International Food Research Journal 26(6): 1781-1788 (December 2019) Journal homepage: http://www.ifrj.upm.edu.my A comparison of the production of polyphenol contents and the expression of genes involved in Vietnamese tea cultivars 1Hoang, T. T. Y., 2Luu, H. L., 2Nguyen, T. L., 3Duong, T. D., 4,5Nguyen, H. D. and 2*Huynh, T. T. H 1Thai Nguyen University of Sciences, Thai Nguyen University, Thai Nguyen Province 24000, Vietnam 2Institute of Genome Research, Vietnam Academy of Science and Technology (VAST), Hanoi 100000, Vietnam 3Thai Nguyen University of Agriculture and Forestry, Thai Nguyen University, Thai Nguyen Province 24000, Vietnam 4Advanced Centre for Bioorganic Chemistry, Institute of Marine Biochemistry, VAST, Hanoi 100000, Vietnam 5University of Science and Technology of Hanoi, VAST, Hanoi 100000, Vietnam Article history Abstract Received: 19 June, 2019 Tea (Camellia sinensis) is a popular health beverage which is consumed all over the world Received in revised form: due to its good aroma and taste. Tea consumption is also considered to reduce the risk of 16 September, 2019 several diseases in humans, including cardiovascular diseases, diabetes and cancers. Recent Accepted: 25 September, 2019 studies have shown that polyphenols derived from tea may contribute to the majority of these pharmaceutical properties. Among all the tea polyphenols, catechins are the main components that include (−)-epicatechin (EC), (−)-epicatechin gallate (ECG), (−)-epigallocatechin (EGC), (−)-epigallocatechin-3 gallate (EGCG), (+)-catechin (C), (−)-catechin gallate (CG), (−)-gallocatechin (GC), and (−)-gallocatechingallate (GCG). In the present work, four Keywords catechins (C, EGC, ECG, and EGCG) and two anthocyanidins (cyanidin 3-O-glucoside and delphinidin 3-O-glucoside) in two Vietnamese tea cultivars, Trungduxanh and Trungdutim, were Catechin LAR quantitatively detected by high-performance liquid chromatography. The total catechin content ANR in Trungduxanh was generally higher than that in Trungdutim. By contrast, the concentrations Trungduxanh of the two anthocyanidins were lower in Trungduxanh than that in Trungdutim, suggesting Trungdutim that Trungdutim tea accumulates anthocyanins to produce purple colour in buds and leaves, rather than converting them into catechins. Real-time PCR was also performed to analyse the expression levels of leucocyanidin reductase (LAR) and anthocyanidin reductase (ANR) genes, which are involved in catechin biosynthesis. In accordance with the HPLC analysis, the qPCR results showed that the transcripts of both genes in Trungduxanh tea were more abundant than in Trungdutim tea. © All Rights Reserved Introduction countries, black tea (fermented) is favoured in the Western countries, and oolong tea (semi-fermented) Tea (Camellia sinensis) is one of the most widely is consumed in southern China (Khan and Mukhtar, consumed beverages. It was first discovered in China 2007). Vietnam is one of the largest tea producing about 2700 BC, after which it spread throughout the and exporting countries in the world. In Vietnam, world. Recently, tea in China has been classified tea is grown mainly in the Northern midland and into seven primary types based on the different mountainous regions, and the Central Highlands. processing methods: green tea, black tea, oolong tea, According to the General Statistics Office of Vietnam white tea, yellow tea, aged pu-erh and ripened pu- (http://www.gso.gov.vn), the total tea growing area erh tea (Yi et al., 2015). Among these, the former had reached 131.5 thousand hectares with an output three types are more popular than the others. Green of 1,033.6 thousand tons in 2016. In the same year, tea (non-fermented) is commonly consumed in Asian Vietnam tea export reached 138 thousand tons which were valued at USD 228 million. *Corresponding author. Email: [email protected] 1782 Hoang et al./IFRJ 26(6) : 1781-1788 Polyphenols that comprise approximately 30% anthocyanins possess several valuable bioactive - 42% of green tea dry weight are responsible for compounds such as antioxidants (Bae and Suh, 2007), tea’s health benefits. Among polyphenols, catechins anticarcinogens (Lee et al., 2009), and antimicrobials (also known as flavan-3-ols) are the most abundant (Viskelis et al., 2009). substances with a concentration of 70% of the High-performance liquid chromatography total tea polyphenol content. Catechins consist (HPLC) is a useful method for the quantitative of two main types, epicatechins and epicatechin determination of polyphenol contents in different epimers. Epicatechins include (−)-epicatechin (EC), tea types. Numerous studies have been conducted (−)-epicatechin gallate (ECG), (−)-epigallocatechin to improve the separation efficiency and to (EGC), and (−)-epigallocatechin-3 gallate (EGCG); simultaneously determine the polyphenol content and epicatechin epimers consist of (+)-catechin (C), in green, black, oolong and pu-erh teas (Yashin et (−)-catechin gallate (CG), (−)-gallocatechin (GC), al., 2015). The efficiency of HPLC analysis is also and (−)-gallocatechin gallate (GCG). Along with dependent on the method of extraction, the type of caffeine and several amino acids, these catechins solvent used, and the method of sample preparation contribute to the quality and taste of tea (Yamamoto (Lorenzo and Munekata, 2016). et al., 1997). Catechins are known for their potential Several studies have investigated the biosynthetic health benefits, including antioxidant activities pathway of catechins and anthocyanins in the tea (Shahidi et al., 2008), prevention or reduction of some plant using genetic and biochemical approaches over types of cancer (Beltz et al., 2006), immune system the recent few decades. Some genes involved in the improvement, and prevention of cardiovascular biosynthesis of catechins and anthocyanins have disease, diabetes and dental decay (Hamilton- been discovered and characterised (Punyasiri et al., Miller, 2001; Yang et al., 2004; Nagao et al., 2007). 2004). A recent study had elucidated the biosynthetic Furthermore, tea catechins have also been widely and regulatory mechanisms of catechins in tea using used in food and cosmetic industries. a combination of transcriptomic analysis and HPLC In addition to catechins, anthocyanins are (Zhang et al., 2018). Key enzymes that directly catalyse important biological compounds in plants. More catechin producing reactions are leucoanthocyanidin than 670 anthocyanins have been identified in nature, reductase (LAR) and anthocyanidin reductase (ANR). among which the following six anthocyanins are LAR is responsible for the formation of catechins primarily found in fruits and vegetables: pelargonidin, and gallocatechins, and ANR plays an important cyanidin, peonidin, delphinidin, petunidin and role in the production of epicatechins (Figure 1) malvidin. Anthocyanins contribute to the colours of (Liu et al., 2015; Zhang et al., 2018). Furthermore, numerous plants, such as grapes (Rivero-Pérez et al., anthocyanins share their precursors with catechins. 2008), berries (Nicoué et al., 2007), and Hibiscus In particular, leucoanthocyanins can be converted flowers (Lo et al., 2007). Studies have reported that into anthocyanins by anthocyanidin synthase (ANS) Figure 1. Putative biosynthetic pathways of catechins and anthocyanins in tea (C. sinensis) leaves (Liu et al., 2015; Zhang et al., 2018). PAL: phenylalanine ammonia-lyase; C4H: cinnamate 4-hydroxylase; 4CL: 4-coumarate CoA ligase; CHS: chalcone synthase; CHI: chalcone isomerase; F3H: flavanone 3-hydroxylase; F3’H: flavonoid 3’-hydroxylase; F3’5’H: flavonoid 3’,5’-hydroxylase; DFR: dihydroflavonol 4-reductase; LAR: leucocyanidin reductase; ANR: anthocyanidin reductase; ANS: anthocyanidin synthase; FGS: flavan-3-ol gallate synthase; UFGT: anthocyanidin 3-O-glucosyltransferase. Hoang et al./IFRJ 26(6) : 1781-1788 1783 or into non-epicatechins by LAR. Anthocyanins such a quaternary HPLC Agilent 1260 pump and a single as cyanidin and delphinidin are catalysed by ANR to quadrupole Agilent 6120 mass spectrometer. The form epicatechins (Liu et al., 2015). extract was injected at different concentrations into a In the present work, the contents of two reversed phase Eclipse XDB C18 column (250 × 4.6 anthocyanins and four catechins that significantly mm, 5 μm) with an XDB C18 guard column (Agilent contribute to the valuable properties of tea were Technologies Inc., USA). Mobile phases consisted analysed in two Vietnamese local tea cultivars, of 1.0% acetic acid in water (v/v) (eluent A) and a Trungduxanh and Trungdutim. Trungduxanh gradient of acetonitrile (ACN) in methanol from 95/5 belongs to the green-coloured tea cultivar, whereas to 5/95 (v/v) for 45 min (eluent B). Detection was Trungdutim is a purple tea that has purple buds and carried out at 280 nm. The flow rate was set at 1.0 mL/ young leaves. The present work also investigated min. The column was maintained at a temperature of the relationship between the expression of LAR and 25°C. The sample injection volume was 5 µL, and all ANR genes and catechin accumulation. The results samples were run in triplicate. of the present work could provide more valuable information about these tea cultivars and support to RNA isolation and quantitative real-time PCR enhance tea conservation and planting activities. Total RNA was extracted from the bud and young leaf samples using the GeneJET RNA Purification Materials and methods Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instruction. The integrity and quality