(Lithocarpus Polystachyus Rehd.) Based Upon Location, Harvesting Time, Leaf Age

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, 5m , 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). Fraction P3-9-8 (26.0 g) A Suiyuan Jianxi April, 2014 Mature B Shaoyan Hunan April, 2014 Mature passed through a silica gel column chromatography eluted with CHCl3/Acetone [60:40, 50:50, 40:60, Juan Yang et al., J.Chem.Soc.Pak., Vol. 40, No. 01, 2018 160

35:65, 30:70 and 20:80 (vol/vol) at 1.5 L each] to Determination of compounds by RP-HPLC-UV afford fractions P3-9-8-1-P3-9-8-5. Fraction P3-9-8-3 (3.9 g) passed through a Sephadex LH-20 column The HPLC analysis was performed on a chromatography eluted with MeOH to obtain Agilent Technologies Agilent 1200 liquid fractions P3-9-8-3-1-P3-9-8-3-6. Fraction P3-9-8-3-3 chromatograph equipped with a Agilent UV detector (151.2 mg) was separated by HPLC using 19% (Agilent Technologies Co. Ltd., Palo Alto, America), MeCN/H2O as the mobile phase and the flow rate of along with a Kromasil C18 reverse-phase column 2.2 mL/min to obtain compound 4 (86.2 mg). (200×4.6 mm inner diameter, 5 μm) protected with a guard column. The selection of the mobile phases Preparation of Sweet Tea samples for HPLC analysis was done on the basis of previous experiments and modified [20, 22]. The mobile phase was composed The dried powder of L. polystachyus Rehd. of 0.1% glacial acetic aqueous solution (solvent A) leaves (0.3 g) was extracted with 20 mL of aqueous and methanol (solvent B) at a flow rate of 1.0 methanol (30:70, v/v) solution in an ultrasonic bath mL/min. Gradient elution was performed as follows: ( 500 W, 50 kHz) for 30 min. After vacuum filtration, 0−10 min, 35−40% B; 10−35 min, 40−65% B; 35−40 the obtained residue was extracted again with 10 mL min, 65−80% B. The sample injection volume was 10 fresh solvent using the same conditions, and the μL and the column temperature was controlled at 30 extraction of the obtained residue was repeated twice. oC. The detection wavelength was 257 nm for the The filtered solution was concentrated to dryness in eight flavonoids. All solutions were filtered through a vacuo and then transferred to a 25 mL volumetric 0.45 μm membrane filter before direct injecting into flask, next diluted with methanol to given volume. the HPLC system. Typical HPLC chromatogram The sample solutions were filtered through 0.45 μm traces of standards and ST extract samples are shown membrane filter prior to injection for HPLC analysis. in Fig. 1. Three parallel analysis was to avoid false positive.

Fig. 1: RP-HPLC chromatograms at 257 nm showing the flavonoid compounds of ST from cultivars collected in April 2014. (D) Combined standard solution, (A) Jiangxi sweet tea, (B) Hunan sweet tea (C) Yunnan sweet tea. Key to peak identification: (1) 3-hydroxy phlorizin; (2) phlorizin; (3) kaempferol-3-O-β-D-glucoside; (4) trilobatin; (5) isoquercitrin; (6) quercitrin; (7) quercetin; (8) phloretin. Juan Yang et al., J.Chem.Soc.Pak., Vol. 40, No. 01, 2018 161

Results and Discussion J = 12.3 Hz, H-5′′), 3.73 (1H, dd, J = 12.3, 5.0 Hz, H-6′′a), 3.30-3.52 (6H, m, H-2′′, 3′′, 4′′, 6′′b, α), 2.82 (2H, t, J = 13 Identification of sweet compounds 7.6 Hz, H-β). C-NMR (100 MHz, CD3OD) δ (ppm): 206.7 (C=O), 167.5 (C-4′), 165.9 (C-6′), 162.3 (C-2′), Four sweet compounds were isolated from 146.0 (C-4), 144.2 (C-3), 134.7 (C-1), 120.8 (C-6), 116.7 the aqueous methanol extracts of ST by repeated (C-2), 116.3 (C-5), 106.7 (C-1′), 102.0 (C-1′′), 98.4 (C-5′), column chromatography. The chemical structure of 95.4 (C-3′), 78.5 (C-5′′), 78.3 (C-3′′), 74.7 (C-2′′), 71.0 four compounds was determined by spectroscopic (C-4′′), 62.4 (C-6′′), 46.9 (C-α), 31.1 (C-β) [25]. analysis (Table-2 and 3). Phlorizin (2). White powder; C21H24O10; 1 13 • - 1 Table-2: H-NMR and C-NMR signals of the ESI -MS m/z 435 [M - H] ; H-NMR (400 MHz, CD3OD) phlorizin, trilobatin and 3-hydroxyl-phlorizin (in δ (ppm): 7.06 (2H, d, J = 8.4 Hz, H-2, 6), 6.68 (2H, d, J = CD3OD). 8.4 Hz, H-3, 5), 6.18 (1H, d, J = 2.2 Hz, H-5′), 5.96 (1H, phlorizin trilobatin 3-hydroxyl-phlorizin No. d, J = 2.2 Hz, H-3′), 5.04 (1H, d, J = 7.2 Hz, H-1′′), 3.91 δH δC δH δC δH δC C-1 133.9 133.8 134.7 (1H, d, J = 12.2 Hz, H-5′′), 3.71 (1H, dd, J = 12.2, 5.3 Hz, C-2 7.06(d) 130.4 7.06(d) 130.3 6.70(d) 116.7 H-6′′a), 3.30-3.47 (6H, m, H-2′′, 3′′, 4′′, 6′′b, α), 2.87 (2H, C-3 6.68(d) 116.1 6.71(d) 116.1 144.2 t, J = 7.7 Hz, H-β). 13C-NMR (100 MHz, CD OD) δ C-4 156.4 156.5 146.0 3 C-5 6.68(d) 116.1 6.71(d) 116.1 6.66(d) 116.3 (ppm): 206.5 (C=O), 167.6 (C-4′), 165.9 (C-6′), 162.3 C-6 7.06(d) 130.4 7.06(d) 130.3 6.56(dd) 120.8 (C-2′), 156.4 (C-4), 133.9 (C-1), 130.4 (C-2, 6), 116.1 C=O 206.5 207.0 206.7 C-α 47.0 47.6 46.9 (C-3, 5), 106.7 (C-1′), 102.0 (C-1′′), 98.3 (C-5′), 95.4 C-β 2.87(t) 30.8 2.87(t) 31.2 2.82(t) 31.1 (C-3′), 78.5 (C-5′′), 78.4 (C-3′′), 74.7(C-2′′), 71.0 (C-4′′), C-1′ 106.7 106.9 106.7 C-2′ 162.3 165.4 162.3 62.4 (C-6′′), 47.0 (C-α), 30.8 (C-β) [26, 27]. C-3′ 5.96(d) 95.4 6.11(s) 96.4 5.96(d) 95.4 C-4′ 167.6 165.0 167.5 Kaempferol-3-O-β-D-glucoside (3). Yellowish C-5′ 6.18(d) 98.3 6.11(s) 96.4 6.18(d) 98.4 • - 1 C-6′ 165.9 165.4 165.9 powder; C21H20O11; ESI -MS m/z 447 [M - H] ; H-NMR Glc-1 5.04(d) 102.0 4.96(d) 101.1 5.05(d) 102.0 (400 MHz, C5D5N) δ (ppm): 8.45 (2H, d, J = 8.7 Hz, H-2′, Glc-2 74.7 74.6 74.7 Glc-3 78.4 77.9 78.3 6′), 6.72 (1H, d, J = 2.1 Hz, H-8), 6.70 (1H, d, J = 2.1 Hz, Glc-4 71.0 71.1 71.0 H-6), 6.36 (2H, d, J = 8.7 Hz, H-3′, 5′). 13C-NMR (100 Glc-5 3.91(d) 78.5 3.93(d) 78.3 3.91(d) 78.5 Glc-6 62.4 62.3 62.4 MHz, C5D5N) δ (ppm): 178.8 (C-4), 166.0 (C-7), 162.8 (C-5), 161.7 (C-4′), 157.6 (C-9), 157.4 (C-2), 135.1 (C-3), Table-3: 1H-NMR and 13C-NMR signals of the 131.9 (C-2′, 6′), 122.0 (C-1′), 116.1 (C-3′, 5′), 105.3 (C-10), 103.9 (C-1′′), 99.9 (C-6), 94.7 (C-8), 79.1 (C-3′′), kaempferol-3-O-β-D-glucoside (in C5D5N). No. kaempferol-3-O-β-D-glucoside 78.6 (C-2′′), 76.2 (C-5′′), 71.5 (C-4′′), 62.6 (C-6′′) [28]. δH δC C-2 157.4 C-3 135.1 Trilobatin (4). Colorless oily matter; C21H24O10; • - 1 C-4 178.8 ESI -MS m/z 435 [M - H] ; H-NMR (400 MHz, CD3OD) C-5 162.8 δ (ppm): 7.06 (2H, d, J = 8.3 Hz, H-2, 6), 6.71 (2H, d, J = C-6 6.70(d) 99.9 C-7 166.0 8.3 Hz, H-3, 5), 6.11 (2H, s, H-3′, 5′), 4.96 (1H, d, J = 7.2 C-8 6.72(d) 94.7 Hz, H-1′′), 3.93 (1H, d, J = 12.2 Hz, H-5′′), 3.73 (1H, dd, C-9 157.6 C-10 105.3 J = 12.2, 5.2 Hz, H-6′′a), 3.32-3.51 (6H, m, H-2′′, 3′′, 4′′, C-1′ 122.0 6′′b, α), 2.87 (2H, t, J = 8.0 Hz, H-β). 13C-NMR (100 C-2′ 8.45(d) 131.9 C-3′ 6.36(d) 116.1 MHz, CD3OD) δ (ppm): 207.0 (C=O), 165.4 (C-2′, 6′), C-4′ 161.7 165.0 (C-4′), 156.5 (C-4), 133.8 (C-1), 130.3 (C-2, 6), C-5′ 6.36(d) 116.1 116.1 (C-3, 5), 106.9 (C-1′), 101.1 (C-1′′), 96.4 (C-3′, 5′), C-6′ 8.45(d) 131.9 Glc-1 103.9 78.3 (C-5′′), 77.9 (C-3′′), 74.6 (C-2′′), 71.1 (C-4′′), 62.3 Glc-2 78.6 (C-6′′), 47.6 (C-α), 31.2 (C-β) [26]. Glc-3 79.1 Glc-4 71.5 Glc-5 76.2 Quantitative analysis of the compounds Glc-6 62.6 In our HPLC system, 3-hydroxy phlorizin, 3-hydroxyl-phlorizin (1). White powder; isoquercitrin, phlorizin, quercitrin, C H O ; ESI•-MS m/z 451 [M - H]-; 1H-NMR (400 21 24 11 kaempferol-3-O-β-D-glucosided, trilobatin, quercetin, MHz, CD OD) δ (ppm): 6.70 (1H, d, J = 1.9 Hz, H-2), 3 and phloretin eluted within the following retention time 6.66 (1H, d, J = 8.1 Hz, H-5), 6.56 (1H, dd, J = 8.1, 1.9 ranges: 13.08-13.25, 15.76-15.96, 18.25-18.41, Hz, H-6), 6.18 (1H, d, J = 2.2 Hz, H-5′), 5.96 (1H, d, J = 19.74-19.91, 20.23-20.50, 22.27-22.57, 26.46-26.64 and 2.2 Hz, H-3′), 5.05 (1H, d, J = 7.2 Hz, H-1′′), 3.91 (1H, d, 2 29.77-29.97 min, respectively. R values for the Juan Yang et al., J.Chem.Soc.Pak., Vol. 40, No. 01, 2018 162 least-squares regression equations fitted to the standard Hunan. Curiously, the results from phytochemical curves were as follows: 3-hydroxy phlorizin (1) (0.9832), quantifications of ST collected from Hunan showed that it isoquercitrin (5) (0.9997), phlorizin (2) (0.9980), has a higher flavonoids content than that of the previous quercitrin (6) (0.9999), kaempferol-3-O-β-Dglucosided (3) study [21, 29]. Both of phlorizin and trilobatin are known (0.9999), trilobatin (4) (0.9998), quercetin (7) (0.9994) for their anti-diabetic activity and used as natural and phloretin (8) (0.9998). Limits of quantitation were as sweeteners. Owing to numerous other pharmacological follows: 1 (378 ng/mL), 2 (125 ng/mL), 3 (75 ng/mL), 4 properties [16, 17, 30], so they are likely to improve (166 ng/mL), 5 (45 ng/mL), 6 (38 ng/mL), 7 (89 ng/mL) health condition. and 8 (151 ng/mL). The inter- and intra-day precisions were determined by analyzing the mixed standard Effect of seasonal variation and maturation degree on solution five times, respectively. The intra- and inter-day flavonoid constituents accuracy was from 97% to 101%, and the RSDs of samples were lower than 3.2%. The recoveries of The variation of flavonoids content in ST isoquercitrin, quercitrin, kaempferol-3-O-β-D-glucoside, collected from Yunnan during the different maturation quercetin, phloretin, 3-hydroxy phlorizin, phlorizin and stages and different harvesting dates was measured and trilobatin were 98.80 %, 99.12 %, 101.56 %, 98.36 %, compared. The variation of flavonoids content in ST at 101.15 %, 97.95 %, 99.58 % and 98.62 %. Results different harvesting dates was significantly enormous indicated that the HPLC had good precision, accuracy (Table-5). Trilobatin was predominant constituent when and repeatability for determination of the markers in the ST was harvested in November, followed by phlorizin, samples. It was clear that the developed method was 3-hydroxy-phloridzin, kaempferol-3-O-β-D-glucoside, reliable and accurate for measurement of the eight isoquercitrin, phloretin, quercitrin and quercetin. Whereas flavonoids in ST. the phlorizin was predominant constituent in ST harvested in March. The content of trilobatin in ST was Effect of different geographical locations on flavonoids highest, reaching 279.74mg/g. He et al. [29] determined constituent the content of trilobatin in ST from different growth sites. The study indicated that the content of trilobatin was The mature ST cultivated in three different locations 114.24–272.35mg/g. The result was approximately (Table-1) was available for comparison. The results consistent with reported reference. The following result showed that the content of flavonoid compounds was indicated that the flavonoid compounds in ST at different markedly different in Sweet Tea collected in the April maturition degree changed only quantitatively (Tab. 5). 2014 (Table-4). The predominant compound was The total flavonoids concentration was highest in the trilobatin, comprising 24.36 % of dry ST extract in Hunan, early stages of leaf growth, declining as the leaves and 18.38 % of dry ST extract in Jiangxi, whereas the maturing. The result was in agreement with the reported phlorizin was the most abundant compound, comprising reference. Li et al. [31] determined the content of 20.83 % of dry ST extract in Yunnan. Quercetin and dihydrocharcone from Lithocarpus polystarch Rehd., and phloretin were not found in measurable quantities in ST the result indicated that the content of dihydrocharcone gathered in Yunnan and Hunan. 3-hydroxy phlorizin was gradually decreased with the increasing of growth time. not found in measurable quantities in ST collected from

Table-4: Concentration (mg/g dry weight) for flavonoid compounds in ST from different locatins (means ± SD) No. 3-hydroxy phlorizin Isoquercitrin Phlorizin Quercitrin Kaempferol-3-O-β-D-glucoside Trilobatin Quercetin Phloretin C5 42.63±3.16 6.36±0.45 208.29±4.92 5.46±0.28 4.07±0.29 33.59±2.82 ND ND A 16.13±1.20 3.58±0.18 11.58±0.42 0.92±0.18 2.02±0.13 183.84±5.18 0.53±0.13 0.27±0.11 B ND 2.52±0.16 0.61±0.24 1.23±0.14 0.65±0.18 243.67±5.44 ND ND ND - not detected. Results indicate averages ± standard deviations for triplicate analyses.

Table-5: Concentraion (mg/g dry weight) of flavonoid compounds in ST at different stages of maturity and different harvesting time (means±SD) No. 3-hydroxy phlorizin Isoquercitrin Phlorizin Quercitrin Kaempferol-3-O-β-D-glucoside Trilobatin Quercetin Phloretin C1 23.44±1.11 11.29±1.21 22.34±0.92 5.46±0.28 4.07±0.29 279.74±2.82 ND ND C2 18.04±1.08 8.96±0.14 11.44±0.26 1.92±0.21 6.45±0.32 257.52±4.23 0.56±0.15 1.37±0.20 C3 14.71±0.33 7.85±0.22 13.65±0.22 1.27±0.16 5.85±0.20 264.60±10.48 0.30±0.11 1.55±0.21 C4 49.18±3.90 6.36±0.18 208.31±8.45 4.30±0.33 3.87±0.14 30.94±3.66 0.15±0.10 0.44±0.16 C5 42.63±3.16 6.36±0.45 208.29±4.92 5.46±0.28 4.07±0.29 33.60±2.82 ND ND C6 62.81±1.77 4.14±0.16 143.12±3.86 3.38±0.22 1.45±0.11 4.87±1.43 ND ND ND - not detected. Results indicate averages ± standard deviations for triplicate analyses. Juan Yang et al., J.Chem.Soc.Pak., Vol. 40, No. 01, 2018 163

Conclusions identification of flavonoid phloridzin from the crude extract of sweet tea (Lithocarpus The results of this study revealed significant polystachyus Rehd.), Molecules., 12, 552 (2007). differences in the quantitative of flavonoids in ST 8. Y. Zhao, X. Li, X. Zeng, S. Huang, S. Z. Hou collected in Yunnan, Hunan and Jiangxi, and the and X. P. Lai, Characterization of phenolic content of some individual compound varied constituents in Lithocarpus polystachyus, Anal. considerably. The current results confirm the fact that Methods., 6, 1359 (2014). flavonoids content is closely related with the 9. H. Q. Dong, Z. X. Ning, Z. X. Cui, L. Li and L. J. geographical origin of the ST. Moreover, according Yu, Microwave-assisted extraction of flavonoids to the observed compositional changes related to from tender leaves of Lithocarpus polystachyus maturation degree and harvest time of ST, the Rehd., Chin. Soc. Agr. Eng., 23, 213 (2007). quantity of flavonoids was markedly different in ST 10. W. Zhou, G. Shui and Z. Tian, Study on sweet growing in the same location but harvested in components of Hunan sweet tea, Food. Sci., 13, different time or different maturation degree. 17 (1992). 11. K. F. Xiao and X. F. Liao, Isolation and The reported huge fluctuation of the content structural identification of a flavonoid from of individual flavonoid in ST is influenced by leaf Lithocarpus polysachy Rehd., Chem. Ind. Forest. age, location and harvest time. Thus, it should be Prod., 26, 85 (2006). carefully considered when the ST is used to be a 12. X. Li, Y. Zhao, S. Z. Hou, S. Huang, W. Q. source of sweetener for food additives, healthcare Yang and X. P. Lai, Identification of the products, or any other purposes. It can therefore be bioactive components of orally administered concluded that the artificial cultivation of L. Lithocarpus polystachyus Rehd. and their polystachyus Rehd. is necessary to ensure its optimal metabolites in rats by liquid chromatography quality and suitability for specific applications. coupled to LTQ Orbitrap mass spectrometry, J. Chromatogr. B., 962, 37 (2014). Acknowledgements 13. C. J. Zhou, S. Huang, J. Q. Liu, S. Q. Qiu, F. Y. Xie, H. P. Song, Y. S. Li, S. Z. Hou and X. P. This work was supported by the National Lai, Sweet tea leaves extract improves leptin Natural Science Foundation of China (No. 31260399 resistance in diet-induced obese rats, J. and No.31460430), and Yunnan Innovation Grant Ethnopharmacol., 145, 386 (2013). (No. 2011CI078), which are gratefully 14. T. D. Bernonville, S. Guyot, J. P. Paulin, M. acknowledged. Gaucher, L. Loufrani, D. Henrion, S. Derbre, D. Guilet, P. Richomme, J. F. Dat and M. N. Brisset, References Dihydrochalcones: implication in resistance to oxidative stress and bioactivities against 1. A. Bassoli and L. Merlini, Sweeteners | intensive, advanced glycation end-products and Encyclopedia of food sciences and nutrition, 2nd vasoconstriction, Phytochemistry., 71, 443 ed., p. 5688 (2003). (2010). 2. B. Caballero, Dietary sucrose and disease, 15. J. R. L. Ehrenkranz, N. G. Lewis, C. R. Kahn Encyclopedia of human nutrition, 3rd ed., p. 231 and J. Roth, Phloridzin: a review, (2013). Diabetes-Metab. Res., 21, 31 (2005). 3. R. H. Lustig, L. A. Schmidt and C. D. Brindis, 16. H. Q. Dong, M. Li, F. Zhu, F. L. Liu and J. B. The toxic truth about sugar, Nature., 482, 27 Huang, Inhibitory potential of trilobatin from (2012). Lithocarpus polystachyus Rehd against 4. B. M. Popkin and S. J. Nielsen, The Sweetening α-glucosidase and α-amylase linked to type 2 of the World's Diet, Obes. Res., 11, 1325 diabetes, Food. Chem., 130, 261 (2012). (2003 ). 17. Y. Zhang, Z. X. Ning and H. Q. Dong, Inhibitory 5. M. Kroger, K. Meister and R. Kava, Low-calorie potential of trilobatin from Lithocarpus sweeteners and other sugar substitutes: a review polystachyus Rehd against key enzymes linked to of the safety issues, Compr. Rev. Food. Sci. F., 5, type II diabetes and its antioxidant activity, Food. 35 (2006). Sci., 32, 32 (2011). 6. M. R. Weihrauch and V. Diehl, Artiﬁcial 18. M. A. Devi and N. P Das, In vitro effects of sweeteners-do they bear a carcinogenic risk, Ann. natural plant polyphenols on the proliferation of Oncol., 15, 1460 (2004). normal and abnormal human lymphocytes and 7. H. Q. Dong, Z. X. Ning, L. J. Yu, L. Li, L. C. their secretions of interleukin-2, Cancer. Lett., Lin and J. B. Huang, Preparative separation and 69, 191 (1993). Juan Yang et al., J.Chem.Soc.Pak., Vol. 40, No. 01, 2018 164

19. M. Kern, G. Pahlke, K. K. Balavenkatraman, F. 25. H. X. Jin, Y. Zhao, X. P. Lai, J. N. Chen and S. D. Bohmer, and D. Marko, Apple polyphenols Huang, Isolation of chemical constituents of affect protein kinase C activity and the onset of Lithocarpus polystachyus Rehd., Chin. Tradit. apoptosis in human colon carcinoma cells, J. Agr. Pat. Med., 34, 2362 (2012). Food. Chem., 55, 4999 (2007). 26. R. L. Nie, T. Takashi, Z. Jun and T. Osamu, 20. C. Puel, A. Quintin, J. Mathey, C. Obled, M. J. Phlorizin and trilobatin, sweet Davicco and P. Lebecque, Prevention of bone dihydrochalcone-glucosides from leaves of loss by phloridzin, an apple polyphenol, in Lithocarpus litseifolius (hance) Rehd. (Fagaceae), ovariectomized rats under inflammation Agr. Biol. Chem., 46, 1933 (1982). condition, Calcified. Tissue. Int., 77, 311 (2005). 27. X. Y. Xu, H. H. Xie, J Hao, Y. M. Jiang and X. 21. S. H. Li, X. J. Wu, Y. Y. Niu, J. Zhang and J. H. Y. Wei, Flavonoid glycosides from the seeds of Cun, Determination of phloridzin in Lithocarpus Litchi chinensis, J. Agr. Food. Chem., 59, 1205 polystarch Rehd. by HPLC method, Sci. Tech. (2011). Food. Ind., 30, 327 (2009). 28. Q. Xie, W. J. Wang, G. Q. Li, Y. Wang, X. Q. 22. Y. Zhao, H. X. Jin, S. Huang and X. P. Lai, Zhang and W. C. Ye, Chemical constituents Simultaneous Determination of Four Flavonoid from the leaves of Hunteria zeylanica, J. Jinan. Glycosides in Extracts of Folium Lithocarpi Univ.(Nat. Sci.), 34, 122 (2013). Polystachyi by HPLC, Tradit. Chin. Drug. Res. 29. C. N. He, Y. Peng, W. Xiao, Y. L. Hu and P. G. Clin. Pharm., 24, 88 (2013). Xiao, Quick determination of five sweet 23. Y. S. Sun, W. Li and Z. B. Liu, Preparative constituents in Duosuike Tiancha by RSLC, isolation, quantification and antioxidant activity China. J. Chin. Mater. Med., 37, 961 (2012). of dihydrochalcones from Sweet Tea 30. Y. Katsuda, T. Sasase, H. Tadaki, Y. Mera and Y. (Lithocarpus polystachyus Rehd.), J. Motohashi, Contribution of hyperglycemia on Chromatogr. B., 1002, 372 (2015). diabetic complications in obese type 2 diabetic 24. X. Li, Y. Zhao, S. Z. Hou, S. Huang, W. Q. SDT fatty rats: effects of SGLT inhibitor Yang, X. P. Lai and X. Zeng, Identification of phlorizin, Exp. Anim., 64, 161 (2015). the bioactive components of orally administered 31. S. H. Li, X. J. Wu and Y. Y. Niu, Detemination Lithocarpus polystachyus Rehd. and their of dihydrocharcone from Lithocarpus polystarch metabolites in rats by liquid chromatography Rehd., Food. Sci. Tech., 35, 281 (2010). coupled to LTQ Orbitrap mass spectrometry, J. Chromatogr. B., 962, 37 (2015).