This study identified the phytochemical and antioxidant activity of mulberry fruit at the seven different stages of ripening. The sample used for the study came from a single source, a single area and a single season. The different ripening stages with different colors, phytochemical compounds and biological activities will be discussed in the following sections.
3.1. Changes in Color, Total Phenolic Content, Total Flavonoid Content and Vitamin C of Mulberry Fruit during Ripening
The color of the skin of the fruit was identifying during the seven stages of ripening. The color of each stage was measurement with a color meter, as shown in
Table 2. The brightness is indicated by the
L* value when compared to positive and negative values; the redness, greenness and positive and negative values were identified by the
a* values, while the yellowness and blueness were determined by the
b* values. The M0-M1 had the highest
L* value (brightness), while the negative value of the
a* value showed a green color. On the other hand, in the next step of the ripening middle stage, the red color increased, as shown in M2–M3. It shows a positive
a* value from −6.3 to 13.3, while the
L* and
b* values decreased. The last of the ripening stages were M4-M7, which showed violet and black colors. The color meter showed a decrease in the
L*,
a* and
b* values, as shown by the decreased bright red and yellow colors. In contrast, the M4-M7 stages showed positive
L*,
a* and
b* values, as shown by the purple to dark purple color, which supports a previous study reporting that mulberry fruit that had a purple color were sources of anthocyanin [
7]. The total color difference (Δ
E) is a combination of parameters
L*,
a* and
b* values. This result indicates that the extent of color change during ripening of M4 and M5 was not significantly different (
p < 0.05) with seven stages of fruit ripening. However, other stages of mulberry ripening were different compared with the initial stage (M0). This result can be explained by the ripening of the mulberry fruit as the color changes from green to purple, which is often connected with the formation of anthocyanins, suggesting a modification of pigment density in the surface tissues and the breakdown of chlorophylls and carotenoids [
5,
25,
26,
27].
The total phenolic content (TPC) of mulberry ripening over the seven stages is shown in
Table 2. The TPC was found to be in a range of 23.2–64.7 mgGAE/gDW, with M7 having the highest TPC, followed by M6, M5, M4 and M0 having the lowest. These results could be explained by M7 having high phenolic acid from the formation of anthocyanins in the last ripening stage. In the first ripening stage (M0–M2), anthocyanins were absent; instead, there was a higher vitamin C level. This was similar to a previous study reporting that TPC increased from unripened to fully-ripened stages [
12,
28]. For the TFC of mulberry with different ripening stages, it was found to be highest in M7 (4.75 mgRE/gDW), follow by M6 (4.27 mgRE/gDW), M5 (4.21 mgRE/gDW) and M4 (4.13 mgRE/gDW), while M0 (2.95 mgRE/gDW) had the lowest TFC (
Table 2). In this regard, the value of M7 was higher than those reported for mulberry fruits for Pakistan mulberry cultivars (
Morus macroura: 2.49 mg CE/g) [
28], Korean mulberry in five cultivars (0.06 to 0.65 mg CE/g) [
29] and China mulberry in 13 cultivars of black mulberry fruit (
M. nigra) (1.21 RE/g to 2.86 mg RE/gDW ) [
14]. Conversely, Pakistan mulberry cultivars (
Morus nigra: 10.2 mg CE/g) [
28] and Turkey mulberry (7.0 mg CE/gDW) [
30] were reported as other countries of mulberry fruits with higher results than in the present study. In this regard, we can admit that the difference in TPC and TFC in mulberry fruit could be related to the ecological condition during harvest, genotype, growth condition, genetic diversity and maturity stage of the fruit [
1,
31,
32,
33].
The most important vitamins for human nutrition are commonly considered to be found in fruits and vegetables, particularly vitamin C [
34]. The vitamin C content of fruits varies according to variety, species and stage of development [
35]. The results are shown in
Table 2. It was found that the amount of vitamin C was significantly different (
p < 0.05) in mulberry fruit within the seven stages of ripening, ranging from 1.6 to 12.3 mg/gDW (
Table 2). The highest vitamin C values were found in the M2 ripening stage, which were 12.3 mg/gDW, followed by M1 (8.8 mg/gDW) and M3 (3.2 mg/gDW). The previously reported vitamin C content of Chinese mulberry fruit was 2.5 mg/g DW [
36], and that of the Egyptian black mulberry was 1.3 mg/gFW [
37]. These contents were lower than those found in the present study, at 4.9 and 9.4-fold, respectively. Vitamin C functions as an antioxidant and a normal daily consumption of 250–500 mg is recommended for antioxidant activity and removal of free radicals [
38].
3.3. Phenolic Acid and Flavonoid Compounds by HPLC
Individual phenolic acid and flavonoid analyses were performed by HPLC. The results show that the content of total phenolic acids was significantly different (
p < 0.05) at different ripening stages, as shown in
Table 3. The total phenolic acid content increased from M0 to M6, ranging from 903 to 3599 µg/g DW, and decreased slightly at M7 (3358 µg/g DW). Some individual phenolic acids increased, particularly vanillic acid, which was found in mulberry fruit during its ripening stages. The initial amount of vanillic acid was increased by 93% to the highest content in M6. Additionally, protocatechuic acid, caffeic acid, syringic acid and cinnamic acid increased with ripeness, as shown by 6%, 28%, 10% and 6%, respectively. These increased from the lowest content (M0) of individual phenolic acid found in the ripening stages. In contrast, gallic acid, p-hydroxybenzoic acid, chlorogenic acid, p-coumaric acid and sinapic acid decreased with the ripening stages, while ferulic acid showed moderate change. These results are similar to those previously reported; phenolic acids were significantly increased from young to mature stages in ivorian gnangnan (
Solanum indicum L.) berries [
28] and pumpkins (
Cucurbita moschata Duchesne) [
43].
The flavonoid compounds (rutin, catechin, quercetin, apigenin, myricetin and kaempferol) in mulberry fruit within the different ripening stages are shown in
Table 3. The results show that the total flavonoid compounds were found in the range of 1994–9566 µg/g DW for M0 and M4. The predominant flavonoid was catechin and it had the highest content in all ripening stages, while the content of kaempferol was lower than the other samples found in the M0 (22 µg/g DW) ripening stage. Catechin, which belongs to the flavonoid compound group, tended to initially increase to its highest in M4 and then decrease to M7. Similar data were reported by Xin et al. [
44] that catechin was the major compound in mulberry fruit of all maturity levels in
M. alba ‘Zhenzhubai’ and
M. alba ‘Da10′, and that the active expression of anthocyanidin reductase or leucoanthocyanidin reductase genes in mulberry fruit during maturation caused their contents to increase and decrease [
44]. Individual flavonoid compounds found in plants, including mulberry fruit, have been shown to have anti-atherosclerotic, anti-inflammatory, anti-tumor, anti-thrombogenic, anti-osteoporotic, anti-bacterial, anti-viral, anti-fungal, anti-oxidant, anti-platelet, anti-thrombotic action and anti-allergic properties [
45,
46].
The PCA score plot of phenolic acid content and ripening stages was separated into different blocks and responsible for 54.86% and 44.26% of the total variance of PCA1 and PCA2, respectively (
Figure 3a). Furthermore, the ripening stages were variable vectors (seven ripening stages: M0–M7) placed at a slight distance from one another, indicating a strong correlation between them. In addition, M0, M1 and M2 were correlated with the contents of gallic acid, p-hydroxybenzoic acid and chlorogenic acid, while M3, M4, M5, M6 and M7 were correlated with the content of vanillic acid and caffeic acid. This indicates that the content of individual phenolic acids relates to the formation of phenolic acids at different stages, which is similar to a previous study reporting that the first stage of ripening is the initial synthesis of phytochemical compounds and the next stage of ripening changes chemical compounds in mulberry fruits during maturation [
47].
The PCA score plot successfully separated the samples for flavonoids content into different blocks, with the first two components (PC1 and PC2) accounting for 99.15% and 0.72% of the total variance, respectively (
Figure 3b). Ripening content and flavonoid compounds were investigated. The results show that apigenin, quercetin and myricetin were the predominant compounds found in all samples, as shown on the left half of the plot, while rutin and catechin were separate groups on the right half and upper half, respectively. Additionally, the ripening of M0 to M7 is displayed on the right half of the plot and all mulberry ripening correlated with catechin content as shown by the highest content of catechin in all ripening stages is shown in
Table 3. According to the results, the initial ripening was found to have a low concentration of flavonoids due to an increase in flavonoid formation in fruits during maturation [
48].
3.4. Amino Acid Contents by LC/MS/MS
Amino acids are abundant in some fruits. During the ripening stages of fruit, the amino acid levels increase due to the ripening inducing dramatic changes in the protein and metabolite complex network [
49]. Similar to our result, the total amino acids found were in the range 2401–4569 µg/g DW (
Table 4). The ripening stages M6 and M5 were shown to have the highest amounts with no significant differences (
p < 0.05). Meanwhile, M0 had lower than total amino acid content at about half that of M6 and M5. The predominant amino acid in the ripening stages of mulberry fruit was asparagine. In all seven ripening stages, the amount was greater than the other amino acid types. Our study indicates that the amino acid contents in mulberry fruit are influenced by the different ripening stages. When comparing the amount of amino acids with M0, five groups were identified as follows: proline was increased in group 1; arginine, aspartic acid, glutamine, glutamic acid, histidine, lysine, methionine, phenylalanine, serine, threonine and tryptophan increased to the last stage and were moderately decreased in group 2; tyrosine, isoleucine and leucine were slightly changed in group 4; and cysteine was not detected in sample in group 5. The change in the amino acid contents was similar to that previously reported. Boggio et al. [
50] and Sorrequieta et al. [
51] found that the free amino acid contents in tomato pericarp increased markedly during the ripening stage due to metabolizing enzymes that occurred during the ripening process. However, the amount of chemical compounds, including amino acids, is influenced by harvesting conditions, genotype, growth conditions, genetic diversity and fruit maturity stage [
1,
31,
32,
33]. Therefore, further studies on this aspect are needed for other plants.
The PCA analysis is shown on a score plot of amino acid content and ripening stages. The result was the separation of the display responsible for 88.75% (PCA1) and 11.04% (PCA2) into the different blocks. The results show that Phe, Gln, Lys, Tyr and Asn were abundant in all ripening mulberry fruits and correlate with ripening due to being located at the right half of the block. Furthermore, because of the variable factors displayed at a slight distance from one another, all ripening (M0–M7) was in correlation between them, as shown in
Figure 4. These findings explain that the stage of ripening causes the different phytochemicals in each stage, while some individual amino acids, such as Tyr and Asn, can be found in all ripening stages, as shown in
Figure 4, and have a high content in all ripening (
Table 4).
3.5. Change in γ-Aminobutyric and Anthocyanin in Different Ripening Stages of Mullberry Fruit
γ-aminobutyric acid, or GABA, is a non-protein amino acid that is found in animals, bacteria and plants [
52]. Several previous studies have found GABA in plants like tea, tomato, tobacco and mulberry [
52]. In this study, GABA was found in all stages of maturation with a significantly different (
p < 0.05) range from 94–273 g/gDW in M0 to M7 (
Table 5). The trend of GABA content increased when the ripeness level increased. The GABA content of the M7 sample was higher than that of the initial ripening stage (M0) by 12.8 times. Previous studies found that the highest amount of GABA was found in the green stage relative to the amino acid content in tomato [
53,
54]. However, the content of GABA that occurred was carbolized in the mitochondrial matrix called the GABA shunt, which happened rapidly during the ripening stages [
54,
55]. Additionally, the GABA level was determined by several factors, such as species, variety, environmental conditions, stress during cultivation and even post-harvest treatments, growth development and ripening stage [
52,
56]. With respect to GABA, it has been reported as a bioactvie compound with health benefits, including antioxidant, neuroprotective, neurological disorder prevention, anti-hypertensive, anti-diabetic, anti-cancer, anti-inflammatory, antimicrobial, anti-allergic, hepatoprotective and renoprotective [
57].
Anthocyanin is widely found in natural material colorants, as shown by a purple color, and has antioxidant, anti-cancer, anti-diabetic, anti-inflammatory and antibacterial properties [
58]. Anthocyanin compounds are useful in food as a natural food colorant, and are also used as purple coloring in cosmetics and medicinal plants. Anthocyanin in mulberry fruit has been reported and the major individual anthocyanin was cyanidin-3-O-glucoside [
1]. In this study, it was reported that cyaninin-3-O-glucoside (C3G) was found in all maturity stages of mulberry fruit, while peonidin-3-glucoside (P3G) was also present (
Table 5). Additionally, the C3G content was greater than P3G in all samples, confirming previous findings that C3G is abundant in mulberry fruit [
59]. The amount of C3G was significantly different (
p < 0.05), with a range from 2–1902 mg/100 gDW in M0 and M6, and dramatically decreased in M7 (1671 mg/100 gDW). The initial amount of C3G in mulberry fruit rapidly increased during the ripening stage from M0 to M6 due to the anthocyanin biosynthesis pathway, which was possibly induced at a position relatively far downstream from the late stage reactions catalyzed by enzymes, including dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS) or anthocyanidin 3-O-glucosyltransferase in the latter stages of development (UFGT) [
9]. C3G is obvious in fruit as a red hue. In mulberry fruit, M2 had a red color on the fruit skin, which indicates that the C3G content was moderately increased and then decreased (M7), similar to the peonidin-3-O glucoside content.
3.6. Antioxidant Activity
The results of the antioxidant activity with mulberry fruit during all seven ripening stages are shown in
Figure 5. The objective of the antioxidant study was to evaluate the amount of DPPH radicals that were scavenged using the Trolox equivalents on centration (TE) and the FRAP valuation (mg FeSO
4/g DW). The chemical DPPH is a free radical. The color of the DPPH has been used for a long time to evaluate the capacity to scavenge free radicals. When antioxidants interact with the DPPH radical, which is detected using a microplate reader, they cause a color shift from purple to yellow, measured by absorbance at 517 nm. The result shows that the radical scavenging activity of DPPH was found to be highest in the last ripening stage (M7) at 19.8 mgTE/gDW and lowest in M3 (7.98 mg TE/gDW) (
Figure 5a). These results indicate that the values of DPPH radical-scavenging activity of mulberry fruit during the seven ripening stages are increased compared with the initial stage (M0) due to the synthesis of some phytochemicals, especially anthocyanin, when leveled up. As reported by previous authors, the high capacity of anthocyanin can scavenge the radical of DPPH [
1,
58]. The green ripening stage (M0-M3) had higher vitamin C content compared with other ripening stages. These compounds, also found in other fruit, perform as natural antioxidants. Moreover, these reports of anti-oxidant activity were assessed using the FRAP method to determine how effectively they reduced molecules that function by donating an atom from hydrogen to break the chain of the free radicals [
60], as shown in
Figure 5b. The results were similar to the DPPH activity. The highest amount of FRAP was in the last ripening stage M7 (22 mg FeSO
4/gDW) and the lowest was in M3 (7 mg FeSO
4/gDW). The antioxidant with DPPH and FRAP assay displayed a similar pattern to the total phenolic content (TPC) and total flavonoid content (TFC) in the mulberry fruit ripening stage. This result indicates that the TPC and TFC are positively correlated with antioxidants with the DPPH and FRAP assays (
Table 6), previously supported by this report [
15]. Nevertheless, the relationship between DPPH/FRAP and TPC/TFC is not always compatible due to some of the phenolic acids and flavonoid compounds having stronger reducing power than others [
4].