Deciphering the nutritive and antioxidant properties of Malay cherry (Lepisanthes alata) fruit dominated by ripening effects

In this study, Malay cherry fruit were explored for the changes in their nutritive and phenolic compositions upon ripening (unripe and ripe stages). Nutritive compositions (sugars, proteins, and fats) of the fruit increased, whilst organic acids of the fruit decreased in ripe fruit. Twenty-eight non-anthocyanin phenolics of the fruit were identified by the high-performance liquid chromatography-high resolution-time of flight mass spectrometry (HPLC-HR-TOF/MS2). Among them, quercetin-3-O-rutinoside and quercetin-3-O-glucoside are dominant species in the unripe fruit, and four more phenolics are shown in the ripe fruit. Additionally, seventeen anthocyanins were solely identified in the ripe fruit. This could be the signature phenolic profile of Malay cherry fruit. The total phenolics and total proanthocyanidins of the fruit significantly decreased upon ripening. Consistently, antioxidant capacities of the fruit also decreased upon ripening. Our results suggest unripe fruit are good sources of phenolic antioxidants that are worthwhile for utilisation as functional food sources.


Introduction
From ancient to modern times, ripe fruit are more acceptable to human beings than unripe ones. This could be simply because the ripe fruit always give a more enjoyable sensory experience. Knowledge of nutrients (sugars, organic acids, proteins, and fats) and phenolics in ripe and unripe fruit is likely to be ignored despite their large differences. McCune et al. revealed the fact that nutrients and phenolics of fruit are dominated by their ripening stages. 1 A growing body of literature has, in fact, demonstrated that ripe sweet cherry, 2 red raspberry, 3 and blueberry 4 possess higher total phenolic contents (TPC) than unripe ones; however, the contrary was observed in the TPC of black raspberry, 3 strawberry, 3 and blackberry. 5 . In other words, the changes in the TPC of ripe fruit are unpredictable. Therefore, an individual study is necessary to understand the nutritive and phenolic changes in a specic fruit upon ripening.
The ripening of fruit includes three distinct stages: unripe, veraison, and ripe stages. As the nutritive and phenolic compositions of fruit vary largely at the intermediate stage, it is hard to be tracked. Therefore, the two steady stages (unripe and ripe) of fruit are expected to be further studied. 6 Fruit are great sources of phenolic antioxidants that are believed to have wide spectrum of health benets. This is especially so for berries and cherries. The berries are rich in phenolic antioxidants of diverse chemical structures and health promotion effects. 7 We have witnessed the market penetration of acai berry from South America to become mainstream health foods because of their high antioxidant contents. 8 Similarly, tart cherries are also known to contain high phenolic antioxidants with anti-inammatory properties that can help reducing pain and accelerating strength recovery aer exercise. 9 It is well known that the phenolics of cherries support the potential preventive health benets of cherry intake in relation to cancer, cardiovascular disease, inammatory disease, diabetes, and Alzheimer's disease. 1 There are many more small fruit, cultivated or wild, that receive little or no attention in scientic community and their potential values await to be uncovered as functional food ingredients. This is especially so in tropical region where there are plenty of exotic fruit. One such example is Lepisanthes alata, commonly known as Malay cherry. L. alata is a tropical species of Sapindaceae native to Southeast Asia. The fruit of Malay cherry are sub-globose shaped (2-4 cm) with the colour from green to dark reddish purple as fruit ripen. The pulp-with-peel portions of the ripe fruit are eaten fresh with fairly sweet taste. 10 While the Malay cherry has long been popular in Singapore as a landscape tree, it attracted international interest resulting from our rst report. 11 We are intrigued by this tree because we found that the fruit and leaf proanthocyanidins showed potent inhibitory activity against starch hydrolases. 11 According to our preliminary study, Malay cherry fruit are rich in not only proanthocyanidins, but also in other phenolic compounds (polyphenols and simple phenolics). Among these phenolic compounds, a continuously vast interest has been focused on anthocyanins and polyphenols due to their relatively high antioxidant capacities and corresponding health benets. 2,12 However, in terms of Malay cherry fruit, there is virtually no information on the prole of phenolic compounds, not to mention phenolic changes upon ripening. To our best knowledge, this is the rst study to evaluate the effect of ripening on the nutritive and antioxidant properties of Malay cherry fruit.
From a food product design standpoint, nutritive and phenolic compositions, which largely determine the functionality of foods, can be modulated by ripening. Therefore, it is a prerequisite to be clear on the nutritive and phenolic compositions of the unripe and ripe fruit of Malay cherry. As well, additional understandings of the changes in total phenolics, total anthocyanins, total proanthocyanidins, and antioxidant capacities of these fruit were of great importance. Therefore, the aim of this study was to investigate the above descriptions. More specically, unripe and ripe fruit were subjected to assess their antioxidant capacities and consequently identify the major phenolic compounds within the fruit.
The fresh Malay cherry fruit were sampled from 20 multiple trees at unripe (green) and ripe (dark reddish purple) stages on 9 and 23 October in Singapore, respectively. The sampling area is a 1 km radius around 1.449 N 103.820 E in Sembawang district of Singapore. Over 100 fruit were sampled at each stage for further study. Aer removal of the seeds of fruit, only the pulp-with-peel portions of fruit were used in this study. The phytonutrient contents in the seeds will be studied separately.

Physicochemical analysis
2.2.1 Nutritive compositions. The moisture contents of fresh unripe and ripe fruit were determined according to AOAC Official Method 930.04. The fresh fruit were lyophilised using an advantage benchtop tray lyophiliser (the VirTis Company, Inc., Gardiner, NY) and ground into ne powders with a mini blender (DM-6, YOUQI, Changhua, Taiwan). The ne powders obtained were stored at À20 C until further determinations. The ash, protein, fat, insoluble dietary bre, and soluble dietary bre contents of fruit powders were determined according to AOAC Official Method 930.05, 977.02, 954.02, and 985.29. 13 The total dietary bre of fruit powders was calculated as combined values of the insoluble and soluble dietary bres.
The sugar contents and compositions of unripe and ripe fruit were evaluated as follows. The defatted and lyophilised fruit powder (1.0 g) was ultrasonicated with aqueous ethanol (10.0 mL, 80%, v/v) for 30 min. The slurry was centrifuged at 12 074 g for 10 min at 4 C to get the supernatant followed by evaporation at 40 C. The concentrated sugar extracts were diluted with deionised water in the ratio of 1 : 1 to get the ethanol-free sugar extracts (4 mL) for the following measurement. The total soluble solids (TSS) of the sugar extracts were measured using a digital RX-5000a refractometer (ATAGO, Tokyo, Japan) and calibrated using deionised water at 20 C. The TSS was expressed as percent sucrose. The reducing sugar contents of the sugar extracts were measured using the DNSA assay and expressed as milligram maltose equivalent per gram of dry weight of fruit, according to our previous study. 14 In brief, 100 mL of DNSA reagent was mixed with 100 mL of sugar extracts or maltose standard solutions (0 to 1.5 g L À1 ) in a 96-well microplate and boiled for 5 min. 100 mL of the cooling mixture was transferred to another 96-well microplate for measuring the absorbance at 540 nm with a Synergy HT microplate reader (Biotek Instruments Inc., Winooski, VT). The sugar compositions of the sugar extracts were analysed by a ultrafast liquid chromatograph Prominence system coupled with a LTII evaporative light scattering detector (Shimadzu, Kyoto, Japan), according to a literature method. 15 Serial diluted sugar extracts (10 mL) or standards (fructose, glucose, and sucrose ranging from 0.1 to 10.0 g L À1 ) were ltered through a regenerated cellulose lter (0.45 mm) and injected into a Zorbax carbohydrate column (4.6 Â 150 mm, 5 mm) with a guard column made by the same materials (Agilent, Palo Alto, CA). Mobile phase was acetonitrile (80%, v/v) at a ow rate of 1.4 mL min À1 for 23 min. The column oven temperature was set at 40 C. Detector was set at 40 C, gain 5, and pressure of 350 kPa. Sugars in the sample were identied by matching the retention times of standards and their concentrations were calculated by peak areas of the standard curves of the respective sugars. The standard curves were plotted with R 2 greater than 0.99. The total sugar contents of fruit were calculated as combined values of fructose, glucose, and sucrose.
The organic acids of unripe and ripe fruit were quantied as follows. The fruit powder (1.0 g) was ultrasonicated with deionised water (10.0 mL) for 30 min. The slurry was centrifuged at 12 074 g for 10 min at 4 C to get the supernatant. The supernatant was ltered and diluted with deionised water up to 25 mL in a volumetric ask. Organic acids of the solution were identied and quantied by ultrafast liquid chromatograph Prominence system coupled with a photodiode array detector (PDA, Shimadzu, Kyoto, Japan), according to a literature method. 15 The solution (10 mL) was injected into a Supelcogel C-610H ion exchange column (7.8 mm Â 300 mm, Supelco, Inc., Bellefonte, PA). The mobile phase was 0.10% H 2 SO 4 at an isocratic ow rate of 0.40 mL min À1 for 50 min at 40 C. Standards were prepared with serial concentrations of malic, succinic, and citric acids at 0.02-10.00 g L À1 . The absorbance was monitored at 210 nm. The concentrations of respective standards were calculated from calibration curves. All the curves had good linearity t (R 2 > 0.99). A FE20 K pH meter (Mettler Toledo, Switzerland) was used for pH measurements of unripe and ripe fruit.
2.2.2 Colour, dimension, and weight measurements. The colours of fresh fruit peel were measured using a CM-5 spectrophotometer (Konica Minolta, Tokyo, Japan) equipped with a D65 illuminant based on the CIE 1976 (L*, a*, b*) colour space. The specular component excluded (SCE) mode with a 3 mm Petri dish was used for all measurements. The dimension (length and diameter) and weight measurements of intact fresh unripe and ripe fruit were carried out.

Extraction and purication of phenolic compounds
The unripe and ripe fruit powders (20.0 g) were separately extracted with methanol (80%, v/v, 2 Â 100 mL) for 2 h by shaking on a vortex shaker. Each slurry was centrifuged and then each supernatant was evaporated at 40 C to obtain crude extracts for solid-phase extraction (SPE).
The phenolic compounds were puried by SPE according to a literature method. 16 The C18 Sep-Pak cartridges (Waters, Wexford, Ireland) were preconditioned sequentially with ethyl acetate (10 mL), methanol (10 mL), and 0.01 M HCl (15 mL). The crude extract (1 mL, approximately 0.5 g L À1 ) was loaded on the C18 cartridge that was eluted with 0.01 M HCl (15 mL). The adsorbed non-anthocyanin phenolics were eluted with ethyl acetate (40 mL). The adsorbed anthocyanins were then eluted with acidic methanol (0.1% HCl in methanol, v/v) until the eluent turned colourless. All eluents were separately evaporated at 40 C and ltered for characterisation and antioxidant capacity.
For the analysis of non-anthocyanin phenolics, the mixture were eluted with the ternary mobile phases consisting of A (50 mM aqueous ammonia acetate, pH 3.6), B (20% A in acetonitrile, v/v, pH 3.6), and C (200 mM acetate acid, pH 2.6). Elution programme started with 14% B and 86% C, changing to 16.5% B and 83.5% C at 12.5 min, 25% B and 75% C at 17.5 min, 80% B and 20% C at 40 min, and washing with 100% A for another 20 min. The ow rate and temperature were set at 1.0 mL min À1 and 25 C. Anthocyanins were eluted with the binary mobile phases consisting of A (acetonitrile) and B (acidic water). Mobile phase B was prepared with 10% acetic acid and 5% acetonitrile, by volume. Elution programme started with 100% B for 5 min, decreasing to 80% B at 20 min, 60% B at 25 min, ramping up to 100% B at 30 min, and holding for 5 min at a ow rate of 1.0 mL min À1 at 25 C.
HR-TOF/MS 2 analyses were performed using a TOF mass spectrometer via electrospray ionisation (ESI) interface and controlled by Compass Data Analysis soware. Mass spectra were acquired in negative mode for non-anthocyanin phenolics and in positive mode for anthocyanins with the range of m/z 50-1500. MS calibration standard was performed using sodium acetate. The MS 2 collision gas was nitrogen. The negative ion ESI parameters were capillary voltage 3500 V, dry gas temperature 200 C, dry gas ow 7.0 L min À1 , and nebuliser 3.0 bar. The positive ion ESI parameters were capillary voltage 4500 V, dry gas temperature 200 C, dry gas ow 7.0 L min À1 , and nebuliser 3.0 bar.

Quantication of total phenolic, anthocyanin, and proanthocyanidin contents
The unripe and ripe fruit powder (1.0 g) was separately extracted with methanol (80%, v/v, 2 Â 5 mL) for 2 h and centrifuged to get the supernatant. The supernatant was ltered and diluted with 80% methanol up to 25 mL in a volumetric ask. The solution was used for further analysis. The total phenolic content (TPC) of fruit was measured using the Folin-Ciocalteu assay with slight modications. 17 The solutions with serious dilutions (20 mL) were mixed with deionised water (90 mL) and Folin-Ciocalteu reagent (10 mL) in a 96-well plate and incubated for 5 min at room temperature in the dark. The Na 2 CO 3 solution (80 mL, 75 g L À1 ) was added to the mixture and incubated for another 2 h. The absorbance was captured at 765 nm using the microplate reader. A calibration curve of gallic acid was constructed yielding a linear correlation (y ¼ 4.8595x + 0.0047) with a high R 2 value of 0.99. The TPC of fruit was expressed as milligram gallic acid equivalent per gram of dry weight of fruit.
The total anthocyanin content (TAC) of fruit was separately measured according to the pH differential method with slight modications of a reported method. 18 Potassium chloride buffer (0.025 M, pH 1.0) and sodium acetate buffer (0.4 M, pH 4.5) were prepared for dilution. Two diluted extracts (20 mL) were mixed with corresponding buffer (180 mL) in a 96-well plate and the absorbance of each well was read at 510 nm and 700 nm using the microplate reader. Absorbance was calculated with the aid of eqn (1) below. The TAC of fruit was calculated using following eqn (2) and expressed as milligram cyanidin-3-Oglucoside equivalent per gram of dry weight of fruit.
The total proanthocyanidin content (TPAC) of fruit was measured using the DMAC assay with slight modications. 19 The extracts were serial diluted with a mixture [80% of ethanol (91%): 20% of deionised water, v/v] and pipetted (70 mL) into a 96-well plate to mix with fresh DMAC solution (210 mL, 0.1% DMAC in acidied ethanol, w/v). The acidied ethanol was prepared by mixing 75% of ethanol (91%), 12.5% of deionised water, and 12.5% of HCl (36%), by volume. The absorbance was read for 30 min using the microplate reader at 640 nm at 25 C. The maximum absorbance was calculated using a predetermined procyanidin A2 calibration curve (y ¼ 24.303x + 0.0151) with R 2 value of 0.99, where y represents the absorbance value and x represents the procyanidin A2 concentration (g L À1 ). The results were expressed as milligram procyanidin A2 equivalent per gram of dry weight of fruit.

Antioxidant capacity analysis
Antioxidant capacity was measured according to a reported ORAC assay. 20 The ORAC assay was carried out on a Synergy HT microplate uorescence reader. The potassium phosphate buffer (75 mM, pH 7.4) was prepared with KH 2 PO 4 and K 2 HPO 4 . 20 mL of the serial-diluted sample or Trolox was mixed with 160 mL of uorescein solution (8.16 Â 10 À5 mM in buffer) in a 96well plate. 20 mL of AAPH solution (153 mM in buffer) was automatically added into the plate to quench the uorescence. The uorescence was measured every minute for 2 h at 37 C. The excitation wavelengths vary from 465 to 505 nm, and emission wavelengths vary from 505 to 555 nm.

Statistical analysis
Statistical analyses were carried out with an independent sample t-test and one-way analysis of variance (ANOVA) using IBM SPSS Statistics V. 22.0 (IBM Corporation, Armonk, NY). All analyses were performed in triplicate and results were expressed as mean AE standard deviation. The signicance level was set at 0.05.

Physicochemical analysis
The physicochemical parameters of unripe and ripe fruit are listed in the Table 1. Among the nutritive compositions of fruit, the moisture contents (79.5% and 80.7%) accounted for the largest portion of the unripe and ripe fruit, which were no signicant difference (p > 0.05 by t-test) as fruit ripen. Among the other compositions, the total dietary bre was responsible for the majority of dried fruit, which was 59% of unripe fruit and 58% of ripe fruit. The total dietary bre of Malay cherry fruit was higher than most of the fruit. For example, it was around 5 times higher than that of ripe apples. 21 Dietary bre is a necessary nutrient in a healthy diet, because it can ease constipation, reduce harmful substance levels (e.g. cholesterol and heavy metals), and prevent large intestine cancer, obesity, diabetes, and coronary heart diseases. 22 Although there was no signicant difference in the total dietary bre, it is worth mentioning that the soluble dietary bre signicantly (p < 0.05) increased from 13.5% to 16.2% as fruit ripen. However, the insoluble dietary bre signicantly decreased from 45.7% to 41.4% as fruit ripen, which may due to the partial degradation of cellulose in the plant cell wall. The cellulose, a main insoluble bre in fruit, could be digested by cellulase into monosaccharides, thereby soening the fruit during ripening. 23 There was a rise in the TSS of fruit from 12.76% to 18.71% of ripen fruit. As a result, the total sugar signicantly increased as fruit ripen. The total protein and total fat slightly increased during fruit ripening. Total ash contents did not experience a signicant change as fruit ripen. The total carbohydrate can be estimated as the summation of total sugar, total dietary bre, and total starch (starch was not detectable in the fruit).
The main sugars found in the Malay cherry fruit were fructose, glucose, and sucrose. The fructose level was always higher than glucose and sucrose in response to various stresses. 24 As fruit ripen, the fructose and glucose signicantly increased from 22.7 and 17.8 to 29.6 and 23.4 mg g À1 dry weight of fruit, while the sucrose content slightly increased from 18.6 to 20 mg g À1 dry weight of fruit. Therefore, the total sugar and reducing sugar (fructose and glucose) of fruit experienced signicant increases as fruit ripen. Similarly, an increase in sugars (fructose, glucose, total sugar, and reducing sugar) as sweet cherries ripen. 25 The principle organic acid was malic acid in Malay cherry fruit, while the second and the third major acids were succinic and citric acids. This nding was agreement with the organic acids in sweet cherries. 26 The three organic acids were high in unripe fruit and decrease in ripe fruit. Malay cherry fruit reected a signicant increase in pH as fruit ripen. In conclusion, the major compositions changes in ripening fruit are a signicant increase in reducing sugar and a signicant fall in organic acids.
The green colour of unripe fruit was as follows: L* ¼ 60, a* ¼ À7.8, b* ¼ 30. The dark reddish-purple colour of ripe fruit (L* ¼ 24.4, a* ¼ 10, b* ¼ 2.4) was from anthocyanins, which are pigments in the plants. Moreover, during ripening, the fruit dimensions increased from 2.6 to 2.8 cm in length and from 2.2 to 3.0 cm in diameter; the fruit weight increased from 7.5 to 16 g.

Structural characterisation of anthocyanins
The anthocyanin pigments found only in the ripe fruit were characterised by HPLC-HR-TOF/MS 2 spectroscopy (Fig. 1A and B) and the corresponding MS prole data are summarised in Table 2. The chemical structures of anthocyanins are shown in the Fig. 3.
Peak    , which was consistent with and tentatively identied as cyanidin-3-O-rutinoside. 27 The peak A18 has fragmentation pattern cyanidin-3-O-rutinoside with one fragment at m/z 287, and thus it was tentatively assigned as cyanidin The indicator of fruit ripening is reddish purple colour development, which results from the accumulation of anthocyanins. In sweet cherries, cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside were found as major anthocyanins and exerted high antioxidant capacities, while tart cherries were rich in cyanidin-3-O-glucosylrutinoside and cyanidin-3-O-rutinoside. 29 However, more varieties of anthocyanins were found in Malay cherry fruit. The subtle structural variations of anthocyanins may dramatically vary their bioactivity. Therefore, it is of great interest to study the health promoting functions of the anthocyanins from Malay cherry fruit.

Structural characterisation of non-anthocyanin phenolics
The MS spectra and HPLC chromatogram of non-anthocyanin fraction from unripe and ripe Malay cherry fruit are shown in    Table 2. The chemical structures of identied compounds in Malay cherry fruit are shown in Fig. 3. Among the identied compounds, majority of them are avonoids along with some nonphenolic compounds and unknown structures.
3.3.6 Other phenolic compounds. Peak P4 gave an anionic m/z 467 [M À H] À (calculated for C 21 H 23 O 12 , 467.1195) and a fragment m/z 287 [M À H À 180] À (due to the loss of a glucoside moiety and a water molecule), which was tentatively iden-tied as mangiferdiol. The mangiferdiol is a mangiferin analogue. The xanthone glucoside mangiferin belongs to polyphenols. 35 Peak P6 produced an anionic m/z 293 [M À H] À (calculated for C 19 H 17 O 3 , 293.1183) and a fragment m/z 113 [M À H À 180] À , which was tentatively identied as 3-isopentadienyl-3 0 ,4,5 0 -trihydroxystilbene. 36 Peaks P13, P15, P20, and P33 were tentatively characterised as verbasoside, primulaverin, neobavaisoavone, and astringin, respectively based on the HRMS peak for molecular ions and their fragmentation patterns. 3.3.7 Nonphenolic compounds. Peak P3 produced an anionic m/z 383 [M À H] À and its chemical formula was calculated as C 15 H 28 O 11 . The peak P3 was tentatively identied as methyl 4-O-galactopyranosyl-2,3-di-O-methylgalactopyranoside. Peaks P12 and P14 produced a parent ion at m/z 401 [M À H] À . The parent ion yielded a fragment at m/z 269 [M À H À 132] À , which was due to the loss of a pentoside moiety (132 Da). They were tentatively identied as benzyl alcohol-hexoside-pentoside isomers. 37 Peak P22 was tentatively characterised as primeveroside. Peak P24 was tentatively characterised as jasminoside R. Peak P39 was tentatively identied as oxylipin, pinellic acid. 38 3.4 Quantication of total phenolic, anthocyanin, and proanthocyanidin contents Table 3 shows the TPC, TAC, and TPAC of unripe and ripe fruit. The TPC of ripe fruit (36.5 AE 0.3 mg gallic acid equivalent per g of dry weight of fruit) showed signicantly (p < 0.05 by t-test) lower than that of unripe fruit (92 AE 3 mg gallic acid equivalent per g of dry weight of fruit). A signicant fall also can be found in the TPAC as fruit ripen. The polyphenols, especially proanthocyanidins, are responsible for the bitter and astringent tastes. 39 As fruit ripen, the decrease in bitterness and astringency in fruit can explain the decrease in TPC and TPAC. 40 In addition, the higher TPC and TPAC in unripe fruit can be attributed to the higher rates of metabolite biosynthesis and protection against invasive actions of external organisms (i.e. diseases and insect pests) and adverse environmental conditions (i.e. UV light) at the earlier age of fruit. 41 Similarly, the decreases in TPC and TPAC as fruit ripen were reported in dates, apples, 42 and grapes. 43 However, the TPC of Malay cherry fruit was higher than most of other fruit, such as tart cherries, sweet cherries, and blueberries, and thus Malay cherry fruit can be served as rich sources of phenolic compounds in a healthy diet. 44 Anthocyanins are synthesised as fruit ripen, resulting in the development of a reddish purple colour. Consistent with the visual colour change, TAC increased markedly from unripe (not detected) to ripe stages (1.32 AE 0.02 mg cyanidin-3-O-glucoside equivalent per g of dry weight of fruit).

Antioxidant capacity
The results for antioxidant capacities of unripe and ripe fruit are shown in Table 3. The crude extracts of unripe and ripe fruit were puried using SPE and obtained three fractions, which were water fraction, non-anthocyanin phenolics, and anthocyanins in sequence. The non-anthocyanin phenolics of unripe fruit had the signicantly (p < 0.05 by ANOVA) highest antioxidant capacities at ORAC value of 4584 mmol Trolox equivalent per g dry weight of fraction, followed by the non-anthocyanin phenolics of ripe fruit (2254 mmol Trolox equivalent per g of dry weight of fruit). The anthocyanins of ripe fruit had medium antioxidant capacity (1031 mmol Trolox equivalent per g of dry weight of fruit). The crude extracts, water fraction, and anthocyanins of unripe fruit had low antioxidant capacities. Therefore, it shows that the non-anthocyanin phenolics were the main antioxidants in Malay cherry fruit, and the non-anthocyanin phenolics of unripe fruit showed signicantly higher antioxidant capacity than ripe fruit. The above ndings implied that the TPC of Malay cherry fruit generally decreased as fruit ripen. In many cases, antioxidant capacity, as well as TPC of berries, such as blueberry 45 and strawberry, 46 tended to decrease as fruit ripen.

Conclusions
From our results, nutritive compositions (sugars, proteins, and fats) of Malay cherry fruit increased, whilst organic acids of fruit decreased upon ripening. The antioxidant capacities of fruit decreased, as the polyphenols of fruit decreased upon ripening. It is apparent that Malay cherry fruit has high bre contents and is rich in polyphenolic antioxidants of diverse structures motifs that are known to have health promotion activity. Therefore, Malay cherry fruit are a good addition to the family of cherries and berries that have been considered as superfoods for human health. The ripening of Malay cherry fruit would be of particular interest to the food industry, as different agricultural practices could obviously affect the levels of benecial effects that could be obtained from consuming their polyphenolic extracts of different ripening stages. It warrants further study on cellular and animal models, to establish scientic evidence of the bioactivity of the polyphenolic compounds extracted from Malay cherry fruit, particularly the unripe fruit.

Conflicts of interest
There are no conicts to declare.