Variation in Levels of Flavonols Myricetin, Quercetin and Kaempferol—In Kenyan Tea ( Camellia sinensis L.) with Processed Tea Types and Geographic Location

The aim of this study was to determine the levels of myricetin, quercetin, and kaempferol in green and black Kenyan types in relation to geographic location of production. Tea samples were extracted before chromatographic separations with a Shimadzu 20A HPLC coupled with PDA. Flavonol levels were quercetin > kaempferol > myricetin. Teas from east of rift, had quercetin 1.25 - 1.83 mg/g and 1.29 - 1.71 mg/g for green and black types, respectively. Kaempferol levels were between 1.28 - 1.72 mg/g in green and 1.36 - 1.76 mg/g in black tea. Myricetin ranged at 0.40 - 0.79 mg/g green and 0.12 - 0.38 mg/g black tea types. Total flavonols in green tea were highest at 4.28 mg/g while black tea was 3.83 mg/g. These trends agree well with those observed in teas west of the rift. For tea types, myricetin and kaempferol showed a significant difference (P ≤ 0.05) between green and black teas. Total flavonols showed no significant difference. Kenyan teas are a potential dietary source of flavonols myricetin, quercetin, and kaempferol as evidenced by the significant quantities recorded in this study.


Introduction
Tea (Camellia sinensis) is a beverage consumed hot or iced for both refreshment and health benefits since ancient times [1]. The beverage is derived from tender tea shoots that contain a full complement of enzymes, biochemical interme- been identified by chromatography [5]. However, only eight have been shown to be present in tea in significant quantities [6]. Furthermore, previous studies focused specifically on the most abundant catechins epigallocatechin gallate (EGCG) and epigallocatechin (EGC), which constitute more than 70% of the total amount of all catechins [7] [8]. Caffeine a central nervous system (CNS) stimulant of the methyl xanthine class is odorless, has a bitter taste and is highly soluble in hot water. Additionally, processed tea leaves contain 3% caffeine by weight, although this can range from 1.4% to 4.5%. Consumption of caffeine has overtime elicited health concerns. However, adverse health impacts to humans depend on factors such as the levels and rate of consumption as well as mental and physical health conditions [9] [10]. Green tea extracts (GTEs) are common ingredients among dietary supplements marketed for weight loss and management [11]. Furthermore, like diet and exercise, they play important roles in ameliorating metabolic syndrome [12].
Besides the polyphenols and alkaloids, tea has a complex chemical composition, containing over 2000 components that include, carotenoids, lignans, amino acids (including L-theanine), vitamins, minerals and trace elements [13]. Although mankind has been drinking tea for more than 5000 years, determination of its chemical composition has not been intensive until recent decades after abundance of scientific data has shown a positive effect of tea on human health [14] [15].
Flavonol glycosides in tea leaves have been quantified as aglycones-myricetin, quercetin, and kaempferol [16]. Figure 1 shows the chemical structures of myricetin, quercetin, and kaempferol. Presence of myricetin, quercetin, and kaempferol has been reported in fruits and vegetables for a long time [17]. Furthermore, these aglycones are also found in abundance in onions, grapes, berries, cherries, broccoli, and citrus fruits, possess protective abilities against tissue injury induced by various drug toxicities [18] [19] [20]. Additionally, are important bioflavonoids having beneficial effects which include cardiovascular protection, anticancer, antitumor, anti-ulcer, anti-allergy, anti-viral, anti-inflammatory activity, anti-diabetic, gastro protective effects, antihypertensive, and immunomodulatory [21] [22] [23]. Documented  (6) and Nandi hills and Western highlands (7) [26] [27] which are predominantly in the eastern (Mt Kenya region) and western (Lake Victoria basin) sides of the great rift valley. It is importantly mentioned here that apigenin, the only flavone identified in tea, and its glycosides, have also been detected in tea however, as very small fraction of the tea polyphenols. More recently, 19 O-glycosylated flavonols, 7 C-glycosylated flavones, 28 acylated glycosylated flavonols, and 3 flavonols have been identified from green and fermented teas using liquid chromatography.
The processed teas including; white tea, yellow tea, green tea, Oolong tea, black tea, purple tea and post fermented tea which are subjected to varying processing conditions in order to attain specific levels of oxidation. For green tea, leaves are plucked, steamed and dried to stop enzymatic activities that cause oxidation while the black tea is allowed to undergo the enzymatic oxidation process supported by polyphenol oxidase inherent in tea leaves. Studies have shown that biochemical compositions of teas sourced from the different regions have distinct levels identifiable with that specific regions [28]. Composition of the same type of tea may vary significantly depending on the place where it was grown mainly due to the cultivar type, soil, climate, altitude and precipitation patterns [29]. The other factors are agronomic practice, processing technologies used and how well the finished products are stored [30]. According to a research conducted by Muthumani et al; 2013 altitude significantly affected the catechin composition of green tea leaves [31]. In another study, black teas from higher altitude showed higher levels of theaflavins and aroma composition while crude fibre remained unchanged by variations in altitude [32] [33].
In analysis of plant composition, several challenges are normally encountered mainly due to the chemical complexity in the plant matrix and the physical nature of the plant materials [34]. High-temperature liquid-solid extraction is mostly applied to isolate the targeted biochemicals to be determined [35]. Other Open Journal of Applied Sciences extraction methods that have been used include, ultrasonic, microwave-assisted, solid-liquid extraction at ambient temperature, and elevated pressure supercritical extraction with carbon dioxide [36]. Solvents, such as chloroform, methanol, ethanol and acetone or mixtures of these and water are used for extraction [37].
For identification of the extracted aglycones, preparative thin layer chromatography (TLC), reverse phase-high performance liquid chromatograph (RP-HPLC), micellar liquid chromatography and gas chromatograph coupled mass spectrometer (GC-MS) are used [38] [39]. The data generated in this study will be used as a pointer to the potency of the Kenyan tea as dietary source of flavonol glycoside aglycones of quercetin, myricetin, and kaempferol which are versatile antioxidants. Moreover, unique levels of these biochemicals ascertained in specific tea cultivars can lead to development of specialty tea products a milestone in value addition chain and product diversification.

Tea Samples
Green tea leaves were sourced from farmers in the seven Kenya Tea Development Agency (KTDA) zoned regions (Aberdare ranges (1 and 2), Mt Kenya (3), Mt Kenya and Nyambene hills (4), Kericho highlands (5), Kisii highlands (6) and Nandi hills and Western highlands (7)). In each of the regions two catchment areas were randomly identified for sample collection with sampling being done in triplicate. All the samples were well packed in brown aluminium lined bags ready for laboratory determinations.

Reagents and Chemicals
Acetonitrile, acetic acid (both HPLC grade), hydrochloric acid, methanol, ethanol (both of analytical grade), the standards of quercetin, kaempferol and myricetin were purchased from Sigma-Aldrich (Germany) through Kobian suppliers.
Double distilled water was prepared in the laboratory.

Preparation of Test Samples
To ensure homogeneity, the samples were ground in accordance to ISO 1572 [40] recommendations and stored in well-sealed containers for protection against light to avoid oxidation of the biochemicals and weakened aroma.

Determination of Dry Matter Content
2.0 ± 0.01 g of the sample was weighed into aluminium dishes and heated in an oven at 103˚C ± 2.0˚C for 4 hours to obtain constant weight. The samples were weighed again and moisture content determined by subtracting the final weight from the initial weight, computed and expressed as a percent.

Extraction of Flavonols Myricetin, Quercetin and Kaempferol
1 g of well powdered tea leave sample was weighed into a 250 mL round bot-Open Journal of Applied Sciences tomed flask. 40 mL of 60% aqueous methanol was added followed by 5 mL of 6M hydrochloric acid (HCL). The mixture was refluxed for 2 hours at the boiling temperature before filtering in a 50 mL volumertric flask. On cooling, the filtrate was made to mark with 60% methanol in water. A 0.45 µ membrane filter was used to filter the sample before injecting into the HPLC.

HPLC Instrumentation and Conditions
The

Statistical Analysis of Data
All statistical analysis was carried out using SAS ® V 9.1 (SAS.2002) for windows statistical software. Analysis of variance (ANOVA) was used to determine the means, coefficient of variation (CV) and any differences between the samples.
Least Significance Difference (LSD) was used to separate means. The probability limit was set at P ≤ 0.05 significant level. Results of the parameter determined were expressed as a mean of the triplicate determinations. Graphical representation of means was done using excel for windows.

Quantitative Levels of Myricetin, Quercetin and Kaempferol in Kenyan Tea
Flavonoids are a group of plant secondary metabolites characterized by a diphenyl propane structure (C 6 -C 3 -C 6 ) [41] and are common constituents of fruits, vegetables and some beverages [42]. The major flavonols present in tea leaves hardly exceeds 3% of the water-soluble solids in tea [43]. The current study has made an attempt to determine the levels of myricetin, quercetin, kaempferol and total flavonol in green and black tea types sourced from different tea growing re-    [46]. Furthermore, other researchers Peterson et al. working on green tea [47] and Luximon-Rammaetal on black tea [48] reported on levels of flavonols that to a greater extend are in agreement with the findings of the study.

Effects of Processing on Levels of Myricetin, Quercetin and Kaempferol in Kenyan Tea
The phytochemical composition of tea leaves is affected by factors such as the growing regions, climatic conditions, cultivars, brewing techniques, agronomic practices and processing conditions [49]. It has been observed that in leaf processing, levels of catechins and polyphenols increase slightly during the withering stage. In black tea manufacture there is a higher loss of catechins due to the conversion of phenolic compounds to theaflavins and thearubigins. In general, fermentation is one of the stages in processing that much emphasis is focused aimed at changing the ingredients in agro-food processing [50].

Variation on Levels of Myricetin, Quercetin and Kaempferol in Kenyan Tea with Geographic Location
The composition of bioactive compounds, as already highlighted, might be influenced by several parameters associated with plant growth. Additional factors include, genetics train, climatic conditions, soil profile, growth altitude and plucking season [53]. Studies have demonstrated that higher elevation is associated with higher tea quality based on levels of catechins, other polyphenols and caffeine [45]. It is reported that there is increase in favorable aromatic compounds

Conclusion
Myricetin, quercetin, and kaempferol have been found in significant amounts in Additionally, the flavonol profile can be effectively used as a mark of origin for the high quality Kenyan tea products. Given that Kenyan teas have shown great potential as sources of the flavonol myricetin, quercetin and kaempferol, there is a need to carry out further research to establish the content of these biochemicals in known superior Kenyan tea cultivars especially the newly released types that score high in quality and yields. This will include cultivars such as TRFK 6/8, Ejulu, EPK-TN14-3, AHP S15/10,