Volatilomic Signatures and Antioxidant Activities of red Mandarin ( Citrus reticulata ‘Dahongpao’) Essential Oils from Peel,Flower,and Leaf

Abstract

Objective

Citrus reticulata cv. ‘Dahongpao’ is an ancient Chinese cultivar whose by-products, peel, flower, and leaf remain underexplored. This study aimed to conduct a comprehensive comparative characterization of the volatilomic profiles, odor-active signatures, and antioxidant activities of the essential oils (EOs) extracted from these three tissues to establish a scientific basis for their differentiated valorization.

Methods

Essential oils were obtained from the three tissues using ultrasound-assisted hydrodistillation. The volatile metabolomes were analyzed using headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry. Antioxidant capacities were evaluated using spectrophotometric assays to determine total phenolic content, ferric reducing antioxidant power, and scavenging activities against superoxide and hydroxyl radicals.

Results

A total of 985 volatile metabolites were identified, dominated by terpenoids (25.99%), esters (15.63%), and heterocyclic compounds (9.44%). Tissue-specific chemical signatures were observed: peel EOs were enriched in terpenoids such as α-citral and (1α,3β,4β)-p-menthane-3,8-diol; flower EOs accumulated oxygenated monoterpene like linalool and nerolidol; leaf EOs were characterized by green-note aldehydes and alcohols, including (Z)-3-hexen-1-ol and phytol. Antioxidant profiling revealed functional divergence: leaf EO showed the highest superoxide scavenging, flower EO exhibited the strongest hydroxyl radical scavenging and reducing power, while peel EO had the highest total phenolic content. Differential metabolite analysis highlighted extensive divergence, with 834 metabolites differing between peel and flower EOs (815 upregulated in peel). Notably, 2-Methoxy-3,5-dimethylpyrazine, a potent odorant with a 0.4 ppb threshold was significantly enriched in peel EO, along with high-impact volatiles like α-citral, 2,4-decadienal, and 2-octanol acetate.

Conclusion

Red mandarin tissues possess distinct metabolite architectures. The peel is a rich reservoir of phenolics and high-impact odorants, making it a prime candidate for the food and fragrance industries. The flower and leaf EOs harbor unique aromatic and bioactive traits suitable for specialized nutraceutical or cosmetic applications. These findings strongly support the differentiated, value-added utilization of all three by-products.

Introduction

Citrus essential oils (EOs) are complex mixtures of volatile organic compounds primarily obtained from the peel, leaves, and flowers of citrus species. These natural products are widely used in the food, beverage, cosmetics, and pharmaceutical industries due to their aromatic properties and multifaceted biological activities, including antimicrobial, antioxidant, anti-inflammatory, and anxiolytic effects.^1,2 Their natural origin and bioactivity have made them attractive candidates to replace synthetic additives, aligning with increasing consumer demand for clean-label and sustainable ingredients. ³

Among citrus species, Citrus reticulata Blanco (mandarin orange) exhibits significant phytochemical diversity, with volatile compositions influenced by cultivar, geography, tissue type, and developmental stage.^4,5 C. reticulata cv. ‘Dahongpao’ (hereafter referred to as red mandarin) is a historically significant landrace grown in the Three Gorges region of China. It is one of the oldest known citrus cultivars, with over 4000 years of cultivation history; this long period of adaptation makes it a promising source of unique phytochemical traits relevant to contemporary applications. In modern agriculture, this cultivar remains economically important, yet substantial amounts of its biomass, including pruned leaves, thinned flowers, and discarded peels are underutilized or wasted.

Phytochemical investigations of C. reticulata have revealed a rich array of volatile and semi-volatile metabolites, including monoterpenes (eg, limonene, γ-terpinene, α-pinene), sesquiterpenes (eg, β-caryophyllene), aldehydes (eg, decanal, octanal), alcohols (eg, linalool, nerol), and esters (eg, ethyl acetate, methyl anthranilate), many of which contribute to the species’ distinct citrus aroma and bioactivities.^6–9 Limonene, the dominant monoterpene in citrus peel oils, has been widely studied for its antioxidant, antimicrobial, and anti-carcinogenic properties.^10,11 Other components like linalool and β-caryophyllene possess anti-inflammatory and anxiolytic effects, making citrus EOs attractive candidates for functional food and therapeutic applications.^12–14

Plant by-products such as flower, leaf, and peel tissues represent valuable yet often overlooked sources of bioactive volatiles.^15–17 Flowers are typically discarded during thinning practices to improve fruit set,^18,19 while pruned leaves and peels are left unused or composted. ²⁰ Recent studies have demonstrated that these tissues contain abundant volatile constituents, including terpenoids, aldehydes, alcohols, and esters, which can contribute to aroma, defense, and antioxidant activity.^21,22 However, systematic characterization of tissue-specific volatile metabolomes in red mandarin remains scarce.

Efforts have been made to valorize citrus processing by-products.^23–25 For instance, citrus peels are already used in the extraction of flavonoids (eg, hesperidin, naringin), pectin, and essential oils in the nutraceutical and cosmetic industries. ²⁶ However, comparatively less attention has been paid to the systematic utilization of flowers and leaves, which are also rich in aromatic compounds. Studies have indicated that C. reticulata flowers contain significant levels of nerolidol and cis-jasmone,^27,28 while the leaves are sources of green note volatiles like (Z)-3-hexen-1-ol and phytol, ²⁹ all of which possess antioxidant or bioactive functions. ³⁰ These compounds not only enhance organoleptic quality but also hold potential for use as natural preservatives, aromatherapy agents, or active ingredients in botanical pesticides.

Volatilomics, an emerging branch of metabolomics enables high-resolution profiling of volatile organic compounds (VOCs) using analytical platforms such as gas chromatography-mass spectrometry (GC-MS). When combined with multivariate statistical tools, such as principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA), and KEGG pathway mapping, volatilomics can reveal key metabolites and their biochemical roles. ³¹ While studies exist on citrus by-products, they often focus on a single tissue, typically the peel. Consequently, a holistic understanding of the metabolic specialization across different plant parts of a single cultivar is missing. A systematic, comparative analysis is crucial to uncover the unique chemical signatures of flowers and leaves alongside the peel, which is essential for guiding their targeted and differentiated valorization. Despite its potential, comprehensive volatilomic studies targeting multiple tissues in red mandarin are lacking. Therefore, this study provides a comprehensive, comparative overview of the volatilomic profiles, odor -impact potential, and antioxidant activities of the peel, flower, and leaf of red mandarin. We hypothesize that each tissue possesses a distinct chemical and functional profile, providing a scientific basis for developing unique, value-added products from these underutilized biomass sources.

Materials and Methods

Plant Materials and Sampling

Fresh samples of C. reticulata cv. ‘Dahongpao’ (red mandarin) were collected from the citrus production base in Dazhou Town, Wanzhou District, Chongqing, China. Tissues included mature fruit peel (PE), flowers (FL) collected during full bloom in April, and leaves (BL) collected during routine pruning in January. Each tissue type was sampled in triplicate and processed immediately or stored at −80 °C prior to extraction.

Essential Oil Extraction

To obtain the necessary yields of bulk essential oil for subsequent functional antioxidant assays, EOs were extracted using ultrasound-assisted hydro-distillation following Chen et al. ³² For each tissue type, 500 g of fresh sample was weighed. Samples were first soaked in ascorbic acid (Macklin, Shanghai, China) solution for 30 min to inhibit oxidation, rinsed with distilled water, and surface-dried in a forced-air oven at 40 °C for one hour to remove excess moisture. To maximize extraction efficiency from the tougher tissues, peels and leaves were ground into a coarse powder (passing through a 40-mesh screen) using a laboratory plant grinder (Model FZ102, Taisite Instrument Co., Ltd, Hebei, China). In contrast, the delicate flower tissues were kept intact to prevent mechanical damage and the potential loss of their most volatile aroma compounds during processing.

Samples were subjected to ultrasonic treatment (100 W, 40kHZ) in an ultrasonic bath (Jiangxi Tianwo Machinery Technology Co., Ltd, Jiangxi, China) for 20 min, followed by hydro-distillation in a round-bottom flask with distilled water at a 1:2 (w/v) ratio. Distillation was conducted at 100 °C for 20 min. The resulting oil-water mixture was separated using a separatory funnel (Jiangsu San-Aisi Scientific Instrument Co., Ltd, Jiangsu, China), and the oil fraction was dried over anhydrous sodium sulfate (analytical grade, Ghtech, Guangdong Guanghua Sci-Tech Co., Ltd, Guangdong, China) and stored in amber vials at 4 °C until analysis.

GC-MS-Based Volatile Metabolomics

To achieve a comprehensive, high-resolution chemical characterization of the volatilome, headspace solid-phase microextraction (HS-SPME) was employed. ³³ This analytical technique offers superior sensitivity for detecting trace-level compounds and minimizes the potential for thermal degradation artifacts that can occur during hydrodistillation, thereby providing a more accurate representation of the tissue's native volatile profile.

Sample Preparation and SPME Extraction

Essential oil aliquots (100 µL) were diluted in 900 µL of isopropanol (HPLC grade, Macklin, Shanghai, China). A 20 µL internal standard (2-octanone, 10 µg/mL in isopropanol; Macklin, Shanghai, China) was added. Volatile compounds were extracted using headspace solid-phase microextraction (HS-SPME) with a 120 µm DVB/CWR/PDMS fiber (Agilent). This tri-phase fiber was selected for its broad-spectrum affinity, enabling the capture of a wide range of volatile and semi-volatile compounds of varying polarities, which is ideal for a comprehensive exploratory analysis. Extraction was performed at 60 °C for 15 min following a 5 min equilibration with agitation.

GC-MS Analysis

Analyses were conducted on an Agilent 8890 GC system coupled to a 7000D triple quadrupole mass spectrometer by Norminkoda Biotechnology Co., Ltd. (Wuhan, China). Separation was achieved using a DB-5MS capillary column (30 m × 0.25 mm × 0.25 µm). Oven temperature programming was as follows: initial hold at 40 °C for 3.5 min; ramp at 10 °C/min to 100 °C; 7 °C/min to 180 °C; and 25 °C/min to 280 °C with a final hold for 5 min. Carrier gas was helium at 1.2 mL/min. Electron ionization (EI) was used at 70 eV. Detection was performed in selected ion monitoring (SIM) mode. This widely targeted volatilomics approach was chosen over full-scan mode to enhance analytical sensitivity and selectivity.^2,31 By monitoring specific ions for a comprehensive list of known and expected citrus volatiles, this method improves the signal-to-noise ratio, enabling the accurate detection of low-abundance compounds critical to the overall aroma and bioactivity profile.

Raw mass spectral data were processed using Agilent MassHunter software. Compound identification was performed by matching spectra to an in-house MS library and retention indices (RI) with literature values. Quantification was based on peak area normalization against the internal standard. All metabolite data were normalized using unit variance scaling prior to multivariate statistical analyses.

Antioxidant Activity Assays

Total Phenolic Content

Total phenolics were determined using the Folin–Ciocalteu method ³⁴ (kit NM-W-0119, Norminkoda Biotechnology, Wuhan). Briefly, 0.1 g of EO extract was mixed with 1.5 mL of 60% ethanol and extracted at 60 °C for 2 h. After centrifugation (12,000 rpm, 10 min), supernatants were reacted with Folin reagent and sodium carbonate. Absorbance was read at 750 nm using a microplate reader. Results were expressed as mg gallic acid equivalents per mL extract (mg/mL).

Ferric Reducing Antioxidant Power (FRAP)

Total antioxidant capacity was assessed using the FRAP method ³⁵ (kit NM-W-0109, Norminkoda). A 0.1 g sample was homogenized with 1 mL cold extraction buffer and centrifuged. The supernatant was reacted with Fe3+-TPTZ chromogenic solution, and absorbance was measured at 590 nm after 10 min incubation at 25 °C. Antioxidant capacity was calculated from a Fe2 + standard curve and expressed in µmol/mL.

Superoxide Anion Radical Scavenging Activity

The assay followed the pyrogallol autoxidation method ³⁶ (kit NM-W-0114, Norminkoda). Samples (0.1 g) were homogenized in 1 mL of 80% ethanol and centrifuged. Supernatants were incubated with pyrogallol solution, and the reduction in absorbance at 320 nm was recorded. Scavenging activity was calculated as:

$$Superoxidescavenging(\text{\%})=\left[1-{\displaystyle \frac{A_{sample}-A_{control}}{A_{blank}}}\right]\times 100\text{\%}$$

Hydroxyl Radical Scavenging Activity

The hydroxyl radical assay (kit NM-W-0115, Norminkoda) was based on the Fenton reaction. ³⁷ Extracts (0.1 g) were homogenized, and supernatants were reacted with salicylic acid and Fe2+/H2O2. The absorbance of the resultant-colored complex was measured at 510 nm. Scavenging activity was calculated using:

$$HydroxylRedicalScavenging(\text{\%})={\displaystyle \frac{[A_{blank}-(A_{sample}-A_{control})]}{A_{blank}}\times 100\text{\%}}$$

Statistical and Multivariate Analysis

All experiments were conducted in triplicate. Data were expressed as mean ± standard deviation. Principal component analysis (PCA), hierarchical clustering, and orthogonal partial least squares-discriminant analysis (OPLS-DA) were performed using R (v4.1.2) and MetaboAnalystR. Differential metabolites were selected based on VIP > 1 and |Log2FC| ≥ 1. KEGG enrichment analysis was conducted using the KEGG Compound and Pathway databases.

Results

Antioxidant Activities of Red Mandarin Essential Oils

To evaluate the antioxidant potential of red mandarin essential oils (EOs), we assessed their radical scavenging activities and total phenolic content across three different tissues: peel (PE), flower (FL), and leaf (BL). Four biochemical parameters were measured: superoxide anion radical scavenging activity, hydroxyl radical scavenging activity, ferric reducing antioxidant power (FRAP), and total phenolic content (TPC).

Among the three tissue-derived EOs, leaf oil exhibited the highest superoxide scavenging activity, averaging 32.22%, followed closely by peel (31.69%) and flower oil (31.44%) (Figure 1A). In contrast, flower EO showed the highest hydroxyl radical scavenging activity, with a mean value of 63.72%, compared to peel (62.72%) and leaf (61.35%) EOs (Figure 1B).

Figure 1.

Pictorial Description of (A) Leaves, (B) Peel, and (C) Flower Tissues and Antioxidant Properties of Essential Oils Extracted from Different Tissues of C. reticulata cv. ‘Dahongpao’. The Violin Plots Illustrate the Data Distribution for Each Tissue Type (BL = Leaf, FL = Flower, PE = Peel), Where the Width of the Shape Represents the Data's Probability Density. the Inner box Plot Shows the Median (Center Line) and the Interquartile Range. (D) Superoxide Anion Radical Scavenging Activity. (E) Hydroxyl Radical Scavenging Activity. F) Ferric Reducing Antioxidant Power (FRAP). G) Total Phenolic Content (TPC).

FRAP values also varied significantly among tissues, indicating differences in reducing capacity (Figure 1C). Flower EO demonstrated the strongest reducing power with an average FRAP value of 0.843 μmol/mL, whereas peel and leaf oils showed lower values of 0.680 and 0.669 μmol/mL, respectively.

Notably, peel EO had the highest total phenolic content (0.272 mg/mL), followed by leaf (0.245 mg/mL), while flower EO contained the lowest phenolic concentration (0.063 mg/mL) (Figure 1D). This pattern suggests that although flower EO exhibits stronger reducing and hydroxyl radical scavenging capacity, its phenolic content is comparatively low, implying that other non-phenolic antioxidants may contribute to its activity.

These results collectively highlight the tissue-specific antioxidant properties of red mandarin EOs and indicate that peel oil is particularly rich in phenolic antioxidants, whereas flower oil may be enriched with other classes of bioactives contributing to its superior hydroxyl scavenging efficiency.

Metabolite Profiling and Quality Control

A total of nine essential oil samples derived from peel (PE), flower (FL), and leaf (BL) tissues of red mandarin were analyzed using headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry (HS-SPME-GC-MS). Metabolomic profiling yielded 985 unique volatile metabolites, as identified against a self-constructed reference database incorporating retention indices, MS spectral libraries, and internal standards.

Quality control (QC) assessment demonstrated high data reliability and reproducibility. The total ion current (TIC) chromatograms of QC mixed samples showed strong overlap in retention times and signal intensities, confirming stable instrument performance (Figure S1). Moreover, the coefficient of variation (CV) distribution indicated that over 85% of detected metabolites in QC samples had CV values below 0.5, and more than 75% below 0.3, ensuring the robustness of the analytical pipeline (Figure S2).

Unsupervised principal component analysis (PCA) revealed clear group separation among tissue types (Figure 2A). The first two principal components—PC1 and PC2—explained 86.02% and 11.86% of the total variance, respectively, effectively capturing the key differences in volatile profiles among peel, flower, and leaf samples. Hierarchical clustering analysis (HCA) further supported this pattern, clustering biological replicates closely together and emphasizing strong within-group similarity and distinct between-group variation. Consistent with these findings, Pearson correlation analysis showed exceptionally high similarity across all samples, with correlation coefficients exceeding 0.97, confirming the robustness and reproducibility of the metabolic profiling data (Figure 2B). Moreover, accumulation pattern of metabolites in three tissues suggested lowest accumulation for most of the metabolites in flower tissues compared to leaf and peel tissues (Figure 2C), illustrating the tissue-specific accumulation patterns. While most metabolite classes were detected across all tissues, their relative abundances were markedly different. Specifically, peel (PE) tissues exhibited the highest overall abundance, showing strong enrichment in classes such as terpenoids, esters, and heterocyclic compounds. In contrast, flower (FL) tissues displayed the lowest overall accumulation for most classes, indicating a more specialized and targeted volatilome. Leaf (BL) tissues presented an intermediate. A detailed quantitative analysis and discussion of these class-specific differences are presented in the subsequent sections.

Figure 2.

Multivariate and Correlation-Based Visualization of Volatile Metabolite Distribution in Red Mandarin Tissues. (A) Principal Component Analysis (PCA) Plot (B) Pearson Correlation Heatmap Depicting Pairwise Correlation Coefficients Among all Biological Replicates Based on Volatile Metabolite Profiles. (C) Heatmap of Z-score Normalized Metabolite Abundance, Where Rows Represent Individual Metabolites and Columns Represent Samples. Red and Green Indicate High and Low Relative Abundance, Respectively.

Metabolite Identification and Classification

A comprehensive GC-MS analysis identified a total of 985 volatile metabolites across essential oil samples derived from the peel, flower, and leaf tissues of red mandarin (Table S1 and S2). These compounds were distributed across a wide range of chemical classes (Figure 3A). Terpenoids were the most abundant, accounting for 25.99% of the total volatiles, followed by esters (15.63%), ketones (9.75%), heterocyclic compounds (9.44%), alcohols (7.72%), and aldehydes (7.21%). Other classes, including phenols, acids, aromatics, amines, and ethers, were also detected in smaller proportions.

Figure 3.

Comparative Analysis of Volatile Metabolite Profiles Across red Mandarin Tissues. (A) Donut Plot Showing the Chemical Classification of 985 Identified Volatile Metabolites from Peel, Flower, and Leaf Essential Oils of C. reticulata cv. ‘Dahongpao’. (B) Bar Graph Summarizing the Number of Significantly Different Metabolites Identified in Three Pairwise Comparisons: FL Versus BL, PE Versus BL, and PE Versus FL. (C) Venn Diagram Illustrating the Overlap and Specificity of Differential Metabolites among the Three Comparisons.

The distribution of these metabolite classes reflected strong tissue-specific patterns. Peel-derived essential oils were especially rich in terpenoids and esters, compounds widely known for their aromatic and bioactive properties. The flower oils contained a high abundance of oxygenated sesquiterpenes such as linalool and nerolidol, which are associated with floral fragrance and antioxidant functions. In contrast, the essential oils extracted from leaf tissue exhibited a relatively lower diversity of volatiles and were dominated by green-note aldehydes and unsaturated alcohols, including (Z)-3-hexen-1-ol and phytol, which are typically associated with herbaceous aromas and plant defense.

These observations suggest that the metabolic profiles of red mandarin tissues are highly specialized, likely reflecting their distinct physiological and ecological roles. Peel tissue, serving as the first barrier against environmental threats, appears to be chemically fortified, while flower tissue maximizes its scent profile to attract pollinators. Leaf tissue, in contrast, demonstrates a profile consistent with vegetative defense and stress signaling.

Differential Metabolite Analysis

To further investigate tissue-specific metabolite differences, pairwise comparisons were conducted between tissue types using OPLS-DA modeling and univariate statistics (Figure 3B). The most extensive metabolic divergence was observed between peel and flower tissues, where a total of 834 metabolites were found to be significantly different. Among them, 815 were upregulated in peel tissue, highlighting the peel as a chemically dense and compositionally diverse reservoir of volatiles. This comparison underscores the peel's specialized role in producing high concentrations of aromatic and protective compounds.

Comparison between flower and leaf tissues revealed 825 significantly altered metabolites, all of which were up-accumulated in flower essential oil (FLO). This complete upregulation suggests a metabolic intensification in flower tissues, consistent with their biological function in reproductive signaling through scent emission. On the other hand, the comparison between peel and leaf essential oils showed a smaller number of differential metabolites, with 147 significantly altered compounds, of which 106 were up accumulated in peel essential oil (POL) and 41 were do-accumulated. This indicates that while both tissues contain terpenoid compounds, the peel expresses a richer and more diverse volatile profile.

Taken together, these differential analyses confirm that essential oil extracted from each tissue type exhibits a distinct volatile metabolic signature. The flower essential oil is dominated by floral and aromatic components, the peel is enriched with terpenoids and esters contributing to both aroma and antimicrobial properties, and the leaf oil reflects a simpler volatile profile with green and herbaceous characteristics. These findings offer important insights into the tissue-specific roles of volatile organic compounds in red mandarin and provide a biochemical basis for targeted utilization of floral, foliar, and peel-derived essential oils in flavor, fragrance, and therapeutic applications.

To further dissect the distribution of differentially expressed metabolites among tissues, a Venn diagram was constructed based on the significant volatile compounds identified in three pairwise comparisons: FL versus BL (flower vs leaf), PE versus BL (peel vs leaf), and PE versus FL (peel vs flower) (Figure 3C). A total of 126 metabolites were commonly differentially expressed in all three comparisons, indicating a shared core of volatile changes that consistently distinguish tissues regardless of their origin. These shared metabolites likely play central roles in defining the global volatile architecture across floral, foliar, and fruit tissues.

Each comparison also revealed a number of uniquely altered metabolites. The FL versus BL comparison showed the greatest specificity, with 71 unique metabolites and 614 additional compounds shared exclusively with PE versus FL, emphasizing the metabolic uniqueness of flower-derived essential oils. The PE versus FL group displayed 91 unique metabolites, confirming substantial chemical divergence between peel and flower tissues. In contrast, PE versus BL showed limited exclusivity, with only 4 uniquely differential metabolites, suggesting that the peel and leaf share a relatively similar volatile baseline compared to the more specialized flower tissue.

Only a small number of metabolites were shared exclusively between FL versus BL and PE versus BL (14 compounds), or between PE versus BL and PE versus FL (3 compounds). This limited overlap further reinforces the finding that most differential volatiles are either broadly regulated or specific to comparisons involving flower tissue.

Altogether, the Venn diagram illustrates a clear stratification of the volatile metabolome, with flower tissues exhibiting the highest degree of metabolic specialization. These findings point toward the flower and peel as metabolically rich and distinct organs, offering promising targets for functional characterization and application in fragrance, flavor, and therapeutic development.

Key Differential Metabolites Across Tissues

To better understand tissue-specific metabolic specialization, we examined both up- and down-accumulated volatile metabolites in three pairwise comparisons: PE versus BL (peel vs leaf), PE versus FL (peel vs flower), and FL versus BL (flower vs leaf). The results revealed distinct chemical signatures, class-specific trends, and candidate compounds of interest for targeted utilization (Table S1).

In the PE versus BL comparison, PEO showed a strong enrichment of volatile compounds spanning ether, heterocyclic compounds, ketones, and aldehydes (Figure 4A, 4B, S3A). Notable up-accumulated metabolites included Propane, 1,1-diethoxy- (ether, Log₂FC = 2.61), 2-Methoxy-3,5-dimethylpyrazine and Benzofuran, 4,7-dimethyl- (heterocyclics), 4-Methyl-5-nonanone (ketone), and Nonanal (aldehyde) (Figure 4C). These volatiles are associated with citrus fragrance, antimicrobial activity, and aromatic complexity. Conversely, several metabolites were significantly down-accumulated in the peel compared to leaf, including Pyrazine, 2-methoxy-3-(1-methylethyl)- and 2-Piperidinimine (both heterocyclic compounds), Decane, 2,4-dimethyl- and 1-Undecyne (hydrocarbons), and Benzenamine, N,N-dimethyl- (amine). These results reflect the peel's selective accumulation of desirable aroma-active volatiles.

Figure 4.

Comparative Profiling of Differentially Accumulated Volatile Metabolites (DAMs) Across Red Mandarin Tissues. (A–C) Results from the PE versus BL (Peel vs Leaf) Comparison. (A) Heatmap Showing Hierarchical Clustering of DAMs Between Peel and Leaf Tissues Based on Normalized Metabolite Intensities. (B) Variable Importance in Projection (VIP) Scores from OPLS-DA Identifying the Most Influential Up- and Down-Accumulated Metabolites Contributing to Group Separation. (C) Top 10 DAMs Ranked by Absolute Log₂ Fold-Change Values. (D–F) Results from the PE versus FL (Peel vs Flower) Comparison. (D) Heatmap Displaying Tissue-Specific Clustering of DAMs. (E) VIP Plot Showing Highly Discriminative Metabolites with Both Up- and Down-Accumulated Compounds Contributing to Model Separation. (F) Bar Graph of the Top 10 Most Significantly Altered Metabolites. (G–I) Results from the FL versus BL (Flower vs Leaf) Comparison. (G) Heatmap Showing Relatively Modest Variation in Metabolite Accumulation Between Flower and Leaf Tissues. (H) VIP Scores from OPLS-DA. (I) Top 10 DAMs Showing Significant Down-Accumulation in Flower Tissue Relative to Leaves.

In the PE versus FL comparison, PEO exhibited dramatic enrichment of terpenoids, aldehydes, and esters (Figure 4D, 4E, and S3B). Among the top up-accumulated compounds were (1Alpha,3beta,4beta)-p-menthane-3,8-diol (terpenoid, Log₂FC = 6.88), 2,4-Decadienal (E,E)- (aldehyde, Log₂FC = 6.69), 2-Octanol, acetate (ester, Log₂FC = 6.63), and α-Cuprenene (terpenoid, Log₂FC = 6.31) (Figure 4F). These high-impact volatiles are well-known for their fragrance intensity and potential bioactivity. On the other hand, several esters and heterocyclic compounds were significantly downregulated in the PEO, including L-Leucine, ethyl ester, (E)-2-Methylbut-2-en-1-yl methacrylate, and 2-Piperidinimine. These findings confirm that while the peel has the richest overall volatile load, certain metabolites may be preferentially retained or biosynthesized in flower tissue.

Interestingly, in the FL versus BL comparison, while 825 metabolites were significantly different between the tissues, none met the stringent combined statistical threshold (VIP > 1 and |Log2FC| ≥ 1) for upregulation in flower tissue relative to leaf. This statistical nuance reflects the highly conservative metabolome of leaf tissue rather than a lack of important compounds in the flower. Indeed, the flower's profile is qualitatively rich and distinct, defined by key floral compounds and a significant down-accumulation of many metabolites found at high levels in the leaf (Figure 4G and 4H). However, the FLO did show significant down-accumulation of several terpenoids, including α-Cuprenene, (+)-epi-Bicyclosesquiphellandrene, and 1H-Benzocycloheptene derivatives—all significantly higher in BLO (Figure 4I and S3C). These trends suggest that although flower tissue yields a lower total number of unique metabolites, it may still contribute key volatile components with desirable floral notes and aromatic complexity, especially in specific esters and oxygenated terpenoids not found at high levels in other tissues.

Taken together, the peel emerges as the most promising tissue for the extraction of functionally and aromatically potent compounds, particularly in the terpenoid and aldehyde categories. However, flower-derived essential oil, despite a lower overall yield, harbors specific metabolites—especially esters and oxygenated aromatics—that may be valuable in applications requiring delicately scented or bioactive volatiles. These findings underscore the potential for integrated and tissue-specific valorization strategies in citrus by-product utilization.

Common Differentially Accumulated Metabolites Across Tissue Comparisons

A total of 126 volatile metabolites were identified as commonly differentially accumulated across all three pairwise tissue comparisons—PE versus BL, PE versus FL, and FL versus BL. These overlapping DAMs represent a shared metabolic signature that consistently distinguishes peel, flower, and leaf essential oils, regardless of comparison direction. Classification of these metabolites revealed broad chemical diversity, with terpenoids (19.0%), esters (15.9%), ketones (10.3%), heterocyclic compounds (9.5%), alcohols (7.1%), aldehydes (6.3%), hydrocarbons (6.3%), phenols (5.6%), and smaller proportions of acids, aromatics, amines, ethers, nitrogen-containing and sulfur-containing volatiles. This diverse composition highlights the multifunctional nature of citrus volatiles in aroma, defense, and ecological signaling.

Most of the overlapping DAMs displayed consistent tissue-biased accumulation patterns, with highest expression typically observed in PEO, moderate levels in FLO, and lowest in BLO. For example, 1H-1,2,3-Triazole, a heterocyclic compound, and Cycloheptanone, a ketone, were strongly enriched in peel tissue, showing substantial log₂ fold changes in both PE versus FL and PE versus BL comparisons. Similarly, Butanoic acid, butyl ester, classified as an ester, exhibited the highest accumulation in peel with log₂FC values of 3.71 in PE versus FL and 1.22 in PE versus BL, suggesting it plays a major role in defining peel aroma characteristics. Other metabolites such as Phenol, 3-fluoro- and 2-Octanol also followed this accumulation trend, further supporting the high metabolic activity and volatile density of peel tissue.

Interestingly, although FLO yielded fewer overall unique metabolites than PEO, several of these core DAMs—particularly esters and alcohols—also showed moderate enrichment in flower relative to leaf, suggesting a contribution to floral fragrance complexity. These shared DAMs, therefore, not only serve as robust chemical markers for tissue differentiation but also reflect the conserved metabolic backbone of citrus volatiles across organ types. Their structural diversity, consistent statistical significance, and high VIP scores in multivariate models underscore their importance in both functional profiling and targeted utilization of citrus essential oils.

Odor Impact Potential of Volatile Metabolites

In addition to statistical and multivariate significance, the sensory relevance of volatile metabolites was evaluated by examining their odor descriptors and odor thresholds (Table S1). Odor threshold refers to the minimum concentration at which a compound becomes perceptible to the human nose; therefore, compounds with low thresholds can exert strong aromatic influence even when present in trace amounts. From the dataset, several volatiles with exceptionally low odor thresholds were identified, indicating their likely disproportionate contribution to the overall scent profile of red mandarin.

The compound with the lowest recorded threshold was 2-Methoxy-3,5-dimethylpyrazine, with a value of just 0.4 parts per billion (ppb). This heterocyclic compound is known for its intense “bread” or “mousy” aroma and is commonly associated with roasted, earthy, or cereal-like notes. It was detected at significantly higher levels in peel tissues compared to flower and leaf, suggesting a potential role in the distinctive base aroma of citrus peel essential oil.

Also notable was 1-Nonen-3-one, a compound with a “pungent” and “mushroom-like” scent, and an odor threshold of 1 ppb. Though less commonly reported in citrus studies, its potent olfactory character implies that even small concentration differences between tissues could have a perceptible impact on overall fragrance. Similarly, 2-Furfurylthiol, with a threshold of 6 ppb, exhibited classic “sulfury”, “roasted”, and “coffee-like” aromas, and is widely recognized in sensory research for its intensity and presence in cooked or toasted profiles. These sulfur-containing volatiles may contribute to the warm and complex notes often found in mature citrus oils.

Other highly potent odorants include 2(5H)-Furanone, 5-ethyl-3-hydroxy-4-methyl- (threshold: 2 ppb), known for sweet, caramel, and maple-like scents; Pentanoic acid, 2-methyl-, ethyl ester (3 ppb), described as “green” and “fruity”; and p-Xylene and Diethyl disulfide, both of which contribute gassy, garlic, or floral overtones depending on context.

These odorants span multiple chemical classes including pyrazines, ketones, esters, and sulfur compounds, reinforcing that no single compound class dominates aroma impact potential. Interestingly, many of these high-impact volatiles were significantly upregulated in peel tissue, further supporting the conclusion that citrus peel contains not only the highest quantity and diversity of volatiles but also the greatest density of sensory-active compounds.

Taken together, these findings highlight a core set of low-threshold, high-odor-impact metabolites that define the aromatic character of red mandarin tissues. Their presence, particularly in the peel, suggests targeted opportunities for high-value fragrance extraction and underscores the need to integrate sensory data when interpreting metabolomic profiles.

Discussion

This study provides a comprehensive comparative analysis of the volatile metabolomes and antioxidant activities of essential oils derived from the peel, flower, and leaf tissues of Citrus reticulata cv. ‘Dahongpao’. The findings reveal distinct tissue-specific chemical signatures and bioactivities, highlighting the potential for valorizing these often-underutilized agricultural by-products, aligning with the growing demand for natural and sustainable ingredients.

Our investigation into the functional bioactivities revealed a clear divergence among the tissues, suggesting different underlying chemical mechanisms. The peel essential oil (PEO) exhibited the highest total phenolic content (TPC), which correlates well with its potent antioxidant capacity previously documented in other citrus extracts. ³⁸ Phenolic compounds are well-known radical scavengers, and their abundance in the peel—the plant's primary protective barrier—is a logical evolutionary adaptation.

Intriguingly, the flower essential oil (FLO) displayed the strongest hydroxyl radical scavenging and ferric reducing power (FRAP), despite having the lowest TPC. This apparent paradox strongly suggests that its antioxidant activity is driven by non-phenolic constituents. Oxygenated monoterpenes like linalool, which was abundant in FLO, are known for their antioxidant properties^12,39 and likely play a primary role. This uncoupling of bioactivity from phenolic content highlights a distinct functional profile for floral tissues. In contrast, the leaf essential oil (BLO) showed the highest specific activity against superoxide anions. This unique capability may be attributed to its characteristic “green leaf volatiles,” such as (Z)-3-hexen-1-ol, or other minor compounds not prominent in peel or flower EOs.^40,41

The volatilomic analysis, conducted via HS-SPME-GC-MS, provided a high-fidelity snapshot of the aroma-relevant chemical space of each tissue. The dominance of terpenoids in the overall profile is consistent with the known composition of citrus oils.^6,42 However, the dramatic upregulation of 815 metabolites in PEO compared to FLO underscores the peel's role as an exceptionally complex chemical reservoir. This profile was not only quantitatively rich but also qualitatively unique, featuring a high density of potent, low-threshold odorants. For instance, the identification of high-impact odorants. 2-Methoxy-3,5-dimethylpyrazine, with its exceptionally low odor threshold (0.4 ppb) and “bread” or “mousy” aroma, was found at higher levels in peel tissue. Alkylmethoxypyrazines are potent aroma compounds known in various foods and beverages, contributing roasted, earthy, or vegetative notes even at trace concentrations.^43–45 Similarly, 1-Nonen-3-one (“pungent,” “mushroom-like,” 1 ppb threshold) and 2-Furfurylthiol (“sulfury,” “roasted,” 6 ppb threshold) contribute significantly to the aroma profile. The higher density of such sensory-active compounds in PEO reinforces its value for fragrance and flavor applications.

Moreover, leaf essential oil (BLO) was characterized by “green note” aldehydes and unsaturated alcohols, such as (Z)-3-hexen-1-ol and phytol, which are typical of vegetative tissues and often associated with plant defense and herbaceous aromas.^40,41 In contrast, the FLO profile was defined by a more targeted accumulation of floral-scented compounds like linalool and nerolidol, consistent with its biological role in attracting pollinators.^42,46,47 The complete upregulation of the flower's volatilome when compared to the leaf signifies a profound metabolic intensification directed toward reproductive signaling. This makes FLO a prime candidate for applications requiring a delicate, specific floral scent profile, such as in high-end perfumery.

The dominance of terpenoids (25.99%) in the overall volatile profile is consistent with the general composition of citrus essential oils, where compounds like limonene, γ-terpinene, and α-pinene are frequently reported as major constituents, particularly in peel oils.^6,48 Our finding that peel essential oil (PEO) was particularly rich in terpenoids and esters aligns with extensive literature. For instance, limonene, often the most abundant monoterpene in citrus peel oils, is well-documented for its aromatic properties and diverse bioactivities, including antioxidant and antimicrobial effects.^10,11

The identification of 126 commonly differentially accumulated metabolites across all three pairwise comparisons indicate a core set of volatiles that define the fundamental chemical architecture of red mandarin tissues, albeit expressed at different levels. The consistent pattern of highest accumulation in PEO, moderate in FLO, and lowest in BLO for many of these core DAMs, such as 1H-12,3-Triazole and Cycloheptanone, further emphasizes the peel's metabolic richness.

It is crucial to acknowledge the limitations inherent in our analytical approach. The study employed two distinct workflows: functional assays were performed on bulk essential oil obtained via hydrodistillation, while chemical profiling was conducted on the headspace of diluted EOs using HS-SPME. Consequently, the volatile profile captured by HS-SPME is not fully representative of the bulk oil's composition, particularly under-representing less volatile or non-volatile compounds that may significantly contribute to antioxidant activity. Therefore, a direct causal link between the abundance of a specific volatile compound detected via HS-SPME and the total antioxidant activity of the bulk oil cannot be established from our data. The two datasets should be interpreted as complementary characterizations of the tissues: the HS-SPME data provides a high-fidelity snapshot of the aroma-relevant volatilome, while the bioassays reflect the integrated functional potential of the entire hydrodistilled oil. Future studies employing direct injection GC-MS or LC-MS would be necessary to comprehensively profile the less volatile components and establish more direct structure-activity relationships.

Future research could delve into the synergistic effects of these complex volatile mixtures, explore other bioactivities (eg, antimicrobial, anti-inflammatory), and investigate the influence of different extraction methods or seasonal variations on the phytochemical profiles. Furthermore, sensory panel evaluations would be invaluable to correlate the instrumental volatile data presented here with human perception of aroma for each tissue type, bridging the gap between chemical profiles and consumer experience.

Conclusion

In conclusion, this study successfully demonstrates the distinct chemical and antioxidant profiles of essential oils from ‘Dahongpao’ red mandarin peel, flower, and leaf. The data strongly support the differentiated valorization of these tissues. Specifically, peel EO, with its dense profile of odorants and phenolics, is a prime candidate for creating natural flavorings in the beverage industry or as an active ingredient in functional food preservatives. Flower EO, with its delicate floral aroma and anxiolytic compounds like linalool, is highly suitable for high-end perfumery and aromatherapy products. Finally, leaf EO, with its characteristic ‘green’ notes and potent superoxide scavenging ability, could be developed for use in cosmetic formulations or as a natural agent in air care products.

Supplemental Material

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Supplemental material, sj-jpeg-1-npx-10.1177_1934578X251382350 for Volatilomic Signatures and Antioxidant Activities of red Mandarin (Citrus reticulata ‘Dahongpao’) Essential Oils from Peel, Flower, and Leaf by Wenling Zhang, Haibing Zhang, Xingyu Chen, Teng Liu, Tingting Feng, Peipei Zhu and Qingyu Nie in Natural Product Communications

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Supplemental material, sj-jpeg-2-npx-10.1177_1934578X251382350 for Volatilomic Signatures and Antioxidant Activities of red Mandarin (Citrus reticulata ‘Dahongpao’) Essential Oils from Peel, Flower, and Leaf by Wenling Zhang, Haibing Zhang, Xingyu Chen, Teng Liu, Tingting Feng, Peipei Zhu and Qingyu Nie in Natural Product Communications

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Supplemental material, sj-jpg-3-npx-10.1177_1934578X251382350 for Volatilomic Signatures and Antioxidant Activities of red Mandarin (Citrus reticulata ‘Dahongpao’) Essential Oils from Peel, Flower, and Leaf by Wenling Zhang, Haibing Zhang, Xingyu Chen, Teng Liu, Tingting Feng, Peipei Zhu and Qingyu Nie in Natural Product Communications

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Supplemental material, sj-xlsx-4-npx-10.1177_1934578X251382350 for Volatilomic Signatures and Antioxidant Activities of red Mandarin (Citrus reticulata ‘Dahongpao’) Essential Oils from Peel, Flower, and Leaf by Wenling Zhang, Haibing Zhang, Xingyu Chen, Teng Liu, Tingting Feng, Peipei Zhu and Qingyu Nie in Natural Product Communications

Supplemental Material

sj-xlsx-5-npx-10.1177_1934578X251382350 - Supplemental material for Volatilomic Signatures and Antioxidant Activities of red Mandarin (Citrus reticulata ‘Dahongpao’) Essential Oils from Peel, Flower, and Leaf

Supplemental material, sj-xlsx-5-npx-10.1177_1934578X251382350 for Volatilomic Signatures and Antioxidant Activities of red Mandarin (Citrus reticulata ‘Dahongpao’) Essential Oils from Peel, Flower, and Leaf by Wenling Zhang, Haibing Zhang, Xingyu Chen, Teng Liu, Tingting Feng, Peipei Zhu and Qingyu Nie in Natural Product Communications