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. 2026 Feb 7;34:103641. doi: 10.1016/j.fochx.2026.103641

Analysis of the dynamic metabolic changes during the ripening of wolfberry (Lycium barbarum L.) fruits by theta-nano-extractive electrospray ionization mass spectrometry

Ying Liu a,b, Linlin Yu c, Shuxiang Yan a,b, Xiaowei Fang b, Tenggao Zhu b,d, Huanwen Chen a,b, Dong Wu a,b,⁎
PMCID: PMC12914441  PMID: 41717382

Abstract

Metabolic changes during the ripening of wolfberry (Lycium barbarum L.) fruits reflect their quality and pharmacological properties. To decipher the metabolic evolution underlying this physiological process, a systematic analysis of wolfberry fruits at different ripening stages (7–35 days after anthesis) was conducted by theta-nano-extractive electrospray ionization mass spectrometry. Multivariate analysis effectively discriminated the various stages and identified proline, γ-aminobutyric acid, arginine, and N1-caffeoyl-N10-dihydrocaffeoyl spermidine as potential differential metabolites. Polyamine and flavonoid metabolism were significantly activated during the early ripening stages, leading to the rapid accumulation of antioxidant components such as spermidine, rutin, and chlorogenic acid. In the late ripening stages, amino acid metabolism was markedly enhanced, resulting in a substantial increase in betaine, serine, and threonine. This study elucidates the key stages and metabolic foundation of quality formation in wolfberry fruits at the metabolomic level, providing a theoretical basis for determining the optimal harvest time and regulating nutritional quality.

Keywords: Lycium barbarum, Fruit ripening, Nutritional quality, Medicinal efficacy, Metabolic profiling

Graphical abstract

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Highlights

  • •

    Rapid analysis of wolfberry metabolites achieved using novel theta-nano-EESI-MS.

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    PCA and PLS-DA effectively distinguished wolfberries at five ripening stages.

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    Early ripening stage drove polyamine/flavonoid metabolite accumulation.

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    Late ripening stage enhanced amino acid metabolite accumulation.

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    Proline, GABA, arginine, and NCNDSP were identified key differentials in wolfberry ripening.

1. Introduction

Wolfberry (Lycium barbarum L.) fruit, a member of the Solanaceae family, is renowned for its exceptional nutritional and pharmacological value. Predominantly cultivated in northwest China and the Mediterranean region, it contains an abundance of metabolic compounds, including various amino acids, phenolic acids, flavonoids, polyamines, betaine, and polysaccharides (Hu et al., 2025). These compounds underpin its diverse pharmacological effects, such as antioxidation, anti-inflammatory, hepatoprotective, and neuroprotective, leading to its widespread use in traditional Chinese medicine, functional foods, and dietary supplements (Hu et al., 2025). The quality and potency of these health-beneficial compounds are intrinsically linked to the fruit's ripening process.

The ripening process of wolfberry fruits includes five stages: expanding young fruit stage (S1), green fruit stage (S2), early yellowing stage (S3), late yellowing stage (S4), and ripening stage (S5). During this process, the types and concentrations of key metabolites undergo dynamic changes. For instance, studies have shown that the levels of fructose, glucose and sucrose gradually increase, contributing to the fruit's sweetness, while the levels of xylitol and maltose decrease (Qian and Yang, 2017). The concentration of organic acids like citric acid, quinic acid, and tartaric acid, crucial for fruit flavor, often peaks in the middle stages before declining (Zhao et al., 2015). Furthermore, the concentrations of key pharmacological components such as phenylalanine, tryptophan, and quercetin were reported to be highest during the late stages of ripening (Xie et al., 2023). However, despite extensive profiling studies, current analytical approaches remain time-consuming, which limits real-time monitoring of ripening-associated metabolites. Many established analytical techniques, while powerful for targeted compound identification, are inherently limited in their ability to provide rapid, sequential metabolic detection of a dynamic biological process.

Currently, methods such as low-temperature headspace-trap gas chromatography coupled with mass spectrometry (MS) (Chen et al., 2015), matrix-assisted laser desorption/ionization MS imaging (MALDI-MSI) (Zhao, Zhang, & Shi, 2021), ultra-performance liquid chromatography-quadrupole time-of-flight MS (UPLC-Q-TOF-MS) (Ahad et al., 2020), and nuclear magnetic resonance (Zhou et al., 2016) have been successfully employed to analyze specific compounds in wolfberry fruits, including volatile organic compounds, betaine, and spermidine. However, their typical operational workflows, which often involve chromatographic separation or multi-step procedures, can extend the analysis time per sample, and put out of achieving dynamic and real-time monitoring of biological processes. This makes it challenging to efficiently capture the rapid metabolic transitions occurring during fruit ripening with high temporal resolution. Therefore, there is a clear need for analytical strategies that can deliver rapid analytical cycles and generate rich metabolic fingerprints to effectively monitor the temporal dynamics of metabolite changes during wolfberry fruit ripening.

To address this gap, we turned to ambient MS. Nano-electrospray ionization MS (nano-ESI-MS) is notable for its high sensitivity, operating with a low-pore needle, a low spray flow rate with no need for auxiliary gas, and producing small, stable spray particles. These attributes allow for the detection of nanovolume samples and rapid acquisition of information from a single sample (Qu et al., 2023), leading to its widespread application in biology, food science, and medicine. For instance, it has been used to analyze amino acids, caffeine, and catechins in Lushan Yunwu tea (Yan et al., 2024), phenylalanine, proline, and aspartate in high-sugar honeys (Yu et al., 2024), and alkaloids in Aconitum (Wei et al., 2024). While powerful, conventional nano-ESI-MS typically requires samples in a purified solution that can limit its speed for complex matrices. To address this limitation, nano-extractive ESI-MS (nano-EESI-MS) was developed. Nano-EESI-MS enables the direct ionization and detection of components from complex samples, offering high sensitivity, rapid analysis, and good accuracy. A notable application is the noninvasive direct sampling EESI-MS method, which utilized disposable acupuncture needles and manual spray emitters to analyze metabolites from fish epidermal mucus (Wu et al., 2024). However, a key challenge with such manual or semi-automatic EESI setups is the potential for unstable signal due to inconsistent extractant introduction, which hinders reliable and continuous monitoring. This limitation underscores the need for a more robust and automated approach. The recent innovation of the theta-nano-extractive ESI-MS (theta-nano-EESI-MS) represents a significant step forward. This technique employs a dual-channel nanocapillary to simultaneously and stably introduce the sample and extractant, replacing conventional configurations (Qu et al., 2023). This design ensures efficient ionization and, crucially, a stable spray and consistent signal, thereby overcoming the major drawback of earlier nano-EESI-MS. By providing stable operation, theta-nano-EESI-MS offers a simplified, sensitive and widely applicable platform for complex matrix analysis, making it ideally suited for the dynamic monitoring of metabolic changes during biological processes.

In this study, wolfberry fruits harvested at 7, 14, 21, 28, and 35 days after anthesis were used as experimental materials. Theta-nano-EESI-MS was employed for the first time to monitor the dynamic changes of amino acids, sugars, organic acids, flavonoids, and alkaloids in five ripening stages of wolfberry fruits. Our findings not only provide a theoretical basis for scientific cultivation and optimal harvesting, but demonstrate the powerful potential of theta-nano-EESI-MS as a robust tool for real-time metabolite analysis during dynamic biological processes like fruit ripening.

2. Materials and methods

2.1. Samples and reagents

The experimental material was “Ningqi No. 1” variety of wolfberry fruit (Lycium barbarum L.), harvested from trees located in Changlin Village, Hongshui Town, Jingtai County, Baiyin City, Gansu Province. For each of the five ripening stages, (7, 14, 21, 28, and 35 days after anthesis, representing the S1 − S5, respectively), disease-free, pest-free, and undamaged fruits were randomly collected from multiple, different trees in the same orchard. All samples were immediately frozen in liquid nitrogen and stored at −80 °C.

Methanol (gradient grade for liquid chromatography, contains ≤100%) and glucose were purchased from Aladdin Biochemical Technology Co., Ltd., (Shanghai, China). Glycine, alanine, γ-aminobutyric acid, serine, proline, threonine, N-methyl-L-proline, aspartate, asparagine, lysine, glutamate, methionine, phenylalanine, arginine, tyrosine, tryptophan, valine, L-homoserine, glutamine, p-coumaric acid, malic acid, gallic acid, citric acid, myricetin, 1, 4-butanediamine, spermine, spermidine, and rutin were purchased from Jiangxi Apsdale Biotechnology Co., Ltd., (Nanchang, China).

2.2. Soluble sugars, polysaccharides, total phenolics, and total flavonoids in wolfberry fruits

Samples of wolfberry fruits at different ripening stages were randomly collected to measure growth, soluble sugars, total polysaccharides, total phenolics, and total flavonoids content. The horizontal and longitudinal diameters were measured with a vernier caliper (E04–150, Asone Co., Ltd., Japan), and the fresh and dry weights were determined with an electronic analytical balance (ME104, Mettler Toledo Instruments Shanghai Co., Ltd., Shanghai, China). For dry weight measurement, the fruits were dried at 80 °C for 48 h in an electric thermostatic drying oven (Boxun Co., Ltd., Shanghai, China) until constant weight was achieved.

Additionally, a 0.10 g fresh sample of wolfberry fruits was ground by an automatic freezing and grinder instrument (JXFSTPRP-CL, Jingxin Co., Ltd., Shanghai, China) at 50 Hz for 2 min. Then, 2 mL of 70% methanol was added, the mixture was vortex-mixed vigorously for 30 s to ensure homogenized, then extracted by a high-power ultrasonic cleaner (KQ-800KDE, Kunshan Ultrasonic Instrument Co., Ltd., Jiangsu, China) at 50 °C for 30 min, then centrifuged at 10,000 rpm for 10 min at 4 °C by high-speed centrifuge (SCI-12, Shanghai Kehuai Instrument Co., Ltd., Shanghai, China). The filtered residue was repeated once, and the supernatants were combined to a constant volume of 5 mL. The solution was used to determine soluble sugars, total polysaccharides, total phenolics, and total flavonoids in the wolfberry fruits. The content of soluble sugars was measured by anthrone colorimetric method (Wu et al., 2022). The different concentrations of glucose solution were used as standards (x, μg/mL), and their absorbance values (y) were measured at 620 nm, then obtained the regression curve. The soluble sugars content was calculated by the curve in wolfberry fruits. The total polysaccharides content was measured by the phenol‑sulfuric acid method (Yue et al., 2022). Standard solutions of varying glucose concentrations (x, μg/mL) and their absorbance values (y) were measured at 490 nm, then obtained the regression curve, the total polysaccharides content in wolfberry fruits was calculated by the curve. The total phenolics content was determined by the Folin-Ciocalteu method (Ainsworth & Gillespie, 2007). Solutions of varying concentrations of gallic acid were employed as standard solutions (x, μg/mL), and the absorbance values (y) were measured at 760 nm to obtained the regression curve. Then the total phenolics content in wolfberry fruits was calculated by the curve. The content of total flavonoids was determined by NaNO2-Al(NO3)3-NaOH colorimetric method (Wang et al., 2022). Standard solutions of varying concentrations of rutin (x, μg/mL) were used, and the absorbance values (y) were measured at 510 nm. From this, the regression curve was constructed. Then the total flavonoids content in wolfberry fruits was calculated by the curve.

2.3. Sample preparation of wolfberry fruits

Following the methods described by Yang et al. (2024) and Do et al. (2024), fresh wolfberry fruit samples were ground for 2 min. Then 0.10 g sample were accurately weighed and transferred to a 2 mL centrifuge tube with 1 mL of extraction solvent (methanol: water = 7:3, v/v), vortexed to mix, and ultrasonically extracted at 50 °C for 30 min. The mixture was then centrifuged at 10,000 rpm at 4 °C for 10 min. The filtered residue was repeated once, and the supernatant from the two extractions was combined and the final volume was fixed to 2 mL, then filtered through a 0.22 μm nylon filter (Waters Associates, Milford, MA). This entire process, from fruit harvesting to extract preparation, was independently repeated three times (n = 3 biological replicates). For each biological replicate, multiple technical replicate extracts were prepared to ensure robust and comprehensive data acquisition during MS analysis.

2.4. Theta-nano-EESI-MS analysis

The theta-nanocapillaries (Gaoss Union Technology Co., Ltd., Wuhan, China) were fabricated from a glass capillary with an inner diameter of 0.78 mm and an outer diameter of 1.00 mm using a horizontal program-controlled microelectrode puller (P-1000 Micropipette Puller, USA). And the theta-nanocapillary was imaged under the LEICA DMi8 inverted biomicroscope (Leica Microsystems Trading Co., Ltd., Shanghai, China), showing a tip inner diameter of 0.5 μm (Fig. S1). The experimental scheme for theta-nano-EESI-MS was shown in Fig. 1, a long platinum electrode was inserted into one end of the theta-nanocapillary filled with 70% methanol, while the other end contained wolfberry fruit extract. A specific high voltage was applied to the opposite end of the platinum electrode, and a 10 Gohm resistor was connected. Electrical resistance has been shown to enhance Nano-ESI-MS stability (Wu et al., 2022). Under the electric field, the nanocapillary tip generates charged droplets carrying the analyte for MS detection. The distance between the sample outlet and the MS inlet was set to 5 mm. The electrospray voltage (0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, and 1.2 kV) and extraction (methanol: water = 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%, v/v) were optimized. The ion source temperature, the ion source voltage, and the ion transfer tube voltage were automatically adjusted by LTQ-MS software system, taking the signal intensity of base peak (m/z 796) as the optimization indicator. The compounds were analyzed by their product ions (m/z) obtained by tandem MS (MS2) from collision-induced dissociation (CID). The isolation width was 1–1.5 Da, and the normalized collision energy was 10–40%. The nano-EESI was developed in our laboratory, and the LTQ-XL linear ion trap MS was equipped with Xcalibur data processing system (Thermo Scientific, Waltham, USA).

Fig. 1.

Fig. 1

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The experimental scheme of theta-nano-EESI-MS for high-throughput, sensitive, and rapid analysis of wolfberry fruits. (A) The experimental scheme of theta-nano-EESI-MS. (B) Schematic diagram of the remote end of theta-nanocapillary. Note: HV represents high voltage.

2.5. Statistical analysis

MS data were acquired from the technical replicate extracts derived from the three independent biological replicates (n = 3). The raw mass data were pre-processed with Microsoft Excel (Office 2016, Microsoft, USA), and values were presented as means ± standard deviation (SD). One-way ANOVA and Tukey's test were performed by SPSS software (version 19.0, IBM Corp., Armonk, NY, USA) to determine the significant differences (p < 0.05). Principal component analysis (PCA) of raw mass data was performed in Matlab R2016a software (version 7.0, Mathworks, Inc., Natick, USA). Partial least squares discriminant analysis (PLS-DA) and variable importance in the projection (VIP) were performed by Simca software (version 14.0, Umetrics, Malmo, Sweden). Heatmap of metabolites was performed by TBtools software (version 2.09, South China Agricultural University, China). The volcano map (the Fold Change threshold was set at Log2 (FC) ≥ 1, and the p-value threshold was set at p < 0.05) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway was analyzed by MetaboAnalyst 6.0 (https://www.metaboanalyst.ca/).

3. Results and discussion

3.1. Growth and bioactive component accumulation during ripening in wolfberry fruits

Wolfberry fruits are ellipsoidal shape, as shown in Fig. 2A. As the ripen, their color changes from green (S1, S2), to light yellow-green (S3), light orange (S4), and finally bright orange-red (S5). Regarding the weight of wolfberry fruits, both the dry weight and the fresh weight showed an upward trend during ripening, peaking at S5 with values of 0.19 ± 0.01 g and 1.02 ± 0.03 g per fruit, respectively. However, there was no significant difference in fruit moisture content for five ripening stages (Fig. 2B). Additionally, the horizontal diameter and longitudinal diameter were highest in S5 (with values of 0.77 ± 0.04 cm and 1.85 ± 0.04 cm, respectively), showing an upward trend (Fig. 2C). This indicates gradual biomass accumulation of wolfberry fruits during ripening. Total polysaccharides content was calculated by the linear regression equation (y = 0.0364x + 0.0080, R2 = 0.9964), with content peaking at S5 (35.60 ± 3.86 mg/g fresh weight, FW), which was a 303.44% increase compared to S1. Studies have showed that polysaccharides content was positively correlated with the ripening (Zheng et al., 2024). For the soluble sugars content, the regression equation (y = 0.0081x + 0.0213, R2 = 0.9965) was calculated. There was no significant difference between the S1 and S2, but it was significantly accumulated at S3, and peaked at S5 (70.81 ± 6.03 mg/g FW), which represents a 197.12% increase compared to S1 (Fig. 2D). Zhao et al. (2015) reported that total sugars content in wolfberry fruits ranged from 13.03 to 76.86 mg/g FW and peaked at 20 days after anthesis, similar with our findings. Additionally, for the total phenolics, the regression equation (y = 0.6043 – 0.0087x, R2 = 0.9952) was calculated. And for the total flavonoids, the regression equation (y = 0.1189 – 0.0076x, R2 = 0.9969) was calculated. The contents of total phenolics and total flavonoids were highest in S5 (with values of 3.50 ± 0.22 mg/g FW and 2.91 ± 0.10 mg/g FW, respectively), increasing by 84.00% and 98.05% compared to S1, respectively (Fig. 2E). Fatchurrahman et al. (2022) measured the total phenolics content of fresh wolfberry fruits as 2.995 ± 0.051 g/kg FW by Folin-Ciocalteu reagent method, consistent with our findings. Furthermore, Ilić et al. (2024) assessed the total flavonoids content in dried wolfberry fruits from different origins, reporting an average of 0.94 mg/g dry weight (DW), slightly lower than our results.

Fig. 2.

Fig. 2

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Physiological characteristics of wolfberry fruits at five ripening stages. (A) Morphological characteristics of wolfberry fruits. (B) Fresh weight, dry weight, and moisture content of wolfberry fruits. (C) Horizontal diameter and longitudinal diameter. (D) The contents of soluble sugar and polysaccharide. (E) The contents of total phenolic and total flavonoid. Note: a, b, c, d, and e indicate significant differences (p < 0.05). S1, S2, S3, S4, and S5 represent the fruits harvested 7, 14, 21, 28, and 35 days after anthesis, respectively.

3.2. Theta-nano-EESI-MS analysis of wolfberry fruits at five ripening stages

Wolfberry fruit metabolites were analyzed by theta-nano-EESI-MS with m/z 796 (base peak) selected as the optimization index. The electrospray voltage (0.5–1.2 kV) was optimized at 0.8 kV, which yielded the highest signal intensity (Fig. S2A). Aqueous methanol solution was optimized to 70% (v/v) within the range of 10–100%, yielding the highest signal intensity (Fig. S2B). After the automatic optimization, the ion source temperature was set to 200 °C. In positive ion mode, the ion source voltage was set to 33 V, the ion transfer tube voltage was set to 55 V. In negative ion mode, the ion source voltage and the ion transfer tube voltage were − 35 V and − 55 V, respectively. Based on the optimized experimental parameters, the theta-nano-EESI-MS fingerprints of wolfberry fruits were directly recorded in merely 20 s per sample. In positive ion mode, major ion peaks were observed, including m/z 796 (derivative of N1-caffeoyl-N10-dihydrocaffeoyl spermidine, NCNDSP), m/z 156 (betaine + K), m/z 175 (arginine), m/z 118 (betaine), and m/z 219 (monosaccharide + K) (Fig. 3 A). And the signal intensities of m/z 219, m/z 175, and m/z 796 gradually increased during the process from S1 to S5, while the signal intensities of m/z 472 and m/z 634 were gradually decreased. Additionally, m/z 104 (γ-aminobutyric acid) was detected in both S4 and S5 (Fig. S3). In negative ion mode, m/z 191 (citric acid) was the base peak. Major ion peaks included m/z 133 (malic acid), m/z 353 (chlorogenic acid), m/z 609 (rutin), and m/z 317 (myricetin), as shown in Fig. 3B. The signal intensities of m/z 133 and m/z 609 were the highest in the S1, and gradually decreased during the process from S1 to S5 (Fig. S4).

Fig. 3.

Fig. 3

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MS analysis of wolfberry fruit samples. (A) Theta-nano-EESI-MS fingerprint of S3 in positive ion mode; (B) Theta-nano-EESI-MS fingerprint of S1 in negative ion mode; (C) Theta-nano-EESI-MS2 fingerprint of the precursor ions at m/z 958; (D) Theta-nano-EESI-MS2 fingerprint of the precursor ions at m/z 796; (E) Theta-nano-EESI-MS2 fingerprint of the precursor ions at m/z 634; (F) Theta-nano-EESI-MS2 fingerprint of the precursor ions at m/z 472; (G) Proposed fragmentation pathway for precursor ions: (1) Structural formula of the precursor ion; (2) Structural formula of the m/z 310 fragment; (3) Structural formula of the m/z 220 fragment; (H) Heatmap analysis of intensities of peaks at m/z 958, m/z 796, m/z 634, and m/z 472 collected by theta-nano-EESI-MS in positive ion mode from wolfberry fruits at five ripening stages. Note: S1, S2, S3, S4, and S5 represent the fruits harvested 7, 14, 21, 28, and 35 days after anthesis, respectively.

The data for MS2 was acquired by CID. By integrating standards, databases, and literature, 50 compounds in wolfberry fruits were identified, including 19 amino acids, 12 organic acids, 7 flavonoids, 3 alkaloids, 3 polyamines, 1 phenylpropanoid, 1 vitamin, and 4 additional compounds (Table S1). In positive ion mode, the protonated ion at m/z 89 was confirmed to be 1, 4-butanediamine (C4H12N2), which generated a major fragment at m/z 72 in MS2 by the loss of NH3. This fragmentation behavior of MS2 was identical for the 1, 4-butanediamine standard compound (Fig. S5A). Then the m/z 118 generated major fragments of m/z 59 and m/z 58, by the loss of C2H3O2 and C3H10N, respectively. This MS2 fragmentation behavior was identical to that of the betaine (C5H11NO2) compound (Fig. S5B). Also, m/z 175 (arginine) generated major fragments of m/z 158, m/z 157, m/z 130, and m/z 116, by the loss of NH3, H2O, COOH, and CH5N3, respectively (Fig. S5C). The m/z 319 (myricetin) generated a major fragment of m/z 257, by the loss of C2H6O2 (Fig. S5D). Additionally, m/z 472 was identified as NCNDSP, with hydrogen substituting R1, R2, R3, and R4 in its structure showing in Fig. 3G(1) (Yang et al., 2024). As shown in Fig. 3F, the sequential loss of an OH group, one caffeoyl group, and one dihydrocaffeoyl group from m/z 472 (NCNDSP) resulted in the formation of ion fragment peaks at m/z 455, m/z 310 (N10-dihydrocaffeoyl spermidine, Fig. 3G(2)), and m/z 220 (N1-caffeoyl spermidine, Fig. 3G(3)), respectively. The structure of NCNDSP contained multiple hydroxyl groups, which were potential sites for glycosylation. The peaks at m/z 958 (Fig. 3C), m/z 796 (Fig. 3D), and m/z 634 (Fig. 3E) corresponded to NCNDSP with three, two, and one hydroxyl glycosylation (m/z 162) groups, respectively. Additionally, m/z 382 in Fig. 3C−E corresponded to the R3 or R4 glycosylation in the fragment ion m/z 220 (Yang et al., 2024). Therefore, the m/z 634, m/z 796, and m/z 958 were inferred to be dicaffeoylspermidine derivatives (DCSPDs) (Zhou et al., 2016). To understanded the metabolism of DCSPDs in wolfberry fruits, heatmap analysis was performed using the characteristic ion signal intensities of m/z 958, m/z 796, m/z 634, and m/z 472. The level of m/z 472 was highest in S1, but decreased gradually as the wolfberry fruits ripened due to increased glycosylation (Zhao et al., 2015). Similarly, the level of m/z 634 increased at S2 and then decreased gradually. The levels of m/z 796 and m/z 958 increased progressively with ripening, accumulated progressively with the ripening of wolfberry fruits, peaked at S5 (Fig. 3H). In the negative ion mode, malic acid (m/z 133) generated a major fragment of m/z 115 by the loss of H2O (Fig. S5E). Then salicylic acid (m/z 137) lost fragments of H2O and CO2, generating peaks at m/z 119 and m/z 93, respectively (Fig. S5F). And spermidine (m/z 144) lost fragments of NH4 and C2H6N, resulting in peaks at m/z 126 and m/z 100, respectively (Fig. S5G). Also, citric acid (m/z 191) generated major fragments of m/z 173 and m/z 111, by the loss of H2O and C2H6O2, respectively (Fig. S5H). Rutin (m/z 609) lost the rutinosyl moiety during deglycosylation, producing a major fragment at m/z 301, corresponding to the aglycone fraction (Fig. S5I). The chemical assignment for the other forty compounds were identified in a similar way in wolfberry fruits by theta-nano-EESI-MS2, including glycine, alanine, oxalic acid, γ-aminobutyric acid, serine, proline, threonine, taurine, N-methyl-L-proline, fluorouracil, asparagine, aspartate, trigonelline, lysine, glutamate, cinnamic acid, methionine, p-coumaric acid, phenylalanine, tyrosine, tryptophan, baicalein, quercetin, N-feruloyltramine, N-feruloyloctopamine, isoquercitrin, malonic acid, fumaric acid, valine, succinic acid, L-homoserine, glutamine, 2-hydroxycinnamic acid, gallic acid, vitamin C, spermine, chlorogenic acid, uridine diphosphate glucose, kaempferol-3-O-rutinoside, and narcissoside (Fig. S5J-AY). Protti et al. (2017) found quercetin, myricetin, rutin, and chlorogenic acid in the wolfberry fruits by LC-MS/MS. Zhao et al. (2015) found citric acid and malic acid in the wolfberry fruits by HPLC. Furthermore, Lu et al. (2021) found γ-aminobutyric acid and arginine in wolfberry fruits. These outcomes were in agreement with the findings of the current study.

3.3. Multivariate analysis and metabolic characterization of wolfberry fruits during ripening

PCA was performed to visualize the molecular differences among wolfberry fruits at five ripening stages. The results of PCA showed a clear discrimination among the five ripening stages of wolfberry fruits depending on the metabolite profiles. In positive ion mode, the eigenvalues for PC1, PC2, and PC3 of PCs (96.9% of the total variance for modeling) were 80.1%, 12.2%, and 4.6%, respectively (Fig. 4A). Simultaneously, supervised analysis of PLS-DA of five ripening stages of wolfberry fruits showed R2 and Q2 values of 0.99 and 0.98 in positive ion mode (Fig. 4B), indicating a strong interpretive and predictive capabilities. Similarly, in negative ion mode, three PCs (PC1 eigenvalue 73.0%, PC2 eigenvalue 22.1%, and PC3 eigenvalue 3.6%) representing about 98.7% of the total variance were selected for modeling (Fig. S6A). For PLS-DA, the R2 and Q2 were 0.99 and 0.98 (Fig. S6B), respectively. Previous studies have also shown differences in metabolites of wolfberry fruits under different ripening stages (Zheng et al., 2024). Meanwhile, the potential differential metabolites were obtained by VIP values analysis (Fig. 4C) and volcano plot (Fig. 4D). The results showed that there are 33 compounds with VIP values > 1 (Table S1), including proline (1.67), betaine (1.53), chlorogenic acid (1.49), phenylalanine (1.48), arginine (1.30), p-coumaric acid (1.29), citric acid (1.20), γ-aminobutyric acid (1.17), and salicylic acid (1.11). In the volcano plot analysis of S1 and S5, twenty-nine metabolites were selected, including 23 up-regulated and 6 down-regulated metabolites (Table S2). Notably up-regulated metabolites included NCNDSP, γ-aminobutyric acid, N-methyl-L-proline, chlorogenic acid, salicylic acid, spermine, spermidine, and rutin. Conversely, proline, betaine, arginine, and lysine were down-regulated, potentially related to functional regulation during ripening in wolfberry fruits (Zhao, Zhang, Liu, et al., 2021). These results showed that these compounds could serve as differential metabolites to distinguish the wolfberry fruits of different ripening stages.

Fig. 4.

Fig. 4

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Multivariate analysis of fingerprints of wolfberry fruits at five ripening stages by theta-nano-EESI-MS. (A) PCA of wolfberry fruits at five ripening stages. (B) PLS-DA of wolfberry fruits at five ripening stages. (C) Heatmap and VIP-value of compounds in wolfberry fruits at five ripening stages. (D) Volcano plot analysis of 49 compounds identified in wolfberry fruits for S1 and S5. Note: GABA represents γ-aminobutyric acid; UDPG represents uridine diphosphate glucose. Betaine and [Betaine + K] belong to the same compound. S1, S2, S3, S4, and S5 represent the fruits harvested 7, 14, 21, 28, and 35 days after anthesis, respectively. Redder colors represent a higher compound content, while bluer colors represent a lower compound content.

The heatmap showed that the levels of 49 compounds in wolfberry fruits at different ripening stages (Fig. 4C). The results showed that the total content of amino acids and flavonoids increased with ripening, while organic acids and alkaloids decreased. The polyamine content initially increased and then decreased, peaking at S2 (Fig. 4C). Betaine, the main active compound in the wolfberry fruits, was used as the standard for content determination of Lycium barbarum L. according to the Pharmacopoeia of the People's Republic of China. It showed an accumulation trend and increased during the late ripening stage (S4 − S5). As a quaternary ammonium alkaloid, betaine exhibits various pharmacological effects, including anti-fatty liver, antihypertensive, and antioxidative properties (Gao et al., 2017). Its accumulation during fruit ripening correlated with the rich pharmacological effects of wolfberry fruits. Zhao et al. (2021) also observed a gradual increase in betaine signal intensity during the ripening of wolfberry fruits by MALDI-MSI.

Amino acids are essential as nitrogen storage forms and functional molecules, serving as precursors for biosynthesis of many compounds (The et al., 2024). During the ripening of wolfberry fruits, the content of amino acids exhibited an overall upward trend, though there were variations in the content changes among different types of amino acids. Specifically, aspartic acid and glutamate, which peaked at S2, function as nitrogen transporters, facilitating the transport of nitrogen sources to young fruits and playing a role in nitrogen assimilation and protein synthesis (The et al., 2024). Additionally, the content of essential amino acids, including lysine, methionine, and phenylalanine, peaks at S5. This may be related to the flavor and nutritional quality of the wolfberry fruits (Lu et al., 2021). However, proline, a functional amino acid, played a key role in regulating osmosis (Zhao, Zhang, Liu, et al., 2021), increased at S4 to maintain the osmotic balance of wolfberry fruits, slightly decreased at S5. Similarly, γ-aminobutyric acid was rapidly synthesized at S4, which may be associated with the regulation of cell density during the ripening of wolfberry fruits (Pan et al., 2022).

Flavonoids are significant secondary metabolites in wolfberry fruits, exhibiting antioxidation, free radical scavenging, and antibacterial effects (Gao et al., 2017). Our results showed that the levels of myricetin, quercetin, and baicalein peaked at S4 and slightly decreased at S5. Rutin, kaempferol-3-O-glucoside, and narcissin were high in S1 and S2, then gradually decreased, likely due to the hydrolysis of flavonoid glycosides to aglycones (e.g., hydrolysis of rutin to quercetin). Overall, the level of flavonoids tended to accumulate during wolfberry fruit ripening. Moreover, the organic acids in wolfberry fruits are crucial compounds affecting their flavor and nutritional value (Mikulic-Petkovsek et al., 2012). Citric and malic acids, the main organic acids, increased in the early ripening stages, peaked at S3, and then progressively declined (Mikulic-Petkovsek et al., 2012). Other organic acids like oxalic, salicylic, succinic, and fumaric acids rapidly synthesized in the early ripening stages but decreased as the fruit ripening, reaching their lowest levels at S5. The swift synthesis of organic acids in the initial stages of fruit development aided in enhancing fruit firmness and preventing rot, whereas the reduction of organic acids during the ripening phase increased the sugar-acid ratio. A lower acid content and higher sugar content contribute to a sweeter taste in the fruits. Our findings that the total sugar level increase significantly during ripening, the accumulation of betaine and specific flavonoids in the late stages (S4 − S5), and the main organic acids, peaking at the middle stage (S3) and then declining, are consistent with the reported in previous, rigorous UPLC-MS-based study on wolfberry fruit ripening (Xie et al., 2023). This agreement not only confirms the reliability of theta-nano-EESI-MS but, more importantly, highlights its capability to successfully capture the full metabolic dynamics aligned with conventional methods. Thereby, theta-nano-EESI-MS provides a powerful complement to established LC-MS workflows, offering a rapid and efficient tool for metabolic analysis of dynamic biological processes such as fruit ripening.

3.4. Quantitative analysis and methodological validation

Twelve compounds in wolfberry fruits at different ripening stages were accurately quantified by theta-nano-EESI-MS, including alanine, γ-aminobutyric acid, serine, proline, aspartic acid, asparagine, lysine, glutamic acid, arginine, citric acid, malic acid, and myricetin. The linearity, limit of detection (LOD), limit of quantification (LOQ), recovery, repeatability, and precision of twelve standards were evaluated (Table S3). The correlation coefficients for the twelve compounds ranged from 0.9947 to 0.9999, indicating that the regression curves for each standard within the linear range were highly correlated, and the regression fitting was excellent under the suitability of the linear range (0.01–100 μg/mL). The LOD was low (0.02–2.29 ng/mL), and LOQ values ranged from 0.06 to 6.94 ng/mL. The recovery ranged from 84.41% to 105.97%, demonstrating that theta-nano-EESI-MS technology provided good repeatability and high accuracy. The relative standard deviation (RSD) of the signal intensity for the characteristic fragment ions generated by CID of each standard ranged from 0.48% to 4.92%, confirming the good stability of the method.

There was the content of twelve compounds in wolfberry fruits at five ripening stages (Table 1). The total amount of twelve compounds showed a gradual upward trend, indicating that the metabolites of wolfberry fruits accumulated during the ripening process. Compared to S1 (1441.84 ± 19.48 μg/g), S5 (6332.44 ± 126.21 μg/g) increased by 339.24%. Additionally, amino acids increased gradually during the ripening process. Specifically, the level of γ-aminobutyric acid was highest, reaching the peaking at S4 (1779.16 ± 9.08 μg/g) compared to S1 (318.61 ± 1.64 μg/g), increased by 458.41%. Moreover, the level of aspartic acid was relatively low, peaked at S2 (72.96 ± 0.85 μg/g), then decreased at S5 (28.97 ± 0.39 μg/g). Furthermore, the contents of proline and asparagine were both peaked at S4 with 1349.73 ± 9.55 and 1492.37 ± 68.93 μg/g, respectively. Lu et al. (2021) found the contents of proline (566.5 ± 157.6 mg/100 g) and asparagine (329.5 ± 111.5 mg/100 g) in wolfberry fruits were high by UHPLC-MS/MS. The concentrations of citric acid (1276.89 ± 16.86 μg/g) and malic acid (589.68 ± 7.75 μg/g) were peaked in S3, then gradually declined to their lowest levels in S5 at 583.90 ± 5.42 and 280.74 ± 4.01 μg/g, respectively. Donno et al. (2015) showed that fresh wolfberry fruits contain 254.09 mg/100 g FW of citric acid and 601.43 mg/100 g FW of malic acid by HPLC. Furthermore, Pires et al. (2018) found that dried wolfberry fruits contain 1.29 g of citric acid per 100 g of DW. Additionally, the content of myricetin was lowest at S1 (2.21 ± 0.12 μg/g). As the fruit ripening progressed, it significantly increased by 869.82% at S4 (194.44 ± 0.93 μg/g).

Table 1.

Contents (μg/g) of twelve compounds in wolfberry fruits at five ripening stages by theta-nano-EESI-MS.

Compound S1 S2 S3 S4 S5
Alanine 61.23 ± 0.25e 257.65 ± 8.14d 283.82 ± 9.13c 320.11 ± 5.27b 793.65 ± 10.23a
γ-Aminobutyric acid 318.61 ± 1.64d 570.51 ± 14.60c 770.33 ± 17.19c 1779.16 ± 9.08a 1372.83 ± 13.87b
Serine 27.64 ± 1.00e 72.13 ± 1.61d 111.26 ± 19.89c 160.97 ± 6.07b 345.31 ± 6.81a
Proline 9.14 ± 0.76e 213.38 ± 3.80d 395.60 ± 3.80c 1349.73 ± 9.55a 771.43 ± 13.69b
Aspartic acid 42.45 ± 0.75b 76.95 ± 4.30a 72.96 ± 0.85a 31.73 ± 0.18c 28.97 ± 0.39c
Asparagine 69.51 ± 3.73d 554.73 ± 5.31c 1257.70 ± 31.63b 1492.37 ± 68.93a 1392.29 ± 60.66a
Lysine 83.29 ± 1.55c 76.45 ± 8.22c 80.55 ± 7.96c 184.98 ± 2.44b 203.55 ± 2.69a
Glutamate 98.84 ± 2.24c 215.20 ± 3.05a 176.41 ± 1.12b 169.09 ± 3.35b 60.05 ± 0.73d
Arginine 108.42 ± 0.59e 124.62 ± 0.33d 236.54 ± 1.64c 371.26 ± 4.12a 355.99 ± 5.90b
Myricetin 2.21 ± 0.12e 102.75 ± 1.58d 122.04 ± 2.63c 194.44 ± 0.93a 143.73 ± 1.81b
Malic acid 166.29 ± 3.48e 184.82 ± 7.94d 589.68 ± 7.75a 370.05 ± 4.13b 280.74 ± 4.01c
Citric acid 454.21 ± 3.37e 472.58 ± 6.81d 1276.89 ± 16.86a 850.35 ± 7.60b 583.90 ± 5.42c
Total 1441.84 ± 19.48d 2921.77 ± 65.69c 5373.78 ± 120.45a 7274.24 ± 121.65b 6332.44 ± 126.21e
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Note: S1, S2, S3, S4, and S5 represent the fruits harvested 7, 14, 21, 28, and 35 days after anthesis, respectively. Values are means ± standard deviation (SD). Different lowercase letters a, b, c, d, and e in the same row indicate significant differences based on p < 0.05.

3.5. Metabolic network and pathway enrichment analysis during wolfberry fruit ripening

The metabolites of wolfberry fruits were analyzed using KEGG database (Fig. S7A), and 13 pathways were significantly enriched (Fig. S7B), including alanine, aspartate, glutamate metabolism, arginine biosynthesis, and the tricarboxylic acid (TCA) cycle, with profound implications for fruit quality and bioactivity. Furthermore, primary and secondary metabolic pathways were mapped in five ripening stages (Fig. 5). The marked activation of alanine, aspartate, and glutamate metabolism, with a significance level of −log10 (p-value) = 8.08, along with the TCA cycle, during early maturation stages establishes the metabolic foundation for energy production and biosynthesis. Alanine was enzymatically converted to pyruvate by alanine aminotransferase, subsequently entering the TCA cycle. Aspartate was metabolized into oxaloacetate via aspartate transaminase, which could either be utilized in the TCA cycle or further transformed into asparagine. Glutamate underwent transamination or deamination to produce α-ketoglutarate (α-KG), which contributed to the energy supply of the TCA cycle. Alternatively, glutamate can be decarboxylated by glutamate decarboxylase to form γ-aminobutyric acid or combined with ammonia by glutamine synthetase to produce glutamine. Aspartate and glutamate, as nitrogen sources and protein precursors, were significantly accumulated in S2, collectively coordinating nitrogen metabolism (The et al., 2024), enhancing oxidative energy supply of the TCA cycle, and promoting the biosynthesis of biomolecules during the ripening of wolfberry fruits. These pathways generate essential intermediates, including pyruvate, oxaloacetate, and α-KG, that not only fuel energy metabolism but serve as precursors for synthesizing lipids, proteins, and nucleic acids in organisms (Cai et al., 2023; Cappel et al., 2019). The dynamic regulation of these pathways directly corresponds to the development of nutritional and pharmacological properties in ripening fruits. Besides, the coordinated upregulation of arginine and proline metabolism, coupled with betaine accumulation in late ripening stages (S4 − S5), enhances osmoprotection and oxidative stress resistance, crucial mechanisms for maintaining cellular integrity and bioactive compound preservation in mature fruits (Liu et al., 2024). Notably, the significant increase in glycine, serine, and threonine levels during late ripening stages (S4 − S5) reflects enhanced betaine metabolism, contributing to the fruits' osmoprotective capacity and nutritional value. Concurrently, glyoxylate and dicarboxylate metabolism (−log10 (p-value) = 5.05) directly converts acetyl-CoA into oxaloacetate, bypassing the decarboxylation step of the TCA cycle and ensures efficient utilization of carbon sources (Chew et al., 2019).

Fig. 5.

Fig. 5

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Heatmap of the synthesis pathways for related compounds, including amino acids, polyamine and flavonoids, at five ripening stages of wolfberry fruits. Note: The compound where the dotted line is located was not identified in this experiment. S1, S2, S3, S4, and S5 represent the fruits harvested 7, 14, 21, 28, and 35 days after anthesis, respectively. Redder colors represent a higher compound content, while bluer colors represent a lower compound content.

Furthermore, isocitrate lyase cleaved isocitric acid into succinic acid and glyoxylate (Qi et al., 2021), while malic acid synthase catalyzed the reaction of glyoxylate with acetyl-CoA to form malic acid. Malic acid then entered the TCA cycle, replenishing its intermediates (Kunze & Hartig, 2013). Additionally, malic acid, fumaric acid, and oxaloacetate were all intermediates of the TCA cycle (Zhang et al., 2023), and were involved in glyoxylate and dicarboxylate metabolism. Citric acid, malic acid, and succinic acid were the primary dicarboxylic acids found in wolfberry fruits (Mikulic-Petkovsek et al., 2012). The levels of these dicarboxylic acids significantly increased during stages S2 or S3, enhancing glyoxylate and dicarboxylate metabolism and promoting the TCA cycle. This ensured an adequate energy supply and carbon recycling during the wolfberry fruit ripening.

The progressive enhancement of polyamine biosynthesis, particularly the conversion of arginine to 1, 4-butanediamine, spermidine and spermine via the ornithine pathway, directly correlates with the development of health-promoting properties. Specifically, arginase catalyzed the conversion of arginine to ornithine, which was subsequently transformed into 1, 4-butanediamine by ornithine decarboxylase. And 1, 4-butanediamine was then converted to spermidine, and spermine was ultimately synthesized by spermine synthetase (Zhao et al., 2024). The arginine content significantly increased in S3 and peaked in S5, thereby enhancing the polyamine metabolism in wolfberry fruits. These nitrogenous compounds, which accumulate significantly as ripening progresses, are well-established contributors to the antioxidant, anti-inflammatory, anti-cancer, and anti-angiogenic activities characteristic of mature wolfberry fruits (Long et al., 2024). As the fruits ripened, the content of amino acids increased, with some redirected to secondary metabolites to bolster downstream metabolic pathways. For instance, the phenylpropanoid metabolism (−log10 (p-value) = 2.10) activation during late maturation drives substantial flavonoid production, including quercetin, myricetin, and rutin (Zhao et al., 2024). The increased phenylalanine availability at S4 − S5 stages provides critical precursors for synthesizing these polyphenolic compounds, which are principal determinants of the fruits' antioxidant capacity and associated health benefits. This comprehensive metabolic programming throughout ripening orchestrates the synchronized development of wolfberry fruits' characteristic sweetness, nutritional density, and pharmacological potential, positioning the fruit as a valuable source of dietary antioxidants and health-promoting compounds.

4. Conclusion

In this study, for the first time, the metabolic changes in wolfberry fruits at 7, 14, 21, 28, and 35 days after anthesis were systematically analyzed using theta-nano-EESI-MS. Our findings demonstrated that early ripening stages were characterized by enhanced alanine, aspartate, and glutamate metabolism, together with an activated TCA cycle, leading to substantial accumulation of aspartate, glutamate, citric acid, malic acid, and succinic acid. These shifts established the biosynthetic foundation for energy supply and organic acid balance. In contrast, the late ripening stages exhibited intensification of phenylalanine metabolism and glycine, serine, and threonine metabolism, driving the rapid accumulation of health-related metabolites such as betaine, glycine, serine, threonine, quercetin, myricetin, and baicalein. These temporal metabolic patterns illuminated the physiological basis for the development of wolfberry fruit's characteristic taste, nutritional value, and bioactive potential. Looking forward, the rapid analysis speed and low sample consumption demonstrated in this study shows promising potential for adaptation to portable MS systems. Theta-nano-EESI-MS could enable real-time, in-field quality assessment (e.g., determination of optimal harvest time, and preliminary screening of bioactive compounds) directly in the field or at processing facilities. Significantly, theta-nano-EESI-MS expands the practical applications of this approach in fruit quality control and phytochemical research.

CRediT authorship contribution statement

Ying Liu: Writing – original draft, Methodology, Investigation, Data curation, Conceptualization. Linlin Yu: Writing – review & editing, Formal analysis. Shuxiang Yan: Writing – review & editing, Methodology, Funding acquisition. Xiaowei Fang: Writing – review & editing, Methodology. Tenggao Zhu: Writing – review & editing, Methodology. Huanwen Chen: Writing – review & editing, Methodology, Funding acquisition. Dong Wu: Writing – review & editing, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was financially supported by The National Natural Science Foundation of China (22504047, 22304065), Science and Technology Plan Project of Jiangxi Provincial Administration of Traditional Chinese Medicine (2025022501), The Key Project at Central Government Level: The Ability Establishment of Sustainable Use for Valuable Chinese Medicine Resources (2060302), and Doctoral Fund of Jiangxi University of Chinese Medicine (2024BSZR058).

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.fochx.2026.103641.

Appendix A. Supplementary data

Supplementary material

mmc1.docx (2.2MB, docx)

Data availability

Data will be made available on request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary material

mmc1.docx (2.2MB, docx)

Data Availability Statement

Data will be made available on request.

Articles from Food Chemistry: X are provided here courtesy of Elsevier

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