Tea polyphenols and catechins have gained prominence in determination of tea quality. Tea leaves contain large amounts of catechins and their derivatives and these have shown positive effects on human health. An experiment was carried out to determine the level of catechins and polyphenols in 20 new clones found in two distinct tea growing areas. The trial comprising twenty clones was established in two distinct sites in Kenya using randomized complete block design with three replicates and consisted of 10 popular cultivars widely cultivated in the Lake Victoria Basin and additional 10 newly improved clones. Tea leaf samples were collected across two seasons; dry and wet seasons. Tea quality parameters entailing catechins, caffeine and total polyphenols was generated for the 20 clones across the two seasons and sites. The ISO 14502-1 and ISO 14502-1:2005 procedures were used for polyphenol and catechin analyses, respectively. The ANOVA results generated indicated that the level of catechins and polyphenols in tea were significantly different (p < 0.05) and varietal dependent. Generally, the assam varieties had higher levels of catechins and polyphenols than the chinary and cambod varieties, indicating why they are popularly cultivated in the East African region and whose tea quality is highly regarded. Furthermore, the results from principle component analysis (PCA) grouped the clones into three major clusters with one comprising of high quality assam varieties, a mixture of moderate quality assam, cambod and chinary varieties and low catechin content assam anthocyanin-rich variety. It is concluded that the level of catechins in tea plants are varietal dependent, although environmental stress has effects on the synthesis of flavanoids. The results demonstrated that the differences in the accumulation of catechins among the tea clones could be important for the discrimination of clonal diversity.
Tea, camellia sinensis (L) O. Kuntze, the most popular universal beverage contains a wide range of the Flavanoids [
Given the importance of tea to the economy of many countries and human health, the use of suitable tea cultivars that are well endowed in both yield and phytochemicals like flavonoids is paramount. The tea improvement programme at the Tea Research Foundation of Kenya (TRFK) has resulted in many new tea varieties, but exploitation of only a few popular clones accounting for about 60% of tea in the country may have resulted in narrowing of the clones which could be exploited for the commercializable germplasm [
The trial comprising twenty clones was established in April 2009 in two major tea growing areas: at the Timbilil Estate of the TRFK, Kericho and Kipkebe in Sotik Ltd (
The trial consisting of 10 popular cultivars widely cultivated in the Lake Victoria Basin covering Kenya, Rwanda, Uganda and Tanzania and an additional 10 newly improved clones was initiated in 2009 as one of two separate studies entailing “Evaluation of tea cultivars to different growing environments in East African countries” and “Evaluation of response of clone TRFK 6/8 to nitrogen rates and harvesting intervals in East African countries” [
About 500 g of fresh shoots in form of two leaves and a bud were plucked from each of the clonal entries and placed in appropriately labelled khaki bags. These were then transferred into a cool box containing ice packs and transported to the lab for processing. The clones were sampled twice under different environmental conditions (wet and dry seasons) as shown in
Catechin analysis was done following the protocol and the procedure described by the International Organization for Standardization (ISO) 14502-1:2005. Two hundred milligrams of ground samples were extracted with 5 ml of warm (70˚C) 70% methanol. The mixture was then warmed in a water bath (70˚C) for 10 minutes and vortexed in between after five minutes interval and then cooled. The cool mixture was centrifuged at 3500 rpm for 10 minutes, and the extract was decanted into a 10 ml cylinder. The samples were then reextracted and the extract decanted to the initial 10 ml cylinder topped-up to 10 ml. One millilitre of the extract was diluted 5-fold using stabilizer reagent and passed through a 0.5 mm pore filter before injection into the HPLC C-6 column for catechin analysis. The mobile phase A consisted of 9% (volume fraction) acetic acid, and mobile phase B was made of 80% (volume fraction) acetonitrile. The flow rate was controlled at 1.0 ml/min, with an injection volume of 20 ml. The column was operated at 40˚C and UV spectra peaks were detected at 278 nm. The chromatographic peaks in the samples were identified by comparing their retention times with chemical standards used.
For polyphenols analysis, the method described by the International Organization for Standardization (ISO) 14502-1 was used for polyphenols. 1.0 ml of the garlic acid standard solutions were transferred into separate glass tubes labelled A, B, C, D and E in triplicates. Subsequently, 1.0 ml of the diluted sample extract was transferred into separate triplicate tubes. 5.0 ml of 10% Folin-Ciocalteu’s reagent was added. Then immediately, 4.0 ml of a sodium carbonate solution (7.5% w/v) was added. The tubes were vortexed and then allowed to stand at room temperature for 60 min before absorbance was determined at 765 nm against the blanks using a spectrophotometer.
The various catechins and polyphenols contents in the clones were analyzed using M-Stat-C statistical software for ANOVA. Principle component analysis (PCA) on the concentration of catechins in different clones was done using GENSTAT software [
The catechins were eluted in the following order Gallic Acid (GA), Epigallo catechin (EGC), Caffeine (Caff), Epicatechin (EC), Epigallocatechingalate (EGCG), Catechin(C) and Epicatechin galate (ECG). An example of eluted catechins following HPLC analysis is presented in
A recapitulation of mean squares of the measured parameters is shown in
The TP contents in the 20 clones presented in
NB: Asterisks *, **, and *** denote significance level at p < 0.05, p < 0.01 and p < 0.001 respectively.
TRFK 12/12, had significantly higher levels of TP at 24.52%, while clone TRFK 301/6 had the least levels, with a mean of 20.14%. However, clones TRFK 31/11, TRFK 371/ 3, BBK 35, had similar TP content at 24.02%, 23.92%, and 23.80% respectively. There was no notable differences in the mean total polyphenols across four environments for clones TRFK 301/4, TRFK 430/90, and TRFK 303/1199.
At the Timbilil site, clone TRFK 371/3 had the highest level of TP at 26.49% during wet season, while clone EPK C12 had the least content of polyphenols at 19.81%. In the dry season, clone TRFK 303/1199 had the highest level of polyphenols of 23.84%, while clone TRFK 301/6 had the least at 18.32%. Although most of the clones showed some slight drop in total polyphenols from wet to dry seasons, the drop by clone TRFK 6/8 and TRFK 371/3 of 6% was rather drammatic.
At the Kipkebe site, clone TRKF 12/12 had the highest level of TP at 25.17% during the wet season, while clone TRFK 301/6 had the least at 19.81%. Clone TRFK 31/11 had the highest level polyphenols of 26.89%, while clone TRFK 301/6 had the least at 20.94% during dry season. Interestingly, clone TRFK 306 had the least level of TP during both seasons. It is worth noting that, unlike for Timbilil, the level of total polyphenols in Kipkebe rose on average from wet to dry season. Clones TRFK 100/5, TRFK 31/11 and TRFK 371/3 appeared to have responded more positively by the onset of stress visited by the dry season.
The total catechin content in the 20 clones presented in
At the Timbilil site, clone TRFK 371/3 had the highest total catechins at 18.85%, while clone TRFK 306 had the least at 9.23% in wet season. In the dry season clone TRFK 6/10 had the highest catechins content at 18.05%, while clone TRFK 306 had the least at 10.63%. Although most of the clones showed significant decrease in total catechins during dry season, it is worth noting that clones TRFK 100/5, TRFK 6/8, and TRFK 12/19 showed a more decrease at the onset of water stress.
At the kipkebe site, clone TRFK 12/19 had the highest level of catechins at 20.27%, while clone TRFK 306 had the least at 10.27%, during wet season. During dry season, clone TRFK 303/1199 had the highest catechin content at 20.11%, while TRFK 306 had the least at 9.94%. The drastic decrease in total catechin content for clones TRFK 12/19, TRFK 11/4, TRFK 7/3 and TRFK12/12 during dry season is notable. Conversly, clone TRFK 430/90 showed a considerable increase in total catechins due to water stress. Thus, the clones were not affected in the same way by the changes in weather.
Garlic Acid content among the 20 clones varied signifycantly (p < 0.05) (
At the Timbilil site wet season, clone TRFK 301/4 the highest GA content while, clone TRFK 12/19, had the least in both seasons (
had the least in both seasons. There was significant decline in GA content as a result of water stress for all the clones except for clones TRFK 6/8, BBK35 and TRFK 31/11 which had significant increase. It is noteworthy that clones TRFK 430/90 and TRFK 371/3 were not affected by the changes in environment.
EGC content varied significantly with sites and seasons for the 20 clones presented in
TRFK 430/90 had the highest EGC content at 6.36%,while clone TRFK 31/11 had the least at 2.63%. All the clones except for clones TRFK 301/6, TRFK 303/577, TRFK 12/19, TRFK 11/4, TRFK 6/8 and TRFK 371/3 had a increase in EGC content during dry season.
At Kipkebe site, clone TRFK 301/5 had the highest EGC content at 6.76%, while clone TRFK 306 had the least at 1.26% during the wet season. In the dry season, clone TRFK 303/577 had the highest EGC content at 6.18% while clone TRFK 306 had the least at 2.59%. although most of the clones had an increase in EGC content in dry season, clone TRFK 306 and TRFK 430/90 had a drastic increase during dry season.
As shown in
season. Considering the means, clone TRFK 306 had the highest catechin content at 0.57%, while clone TRFK 31/11 had the least at 0.26%. Clones TRFK 6/10 and TRFK 301/6; clones TRFK 303/577 and TRFK 12/19; clones EPK C12 and TRFK 11/4; and clones TRFK 7/3, TRFK100/5 and TRFK 430/90 had equal catechin contents. At the Timbilil site, wet season, clone TRFK 12/12 had the highest catechin (+C) at 0.72%, while clone 31/11 had the least at 0.12%. Conversely during the dry season, clone TRFK 306 had the highest (+C) at 0.74% while clone TRFK 31/11 had the least at 0.17%. At the Kipkebe site, wet season, clone TRFK 303/577, had the highest catechin at 0.71%, while clone TRFK 301/5 had the least at 0.18%. In dry season, clone TRFK 306 had the highest catechin content, while clone TRFK 301/6 had the least at 0.22%. It is worth noting that most of the clones had lower catechins during dry season. However, clone TRFK 306 had considerable increase in catechin content during the dry season for both sites.
The level of caffeine varied considerably among the 20 clones presented in
There was a substantial variation (p < 0.05) in EC content among the 20 clones presented in
NS: denotes non-significant.
0.89%. On the other hand, during the dry season, clone TRFK 301/6 had the highest EC content at 4.91%, while clone TRFK31/8 had the least at 1.02%. At this site, most of the clones had an increase in EC content. It is worth noting that at the two sites, Cambod clones TRFK301/5, TRFK 301/4 and TRFK 301/6 generally had high EC content than the Assam clones.
EGCG content in the 20 clones presented in
the clones had a decline in their EGCG content during the dry season, although clones TRFK 6/8 and TRFK 31/11 showed more decrease. Nevertheless, at the Kipkebe site, clone TRFK 31/11 had the highest EGCG content at 10.38%, while clone TRFK 6/8 had the least at 5.93% during wet season. During the dry season, clone 31/11 had the highest EGCG content at 10.68%, while clone TRFK 301/6 had the least at 3.33%. Majority of the clones had EGCG content decline during the dry season.
There was significant variation in ECG content in the 20 clones presented in
The principle component analysis grouped the clones into three clusters (
The principle component analysis on the total polyphenols presented in
The results from catechin analysis indicate that the level of biochemical components in tea is varietal dependent. This is an indication that each individual clone is unique in the level of catechins. Most of the clones used showed some level of association between the total polyphenols and soil water content. There was lower polyphenols content in dry season compared to the wet season in most of the clones. This was expected because water is one of the raw materials for photosynthesis and it has direct impacts on organic synthesis of plants of both the primary and the secondary metabolites. The leaf is the major source of photo assimilates, which yields the major precursors of secondary metabolism such as malonylCoA and coumaroyl-CoA which are dependent on the light and the soil water content [
The clones had high level of caffeine during the dry season. This is because accumulation of secondary metabolites such as caffeine by plants is known to be a form of defence mechanism adopted by plants. This mechanism helps plants respond to and adapt to environmental stresses [
The new improved clones had high levels of catechins like the other commercialized clones used in this study except for clone TRFK 306. The low levels of catechins in fresh leaves of clone TRFK 306 could be attributed to the up regulation of the anthocyanin synthase in biosynthetic pathway for anthocyanins rather than the leucoanthocyanin reductase in catechin biosynthetic pathway probably due to environmental stimuli [
The catechin content in some of the clones was not
significantly affected by seasonal variation. This could be attributed to the slight changes in the catechin levels in individual clones, resulting from seasonal variation due to the principal amounts of the specific catechins in the leaf. However, organic compounds that are synthesized in small quantities, such as C, ECG, EGC and EC, could be expected to exhibit significant changes when the plant is under stress, but this was not the case for the results of some of the clones from this study. This observation rules out the influence of principal amount of catechins in plants as the basis of the differential water stress response of the plants. The results obtained from this study are corroborated by earlier result which indicated that the changes in soil water content did not have effects on the level of catechins [
On the other hand, the differences in the levels of catechins in the different clones could be attributed to the up regulation or down regulation of the enzyme flavanone 3-hydroxylase. Flavanone 3-hydroxylase (F3H) is an abundant enzyme in tea leaves that catalyses the stereo specific hydroxylation of (2S)-naringenin to form (2R, 3R)-dihydrokaempferol. In a study carried out to determine the concentration of catechins, and CsF3H gene expression found a positive correlation between the concentration of catechins, and CsF3H gene expression in leaves at different developmental stage. CsF3H expression was down-regulated in response to drought, abscisic acid and gibberellic acid treatment, but up-regulated in response to wounding. The concentration of catechins paralleled the expression data. In addition, they also reported that drought decreased the level of catechins in tea [
From principle component analysis, a clear structure was observed within the different clones of tea with the identification of different clusters based on the catechins and polyphenols shown in Figures 2 and 3. The Assam teas have higher catechins content compared to the Cambod teas, which have moderate catechins. This could be attributed to the unique catechin profile of Cambod varieties. The Cambod clones were low in EGCG, medium low in EGC and high in EC and ECG content. These results are similar to the results obtained in a preceding study [
From this study it can be concluded that the level of catechins in tea plants are varietal dependent. Environmental stress has effects on the synthesis of Flavanoids as well as the levels of caffeine in plants. The results presented indicated that the differences in the accumulation of catechins among the tea clones, and by extension important for the discrimination of clonal diversity. The determination of the influence of wider climatic conditions on the biosynthesis and the content of Flavanoids could be explored as the subjects for further research.