采用气质联用技术和高效液相色谱对布朗山古树普洱晒青茶挥发性成分和生物活性成分进行比较分析

Comparison of volatile profiles and bioactive components of sun-dried Pu-erh tea leaves from ancient tea plants on Bulang Mountain measured by GC-MS and HPLC # # 采用气质联用技术和高效液相色谱对布朗山古树普洱晒青茶挥发性成分和生物活性成分进行比较分析 1 2 first-author 1 2 1 2 1 2 1 2 1 2 1 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:yanliang879@163.com †E-mail:yanliang879@163.com yanliang879@163.com College of Tea (Pu’er), West Yunnan University of Applied Sciences, Pu’er 665000, China College of Tea (Pu’er), West Yunnan University of Applied Sciences, Pu’er 665000, China Pu’er Institute of Pu-erh Tea, Pu’er 665000, China Pu’er Institute of Pu-erh Tea, Pu’er 665000, China To explore the volatile profiles and the contents of ten bioactive components (polyphenols and caffeine) of sun-dried Pu-erh tea leaves from ancient tea plants on Bulang Mountain, 17 samples of three tea varieties were analyzed by headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) and high-performance liquid chromatography (HPLC). A total of 75 volatile components were tentatively identified. Laomaner (LME), Laobanzhang (LBZ), and other teas on Bulang Mountain (BL) contained 70, 53, and 71 volatile compounds, respectively. Among the volatile compounds, alcohols (30.2%–45.8%), hydrocarbons (13.7%–17.5%), and ketones (12.4%–23.4%) were qualitatively the most dominant volatile compounds in the different tea varieties. The average content of polyphenol was highest in LME (102.1 mg/g), followed by BL (98.7 mg/g) and LBZ (88.0 mg/g), while caffeine showed the opposite trend, 27.3 mg/g in LME, 33.5 mg/g in BL, and 38.1 mg/g in LBZ. Principal component analysis applied to both the volatile compounds and ten bioactive components showed a poor separation of samples according to varieties, while partial least squares-discriminant analysis (PLS-DA) showed satisfactory discrimination. Thirty-four volatile components and five bioactive compounds were selected as major discriminators (variable importance in projection (VIP) >1) among the tea varieties. These results suggest that chromatographic data combined with multivariate analysis could provide a useful technique to characterize and distinguish the sun-dried Pu-erh tea leaves from ancient tea varieties on Bulang Mountain. 目的 2 为布朗山不同品种古树普洱晒青茶香气物质和活性成分(多酚类和咖啡碱)深入研究提供理论依据. 创新点 2 首次采用气质联用技术(GC-MS)和高效液相色谱(HPLC)结合化学计量学对布朗山不同品种的古树普洱晒青茶进行研究,探究不同品种古树晒青茶成分之间的差异性. 方法 2 采用顶空固相微萃取法(HS-SPME)结合GC-MS对香气成分进行鉴定;采用70%甲醇溶液对活性成分进行富集,使用HPLC进行分析;结合主成分分析(PCA)和偏最小二乘判别分析(PLS-DA)对不同品种的古树普洱晒青茶进行区分并对区分起主要作用的物质进行筛选. 结论 2 三个品种的古树普洱晒青茶(老曼峨、老班章和布朗)香气成分组成及含量差异较大,但主要成分均为醇类、碳氢类和酮类化合物.老曼峨中多酚类化合物含量较高,其次为布朗和老班章,相比于多酚类,咖啡碱含量表现出了相反的趋势.相比于PCA分析,PLS-DA分析对三个品种的古树普洱晒青茶均显示出了较好的区分效果.变量投影重要性准则(VIP)分析结果显示34种香气和5种生物活性成分对不同品种的古树普洱晒青茶区分贡献较大. Sun-dried Pu-erh tea Ancient tea plant Bulang Mountain Volatile compound Bioactive component 普洱晒青茶 古茶树 布朗山 挥发性成分 生物活性成分 Introduction 1 Bulang Mountain is located in Menghai County, Xishuangbanna Dai Autonomous Prefecture, Yunnan Province, China, near the Chinese–Burmese border. Bulang Mountain, with an area of 1016.34 km 2 , is a famous Pu-erh tea-producing area and is one of the best preserved areas of ancient tea plants. There are 600 ha of gardens containing the Laomaner (LME), Laobanzhang (LBZ), and other ancient tea plants (BL) of the area. The tea plants are distributed mainly at altitudes of 1300–1900 m (Tao et al., 2013 2013 ). The teas in Bulang Mountain are characterized by their unique aroma, similar to the sweet smell of malt sugar. They are yellow or green and have an immediate sweet taste followed by mellow and bitter tastes. They are very astringent (Xia et al., 2012 2012 ). The plant stems are strong and bear numerous hairs. As a valuable resource, the ancient teas in Bulang Mountain have been consistently pursued by tea traders and Pu-erh tea enthusiasts worldwide. Food flavor mainly includes taste and aroma. Non-volatile compounds usually contribute to taste, while volatile components contribute to aroma (Lin et al., 2012 2012 ; Du et al., 2014 2014 ). The volatile components of sun-dried Pu-erh tea leaves from ancient tea plants are similar to those of common Pu-erh green or Pu-erh raw tea leaves and include mainly alcohols, hydrocarbons, aldehydes, ketones, and esters (Lv et al., 2015 2015 ). However, the relative volatile composition, concentrations, and perception thresholds of individual components give sun-dried Pu-erh tea leaves of these ancient tea plants their distinctive aroma. The analysis of the volatile components of Pu-erh green teas has been used for various purposes including distinguishing between teas from different mountains (Wu et al., 2016 2016 ) and differentiating between varieties and quality grades (Lv et al., 2015 2015 ). The non-volatile components of the tea leaves include polyphenols, alkaloids, polysaccharides, free amino acids, and proteins. Among these, polyphenol and alkaloid bioactive components have been well studied, including gallic acid, (+)-catechin, (−)-gallocatechin, (−)-catechin gallate, (−)-epigallocatechin, (−)-epicatechin gallate, (−)-epicatechin, (−)-epigallocatechin gallate, caffeine, and (−)-gallocatechin gallate (Fan et al., 2017 2017 ). Up to date, scientific information on volatile constituents, polyphenols, and alkaloids of different varieties of Pu-erh tea from ancient tea plants on Bulang Mountain is rather scarce (Cai et al., 2014 2014 ). Therefore, an in-depth study is necessary to explore their volatile profiles and bioactive component characteristics. The aim of this work was to chemically characterize the volatile components and ten bioactive components of Pu-erh tea leaves from ancient tea plants on Bulang Mountain using headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) and high-performance liquid chromatography (HPLC). Principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) were used to investigate the relationships between the samples. To our knowledge, no similar publications are available in the literature. Materials and methods 1 Samples and chemicals 2 Seventeen sun-dried Pu-erh tea samples belong to three tea varieties, including five LME, six LBZ, and six BL samples (Table 1 1 ), which were identified by the Pu’er Institute of Pu-erh tea (Pu’er, Yunnan, China). Those samples were harvested in 2017 and kept in a dry environment. The samples were ground to pass through a 30–60 mesh and sealed before use. Sodium chloride (99.5%, chromatographic grade, the same below), linalool (98.0%), α-terpineol (98.0%), geraniol (99.0%), nerol (99.0%), α-ionone (92.0%), β-ionone (97.0%), ( E )-nerolidol (≥85.0%), methyl salicylate (98.0%), and phytol (95.0%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). A homologous series of n -alkanes solutions (C 8 ‒C 32 ) was purchased from AccuStandard (USA) and used to calculate the retention index (RI) of each compound. Gallic acid, (+)-catechin, (−)-gallocatechin, (−)-catechin gallate, (−)-epigallocatechin, (−)-epicatechin, (−)-epicatechin gallate, (−)-epigallocatechin gallate, (−)-gallocatechin gallate, and caffeine were purchased from the Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The purity of all these standards was above 95%. Acetonitrile, methanol, and phosphoric acid (HPLC grade) were purchased from Macklin (Shanghai, China). Ultra-pure water was obtained from a Milli-Q purification system (Millipore, Bedford, USA). Sample ID Name Region Harvest time  LME1–LME5 Laomaner tea Bulang Mountain Apr. 2017  LBZ1–LBZ6 Laobanzhang tea Bulang Mountain Apr. 2017  BL1–BL6 Other sun-dried Pu-erh tea of ancient tea plants Bulang Mountain Apr. 2017 HS-SPME of samples 2 SPME is an equilibrium technique. Many experimental parameters affect this equilibrium. To ensure good-quality SPME extraction, optimization was needed (He et al., 2016 2016 ). In this study, the effects of major factors, such as extraction fiber, extraction temperature, extraction time, and the amount of water, on extraction efficiency were investigated comprehensively before the experiment. Through comparison of the total peak area, the optimal extraction process was as follows: 4.0 g of tea sample was transferred into a 100-mL SPME headspace vial, following the addition of 4.0 g of sodium chloride and 16 mL of water, and then the vial was sealed using a polytetrafluoroethylene (PTFA) septum. The vial was then placed on a magnetic stirring apparatus at 300 r/min and equilibrated at 60 °C for 5 min. A 65-µm polydimethylsiloxane (PDMS)/divinylbenzene (DVB) fiber (Supelco Inc., Bellefonte, USA) was exposed to the headspace over the sample for 60 min. After extraction, the fiber was exposed in the GC-MS injector at 250 °C for 4 min and then the headspace was immediately analyzed by GC-MS. Volatile component analysis by GC-MS 2 Volatile components were analyzed according to the method described by Lv et al. ( 2015 2015 ) with some modifications. After the extraction procedure, the SPME device was removed from the SPME vial and sent for GC analysis. An Agilent 7890A GC directly interfaced with an Agilent 5975C mass selective detector quadrupole MS instrument (Agilent Technologies, Palo Alto, CA, USA) was used. An HP-5MS capillary column (30 m×0.25 mm i.d., 0.25 µm film thickness) from Agilent Technologies was used. Experimental conditions of the GC procedure were as follows: the initial temperature was 50 °C, increased to 150 °C at a rate of 2 °C/min, and then ramped at 10 °C/min to 250 °C, maintained for 5 min. The total GC runtime was 65 min. The temperature of the sample injection port was 250 °C and the injection mode was splitless. The carrier gas (He, percentage purity > 99.9%) was in constant-flow mode, with a flow velocity of 0.8 mL/min. The MS was operated in an electron-impact mode at 70 eV. The temperatures of the interface (280 °C), ion source (230 °C) and quadrupole (150 °C) were all at default settings for GC-MS. The acquisition mode was full-scan (from 35 to 350 atomic mass units (aum)), and the solvent delay time was 3.0 min. The volatile compounds were identified as previously described (Wang et al., 2017 2017 ), and peaks were deconvoluted using AMDIS software (NIST, USA). Identification was based on mass matching with the NIST 11.L and WILEY 07 mass spectra databases and/or comparison against standards where available. The RIs of target components were obtained using n -alkanes (C 8 –C 32 ) under the same GC-MS procedure. The calculated RI was compared with the reported RI. The percentage amount of target compound was calculated by the percentage of peak area of target compound relative to the total peak area of all compounds. Polyphenols and alkaloids analyzed by HPLC 2 The HPLC analysis was based on a slightly modified method described by Wang et al. ( 2016a 2016a ). Each sample (about 0.2 g) was placed into a 10-mL centrifuge tube, and 4 mL of preheated 70% methanol was added. The sample was extracted for 10 min in a 70 °C water bath, and then the extract was centrifuged at 3500 r/min for 10 min. The supernatant was transferred into a 50-mL volumetric flask, and the sample was extracted again using the above process. Finally, the extracts were combined and made up to 50 mL with ultrapure water. The solution was filtered through a 0.45-µm membrane prior to injection into the HPLC system. The polyphenols and alkaloids were analyzed using an Agilent 1260 series HPLC system, including a G1311C quaternionic pump, a G1329B autosampler, a G1316A thermostatted column compartment, and a G1315D DAD detector (Agilent Technologies) to acquire quantitative chromatograms. A Phenomenex Synergi Hydro-RP 80A-C18 column (4 μm, 4.6 mm×250 mm) was used. Acetonitrile (solvent A) and 0.05% phosphoric acid aqueous solution (solvent B) were used as the mobile phase at a flow rate of 1 mL/min. The gradient program was set as follows: 0–50 min, linear gradient 2%–25% A; 50–55 min, linear gradient 25%–2% A. The column temperature was 30 °C and the injection volume was 10 μL. Nine polyphenol compounds and caffeine were simultaneously detected at 210 nm. Each target compound was identified by comparing its retention time with that of a standard substance. All samples were tested in triplicate. Statistical analysis 2 PCA and PLS-DA were performed using XLSTAT v. 2016 (Addinsoft, New York, NY, USA) and SIMCA-P 12 software (Umetrics, Umea, Sweden), respectively, to investigate the relationships among the 17 tea samples. Duncan’s multiple range tests were used for the significance of differences ( P ≤0.05) among the tea samples using SPSS statistics 17.0 software (SPSS Inc., Chicago, IL, USA). Results and discussion 1 Recovery and repeatability of the HS-SPME method 2 We investigated the intra-day (five replicates within one day) and inter-day repeatability (one replicate on each of five consecutive days) of nine major compounds selected, to examine the repeatability and accuracy of the established HS-SPME-GC-MS method. To investigate recovery rates, two levels (2 and 8 µg) of mixed standard were added before and after HS-SPME. The repeatability and recovery rates of the HS-SPME method are presented in Table 2 2 . The HS-SPME method showed satisfactory repeatability. The intra-day relative standard deviation (RSD) ranged from 3.36% to 6.71%. As an example, detailed total ion chromatograms and peak areas of the nine compounds are shown in Fig. S1 S1 and Table S1 S1 , respectively. The inter-day RSD ranged from 4.19% to 7.04%. Satisfactory accuracy was achieved, with recoveries of low-level compounds ranging from 72.7% to 93.6% and recoveries of high-level compounds ranging from 70.6% to 106.4%. These results indicate that this technique is suitable for the analysis of volatile compounds of Pu-erh tea leaves. RI: retention index 2 Compound 2 RI 2 Intra-day repeatability (%) ( n =5) 2 Inter-day repeatability (%) ( n =5) 2 Recovery (%) Spiked 2 μg ( n =3) Spiked 8 μg ( n =3) Linalool 1101 4.81 5.63 85.34 88.79 α-Terpineol 1192 4.07 4.19 79.37 77.15 Methyl salicylate 1195 6.47 6.68 93.62 89.54 Nerol 1230 3.36 5.35 87.38 90.12 Geraniol 1256 5.57 6.18 92.16 106.39 α-Ionone 1429 4.12 5.31 90.29 88.74 β-Ionone 1487 4.75 4.29 79.36 83.55 ( E )-Nerolidol 1569 6.71 7.04 72.69 70.57 Phytol 2116 6.04 6.84 88.42 84.69 Volatile component analysis 2 To investigate the aroma characteristics, HS-SPME-GC-MS was used for detection and identification of the volatile components of the tested tea samples. The representative total ion chromatograms of Pu-erh tea leaves from the three varieties of ancient trees are shown in Fig. 1 1 . The relative contents of identified compounds are shown in Table S2 S2 . The average contents of identified compounds of the same kind of tea samples as well as their standard deviation are summarized in Table 3 3 . 1 The calculated retention index (RI) on HP-5MS. 2 The RI values found in the literature (on HP-5MS), which were provided by the NIST Chemistry WebBook ( http://webbook.nist.gov/chemistry http://webbook.nist.gov/chemistry ). 3 The content of volatile compounds is represented as mean value±standard deviation (SD), and different letters indicate significant differences ( P < 0.05, ANOVA, Duncan’s multiple range test). 4 Odor descriptions of odor-active compounds identified in the literature. LME: Laomaner; LBZ: Laobanzhang; BL: other ancient tea plants on Bulang Mountain; ID: identification; MS: mass spectrometry; STD: standard. References: 1, Du et al., 2014 2014 ; 2, Xu et al., 2015 2015 ; 3, He et al., 2016 2016 ; 4, Wang et al., 2017 2017 ; 5, Burdock, 2010 2010 ; 6, Zhu et al., 2008 2008 ; 7, Wang et al., 2008 2008 ; 8, Lv et al., 2012 2012 ; 9, Schuh and Schieberle, 2006 2006 ; 10, Kumazawa and Masuda, 2002 2002 2 Compound 2 ID 2 RI 1 2 RI 2 3 Average relative percentage content (%) 3 2 Odor note 4 2 Reference LME LBZ BL 1-Octen-3-ol MS, RI 982 986 0.782±0.371 a 0.476±0.532 a 0.880±0.849 a Mushroom 1, 2 β-Myrcene MS, RI 991 992 0.658±0.084 a 0.315±0.489 a 0.299±0.329 a Sweet, floral 2 d-Limonene MS, RI 1028 1030 1.807±0.495 a 0.630±0.525 b 1.052±0.568 b Lemon-like 3, 4 ( Z )-Ocimene MS, RI 1037 1039 0.526±0.089 a 0.510±0.399 a 0.329±0.267 a Herbaceous 5 ( Z )-2-Octen-1-ol MS, RI 1062 1066 0.455±0.324 a 0.000±0.000 a 0.296±0.508 a Fatty 5 1-Octanol MS, RI 1064 1068 0.198±0.275 ab 0.000±0.000 b 0.584±0.557ʏacһ Orange, rose 5 Linaloloxide I MS, RI 1068 1062 0.699±0.962 a 0.256±0.292 a 0.954±1.082 a Floral, woody 1, 4 Linaloloxide II MS, RI 1072 1074 1.660±1.435 ab 0.766±0.945 b 2.561±1.351ʏacһ Floral, woody 1, 4 Linalool MS, RI, STD 1101 1106 16.749±3.678 a 12.500±4.511 a 17.014±4.681 a Floral, woody, fruity 1, 4 Hotrienol MS, RI 1106 1109 2.041±0.377 a 0.510±0.576 b 1.937±1.277 a Musty 2, 6 Phenylethyl alcohol MS, RI 1118 1114 0.071±0.158 a 0.000±0.000 a 0.091±0.143 a Honey, spice, rose, lilac 1, 4 l-Menthone MS, RI 1160 1166 0.592±0.223 a 0.000±0.000 b 0.460±0.275 a Mint 5 Benzyl acetate MS, RI 1165 1165 1.488±0.588 a 0.617±0.833 b 1.638±0.536 a Jasmine-like 7 Linaloloxide III MS, RI 1169 1171 2.857±0.447 a 2.280±1.746 a 3.329±1.106 a Floral, woody 2, 8 (−)-4-Terpineol MS, RI 1174 1175 0.572±0.433 a 0.000±0.000 b 0.325±0.310 ab Fruity, sweet 3 α-Terpineol MS, RI, STD 1192 1190 7.018±1.686 a 5.903±1.414 a 6.430±1.140 a Lilac-like 1, 3 Methyl salicylate MS, RI, STD 1195 1193 0.806±0.404 a 0.217±0.531 a 0.895±0.696 a Mint, wintergreen-like 4, 7 Estragole MS, RI 1199 1196 0.469±0.291 a 0.000±0.000 b 0.305±0.197 a Anise-like 1 Safranal MS, RI 1207 1205 0.273±0.261 a 0.000±0.000 b 0.257±0.216 a Herbaceous, sweet 3, 4 β-Cyclocitral MS, RI 1213 1216 0.268±0.406 a 0.452±0.518 a 0.279±0.488 a Floral, fruity 1, 4 Nerol MS, RI, STD 1230 1232 2.068±0.411 a 0.303±0.340 b 1.825±1.009 a Rose-like, sweet 1, 3, 4 5-Methyl-isothiazole MS, RI 1247 0.456±0.481 a 0.451±0.659 a 0.822±0.311 a Unpleasant odor 5 Geraniol MS, RI, STD 1256 1258 4.879±1.012 a 2.973±1.199 a 4.249±2.247 a Rose, geranium 1, 7 Nonanoic acid MS, RI 1278 1272 1.329±0.373 a 0.919±0.566 a 1.359±0.685 a Fatty 5 2-Methyl-naphthalene MS, RI 1298 1299 0.477±0.342 a 0.359±0.565 a 0.083±0.204 a Herbal-like 2 Indole MS, RI 1303 1300 0.315±0.373 a 0.805±1.033 a 0.489±0.506 a Unpleasant flavor 1, 3 1-Methyl-naphthalene MS, RI 1312 1315 0.162±0.268 a 0.000±0.000 a 0.343±0.386 a Unpleasant flavor 3 Geranic acid MS, RI 1354 1355 0.180±0.246 a 0.000±0.000 a 0.083±0.203 a Floral, sweet 5 3-Methyl-tridecane MS, RI 1372 1369 0.190±0.268 a 0.259±0.315 a 0.000±0.000 a 3,5-Dimethyldodecane MS, RI 1373 0.216±0.484 a 0.612±0.650 a 0.846±1.578 a ( Z )-3-Hexenyl hexanoate MS, RI 1381 1381 0.306±0.183 a 0.566±0.558 a 0.339±0.314 a Fruity, green 3, 6 β-Damascenone MS, RI 1385 1388 0.202±0.312 a 0.000±0.000 a 0.065±0.160 a Apple, rose, honey 6, 9 Hexyl hexanoate MS, RI 1386 1385 0.000±0.000 a 0.000±0.000 a 0.143±0.222 a Herbaceous-like 5 ( E )-2-Hexenyl hexanoate MS, RI 1392 1391 0.000±0.000 a 0.000±0.000 a 0.072±0.115 a Fruity, green 5 ( Z )-Jasmone MS, RI 1397 1396 0.000±0.000 a 1.744±2.126 b 0.000±0.000 a Fruity, jasmine 10 Cyclododecanone MS, RI 1407 0.000±0.000 a 0.000±0.000 a 0.312±0.353 b β-Caryophyllene MS, RI 1418 1417 0.475±0.318 a 0.724±0.679 a 0.558±0.327 a Clove-like 3, 4 α-Ionone MS, RI, STD 1429 1427 0.739±0.189 a 2.056±0.313 ab 3.326±3.035 b Wood, violet 1, 4 Geranyl acetone MS, RI 1454 1457 2.065±1.174 a 4.074±1.810 b 3.288±1.160 ab Magnolia, green, fruity 1, 4 ( Z )-β-Farnesene MS, RI 1458 1457 0.158±0.220 a 0.145±0.355 a 0.330±0.590 a Floral, green 5 Dehydro-β-ionone MS, RI 1484 1485 1.460±2.359 a 0.658±0.561 a 0.241±0.209 a Woody, fruity 5 β-Ionone MS, RI, STD 1487 1485 2.628±1.265 a 7.373±1.004 b 1.709±1.880 a Woody, fruity 1, 9 α-Farnesene MS, RI 1507 1510 0.489±0.365 a 0.963±0.897 a 0.690±0.404 a Fruity 3, 4 Butylated hydroxytoluene MS, RI 1516 1518 0.695±0.673 a 1.791±2.401 a 1.229±0.767 a Faint musty 1 α-Amorphene MS, RI 1517 1516 0.038±0.085 a 0.000±0.000 a 0.048±0.119 a 5-Pentylresorcinol MS, RI 1531 2.667±1.786 ab 1.133±2.776 a 4.519±2.555 b Dihydroactinidiolide MS, RI 1539 1535 1.213±0.618 a 3.180±0.988 b 1.306±0.360 a Coumarin, musky 3, 8 α-Calacorene MS, RI 1543 1542 0.502±0.403 a 0.382±0.603 a 0.361±0.572 a Cyclopentadecane MS, RI 1549 0.279±0.385 a 0.242±0.594 a 0.103±0.253 a 4-Methyl-pentadecane MS, RI 1559 0.113±0.253 a 0.283±0.467 a 0.741±0.197 b E -Nerolidol MS, RI, STD 1569 1562 1.519±1.226 a 1.591±1.824 a 2.221±1.364 a Rose-like 3, 8 3-Methyl-pentadecane MS, RI 1571 1570 0.293±0.656 a 1.348±1.151 a 0.402±0.632 a Farnesyl acetaldehyde MS, RI 1580 0.318±0.712 a 0.000±0.000 a 0.598±0.964 a Diethyl phthalate MS, RI 1602 1603 0.040±0.089 a 0.000±0.000 a 0.000±0.000 a Cedrol MS, RI 1605 1607 0.208±0.466 a 0.000±0.000 a 0.639±0.992 a Woody 1 Benzophenone MS, RI 1621 1625 0.616±0.858 a 0.248±0.385 a 0.079±0.195 a Rose-like 2 τ-Muurolol MS, RI 1643 1648 0.000±0.000 a 0.000±0.000 a 0.255±0.625 a α-Cadinol MS, RI 1658 1663 0.585±0.448 a 0.000±0.000 b 0.216±0.529 ab Spicy 5 2,2',5,5'-Tetramethyl-1,1'-biphenyl MS, RI 1665 1668 2.900±1.532 a 3.727±1.345 a 2.006±1.823 a 4'-Benzylacetophenone MS, RI 1697 1.549±0.717 a 1.911±1.078 a 0.986±0.838 a 2,6,10,14-Tetramethyl-pentadecane MS, RI 1705 1703 2.443±1.545 a 2.569±1.327 a 1.573±1.396 a 3-Methyl-heptadecane MS, RI 1771 1771 0.696±0.559 a 0.161±0.396 a 0.176±0.277 a 2,6,10,14-Tetramethyl-hexadecane MS, RI 1808 1810 1.973±0.693 a 1.553±1.366 a 1.132±0.629 a 3-Octadecyne MS, RI 1833 2.158±1.482 a 2.734±3.472 a 2.595±2.478 a 6,10,14-Trimethyl-2-pentadecanone MS, RI 1839 1838 2.515±1.094 a 5.295±2.900 a 3.307±1.684 a Floral, jasmine 1 Caffeine MS, RI 1842 1842 2.349±1.564 ab 4.832±1.736 a 2.117±2.371 b Diisobutyl phthalate MS, RI 1869 1871 1.363±1.326 a 1.145±0.974 a 0.319±0.501 a Methyl palmitate MS, RI 1927 1928 0.901±0.600 a 1.066±0.830 a 0.704±0.372 a Ester-like 5 Isophytol MS, RI 1947 1948 1.049±1.442 a 0.000±0.000 b 0.359±0.166 ab Floral, herbal, green 1 n Hexadecanoic acid MS, RI 1965 1964 1.997±0.987 a 1.661±0.969 a 1.326±0.890 a Dibutyl phthalate MS, RI 1969 1965 0.790±0.280 a 2.165±0.960 a 1.326±1.696 a Methyl linoleate MS, RI 2095 2092 0.091±0.127 a 0.245±0.267 a 0.147±0.132 a Methyl linolenate MS, RI 2098 2101 0.030±0.068 a 0.000±0.000 a 0.156±0.116 b Phytol MS, RI, STD 2116 2119 1.449±0.529 a 2.614±3.154 a 1.585±0.993 a Floral, balsam 1, 4 Oleic acid MS, RI 2143 2140 0.109±0.244 a 0.000±0.000 a 0.000±0.000 a Fresh cut grass like 5 4 Alcohols 44.866±6.566 a 30.177±14.142 b 45.757±11.229 a 4 Hydrocarbons 16.560±2.444 a 17.523±6.163 a 13.672±4.707 a 4 Ketones 12.369±1.426 a 23.363±6.380 b 13.777±3.705 a 4 Esters 7.032±1.351 a 9.204±1.768 b 7.048±1.302 a 4 Acids 3.616±1.367 a 2.580±0.976 a 2.768±0.373 a 4 Nitrogen compounds 3.121±2.045 a 6.089±2.766 a 3.429±2.377 a 4 Aldehydes 0.860±0.665 a 0.452±0.518 a 1.135±1.364 a 4 Phenols 3.362±2.419 a 2.924±3.172 a 5.748±2.934 a 4 Methoxy-phenolic compounds 0.469±0.291 a 0.000±0.000 b 0.305±0.197 a Among 75 volatile components tentatively identified according to their mass spectra and RI, 70, 53, and 71 volatile compounds were identified in LME, LBZ, and BL, respectively. These compounds included alcohols, hydrocarbons, ketones, esters, acids, nitrogen compounds, aldehydes, phenols, and methoxy-phenolic compounds. Nineteen alcohols were identified in all tea samples. The relative content of total alcohols ranged from 30.2% to 45.8%, the lowest in LBZ and the highest in BL. Linalool, α-terpineol, geraniol, linalool oxide, hotrienol, phytol, ( E )-nerolidol, and nerol were the most abundant alcohols. These principal volatile compounds found in green teas contribute greatly to the flavor of the tea. Linalool has been identified as one of the key aroma compounds in Pu-erh raw tea, green tea, black tea, and tea infusions, and contributes citrus and floral aroma notes. α-Terpineol provides “lilac-like” and “sweet” aroma notes. Nerol, geraniol, and ( E )-nerolidol provide “rose-like” aroma notes. Geraniol is an extremely potent volatile with a threshold of 10 µg/kg, and is present at trace levels. Geraniol has been identified as the key component of black tea, greatly contributing to the tea flavor (Shi et al., 2014 2014 ). The results were consistent with those of Dianhong tea (Wang et al., 2017 2017 ). ( E )-Nerolidol is a sesquiterpene, which exists in many teas as a main aroma compound, especially oolong tea, at relatively high content, and can be regarded as one of the key aroma components for the high-quality flavor of oolong tea (Wang et al., 2016b 2016b ). Linalool oxides have a “floral” and “woody” odor (Alasalvar et al., 2012 2012 ). Hotrienol adds a “musty” aroma note and is one of the major compounds in oolong tea (Pripdeevech and Machan, 2011 2011 ). Phytol has a “floral” and “balsam” odor and is a volatile compound common in many plants (Byju et al., 2013 2013 ). Twenty hydrocarbon compounds, including saturated and unsaturated hydrocarbons, were identified in tea samples. Saturated hydrocarbons usually have low odor notes, whereas unsaturated hydrocarbons have high odor notes and contribute more to the overall flavor of tea (Wang et al., 2016b 2016b ). Unsaturated hydrocarbons, such as D -limonene, are regarded as the prominent components of green tea volatile and are described as having “lemon-like” aroma notes (He et al., 2016 2016 ). Moreover, another important aroma compound ( Z )-ocimene offers a strong sense of “warm herbaceous” aroma. β-Myrcene has “sweet” and “floral” aroma notes. β-Caryophyllene has a “woody-spicy” and “clove-like” aroma. α-Farnesene provides “fruity” aroma and has been reported as the main flavor component of oolong tea (Lin et al., 2013 2013 ). Eleven ketones were detected in all tea samples, with β-ionone, 6,10,14-trimethyl-2-pentadecanone, geranyl acetone, dehydro-β-ionone, and α-ionone predominant. β-Ionone has pleasant violet fragrance and complex woody and fruity aroma notes. It has been reported as the dominant aroma compound in various teas (Gulati and Ravindranath, 1996 1996 ). 6,10, 14-Trimethyl-2-pentadecanone was described as having a “floral” and “jasmine” odor in Kangra tea and Pu-erh tea, and was found in all tea leaf samples. Geranyl acetone plays an important role in the aroma of Pu-erh and Dianhong teas (Xu et al., 2015 2015 ; Wang et al., 2017 2017 ). Dehydro-β-ionone has “woody” and “fruity” aroma notes. α-Ionone was detected in all tea samples and has been regarded as an important aroma compound in infusions of Dianhong tea (Wang et al., 2017 2017 ). Twelve esters were identified in tea samples, of which dihydroactinidiolide, benzyl acetate, methyl salicylate, ( Z )-3-hexenyl hexanoate, diisobutyl phthalate, methyl palmitate, and dibutyl phthalate were predominant. Dihydroactinidiolide in Pu-erh tea was described as “smelling like coumarin” and “musky”, and from Eleocharis coloradoensis is known to be a potent growth inhibitor (Stevens and Merrill, 1981 1981 ). Benzyl acetate provides “jasmine-like” aroma notes. Methyl salicylate has been reported in various teas and is recognized as an important compound for the formation of overall tea aroma (Lv et al., 2014 2014 ). ( Z )-3-Hexenyl hexanoate was previously reported in oolong tea (Kawakami et al., 1995 1995 ). Diisobutyl phthalate and dibutyl phthalate were reported in Pu-erh tea (Du et al., 2016 2016 ). Methyl palmitate is produced from the esterification reaction of hexadecanoic acid and was detected in rooibos tea (Kawakami et al., 1993 1993 ). Acids, namely nonanoic acid, n -hexadecanoic acid, geranic acid, and oleic acid, were detected in all tea samples. Branched-chain acids are usually generated from the degradation of branched-chain amino acids, whereas straight-chain acids are generated from the degradation of fatty acids. Nonanoic acid provides “fatty” aroma notes, and was once identified in Pu-erh tea (He et al., 2016 2016 ), whereas oleic acid provides an aroma like fresh cut grass. Geranic acid gives “floral” and “sweet” aroma notes. The nitrogen compounds in the tea samples were 5-methylisothiazole, indole, and caffeine. 5-Methylisothiazole and indole have an unpleasant odor, and caffeine affects mainly the taste characteristics of the tea. Aldehydes, including safranal, β-cyclocitral, and farnesyl acetaldehyde, were identified in the tea samples. Safranal provides a “herbaceous” and “sweet” aroma note and has been reported as a character aroma in infusions of Zijuan tea (He et al., 2016 2016 ). β-Cyclocitral has “floral” and “fruity” aroma notes, which is identified as an major odorant contributing to the aroma of Zijuan and Pu-erh green teas (He et al., 2016 2016 ). Farnesyl acetaldehyde was first reported in Pu-erh green tea. Aldehydes usually have lower odor thresholds than their homologous alcohols. Two phenols, namely 5-pentylresorcinol and butylated hydroxytoluene, were detected in all samples. These compounds were previously identified in various tea samples (Kumazawa and Masuda, 2002 2002 ; Yao et al., 2005 2005 ). Estragole is a methoxyphenolic compound found in tea samples and was previously reported in ripened Pu-erh tea leaves (Du et al., 2014 2014 ). Polyphenols and alkaloids 2 Comparisons of the polyphenols and alkaloids identified in the tea leaf samples are reported in Tables 4 4 and S3. The total polyphenol content ranged from 84.5 to 111.4 mg/g in LME, from 79.5 to 98.4 mg/g in LBZ, and from 93.2 to 104.8 mg/g in other sun-dried Pu-erh tea leaves (BL) from the ancient tea plants. The average content of caffeine was higher in LBZ teas (range, 28.9–43.6 mg/g; mean, 38.1 mg/g) than in BL (range, 24.8–42.2 mg/g; mean, 33.5 mg/g) and LME (range, 23.6–35.2 mg/g; mean, 27.3 mg/g) teas. The possible reason is differences among the varieties of the ancient tea plants. GA: gallic acid; GC: (−)-gallocatechin; EGC: (−)-epigallocatechin; C: (+)-catechin; EC: (−)-epicatechin; EGCG: (−)-epigallocatechin gallate; GCG: (−)-gallocatechin gallate; ECG: (−)-epicatechin gallate; CG: (−)-catechin gallate; CAF: caffeine. Different letters indicate significant differences ( P < 0.05, ANOVA, Duncan’s multiple range test) 3 Compound 8 Polyphenol or alkaloid content (mg/g) 2 LME 2 LBZ 2 BL Mean Range Mean Range Mean Range 9 Polyphenols  GA 8.604 a 8.074–9.140 7.405 b 6.735–7.921 8.546 a 7.218–9.243  GC 3.858 a 3.151–4.368 2.670 b 1.863–3.523 2.785 b 2.305–3.054  EGC 7.221 a 6.058–9.271 4.088 b 2.257–5.483 5.930 a 4.807–7.344  C 5.630 a 3.864–7.940 7.049 a 5.401–10.064 5.720 a 4.494–8.339  EC 16.063 a 11.467–22.540 16.464 a 12.546–21.420 16.141 a 11.843–19.060  EGCG 25.899 a 22.878–30.573 16.462 b 12.870–21.231 23.912 a 20.645–26.749  GCG 5.417 a 4.410–7.243 4.539 a 3.842–6.086 4.314 a 2.269–5.821  ECG 25.775 a 18.560–30.069 24.325 a 20.676–29.881 27.998 a 24.373–31.039  CG 3.642 a 2.545–5.603 4.978 a 3.374–7.930 3.377 a 1.422–5.536  Total content 102.109 a 84.463–111.424 87.980 b 79.499–98.393 98.723 a 93.155–104.757 9 Alkaloids  CAF 27.300 a 23.597–35.210 38.100 b 28.908–43.566 33.516 ab 24.831–42.222 Relationships among sun-dried Pu-erh teas 2 To identify the relationships among sun-dried Pu-erh teas from ancient tea plants, PCA and PLS-DA were performed using the detection data from GC-MS and HPLC, respectively. The results showed that the first two principal components of the PCA applied to the volatile and bioactive component data accounted for 36.2% and 57.4% of the total variance, respectively. The varieties were poorly separated (Fig. 2 2 ), especially for the PCA score plot of the volatile data in which good discrimination was observed only between LME and LBZ teas. The overlaps observed between LME and BL teas, and between LBZ and BL teas suggest that they have some similar chemical characteristics. As for the PCA score plot of the bioactive component data, a trend showing discrimination among samples along PC1 is apparent (score plot, Fig. 2b 2b ). LME teas were located on the right hand side of the plot and were well separated from LBZ, and BL teas were located between LME and LBZ teas. (a) PCA score plot of volatile data; (b) PCA score plot of data for ten bioactive compounds PLS-DA was carried out using 75 volatile and 10 bioactive components. Different teas were well separated in the two scatter plots (Figs. 3a and 3b 3a and 3b ). Cross-validation results ( R 2 =0.389 and Q 2 =−0.187 using volatile components; R 2 =0.225 and Q 2 =−0.269 using bioactive components) showed that there was no over-fitting in the models. The 17 tea samples were well distributed and showed the possibility of outliers, groups, similarities, and other patterns in the data. To explain the relationships between variables and tea samples, loading scatter plots were performed (Figs. 3c and 3d 3c and 3d ). The volatile components, D -limonene (V3), L -menthone (V12), estragole (V18), nerol (V21), cyclododecanone (V36), α-ionone (V38), β-ionone (V42), dihydroactinidiolide (V47), 4-methyl-pentadecane (V50), and methyl linolenate (V73) had high-loading values. Among the bioactive components, (−)-gallocatechin, (−)-epigallocatechin gallate, and (−)-epigallocatechin had higher-loading values. These components contributed most to the discrimination between the varieties of sun-dried Pu-erh tea leaves. To weigh the importance of the effect of each variable on discrimination, variable importance in projection (VIP) plots (Figs. S2a and S2b), which summarize the importance of each variable in explaining X and correlate each variable to Y , were constructed. According to the VIP plots, 34 volatile compounds and 5 bioactive components had VIP values greater than 1.0 (Table S4 S4 ), which means that these variables were primarily responsible for the discrimination of the three tea cultivar samples. Among them, 4-methyl-pentadecane (VIP value=1.590), β-ionone (1.495), D -limonene (1.425), L -menthone (1.423), methyl linolenate (1.411), and (−)-gallocatechin (1.410) had the largest VIP values, consistent with the analytical results of Duncan’s multiple range tests (Tables 3 3 and 4 4 ). (a) PLS-DA score plot of volatile data; (b) PLS-DA score plot of bioactive compound data; (c) PLS-DA loading scatter plot of volatile data; (d) PLS-DA loading scatter plot of bioactive compound data. GA: gallic acid; GC: (−)-gallocatechin; EGC: (−)-epigallocatechin; C: (+)-catechin; EC: (−)-epicatechin; EGCG: (−)-epigallocatechin gallate; GCG: (−)-gallocatechin gallate; ECG: (−)-epicatechin gallate; CG: (−)-catechin gallate; CAF: caffeine. V1–V75 are shown in Table S2 Conclusions 1 This study presents the characterization of aroma profiles by HS-SPME-GC-MS and analysis of the contents of ten bioactive components (polyphenols and alkaloids) by HPLC of sun-dried Pu-erh leaves from ancient tea plants from Bulang Mountain. Chemometric methods, including unsupervised PCA and supervised PLS-DA, were used to distinguish the tea samples according to their varieties. PLS-DA was proven to be satisfactory in distinguishing the different varieties of the tea samples according to their volatile compounds and bioactive components. Major contributors to odor and taste were selected to distinguish the different tea varieties based on VIP values. This study suggested that HS-SPME-GC-MS and HPLC combined with multivariate data analysis are sensitive and ideal methods for characterizing and distinguishing the different varieties of Pu-erh tea from Bulang Mountain. Further investigations on Pu-erh tea leaves from these ancient plants are recommended. References C Alasalvar , B Topal , A Serpen , 等 . Flavor characteristics of seven grades of black tea produced in Turkey . J Agric Food Chem , 2012 . 60 ( 25 ): 6323 - 6332 . 22642545 DOI: 10.1021/jf301498p http://doi.org/10.1021/jf301498p . Flavor characteristics of seven grades of black tea produced in Turkey GA Burdock . Fenaroli’s Handbook of Flavor Ingredients, 6th Ed , 2010 . CRC Press, Florida, USA , . Fenaroli’s Handbook of Flavor Ingredients, 6th Ed K Byju , V Anuradha , E Rosmine , 等 . Chemical characterization of the lipophilic extract of Hydrilla verticillata : a widely spread aquatic weed . J Plant Biochem Biotechnol , 2013 . 22 ( 3 ): 304 - 311 . DOI: 10.1007/s13562-012-0159-5 http://doi.org/10.1007/s13562-012-0159-5 . Chemical characterization of the lipophilic extract of Hydrilla verticillata: a widely spread aquatic weed L Cai , MZ Liang , LF Xia , 等 . Analysis of fingerprint for local old-plant tea variety “Laobanzhang” by HPLC . Hunan Agric Sci , 2014 . ( 8 ): 10 - 11 . 10 (in Chinese) DOI: 10.3969/j.issn.1006-060X.2014.08.003 http://doi.org/10.3969/j.issn.1006-060X.2014.08.003 . Analysis of fingerprint for local old-plant tea variety “Laobanzhang” by HPLC LP Du , JX Li , W Li , 等 . Characterization of volatile compounds of Pu-erh tea using solid-phase microextraction and simultaneous distillation-extraction coupled with gas chromatography-mass spectrometry . Food Res Int , 2014 . 57 61 - 70 . DOI: 10.1016/j.foodres.2014.01.008 http://doi.org/10.1016/j.foodres.2014.01.008 . Characterization of volatile compounds of Pu-erh tea using solid-phase microextraction and simultaneous distillation-extraction coupled with gas chromatography-mass spectrometry LP Du , LJ Ma , Y Qiao , 等 . Determination of phthalate esters in teas and tea infusions by gas chromatography-mass spectrometry . Food Chem , 2016 . 197 1200 - 1206 . 26675858 DOI: 10.1016/j.foodchem.2015.11.082 http://doi.org/10.1016/j.foodchem.2015.11.082 . Determination of phthalate esters in teas and tea infusions by gas chromatography-mass spectrometry DM Fan , K Fan , CP Yu , 等 . Tea polyphenols dominate the short-term tea ( Camellia sinensis ) leaf litter decomposition . J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 2017 . 18 ( 2 ): 99 - 108 . DOI: 10.1631/jzus.B1600044 http://doi.org/10.1631/jzus.B1600044 . Tea polyphenols dominate the short-term tea (Camellia sinensis) leaf litter decomposition A Gulati , SD Ravindranath . Seasonal variations in quality of Kangra tea ( Camellia sinensis (L) O Kuntze) in Himachal Pradesh . J Sci Food Agric , 1996 . 71 ( 2 ): 231 - 236 . DOI: 10.1002/(SICI)1097-0010(199606)71:2<231::AID-JSFA573>3.0.CO;2-Y http://doi.org/10.1002/(SICI)1097-0010(199606)71:2<231::AID-JSFA573>3.0.CO;2-Y . Seasonal variations in quality of Kangra tea (Camellia sinensis (L) O Kuntze) in Himachal Pradesh CJ He , XM Guo , YM Yang , 等 . Characterization of the aromatic profile in “Zijuan” and “Pu-erh” green teas by headspace solid-phase microextraction coupled with GC-O and GC-MS . Anal Methods , 2016 . 8 ( 23 ): 4727 - 4735 . DOI: 10.1039/C6AY00700G http://doi.org/10.1039/C6AY00700G . Characterization of the aromatic profile in “Zijuan” and “Pu-erh” green teas by headspace solid-phase microextraction coupled with GC-O and GC-MS M Kawakami , A Kobayashi , K Kator . Volatile constituents of Rooibos tea ( Aspalathus linearis ) as affected by extraction process . J Agric Food Chem , 1993 . 41 ( 4 ): 633 - 636 . DOI: 10.1021/jf00028a023 http://doi.org/10.1021/jf00028a023 . Volatile constituents of Rooibos tea (Aspalathus linearis) as affected by extraction process M Kawakami , SN Ganguly , J Banerjee , 等 . Aroma composition of oolong tea and black tea by brewed extraction method and characterizing compounds of Darjeeling tea aroma . J Agric Food Chem , 1995 . 43 ( 1 ): 200 - 207 . DOI: 10.1021/jf00049a037 http://doi.org/10.1021/jf00049a037 . Aroma composition of oolong tea and black tea by brewed extraction method and characterizing compounds of Darjeeling tea aroma K Kumazawa , H Masuda . Identification of potent odorants in different green tea varieties using flavor dilution technique . J Agric Food Chem , 2002 . 50 ( 20 ): 5660 - 5663 . 12236694 DOI: 10.1021/jf020498j http://doi.org/10.1021/jf020498j . Identification of potent odorants in different green tea varieties using flavor dilution technique J Lin , Y Dai , YN Guo , 等 . Volatile profile analysis and quality prediction of Longjing tea ( Camellia sinensis ) by HS-SPME/GC-MS . J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 2012 . 13 ( 12 ): 972 - 980 . DOI: 10.1631/jzus.B1200086 http://doi.org/10.1631/jzus.B1200086 . Volatile profile analysis and quality prediction of Longjing tea (Camellia sinensis) by HS-SPME/GC-MS J Lin , P Zhang , ZQ Pan , 等 . Discrimination of oolong tea ( Camellia sinensis ) varieties based on feature extraction and selection from aromatic profiles analysed by HS-SPME/GC-MS . Food Chem , 2013 . 141 ( 1 ): 259 - 265 . 23768356 DOI: 10.1016/j.foodchem.2013.02.128 http://doi.org/10.1016/j.foodchem.2013.02.128 . Discrimination of oolong tea (Camellia sinensis) varieties based on feature extraction and selection from aromatic profiles analysed by HS-SPME/GC-MS HP Lv , QS Zhong , Z Lin , 等 . Aroma characterisation of Pu-erh tea using headspace-solid phase microextraction combined with GC/MS and GC-olfactometry . Food Chem , 2012 . 130 ( 4 ): 1074 - 1081 . DOI: 10.1016/j.foodchem.2011.07.135 http://doi.org/10.1016/j.foodchem.2011.07.135 . Aroma characterisation of Pu-erh tea using headspace-solid phase microextraction combined with GC/MS and GC-olfactometry SD Lv , YS Wu , CW Li , 等 . Comparative analysis of Pu-erh and Fuzhuan teas by fully automatic headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry and chemometric methods . J Agric Food Chem , 2014 . 62 ( 8 ): 1810 - 1818 . 24512533 DOI: 10.1021/jf405237u http://doi.org/10.1021/jf405237u . Comparative analysis of Pu-erh and Fuzhuan teas by fully automatic headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry and chemometric methods SD Lv , YS Wu , YZ Song , 等 . Multivariate analysis based on GC-MS fingerprint and volatile composition for the quality evaluation of Pu-erh green tea . Food Anal Methods , 2015 . 8 ( 2 ): 321 - 333 . DOI: 10.1007/s12161-014-9900-0 http://doi.org/10.1007/s12161-014-9900-0 . Multivariate analysis based on GC-MS fingerprint and volatile composition for the quality evaluation of Pu-erh green tea P Pripdeevech , T Machan . Fingerprint of volatile flavour constituents and antioxidant activities of teas from Thailand . Food Chem , 2011 . 125 ( 2 ): 797 - 802 . DOI: 10.1016/j.foodchem.2010.09.074 http://doi.org/10.1016/j.foodchem.2010.09.074 . Fingerprint of volatile flavour constituents and antioxidant activities of teas from Thailand C Schuh , P Schieberle . Characterization of the key aroma compounds in the beverage prepared from Darjeeling black tea: quantitative differences between tea leaves and infusion . J Agric Food Chem , 2006 . 54 ( 3 ): 916 - 924 . 16448203 DOI: 10.1021/jf052495n http://doi.org/10.1021/jf052495n . Characterization of the key aroma compounds in the beverage prepared from Darjeeling black tea: quantitative differences between tea leaves and infusion J Shi , L Wang , CY Ma , 等 . Aroma changes of black tea prepared from methyl jasmonate treated tea plants . J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 2014 . 15 ( 4 ): 313 - 321 . DOI: 10.1631/jzus.B1300238 http://doi.org/10.1631/jzus.B1300238 . Aroma changes of black tea prepared from methyl jasmonate treated tea plants KL Stevens , GB Merrill . Dihydroactinidiolide–a potent growth inhibitor from Eleocharis coloradoensis (Spikerush) . Experientia , 1981 . 37 ( 11 ): 1133 DOI: 10.1007/BF01989879 http://doi.org/10.1007/BF01989879 . Dihydroactinidiolide–a potent growth inhibitor from Eleocharis coloradoensis (Spikerush) H Tao , ZR Li , F Li . Development plan and countermeasures of tea industry of Bulang Mountain . Agric Technol , 2013 . ( 7 ): 231 (in Chinese) DOI: 10.3969/j.issn.1671-962X.2013.07.193 http://doi.org/10.3969/j.issn.1671-962X.2013.07.193 . Development plan and countermeasures of tea industry of Bulang Mountain C Wang , CX Zhang , CF Shao , 等 . Chemical fingerprint analysis for the quality evaluation of Deepure instant Pu-erh tea by HPLC combined with chemometrics . Food Anal Methods , 2016 . 9 ( 12 ): 3298 - 3309 . DOI: 10.1007/s12161-016-0524-4 http://doi.org/10.1007/s12161-016-0524-4 . Chemical fingerprint analysis for the quality evaluation of Deepure instant Pu-erh tea by HPLC combined with chemometrics C Wang , SD Lv , YS Wu , 等 . Study of aroma formation and transformation during the manufacturing process of Biluochun green tea in Yunnan Province by HS-SPME and GC-MS . J Sci Food Agric , 2016 . 96 ( 13 ): 4492 - 4498 . 26858163 DOI: 10.1002/jsfa.7663 http://doi.org/10.1002/jsfa.7663 . Study of aroma formation and transformation during the manufacturing process of Biluochun green tea in Yunnan Province by HS-SPME and GC-MS C Wang , CX Zhang , YW Kong , 等 . A comparative study of volatile components in Dianhong teas from fresh leaves of four tea cultivars by using chromatography-mass spectrometry, multivariate data analysis, and descriptive sensory analysis . Food Res Int , 2017 . 100 267 - 275 . 28873687 DOI: 10.1016/j.foodres.2017.07.013 http://doi.org/10.1016/j.foodres.2017.07.013 . A comparative study of volatile components in Dianhong teas from fresh leaves of four tea cultivars by using chromatography-mass spectrometry, multivariate data analysis, and descriptive sensory analysis LF Wang , JY Lee , JO Chung , 等 . Discrimination of teas with different degrees of fermentation by SPME-GC analysis of the characteristic volatile flavour compounds . Food Chem , 2008 . 109 ( 1 ): 196 - 206 . 26054281 DOI: 10.1016/j.foodchem.2007.12.054 http://doi.org/10.1016/j.foodchem.2007.12.054 . Discrimination of teas with different degrees of fermentation by SPME-GC analysis of the characteristic volatile flavour compounds YS Wu , SD Lv , C Wang , 等 . Comparative analysis of volatiles difference of Yunnan sun-dried Pu-erh green tea from different tea mountains: Jingmai and Wuliang mountain by chemical fingerprint similarity combined with principal component analysis and cluster analysis . Chem Cent J, 10:11 , 2016 . DOI: 10.1186/s13065-016-0159-y http://doi.org/10.1186/s13065-016-0159-y . Comparative analysis of volatiles difference of Yunnan sun-dried Pu-erh green tea from different tea mountains: Jingmai and Wuliang mountain by chemical fingerprint similarity combined with principal component analysis and cluster analysis LF Xia , MZ Liang , L Wang , 等 . Studying on the quality of Menghai’s sunny dried tea . Chin Agric Sci Bull , 2012 . 28 ( 16 ): 239 - 244 . (in Chinese) DOI: 10.3969/j.issn.1000-6850.2012.16.043 http://doi.org/10.3969/j.issn.1000-6850.2012.16.043 . Studying on the quality of Menghai’s sunny dried tea YQ Xu , C Wang , CW Li , 等 . Characterization of aroma-active compounds of Pu-erh tea by headspace solid-phase microextraction (HS-SPME) and simultaneous distillation-extraction (SDE) coupled with GC-olfactometry and GC-MS . Food Anal Methods , 2015 . 9 ( 5 ): 1188 - 1198 . DOI: 10.1007/s12161 http://doi.org/10.1007/s12161 . Characterization of aroma-active compounds of Pu-erh tea by headspace solid-phase microextraction (HS-SPME) and simultaneous distillation-extraction (SDE) coupled with GC-olfactometry and GC-MS SS Yao , WF Guo , Y Lu , 等 . Flavor characteristics of lapsang souchong and smoked lapsang souchong, a special Chinese black tea with pine smoking process . J Agric Food Chem , 2005 . 53 ( 22 ): 8688 - 8693 . 16248572 DOI: 10.1021/jf058059i http://doi.org/10.1021/jf058059i . Flavor characteristics of lapsang souchong and smoked lapsang souchong, a special Chinese black tea with pine smoking process M Zhu , E Li , H He . Determination of volatile chemical constitutes in tea by simultaneous distillation extraction, vacuum hydrodistillation and thermal desorption . Chromatographia , 2008 . 68 ( 7-8 ): 603 - 610 . DOI: 10.1365/s10337-008-0732-1 http://doi.org/10.1365/s10337-008-0732-1 . Determination of volatile chemical constitutes in tea by simultaneous distillation extraction, vacuum hydrodistillation and thermal desorption Special thanks are given to the Tianxing Pu-erh Tea Industry Co., Ltd. (Pu’er, China), Chunli Pu-erh Tea Shop (Pu’er, China), and Gang LI (tea grower of Laobanzhang village, Xishuangbanna, Yunnan, China) for samples and support. Compliance with ethics guidelines Wen-jie ZHANG, Cong LIU, Rui-juan YANG, Ting-ting ZHENG, Miao-miao ZHAO, Li MA, and Liang YAN declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1800183 ,, 2018-03-27 2018-06-17 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2019 2019 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 07 20