Effect of fertilizer application on tea plant productivity and phytochemicals in prepared black tea

Abstract

Different applications of the fertilizers were carried out to study their effect on tea plant productivity and phytochemicals in prepared black tea. Weight and dimensions of the bud, first and second leaf of each variety was taken for this purpose. Organic application (T1) 200 g vermicomposting per bush and inorganic application (T2) which is a combination of 2.5 g urea, 5 g Di-ammonium phosphate and 1 g potash was applied per bush. Weights of the bud was found higher in organic treated Ambari (0.061 g), Phubtsering 312 (0.056 g), Takda 78 (0.069 g) but in Banekbern 157 (0.046 g) and Gumti (0.07 g) inorganic fertilizer treatment increased the weight. The weight of first leaves ranged from 0.04 g to 0.115 g for the experimental samples. The weight of first leaves of PB 312, Banekbern 157 and Gumti varieties was observed to have greater for inorganic while for AV2 and Takda 78 weight of first leaf was observed to be greater for organic treatment. The weight of second leaf was found greater for inorganic treatment for all varieties which ranged from 0.115 g to 0.335 g in all samples of tea. In all varieties length was found to be increased in inorganic treatments. In all varieties, inorganic practice has positive effect in the growth and yield of the leaves. The fresh leaves and black tea prepared from the leaves of organic treatments had better tea quality in reference to TPC, TFC and DPPH radical scavenging activity. The caffeine content was not significantly different (p > 0.05) for the organic and inorganic treatment for both fresh leaves and prepared tea. Black tea prepared by organic treated leaves from Gumti and AV2 were superior to other samples regarding the phytochemical properties. Inorganic treatments increased the yield and growth but the quality of tea was increased by organic fertilizers.

Keywords:

• Tea
• Fertilizers
• Phytochemicals
• Black tea
• Organic and Inorganic

1. Introduction

Tea plant Cammelia sinensis (L.) is an important cash crop in tropical and subtropical areas (Meegahakumbura et al., 2018). It is source of very refreshing and popular drink that is hot aqueous infusion of its dried leaves (KC et al., 2020). Tea plant require appreciable amount of both macro and micro nutrients for proper growth and development. The minerals like nitrogen, potassium, phosphorous and magnesium are essential nutrients and their deficiency lead to the adverse effect on the yield and the quality of tea (Islam et al., 2017). These essential nutrients could be derived from fertilization. Fertilization, both organic and inorganic, is an important agricultural practice to increase crop yields, improve plant growth and development and maintain soil fertility by supplying nutrients to the plants (Qiu et al., 2014). The high fertilizer cost reduces the net returns and profits to the farmer. Also, the uncontrolled use of inorganic fertilizers leads to soil quality and water degradation through surface runoff and leaching. A better nutrient management strategy will be beneficial to farmers saving the high cost imposed on fertilizer and enable the production in a sustainable manner (Mokaya, 2016).

In the study conducted by Gao et al. (Gao et al., 2020) it was found that the potassium content and organic matters were increased as the chemical fertilizer, that is, urea was decreased in an appropriate amounts from 85 to 40% in tea thus improving the fertility of soil of tea gardens; also the reduction of chemical fertilizer were reported to increase polyphenols content and water extract in tea leaves, which improve the Oolong tea infusion. The caffeine content was also reported to decrease as the chemical fertilizer in the tea garden was decreased (Gao et al., 2020), which could be beneficial for producing low-caffeine tea. Similarly, Islam et al. (Islam et al., 2017) reported that the application of organic fertilizer improved the plant’s height, number of leaves and branches, and organic matter. Organic fertilizers can be used in place of chemical fertilizers because they have been found to improve soil fertility, water holding capacity, and structure, while chemical fertilizers may degrade the soil’s physical properties and fertility. Contradictory results regarding effect on caffeine content was reported (Gao et al., 2020), (Tabu et al., 2015). It was reported that there was an increase in amino acid and decrease in catechins in tea due to increased nitrogen source. Chemical and organic fertilizers’ effects on the growth of the tea plant and the quality of black tea have not yet been studied in Nepal. Thus, this study was performed to fulfill this gap and also this study helps to suggest the better fertilizer and their proper use to increase the quality and productivity of tea.

2. Materials and Methods

2.1. Research site selection

Tea gardens of Nepal Tea and Coffee Development Board (NTCDB), Hile, Dhankuta (27°02ʹ05.9”N, 87°19ʹ10.7”E) were selected for the application of the organic and inorganic fertilizers in tea bushes. In this region, the average annual rainfall is 135.21 mm and the average relative humidity is 55.82%. Similarly the average annual high temperature 33.28°C while the average low temperature 23.39°C Different varieties selected for the research were AV2, Takda 78, Phubtsering 312 (PB-312), Gumti and Banekbern 157 (Bb-157). Systematically arranged plots for the bush of each variety were selected.

2.2. Collection of tea leaves

Collection of tea clones were based on the survey (questionnaire to tea farmers of the Hile area) and the most cultivated five clones were taken for the study and maintained cold chain up to the laboratory in ice box to preserve the samples prior to analysis. Five confirmed varieties (having highest consumption) were taken as sample. Sample size was 100 leaves if the plot is systematic and 1–2 leaves in each bush if the plot is random (TRI, 2009).

2.3. Application of the fertilizers

Experimental plots were subjected to the fertilizer application (Table 1) by ring method on monthly basis at 9:00 to 11:30 AM from December 2019, after the light pruning (LP) where the tea bushes were trimmed at 18 inch from the ground level. In all flush seasons: required plucking was done. In December 2020, level of skiff (LOS) was done where the tea bushes were trimmed at 26 inch from the ground level. In the month of April in 2021, plucking was done for the data collection and the experiments.

Table 1. Fertilizer mixtures

Fertilizer mix	Application
Organic application (T1)	200 g vermicomposting per bush
Inorganic application (T2)	(urea 2.5 g + 5 g Di-ammonium phosphate (DAP) + 1 g Potash) per bush

2.4. Measurement of physical parameters of tea (bud, 1st leaf and 2nd leaf)

The average value of each sets of 20 specimens (bud, 1st leaf and 2nd leaf) of each clone were taken to measure length, breadth and weight.

2.5. Preparation of tea

The collected samples of tea leaves were processed for black tea in National Commercial Agriculture Research Program (NCARP), Pakhribas, Dhankuta and analyzed for total polyphenol content, total flavonoid content, caffeine content and DPPH activity in Central Campus of Technology Dharan and Nilgiri College, Itahari. Orthodox black tea was prepared as described by Kc et al. (KC et al., 2020). Fine plucking (containing bud to second leaf) was carried out by hand. Withering was carried out for 12–15 hrs with 60–65% wither. Tea rolling was done for 25–30 mins followed by 90 mins fermentation at 28°C. Then cabinet dryer was used to dry the tea at 110°C for 20–25 mins till moisture reached 5–6%.

2.5.1. Extraction of phytochemicals

Phytochemicals from prepared tea and leaves were extracted using methanol as described by Punyasiri et al. (Punyasiri et al., 2015) with a slight modification. 10 g of samples will be steeped in 80%, 100 ml methanol at 66°C for 10 min. Then it was cooled to room temperature and homogenized for 3 min using grinder. Subsequently, it was filtered using Whatman no. 41 and the residue was re-extracted following the above procedure. The extract was stored in a screw capped bottle at 4 ± 2°C until analysis. 10 ml of extract was evaporated, dried at 80°C and the residue was measured to know its concentration.

2.5.2. Determination of total phenolic content (TPC)

TPC of the sample was determined using spectrophotometric method with some modifications (Jaradat et al., 2015). 0.5 ml of plant extract, 2.5 ml of 10% Folin-Ciocalteu’s reagent and 2.5 ml of 7.5% of Na₂CO₃ was mixed and it was incubated at 45°C for 45 min. The absorbance at 765 nm on UV-Vis spectrophotometer for each samples was analyzed in triplicate. The test results was correlated with standard gallic acid curve and TPC was expressed as mg Gallic acid equivalent (mg GAE/g) of dry matter in extract.

2.5.3. Determination of total flavonoid content (TFC)

TFC of the sample was determined using a modified Aluminum chloride assay method (Barek et al., 2015). 2 ml of the methanolic extract was mixed with 0.2 ml of NaNO₂ (5%, w/v) and after 5 min, 0.2 ml of AlCl₃ (2%, w/v) was added and allowed to stand for 6 min. This followed addition of 2 ml of 1 N NaOH and finally volume was made up to 5 ml. After holding for 15 min at room temperature the absorbance was measured at 510 nm on UV-Vis spectrophotometer. The test result was correlated with standard Quercetin curve and TFC was expressed as mg quercetin equivalents (mg QE/g) of dry matter in extract.

2.5.4. Determination of DPPH radical scavenging activity

The DPPH radical scavenging activities (antioxidant activities) of the extracts were determined as per the method described by Vignoli et al. (Vignoli et al., 2011). 1 ml of the extract was mixed with 2 ml of DPPH (0.004% in methanol, corresponding to 100 μM) incubated at 37°C in dark (wrapped with aluminum foil) for 20 min (for completion of reaction) before spectrophotometric analysis. The absorbance was measured at 517 nm on UV-Vis spectrophotometer after 30 min incubation in the dark. Finally, percentage scavenging activity was determined using following equation:

$\mathrm{S}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}\left(\mathrm{\%}\right)=\hspace{0.17em}\frac{\mathrm{A}\mathrm{c}-\mathrm{A}\mathrm{s}}{\mathrm{A}\mathrm{c}}\times 100$

Ac represents absorbance of control, and As stands for absorbance of the test sample.

2.6. Statistical analysis

The experiments were conducted in triplicate and were analyzed by one- way analysis of variance (ANOVA) and t-test by using software GenStat Release 12.1 (Copyright 2009, VSN International Ltd.) and for significant difference at 5%, means were separated using Tukey test. R studio was used for the correlation plot and PCA biplot.

3. Results and discussions

3.1. Effect of fertilizer application of tea plants

3.1.1. Bud

The length and breadth of buds for organic (T1) and inorganic treatments (T2) were found significantly different (<0.05) for tea plants (Figure 1, Figure 2 and Table 2).

Figure 1. The length of buds of different tea varieties under organic (T1) and inorganic treatments (T2).

* Bars sharing similar letters are not significantly different by LSD at p > 0.05

Figure 2. The breadth and weight of buds of different tea varieties under organic (T1) and inorganic treatments (T2).

* Bars sharing similar letters are not significantly different by LSD at p > 0.05

Table 2. T test for the physical properties of tea plant with organic and inorganic treatments

Tea varieties	Leaf specimens	Bud		1st leaf		2nd leaf
	Physical parameters	/t-value/	p-value	/t-value/	p-value	/t-value/	p-value
AV2	Weight (g)	5.883484	0.004171*	3.333974	0.044587*	23.2517	2.03E-05*
	Length (cm)	11.9182	0.00127*	2.288618	0.149311	106.75	1.81E-06*
	Breadth (cm)	2.65165	0.056884	8.812002	0.000915*	7.397576	0.005109*
PB-312	Weight (g)	4.52267	0.010637*	3.87298	0.017948*	21.9107	0.000208*
	Length (cm)	16.3565	0.000497*	19.4155	0.000298*	29.4553	7.91E-06*
	Breadth (cm)	2	0.139326	13.0052	0.00586*	8.10931	0.001257*
Takda 78	Weight (g)	5.753965	0.010429*	18.97857	4.54E-05*	11.555	0.00032*
	Length (cm)	15.411	0.000594*	10.95592	0.000394*	8.85438	0.000898*
	Breadth (cm)	8.12624	0.001247*	9.888666	0.000587*	6.14701	0.003552*
Bb-157	Weight (g)	4.54344	0.045185*	13.75	0.000162*	30.7439	7.56E-05*
	Length (cm)	1	0.391002	45.1668	2.39E-05*	19.9189	0.000277*
	Breadth (cm)	2.44949	0.070484	17.6392	6.07E-05*	17.6392	6.07E-05*
Gumti	Weight (g)	10	0.002128*	7.3413	0.001833*	56.0811	6.05E-07*
	Length (cm)	4.810702	0.040597*	8.140806	0.001239*	25.1029	0.000139*
	Breadth (cm)	1.73925	0.156975	6.25	0.003341*	8.75	0.00094*

*Significant at p < 0.05, E denotes the exponential

However, Takda78 was only the variety whose bud breadth was significantly affected by fertilizer application. The length of bud of Gumti variety was observed longer as compared to the other variety for organic treatment while in inorganic length of bud of PB-312 and Takda 78 were observed greater than other varieties. The breadth of Takda 78, Bb-157 and Gumti were observed wider for inorganic treatment while on organic treatment the breadth of Bb-157 and Gumti were observed wider as compared to other varieties. There was no significant difference in length of bud of all varieties except Bb-157 (Table 2). Similarly, weight of bud for organic and inorganic treatments were found significantly different (P < 0.05) for all the varieties of tea as shown in Figure 2. The weight of bud was observed higher for Gumti for inorganic treatment and Takda 78 for organic treatment than other varieties. AV2, PB-312 and Takda 78 was observed to have higher weight of bud for organic treatment while Bb-157 and Gumti weight of bud was observed to be higher for inorganic treatment.

Mineralization process of the fertilization release the nutrient elements source that can be utilized by plant for growth and development. But the process of mineralization take longer time for organic fertilizers as compared to inorganic fertilizers, as it releases nutrients more slowly (Purbajanti et al., 2019). Also, organic fertilizers contain a low levels of nutrients thus require in large amount to overcome deficiencies than that of inorganic fertilizers. While the inorganic fertilizers are loaded with high levels nutrients which are rapidly available for uptake by plant (Devkota et al., 2021). This might be the reason for the slower growth of bud length and width in organic fertilizers and faster in inorganic fertilizers.

3.1.2. First leaf

The length and breadth of first leaf for organic (T1) and inorganic treatments (T2) were found significantly different (<0.05) for all the varieties of tea (Figure 3 and Table 2).

Figure 3. The length, breadth and weight of first leaf of different tea varieties under organic (T1) and inorganic treatments (T2).

* Bars sharing similar letters are not significantly different by LSD at p > 0.05

The length of first leaf of PB-312 variety was observed longer as compared to the other variety for both organic and inorganic treatment. The breadth of first leaf of Bb-157 was observed wider for inorganic treatment while on organic treatment the breadth of AV2 and Gumti were observed wider as compared to other varieties. Similarly, weight of first leaf for organic and inorganic treatments were found significantly different (<0.05) for all the varieties of tea as shown in Figure 3. The weight of first leaf was observed higher for PB-312 for both inorganic and organic treatment than other varieties. PB-312, Bb-157 and Gumti varieties was observed to have higher weight of first leaf for inorganic treatment than that of organic treatments while for AV2 and Takda 78 weight of first leaf was observed to be higher for organic treatment. Moreover, AV2 was only variety which didn’t show significant difference in length of first leaf due to fertilizer application (Table 2).

The release of essential nutrients by organic fertilizers is very slow as compared to inorganic fertilizers which results in decrease of sulphur and phosphorous concentrations in the leaves, thus decreasing the growth and yield (Heeb et al., 2006). This might be the reason for the less growth in length, breadth of first leaves and less weight for organic treatment than inorganic treatment. Pavlou et al. (Pavlou et al., 2007) reported the highest level of nitrate for the inorganic fertilization treatments initially of lettuce for all the seasons, but the residual nitrogen, potassium and phosphorous was found to be enhance in the soil treated with organic fertilizers as compared to that of inorganic fertilization. This might be applicable to tea leaves also.

3.1.3. Second leaf

The length and breadth of second leaves for organic (T1) and inorganic treatments (T2) were found significantly different (<0.05) for all the varieties of tea as shown in Figure 4. The length of second leaves of PB-312 variety was observed longer as compared to the other variety while breadth of second leaves for variety Bb-157 was found to be wider as compared to other varieties. It was observed that the length and breadth was higher for inorganic treatment than that of organic treatment for all variety except AV2. Similarly, weight of second leaves for organic and inorganic treatments were found significantly different (<0.05) for all the varieties of tea (Figure 4). The weight of second leaves was observed higher for PB-312 than other varieties. All the varieties was observed to have higher weight of second leaves for inorganic treatment than that of organic treatments.

Figure 4. Caffeine (%) content of different varieties of (a) fresh leaves and (b) black tea under organic (T1) and inorganic treatments (T2).

* Bars sharing similar letters are not significantly different by LSD at p > 0.05. Alphabets “a-d” is used for fresh leaves while “m-p” is used for black tea.

Mokaya (Mokaya, 2016) observed that the length and width of second leaves of tea plant was higher for the inorganic fertilizers than that of organic fertilizer. This was similar to the result found in our study. Qiu et al. (Qiu et al., 2014) also state that the yield of tea was higher for the mixed fertilizers than that of single organic fertilizers. The inorganic fertilizers are rich in micro-nutrients such as S, Na, Zn, P, K and Mg which helps in plant and also helps to increase plant yields as compared to that of organic fertilizers (Kanton et al., 2016). Organic fertilizers did not increase the average length, width and yield of the second leaf as compared to that of inorganic fertilizers. This could be led to the result that the organic fertilizer needed longer time to release nutrients necessary for the plant growth; and also the growth was related to climatic condition, at different season different pattern of growth of second leaves of tea plant was observed at Kenya reported by (Mokaya, 2016). So, in this study the growth of second leaves might be affected by the micro-nutrient present inorganic fertilizers and climatic conditions which was required for the proper release of nutrients by organic fertilizers.

3.2. Effect on the phytochemicals of the tea leaves

3.2.1. Caffeine

The caffeine content of tea leaves of varieties AV2, Bb-157, Takda 78 and PB-312 under organic and inorganic treatments were not significantly difference (>0.05) from each other (Figure 4 and Table 3). Similar results were observed for caffeine content in black tea while Bb-157 was found significantly different (<0.05) between organic and inorganic treatments. The highest caffeine content was observed for AV2 variety with 3.4% for organic treatment and 3.3% for inorganic treatment. The caffeine content of black tea was found to be lower than that of fresh leaves. For black tea from organic treated Gumti variety, the caffeine content was higher for organic treatment than inorganic treatment. For black tea, the highest caffeine content was observed in AV2 variety with 3.22% for organic treatment while the least amount of 2.17% was observed in organic treated Bb-157. However, caffeine content was found to be high in the nitrogenous fertilized tea leaves, indicating the stress in plants (Jahan et al., 2022). The caffeine content of black tea ranges from 1–5% by dry weight (Kanton et al., 2016). The tea caffeine content depends upon the fertilizer application, plucking methods, and maturity and fermentation periods. The concentration of caffeine is higher in leaf tissue than stem tissue. Also coarser plucking would lower the caffeine content of tea than that of fine plucking (Cloughley, 1982). These might be the reason for the variation of caffeine in black tea produced from different varieties. In both fertilizers the same level of caffeine content was observed in our study. Pham et al. (Pham et al., 2019) reported the caffeine content of black tea was lower than the green tea and fresh leaves. Same as in our study also caffeine content of black tea was reported lower than fresh leaves. Brewing methods, processing styles, terroir, age of tea leaves and the cultivars of the tea plant are major factors for the variation of caffeine content in tea brew. Similarly, chloroplast produces caffeine that protects the new growths of plant from insect attacks (Drew, 2019). There was no significant effect of organic and inorganic fertilizers in caffeine content of both tea leaves and black tea. Almost all researches performed previously by different researchers did not support our result about fertilization type effect on caffeine content of tea. The higher amount of caffeine content was reported in tea plants which was supplied with ammonium fertilizers (S-X et al., 2013). Also, Tabu et al. (Tabu et al., 2015) reported the high amount of caffeine in tea infusion prepared from the tea grown in inorganic fertilizers than that of cow manure.

Table 3. T test for the chemical properties of tea leaves and its black tea with organic and inorganic treatments

Variety	Parameters	/t-value/ between T1 and T2 for leaves	p-value	/t-value/ between T1 and T2 for black tea	p-value
Bb-157	Total Flavonoid content (mg QE/g dry extract)	7.16	0.00201*	44.18	1.6E-06*
	Total Polyphenols content (mg GAE/g dry extract)	62.98	3.81E-07*	41.57	3.1E-05*
	% DPPH inhibition)	11.79	0.0003*	18.10	0.00037*
	Caffeine (%)	0.069	0.947	3.595	0.036*
AV2	Total Flavonoid content (mg QE/g dry extract)	77.81	4.68E-06*	73.45	5.56E-06*
	Total Polyphenols content (mg GAE/g dry extract)	123.55	2.57E-08*	88.95	4.58E-08*
	% DPPH inhibition	5.64	0.01105*	3.34	0.044*
	Caffeine (%)	2.658	0.076	2.17	0.161
Takda 78	Total Flavonoid content (mg QE/g dry extract)	23.08	2.08E-05*	2.13	0.001 *
	Total Polyphenols content (mg GAE/g dry extract)	122.626	1.20E-06*	14.36	0.004*
	% DPPH inhibition	14.6	0.0007*	14.01	0.00015*
	Caffeine (%)	0.25	0.81	1.6	0.18
PB-312	Total Flavonoid content (mg QE/g dry extract)	50.05911	9.53E-07*	38.42	3.88E-05*
	Total Polyphenols content (mg GAE/g dry extract)	194.7434	4.17E-09*	161.58	8.8E-09*
	% DPPH inhibition	4.71407	0.01807*	2.78	0.049*
	Caffeine (%)	0.079	0.94	0.75	0.49
Gumti	Total Flavonoid content (mg QE/g dry extract)	39.04405	2.57E-06*	113.77	1.5E-06*
	Total Polyphenols content (mg GAE/g dry extract)	115.9107	1.42E-06*	111.4	3.89E-08*
	% DPPH inhibition	26.49	1.2E-05*	39.35	2.49E-06*
	Caffeine (%)	3.11	0.052	2.29	0.08

*Significant at p < 0.05, QE = Quercetin equivalent, GAE = Gallic acid equivalent, E denotes the exponential

3.2.2. Total Phenolic Content (TPC)

The total phenolic content of organic and inorganic treatments were observed significantly difference (<0.05) from each other for all the varieties of tea leaves and black tea (Figure 5 and Table 3). The TPC of organic treatment was observed to be higher than that of inorganic treatments for all varieties of tea leaves except Takda 78 and similar result was observed for black tea as well. In inorganic treatment Takda 78 variety with 632.58 mg GAE/g dry extract was reported to have higher TPC in tea leaves than others while for organic treatment Gumti variety was reported to have higher TPC with 620.58 mg GAE/ g dry extract than others. The TPC of black tea was found to be lower than that of fresh leaves. In inorganic treatment black tea from Takda 78 variety with 563.6 mg GAE/g dry extract was reported to have higher TPC than others while for organic treatment black tea from Gumti variety was reported to have higher TPC with 600.3 mg GAE/ g dry extract than others. The reason to decrease of TPC in black tea might be due to the disruption of the compartments of the cell during crushing brings the phenolic compounds present in vacuole in contact with polyphenol oxidases present in plastid thus resulting in oxidation of phenolics (Vastrad et al., 2022). Ruan et al. (Ruan et al., 1999) reported that potassium increased the polyphenols in tea leaves while theaflavins and thearubigins, were increased in brewed tea, Tabu et al. (Tabu et al., 2015) reported that the two major phenolic compounds of tea theaflavin and thearubigin were found to be affected by fertilization process. The inorganic fertilization treatment reduced the theaflavin content of tea while thearubigin was not affected. Tabu et al., (Tabu et al., 2015) stated that increase in nitrogen content increases free amino acid content in leaves of tea but may reduce catechins production. So, overall effect on phenolic content was increased. Similar result was observed in our study also. Similarly, Ibrahim et al. (Ibrahim et al., 2013) also reported in increase of total phenolics in Labisia pumila with organic fertilizers treatment than that of inorganic over the end of 15 weeks. According to Ibrahim et al. (Ibrahim et al., 2013), total polyphenols content in Labisia pumila Benth was enhanced by 12% in the treatment with organic fertilizer as compared to inorganic fertilization. A similar increment could be also observed for tea. The inorganic fertilizers provide readily available nitrogen sources which might favors development and growth of plant but does not favor the production of phytochemicals and other secondary metabolites. But due to the absence of readily accessible nutrients in organic treatment, which provide the nutrient more slowly than inorganic fertilizers, might leads to the exposure of plant in more stressful situations, thus initiating the production of secondary metabolites such as polyphenols in plants (Devkota et al., 2021),(Mastura et al., 2017).

Figure 5. TPC (mg GAE/g dry extract) of different varieties of (a) fresh leaves and (b) black tea under organic (T1) and inorganic treatments (T2).

* Bars sharing similar letters are not significantly different by LSD at p > 0.05. Alphabets “a-j” is used for fresh leaves while “m-r” is used for black tea.

3.2.3. Total Flavonoid content (TFC)

The total flavonoid content of organic (T1) and inorganic treatments were observed significantly difference (<0.05) from each other for all the varieties of tea leaves and black tea (Figure 6 and Table 3). The TFC of organic treatment was observed to be higher than that of inorganic treatments for all varieties of tea leaves. In inorganic treatment Banekbern 157 variety was reported to have greater TFC in tea leaves with 293.12 mg QE/g dry extract than others while for organic treatment Takda 78 variety with 358.74 mg QE/g dry extract was reported to have higher TFC than others. The TFC content of black tea was found to be lower than that of fresh leaves. In the study of the Bangladesh, the quality of tea leaves was severely affected, where contents of total flavonoids, polyphenols, soluble solids, vitamin C, vitamin B1, and vitamin B2 were reduces which might be due to the potential stress due to nitrogenous fertilization (Jahan et al., 2022). The TFC of black tea with organic treatment was observed to be higher than that of inorganic treatments for all varieties of black tea samples. Inorganic treated black tea from Takda 78 was reported to have higher TFC with 219.5 mg QE/g dry extract than others while for organic treatment black tea from Gumti variety had higher TFC 307.4 mg QE/g dry extract. The fermentation or oxidation process during black tea manufacturing results in the oxidation of flavonoids present in the tea leaves due to the release of polyphenol oxidase. It not only transforms the flavonoids but also gives the taste and color to the product (Dwyer & Peterson, 2013). This might be the reason of decrease in TFC of black tea than that of tea leaves. Saikia and Upadhyaya (Saikia & Upadhyaya, 2011) reported the highest level of total flavonoids and phenol in the root of plant Asparagus racemosus grown in vermicompost treated soil. Similar type of result was observed in our study of tea leaves. Chaudhai and Jamatia (Chaudhuri & Jamatia, 2021) also reported the higher flavonoid contents on the tea leaves by the treatment with rubber leaf vermicompost fertilizers to the soil. Elevated levels of phenolic compounds and flavonoid content was found in organically grown crops than conventionally grown crops (Bagchi et al., 2015). According to Ibrahim et al. (Ibrahim et al., 2013) total flavonoids content in Labisia pumila Benth was enhanced by 22% in the treatment with organic fertilizer as compared to inorganic fertilization. Similar increment could be also observed for tea. The inorganic fertilizers provide readily available nitrogen sources which might favors development and growth of plant but does not favor the production of secondary metabolites (Mastura et al., 2017).

Figure 6. TFC (mg QE/g dry extract) of different varieties of (a) fresh leaves and (b) black tea under organic (T1) and inorganic treatments (T2).

* Bars sharing similar letters are not significantly different by LSD at p > 0.05. Alphabets “a-h” is used for fresh leaves while “m-t” is used for black tea.

3.2.4. DPPH free radical scavenging activity

The % DPPH scavenging activity of organic and inorganic treatments were observed significantly difference (<0.05) from each other for all the varieties of tea leaves (Figure 7 and Table 3). The % DPPH scavenging activity of organic treatment was observed to be higher than that of inorganic treatments for Bb-157 and Gumti varieties of tea leaves while in AV2, Takda 78 and PB-312 the inorganic treatment were shown to have higher % DPPH scavenging activity. In inorganic treatment leaves of Takda 78 variety was reported to have greater % DPPH scavenging activity with 66.5% than others while for organic treatment Banekbern 157 and AV2 variety with 50.89% and 51.27%, respectively. Moreover, free-radical scavenging activity (DPPH), was higher in the nitrogenous fertilized tea leaves, indicating the stress in plants (Jahan et al., 2022). The % DPPH scavenging activity of black tea for organic treatment was observed to be lower than that of inorganic treatments for black tea from all varieties. For organic and inorganic treatments, black tea from Gumti variety was reported to have higher % DPPH scavenging activity with 49.43% and 55.5%, respectively.

Figure 7. % DPPH scavenging activity of different varieties of (a) fresh leaves and (b) black tea under organic (T1) and inorganic treatments (T2).

* Bars sharing similar letters are not significantly different by LSD at p > 0.05. Alphabets “a-f” is used for fresh leaves while “m-s” is used for black tea.

According to KC et al. (KC et al., 2020) the radical scavenging activity in the black tea found in Nepal ranged from 21.99 to 76.32 (μg/mL). The similar range was observed in our study too. Also, Kodama et al., (Kodama et al., 2010) reported the DPPH scavenging activity of green tea infusion was in the range of 23 a 131 mmoles equivalents. This was also almost similar to result of obtained in our study. Fernando and Soysa (Fernando & Soysa, 2015) observed the significant correlation between the antioxidant capacity of CTC black tea with the level of flavonoids, EGCG and TPC. For caffeine a weak correlation was observed with antioxidant activity of tea. The antioxidant activity variance for different treatments (organic and inorganic) in our study might be due to the slow diffusion of polyphenols and flavonoids in tea extract compared to caffeine which diffuse faster in tea extract (Fernando & Soysa, 2015). Gramza et al. (Gramza et al., 2005) discussed about the ability of DPPH free radical scavenging was also due to the pheophytin and chlorophyll content present in tea leaves. It was confirmed that hydrogen can be delivered for DPPH radical by chlorophyll as well as for scavenging the radicals formed during lipid oxygenation. Since, chlorophyll content differs for the different tea varieties and lost during the enzymatic oxidation (Ošťádalová et al., 2015). Thus, we could say that DPPH scavenging activity in our study differs due to the difference in phytochemicals and chlorophyll content of different varieties of tea.

3.3. Correlation plot for the chemical attributes in black tea

The TPC and DPPH of tea leaves and black tea both were positively correlated with correlation coefficient of 0.411 while strong correlation (correlation coefficient of 0.83) was observed between TPC and TFC (Figure 8). While TFC and DPPH were positively correlated with correlation coefficient 0.416. Also, a positive correlation was observed with caffeine for DPPH, TPC and TFC. Similar results were observed by KC et al. (KC et al., 2020) By comparing the correlation coefficients, it is possible to suggest that phenolic and flavonoids are highly responsible for the antioxidant activity rather than caffeine. Phenolics and flavonoid molecules can donate hydrogen atom to free radicals and their structural composition aid on the free radical scavenging activity.

Figure 8. Correlation plot between TFC, TPC, % DPPH scavenging activity, and caffeine content of black tea.

3.4. Principle component analysis (PCA)

PCA was done to select the optimum sample of black tea prepared by organic treated and inorganic treated leaves of different varieties where the first principal component (Dim.1) was responsible for 54.5% variation with eigen value of 2.18 while second principal component (Dim.2) was reported to 28% variation with eigen value of 1.12 these two component together account for 82.54 percent variations (Figure 9a). The shape palette can deal with a maximum of 6 discrete values because more than 6 becomes difficult to discriminate. But total ten samples with organic and inorganic treated for five varieties are present. So, instead of dealing manually, tea prepared from inorganic fertilizer treated leaves were excluded and PCA was done again. First principal component (Dim.1) was responsible for 66.2% variation with eigen value of 2.64 while second principal component (Dim.2) was reported to 29.5% variation with eigen value of 1.17 these two component together account for 95.63 percent variations (Figure 9b). The distribution of different tea samples was influenced by TFC, TPC, DPPH radical scavenging activity and caffeine content. Among these samples, the samples coded as GT1 and AT1 were positioned on the first quadrant (Figure 9b) which illustrates black tea prepared by organic treated leaves from Gumti and AV2 were superior to other samples regarding the phytochemical properties. Similar result was demonstrated by Sun et al. (Sun et al., 2021) where application of cow manure in soils changed the metabolic characteristics of tea shoots and improved tea quality.

Figure 9. Bi-plot distributions of different samples with grouping TFC, TPC, % DPPH scavenging activity, and caffeine content (a) black tea under organic and inorganic treatments (b) black tea under organic treatment only. Here, A, B, G, P, and T denote AV2, Bb-157, Gumti, PB-312, and Takda 78, respectively, while T1 and T2 denote organic and inorganic, respectively.

4. Conclusion

Natural tea farming promotes and enhances biodiversity, organic cycles, soil biological activity by the means of control practices that repair, maintain, and enhance ecological harmony. The organic tea production device is not the same as organic tea production in reference to productivity and adequate quality. Despite the fact, inorganic practice elevated the productivity of all the varieties; Ambari, Takda 78, Phubtsering 312, Gumti and Banekbern 157, but in terms of phytochemicals, organic practice was found to be superior. Although there was no significant difference in the caffeine content of prepared black tea from the leaves of organic and inorganic practice, total polyphenols, flavonoids and DPPH free radicals scavenging activities were superior in black tea from the organic leaves. For this reason, the natural practice must be accomplished with elevated productivity and quality.

Acknowledgments

The authors would like to acknowledge, University Grants Commission, Nepal, National Commercial Agriculture Research Program (NCARP), Pakhribas, Dhankuta, Nilgiri College, Itahari and Nepal Tea and Coffee Development Board, Hile for providing support for this research work.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The authors received no direct funding for this research.

Notes on contributors

Lila Devi Shiwakoti

Lila Devi Shiwakoti is an Agriculture Technician in Nepal Tea and Coffee Development Board, Nepal. She has worked in Panitar-Ilam, Hile-Dhankuta, Jasbire-Ilam and Birtamod-Jhapa. Besides providing the suggestions and trainings on pre-harvest and post-harvest practices of tea to the farmers and processors, she regularly visits tea gardens and factories for improvement in tea sectors of Nepal. She is also associated in the research team of Yadav KC-Assistant Professor who is involved in tea research. Most of the farmers are in dilemma considering the quality and productivity of the tea with organic and inorganic practices. This research aims to convince the farmers for organic practices with the scientific data.

Lila Devi Shiwakoti Conceptualization, Methodology, Data curation, Writing- draft preparation

Krishna Chalise Data curation, writing, reviewing

Krishna Chalise

Krishna Chalise Data curation, writing, reviewing

Pankaj Dahal

Pankaj Dahal Writing-original draft preparation, reviewing

Ramesh Shiwakoti

Ramesh Shiwakoti Methodology, Writing- reviewing and editing

Nirat Katuwal

Nirat Katuwal Methodology, Writing-reviewing and editing

Yadav Kc

Yadav KC Conceptualization, Methodology, Data curation, Supervision, Writing-critical revision of the manuscript