Rhodomyrtus tomentosa Fruits in Two Ripening Stages: Chemical Compositions, Antioxidant Capacity and Digestive Enzymes Inhibitory Activity

Rhodomyrtus tomentosa fruit (RTF) has been known as a food source with multiple health-care components. In this work, nutrition characteristics, free and bound phenolic profiles, antioxidant properties in vitro and digestive enzymes inhibitory activities of un-fully mature RTF (UM-RTF) and fully mature RTF (FM-RTF) were evaluated for the first time. Results verified that high levels of energy, ascorbic acid, organic acids and total phenolics were observed in FM-RTF. Moreover, FM-RTF had significant higher total phenolic content (TPC), but significantly lower total flavonoid content (TFC) than UM-RTF. In addition, twenty phenolic compounds in RTF were identified by high performance liquid chromatography–electrospray ionization–quadrupole time-of-flight tandem mass spectrometry (HPLC-ESI-qTOF-MS/MS) method. Quantitative analysis results indicated that gallic acid, ellagic acid and astragalin were the predominant free phenolics, while gallic acid and syringetin-3-O-glucoside were dominant in bound phenolic fractions. In contrast, higher contents of phenolics were observed in FM-RTF. The results also confirmed that FM-RTF exhibited higher antioxidant activities and digestive enzymes inhibitory activities than UM-RTF. Strong inhibitory ability on α-glucosidase was found in RTF, while bound phenolics showed a stronger α-amylase inhibitory effect than free phenolics. Moreover, the interaction between the main phenolic compounds and α-glucosidase/α-amylase was preliminary explored by molecular docking analysis. The results provided valuable data about the chemical compositions and biological potential of R. tomentosa fruits in both maturation stages studied.

It has been found that the ripeness of fruits has a great influence on the nutritional characteristics, chemical components and biological properties [7,8]. Normally, total soluble solids, amino acids, sugars, phenolics, anthocyanins and bio-activities in berry fruits tend to increase during ripening [9][10][11]. In the folk medicine, un-fully mature RTF (UM-RTF) is generally used to treat dysentery or diarrhea. In contrast, fully mature RTF (FM-RTF), is considered as a diet source rich in natural antioxidants [5,6,10]. Hence, understanding the changes in chemical compositions is of great importance for determining the best ripening stage for fruits with better quality and nutritional value [12][13][14]. Lai et al. (2013) reported that RTF extract consisted of phenolic acids, flavonols, anthocyanins, ellagitannins and stilbenes. Among them, piceatannol is a dominant phenolic compound [9]. Currently, studies on R. tomentosa are mainly concentrated on the phytochemicals from its leaves, flowers and stems owing to their antioxidant, anti-bacterial, anti-inflammatory properties and DNA damage prevention effects [15][16][17][18]. A comprehensive analysis of the nutritional characteristics, chemical composition and bio-activities of RTF during the ripening stage has not yet been carried out.
Taking into account the medicinal value of this edible fruit, this study aims to analyze nutritional compositions, free and bound phenolic compounds of RTF in two ripening stages. Additionally, the antioxidant activities in vitro and digestive enzymes inhibitory ability of free and bound phenolic fractions of UM-RTF and FM-RTF were also investigated. More importantly, the digestive enzyme inhibitory activities of the main phenolic compounds were revealed by molecular docking analysis. This preliminary study may highlight the potential valuable of this unexploited fruit, thus promoting its development and application in the food industry.

Nutritional Assessment
The AOAC method was adopted to determine the contents of protein, carbohydrates and ash of UM-RTF and FM-RTF [19]. The calculation of total energy was implemented according to the formula: Energy (kcal) = 4 × (g protein + g carbohydrates) + 9 × (g fat) [20].
Free sugars of UM-RTF and FM-RTF were extracted by the procedure described in [19]. The contents of free sugars of UM-RTF and FM-RTF were determined by a 1260 HPLC system equipped with a refraction index detector. Prior to HPLC analysis, the extracts were filtered using 0.45 µm Whatman nylon filters. The free sugars were quantified by the internal standard method and the results were expressed as mg/g DW [21].
The contents of organic acids of UM-RTF and FM-RTF were measured using a 1260 HPLC system coupled with a photodiode array detector by using the method reported by Pereira et al. [22]. The free organic acids were quantified by the internal standard method and results were expressed as µg/g DW.
The contents of free amino acids of UM-RTF and FM-RTF were analyzed by the method by Song et al. [23]. The separation of the free amino acids of the extracts was conducted using an L-8900 automatic amino acid analyzer (Hitachi Co. Ltd., Tokyo, Japan), equipped with a 2622 Hitachi custom ion-exchange resin column (60 × 4.6 mm, 5 µm, Tokyo, Japan). Briefly, the samples (1.0 g) were extracted with 20 mL 1% sulfo salicylic acid under ultrasonic powder of 320 W for 30 min. The chromatographic conditions were in agreement with the methods described by Song et al. [23]. The contents of free amino acids were calculated by the internal standards method, and results were expressed as mg/g DW.

Extraction of Free and Bound Phenolic Fractions
Different phenolic fractions were obtained according to the method of Wang et al. 2019 [24]. Figure S1 showed the flow diagram of extraction and analysis for UM-RTF and FM-RTF. Briefly, RTF powder (2 g) was mixed with 10 mL of 70% ethanol/water (v/v) in a 15-mL Eppendorf tube, followed by extraction twice in an ultrasonic bath of 320 W at 50 • C for 30 min. After that, centrifugal treatment was conducted at 5000× g for 10 min at 4 • C, and the combined filtrate was evaporated to dryness in vacuum at 30 • C. The dryness was resolved in 5 mL of 50% ethanol/water (v/v) to obtain free phenolic fractions. After free phenolic extraction, the residues were dried to a fixed weight at 50 • C, then used to extract the bound phenolics. The above residue (1 g) was soaked in 40 mL of 2 M NaOH at 30 • C for 2 h in nitrogen. After that, pH value of the hydrolysate was adjusted to 2 by using 6 M HCl. The mixture was degreased three times with 50 mL hexane. Then, the supernatant was extracted three times with 15 mL of diethyl ether/ethyl acetate (1:1, v/v) using the method by Wang et al. [24]. The combined extraction was evaporated to dryness in vacuum at 30 • C. The dryness was reconstituted in 5 mL of 50% ethanol to obtain bound phenolic fractions. Free and bound phenolic fractions were stored at −20 • C for later use.

Measurement of Phenolic and Flavonoid Contents
The determination of phenolic content in free and bound phenolic fractions was carried out using the Folin-Ciocalteau method by Wu et al. [25]. Gallic acid with concentration ranging from 0.1-1.0 mg/mL was used as the standard. Results were expressed in mg gallic acid equivalents per g sample in dry weight (mg GAE/g DW). The content of free and bound flavonoid was measured using the reported method of Li et al. [26] by taking rutin as the standard. Results were expressed in mg rutin equivalents per g sample in dry weight (mg RE/g DW).

HPLC-ESI-qTOF/MS and HPLC-DAD Analysis
The phenolic compositions were identified using an Agilent 1260 HPLC system coupled with a high-resolution time-of-flight (HR-qTOF) mass detector and an electrospray ionization (ESI) source. An Aligent Zorbax Eclipse C18 plus column was adopted to identify the compounds. Two mobile phases consisted of acetonitrile-0.1% formic acid (A) and water-formic acid (B). The gradient elution program was as follows: 0-5 min, 15% A; 5-30 min, 15-35% A; 30-40 min, 35-50% A; 40-45 min, 80% A; 45-50 min, 15% A at a flow rate of 0.8 mL/min. The other chromatogram conditions were as follows: injection volume of 10 µL, column temperature of 30 • C;, and the detection wavelength ranging from 200 to 600 nm. The ESI source conditions were referred to our previously described method [27,28]. Bruker compass DataAnalysis software was used to acquire MS data. The identified compounds were quantified using HPLC-DAD method, and the chromatographic conditions were consistent with the above developed HPLC-ESI-qTOF-MS/MS. The contents of the main compounds were expressed in µg/g DW.

Antioxidant Activity
The DPPH and ABTS + assays were conducted following the previous protocols by Wang et al. [24]. The scavenging results of DPPH and ABTS + were expressed as micromole trolox equivalents per gram dried weight (µmol TE/g DW). The determination of OH − scavenging ability was performed using the method proposed by Wang et al. [24], and the result were expressed in µmol TE/g DW as well. The FRAP assay was conducted following the same method in [29]. The FRAP value was expressed as micromoles of ferrous sulfate equivalents (Fe(II)SE) per gram of dried weight (µM Fe(II)SE/g DW).
2.7. Digestive Enzyme Inhibition Activities 2.7.1. α-Glucosidase Inhibitory Activity The α-glucosidase inhibitory activity (α-GIA) of the extracts was measured using the method by Li et al. [26]. In short, 1.0 U/mL α-glucosidase (50 µL) dissolving in 0.01 M PBS (pH 6.9), 50 µL of the diluted sample extracts (5,10,15,20,30,60,80, 120 µg/mL dissolving in 20% ethanol) and 100 µL of 0.01 M PBS (pH 6.9) were added to an Eppendorf tube and placed in a water bath for 10 min at 37 • C, followed by addition of 5.0 mM p-NPG solution (100 µL) for another 20 min of incubation at 37 • C. After that, 300 µL of 0.2 M Na 2 CO 3 solution was added to terminate the reaction. Finally, the absorbance of the reaction was determined at 450 nm using a microplate reader (SpectraMax M5 Molecular Device, CA, USA). The α-glucosidase inhibition rate of the sample extracts was calculated by Equation (1):

α-Amylase Inhibition Activity
The determination of α-amylase inhibitory activity (α-AIA) was carried out using the method by Zhu et al. (2019) with slight modifications [30]. First, 100 µL of 0.5 U/mL α-amylase in PBS (pH = 6.9) was mixed thoroughly with 50 µL of the diluted sample extracts (1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0 mg/mL in 20% ethanol) or the phenolic standards, followed by incubation at 37 • C; for 10 min and addition of soluble starch solution (100 µL) for another 20 min of incubation at 37 • C;. After that, 200 µL of saturated Na 2 CO 3 solution was added to terminate the reaction. Finally, the absorbance at 490 nm was determined by using a microplate reader (SpectraMax M5 Molecular Device, CA, USA). The α-amylase inhibition rate of the sample extracts was calculated by Equation (1).

Molecular Docking Analysis
The binding mechanisms between the major phenolic compounds and the receptors can be explained by molecular docking simulation method reported by Li et al. [26]. The 2D structure documents of the major phenolics compounds in NFE and acarbose were downloaded from the website (http://zinc.docking.org/, accessed on 12 July 2022). The 3D structures of α-glucosidase (PDB ID: 3A4A) and α-amylase (PDB ID: 1PPI) were acquired from the Protein Data Bank website (http://www.rcsb.org/pdb, accessed on 12 July 2022). Molecular docking in Surflex-Dock Geom (SFXC) mode were carried out using SYBYL-X 2.0 software. Before molecular docking analysis, α-glucosidase and α-amylase were treated by removing the ligands and water molecules and adding CHARMM force field and polar hydrogen. In order to obtain the optimal binding mode, the free energy minimization under the CHARMM force field was selected for docking. The docking parameters (Cscore, T-score, interaction force types, hydrogen bonds distances and interaction sites) were gained from the molecular docking.

Statistic Analysis
All experimental tests were conducted in triplicate and the results were expressed as mean ± standard deviation. The experimental data were evaluated by Statistic software version 19.0. One-way analysis of variance (ANOVA) followed by Tukey's HSD test and two-way ANOVA were conducted for significant difference analysis. The difference was considered significant when p < 0.05. Table 1 shows the nutritional composition of UM-RTF and FM-RTF. The statistical analysis verified that parameters for the UM-RTF and FM-RTF were significantly different (p ≤ 0.05). The FM-RTF showed the highest concentrations of moisture (32.43%), carbohydrates (65.29%), ascorbic acid (2.57 mg/g DW) and energy (81.23%), while UM-RTF exhibited the highest content of ash (19.87%). It could be observed that protein content of RTF was about 38.78-40.15 mg/g DW, which agreed with the result of Lai et al. [1]. The total organic acids content of FM-RTF (2273.84 µg/g DW) was significantly higher than that of UM-RTF (1707.64 µg/g DW). Seven organic acids were identified in RTF. The main organic acids measured in FM-RTF were malic acid (829.58 ± 7.62 µg/g DW), succinic acid (468.01 ± 43.35 µg/g DW), acetic acid (389.06 ± 30.57 µg/g DW), oxalic acid (335.14 ± 3.65 µg/g DW), pyruvic acid (211.92 ± 15.15 µg/g DW) and D-galacturonic acid (42.69 ± 3.61). The contents of most organic acids in FM-RTF were higher than those in UM-RTF. So far, there is a lack of primary data on the organic acids of RTF. Regarding the free sugar profiles, the main sugar found in the RTF was glucose (124.55 ± 1.79 mg/g DW for FM-RTF, 137.13 ± 4.96 mg/g DW for UM-RTF). No significant difference in total sugar contents was observed in two ripening stages of RTF. The amino acids content of RTF in two ripening stages was insignificantly different. The total amino acids content was in the range 15.27-16.21 mg/g DW. About 15 amino acids existed in RTF. Glutamic acid, L-arginine, tyrosine and leucine were the dominant amino acids of UM-RTF and FM-RTF, which were in line with the reports of Lai et al. (2015) [1]. Compared with the recommended daily intake, the RTF is a relatively good fruit source rich in amino acids [1,2]. FM-RTF presented a TPC (31.53 ± 1.36 mg GAE/g DW), which is lower than that in the study of Lai et al. [1] was significantly higher than that in the work of Huang et al. [31]. This may be caused by genetic variations of the samples. In addition, it can be observed that free phenolic content (12.43 mg GAE/g DW) and bound phenolic content (17.38 mg GAE/g DW) in FM-RTF were higher than those in UM-RTF. However, the opposite result was observed for TFC. UM-RTF had higher TFC (9.87 mg RE/g DW), free flavonoid content (5.32 mg RE/g DW) and bound flavonoid content (3.17 mg RE/g DW) than FM-RTF. Compared with other berry fruits, RTF is a better source rich in dietary polyphenols [1,2,31].

Identification and Quantification of Phenolic Compositions
The phenolic profiles of different fractions in two ripening stages were identified by HPLC-ESI-qTOF-MS/MS method ( Figure 1). Table 2 presents the retention time, λ max , parent ion, main fragment ions and tentative identification. Seventy compounds were identified, consisting of eight phenolic acids (peaks 1-5, 11, 12 and 15), seven flavonoids/anthocyanins compounds (peaks 6, 7, 10, 13, 14, 18, and 20), two other compounds (Peaks 8 and 16) and three unknown compounds (peaks 9, 17 and 19).   [2,9]. Peaks 17 and 19 can be preliminary inferred as flavonoids compounds by analyzing their typical UV-vis spectral characteristics (λ max at 254 and 350 nm). Peak 20 can be preliminary determined as kaempferol glycoside owing to its MS 2 fragment ion at m/z at 287.04 [C 15 H 10 O 6 + H] + (loss of kaempferol aglycone). It can be observed from Table 3 that the free phenolics fractions had a wider range of phenolic compositions than bound phenolics fractions. For free phenolic fractions, FM-RTF also showed higher individual phenolic contents than UM-RTF. For FM-RTF, the highest content of astragalin (307.92 ± 5.00 µg/mL) was found in free phenolic fractions. Regardless of UM-RTF or FM-RTF, gallic acid, p-hydroxycinnamic acid, ellagic acid and astragalin were dominant phenolic compounds in free phenolic fraction. In contrast, gallic acid and syringetin-3-O-glucoside were dominant phenolic compounds in bound phenolic fraction. For UM-RTF and FM-RTF, the content of gallic acid reached to 717.24 ± 30.95 µg/mL and 507.18 ± 27.07 µg/mL in bound phenolic fraction. Wang et al. (2022ab) found high levels of gallic acid, protocatechuic acid, ellagic acid and astragalin in RTF extracts, which agreed with our study [5,6]. Zhao et al. [2] revealed that gallic acid, ellagic acid, astragalin, piceatannol, and resveratrol were the main phenolic compounds in RTF. So far, there are few studies on free and bound phenolics of RTF.

Conclusions
In this work, the nutrition characteristics, free and bound phenolic profiles, antioxidant properties in vitro and digestive enzymes inhibitory activities of RTF in both maturation stages were evaluated for the first time. Compared with UM-RTF, FM-RTF had higher contents of energy, ascorbic acid, organic acids and total phenolics than UM-RTF but lower TFC. In addition, gallic acid, ellagic acid and astragalin were the predominant free phenolics, while gallic acid and syringetin-3-O-glucoside were dominant in bound phenolic fractions. Regardless of UM-RTF or FM-RTF, stronger antioxidant activities and α-glucosidase inhibition ability were observed in free/bound phenolic fractions, while bound phenolic fractions showed stronger α-amylase inhibitory effect than free phenolics fractions. Strong correlations can be found between the phytochemical compositions and the bio-activities investigated. Furthermore, the interactions between the main phenolics in RTF and α-glucosidase/α-amylase were also analyzed by molecular docking. This study highlighted the potential value of unexploited R tomentosa fruits in two ripening stages, thus promoting its development and application in the food industry.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/antiox11071390/s1, Figure S1: The flow diagram of extraction and analysis for Rhodomyrtus tomentosa fruit (RTF) in two ripening stages. Figure S2: Heatmap analysis of correlation matrix between the major compounds and the bioactivities of RTF.

Conflicts of Interest:
The authors declare no conflict of interest.