Catechins (flavan-3-ols) are polyphenolic plant secondary metabolites that have been strongly associated with a wide variety of beneficial health effects in vitro, in vivo and clinically. This study reports findings on the content of catechins in tea seed oil (TSO) extracted by Soxhlet extraction from seeds of different clones of Kenyan tea. Extraction of catechins from the crude oils was achieved by sequential liquid-liquid extraction (LLE) using methanol and quantified by reverse phase High Performance Liquid Chromatography (RP-HPLC). Results obtained revealed that all the crude test oils contained catechins, with oils extracted from clones TRFK K-Purple and GW-Ejulu having the highest total catechin content of 9.8 ± 0.25 and 9.0 ± 0.83 (×10 -3% flavonoids) respectively. Statistically significant differences (p < 0.05) were evident in the total catechin contents of crude oils extracted from tea seeds with those extracted from corn, sunflower and soybean seeds. Moreover, clonal variations were evident, as the total catechin contents of oils extracted from clones TRFK K-Purple and GW-Ejulu were statistically different (p < 0.05) from those extracted from clones TRFK 301/3, TRFK 301/4, TRFK 301/5, TRFK 306, TRFK 91/1 and TRFCA SFS 150. Thus, the current findings strongly suggest that oils from seeds of Kenyan tea cultivars can be a potential source of potent natural antioxidants.
The tea plant is mainly cultivated for its prolific vegetative growth [
Tea is widely consumed worldwide [
Seeds of different plants usually contain various antioxidants. Indeed, it has recently been reported that the naturally occurring active compounds in tea seeds are different from those in other kinds of oil seeds as is the case of saponin [
Kenya has continued to be a producer of processed tea at the primary level with very little product differentiation and value addition, over 95% of which is sold in bulk in the export market. Owing to global overproduction of tea, the profitability of the Kenyan tea enterprise has declined in the recent past. Thus, the need to diversify tea products in a bid to reshape the future of this key industry cannot be understated. Indeed, the need for researchers and tea industry stakeholders in Kenya to exploit TSO as an avenue to ease on the current dwindling returns to tea farmers resulting from global overproduction of black tea is the need of the day. With this in mind, the current research seeks to quantify the total catechin content in TSO extracted from seeds of selected Kenyan tea clones, with the expectation that findings from the research will immensely contribute to the development of diversified products from the tea plant, hence enhancing sustainability and profitability of the local tea industry.
Authentic catechin standards viz., (+)-catechin (C), (-)-epicatechin (EC), epigallocatechin (EGC), (-)-epicate- chingallate (ECG) and (-)-epigallocatechingallate (EGCG) were purchased from Sigma Aldrich, Germany. Also, the following chemicals were used; n-hexane (≥99.5%), methanol (≥99.5%), ethylenediaminetetraacetic acid (EDTA ≥ 99.1%), acetonitrile (HPLC grade), ascorbic acid (≥99.0%) and nitrogen gas (99.9%). Double distilled water was used in all dilutions throughout the study.
Mature and healthy tea seeds from eight randomly selected clones of tea viz., TRFK 301/3, TRFK 301/4, TRFK 301/5, GW-Ejulu, K-Purple, TRFK 306, TRFK 91/1 and TRFCA SFS 150, were harvested in triplicates from mature, healthy tea plants (
Sun-dried tea seeds were manually de-husked and the husks carefully separated from the kernels (
(
Extraction of catechins from the crude oils was achieved by dissolving 2.0 ± 0.001 g of oil in 2.0 mL of n-hexane in 10 mL graduated extraction tubes. The mixture was thoroughly mixed using a vortex mixer (VM-1000, Digisystem Laboratory Instruments Inc., Taiwan) for one minute followed by liquid-liquid extraction (LLE) using 4.0 mL of 80:20 v/v methanol: water mixture in order to assay the polar fraction. The final mixture was vortexed for 10 seconds followed by centrifugation for 10 minutes at 3500 revolutions per minute (rpm), using a digital high speed universal centrifuge (HSCEN-204, M.R.C Ltd., Israel) fitted with a rotor (RA-1512S, M.R.C Ltd., Israel). The supernatant (methanolic extract) was decanted into a clean graduated tube and the same sample was subjected to a second and third extractions only with the addition of 4.0 mL of the 80:20 v/v methanol:water mixture; each time, the supernatant was collected in the same graduated tube for each replicate of each sample. The methanolic extracts were stored refrigerated at −15˚C ± 0.5˚C until the time of analysis.
The catechins content in the methanolic extracts of the crude oils were quantitatively estimated by Reverse Phase High Performance Liquid Chromatography (RP-HPLC). 1.0 mL of the methanolic extract was accurately transferred into a clean and dry graduated sample tube and diluted to 5.0 mL with a stabilizing solution consti- tuting of 10% v/v acetonitrile, 500 µg∙mL−1 ethylenediaminetetraacetic acid (EDTA) and 10 mg∙mL−1 ascorbic acid in the ratio 2:1:1, diluted five times. The solution was then filtered through a 0.45 µm nylon membrane filter and transferred into sample vials. The chromatographic set-up comprised of a Shimadzu LC 20 AT HPLC system fitted with a SIL 20A auto-sampler, an SPD-20 UV-Visible detector, a class LC10 chromatography work- station and a Gemini 5 µm C6 Phenyl 110Å, 250 mm × 4.6 mm i.d (Phenomenex, Torrance, CA, USA) separation column.
A binary gradient elution was carried out using two solvent systems, eluents A and B, constituting of acetonitrile/acetic acid/double distilled water in the ratios 9:2:89 and 80:2:18 v/v/v respectively. The eluent composition for the binary gradient condition started at 100% eluent A for 10 minutes, then over 15 minutes, a linear gradient to 68% eluent A, 32% eluent B and held at this composition for 7 minutes. The condition was then reset to 100% eluent A and allowed to equilibrate for 10 minutes before the next injection. The flow rate of the eluents, injection volume, column temperature and λmax were 1.0 µL∙min−1, 20 µL, 35˚C ± 0.5˚C and 278 nm, respectively. Identification of individual catechins was carried out by comparing the retention times of the sample peaks with those of the authentic catechin standards and caffeine analysed under similar conditions. Quantitation of catechins and caffeine were performed using the caffeine calibration curve obtained (
Data obtained were subjected to analysis of variance (ANOVA) using MSTAT statistical package version 2.10 at p < 0.05. The least significant difference (LSD) test was used in mean separation where statistically significant differences were recorded. Data are tabulated as means of the triplicate determinations ± standard deviation.
The caffeine calibration curve obtained (y = 50617x + 473.6) and used in the quantitation of the individual ca-
techin contents in the methanolic extracts of the crude oils demonstrated adequate linearity, r2 = 0.9999 (
The order of elution of the catechins was EGC, +C, EC, EGCG and ECG.
The total catechin contents of the crude tea and control oils were as given in
Reference [
Tea clone | Total catechin content (×10−3) |
---|---|
TRFK 301/3 | #b6.4 ± 0.34 |
TRFK 301/4 | b4.8 ± 1.09 |
TRFK 301/5 | b4.9 ± 0.41 |
GW-Ejulu | a9.0 ± 0.83 |
TRFK K-Purple | a9.8 ± 0.25 |
TRFK 306 | b5.9 ± 1.70 |
TRFK 91/1 | b5.0 ± 1.11 |
TRFCA SFS 150 | b5.1 ± 0.48 |
*Corn | c1.9 ± 0.21 |
*Sunflower | c0.7 ± 0.24 |
*Soybean | c1.6 ± 0.66 |
Overall mean | 5.01 |
CV (%) | 19.1 |
LSD | 1.6 |
*Samples used as controls; CV: coefficient of variation; LSD: Least Significant Difference; #Values preceded with the same superscript letter are not statistically different at (p ≤ 0.05).
pherol compounds. However, the current data also demonstrates the presence of catechins in the three vegetable oils (corn, soybean and sunflower) used as controls. Earlier, the current authors reported that TSO from clone GW- Ejulu had the highest total polyphenolic content (0.043 mg∙L−1 gallic acid) as well as antioxidant capacity (20.6% 2,2-diphenyl-1-picrylhydrazyl, DPPH, radical scavenging activity [
Prior to the reports by [
Findings of this study clearly demonstrate the presence of catechins in all crude oils extracted from the tea seeds obtained from eight clones of Kenyan tea. These polyphenolic compounds have been reported to possess potent antioxidant activity thought to account for their protective role against a number of diseases. Thus, TSO is a promising source of natural antioxidants, as well as a potential source of diversified products from the tea plant and extra income for tea growers.
The authors confirm that this article content has no conflict of interest.
The authors acknowledge the Tea Research Institute (TRI) formerly the Tea Research Foundation of Kenya (TRFK) for funding this project. This article is published with the permission of the director, TRI.