Abstract

Tea harvested in North-Eastern parts of India is renowned for high quality tea including various therapeutic potentials like antioxidant and antimicrobial properties. Tea samples collected from Darjeeling, Assam and Jalpaiguri gardens are subjected for metabolite quality analysis processes. The geographic factors including soil characters can influence the qualities of tea according to their inherent secondary metabolites. One of the major classes of tea secondary metabolite is tea flavonoids responsible for tea quality parameters including Rutin, Quercetin, Epigallocatechin gallate, Tannic acid, Gallic acid etc. Spectrophotometric, Spectrofluorimetric scan analysis, HPLC, FTIR analysis characterize the differential contents of different tea flavonoids of different origin. The results suggest that tea extracts from Assam, Darjeeling, and Jalpaiguri possess valuable bioactive secondary metabolites to impart antimicrobial and antioxidant activities. The variations in these properties can be attributed to a combination of factors, including soil characteristics and cultivation practices of the different origin of Northeastern Indian tea gardens.

Keywords:Tea; Metabolites; Antioxidant; Antimicrobial; FTIR; HPLC; Quercetin; Rutin

Introduction

Tea is one of the most widely consumed beverages in the world. It is a rich source of bioactive compounds, including Epigallocatechin Gallate (EGCG), rutin, quercetin, gallic acid, and tannic acid, which have been extensively studied for their potential health benefits. Tea plant (Camellia sinensis) belongs to genus Camellia L. and family Theaceae. Compared with other plants, secondary metabolites of tea plants not only endow tea with unique therapeutic quality, but also benefit human health. As an important economic plant, tea has been studied in many fields, including health, food production, and culture. These metabolites possess antioxidant, antimicrobial and anti-inflammatory properties, which may contribute to reducing the risk of chronic diseases such as cardiovascular disease, cancer, and neurodegenerative disorders. The tea plant is a perennial and economically significant crop and boasts a productive lifespan of up to 100 years [1-2]. Research has highlighted the critical factors influencing peak tea yields, including genotype, environment, management practices, and their interactions. EGCG has garnered significant attention for its potential anti-cancer effects [3-4], with studies suggesting its ability to inhibit tumour growth and metastasis. Rutin and quercetin, flavonoids with antioxidant and anti-inflammatory properties, have been shown to improve blood circulation and reduce the risk of heart disease. Gallic acid and tannic acid, polyphenols known for their astringent taste, have demonstrated antioxidant and anti-inflammatory activities as well [5-6].

From a human perspective, secondary metabolites act as important components for economic crop quality. Various secondary metabolites contribute to the quality and function of tea [7]. During tea plant growth (preharvest stage) and the tea manufacturing process (postharvest stage), many stresses are used to “modify” tea metabolites [8-10]. At the preharvest stage, shading treatment (abiotic stress) has been shown to enhance free amino acids and aromatic aroma compounds, and reduce catechins [11-13].

Furthermore, tea leaf attacked by tea green leafhoppers (biotic stress) increases honey-fruit aroma compounds [8] [14]. Compared with other economic crops, the postharvest manufacturing process is an important stage for improving tea quality, especially regarding tea aroma. A comprehensive investigation of aroma compound formation at this stage showed that jasmonic acid (JA)–key genes–characteristic aroma compounds was the main regulatory route [15-19]. To date, much research has been conducted to clarify the mechanism involved in improving tea quality with stresses (Figure 1).

This study aims to investigate the geographical variation and altitudinal influence on tea metabolite profiles and associated soil parameters. By employing a combination of analytical techniques, including Fourier-Transform Infrared Spectroscopy (FTIR), Ultraviolet-Visible (UV-Vis) Spectroscopy, High-Performance Liquid Chromatography (HPLC), Thin layer chromatography (TLC), and Fluorimetry, we will characterize the changes in metabolite composition and explore the underlying mechanisms influenced by environmental factors.

The specific objectives of this research are to identify and quantify key tea metabolites, such as EGCG, rutin, quercetin, gallic acid, and tannic acid, in tea samples collected from different geographical locations and altitudes; investigate the effects of varying soil properties on tea metabolite production; explore the interplay between geographical factors, soil characteristics, and tea plant physiology in determining metabolite profiles; and provide insights into the potential implications of geographical variations in tea metabolites for the quality and health benefits of tea. The amount of these compounds in tea can vary depending on factors such as the type of tea, growing conditions, and processing methods [20][1] [21-23]. Therefore, consuming a variety of teas can help maximize the potential health benefits associated with these bioactive compounds.

By addressing these objectives, this research will contribute to a deeper understanding of tea plant biology and provide valuable information for tea production and quality control, particularly in identifying regions with optimal conditions for cultivating tea with desired metabolite profiles. Furthermore, this study may have broader implications for the development of functional foods and nutraceuticals derived from tea. By understanding the factors that influence the production of bioactive compounds in tea, researchers can explore strategies to enhance their levels and develop products with targeted health benefits. Additionally, this research may contribute to the development of sustainable tea production practices, as it can help identify regions with optimal growing conditions and inform the development of management strategies that minimize environmental impact.

Experimental Procedures

Origin of tea samples

Tea plants (Camellia sinensis) from three different places were analyzed in this study. Located Dibrugarh (27.4705° N, 94.9125° E), Assam; Balasun (26.8606°N 88.2357°E), Darjeeling; VVtea garden (26.5683° N, 88.5396° E) Fatapukur, Jalpaiguri, India. Fresh tea leaves TV 25 were plucked at these places between June 20, 2024, and June 26, 2024, immediately kept in dry ice, sent to St. Xavier’s College (Autonomous), Kolkata, and stored at − 80 °C until further analysis.

Extraction of Tea leaf samples

Tea leaf samples from Assam, Jalpaiguri, and Darjeeling were extracted by maceration technique in dimethyl sulfoxide (DMSO) solvent. Leaf samples dried, ground, and homogenized to ensure uniform particle size. 50 grams of each sample were weighed and placed in separate centrifuge tubes. 10 millilitres of DMSO were added to each tube, followed by vigorous homogenization using a mortar pestle. The homogenized mixtures were then centrifuged at 5°C for 5 minutes at 10,000 rpm to separate the solid residue from the extracted compounds. The supernatant, containing the extracted tea leaf compounds, was carefully transferred to clean Eppendorf tubes for further analysis. This modified maceration technique offers several advantages over traditional extraction methods [22][1][16]. The direct homogenization and centrifugation steps streamline the process, reducing the risk of sample degradation and minimizing the time required for extraction. By using a small volume of DMSO, the technique is suitable for extracting bioactive compounds from limited quantities of tea leaf samples. Moreover, the absence of additional filtration or concentration steps simplifies the workflow and enhances efficiency.

The extracted tea leaf samples can now be subjected to various analytical techniques to identify and quantify the specific bioactive compounds present. These compounds may include polyphenols, alkaloids, flavonoids, and other compounds with potential health benefits. Further analysis can help elucidate the unique characteristics of tea leaf extracts from different regions and provide valuable insights into their potential applications for various health related and medical applications [8][4][7].

UV-Visible Spectrophotometric Analysis of Tea Polyphenols and Flavonols

To characterize the polyphenolic compounds, present in the extracted tea leaf samples, UV-visible spectrophotometric scanning was performed. This technique allows for the qualitative and quantitative analysis of these compounds based on their absorption of light in the ultraviolet and visible regions of the spectrum.

Standard solutions of tannic acid, gallic acid, quercetin, epigallocatechin gallate (EGCG), and rutin were prepared at known concentrations of 1mg/ml and used as reference standards. These compounds are representative of the major classes of polyphenols found in tea leaves, including tannins, flavonoids, and flavanols. The extracted tea leaf samples were diluted with dimethyl sulfoxide (DMSO) to a suitable concentration and scanned in a UV-visible spectrophotometer over a wavelength range of 200- 400 nm. The absorbance spectra of the samples were compared to the spectra of the standard solutions to identify and quantify the specific polyphenols present. The characteristic absorption peaks of polyphenols in the UV-visible region are primarily due to their aromatic structures. Tannins and flavonoids typically exhibit absorption maxima between 260 and 280 nm, [24-25], while flavanols like EGCG often show absorption peaks around 270-280 nm and 360-370 nm. By comparing the absorption spectra of the tea leaf samples to the standard solutions, it is intended to identify the presence of specific polyphenols and estimate their relative concentrations. This information is valuable for understanding the biomolecule profile and potential health benefits of tea and its constituent compounds.

Characterization of Tea Polyphenols including Flavonoids Using FTIR Spectrometry

Fourier-Transform Infrared (FTIR) spectroscopy was employed to characterize the functional groups present in the extracted tea leaf samples. This technique provides a fingerprintlike spectrum of the sample, revealing the presence of specific chemical bonds. To establish a reference point for the interpretation of the obtained spectra, standard compounds known for their polyphenol and flavonol content were also analyzed: tannic acid, gallic acid, quercetin, epigallocatechin gallate (EGCG), and rutin. The tea leaf extracts and standard compounds were prepared in dimethyl sulfoxide (DMSO) solvent, a suitable medium for FTIR analysis. The samples were then scanned in the wavenumber range of 4000 cm⁻¹ to 1000 cm⁻¹. The FTIR spectra of the samples and standards were compared to identify characteristic peaks associated with polyphenols and flavonols.

Key regions of interest in the FTIR spectra for the identification of polyphenols and flavonols include:
* 3400-3200 cm⁻¹: This region is indicative of O-H stretching vibrations, typically associated with phenolic hydroxyl groups.
* 1700-1600 cm⁻¹: This region is associated with carbonyl (C=O) stretching vibrations, often found in flavonoids and tannins.
* 1600-1500 cm⁻¹: This region is associated with aromatic C=C stretching vibrations, characteristic of polyphenols and flavonoids.
* 1200-1000 cm⁻¹: This region is associated with C-O stretching vibrations, often found in phenolic compounds.

By comparing the FTIR spectra of the tea leaf extracts to the spectra of the standard compounds, it was possible to identify the presence and relative abundance of specific polyphenols and flavonols in the samples. This information can be valuable for understanding the antioxidant properties and other bioactive components of tea extracts. Furthermore, FTIR spectroscopy can be used to assess the quality and consistency of tea products based on their polyphenol and flavonol content.

Spectrofluorimetric Analysis of Tea Polyphenols and Flavonols

To further characterize the polyphenolic compounds in the extracted tea leaf samples, spectrofluorimetric analysis was conducted. This technique measures the fluorescence emitted by a compound when it is excited by light of a specific wavelength [12][10]. Standard solutions of tannic acid, gallic acid, quercetin, epigallocatechin gallate (EGCG), and rutin were prepared at known concentrations of 1 mg/ml. The excitation wavelength was set to 270 nm for all standards except for tannic acid, which was excited at 275 nm and rutin, which was excited at 256 nm. The extracted tea leaf samples were also diluted with dimethyl sulfoxide (DMSO) and analyzed using the same spectrofluorimetric conditions. The scan was done twice for each sample and for studying the presence of rutin in the samples, the excitation wavelength was set to 256 nm, while for the other standards, 270 nm was used. The emission wavelength was scanned from 270 nm to 620 nm (and from 300 nm to 600 nm the second time to avoid interference from the excitation wavelength.).

The fluorescence spectra of the samples and standards were compared to identify the presence of specific polyphenols and to estimate their relative concentrations. Polyphenols exhibit characteristic fluorescence emission spectra, which can be used to differentiate between different classes of compounds. By combining UV-visible spectrophotometry and spectrofluorimetric analysis, a more comprehensive characterization of the polyphenolic compounds in the tea leaf samples could be achieved. This approach allows for the identification and quantification of both absorbing and fluorescent polyphenols, providing valuable insights into the composition and potential health benefits of tea.

HPLC Analysis of Tea Polyphenols and Flavonols compound

To comprehensively characterize the polyphenolic compounds, present in the extracted tea leaf samples, high-performance liquid chromatography (HPLC) analysis was conducted. HPLC is a powerful technique for separating and quantifying individual components in a complex mixture. A reverse-phase C18 column was employed for the separation of polyphenols. The mobile phase consisted of a gradient of acetonitrile and milli-Q water, with increasing concentrations of acetonitrile over time to elute compounds of varying polarity. The extracted tea leaf samples and standard solutions of tannic acid, gallic acid, quercetin, epigallocatechin gallate (EGCG), and rutin were injected into the HPLC system. The eluted compounds were detected using a UV-visible detector set at 285 nm, a wavelength at which many polyphenols exhibit strong absorbance.

The retention times of the peaks in the chromatograms were compared to those of the standards to identify the individual polyphenolic compounds. The peak areas were used to quantify the concentration of each compound based on a calibration curve constructed using the standard solutions. HPLC provides a high-resolution separation of polyphenols, allowing for the identification and quantification of individual compounds even in complex mixtures. This technique is particularly useful for determining the relative abundance of different polyphenols in tea leaves and for comparing the polyphenolic profiles of various tea varieties. By combining HPLC with UV-visible detection, it is possible to obtain detailed information about the composition of polyphenols in tea and to assess the potential health benefits associated with these compounds [24-25].

Total Free Amino Acids Assay

To determine the total free amino acids content in the extracted tea samples, a spectrophotometric method using ninhydrin was employed. Ninhydrin reacts with primary and secondary amino acids to produce a purple-colored complex. The intensity of this color, measured at 570 nm, is directly proportional to the concentration of amino acids present in the sample.

For the assay, 1 ml of ninhydrin solution (3.5 mg/ml in ethanol) was added to 5 ml of the extracted tea sample (dissolved in DMSO). The mixture was incubated at 100°C for 5-7 minutes to facilitate the reaction between ninhydrin and amino acids. This elevated temperature accelerates the reaction rate, ensuring complete derivatization of the amino acids. The formation of the purple-colored complex, known as Ruhemann’s purple, is a result of the condensation reaction between the amino group of the amino acid and the carbonyl groups of ninhydrin. Alanine is taken as standard (1mg/ml).

After cooling to room temperature, the absorbance of the resulting solution was measured at 570 nm using a spectrophotometer. The intensity of the absorbance is directly related to the concentration of amino acids in the sample. By comparing the absorbance of the samples to a standard curve, the concentration of total free amino acids can be determined. This assay provides a reliable and quantitative method for assessing the amino acid composition of tea samples, which is important for understanding their nutritional value and potential health benefits.

Antimicrobial Activity of Tea Metabolites

To assess the antimicrobial activity of the extracted tea leaf samples, a well diffusion assay was employed. This method involves creating wells in agar plates seeded with bacterial cultures and then adding the test samples to these wells. The diffusion of the antimicrobial compounds from the wells into the surrounding agar can inhibit bacterial growth, forming zones of inhibition. The bacterial strains used in this study were Escherichia coli and Bacillus species, representing Gram-negative and Gram-positive bacteria, respectively. These strains were chosen to evaluate the broad-spectrum antimicrobial potential of the tea metabolites. Ampicillin, a well-established antibiotic, was used as a positive control to compare the antimicrobial activity of the tea samples.

The extracted tea leaf samples were dissolved in dimethyl sulfoxide (DMSO) to ensure proper solubility and dispersion of the bioactive compounds. DMSO was also used as a negative control to assess its potential antimicrobial effects. Agar plates were prepared containing Mueller-Hinton broth, a nutrient-rich medium suitable for bacterial growth. The bacterial strains were then inoculated onto the agar plates and allowed to grow overnight to form a confluent lawn. Wells were punched into the agar plates using a sterile cork borer. The extracted tea samples and ampicillin were added to the wells in known concentrations. The plates were incubated at an appropriate temperature (e.g., 37°C) for a specific duration (e.g., 24 hours). After incubation, the zones of inhibition around the wells were measured and compared. The diameter of the zones of inhibition can be used to assess the antimicrobial potency of the tea metabolites relative to the positive control. By comparing the zones of inhibition produced by the tea samples to the positive control, it is possible to determine whether the tea metabolites possess significant antimicrobial activity against the selected bacterial strains. This information can contribute to understanding the potential therapeutic applications of teaderived compounds in combating bacterial infections.

Characterization of Antioxidant Properties of Extracted Tea Samples Using Spectrophotometric Assay

To evaluate the antioxidant properties of the extracted tea leaf samples, a spectrophotometric assay using 2,2-diphenyl- 1-picrylhydrazyl (DPPH) free radical was employed. The DPPH radical is a stable free radical that exhibits a characteristic purple color. When DPPH encounters an antioxidant compound, it undergoes a reduction, resulting in a loss of its characteristic color. The degree of color change directly correlates with the antioxidant activity of the sample.

For the assay, 1 mL of each extracted tea sample in dimethyl sulfoxide (DMSO) was mixed with 1 mL of a 0.04 mg/mL DPPH solution in DMSO. The mixture was incubated in the dark at room temperature for 30 minutes to allow for complete interaction between the DPPH radical and the antioxidant compounds present in the tea samples. Subsequently, the absorbance of the mixture was measured at 517 nm using a spectrophotometer.

Additionally, 2 mL of the DPPH solution was used as a positive control or standard, representing the maximum absorbance value in the absence of any antioxidant activity.

The decrease in absorbance at 517 nm compared to the positive control indicates the scavenging of DPPH radicals by the antioxidant compounds in the tea samples. A higher reduction in absorbance corresponds to a greater antioxidant activity. The results obtained from this assay can be used to compare the antioxidant capacities of the tea samples from different regions (Assam, Jalpaiguri, and Darjeeling) and to identify potential correlations between antioxidant activity and other factors such as tea variety, cultivation conditions, or processing methods.

Colony Forming Unit Assay for Soil Samples

To determine the microbial activity within the soil samples associated with tea cultivation at varying altitudes, a colonyforming unit (CFU) assay was conducted. This method quantifies the number of viable microorganisms present in each sample by counting the distinct colonies formed on a nutrient-rich medium. [6]. Soil samples were initially prepared by suspending 250 mg of each sample in 10 mL of distilled water, creating a stock solution. Serial dilutions (1:10, 1:100, and 1:1000) were then prepared from the stock solution to ensure accurate colony counts within a manageable range. A volume of 200 μL of each dilution, including the undiluted stock solution, was plated onto separate autoclaved nutrient agar plates. Nutrient agar provides essential nutrients for microbial growth, allowing colonies to develop. The plates were incubated at an optimal temperature (typically 37°C) for a specific duration (e.g., 24-48 hours) to allow microbial growth.

Following incubation, visible colonies were counted on each plate. The number of colonies formed on each plate was multiplied by the corresponding dilution factor to determine the total number of CFUs present in the original soil sample. This calculation provides a quantitative measure of microbial abundance and diversity within the soil samples. By comparing the CFU counts among soil samples collected from different altitudes, this assay can help elucidate the influence of altitude on microbial activity within tea-cultivating soils. Factors such as temperature, oxygen availability, and nutrient composition may vary across different altitudes, impacting microbial populations and their metabolic processes.

Nutrient estimation of soil samples

To assess the micronutrient and macronutrient content of soil samples collected from tea-cultivating regions in Assam, Jalpaiguri, Darjeeling, and Nagaland, a total of 12.5 grams of soil from each location was mixed with 25 millilitres of water and filtered. The resulting filtrate was analyzed using the Soil Saathi app, which employs radioimmunoassay (RIA) technology.

Dedicated liquid reagents were used to determine the concentrations of magnesium, calcium, sulfur, zinc, iron, and manganese. These reagents were added to the soil samples in RIA vials and incubated for 30 minutes before analysis. For nitrogen, phosphorus, potassium, and organic carbon, capsules containing reagent powder were added to the samples and incubated for 5 minutes.

Upon incubation, the Soil Saathi app was used to estimate the nutrient content of each soil sample. This innovative application utilizes RIA to accurately measure the specific concentrations of the target nutrients. By employing this method, we were able to comprehensively characterize the nutrient profiles of soils from diverse tea-cultivating regions, providing valuable insights into the potential limitations and nutrient requirements for sustainable tea production in these areas.

Statistical analysis:

All the results and the correlations were tested (regression analysis) with ANOVA by SPSS analysis software version 20.0.

Results and Discussion

Analysis of tea metabolites by UV-Vis Spectrophotometer (UVVis)

The absorption spectra of tea extracted and the standards in dimethyl sulfoxide (DMSO) are recorded at room temperature using a UV-VIS spectrophotometer are displayed in Figure 2. Figure 3 demonstrates how the absorption peaks that describe the tea from each of the three places are not exact, but rather exhibit subtle variations. Another prominent difference was that the peak observed in DMSO solvent was quite broad as DMSO, a highly polar solvent with strong hydrogen bonding capabilities and high viscosity, can lead to broader UV-Vis peaks compared to other solvents. These factors contribute to increased collisional broadening and solvent-solute interactions, which can disrupt the vibrational energy levels of the analyte, resulting in broader spectral lines. This phenomenon is particularly noticeable in DMSO due to its unique solvent properties.

The characteristics of tea extract from Dibrugarh, Assam (grown at 358 to 423 ft height) can be identified by two main absorption peaks at 233 and 370 nm in DMSO. As the sample shows peaks at 370 and 233 nm, it suggests similarity to quercetin and rutin’s carbonyl group (peaking at 371 nm), contains aromatic rings (233 nm), and has a chromophore that absorbs at longer wavelengths (370 nm). It likely has rutin or quercetin in it.

Two primary absorption peaks, at 229.5 nm and 370.5 nm, are characteristic of Balasun tea extract in DMSO, which is grown at an elevation of around 365 to 1,375 meters. The signal at 229.5 nm is frequently linked to proteins and aromatic amino acids. It might be a sign of the presence of chemicals in tea like theanine, which has a soothing effect. Often associated with flavonoids, a kind of polyphenols present in tea, is the peak at 370.5 nm. It can indicate the existence of substances like quercetin, rutin.

Two primary absorption peaks, at 234.0 nm and 371.5 nm, are present in the DMSO tea extract of Fatapukur VVtea, which is grown at an elevation of around 328.08 feet. The following putative substances were identified by comparing these peaks to the reference spectra and therefore suggests that the peak at 234.05 nm is compatible with the distinctive absorption of EGCG, and the peak at 371.5 nm suggests the probable presence of rutin.

Fourier Transform Infrared (FTIR) analysis of tea samples

Fourier-Transform Infrared Spectroscopy (FTIR) analysis of the tea extracts revealed distinctive spectral patterns consistent with the presence of bioactive compounds. The observed spectral features were compared to reference spectra of rutin and tannic acid (Figure 4). Based on this comparison, the FTIR analysis suggests the potential presence of some of these compounds in the varied tea samples.

FTIR analysis of the tea extract from Dibrugarh, Assam (Figure 5a) revealed distinctive spectral patterns indicative of the presence of specific functional groups. Prominent peaks were observed at 3426.66 cm⁻¹, 1659.35 cm⁻¹, and 1315.20 cm⁻¹, providing valuable insights into the composition of the tea sample. The broad peak at 3426.66 cm⁻¹ is characteristic of O-H stretching vibrations, suggesting the presence of hydroxyl groups. This is consistent with the structure of both rutin and tannic acid, which contain multiple hydroxyl groups. The peak at 1659.35 cm⁻¹ is indicative of C=O stretching vibrations. This could be attributed to conjugated C=O stretching in aromatic rings or C=O stretching in ester linkages, both of which are present in rutin and tannic acid. The peak at 1315.20 cm⁻¹ is less specific but could be related to aromatic C-H bending vibrations or, less likely, C-N stretching in some tea components.

FTIR analysis of the tea extract from Balasun and Jalpaiguri (Figure 5b) revealed distinctive spectral patterns indicative of the presence of specific functional groups. Prominent peaks were observed at 3428.31 cm⁻¹, 1658.78 cm⁻¹, and 1014.40 cm⁻¹, providing valuable insights into the composition of the tea sample. The broad peak at 3428.31 cm⁻¹ is characteristic of O-H stretching vibrations, suggesting the presence of hydroxyl (OH) groups. This is consistent with the structure of several tea metabolites, including rutin, tannic acid, and gallic acid. The peak at 1658.78 cm⁻¹ is indicative of C=O stretching vibrations, likely associated with carbonyl groups in aromatic rings or ester linkages. This is consistent with the presence of compounds like rutin and tannic acid. The peak at 1014.40 cm⁻¹ is less specific but could be related to C-H bending vibrations in aromatic rings or C-O stretching vibrations in alcohols or ethers. Based on these FTIR observations, the tea extract is likely to contain both rutin and tannic acid.

The FTIR spectrum of the tea sample from Fatapukur, Jalpaiguri (Figure 5c) exhibited prominent peaks at 3432.51 cm⁻¹, 1658.83 cm⁻¹, 1315.34 cm⁻¹, and 1016.07 cm⁻¹. These peaks suggest the presence of hydroxyl (OH) groups, carbonyl groups, aromatic C-H bending vibrations, and C-O stretching vibrations. Based on these findings, the potential compounds in the tea sample also probably contains both rutin and tannic acid.

Spectrofluorimetric analysis of tea extracts

Fluorometric analysis of the tea extracts revealed distinct fluorescence emission spectra, indicative of the presence of specific bioactive compounds. The observed fluorescence intensities and emission wavelengths were compared to reference spectra of known tea metabolites to identify potential compounds in the samples.

Spectrofluorimetric analysis of the tea extract from Dibrugarh, Assam, revealed a prominent emission peak at 540.6 nm and 512 nm. This emission peak is consistent with the fluorescence characteristics of EGCG, rutin, and gallic acid, all of which exhibit emission maxima in the 540-550 nm range. However, the lack of a significant emission peak around 360-370 nm, which is characteristic of EGCG, suggests that EGCG may be present in lower concentrations or absent in this particular tea sample. The observed emission peak at 540.6 nm is most likely due to a combination of rutin and gallic acid. Rutin has been reported to exhibit emission peaks around 513 nm and 551 nm, while gallic acid has been shown to emit fluorescence around 540 nm. The observed emission peak at 540.6 nm could be a result of the overlapping fluorescence of these two compounds (Figure 6).

Spectrofluorimetric analysis of the Balasun, Darjeeling tea extract revealed an emission maximum at 500.6 nm. This emission peak is most consistent with the emission spectra of rutin and tannic acid, both of which exhibit emission maxima around 550 nm. However, the observed shift in the emission maximum to 500.6 nm could be attributed to factors such as matrix effects, concentration differences, or environmental conditions.

The Fatapukur VVtea tea extract exhibited an emission maximum at 500.4 nm. This emission peak is also most consistent with the emission spectra of rutin and tannic acid, both of which exhibit emission maxima around 550 nm. However, the observed shift in the emission maximum to 500.4 nm could be attributed to factors such as matrix effects, concentration differences, or environmental conditions.

HPLC Analysis of Tea Metabolites

High-Performance Liquid Chromatography (HPLC) was employed to analyze the bioactive compounds present in tea extracts from various regions. This technique allows for the separation, identification, and quantification of individual components within a complex mixture. By comparing the HPLC chromatograms of different tea extracts, we can gain insights into the variations in metabolite composition and potential health benefits associated with each region.

The retention times of the analytes were compared to those of reference standards: gallic acid (3.042 minutes), tannic acid (3.742 and 6.828 minutes), EGCG (3.820 minutes), quercetin (1.290, 1.734, 1.907, 2.235, and 3.456 minutes), and rutin (2.380, 6.500, and 6.850 minutes). These reference retention times will serve as a basis for interpreting the HPLC data obtained from the tea extracts.

HPLC analysis of the Fatapukur tea extract revealed prominent peaks at 1.435, 2.433, and 7.255 minutes, which align with the retention times of gallic acid (3.042 minutes), rutin (2.380 and 6.500 minutes), and tannic acid (3.742 and 6.828 minutes), respectively. These findings suggest the presence of these bioactive compounds in the Fatapukur tea extract (Figure 7).

HPLC analysis of the Balasun tea extract revealed prominent peaks at 2.174 and 7.237 minutes, suggesting the presence of gallic acid and tannic acid. While the observed retention times for these compounds differed slightly from the reference standards, the overall similarity indicates their likely presence in the Balasun tea extract. However, further analysis is required to confirm the presence of other compounds, such as EGCG, rutin, and quercetin, and to quantify the specific levels of all identified metabolites. The observed differences in retention times could be attributed to matrix effects, concentration variations, or other factors influencing the chromatographic behavior of the analytes (Figure 8).

HPLC analysis of the Assam tea extract revealed prominent peaks at 1.959, 2.749, 3.571, 5.343, 5.874, 6.070, and 6.503 minutes. By comparing these retention times to the reference standards, the following potential compounds were identified:
• Gallic acid: The peak at 1.959 minutes aligns with the retention time of gallic acid.
• Rutin: The peak at 2.749 minutes is consistent with the retention time of rutin.
• Tannic acid: The peak at 6.503 minutes suggests the presence of tannic acid.
• EGCG: While the reference retention time for EGCG (3.820 minutes) is not an exact match, the peak at 3.571 minutes could potentially correspond to EGCG, given the variability in retention times that can occur due to factors such as matrix effects and column conditions.
• Quercetin: The presence of multiple peaks in the Assam tea extract (1.290, 1.734, 1.907, 2.235, and 3.456 minutes) suggests the possibility of quercetin.

The HPLC analysis suggests the presence of gallic acid, rutin, and potentially EGCG and quercetin in the Assam tea extract. These compounds are known for their antioxidant and bioactive properties, contributing to the potential health benefits of tea.

Antioxidant Assay of tea extracts

The provided data demonstrates the antioxidant potential of various tea extracts using the DPPH (2,2-diphenyl-1- picrylhydrazyl) assay. The antioxidant potential is expressed as a percentage of the control value % reduction of DPPH/mg fresh wt., which represents the initial absorbance of the DPPH solution.

The antioxidant potential of various tea extracts was evaluated using the DPPH assay. All tea extracts exhibited significant antioxidant activity, with Fatapukur Untreated demonstrating the highest potential (81.152±0.01%). Balasun and Assam tea extracts also showed notable antioxidant activity, with values of 77.161±0.02% and 69.711±0.01%, respectively. These findings highlight the potent antioxidant properties of tea-based compounds and their potential contribution to overall health benefits. Further research is warranted to isolate and characterize the specific compounds responsible for the antioxidant activity and to evaluate their efficacy in various biological systems.

The average antioxidant potential of the tea samples was calculated to be 82.006±0.02 V%. This suggests that all the tea extracts possess notable antioxidant properties.

Antimicrobial Assay of tea extracts on Escherichia coli and Bacillus subtilid

The well diffusion method was employed to evaluate the antimicrobial activity of Fatapukur VVtea tea extract, DMSO, and ampicillin against both Escherichia coli and Bacillus subtilis. A clear zone of inhibition was observed around the Fatapukur tea extract well against both bacterial strains (Figure 9b and 10b) indicating the presence of antimicrobial compounds. The zone of inhibition measured 1.4±0.001 cm for Escherichia coli and 1.3±0.001 cm for Bacillus subtilis. In contrast, DMSO did not exhibit any antimicrobial activity, while ampicillin displayed potent inhibition. These findings suggest the potential of Fatapukur tea extract as a natural antimicrobial agent with broad-spectrum activity against both Gram-negative and Gram-positive bacteria.

The well diffusion method was also employed to evaluate the antimicrobial activity of Assam tea extract against both Bacillus subtilis and Escherichia coli. A clear zone of inhibition was observed (Figure 9a and 10a) around the Assam tea extract against both bacterial strains, indicating the presence of antimicrobial compounds. The zone of inhibition was 1.3 cm for Bacillus subtilis and 1.2 cm for Escherichia coli. In contrast, DMSO did not exhibit any antimicrobial activity. These findings suggest the potential of Assam tea extract as a natural antimicrobial agent with broadspectrum activity.

Lastly, well-diffusion method revealed that Balasun tea extract exhibited (Figure 9c and 10c) antimicrobial activity against both Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria. A zone of inhibition of 1.3±0.01 cm was observed against Bacillus subtilis, while a zone of 1.5±0.01 cm was observed against Escherichia coli. In contrast, DMSO showed no antimicrobial activity, and ampicillin displayed potent inhibition against both bacteria. These findings suggest the potential of Balasun tea extract as a natural antimicrobial agent with broadspectrum activity.

Analysis of Free Amino Acid Content in Tea Samples

The analysis of free amino acid content in tea samples provides valuable insights into the nutritional composition and potential health benefits of tea. Amino acids are essential building blocks of proteins and play crucial roles in various biological processes [8]. By quantifying the levels of free amino acids in tea, we can assess its nutritional value and explore its potential applications in food and pharmaceutical industries. The absorbance values obtained at 570 nm for the tea samples indicate variations in their total free amino acid content and compared with known amino acid alanine content (1mg/ml). Balasun exhibited the highest content (0.38±0.001mg/ml), suggesting the highest concentration of free amino acids. Fatapukur and Assam tea samples showed lower values (0.25±0.002mg/ml and 0.28±0.002mg/ml respectively). These findings suggest that the amino acid composition of tea can vary depending on factors such as soil conditions, climate, and cultivar.

Macronutrient and micronutrient content of soil from tea gardens

The evaluation of macronutrient and micronutrient content in tea samples from Assam, Darjeeling, and Jalpaiguri is crucial for understanding the factors influencing tea quality and composition. Variations in soil conditions, climate, and agricultural practices across these regions can significantly impact the nutrient uptake and accumulation in tea plants. By analyzing the macronutrient and micronutrient profiles of tea samples from different locations, we can gain insights into the relationship between soil composition, tea plant physiology, and the production of bioactive compounds in tea. These findings can inform sustainable tea cultivation practices, optimize nutrient management, and contribute to the production of high-quality tea with desired nutritional and functional properties. Following Table 1 shows the amount of macro and micronutrients found in the soil of Assam, Darjeeling and Jalpaiguri, these results are further corelated with the amount of tea metabolites identified.

Colony-Forming Unit (CFU) Counts of soils from various tea gardens

The bacterial colony counts obtained from soil samples collected from Assam, Darjeeling (Balasun subsoil), and Jalpaiguri (Fatapukur) demonstrate variations in microbial populations (Figures 11, 12 & 13).
• Assam (Dibrugarh): Colony count (1:1000) dilution- 284 x 4 = 1134 CFU/mL (approximately).
• Jalpaiguri (Fatapukur): Colony count (1:1000) dilutions- 297 X 4 = 1188 CFU/mL (approximately).
• Darjeeling (Balasun): Colony count (1:1000) dilutions- 318 X 4 = 1272 CFU/mL (approximately).

Darjeeling (Balasun) soil exhibited the highest bacterial colony count, suggesting a relatively higher microbial diversity or abundance compared to the other regions. Similar Counts: Assam and Jalpaiguri soils displayed comparable bacterial counts, indicating similar levels of microbial activity.

Conclusion

The present study investigated the influence of geographical factors and soil characteristics on the metabolite composition and biological activities of tea extracts from Assam, Jalpaiguri, and Balasun. The results obtained from UV-Vis spectroscopy, FTIR analysis, spectrofluorimetric analysis, antimicrobial assays, HPLC analysis, and total free amino acid (TFAA) determination provide valuable insights into the interrelationships between these factors. The observed variations in tea metabolite profiles among the different regions can be attributed to variations in soil characteristics. Soil composition, including factors such as pH, organic matter content, and nutrient availability, can influence the uptake and accumulation of specific elements and compounds by tea plants. These, in turn, can affect the biosynthesis of secondary metabolites, such as polyphenols and amino acids.

The UV-Vis and FTIR analyses revealed distinct spectral patterns in the tea extracts, suggesting the presence of specific functional groups and compounds. The observed peaks were consistent with the presence of compounds such as gallic acid, rutin, tannic acid, and potentially EGCG and quercetin. These compounds are known for their antioxidant and antimicrobial properties. Fluorometric analysis indicated variations in the emission intensity of the tea extracts, suggesting differences in the concentration or properties of fluorescent compounds.

HPLC analysis confirmed the presence of several bioactive compounds in the tea extracts, including gallic acid, rutin, tannic acid, and potentially EGCG and quercetin. The relative abundance of these compounds may vary among the different regions due to variations in soil characteristics, climate, and cultivation practices.

Based on the HPLC analysis, the following observations can be made regarding the relative abundance of bioactive compounds in the tea extracts from Assam, Balasun, and Jalpaiguri:
Gallic Acid: While gallic acid was detected in all three tea extracts, Balasun and Assam tea extracts exhibited higher levels of this compound compared to Fatapukur VVtea tea extract.
Tannic Acid: All three tea extracts contained tannic acid, with similar levels observed in Balasun and Fatapukur tea extracts.
Assam tea extract may have slightly lower levels of tannic acid. EGCG: EGCG was detected in all three tea extracts, with the highest levels observed in Fatapukur VVtea tea extract.
Quercetin: Quercetin was detected in all three tea extracts, with similar levels observed in Balasun and Assam tea extracts. Fatapukur tea extract may have slightly lower levels of quercetin.
Rutin: The HPLC analysis revealed potential evidence for the presence of rutin in all three tea extracts (Fatapukur, Balasun, and Assam). While the observed retention times varied slightly from the reference standard, the data suggest that rutin may be present in varying concentrations in these samples.

The analysis of total free amino acids revealed variations among the tea samples. Balasun tea exhibited the highest concentration of free amino acids, followed by Jalpaiguri and Assam teas. These differences could be attributed to factors such as soil nutrient availability and tea cultivar. The results suggest that while all three tea extracts contain a similar profile of bioactive compounds, there are variations in the relative abundance of these compounds. Balasun tea extract appears to be relatively rich in gallic acid and tannic acid, while Fatapukur tea extract may be higher in EGCG and quercetin.

The antimicrobial assays demonstrated that tea extracts from all three regions exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria. This suggests the presence of bioactive compounds in tea that can inhibit the growth of microorganisms. The observed differences in antimicrobial activity among the tea extracts could be attributed to variations in the concentration of antimicrobial compounds or their synergistic interactions.

Antimicrobial Activity: The correlation between total metabolite content and antimicrobial activity was found to be 0.77, indicating a strong positive correlation. This suggests that regions with higher levels of total metabolites tend to have higher antimicrobial activity in their tea extracts.

Antioxidant Potential: The correlation between total metabolite content and antioxidant potential was found to be 0.52, indicating a moderate positive correlation. This suggests that there is a positive association between the number of metabolites and the antioxidant potential of tea extracts, but the relationship is not as strong as the one observed for antimicrobial activity.

The analysis reveals that Fatapukur tea extract, with its higher total metabolite content, exhibits the most pronounced antimicrobial and antioxidant activities. Balasun tea extract, despite having a lower total metabolite content, still demonstrates significant bioactivity. Assam tea extract, while exhibiting lower total metabolite levels compared to Fatapukur, maintains moderate antimicrobial and antioxidant properties. These findings highlight the regional variations in tea extract composition and their potential implications for health benefits.
• Nitrogen: Nitrogen showed a strong positive correlation with both antimicrobial activity and antioxidant potential, suggesting that higher nitrogen levels in the soil may contribute to the production of bioactive compounds with these properties.
• Organic Carbon: Organic carbon also exhibited a positive correlation with antimicrobial activity and antioxidant potential, indicating its importance in soil health and tea quality.
• Phosphorus: Phosphorus showed moderate positive correlations with antimicrobial activity and antioxidant potential, suggesting its role in influencing tea extract properties.
• Potassium: Potassium exhibited moderate positive correlations with antimicrobial activity and antioxidant potential, indicating its potential influence on tea quality.

The correlation analysis (regression analysis) and Table 2 suggests that soil macronutrients, particularly nitrogen and organic carbon, are positively correlated with the antimicrobial and antioxidant properties of tea extracts. These findings highlight the importance of soil health and nutrient management in tea cultivation for producing high-quality tea with desirable functional properties. The data on micronutrient content in the soils of Assam, Darjeeling (Balasun), and Jalpaiguri was further analyzed with the previously obtained results for antimicrobial activity, antioxidant potential, and metabolite composition of the tea extracts.

The comprehensive analysis of tea extracts from Assam, Darjeeling (Balasun), and Jalpaiguri has revealed a complex interplay between soil characteristics, metabolite composition, and biological activities.
• Soil nutrients: Variations in soil macronutrients (nitrogen, phosphorus, potassium) among the regions influence the growth and development of tea plants, potentially impacting the production of bioactive compounds. Micronutrients such as magnesium, calcium, sulfur, iron, manganese, and copper also play a role in tea plant health and metabolite production.
• Tea Metabolite Composition: The tea extracts analyzed contained a variety of bioactive compounds, including gallic acid, rutin, tannic acid, EGCG, and quercetin. The relative abundance of these compounds varied among the different tea extracts.
• Antimicrobial Activity: All tea extracts exhibited antimicrobial activity against both Gram-positive and Gramnegative bacteria. Fatapukur VVtea tea extract demonstrated the highest antimicrobial activity, followed by Balasun and Assam tea extracts.
• Antioxidant Activity: All tea extracts exhibited significant antioxidant properties, with Fatapukur VVtea tea extract showing the highest potential.
• Total Free Amino Acids: Balasun tea extract was found to have the highest concentration of free amino acids, followed by Jalpaiguri and Assam tea extracts.

Overall, the results suggest that tea extracts from Assam, Darjeeling, and Jalpaiguri possess valuable bioactive properties, including antimicrobial and antioxidant activities. The variations in these properties can be attributed to a combination of factors, including soil characteristics and cultivation practices.

Acknowledgement

The project is done with the help of the intramural fund disbursed by St. Xavier’s College, Kolkata (Project Reference no. (IMSXC2023-24/002).

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