Juan Yang et al., J.Chem.Soc.Pak., Vol. 40, No. 01, 2018 158 Identification and Quantitative Evaluation of Major Sweet Ingredients in Sweet Tea (Lithocarpus polystachyus Rehd.) Based Upon Location, Harvesting Time, Leaf Age Juan Yang, Yuyi Huang, Zhi Yang, Chao Zhou and Xujia Hu* Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, P.R. China. [email protected]* (Received on 11th April 2016, accepted in revised form 13th September 2017) Summary: Lithocarpus polystachyus Rehd. (Sweet Tea) is a traditional sweet tea plant. Flavonoids are the essential sweet active constituents of Sweet Tea, and they have direct relationship with the sweet tonic beverage and traditional herb. In this work, the chemical components of the sweeteners isolated from Sweet Tea were identified as 3-hydroxy phlorizin (1), phlorizin (2), kaempferol-3-O-β-D-glucoside (3) and trilobatin (4) by ESI•-MS and NMR analyses. Quantitative analysis of the flavonoid compounds in Sweet Tea from Yunnan province of China indicated their content was influenced by harvesting time and leaf age. Phlorizin and trilobatin were identified as principal flavonoids in Sweet Tea. The highest concentration of trilobatin was in April, and the highest concentration of phlorizin was in November. The flavonoids content was the highest in young leaves and decreased at mature stage. Moreover, considerable variations of flavonoids content in Sweet Tea from three locations (Yunnan, Hunan, Jiangxi) were observed. It was concluded that the quality of Sweet Tea is greatly influenced by harvesting time, location and leaf age. Therefore, the artificial cultivation of Sweet Tea is necessary to ensure its optimal quality and suitability for specific applications. Keywords: Sweet active components; Lithocarpus polystachyus Rehd.; Phlorizin; Trilobatin; NMR; Artificial cultivation. Introduction For decades, sugar has been the vital the Sweet Tea [9–13]. Trilobatin, phloridzin and sweetener in human’s daily diet, accounting for a 3-hydroxy-phlorizin were identified as the primary great proportion of the daily energy intake with little bioactive substances in L. polystachyus Rehd. Studies nutritional value [1, 2]. However, the intake of high have indicated that phlorizin restrained the intestinal glucose is closely associated with some adverse absorption of glucose, leading to the normalization of health conditions, such as diabetes and obesity, thus blood glucose and total decrease of glycaemia in it has led to a strong demand for completely or partly animal models [14, 15]. Moreover, trilobatin showed replacing sugar in foods by low-calorie sweeteners [3, inhibitory effect against α-glucosidase and medium 4]. Sugar-free or low calorie foods and beverages are inhibitory effect against α-amylase with less side becoming increasingly popular in many countries [5]. effects. Some studies indicated that trilobatin has Sweeteners mainly include natural sweeteners and potential anti-diabetic effect [16, 17]. Phloretin artificial sweeteners. Natural sweetners had higher possessed anti-tumor activity and can suppress security, better taste than artificial sweeteners, and human leukemia cell growth[18, 19]. Phloridzin and artificial sweeteners had a potential risk of cancer [6]. phloretin provided protection against Many studies have shown that some dihydrochalcone ovariectomy-induced osteopenia under inflammation glycosides are natural low-calorie sweeteners, such as state owing to possible anti-inflammatory activity the phlorizin, trilobatin, 3-hydroxy-phlorizin, and so [20]. In addition, ST was found to be a potential on. At present most of the phlorizin applied to suitable source of sweetener for patients with business is isolated from the root of apple trees, but diabetes. However, many studies showed that the its application has been largely restricted because of content of bioactive components are largely high production cost. influenced by the raw plant material, such as location, climate, harvesting time and freshness. Currently, Lithocarpus polystachyus Rehd., a perennial many studies have determined the content of shrub, is widely distributed in the mountainous areas bioactive compounds using high-performance liquid of southern China. Its leaves, called Sweet Tea (ST), chromatography (HPLC)-based analysis methods [21, can be reaped two or three times a year and have 22, 23]. The aim of the present study was to isolate been used as a source of sweet food and traditional and identify the main sweet compounds in ST. In herb [7, 8]. Studies have showed dihydrochalcones addition, the compounds isolated from ST were used had anti-obesity effect and its content reached 7% in to quantitatively evaluate the quality of ST. The *To whom all correspondence should be addressed. Juan Yang et al., J.Chem.Soc.Pak., Vol. 40, No. 01, 2018 159 influence of location, harvesting time and leaf age for Extraction and isolation the individual flavonoid compound was determined. The air-dried and powdered ST (4,700.0 g) Experimental collected from Yunan in April 2014 was extracted three times with 70% MeOH (10 L×3) at room Chemicals and reagents temperature (25-32 oC) for 3 days each time, then combined, and concentrated MeOH extract (1,145.0 g) The standards of isoquercitrin, quercetin and was partitioned with petroleum ether (25.2 g), EtOAc phloretin were purchased from Weikeqi Standards Co. (612.0 g) and n-BuOH (385.0 g) (each solvent 3×3 L). (Chengdu, China). Quercitrin was from Pufeide The ethyl-acetate fraction (612.0 g) was subjected to Bio-Technology Co. (Chengdu, China). Phlorizin, macroporous resin column chromatography eluted kaempferol-3-O-β-Dglucosided, 3-hydroxy phlorizin with MeOH/H2O [0:100, 10:90, 30:70, 50:50, 70:30, and trilobatin were purified in our laboratory, and the 90:10, and 100:0 (vol/vol) at each solvent 3×4000 method of isolation is described below. Methanol mL, respectively] to gain fractions P1-P9 after used in HPLC analysis was chromatographic grade pooling according to their TLC profiles. Fraction P3 from Xingke Co. (Shanghai, China). Other chemicals (217.2 g) was passed through silica gel column and solvents were analytical grade. chromatography, eluted with CHCl3/MeOH [100:5, Apparatus 100:7.5, 90:10, 85:15, 75:25, and 50:50 (vol/vol), at 9.5, 8.3, 8.0, 9.1, 1.2, 1.2, and 1.4 L, respectively] to 1H-NMR and 13C-NMR spectra were gain fractions P3-1-P3-10. Fraction P3-9 (83.3 g) recorded with Bruker AM-400 spectrometers. passed through a Develosil ODS column Coupling constants were expressed in hertz, and chromatography and eluted with MeOH/H2O [25:75, chemical shifts were given with tetramethylsilane as 30:70, 45:55, 55:45, 65:35, 80:20, and 100:0 internal standard. The HPLC analysis was performed (vol/vol), at 20 L each] to gain fractions on a Agilent Technologies Agilent 1200 liquid P3-9-1-P3-9-9. Fraction P3-9-5 (14.3 g) passed chromatograph equipped with a Agilent UV detector through a silica gel column chromatography, eluted (Agilent Technologies Co. Ltd., Palo Alto, America), with a gradient of CHCl3/Acetone [70:30, 60:40, along with a reverse-phase column (elite kromasil 50:50, 40:60, 30:70 and 20:80 (vol/vol) at 1 L each] C18, 5m , 250 × 4.6mm). Column chromatography to afford fractions P3-9-5-1-P3-9-5-4. Fraction was performed over silica gel (300-400 mesh, P3-9-5-2 (3.0 g) was submitted to Sephadex LH-20 200-300 mesh, 100-200 mesh, 80-100 mesh, Qingdao column chromatography eluted with acetone to yield Haiyang Chemical Co., Ltd., Qingdao, China), compound 2 (368.0 mg). Fraction P3-9-6 (300.0 mg) LiChroprep RP-18 reversed-phase column (40-63 µm, passed through a Sephadex LH-20 column Merck Co., Darmstadt, Germany), Sephadex LH-20 chromatography eluted with MeOH to gain fractions (40-70 µm, Amersham Pharmacia Biotech AB Co., P3-9-6-1-P3-9-6-3. Fraction P3-9-6-2 (118.8 mg) was Uppsala, Sweden), and Macroporous resin D101 subjected to Sephadex LH-20 column (Haiguang chemical co., Ltd., Tianjin, china). chromatography eluted with acetone to offer compound 3 (93.4 mg). Fraction P3-9-3 (28.6 g) was Plant material subjected to silica gel column chromatography eluted The samples of Sweet Tea (ST) used in this with CHCl3/Acetone [60:40, 50:50, 40:60, 35:65, study were collected from the leaves of L. 30:70, and 20:80 (vol/vol) at 9.0, 11.0, 12.0, 16.0, Polystachyus Rehd. in Wenshan Yunnan Province, 10.0, and 7.5 L, respectively] to gain fractions Suiyuan Jiangxi Province and Shaoyan Hunan P3-9-3-1-P3-9-3-12. Fraction P3-9-3-5 (2.1 g) passed Province (Table-1). The leaves of Sweet Tea (ST) through a silica gel column chromatography eluted o were dried at 40 C in an oven, ground to powder with CHCl3/EtOAc [1:15, 1:20, 1:30, 1:40, 1:80, and with a tissue grinder and passed a 1.2-mm size mesh, 1:120 at 500 mL each] to give fractions then stored at 4 oC until analysis. P3-9-3-5-1-P3-9-3-5-8. Fraction P3-9-3-5-5 (218.2 mg) was subjected to C8 column chromatography Table-1: Origins of the collected ST. eluted with MeOH/H2O [10:90, 20:80, 25:75, 30:70 No. Origin Harvesting time Maturity stage and 40:60 (vol/vol) at 1 L each] to attain fractions C1 Wenshan Yunnan November, 2014 Young C2 Wenshan Yunnan November, 2014 Mature P3-9-3-5-5-1-P3-9-3-5-5-6. Fraction P3-9-3-5-5-2 C3 Wenshan Yunnan November, 2014 Old (138.6 mg) passed through a Sephadex LH-20 C4 Wenshan Yunnan April, 2014 Young C5 Wenshan Yunnan April, 2014 Mature column chromatography eluted with MeOH to supply C6 Wenshan Yunnan April, 2014 Old compound 1 (64.6 mg).