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Metabolomic analysis of acerola cherry (Malpighia emarginata) fruit during ripening development via UPLC-Q-TOF and contribution to the antioxidant activity
Journal Pre-proofs Metabolomic analysis of acerola cherry (Malpighia emarginata) fruit during ripening development via UPLC-Q-TOF and contribution to the antioxidant activity Mingfeng Xu, Chenjia Shen, Han Zheng, Yunsheng Xu, Changfeng Xue, Beiwei Zhu, Jiangning Hu PII: DOI: Reference:
S0963-9969(19)30801-4 https://doi.org/10.1016/j.foodres.2019.108915 FRIN 108915
To appear in:
Food Research International
Received Date: Revised Date: Accepted Date:
15 July 2019 7 December 2019 15 December 2019
Please cite this article as: Xu, M., Shen, C., Zheng, H., Xu, Y., Xue, C., Zhu, B., Hu, J., Metabolomic analysis of acerola cherry (Malpighia emarginata) fruit during ripening development via UPLC-Q-TOF and contribution to the antioxidant activity, Food Research International (2019), doi: https://doi.org/10.1016/j.foodres.2019.108915
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Metabolomic analysis of acerola cherry (Malpighia emarginata) fruit during ripening development via UPLC-Q-TOF and contribution to the antioxidant activity Mingfeng Xu
a,b
, Chenjia Shen b, Han Zheng a, Yunsheng Xu c, Changfeng Xue c,
Beiwei Zhu a, Jiangning Hua,*
a School b
of Food Science and Technology, Dalian Polytechnic University, Dalian 116034, China
College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036,
China; c
School of Food Science and Engineering, Hainan Tropical Ocean University, Sanya 572022,
China;
Running title: Metabolomic
analysis of acerola cherry during ripening
development
* To Corresponding authors: Telephone No: +86-411-86318731, E-mail address: [email protected] (J-N Hu);
ABSTRACT: Acerola cherry (Malpighia emarginata D.C.) is a tropical fruit of great economic and nutritional value due to its high content of vitamin C. However, there is little information available about which ripening stage of Acerola cherry can provide the best nutrients. In the current study, the chemical variation at two developmental stages
(immature and mature) were investigated by metabolic profiling, and the
biological properties of Acerola cherry and its antioxidant assays at four developmental stages were measured, respectively. Through comprehensive metabolites analysis via ultra-high-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry system (UPLC-QTOF), 1,896 annotated metabolite features were obtained, and 133 metabolites were finally identified according to the MS/MS fragments compared with these standards in in-house database. Statistically differences in the levels of amino acids, flavonoids, lipids, terpenoids and ascorbic acids were found between mature and immature fruits. Interestingly, most of differential accumulated amino acids, flavonoids, lipids, and terpenoids predominantly accumulated in the mature fruits and ascorbic acid predominantly accumulated in the immature fruits. On the other hand, their antioxidant activities were compared. The alcoholic extract of immature acerola fruit possessed better scavenging ability of DPPH and ABTS than the mature one. The well correlations were found between the antioxidant potential with its content of ascorbic acid ( r = 0.9803 and 0.9897, respectively ). In conclusion, Acerola cherry showed very different metabolite profile and antioxidant activities during the fruit ripening development. The maturity of Acerola cherry has to be considered when it is being used for health food products.
KEYWORDS: metabolome; fruit ripening; UPLC-Q-TOF; ascorbic acid; antioxidant activity
1. Introduction Malpighia emarginata DC., commonly known as Acerola cherry or Barbados cherry, is native to South and Central America, and also cultivated in tropical or subtropical climate zones and south areas of China (Leffa et al., 2014; Silva, Duarte, & Barrozo, 2019). With increasing health awareness across the world, currently Acerola becomes popular due to its high antioxidant activity and its substantial amounts of ascorbic acid and other bioactive compounds including amino acids (AA), β-carotenoids (CA), phenolic compounds (PC) and minerals. Many studies showed that the antioxidant activity of Acerola extract was much higher as compared to polyphenols-rich extracts of other fruits, such as açai, cherry, grape, mango and strawberry (Chang, Alasalvar, & Shahidi, 2018). Due to the high concentration of these compounds, acerola fruits can be good candidates for the development of novel functional foods with promising effects on human health (Belwal et al., 2018; Cappato et al., 2018). Various commercial products containing Acerola are being used as dietary supplements. The global Acerola consumption market is estimated to reach US &17.5 billion by 2026 with 8.5% of compound annual growth rate (Belwal et al., 2018). Usually, Acerola cherry tends to be consumed as fresh fruit. The fresh fruit is considered rich in nutrients and contributes to the human health benefit. In this globalized marketed context, how to keep the fruits with high quality during long distance transportation or long time storage is a tough problem. It is known that excessive softening is a common cause of postharvest losses (Cai et al., 2019). A complex biological process after postharvest in Acerola cherry occurs with a notable influence on the human diet. For instance, vitamin C content in Acerola fruits was reported to change during ripening stages. High content of Vitamin C was found in immature fruits (1.9 g/100 g of juice) decreased significantly during ripening (0.97 g/100 g of juice in mature fruits) (Stafussa et al., 2018). The quantity and the composition of phenolic compounds also depend upon the ripening stage (Belwal et al., 2018). Proanthocyanidins were the major phenolic compounds in immature fruits
but during ripening, the amount of these compounds decreased and anthocyanins were the main polyphenolic compounds in mature fruits. Its chemical composition depends on cultivar, environmental conditions, and on the stage of fruit maturity (De Rosso & Mercadante, 2007). Comprehensive understanding of the change of nutrients during the fruit ripening is necessary. At present, metabolomics is important for the identification of metabolic signatures or patterns associated with various conditions (Lee, Jang, Choi, Joo, & Jeong, 2017). Metabolic changes during fruit development and ripening were observed to reveal physiological and nutritional traits that enhance fruit quality (Zheng et al., 2018). Small metabolites are important contributors to fruit flavor (Pinsorn et al., 2018). Primary metabolites including sugars and organic acids, in particular, are involved in sweetness and acidity taste formation. Amino acids are also related to fruit quality traits, by acting as precursors of volatile compounds involved in fruit aromas, and as substrates for polyphenol oxidases associated with the browning reaction of fruit (Cascia et al., 2013). Thus, metabolomics has been used to identify biomarkers associated with fruit quality (Monti et al., 2016). Much scientific literature abounds on the analyses of Acerola cherry. However, very few reports are available on the use of metabolomics as a tool for quality evaluations. To the best of our knowledge, there is no report about the comparative analyses of chemical composition together with its antioxidant activity of Acerola cherry from different stages of ripening. This study examined the differences between Acerola cherry from two different stages of ripening at the metabolite level using an untargeted UPLC-Q-TOF-based metabolomics approach and multivariate statistical tools. Our study brings to the forefront the essence and predictive power of metabolomics in detecting ripeing stages of Acerola cherry based on the levels of their metabolites and antioxidant activity . 2. Materials and methods 2.1. Plant materials and sampling Cultivated M. emarginata fruits were collected at Dahailan planting base (E 110°
37.70′, N 19°54.70′), Haikou, Hainan province, China, in early May 2018. The mean values for maximum and minimum temperature (℃) for the month of May 2018 were about 31.7 and 25.3, respectively. The total rainfall of the month of May 2018 was 181.8 mm. The fruit samples were harvested at four stages of maturity: immature stage (fully green and hard, 20 days after full bloom, IM); (2) pre-mature stage (green–reddish and partially hard, 24 days after full bloom, PM); (3) mature stage (red and partially soft, 28 days after full bloom MAT) and (4) over-mature (red and soft, 32 days after full bloom, OM). The fruits were harvested in the morning and immediately washed with tap water. Next, the seeds were manually removed and the pulp was homogenized using a blender for 1 min at room temperature and kept frozen at -80 °C until further use. 2.2. Metabolite extraction and pretreatment The collected samples from two stages of maturity (IM and MAT, 25 mg each, n = 10) were first thawed on ice, and transferred to a 1.5 mL centrifuge tube with 1 mL of extraction solution (50 % methanol). It was vortexed at room temperature in the dark for 10 min, and subjected to ultrasonication for 5 min on ice. The extraction mixture was then stored overnight at -20℃. After centrifugation at 4,000 g for 20 min at 4 ℃, the supernatant was transferred into a new tube. The extraction solution was collected, vacuum-dried and resuspended in 50% methanol. Meanwhile, a quality control (QC) pooled sample was prepared by mixing an equal volume of each extraction sample from each species. All the samples were stored at -80℃ prior to UPLC-MS/MS analysis. 2.3. Untargeted metabolomic analysis An ultra performance liquid chromatography (UPLC) system (SCIEX, UK) was used. An ACQUITY UPLC BEH Amide column (2.1 × 100 mm, 1.7 μm; Waters， UK) maintained at 35 ℃ was used. The mobile phase consisted of solvent A (IPA:ACN = 9:1 + 0.1% formic acid) and solvent B (aqueous solution with 25 mM ammonium acetate and 25 mM NH4H2O). The linear gradient elution procedures were set as follows: 0 ~ 0.5 min, 95% solvent A; 0.5 ~ 9.5 min, 95% to 65% solvent A; 9.5
~ 10.5 min, 65% ~ 40% solvent A; 10.5 ~ 12.0 min, 40% solvent A; 12.0 ~ 12.2 min, 40% ~ 95% solvent A; 12.2 ~ 15.0 min, 95% solvent A. The flow rate was 0.5 mL/min and the volume injected was 4 μL. A high-resolution tandem mass spectrometer SCIEX Triple-TOF-5600 plus (Applied Biosystems) was used to detect metabolites. The sheath gas flow was set at a pressure of 30 psi, ion source gas 1 and ion source gas 2 were both set at 60 psi, and the interface heater temperature was set at 650 ℃. The ion spray voltages were set at 5000 V and - 4500 V for the positive and negative ion modes, respectively. The mass spectrometry data were acquired in information-dependent acquisition (IDA) mode. The TOF mass range was from 60 to 1,200 Da. During the acquisition, the mass accuracy was calibrated every 20 samples. Furthermore, in order to evaluate the system stability of UPLC-MS/MS analysis, a QC sample was analyzed right after every 10 samples. 2.4. Bioinformatic analysis of the untargeted metabolomic datasets The acquired MS data pretreatments including peak detection, peak grouping, retention time correction, second peak grouping, and annotation of isotopes and adducts were performed by using the XCMS software. LC-MS raw data files were converted into mz.XML format using the Proteo Wizard MS Convert tools and then processed by the XCMS, CAMERA and metaX toolbox implemented with the R software. Each ion was identified by the m/z data and retention time (RT). Intensities of each peak were recorded and a three dimensional matrix containing arbitrarily assigned peak indices (retention time-m/z pairs), sample names (observations) and ion intensity information (variables) was generated. The online KEGG, HMDB databases were used to annotate the metabolites by matching the exact molecular mass data (m/z) of the samples with those from the databases. The intensities of peak data were further preprocessed by an in-house software metaX. Those features that were detected in less than 50% of the QC samples or 80% of the biological samples were removed, and the remaining peaks with missing values were imputed with the k-nearest neighbor algorithm to further improve the data quality. Principal component analysis (PCA)
was performed for outlier detection and batch effect evaluation using the pre-processed dataset. Quality control-based robust LOESS signal correction was fitted to the QC data with respect to the order of injection to minimize signal intensity drift over time. In addition, variables were then filtered to remove those with a mean intensity that was lower than twice the mean intensity in reagent blanks, or variables with a coefficient of variation in the QC samples above 30%. 2.5. Quantitative analysis of ascorbic acid To quantify ascorbic acid content, 0.5 g of sample and 5 mL of 5% metaphosphoric acid solution were placed into a 15 mL centrifuge tube. The tube was sonicated for 3 min and centrifuged at 7000 rpm for 10 min at 5 ℃. Samples were then filtered (0.22 mm) prior to HPLC analysis (da Silva Barros et al., 2019; Ribeiro et al., 2018). Analyses were performed using an Agilent 1260 High performance liquid chromatography system with a DAD UV-vis absorption detector (Agilent, Santa Clara, CA). The column used was a reverse C18 (Sunfire RP-18, 5 μm, miford, Massachusetts, USA). The analysis was performed under isocratic mode at a flow rate of 0.8 mL/min with a detection of 254 nm, using a mobile phase of water with 50 mM KH2PO4 and methanol in a relative proportion of 96:4 (v/v), and the column temperature was 25 ℃. Ascorbic acid was identified by comparing the retention time of the sample peak with that of the ascorbic acid standard. Quantification was carried out using external standardization and results were expressed as mg/g FW. 2.6. Extraction and determination of total phenolic content (TPC) The extraction and fractionation of the phenolic compounds were achieved according to the method described in F. Jimenez-Aspee with necessary modifications (Jimenez-Aspee et al., 2016). Briefly, 10 grams of the products were extracted three times with 100 mL of methanol under stirring in an ultrasonic bath for 30 min at room temperature. Then, the samples were centrifuged at 12,000 ×g during 20 min, and then vacuum filtered to separate the extracted phenolics from residue. The concentrated extract was dissolved to a volume of 10 mL with methanol. The extracts were mixed
with diluted Folin-Ciocalteu reagent and 15% sodium carbonate. Absorption at 765 nm was measured in a microplate reader after incubation for 30 min at room temperature, and results were expressed as mg gallic acid equivalents per gram of fresh weight of fruit (mg GAE/g FW). 2.7. Phenolic determination by HPLC Phenolic constituents of the extracts were determined using a HPLC method of Bittencourt with some modifications (Bittencourt et al., 2015). Briefly, 10 μg of the extracts were dissolved in 1 mL of methanol, and the solution was filtered through a 0.2 μm PVDF target syringe filter. The phenolic compounds were quantified using Shimadzu HPLC system (SCL-10 A vp, Shimadzu Corporation, Kyoto, Japan) equipped with a UV detector, and absorbance was monitored at 325 nm. Sunfire RP-C18 ODS (Miford, Massachusetts, USA) column at a maintained temperature (35 ℃) was used. Gradient solvents of water−formic acid (99:1, v/v) (solvent A) and acetonitrile (solvent B) in HPLC grade were used to elute the sample at a flow rate of 1.0 mL/min. Total run time was 35 min, and individual phenolic compounds were quantified by comparing with the corresponding standards. The TP content results were expressed as mg gallic acid equivalents per gram of fresh weight of fruit (mg GAE/g FW). 2.8. Determination of the antioxidant activity (ABTS test) and free radical scavenging activity (DPPH assay). The antioxidant activity of the extracts, on the basis of the scavenging activity of the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical, was carried out by the method of Bannour (Bannour et al., 2017). Antioxidant activity was determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging method. A 0.5 ml of plant extract was added to 2.5 ml of a 0.004% ethanol solution of DPPH. Absorbance at 517 nm was determined after 30 min and trolox was used as standard. A calibration curve was prepared, and different Trolox concentrations (standard trolox solutions ranging from 10 to 320 μM) were also evaluated against the radical. Antioxidant
activity was expressed as Trolox equivalent antioxidant capacity (TEAC), micromoles of Trolox per gram FW. ABTS+ assay was determined according to the method described by Yun et al. (Yun et al., 2018). ABTS+ radical cation was produced by mixing 7 mM ABTS solution with 2.45 mM potassium persulfate and the mixture was allowed to stand in the dark at room temperature. The ABTS+ solution was diluted with ethanol to an absorbance of 0.70 pounds were quantified by 5 μL of the sample or trolox standard was added to 2 mL of diluted ABTS+ solution. The absorbance at 734 nm was measured. The antioxidant activity was expressed as Trolox equivalent antioxidant capacity (TEAC), micromoles of Trolox per gram FW. 2.9. Statistical analysis. For the untargeted metabolomic analysis, Wilcoxon tests were carried out to examine metabolic differences between every two samples. The principal component analysis (PCA) was conducted using the metaX to discriminate different variables between groups. The VIP value was calculated and a VIP cut-off value of 1.0 was used to select important features. The quantification results of ascorbic acid, phenolic compounds and antioxidant activity are presented as mean ± standard deviation. The P values were adjusted for multiple testing correction by false discovery rate (FDR; Benjamini – Hocway analysis of variance) was carried out to compare the content differences between immature and mature fruits. and discussion 3. Results and discussion 3.1. Untargeted metabolite profiling of the mature and immature fruits To explore global metabolic variations, an untargeted approach was applied (N = 10), identifying 1,896 annotated metabolite features from 8,054 ion features (Table S1), and finally 133 metabolites were identified according to the MS/MS fragments compared with these standards in in-house database. To check the quality of MS data from different sample groups, several quality control parameters, including total ion
chromatograms (Fig. S1), the m/z width and retention-time width (Fig. S2), were generated and analyzed, suggesting a high degree of overlap and a good sample preparation process. Furthermore, a principal component analysis (PCA) was performed. Our data showed that PC1 and PC2 were responsible for 33.32% and 11.03% of the variation, respectively, indicating a clear separation between the metabolite features from mature and immature fruits (Fig. 1b). An overview of the metabolite profiling of acerola cherry fruits during different mature stages is shown in a heatmap (Fig. 1c). Previous studies have identified a number of metabolites, such as vitamins, polyphenols, polysaccharides, carotenoids and bioflavonoids, of Acerola cherry (Barros et al., 2019; Santos, Rodrigues, & Fernandes, 2018). A traditional phytochemical analysis could only focus on a small number of metabolites belonging to one or several particular categories (Kawaguchi, Tanabe, & Nagamine, 2007). In our study, a large number of metabolite features belonging to various categories were identified, giving us an opportunity to clarify the regulation mechanism of nutritional ingredients in Acerola cherry.
3.2. Identification of differentially accumulated metabolite features (DAMFs) between the mature and immature fruits To reveal the variations in abundance levels of metabolite features between mature and immature fruits, all the identified metabolite features were quantified. Analysis of two important quality control parameters, including normalized intensity (Fig. S3) and coefficient of variation (Fig. S4), showed a high repeatability of the MS data. Statistical analysis identified 1000 significant DAMFs, including 684 up- and 316 down-regulated metabolite features, were identified in the mature fruits compared with the immature fruits (Fig. 2a and b). Most of the identified metabolite features were assigned into various major metabolic categories, such as alkaloids, amino acids, VCs, carotenoids, flavonoids, hormones, lipids, phenylalanines, phenylpropanoids, saccharides, steroids, terpenoids, and ubiquinones (Fig. 2c and Table S2).
Furthermore, the number of mature- and immature-predominantly accumulated metabolite features belonging to each category was analyzed. Interestingly, most of differential
accumulated
amino
acids,
flavonoids,
lipids,
and
terpenoids
predominantly accumulated in the mature fruits rather than in immature fruits. Additionally, all differential accumulated ascorbic acids predominantly accumulated in the immature fruits (Fig. 2d). During the ripening process, there was a dynamic variation in fruit composition. For example, the early ripening phase of dates enriched in hormones, amines, tannins, sucrose, and the later ripening phase of dates enriched in phenylpropanoids and volatiles (Diboun et al., 2015). In guava fruits, sucrose, fructose, serine and citric acid were particularly related to the ripening process (Lee, Choi, Cho, & Kim, 2010). In addition, differences in bioactive compounds, such as ascorbic acid, total phenolics, carotenoids and antioxidant activity, between mature green and red stages of pepper at harvest have been reported (Jang, Jung, Lee, Choi, & Lee, 2015). However, few studies have been done to reveal the changes in nutritional ingredients during the ripening process. Here, untargeted metabolomic analysis helps us to systematically investigate the metabolic variations between mature and immature fruits of Acerola cherry.
3.3. Untargeted metabolomics analysis reveals the variations in antioxidants A large number of antioxidants were identified in the acerola cherry fruits. For carotenoids, phytoenes, including prephytoene-PP and phytofluene, lutein, lycopene and β-carotene were identified. Among these carotenoids, prephytoene-PP and lutein predominantly accumulated in the mature fruits rather than in immature fruits. For phenolics, 2 chlorogenic acids, 8 anthocyanins, 12 flavones, 6 isoflavonoids, 3 lignans, 6 phenolic acids, and 5 polyphenols, were identified. Quantitative analysis showed that 2 chlorogenic acids, including chlorogenic acid and isochlororgenic acid, 4 anthocyanins, including cyanidin, delphinidin-3β-D-glucoside, phloretin, peonidin, most of the identified flavones, such as genistein, luteolin, kaempferol, apigenin
7-glucoside, quercitrin, astragalin, kaempferol 3-O-β-glucoside, vitexin, malonylapiin and
isovitexin,
3
isoflavonoids,
including
6,7,4'-Trihydroxyisoflavone,
2,4',7-Trihydroxyisoflavanone, and 2',4,4',6'-Tetrahydroxychalcone, and 1 lignan (sesaminol), predominantly accumulated in the mature fruits rather than in immature fruits. Meanwhile, 3 anthocyanins, including peonidin 3,5-diglucoside, tulipanin, and cyanidin-3-(p-coumaroyl-glucoside), 2 flavones, including apigenin and rutin, and 3 phenolic
acids,
including
trans-3-hydroxycinnamic
acid,
ferulic
acid
and
feruloylglycine, predominantly accumulated in the immature fruits rather than in mature fruits (Fig. 3). 3.4. Untargeted metabolomics analysis reveals the variations in nutrient substances Moreover, a series of nutrient substances, including 12 amino acids, 12 saccharides, 14 fatty acids and ascorbic acid, were identified. Most of the identified amino acids highly accumulated in the mature fruits and ascorbic acid highly accumulated in the immature fruits. For saccharides, 2-dehydro-3-deoxy-L-fuconate, 2-dehydro-3-deoxy-L-rhamnonate,
D-fructose
and
D-mannose
predominantly
accumulated in the mature fruits and cellobiose, trehalose and isomaltose predominantly accumulated in the immature fruits. No significant variations in fatty acid contents were observed between the mature and immature fruits (Fig. 4). The nutritional quality of Acerola cherry fruits is closely correlated with the presence of soluble sugars, organic acids, amino acids, and some major secondary metabolites. These compounds play an important role in maintaining fruit quality and nutritive value. The heatmap showed that the metabolites have a differential distribution during development and maturation. Almost all amino acids exhibited significance increases during fruit development except L-Lysine and L-Phenylalanine, and these contributed to the formation of fruity, sweaty, and peach-like flavour in Acerola fruits. Saccharides were the metabolites that exhibited a high degree of variance during fruit development, while fruit ripening was characterized by the increases in levels of L-Rhamnono-1,4-lactone, fructose, mannose, which comprised the major soluble sugars in Acerola fruit. Similar results
have been reported by Oms-Oliu (Oms-Oliu et al., 2011). No significant variations in fatty acid contents were observed between the mature and immature fruits. Carotenoids are another important bioactive compound class present in Acerola. Mature fruits showed higher carotenoid content as compared to immature and half-mature fruits. Carotenoid content was also influenced by the genotypes, sunlight exposure, harvesting season and different stages of maturity (Belwal et al., 2018). 3.5. Quantitative analysis confirms the variations in ascorbic acid between mature and immature fruits. To precisely determine the content differences of ascorbic acid between mature and immature fruits, the content was quantified using a HPLC method. The untargeted metabolomics analysis indicated that ascorbic acid was predominantly accumulated in immature fruits, and the direct quantification with an authentic standard of ascorbic acid confirmed this phenomenon. The content of ascorbic acid was severely influenced by the maturation process. Significant decline was observed from immature to over-mature (Fig. 5). The content of ascorbic acid in immature fruit (23.86 ± 2.35 mg/g FW) was 1.9-fold greater than that in mature one (12.25 ± 1.61 mg/g FW). Similar to current work, Mezadri et al. reported levels of 9.52 and 17.7 g of ascorbic acid kg-1 of pulp (Mezadri, Villano, Fernandez-Pachon, Garcia-Parrilla, & Troncoso, 2008). Ascorbic acid levels vary considerably according to degree of maturation, conditions of cultivation, storage and processing. However, it is important to highlight all stages of maturity exceed the recommended vitamin C intake showing that they are outstanding sources for human diet. The consumption of approximately 10 g of fruits has already reached the daily recommendation of vitamin C. 3.6. Total phenolic and individual phenolic compounds between mature and immature fruits The TPCs of Acerola at four different maturity stages are shown in Fig. 5. The stage of maturity had a significant influence on the TPCs in Acerola cherry. During fruit ripening process, a linear pattern of increase was observed for TPCs ,while it is
followed by a decline when over-mature (Fig. 5). The sum of TPCs of Acerola sharply increased from 14.21 ± 1.81 mg GAE/g FW at immature stage to 24.48 ± 2.26 mg GAE/g FW at mature stage (p < 0.05), but decline to 21.42 ± 1.91 mg GAE/g FW when it is over-mature. Fig. 6a shows a chromatogram of an acerola pulp containing numerous peaks. A total of six major phenolic compounds were identified. A1-A7 was identified as (1) chlorogenic acid (2) p-coumaric acid, (3) ferulic acid, (4) kaempferol, (5) luteolin, (6) rutin, and (7) apigenin because of those spectral data and retention time using standard as reference. The concentration of (1)chlorogenic acid, (4) kaempferol, (5) luteolin, (6) rutin, and (7) apigenin in the mature fruit were higher than those in immature. By contrast, the concentration of (2) p-coumaric acid, (3) ferulic acid tended to decrease in more ripe fruits. This agreed well with the data reported by other authors, indicating decreases of ferulic acid with the developmental stages (Wang et al., 2016). The representative HPLC chromatograms of ascorbic acid between mature and immature fruits are shown in Fig. 6. Phenolic compounds, as secondary metabolites, are a large group of molecules widely distributed in fruits and vegetables (Acero, Gradillas, Beltran, Garcia, & Munoz Mingarro, 2019). In Acerola cherry, the TPCs increased with maturity stage, which was in agreement with those reported previously for Acerola and other fruits (Sulas, Petretto, Pintore, & Piluzza, 2017). However, the quantities of TPC obtained from this study were lower than the previously published results (Mishra, Mishra, & Mahanta, 2014). This might be mainly attributed to the differences in the cultivar and sources of the materials, as well as the genus, cultivar, species, ripening stage, soil and climate. The developmental stages not only had a significant impact on the total polyphenol concentration, but a large number of individual phenolic compounds also varied. It was obviously that most of the chromatography peaks were higher in mature stage than immature one. This agreed well with the data from the metabolic result.
3.7. Antioxidant activity Antioxidant mechanisms in biological tissues are extremely complex, and there is
not one method that can provide unequivocal results. Thus the antioxidant capacity of Acerola was evaluated based on DPPH and ABTS radical scavenging activity, as shown in Fig. 5. The DPPH and ABTS radical scavenging activity in immature stage was 180.52 ± 15.41 μM TE/g DW and 141.26 ± 10.12μM TE/g DW, respectively. These values reduced by 39.8% (108.66 ± 8.91 μM TE/g DW) for DPPH and 36.0% (90.41 ± 8.8 μM TE/g DW) for ABTS. In biological analysis, the variation between mature and immature Acerola fruit in antioxidative effect has been reported. Phenolics were considered as one of the ingredients in Acerola fruit contributing to this effect, yet whether the levels of Phenolics were correlated with the activity is still debatable. Mature fruit containing significantly higher amount of flavonoidic compounds showed better antioxidative activity than that of the mature one. In contrast, our study demonstrated that the antioxidative activity did not show significant correlation with the amount of Phenolics. The highly positive correlation between DPPH and ABTS suggests that both methods are suitable to measure the antioxidant capacity of acerola. A high positive correlation was found between the ascorbic acid (vitamin C) content and DPPH (r=0.9830) as well as a high positive correlation was identified between ascorbic acid content and ABTS (r=0.9897). The reduction in antioxidant activities during acerola development may be associated with apparent decrease in quantity of vitamin C in the fruit. Although phenolic compounds are known as antioxidant compounds, their increase during fruit development constituted only small proportion of total in fruit, hence the change in vitamin C a much more significant influence than phenolic compounds on acerola antioxidant capacity. The ABTS and DPPH radical scavenging activities were used to compare the antioxidant activity between mature and immature acerola cherry extracts. The results showed that during the maturity process both the DPPH and ABTS radical scavenging activities decreased from immature to mature stage, suggesting that ascorbic acid has a significant contribution to the antioxidant capacity of the acerola.
4. Conclusion In this study, we investigated their chemical and biological properties by metabolic profiling and antioxidant assays. In LC-MS profiling, 1,896 annotated metabolite features were obtained, and 133 metabolites were finally identified according to the MS/MS fragments compared with these standards in in-house database. Statistically differences in the levels of amino acids, flavonoids, lipids, terpenoids and ascorbic acids were found between mature and immature fruits. In parallel, their antioxidant activities were compared. The alcoholic extract of immature acerola fruit possessed better DPPH effect than the mature one. The ascorbic acid was highly correlated with antioxidant potential and the metabolite level is higher in immature than that of mature. In this regard Acerola was found to be rich in bioactive compounds especially ascorbic acid and polyphenolics and showed varied health beneficial effects. Hence, Acerola is anticipated to be a good candidate for dietary supplements and functional food manufacturers looking to the development of new products. Acknowledgments This work is Financial supported by the National Key R&D Program of China (2018YFC0311205), the Hangzhou Science and Technology Development Program (20191203B01) and the Zhejiang Science and Technology Project (2018C02053). References Acero, N., Gradillas, A., Beltran, M., Garcia, A., & Munoz Mingarro, D. (2019). Comparison of phenolic compounds profile and antioxidant properties of different sweet cherry (Prunus avium L.) varieties. Food Chemistry, 279, 260-271. Bannour, M., Fellah, B., Rocchetti, G., Ashi-Smiti, S., Lachenmeier, D. W., Lucini, L., & Khadhri, A. (2017). Phenolic profiling and antioxidant capacity of Calligonum azel Maire, a Tunisian desert plant. Food Research International, 101, 148-154. Barros, B. R. S., Barboza, B. R., Ramos, B. A., Moura, M. C., Coelho, L., Napoleao, T. H., Correia, M. T. S., Paiva, P. M. G., Cruz Filho, I. J. D., Silva, T. D. D., Lima, C. S. A., & Melo, C. M. L. (2019). Saline extract from Malpighia emarginata DC leaves showed higher polyphenol presence, antioxidant and antifungal activity and promoted cell proliferation in mice splenocytes. Anais
da Academia Brasileira de Ciências, 91 (1), e20190916. Belwal, T., Devkota, H. P., Hassan, H. A., Ahluwalia, S., Ramadan, M. F., Mocan, A., & Atanasov, A. G. (2018). Phytopharmacology of Acerola (Malpighia spp.) and its potential as functional food. Trends in Food Science & Technology, 74, 99-106. Bittencourt, M. L. F., Ribeiro, P. R., Franco, R. L. P., Hilhorst, H. W. M., de Castro, R. D., & Fernandez, L. G. (2015). Metabolite profiling, antioxidant and antibacterial activities of Brazilian propolis: Use of correlation and multivariate analyses to identify potential bioactive compounds. Food Research International, 76 (Pt 3), 449-457. Cai, J., Chen, T., Zhang, Z., Li, B., Qin, G., & Tian, S. (2019). Metabolic dynamics during loquat fruit ripening and postharvest technologies. Frontiers in Plant Science, 10, 619. Cappato, L. P., Ferreira, M. V. S., Moraes, J., Pires, R. P. S., Rocha, R. S., Silva, R., Neto, R. P. C., Tavares, M. I. B., Freitas, M. Q., Rodrigues, F. N., Calado, V. M. A., Raices, R. S. L., Silva, M. C., & Cruz, A. G. (2018). Whey acerola-flavoured drink submitted Ohmic Heating: Bioactive compounds, antioxidant capacity, thermal behavior, water mobility, fatty acid profile and volatile compounds. Food Chemistry, 263, 81-88. Cascia, G., Bulley, S. M., Punter, M., Bowen, J., Rassam, M., Schotsmans, W. C., Larrigaudiere, C., & Johnston, J. W. (2013). Investigation of ascorbate metabolism during inducement of storage disorders in pear. Physiol Plantarum, 147 (2), 121-134. Chang, S. K., Alasalvar, C., & Shahidi, F. (2018). Superfruits: Phytochemicals, antioxidant efficacies, and health effects - A comprehensive review. Critical Reviews in Food Science and Nutrition, 1-25. da Silva Barros, B. R., do Nascimento, D. K. D., de Araújo, D. R. C., da Costa Batista, F. R., de Oliveira Lima, A. M. N., da Cruz Filho, I. J., de Oliveira, M. L., & de Melo, C. M. L. (2019). Phytochemical analysis, nutritional profile and immunostimulatory activity of aqueous extract from Malpighia emarginata DC leaves. Biocatalysis and Agricultural Biotechnology, 101442. De Rosso, V. V., & Mercadante, A. Z. (2007). The high ascorbic acid content is the main cause of the low stability of anthocyanin extracts from acerola. Food Chemistry, 103 (3), 935-943. Diboun, I., Mathew, S., Al-Rayyashi, M., Elrayess, M., Torres, M., Halama, A., Meret, M., Mohney, R. P., Karoly, E. D., Malek, J., & Suhre, K. (2015). Metabolomics of dates (Phoenix dactylifera) reveals a highly dynamic ripening process accounting for major variation in fruit composition. BMC Plant Biology, 15, 291. Jang, Y. K., Jung, E. S., Lee, H. A., Choi, D., & Lee, C. H. (2015). Metabolomic characterization of hot pepper (Capsicum annuum "CM334") during fruit development. Journal of Agricultural and Food Chemistry, 63 (43), 9452-9460. Jimenez-Aspee, F., Theoduloz, C., Vieira, M. N., Rodriguez-Werner, M. A., Schmalfuss, E., Winterhalter, P., & Schmeda-Hirschmann, G. (2016). Phenolics from the patagonian currants Ribes spp.: Isolation, characterization and cytoprotective effect in human AGS cells. Journal of Functional Foods, 26, 11-26. Kawaguchi, M., Tanabe, H., & Nagamine, K. (2007). Isolation and characterization of a novel flavonoid possessing a 4,2''-glycosidic linkage from green mature acerola (Malpighia emarginata DC.) fruit. Bioscience Biotechnology and Biochemistry, 71 (5), 1130-1135. Lee, S., Choi, H. K., Cho, S. K., & Kim, Y. S. (2010). Metabolic analysis of guava (Psidium guajava L.) fruits at different ripening stages using different data-processing approaches. Journal of Chromatography B-Analytical Technologies in the Biomedical Life Sciences, 878 (29), 2983-2988.
Lee, S., Jang, W. J., Choi, B., Joo, S. H., & Jeong, C. H. (2017). Comparative metabolomic analysis of HPAC cells following the acquisition of erlotinib resistance. Oncology Letters, 13 (5), 3437-3444. Leffa, D. D., da Silva, J., Daumann, F., Dajori, A. L. F., Longaretti, L. M., Damiani, A. P., de Lira, F., Campos, F., Ferraz, A. D. F., Correa, D. S., & de Andrade, V. M. (2014). Corrective effects of acerola (Malpighia emarginata DC.) juice intake on biochemical and genotoxical parameters in mice fed on a high-fat diet. Mutation Research-Fundamental and Molecular Mechanisms of Mutagenesis, 770, 144-152. Mezadri, T., Villano, D., Fernandez-Pachon, M. S., Garcia-Parrilla, M. C., & Troncoso, A. M. (2008). Antioxidant compounds and antioxidant activity in acerola (Malpighia emarginata DC.) fruits and derivatives. Journal of Food Composition and Analysis, 21 (4), 282-290. Mishra, P., Mishra, S., & Mahanta, C. L. (2014). Effect of maltodextrin concentration and inlet temperature during spray drying on physicochemical and antioxidant properties of amla (Emblica officinalis) juice powder. Food and Bioproducts Processing, 92 (C3), 252-258. Monti, L. L., Bustamante, C. A., Osorio, S., Gabilondo, J., Borsani, J., Lauxmann, M. A., Maulion, E., Valentini, G., Budde, C. O., Fernie, A. R., Lara, M. V., & Drincovich, M. F. (2016). Metabolic profiling of a range of peach fruit varieties reveals high metabolic diversity and commonalities and differences during ripening. Food Chemistry, 190, 879-888. Oms-Oliu, G., Hertog, M. L. A. T. M., Van de Poel, B., Ampofo-Asiama, J., Geeraerd, A. H., & Nicolaï, B. M. (2011). Metabolic characterization of tomato fruit during preharvest development, ripening, and postharvest shelf-life. Postharvest Biology and Technology, 62 (1), 7-16. Pinsorn, P., Oikawa, A., Watanabe, M., Sasaki, R., Ngamchuachit, P., Hoefgen, R., Saito, K., & Sirikantaramas, S. (2018). Metabolic variation in the pulps of two durian cultivars: Unraveling the metabolites that contribute to the flavor. Food Chemistry, 268, 118-125. Ribeiro, H. L., Oliveira, A. V., Brito, E. S., Ribeiro, P. R. V., Souza Filho, M. S. M., & Azeredo, H. M. C. (2018). Stabilizing effect of montmorillonite on acerola juice anthocyanins. Food Chemistry, 245, 966-973. Santos, V. O., Rodrigues, S., & Fernandes, F. A. N. (2018). Improvements on the stability and vitamin content of acerola juice obtained by ultrasonic processing. Foods, 7 (5). Silva, P. B., Duarte, C. R., & Barrozo, M. A. S. (2019). A novel system for drying of agro-industrial acerola (Malpighia emarginata D. C.) waste for use as bioactive compound source. Innovative Food Science & Emerging Technologies, 52, 350-357. Stafussa, A. P., Maciel, G. M., Rampazzo, V., Bona, E., Makara, C. N., Demczuk, B., & Haminiuk, C. W. I. (2018). Bioactive compounds of 44 traditional and exotic Brazilian fruit pulps: phenolic compounds and antioxidant activity. International Journal of Food Properties, 21 (1), 121-133. Sulas, L., Petretto, G. L., Pintore, G., & Piluzza, G. (2017). Bioactive compounds and antioxidants from a Mediterranean garland harvested at two stages of maturity. Natural Product Research, 31 (24), 2941-2944. Wang, B., Huang, Q., Venkitasamy, C., Chai, H., Gao, H., Cheng, N., Cao, W., Lv, X., & Pan, Z. (2016). Changes in phenolic compounds and their antioxidant capacities in jujube (Ziziphus jujuba Miller) during three edible maturity stages. LWT - Food Science and Technology, 66, 56-62. Yun, D. Y., Kang, Y. G., Kim, E. H., Kim, M., Park, N. H., Choi, H. T., Go, G. H., Lee, J. H., Park, J. S., & Hong, Y. S. (2018). Metabolomics approach for understanding geographical dependence of soybean leaf metabolome. Food Research International, 106, 842-852.
Zheng, X., Liu, F., Shi, X., Wang, B., Li, K., Li, B., & Zhuge, B. (2018). Dynamic correlations between microbiota succession and flavor development involved in the ripening of Kazak artisanal cheese. Food Research International, 105, 733-742.
Additional files Additional file 1: Fig. S1. The total ion chromatograms of all the samples. (DOCX 373 kb) Additional file 2: Fig. S2 The m/z width (a) and retention-time width (b) of all the samples. (DOCX 609 kb) Additional file 3: Fig. S3 The normalized intensity of the MS data. (DOCX 50 kb) Additional file 4: Fig. S4 The coefficient of variation of the data from different samples. (DOCX 86 kb) Additional file 5: Table S1. Detail information of all identified metabolites.(XLSX 4718 kb) Additional file 6: Table S2. Identification and classification of all metabolites. (XLSX 531 kb)
Fig. 1. Untargeted metabolite profiling reveals the metabolites in the wild cherry fruits. (a) Pictures of the mature and immature wild cherry fruits. (b) The PCA data of the samples from mature and immature wild cherry fruits. Red indicated the mature fruits and green indicated the immature fruits. (c) A heatmap of the metabolites identified in the metabolomes of the two fruit samples (n = 10). The heatmap scale ranges from -4 to +4 on a log2 scale.
Figure 2 (a)
(c)
Alkaloids
128
Carotenoids
5
VC
10
-4 0 4 Log2(MAT/IMAT)
12
Carotenoid
Flavonoid
83 18
Hormone
Lipids
205
77
82
Hormones
Up Dn None
8
20
18
Flavonoids
0
Ascorbic acid
58
Amino acids
15
Amino acid
0.8
0.3
-0.2
-log10 (Q value)
(d)
Normalized ion strength -1 -0.7 -0.2 0 0.2 0.7 1 -0.7
-1.2
T test 20
Lipid
15
49
(b)
600
400
316
300
100
0 Dn
Saccharides
191
Terpenoid
16
54 Ubiquinones
200
21
68 26
Terpenoids
Number
500
42 Steroids Phenylpropanoids
684
700
Saccharides Phenylalanines
16 800
Up
17 272
Down
None
Up
Fig. 2. Identification of the DAMs between the mature and immature fruits. (a) Significance analysis of the DAMs between the mature and immature fruits by Volcanoplot. (b) The number of up- and down-regulated metabolites in the mature fruits compared with the immature fruits. (c) The relative abundances of the metabolites belonging to various major metabolic categories. (d) The numbers of upand down-regulated metabolites in different metabolic categories were shown in pies.
Figure 3 -4
Phytoene
Carotenoids
Lutein Lycopene β-Carotene Chlorogenic acid
Anthocyanins
Flavone and flavonol
Phenolics Isoflavonoid
Lignans
Phenolic acids
Polyphenols
0 Mature
4 Immature Prephytoene diphosphate Phytofluene Lutein Lycopene β-Carotene Chlorogenic acid Isochlorogenic acid Cyanidin Delphinidin Delphinidin-3β-D-glucoside Phloretin Peonidin Peonidin 3,5-diglucoside Tulipanin Cyanidin-3-(p-coumaroyl-glucoside) Apigenin Genistein Luteolin Kaempferol Apigenin 7-glucoside Quercitrin Astragalin Kaempferol 3-O-β-glucoside Vitexin Isovitexin Rutin Malonylapiin Formononetin Isoformononetin 7,8,4'-Trihydroxyflavone 6,7,4'-Trihydroxyisoflavone 2,4',7-Trihydroxyisoflavanone 2',4,4',6'-Tetrahydroxychalcone Sesaminol Lariciresinol Secoisolariciresinol monoglucoside 4-Hydroxycinnamic acid trans-3-Hydroxycinnamic acid 3,4-Dihydroxyhydrocinnamic acid Ferulic acid 4-p-Coumaroylquinic acid Feruloylglycine Isopimpinellin Vanillin Methoxyacetophenone Mellein Catechol
Fig. 3. Variations in the abundances of antioxidants between the mature and immature fruits. A heatmap of the relative amounts of antioxidants, including carotenoids and phenolics. The heatmap scale ranges from -4 to +4 on a log2 scale.
Figure 4
-4
Amino acids
Saccharides
Fatty acids
Ascorbic acid
0 Mature
4 Immature L-Serine L-Proline L-Valine L-Leucine L-Isoleucine L-Glutamine L-Lysine L-Methionine L-Histidin L-Phenylalanine L-Asparagine L-Arginine 2,4-Diketo-3-deoxy-L-fuconate L-Rhamnono-1,4-lactone 2-Dehydro-3-deoxy-L-fuconate 2-Dehydro-3-deoxy-L-rhamnonate D-Fructose D-Mannose L-Sorbose alpha-D-Glucose D-Allose Cellobiose Trehalose Isomaltose 1,6-D mannobiose linoleate Hexadecanoic Icosapentaenoic Icosadienoic Arachidonate Icosenoic Docosadienoic Docosahexaenoic Tetracosanoic Octanoic Dodecanoic Tetradecanoic (9Z)-Hexadecenoic Octadecanoic Ascorbate
Fig. 4. Variations in the abundances of nutrients between the mature and immature fruits. A heatmap of the relative amounts of nutrients, including amino acids, saccharides, fatty acids and ascorbic acid. The heatmap scale ranges from -4 to +4 on a log2 scale.
Fig. 5
Fig. 5. Concentrations of bioactive compounds (Vc, TPc) and antioxidant activity measured by assays of DPPH and ABTS Vc=Vitamin C, TPc=Total Phenolic compounds, ABTS=ABTS scavenging activity, DPPH=DPPH radical scavenging activity
Fig. 6. Chromatogram of acerola at mature (a) and immature (b) stages at 325 nm (1)chlorogenic acid (2) p-coumaric acid, (3) ferulic acid, (3) quercetin, (4) kaempferol, (5) luteolin, (6) rutin, and (7) apigenin
Graphical Abstract
Highlights
Through comprehensive metabolites analysis, 1896 annotated metabolites were obtained. Statistically differences in the levels of amino acids, flavonoids, lipids, terpenoids and ascorbic acids were found between mature and immature fruits. The well correlations were found between the antioxidant potential with its content of ascorbic acid. The maturity of Acerola cherry has to be considered when it is being used for health food products.
Author Contributions Section: Mingfeng Xu: experiment. Chenjia Shen and Han Zheng: Data curation, Writing-Original draft preparation. Yunsheng Xu and Changfeng Xue: samples collection. Beiwei Zhu: WritingReviewing and Editing. Jiangning Hu: Supervision.
S0963-9969(19)30801-4 https://doi.org/10.1016/j.foodres.2019.108915 FRIN 108915
To appear in:
Food Research International
Received Date: Revised Date: Accepted Date:
15 July 2019 7 December 2019 15 December 2019
Please cite this article as: Xu, M., Shen, C., Zheng, H., Xu, Y., Xue, C., Zhu, B., Hu, J., Metabolomic analysis of acerola cherry (Malpighia emarginata) fruit during ripening development via UPLC-Q-TOF and contribution to the antioxidant activity, Food Research International (2019), doi: https://doi.org/10.1016/j.foodres.2019.108915
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Metabolomic analysis of acerola cherry (Malpighia emarginata) fruit during ripening development via UPLC-Q-TOF and contribution to the antioxidant activity Mingfeng Xu
a,b
, Chenjia Shen b, Han Zheng a, Yunsheng Xu c, Changfeng Xue c,
Beiwei Zhu a, Jiangning Hua,*
a School b
of Food Science and Technology, Dalian Polytechnic University, Dalian 116034, China
College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036,
China; c
School of Food Science and Engineering, Hainan Tropical Ocean University, Sanya 572022,
China;
Running title: Metabolomic
analysis of acerola cherry during ripening
development
* To Corresponding authors: Telephone No: +86-411-86318731, E-mail address: [email protected] (J-N Hu);
ABSTRACT: Acerola cherry (Malpighia emarginata D.C.) is a tropical fruit of great economic and nutritional value due to its high content of vitamin C. However, there is little information available about which ripening stage of Acerola cherry can provide the best nutrients. In the current study, the chemical variation at two developmental stages
(immature and mature) were investigated by metabolic profiling, and the
biological properties of Acerola cherry and its antioxidant assays at four developmental stages were measured, respectively. Through comprehensive metabolites analysis via ultra-high-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry system (UPLC-QTOF), 1,896 annotated metabolite features were obtained, and 133 metabolites were finally identified according to the MS/MS fragments compared with these standards in in-house database. Statistically differences in the levels of amino acids, flavonoids, lipids, terpenoids and ascorbic acids were found between mature and immature fruits. Interestingly, most of differential accumulated amino acids, flavonoids, lipids, and terpenoids predominantly accumulated in the mature fruits and ascorbic acid predominantly accumulated in the immature fruits. On the other hand, their antioxidant activities were compared. The alcoholic extract of immature acerola fruit possessed better scavenging ability of DPPH and ABTS than the mature one. The well correlations were found between the antioxidant potential with its content of ascorbic acid ( r = 0.9803 and 0.9897, respectively ). In conclusion, Acerola cherry showed very different metabolite profile and antioxidant activities during the fruit ripening development. The maturity of Acerola cherry has to be considered when it is being used for health food products.
KEYWORDS: metabolome; fruit ripening; UPLC-Q-TOF; ascorbic acid; antioxidant activity
1. Introduction Malpighia emarginata DC., commonly known as Acerola cherry or Barbados cherry, is native to South and Central America, and also cultivated in tropical or subtropical climate zones and south areas of China (Leffa et al., 2014; Silva, Duarte, & Barrozo, 2019). With increasing health awareness across the world, currently Acerola becomes popular due to its high antioxidant activity and its substantial amounts of ascorbic acid and other bioactive compounds including amino acids (AA), β-carotenoids (CA), phenolic compounds (PC) and minerals. Many studies showed that the antioxidant activity of Acerola extract was much higher as compared to polyphenols-rich extracts of other fruits, such as açai, cherry, grape, mango and strawberry (Chang, Alasalvar, & Shahidi, 2018). Due to the high concentration of these compounds, acerola fruits can be good candidates for the development of novel functional foods with promising effects on human health (Belwal et al., 2018; Cappato et al., 2018). Various commercial products containing Acerola are being used as dietary supplements. The global Acerola consumption market is estimated to reach US &17.5 billion by 2026 with 8.5% of compound annual growth rate (Belwal et al., 2018). Usually, Acerola cherry tends to be consumed as fresh fruit. The fresh fruit is considered rich in nutrients and contributes to the human health benefit. In this globalized marketed context, how to keep the fruits with high quality during long distance transportation or long time storage is a tough problem. It is known that excessive softening is a common cause of postharvest losses (Cai et al., 2019). A complex biological process after postharvest in Acerola cherry occurs with a notable influence on the human diet. For instance, vitamin C content in Acerola fruits was reported to change during ripening stages. High content of Vitamin C was found in immature fruits (1.9 g/100 g of juice) decreased significantly during ripening (0.97 g/100 g of juice in mature fruits) (Stafussa et al., 2018). The quantity and the composition of phenolic compounds also depend upon the ripening stage (Belwal et al., 2018). Proanthocyanidins were the major phenolic compounds in immature fruits
but during ripening, the amount of these compounds decreased and anthocyanins were the main polyphenolic compounds in mature fruits. Its chemical composition depends on cultivar, environmental conditions, and on the stage of fruit maturity (De Rosso & Mercadante, 2007). Comprehensive understanding of the change of nutrients during the fruit ripening is necessary. At present, metabolomics is important for the identification of metabolic signatures or patterns associated with various conditions (Lee, Jang, Choi, Joo, & Jeong, 2017). Metabolic changes during fruit development and ripening were observed to reveal physiological and nutritional traits that enhance fruit quality (Zheng et al., 2018). Small metabolites are important contributors to fruit flavor (Pinsorn et al., 2018). Primary metabolites including sugars and organic acids, in particular, are involved in sweetness and acidity taste formation. Amino acids are also related to fruit quality traits, by acting as precursors of volatile compounds involved in fruit aromas, and as substrates for polyphenol oxidases associated with the browning reaction of fruit (Cascia et al., 2013). Thus, metabolomics has been used to identify biomarkers associated with fruit quality (Monti et al., 2016). Much scientific literature abounds on the analyses of Acerola cherry. However, very few reports are available on the use of metabolomics as a tool for quality evaluations. To the best of our knowledge, there is no report about the comparative analyses of chemical composition together with its antioxidant activity of Acerola cherry from different stages of ripening. This study examined the differences between Acerola cherry from two different stages of ripening at the metabolite level using an untargeted UPLC-Q-TOF-based metabolomics approach and multivariate statistical tools. Our study brings to the forefront the essence and predictive power of metabolomics in detecting ripeing stages of Acerola cherry based on the levels of their metabolites and antioxidant activity . 2. Materials and methods 2.1. Plant materials and sampling Cultivated M. emarginata fruits were collected at Dahailan planting base (E 110°
37.70′, N 19°54.70′), Haikou, Hainan province, China, in early May 2018. The mean values for maximum and minimum temperature (℃) for the month of May 2018 were about 31.7 and 25.3, respectively. The total rainfall of the month of May 2018 was 181.8 mm. The fruit samples were harvested at four stages of maturity: immature stage (fully green and hard, 20 days after full bloom, IM); (2) pre-mature stage (green–reddish and partially hard, 24 days after full bloom, PM); (3) mature stage (red and partially soft, 28 days after full bloom MAT) and (4) over-mature (red and soft, 32 days after full bloom, OM). The fruits were harvested in the morning and immediately washed with tap water. Next, the seeds were manually removed and the pulp was homogenized using a blender for 1 min at room temperature and kept frozen at -80 °C until further use. 2.2. Metabolite extraction and pretreatment The collected samples from two stages of maturity (IM and MAT, 25 mg each, n = 10) were first thawed on ice, and transferred to a 1.5 mL centrifuge tube with 1 mL of extraction solution (50 % methanol). It was vortexed at room temperature in the dark for 10 min, and subjected to ultrasonication for 5 min on ice. The extraction mixture was then stored overnight at -20℃. After centrifugation at 4,000 g for 20 min at 4 ℃, the supernatant was transferred into a new tube. The extraction solution was collected, vacuum-dried and resuspended in 50% methanol. Meanwhile, a quality control (QC) pooled sample was prepared by mixing an equal volume of each extraction sample from each species. All the samples were stored at -80℃ prior to UPLC-MS/MS analysis. 2.3. Untargeted metabolomic analysis An ultra performance liquid chromatography (UPLC) system (SCIEX, UK) was used. An ACQUITY UPLC BEH Amide column (2.1 × 100 mm, 1.7 μm; Waters， UK) maintained at 35 ℃ was used. The mobile phase consisted of solvent A (IPA:ACN = 9:1 + 0.1% formic acid) and solvent B (aqueous solution with 25 mM ammonium acetate and 25 mM NH4H2O). The linear gradient elution procedures were set as follows: 0 ~ 0.5 min, 95% solvent A; 0.5 ~ 9.5 min, 95% to 65% solvent A; 9.5
~ 10.5 min, 65% ~ 40% solvent A; 10.5 ~ 12.0 min, 40% solvent A; 12.0 ~ 12.2 min, 40% ~ 95% solvent A; 12.2 ~ 15.0 min, 95% solvent A. The flow rate was 0.5 mL/min and the volume injected was 4 μL. A high-resolution tandem mass spectrometer SCIEX Triple-TOF-5600 plus (Applied Biosystems) was used to detect metabolites. The sheath gas flow was set at a pressure of 30 psi, ion source gas 1 and ion source gas 2 were both set at 60 psi, and the interface heater temperature was set at 650 ℃. The ion spray voltages were set at 5000 V and - 4500 V for the positive and negative ion modes, respectively. The mass spectrometry data were acquired in information-dependent acquisition (IDA) mode. The TOF mass range was from 60 to 1,200 Da. During the acquisition, the mass accuracy was calibrated every 20 samples. Furthermore, in order to evaluate the system stability of UPLC-MS/MS analysis, a QC sample was analyzed right after every 10 samples. 2.4. Bioinformatic analysis of the untargeted metabolomic datasets The acquired MS data pretreatments including peak detection, peak grouping, retention time correction, second peak grouping, and annotation of isotopes and adducts were performed by using the XCMS software. LC-MS raw data files were converted into mz.XML format using the Proteo Wizard MS Convert tools and then processed by the XCMS, CAMERA and metaX toolbox implemented with the R software. Each ion was identified by the m/z data and retention time (RT). Intensities of each peak were recorded and a three dimensional matrix containing arbitrarily assigned peak indices (retention time-m/z pairs), sample names (observations) and ion intensity information (variables) was generated. The online KEGG, HMDB databases were used to annotate the metabolites by matching the exact molecular mass data (m/z) of the samples with those from the databases. The intensities of peak data were further preprocessed by an in-house software metaX. Those features that were detected in less than 50% of the QC samples or 80% of the biological samples were removed, and the remaining peaks with missing values were imputed with the k-nearest neighbor algorithm to further improve the data quality. Principal component analysis (PCA)
was performed for outlier detection and batch effect evaluation using the pre-processed dataset. Quality control-based robust LOESS signal correction was fitted to the QC data with respect to the order of injection to minimize signal intensity drift over time. In addition, variables were then filtered to remove those with a mean intensity that was lower than twice the mean intensity in reagent blanks, or variables with a coefficient of variation in the QC samples above 30%. 2.5. Quantitative analysis of ascorbic acid To quantify ascorbic acid content, 0.5 g of sample and 5 mL of 5% metaphosphoric acid solution were placed into a 15 mL centrifuge tube. The tube was sonicated for 3 min and centrifuged at 7000 rpm for 10 min at 5 ℃. Samples were then filtered (0.22 mm) prior to HPLC analysis (da Silva Barros et al., 2019; Ribeiro et al., 2018). Analyses were performed using an Agilent 1260 High performance liquid chromatography system with a DAD UV-vis absorption detector (Agilent, Santa Clara, CA). The column used was a reverse C18 (Sunfire RP-18, 5 μm, miford, Massachusetts, USA). The analysis was performed under isocratic mode at a flow rate of 0.8 mL/min with a detection of 254 nm, using a mobile phase of water with 50 mM KH2PO4 and methanol in a relative proportion of 96:4 (v/v), and the column temperature was 25 ℃. Ascorbic acid was identified by comparing the retention time of the sample peak with that of the ascorbic acid standard. Quantification was carried out using external standardization and results were expressed as mg/g FW. 2.6. Extraction and determination of total phenolic content (TPC) The extraction and fractionation of the phenolic compounds were achieved according to the method described in F. Jimenez-Aspee with necessary modifications (Jimenez-Aspee et al., 2016). Briefly, 10 grams of the products were extracted three times with 100 mL of methanol under stirring in an ultrasonic bath for 30 min at room temperature. Then, the samples were centrifuged at 12,000 ×g during 20 min, and then vacuum filtered to separate the extracted phenolics from residue. The concentrated extract was dissolved to a volume of 10 mL with methanol. The extracts were mixed
with diluted Folin-Ciocalteu reagent and 15% sodium carbonate. Absorption at 765 nm was measured in a microplate reader after incubation for 30 min at room temperature, and results were expressed as mg gallic acid equivalents per gram of fresh weight of fruit (mg GAE/g FW). 2.7. Phenolic determination by HPLC Phenolic constituents of the extracts were determined using a HPLC method of Bittencourt with some modifications (Bittencourt et al., 2015). Briefly, 10 μg of the extracts were dissolved in 1 mL of methanol, and the solution was filtered through a 0.2 μm PVDF target syringe filter. The phenolic compounds were quantified using Shimadzu HPLC system (SCL-10 A vp, Shimadzu Corporation, Kyoto, Japan) equipped with a UV detector, and absorbance was monitored at 325 nm. Sunfire RP-C18 ODS (Miford, Massachusetts, USA) column at a maintained temperature (35 ℃) was used. Gradient solvents of water−formic acid (99:1, v/v) (solvent A) and acetonitrile (solvent B) in HPLC grade were used to elute the sample at a flow rate of 1.0 mL/min. Total run time was 35 min, and individual phenolic compounds were quantified by comparing with the corresponding standards. The TP content results were expressed as mg gallic acid equivalents per gram of fresh weight of fruit (mg GAE/g FW). 2.8. Determination of the antioxidant activity (ABTS test) and free radical scavenging activity (DPPH assay). The antioxidant activity of the extracts, on the basis of the scavenging activity of the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical, was carried out by the method of Bannour (Bannour et al., 2017). Antioxidant activity was determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging method. A 0.5 ml of plant extract was added to 2.5 ml of a 0.004% ethanol solution of DPPH. Absorbance at 517 nm was determined after 30 min and trolox was used as standard. A calibration curve was prepared, and different Trolox concentrations (standard trolox solutions ranging from 10 to 320 μM) were also evaluated against the radical. Antioxidant
activity was expressed as Trolox equivalent antioxidant capacity (TEAC), micromoles of Trolox per gram FW. ABTS+ assay was determined according to the method described by Yun et al. (Yun et al., 2018). ABTS+ radical cation was produced by mixing 7 mM ABTS solution with 2.45 mM potassium persulfate and the mixture was allowed to stand in the dark at room temperature. The ABTS+ solution was diluted with ethanol to an absorbance of 0.70 pounds were quantified by 5 μL of the sample or trolox standard was added to 2 mL of diluted ABTS+ solution. The absorbance at 734 nm was measured. The antioxidant activity was expressed as Trolox equivalent antioxidant capacity (TEAC), micromoles of Trolox per gram FW. 2.9. Statistical analysis. For the untargeted metabolomic analysis, Wilcoxon tests were carried out to examine metabolic differences between every two samples. The principal component analysis (PCA) was conducted using the metaX to discriminate different variables between groups. The VIP value was calculated and a VIP cut-off value of 1.0 was used to select important features. The quantification results of ascorbic acid, phenolic compounds and antioxidant activity are presented as mean ± standard deviation. The P values were adjusted for multiple testing correction by false discovery rate (FDR; Benjamini – Hocway analysis of variance) was carried out to compare the content differences between immature and mature fruits. and discussion 3. Results and discussion 3.1. Untargeted metabolite profiling of the mature and immature fruits To explore global metabolic variations, an untargeted approach was applied (N = 10), identifying 1,896 annotated metabolite features from 8,054 ion features (Table S1), and finally 133 metabolites were identified according to the MS/MS fragments compared with these standards in in-house database. To check the quality of MS data from different sample groups, several quality control parameters, including total ion
chromatograms (Fig. S1), the m/z width and retention-time width (Fig. S2), were generated and analyzed, suggesting a high degree of overlap and a good sample preparation process. Furthermore, a principal component analysis (PCA) was performed. Our data showed that PC1 and PC2 were responsible for 33.32% and 11.03% of the variation, respectively, indicating a clear separation between the metabolite features from mature and immature fruits (Fig. 1b). An overview of the metabolite profiling of acerola cherry fruits during different mature stages is shown in a heatmap (Fig. 1c). Previous studies have identified a number of metabolites, such as vitamins, polyphenols, polysaccharides, carotenoids and bioflavonoids, of Acerola cherry (Barros et al., 2019; Santos, Rodrigues, & Fernandes, 2018). A traditional phytochemical analysis could only focus on a small number of metabolites belonging to one or several particular categories (Kawaguchi, Tanabe, & Nagamine, 2007). In our study, a large number of metabolite features belonging to various categories were identified, giving us an opportunity to clarify the regulation mechanism of nutritional ingredients in Acerola cherry.
3.2. Identification of differentially accumulated metabolite features (DAMFs) between the mature and immature fruits To reveal the variations in abundance levels of metabolite features between mature and immature fruits, all the identified metabolite features were quantified. Analysis of two important quality control parameters, including normalized intensity (Fig. S3) and coefficient of variation (Fig. S4), showed a high repeatability of the MS data. Statistical analysis identified 1000 significant DAMFs, including 684 up- and 316 down-regulated metabolite features, were identified in the mature fruits compared with the immature fruits (Fig. 2a and b). Most of the identified metabolite features were assigned into various major metabolic categories, such as alkaloids, amino acids, VCs, carotenoids, flavonoids, hormones, lipids, phenylalanines, phenylpropanoids, saccharides, steroids, terpenoids, and ubiquinones (Fig. 2c and Table S2).
Furthermore, the number of mature- and immature-predominantly accumulated metabolite features belonging to each category was analyzed. Interestingly, most of differential
accumulated
amino
acids,
flavonoids,
lipids,
and
terpenoids
predominantly accumulated in the mature fruits rather than in immature fruits. Additionally, all differential accumulated ascorbic acids predominantly accumulated in the immature fruits (Fig. 2d). During the ripening process, there was a dynamic variation in fruit composition. For example, the early ripening phase of dates enriched in hormones, amines, tannins, sucrose, and the later ripening phase of dates enriched in phenylpropanoids and volatiles (Diboun et al., 2015). In guava fruits, sucrose, fructose, serine and citric acid were particularly related to the ripening process (Lee, Choi, Cho, & Kim, 2010). In addition, differences in bioactive compounds, such as ascorbic acid, total phenolics, carotenoids and antioxidant activity, between mature green and red stages of pepper at harvest have been reported (Jang, Jung, Lee, Choi, & Lee, 2015). However, few studies have been done to reveal the changes in nutritional ingredients during the ripening process. Here, untargeted metabolomic analysis helps us to systematically investigate the metabolic variations between mature and immature fruits of Acerola cherry.
3.3. Untargeted metabolomics analysis reveals the variations in antioxidants A large number of antioxidants were identified in the acerola cherry fruits. For carotenoids, phytoenes, including prephytoene-PP and phytofluene, lutein, lycopene and β-carotene were identified. Among these carotenoids, prephytoene-PP and lutein predominantly accumulated in the mature fruits rather than in immature fruits. For phenolics, 2 chlorogenic acids, 8 anthocyanins, 12 flavones, 6 isoflavonoids, 3 lignans, 6 phenolic acids, and 5 polyphenols, were identified. Quantitative analysis showed that 2 chlorogenic acids, including chlorogenic acid and isochlororgenic acid, 4 anthocyanins, including cyanidin, delphinidin-3β-D-glucoside, phloretin, peonidin, most of the identified flavones, such as genistein, luteolin, kaempferol, apigenin
7-glucoside, quercitrin, astragalin, kaempferol 3-O-β-glucoside, vitexin, malonylapiin and
isovitexin,
3
isoflavonoids,
including
6,7,4'-Trihydroxyisoflavone,
2,4',7-Trihydroxyisoflavanone, and 2',4,4',6'-Tetrahydroxychalcone, and 1 lignan (sesaminol), predominantly accumulated in the mature fruits rather than in immature fruits. Meanwhile, 3 anthocyanins, including peonidin 3,5-diglucoside, tulipanin, and cyanidin-3-(p-coumaroyl-glucoside), 2 flavones, including apigenin and rutin, and 3 phenolic
acids,
including
trans-3-hydroxycinnamic
acid,
ferulic
acid
and
feruloylglycine, predominantly accumulated in the immature fruits rather than in mature fruits (Fig. 3). 3.4. Untargeted metabolomics analysis reveals the variations in nutrient substances Moreover, a series of nutrient substances, including 12 amino acids, 12 saccharides, 14 fatty acids and ascorbic acid, were identified. Most of the identified amino acids highly accumulated in the mature fruits and ascorbic acid highly accumulated in the immature fruits. For saccharides, 2-dehydro-3-deoxy-L-fuconate, 2-dehydro-3-deoxy-L-rhamnonate,
D-fructose
and
D-mannose
predominantly
accumulated in the mature fruits and cellobiose, trehalose and isomaltose predominantly accumulated in the immature fruits. No significant variations in fatty acid contents were observed between the mature and immature fruits (Fig. 4). The nutritional quality of Acerola cherry fruits is closely correlated with the presence of soluble sugars, organic acids, amino acids, and some major secondary metabolites. These compounds play an important role in maintaining fruit quality and nutritive value. The heatmap showed that the metabolites have a differential distribution during development and maturation. Almost all amino acids exhibited significance increases during fruit development except L-Lysine and L-Phenylalanine, and these contributed to the formation of fruity, sweaty, and peach-like flavour in Acerola fruits. Saccharides were the metabolites that exhibited a high degree of variance during fruit development, while fruit ripening was characterized by the increases in levels of L-Rhamnono-1,4-lactone, fructose, mannose, which comprised the major soluble sugars in Acerola fruit. Similar results
have been reported by Oms-Oliu (Oms-Oliu et al., 2011). No significant variations in fatty acid contents were observed between the mature and immature fruits. Carotenoids are another important bioactive compound class present in Acerola. Mature fruits showed higher carotenoid content as compared to immature and half-mature fruits. Carotenoid content was also influenced by the genotypes, sunlight exposure, harvesting season and different stages of maturity (Belwal et al., 2018). 3.5. Quantitative analysis confirms the variations in ascorbic acid between mature and immature fruits. To precisely determine the content differences of ascorbic acid between mature and immature fruits, the content was quantified using a HPLC method. The untargeted metabolomics analysis indicated that ascorbic acid was predominantly accumulated in immature fruits, and the direct quantification with an authentic standard of ascorbic acid confirmed this phenomenon. The content of ascorbic acid was severely influenced by the maturation process. Significant decline was observed from immature to over-mature (Fig. 5). The content of ascorbic acid in immature fruit (23.86 ± 2.35 mg/g FW) was 1.9-fold greater than that in mature one (12.25 ± 1.61 mg/g FW). Similar to current work, Mezadri et al. reported levels of 9.52 and 17.7 g of ascorbic acid kg-1 of pulp (Mezadri, Villano, Fernandez-Pachon, Garcia-Parrilla, & Troncoso, 2008). Ascorbic acid levels vary considerably according to degree of maturation, conditions of cultivation, storage and processing. However, it is important to highlight all stages of maturity exceed the recommended vitamin C intake showing that they are outstanding sources for human diet. The consumption of approximately 10 g of fruits has already reached the daily recommendation of vitamin C. 3.6. Total phenolic and individual phenolic compounds between mature and immature fruits The TPCs of Acerola at four different maturity stages are shown in Fig. 5. The stage of maturity had a significant influence on the TPCs in Acerola cherry. During fruit ripening process, a linear pattern of increase was observed for TPCs ,while it is
followed by a decline when over-mature (Fig. 5). The sum of TPCs of Acerola sharply increased from 14.21 ± 1.81 mg GAE/g FW at immature stage to 24.48 ± 2.26 mg GAE/g FW at mature stage (p < 0.05), but decline to 21.42 ± 1.91 mg GAE/g FW when it is over-mature. Fig. 6a shows a chromatogram of an acerola pulp containing numerous peaks. A total of six major phenolic compounds were identified. A1-A7 was identified as (1) chlorogenic acid (2) p-coumaric acid, (3) ferulic acid, (4) kaempferol, (5) luteolin, (6) rutin, and (7) apigenin because of those spectral data and retention time using standard as reference. The concentration of (1)chlorogenic acid, (4) kaempferol, (5) luteolin, (6) rutin, and (7) apigenin in the mature fruit were higher than those in immature. By contrast, the concentration of (2) p-coumaric acid, (3) ferulic acid tended to decrease in more ripe fruits. This agreed well with the data reported by other authors, indicating decreases of ferulic acid with the developmental stages (Wang et al., 2016). The representative HPLC chromatograms of ascorbic acid between mature and immature fruits are shown in Fig. 6. Phenolic compounds, as secondary metabolites, are a large group of molecules widely distributed in fruits and vegetables (Acero, Gradillas, Beltran, Garcia, & Munoz Mingarro, 2019). In Acerola cherry, the TPCs increased with maturity stage, which was in agreement with those reported previously for Acerola and other fruits (Sulas, Petretto, Pintore, & Piluzza, 2017). However, the quantities of TPC obtained from this study were lower than the previously published results (Mishra, Mishra, & Mahanta, 2014). This might be mainly attributed to the differences in the cultivar and sources of the materials, as well as the genus, cultivar, species, ripening stage, soil and climate. The developmental stages not only had a significant impact on the total polyphenol concentration, but a large number of individual phenolic compounds also varied. It was obviously that most of the chromatography peaks were higher in mature stage than immature one. This agreed well with the data from the metabolic result.
3.7. Antioxidant activity Antioxidant mechanisms in biological tissues are extremely complex, and there is
not one method that can provide unequivocal results. Thus the antioxidant capacity of Acerola was evaluated based on DPPH and ABTS radical scavenging activity, as shown in Fig. 5. The DPPH and ABTS radical scavenging activity in immature stage was 180.52 ± 15.41 μM TE/g DW and 141.26 ± 10.12μM TE/g DW, respectively. These values reduced by 39.8% (108.66 ± 8.91 μM TE/g DW) for DPPH and 36.0% (90.41 ± 8.8 μM TE/g DW) for ABTS. In biological analysis, the variation between mature and immature Acerola fruit in antioxidative effect has been reported. Phenolics were considered as one of the ingredients in Acerola fruit contributing to this effect, yet whether the levels of Phenolics were correlated with the activity is still debatable. Mature fruit containing significantly higher amount of flavonoidic compounds showed better antioxidative activity than that of the mature one. In contrast, our study demonstrated that the antioxidative activity did not show significant correlation with the amount of Phenolics. The highly positive correlation between DPPH and ABTS suggests that both methods are suitable to measure the antioxidant capacity of acerola. A high positive correlation was found between the ascorbic acid (vitamin C) content and DPPH (r=0.9830) as well as a high positive correlation was identified between ascorbic acid content and ABTS (r=0.9897). The reduction in antioxidant activities during acerola development may be associated with apparent decrease in quantity of vitamin C in the fruit. Although phenolic compounds are known as antioxidant compounds, their increase during fruit development constituted only small proportion of total in fruit, hence the change in vitamin C a much more significant influence than phenolic compounds on acerola antioxidant capacity. The ABTS and DPPH radical scavenging activities were used to compare the antioxidant activity between mature and immature acerola cherry extracts. The results showed that during the maturity process both the DPPH and ABTS radical scavenging activities decreased from immature to mature stage, suggesting that ascorbic acid has a significant contribution to the antioxidant capacity of the acerola.
4. Conclusion In this study, we investigated their chemical and biological properties by metabolic profiling and antioxidant assays. In LC-MS profiling, 1,896 annotated metabolite features were obtained, and 133 metabolites were finally identified according to the MS/MS fragments compared with these standards in in-house database. Statistically differences in the levels of amino acids, flavonoids, lipids, terpenoids and ascorbic acids were found between mature and immature fruits. In parallel, their antioxidant activities were compared. The alcoholic extract of immature acerola fruit possessed better DPPH effect than the mature one. The ascorbic acid was highly correlated with antioxidant potential and the metabolite level is higher in immature than that of mature. In this regard Acerola was found to be rich in bioactive compounds especially ascorbic acid and polyphenolics and showed varied health beneficial effects. Hence, Acerola is anticipated to be a good candidate for dietary supplements and functional food manufacturers looking to the development of new products. Acknowledgments This work is Financial supported by the National Key R&D Program of China (2018YFC0311205), the Hangzhou Science and Technology Development Program (20191203B01) and the Zhejiang Science and Technology Project (2018C02053). References Acero, N., Gradillas, A., Beltran, M., Garcia, A., & Munoz Mingarro, D. (2019). Comparison of phenolic compounds profile and antioxidant properties of different sweet cherry (Prunus avium L.) varieties. Food Chemistry, 279, 260-271. Bannour, M., Fellah, B., Rocchetti, G., Ashi-Smiti, S., Lachenmeier, D. W., Lucini, L., & Khadhri, A. (2017). Phenolic profiling and antioxidant capacity of Calligonum azel Maire, a Tunisian desert plant. Food Research International, 101, 148-154. Barros, B. R. S., Barboza, B. R., Ramos, B. A., Moura, M. C., Coelho, L., Napoleao, T. H., Correia, M. T. S., Paiva, P. M. G., Cruz Filho, I. J. D., Silva, T. D. D., Lima, C. S. A., & Melo, C. M. L. (2019). Saline extract from Malpighia emarginata DC leaves showed higher polyphenol presence, antioxidant and antifungal activity and promoted cell proliferation in mice splenocytes. Anais
da Academia Brasileira de Ciências, 91 (1), e20190916. Belwal, T., Devkota, H. P., Hassan, H. A., Ahluwalia, S., Ramadan, M. F., Mocan, A., & Atanasov, A. G. (2018). Phytopharmacology of Acerola (Malpighia spp.) and its potential as functional food. Trends in Food Science & Technology, 74, 99-106. Bittencourt, M. L. F., Ribeiro, P. R., Franco, R. L. P., Hilhorst, H. W. M., de Castro, R. D., & Fernandez, L. G. (2015). Metabolite profiling, antioxidant and antibacterial activities of Brazilian propolis: Use of correlation and multivariate analyses to identify potential bioactive compounds. Food Research International, 76 (Pt 3), 449-457. Cai, J., Chen, T., Zhang, Z., Li, B., Qin, G., & Tian, S. (2019). Metabolic dynamics during loquat fruit ripening and postharvest technologies. Frontiers in Plant Science, 10, 619. Cappato, L. P., Ferreira, M. V. S., Moraes, J., Pires, R. P. S., Rocha, R. S., Silva, R., Neto, R. P. C., Tavares, M. I. B., Freitas, M. Q., Rodrigues, F. N., Calado, V. M. A., Raices, R. S. L., Silva, M. C., & Cruz, A. G. (2018). Whey acerola-flavoured drink submitted Ohmic Heating: Bioactive compounds, antioxidant capacity, thermal behavior, water mobility, fatty acid profile and volatile compounds. Food Chemistry, 263, 81-88. Cascia, G., Bulley, S. M., Punter, M., Bowen, J., Rassam, M., Schotsmans, W. C., Larrigaudiere, C., & Johnston, J. W. (2013). Investigation of ascorbate metabolism during inducement of storage disorders in pear. Physiol Plantarum, 147 (2), 121-134. Chang, S. K., Alasalvar, C., & Shahidi, F. (2018). Superfruits: Phytochemicals, antioxidant efficacies, and health effects - A comprehensive review. Critical Reviews in Food Science and Nutrition, 1-25. da Silva Barros, B. R., do Nascimento, D. K. D., de Araújo, D. R. C., da Costa Batista, F. R., de Oliveira Lima, A. M. N., da Cruz Filho, I. J., de Oliveira, M. L., & de Melo, C. M. L. (2019). Phytochemical analysis, nutritional profile and immunostimulatory activity of aqueous extract from Malpighia emarginata DC leaves. Biocatalysis and Agricultural Biotechnology, 101442. De Rosso, V. V., & Mercadante, A. Z. (2007). The high ascorbic acid content is the main cause of the low stability of anthocyanin extracts from acerola. Food Chemistry, 103 (3), 935-943. Diboun, I., Mathew, S., Al-Rayyashi, M., Elrayess, M., Torres, M., Halama, A., Meret, M., Mohney, R. P., Karoly, E. D., Malek, J., & Suhre, K. (2015). Metabolomics of dates (Phoenix dactylifera) reveals a highly dynamic ripening process accounting for major variation in fruit composition. BMC Plant Biology, 15, 291. Jang, Y. K., Jung, E. S., Lee, H. A., Choi, D., & Lee, C. H. (2015). Metabolomic characterization of hot pepper (Capsicum annuum "CM334") during fruit development. Journal of Agricultural and Food Chemistry, 63 (43), 9452-9460. Jimenez-Aspee, F., Theoduloz, C., Vieira, M. N., Rodriguez-Werner, M. A., Schmalfuss, E., Winterhalter, P., & Schmeda-Hirschmann, G. (2016). Phenolics from the patagonian currants Ribes spp.: Isolation, characterization and cytoprotective effect in human AGS cells. Journal of Functional Foods, 26, 11-26. Kawaguchi, M., Tanabe, H., & Nagamine, K. (2007). Isolation and characterization of a novel flavonoid possessing a 4,2''-glycosidic linkage from green mature acerola (Malpighia emarginata DC.) fruit. Bioscience Biotechnology and Biochemistry, 71 (5), 1130-1135. Lee, S., Choi, H. K., Cho, S. K., & Kim, Y. S. (2010). Metabolic analysis of guava (Psidium guajava L.) fruits at different ripening stages using different data-processing approaches. Journal of Chromatography B-Analytical Technologies in the Biomedical Life Sciences, 878 (29), 2983-2988.
Lee, S., Jang, W. J., Choi, B., Joo, S. H., & Jeong, C. H. (2017). Comparative metabolomic analysis of HPAC cells following the acquisition of erlotinib resistance. Oncology Letters, 13 (5), 3437-3444. Leffa, D. D., da Silva, J., Daumann, F., Dajori, A. L. F., Longaretti, L. M., Damiani, A. P., de Lira, F., Campos, F., Ferraz, A. D. F., Correa, D. S., & de Andrade, V. M. (2014). Corrective effects of acerola (Malpighia emarginata DC.) juice intake on biochemical and genotoxical parameters in mice fed on a high-fat diet. Mutation Research-Fundamental and Molecular Mechanisms of Mutagenesis, 770, 144-152. Mezadri, T., Villano, D., Fernandez-Pachon, M. S., Garcia-Parrilla, M. C., & Troncoso, A. M. (2008). Antioxidant compounds and antioxidant activity in acerola (Malpighia emarginata DC.) fruits and derivatives. Journal of Food Composition and Analysis, 21 (4), 282-290. Mishra, P., Mishra, S., & Mahanta, C. L. (2014). Effect of maltodextrin concentration and inlet temperature during spray drying on physicochemical and antioxidant properties of amla (Emblica officinalis) juice powder. Food and Bioproducts Processing, 92 (C3), 252-258. Monti, L. L., Bustamante, C. A., Osorio, S., Gabilondo, J., Borsani, J., Lauxmann, M. A., Maulion, E., Valentini, G., Budde, C. O., Fernie, A. R., Lara, M. V., & Drincovich, M. F. (2016). Metabolic profiling of a range of peach fruit varieties reveals high metabolic diversity and commonalities and differences during ripening. Food Chemistry, 190, 879-888. Oms-Oliu, G., Hertog, M. L. A. T. M., Van de Poel, B., Ampofo-Asiama, J., Geeraerd, A. H., & Nicolaï, B. M. (2011). Metabolic characterization of tomato fruit during preharvest development, ripening, and postharvest shelf-life. Postharvest Biology and Technology, 62 (1), 7-16. Pinsorn, P., Oikawa, A., Watanabe, M., Sasaki, R., Ngamchuachit, P., Hoefgen, R., Saito, K., & Sirikantaramas, S. (2018). Metabolic variation in the pulps of two durian cultivars: Unraveling the metabolites that contribute to the flavor. Food Chemistry, 268, 118-125. Ribeiro, H. L., Oliveira, A. V., Brito, E. S., Ribeiro, P. R. V., Souza Filho, M. S. M., & Azeredo, H. M. C. (2018). Stabilizing effect of montmorillonite on acerola juice anthocyanins. Food Chemistry, 245, 966-973. Santos, V. O., Rodrigues, S., & Fernandes, F. A. N. (2018). Improvements on the stability and vitamin content of acerola juice obtained by ultrasonic processing. Foods, 7 (5). Silva, P. B., Duarte, C. R., & Barrozo, M. A. S. (2019). A novel system for drying of agro-industrial acerola (Malpighia emarginata D. C.) waste for use as bioactive compound source. Innovative Food Science & Emerging Technologies, 52, 350-357. Stafussa, A. P., Maciel, G. M., Rampazzo, V., Bona, E., Makara, C. N., Demczuk, B., & Haminiuk, C. W. I. (2018). Bioactive compounds of 44 traditional and exotic Brazilian fruit pulps: phenolic compounds and antioxidant activity. International Journal of Food Properties, 21 (1), 121-133. Sulas, L., Petretto, G. L., Pintore, G., & Piluzza, G. (2017). Bioactive compounds and antioxidants from a Mediterranean garland harvested at two stages of maturity. Natural Product Research, 31 (24), 2941-2944. Wang, B., Huang, Q., Venkitasamy, C., Chai, H., Gao, H., Cheng, N., Cao, W., Lv, X., & Pan, Z. (2016). Changes in phenolic compounds and their antioxidant capacities in jujube (Ziziphus jujuba Miller) during three edible maturity stages. LWT - Food Science and Technology, 66, 56-62. Yun, D. Y., Kang, Y. G., Kim, E. H., Kim, M., Park, N. H., Choi, H. T., Go, G. H., Lee, J. H., Park, J. S., & Hong, Y. S. (2018). Metabolomics approach for understanding geographical dependence of soybean leaf metabolome. Food Research International, 106, 842-852.
Zheng, X., Liu, F., Shi, X., Wang, B., Li, K., Li, B., & Zhuge, B. (2018). Dynamic correlations between microbiota succession and flavor development involved in the ripening of Kazak artisanal cheese. Food Research International, 105, 733-742.
Additional files Additional file 1: Fig. S1. The total ion chromatograms of all the samples. (DOCX 373 kb) Additional file 2: Fig. S2 The m/z width (a) and retention-time width (b) of all the samples. (DOCX 609 kb) Additional file 3: Fig. S3 The normalized intensity of the MS data. (DOCX 50 kb) Additional file 4: Fig. S4 The coefficient of variation of the data from different samples. (DOCX 86 kb) Additional file 5: Table S1. Detail information of all identified metabolites.(XLSX 4718 kb) Additional file 6: Table S2. Identification and classification of all metabolites. (XLSX 531 kb)
Fig. 1. Untargeted metabolite profiling reveals the metabolites in the wild cherry fruits. (a) Pictures of the mature and immature wild cherry fruits. (b) The PCA data of the samples from mature and immature wild cherry fruits. Red indicated the mature fruits and green indicated the immature fruits. (c) A heatmap of the metabolites identified in the metabolomes of the two fruit samples (n = 10). The heatmap scale ranges from -4 to +4 on a log2 scale.
Figure 2 (a)
(c)
Alkaloids
128
Carotenoids
5
VC
10
-4 0 4 Log2(MAT/IMAT)
12
Carotenoid
Flavonoid
83 18
Hormone
Lipids
205
77
82
Hormones
Up Dn None
8
20
18
Flavonoids
0
Ascorbic acid
58
Amino acids
15
Amino acid
0.8
0.3
-0.2
-log10 (Q value)
(d)
Normalized ion strength -1 -0.7 -0.2 0 0.2 0.7 1 -0.7
-1.2
T test 20
Lipid
15
49
(b)
600
400
316
300
100
0 Dn
Saccharides
191
Terpenoid
16
54 Ubiquinones
200
21
68 26
Terpenoids
Number
500
42 Steroids Phenylpropanoids
684
700
Saccharides Phenylalanines
16 800
Up
17 272
Down
None
Up
Fig. 2. Identification of the DAMs between the mature and immature fruits. (a) Significance analysis of the DAMs between the mature and immature fruits by Volcanoplot. (b) The number of up- and down-regulated metabolites in the mature fruits compared with the immature fruits. (c) The relative abundances of the metabolites belonging to various major metabolic categories. (d) The numbers of upand down-regulated metabolites in different metabolic categories were shown in pies.
Figure 3 -4
Phytoene
Carotenoids
Lutein Lycopene β-Carotene Chlorogenic acid
Anthocyanins
Flavone and flavonol
Phenolics Isoflavonoid
Lignans
Phenolic acids
Polyphenols
0 Mature
4 Immature Prephytoene diphosphate Phytofluene Lutein Lycopene β-Carotene Chlorogenic acid Isochlorogenic acid Cyanidin Delphinidin Delphinidin-3β-D-glucoside Phloretin Peonidin Peonidin 3,5-diglucoside Tulipanin Cyanidin-3-(p-coumaroyl-glucoside) Apigenin Genistein Luteolin Kaempferol Apigenin 7-glucoside Quercitrin Astragalin Kaempferol 3-O-β-glucoside Vitexin Isovitexin Rutin Malonylapiin Formononetin Isoformononetin 7,8,4'-Trihydroxyflavone 6,7,4'-Trihydroxyisoflavone 2,4',7-Trihydroxyisoflavanone 2',4,4',6'-Tetrahydroxychalcone Sesaminol Lariciresinol Secoisolariciresinol monoglucoside 4-Hydroxycinnamic acid trans-3-Hydroxycinnamic acid 3,4-Dihydroxyhydrocinnamic acid Ferulic acid 4-p-Coumaroylquinic acid Feruloylglycine Isopimpinellin Vanillin Methoxyacetophenone Mellein Catechol
Fig. 3. Variations in the abundances of antioxidants between the mature and immature fruits. A heatmap of the relative amounts of antioxidants, including carotenoids and phenolics. The heatmap scale ranges from -4 to +4 on a log2 scale.
Figure 4
-4
Amino acids
Saccharides
Fatty acids
Ascorbic acid
0 Mature
4 Immature L-Serine L-Proline L-Valine L-Leucine L-Isoleucine L-Glutamine L-Lysine L-Methionine L-Histidin L-Phenylalanine L-Asparagine L-Arginine 2,4-Diketo-3-deoxy-L-fuconate L-Rhamnono-1,4-lactone 2-Dehydro-3-deoxy-L-fuconate 2-Dehydro-3-deoxy-L-rhamnonate D-Fructose D-Mannose L-Sorbose alpha-D-Glucose D-Allose Cellobiose Trehalose Isomaltose 1,6-D mannobiose linoleate Hexadecanoic Icosapentaenoic Icosadienoic Arachidonate Icosenoic Docosadienoic Docosahexaenoic Tetracosanoic Octanoic Dodecanoic Tetradecanoic (9Z)-Hexadecenoic Octadecanoic Ascorbate
Fig. 4. Variations in the abundances of nutrients between the mature and immature fruits. A heatmap of the relative amounts of nutrients, including amino acids, saccharides, fatty acids and ascorbic acid. The heatmap scale ranges from -4 to +4 on a log2 scale.
Fig. 5
Fig. 5. Concentrations of bioactive compounds (Vc, TPc) and antioxidant activity measured by assays of DPPH and ABTS Vc=Vitamin C, TPc=Total Phenolic compounds, ABTS=ABTS scavenging activity, DPPH=DPPH radical scavenging activity
Fig. 6. Chromatogram of acerola at mature (a) and immature (b) stages at 325 nm (1)chlorogenic acid (2) p-coumaric acid, (3) ferulic acid, (3) quercetin, (4) kaempferol, (5) luteolin, (6) rutin, and (7) apigenin
Graphical Abstract
Highlights
Through comprehensive metabolites analysis, 1896 annotated metabolites were obtained. Statistically differences in the levels of amino acids, flavonoids, lipids, terpenoids and ascorbic acids were found between mature and immature fruits. The well correlations were found between the antioxidant potential with its content of ascorbic acid. The maturity of Acerola cherry has to be considered when it is being used for health food products.
Author Contributions Section: Mingfeng Xu: experiment. Chenjia Shen and Han Zheng: Data curation, Writing-Original draft preparation. Yunsheng Xu and Changfeng Xue: samples collection. Beiwei Zhu: WritingReviewing and Editing. Jiangning Hu: Supervision.