LC-MS grade methanol, ethanol, acetonitrile, formic acid, 0.1% (w/v) poly(L-lysine) hydrobromide solution, and gelatin were obtained from Sigma-Aldrich (Shanghai, China). Analytical grade ammonium acetate, chloral hydrate, phloroglucinol, iodine, sulfuric acid, and ethanol were obtained from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). The optimum cutting temperature compound was obtained from Leica (Nussloch, Germany). Water was purified with a Milli-Q filtration system (Millipore, Bedford, MA, USA). ZSS at different developmental stages—white maturity (S1), firm ripening (S2), and full ripening (S3)-was provided by Wennuanhe, Xingtai City, Hebei Province, China.

Standard solutions of various terpenoids, flavonoids, alkaloids, fatty acids, amino acids, and other low molecular weight compounds, including jujuboside A, jujuboside B, sanjoinenine, spinosin, 6’’’-feruloylspinosin, apigenin-6-glucosyl-7-O-methyl ether (AGOME), vicenin-2, 6’’-p-coumaroylspinosin, 6’’’-sinapoylspinosin, (2S, 3S)-N-[(1S)-1-carbamoyl-3-menthyl-butyl]-2-[[(2S)-2-dimethylamino-3-phenyl-propanoyl]amino]-3-(4-formylphenoxy)-4-methyl-valeramide (CMBDPM), DL-nuciferine, amphibine D, ortho-nornuciferine, glutamic acid, proline, lysine, phenylalanine, methionine, L-isoleucine, arginine, serine, (S)-coclaurine, eleutheroside A, adenosine, and catechin, were prepared at a concentration of 1 mmol in deionized water for MALDI-TOF MSI detection. To prepare the mixed standard solution, 25 low molecular weight compounds were dissolved in deionized water at a concentration of 10 mM and diluted to the desired concentrations. One microliter of each standard solution was spotted on an AnchorChip target plate for MALDI-TOF MS detection.

ZSS tissues at different developmental stages were cryo-sectioned at −20°C into 20-μm thick slices using a Leica CM1860 cryostat (Leica Microsystems, Wetzlar, Germany). The serial tissue slices were then immediately thaw-mounted on the conductive sides of indium tin oxide-coated microscope glass slides (Bruker Daltonics, Billerica, MA, USA) (batch number 20220727A, specific resistance 6 Ω).

MALDI matrices were applied using an ImagePrep electronic matrix sprayer (Bruker Daltonics, Bremen, Germany). The detection matrix was 12 mg/mL 2-MBT in methanol: water (4:1, v/v) containing 0.2% trifluoroacetic acid. The matrix spraying was conducted according to the instrument manufacturer’s method with some modifications (Ma et al., 2024; Du et al., 2020). During the matrix spraying, the incubation time was 30 s, the wetness was 40%, and other parameters were set to default. Sprayed tissue sections were prepared for MALDI-MSI analysis, and an Epson Perfection V550 Photo Scanner (Epson (China) Co., Ltd., Beijing, China) was used to capture optical images of the tissue sections.

The detection matrix was 12 mg/mL 2-MBT in methanol: water (4:1, v/v) containing 0.2% trifluoroacetic acid. The matrix spraying was conducted according to the instrument manufacturer’s method with some modifications (Ma et al., 2024; Du et al., 2020). During the matrix spraying, the incubation time was 30 s, the wetness was 40%, and other parameters were set to default. Sprayed tissue sections were prepared for all profiling and imaging experiments were performed using an Autoflex Speed MALDI-TOF/TOF-MS (Bruker Daltonics, Billerica, MA, USA). The MALDI source was equipped with a 2,000 Hz solid-state Smartbeam Nd: YAG UV laser (355 nm, Azura Laser AG, Bremen, Germany). Mass spectra were acquired over a mass range of m/z 100 to 1,500 in positive-ion mode with broadband detection, obtained from 40 scans at 500 laser shots each. For imaging data acquisition, 100 μm laser raster step sizes were used to detect endogenous low molecular weight compounds in the ZSS tissue sections, with each scan (pixel) accumulated from 500 laser shots.

Mass calibration employed a standard mixture for external calibration, comprising peptides with defined m/z values: bradykinin 1–7 ([M+H]⁺, m/z=757.40), angiotensin II ([M+H]⁺, m/z=1046.54), angiotensin I ([M+H]⁺, m/z=1296.69), substance P ([M+H]⁺, m/z=1347.74), and bombesin ([M+H]⁺, m/z=1619.82). Internal calibration referenced the matrix ion 2-MBT ([M+H]⁺, m/z=167.99) and a peptide standard ([M+H]⁺, m/z=1349.69), with the intensity of the peptide standard normalizing the spectral data. Calibration was conducted in cubic-enhanced mode to ensure the accuracy of mass measurements. The cubic-enhanced mode was chosen for both external and internal mass calibration processing. The data were based on three biological replicates, and each biological replicate corresponded to three technical replicates.

Using Bruker’s FlexImaging 4.1 software (https://www.bruker.com/zh/services/software-downloads.html), a correction pen was used to mark the “teaching points” (typically three points) around a tissue section for correct UV laser positioning for spectral acquisition. The MALDI-TOF mass spectra were processed with total ion current normalization, and the signal intensity of each imaging data point was represented as normalized intensity. MALDI-MSI analysis, and an Epson Perfection V550 Photo Scanner (Epson (China) Co., Ltd., Beijing, China) was used to capture optical images of the tissue sections.

After data collection using MALDI-TOF-MSI, the spectral files were processed using FlexAnalysis 3.4 software (Bruker Daltonics GmbH, Bremen, Germany) on cryo-sectioned tissue. In each sample’s measured area, regions such as cotyledon, radicle, and endosperm were defined according to the botanical structure of ZSS. Regions of interest containing features like the cotyledon, radicle, and endosperm were identified. Average spectra from these regions were used to set peak integration ranges. External and internal calibrations were conducted during acquisition to correct for instrumental issues. Post-export, m/z values were aligned across replicates to match compounds across samples. The aligned data were integrated into a CSV file. SPSS26.0 software (https://www.ibm.com/cn-zh/spss) was used for data analysis, and the statistical results were expressed as x̄ ± s. In order to evaluate the significant differences between groups, Duncan’s new multiple range test (DNMRT) was used to compare the mean values of different groups on the basis of the data conforming to normal distribution and homogeneity of variance. First, arrange all the averages in descending order. The maximum average is then marked as a and compared with the adjacent average. If the difference is not significant, the adjacent mean is also marked as a; If significant, the next mean is labeled b. This process is repeated until all averages are marked. Finally, the mean of the same markers indicated no significant difference, while the mean of different markers indicated significant difference, and the significance level was set at P < 0.05. Use GraphPadPrism9 software (https://www.graphpad.com/scientific-software/prism/www.graphpad.com/scientific-software/prism/) to draw the column chart. Prior to multivariate analysis, the data were normalized using mean-centering and unit variance scaling. PCA and OPLS-DA were then applied using SIMCA 14.1 (https://www.sartorius.com/en/products/process-analytical-technology/data-analytics-software/mvda-software/simca) to differentiate samples with various quality parameters and identify potential quality-associated markers to differentiate samples and identify quality markers (Hou et al., 2022).

The morphological analysis of Ziziphus jujube Mill. var. spinosa fruit at different developmental stages revealed distinct characteristics ( Figure 1 ). At the white maturity (S1) stage, the fruit surface was green, with smaller flesh and a hard texture. At the firm ripening (S2) stage, the fruit was larger, mostly green with some red, and had a firmer texture. At the full ripening (S3) stage, the fruit surface was completely red, and the flesh was soft and fragrant. The extrinsic feature of the ZSS seed is oblate and oval with a raised vertical line in the middle. The seed surface at S1 is green and comparatively shriveled, at S2 is yellow to yellow-brown, and at S3 is relatively plump and shiny with a purplish-red or purplish-brown hue. To further understand the internal structure of ZSS, this research freeze-dried and sliced ZSS in different periods ( Figure 1 ). The results of sectioning showed that the tissues of ZSS were endosperm, radicle, and cotyledon from outside to inside.

The integrated metabolite profiles of ZSS at different growth stages were generated using MALDI-MSI ( Supplementary Figure 1 ). Positive ions were mainly detected in the m/z 100-1200 range. By screening ions based on ZSS standards, literature reviews, and other databases, 25 components, including flavones, saponins, alkaloids, and amino acids, were selected for MALDI-MSI analysis. These compounds, representative of four different classes of bioactive components (terpenoids, flavonoids, alkaloids, and amino acids), are detailed in Table 1 .

MALDI imaging revealed the spatial distribution of terpenoids such as jujuboside A (m/z=1245.566, [M+K]⁺), jujuboside B (m/z=1045.558, [M+H]⁺), and sanjoinenine (m/z=490.270, [M+H]⁺) in ZSS. Jujuboside A was mainly concentrated in the radicle, while jujuboside B and sanjoinenine were primarily found in the cotyledons and endosperm ( Figure 2 ).

The results indicated that jujuboside A content increased by 63.36% in S3 compared to S1, and by 22.34% in S2 compared to S1. This suggests that jujuboside A accumulation increases with plant growth. Jujuboside B content was 462.55% higher in S3 and 76.81% lower in S2 than in S1. Sanjoinenine content decreased by 33.37% in S2 compared to S1, but increased by 12.16% in S3 compared to S2 ( Table 1 ). These results indicate that jujuboside B and sanjoinenine accumulation first decreases and then increases with growth.

MALDI imaging also mapped the spatial distribution of flavonoids such as spinosin (m/z=647.137, [M+K]⁺), 6’’’-feruloylspinosin (m/z=785.228, [M+H]⁺), AGOME (m/z=469.111, [M+Na]⁺), vicenin-2 (m/z=617.147, [M+Na]⁺), 6’’-p-coumaroylspinosin (m/z=791.216, [M+Na]⁺), 6’’’-sinapoylspinosin (m/z=799.244, [M+H]⁺), and CMBDPM (m/z=539.323, [M+H]⁺). Spinosin was mainly concentrated in the radicle and endosperm, with content gradually increasing. 6’’’-feruloylspinosin was mainly found in the cotyledons. AGOME was distributed in the cotyledons and endosperm. Vicenin-2 was located primarily in the radicle and endosperm, while 6’’-p-coumaroylspinosin and 6’’’-sinapoylspinosin were mainly in the endosperm, and CMBDPM was mainly in the radicle ( Figure 3 ).

Comparing S3 to S1, spinosin increased by 324.61%, 6’’’-feruloylspinosin by 267.66%, and AGOME by 42.15%. In S2, spinosin increased by 156.50%, 6’’’-feruloylspinosin by 110.85%, and AGOME by 15.06% compared to S1. This indicates that spinosin, 6’’’-feruloylspinosin, and AGOME gradually accumulate over time during plant growth. S3 also showed a 36.09% increase in vicenin-2, a 27.46% increase in 6’’-p-coumaroylspinosin, and a 60.63% increase in 6’’’-sinapoylspinosin compared to S1. In S2, vicenin-2 was 8.04% lower, 6’’-p-coumaroylspinosin was 6.44% lower, and 6’’’-sinapoylspinosin was 60.63% lower compared to S1. These results suggest that the accumulation of vicenin-2, 6’’-p-coumaroylspinosin, and 6’’’-sinapoylspinosin first increases over time. CMBDPM content in S3 was 16.54% lower than in S1, while in S2, it increased by 5.96%, indicating that CMBDPM increases during growth and development.

MALDI imaging of ZSS identified the spatial distribution of several alkaloids, including dl-nuciferine (m/z=296.164, [M+H]⁺), amphibine D (m/z=632.381, [M+H]⁺), ortho-nornuciferine (m/z=304.131, [M+Na]⁺), and (S)-coclaurine (m/z=324.099, [M+K]⁺). dl-nuciferine was primarily concentrated in the radicle, amphibine D in the endosperm, and ortho-nornuciferine in both the endosperm and cotyledon. Initially, ortho-nornuciferine was widely distributed in the endosperm and cotyledons at S1. However, its content gradually increased in the endosperm and decreased in the cotyledons at S2 and S3. (S)-coclaurine is mainly distributed in the cotyledons, and gradually decreases over time, which was very low when present at S3 ( Figure 4 ).

Specifically, S3 showed an 18.24% increase in dl-nuciferine, a 49.81% decrease in amphibine D, and a 36.74% decrease in ortho-nornuciferine compared to S1. In S2, dl-nuciferine increased by 15.71%, amphibine D by 63.73%, and ortho-nornuciferine by 8.96% compared to S1 ( Table 1 ). These results indicate that dl-nuciferine content increases in the organism, with the fastest growth occurring between S1 and S2. Amphibine D content decreases initially and then increases, while Ortho-nornuciferine content gradually decreases, with the fastest decrease occurring between S2 and S3. S3 showed 65.43% reduce in (S)-coclaurine compared to S1. In S2, (S) -coclaurine was reduced by 59.00% compared to S1.

MALDI imaging also revealed the spatial distribution of four essential amino-acids: lysine (m/z=185.069, [M+K]⁺), phenylalanine (m/z=166.086, [M+H]⁺), methionine (m/z=172.040, [M+Na]⁺), and L-isoleucine (m/z=170.058, [M+K]⁺) ( Figure 5 ). Methionine was mainly distributed in cotyledons and radicles, with content first decreasing and then increasing. Lysine and phenylalanine were Mainly distributed in the cotyledons, with content gradually increasing. L-isoleucine is distributed throughout the ZSS, especially high in the endosperm ( Figure 5 ).

Compared to S1, S3 showed reductions of 31.42% in methionine, and 25.45% in L-isoleucine. S2 showed reductions of 23.35% in methionine, and 3.61% in L-isoleucine, compared to S1. These findings indicate that the content of methionine and L-isoleucine generally decreases over time. Conversely, compared to S1, S3 showed increases of 514.13% in lysine, and 582.77% in phenylalanine. S2 showed increases of 296.92% in lysine, and 192.94% in phenylalanine compared to S1. This indicates that both of these amino acids increase during plant growth ( Table 1 ).

MALDI imaging also revealed the spatial distribution of seven non-essential amino acids: glutamic acid (m/z=186.016, [M+K]⁺), proline (m/z=138.052, [M+Na]⁺), arginine (m/z=175.119, [M+H]⁺), serine (m/z=106.050, [M+H]⁺), eleutheroside A (m/z=577.446, [M+H]⁺), adenosine (m/z=306.060, [M+K]⁺), and catechin (m/z=291.086, [M+H]⁺) ( Figure 6 ). Glutamic acid was mainly distributed in the cotyledons, with content increasing over time. Proline was distributed in cotyledons and radicles, with content first decreasing and then increasing. Arginine was the highest throughout the seed and in cotyledons at S1 initially, but gradually decreases, becoming concentrated in the radicle. Serine was mainly distributed in the endosperm, with content first decreasing and then increasing. (S)-coclaurine was mainly distributed in the cotyledons, with content decreasing over time, and becoming very low by S3. Eleutheroside A, adenosine, and catechin were mainly distributed in the cotyledons and endosperm, with content first decreasing and then increasing.

Compared to S1, S3 showed reductions of 45.64% in proline, 98.72% in arginine, 33.23% in serine, and 65.43% in (S)-coclaurine. S2 showed reductions of 45.30% in proline, 96.56% in arginine, 17.96% in serine, and 59.00% in (S)-coclaurine compared to S1. These findings indicate that the content of these compounds generally decreases over time. Conversely, compared to S1, S3 showed increases of 732.92% in glutamic acid. S2 showed increases of 192.94% in phenylalanine compared to S1. This indicates that glutamic acid increase during plant growth. S3 also showed increases of 15.97% in eleutheroside A, 70.43% in adenosine, and 14.80% in catechin compared to S1. S2 showed increases of 18.34% in eleutheroside A, 74.53% in adenosine, and 43.05% in catechin compared to S1 ( Table 1 ). Thus, the content of eleutheroside A increases first and then decreases, while adenosine and catechin decrease first and then increase.

To harness the capabilities of mass spectrometry imaging for detailed analysis of ZSS quality, ZSS samples representing different growth phases and characterized by quality attributes were examined using MALDI-MSI. Despite commonalities in the spatial distribution of bioactive ingredients across samples, differences in relative signal intensity were observed in smaller tissue regions. The OPLS-DA technique was employed to differentiate ZSS samples from different growth periods and identify key components contributing to these distinctions.

The OPLS-DA models, constructed using MSI data from the cross-sectional analysis of the full spectral range (m/z 100 to 1,200), elucidated the variations in components during different growth phases. Both Principal Component Analysis (PCA) and OPLS-DA identified disparities in the compounds as ZSS progressed through growth stages ( Figures 7A, B ). The independent variable model parameter (R2X) and dependent variable model parameter (R2Y) were both greater than 0.5, indicating good model stability and predictive ability. The permutation test plot showed that the model did not overfit and had good predictive ability ( Figure 7C ).

The Very Important Projection (VIP) values for 6’’’-sinapoylspinosin, catechin, vicenin-2, 6’’-p-coumaroylspinosin, jujuboside B, amphibine D, adenosine, sanjoinenine, and proline were all greater than 1 ( Figure 7D ). Therefore, these components could be used as potential quality-associated markers to distinguish ZSS growth at different periods.

This research presents an in-depth analysis of the relative peak areas of nine bioactive constituents with Variable Importance in Projection (VIP) values exceeding unity, scrutinizing their concentration fluctuations throughout the ripening process of ZSS ( Figure 8 ). The bar chart data starkly delineate the trend, illustrating an initial decline followed by an ascent in the content of these bioactive components.

In the 2020 edition of the Chinese Pharmacopoeia, jujuboside A and spinosin are stipulated as the quality marker compounds for the acid ZSS. This research, through meticulous peak area analysis, uncovers a consistent increase in the levels of these two constituents throughout the entire growth phase of ZSS.