivalent (TE)/g extract.

Determination of Enzyme Inhibitory Activities
The inhibitory activities were determined using the key enzymes relevant to obesity (lipase) and diabetes (α-amylase and α-glucosidase). The enzyme assays were performed using the well-established protocols as previously described [6,24] on the 96-well UVvisible microplate reader. The dried extracts were dissolved in 10% (v/v) DMSO before performing enzyme inhibitory assays. All chemicals and reagents were received from Sigma-Aldrich (St. Louis, MO, USA).
The inhibitory activities were expressed as percentage of inhibition using the following equation: where A is the initial velocity of the control reaction with enzyme (control), a is the initial velocity of the control reaction without enzyme (control blank), B is the initial velocity of the enzyme reaction with extract (sample), and b is the initial velocity of the reaction with extract but without enzyme (sample blank).

Determination of Mutagenicity and Anti-Mutagenicity Using Ames Test
Mutagenicity testing was performed using Salmonella typhimurium tester strain, TA98, which is sensitive to frameshift mutations, as previously described [25]. In brief, the fruit extracts (100-300 mg/plate) were mixed with TA98 (provided by Dr. W. Kusamran from the National Cancer Institute, Ministry of Public Health, Bangkok, Thailand) at 2 × 10 9 CFU/mL, phosphate buffer and S9 mix (mouse liver homogenate, Sigma-Aldrich, St. Louis, MO, USA). The contents were then mixed with top agar and poured on minimal agar plates followed by incubation at 37 • C. After 48 h, the number of histidine revertant colonies of each plate was counted, and 2-aminofluorene (2-AF, 1 µg/plate) was used as a positive control. All experiments were performed at least in triplicate.
Anti-mutagenicity testing was performed using the active growth of TA98 at 2 × 10 9 CFU/mL. This was mixed with each indirect standard mutagen, including tryptophan pyrolysis products (Trp-P1 at 50 ng/plate or Trp-P2 at 20 ng/plate) and 2-amino-3,8dimethylimidazo [4,5-f]quinoxaline (MeIQx) at 25 ng/plate, phosphate buffer and S9 mix. The contents were then mixed with top agar and poured on minimal agar plates followed by incubation at 37 • C for 48 h. Then, the number of histidine revertant colonies on each plate was determined. The percentage of inhibition was calculated using the following equation: where C 0 is the number of revertant colonies from the positive control per plate, C 100 is the number of spontaneous revertant colonies from the negative control per plate, and S is the number of revertant colonies per plate induced by mutagen in the presence of the fruit extract. Percentage of inhibition was strong when it was higher than 60%, 60-41% was moderate, 40-21% was weak, and <20% had a negligible effect [26].
Cytotoxicity was analyzed using a water-soluble tetrazolium salt (WST-1) assay. Concentrations of 1 × 10 4 exponentially growing Raji cells were seeded in RPMI 1640 and cultured per well of a 96-well plate for 24 h. Before testing, cells were washed with phosphate buffer saline (PBS) and treated with the fruit extract up to 200 µg/mL. After 48 h, tetrazolium salt from the Cell Proliferation Reagent WST-1 kit (Merck, Darmstadt, Germany) was added and incubated at 37 • C in the dark for an hour. Then, the amount of cell viability, which was represented by the amount of formazan formation, was determined using a microplate reader (Infinite ® 200 PRO, Tecan, Männedorf, Switzerland) at 440 nm.
Screening of chemopreventive properties was determined using a phenotypic screening assay. Concentrations of 1 × 10 5 log phase cells were cultured per well of a 6-well plate in RMPI 1640 containing 10% (v/v) fetal calf serum, pyruvate (1 mM), penicillin (100 U/mL), streptomycin (100 µg/mL), 10 mM sodium butyrate and 65 nM 12-O-tetradecanoyl-phorbol-13-acetate (TPA or PMA, Merck, Darmstadt, Germany) with or without the fruit extract as described [27]. Cells were then incubated for the next 48 h at 37 • C. Deformed Raji cells (tree branch-like, dilation and flatness) were scored under a phase contrast microscope with a DP74 camera (IX83, Olympus, Tokyo, Japan). Counting was conducted randomly from ten areas covering at least 500 cells.

Statistical Analysis
All experiments were performed in triplicate (n = 3) or as indicated otherwise, and the results were expressed as mean ± standard deviation (SD). The significant differences between values were determined at p < 0.05 using one-way analysis of variance (ANOVA), followed by Duncan's multiple comparison test (more than two data) or Student's unpaired t-test (two data) from the statistical package for the social sciences (version 18 for Windows, SPSS Inc., Chicago, IL, USA).
Principal component analysis (PCA) and hierarchical cluster analysis (HCA) of TPCs, antioxidant activities, and enzyme inhibitory activities were determined using XLSTAT ® Trial (Addinsoft Inc., New York, NY, USA).

Phytochemical Analysis
Phenolic profiles including phenolic acids and flavonoids of eight fruit extracts including Psidium guajava 'Kimju', Psidium guajava 'Keenok', Ananas comosus 'Pattavia', Ananas comosus 'Phulae', Durio zibethinus 'Chanee', Durio zibethinus 'Monthong', Carica papaya 'Khaekdum' and Mangifera indica 'Namdokmai' were analyzed using high-performance liquid chromatography (HPLC) ( Table 1). Results indicated that among phenolic acids, the highest amounts of gallic acid and 4-hydroxybenzoic acid were detected in M. indica 'Namdokmai', while the highest ferulic acid content was detected in A. comosus 'Pattavia'. The highest content of caffeic acid was detected in A. comosus 'Phulae', and p-coumaric acid in D. zibethinus 'Chanee'. The fruit extract of P. guajava 'Kimju' possessed the highest contents of syringic acid and chlorogenic acid, while the highest vanillic acid content was found in D. zibethinus 'Monthong'. Sinapic acid was only detected in C. papaya 'Khaekdum'. Among the fruit extracts, C. papaya 'Khaekdum' exhibited the greatest variety of phenolic acids, while only one phenolic acid was detected in P. guajava 'Keenok'. A. comosus 'Phulae' contained the greatest variety of flavonoids with hesperidin the highest. A. comosus 'Phulae' contained the highest contents of naringenin and apigenin, while kaempferol was only detected in this extract. Luteolin was mostly found in A. comosus 'Pattavia', with quercetin the highest in P. guajava 'Kimju'. Trace amounts of isorhamnetin were only found in both cultivars of P. guajava, while myricetin was only detected in both cultivars of A. comosus. Interestingly, no flavonoid was detected in both cultivars of D. zibethinus.
Comparing between cultivars, P. guajava 'Kimju' exhibited greater varieties and quantities of phenolics than 'Keenok' cultivar, leading to greater TPCs and TFCs (1.2 and 2.5 times greater, respectively). Even though A. comosus 'Phulae' exhibited greater varieties of phenolics than 'Pattavia' cultivar, the later exhibited 3.2 and 5.1 times higher TPCs and TFCs, respectively than the former. Similar results were observed in D. zibethinus, whereby 'Monthong' cultivar exhibited a greater variety of phenolics but lower contents than 'Chanee', which exhibited 1.3 and 28.7 times higher TPCs and TFCs, respectively than 'Monthong' cultivar.

Antioxidant Activities
Antioxidant activities were performed using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, ferric reducing antioxidant power (FRAP) and oxygen radical antioxidant capacity (ORAC) assays to investigate different antioxidant mechanisms of the fruit extracts. The DPPH radical scavenging and FRAP assays follow single electron transfer (SET) mechanism, while the ORAC assay follows the hydrogen atom transfer (HAT) mechanism.

Enzyme Inhibitory Activities
Inhibitory activities of the key enzymes relevant to obesity (lipase) and diabetes (αamylase and α-glucosidase) were tested to investigate the potential biological functions of the fruit extracts. Lipase, a lipid degrading enzyme, is one of the anti-obesity drug targets to control the availability of excessive fat before absorption into the body. Likewise, αamylase and α-glucosidase are also targeted to control diabetes since they are carbohydrate degrading enzymes.

Mutagenicity and Anti-Mutagenicity Analysis of Fruit Extracts
One hallmark of cancer is genomic instability driven by DNA mutations [28]. Several studies have shown that consumption of vegetables and fruits might be associated with a reduction in cancer risk [29,30], due to their rich nutritive values and phytochemicals that play a role as antimutagenic agents [31]. As shown in Table 1, all eight fruit extracts contained various phytochemicals; thus, both the mutagenic and antimutagenic properties of the extracts were tested using the Salmonella typhimurium/microsome assay (Ames test) to determine their mutagenic and antimutagenic potentials. Advantages of this assay include rapid and representative metabolic activation with the addition of liver homogenate (S9 mix). S. typhimurium strain TA98 was used as a bacterial model. Results indicated that TA98 displayed a spontaneous background of revertant colonies (negative control) in the presence of the S9 mix ranging from 25-37 colonies (Table 4), and in the normal range of this strain [32]. The revertant colonies numbered 1252 after exposure to 2-aminofluorene (2-AF, a potent mutagen), implying that the tested strain was sensitive to detect mutagens with metabolic activation. Three extract concentrations (100, 200 and 300 mg/plate) that did not alter bacterial growth (data not shown) were used in the assay. Data showed no induction of revertant colonies when TA98 was exposed to all ranges of fruit extracts in the presence of metabolic activation, while dose-dependent was absent (Table 4), indicating that the fruit extracts were not mutagenic in this condition.

Chemopreventive Properties of Fruit Extracts
DNA mutation is an initiation step in carcinogenesis. As shown in Figure 1, all fruit extracts reduced chemical-mediated DNA mutations. Hence, it was interesting to examine  All data are expressed as mean ± standard deviation (SD) of triplicate experiments (n = 3). 2-Aminofluorene (2-AF, 1 µg/plate) was used as a positive control, and revertant colonies were 1252 ± 112.

Chemopreventive Properties of Fruit Extracts
DNA mutation is an initiation step in carcinogenesis. As shown in Figure 1, all fruit extracts reduced chemical-mediated DNA mutations. Hence, it was interesting to examine whether the extracts exhibited chemopreventive properties. Here, the phenotypic screening assay or anti-deforming assay in Raji cells was utilized. Raji cells are human lymphoblastoid cells with Epstein Barr Virus (EBV) early antigen (EA) integrated into the genome [34]. Under normal conditions, the EA is not expressed, leading to unchanged cell characteristics. Tumor-promoting agents like 12-O-tetradecanoylphorbol13-acetate (TPA) and sodium butyrate induce EA-mediated morphological changes or deformed cells. These morphological changes (tree branch-like, dilation and flatness) render cancer-like Raji cells. Supplementary Figure S5 shows the cell morphology of Raji cells under both positive and negative conditions. Hence, inhibition of Raji morphological changes was used as a readout for inhibitory properties of cancer promotion of the extracts [34].
First, the cytotoxicity of the fruit extracts toward Raji cells was studied using a watersoluble tetrazolium salt (WST-1) assay. The cells were treated with various concentrations of fruit extracts (0-200 µg/mL) for 48 h. Results indicated that, except for M. indica 'Namdokmai' at 100 and 200 µg/mL, fruit extracts were not cytotoxic to Raji cells ( Figure 2). Chemopreventive properties of the fruit extracts were further investigated via phenotypic screening assay. In the positive control, Raji cells were deformed at 28 cells from 500 cells after treatment with cancer-promoting compounds, TPA and sodium butyrate ( Figure 3). These results concurred with a previous report [27]. Compared to the positive control, A. comosus 'Pattavia', A. comosus 'Phulae', D. zibethinus 'Chanee' and D. zibethinus 'Monthong' exhibited the same quantity of deformed cells as a positive control, suggesting that these four extracts lacked chemopreventive properties determined by this assay. Interestingly, P. guajava 'Kimju', P. guajava 'Keenok', C. papaya 'Khaekdum' and M. indica 'Namdokmai' significantly inhibited deformation of Raji cells. Although we did not obtain a dosedependent effect, the data implied the chemopreventive effect of the tested fruit extracts. M. indica 'Namdokmai' exhibited cytotoxicity at 100 and 200 µg/mL; therefore, at these doses we were unable to exclude the possibility of cell death effect. However, a safe dose of M. indica 'Namdokmai' at 50 µg/mL (Figures 3 and 4) strongly inhibited the formation of deformed cells, indicating that among the eight extracts, M. indica 'Namdokmai' was the most potent inhibitor for cancer promotion. Figure 4 shows an example of Raji cells treated with TPA and A. comosus 'Phulae' or M. indica 'Namdokmai', while Supplementary Figure  S6 shows representative images of Raji cells treated with other fruit extracts.     A.

Correlation by Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA)
To determine the relationship between fruit extracts and TPCs, antioxidant properties (DPPH radical scavenging, FRAP and ORAC activities) and enzyme inhibitory activities (lipase, α-amylase and α-glucosidase), principal component analysis (PCA) and hierarchical cluster analysis (HCA) were employed. These techniques are suitable for the evaluation of various factors within one step compared to the Pearson correlation.
After the first PCA analysis, TFCs were separated from the total variables determined by Bartlett's sphericity test (data not shown), indicating that TFCs did not correlate with all variables. Thus, the second analysis excluded TFCs from the PCA analysis. Figure 5 illustrates the relationship among observations (taxa of fruit extracts), variables (TPCs, DPPH radical scavenging activities, FRAP activities, ORAC activities, lipase inhibitory

Correlation by Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA)
To determine the relationship between fruit extracts and TPCs, antioxidant properties (DPPH radical scavenging, FRAP and ORAC activities) and enzyme inhibitory activities (lipase, α-amylase and α-glucosidase), principal component analysis (PCA) and hierarchical cluster analysis (HCA) were employed. These techniques are suitable for the evaluation of various factors within one step compared to the Pearson correlation.
After the first PCA analysis, TFCs were separated from the total variables determined by Bartlett's sphericity test (data not shown), indicating that TFCs did not correlate with all variables. Thus, the second analysis excluded TFCs from the PCA analysis. Figure 5 illustrates the relationship among observations (taxa of fruit extracts), variables (TPCs, DPPH radical scavenging activities, FRAP activities, ORAC activities, lipase inhibitory activities, α-amylase inhibitory activities and α-glucosidase inhibitory activities) as a biplot (combined between observations and variables). The first two axes (PC1 and PC2) covered 89.49% of the total variables. PC1 (74.04%) contained TPCs, DPPH radical scav-enging activities, FRAP activities, ORAC activities, α-amylase inhibitory activities and α-glucosidase inhibitory activities, while PC2 (15.49%) contained only lipase inhibitory activities. Interestingly, M. indica 'Namdokmai', P. guajava 'Kimju' and P. guajava 'Keenok' were located in PC1, indicating that these three extracts exhibited high TPCs, DPPH radical scavenging activities, FRAP activities, ORAC activities, α-amylase inhibitory activities and α-glucosidase inhibitory activities. On the other hand, A. comosus 'Pattavia', A. comosus 'Phulae', D. zibethinus 'Chanee' and M. indica 'Namdokmai' were projected in PC2, indicating that they were potent anti-lipase inhibitors. M. indica 'Namdokmai' was the only extract projected in both PCs, and it was the most potent fruit extract against antioxidant, obesity and diabetes, possibly due to its high TPCs. Interestingly, D. zibethinus 'Chanee' and D. zibethinus 'Monthong' were located in PC2 but in the opposite direction, indicating that different cultivars could affected biological activities, particular lipase inhibition. activities, α-amylase inhibitory activities and α-glucosidase inhibitory activities) as a biplot (combined between observations and variables). The first two axes (PC1 and PC2) covered 89.49% of the total variables. PC1 (74.04%) contained TPCs, DPPH radical scavenging activities, FRAP activities, ORAC activities, α-amylase inhibitory activities and α-glucosidase inhibitory activities, while PC2 (15.49%) contained only lipase inhibitory activities. Interestingly, M. indica 'Namdokmai', P. guajava 'Kimju' and P. guajava 'Keenok' were located in PC1, indicating that these three extracts exhibited high TPCs, DPPH radical scavenging activities, FRAP activities, ORAC activities, α-amylase inhibitory activities and αglucosidase inhibitory activities. On the other hand, A. comosus 'Pattavia', A. comosus 'Phulae', D. zibethinus 'Chanee' and M. indica 'Namdokmai' were projected in PC2, indicating that they were potent anti-lipase inhibitors. M. indica 'Namdokmai' was the only extract projected in both PCs, and it was the most potent fruit extract against antioxidant, obesity and diabetes, possibly due to its high TPCs. Interestingly, D. zibethinus 'Chanee' and D. zibethinus 'Monthong' were located in PC2 but in the opposite direction, indicating that different cultivars could affected biological activities, particular lipase inhibition. To further evaluate the extracts, the HCA was employed as shown in Figure 6. The vertical axis represents dissimilarity between the groups. If the values are large, they are considered to be in different groups. The HCA was divided into two groups. Cluster 1 consisted of A. comosus 'Phulae', D. zibethinus 'Chanee', D. zibethinus 'Monthong' and C. papaya 'Khaekdum', while cluster 2 contained P. guajava 'Kimju', P. guajava 'Keenok', A. comosus 'Pattavia', D. zibethinus 'Chanee' and M. indica 'Namdokmai'. Both HCA and PCA were well-correlated. Cluster 1 represented extracts harboring moderate to poor activities, while cluster 2 contained extracts with moderate to high activities. To further evaluate the extracts, the HCA was employed as shown in Figure 6. The vertical axis represents dissimilarity between the groups. If the values are large, they are considered to be in different groups. The HCA was divided into two groups. Cluster 1 consisted of A. comosus 'Phulae', D. zibethinus 'Chanee', D. zibethinus 'Monthong' and C. papaya 'Khaekdum', while cluster 2 contained P. guajava 'Kimju', P. guajava 'Keenok', A. comosus 'Pattavia', D. zibethinus 'Chanee' and M. indica 'Namdokmai'. Both HCA and PCA were well-correlated. Cluster 1 represented extracts harboring moderate to poor activities, while cluster 2 contained extracts with moderate to high activities.

Discussion
Non-communicable diseases (NCDs) such as cancer, diabetes, hypertension and cardiovascular diseases are critical health problems with long-term risk effects including smoking, alcohol consumption, physical inactivity and especially, unhealthy diets [35]. These factors generate oxidative stress, which plays a significant role in NCD progression [36]. Fruit and vegetable consumption has been proven to ameliorate NCD morbidity and mortality, with reduced side effects compared to synthetic drugs [37]. These health properties are generated by phytochemicals such as polyphenolic compounds, tocopherols and carotenoids [38]. Investigation of the phytochemicals showing antioxidant activities, enzyme inhibitory activities, antimutagenic and chemopreventive properties of eight tropical fruit extracts including Psidium guajava 'Kimju', Psidium guajava 'Keenok', Ananas comosus 'Pattavia', Ananas comosus 'Phulae', Durio zibethinus 'Chanee', Durio zibethinus 'Monthong', Carica papaya 'Khaekdum' and Mangifera indica 'Namdokmai' indicated that (i) M. indica 'Namdokmai' exhibited the highest total phenolic contents (TPCs) with 4hydroxybenzoic acid and gallic acid the most abundant phenolics, (ii) high TPCs in M. indica 'Namdokmai' led to high antioxidant activities and enzyme inhibitory potentials, (iii) all tested tropical fruit extracts were not genotoxic and acted as anti-mutagenic compounds against known mutagens, and (iv) M. indica 'Namdokmai' was the most potent extract in suppression of cancer promotion.
Phenolics found in M. indica 'Namdokmai' were mainly 4-hydroxybenzoic acid and gallic acid. Results concurred with a previous study on M. indica L. cv. Ataulfo, suggesting that gallic acid was also a predominant phenolic (39%), while smaller amounts of 4-hydroxybenzoic acid were detected [39]. Five commercially available mango cultivars including Kent, Tommy Atkins, Keitt, Haden and Ataulfo from Mexico, Peru, Brazil and Ecuador exhibited TPCs ranging from 19.5 to 130.8 mg gallic acid equivalent (GAE)/100 g

Discussion
Non-communicable diseases (NCDs) such as cancer, diabetes, hypertension and cardiovascular diseases are critical health problems with long-term risk effects including smoking, alcohol consumption, physical inactivity and especially, unhealthy diets [35]. These factors generate oxidative stress, which plays a significant role in NCD progression [36]. Fruit and vegetable consumption has been proven to ameliorate NCD morbidity and mortality, with reduced side effects compared to synthetic drugs [37]. These health properties are generated by phytochemicals such as polyphenolic compounds, tocopherols and carotenoids [38]. Investigation of the phytochemicals showing antioxidant activities, enzyme inhibitory activities, antimutagenic and chemopreventive properties of eight tropical fruit extracts including Psidium guajava 'Kimju', Psidium guajava 'Keenok', Ananas comosus 'Pattavia', Ananas comosus 'Phulae', Durio zibethinus 'Chanee', Durio zibethinus 'Monthong', Carica papaya 'Khaekdum' and Mangifera indica 'Namdokmai' indicated that (i) M. indica 'Namdokmai' exhibited the highest total phenolic contents (TPCs) with 4hydroxybenzoic acid and gallic acid the most abundant phenolics, (ii) high TPCs in M. indica 'Namdokmai' led to high antioxidant activities and enzyme inhibitory potentials, (iii) all tested tropical fruit extracts were not genotoxic and acted as anti-mutagenic compounds against known mutagens, and (iv) M. indica 'Namdokmai' was the most potent extract in suppression of cancer promotion.
Phenolics found in M. indica 'Namdokmai' were mainly 4-hydroxybenzoic acid and gallic acid. Results concurred with a previous study on M. indica L. cv. Ataulfo, suggesting that gallic acid was also a predominant phenolic (39%), while smaller amounts of 4-hydroxybenzoic acid were detected [39]. Five commercially available mango cultivars including Kent, Tommy Atkins, Keitt, Haden and Ataulfo from Mexico, Peru, Brazil and Ecuador exhibited TPCs ranging from 19.5 to 130.8 mg gallic acid equivalent (GAE)/100 g fresh weight (FW) [40,41]. Interestingly, TPCs tended to increase with longer harvesting time or when the mango ripened [39,40]. Thus, high TPCs detected in M. indica 'Namdokmai' might be due to its ripening stage.
High phenolics in M. indica 'Namdokmai' also led to high lipase inhibitory activities. A previous study on seven M. indica varieties including Amrapali, Fazli, Golapkhas, Gopalbhog, Himsagar, Langra and Mohanbhog from India suggested that their methanolic extracts exhibited lipase inhibition, with half-maximal inhibitory concentration (IC 50 ) ranging 1.5-5.2 mg/mL [44]. The phenolics detected in these mangoes as gallic acid and 4-hydroxy benzoic acid also acted as strong anti-lipase inhibitors, with IC 50 values of 0.08 and 0.16 mg/mL, respectively [44]. Gallic acid also acted as a competitive inhibitor against α-glucosidase, with IC 50 value of 220.12 µg/mL, which was stronger than acarbose, an antidiabetic drug with IC 50 value of 823 µg/mL [45]. Gallic acid in combination with acarbose (3:1 ratio) also effectively inhibited α-amylase (82.2% inhibition), which was stronger than gallic acid (49% inhibition with 25 µM) but similar to acarbose (82.8% inhibition with 25 µM) [46]. These results suggested the reduction in usage of the synthetic drug with synergistic effect with natural bioactive compound.
Carcinogenesis is a multi-step physiological process equipped with initiation, promotion and progression [51]. This study revealed that four out of eight fruit extracts including P. guajava 'Kimju', P. guajava 'Keenok', C. papaya 'Khaekdum' and M. indica 'Namdokmai' possessed chemopreventive effects against TPA-mediated Raji deformation. M. indica 'Namdokmai' was determined as the most potent extract. A tumor-promoting agent, 12-Otetradecanoyl-phorbol-13-acetate (TPA) activated the mitogen-activated protein kinases pathway (MAPK) via phosphorylation of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) and p38 [52], which leads to Epstein Barr virus (EBV) expression and eventually cell deformation. Thus, one hypothesis postulated from a previous finding suggested that phenolics might contribute to inhibition of Raji deformation. For example, a polyphenol, resveratrol (3,4 ,5-trihydroxy-trans-stilbene), suppressed the phosphorylation of ERK1/2 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) pathway when Raji cells were exposed to TPA [52]. As previously stated, M. indica 'Namdokmai' was rich in 4-hydroxybenzoic acid and gallic acid. It was previously found that gallic acid isolated from Peltophorum pterocarpum effectively inhibited deformation of Raji cells [53]. Moreover, gallic acid in various studies showed the same trend on MAPK inhibition; for example, gallic acid at 25-75 µM suppressed ERK1/2 phosphorylation in osteosarcoma cells [54], and MAPK involving proteins ERK1/2 and c-Jun NH 2 -terminal kinase 1 and 2 (JNK1/2) showed reduced phosphorylation after treatment with gallic acid at 30-60 µM in oral cancer cells [55]. In our study, 200 µg/mL of M. indica 'Namdokmai' contained 1.41 µM of gallic acid, indicating that gallic acid might not solely contribute to the chemopreventive properties observed in Figures 3 and 4; however, the synergistic effects between phenolics and flavonoids must not be neglected [56]. Among the four fruit extracts with anti-deformation effect, three including P. guajava 'Kimju', P. guajava 'Keenok' and M. indica 'Namdokmai' possessed high antioxidant activities. TPA is well-characterized as a tumor-promoting agent but also acts as both an oxidative stress and inflammation inducer [57]. Therefore, these three extracts might exert their chemopreventive effects by quenching TPA-induced oxidative stress. It remains unclear how C. papaya 'Khaekdum' exhibits chemopreventive properties via the anti-deformation method because the phenolic and flavonoid contents were moderately low. Further determinations on more phytochemical profiles are required to clarify this issue.
In conclusion, other than being nutritionally rich, data from our study both enhance and promote fruit consumption and functional food development. M. indica 'Namdokmai', a famous cultivar of mango in Thailand, is a rich source of phytochemicals, antioxidants, and NCDs-relevant key enzyme inhibitors, in which consumption of this fruit could possibly reduce DNA mutations. Furthermore, our results showed that different cultivars effectively contributed to phytonutrients and biological activities.