Accepted Manuscript

Antioxidant capacity and major polyphenols composition of teas as affected by

geographical location, plantation elevation and leaf grade

Chuang Zhang, Claire Li-Chieh Suen, Chao Yang, Siew Young Quek

PII: S0308-8146(17)31602-3
DOI: https://doi.org/10.1016/j.foodchem.2017.09.126
Reference: FOCH 21795

To appear in: Food Chemistry

Received Date: 14 March 2017

Revised Date: 24 September 2017
Accepted Date: 26 September 2017

Please cite this article as: Zhang, C., Li-Chieh Suen, C., Yang, C., Young Quek, S., Antioxidant capacity and major
polyphenols composition of teas as affected by geographical location, plantation elevation and leaf grade, Food
Chemistry (2017), doi: https://doi.org/10.1016/j.foodchem.2017.09.126

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1 Antioxidant capacity and major polyphenols composition of teas as affected by

2 geographical location, plantation elevation and leaf grade

3 Chuang Zhanga, Claire Li-Chieh Suena, Chao Yanga, Siew Young Queka*

4 a
Food Science, School of Chemical Sciences, The University of Auckland, Private Bag 92019,

5 Auckland 1142, New Zealand

7 *Corresponding author.

8 E-mail addresses: sy.quek@auckland.ac.nz (Siew Young Quek)

9 Add: Building 302, Science Centre, The University of Auckland, 23 Symonds Street,

10 Auckland 1010, New Zealand

11 Tel: +64 9 373 7599 Ext 85852

12 Fax: +64 9 373 7422

13

1
14 Abstract
15 Tea polyphenols has been a topic of discussion due to their health benefits. Nevertheless,

16 detailed study on the antioxidant capacity and polyphenols contents of teas in relation to

17 factors including geographical locations, plantation elevations and leaf grades have been

18 limited. In this study, 53 tea samples were analysed to determine the individual and total

19 catechins and theaflavins contents by HPLC and the total antioxidant capacity by Oxygen

20 Radical Absorbance Capacity (ORAC) methods. Results show that the polyphenols

21 (catechins and theaflavins) contents were significantly influenced by plantation location.

22 Black tea from low plantation elevation contained 22 to 28% more polyphenols than those

23 from high elevation. Small tea leaves had up to 15% more polyphenols than larger leaves

24 from similar elevation. The results were further confirmed by Principal Composition Analysis

25 (PCA), which grouped the black and green tea samples into 3 different clusters, respectively.

26

27 Keywords: tea polyphenols, catechins, theaflavins, antioxidant capacity

2
28 1. Introduction

29 Tea consumption has a long history in popular in some Asian, South American and European

30 countries, as a beverage and as herbal medicine. Since the last decades, tea has emerged as a

31 popular source of dietary antioxidants and its effects are being investigated by in vitro and in

32 vivo methods (Yang et al., 2016). Black tea and green tea are two main types of tea, and their

33 antioxidant effects are thought to be contributed by polyphenols (Vinson & Dabbagh, 1998).

34 Tea polyphenols includes flavonols (quercetein, kaempferol, myricetin), flavan-3-ols

35 (catechins and theaflavins), and a small amount of purine alkaloids (caffeine and

36 theobromine), gallic acid derivatives (gallic acid, 5-galloylquinic acid), and

37 hydroxycinammate quinic esters (caffeoylquinic acids) (Del Rio et al., 2004). Among them,

38 catechins and theaflavins are the two common indices used to determine the antioxidant

39 ability of tea.

40

41 Antioxidants are compounds that can reduce, slow down or prevent oxidation process (Kaur

42 & Kapoor, 2001). Antioxidant compounds can reduce radicals produced from oxidation

43 reactions in human body, thus contribute to its anti-carcinogenic properties (Rodrigo et al.,

44 2011). Many studies have found that tea antioxidant compounds have a promising effect on

45 various cancer cells in vitro, as well as protecting DNA from being damaged by free radicals

46 (Ježovičová et al., 2016; Yang et al., 2016; Friedman et al., 2007).

47

48 Tea catechins have two geometrical isomers (trans-catechins and cis-epicatechins), and each

49 isomer has two optical isomers: (+)-catechin and (–)-catechin; (+)-epicatechin and (–)-

50 epicatechin. (–)-catechin can be turned into (–)-catechin-3-gallate, epicatechin-3-gallate, (–)-

51 epigallocatechin-3-gallate, and (–)-gallocatechin-3-gallate by esterification with gallic acid.

52 By oxidative coupling, different catechins can form four types of theaflavins including

3
53 theaflavin (TF), theaflavin-3-gallate (TF3G), theaflavin-3’-gallate (TF3’G) and theaflavin-3,

54 3’-digallate (TF33’G) (Friedman et al., 2005).

55

56 Catechins are the primary polyphenols present in fresh tea leaves, and are mostly preserved in

57 green tea (Graham, 1992; Astill et al., 2001). However, during processing black tea, most

58 catechins in fresh green tea leaves are oxidised to form theaflavins through an oxidative

59 fermentation process (Graham, 1992). This process involves enzymatic browning by

60 polyphenol oxidase (Sharma et al., 2009) where the primary substrate of the enzyme is o-

61 dihydric phenols, i.e., catechins in tea leaves (Graham, 1992). This is a complex process

62 involves multi-step reaction pathway (Tanaka et al., 2001). Theaflavins contribute to the dark

63 and reddish colour of black tea, as well as the astringent and bitter taste, which is also an

64 important characteristic of black tea.

65

66 Determination of total catechins and theaflavins have been normally used to describe the

67 quality of green and black tea. However, individual catechin and theaflavin especially (–)-

68 epigallocatechin gallate (EGCG) and TF33’G are being used to evaluate the quality of green

69 and black tea (El-Shahawi, 2012). EGCG is the main catechin in green tea and has been

70 found to have anticarcinogenic property (Chowdhury et al., 2016). The advantages of EGCG

71 as anti-cancer remedy are due to its safety, bioavailability and low cost (Singh et al., 2011). It

72 has been reported to be a powerful polyphenolic antioxidant, which is proposed to prevent

73 oxidative damage of healthy cells and inhibit the inflammatory procedures that cause

74 transformation, hyperproliferation and suppression of cancer (Wang et al., 2016; Yang et al.,

75 2016; Thawonsuwan et al., 2010). EGCG has also been reported to have other beneficial

76 effects on diabetes, stroke and obesity (Oršolić et al., 2013; Zhang et al., 2016; Sergent et al.,

77 2012).

4
78

79 TF33’G, one of the theaflavins, is a polymerized and oxidized product from catechins in

80 black tea. Studies have showed that TF33’G has powerful anti-cancer and antioxidant

81 properties (Gao et al., 2016; Kimutai et al., 2016). In recent study, Ying et al. (2016) verified

82 that TF33’G has even more potent anti-tumour activity compared to EGCG in inhibiting

83 women ovarian carcinoma OVCAR-3 cell-induced angiogenesis via Akt and Notch-1

84 pathways. A positive and significantly high correlation between TF33’G and antioxidant

85 activity has also been found in black tea (Kimutai et al., 2016).

86

87 In addition to derivatization during tea processing, the concentrations of catechins and

88 theaflavins in green and black teas may be influenced by other factors such as tea tree variety,

89 growing environment, manufacturing conditions, etc. Many studies have been conducted on

90 the antioxidants in tea (Damiani et al., 2014; Koczka et al., 2016), but few study aimed to

91 analyse the diversity of tea antioxidant activity affected by different geographical locations,

92 plantation elevation, leaf size and especially leaf grade. New Zealand, has diversified tea-

93 consuming ethnics, with various tea types and brands from different parts of the world. This

94 provides a convenience platform to study tea properties from a wide range of samples.

95

96 This study is therefore aimed to investigate factors (geographical and plantation locations,

97 plantation elevations and leaf grades) that affect antioxidant capacity. It also aims to study the

98 contents of the major individual catechins and theaflavins of the 53 samples sourced from

99 different countries and areas.

100

101 2. Materials and methods

102 2.1 Materials

5
103 A total of 53 black tea and green tea samples were analysed in this study. Among which, 48

104 of them were kindly given by Bell Tea and coffee Company Ltd. (Auckland, New Zealand),

105 4 Longjing tea samples were obtained from Meichun tea estate (Hangzhou, China) and one

106 additional sample was obtained from Aaah Tea (Auckland, New Zealand). All the standards

107 used were of HPLC grade including, (–)-Catechin (C); (–)-catechin gallate (CG); (–)-

108 epicatechin gallate (ECG); (–)-gallocatechin (GC); (–)-epigallocatechin (EGC); (–)-

109 gallocatechin gallate (GCG); (–)-EGCG which were purchased from Sigma-Aldrich (St.

110 Louis, USA). The mixed theaflavins were purchased from Sigma-Aldrich (St. Louis, USA)

111 which contains TF, TF3G, TF3’G, TF33’G. Other HPLC grade chemicals used are as follow:

112 formic acid from BDH Chemical Ltd., Co (Poole, England); acetonitrile from Romil Ltd., Co

113 (Cambridge, England); acetone, ethanol and methanol from Burdick and Jackson Ltd., Co

114 (Muskegon, USA). Milli-Q water was used in the experiments quality.

115

116 2.2 Extraction and preparation of tea samples

117 Extraction methods were based on the experiment performed by Friedman et al. (2006). A

118 total of 1.5 g of dried tea leaves were weighed into a conical flask. Fifty mL of 80% ethanol

119 was added and incubated for 15 min capped with cold finger condensers to prevent solvent

120 evaporation in a 60 °C water bath (Ratek Shaking Water bath SWB, Australia). The extracted

121 solvents were placed into another collection conical flask. The residue tea leaves were further

122 extracted for the second time under the same condition. The total 100 mL extraction

123 supernatants were then mixed and centrifuged at 2000 rpm, 4 ℃ for 20 min to subside solid

124 remains.

125

126 One mL of supernatant was then transferred into a 1.5 mL Eppendorf snap-cap

127 microcentrifuge vial to evaporate the solvent using an Eppendorf vacuum concentrator

6
128 (Eppendorf 5301, Germany) at 30 °C. The dried extract were then sealed and stored in the

129 freezer at -20 °C (Fisher & Paykel, NZ). All tests were performed in triplicates. Prior to

130 HPLC analysis, extracts from each Eppendorf vial were reconstituted with 1 mL of 80%

131 methanol and then sonicated in an ultrasonic bath (Elma Transsonic T460, Germany) until all

132 solids were dissolved. The sample was then filtered with a 0.45 μm Millipore hydrophobic

133 syringe filter (USA). An aliquot of 200 μL was diluted to 1000 μL with 80% methanol in a 2

134 mL HPLC glass vial for a well-prepared use of HPLC analysis.

135

136 2.3 HPLC protocol

137 Catechin and theaflavin were determined by HPLC according to Del Rio et al. (2004). Briefly,

138 chromatographic separation was performed on a HP 1100 HPLC (Agilent Technologies,

139 Wilmington, DE, USA) equipped with a C12 reverse phase column (4.6 mm × 250 mm; 4 μm,

140 Agilent Technologies, Wilmington, DE, USA) with auto-sampler and photodiode array

141 detector. Two mobile phases: A, acetonitrile, and B, 1% formic acid in water (v/v), were used

142 and elution was at a flow rate of 1.0 mL min-1. The mobile phase was programmed

143 consecutively in linear gradients as follows: 0 min, 5% A (95% B); 5 min, 10% A (90% B);

144 40 min, 25% A (75% B); 45 min, 30% A (70% B); 55 min, 30% A (70% B); 60 min: 5% A

145 (95% B); 65 min: 5% A (95% B). The UV λmax of 280 nm was used for catechins, and 365

146 nm was used for theaflavins detection. The injection volume was 20 μL. Individual catechin

147 and theaflavin compound were determined using standards as mentioned in section 2.1.

148

149 2.4 Oxygen-radical absorbance capacity (ORAC) assay

150 The ORAC assay was adapted from the methods of Huang et al. (2002) and Bisby et al.

151 (2008). The reaction was performed in phosphate buffer (75 mM, pH 7.4) in 96 well plates.

152 Twenty-five μL of tea extract and 150 μL fluorescein (8.4 × 10-5 mM) were mixed in the 96

7
153 well plate and pre-incubated for 5 min at 37 °C, followed by addition of 25 μL of AAPH

154 solution (0.3072 M). The plate was then placed in the plate reader (PerkinElmer 2300

155 EnSpire multilabel reader) and measured at 485 nm and 520 nm respectively. The control

156 was consisted of 25 μL phosphate buffer. Trolox solutions of 0, 10, 20, 30, 40, 50, 100 mg/L

157 was used to construct the standard curve for the ORAC assay (R2=0.999). The ORAC values

158 were expressed as μmoL Trolox equivalents/g (TE/g) of dried tea using the standard curve

159 established previously. All samples were analysed in triplicate.

160

161 2.5 Statistical analysis

162 Data were reported as mean value ± standard deviation as calculated by Microsoft Excel.

163 ANOVA, post hoc tests, Bivariate correlation and PCA of the data were conducted using

164 SPSS Statistics 23. Data were considered significantly different when p < 0.05.

165

166 3. Results and Discussion

167 3.1 Effects of geographical location and tea plantation

168 3.1.1 Black tea

169 A total of 19 black tea samples sourced from 7 different countries were studied to observe the

170 effects of geographical location. From Table 1, the catechins contents in the black tea

171 samples were found to be in the range of 28.1 mg/g (Lado, Vietnam) to 104.6 mg/g

172 (Semugih/Jolotigo, Java). GC was the major catechin compound present in the black tea

173 samples, ranging from 40% to 75% of all catechins present in black teas studied. This result

174 is in agreement with previous studies (Astill et al., 2001; Friedman et al., 2006). Table 1 also

175 reveals that theaflavins is the major antioxidant compound in the black tea samples with

176 concentration ranging from 28.6 mg/g (Lapsang Souchong, China) to 184.3 mg/g (Taveta,

177 Africa). TF, TF3G, TF3’G and TF33’G were all present in the black tea samples except

8
178 Yunnan 2 and Lopsang Souchong both were from China. Comparing with the data from

179 literatures (Yao et al., 2006; Friedman, et al., 2006), this study showed samples with

180 relatively higher theaflavin content, up to 184.3 mg/g. These results could be attributed to the

181 extraction method and samples used in different studies. Firstly, the extractability of

182 theaflavin is highly dependent on the extracting solvents. Most of the studies had used water

183 for extraction, however, it has been found that water extraction could be 10 times less

184 effective than ethanol for extracting theaflavins (Friedman, et al., 2006). Secondly, in many

185 of the studies (Peterson et al., 2004; Astill et al., 2001; Venditti et al., 2010), the tea samples

186 analysed were blended samples available commercially. Blended teas were reported to have

187 lower catechin content and could have up to 50% less theaflavin than the unblended tea

188 (Peterson et al., 2004). Thirdly, the freshness of the tea samples could be an important

189 contributing factor. Freshly produced tea was reported to have higher theaflavin content than

190 tea that has been stored for a long time after being commercially packed (Yao et al., 2006).

191 For this study, samples were sourced directly from the tea plantation at the same season, and

192 ethanol was applied as the extraction solvent. This may explain the higher levels of theaflavin

193 found in current study. The average ratio of theaflavin to catechin in the samples was 7 to 3,

194 which was in agreement with the findings from literature based on similar HPLC protocol

195 (Del Rio et al., 2004).

196

197 A closer look at all the tea samples reveals that samples from Java (Semugih/Jolotigo) and

198 Africa (Taita) both have higher content of GC (57.6 and 45.2 mg/g) and EGCG (16.7 and

199 11.3 mg/g) as well as total catechins comparing with other samples, which may explain the

200 higher theaflavins (149.3 and 173.3 mg/g) in these samples.

201

9
202 The antioxidant capacities as presented by ORAC value in Table 1 of the black tea samples

203 were significantly influenced by geographical location (p < 0.05). The ORAC antioxidant

204 capacity was ranged from 584.8 μmol TE/g (Keemum, China) to 1529.1 μmol TE/g (Bah

205 butong, Sumatra). The ORAC values of the 4 Chinese tea samples were obviously lower than

206 the samples from Africa, Java, Papua New Guinea and Sumatra, which were more closely

207 resembled those from Vietnam. These patterns were positively correlated to the total

208 catechins and theaflavins as analysed by HPLC.

209

210 The Pearson correlation between the polyphenols contents (total catechins and theaflavins)

211 and the ORAC values of the black teas from different countries was 0.856 (p < 0.01),

212 indicating that the total catechins and theaflavins contents as determined by HPLC could be

213 used to predict the ORAC values of the tea samples. While analysing the effect of catechins

214 and theaflavins, it was found that the Pearson coefficient for the relationship between the

215 total theaflavins and ORAC value was higher (0.845) than that of the total catechins and

216 ORAC value, however, both relationships were statistically significant (p < 0.01). The above

217 results may indicate that theaflavins contributed to a higher antioxidant capacity than

218 catechins in the black tea samples. This results are reasonable because theaflavin is present at

219 a higher content in black tea.

220

221 3.1.2 Green teas

222 A total of 28 green tea samples (24 samples from Sri Lanka and 4 samples from China) were

223 studied to observe the effects of tea plantation. As expected, theaflavins content was not

224 detected in some green tea samples or present at very low content (Table 2). Catechins

225 content was found to range from 143.6 to 282.6 mg/g in all samples, which was similar to the

226 previous results reported by Astill et al. (2001). Table 2 also shows that EGCG was the main

10
227 catechins in the samples and Kotagala (Chunmee (CH)) and Melfort (Gunpowder Extra

228 Special (GP EX SP)) samples had the highest EGCG contents among all the 28 samples

229 (111.1 and 107.1 mg/g, respectively). The EGCG contents of the green tea samples in the

230 area of Glassaugh and Oliphant were lower than other areas with 67.5 and 52.1 mg/g in

231 average, and the total catechins were at the low side too (Table 2). Results also show that

232 theaflavins was only present in tea samples from these two plantations except one sample

233 (Young Hyson grade 2 (YH2), big leaf). This suggests that the Oliphant and Glassaugh

234 plantations may employ a different processing method which involves partial fermentation of

235 the fresh tea leaves, as a result, theaflavins was formed during fermentation process (Graham,

236 1992). Unlike the theaflavins contents, which mainly related to the post-harvest processing of

237 the tea, the amount of catechins in the final product is primarily dependent on the

238 environment where the plant grows (Wei et al., 2011). Nevertheless, the results from current

239 study shows that both processing method and environment could account for the difference in

240 catechins contents in the samples.

241

242 In terms of antioxidant capacity, the ORAC values for green teas were between 1436.1 to

243 2824.4 μmol TE/g (Table 2), which is similar to the results of Henning et al. (2003).

244 Generally, the ORAC values showed a similar trend as in the polyphenols contents (total

245 catechins and theaflavins). There was a significant relationship between the ORAC value and

246 the polyphenols contents (total catechins and theaflavins) in the green tea samples as shown

247 by Pearson coefficient (0.884, p < 0.01).

248

249 3.2 Effects of tea plantation elevation and leaf size

250 Tea can be grown at various plantation elevations ranging from sea level to above 2,700

251 metres (Owuor et al., 2008). The difference in growth elevation could subject the plant to

11
252 various environmental factors, such as humidity, sunlight intensity and temperature,

253 subsequently influencing the amount of antioxidants present (Berry & Bjorkman, 1980). To

254 evaluate the effect of plantation elevation on the polyphenols contents, tea samples from three

255 different plantation elevations were categorised by using common industrial standards (low

256 elevation, sea level and up to 800 metres; medium elevation, 800 metres to 1,200 metres; and

257 high elevation, 1,200 metres and above) and the results were compared. Samples with two

258 different leaf sizes were also compared at these plantation elevations. Big leaf size referred to

259 leaf with Broken Orange Pekoe (BOP) grade, while small leaf size referred to leaf with

260 Broken Orange Pekoe Fannings (BOPF) grade.

261

262 Table 3 shows the polyphenols contents (total catechins and theaflavins as determined by

263 HPLC) of black tea samples obtained from different plantation elevation. Samples from the

264 low elevation group showed the highest amount of antioxidants, followed by the medium

265 elevation group and lastly the high elevation group. This trend was observed for both the two

266 different leaf size samples. The HPLC results in Table 3 consistently show that the

267 polyphenols contents varied depending on plantation elevation, where the lower elevation tea

268 samples had the highest amount of catechins and theaflavins. Among the catechins (Table 4),

269 GC was the major catechin ranging from 51.1 mg/g (Adisham, Pekoe) to 102.4 mg/g

270 (Adisham, BOPF), while the contents of the four different theaflavins were quite similar.

271

272 At similar plantation elevation, samples with larger leaf size consistently had lower

273 antioxidant capacity (Table 3). The trend between plantation elevation and antioxidant

274 capacity (represented by the ORAC value) was the same as the polyphenols contents data

275 obtained by the HPLC. However, the differences of antioxidant activities between different

276 elevations were not as prominent, especially for the small sized leaves samples.

12
277

278 3.3 Effects of processed leaf grade

279 Comparing to other tea species, tea leaf grading is a key quality index for tea. This include

280 the measurement of leaf size and quality, the processing method and the shape of the leaf.

281 The leaf grading of green tea provides information on the appearance of green teas which is

282 traditionally considered as an important factor for consumer when buying teas. To evaluate if

283 tea leaf grade affecting the polyphenols contents, a total of 23 green tea samples with

284 different leaf grade were sourced from Sri Lanka for this study. All the samples were

285 categorised into six grades: gunpowder (GP), hyson, orange pekoe (OP), standard leaf (SL),

286 fannings (FNGS), and CH. Within each category, subtle sub-grades such as gunpowder and

287 gunpowder special were ignored for the purpose of this study as the sub-grades were similar

288 and sometimes did not reflect a different grade or different quality within the grade.

289

290 Results (Table 4) show that EGCG was the major catechin with the concentration ranging

291 from 41.0 (Orange Pekoe A (OPA) (2) grade) to 111.1 mg/g (CH (1) grade), following by

292 EGC and GC. From the HPLC data, there was significant evidence to show that the total

293 catechin content was affected by the leaf grade (p < 0.05). Post hoc test also showed that

294 catechin content in GP and CH grade were significantly different from all the other samples

295 (p < 0.05), but there was no significant difference between those two grades. Theaflavins, on

296 the other hand, was only present in samples from certain plantations (Glassaugh, Kotagala

297 and Oliphant), and was detected from FNGS, OP, SL samples. However, it was completely

298 absent from the CH and GP samples.

299

300 The processing method of different leaf grades was suggested to have influenced the amount

301 of antioxidants in the green tea leaves. Catechins contents in fresh leaves were the highest,

13
302 then degraded gradually during processing and fermentation during the production of black

303 tea, to form theaflavins (Tanaka & Kouno, 2003). The high catechins contents in GP grade

304 leaves can be explained by the processing method where leaves were rolled into a tight ball

305 after being steamed (Takeo, 1992). Typically, this was also the process to produce the fine

306 sensory quality of the gunpowder teas as the tightly packed ball prevented loss of aroma and

307 flavour volatiles. This may also prevent the loss of catechins through the processing which

308 involved drying process. By comparison, FNGS and SL undergo a much more intense

309 processing method (Hampton, 1992).

310

311 The range of ORAC values was between 1844.5 (Hyson) and 2737.2 µmol TE/g (GP EX SP)

312 (Table 4). Results indicate there was no obvious pattern when the data was categorised under

313 the factor leaf grade, and no significant evidence to suggest that leaf grade influenced the

314 total antioxidant capacity. Results show big variation of the ORAC values within each tea

315 grade. For example, the differences between the GP grade tea with the lowest ORAC value

316 (GP grade 1 and sample number 2) and the highest ORAC value (GP EX SP) was 25.1%.

317 Similarly, differences between the hyson grade tea with the lowest ORAC value (Hyson) and

318 the highest ORAC value (Young Hyson (YH)) was 24.8%. Post hoc analysis showed no

319 evidence that any one of the tea grades was significantly different from another (p > 0.05).

320 The Bivariate correlation test showed that the Pearson correlation between polyphenols

321 contents (contents of catechins and theaflavins by HPLC) and the ORAC values of green tea

322 samples was 0.985 (p < 0.01), indicating the total contents of catechins and theaflavins as

323 determined by HPLC could be used to predict the total antioxidant capacity of the samples.

324

325 3.4 The relationship of tea samples as affected by different factors

14
326 Based on the PCA results, the variances of the first three PCs of black tea were 47.60%,

327 22.75% and 9.78%, while those for the green tea were 52.37%, 24.18% and 13.87%. The

328 cumulative variance of the first three PCs of black and green tea were 80.13% and 90.42%,

329 respectively, indicating these data could represent all the results as the cumulative variance

330 was over 80%.

331

332 From Fig. 1A, the 25 black tea samples could be divided into 3 groups, with one sample from

333 China appeared (No.7) as an independent outlier. Group I composed of 17 black tea samples,

334 including seven out of the eight samples from Sri Lanka (No.12, 13, 20, 21, 23, 24 and 25),

335 all the four black tea samples from Sumatra (No.14 to 17), the two black tea samples from

336 Africa (No. 1 and 2), two out of three samples from Java (No. 9 and 10), and the samples

337 from Papua New Guinea (No. 11) and Argentine (No. 3). Group II consisted of five black tea

338 samples, including three out of the four samples (No. 4 to 6) from China and the two

339 Vietnamese samples (No. 18 and 19). Group III consisted of two samples from Java (No. 8)

340 and Sri Lanka (No. 22). Further analysis on Fig. 1A indicated that most of the teas were

341 closely clustered in either one of the three groups of the PCs space except the Yunnan 2

342 sample (No. 7). In the PCs space, the black tea of Yunnan 2 from China with the grade of

343 BOP was far from the other three Chinese black tea samples which were in group II (No. 4 to

344 6). This may due to sample No. 7 had a different leaf grade (BOP) and leaf size (Medium)

345 comparing to the other three black tea samples from China (No. 4 to 6). Another slightly

346 deviated sample in the PCs space, No. 8 from Java in group III, was located different from

347 the other two Java samples in group III (No. 9, 10), had a different leaf grade of FNGS

348 compared to the other samples with the leaf grade of BOPF. These results show that leaf

349 grade and size are indeed the two important factors that may affect the composition of the

350 polyphenols in black teas from the same country.

15
351

352 From Fig. 1B, the 28 green tea samples appeared to be grouped into 3 groups, with one

353 sample from Sri Lanka (No. 30) appeared as an independent outlier. Group I consisted of

354 eleven Sri Lanka green tea samples, among those five out of seven samples were from

355 Glassaugh (No. 26, 27, 28, 31 and 32), three out of the seven samples were from Kotagala

356 (No. 33, 37 and 38), two out of the five samples were from Oliphant (No. 46 and 48) and one

357 out of the five sample (No. 41) was from Melfort. Group II contained all the 4 Chinese

358 Longjing tea samples (No. 50 to 53) and four Sri Lanka samples including the other three

359 samples from Oliphant (No. 45, 47 and 49) and one sample from Glassaugh (No. 29). Group

360 III consisted of 8 Sri Lanka green tea samples, including four out of the five samples from

361 Melfort (No. 40, 42, 43 and 44), and the rest of the four samples from Kotagala. Further

362 analysis on Fig. 1B reveals that 83% of the Sri Lanka green tea samples were scattered in

363 group I and III, while all the Chinese tea samples were distributed in group II. Comparing the

364 locations of the samples in the three dimension space, group I and III were closer at the

365 bottom, while all the 4 Longjing tea samples located at the top in group II, seem to have a

366 distant from most of the Sri Lanka tea samples. From Fig. 1B, specifically, sample No. 29

367 with small leaf size from Glassaugh was located in group II away from the other 5 Glassaugh

368 samples with large leaf size in group I. This indicates that the factor of tea leaf size played a

369 significant role in differentiating the six samples from the same tea estate. From these results,

370 we can also see that samples with GP1, GP2, Gunpowder Special (GP SP) and CH leaf grade

371 (No. 34-36 and 39) from Kotagala and samples with leaf grade of GP1, GP2, GP EX SP and

372 CH from Melfort, were closely clustered in group III of the PCs space. This suggests the

373 similarity of the green tea samples with the general leaf grade of GP and CH from Kotagala

374 and Melfort in Sri Lanka. The results are in agreement with the data of total catechins and

375 total antioxidant capacity by ORAC in section 3.3.

16
376

377 4. Conclusions

378 Consistent with literature, black tea antioxidants were found to be predominately consisted of

379 theaflavins with the ratio of theaflavins to catechins of 7:3. Both catechins and theaflavins

380 contents of black teas as determined by HPLC and ORAC were significantly influenced by

381 the factor of plantation location (p < 0.05) as follow: Low elevation > Medium elevation >

382 High elevation. Up to 15% more polyphenols were extracted from smaller sized leaves than

383 the larger sized leaves using the same sample processing method. Plantation estates and tea

384 leaf grades were found to affect the polyphenols contents in green tea significantly (p < 0.05).

385 The green tea samples in this study consisted of 87% to 100% catechins, ranging between

386 143.6 and 282.6 mg/g. Only samples from Glassaugh estate and Oliphant estates contain

387 theaflavins (average of 12.5 mg/g). Green tea samples with GP and CH grades contained

388 more catechins compared with other grades. The PCA analysis confirmed that the

389 composition of tea polyphenols could be effected by geographical and tea plantation location,

390 leaf grade and leaf size. Overall, the results from this study could serve as a reference to

391 consumers when purchasing teas for consumption. Further studies can be conducted to

392 investigate the mechanism between antioxidant capacity and the factors as discussed above.

393 Contribution of other polyphenols compounds to the antioxidant capacity assay can be further

394 investigated, including thearubigins, ascorbic acid and chlorophyll.

395

17
396 Acknowledgement

397 The authors wish to acknowledge Bell Tea & Coffee Company for providing the majority of

398 the samples, Drs Laurence Eyres and Sally Xiong for discussion and technical advice and the

399 Foundation of Research Science & Technology (FRST) for the funding.

400

401 Appendices

402 The information of all the 53 tea samples including tea type, country of origin, plantation

403 location, leaf grade and relative leaf size are shown in Table A1.

404

405 Figure A1 shows all the tea samples used in this study (employs 1 cm as the measuring scale).

406 Dry tea leaves within 0.2 cm is defined as Small, 0.2 to 0.5 cm as Medium, larger than 0.5 cm

407 as Large.

18
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520

23
521

522 Fig. 1 PCA score plots (PC1 × PC2 × PC3) for black and green tea samples.

523 Fig. A1 Appearance of the tea samples.

524

525

526

24
 4 7 3
1 II
53
45
52
3 29
2
47
1 III
22
2 49
51
1
PC3 (9.78%)

PC3 (13.87%)
8 30
1 II 1 I 1 III
10
1 6 50
35
14 15 20
5 0 26 42
17 2 1 I 34
44
0 19 28 40 39
18 4 36
16 13 41 43
24 25 37 33
11 23 32
38
9
3 -1 46
27
21 48
-1 1
12 4 31
-5 3 3
-4 -5 4 2
2 -
-3 1 -3 1
-2 -2
-1 0 ) -1 0 )
PC1
0
-1 .7 5% PC1 0 8%
1
4.1
1 -1
( 47.6 22 ( 52.3
0%)
2 -2
2( 7% )
2 -2 2(
2
3 -3 PC A
3
PC B
527 4 4 -3

528
529

530

25
531
532

26
533 Table 1 Polyphenols contents and antioxidant capacities of black tea from different countries

Catechins (mg/g) Theaflavins (mg/g) OR

AC
Total Total Val
Gra Catechin Theafl ue
Origin 1 EGC GC T TF TF TF3
de GC EGC C ECG CG s avins μmo
G G (mg/g) F 3G 3’G 3’G (mg/g) l
TE/
g
11 968.
28.1 ± .1 26. 17.9 72.6 ± 2±
19.3 2.2 ± 0.2 ± 1.7 ± 2.8 ± 0.0 ± 1.9 ± 17.4
Vietnam (Lado) FD a ± 2± ± d 50.8
± 1.1 0.0 0.0 0.0 0.1 0.0 0.3 1.5 ± 0.6 4.5
2. 0.9 0.6 de
4
12 783.
39.2 ± .5 17. 13.7 58.3 ± 9±
Vietnam (Minh 29.4 1.0 ± 1.1 ± 2.5 ± 2.9 ± 0.6 ± 1.7 ± 14.7
FD b ± 4± ± c 20.6
Anh) ± 1.7 0.0 0.0 0.1 0.1 0.1 0.1 2.1 ± 0.5 3.0
0. 1.5 0.9 bc
1
31 1213
69.0 ± .7 44. 42.0 157.4 ± .8 ±
Sri Lanka BOP 45.5 6.0 ± 4.4 ± 4.6 ± 3.2 ± 2.9 ± 2.4 ± 39.3 fg
f ± 4± ± h 5.9
(Watawala 1) F ± 1.2 0.4 0.3 0.7 0.1 0.5 0.1 3.3 ± 2.9 10.9
2. 2.0 4.0 h
0
28 1224
68.1 ± .9 43. 35.7 138.8 ± .3 ±
Sri Lanka 38.3 5.3 ± 6.1 ± 6.7 ± 4.2 ± 5.5 ± 2.0 ± 31.1 f
BOP f ± 1± ± fg 12.7
(Watawala 2) ± 1.3 1.1 0.6 1.2 1.6 0.6 0.4 6.8 ± 0.3 2.6
0. 0.8 0.6 gh
9
36 1265
54.4 ± .5 57. 41.7 173.5 ± .8 ±
Papua New 26.0 12.1 3.1 ± 4.3 ± 3.9 ± 1.8 ± 3.2 ± 38.1 g
PF1 c ± 2± ± ij 6.4
Guinea ± 1.2 ± 2.8 0.5 0.4 0.7 0.4 0.2 6.2 ± 2.2 11.9
2. 3.8 3.1 h
8
28 1072
63.7 ± .9 31. 27.7 114.4 ± .3 ±
Sumatra BOP 36.9 2.6 ± 3.9 ± 7.6 ± 3.3 ± 6.8 ± 2.6 ± 26.6
def ± 2± ± e 65.8
(Tobasari) F ± 0.3 0.0 0.5 0.2 0.1 0.1 0.0 1.2 ± 0.5 1.1
0. 0.3 0.1 ef
2
32 1529
78.4 ± .1 37. 33.0 132.9 ± .1
Sumatra (Bah BOP 47.0 3.9 ± 4.8 ± 8.5 ± 3.4 ± 8.0 ± 2.8 ± 30.3
g ± 5± ± f ±79.
Butong) F ± 1.2 0.1 1.1 0.9 0.1 0.4 0.1 3.9 ± 0.4 2.6
0. 1.3 0.5 j
4 3
32 1107
57.0 ± .5 33. 29.5 128.0 ± .9 ±
Sumatra BOP 32.4 6.1 ± 3.4 ± 5.2 ± 4.1 ± 3.7 ± 2.1 ± 32.9
cde ± 1± ± f 51.9
(Sidamanik) F ± 2.0 0.4 0.3 0.7 1.6 1.1 0.6 6.7 ± 3.8 12.3
6. 1.1 1.0 efg
4
28 1221
46.0 ± .8 59. 39.2 154.4 ± .8 ±
Sumatra (Kayu 22.9 4.5 ± 4.2 ± 4.5 ± 3.3 ± 3.3 ± 3.3 ± 27.3
F3 b ± 1± ± h 100.
Aro) ± 0.9 0.8 0.9 0.2 0.1 0.1 0.1 3.1 ± 0.7 3.9
0. 2.4 0.7 fgh
1 2
42 1263
Java 104.6 ± .1 37. 31.8 149.3 ± .2 ±
FN 57.6 4.5 ± 5.2 ± 16.7 4.7 ± 13.7 2.2 ± 38.4
(Semugih/Joloti h ± 0± ± gh 85.3
GS ± 2.2 0.9 2.5 ± 2.4 0.8 ± 1.5 0.0 10.3 ± 1.9 4.7
go) 2. 0.6 0.1 gh
1
30 1226
60.1 ± .3 37. 39.4 146.8 ± .2 ±
BOP 36.2 9.2 ± 2.8 ± 3.4 ± 4.2 ± 2.0 ± 2.3 ± 39.3 f
Java (Senorati) cdef ± 8± ± gh 20.0
F ± 1.3 0.2 0.5 0.0 0.7 0.0 0.1 2.8 ± 1.8 8.1
2. 1.1 3.2 gh
0
28 1240
64.4 ± .5 49. 39.3 151.8 ± .2 ±
BOP 25.6 3.8 ± 5.9 ± 13.3 3.9 ± 9.0 ± 2.9 ± 34.8
Java (Kaligua) def ± 2± ± h 10.0
F ± 1.8 0.1 0.3 ± 0.4 0.0 0.5 0.2 3.3 ± 1.3 5.1
1. 1.1 1.7 gh
0
15 681.
55.3 ± .3 10. 10.8 46.7 ± 7±
LEA 31.4 3.9 ± 1.8 ± 4.2 ± 3.5 ± 9.2 ± 1.3 ± 10.5
China (Yunnan) cd ± 1± ± b 12.1
F ± 0.7 0.3 0.1 1.5 0.3 1.2 0.0 4.1 ± 0.4 2.0
0. 0.6 0.1 ab
9
970.
39.6 ± 11. 13.3 37.9 ± 9±
China (Yunnan 17.9 4.4 ± 1.5 ± 2.2 ± 3.4 ± 3.9 ± 6.3 ± N 12.8
BOP b 8± ± ab 34.8
2) ± 1.3 0.3 0.3 0.3 0.5 0.2 0.7 3.6 D ± 0.9 3.1
1.3 0.9 de

12 548.
43.2 ± .1 9.3 12.3 45.6 ± 8±
China LEA 28.0 4.0 ± 2.7 ± 1.9 ± 3.1 ± 2.3 ± 1.2 ± 11.9
b ± ± ± b 25.8
(Keemun) F ± 1.2 1.1 0.4 0.0 0.1 0.0 0.0 2.8 ± 0.1 1.8
0. 1.3 0.2 a
2

27
 890.
30.9 ± 9.4 9.3 28.6 ± 9±
China (Lapsang LEA 16.1 3.0 ± 2.0 ± 2.8 ± 2.8 ± 2.7 ± 1.5 ± N 9.9 ±
a ± ± a 32.0
Souchong) F ± 1.4 0.6 0.7 0.7 0.1 0.0 0.4 3.9 D 0.7 6.7
4.1 1.9 cd

29 1174
61.9 ± .3 53. 41.8 170.3 ± .7 ±
Argentina (Las 29.5 10.6 5.6 ± 6.3 ± 4.5 ± 3.0 ± 2.4 ± 45.3 f
F2 cdef ± 9± ± i 21.9
Marias) ± 0.5 ± 2.8 0.9 0.3 0.5 0.7 0.0 5.7 ± 0.1 2.8
0. 2.0 0.7 g
0
37 1456
65.8 ± .7 53. 49.4 184.3 ± .3 ±
41.9 5.3 ± 5.1 ± 4.4 ± 4.4 ± 1.6 ± 3.1 ± 43.6 i
Africa (Taveta) PF1 ef ± 6± ± j 67.0
± 2.8 1.3 0.3 0.1 1.9 0.7 0.7 7.8 ± 1.6 5.9
2. 0.8 0.8 j
7
37 1376
78.3 ± .4 49. 44.5 173.3 ± .4 ±
45.2 3.4 ± 5.6 ± 11.3 3.7 ± 6.1 ± 3.0 ± 42.4
Africa (Taita) PF1 g ± 0± ± ij 56.0
± 1.9 0.4 0.5 ± 0.4 0.0 0.7 0.1 4.0 ± 1.4 12.1
4. 3.5 2.3 hi
9
534
535 1
FD: Fanning Dust, BOPF: Broken Orange Pekoe Fannings, BOP: Broken Orange Pekoe, PF1: Pekoe Fannings grade 1, F3:
536 Fannings grade 3, FNGS: Fannings, LEAF: rolled whole tea leaves, F2: Fannings grade 2.
537
538 Values in the same column with the different letters differ significantly (p < 0.05).

539

540

28
 ORA
Total C
Total
Gra EGC T TF TF3 TF3 Theafla Valu
GC EGC C GCG ECG CG Catechins
de1 G F 3G ’G 3’G vins e
(mg/g)
(mg/g) μmol
TE/g
Glassa OPA 27.5 ± 56.7 ± 6.3 ± 73.1 ± 5.3 ± 21.7 ± 2.5 ± 193.1 ± N 5.8 ND ND 5.8 ± 1937.
ugh 0.8 7.3 0.7 6.8 2.2 0.9 0.2 def D ± b 8±
18.9 0.1
(Sri 0.1 d
15.0
Lanka ef
)
OPA 4.4 ± 56.9 ± 5.1 ± 62.7 ± 3.2 ± 19.9 ± 2.3 ± 154.5 ± N 6.3 ND ND 6.3 ± 1544.
2 0.3 4.3 0.1 11.3 0.0 1.5 0.2 ab D ± b 4±
17.7 0.2 ab
0.2 5.0
OP 19.9 ± 52.9 ± 5.5 ± 66.1 ± 4.7 ± 22.0 ± 2.3 ± 173.4 ± N 6.2 ND ND 6.2 ± 1733.
2.2 1.2 0.7 6.6 0.3 2.1 0.3 bcd D ± b 9±
13.4 0.8
0.8 112.0
bcd

FNG 17.3 ± 68.4 ± 5.6 ± 71.4 ± 4.7 ± 22.7 ± 2.6 ± 192.7 ± N 14.8 3.7 3.3 ± 21.8 ± 1927.
S 0.3 3.2 0.1 1.2 0.2 0.5 0.0 def D ± ± 0.0 g 9±
5.5 0.2
0.1 0.1 d
11.0
ef

YH2 28.2 ± 59.5 ± 6.1 ± 80.1 ± 4.8 ± 25.6 ± 2.9 ± 207.2 ± N 7.2 3.4 ND 10.6 ± 2071.
SM 1.5 2.9 0.4 1.2 0.3 0.2 0.0 fgh D ± ± d 4±
6.5 1.9
1.8 0.1 fg
52.0
YH2 12.4 ± 63.0 ± 4.2 ± 59.0 ± 3.3 ± 15.5 ± 1.9 ± 159.3 ± N ND ND ND a 1532.
ab
ND
BIG 0.4 6.8 0.3 1.1 0.0 0.4 0.0 9.0 D 5±
a
11.1
b

SL 9.4 ± 61.9 ± 5.3 ± 60.0 ± 3.5 ± 19.3 ± 2.1 ± 161.5 ± N 6.3 ND ND 6.3 ± 1613.
2.4 6.4 0.1 0.7 0.3 0.7 0.1 abc D ± b 5±
10.7 0.3
0.3 a
13.7
bc

Kotag BOP 44.0 ± 41.2 ± 4.8 ± 80.0 ± 3.7 ± 24.0 ± 3.4 ± 201.1 ± N ND ND ND a 2011.
efgh
ND
ala 8.8 1.7 0.7 0.1 0.2 0.6 0.1 12.2 D 2±
(Sri ef
54.6
Lanka g
)
GP1 53.2 ± 36.9 ± 5.6 ± 96.7 ± 4.5 ± 31.1 ± 4.4 ± 232.4 ± N ND ND ND a 2324.
ND
2.8 0.9 0.1 0.1 0.3 0.7 0.2 ijk D 3±
5.1 hi
27.0
GP2 53.1 ± 41.9 ± 6.4 ± 98.3 ± 6.3 ± 31.0 ± 4.4 ± 241.4 ± N ND ND ND a 2412.
ND
1.5 2.0 0.3 3.1 2.0 1.5 0.1 jkl D 3±
10.5 hi
84.6
j

GP 48.8 ± 39.3 ± 5.3 ± 86.9 ± 4.2 ± 30.5 ± 4.3 ± 219.3 ± N ND ND ND a 2191.

ND
SP 3.2 3.2 0.3 5.7 0.4 1.1 0.4 ghij D 6±
14.3
g
73.0
h

HYS 39.5 ± 41.2 ± 4.6 ± 81.3 ± 3.5 ± 23.8 ± 3.1 ± 197.0 ± N ND ND ND a 1970.
ND
ON 7.5 1.9 0.1 1.1 0.1 1.4 0.4 defg D 8±
12.5
113.1
defg

YH2 35.4 ± 40.6 ± 4.8 ± 76.3 ± 3.7 ± 22.7 ± 3.1 ± 186.6 ± N ND ND ND a 1864.
ND
2.0 0.1 0.0 0.5 0.2 0.1 0.1 cdef D 7±
3.0
111.6
cdef

CH 66.8 ± 44.5 ± 6.5 ± 111.1 5.0 ± 35.3 ± 5.5 ± 274.7 ± N ND ND ND a 2746.

ND
2.6 3.3 0.7 ± 4.2 0.6 0.9 0.1 n D 1±
12.4 l
112.9
Melfo CH 81.6 ± 40.7 ± 7.2 ± 97.5 ± 4.8 ± 27.0 ± 4.7 ± 263.5 ± N ND ND ND a 2635.
ND
rt 4.6 1.7 0.2 0.3 0.3 0.8 0.3 mn D 0±
8.2l jk
(Sri 66.4
Lanka l
)
SP 59.9 ± 38.6 ± 5.5 ± 75.7 ± 4.6 ± 21.5 ± 3.3 ± 209.1 ± N ND ND ND a 2060.
ND
HYS 4.1 4.1 0.0 8.5 0.1 1.2 0.1 fghi D 4±
18.1
ON fg
94.6
GP1 81.0 ± 39.7 ± 6.0 ± 101.9 5.4 ± 30.3 ± 5.7 ± 270.0 ± N ND ND ND a 2701.
ND
0.6 1.8 0.2 ± 1.3 0.0 0.7 0.2 mn D 2±
4.8
kl
74.8
GP 60.0 ± 48.1 ± 6.0 ± 98.3 ± 4.6 ± 27.8 ± 4.0 ± 248.8 ± N ND ND ND a 2488.
ND
SP 4.4 6.2 0.7 9.4 0.4 3.1 0.3 klm D 2±
24.5
i
100.1

29
 jk

GP 84.7 ± 41.8 ± 6.5 ± 107.1 5.4 ± 31.2 ± 5.9 ± 282.6 ± N ND ND ND a 2824.

ND
EX 2.2 0.1 0.1 ± 5.3 0.1 1.3 0.5 n D 4±
9.6 l
SP 100.7
Oliph FNG 11.8 ± 69.4 ± 5.6 ± 59.4 ± 4.6 ± 23.3 ± 2.6 ± 176.7 ± N 18.8 5.0 3.4 ± 27.2 ± 1766.
ant S 1.4 3.7 0.2 2.5 0.2 1.0 0.2 bcde D ± ± 0.2 h 4±
9.2 1.1
(Sri 0.7 0.2 b
70.4
Lanka cde
)
SL 11.1 ± 78.2 ± 4.7 ± 52.8 ± 3.3 ± 19.5 ± 2.0 ± 171.6 ± N 8.9 ND ND 8.9 ± 1716.
1.1 3.4 0.8 3.7 0.1 0.7 0.1 bcd D ± c 2±
9.9 0.5
0.5 b
82.5
cd

OPA 17.7 ± 67.1 ± 5.4 ± 57.7 ± 4.4 ± 22.0 ± 2.2 ± 176.5 ± N 13.1 3.8 2.8 ± 19.7 ± 1766.
1.7 0.9 0.3 3.3 0.9 1.3 0.1 bcde D ± ± 0.3 f 1±
8.5 2.4
1.9 0.2 b
61.3
cde

OPA 9.2 ± 69.6 ± 3.7 ± 41.0 ± 3.1 ± 15.2 ± 1.8 ± 143.6 ± N 6.8 ND ND 6.8 ± 1436.
2 0.5 10.3 0.3 3.8 0.1 1.1 0.1 a D ± b 1±
16.2 1.0 a
1.0 50.2
YH 12.1 ± 71.1 ± 4.2 ± 49.4 ± 3.6 ± 18.5 ± 2.1 ± 161.0 ± N 12.0 3.6 2.7 ± 18.3 ± 1611.
0.8 0.9 0.2 4.0 0.1 0.8 0.2 abc D ± ± 0.4 e 4±
7.0 2.2
1.8 0.0 a
90.8
bc

Longji Grad 34.9 ± 8.0 ± 3.7 ± 86.5 ± 5.3 ± 21.2 ± 1.7 ± 161.3 ± N ND ND ND a 2090.
ND
ng e1 3.4 0.6 0.2 10.4 1.2 1.9 0.1 abc D 4±
17.8
(Chin fg
71.9
a) Grad 38.9 ± 8.8 ± 5.0 ± 84.0 ± 6.9 ± 19.4 ± 1.5 ± 164.5 ± N ND ND ND a 1954.
ND
e2 2.9 1.4 0.4 9.5 0.9 1.7 0.0 abc D 0±
16.8
100.6
defg

Grad 39.6 ± 9.5 ± 5.0 ± 96.7 ± 9.4 ± 23.6 ± 1.7 ± 185.5 ± N ND ND ND a 1844.
ND
e3 4.5 1.6 0.2 11.6 1.1 1.6 0.1 cdef D 4±
20.7 c
82.5
def

Grad 45.6 ± 17.7 ± 4.1 ± 92.9 ± 10.6 ± 22.9 ± 1.7 ± 195.5 ± N ND ND ND a 1644.
defg
ND
e4 4.8 1.9 0.2 9.9 1.2 2.8 0.2 21.0 D 8±
a
70.1
bc

541 Table 2 Polyphenols contents and antioxidant capacities of green tea from different plantation locations
542
543 1
OP: Orange Pekoe, OPA: Orange Pekoe A, OPA2: Orange Pekoe A grade 2, FNGS: Fannings, YH: Young Hyson, YH2:
544 Young Hyson grade 2, SL: Standard leaf, BOP: Broken Orange Pekoe, BOPF: Broken Orange Pekoe Fannings, GP1:
545 Gunpowder grade 1, GP2: Gunpowder grade 2, GP SP: Gunpowder Special, GP EX SP: Gunpowder Extra Special, CH:
546 Chunmee, SP HYSON: Special HYSON.
547
548 Values in the same column with the different letters differ significantly (p < 0.05).

549

30
550 Table 3 Polyphenols contents and antioxidant capacities of black tea as affected by plantation elevation

Catechins (mg/g) Theaflavins (mg/g) OR

AC
Total Val
1 Total
Gr Theafla ue
Origin Catechins
ade GC EGC C
EGC GC
ECG CG
T TF TF3 TF3 vins μmo
G G (mg/g) F 3G ’G 3’G (mg/g) l
TE/
g
Sri BOP 57.6 ± 3.3 ± 4.4 ± 4.0 ± 3.1 ± 4.8 ± 1.5 ± 133.0 ± 28 26.7 33.3 33.8 123.8 ± 1308
Lanka F 7.5 0.1 1.2 0.6 0.2 0.7 0.1 b .0 ± ± 4.3 ± 1.8 b .4 ±
13.5 12.1
(Homad ± 2.5 78.9
ola) 1. a
7
Sri Peko 95.6 ± 3.7 ± 4.2 ± 16.9 ± 3.2 ± 1.8 ± 2.5 ± 127.9 ± 23 29.2 24.0 20.9 97.3 ± 1257
Lanka e 12.3 0.3 0.2 0.5 0.3 0.6 0.1 b .2 ± ± 1.7 ± 1.2 a .2 ±
14.3 7.4
(Homad ± 3.3 104.
ola 2) 1. a
1
2
Sri BOP 56.6 ± 3.1 ± 4.1 ± 9.6 ± 3.3 ± 6.7 ± 1.7 ± 85.1 ± 35 31.9 33.2 31.4 131.4 ± 1273
Lanka F 8.6 0.6 2.2 3.4 0.1 3.4 0.1 a .0 ± ± 8.3 ± 3.8 b .5 ±
18.4 20.5
(Kenilw ± 1.1 67.0
orth) 7. a
3
Sri Peko 72.0 ± 3.5 ± 3.9 ± 18.9 ± 3.6 ± 14.1 ± 2.4 ± 118.4 ± 21 24.0 19.2 20.3 85.4 ± 1138
Lanka e 3.9 0.4 1.9 4.3 0.2 1.7 0.2 b .9 ± ± 1.0 ± 1.8 a .9 ±
12.6 7.1
(Kenilw ± 2.3 44.9
orth 2) 2. a
0
Sri BOP 102.4 2.9 ± 3.8 ± 9.3 ± 3.2 ± 9.4 ± 2.0 ± 78.7 ± 33 29.1 31.0 30.3 121.8 ± 1249
Lanka F ± 8.3 0.4 2.2 0.8 0.2 1.5 0.1 a .4 ± ± 2.1 ± 1.9 b .2 ±
10.4 10.3
(Adisha ± 2.2 20.7
m) 5. a
9
Sri Peko 51.1 ± 3.0 ± 5.3 ± 5.8 ± 3.3 ± 5.1 ± 1.5 ± a 23 22.4 24.0 23.9 94.1 ± 1107
75.1 ± 6.9 a
Lanka e 3.2 0.9 1.4 0.7 0.1 0.6 0.0 .8 ± ± 1.8 ± 1.7 6.1 .7 ±
(Adisha ± 2.4 35.0
m 2) 0. a
2
551
552 1
BOPF (small leaf size): Broken Orange Pekoe Fannings, Pekoe (large leaf size).
553
554 Values in the same column with the different letters differ significantly (p < 0.05).

555

556

31
557 Table 4 Polyphenols contents and antioxidant capacities of green tea with different leaf grades

Catechins (mg/g) Theaflavins (mg/g) ORA

Total
Total C
Theafla
Grade1 Catechins T TF3 TF3 TF33 Value
GC EGC C EGCG GCG ECG CG vins
(mg/g) F G ’G ’G μmol
(mg/g)
TE/g
2572.5
66.8 ± 44.5 ± 6.5 ± 111.1 ± 5.0 ± 35.3 ± 5.5 ± 274.7 ± N a
CH (1) j ND ND ND ND ±
2.6 3.3 0.7 4.2 0.6 0.9 0.1 12.4 D hi
78.9
2007.5
±
81.6 ± 40.7 ± 7.2 ± 97.5 ± 4.8 ± 27.0 ± 4.7 ± ij N a ab
CH (2) 263.5 ± 8.2 ND ND ND ND 87.9
4.6 1.7 0.2 0.3 0.3 0.8 0.3 D
c

2505.1
192.7 ± 14.8 21.8 ± ±
FNGS 17.3 ± 68.4 ± 5.6 ± 71.4 ± 4.7 ± 22.7 ± 2.6 ± N 3.7 ± 3.3 ± g
cde ± f 127.9
(1) 0.3 3.2 0.1 1.2 0.2 0.5 0.0 5.5 D 0.1 0.0 0.2
0.1 hi

2226.3
176.7 ± 18.8 27.2 ± ±
FNGS 11.8 ± 69.4 ± 5.6 ± 59.4 ± 4.6 ± 23.3 ± 2.6 ± N 5.0 ± 3.4 ± c
bcd ± g 143.2
(2) 1.4 3.7 0.2 2.5 0.2 1.0 0.2 9.2 D 0.2 0.2 1.1
0.7 def

2162.9
171.6 ± 8.9 8.9 ± ±
11.1 ± 78.2 ± 4.7 ± 52.8 ± 3.3 ± 19.5 ± 2.0 ± N b
SL (1) bc ± ND ND c 102.9
1.1 3.4 0.8 3.7 0.1 0.7 0.1 9.9 D 0.5
0.5 cde

6.3 2605.5
9.6 ± 61.9 ± 5.3 ± 60.0 ± 3.5 ± 19.3 ± 2.1 ± 161.7 ± N 6.3 ±
SL (2) ab ± ND ND b ±
2.4 6.4 0.1 0.7 0.3 0.7 0.1 10.7 D 0.3 hi
0.3 56.8
2275.3
173.4 ± 6.2 6.2 ± ±
19.9 ± 52.9 ± 5.5 ± 66.1 ± 4.7 ± 22.0 ± 2.3 ± N de
OP bc ± ND ND b 36.8
2.2 1.2 0.7 6.6 0.3 2.1 0.3 13.4 D 0.8
0.8 fg

2462.1
176.5 ± 13.1 19.7 ± ±
OPA 17.7 ± 67.1 ± 5.4 ± 57.7 ± 4.4 ± 22.0 ± 2.2 ± N 3.8 ± 2.8 ± fg
bcd ± e 48.2
(1) 1.7 0.9 0.3 3.3 0.9 1.3 0.1 8.5 D 0.2 0.3 2.4
1.9 h

6.8 2560.3
OPA 9.2 ± 69.6 ± 3.7 ± 41.0 ± 3.1 ± 15.2 ± 1.8 ± 143.6 ± N 6.8 ±
a ± ND ND b ±
(2) 0.5 10.3 0.3 3.8 0.1 1.1 0.1 16.2 D 1.0 hi
1.0 65.8
2288.5
193.1 ± 5.8 5.8 ± ±
OPA2 27.5 ± 56.7 ± 6.3 ± 73.1 ± 5.3 ± 21.7 ± 2.5 ± N d
cde ± ND ND b 127.6
(1) 0.8 7.3 0.7 6.8 2.2 0.9 0.2 18.9 D 0.1
0.1 efg

2098.8
154.5 ± 6.3 6.3 ± ±
OPA2 4.4 ± 56.9 ± 5.1 ± 62.7 ± 3.2 ± 19.9 ± 2.3 ± N bc
ab ± ND ND b 15.9
(2) 0.3 4.3 0.1 11.3 0.0 1.5 0.2 17.7 D 0.2
0.2 d

1844.5
39.5 ± 41.2 ± 4.6 ± 81.3 ± 3.5 ± 23.8 ± 3.1 ± 197.0 ± N a
Hyson de ND ND ND ND ±
7.5 1.9 0.1 1.1 0.1 1.4 0.4 12.5 D a
128.4
2453.2
161.0 ± 12.0 18.3 ± ±
12.1 ± 71.1 ± 4.2 ± 49.4 ± 3.6 ± 18.5 ± 2.1 ± N 3.6 ± 2.7 ± fg
YH ab ± e 71.5
0.8 0.9 0.2 4.0 0.1 0.8 0.2 7.0 D 0.0 0.4 2.2
1.8 h

2014.1
186.6 ± ±
35.4 ± 40.6 ± 4.8 ± 76.3 ± 3.7 ± 22.7 ± 3.1 ± N a ab
YH2 cde ND ND ND ND 57.6
2.0 0.1 0.0 0.5 0.2 0.1 0.1 3.0 D
c

1945.8
YH2 12.4 ± 63.0 ± 4.2 ± 59.0 ± 3.3 ± 15.5 ± 1.9 ± 159.3 ± N a
ab ND ND ND ND ±
(BIG) 0.4 6.8 0.3 1.1 0.0 0.4 0.0 9.0 D ab
85.2
2388.7
207.2 ± 7.2 10.6 ± ±
YH2 28.2 ± 59.5 ± 6.1 ± 80.1 ± 4.8 ± 25.6 ± 2.9 ± N 3.4 ± ef
ef ± ND d 13.3
(SM) 1.5 2.9 0.4 1.2 0.3 0.2 0.0 6.5 D 0.1 1.9
1.8 gh

2117.0
60.0 ± 38.6 ± 5.5 ± 75.7 ± 4.6 ± 21.5 ± 3.3 ± 209.2 ± N ±
a bc
HY SP ef ND ND ND ND 46.9
4.1 4.1 0.0 8.5 0.1 1.2 0.1 18.1 D
d

53.2 ± 36.9 ± 5.6 ± 96.7 ± 4.5 ± 31.1 ± 4.4 ± 232.4 ± N a 2561.1

GP1 (1) gh ND ND ND ND
2.8 0.9 0.1 0.1 0.3 0.7 0.2 5.1 D ±

32
 hi
13.1
2048.1
81.0 ± 39.7 ± 6.0 ± 101.9 ± 5.4 ± 30.3 ± 5.7 ± N ±
j a abc
GP1 (2) 270.0 ± 4.8 ND ND ND ND 2.0
0.6 1.8 0.2 1.3 0.0 0.7 0.2 D
d

53.1 ± 41.9 ± 6.4 ± 98.3 ± 6.3 ± 31.0 ± 4.4 ± 241.4 ± N a 2571.4

GP2 h ND ND ND ND hi
1.5 2.0 0.3 3.1 2.0 1.5 0.1 10.5 D ± 9.8
2202.2
219.3 ± ±
GP SP 48.8 ± 39.3 ± 5.3 ± 86.9 ± 4.2 ± 30.5 ± 4.3 ± N a cd
fg ND ND ND ND 50.5
(1) 3.2 3.2 0.3 5.7 0.4 1.1 0.4 14.3 D
e

2282.6
248.8 ± ±
GP SP 60.0 ± 48.1 ± 6.0 ± 98.3 ± 4.6 ± 27.8 ± 4.0 ± N a de
hi ND ND ND ND 35.4
(2) 4.4 6.2 0.7 9.4 0.4 3.1 0.3 24.5 D
fg

GP EX 84.7 ± 41.8 ± 6.5 ± 107.1 ± 5.4 ± 31.2 ± 5.9 ± j N a 2737.2

282.6 ± 9.6 ND ND ND ND i
SP 2.2 0.1 0.1 5.3 0.1 1.3 0.5 D ± 46.5
558
559 1
CH: Chunmee, FNGS: Fannings, SL: Standard leaf, OP: Orange Pekoe, OPA: Orange Pekoe A, OPA2: Orange Pekoe A
560 grade 2, YH: Young Hyson, YH2: Young Hyson grade 2, HY SP: Hyson Special, GP1: Gunpowder grade 1, GP2:
561 Gunpowder grade 2, GP SP: Gunpowder Special, GP EX SP: Gunpowder Extra Special.
562
563 Values in the same column with the different letters differ significantly (p < 0.05).

564

565

33
566 Table A1 Information of tea samples
Sample Relative
Type Country of origin plantation location Leaf Grade1
number Leaf size

1 Black Tea Africa Taveta PF1 Small

2 Black Tea Africa Taita PF1 Small
3 Black Tea Argentina Las Marias F2 Small
4 Black Tea China Keemun tea LEAF Medium
5 Black Tea China Lapsan Souchong tea LEAF Large
6 Black Tea China Yunnan 1 LEAF Large
7 Black Tea China Yunnan 2 BOP Medium
8 Black Tea Java Senorati FNGS Small
9 Black Tea Java Semugih/Jolotigo BOPF Small
10 Black Tea Java Kaligua BOPF Small
11 Black Tea Papua New Guinea Avianp PF1 Small
12 Black Tea Sri Lanka Watawala 1 BOPF Small
13 Black Tea Sri Lanka Watawala 2 BOP Medium
14 Black Tea Sumatra Tobasari BOPF Medium
15 Black Tea Sumatra Bah Butong BOPF Medium
16 Black Tea Sumatra Sidamanik BOPF Medium
17 Black Tea Sumatra Kayo Aro F3 Small
18 Black Tea Vietnam Lado FD Small
19 Black Tea Vietnam Minh Anh FD Small
20 Black Tea Sri Lanka Homadola Pekoe Large
21 Black Tea Sri Lanka Homadola BOPF Small
22 Black Tea Sri Lanka Kenilworth Pekoe Large
23 Black Tea Sri Lanka Kenilworth BOPF Small
24 Black Tea Sri Lanka Adisham Pekoe Large
25 Black Tea Sri Lanka Adisham BOPF Small
26 Green Tea Sri Lanka Glassaugh OPA Large
27 Green Tea Sri Lanka Glassaugh OPA2 Large
28 Green Tea Sri Lanka Glassaugh OP Large
29 Green Tea Sri Lanka Glassaugh FNGS Small
30 Green Tea Sri Lanka Glassaugh YH2 (small leaf) Large
31 Green Tea Sri Lanka Glassaugh YH2 (big leaf) Large
32 Green Tea Sri Lanka Glassaugh SL Large
33 Green Tea Sri Lanka Kotagala BOP Small
34 Green Tea Sri Lanka Kotagala GP1 Large
35 Green Tea Sri Lanka Kotagala GP2 Large
36 Green Tea Sri Lanka Kotagala GP SP Large
37 Green Tea Sri Lanka Kotagala Hyson Large
38 Green Tea Sri Lanka Kotagala YH2 Large
39 Green Tea Sri Lanka Kotagala CH Medium
40 Green Tea Sri Lanka Melfort CH Large
41 Green Tea Sri Lanka Melfort HY SP Large
42 Green Tea Sri Lanka Melfort GP1 Large
43 Green Tea Sri Lanka Melfort GP SP Large

34
 44 Green Tea Sri Lanka Melfort GP EX SP Large
45 Green Tea Sri Lanka Oliphant FNGS Medium
46 Green Tea Sri Lanka Oliphant SL Large
47 Green Tea Sri Lanka Oliphant OPA Large
48 Green Tea Sri Lanka Oliphant OPA2 Large
49 Green Tea Sri Lanka Oliphant YH Large
50 Green Tea China Longjing LJ1 Large
51 Green Tea China Longjing LJ2 Large
52 Green Tea China Longjing LJ3 Large
53 Green Tea China Longjing LJ4 Large
567
568 1
PF1: Pekoe Fannings grade 1, F2: Fannings grade 2, F3: Fannings grade 3, LEAF: rolled whole tea leaves, BOP: Broken
569 Orange Pekoe, BOPF: Broken Orange Pekoe Fannings, FNGS: Fannings, FD: Fanning Dust, OP: Orange Pekoe, OPA:
570 Orange Pekoe A, OPA2: Orange Pekoe A grade 2, CH: Chunmee, GP1: Gunpowder grade 1, GP2: Gunpowder grade 2, GP
571 SP: Gunpowder Special, GP EX SP: Gunpowder Extra Special, YH: Young Hyson, YH2: Young Hyson grade 2, HY SP:
572 Hyson Special, SL: Standard leaf, LG (1-4): Longjing Grade (1-4).

573

574

35
575 Highlights

576  Polyphenols contents in teas are significantly affected by plantation location.

577  Black tea from low elevation has > 20% polyphenols than higher elevation.

578  Small leaves has up to 15% more polyphenols than lager leaves.

579  Leaf grade has an effect on total catechins content and antioxidant capacity.

580

581

582

36