Investigating Changes in Flavonoid Metabolite Profiles in Coconut Water with LC-MS/MS

While the meat and water of the coconut have garnered significant attention for their richness in healthful flavonoids, the dynamics of flavonoid metabolites in coconut water during different developmental stages remain poorly understood. Research conducted by the Chinese Academy of Tropical Agricultural Sciences (Wenchang, China) investigated the changes in flavonoid metabolite profiles in coconut water from two varieties, Wenye No.5 (W5) and Hainan local coconut (CK), across six developmental stages, utilizing liquid chromatography-tandem mass spectrometry (LC–MS/MS) in their work. Their findings have repercussions in yielding coconut-based products offering superior nutritional and functional properties. LCGC International spoke to Wenrao Li and Chengxu Sun, corresponding authors of the paper that resulted from this work, about their team’s findings.

To what do you owe the recent popularity surge in coconut water?

The popularity of coconut water can be mainly attributed to its unique health benefits and refreshing taste. As a natural beverage, coconut water is rich in electrolytes, minerals, and vitamins, which help maintain the body's hydration balance and promote health. Its low-sugar, additive-free characteristics also meet the modern consumers' pursuit of a healthy lifestyle, especially enthusiasts and health-conscious consumers. In addition, the refreshing taste and natural sweetness of coconut water provide a pleasant drinking experience. In the context of multiculturalism, coconut water has also become a symbol of tropical charm, attracting the favor of many consumers. With the diversification of market channels, the purchase of coconut water has become more convenient, usually sold in the form of easy-to-open coconut fruits or mixed coconut water, which also meets the needs of busy urban dwellers seeking a convenient lifestyle, further promoting its popularity and acceptance.

What exactly are flavonoids, and why are they important?

Flavonoids are a class of important plant secondary metabolites that are widely present in a variety of food plants such as fruits, vegetables, legumes, and tea. They exist in the form of conjugates (glycosides) or free forms (aglycones). Flavonoids are important because they possess a range of biological activities, such as antioxidant, anti-inflammatory, anticancer, hypoglycemic, and cardioprotective effects, which have significant benefits for human health. For instance, their antioxidant action can help eliminate free radicals in the human body, reducing oxidative stress and cellular damage; their anti-inflammatory action can inhibit inflammatory responses and alleviate discomfort caused by inflammation; their anticancer action can prevent the formation and spread of tumor cells. Additionally, flavonoids contribute to lowering blood pressure, improving blood circulation, and reducing the risk of cardiovascular diseases. Therefore, flavonoids have received extensive attention and research in fields such as food science, nutrition, and medicine. Although flavonoids have shown potential health benefits in laboratory studies and some observational studies, more research is needed to fully understand their effects on human health. Thus, it is encouraged for people to consume a diet rich in fruits and vegetables, which are natural sources of flavonoids, to promote overall health.

In your paper (1), you state that there is a scarcity of reports on flavonoids found in coconut water. Why do you believe this is the case?

Firstly, as the juice of a tropical fruit, traditional research on coconut water has focused more on nutritional components, taste, and physiological functions, with relatively less study on specific chemical components, especially flavonoids. Secondly, the content of flavonoids in coconut water is relatively low, and the forms in which flavonoids exist are complex, increasing the difficulty of detection and research. Traditional analytical methods cannot accurately and comprehensively detect the flavonoid components in coconut water. Therefore, it is with the application of modern biotechnological methods that the role of flavonoids in coconut water has gradually been revealed to people. Although coconut water, as a natural healthy beverage, has broad prospects for research and application, research on its flavonoid components still needs to be further strengthened, especially in terms of optimal amounts and specific health outcomes, to gain a more comprehensive understanding of its biological functions and nutritional value.

Your team employed the metabolomics approach using liquid chromatography-tandem mass spectrometry (LC–MS/MS) to investigate the changes in flavonoid metabolite profiles in coconut water. What was it about these techniques that made you select them for your analysis and what does the term metabolomics approach refer to in this context?

As we gradually recognized the potential role of flavonoid substances in our research on coconut water, we first attempted to use traditional physiological methods and detected slight variations in the total amount, which excited us. However, we could not precisely understand the changes and effects of flavonoids until we learned about LC–MS/MS technology in other related studies. LC–MS/MS combines the high separation capability of liquid chromatography with the high sensitivity and selectivity of mass spectrometry. Moreover, the multi-stage mass analysis function of tandem mass spectrometry further enhances the specificity of the analysis, making it possible to accurately identify and quantify target compounds in complex matrices. This method, by systematically analyzing the flavonoid metabolite profile in coconut water, can provide insights into the biological characteristics and quality changes of coconut water, offering a scientific basis for the development and utilization of coconut water. Therefore, LC–MS/MS technology, with its outstanding analytical performance, has become the ideal choice for studying the changes in the flavonoid metabolite profile of coconut water, and metabolomic methods, based on this, provide strong support for in-depth research on coconut water.

Briefly state your findings in this study.

This study compared the changes in flavonoid metabolites in the coconut water of coconut varieties CK (indigenous variety in China) and W5 (introduced from Vietnam) during various stages of growth. It was found that the content of flavonoids significantly decreased with fruit maturation, especially after peaking at the age of two months and then gradually decreasing. The CK variety had a higher content of flavonoids than W5, and the changes were more dramatic, indicating that there are essential differences between two varieties in flavonoid synthesis and regulation. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the differential metabolites were mainly enriched in the flavonoid biosynthesis pathway (ko 00941) and flavonoid and flavonol biosynthesis pathway (ko 00944), with key metabolites such as kaempferol-3-O-glucoside and catechin accumulation varying by variety and developmental stage. The study not only enhances the understanding of the differential metabolism of flavonoids in coconut water but also provides clues for the study of flavonoid metabolic pathways and functions, which is helpful for the improvement of coconut varieties and quality enhancement. However, the study is limited by the small sample size and insufficient analysis of environmental factors, and the specific mechanisms of flavonoids in coconut development still need further exploration. Future research should focus on these aspects.

Do your findings correlate with what you had hypothesized?

When we obtained the research results, we realized that it was consistent with our initial hypothesis. We hypothesized that there would be a certain regularity in the flavonoid metabolites present in coconut water at different ages. Using LC–MS/MS technology, we actually detected a variety of flavonoid metabolites in coconut water at different ages, and knew their content, distribution, and patterns of change, ultimately arriving at the aforementioned findings. These discoveries not only validated the reasonableness of the hypothesis but also further enriched our understanding of flavonoid metabolites in coconut water. Together, our hypothesis and findings form a comprehensive framework for the study of flavonoid metabolites in coconut water.

Was there anything particularly unexpected that stands out from your perspective?

Throughout our entire research process, we indeed encountered results that we did not anticipate. For instance, we discovered some previously unforeseen flavonoid metabolites. Although we had a certain understanding of the types of flavonoids in coconut water, the high sensitivity and resolution of LC–MS/MS technology allowed more unknown metabolites to come into view. While based on previous studies, we speculated that the content and proportion of flavonoid metabolites in coconut water might vary by variety (origin), when the actual results were presented to us, we found that the degree of difference exceeded our predictions. Particularly, the significant differences in the flavonoids content of exotic species. In addition, during the data analysis process, we discovered some complex interactions and correlations among flavonoid metabolites, relationships that we did not anticipate. It reveals new mechanisms or regulatory pathways of flavonoid metabolism in coconut water. These findings will provide new perspectives and ideas for the in-depth study of coconut water.

Were there any limitations or challenges you encountered in your work?

During the research process, we did indeed encounter some challenges. For instance, the complexity of coconut water samples is a significant issue, as it contains a variety of components that can interfere with target substances in LC–MS/MS analysis, making it difficult to accurately separate and detect flavonoid metabolites. Secondly, the pre-treatment process of coconut water samples is also fraught with challenges. The content of flavonoid metabolites in coconut water is low, and they are susceptible to degradation due to factors such as light and heat. Therefore, great care must be taken during the extraction and purification process to avoid loss or alteration of the target compounds. Additionally, data analysis and result validation is a daunting task. Due to the multitude of flavonoid metabolites in coconut water, and the potential presence of isomers, careful analysis of mass spectrometry data and combined with other information for comprehensive judgment is required. These limitations and challenges have prompted us to continuously optimize experimental methods, in order to gain a deeper understanding of the patterns and biological functions of flavonoid metabolites in coconut water.

What best practices can you recommend in this type of analysis for both instrument parameters and data analysis?

In terms of instrument parameters and data analysis, we have the following suggestions: First, select instruments with high resolution and high sensitivity, such as high-resolution mass spectrometers (for example, liquid chromatography-mass spectrometry [LC–MS] or gas chromatography-mass spectrometry [GC–MS]) and nuclear magnetic resonance (NMR) devices, to ensure accurate detection of minor changes in metabolites and to ensure that the selected instruments have good compatibility for the smooth separation and detection of metabolites. Second, in terms of data analysis, preprocess the raw data, including noise reduction, baseline correction, spectral peak detection and identification, to improve data quality. Convert spectral information into a unified data set and perform sample normalization and variable scale scaling for data preprocessing, so as to proceed with subsequent statistical analysis. Third, associate the screened biomarkers with biological metabolic pathways or metabolic networks, and reveal the overall metabolic dynamic changes of the organism from the perspective of metabolic networks or metabolic pathways.

Do you imagine this technique being used to detect changes in flavonoid metabolite profiles in other beverages?

LC–MS/MS technology can indeed be used to detect changes in flavonoid metabolites in other beverages. This technique is renowned for its high sensitivity, high resolution, and high specificity, making it highly suitable for the separation and detection of trace components in complex matrices. In beverage analysis, LC–MS/MS can accurately identify and quantify various flavonoid metabolites, including flavones, flavonols, and isoflavones. The content and distribution of these flavonoid metabolites in beverages may be influenced by a variety of factors, such as the source of raw materials, processing techniques, and storage conditions. Through the use of LC–MS/MS technology, we can gain a deeper understanding of the mechanisms by which these factors influence the metabolites, providing a scientific basis for quality control and standardized production of beverages. Therefore, LC–MS/MS technology has a broad application prospect in the field of flavonoid metabolite analysis in beverages.

Can you please summarize the feedback that you have received from others regarding this work?

We have received positive feedback from peers and experts. They generally believe that this work is of significant importance for gaining a deeper understanding of the biological characteristics and quality changes of coconut water. Our results have revealed the complexity and diversity of flavonoid metabolites in coconut water, providing a new perspective for the development and utilization of coconut water. At the same time, they also pointed out some highlights of our work, such as the rationality of the experimental design and the rigor of data analysis. These feedback comments not only affirm our work but also provide necessary references for our future research directions. Overall, these feedback comments demonstrate the innovation and practicality of our research in the field of flavonoid metabolites in coconut water, offering new ideas and methods for researchers in related fields.

What are the next steps in this research and are you planning to be involved in improving this technology?

Regarding the next steps in using LC–MS/MS technology to study the flavonoid metabolite profile in coconut water, the focus is mainly on the following aspects: First, further optimization of experimental conditions and methods is planned to enhance the detection sensitivity and accuracy of flavonoid metabolites in coconut water. Second, the sample size will be expanded to perform genome resequencing on coconuts at different developmental stages, studying the differences between the genomes of coconuts from different varieties and origins, as well as the relationship between these differences and the growth and development of coconuts. Third, a joint analysis of the coconut genome and metabolome will be conducted to explore the relationship between the genome and metabolites at different developmental stages. We plan to continue in-depth research on the application of LC–MS/MS technology in the detection of flavonoid metabolites, committed to improving the practicality and accuracy of the technology, and contributing to research in the fields of food science and nutrition.

Reference

1. Hou, M.; John Martin, J. J.; Song, Y.; Wang, Q.; Cao, H.; Li, W.; Sun, C. Dynamics of Flavonoid Metabolites in Coconut Water Based on Metabolomics Perspective. Front. Plant Sci. 2024, 15, 1468858. DOI: 10.3389/fpls.2024.1468858

Wenrao Li (above), Chengxu Sun (below), and their team members have been dedicated to researching the breeding of germplasm resources, physiological ecology, plant metabolomics, genomics, and digital agriculture of tropical palm crops. They also focus on the root functions of invasive plants, their interactions with the environment, and their metabolic mechanisms. As their main research material, coconuts have made significant progress in the breeding of new varieties, non-destructive detection and identification, and the metabolic mechanisms of quality formation. The team has obtained five Chinese invention patents, including a method for identifying coconut cold tolerance based on digital models. In addition, they have been granted three new coconut varieties (Wen Ye No. 4, Wen Ye No. 5, and Wen Ye No. 6) and two new variety protection rights for coconut-like plants. They have successfully planned the "Wenchang Coconut" as a regional brand for agricultural products with geographical indications, which is currently showing a good development trend and enjoys a high level of market recognition. Currently, they are committed to the design and development of functional coconut water products. Photos courtesy of Li and Sun.