Phytochemical composition and health-enhancing properties of Oryza sativa L. leaf tea

1Department of Agro-industry, Faculty of Agriculture and Technology, Rajamangala University of Technology Isan, Surin 32000, Thailand 2Department of Pharmaceutical Botany, Faculty of Pharmacy, Van Yuzuncu Yil University, Van 65090, Turkey 3Department of Medical Services and Techniques, Vocational School of Health Services, University of Bingol, Turkey 4Food Science and Technology, School of Chemical Sciences and Engineering, University of New South Wales, Sydney, NSW 2052, Australia


Introduction
Herbal teas are a rich source of plant-derived phytochemicals that contribute towards lowering the incidences of chronic diseases [1]. Green tea leaf (Camelia sinensis (L.) Kuntze) is the classic example of a plant-based beverage that, due to its health properties, become extensively utilized in nutraceutical and functional foods industries [2]. Catechins -the dominant phenolic compounds of green tea, are responsible for its distinctive sensory [2] and health promoting properties, including antioxidant and antimicrobial [3], alleviation of clinical features of metabolic syndrome, memory disorders [4], reduction of pathological angiogenesis [5] and cancer prevention [6].
Locally grown botanicals are utilized for the production of herbal teas that display comparable antioxidant capacities to C. sinensis leaf tea [7]. In agreement, herbal teas obtained from rooibos, rosemary, lemongrass, mulberry, bamboo, lotus, peppermint, persimmon and mate are rich sources of antioxidant and antimicrobial compounds [3]. Among numerous water lily cultivars, the 'Conqueror' and 'Virginia' produced teas with an outstanding fragrance, the highest total polyphenols content and superior antioxidant capacities [8].
Recently the Thai government initiated the "One District One Product" program. This initiative represents an important strategy to support local communities through the promotion of their products and sustainable utilization of the local flora. Under this program new plant sources for production of functional tea are evaluated. Rice (Oryza sativa L.) is the staple food crop of Thailand [9] with the largest areas of rice cultivation being the central plains and Northeastern Region [10]. Due to a high market demand for jasmine rice its cultivation in Northeastern Region is growing. The unique scent of jasmine rice can be captured in young leaf tea. The present project aimed at the development of a functional tea obtained from young jasmine rice leaf characterized by unique sensory and health-promoting properties.

Antioxidant capacity
Total phenolic content (Folin -Ciocalteu assay) and ferric reducing antioxidant power (FRAP) assay: The levels of total phenolics (TP) and total reducing capacities of extracts were assayed as described previously [12].
Free radical scavenging activity (DPPH assay): Free radical scavenging activity was assayed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method according to Sharma and Bhat [13]. Briefly, lyophilized tea extracts were dissolved in 80% acetone (concentration range 0.01 to 10.0 mg/mL) and 0.06 mM of DPPH was dissolved in 100% methanol. The DPPH radical solution in methanol (3.5 mL, 0.06 mM) was mixed well with 100 μL of tea extract solutions. The mixture was then incubated for 30 min in the dark at room temperature. The absorbance was measured at 517 nm using a spectrophotometer (Libra S22, Holliston, MA, USA). The half effective concentration required for 50% decrease in absorbance of DPPH radicals (EC 50 ) was calculated and expressed as mg DW of lyophilized tea extract per mL of the solution (mg/mL).
Oxygen radical scavenging capacity (ORAC) assay: The ORAC assay was performed as previously described [11]. The antioxidant capacity of the samples was expressed as μmol trolox equivalent per gram of dry weight of lyophilized extract (μmol TE/g DW) based on a trolox standard curve.

Sugar determination
Reducing sugars and total sugars were measured as reported by AOAC [14]. All results were expressed as mg of glucose equivalent per gram dry weight (mg G E/g DW) of lyophilized extract.

Identification of phenolic compounds by liquid chromatographydiode array-mass spectrometry (LC-DAD-MS/MS):
Phenolic compounds were identified with the help of a Quantum triple stage quadrupole (TSQ) mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) as described by Dalar and Konczak [15] with the exception that the gradient elution was modified as follows: 0% solvent B for 2 min, then 40% B for 6 min, following 60% of B for 8 min and 100% B for 4 min.
Quantification of phenolic compounds by high performance liquid chromatography-diode array detector (HPLC-DAD): Phenolic compounds were quantified according to Dalar and Konczak [16]. A modified linear gradient elution was applied of solvent B from 0 to 20% over 3 min, to 40% over 5 min, to 60% over 5 min, to 100% over 5 min and the elution was held at the rate of 100% B for another 5 min.

Identification of volatile compounds
Head space solid phase microextraction (HS-SPME) procedure: Two grams of lyophilized plant extracts were weighed into 40 mL vials to undergo a solid-phase microextraction (SPME) procedure. The procedure was carried out using divinyl benzene/ carboxen/polydimethylsiloxane fiber, with 50/30 mL film thickness, preconditioned before analysis. The vials were equipped with ''mininert'' valves and kept at 35°C with permanent internal stirring. The samples were left to equilibrate for 30 min. Next, the SPME fiber was placed in the headspace for 40 min. Then the fiber was introduced into GC injector and left for 3 min to thermal desorption to increase the volatility of compounds. The microextraction procedure was optimized according to Verzera and collaborators [17].

Gas chromatography mass spectrometry (GC-MS) analysis
The analysis was carried out by gas chromatography mass spectroscopy as described by Dool and Kratz [18]. Fatty acids and volatile compounds were analyzed on Varian 3800 gas chromatograph as described by Uzun and collaborators [19].

Enzyme inhibitory activities
Alpha-amylase inhibitory activity: Inhibition of α-amylase was conducted using the Caraway-Somogyi iodine/potassium iodide (IKI) method as described by Dalar and collaborators [11]. The α-amylase inhibitory activity was expressed as micromoles of acarbose equivalents per gram dry weight of lyophilized extract (μM A E/g DW).
Lipase inhibitory activity: The lipase inhibitory activity assay was performed according to Dalar and collaborators [11] using 4-methylumbelliferyl oleate (0.1 mM) as a substrate. The lipase inhibition was reported in micromoles of orlistate equivalents (μM olistate E/g DW of lyophilized tea extract).

Statistical analysis
The mean of results and standard deviations (SD) were calculated based on at least three independent evaluations (n=3). The half effective concentration (EC 50 ) value was calculated from the corresponding dose inhibition curve according to their best fit shapes based on at least four reaction points using Microsoft Excel. Statistical correlation analyses were performed using Graphpad Prism 5 (Graphpad Software, CA, USA).

Yield, sugar content and antioxidant capacities of lyophilized tea extracts
Samples of dry tea (KM, HN and GT) were extracted sequentially using solvents with increasing polarity level: 1) acidified acetone, 2) acidified ethanol and 3) acidified MilliQ water. This approach has been suggested to achieve the maximum recovery of phytochemicals and good compounds differentiation during extraction from unknown plant sources [20].
The highest product yield (28.9%) was recorded for GT acetonebased extract ( Table 1). The yields of water-based extracts obtained from KM and HN rice leaf teas were 2.5-to 2.0-fold that of GT water extract yield indicating comparatively higher levels of polar compounds in rice teas. All three extracts of KM rice tea contained the highest level of total sugar for each category of extract (acetone-, ethanol, or water-based) and were followed by GT extracts. The lowest total sugars contained water-and acetone-based HN tea extracts ( Table 1). The water-based GT extract and ethanol-and acetone-based KM extracts contained the highest level of reducing sugars for each extract category (Table 1). These levels of reducing sugars are comparable or higher than those of Eryngium bornmuelleri leaf tea extracts (acetone-based extract: 71.1±0.6 mg G E/g DW, ethanol-based: 49.0±0.9 mg G E/g DW, waterbased: 37.0±1.1 mg G E/g DW [11]. The antioxidant potential of sequential leaf tea extracts was evaluated using four reagent-based assays, including total phenolics (TP, Folin-Ciocalteu method), 1,1-diphenyl-2-picrylhydrazyl (DPPH•) free radical assay, total reducing capacity (ferric reducing capacity power, FRAP) and oxygen radical absorbance capacity (ORAC).
The highest levels of total phenolics were found in acetone-based extracts of all teas (Table 1). These levels were approximately 3-times higher than those of the respective ethanol-based extracts. The least amount of phenolic compounds contained water-based extracts, with the exception of KM. In summary, the highest TP contained green tea, followed by KM and HN tea ( Table 1).
The DPPH free radical assay allows to measure the amount of lyophilized extract required to obtain 50% decrease in absorbance of DPPH free radicals in reaction solution (expressed as EC 50 ). The highest DPPH radical scavenging capacity exhibited the acetone-based extracts of all teas (Table 1). This result correlates well with the highest levels of total phenolic compounds detected in the same extracts ( Table 1). The lowest DPPH radical scavenging ability within their extract category exhibited HM-acetone-, KM ethanol-and KM water-based extract. The HN acetone-based and ethanol-based extracts had comparable DPPH free radical scavenging ability and were slightly more potent than HN water extract (Table 1). Similar tendency was observed for the FRAP and ORAC values; among all the extracts acetone-based exhibited the highest values and were followed by ethanol-and waterbased extracts (Table 1). Superior FRAP and ORAC values in each category of extracts were obtained for green tea (Table 1). Among the acetone-based extracts KM performed better than HN, however among the water-based extracts opposite result was observed (Table 1). Both acetone-based jasmine rice leaf tea extracts (KM and HN) and the reference sample of green tea (GT) exhibited higher ORAC and FRAP values than those of Eryngium bornmuelleri leaf tea extracts obtained through the same extraction process. However, the ORAC and FRAP values of the ethanol-and water-based extracts evaluated in this study were comparable to those of the respective ethanol-and water-based Eryngium bornmuelleri leaf tea extracts [11]. These results suggest that with regards to antioxidant capacity jasmine rice leaf teas are inferior to green tea, and they are comparable or superior to other traditionally utilized ethnic teas prepared from locally grown botanicals.

Identification and quantification of phenolic compounds present in tea extracts
Identification of phenolic compounds in sequential extracts obtained from rice leaf tea was carried out by high performance liquid chromatography (HPLC) and liquid chromatography mass spectrometry (LC-MS) analyses ( Table 2). The HPLC profiles of acetone-and ethanol-based extracts of both jasmine rice leaf teas were very similar with the exception that the concentration of phenolic compounds in acetone-based extract was higher (Figures 2 and 3). The majority of compounds present in KM and HN teas were visible on HPLC chromatogram at 326 nm wavelength (Figure 2). The spectral characteristics of HPLC peaks revealed that chlorogenic acids (4and 5-chlorogenic acid) are the dominating phenolic compounds of both jasmine rice teas. The majority of compounds present in green tea extracts were found at 280 nm with gallic acid, catechin and epigallocatechin identified ( Figure 3C). Gallic acid was also the dominating phenolic compound in KM water extract ( Figure 4A)    Figure 4B). In agreement with the HPLC chromatograms the mass spectroscopy data confirmed that hydroxycinnamic acids were the major phenolic compounds identified in both jasmine rice leaf teas ( Table 2). Chlorogenic acid and p-coumaric acid were the major phenolic compounds of KM acetone and ethanol extracts representing, respectively, 36% and 34% of total phenolics (Table 2). Chlorogenic acid identified in HN ethanol and water extracts accounted for, respectively, 59% and 54% of total phenolics. Significant amounts of gallic acid and catechin were also detected ( Table 2). The dominating compounds of GT extracts were catechin and epigallocatechin gallate (Figures 2-4). Dai and Mumper [20] reported that ethanol is an efficient extraction solvent of low molecular weight polyphenols, while aqueous acetone is more suitable for the extraction of high molecular weight flavanols. This study identified similar phenolic composition of both, ethanol and acetone jasmine rice leaf tea extracts (Table 2). These results suggest that in the evaluated jasmine rice leaf teas high molecular weight compounds are present at very low levels or are absent. Similar results were reported for Eryngium bornmuelleri leaf tea [11].

Identification of volatile compounds by gas chromatography mass spectroscopy (GCMS)
Volatile compounds are required in food development as they provide a pleasant scent. Moreover, they exhibit pharmacological activities through their antioxidant, anti-inflammatory, anti-cancer and anti-obesity properties [21,22]. Jasmine rice is known for its attractive aroma, therefore the presence of volatile compounds in jasmine rice leaf teas was investigated. The GC-MS profiles of KM, HN and GT volatiles revealed the presence of similar compounds in all teas ( Figures  5-7) (Tables 3 and 4). Tridecane was the predominant volatile of KM acetone extract, followed by 2,6-Di-tert-butylphenol and pentadecane. In the KM ethanol extract dominated caryophyllene and 2,6-Di-tertbutylphenol. Five compounds were identified in KM water extract: eicosane, 3-ethyl-5-(2-ethylbutyl) octadecane, etracosane, heneicosane 11-(1-ethylpropyl) and dodecane. Traces of the same compounds were also found in acetone-and ethanol-based extracts (Table 3). Dihydroactiniolide was found in HN acetone extract, while dodecane isomer and dodecane were detected in ethanol-and water-based extract, respectively (Table 3). Three volatile compounds (tridecane, dodecane and 2,6-Di-tert-butylphenol) were found in HN and KM water-based extracts. Five volatile compounds, including tridecane, 1-dodecene, dodecane, 1-tetradecene and 2,6-di-tert-butylphenol were identified in GT acetone extract. Green tea ethanol-and water-based extracts comprised, respectively, 3 compounds (tridecane, dodecane, α-fenchylacetate) and 4 compounds (dodecane, 1-tetradecene, 2,6-ditert-butylphenol, eicosane) ( Table 4). The results revealed that similar volatile compounds are present in KM and HN water-based extracts. This finding suggests that these volatile compounds will be released into tea during the infusion process and may influence its sensory properties and consumers' preferences.

Identification of fatty acid composition
In a life cell the fatty (carboxylic) acids from foods are utilized in conversion of biochemical energy from nutrients into adenosine triphosphate (ATP). The GCMS analysis of fatty acids revealed the presence of similar fatty acids in all evaluated teas (Table 5) Table 3). The ethanol-based KM and HN extracts also contained α-linolenic acid, palmitic acid and linoleic acid. Traces of stearic acid were also present. The water extracts contained α-linolenic acid, palmitic acid and linoleic acid. Similar fatty acids composition and content was found in GT extracts with the exception of vaccenic acid which was detected in acetone and ethanol extracts and stearic acid detected in water extract. According to Kobayashi and collaborators [23], presence of the major fatty acid derivatives: C6 and C9 alcohols and aldehydes in green tea is the key contributor to its fresh odor.

Enzyme inhibitory activities
Obesity -one of the greatest threats to global health is currently treated with synthetic inhibitors of digestive enzymes, such as acarbose and orlistat. Because an uptake of synthetic inhibitors may lead to unwanted side effects, the attention of researchers is directed to natural compounds from plant sources that provide safer alternative. Within  this study enzyme-inhibitory activities of jasmine rice leaf teas were evaluated.

Alpha-amylase and pancreatic lipase inhibitory activities
The extracts of KM and HN tea inhibited the activity of α-amylase in a dose response manner (data not shown). The inhibitory activity of KM acetone extract was 2.3-fold that of KM ethanol-and 9.8-fold that of KM water-based extract. The acetone and ethanol extracts of HN exhibited comparable α-amylase inhibitory activities and were 6.6-fold those of HN water extract. Alpha-amylase inhibitory activities of water extracts obtained from all evaluated samples were comparable (Table 6).
Acetone extract of KM as well as acetone extract of GT exhibited the strongest and similar pancreatic lipase inhibition. The inhibitory activity of HN acetone extract was 3.5-fold lower ( Table 6). Similar low inhibitory activities towards pancreatic lipase showed ethanol and water extracts of KN and HN. Lipase inhibitory activities of jasmine rice leaf teas were lower than those of Eremochloa ophiuroides (centipede grass) leaf methanolic extracts (IC 50 of 33.6±2.0 μM equal to 0.019±0.001 mg/ mL) [24].
Chlorogenic acid possesses pronounced α-amylase inhibitory properties [24]. The presence of chlorogenic acid at high levels in ethanol-and acetone-based extracts of jasmine rice leaf teas may be responsible for the enzyme inhibition observed in this study. Lower levels of chlorogenic acid in KM and HN water-based extracts correlate well with the lower IC 50 values ( Table 6). Structure of dietary polyphenols influences their α-amylase inhibitory effect (Xiao et al., 2013). This effect increases with the presence of hydroxylation of flavonoids and/or presence of an unsaturated 2,3-bond in conjugation with a 4-carbonyl group. Glycosylation of flavonoids as well as methylation and methoxylation weakens the inhibitory effect [25,26]. In agreement, in the present study the chlorogenic acid rich extracts obtained from KM and HN rice teas exhibited pronounced enzyme-inhibitory activities.