Chemical Composition and Biological Activities of Torreya grandis Kernels: Characteristics of Polymethylene-Interrupted Fatty Acids and Polyphenolic Compounds and Their Potential Health Effects

Abstract

Torreya grandis kernels, with their long cultivation history and significant economic value, have gained attention for their characteristic chemical components. This review systematically evaluates recent research on the chemical constituents and biological activities of T. grandis kernels. The key highlights include the following. (1) Chemical composition: This review details their unique fatty acid profile, particularly the high content of unsaturated fatty acids and rare polymethylene-interrupted polyunsaturated fatty acids such as sciadonic acid. It also examines polyphenolic compounds (flavonoids, phenolic acids, and biflavonoids like kayaflavone) and volatile components dominated by D-limonene. Other constituents, such as proteins, amino acids, vitamins, and minerals, are covered. Advanced analytical techniques (Gas Chromatography–Mass Spectrometry, GC-MS; Liquid Chromatography–Tandem Mass Spectrometry, LC-MS/MS) for component identification are discussed. (2) Biological activities: This review summarizes the major biological activities of T. grandis kernel extracts and key components. These include antioxidant effects (via the polyphenol-mediated NF-E2-related factor 2 (Nrf2) pathway), anti-inflammatory properties (via polymethylene-interrupted polyunsaturated fatty acids, PMI-PUFAs, inhibition of 5-LOX, and polyphenol regulation of NF-κB), and cardiovascular protection (potentially involving the AMPKα/SREBP-1c pathway). Research on gut microbiota regulation and enzyme inhibition is also outlined. (3) Research gaps and prospects: This review critically analyzes the limitations in the current research, including mechanism elucidation, component interactions, bioavailability, and safety assessment (especially the lack of human studies). Future research directions should focus on multiomics integration, structure–activity relationship analysis, standardization, and rigorous clinical evaluation. This review provides a theoretical reference for understanding the scientific value of T. grandis kernels and promoting their sustainable development.

1. Introduction

Torreya grandis (abbreviated as T. grandis) is a unique gymnosperm tree species native to China, with a long cultivation history dating back over 1500 years [1,2]. This species is mainly distributed in the subtropical hilly regions of southeastern China, particularly holding important cultural and economic status in Shaoxing City, Zhejiang Province [3,4]. The T. grandis industry has an annual production of approximately 9000 tons, with a market value exceeding CNY 1.7 billion, becoming an important pillar for regional economic development and rural revitalization [5,6,7]. Notably, advanced cultivation techniques, such as intelligent monitoring and precision management, have demonstrated key roles in improving cultivation efficiency, enhancing kernel quality, and promoting sustainable production [8].

T. grandis kernels have attracted attention due to their rich and unique nutritional components and phytochemicals, including characteristic fatty acids (such as specific polyunsaturated fatty acids), polyphenols, vitamins, minerals, and special proteins [9]. Based on these components, various value-added products have been successfully developed, including high-end edible oils, functional foods, and traditional Chinese medicines [10,11,12]. Their distinctive flavor characteristics have enhanced market acceptance, while increasing consumer interest in plant-based nutrition, natural bioactive components, and sustainable food systems has further stimulated market interest in T. grandis in recent years [13,14,15].

Studies have shown that T. grandis kernels are rich in various bioactive components, including flavonoids, lignans, and diterpene compounds, which are believed to exert multiple health effects through antioxidant, anti-inflammatory, and anti-tumor mechanisms [16]. Their antioxidant activity helps reduce the risk of chronic diseases [17,18,19], while their anti-inflammatory properties play a key role in preventing and managing inflammation-related diseases [20,21,22,23]. A prominent feature of T. grandis kernels is their unique fatty acid composition, particularly the high content of polymethylene-interrupted polyunsaturated fatty acids (PMI-PUFAs), which exhibit significant biological activity in regulating inflammatory responses and lipid metabolism [24]. Therefore, in-depth research on the chemical components and biological activities of T. grandis kernels not only elucidates their nutritional value but also establishes a solid scientific foundation for their development as functional foods and potential therapeutic agents.

The current research primarily focuses on three areas: (1) comprehensive chemical component characterization using advanced analytical platforms and multiomics approaches; (2) the evaluation of biological activities through in vitro and in vivo experimental models; and (3) the investigation of action mechanisms at the molecular level to elucidate structure–activity relationships and regulatory pathways. Despite significant progress, several challenges remain: the exact mechanisms of action for some bioactive components are yet to be clarified, potential synergistic or antagonistic interactions between different components require further exploration, and their application potential in functional foods and natural medicine development needs more systematic evaluation [25].

Therefore, this review aims to systematically compile and evaluate recent research progress on the major chemical components of T. grandis kernels (focusing on fatty acids, polyphenols, and volatile compounds), their compositional characteristics, analytical methods, and related biological activities (with emphasis on antioxidant, anti-inflammatory, and cardiovascular protective effects). Additionally, this review thoroughly discusses the potential molecular mechanisms of these activities, critically analyzes the limitations and gaps in the current research, and prospects the future of high-value utilization in functional foods and natural medicines, providing scientific references and a theoretical basis for subsequent basic research, product development, and quality control.

The systematic literature search for this review was primarily based on databases such as PubMed and Web of Science. To ensure comprehensiveness and representativeness, the cited literature was mainly obtained through systematic searches of PubMed, Web of Science, and other databases. The search strategy combined subject terms and free words, with the main search terms including “T. grandis”, “chemical composition”, “fatty acids”, “polyphenols”, “flavonoids”, and others. Selection priority was given to the literature published in the past decade, with a focus on peer-reviewed original research papers and high-quality reviews. The following criteria were applied when selecting specific literature and discussing compounds: (1) employment of recognized, reliable, and modern analytical techniques; (2) high reliability of research results with detailed data; (3) high relevance to the key chemical categories of T. grandis kernels (such as unique PMI-PUFAs and polyphenols); (4) revelation of compounds with high content, novel structure, or important potential biological activities; and (5) contribution to elucidating the effects of factors such as variety, cultivation conditions, processing, or extraction methods for chemical components. Representative research findings that met the above criteria and provided substantial information were included in this review for focused introduction and analysis.

2. Chemical Constituents of T. grandis Kernels

Comprehensive analysis indicates that T. grandis kernels contain diverse chemical components, many of which possess biological activities. Advanced analytical techniques, particularly Gas Chromatography–Mass Spectrometry (GC-MS) and Liquid Chromatography–Tandem Mass Spectrometry (LC-MS/MS), have been widely applied to identify their major chemical constituents, including fatty acids, polyphenols, volatile compounds, and amino acids [18,22]. These techniques not only enable qualitative and quantitative analysis of target compounds but also provide critical chemical information for elucidating structure–activity relationships and expanding functional applications.

However, while previous studies have made progress in revealing the overall profile of these compounds, they often focused on the isolation and characterization of individual or certain classes of compounds. Systematic research on complex interactions between different components (such as synergistic or antagonistic effects) and their regulatory mechanisms within the overall metabolic network remains insufficient. Particularly noteworthy is that the dynamic evolution patterns and molecular regulatory networks of chemical components during the unique after-ripening process of T. grandis kernels still require in-depth analysis [26]. Additionally, the specific effects and mechanisms of environmental factors (such as climate, soil, and altitude) and cultivation management practices (such as fertilization) on the accumulation patterns of chemical components in the kernels are important research directions that need strengthening [27]. This knowledge is crucial for achieving directional cultivation and high-value utilization of T. grandis resources.

The aforementioned research gaps highlight the necessity of the future adoption of integrated approaches combining multiple omics technologies (such as metabolomics, genomics, transcriptomics) with physiological studies, aiming to comprehensively reveal the complex phytochemical profile of T. grandis kernels. Based on this background, this section will systematically elaborate on the major chemical component categories reported in T. grandis kernels, focusing on their structural characteristics, content levels, extraction and analytical methods of representative compounds, and potential biological significance.

2.1. Fatty Acids

The lipid composition of T. grandis kernels has significant characteristics, most notably, its extremely high content of unsaturated fatty acids (UFAs). A study on T. grandis kernel samples from three different locations in Zhejiang Province, China (Fuyang, Zhuji, and Fengqiao), where 30 g of kernel powder from each location was used for oil extraction and the three extracted oil samples were repeatedly analyzed, showed [28] that the total oil content ranged from 51.52% to 55.64%. This value is significantly higher than conventional oil crops, such as sunflower seeds (which typically have oil contents in the range of 35%–50%) [29]. More importantly, the study determined that unsaturated fatty acids accounted for 87.95% to 94.25% of the total fatty acid content in T. grandis kernels [28], making them an important source of bioactive lipids.

As shown in

Table 1. Chemical composition of fatty acids in T. grandis kernels. Content data presented as percentage of total fatty acids. The data were obtained from GC-MS analysis.

Fatty Acid Name	Formula	Content (%)
Oleic Acid	C18:1	31.32–37.87
Linoleic Acid	C18:2	35.22–37.74
Palmitic Acid	C16:0	13.05–20.73
Sciadonic acid	C20:3 Δ5,11,14	9.13–12.96
Hexadecatrienoic Acid	C16:3	9.53
Heptadecanoic Acid	C17:0	1.78–6.31
Eicosatrienoic Acid	C20:3	1.66–6.31
Stearic Acid	C18:0	2.76–3.54
Arachidic Acid	C20:0	1.62
Ethyl linoleate	C18:2n−6	1.14
Eicosadienoic Acid	C20:2	0.22–0.88
Eicosenoic Acid	C20:1	0.16–0.50
Myristic Acid	C14:0	Not Detected
Palmitoleic Acid	C16:1	Not Detected

All the above data references originate from [28].

, the main fatty acid components of T. grandis kernels include linoleic acid (LA, C_18:2Δ^9,12; approximately 35.82%) and oleic acid (OA, C_18:1Δ⁹; approximately 30.37%). Particularly noteworthy is that T. grandis kernels contain a considerable amount of a rare PMI-PUFA—sciadonic acid (cis-5,11,14-eicosatrienoic acid, C_20:3Δ^5,11,14)—with a content ranging from 9.13% to 12.96%, according to different research reports [28], as shown in Figure 1a. The presence of sciadonic acid and its relatively high content is a key chemical characteristic that distinguishes T. grandis kernel oil from the vast majority of common vegetable oils, endowing it with unique nutritional value and bioactive potential. This unique fatty acid composition, especially the combination of high content of unsaturated fatty acids and special PMI-PUFAs (such as sciadonic acid), is considered the material basis for the potential application value of T. grandis oil in promoting cardiovascular health, regulating lipid metabolism, and inhibiting inflammatory responses [30,31,32]. Sciadonic acid possesses an atypical polymethylene-interrupted double bond system, a structural feature that makes it different in metabolism and bioactivity from common n-3 or n-6 series polyunsaturated fatty acids (PUFAs). The detailed fatty acid composition of T. grandis kernels is shown in Figure 1b.

It is worth noting that T. grandis oil not only has an outstanding content of PMI-PUFAs, but its high content of linoleic acid and oleic acid themselves are also important dietary fatty acids for maintaining healthy blood lipid levels, especially when they replace saturated fatty acid intake [33]. Therefore, the potential health benefits of T. grandis oil likely stem from the synergistic effects of its various unsaturated fatty acid components, rather than solely from the contribution of sciadonic acid. Future research should more systematically evaluate the overall biological effects of this unique fatty acid profile.

Understanding the biosynthetic pathways of PMI-PUFAs, which are relatively common unique fatty acids in gymnosperms, is crucial for elucidating their biological significance and developing potential applications. T. grandis belongs to conifers, and in conifers, the diversity of PUFAs mainly depends on the synergistic action of fatty acid desaturases and elongases [34,35]. PMI-PUFAs typically contain atypical double bond positions (such as Δ⁵), and their synthesis particularly requires “front-end desaturases” acting on elongated fatty acyl substrates, such as Δ⁵- and Δ⁶-desaturases [36,37,38,39,40]. The Δ⁵-desaturation pathway is known to be very active in various conifers (especially in Pinaceae) and is responsible for synthesizing characteristic fatty acids such as pinolenic acid (C_18:3Δ^5,9,12) [41,42].

Recent assembly and analysis of the T. grandis genome [43] have brought breakthrough progress in revealing its unique PMI-PUFA biosynthesis mechanism. The study successfully identified two key enzyme genes directly responsible for synthesizing the signature PMI-PUFA of T. grandis kernels—sciadonic acid. These two enzymes are as follows:

1. C₁₈ Δ⁹-elongase: This enzyme may be responsible for elongating C₁₈ substrates, such as linoleic acid (C_18:2Δ^9,12), to produce corresponding C₂₀ intermediates (e.g., eicosadienoic acid C_20:2Δ^11,14).

2. C₂₀ Δ⁵-desaturase: This enzyme subsequently acts on the C₂₀ intermediate, introducing a critical Δ⁵ double bond at the C₅ position, ultimately forming sciadonic acid [43].

Starting from the structure of sciadonic acid (C_20:3Δ^5,11,14) and its inferred precursor linoleic acid (C_18:2Δ^9,12), its biosynthetic pathway exhibits high specificity: first, the Δ⁹-elongase extends the carboxyl end of linoleic acid by two carbon atoms, forming C_20:2Δ^11,14, followed by the Δ⁵-desaturase introducing a double bond at the C₅ position. This highly specific enzymatic reaction sequence explains why sciadonic acid is relatively rare in nature [41].

Notably, the orthologous genes of these two key genes exist in multiple plant lineages but appear to be absent in angiosperms, revealing the unique evolutionary trajectory of the sciadonic acid biosynthetic pathway in gymnosperms and other non-flowering plants. Further structural and functional analysis indicated that the conserved histidine-rich motifs in the T. grandis Δ⁵-desaturase are crucial for its catalytic activity [43].

Besides these core biosynthetic enzymes, comparative transcriptomic studies on different T. grandis cultivars and kernel developmental stages have identified numerous candidate genes potentially involved in regulating overall lipid metabolism and fatty acid profiles. Zhang et al. [31] compared oil and fatty acid content changes during kernel development between two T. grandis varieties, and through transcriptome analysis, they identified 139 candidate genes related to fatty acid biosynthesis, elongation, and metabolism. Ding et al. [32] conducted transcriptome analysis on 10 T. grandis landraces, identifying 175 genes related to lipid biosynthesis, and predicted genes (such as BCC, FATA, and LPAT) and regulatory factors (such as TgWRI1 and TgFUS3) that might play key roles in oil accumulation. These genes encode other desaturases, elongases, acyltransferases, oil body proteins, and key transcription factors that potentially regulate oil accumulation and composition (such as homologs of WRI1, FUS3, and LBD40) [24]. However, the exact functions and interactions of these candidate genes in the T. grandis PMI-PUFA synthesis and lipid accumulation regulatory network still await further experimental verification. Transcriptome data provide numerous potential regulatory targets for in-depth research. For example, WRI1 and FUS3 are known key regulatory factors in plant oil synthesis [44], and their high expression in T. grandis may directly drive its high oil content. An in-depth study of how these transcription factors coordinately regulate the expression of the aforementioned Δ⁹-elongase and Δ⁵-desaturase genes will be key to understanding the unique oil accumulation pattern of T. grandis. Additionally, the expression levels of oil-body-protein-related genes may also affect the final oil storage and stability [45].

PMI-PUFAs, due to their unique structural features, exhibit different metabolic pathways and biological activities compared to common polyunsaturated fatty acids. Their potential health benefits are primarily mediated through several key mechanisms [46]:

1. Cell membrane modification: PMI-PUFAs can be incorporated into cellular phospholipid membranes. This incorporation can alter membrane fluidity, lipid raft structures, and the function of membrane-bound proteins, thereby affecting cell signaling transduction and transmembrane transport processes [47].

2. Modulation of eicosanoid pathways: PMI-PUFAs can act as competitive inhibitors or alternative substrates, interacting with key enzymes (such as cyclooxygenase COX and lipoxygenase LOX) involved in arachidonic acid (AA) metabolism. By competing with AA for binding these enzymes, PMI-PUFAs can alter the types and proportions of eicosanoids produced (such as prostaglandins, leukotrienes, etc.), typically tending to reduce the generation of pro-inflammatory mediators derived from AA [48].

3. Gene regulation via nuclear receptors: An important mechanism involves the interaction of PMI-PUFAs with nuclear receptors, particularly the Peroxisome Proliferator-Activated Receptor (PPAR) family (including the α, δ/β, and γ subtypes). These receptors are lipid-activated transcription factors [49,50]. When bound to ligands, such as specific fatty acids, PPARs regulate the expression of numerous target genes, which are broadly involved in lipid metabolism, glucose homeostasis, inflammatory responses, and cell differentiation, and this regulation often has tissue specificity [51,52].

Specifically, studies have shown that certain unsaturated fatty acids, possibly including PMI-PUFAs derived from sources such as T. grandis, can act as PPARγ agonists. Research on adipocytes has demonstrated that the activation of PPARγ by such fatty acids can upregulate the expression and secretion of adiponectin [53]. Adiponectin is an important adipokine known to enhance insulin sensitivity and possess significant anti-inflammatory properties [54]. Therefore, the PPARγ–adiponectin axis constitutes a key molecular bridge connecting these special fatty acids with their potential metabolic health benefits.

Considering the unique structure of sciadonic acid, its binding affinity and activation efficiency for PPARs (especially PPARγ) may differ from common PUFAs, which requires further experimental verification. If sciadonic acid can indeed effectively activate PPARγ and upregulate adiponectin, then T. grandis oil may have unique advantages in improving insulin resistance and related metabolic syndromes. Additionally, the effects of PMI-PUFAs on COX/LOX enzyme activity, as well as their specific integration into cell membranes and effects on membrane function, are also directions worthy of in-depth study in the future, which may collectively explain their multifaceted biological activities.

The extraction of T. grandis kernel oil is a key step in its processing and utilization. Different extraction methods not only affect the oil yield but also significantly impact the quality, flavor, and retention of nutritional components (especially heat-sensitive and easily oxidized components), as well as production costs and environmental sustainability [55,56]. Therefore, selecting appropriate extraction techniques is crucial for achieving high-value utilization of T. grandis kernel resources. The main methods currently applied to or with potential application value for T. grandis kernel oil extraction and their comparative characteristics are shown in

Table 2. Comparison of different extraction methods for T. grandis seed kernel oil.

Extraction Method	Principle	Advantages	Disadvantages
Pressing	Physical/mechanical pressure squeezes oil out. Includes cold and hot pressing.	Cold pressing: Simple, relatively low-cost [55]; solvent-free, good quality [57]. Hot pressing: Higher yield than cold pressing [57].	Cold pressing: Low yield, high residual oil in cake [55,57]. Hot pressing: High temperature can damage nutrients/quality [57].
Solvent Extraction	Uses organic solvents to dissolve and extract oil and then removes solvent by distillation.	High yield, mature technology, suitable for industrial scale [55,58].	Risk of solvent residue [58,59]; high safety requirements [55]; high-temp. desolventizing affects quality [58]; environmental pollution [55].
SFE-CO2	Uses supercritical CO2 as a selective solvent; separates oil by reducing pressure.	No solvent residue, pure product [56,59]; low temp., protects active compounds, high-quality [55,56]; environmentally friendly [55,56].	High equipment and operating costs [55,56]; relatively complex operation [55].
Aqueous Enzymatic Extraction	Uses enzymes in water to break down cell structures and release oil.	Solvent-free, eco-friendly, high safety [55,60]; mild conditions protect active compounds [60]; can co-extract oil and protein [60].	Often low yield [55,61]; high enzyme cost [60]; long reaction time [61]; emulsification makes separation difficult [55,60]; complex process [61].
Subcritical Fluid Extraction	Uses subcritical low-carbon alkanes (e.g., butane, propane) as solvent; separates by depressurization.	Lower P/T requirements than SFE-CO2, lower investment [62]; higher efficiency [62,63]; low-residue (testing needed) [63]; lower operating temp [62].	Very high safety risks (flammable solvents) [55,62,63].
Microwave-Assisted Extraction	Microwaves rapidly heat polar molecules, rupturing cells and speeding up oil release into solvent. Often used with other methods.	Significantly shorter time, higher efficiency [55,64]; less solvent needed [64]; lower energy consumption [55].	Risk of local overheating damaging sensitive compounds [55,64]; uniformity challenge [64]; scale-up difficulties [55].
Ultrasound-Assisted Extraction	Ultrasound effects (cavitation, vibration) break cell walls, improving solvent penetration and mass transfer. Often used with other methods.	Higher efficiency, shorter time [55,65]; lower temp. operation protects sensitive compounds [65]; simpler equipment, easier operation/scale-up [55].	High intensity may affect oil stability [55,65]; noise, complex optimization [65]; equipment heating [55].

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2.2. Polyphenols

T. grandis kernels contain a diverse range of polyphenolic compounds, including flavonoids, phenolic acids, tannins, and lignans [28,56,66]. These bioactive molecules exhibit significant antioxidant and anti-inflammatory properties while offering protective effects against various chronic diseases [67].

Recent advances in metabolomics studies have provided new insights into the polyphenolic composition of T. grandis kernels. Through LC-MS/MS analysis, Yan et al. [68] identified 107 different flavonoid compounds, among which 19 showed strong correlations with antioxidant activity. This finding provides critical evidence for the targeted screening and enrichment of functional flavonoids, although the underlying biosynthetic pathways and regulatory mechanisms of these bioactive compounds remain to be fully elucidated.

Comparative analysis among different cultivars has revealed significant variations in the total phenolic content. For instance, the Zhenong 1 cultivar contains 16.92 mg/g total phenols, compared to 8.34 mg/g in the Jiulongshan cultivar kernels [28]. These findings strongly suggest that genetic background is a key determinant in polyphenol accumulation.

Regarding extraction methods, an innovative approach combining deep eutectic solvents with magnetic nanomaterials has shown promising results, achieving flavonoid extraction yields of 12.741 mg/g [43]. However, both the selective enrichment mechanism and challenges in industrial-scale production warrant further investigation.

In an extraction optimization study, Zhu et al. [69] found that 70% ethanol extraction yielded optimal results, with the highest total phenolic content (5.86 mg/g) and flavonoid content (7.78 mg/g). High-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (HPLC-QTOF-MS) analysis identified 19 different compounds, predominantly phenolic acids. This establishes a comprehensive phytochemical profile for future research initiatives.

Despite the progress in identifying polyphenols, particularly flavonoids, in T. grandis kernels (as demonstrated by Yan et al. [68]), research on the bioavailability and metabolic pathways of these specific compounds in humans remains remarkably limited. Understanding these aspects is crucial for evaluating their true health benefits.

Generally, the bioavailability of dietary flavonoids is influenced by multiple factors, including their chemical structure (such as glycosylation patterns and degree of polymerization), food matrix, intake dosage, and the host’s physiological conditions (such as gut microbiota composition and digestive enzyme activity) [70,71]. Most naturally occurring flavonoids exist as glycosides, requiring hydrolysis by intestinal enzymes (such as lactase phlorizin hydrolase) or glycosidases produced by gut microbiota to release their aglycone forms for easier absorption by small intestinal epithelial cells [72]. However, absorption efficiency is typically low, with most ingested flavonoids reaching the colon.

The flavonoid aglycones or their hydrolyzed products absorbed into the body undergo extensive “first-pass metabolism”, primarily in intestinal cells and the liver. This includes Phase I metabolism (such as cytochrome P450 enzyme-mediated oxidation, reduction, and hydrolysis reactions) and Phase II metabolism (mainly conjugation reactions with glucuronic acid, sulfate groups, or methyl groups, namely, glucuronidation, sulfation, and methylation) [70,73]. These metabolic transformations significantly alter the structure, solubility, and biological activity of flavonoid compounds and facilitate their excretion through urine or bile.

Intestinal microbiota play a crucial role in flavonoid metabolism. Flavonoid compounds not absorbed in the small intestine (including glycosides, aglycones, and more complex oligomers or polymers, such as proanthocyanidins, if present in the kernels) are extensively degraded by microbial communities in the colon. Microbes can perform various transformations, including deglycosylation, ester hydrolysis, ring opening (such as C-ring cleavage), dehydroxylation, and demethylation, producing a series of low-molecular-weight phenolic acid metabolites, such as hydroxyphenylacetic acid and hydroxyphenylpropionic acid [72,74]. These microbial metabolites can be absorbed into the systemic circulation, with biological activities that may differ from the parent compounds. They may even reach higher concentrations in the body than their parent compounds and are considered important mediators of many dietary polyphenol health effects [75].

Given the diversity of flavonoid compounds identified in T. grandis kernels [65], it can be expected that they would follow these general patterns of absorption and metabolism in the human body. For instance, potentially present flavan-3-ols (such as catechin and epicatechin), flavonols (such as quercetin and kaempferol glycosides), and flavones (such as apigenin and luteolin glycosides) all have corresponding metabolic pathway studies (though not specific to T. grandis sources). However, precise bioavailability data, specific metabolite profiles, and biological activities of these metabolites for key flavonoid compounds from T. grandis kernels (such as the 19 compounds identified by Yan et al. [65] as strongly correlated with antioxidant activity) remain a knowledge gap. Future research urgently needs to elucidate the in vivo fate of T. grandis kernel flavonoids through in vitro simulated digestion, cell models, animal experiments, and human intervention studies. This will provide crucial evidence for evaluating their potential health value and functional food development.

Among the various flavonoid compounds identified in T. grandis kernels, in addition to common flavones, flavonols, and flavan-3-ols, special attention should be given to the presence of biflavonoids. These compounds consist of two flavonoid monomer units connected through C-C or C-O-C bonds, possessing more complex structures and relatively limited distribution in the plant kingdom. They are mainly found in gymnosperms, ferns, and bryophytes, while being relatively rare in angiosperms [76]. The study by Gao et al. [18] extensively explored the biosynthetic mechanism of a representative biflavonoid in T. grandis: Kayaflavone. Therefore, biflavonoids, represented by Kayaflavone, can be considered as characteristic or relatively unique polyphenolic components in T. grandis kernels with taxonomic significance and potential unique biological activities. This further enriches the chemical uniqueness of T. grandis kernels and may contribute to their distinctive biological activities.

The molecular mechanisms underlying the biological activities of T. grandis polyphenols involve the Keap1-Nrf2-ARE signaling pathway. NRF2 (NF-E2-related factor 2) is a key transcription factor in cellular oxidative stress responses, primarily regulated by Keap1 (Kelch-like ECH-associated protein 1). Antioxidant compounds, including polyphenols, induce conformational changes in Keap1, leading to NRF2 release, nuclear translocation, and binding to antioxidant response elements (AREs). This activates genes encoding phase II detoxification enzymes, enhancing cellular protection against oxidative damage [30,77]. T. grandis polyphenols provide a dual mechanism for cellular protection by both directly scavenging free radicals and simultaneously activating the NRF2/ARE signaling pathway [3,78].

Figure 2 illustrates the regulatory mechanism of the Nrf2 signaling pathway and the intervention effects of T. grandis polyphenols. The left panel depicts the process under basal conditions, where Nrf2 is bound by the Keap1-Cul3-RBX1 complex, leading to its ubiquitination and subsequent proteasomal degradation. The right panel shows that under oxidative stress conditions, polyphenols from T. grandis modify cysteine residues on Keap1, thereby promoting Nrf2 release from the complex. Subsequently, Nrf2 translocates to the nucleus, where it binds to ARE elements together with sMaf proteins, inducing the expression of antioxidant proteins and exerting cytoprotective effects. This mechanism reveals the molecular basis by which T. grandis polyphenols combat oxidative stress through the activation of the Nrf2 signaling pathway.

Cysteine plays an extremely important role in this process. The Keap1 protein contains multiple cysteine residues that are highly reactive and serve as sensors for oxidative stress. Under normal conditions, Keap1 binds to Nrf2 through these cysteine residues, promoting Nrf2 ubiquitination and eventual degradation. In the presence of T. grandis polyphenols, these compounds can react with cysteine residues on Keap1, causing conformational changes in Keap1. This modification of cysteine residues directly leads to Keap1 releasing Nrf2, preventing its degradation and allowing its translocation to the nucleus. It is precisely this cysteine-based “molecular switch” mechanism that enables cells to sense oxidative stress and activate antioxidant defense through Nrf2-mediated pathways. The modification of cysteine residues can be considered the molecular target and key mechanism for the antioxidant activity of T. grandis polyphenols.

2.3. Volatile Compounds

Understanding the volatile compound profile of T. grandis kernels is crucial for characterizing their flavor properties and developing functional products. Comparative GC-MS analysis of different T. grandis cultivars has revealed D-limonene as the predominant characteristic volatile compound. This compound not only contributes a unique citrus aroma but also exhibits significant antioxidant and antimicrobial properties. The D-limonene-dominated fresh citrus and terpenic aroma profile clearly distinguishes T. grandis from numerous other nuts characterized by “nutty” notes (typically derived from pyrazines, aldehydes, etc.) or fatty flavors (such as walnuts, almonds, and peanuts), conferring a unique flavor positioning. The structure–activity relationships of these volatile compounds suggest potential applications as natural preservatives and flavoring agents [66,79,80].

Advanced analytical techniques have enabled the systematic characterization of T. grandis kernel volatiles. Using headspace solid-phase microextraction coupled with gas chromatography–tandem mass spectrometry (HS-SPME-GC-MS/MS), Zhang et al. [15] identified 42 different volatile compounds, including aldehydes, esters, alcohols, ketones, hydrocarbons, and heterocyclic compounds. Hydrocarbon compounds were predominant, accounting for over 40% of the total volatile components, with D-limonene as the major compound (odor threshold: 10 μg/kg). This extremely low odor threshold makes D-limonene a critical determinant of T. grandis kernels’ unique aroma characteristics. Compared to other D-limonene-rich citrus fruits, T. grandis aroma is generally perceived as more mellow and complex, featuring not only bright citrus top notes but also woody, resinous, or subtly sweet fragrances from other volatiles (such as α-pinene, β-pinene, other terpene alcohols, and esters). When compared to pine nuts, which are also coniferous tree seeds, T. grandis exhibits less sharp piney notes and more prominent citrus characteristics, creating a unique balance [26]. Figure 3 illustrates the aroma characteristics of volatile compounds in T. grandis kernels.

However, the complex and unique flavor characteristics of T. grandis kernels are not determined by a single compound, but rather result from the combination and interaction (synergistic or inhibitory) of multiple volatile components in specific proportions. This highlights the importance of future research in establishing comprehensive chemometric models that correlate complete volatile compound profiles with sensory evaluation data, such as Quantitative Descriptive Analysis.

Several research gaps remain in this field. These include the specific effects and kinetic patterns of processing parameters (such as roasting temperature, time, and methods) on the formation and degradation of key volatile components; the biosynthetic pathways and regulatory mechanisms of major flavor compounds; the influence of environmental factors (climate, soil, altitude, etc.) and cultivation management practices on volatile accumulation patterns; and the specific variations in volatile profiles among different cultivars or local varieties and their genetic basis. A deeper understanding of these aspects holds significant importance for flavor quality control, processing optimization, and distinctive cultivar breeding. The chemical composition information for some major volatile compounds in T. grandis kernels is presented in

Table 3. Chemical composition of volatile compounds in T. grandis kernels. The content is presented as a relative percentage of the total volatile compounds determined by GC-MS analysis. The compounds were identified by comparison with mass spectral libraries and retention indices.

Compound Name	Formula	Content (%)	Reference
D-Limonene	C10H16	31.37–46.16	[31]
Carveol	C10H16O	1.67–4.36	[31]
α-Pinene	C10H16	1.63–5.78	[31]
L-Limonene	C10H16	1.99	[69]
2,4-Decadienal	C10H16O	0.60	[69]
Octadecanal	C18H36O	1.22	[69]
Terpinolene	C10H16	1.60–4.97	[12]
Guaiene	C15H24	1.91–4.14	[12]
1,2,3-Trimethoxy-5-methylbenzene	C10H14O3	5.50–12.74	[12]
2,5-Dimethylpyrazine	C6H8N2	1.46–3.90	[12]
Maltol	C6H6O3	0.83–2.04	[12]

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2.4. Other Constituents

T. grandis kernels are notable for their distinctive nutritional composition. As a high-energy food, these kernels contain relatively abundant protein content, reported to range from approximately 7% to 16% [66,71]. The kernels contain approximately 29.8 g/100 g of carbohydrates, 6.8 g/100 g of crude fiber, and 2.9 g/100 g of ash [81], though these values may vary depending on cultivar and analytical methods.

Regarding minerals, the kernels exhibit a diverse profile including various trace elements such as cobalt (0.28–0.50 mg/kg), selenium (52.91–68.71 μg/kg), magnesium, and calcium [31]. The notably elevated selenium content exceeds that of most other nuts [82], suggesting potential unique mineral metabolism mechanisms. These kernels are also rich in essential vitamins, particularly vitamin E, with antioxidant activity [78] (with β-tocopherol being a characteristic component of T. grandis oil [10]), and vitamin B₃ (niacin), which promotes energy metabolism [83]. β-tocopherol not only acts as an antioxidant to improve the oxidative stability of kernel oil and extend shelf life [66], but it may also regulate cellular signaling pathways [10].

The amino acid composition of T. grandis kernels is particularly noteworthy, with essential amino acids accounting for 38%–41% of the total amino acid content [84]. The presence of flavor-active amino acids, especially glutamic acid and aspartic acid, contributes to their characteristic flavor profile [12,15]. Comprehensive analysis by Zhu et al. [69] revealed an average protein content of 12.5 g/100 g and identified 16 different amino acids. Essential amino acids comprised 38.6% of this profile, while medicinal amino acids accounted for over 60%. Aspartic acid, with anti-fatigue properties, and glutamic acid, with neurotrophic effects, are present in significant amounts.

The protein and amino acid compositions vary significantly by geographical origin. Analysis has shown protein contents ranging from 9.44–15.24 g/100 g across different regions, while total amino acid contents vary from 94.29 to 144.62 mg/g [85]. These variations may be attributed to environmental factors, including soil conditions, climate, and cultivation practices. However, the molecular regulatory mechanisms controlling protein and amino acid accumulation remain to be fully elucidated.

Regarding potential antinutritional factors, research has indicated that T. grandis kernels contain components such as phenolic acids and tannins [86]. Tannins are common secondary metabolites in plants, known to potentially bind with proteins at high concentrations, affecting their digestion and absorption, or chelate certain minerals, reducing their bioavailability. As for other common antinutritional factors, such as phytic acid or trypsin inhibitors, there are currently no definitive research data reporting their specific content and potential effects in T. grandis kernels. Therefore, the exact status of these specific antinutritional factors in T. grandis kernels, as well as their content changes and actual effects after traditional processing methods, such as roasting, requires specialized future research for evaluation.

In summary, to more comprehensively understand and utilize the nutritional value of T. grandis kernels, future research should focus on several key areas: (1) elucidating the biosynthetic pathways and molecular regulatory networks controlling the accumulation of key nutritional components (such as specific amino acids, vitamin E, selenium, etc.) during kernel development; (2) systematically studying the in vitro digestibility characteristics and in vivo bioavailability of its proteins; (3) evaluating the effects of different processing methods (such as roasting, boiling, fermentation, etc.) on major nutritional components and potential antinutritional factor content; and (4) developing and establishing standardized quality assessment systems for T. grandis kernel products based on comprehensive chemical composition and nutritional value. Addressing these knowledge gaps is crucial for guiding breeding programs, optimizing processing methods, and developing high-quality T. grandis functional foods and nutritional products.

3. Biological Activities of T. grandis Kernels

The biological activities of T. grandis kernels are diverse and interconnected, as illustrated in Figure 4, which provides a comprehensive overview of their major biological effects and potential applications.

The molecular mechanisms underlying the biological activities of T. grandis kernels are illustrated in Figure 5. The diagram shows two main bioactive component groups: fatty acids and polyphenols. The fatty acid components, including unsaturated fatty acids and PMI-PUFAS, act through pathways involving AMPK receptors, IL-1β receptors, TNF-α receptors, and TLR4, ultimately influencing PPARα, 5-LOX, and NLRP3 inflammasome activity. These pathways lead to the suppression of inflammatory mediators and the regulation of lipogenic and inflammatory genes. Meanwhile, polyphenols, categorized as flavonoids and phenolic acids, influence pathways including the MAPK cascade, NF-κB, and antioxidant response elements, like SOD, GSH-Px, and NRF2. The diagram demonstrates how these molecular interactions result in enhanced antioxidant activity, reduced inflammation, and improved energy expenditure through the regulation of various genes, including FAO genes, lipogenic genes, and inflammatory genes. This complex network of signaling pathways explains the diverse therapeutic potential of T. grandis kernels in addressing oxidative stress, inflammation, and metabolic disorders.

3.1. Antioxidant Activities

Among the various biological properties exhibited by T. grandis kernels, their antioxidant activity has been one of the most extensively studied areas. The major research findings regarding these activities are summarized in

Table 4. Antioxidant activities of T. grandis kernels from different studies.

Sample Type	Parameter	Value	Reference
Kernel extract	DPPH (IC50) Hydroxyl radical (IC50)	120 μg/mL >100 μg/mL	[29]
			[29]
Kernel extract	DPPH (IC50) ABTS (IC50)	0.08 mg/mL 0.07 mg/mL	[52]
			[52]
Kernel extract (70% ethanol)	ABTS (IC50) DPPH (IC50) Total phenolic content Total flavonoid content	0.70 mg/mL 11.48 mg/mL 5.86 mg/g 7.78 mg/g	[41]
			[41]
			[41]
			[41]
Kernel oil (Zhejiang No. 1)	DPPH (IC50)	123.76 ± 0.13 μg/mL	[38]
Homogenized hydrosol	ABTS scavenging rate	84%	[50]
	DPPH scavenging rate	58%	[50]
	Hydroxyl radical scavenging rate	60%	[50]
Kernel oil (2% in high-fat diet)	SOD activity GSH-Px activity T-AOC activity MDA level	↑ * ↑ * ↑ * ↓ *	[10]
			[10]
			[10]
			[10]
Kernel oil	Total phenolic content	11.3 mg/kg	[10]

Note: * p < 0.05 compared to the control group; ↑: significantly increased; ↓: significantly decreased; ABTS: 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); DPPH: 2,2-Diphenyl-1-picrylhydrazyl; SOD: Superoxide Dismutase; GSH-Px: Glutathione peroxidase; T-AOC: Total antioxidant capacity; MDA: Malondialdehyde.

.

In vitro studies demonstrate that T. grandis kernel extracts exert antioxidant effects through multiple mechanisms, including direct scavenging of DPPH and ABTS radicals, reduction of Fe³⁺, and enhancement in endogenous antioxidant defense systems [87]. These activities are primarily attributed to polyphenolic compounds, particularly the flavonoids present in the kernels. Advanced LC-MS/MS analysis has identified 107 different flavonoid compounds, with 19 showing significant correlation with antioxidant activity [24,68].

The antioxidant capacity of T. grandis kernels varies significantly with extraction methods and cultivar types. Ni and Shi [28] reported that kernel extracts exhibited moderate scavenging activities against DPPH and hydroxyl radicals (IC₅₀ values of 120 μg/mL and >100 μg/mL, respectively). In contrast, Wang et al. [85] found that cultivated kernel extracts demonstrated superior DPPH and ABTS radical scavenging activities (IC₅₀ values of 0.08 and 0.07 mg/mL, respectively). These discrepancies may reflect differences in sample sources, extraction protocols, and analytical methods. Notably, the Fe³⁺ reducing capacity of T. grandis kernel extracts was second only to walnut kernels, suggesting a selective antioxidant action pattern operating through specific molecular mechanisms.

Under optimized extraction conditions, Zhu et al. [69] found that 50% ethanol extracts exhibited optimal ABTS radical scavenging activity (IC₅₀ = 0.70 mg/mL), while 70% ethanol extracts showed maximum DPPH radical scavenging activity (IC₅₀ = 11.48 mg/mL). Further research by Ni et al. [88] demonstrated that oil extracted from Zhenong 1 cultivar exhibited potent DPPH radical scavenging activity (IC₅₀ = 123.76 ± 0.13 μg/mL) with concentration-dependent characteristics. Additionally, T. grandis kernels contain β-tocopherol, which provides significant cytoprotective effects against oxidative damage while enhancing oil oxidative stability [10].

Processing technologies can significantly influence antioxidant capacity. Yao et al. [89] demonstrated that kernel aqueous colloids subjected to high-pressure homogenization (undiluted) achieved scavenging rates of 84%, 58%, and 60% for ABTS, DPPH, and hydroxyl radicals, respectively, which were significantly higher than untreated colloids due to improved bioactive compound release.

In vivo studies provide further evidence for antioxidant effects. Xiao et al. [90] found that rats supplemented with 2% T. grandis kernel oil showed significantly increased activities of SOD, GSH-Px, and T-AOC in serum, with decreased MDA levels, indicating enhanced systemic antioxidant capacity. However, their study did not investigate the absorption and metabolism of active components, and long-term consumption was associated with adverse effects, including inflammatory cell infiltration in the liver and intestine, highlighting important considerations for safe application.

Zhang et al. [91] conducted research using a combined metabolomics and transcriptomics approach. The study selected three grafted trees of the T. grandis “Merrillii” cultivar, collecting samples at three key developmental stages of kernel development. Each biological replicate at each stage consisted of 10 kernels mixed from a single tree, with three biological replicates in total. The study identified 124 flavonoids, finding that 9 were highly correlated with kernel antioxidant activity. Through gene expression analysis, key synthetic enzyme genes such as CHS, DFR, and ANS were identified, and further experimental validation showed that overexpression of the TgDFR1 gene significantly enhanced antioxidant activity. Their work provides molecular-level insights into the flavonoid accumulation mechanisms and their functions in T. grandis.

It is noteworthy that T. grandis kernel extracts are complex mixtures of various bioactive components, including polyphenolic compounds and tocopherols. These compounds with different chemical structures may produce synergistic antioxidant effects by acting on different oxidative stress targets, scavenging different types of free radicals, or interacting with each other (for example, one antioxidant regenerating another) [92,93]. Therefore, the overall biological activity of the extract may not simply equal the sum of individual component actions. Elucidating these potential synergistic effects is crucial for comprehensively understanding the antioxidant potential of T. grandis kernels, though specific research in this area remains to be conducted.

Current research limitations mainly include the following: in vitro studies are predominant and in vivo mechanistic studies are severely insufficient, particularly with an extreme lack of pharmacokinetic (absorption, distribution, metabolism, excretion) and bioavailability data for key active compounds (such as flavonoids related to antioxidant activity and β-tocopherol) from T. grandis kernels and their metabolites in animals or humans. Obtaining these data is crucial for verifying whether these compounds can achieve effective concentrations in vivo, exert actual biological functions, and determine safe and effective dosages [94]; the assessment of potential synergistic interactions between different bioactive components remains inadequate; and the lack of standardized activity evaluation systems hinders comprehensive comparative analysis across studies. Future research should prioritize addressing these gaps (particularly pharmacokinetic and bioavailability studies) to fully and scientifically elucidate the antioxidant potential of T. grandis kernels in therapeutic applications.

3.2. Anti-Inflammatory Effects

The anti-inflammatory activity of T. grandis kernels is believed to originate from complex interactions among their multiple bioactive components, with several key mechanisms already elucidated. Among these, PMI-PUFAs play a crucial role in regulating inflammatory mediator production [95]. Zhou et al. [96] provided direct evidence for this using molecular distillation technology. They enriched the sciadonic acid content from 11% to 25% and found that this enriched oil (compared to crude oil) significantly enhanced the inhibition of 5-lipoxygenase (5-LOX) (65% inhibition at 66.7 μg/mL) and exhibited superior anti-inflammatory effects in a mouse ear edema model (63.1% inhibition vs. 29.4% for crude oil). Their work not only clarified the anti-inflammatory potential of PMI-PUFAs through 5-LOX inhibition but also directly compared the efficacy differences between enriched fractions and relatively crude extracts (crude oil), addressing practical concerns about the utility of extracts with different processing degrees.

Meanwhile, the ethanol extract of T. grandis kernels (EST), as a complex plant matrix containing multiple compounds [97], also exhibits systematic anti-inflammatory properties through various pathways. Yao et al. [9] revealed that administering 5 mg/kg body weight of EST daily to hyperuricemic mice for two weeks improved hyperuricemia by enhancing endogenous antioxidant defense systems (increasing SOD and GSH-Px activities) while suppressing the expression of pro-inflammatory mediators (IL-1β, TNF-α, PGE₂). This observed regulation of the oxidative stress–inflammation cascade at specific doses suggests potential applications in metabolic diseases [98]. However, it should be emphasized that the overall effect of EST likely originates from complex synergistic or antagonistic interactions among its multiple components (including, but not limited to, polyphenols, fatty acids, vitamins, and other trace constituents), which may result in significantly different mechanisms of action and efficacy compared to single purified compounds isolated from it. Understanding this “matrix effect” is crucial for accurately evaluating and utilizing the biological activities of plant extracts [99], but the current research on T. grandis extracts in this aspect remains insufficient.

The polyphenolic compounds in T. grandis kernels (content of approximately 9.22–22.16 μg/g) are believed to potentially act synergistically with other components within the kernel matrix to exert anti-inflammatory effects through the regulation of inflammatory signaling pathways (particularly NF-κB) [66,100]. The proposed mechanisms include the inhibition of NF-κB pathway activation and NLRP3 inflammasome assembly, the attenuation of mitochondrial ROS production, the inhibition of MAPK pathway activation to reduce inflammatory cell recruitment, and the downregulation of pro-inflammatory factors through the suppression of COX-2 expression/activity and modulation of arachidonic acid metabolism [100,101]. However, it should be noted that these mechanisms are mostly based on in vitro experiments or inferences from general polyphenol functions. Within the specific and complex system of T. grandis kernels, how various polyphenols precisely interact and their interactions with other important components, such as PMI-PUFAs, still require in-depth experimental elucidation.

Although the mechanism of PMI-PUFAs’ anti-inflammatory action through 5-LOX inhibition has been reported, and polyphenols’ regulation of the NF-κB pathway is a known anti-inflammatory mechanism, the distinctive feature of T. grandis kernels lies in their simultaneous abundance of these two classes of anti-inflammatory components with different targets. Therefore, we hypothesize that the anti-inflammatory effects of T. grandis kernels may result from the multi-pathway, multi-target synergistic action of their PMI-PUFAs and diverse polyphenols (potentially including β-tocopherol and other components). This intrinsic combination of constituents may confer advantages over single-component sources when addressing complex inflammatory conditions. However, research directly demonstrating this synergistic effect and its contribution to the overall extract’s efficacy remains lacking, representing an important direction for future investigation.

Despite progress in elucidating the anti-inflammatory effects of T. grandis kernels, the current research still faces significant limitations that restrict its translation to practical applications. First, our understanding remains very limited regarding how interactions (synergistic, antagonistic, or effects on each other’s stability/bioavailability) among complex components within the extract matrix ultimately determine its anti-inflammatory effects [102]. Second, the precise molecular targets and detailed pathways of many active compounds remain incompletely defined. Most critically, the current research evidence relies primarily on in vitro cell models and basic animal models (such as acute inflammation models). To date, literature searches have not identified any published human clinical trials of T. grandis kernel extracts or their specific anti-inflammatory components (such as enriched PMI-PUFAs or specific polyphenols) to directly verify their anti-inflammatory effects and safety in humans. Similarly, efficacy data from animal models more representative of human chronic inflammatory diseases, as well as pharmacokinetic and bioavailability studies of key active components in vivo, are also lacking. Such studies are crucial for confirming the clinical translation potential of findings from in vitro and basic animal models and determining safe and effective dose ranges in humans. This constitutes the main bottleneck in advancing the current research toward practical applications [103,104].

Therefore, future research directions should include in-depth investigation of interaction mechanisms and molecular targets among active components; rigorous pharmacokinetic and bioavailability studies; efficacy validation in more relevant disease models; and, ultimately, well-designed human clinical trials to evaluate safety and efficacy. Additionally, expanding research to other potential health effects, such as cardiovascular protection, and establishing standardized evaluation systems will be crucial for fully realizing the development value of T. grandis kernels as anti-inflammatory and multifunctional health products.

3.3. Cardiovascular and Cerebrovascular Protective Effects

The cardiovascular and cerebrovascular protective effects of T. grandis kernels are supported by multi-level experimental evidence, exhibiting typical “multi-target, multi-pathway” characteristics. The kernels are rich in unsaturated fatty acids (UFAs, reaching 88.65%), particularly oleic acid (35.38%) and linoleic acid (40.61%) [10], which are components widely recognized for their roles in reducing low-density lipoprotein cholesterol (LDL-C), preventing atherosclerosis, and improving lipid metabolism.

Animal experiments have provided important molecular-level insights for understanding their mechanisms of action, particularly through elucidating key signaling pathways. For instance, Xiao et al. [90] demonstrated, in a 7-week high-fat diet rat model, that feeding with 2% T. grandis kernel oil significantly reduced serum total cholesterol, LDL-C, and blood glucose levels. The study thoroughly investigated the underlying mechanisms, finding close associations with the regulation of the AMP-activated protein kinase α (AMPK α)/sterol regulatory element-binding protein-1c (SREBP-1c) signaling pathway. Specifically, AMPK α activation can phosphorylate SREBP-1c at the Ser372 site, thereby inhibiting SREBP processing and transcriptional activity, ultimately suppressing hepatic lipogenesis, improving lipid profiles, and potentially alleviating insulin resistance and atherosclerosis [105,106,107]. This detailed elucidation of the AMPKα/SREBP-1c pathway provides a reasonable molecular explanation for the lipid-lowering effects of T. grandis kernel oil, representing a strength of the current research.

Furthermore, rare fatty acids (such as eicosatrienoic acid, 6.78%–8.37%) and phytosterols (such as β-sitosterol, 0.90–1.29 mg/g) contained in T. grandis kernel oil have also been proven to have significant lipid-lowering effects [66,87]. These compounds may produce synergistic effects with major unsaturated fatty acids through multiple mechanisms, including regulating cholesterol absorption and modulating membrane fluidity [108].

Despite encouraging mechanistic studies (such as AMPK pathway analysis) and preliminary animal experimental results, several important research limitations remain before these findings can be translated into reliable health recommendations or therapeutic applications, particularly the lack of long-term safety and efficacy data:

(1)

Lack of long-term safety and efficacy evaluation: Current animal studies (such as Xiao et al. [90]) have relatively short durations. While they can reveal short-term effects and partial mechanisms, they are insufficient to simulate scenarios of long-term human consumption or chronic cardiovascular disease prevention/treatment. To date, there are no publicly reported long-term (e.g., 6 months or longer) animal functional studies or chronic toxicity tests following standard guidelines (such as OECD and ICH guidelines) for T. grandis kernels or their extracts. Such long-term studies are crucial for comprehensively evaluating sustained efficacy, identifying potential delayed toxicity or cumulative effects, and determining long-term safe dosage ranges [109]. Given that Xiao et al. [90] mentioned (as described in the anti-inflammatory section) that long-term consumption of T. grandis oil under certain conditions may be associated with liver and intestinal adverse effects, rigorous long-term safety and chronic toxicity assessments are particularly necessary.

(2)

Unclear in vivo processes of active components: Pharmacokinetic data (absorption, distribution, metabolism, excretion) and the bioavailability of key active components (such as specific fatty acids, phytosterols, and their metabolites) remain very limited.

(3)

Incomplete risk–benefit assessment: There is a lack of comprehensive risk–benefit analysis based on different doses, populations, and long-term applications.

(4)

Absence of human evidence: Most critically, there is a complete absence of well-designed randomized controlled trials in humans to directly verify the cardiovascular and cerebrovascular protective effects and safety of T. grandis kernels or their products.

In summary, although T. grandis kernels show preliminary, mechanism-level evidence supporting their potential in improving lipid metabolism and cardiovascular protection, future research must address the above gaps to establish their therapeutic value in this field. This requires conducting systematic long-term animal efficacy and safety studies, chronic toxicity evaluations, pharmacokinetic studies, exploration of new mechanisms, including gut microbiota, and, ultimately, high-quality human clinical trial evidence for support.

3.4. Other Biological Activities

T. grandis kernels exhibit diverse biological activities beyond traditional understanding. For instance, Zhu et al. [69] found in vitro that their 70% ethanol extract demonstrated α-glucosidase inhibitory activity comparable to the clinical antidiabetic drug acarbose (IC₅₀ = 0.60 mg/mL) while also showing significant tyrosinase inhibition (IC₅₀ = 6.60 mg/mL). These preliminary findings suggest potential applications in blood glucose control and skin whitening (inhibiting pigmentation). However, translating in vitro enzyme inhibitory activity to long-term in vivo efficacy and safety represents a significant gap. Their specific molecular targets, in vivo metabolic processes, and effects and safety profiles in relevant long-term animal models (such as diabetic models and long-term skin application models) require thorough investigation.

Regarding gastrointestinal function regulation, T. grandis kernel oil shows significant effects, particularly the discovery of its gut microbiota modulation, which adds research value [16]. This aligns with our current understanding of the gut–brain axis and the central role of microbiota in maintaining intestinal health [110]. In a loperamide-induced slow transit constipation model, Wang et al. [16] revealed that T. grandis oil improves constipation symptoms through multiple mechanisms, including upregulating tight junction proteins (Occludin, Claudin-1, ZO-1) to enhance intestinal barrier function; optimizing 5-HT₃ and 5-HT₄ receptor expression balance to regulate serotonin signaling; improving gut microbiota composition (decreasing Verrucomicrobia and Proteobacteria, increasing Firmicutes and Bacteroidetes); and elevating endothelin-1 levels. This multi-pathway systemic regulation provides a molecular basis for developing natural intestinal function modulators derived from T. grandis. However, it should be noted that the study’s observation period (15 days) may be insufficient to fully evaluate long-term management effects and safety for chronic constipation patients.

The unique component of T. grandis kernels—sciadonic acid (cis-5,11,14-eicosatrienoic acid)—also exhibits remarkable biological activities. Particularly noteworthy are the recent findings indicating its ability to improve glycolipid metabolism by regulating insulin signaling pathways and reshaping the gut microbiome [111,112,113,114], again highlighting the potential pivotal role of gut microbiota in mediating various biological activities of T. grandis. Meng et al. [115] developed a silver ion-assisted urea complexation technique that can enrich sciadonic acid (SA) purity in T. grandis kernel oil fatty acid ethyl esters from approximately 9.95% to as high as 89.3% (recovery rate of ~32%). The study mentioned that sciadonic acid has previously been reported by other research to have various biological activities, including anti-inflammatory and anti-tumor effects [116,117,118,119]. Nevertheless, as emphasized in the related literature [6,120,121], the research on the long-term physiological functions, precise dose–response relationships, and, especially, the safety of long-term consumption (including potential toxic side effects) of special fatty acids like sciadonic acid remains very limited. This represents a key challenge in developing it as a functional food ingredient or pharmaceutical preparation.

In summary, while the current research on these “other biological activities” of T. grandis kernels reveals broad application prospects, it still faces several serious challenges: (1) the systematic regulatory networks of key signaling pathways and their cross-talk mechanisms remain incompletely elucidated; (2) our insufficient understanding of potential interactions and overall effect patterns among different biological activities (for example, the intrinsic connections between antidiabetic activity and intestinal regulation or anti-inflammatory activities); (3) the lack of in vivo pharmacokinetic and bioavailability data for key active compounds (such as specific polyphenols and sciadonic acid) responsible for these diverse activities; (4) the almost complete absence of high-quality clinical evaluation studies for these specific indications (such as adjuvant therapy for type 2 diabetes, improvement in skin pigmentation disorders, and long-term management of chronic constipation); and (5) similar to the limitations in cardiovascular research mentioned earlier, there is also a lack of long-term animal model studies or standardized chronic toxicity tests for these diverse biological activities. For instance, studies systematically evaluating the sustained effects of the long-term administration of T. grandis extracts or sciadonic acid on blood glucose homeostasis and pancreatic function, long-term safety on skin conditions and pigment metabolism, or long-term efficacy and tolerance in chronic constipation animal models have not been reported. Given that these potential applications (such as blood glucose management, chronic constipation treatment, and skin care) often involve long-term or even lifelong use, conducting these long-term efficacy and safety studies (including chronic toxicity) is crucial for confirming their application value and mitigating potential risks [122,123].

Future research should prioritize in-depth exploration of the precise molecular mechanisms of various activities and their interconnections, establish standardized in vitro and in vivo activity evaluation systems, focus on comprehensive preclinical safety and efficacy assessments, including long-term administration models and chronic toxicity studies, validate clinical application value for specific indications through well-designed, evidence-based medical research, and develop quality-controllable novel formulation technologies to fully and safely develop and utilize the multifunctional health potential of T. grandis kernels.

4. Factors Influencing Metabolite Composition and Variability in T. grandis Kernels

The synthesis and accumulation of metabolites (particularly fatty acids, phenolics, terpenoids, and vitamin E) in T. grandis kernels is a physiological process finely regulated by multiple factors [124,125]. The primary influencing factor is genetic background, as different T. grandis cultivars exhibit significant variations in oil content and fatty acid accumulation patterns during kernel development [31]. Additionally, developmental stage plays a crucial role; as kernels progress from formation to growth and maturation, their internal metabolic activities undergo significant changes, with dynamic variations not only in oil and fatty acid components [31] but also in the content of other important nutrients, such as amino acids, and the expression of key genes in related biosynthetic pathways [126].

External environmental conditions are equally important for metabolite accumulation. For instance, post-harvest temperature and humidity conditions significantly affect oil quality during the after-ripening process of T. grandis cultivars, particularly the content of easily oxidizable unsaturated fatty acids [87], indirectly indicating the importance of growth environmental factors on metabolite synthesis and stability. Furthermore, cultivation management practices, such as foliar fertilization during the seed filling stage, have been demonstrated to influence final kernel quality parameters [127]. Although post-harvest treatments (such as after-ripening, drying, and roasting) and storage conditions are not biosynthetic processes per se, they significantly alter the final product’s chemical composition and sensory quality by affecting metabolite transformation (such as flavor compound formation) and degradation (such as oxidation) [79]. Therefore, understanding and integrating an analysis of these genetic, developmental, environmental, and anthropogenic factors and their complex interactions is crucial for guiding T. grandis variety breeding, optimizing cultivation management techniques to enhance specific metabolite content, and improving processing technologies to maintain or enhance product value.

Regarding the inherent heterogeneity in trees, the metabolite composition in T. grandis kernels varies significantly among individuals rather than being completely uniform. This inconsistency primarily stems from the combined effects of genetic and environmental factors. First, as an outcrossing species, T. grandis harbors rich genetic diversity within its natural populations and cultivated varieties. Studies using molecular markers (such as EST-SSR) have confirmed significant genetic differentiation among T. grandis populations from different geographical origins, with most genetic variation existing within populations [127,128]. These differences in genetic background directly result in significant variations in major kernel quality traits (such as oil content and fatty acid components) among different local varieties or populations [24,125]. For example, studies have reported that the relative fat content in T. grandis kernels from different populations can range from 29.36% to 42.35% [127].

Second, environmental factors, including macro-scale geographical and climatic conditions and micro-scale habitat differences, profoundly influence metabolite accumulation. Research has clearly indicated that altitude significantly affects both the external appearance and nutritional quality of T. grandis seeds [129]. Additionally, tree age is an important factor; studies have found that kernels from older T. grandis trees (such as 100 and 1000 years old) contain higher levels of total flavonoids and specific flavonoid monomers compared to kernels from younger trees (10 years old), exhibiting stronger antioxidant activity, which is related to age-dependent gene expression regulation [2]. Therefore, the metabolite composition of T. grandis kernels is the result of complex interactions among genetic background, environmental conditions, developmental stage (including tree age), and cultivation management, exhibiting obvious individual and population heterogeneity. This provides a foundation for discovering superior germplasm resources while also posing challenges for quality control and standardized production.

5. Conclusions and Future Perspectives

This review comprehensively integrates the current research on the chemical composition and biological activities of T. grandis kernels, revealing their refined profile of bioactive compounds and multifaceted potential therapeutic value. Recent analytical advances, including visible-near infrared spectroscopy that achieves over 96% accuracy in non-destructive quality assessment [130], have significantly deepened our understanding of this unique plant resource.

T. grandis kernels exhibit exceptional phytochemical characteristics, primarily manifested in three unique aspects. First, their high unsaturated fatty acid content (87.95%–94.25%) includes a significant proportion of PMI-PUFAs, particularly sciadonic acid (9.13%–12.96%), distinguishing them from conventional oil crops. Second, their polyphenol profile contains 107 identified flavonoid compounds, with 19 showing strong correlation with antioxidant capacity. Third, their volatile components are dominated by D-limonene (31.37%–46.16%), which constitutes their distinctive sensory characteristics and exhibits significant biological activity.

Regarding mechanisms of action, preliminary studies suggest that T. grandis kernels may exert “multi-target, multi-pathway” biological effects through integrated molecular networks. Their antioxidant activity is believed to be primarily mediated by polyphenol-driven Keap1-Nrf2-ARE signaling pathways, while their anti-inflammatory properties are hypothesized to originate from PMI-PUFA-regulated inhibition of 5-lipoxygenase and downstream inflammatory mediators. Cardiovascular protective effects are indicated by research to be primarily achieved through the regulation of the AMPK α/SREBP-1c pathway, which inhibits lipogenesis and alleviates dyslipidemia, possibly supplemented by rare fatty acids’ effects on membrane dynamics and cholesterol metabolism. It must be emphasized that our current understanding of these mechanisms is primarily based on in vitro experiments and preliminary animal model studies, and their exact effects in humans require further verification.

Recent technological innovations in extraction methods, including supercritical CO₂ extraction and enzyme-assisted aqueous extraction with microwave pretreatment, have significantly improved yields and the preservation of bioactive compounds, facilitating the development of higher-quality T. grandis products.

However, several critical knowledge gaps and research limitations remain, indicating that our understanding of T. grandis kernels is still in its infancy and requires interdisciplinary research attention.

Reviewing the existing literature, we interpret that although some compounds (such as oleic acid, linoleic acid, and common flavonoids) also exist in other plant resources, the core value and uniqueness of T. grandis kernels lies in several aspects: (1) containing characteristic or rare components, such as a high content of PMI-PUFAs, sciadonic acid, and unique biflavonoids (such as kayaflavone); (2) these compounds exist in unique proportions and combinations, forming a distinct chemical fingerprint, exemplified by their specific ratios of high unsaturated fatty acids to PMI-PUFAs and their D-limonene-dominated unique aroma profile; and (3) multiple different classes of bioactive components (fatty acids, polyphenols, vitamin E, selenium, etc.) coexist within the same matrix, suggesting the possibility of potential synergistic biological effects. Therefore, the research value of T. grandis kernels should not be underestimated merely due to the ubiquity of some components. Future research should not only continue to identify and quantify their components but also focus on verifying the synergistic effects brought by these unique combinations and analyzing in-depth the exact relationship between their specific chemical profile and observed biological activities. This is crucial for scientifically evaluating the health value of T. grandis kernels and guiding their development as functional foods or natural medicines.

The integration of traditional knowledge with cutting-edge analytical techniques offers new possibilities for developing T. grandis-derived products with potentially enhanced biological activities and consistent quality. However, we must clearly recognize that the current evidence for most biological activities and so-called health effects of T. grandis kernels comes predominantly from in vitro studies and preclinical models. Their actual effects in humans, bioavailability, and long-term safety remain to be confirmed through future large-scale, high-quality clinical studies. Nevertheless, with deeper research and the accumulation of future clinical evidence, T. grandis kernels may demonstrate some application potential in functional foods, nutritional supplements, and natural medicines, perhaps providing potential assistance in improving multiple aspects of metabolic health through their diverse bioactive components. The sustainable development and comprehensive utilization of this unique plant resource not only provide opportunities for scientific research but also suggest the possibility of developing high-value products with potential health benefits while preserving biodiversity. However, this ultimately depends on future rigorous scientific validation, strict safety assessment, and sustainable resource management strategies.