Quantifying Catechins in Endophytic Fungi Isolated from Tea Leaves with High Performance Liquid Chromatography (HPLC)

Tea, Camellia sinensis (L.) Kuntze, is one of the major cash crops of the Indian sub-Himalayan region due to its acceptance as a recreational drink and functional food, as well as in evidence-based pharmacology. Catechins are the major bioactive component in tea, and they are known to offer metabolic health benefits due to the anti-inflammatory and antioxidative effects. Recently, there has been worldwide increased demand for alternate sources of catechins in the nutraceutical, pharmaceutical, and cosmetic industries, as well as the coinciding decline in tea production. A recent study conducted at the Department of Biotechnology of Thapar Institute of Engineering & Technology (Punjab, India) examined the isolation and characterization of catechin-producing endophytic fungi isolated from tea leaves, their chemical characterization, and associated bioactivities. LCGC International spoke to Priyankar Dey of the Thapar Institute of Engineering & Technology about the work done by his group, and the paper that resulted from it.

Your paper (1) is based on your work isolating and characterizing catechin-producing endophytic fungi isolated from tea leaves, their chemical characterization, and associated bioactivities. How prevalent a problem is this?

This work is relevant because it may help meet the increasing need for catechins, which are essential for metabolic health because of their anti-inflammatory and antioxidative qualities. Alternative sources of these bioactive chemicals are desperately needed since climate change is making the drop in tea production worse, particularly in the sub-Himalayan area. The research emphasizes how endophytes, or microorganisms found within plants, may improve host plants' metabolomics and stress tolerance while also having the potential to be a sustainable supply of catechins. These endophytes can create comparable advantageous metabolites because they share metabolic pathways with their host plants. Global cultivation of medicinal plants, including tea, has been significantly impacted by climate and environmental factors, requiring creative approaches to preserve pharmacologically significant phytochemical outputs. Although this tactic is yet unexplored, endophytes from Camellia sinensis (L.) Kuntze may provide a fresh alternative. Finding the endophytes in tea that produce catechins may have an advantage: it might encourage ecologically friendly farming methods and provide a different supply of catechins. However, the natural commensalism required for such advantageous plant-microbe interactions may be hampered by present farming practices, which heavily rely on agrochemicals. This research is important because it may yield new endophytes from C. sinensis that produce catechins. This could change the way we source these compounds for the pharmaceutical, cosmetic, and nutraceutical industries and help traditional agriculture remain sustainable in the face of climate variations.

Do these fungal issues apply to all or most types of plants used in tea mixtures or only to specific types of teas?

Endophytic fungi are commonly found in almost every plant species, regardless of whether these plants grow naturally in the wild or are cultivated in agricultural settings. In many cases, these fungi establish beneficial symbiotic relationships with their host plants. Within this symbiosis, the fungi receive shelter and nutrients from the host plant, which, in return, gains enhanced defense mechanisms against pathogens that might otherwise invade and cause harm. Intriguingly, these endophytes can sometimes acquire genes responsible for the synthesis of secondary metabolites from their host plants. This genetic acquisition enables the fungi to produce bioactive compounds independently, contributing further to their symbiotic role. Nonetheless, it is important to note that not all interactions between endophytic fungi and plants are beneficial; some fungi can become pathogenic, leading to diseases in the host plants. The prevalence of endophytic fungi may be significantly reduced in agricultural systems where plants are regularly treated with high doses of pesticides and fungicides, which inhibit the colonization of these fungi. Consequently, the presence of endophytes in plants such as tea is a common phenomenon, not a rarity.

What are the potential ramifications of their presence to a tea drinker’s health?

Because tea contains bioactive catechins, which are known to have health advantages, it is used both recreational and medical purposes. The new work provides encouraging pathways for the sustainable manufacturing of catechins, even if it may not immediately change the health of tea users. By separating the endophytes that produce catechins from tea leaves, it becomes possible to cultivate these endophytes in large quantities, establishing a reliable supply of these bioactives for pharmacological and therapeutic uses. Moreover, the catechin production from these endophytes might be maximized by using metabolic and genetic engineering to improve catechin biosynthesis. This study helps the sustainable fulfilling of the growing demand in the healthcare industry for products high in catechins. The research tackles both short-term and long-term issues in the business by concentrating on scalable manufacturing techniques for bioactive substances obtained from tea, which makes it easier to produce catechins in large quantities. This strategy might revolutionize the way these priceless chemicals are acquired for widespread medicinal applications by meeting growing therapeutic needs without sacrificing environmental sustainability.

Are there other implications, such as from an economic standpoint?

Tea leaves are currently the most reliable source of bioactive catechins, which are vital for various applications such as evidence-based pharmacology, functional food research, and tea-centric prophylactic strategies. This is particularly true for green tea leaves, given their high catechin concentration. However, the cultivation of tea, essential for catechin extraction, is fraught with numerous economic challenges. Factors such as changing seasonal temperatures and rainfall can lead to unpredictable harvests, while fluctuating market prices can significantly impact growers' income. High production costs and labor shortages further complicate the economic landscape, as do land degradation and pest and disease management issues. Ensuring quality and maintaining compliance with regulatory standards require additional resources, and access to necessary financial and technological support can be limited. Given these constraints, relying solely on conventional tea cultivation to meet the growing demand for catechins is increasingly challenging. As an alternative, endophytic fungal biosynthesis of catechins presents a more economical solution. This method offers sustainable and rapid production capabilities, potentially alleviating many economic constraints associated with tea-derived catechin production and supporting a consistent and scalable supply chain.

What causes the fungus to infect the tea leaves? Are there steps growers can take to prevent the infection?

Endophytic fungi infect host plants primarily to secure a stable environment that provides essential nutrients and protection necessary for their growth and reproduction. By colonizing the internal tissues of plants without causing immediate harm, these fungi gain access to carbohydrates and other nutritional resources produced by the plant. This association often begins when fungal spores enter the plant through stomata, wounds, or root tissues. Within the host, these fungi find a favorable habitat that shields them from external adversities such as extreme weather and predators. In turn, many endophytic fungi offer significant benefits to the host plant, such as enhanced resistance to pests, pathogens, and environmental stresses, creating a symbiotic relationship. This mutually beneficial interaction fosters improved plant health and survival, thereby ensuring the persistence and proliferation of the fungi within the ecosystem.

It is to be noted that all endophytic fungi are not pathogenic. However, plant growers can take several steps to prevent or reduce infections caused by pathogenic endophytic fungi. Firstly, implementing integrated pest management (IPM) strategies can help maintain a balanced ecosystem, reducing the need for excessive chemical use that may disturb beneficial microbes. Regular monitoring of plant health and soil conditions can identify early signs of fungal infection, allowing for timely interventions. Selecting resistant plant varieties and ensuring proper crop rotation can minimize conditions favorable to harmful fungi. Additionally, practicing good sanitation by removing infected plant debris and maintaining optimal growing conditions such as proper irrigation and soil health can further reduce the risk of infection while fostering beneficial microbial communities.

Your team used high performance liquid chromatography (HPLC) and gas chromatography–mass spectroscopy (GC–MS) in their work. What was it about these techniques that made you select them for your analysis?

In the present study, we used HPLC to quantify catechin and epigallocatechin gallate (EGCG) and used GC–MS coupled with Silylation to elucidate the untargeted metabolomic profile of the endophytic fungi. Choosing HPLC to quantify catechin and EGCG in endophytic fungi isolated from tea leaves offered several advantages. HPLC is established for its precision, accuracy, and ability to separate complex mixtures, making it ideal for analyzing catechins and other polyphenolic compounds that coexist in intricate biological matrices of fungal extracts. HPLC is highly sensitive and specific, allowing the detection and quantification of catechin and EGCG even at low concentrations. It is a targeted approach since catechin quantification was based on a standard curve. The HPLC method we followed was well-established, and we have already used this method to quantify catechins in green tea in several of our previous studies. The method also provided quick analysis with high resolution, reducing the time required to obtain comprehensive data. Therefore, HPLC serves as a robust and versatile analytical tool for reliably quantifying catechins and EGCG in tea-derived endophytic fungi, contributing valuable insights into their metabolic activities and potential health benefits.

Choosing GC–MS coupled with silylation for untargeted metabolomics of endophytic fungi from tea leaves offered comprehensive advantages. This method excels in the detection and analysis of a wide range of metabolites, particularly low molecular weight and volatile compounds, by enhancing their volatility and thermal stability through silylation. Silylation effectively derivatizes polar functional groups, facilitating the separation and identification of diverse metabolites, including sugars, amino acids, and organic acids. GC–MS provides high sensitivity, specificity, and resolution, making it ideal for complex biological matrices like fungal extracts. Moreover, its ability to provide detailed mass spectral data enabled accurate structural elucidation and identification of unknown compounds. The robustness and reproducibility of GC–MS with silylation make it a powerful approach for comprehensive metabolomic profiling, offering insights into the metabolomic landscape of endophytic fungi.

Briefly state your findings in this study.

The main objectives of the study were to isolate and characterize catechin-producing endophytic fungi from organically grown tea leaves as an alternative source of catechins, using HPLC and GC-MS for metabolomic profiling and pathway identification. Additionally, the study aimed to evaluate the bioactivities of the fungal extracts, particularly their antioxidant properties and potential prebiotic effects on Lactobacillus species, thereby offering a sustainable solution to the declining tea production due to climate change. The study identified two endophytic fungal isolates, CSPL6 and CSPL5b, characterized as Pseudopestalotiopsis camelliae-sinensis and Didymella sinensis, respectively, based on their morphological and molecular traits. Chemical analysis revealed that both isolates had significant phenolic and flavonoid contents, with CSPL6 and CSPL5b exhibiting high concentrations of catechins and EGCG as determined by HPLC. Untargeted metabolomics identified 79 and 66 metabolites in CSPL6 and CSPL5b, respectively, with distinct enrichment patterns for metabolic pathways related to fatty acid and amino acid metabolism. Both fungal extracts demonstrated antioxidant activities, with CSPL5b showing superior effects in scavenging various radicals compared to CSPL6. Additionally, both extracts exhibited prebiotic effects on Lactobacillus species at low concentrations, enhancing their growth, though higher doses were inhibitory. These findings suggest that CSPL6 and CSPL5b are potential alternative sources for producing bioactive catechins with beneficial antioxidant and prebiotic properties.

Do your findings correlate with what you had hypothesized?

Endophytic fungi are commonly found in wild plants, but their prevalence and diversity can significantly decrease in commercially cultivated cash crops due to the extensive use of pesticides and fungicides. Despite numerous studies attempting to isolate endophytes from tea leaves, none had successfully identified catechin-producing varieties. Addressing this gap, we focused on isolating endophytes from organically grown tea plants, hypothesizing that such conditions would preserve a diverse range of endophytic fungi. Our hypothesis had two main components: first, that endophytes acquire secondary metabolite biosynthetic genes from their host plants through horizontal gene transfer, suggesting that tea-associated endophytes could obtain catechin-biosynthesizing genes from tea plants; second, that organic tea plantations are optimal sites for isolating and characterizing these catechin-producing endophytes. Our study confirmed this hypothesis, successfully isolating catechin-producing endophytes and demonstrating the antioxidant and prebiotic properties of their catechin-rich extracts. This research suggests that these isolated endophytes can serve as a sustainable alternative source of bioactive catechins, reducing reliance on tea plants for these compounds. Thus, our findings present an innovative approach to harnessing the potential of endophytes for producing valuable phytochemicals, offering promising implications for nutraceutical and pharmaceutical applications.

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

The study was driven by a clear hypothesis and followed a straightforward methodology. A notable finding was that while several endophytes were isolated from different parts of the tea plants, not all had the ability to produce catechins. Specifically, endophytes capable of producing catechins were predominantly found in leaf isolates. This suggests that not all fungal endophytes can acquire genes necessary for catechin biosynthesis from the tea plants, although further validation is required to confirm this hypothesis. It was also observed that the identified endophytes, Pseudopestalotiopsis camelliae-sinensis and Didymella sinensis, were not new to tea plants; however, their potential to produce catechins was documented for the first time. Consistent with our hypothesis, this discovery might be attributed to their isolation from organically grown tea plants, which may provide a conducive environment for the acquisition of catechin-producing capabilities.

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

Our study, while providing valuable insights, has several limitations that should be acknowledged. One of the primary limitations is that sampling was conducted at a single tea plantation. This limits our ability to generalize findings about the abundance of catechin-producing endophytes across different geographical locations or environmental conditions. Hence, our observations cannot conclusively determine if the presence of these endophytes is specific to the sampled location or if it reflects a broader trend across various tea-growing regions. Moreover, our efforts to isolate catechin-producing endophytes focused exclusively on tea leaves, as they are known to contain the highest catechin concentrations. However, it is plausible that similar endophytes could reside in other parts of the tea plant, such as roots or stems, which were not explored in this study. Future research should consider these plant parts to potentially uncover more sources of catechin-producing endophytes. Furthermore, while we concentrated on catechin and EGCG, tea plants may host endophytes capable of producing a range of other valuable secondary metabolites, which were not assessed in our study. Exploring these compounds could provide a more comprehensive understanding of the beneficial endophytes present. In terms of methodology, we employed GC–MS for untargeted metabolomics analysis. Nevertheless, incorporating LC–MS-based untargeted metabolomics could have allowed the identification of higher molecular weight, polar secondary metabolites, enriching the chemical profile of our findings. Finally, although we successfully isolated nine distinct endophyte strains from tea leaves, our focus remained on the two isolates that produced catechins. Consequently, the potential bioactivities and metabolic capabilities of the remaining seven isolates remain unexplored, representing an area for future research.

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

In the current study, we employed targeted HPLC analysis for quantification of catechin and EGCG, whereas GC–MS coupled with silylation was utilized for untargeted metabolomic fingerprinting. The HPLC methodology utilized was quite straightforward and an established process. But for the GC–MS analysis, we recommend certain caution that needs to be taken, mostly for analyses that are outsourced, to maintain data quality and reproducibility. A primary challenge is the absence of replicates, as researchers often conduct single runs of pooled samples, which undermines the integrity of the findings. Replicates are essential for verifying data and ensuring trustworthy results, yet, they are frequently overlooked. Another significant issue is the optimization of instrument protocols. Unique characteristics of each sample necessitate tailored optimization, including adjustments in sample preparation and critical instrument settings, such as injector port and transfer line temperature. These adjustments are vital to prevent issues like compound accumulation and misidentification during analysis. Additionally, proper maintenance between runs, including washing the column and managing temperature gradients, is crucial for consistent peak resolution and reliable quantitative evaluations. Data interpretation poses another challenge, as outsourced analyses often provide tentatively identified compounds lacking thorough examination. To validate these identifications, careful analysis of mass fragment patterns is essential, an expertise that only comes from experience. Furthermore, the use of internal standards is crucial for accurate quantification, as variations in ionization efficiencies can mislead interpretations of phytochemical concentrations.The complexity of plant extracts complicates the analysis, as intricate mixtures may interfere with chemical separation, leading to the misidentification of low-abundant compounds. Derivatization is often necessary to make polar, high molecular weight phytochemicals suitable for GC–MS, yet optimizing conditions for this process can be challenging. Ultimately, effective utilization of GC–MS requires a deep understanding of its operational intricacies and meticulous maintenance practices. Many of these recommendations are although well established, but prioritizing these factors will enhance the reliability and replicability of phytochemical studies, leading to more robust scientific findings and ensuring research integrity.

Do you imagine this technique being used to detect fungus in other crops, especially those used for teas and tea blends?

The isolation of endophytic fungi from cash crops such as tea is a well-established practice in scientific research. Numerous studies have documented the isolation of endophytes from a variety of plants, revealing organisms that not only cause diseases but also offer benefits like disease prevention and enhanced tolerance to abiotic stresses for the host plant. Our research emphasizes the significance of organic plantations, as they increase the likelihood of isolating endophytes with favorable traits. This method could be extended to other commercial crops to discover beneficial endophytes. We aim to apply and validate our approach on various organically grown Indian cash crops, hoping to identify alternative sources of valuable secondary metabolites that could benefit agriculture and medicine.

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

The work was published just a week ago, so we have encountered only limited feedback thus far. However, the responses we have received have been overwhelmingly positive, with many praising our experimental approach and methodology. Several colleagues have specifically commended our decision to select organically grown tea as the study material, highlighting its significance over conventionally grown tea in terms of sustainability and health benefits. Additionally, we have received constructive suggestions to consider implementing metabolic engineering strategies aimed at improving fungal strains. We are currently evaluating these recommendations and are excited about the potential for enhancing our research outcomes based on this insightful feedback.

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

Our research operates within an interdisciplinary environment, bringing together experts from various scientific domains to foster innovation and collaboration. We aim to culture the isolated endophytic fungi under a variety of culture conditions while applying different elicitors to enhance catechin production. This strategy not only seeks to diminish our reliance on tea plants as the primary source of catechins but also aims to streamline the mass production process of these valuable compounds, making it more efficient and less time-consuming. Additionally, we plan to assess the potential of these isolates to produce other important phytochemicals, such as epigallocatechin and epicatechin gallate, which hold significant pharmacological value. Finally, by incorporating LC–MS-based methodologies, we hope to explore whether the isolated fungi can generate a broader range of bioactive molecules beyond catechins. Ultimately, our goal is to identify alternative sources of bioactive phytochemicals, contributing to advancements in nutraceuticals and pharmaceuticals while promoting sustainable practices.

References

1. Sidhu, D.; Vasundhara, M.; Dey, P. Chemical Characterization, Pathway Enrichments and Bioactive Potentials of Catechin-Producing Endophytic Fungi Isolated from Tea Leaves. RSC Adv. 2024, 14 (45), 33034–33047. DOI: 10.1039/d4ra05758a

Priyankar Dey is a tenured faculty in the Department of Biotechnology, Thapar Institute of Engineering and Technology (India). He obtained his PhD in Immunopharmacology from the University of North Bengal and did postdoctoral research at The Ohio State University. He is also co-Founder and Director of Fermibio Solutions Pvt. Ltd., which is a microbiome company. His current research centers around defining the intestinal-level 3-M interactions between metabolism, microbiome, and metabolome in relation to non-communicable chronic metabolic conditions like obesity, type II diabetes, and nonalcoholic steatohepatitis. In parallel, he is also engaged in applied research to develop phytochemical-based prophylactic and therapeutic strategies against chronic metabolic disease, especially focusing on intestinal and hepatic health (e.g., dysbiosis, gut barrier dysfunction, LPS/TLR4-response). He is trained in metabolomic and metabonomic techniques. He has secured research grants from DST, SERB, DBT-BIRAC and ICMR worth 45 million INR, authored >90 peer-reviewed articles, 4 book chapters, and 3 patents. He is associate editor of Frontiers in Physiology, Frontiers in Nutrition, and editorial board member of Nutrition Research (Elsevier), Heliyon (Cell Press), Advances in Pharmacological and Pharmaceutical Sciences (Wiley), Nutrients (MDPI). He is currently supervising five PhD students and two MS students. Dey enjoys cooking and reading books.

Photo courtesy of Dey.

For more and updated information, please visit his lab webpage here:

https://sites.google.com/view/3mlab/home?authuser=0