Comprehensive quality evaluation of different types of Gardeniae Fructus (Zhizi) and Shuizhizi based on LC-MS/MS

Gardeniae Fructus (Zhizi) serves as both a medicinal and edible substance and finds widespread use in various industries. There are often two kinds of medicinal materials in the market: Zhizi and Shuizhizi. Typically, Zhizi with small, round fruit is used for medicinal purposes, while Shuizhizi, characterized by large, elongated fruit, is employed for dyeing. Market surveys have revealed a diverse range of Zhizi types, and modern research indicates that Shuizhizi contains rich chemical components and pharmacological activities. In this study, we collected 25 batches of Zhizi and Shuizhizi samples, categorizing them based on appearance into obovate and round fruits, with seven length grades (A–G). Using the ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QQQ-MS/MS) method, we simultaneously quantified 13 main chemical components in fruits of Gardenia species. In addition, we compared the weight percentage of the pericarp, flesh, and seeds parts of samples with different traits, and quantified 13 chemical components in different parts. Results indicated that, aside from a few instances of overlapping fruit size ranges, Shuizhizi generally exhibits larger and longer dimensions than Zhizi. The weight proportion of the Shuizhizi pericarp is often higher than that of the Zhizi pericarp. Quantitative results highlighted significant differences in the chemical component content between Zhizi and Shuizhizi, with Shuizhizi generally containing higher levels of iridoids. The PCA and OPLS-DA analysis distinctly divided Shuizhizi and Zhizi, among which three iridoids, two organic acids, and one flavonoid made significant contributions to their classification. Cluster heatmap analysis also demonstrated complete separation between Zhizi and Shuizhizi, with clear distinctions among Zhizi samples from different origins. The distribution of the 13 chemical components in different Zhizi and Shuizhizi parts remained consistent, with iridoids and pigments concentrated in the seeds and flesh, and two organic acids and one flavonoid enriched in the pericarp. In summary, this study contributes valuable insights for classifying Zhizi and offers guidance on the rational use of Shuizhizi and the different parts of Zhizi.


Introduction
Gardeniae Fructus known as Zhizi in Chinese, serves as a common Chinese herbal medicine and finds applications in pharmaceuticals, food, cosmetics, and dye industries (Chen et al., 2020b;Tian et al., 2022;Jin et al., 2023).Its historical use dates back over 2000 years to the Han Dynasty, when it was documented as both a medicinal and dyeing agent.Traditionally, small, round fruits are designated for medicinal purposes, referred to as Zhizi or Shanzhizi in Chinese.Conversely, large, elongated fruits known as "Fu shi Zhizi" or Shuizhizi are used exclusively for dyeing (Liu and Peng, 2016).Currently, two distinct forms of medicinal materials persist in the market: Zhizi, characterized by smaller fruits, and the slightly larger Shuizhizi, aligning with historical herbal medicine records.Zhizi is derived from the dried ripe fruit of Gardenia jasminoides Ellis (Committee for the Pharmacopoeia of PR China, 2020), while Shuizhizi originates from various sources, primarily from G. jasminoides f. longicarpa Z. W. Xie & M. Okada or G. jasminoides var.grandiflora Nakai (Xie, 1991;Liu et al., 2023).
In recent years, advanced analytical methods such as ultra-flow liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry (UFLC-Q-TRAP-MS/MS) and ultraperformance liquid chromatography (UPLC) have been employed for quantitative analysis and fingerprinting of Zhizi, providing insights into optimal harvesting times and quality evaluations from different regions (Shan et al., 2019;Cao et al., 2021a;Li et al., 2021).Additionally, researchers have utilized ultrahigh-performance liquid chromatography/quadrupole-orbitrap mass spectrometry (UHPLC/ Q-Orbitrap MS) and UHPLC-triple quadrupole-linear ion trap mass spectrometry (UHPLC-QTRAP-MS) methods to identify thirteen differential markers and quantify metabolites in Zhizi and Shuizhizi, aiding in their differentiation (Zhang et al., 2024).
Recently, increasing attention has been given to the relationship between the appearance and quality of Chinese medicinal materials such as Peucedani Radix and Cimicifugae Rhizoma (Chu et al., 2020;Ma et al., 2023).Previous studies on Zhizi indicate a significant correlation between its appearance traits and its quality and medicinal properties.For example, as the color of Zhizi deepens into red, it indicates higher levels of crocin I. Conversely, a more yellow hue signifies had higher concentrations of total phenolic acids and rutin within the fruit (Fu et al., 2020a;Ye et al., 2022a).Another study revealed disparities in the chemical composition of Zhizi across various fruit ridges (Tang et al., 2020).
The existing commodity specification standard for Gardeniae Fructus primarily categorizes it into different grades, based on factors like fruit maturity, fullness, and color (Zhang et al., 2019).Whereas, due to long-term artificial cultivation, the shape, size and other characteristics of the Zhizi have undergone significant variation.Market transactions often rely on the shape and size of Gardeniae Fructus for grade evaluations, where smaller, round fruits are considered higher quality.However, the diversity in Zhizi's characteristics and its impact on quality warrant further research.This paper aims to contribute to this understanding by collecting different types of Zhizi and Shuizhizi, utilizing UHPLC-QQQ-MS/MS for the simultaneous determination of 13 active components (six iridoids, four organic acids, two pigments, and one flavonoid) and analyzing those major active components in the whole fruit and different parts of Zhizi and Shuizhizi.The objective is to comprehensively evaluate the quality of Zhizi and Shuizhizi with different characteristics.
LC-MS-grade methanol and acetonitrile were sourced from Merck (Darmstadt, Germany), while formic acid (LC-MS grade) was acquired from Aladdin (California, United States).Ultrapure water was supplied by a Milli-Q system (Millipore, United States).All the other reagents used were of analytical grade.

Sample collection and classification
Twenty-five batches of Gardenia species samples, comprising nineteen batches of Zhizi samples and six batches of Shuizhizi samples, were collected from the Jiangxi and Fujian provinces.The detailed sample information is provided in Supplementary Table 1.Prof. Huasheng Peng of the National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, authenticated all samples.The samples were categorized based on obovate and round morphological characteristics.Obovate and round fruits were distinguished by the largest fruit diameter's locationupper and middle parts of the whole fruit, respectively.Additionally, all samples were further classified into seven length grades (A-G) based on fruit length, measured from the fruit base (excluding, the fruit stalk) to the top narrowing place.Figure 1 illustrates the samples with different morphological features.

Measurement of fruit length, maximum diameter, weight, and volume
Fifteen fruits were randomly selected per batch.Using a vernier caliper, the length between the base (excluding the stem) and the narrow apex of each fruit, along with the maximum diameter, were measured.Both length and maximum diameter measurements were repeated three times for each fruit.Subsequently, the ratio of the length to the maximum diameter was then calculated for each fruit.
For each batch of the Zhizi samples, random division into three groups, each containing an equal number of seeds, was performed.An electronic balance was used to measure the weight of each group.After filling a beaker with fine sand (120 mesh), the fruits were buried in the sand.The spilled fine sand's volume was determined using a volumetric cylinder, representing the volume of each fruit group.Weight and volume measurements were repeated three times, and the density of each fruit was calculated as the weight-to-volume ratio.
2.3.2Determination of the weight ratio of pericarp, flesh, and seeds in fruit Nine fruits, originating from various geographical regions and possessing different length grades, were randomly selected.These fruits were manually divided into three parts: the pericarp, flesh, and seeds, with three parallels for each part.The weight of each part was measured, and the weight ratio of different parts in relation to the whole fruit was calculated.

Quantitative analysis of chemical compounds 2.4.1 Standard and sample preparation
The standard compounds were accurately weighed and individually dissolved in 70% methanol to prepare stock solutions Fruits of Gardenia species (Zhizi and Shuizhizi) with different morphological traits and parts, as well as the weight ratio pie graphs of different parts of them.(A) Obovate Jiangxi Shuizhizi (5.3 cm < L ≤ 6. of approximately 1.0 mg/mL, except for crocin I and crocin II, which were stored in 0.5 mg/mL stock solutions.These stock solutions were then diluted with 70% methanol to construct calibration curves.The resulting standard solution was stored at -20°C until injection.The dried sample powder (60 mesh), precisely weighed at 0.1 g, was placed in conical flasks and combined with 25 mL of 70% methanol.After weighing, the mixture underwent ultrasonication at 25°C (100 W, 40 kHz) for 30 min.Subsequently, the solutions were cooled to room temperature, and the same solvent was added to each solution to compensate for the weight loss.The supernatant was collected after centrifugation at 5000 r/min for 10 min.Finally, the solution was filtered through a 0.22 µm Millipore filter and stored at 4°C before injection.
All analytes were detected by a Q-TRAP ™ 4500 MS/MS system (AB Sciex, United States) equipped with an electrospray ionization (ESI) source for mass spectrometry detection.Compounddependent MS parameters, including precursor ion (Q1), production (Q3), declustering potential (DP), and collision energy (CE) were designed and optimized using the multiple reaction monitoring (MRM) modes.Other MS parameters were set as follows: source temperature, 500°C; ion source gas 1, 40 psi; ion source gas 2, 40 psi; curtain gas, 40 psi; and dwell time, 50 ms.Data was acquired and processed using AB Sciex Analyst 1.5.2 software (AB Sciex, United States).

Method validation
A series of standard solutions of appropriate concentrations were prepared to construct a calibration curve.The limits of detection (LODs) and quantification (LOQs) were defined using standard solutions as signal-to-noise (S/N) concentrations of 3 and 10, respectively.Testing of the 13 analytes within Sample S5 was repeated six times per day to evaluate the intra-day precision.To assess inter-day precision, the same solution was analyzed for three consecutive days.The repeatability of the method was verified by testing sample solution S5 six times.Aliquots from the same solution were injected at 0, 2, 4, 8, 12, and 24 h, respectively, and the relative standard deviation (RSD) values were calculated to validate stability.In the recovery test, standard analytes were added to six samples taken from S5 at amounts equivalent to those of the samples and then processed and analyzed following the procedure described as described above.

Data analysis
All data were expressed as mean ± standard deviation (Mean ± SD).Statistical evaluation was conducted using a one-way analysis of variance (ANOVA) and the Duncan's multiple range test with IBM SPSS 26.0 Statistics software (SPSS, Inc., United States).Statistically significant differences were considered for a p-value lower than 0.05.Significantly different data (p < 0.05) were denoted by different letters.The histogram and pie graphs were created using Origin 2021 software (OriginLab, United States).Principal component analysis (PCA) scores and orthogonal partial least squares discriminant analysis (OPLS-DA) were performed using SIMCA 14.1 (Umetrics Inc., Sweden) (Hu et al., 2022).A hierarchical clustering heat map was generated using the TBtools (Chen et al., 2020a).
For each fruit, the length, maximum diameter, ratio of length to maximum diameter, weight, volume, and density were considered as indices for analyzing the appearance characteristics.The data underwent analysis of ANOVA, and Duncan's multiple range test results are presented in Table 1.The findings indicated that Shuizhizi was generally longer than Zhizi, although their length ranges overlapped.Significant differences were observed in the length of samples from different length grades.The ratio of fruit length to its maximum diameter served as an indicator of fruit appearance type, with a ratio closer to 1 suggesting a more rounded fruit.Significantly different ratios were observed between obovate and round Zhizi (p < 0.05).Round fruits exhibited a notably smaller ratio of length to maximum fruit diameter compared to obovate fruits.The experimental results supported the reliability of sample classification based on appearance.Additionally, for Shuizhizi, longer samples tended to have a larger ratio of length to maximum fruit diameter and heavier weight.When comparing Shuizhizi with Zhizi of the same length grade, Shuizhizi exhibited a larger volume and lower density than Zhizi.Variations were observed in the weight proportion of pericarp, flesh, and seeds of Gardenia species fruit with different appearance characters and from different regions.Detailed information is presented in Figure 1.Notably, the pericarp weight constituted 25.7% to 33.15% of the total Zhizi fruit weight, while for Shuizhizi, it accounted for 32.78% to 36.32%.The pericarp weight proportion of Shuizhizi tended to be higher than that of Zhizi, while the total weight proportions of the flesh and seeds in Shuizhizi were generally lower than those in Shuizhizi compared to Zhizi.As the length grade of Shuizhizi decreased (Figures 1A-D1), there was a corresponding increase in the weight proportion of seeds and a decrease in the weight proportion of pericarps.The weight proportion of flesh changed with the proportions of pericarps and seeds.When the length ranges of Zhizi and Shuizhizi were the same (Figures 1D1, 2), the weight proportions of the flesh and seeds in Zhizi were higher than those in Shuizhizi, while the weight proportion of the pericarp was lower than that of Shuizhizi.
In various length grades of Zhizi from the Fujian production region (Figures 1D2, E1), shorter lengths correlated with higher weight proportion of seeds and lower weight proportions of flesh and pericarp.Across different length grades of obovate Zhizi from the Jiangxi production region (Figures 1E2, F1), decreasing lengths corresponded to lower weight proportions of seeds and higher weight proportions of flesh and pericarp.At the same length grade (Figures 1E1, 2), Zhizi sourced from Jiangxi showed a higher weight proportion of the pericarp and flesh compared to that from the Fujian.Conversely, the proportion of seeds weight was lower in Jiangxiproduced Zhizi.As the length of the round Zhizi decreased, there was a notable increase in the weight ratio of the pericarp; while the weight ratio of the flesh and seeds remained relatively low (Figures 1F2,  G).Conversely, at identical length grades for obovate and round Zhizi (Figures 1F1, 2), obovate Zhizi exhibited relatively higher ratios of pericarp and flesh, alongside a relatively lower weight ratio of seeds.

Optimization of extraction solvents and UHPLC-MS/MS conditions
In UHPLC-MS/MS experiments, various extraction solvents, including 50% methanol, 70% methanol, and 70% ethanol, were employed for sample extraction.The results revealed that methanol yielded a superior extraction effect compared to ethanol.While the iridoids were more efficiently extracted with the 50% methanol solution, other components exhibited more comprehensive extraction in the 70% methanol solution.Ultrasonic extraction for either 30 or 40 minutes showed no significant difference.After careful consideration, 70% methanol was chosen for extraction, and ultrasonication for 30 minutes was deemed optimal.The optimized mass detection parameters for each analyte and representative chromatogram are presented in Table 2 and Supplementary Figure 2, respectively.

Method validation
In this study, the method's performance was thoroughly validated, encompassing linearity, LOD, LOQ, precision, stability, and recovery.The results are summarized in Table 3.All the standard curves demonstrated excellent linear relationships (r > 0.999) across a relatively broad concentration ranges.The LOD and LOQ for the 13 compounds ranged from 0.0003 to 0.5 µg/mL and 0.0005 to 1.2 µg/mL, respectively.Intra-day precision exhibited RSD values ranging between 0.92% and 4.57%, while inter-day precision ranged from 2.27% to 5.82%.The stability RSD values for the 13 compounds were all below 4.98%, and repeatability was less than 6.03%.Overall recoveries fell within the range of 95.23% to 104.51%, with RSD values below 4.8%.These results affirmed the suitability of the method for the simultaneous quantitative analysis of the 13 compounds in both Zhizi and Shuizhizi.

Comparative content analysis of 13 compounds in Zhizi and Shuizhizi
To further conduct quantitative analysis and investigate potential differences in compound contents between Zhizi and Shuizhizi with distinct appearance traits, we employed the UHPLC-QQQ-MS/MS method.This allowed us to simultaneously determine the levels of 13 chemical components in both Zhizi and Shuizhizi.The targeted components included six iridoids (geniposide, genipin, shanzhiside, geniposidic acid, genipin 1-gentiobioside, and deacetylasperulosidic acid methyl ester), two pigments (crocin I and crocin II), four organic acids (caffeic acid, chlorogenic acid, neochlorogenic acid and protocatechuic acid) and one flavonoid (isoquercitrin).The results depicting the content of compounds in different types of Zhizi and Shuizhizi are illustrated in Figure 2. The detailed content values for the 13 compounds in the 25 batches of Zhizi and Shuizhizi samples can be found in Supplementary Table 2.For each fruit morphological index, different letters in the upper right corner of the same columns denote significant differences (p < 0.05) among different samples.Using in the middle "-" omit the middle letter when there are more than two letters.

Comparative analysis of 13 components in different types of Zhizi and Shuizhizi
Iridoids play a crucial role as chemical constituents of Gardeniae Fructus.Notably, there was no significant difference in geniposide content between Zhizi and Shuizhizi.However, Shuizhizi exhibited a higher content of iridoids, such as geniposidic acid, genipin, shanzhiside, and deacetylasperulosidic acid methyl ester, compared to Zhizi.As the fruit length of the Gardenia species decreased, the protocatechuic acid content decreased.Additionally, a decline in length corresponded to a decrease in the chlorogenic acid content in Zhizi.
In Shuizhizi, the contents of geniposide, geniposidic acid, genipin, and protocatechuic acid in grade A were slightly higher than in other grades.However, the content of deacetylasperulosidic acid methyl ester in grade C surpassed that in grades A and B, and the content of isoquercitrin in grade D was higher than in grades A-C.In addition, the content of crocin I and crocin II in grade C was slightly lower than in grades A, B, and D. When Zhizi and Shuizhizi were in length grade D, the content of genipin 1-gentiobioside in Zhizi surpassed that in Shuizhizi.Conversely, the contents of geniposidic acid, shanzhiside, and deacetylasperulosidic acid methyl ester in Shuizhizi were mostly higher than in Zhizi.
Comparing obovate and round Zhizi with the same length grade, the contents of caffeic acid, crocin I, and crocin II were slightly higher in obovate Zhizi.However, isoquercitrin showed slightly lower content in obovate Zhizi.With decreasing length grades in round Zhizi, the geniposide, shanzhiside, crocin I, and crocin II contents exhibited a decreasing trend, while chlorogenic acid and caffeic acid contents showed increasing trends.Zhizi from Fujian demonstrated a higher content of genipin 1-gentiobioside and protocatechuic acid, but lower contents of geniposidic acid, shanzhiside, deacetylasperulosidic acid methyl ester, caffeic acid, chlorogenic acid, and neochlorogenic acid compared to most Zhizi from Jiangxi.In the E length grade, Zhizi from Fujian exhibited lower contents of shanzhiside, deacetylasperulosidic acid methyl ester, chlorogenic acid, and neochlorogenic acid than Zhizi from Jiangxi.However, the contents of genipin 1-gentiobioside and protocatechuic acid were slightly higher in Zhizi from Fujian.
The quantitative results indicated that most iridoids in Shuizhizi were higher than those in Zhizi.Furthermore, there are differences in the chemical component content between Zhizi from Jiangxi and Fujian.Additionally, a decreasing trend in the protocatechuic acid was noted with decreasing length in both Zhizi and Shuizhizi.

Multivariate statistical analysis
PCA and OPLS-DA, as commonly used multivariate statistical analysis methods, can achieve sample classification.The content of 13 compounds in 25 batches of Zhizi and Shuizhizi was used for PCA analysis, and the results showed that 19 batches of Zhizi and 6 batches of Shuizhizi could be well distinguished (Figure 3A), consistent with prior studies (Cao et al., 2021a).In order to further understand which compounds have a significant contribution in distinguishing between Zhizi and Shuizhizi, those compounds datas were used for OPLS-DA analysis (Figure 3B).In the established statistical model, the R 2 Y, and Q 2 were 0.95 and 0.931, respectively.At the same time, 200 permutation tests were carried out to verify the OPLS-DA model.The vertical intercept values of R 2 and Q 2 were 0.18 and -0.644, respectively.The above data indicated that the established OPLS-DA model had good quality.Through OPLS-DA analysis, each compound can obtain a variable importance of projection (VIP) value.The larger the VIP value, the greater the contribution of the substance to distinguishing different groups.When the VIP value of a compound exceeded 1, it was considered to carry the most relevant information for group classification.The OPLS-DA score plot (Figure 3B) showed that Zhizi and Shuizhizi were completely separated, and the VIP value (Figure 3C) indicated that three iridoids (geniposidic acid, shanzhiside, genipin), two organic acids (chlorogenic acid, protocatechuic acid), and one flavonoids (isoquercetin) had a greater contribution to distinguishing them.
The hierarchical clustering analysis heatmap effectively classifies different samples based on the similarity of their chemical components.
In this study, a hierarchical cluster analysis heatmap was generated for the 13 chemical components across 25 batches of Zhizi and Shuizhizi samples.The results are presented in Figure 3D.The colors in the figure indicate the content of each compound, ranging from low (blue) to high (red).The samples were distinctly grouped into four categories.Group I (S3 and S4) and Group II (S1, S2, S5, and S6) comprised Shuizhizi samples from Jiangxi, Group III (S7-S10) consisted of Zhizi samples from Fujian, and Group IV (S11-S25) included Zhizi samples from Jiangxi.In this study, all Zhizi (Groups III and IV) and Shuizhizi (Groups I and II) samples could be distinguished based on their chemical composition, consistent with the results of PCA and OPLS-DA.Moreover, Zhizi from Jiangxi (Group IV) and Fujian (Group III) were successfully differentiated.Shuizhizi was further divided into two groups, and compared with Group I, Group II exhibited higher levels of genipin 1-gentiobioside, crocin I, and crocin II, along with lower levels of caffeic acid.This distinction might be attributed to the fact that there are two main cultivated varieties of Shuizhizi, whose fruits are often mixed together for use.

Analysis of different parts of Zhizi and Shuizhizi
To delve deeper into the variations in compound content across different parts of Zhizi and Shuizhizi, the UHPLC-QQQ-MS/MS method was employed to simultaneously determine the content of 13 components in distinct parts (pericarp, flesh, and seeds) of both Zhizi and Shuizhizi.
The total content of various compounds in the pericarp, flesh, and seeds of Zhizi and Shuizhizi is depicted in Supplementary Figure 3. Notably, the total contents of six iridoids and two pigments were higher in the flesh and seeds, while lower in the pericarp of both Zhizi and Shuizhizi.Conversely, the total contents of four organic acids and one flavonoid were highest in the pericarp, with lower contents in the flesh and seeds.Specifically, the total content of six iridoids in the seeds of Shuizhizi surpassed that in the Zhizi.Moreover, the total content of the two pigments in the flesh of Zhizi was significantly higher than in Shuizhizi.Additionally, the total contents of the four organic acids and one flavonoid in the pericarp, flesh, and seeds of Zhizi were higher than those in the corresponding parts of Shuizhizi.
The distributions of the 13 compounds in the pericarp, flesh, and seeds of Zhizi and Shuizhizi exhibited a similar pattern, as depicted in Figure 4.For iridoids and pigments, geniposide, genipin 1-gentiobioside, shanzhiside, genipin, and crocin I showed higher contents in the flesh and seeds and lower in the pericarp.Conversely, the contents of geniposidic acid and deacetylasperulosidic acid methyl ester were higher in the pericarp and lower in the flesh and seeds in both Zhizi and Shuizhizi.Genipin 1-gentiobioside demonstrated the highest content in the seeds, followed by the flesh, and the lowest in the pericarp for both Zhizi and Shuihizi.Crocin II exhibited higher content in the pericarp and flesh of Shuizhizi, and the lowest in the seeds.In Zhizi, crocin II content in the flesh was higher than in the pericarp and seeds.The contents of organic acids (chlorogenic acid, neochlorogenic acid, and protocatechuic acid) and one flavonoid (isoquercitrin) were highest in the pericarps of both Zhizi and Shuizhizi, with lower contents in the flesh and seeds.Conversely, caffeic acid content exhibited the following order in Zhizi and Shuizhizi: seeds > flesh > pericarp.Furthermore, the chlorogenic acid, neochlorogenic acid, protocatechuic acid, and isoquercitrin contents in the pericarp of Zhizi were significantly higher than those in Shuizhizi.

Discussion
4.1 Comprehensive analysis of various Zhizi and Shuizhizi types: foundations for Shuizhizi development and utilization Gardeniae Fructus, known as Zhizi, boasts a medicinal and dyeing history of thousands of years in China.Ancient herbalists recognized two distinct appearances of fruits of Gardenia speciesa larger one primarily used for dyeing and a rounder, smaller variant for medicinal purposes.Although most of the fruit morphologies of Zhizi and Shuizhizi are similar, Shuizhizi is generally larger and longer than Zhizi, with occasional size overlap between the two (Chen, 2018), as seen in samples S6, S7, and S8 in this study.Currently, a variety of appearance parameters have been used to differentiate medicinal fruits from different varieties or locations (Huang et al., 2022;Ma et al., 2022).Therefore, beyond solely measuring the length and maximum diameter of the fruits, this study incorporated measurements such as weight, volume, and density to comprehensively analyze the appearance traits of Zhizi and Shuizhizi.This approach provides a more intuitive understanding of their respective visual characteristics.
When the lengths of Zhizi and Shuizhizi were similar, Zhizi exhibited smaller volume and higher density.In addition to these differences in appearance, notable distinctions existed in the chemical composition between Zhizi and Shuizhizi.Some researchers have highlighted that the primary differentiators in Zhizi and Shuizhizi are mainly iridoids (Zhang et al., 2024).Ye et al. employed both non-targeted and targeted data analyses to accurately discern the differences in phytochemicals accumulated in the fruits of three Gardenia species, Shuizhizi and two kinds of Zhizi cultivars (Ye et al., 2022b).The 13 compounds quantified in this study, including six iridoids, four organic acids, two pigments, and one flavonoid effectively differentiated between the medicinal materials of Zhizi and Shuizhizi, as well as between Zhizi sourced from Jiangxi and Fujian (Figure 3).A previous study showed that Gardeniae Fructus with leaner and longer fruit shapes exhibited a lower total content of six phenolic acids and rutin (Ye et al., 2022a).In our study, the protocatechuic acid content decreased with the decrease of length grade for both Zhizi and Shuizhizi.The chlorogenic acid content in Zhizi displayed an upward trend with decreasing length.Zhizi exhibits superior quality characterized by small, round, thin pericarps and red color (Liu and Peng, 2016;Rao et al., 2023).The results of our study indicated the inaccuracy of dividing Gardeniae Fructus grades solely based on length grade.This study provides a reference for commodity specification and grade division of Zhizi, and further investigates the classification of Zhizi and Shuizhizi.
Historical herbal texts mention the medical use of Zhizi while excluding Shuizhizi from medicinal applications.However, ancient prescriptions such as Treatise on Febrile Diseases include references to "Fei Zhizi" and "Da Zhizi" as medicine ingredients, indicating that Shuizhizi was indeed used in ancient medicine (Xu et al., 2020b).Iridoids stand as the primary active components in Gardeniae Fructus, with geniposide, an iridoid, serving as a quality evaluation marker in the Chinese Pharmacopoeia, which stipulates that the content of geniposide in Gardeniae Fructus should not be less than 1.8% (Committee for the Pharmacopoeia of PR China, 2020).In this study, both Zhizi and Shuizhizi met the geniposide content standard outlined in the Chinese Pharmacopoeia, and no significant difference was observed in the geniposide contents of Zhizi and Shuizhizi.In addition, Shuizhizi exhibits a range of other iridoids and pigments.The quantities of six iridoids in Shuizhizi were generally higher than those found in Zhizi, with the exception of geniposide and genipin 1-gentiobioside.Modern pharmacological studies have revealed significant diuretic and antihypertensive effects in the total extract of Shuizhizi and its ethyl acetate extracts (Fu et al., 2020b).Shuizhizi also demonstrates anti-allergic pharmacological activity, with geniposide being one of its main efficacious substances (Wang et al., 2020).Furthermore, certain terpenoids present in Shuizhizi exhibit renoprotective activity (Cao et al., 2021b).These findings provide a foundation for the judicious use of Shuizhizi, apart from its use as a dye, and highlight its potential medical applications.Yet, further research is needed to fully explore its medicinal value.

Implications of chemical component distribution variation in different parts of Gardenia fruit for clinical drug usage
Secondary metabolites in medicinal plants are unevenly distributed across different organs and tissues (Chen et al., 2019;Zhao et al., 2021).In terms of plant fruits, variations in chemical components exist across different parts of the fruit, as observed in Juglans regia L. and Citrus reticulata Blanco (Bourais et al., 2022;Yang et al., 2022).Previous studies have found that representative iridoids (geniposidic acid, genipin 1-gentiobioside, and geniposide) and pigments (crocin I and crocin II) were predominantly concentrated in the kernel of Zhizi, while organic acids and flavonoids (chlorogenic acid and rutin) were mainly distributed in the pericarp of Zhizi (Wu et al., 2014).In this study, the fruits of Zhizi and Shuizhizi were divided into three parts: pericarp, flesh, and seeds.It was observed that the weight ratios of pericarp, flesh, and seeds varied among different character types of Zhizi and Shuizhizi (Figure 1).The weight ratio of Zhizi pericarps was mostly smaller than that of Shuizhizi pericarps, consistent with the notion of favoring thin pericarps for medicinal applications (Rao et al., 2023).Iridoids constitute the key chemical components of Zhizi, exhibiting the highest content and significant biological activity (Chen et al., 2020b).In this study, geniposidic acid and deacetylasperulosidic acid methyl ester were found to be primarily distributed in the pericarp, while geniposide, genipin, genipin 1gentiobioside and shanzhiside were predominantly concentrated in the flesh and seeds.The two pigments were mostly concentrated in the flesh and seeds of Zhizi and Shuizhizi, whereas the four organic acids and one flavonoid mainly accumulated in the pericarps of Zhizi and Shuizhizi.
Previous studies shown that, the distribution differences of compounds in Zhizi might be closely related to the tissue-specific expression of genes involved in the synthesis or regulation of these compounds.Xu et al. found significant differences in the accumulation patterns of geniposide and crocin during fruit ripening in Zhizi (Xu et al., 2023).Moreover, the enrichment degree of iridoids such as geniposide in the kernel was significantly higher than that in the peel at the growth and maturation stage, which was highly consistent with the expression level of predicted genes of main enzyme G8O (8-hydroxygeraniol dehydrogenase) in its metabolic pathway.The high expression of G8Os in the kernel of Zhizi might be the reason for the high content of geniposide in the kernel (Pan et al., 2021).Recently, there have been many studies on the metabolic pathway of crocin biosynthesis in Zhizi (Xu et al., 2020a;Shen et al., 2022).During the development of gardenia fruit, when the fruit color turns red, key enzyme genes involved in the biosynthesis of crocin exhibit significantly high expression levels (Zhang et al., 2023).The accumulation of crocin has guiding significance for determining the appropriate harvesting time of fruits.
Iridoids and pigments constitute the key chemical components of Zhizi, exhibiting the highest content and significant biological activity (Chen et al., 2020b;Wang et al., 2024).In addition, the main components of the two categories exhibited lower distribution in its pericarp, which aligns with the traditional wisdom that emphasizes the superiority of Zhizi with a lighter pericarp.In addition to using the whole fruit as a medicine, the historical use of Gardeniae Fructus's pericarp and kernel (Li et al., 2020) is supported by traditional texts like Lei Gong Pao Zhi Lun, which recommends peeling and whisking Zhizi first and using the kernel for medicinal purposes (Lei, 1985).The ancient Compendium of Materia Medica documents Zhizi's dual role in shelling or removing shells to treat different diseases (Li, 1996).Recent research reveals that the pericarp of Gardeniae Fructus is a potent source of dietary fibers and exhibits hypoglycemic properties (Meng et al., 2021).Findings from this study indicated that Zhizi's flesh and seeds contain higher concentrations of iridoids and pigments, while organic acids and flavonoids are predominantly present in its pericarp.These insights offer a valuable reference for exploring the distinctive components across different parts of Zhizi.Meanwhile, further genetic evidence is necessary to elucidate the underlying mechanisms behind the medicinal differences in its different parts.

Conclusion
This study investigated obovate and round Zhizi and Shuizhizi fruits by categorizing them into seven different grades (A-G) based on their length.Beyond exploring appearance traits and weight distributions of the pericarp, flesh, and seeds in various types of Zhizi and Shuizhizi, an effective UHPLC-QQQ-MS/MS method was developed to simultaneously quantify 13 main chemical components in the entire fruit and its different parts.The findings revealed that, generally, Shuizhizi was larger and longer than Zhizi, albeit with occasional crossovers in fruit size ranges.The weight proportion of the Shuizhizi pericarp was mostly higher than that of the Zhizi pericarp.Quantitative analysis of the 13 main chemical components indicated significant differences between Zhizi and Shuizhizi.PCA, OPLS-DA, and heatmap cluster analysis distinctly divided Shuizhizi and Zhizi, and Zhizi from different regions also showed clear distinctions through heatmap cluster analysis.Except for geniposide and genipin 1-gentiobioside, the contents of other four iridoids in Shuizhizi were generally higher than those in Zhizi.Furthermore, the distribution of the 13 chemical components in different parts of Zhizi and Shuizhizi remained consistent.Most iridoids and pigments were concentrated in the seeds and flesh, while organic acids and a flavonoid were primarily found in the pericarps for both.This study provides insights for classifying commodity specifications and grades of Gardeniae Fructus.Additionally, it offers guidance for the clinical application of Shuizhizi and the scientifically rational utilization of different parts of Zhizi.

Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material.Further inquiries can be directed to the corresponding authors.

TABLE 1
Morphological features of Zhizi and Shuizhizi.

TABLE 2
MS parameters of 13 investigated compounds.

TABLE 3
Method validation of 13 compounds.