Coconut is an important tropical cash crop known for its nutrient-rich meat and water. Coconut water in particular, is a popular natural beverage packed with essential nutrients (Pummer et al., 2001). Coconut water is reported to contain 5-9% total soluble solids (TSS), with over 80% being soluble sugars like glucose, sucrose and fructose (Mahayothee et al., 2016). Additionally, coconut water contains essential components including minerals, amino acids, enzymes, organic acids, fatty acids, vitamins and few phenolic compounds (Kumar et al., 2021). Phenolic compounds are recognized for their bioactive properties, with coconut water containing catechin, salicylic acid, 4-hydroxybenzoic acid, butyric acid, o-coumaric acid, p-coumaric acid, gallic acid and caffeic acid in coconut water (Chang and Wu, 2011; Mahayothee et al., 2016).

Flavonoids as a type of polyphenols widely found in many plants (Kozlowska and Szostak-Wegierek, 2014; Rudrapal and Chetia, 2016), are typically categorized into seven subclasses including, flavonols, flavones, isoflavones, anthocyanins, flavanones, flavanols, and chalcones (Wang et al., 2022). Research has demonstrated that flavonoids are prevalent in plants and play a crucial role in plant growth, reproduction and protection (Tang et al., 2014; Tian et al., 2014; Deng et al., 2019). Flavonoids serve as major pigments in plants and function as antitoxins or antioxidants, protecting plants from biotic and abiotic stress (Cavaiuolo et al., 2013; Iwashina, 2003; Zhang et al., 2020; Pourcel et al., 2007). In the fields of medicine and health, plant-derived flavonoids, as a bioactive compound, have been found to exhibit a broad spectrum of pharmacological effects, including neurological relief, anti-inflammatory, antioxidant, hepatoprotective (Leyva-López et al., 2016; Liu et al., 2017; Al-Khayri et al., 2022; Shen et al., 2022). Furthermore, flavonoids have been shown to help prevent osteoporosis, cancer, and reduces the risk of cardiovascular disease (Imran et al., 2019; Wang et al., 2017; Fernandes et al., 2017; Liu et al., 2021), ultimately having a profound impact on promoting human health and well-being (Tungmunnithum et al., 2018; Batiha et al., 2020; Kumar et al., 2017).

Metabolomics as a key histological technology that focuses on the precise qualitative and quantitative analysis of metabolites, and subsequently performs an in-depth analysis of metabolic pathways and network structures, providing a solid scientific foundation for revealing the metabolic mechanisms underlying the macroscopic phenotypes of different organisms (Fiehn, 2002). In particular, the integration of liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology with chemoinformatics methods has significantly enhanced the ability to screen the diversity of complex metabolites, such as flavonoids, in a high-throughput manner (Akimoto et al., 2017; Tsugawa et al., 2016, 2019).

Coconut water, a natural beverage, that is rich in compounds, but there is a scarcity of reports on flavonoids. The change trend and metabolic mechanism of flavonoids in coconut water at different developmental stages are still unclear. To address this, we employed a combination of LC-MS/MS technique and metabolomics to conduct an in-depth study of flavonoid metabolites in coconut water at different developmental stages of the fruits of two coconut varieties. The aim of this study was to elucidate the characteristics of changes in flavonoid metabolites during the developmental stages of different coconuts and their roles during the ripening process of coconut fruits. The findings on flavonoid synthesis can offer insights into improving coconut quality. Understanding how flavonoids are synthesized in different coconut varieties can provide a scientific basis for enhancing their quality and nutritional value. This research may lead to better methods for heterologous synthesis of flavonoids, contributing to both theoretical knowledge and practical applications in coconut variety improvement.

Flavonoid is one of the important compounds in coconut water, which has an important effect on the growth and development of coconut. The change trend and metabolic mechanism of flavonoid in coconut water at different developmental stages are still unclear. In this study, the related problems were studied by metabonomics method. By examining the flavonoid content and metabolic properties of two coconut varieties (CK and W5) at different stages of fruit development, the results revealed that the total flavonoid content of both varieties exhibited a significant decline with fruit maturation, with the highest content observed at the age of fruit in 2 months. The total flavonoid content of CK varieties was not only numerically higher than that of W5, but also displayed more significant variation ( Figure 1 ), suggesting that CK varieties may possess a more active metabolic mechanism for flavonoid synthesis and regulation, whereas W5 varieties exhibited a more stable metabolic profile. This finding highlighted essential differences in flavonoid metabolism among coconut varieties. Further analysis showed that the total flavonoid content decreased sharply during specific stages of coconut fruit development, such as between 8 and 10 months of age ( Figure 1 ). This phenomenon may be attributed to the slowing down of flavonoid synthesis rates during fruit ripening, the changes in internal chemical composition, and the combination of flavonoids with other compounds (such as free glycoside elements and glycosides) to form more stable and water-soluble glycosidic compounds (such as flavonols, flavonoid glycosides). These glycosidic compounds play a crucial role in plant growth and development (Nabavi et al., 2020; Ojeda et al., 2002; Ferreyra et al., 2007). The metabolic trends of flavonoid compounds in plants have been reported to be diverse. For example, similar decreasing trends in total flavonoid content have been observed in grapes, strawberries, and pecans (Awad et al., 2017; Ferreyra et al., 2007; Jia et al., 2018), whereas contrasting findings have been reported in green beans and mangoes (Huang et al., 2023; Gan et al., 2016; Lu et al., 2019), which highlights the complexity and diversity of flavonoid metabolism across the plant kingdom. As an important secondary metabolite in plants, flavonoids have a more intricate synthesis and metabolism process (Liu et al., 2021). Research has shown that flavonoid biosynthesis in plants is not only tissue-specific, but also regulated by the plant’s developmental stage and influenced by environmental factors (Yang et al., 2018; Shi et al., 2022). Changes in plant chemical composition and bioactivity are strongly correlated with changes in different metabolic stages and climatic factors, such as temperature, soil moisture and rainfall (Yang et al., 2016; Prinsloo and Nogemane, 2018). Drought, high temperature and light exposure affect flavonoid and phenolic content in plants (Carvalho et al., 2010; Zheng et al., 2012; Liu et al., 2011). For instance, high temperatures (30-40°C) inhibit flavonoid biosynthesis, gene expression and enzyme activity (Dela et al., 2003). The sharp decline in flavonoid content between 8 and 10 months of age in the present study may be attributed to the high temperatures during this period. In addition to environmental factors, the differences in the synthesis of different species of flavonoids may also be related to the metabolic pathway, the synthesis of flavonoids involves the metabolic pathway of phenylpropanoid compounds, which involves the catalysis of several enzymes (PAL, C4H, 4CL), which catalyze the deamination of the aromatic amino acid phenylalanine (Phe) to produce coumaric acid (by PAL), then oxidized to 4-coumaric acid (by C4H). Through the addition of coenzyme A, the final activation into p-acyl-CoA (4CL), after a series of complex reactions, the formation of a series of phenolic substances and other secondary metabolites. In addition to environmental factors, differences in the synthesis of flavonoids in different species may be related to metabolic pathways. Flavonoid synthesis involves the metabolic pathway of phenylpropanoids, which involves the catalysis of several enzymes (PAL, C4H, 4CL), These enzymes catalyze the deamination of the aromatic amino acid phenylalanine (Phe) to form coumaric acid (by PAL), which is then oxidized to 4-coumaric acid (by C4H). Then, a series of secondary metabolites such as phenolics are formed through the addition of coenzyme A (CoA), which is finally activated into p - acyl- CoA (4CL), after a series of complex reactions (Nabavi et al., 2020). Flavonoids may also undergo a series of modification (glycosylation, methylation) and translocation processes after synthesis, which also affect the final content and distribution of flavonoids. The Arabidopsis UDP glycosyltransferases, UGT79B2 and UGT79B3, function as anthocyanin rhamnosyltransferases that confer abiotic stress tolerance by regulating anthocyanin accumulation (Li et al., 2017), There may be differences in the activity of these enzymes and substrate availability in different species, leading to differences in the rate of flavonoid synthesis and products. On the other hand, genetic factors also influence the differences in flavonoid content among different varieties, and the expression of genes involved in the flavonoid synthesis pathway varies among different varieties of plants. These genes include genes encoding flavonoid synthases (CHS, CHI, F3H) and transcription factors that regulate the expression of these genes. Mutants of several pro-flavonoid compound synthesis key enzymes corresponding to the key genes CHS, CHI, F3′H, and DFR were isolated in Arabidopsis thaliana, which play an important role in the synthesis of plant flavonoid compounds (Shen et al., 2022); In tobacco, both transcription factors NtMYB12a and NtMYB12b are involved in regulating flavonoid biosynthesis; and in tomato, the transcription factor WD40 protein SlAN11 is coordinated with bHLH and MYB proteins to regulate flavonoid biosynthesis and seed dormancy (Gao et al., 2018); Overexpression of the transcription factor MdWRKY11 in apple promoted the expression of F3H, FLS, DFR, anthocyanin synthase (ANS), and UFGT, which contributed to the increased accumulation of flavonoids and anthocyanins in apple healing tissues (Wang et al., 2018). CsMYB6A and CsUGT72AM1, key genes identified from tea tree, regulate the accumulation of anthocyanins and flavonols in purple leaf tea (He et al., 2018).Differences in gene expression directly lead to differences in the rate and products of flavonoid synthesis, thus affecting the flavonoid content in different varieties. It is hypothesized that the decrease in flavonoid content in this study is related to the conversion of other substances, environmental factors, metabolic pathways and genetic factors.

The flavonoid species and their metabolic properties in coconut water were systematically analyzed by UPLC-MS/MS technique and metabolomics approach, resulting in the detection of a total of 123 flavonoid compounds. The results of PCA and cluster analyses further emphasized this difference, indicating that metabolite accumulation was significantly higher in the CK variety compared to the other periods and in W5 ( Figure 3 ). In the screening of differential metabolites, we identified a series of metabolites that differed significantly between CK and W5 varieties, including catechins and epicatechins, which play key roles in flavonoid biosynthesis. There are four common catechins: (+)-catechin, (-)-catechin, (+)-epicatechin, and (-)-epicatechin. Catechin biosynthesis involves the general phenylpropane pathway, which branches out into specific pathways of flavonoid biosynthesis (Xiong et al., 2013; Yu et al., 2022). Numerous studies have demonstrated that low concentrations of catechins have hormonal effects, promote, and enhance resistance, whereas high concentrations of catechins may be inhibitory (Hong et al., 2015; Prithiviraj et al., 2007; Ahammed et al., 2018). Additionally, low doses of (±) - catechins have been shown to elicit a moderate increase in ROS accumulation in the meristematic tissues of treated plants, which may trigger cell wall loosening, thereby promoting cell division and cell expansion for growth (Ahammed et al., 2023). In this study, we found that metabolites such as (+)-catechin and (-)-epicatechin were detected in the flavonoid biosynthesis pathway of W5 compared to CK and showed a significant down-regulation trend. Furthermore, gallocatechin and (-)-epiafatechin also exhibited significant down-regulation trends during fruit development ( Figure 8 ). Based on these findings, it was inferred that W5 grows and develops better relative to CK and may accumulate more metabolites such as amino acids, proteins and sugars.

Flavonoids from coconut water species have been found to possess medicinal value, based on the results of KEGG functional annotation and enrichment analyses, which showed that flavonoids and flavonols have distinctly different distributions in the two species, with CK containing a higher abundance of flavonoids, while W5 up-regulated the accumulation of metabolites, particularly kaempferol-3-O-glucoside ( Figure 8 ). Notably, Kaempferol-3-O-glucoside has been reported to exhibit a range of biological activities, including antimicrobial, anti-inflammatory, neuro- and cardioprotective, antitumour and antidiabetic properties (Riaz et al., 2018). Additionally, it has been shown to inhibit intestinal α-glucosidase and α–amylase activities (Hong et al., 2013), may act as a potential insulin secretagogue, contributing to glucose homeostasis (Rey et al., 2019). The study showed low expression of several differential metabolites such as catechin, saccharin chalcone, lonicera glycosides, rutin and other metabolites. Notably, the study showed that the expression of catechin changed noticeably, whereas the levels of other flavonoid metabolites remained relatively stable. These findings suggest that kaempferol-3-O-glucoside, and catechin may be a class of differential substances characterized in CK and W5. Differential metabolites were mainly concentrated in the flavonoid and flavonol biosynthesis pathways ( Figure 7 ), highlighting the importance of flavonoids in coconut fruit metabolism and implying that they may play a role in physiological functions and stress responses in the fruit.

The present study compared the changes in flavonoid metabolites in coconut water from CK and W5 varieties at different stages of growth. The results showed that t the flavonoid content decreased significantly with fruit maturation, with a peak at 2 months of age and then gradually decreased. The flavonoid content of CK varieties was higher than that of W5 and varied more drastically, indicating fundamental differences in flavonoid synthesis and regulation between the two varieties. KEGG enrichment analyses showed that differential metabolites were mainly enriched in the flavonoid biosynthesis pathway (ko 00941), the flavonoid and flavonol biosynthesis pathway (ko 00944). Furthermore, the accumulation of key metabolites, such as kaempferol-3-O-glucoside and catechin, varied according to cultivar and developmental stage. This study not only enhanced our understanding of differential metabolism of flavonoids in coconut water, but also provided valuable insights into the study of flavonoid metabolic pathways and functions, which can contribute to the improvement of coconut varieties and quality enhancement. However, the study was limited by the sample size and inadequate analysis of environmental factors, and the specific mechanisms of flavonoids in coconut development require further in-depth exploration. Future studies should focus on these aspects to fully elucidate the complexities of flavonoid metabolism in coconut water.