Widely targeted metabolic analysis revealed the changed pigmentation and bioactive compounds in the ripening Berchemia floribunda (Wall.) Brongn. fruit

Abstract Berchemia plants were important materials for Chinese traditional medicines due to their special secondary metabolites. Unlike the root, stem and leaf tissues, Berchemia floribunda (Wall.) Brongn. fruit was lacked of systematic metabolic investigation. Biochemical analysis found that the total flavonoid and total phenolic content of Berchemia fruit pulp showed a peak value at red ripe stage, and then decreased, but the total anthocyanin content sharply increased along with the coloration. By widely targeted metabolomic analysis, 644 metabolites were identified and categorized into 23 groups mainly including flavonoid, organic acids, amino acids, lipids, phenylpropanoid, nucleotides, alkaloids, carbohydrates, alcohols, anthocyanins & proanthocyanidins, vitamins, terpenes, polyphenols, phenolamides, quinones, indole derivatives, and sterides. Among them, 111 metabolites and 123 metabolites respectively showed up‐ and down‐regulation from break stage to full mature. KEGG enrichment analysis indicated that active secondary metabolism such as biosynthesis of phenylpropanoids, flavonoid, and alkaloids happened during Berchemia fruit ripening. More importantly, Cyanidin‐3‐O‐galactoside and other 3 cyanidins were found to be the predominant pigments in mature Berchemia fruit and increased cyanidins and pelargonidins but decreased anthocyanins might be contributed to the purple pigmentation of Berchemia fruit. Interestingly, 29 pharmaceutical compounds previously reported in other Berchemia tissues were also detected in ripening Berchemia fruit pulp: 8 flavonoid, 2 quinones & sucrose showed up‐regulated accumulation while 6 polyphenols, 5 flavonoid, 3 phenylpropanoid, 2 organic acids, 1 quinones and β‐sitosterol showed down‐regulated accumulation In conclusion, our first comprehensive metabolic fingerprint will promote the further study of B. floribunda fruit and its medical and food application.


| INTRODUC TI ON
The root, stem, vine, leaf and whole plant of some Berchemia (Rhamnaceae) species have been used in Chinese traditional medicines (In Directory of Chinese Materia Medica, 1986;Inoshiri et al., 1987;Kang et al., 2017). These Berchemia plants were reported to relieve pain, act as expectorant, antipyretic and be used for treatment of gall stones, liver disease, rheumatic arthritis, tuberculosis (TB), acute or chronic tracheitis, jaundice, diarrhea and carbuncle (In Directory of Chinese Materia Medica, 1986;Inoshiri et al., 1987;Kang et al., 2017). The Berchemia (Rhamnaceae) comprises 32 deciduous plants worldwide which were mainly located in temperate and tropical areas in Asia. 1 Among them, 18 species and 6 varieties were distributed in south, southwest, central south and east of China (Chen & Dong, 2006). The dried root of Berchemia lineata (L.) DC). was named as Tiebaojin, Huangshanteng, Goujiaoli, Tiyuncao or Laoshucao in traditional Chinese medicine (Wei et al., 2015). Previous researches indicated that the stem, vine and root materials used for Chinese traditional medicine "Tiebaojin" were actually from more than 4 Berchemia Berchemia floribunda (Wall.) Brongn . Although the tissues used for medicine were produced from different plants of Berchemia genera and their medical chemical constituents might be distinct, the dominant metabolites in these materials were commonly flavonoids and flavonoid glycosides, phenols and phenolic glycosides , lignans, quinones and their dimer forms, and terpenes (Wei et al., 2015). At present, many pharmaceutical compounds had been separated from stem, leaves, wood, root, barks and whole plant of these Berchemia genera, but little is known about the chemical constituents of the Berchemia fruits which was used in food coloring and Tibetan medicine (Kang et al., 2017). Brongn. And Berchemia lineata (L.) DC.). In addition, polyphenols such as gallocatechin, catechin, epigallocatechin, epicatechin, protocatechuic acid and protocatechuic acid O-glucoside were found to be abundant in these Berchemia plants. Phenylpropanoid such as ferulic acid, vanillic acid, phillygenin, quinones such as emodin, chrysophanic acid, and aurantio-obtusi, organic acids (4-Hydroxybenzoic acid, syringic acid O-glucoside), and β-sitosterol were also isolated from these Berchemia plants. Although more than 30 metabolites were isolated and investigated in the root, stem, vine, leaf and whole plant of Berchemia plants, limited information is reported about the chemical constituents of Berchemia fruits.
In recent years, liquid chromatography-mass spectrometry (LC-MS)-based metabolomics has been facilitated by the construction of MS2 spectral tag (MS2T) library from the total scan ESI MS/MS data, and the development of widely targeted metabolomic method using MS/MS data gathered from authentic standards (Chen et al., 2013).
In recent years, UPLC-ESI-MS/MS based widely targeted metabolomic method has been widely applied in plant metabolite analysis in maize (Wen et al., 2014), rice Chen et al., 2013;Dong et al., 2014), tomato (Zhu et al., 2018), sweet potato , fig (Wang, Cui, et al., 2017), sesame , strawberry (Fragaria × ananassa) (Paolo et al., 2018), asparaguses (Dong et al., 2019), citrus (Wang et al., 2016Wang, Yang, et al., 2017), potato (Cho et al., 2016), buckwheat , tea (Zheng et al., 2019;Zhu et al., 2020;Wu et al., 2020), wheat (Chen et al., 2020), pepper and other plants (Ginkgo, Meng et al., 2019;Phalaenopsis amabilis, Meng et al., 2020;Qingke, Zeng et al., 2020). In the place of origin, the Berchemia fruits were usually not harvested according to their grade of maturity. The differences in metabolic components of fruits with different ripenesses had not caught enough attention and not been compared. In this study, we analyzed the secondary metabolites of Berchemia floribunda (Wall.) Brongn. fruits from break stage (start coloring) and full-mature stage by a widely targeted metabolomic method using HPLC-ESI-triple quadrupole-linear ion trap. We further screened out and annotated the significantly differently accumulated metabolites (DAM) in Berchemia fruits during the ripening process. Our comprehensive metabolic fingerprint was expected to guide the maturity grading of Berchemia floribunda (Wall.) Brongn. fruits and their further applications in food and pharmaceutical industry.

| Fruit materials
The Gou-er-cha (Berchemia floribunda (Wall.) Brongn.) fruits were harvested from the mountainside at an altitude of 2000 m located in Danba town, Ganzi Tibetan Autonomous Prefecture, Sichuan Province, China. The harvested fruits were immediately taken to the laboratory and graded according to maturity and coloring stages: break (B), red ripe (RP), and full-mature (FM) stage. After the removal K E Y W O R D S anthocyanins, Berchemia floribunda (Wall.) Brongn. fruit, flavonoid, phenylpropanoids, widely targeted metabolic analysis; bioactive compounds of seeds, the pulp of fruit was immediately frozen in liquid nitrogen and stored at −80°C until be used.

| Determinations of total phenolics, flavonoid, and anthocyanin contents
The ethanolic extract used for determination of total phenolics and flavonoid contents were prepared as follows: the frozen sample was ground into powder in liquid nitrogen; 0.1 g powder was added into 3 ml 80% ethanol in 10 ml tuber and then extracted under a ultrasonication for 30 min (with a ice bath to cool); after a centrifugation at 5,000 g for 5 min, the supernatant was transfered 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. The ethanolic extract was stored at amber colored air-tight containers at 4°C.
The total phenolic content (TPC) was determined by the Folin-Ciocalteu method (Pastrana-Bonilla et al., 2003). 0.25 ml ethanolic extract (or standard solution of gallic acid) was added into 5.75 ml deionized water in a 25 ml amber volumetric flask, then mixed with 0.5 ml Folin-Ciocalteau reagent. 2 min later, 1.5 ml 20% Na 2 CO 3 was added and fully mixed. The solution was adjusted to 25 ml using 80% ethanol and kept under dark for 30 min. The optical density of the blue-colored samples was measured at 760 nm. The total phenolic contents were calculated according to the standard curve and expressed as mg gallic acid equivalent (GAE)/g fresh weight. The assay was subjected to three repeats.
The total flavonoid contents were measured using a modified colorimetric method (Jia et al., 1999;Liu et al., 2008). The ethanolic extract solution was diluted by three folds. Then, 1 ml diluted ethanolic extract was added to a test tube containing 4 ml of 80% ethanol. Sodium nitrite solution (5%, 0.5 ml) was added to the mixture and maintained for 6 min. Then, 0.5 ml of 10% aluminum nitrate was added, fully mixed and maintained for 6 min. 0.5 ml of 1 M sodium hydroxide was finally added and fully mixed maintained for 10 min.
The absorbance of the mixture at 510 nm was measured immediately in comparison to a standard curve prepared by rutin. The flavonoid contents were expressed as mg rutin equivalent (RE)/g fresh weight.
The anthocyanin contents were measured and calculated according to a colorimetric method (Fuleki & Francis, 1968).
Note: A 536nm : absorbance at 536 nm; V: constant volume before test, N: dilution times, extinction coefficient for anthocyanin was 98.2, m: sample mass.

| Sample extraction
The frozen pulp was crushed using a mixer mill (MM 400; Retsch, Germany) with a zirconia bead for 1.5 min at 30 Hz. Sample powder of 100 mg was weighted and extracted overnight at 4°C with 1.0 ml 70% aqueous methanol, vortexed for three times during the period to increase the extraction efficiency. After be centrifuged at 10,000 g for 10 min, the supernatant was collected, passed through a Carbon-GCB SPE Cartridge (250 mg, 3 ml, CNWBOND, ANPEL).

| ESI-Q TRAP-MS/MS
The Mass spectrometry was according to the previous reported method for analyzing widely targeted metabolites (Chen et al., 2013).
LIT and triple quadrupole (QQQ) scans were acquired 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, operating in a positive ion mode and controlled by Analyst 1.6.3 software (AB Sciex, Waltham, MA, USA). The ESI source operation parameters were as follows: ion source, turbo spray; source temperature 500°C; ion spray voltage (IS) 5,500 V; ion source gas I (GSI), gas II (GSII), curtain gas (CUR) were set at 55, 60, and 25.0 psi, respectively; the collision gas (CAD) was high.
Instrument tuning and mass calibration were performed with 10 and 100 μM polypropylene glycol solutions in QQQ and LIT modes,

| Qualitative and quantitative analysis of metabolites
After removal of the isotope signal and the repetitive signal, 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.).
The metabolites were quantified using multiple reaction monitoring (MRM) of triple quadrupole mass spectrometry. The ions corresponding to other molecular weight substances were excluded, and the precursor ions of the target substance were screened.
Meanwhile, in the collision cell, the precursor ions were ionized to break and form fragment ions, and the characteristic fragment ions were selected by triple quadrupole filtration. This makes the quantitative results more accurate and repeatable (Fraga et al., 2010). The

| 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.

| Determination of total flavonoid, total phenolic and total anthocyanin contents
The Berchemia fruits showed a yellowish-pink pigmentation at break stage and turned to be red at red ripe stage. It was very interesting to note that the fruit color further turned to be purple black at the full-mature stage (Figure 1). In order to investigate the level of bioactive compounds, we determined the total phenolic, total flavonoid, and total anthocyanin content in pulp of Berchemia fruits at break, red ripe and full-mature stage. As shown in Figure

| The enriched pathways of up-regulated and down-regulated DAMs
The number and percentage of up-regulated and down-regulated DAMs in identified compounds were analyzed (

| The changes of pigment compounds related to coloration of Berchemia fruit
Few large-scale investigation of the pigments in the Berchemia fruit was reported. According to a previous results of TLC, the main pigments in Berchemia fruit were deduced to be pelargonidin 5-glucoside and pelargonidin 3-glucoside-5-rutinoside (Zhou, 2000). The yellowish-pink Berchemia fruits was turned to be red at red ripe stage and then be purple at full mature. In this experiment, sixteen anthocyanins (including 5 cyanidins, 4 peonidins, 3 pelargonidin, 1 petunidin, 1 rosinidin and 1 delphinidin) and 3 proanthocyanidins (including procyanidin A2, procyanidin B2 and procyanidin B3) were detected in Berchemia fruits ( Table 2). The anthocyanin with highest abundance (10 8 -10 9 ) was cyanidin 3-O-galactoside, which was increased by 3.17 folds from B to FM stage. The abundance of cya-

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflicts of interest.  (Bekker et al., 1996), 14

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.