Comparative metabolomics reveals differences in primary and secondary metabolites between “Shixia” and “Chuliang” longan (Dimocarpus longan Lour.) pulp

Abstract Longan was a characteristic fruit for both medicine and food in China, which was rich in primary and secondary metabolites. Comprehensive high‐throughput identification and comparison of metabolites in longan pulp among different varieties were still lacked. “Shixia” (SX) and “Chuliang” (CL) were the biggest major cultivars of longan in China. In this study, the content of total soluble solid, total flavonoid, and total phenolics indicated the difference of sweetness and bioactive compound content between the SX and CL pulp. Through a widely targeted metabolome, a total of 514 metabolites were identified and categorized into 23 groups mainly including flavonoids, amino acids & derivatives, lipids, phenolic acids, nucleotides & derivatives, alkaloids, organic acids and sugars & derivatives. A total of 89 metabolites with significantly differential accumulation (variable importance in projection (VIP) value ≧1, p‐value <.05) over 1.2 fold were found between SX and CL, which were mainly enriched into pathways including flavone and flavonol biosynthesis, glycolysis/gluconeogenesis, and arginine and proline metabolism. Higher leveled hexose and hexose‐phosphate (i.e., β‐D‐glucose, D(+)‐glucose, glucose‐1‐phosphate and glucose‐6‐phosphate), dominant organic acids (i.e., citric acid, succinic acid, D‐malic acid, and citramalate), and essential amino acids (L‐threonine, L‐valine, L‐isoleucine, L‐leucine, L‐phenylalanine and L‐lysine) in SX pulp might be contributed to the taste and flavor difference between SX and CL. Moreover, the greatly differential accumulated secondary metabolites especially flavonoids and phenolic acids might result in different medicinal and nutritional characteristic between SX and CL. In conclusion, this study provided a systemic metabolic basis for understanding the nutritional differences between SX and CL and would help deepen the molecular biology and pharmacology research on characteristic metabolites in longan pulp.


| INTRODUC TI ON
Longan (Dimocarpus longan Lour.), which was originated in China and belongs to the Longan genus of Sapindaceae, is a famous tropical and subtropical fruit (Wu et al., 2007). As an edible fruit and traditional Chinese medicine, longan has been consumed for thousands of years (Zhang et al., 2020). Due to the high leveled primary metabolites including sugars (Chen et al., 2015;Shuai et al., 2016), organic acids (Hu et al., 2006) and amino acids (Dai et al., 2010) as well as abundant secondary metabolites containing polyphenols, flavonoids, alkaloids (Tang et al., 2019), polysaccharides (Yang et al., 2009), vitamins, nucleotides (or nucleosides) (Xiao et al., 2007), tannins, proanthocyanidins and other bioactive compounds (Sheng and Wang, 2010), the longan pulp has been used as a traditional Chinese medicine for a long history to promote blood metabolism, soothe nerves, relieve insomnia, prevent amnesia, extend longevity, cure neural pain and swelling, treat palpitation and serve as anti-hyperglycemic agents in Asian countries (Li et al., 2015;Park et al., 2010;Yang et al., 2008;Yi et al., 2012;Zhang et al., 2020;Zhu et al., 2013).
In recent years, studies on metabolites in longan fruits were emerged and increased. Hu et al. (2006) compared the fruit qualities and analyzed the sugars and organic acids by high-performance liquid chromatography (HPLC) in longan fruits of 12 cultivars and found the significant difference of fruit qualities, soluble solid content and related indexes among cultivars. Chen et al. (2015) analyzed the soluble sugars in the mature fruits of 63 longan germplasms and found that the main sugars including sucrose, fructose, and glucose varied among cultivars. Luo et al. (2018) found that total soluble solid (TSS) content, sugar components, and sucrose/hexose ratio in the longan pulp were different among cultivars. The difference in soluble acid invertase activities might result in the varied hexose/ sucrose ratio in longan fruits among cultivars. Dai et al. (2010) determined the content of amino acids in the pulp of 18 longan varieties using HPLC and found the significant differences in amino acid content among varieties. The content of glutamic acid showed the greatest difference among longan varieties (Dai et al., 2010). Zhang et al. (2018) found that the content of phenolic acids and flavonoids as well as antioxidant capacities in the longan pulp varied among 24 cultivars, and in further suggested that the content of free and combined phenolic acids showed significantly positive correlation with the antioxidant activities . However, the abovementioned literatures were mostly concentrated on analyzing a certain class of metabolites or small scale of metabolites and their physicochemical properties in longan fruits (Sheng and Wang, 2010;Zhang et al., 2020). High-throughput identification and comparison of the metabolic profiles of longan pulp among different varieties were still lacked.
In the last two decades, the metabolomics based on liquid chromatography-mass spectrometry (LC-MS) has been widely applied in metabolome analysis on many fruit crops such as citrus (Wang et al., 2016), fig , tomato (Zhu et al., 2018), strawberry (Fragaria × ananassa) (Paolo et al., 2018), apple (Xu et al., 2020), and litchi (Guo et al., 2019). In this study, the pulp of "Shixia" and "Chuliang" longan were used as materials, and a variety of primary and secondary compounds such as sugar, organic acids, amino acids, alcohols, flavonoids, phenolic acids, nucleotides, anthocyanins, and alkaloids were determined by ultra-high performance liquid chromatography-mass spectrometry (UPLC/MS/MS). We screened out obviously different metabolites accumulated in the maturation process by OPLS-DA and then annotated them using KEGG and enrichment analysis. The results revealed the difference of metabolites in the flesh of the two cultivars, which provided a theoretical basis for the evaluation of their quality and nutritional differences, and were helpful to promote the breeding of new cultivars of high quality.

| Longan fruits
The "Shixia" (SX) and "Chuliang" (CL) longan fruits with commercial maturity were harvested from the same orchard of Institute of Fruit Tree Research in Guangdong Academy of Agricultural Sciences on August 2, 2018, and August 7, 2018, respectively. The harvested fruits were immediately transported to the laboratory. More than 200 "Shixia" and "Chuliang" longan fruits with no disease, no damage and uniform size were selected for sampling. After the removal of seeds, the pulp of fruit was immediately frozen in liquid nitrogen and stored at −80°C until be used.

| Determinations of the content of total soluble solid, total phenolic and flavonoid
The pulp was separated and used for juicing and determination of the total soluble solid (TSS) content using a Brix refractometer (PAL-1, ATAGO Co., Ltd.), and the assay was subjected to three repeats.
The ethanolic extract used for determination of total phenolic content and total flavonoid content were prepared according to our previously reported method . In brief, 3 g powder of sample, ground by liquid nitrogen, was added into 3 ml 80% ethanol and extracted under ultrasonication for 30 min (with an ice bath for cooling). After a centrifugation at 5000 g for 5 min, the supernatant was transferred into a 10 ml volumetric flask. The residue was then extracted twice with 3 ml 80% ethanol as described above.
The combined ethanolic extract in volumetric flask was adjusted to 10 ml using 80% ethanol and stored at −20°C for determining total phenolic content and total flavonoid content.
The total phenolic content (TPC) was determined by the Folin-Ciocalteu method (Bonilla et al., 2003) and the steps according to our previously reported method . The total phenolic content was calculated according to the standard curve and expressed as mg gallic acid equivalent (GAE)/g fresh weight (FW).
The assay was subjected to three repeats.
The total flavonoid content (TFC) was measured using a modified colorimetric method (Jia et al., 1999;Liu et al., 2008) and the steps according to our previously reported method . The absorbance of the mixture at 510 nm was measured immediately in comparison with a standard curve prepared by rutin. The flavonoid content was expressed as mg rutin equivalent (RE)/g FW.

| Sample extraction
The pulp was frozen by liquid nitrogen and crushed using a mixer mill (MM 400; Retsch) with a zirconia bead for 1.5 min at 30 Hz. Sample powder (100 mg) was added to 1.0 ml 70% aqueous methanol and extracted overnight at 4°C. For increasing the extraction efficiency, each sample was vortexed for three times during the period. After a centrifugation at 10,000 g 10 min, the supernatant was collected, fil-

| ESI-Q TRAP-MS/MS
Widely targeted metabolites were analyzed by LIT and triple quadrupole (QQQ) scans using a triple quadrupole-linear ion trap mass spectrometer (Applied Biosystems 6500 QTRAP) . The MS/MS system was equipped with an ESI Turbo Ion-Spray interface and controlled by Analyst 1.6.3 software (AB Sciex). The parameters for operating ESI source were set as follows: ion source, turbo spray; source temperature 500°C; ion spray voltage (IS) 5500 V; ion source gas I (GSI), gas II (GSII), curtain gas (CUR) were set at 55, 60, and 25.0 psi, respectively; high collision gas (CAD). Instrument tuning and mass calibration were performed with 10 and 100 μM polypropylene glycol solutions in QQQ and LIT modes, respectively. QQQ scans were acquired as MRM experiments with collision nitrogen gas set to 5 psi. DP and CE for individual MRM transitions was done with further DP and CE optimization. A specific set of MRM transitions were monitored for each period according to the metabolites eluted within this period.

| Identification and quantitative analysis of metabolites
After the isotope signal and the repetitive signal were removed, the metabolites were qualitative by the secondary spectral information based on the public metabolite database (e.g., MassBank, KNApSAcK...) and the self-built database MetWare database (from Metware Biotechnology Co., Ltd.).
Multiple reaction monitoring (MRM) of triple quadrupole mass spectrometry was employed to quantify each metabolite: only the precursor ions of the target substance were screened and the ionized in the collision cell to break and form fragment ions. The precursor ions and the characteristic fragment ions were selected by triple quadrupole filtration to make more accurate and repeatable quantitative results (Fraga et al., 2010). After the integration and correction of chromatographic peaks using MultiaQuant software 3.0.3 and performed on each mass spectrometry files, the relative content of the corresponding substance (area of each chromatographic peak) was calculated.
2.4.5 | Orthogonal partial least squares discriminant analysis and screening of differential accumulated metabolites The metabolites that were not detected in more than two repeats of either "Shixia" or "Chuliang" were eliminated. The rest metabolites were used for orthogonal partial least squares discriminant analysis (OPLS-DA) (Eriksson et al., 2006). OPLS-DA was performed to eliminate the factors unrelated to sample grouping, the errors between samples and other random errors and was usually used to maximize the differences between groups. Theoretically, the more reliable model was characterized by the values of R 2 Y and Q 2 Y closer to 1. A model was generally considered as reliable if the values of R2Y and Q2Y were greater than 0.5 and the difference between them was less than 0.3. The metabolites with variable importance in projection (VIP) value ≧1, p-value (SX versus CL, t-test) <.05 and |log 1.2 (SX/CL)|≧1 were identified as differential accumulated metabolites (DAMs).

| Statistical analysis
The variance of data was analyzed using SPSS software package release 18.0 (SPSS Inc). Multiple comparisons were performed by one-way ANOVA based on Duncan's multiple range tests, while paired-samples t-tests were performed to test the statistical significance between two samples.

| The content of total soluble solid, total flavonoid and total phenolics
An noteworthy significant difference was found between the TSS content of the SX longan pulp (23.98%) and that of CL longan pulp (22.67%; Figure 1a). This result was consistent with our previous result that the components of TSS as well as sucrose, glucose, and fructose in the mature longan pulp varied among cultivars and showed a significant difference between SX and CL . The total content of flavonoid and phenolic acids, the most abundant secondary metabolites in longan fruit, was also determined and compared between SX and CL longan pulp. The total flavonoid content in mature SX and CL longan pulp was 0.106 and 0.132 mg/g FW, respectively, while the total phenolic content in mature SX and CL longan pulp was 0.844 and 0.776 mg/g FW, respectively (Figure 1b,c). It was worthy to note that the high standard deviation and insignificant difference of total flavonoid and total phenolic content between SX and CL pulp might be resulted from the poor uniformity among individual longan fruits or biological repeats of samples. However, the above results definitely indicated the differences of sweetness and bioactive compounds content between the SX and CL pulp.

| Identification, quantification, and classification of metabolites detected in mature SX and CL longan pulp
In order to make a systematic metabolic comparing between SX and CL longan pulp, a HPLC-ESI-triple quadrupole-linear ion trap (Q-TRAP)-MS analysis was used to identify and quantify the metabolites.
In total, 514 metabolites categorized into 23 groups were detected in F I G U R E 1 The content of total soluble solid (a), total flavonoid (b) and total phenolic (c). Note: ***p-value <.001, two paired t-test both of the pulp samples. Among these metabolites, 345 metabolites were annotated with cpd_id in KEEG COMPOUND database but 169 metabolites were not annotated with any cpd_id (Figure 2a).
The largest group of secondary metabolites identified in the pulp was flavonoid, containing 97 metabolites and accounting for 18.87% of the detected metabolites (Figure 2a

| Significantly differently accumulated metabolites between mature "Shixia" and "Chuliang" pulp
Based on the OPLS-DA analysis and significance test, the metabolites with variable importance in projection (VIP) value ≥1 and 1.2 fold-change (SX versus CL, t-test, p-value <.05) were identified as the significantly differently accumulated metabolites (DAMs) between SX and CL longan pulp at mature stage. Three biological repeats of SX samples were completely separated from those of CL samples by the OPLS-DA analysis (Figure 3a). The evaluation indexes of the model were R 2 Y(cum) = 0.999 and Q 2 Y(cum) = 0.994, which indicated the good reliability and predictability of the model. In total, 89 DAMs containing 33 upregulated metabolites and 56 downregulated metabolites were found between "Shixia" and "Chuliang" longan pulp, while 425 metabolites showed no significant difference. Among the upregulated metabolites, 14, 7, and 12 metabolites showed fold change (FC, SX versus CL) ≧ 2.0, 2.0 > FC ≧ 1.5, and 1.5 > FC ≧ 1.2, respectively. Among  (Table 3).

| Integrative analysis of DAMs in carbohydrate, amino acid and secondary metabolism
Through a mapping of the sugars and acids, it was found that most of the monosaccharides (such as β-D-glucose, D [+]-glucose, and fructose) and their derivatives (such as glucose-1-phosphate, α-Dglucose-1P, β-D-glucose, galactinol, D-myo-inositol, D-sorbitol) were upregulated in SX pulp. Disaccharides (such as sucrose, turanase, isomaltulose, lactose, D-[+]-trehalose, and melibiose) were detected, but were not significant different between SX and CL longan pulp (Figure 4). This result was consistent with the higher TSS and sweetness in SX when compared to CL longan (Figure 1a).

| Metabolic basis of flavor-related quality differences between SX and CL pulp
The ratio of sugar to acid performed a significant influence on fruit flavor (Li et al., 2014;Zheng et al., 2015). Previous reports indicated that the components and content of organic acids in longan pulp varied among varieties. More importantly, the content of organic acids in longan aril mainly including malic acid, oxaloacetic acid, α-ketoglutaric acid, oxalic acid, citric acid, tartaric acid, and quinic acid was far lower than that of organic acids in other fruits such as citrus and apples (Hu et al., 2006). Our results showed that the accumulation of citric acid and quinic acid in SX was higher than that in CL, which was consistent with the experimental results of (Hu et al., 2006). A more comprehensive detection of organic acids, which were composed of 35 members, were presented in this study.
Citric Acid, succinic acid, D-malic acid, and citramalate, which were important intermediate metabolites in TCA cycle, showed the highest abundance in longan pulp and higher level in SX longan pulp. In addition, our previous results indicated that the content of the main sugars (sucrose, glucose, and fructose) in longan pulp were varied among varieties . It was worthy to note that the hexose and hexose-phosphate such as β-D-glucose, D (+)-glucose, glucose-1-phosphate, and glucose-6-phosphate showed the highest abundance among the monosaccharides in longan pulp and the content of these compounds was significant higher in SX longan pulp (Table 1 and Figure 4). These results may explained the difference of flavor between SX and CL longan fruits.
Amino acids were also the important contributors to flavor of fruits (Lu et al., 2017), which were abundant in longan fruit and greatly varied among cultivars (Dai et al., 2010). In total, 70 amino acids and derivatives were detected in SX and CL longan pulp. Dai et al. (2010) found that L-glutamic acid showed the highest abundance in longan pulp. This result was consistent with our result. Dai et al. (2010) also found that all of the six essential amino acids (L-threonine, L-valine, L-isoleucine, L-leucine, L-phenylalanine, and L-lysine) together with three nonessential amino acids (L-arginine, L-glycine and L-proline) showed higher accumulation in SX longan pulp. However, our results showed that most of the essential amino acids (L-tryptophan, L-phenylalanine, L-methionine, L-leucine, Lisoleucine, and L-valine) except L-lysine showed a lower accumulation in SX pulp. Moreover, except L-tyrosine, three nonessential amino acids (L-histidine, L-aspartic acid, and L-serine) was lower accumulated in SX pulp but the other four ones (L-glutamic acid, L-citrulline, L-arginine, and L-proline) showed higher accumulation in SX pulp ( Figure 5). These contrary results of essential amino acids (L-phenylalanine, L-leucine, L-isoleucine and L-valine) between our study and previous report indicated that strict control of preharvest operations and environment were required to make credible comparison of amino acids between two cultivars. In total, the difference of sugars, organic acids, and amino acids between SX and CL together acts as the metabolic basis for the difference of flavor between these two varieties.

F I G U R E 4
Differences in the content of sugar and organic acid between "Shixia" and "Chuliang" longan pulp (p-value <.05, two paired t-test). Note: upward arrow: upregulated; downward arrow: downregulated; gray background: undetected

| Difference of secondary metabolites beneficial to human health between SX and CL pulp
Flavonoids and phenolic acids were the largest two groups of secondary metabolites in longan fruit. Recent studies have shown that polyphenols from longan seeds or flower performed benefits on human health, such as anti-oxidation (Zheng et al., 2009), lowering cholesterol (Liu et al., 2012), lowering blood glucose (Yang et al., 2010), anti-tumor Zhong et al., 2010), antibacterial and anti-inflammatory (Lee et al., 2016). Our results showed that the content of many flavonoids and phenolic acids in pulp was significantly different between SX and CL longan.
The flavonoid biosynthesis was linked with the phenylpropanoid biosynthesis pathway through the naringenin synthesized isosinensetin were also detected in SX and CL longan pulp. Moreover, the typical flavones reported in citrus such as diosmetin, diosmin, vitexin, aglycones of vitexin, isovitexin, luteolin, apigenin, chrysoeriol were also detected in SX and CL longan pulp. It was interesting to note that most of these flavones and their aglycones showed higher accumulation in SX longan pulp ( Figure 6). Therefore, although the TPC and TFC were not significant different between SX and CL longan pulp, the great difference in the abundance of each secondary metabolites especially flavonoids and phenolic acids between SX and CL might lead to a distinct varied medicinal effect.

| CON CLUS ION
"Shixia" and "Chuliang" were the biggest two mainly-planted longan cultivars in China. In this study, a systemic investigation of metabolic difference between SX and CL longan pulp was performed using an LC-MS/MS based widely targeted metabolomics. Totally, 514 primary and secondary metabolites belonging to 23 categories were identified in the pulp of them, among which flavonoids were the most abundant, followed by amino acids, lipids and phenolic acids.
A total of 89 metabolites with significant differential accumulation over 1.2 fold between "Shixia" and "Chuliang" were mainly enriched

F I G U R E 6
Differentially accumulated secondary metabolites between SX and CL longan pulp (p-value <.05, two paired t-test). Note: upward arrow: upregulated; downward arrow: downregulated; gray background: undetected