Principal Component Analysis based Clustering of UPASI Tea Cultivars for their Diversity on Free Radical Scavenging Activity
K.M. Maria John
Tea is one of the popular beverages which immensely have therapeutic activity against various diseases. The biochemical and therapeutic properties of tea was observed to be varying because of its seasonal and varietal difference which will reflect in the quality of the end product. Yield and quality are the major factors determining the profit. So, research is still going on to search high quality tea plant among the huge tea seedling population. This process is basically a time involving (in years) or cost of selection will be high. The present study, grouping of tea plant was made by using total catechins and polyphenols activity against free radicals such as DPPH and Hydroxyl radicals. UPASI-17 registered the highest polyphenol, catechin content as well as its scavenging activity against free radicals than that of other tea cultivars. When considering the catechin and polyphenols scavenging activity, catechin influence higher than that of polyphenols against DPPH and Hydroxyl radicals. IV distinct groups were observed through Principal Compound Analysis (PCA) and dendrogram analysis based on individual clones free radical scavenging activity. By this analysis all clones have high free radical scavenging property was grouped separately. By adapting this method of analysis, it will reduce the time and cost involvement in the screening of new tea varieties from the seedling population and help in the production of high free radical scavenging black tea.
to cite this article:
K.M. Maria John and R. Premkumar, 2012. Principal Component Analysis based Clustering of UPASI Tea Cultivars for their Diversity on Free Radical Scavenging Activity. Research Journal of Phytochemistry, 6: 1-8.
Received: November 02, 2011;
Accepted: January 28, 2012;
Published: February 13, 2012
Tea is one of the major economic crops of India which provides good foreign
exchange to the nation. South India is playing a vital role in the production
and exporting of tea in the world market, particularly Tamil Nadu. Planters
are facing various problems to achieve this essential process for the country.
Yield and quality of tea plants needs to improve every time to stand in the
market. For this important process, screening of tea plants among the huge seedling
population was continued to get good accessions in terms of quality and yield.
Infilling, inter planting, inter row planting and replanting were based on these
selected cultivars to improve the required yield and quality (Muraleedharan,
2005). To identify such high quality plants found to be a big task because
of the huge seedling population of South India (90%). Quality of tea is one
of the major criteria for the high price which immensely contains high antioxidant
capability (Owuor and Obanda, 2007; Nshimiyimana
and He, 2010). A careful screening might be required for the selection in
terms of quality.
It is well known that the crop shoots of tea contained various biochemicals
such as flavonoids, alkaloids and enzymes besides the higher amount of polyphenols
(John et al., 2006a, b).
Therapeutic functions of polyphenols can be listed more like antioxidant, anticancer
and cholesterol content (Yang and Landau, 2000; Mukhtar
and Ahmad, 2000; Davies et al., 2003). One
of the major properties of polyphenol was its scavenging activity against free
radicals in humans (Mudgal et al., 2010; Gupta
et al., 2008; Arnao et al., 1999;
Robards et al., 1999). Because of the potential
damaging nature of free radicals, oxidative stress may cause physiological stress
when antioxidant defences are unable to cope with them (Gill
et al., 2011a; Frankel, 1993).
So, the individual catechin based tea characterization was made by Saravanan
et al. (2005). But in that diversity study, we concentrated only
on individual catechin molecules and not with total polyphenols and its antioxidant
potential. The total polyphenol content in tea was found to be more than 30%
among them catechin which contributes around 20%. Role of other phenolics compounds
in free radical scavenging activity also to be consider when going for characterization.
It will give a clear idea as to which group of plants will have high amount
of free radical scavenging property. To develop such selection criteria an attempt
was made to characterize tea clones on the basis of its free radical scavenging
MATERIALS AND METHODS
Tea shoots comprising two leaves and a bud were harvested every month from UPASI Tea Research Experimental Farm, Valparai was used for the study. To avoid variations, uniformly aged and pruned plants were selected for this study.
Tea shoots collected were subjected to analyse its total catechins and polyphenol
content using spectrometer (Model: Genesys 10 UV) adopting the method reported
by Swain and Hillis (1959) and Dev
Choudhary Goswami (1983).
Scavenging activity: Free radical scavenging activity of OH was carried
out following the procedure of Halliwell and Gutteridge (1981),
whereas DPPH free radical was assayed following the method of Choi
et al. (2002) and Tagashira and Ohtake (1998).
DPPH free radical scavenging activity was analysed by using 0.5 mL of substrate, 1.0 mL of 0.1 mm DPPH in ethanol and 0.5 mL of 50 mm Tris-HCl bufler (pH 7.4), after 30 min of incubation the OD was taken at 517 nm.
OH radical scavenging activity was analysed by adding 0.5 mL substrate with 1.0 mL of EDTA solution and mixed with 1.0 mL of DMSO (0.85%) in 0.1 m phosphate bufler (pH 7.4) to initiate the reaction followed by the addition of 0.5 mL of 0.22% ascorbic acid. After incubation and colour development the absorbance was observed at 412 nm. The difference between control and treatment OD was taken for scavenging activity calculation.
PCA analyses: Based on the data obtained, values were subjected to linear regression analysis and principal component analysis using the Special Software SIMCA P for clustering the tea clones. For PCA analysis total catechins and polyphenols scavenging activity against free radical were considered.
RESULTS AND DISCUSSION
The polyphenol and catechin level in all the UPASI tea clones were examined and presented in Fig. 1. DPPH and hydroxyl free radicals scavenging property of UPASI clones as well as its corresponding catechin and polyphenol values were also analysed and presented. Total polyphenol content was found to be high with UPASI 17 clone followed by UPASI 8 (29.03 and 28.18%) and lowest amount was registered with UPASI 14 followed by UPASI 4 clones respectively (21.55 and 22%). From the same extract the total catechin content was also analysed and the results revealed that UPASI 17 (19.83) registered high catechin content followed by UPASI 3 (18.07), the lowest catechin content was registered with UPASI-16 (14.57) followed by UPASI 25 (14.95). The DPPH and hydroxyl free radical scavenging activity of all the UPASI tea clones were studied. The scavenging activity was found to be high with UPASI-17 followed by UPASI 19 (32.52, 30.39) in case of DPPH radicals. The lowest amount of DPPH scavenger was UPASI-26 and UPASI-27, respectively (27.05 and 27.09). UPASI-17 followed by UPASI-3 (26.4 and 24.71) registered the highest hydroxyl radical scavenging activity. The lowest activity was noticed with UPASI- 16 and UPASI-5 (21.29 and 21.32).
With the available data a PCA and dendrogram analysis was made by using SIMCA
P software. The catechin and polyphenol content of the clones were plotted in
X axis and the free radicals scavenging percentage of the individual clones
were taken in Y axis. While analysed with this conditions IV different groups
had been segregated from the data. The free radical scavenging property of catechin
and polyphenols against DPPH and hydroxyl freeradicals are analysed with PCA
and presented in Fig. 2. The dendrogram of the UPASI clones
polyphenol and catechin content against DPPH and Hydroxyl radicals were presented
in Fig. 3. Based on the PCA clustering analysis and dendrogram
of the clones, four distinct clustering was observed based on the clones polyphenol
and catechin content against DPPH and hydroxyl radical scavenging activity.
Group 1 showed the lowest scavenging activity against free radicals. UP 16,
UP 25, UP 5, UP 4 and UP 14 falls under this group and were found to be average
quality clones released by UPASI Tea Research institute. UP 26, UP 27, UP 22,
UP 13, UP 7, UP 11, UP 18, UP 6 and UP 9 falls under group 2.
|| Total polyphenol and catechin content of UPASI tea clones
||Principle compound analysis (PCA) of UPASI tea clones against
DPPH and Hydroxyl scavenging potential
||Dendrogram of UPASI tea clones based on free radical scavenging
potential against DPPH and Hydroxyl Radicals
All the medium quality clones were clustered under group 3 (UP 1, UP 2, UP
8, UP 10, UP 12, UP 19, UP 20, UP 21 and UP 24). There is no trend on the basis
of jats were observed with the clustering analysis.
A linear regression was plotted against the radical scavenging activity against
polyphenols andcatechins by means of predicted and observed values. Only few
predicted results coincide with the observed results. In DPPH against polyphenols
and catechins only 4 clones such as UPASI 5, 9, 15 and 16 fall under the predicted
line (Fig. 4a).
||Linearity between the catechin and polyphenols content against
Thirteen clones show lower values than the predicted values. Nine of them
were found to have higher value than the observed values. In the case of hydroxyl
radicals, only 3 clones such as UPASI 10, 16 and 25 were plotted in the predicted
line. 14 clones show lower values than the predicted values and 9 of them were
found to have higher value than the observed values (Fig. 4b).
In UP 17, the level of catechin and polyphenols was high resulted with high
free radical scavenging activity was observed. The influence of total catechins
and total polyphenols against DPPH and Hydroxyl radicals were compared and presented
in Fig. 5. Results revealed that the catechin influences more
scavenging activity against free radicalsthan total polyphenol content of the
Free radical scavenging activity of plants is found to be very important for
humans day to day life to overcome certain illness (Choi
et al., 2002; Semalty et al., 2009;
Gill et al., 2011b; Arora
et al., 2011). Tea is one of the major crops which produce various
phenolics components which served as free radical scavengers. Bravo
et al. (2007) studied the antioxiadant activity of tea and compared
with commercially available drinks. Wide variation in terms of such antioxidant
compounds present in tea was observed. Grouping of tea was found to be one of
the best tools to characterise tea plant from seedlings and to develop into
good quality or high yielding accession. Based on our earlier study, we found
that grouping of tea can be done not only with morphological, physiological
and molecular but also can be done with biochemical parameters such as EGCG,
EC, EGC and ECG (Saravanan et al., 2005). The
clustering of tea clones based on free radical scavenging potential was found
to be an important aspect to select a cultivar. Thomas et
al. (2008) studied the diversity among various forms of flavanols in
selected UPASI tea germplasm.
||Effect of total catechins and polyphenols on free radical
While analysing the clusters, the group was not segregated based on the jats
of the tea plants. It clearly indicates that the radical scavenging property
of the clones will not depend upon the jats or its morphology. Based on our
study it indicates that the amount of EGCG was high with UP 17, so the high
free radical scavenging activity was obtained. Amount of polyphenols and catechins
was found to be high in the case of group IV and the free radical scavenging
property was also observed high. This result was coinciding with Muthiah
et al. (2009) stating that the amount of free radicals scavenging
found to be influenced by the catechins content. Borse et
al. (2007) reported that marginal variations in the total catechins
do not interfere with its radical scavenging activity. But, we observed that
marginal variation in polyphenol content of the plant does not influences the
free radical scavenging activity but the catechins influences the activity.
Xu et al. (2004) stated that the epicatechin
molecules are high free radical scavengers among the polyphenols. The UPASI
clones contains high catechin content, had high free radical scavenging activity
and the influence of catechins on free radicals scavenging property was higher
than that of total polyphenols in the plants. The iron toxicity on polyphenol
content in tea was reported by Hemalatha and Venkatesan
(2011). According to John et al. (2006c),
the higher DNA protection activity was observed with high concentration of catechin
molecules. These results coincide with the present work as stated by Saravanan
et al. (2005), the seasonal and clonal influence was observed in
the case of catechin and polyphenols as well as its free radical scavenging
activity. When analysed this data with PCA, it gave IV distinct groups but in
our earlier study by using individual catechin molecules for characterization
produces 5 distinct groups were observed.
In this study, we found that there is no relationship between grouping and
jats. All the groups were segregated based on its free radical content. Gulati
et al. (2009) studied tea catechins as biomarker to study the diversity
of Indian tea clones. Same result was observed in the present study stating
that no relationship between biochemical content and its varietal differences.
Selecting a cultivar based on its antioxidant property might be one of the best
tool because tea is concern for its anti inflammatory properties. So, the ratio
of major secondary metabolites such as polyphenols and catechins played a vital
role in the quality potential of tea (Kottur et al.,
2010), not only for quality of black tea but also for its therapeutic aspects.
It is a simple and reliable technique to screen the tea plants from the huge seedling populations with high therapeutic potential. Tea is not only taken for its simulative activity as beverage but also for its immense therapeutic property. The cultivars screened by this method will provide good source of catechins in made tea leads to good therapeutic activity in the end product.
We are thankful to Dr. P. Mohan Kumar, Director, UPASI Tea Research Foundation, Tea Research Institute, Valparai for his constant encouragement and suggestions.
Arnao, M.B, A. Cano and M. Acosta, 1999.
Methods to measure the antioxidant activity in plant material: A comparative discussion. Free Radic. Res., 31: 89-96.PubMed |
Arora, R., M. Kaur and N.S. Gill, 2011.
Antioxidant activity and pharmacological evaluation of Cucumis melo
var. agrestis methanolic seed extract. Res. J. Phytochem., 5: 146-155.CrossRef | Direct Link |
Gulati, A., S. Rajkumar, S. Karthigeyan, R.K. Sud and D. Vijayan et al
Catechin and catechin fractions as biochemical markers to study the diversity of Indian tea (Camellia sinensis
(L.) O. Kuntze) germplasm. Chem. Biodivers., 6: 1042-1052.CrossRef | PubMed | Direct Link |
Borse B.B., H. Vijay Kumar and L. Jagan Mohan Rao, 2007.
Radical scavenging conserves from unused fresh green tea leaves. J. Agric. Food Chem., 55: 1750-1754.CrossRef |
Choi, C.W., S.C. Kim, S.S. Hwang, B.K. Choi and H.J. Ahn et al
Antioxidant activity and free radical scavenging capacity between Korean medicinal plants and flavonoids by assay-guided comparison. Plant Sci., 163: 1161-1168.CrossRef | Direct Link |
Nshimiyimana, D.S. and Q. He, 2010.
Radical scavenging capacity of rwandan CTC tea polyphenols extracted using microwave assisted extraction. Pak. J. Nutr., 9: 589-593.CrossRef | Direct Link |
Frankel, E.N., 1993.
In search of better methods to evaluate natural antioxidants and oxidative stability in food lipids. Trends Food Sci. Technol., 4: 220-225.CrossRef | Direct Link |
Gill, N.S., S. Kaur, R. Arora and M. Bali, 2011.
Screening of antioxidant and antiulcer potential of Citrullus colocynthis
methanolic seed extract. Res. J. Phytochem., 2: 98-106.CrossRef | Direct Link |
Kottur, G., S. Venkatesan, R.S.S. Kumar and S. Murugesan, 2010.
Diversity among various forms of catechins and its synthesizing enzyme (phenylalanine ammonia lyase) in relation to quality of black tea (Camellia
spp.). J. Sci. Food Agric., 90: 1533-1537.CrossRef | Direct Link |
Gupta, J., Y.H. Siddique, T. Beg, G. Ara and M. Afzal, 2008.
A review on the beneficial effects of tea polyphenols on human health. Int. J. Pharmacol., 4: 314-338.CrossRef | Direct Link |
Halliwell, B. and J.M.C. Gutteridge, 1981.
Formation of a thiobarbituric-acid-reactive substance from deoxyribose in the presence of iron salts: The role of superoxide and hydroxyl radicals. FEBS Lett., 128: 347-352.CrossRef | PubMed | Direct Link |
Hemalatha, K. and S. Venkatesan, 2011.
Impact of iron toxicity on certain enzymes and biochemical parameters of tea. Asian J. Biochem., 6: 384-394.CrossRef | Direct Link |
Bravo, L., L. Goya and E. Lecumberri, 2007.
LC/MS characterization of phenolic constituents of mate (Ilex paraguariensis
, St. Hil.) and its antioxidant activity compared to commonly consumed beverages. Food Res. Int., 40: 393-405.CrossRef | Direct Link |
Choudhary, M.N.D. and M.R. Goswami, 1983.
A rapid method for determination of total polyphenolic matter in tea Camellia sinensis
L. O Kuntze. Two Bud, 30: 59-61.
Muthiah, M.J., J. Thomas, R.R. Kumar and A.K.A. Mandal, 2009.
Studies on radical scavenging activity of tea leaves and effect of additives on activities of black tea liquor. Int. J. Food Sci. Technol., 44: 2070-2074.CrossRef | Direct Link |
John, K.M.M., S.D. Joshi, A.K.A. Mandal, R.R. Kumar, R. Premkumar, 2006.
Potential role of tea catechins in protection of DNA from free radical damage. J. Plantation Crops, 34: 597-600.
John, K.M.M., D. Vijayan, R.R. Kumar and R. Premkumar, 2006.
Factors influencing the efficiency of extraction of polyphenols from Young tea leaves. Asian J. Plant Sci., 5: 123-126.CrossRef | Direct Link |
John, K.M.M., R. Sasikumar, D. Vijayan, M. Saravanan, P.R. Rahul and R.R. Kumar, 2006.
Influence of externally added substrates on total catechin content in tea leaves (Camellia
sp.). Asian J. Plant Sci., 5: 116-119.CrossRef | Direct Link |
Davies, M.J., J.T. Judd, D.J. Baer, B.A. Clevidence and D.R. Paul et al
Black tea consumption reduces total and LDL cholesterol in mildly hypercholesterolemic adults. J. Nutr., 133: 3298S-3302S.Direct Link |
Mudgal, V., N. Madaan, A. Mudgal and S. Mishra, 2010.
Dietary polyphenols and human health. Asian J. Biochem., 5: 154-162.CrossRef | Direct Link |
Mukhtar, H. and N. Ahmad, 2000.
Tea polyphenols: Prevention of cancer and optimizing health. Am. J. Clin. Nutr., 71: 1698s-1702s.Direct Link |
Muraleedharan, N., 2005.
Annual report. UPASI Tea Research Foundation, India, pp: 1-130. http://www.biriz.biz/cay/islah/UPASI_Report.pdf.
Gill, N.S., R. Arora and S.R. Kumar, 2011.
Evaluation of antioxidant, anti-inflammatory and analgesic potential of the Luffa acutangula
Roxb. Var. amara. Res. J. Phytochem., 5: 201-208.CrossRef | Direct Link |
Owuor, P.O. and M. Obanda, 2007.
The use of green tea (Camellia sinensis
) leaf flavan-3-ol composition in predicting plain black tea quality potential. Food Chem., 100: 873-884.CrossRef | Direct Link |
Robards, K., P.D. Prenzeler, G. Tucker, P. Swatsitang and W. Glover, 1999.
Phenolic compounds and their role in oxidative processes in fruits. Food Chem., 66: 401-436.CrossRef | Direct Link |
Saravanan, M., K.M.M. John, R.R. Kumar, P.K. Pius and R. Sasikumar, 2005.
Genetic diversity of UPASI tea clones (Camellia sinensis
(L.) O. Kuntze) on the basis of total catechins and their fractions. Phytochemistry, 66: 561-565.CrossRef | PubMed | Direct Link |
Semalty, M., A. Semalty, G.P. Joshi and M.S.M. Rawat, 2009.
Comparison of in vitro
antioxidant activity of Trigonella foenum-graecum
and T. corniculata
Seeds. Res. J. Phytochem., 3: 63-67.CrossRef | Direct Link |
Swain, T. and W.E. Hillis, 1959.
The phenolic constituents of Prunus domestica
. I. The quantitative analysis of phenolic constituents. J. Sci. Food Agric., 10: 63-68.CrossRef | Direct Link |
Tagashira, M. and Y. Ohtake, 1998.
A new antioxidative 1,3-benzodioxole from Melissa officinalis
. Planta Med., 64: 555-558.CrossRef | Direct Link |
Thomas, J., R.R. Kumar and A.K.A. Mandal, 2008.
Diversity among various forms of ﬂavanols in selected UPASI tea germplasm. J. Plantation Crops, 36: 171-174.
Xu, J.Z., S.Y.V. Yeung, Q. Chang, Y. Huang and Z.Y. Chen, 2004.
Comparison of antioxidant activity and absorption of tea epicatechins with their epimers. Br. J. Nutr., 91: 873-881.CrossRef | PubMed | Direct Link |
Yang, C.S. and J.M. Landau, 2000.
Effects of tea consumption on nutrition and health. J. Nutr., 130: 2409-2412.CrossRef | Direct Link |