Endogenous nicotine was confirmed to be present in tea plants (Camellia sinensis L.) by liquid chromatography-tandem mass spectrometry of tea samples from tea-producing regions in six Asian countries. All samples contained nicotine (0.011–0.694 μg g −1 dry weight). Nicotine contents remained constant during manufacturing of green, oolong and black teas, implying that nicotine is stable against heating, drying, enzymatic oxidation and mechanical damage during processing. Flower buds and seeds of cultivar Yabukita also contained nicotine (0.030–0.041 μg g −1 dry weight). A comparison of two cultivars revealed that higher nicotine contents were found in the black tea cultivar Benifuki. All plant parts of hydroponic Yabukita contained nicotine (0.003–0.013 μg g −1 dry weight). Tea cells cultured in B5 medium as well as roots and stems of tea seedlings contained nicotine levels similar to those of new leaves from field-grown plants. Although the levels of endogenous nicotine in tea plants are extremely low and sample contamination cannot be discounted, these levels exceed the maximum acceptable limit in Japan (0.01 μg g −1 dry weight).

Funding: This work was supported by the Japanese Society for the Promotion of Science Grant-in-Aid for Scientific Research Number 26292010 (AM) and 17H03756 (AM). We carried out this research entirely through public research funds in Japan (JSPS KAKENHI). We did not receive financial assistance for this study from the relevant private company (Mitsui Norin Co. Ltd); Mitsui Norin Co. Ltd provided salaries for authors KT, NH, HH and FN. The specific roles of these authors are articulated in the 'author contributions' section. The funders also did not have any additional role in the study design, data collection, decision to publish, or preparation of the manuscript.

To determine whether endogenous nicotine is present in Japanese green tea, in this study we measured nicotine contents of several types of tea from Japan and five other Asian countries. Nicotine was detected in all analyzed Japanese green tea samples, but the contents were lower than those of black teas. To determine the effect of different manufacturing processes used to generate teas such as green, oolong and black, we next measured nicotine contents using two Japanese cultivars: Yabukita, an important Japanese green tea cultivar, and Benifuki, a newly introduced black tea cultivar and Assam hybrid. We also investigated the nicotine contents of various tea plant organs (leaves, stems, roots, flower buds and seeds) and explored seasonal changes in nicotine contents of field-grown tea plants. The nicotine content of aseptic cultured tea cells was also determined. Finally, the origin of nicotine in tea was considered in the light of these findings.

Tobacco (Nicotiana tabacum) is the only plant in which nicotine biosynthesis has actually been confirmed to occur [ 6 ]. Various authors [ 5 , 6 ] have proposed that the nicotine detected in tea is due to insecticide contamination. Sheen [ 5 ] has suggested that the content of nicotine in tomato is lessened during processing due to enzymatic oxidation and other chemical reactions. In contrast, however, a screening for nicotine in various processed tomato products by Siegmund et al. [ 8 ] has indicated that nicotine is thermally stable and does not degrade or evaporate during processing. In tobacco leaves, various post-harvest reactions during curing degrade nicotine into its nitrogen oxide as well as into cotinine and other alkaloids [ 12 ]. Also in tomato, Siegmund et al. [ 8 ] has observed that nicotine contents in tomato decrease with increasing ripeness and differ between varieties. Taken together, these findings suggest that several factors affect nicotine content in plants. Nevertheless, the above-mentioned assumptions as well as the possibility of nicotine biosynthesis have not been confirmed in plants other than tobacco.

Nicotine, which is an alkaloid compound mainly found in the genus Nicotiana (e.g. tobacco), has been detected in many food crops [ 4 ]. Nicotine acts as an agonist at nicotinic acetylcholine receptors, therefore, it has been used as a pesticide because of its toxicity to organisms. Previous studies have revealed the presence of nicotine not only in solanaceous crops, namely, potato (Solanum tuberosum), tomato (S. lycopersicum), eggplant (S. melongena) and pepper (Capsicum annum), but also in wild mushrooms and cauliflower (Brassica oleracea) [ 5 – 11 ]. These findings suggest that nicotine is widely distributed in plants and fungi. In a survey reported by the European Food Safety Authority (EFSA), nicotine concentrations were measured in 332 tea samples (87 green, 239 black and 6 Chinese white) originating from numerous tea-producing countries [ 4 ]. Nicotine was detected in 274 samples, with contents of 53 samples found to be over the default maximum residue level (MRL) of 0.01 mg kg −1 dry weight (DW). Prior to this EFSA report, other several studies uncovered nicotine in tea at levels ranging from 0 to 28.01 mg kg −1 DW [ 5 – 8 ]. In Japan, where no nicotine-containing pesticides have been listed in the Agricultural Chemicals Regulation Law since 2001, the MRL of nicotine is set at 0.01 mg kg −1 DW. Japanese green tea should thus have no detectable exogenous nicotine. The nicotine content of Japanese green tea was not measured in any of the above-mentioned studies.

Tea, derived from the tea plant (Camellia sinensis L.), is one of the world’s most popular beverages. Various types, such as green, black and/or oolong, are consumed in different localities. Unlike oolong and black teas, green tea is made without the use of withering and enzymatic oxidation (e.g. polyphenol oxidase and peroxidase) processes. Consequently, catechins and ascorbic acid contents of green tea are generally higher than those of other teas [ 1 , 2 ]. Green tea, produced mainly in China, Vietnam and Japan, is classified into Chinese and Japanese styles [ 3 ]. Japanese-style green tea typically differs from Chinese in the way in which the leaves are heated: Japanese-style tea leaves are heated with a steaming machine—which minimizes the deactivation of oxidation enzymes—whereas Chinese tea production involves a parching machine ( S1 Fig ). Sencha, the most common Japanese green tea, plays an important role in Japanese culture. The export volume of Japanese green tea has recently increased greatly. Because these compounds are beneficial to human health, their higher levels in green tea have resulted an increase in green tea production and consumption—up to one-third that of black tea.

Nicotine was quantified on a liquid chromatograph (1100 series, Agilent, CA, USA) equipped with a triple quadrupole mass spectrometer (3200 QTRAP®, Sciex, MA, USA) with a TurboIonSpray® (Sciex, MA, USA) electrospray ionization source operated in positive ionization mode. The chromatographic analysis was performed on a 150 × 2.1 mm InertSustain phenylhexyl column (GL Science, Tokyo, Japan). Acetonitrile (B) and 20 mM ammonium hydrogen carbonate (A) were used as the mobile phase at a flow rate of 0.25 mL min −1 . Elution was performed with the following gradient: initial concentration of 10% B, followed by a 0.1-min linear gradient from 10% to 35% B, a 0.9-min hold at 35% B, a 0.1-min linear gradient from 35% to 50% B, a 1.9-min hold at 50% B, a 2.0-min linear gradient from 50% to 75% B, a 2.0-min hold at 75% B, a 0.1-min gradient from 75% to 10% B, and a final concentration of 10% B for 4.9 min.

Nicotine extraction from tea samples was carried out according to the modified QuEChERS method of Cavalieri et al. [ 16 ]. One gram of powdered tea sample was added to a 50-mL polytetrafluoroethylene (PTFE) centrifuge tube along with 50 μL of nicotine d 3 internal standard (Sigma, MO, USA) at a concentration of 5 μg mL −1 . To this mixture was added 20 mL of 0.1 M ammonia solution followed by incubation for 10 min to allow for swelling of the tea powder. After adding 10 mL acetonitrile, 6 g MgSO 4 and 1.5 g CH 3 COONa, the solution was mixed on a shaker at 3,000 rpm for 5 min (SA300, Yamoto, Tokyo, Japan). Following centrifugation at 5,000 ×g for 4 min, 1.5 mL of the resulting supernatant was transferred to a 2-mL PTFE centrifuge tube containing 50 mg of Primary Secondary Amine (Agilent, CA, USA) and 150 mg of MgSO 4 . The sample was mixed for 1 min and then centrifuged at 18,000 ×g for 5 min. The supernatant was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Nicotine in B5 medium from cultured tea cells was also extracted using the same procedure, except that the final concentration of added ammonia solution was 0.1 M.

Suspension-cultured tea cells (Panel B in S3 Fig ) were prepared from calli derived from anthers of Yabukita [ 14 ]. The cells were subcultured weekly in B5 medium at pH 5.6 in darkness at 25°C on a rotary shaker (100 rpm). Exponentially growing cells were harvested after 9 days by suction filtration, suspended and rinsed three times with a simple solution containing 0.2 mM CaCl 2 at pH 5.6. The cells were frozen in liquid nitrogen and freeze-dried. B5 medium was also collected before and after 9 days cultivation and stored at −20°C until analysis. The samples were freeze-dried, ground and stored in the same manner as described above.

Flower buds of 35-year-old Yabukita tea plants were collected from the tea garden of the Shizuoka Prefecture Tea Research Center (Kikugawa, Shizuoka, Japan) on 5 November 2015. The flower buds were immediately divided into petals, filaments and anthers (Panel A in S3 Fig ). On 26 October 2015, fully mature fruits containing one to four seeds were collected from 20-year-old Yabukita tea plants in a private tea garden in Shizuoka, Japan. After peeling away the pericarp, the outer seed coat was broken with a hammer, and the outer and inner seed coats were carefully removed. The collected flower buds and tea seeds were freeze-dried, ground and stored as described earlier. Because collection of adequate samples for nicotine analysis was difficult, only a single replicate was performed.

New leaves, new stems, mature leaves and roots of Yabukita and Benifuki were harvested from the NIFTS tea garden (Makurazaki, Kagoshima, Japan) on 18 April (first crop season), 18 June (second crop season) and 13 August (third crop season) in 2014. Mature leaves and roots were also harvested on 27 February during the off season in 2015. The roots were collected from the soil layer between hedges at a depth of 10 cm. After washing three times with deionized water, the samples were freeze-dried, ground and stored in the same manner as described above.

New shoots of 30-year-old plants of two tea cultivars (Yabukita and Benifuki) were harvested during the first crop season on 25 April 2015 at the NIFTS tea garden (Makurazaki, Kagoshima, Japan). The shoots were immediately used to manufacture Japanese (Sencha, steaming type) and Chinese (pan-fired tea, panning type) green teas, oolong tea and black teas following the processes shown in S1 Fig . Following processing, samples were collected for nicotine analysis and stored in a freezer at −20°C until tea manufacturing. The manufactured tea samples were then freeze-dried, ground and stored in the same manner as described above.

Thirty-two green tea (C. sinensis var. assamica), 79 black tea (C. sinensis var. sinensis and/or C. sinensis var. assamica) and 1 oolong tea (C. sinensis var. sinensis) sample(s) were subjected to nicotine analysis ( S1 Table ). The Indian tea samples were purchased from 16 and 3 tea estates in Darjeeling and Assam, respectively, by Mitsui Norin Co. Ltd., one of the major tea companies in Japan. With the exception of one estate in Assam from which no autumn sample was obtained, tea collections were made during all four crop seasons (first, second, rain/monsoon and autumn; S4 Fig ). All samples were from organically grown tea leaves received Japanese Agricultural Standard (JAS) certification, i.e. grown without the use of chemicals including insecticides. In the case of the Japanese samples, which were collected by the Tea Research Division of the NARO Institute of Fruit and Tea Science (NIFTS) (Makurazaki, Kagoshima, Japan), the leaves came from a cultivated tea garden where no insecticides (including nicotine) have been used for several decades. Other tea samples were purchased from organic cultivation in local markets of each country. All green teas collected in Japan were typical Japanese types (Sencha), produced using steam, while those from Taiwan were Chinese types (pan-fired). Tea samples were dried in a freeze dryer (FDU-2000, EYELA, Tokyo, Japan) and then ground into a fine powder with a vibrating mill (SA300, Yamato, Tokyo, Japan). The samples were stored in desiccators until nicotine analysis.

Table 3 displays seasonal changes in nicotine contents of new leaves, new stems, mature leaves and roots of Yabukita and Benifuki. In new leaves and stems of Yabukita, nicotine contents during the first crop season (0.010 and 0.011 μg g −1 DW, respectively) were significantly lower than those during the second (0.023 and 0.033 μg g −1 DW) and third (0.022 and 0.036 μg g −1 DW). Nicotine contents in these two organs were highest in Benifuki during the third season (0.036 and 0.032 μg g −1 DW). Although nicotine contents in mature leaves of both cultivars were highest in the off season (0.024 μg g −1 DW in Yabukita and 0.021 μg g −1 DW in Benifuki), the ranges of their seasonal changes were smaller than those of new leaves and new stems. In roots of both cultivars, nicotine contents remained constant (ca. 0.028 μg g −1 DW in Yabukita and 0.030 μg g −1 DW in Benifuki). When averaged over first to third crop seasons, the highest nicotine contents were found in roots.

Discussion

Among the tea samples collected from tea-producing localities in six Asian countries, we found detectable levels of nicotine in Japanese green tea (Table 1). This result reconfirms the presence of nicotine in Japanese green tea. With respect to maximum values among tea-producing regions, nicotine contents of organically grown black tea from Assam and Darjeeling were higher than those of Japanese green tea. In a study of 41 black, 14 oolong, 26 green and 6 white tea samples, conducted with LC-ESI-MS/MS using a modified QuEChERS method, Thräne et al. [17] observed that the highest nicotine contents (> 0.6 μg g−1 DW) were found in samples from India and China. They also reported that tea samples from Japan had contents below 0.19 μg g−1 DW. These results suggest that nicotine contents of tea leaves differ among producing regions and cultivars. In spite of the relatively small number of samples in the present study, we also observed that nicotine contents of black tea (0.024–0.694 μg g−1 DW) were significantly higher than those of green tea (0.011–0.40 μg g−1 DW) (Table 1). This trend is in line with average nicotine contents reported by EFSA 2011 [4] of 0.130 μg g−1 DW (0–0.873 μg g−1 DW) for black tea vs. 0.075 μg g−1 DW for green tea (0–0.440 μg g−1 DW)—although it should be noted that these samples did not include Japanese green tea. In tobacco plants, it is reported that nicotine synthesized in roots accumulates in vacuoles [18] and so also the flavonoids such as catechins are found in the vacuoles in tea [19]. Understanding the significance of these observed differences among producing regions and cultivars will require future investigation of cultivation environments, such as soil conditions and climate, and the introduction of additional cultivars. Thräne et al. [17] found that average nicotine contents seemed to increase with increasing degree of fermentation, although black teas with contents below a LOQ of 0.01 μg g−1 DW were also observed. In contrast, the results of Sheen [5] suggest that nicotine is reduced in tomato via enzymatic oxidation and other chemical reactions during processing. As is well known, tea is classified into three types based on the degree of fermentation: non-fermented (green tea), semi-fermented (oolong tea) and full-fermented (black tea). During the tea manufacturing process, leaves are typically heated by steaming (ca. 100°C), hot air exposure (65–115°C) or by placement on high-temperature pans (ca. 300°C) to diminish oxidative enzyme activities and for drying (S1 Fig). Nicotine contents in tea leaves would thus be expected to change during manufacturing. In this study, however, nicotine contents remained constant regardless of tea type (Sencha, pan-fired, oolong or black). This result suggests that heating and fermentation have no effect on nicotine contents in tea leaves. Similarly, the application of withering, a leaf-damaging process, during the manufacturing of oolong and black teas had no effect on nicotine contents.

A comparison of two cultivars revealed that nicotine contents in C. sinensis var. sinensis ‘Yabukita’ were lower than those of C. sinensis var. sinensis × var. assamica ‘Benifuki’ in new leaves subjected to tea manufacturing processes (Table 2), in new leaves of field-grown tea plants (Table 3) and in new leaves and roots of hydroponically grown tea plants (Table 4). These results suggest that nicotine contents of C. sinensis var. assamica are higher than those of var. sinensis. Because leaves of varieties sinensis and assamica are generally used to make green and black teas, respectively, this implies that a difference exists in nicotine accumulation among tea varieties. In samples of var. assamica from India, however, we detected nicotine contents as low as 0.100 μg g−1 DW, even though the highest value was also detected in an Indian tea sample (0.694 μg g−1 DW; Table 1). A wide range of nicotine contents in Indian black tea has also been observed in the above studies. Two possible explanations for these results can be proposed. One possibility is genetic variation in nicotine synthesis in tea plants; the other is environmental contamination due to various reasons—for example, from smokers (cigarette smoke or nicotine residues on fingers of harvesters) [20] or seepage into ground water from nearby cultivated tobacco plants [21]. Because nicotine was detected in all types of tea samples, the latter possibility seems unlikely but must still be considered.

New tea shoots (new leaves and stems) were harvested several times every year from spring to autumn. In general, plucking was carried out four times annually in India and two or three times in Japan. No clear seasonal trend, such as highest value, could be discerned in the nicotine content of Indian black teas collected from 14 tea estates (S4 Fig). In Japan, nicotine contents of new leaves and stems of field-grown tea plants were highest during second and third crop seasons (Table 3). In tobacco plants, damage to leaves increases nicotine contents, indicating that nicotine biosynthesis is affected in response to mechanical wounding [22–24]. Wang et al. [25] have also reported that excision of tobacco shoot apices causes dramatic increases in nicotine contents in leaves. Before beginning this study, we therefore assumed that wounding caused by plucking of tea new shoots might increase nicotine contents, thereby leading to higher levels in tea leaves during second and third crop seasons compared with the first season. Although this trend was observed in Japanese field-grown plants, no such pattern was uncovered in Indian black tea. In tobacco plants, nicotine biosynthesis is regulated by multiple biotic and abiotic stresses during the growing season, such as drought, heat and herbivore or insect damage [26, 27]. Because the type and degree of stress experienced under field conditions among tea-growing regions was variable, the individual seasonal fluctuation of nicotine content in new tea shoots differed between tea estates. The seasonal change in nicotine contents of new shoots observed in this study consequently suggests that nicotine biosynthesis and degradation is carried out in tea plants.

Leaves, stems and roots as well as seeds and flower buds also contained nicotine (Table 5). In field-grown tea plants, the annual average nicotine content was highest in roots (Table 3). Roots were also the organs with the highest contents of nicotine in tea plants grown hydroponically in a greenhouse and aseptic seedlings (Tables 4 and 5). In tobacco plants, nicotine is synthesized in roots [28], where it remains most abundant until topping [25]. On the basis of nicotine presence in other plant organs besides fruits, Sheen [5] concluded that tomato also biosynthesizes nicotine. Although no other reliable evidence is currently available, our data strongly suggest that nicotine is synthesized in the roots of tea plants. Similar to aseptic seedlings, suspension-cultured tea cells contained nicotine (0.033 μg g−1 DW) (Table 5). These cells were derived from anthers of Yabukita and had been sub-cultured weekly since 1994 in B5 medium under aseptic conditions. Nicotine was not detected in the B5 medium alone. Even when grown under these conditions, which virtually guaranteed the complete exclusion of nicotine contamination from exogenous sources, tea cells contained nicotine. These results strongly support that endogenous nicotine is present in tea plants.

In tobacco plants, nicotine is synthesized from putrescine, a polyamine produced either directly from ornithine in a reaction catalyzed by ornithine decarboxylase (EC 4.1.1.17) or indirectly from arginine in a reaction catalyzed by arginine decarboxylase (EC 4.1.1.17) [29, 30]. The first step in nicotine biosynthesis is the conversion of putrescine to N-methylputrescine, which is catalyzed by putrescine N-methyltransferase [31–35]. Although the final step of nicotine biosynthesis is still not known clearly, recently it was reported that an A622, a member of PIP family of NADPH-dependent reductases, and berberine bridge enzyme like proteins (BBLs) have these function [36]. Besides, the production of nicotine and the expression levels of genes involved in nicotine biosynthesis showed an increase after wounding and jasmonate treatment [37] mediated by the jasmonate signaling cascade, which is regulated defense responses against environmental stresses [38].Wang et al. [25] reported that nicotine content increases in tobacco plants harvest at the upper leaves during cultivation. In general, even in the cultivation of tea, the risk of infestation of insects and pathogenic attacks also increases in the summer season compared with the winter season. Indeed, our results represent that nicotine contents in tea differ in each region and season (Tables 1 and 3), especially their values tends to be high in summer season. In other words, environmental factors related to jasmonate signaling may affect nicotine biosynthesis in tea. The activity and gene expression of these enzymes have not been examined in tea plants. To provide further evidence of endogenous nicotine, molecular-level investigation of nicotine synthesis in tea plants is needed.

Compared with other plant species, e.g. solanaceous; < 100 μg kg−1 FW (fresh weight) on average [8, 10], it was confirmed that the content of nicotine contained in tea was of same level. Based on the EFSA report 2011 [4], intake of nicotine via a cup of tea (1.5 g leaf/150 ml) calculated from our results is 0.008 to 0.500 μg when calculated at a transition rate of 48% (median), and it is 0.017 to 1.041 μg when it is 100%, these were also reported to be of same level (≤ 1 μg day−1) as in solanaceous, and much lower than that via passive smoking (80 μg day−1) or smoking (number of cigarettes × 1 mg day−1) [10]. According to an EFSA report 2011 [4], tea is the major source of ingested nicotine among herbal infusions and spices, but its maximum contribution amounts to only 0.7% of acceptable daily intake. On this basis, EFSA proposed a MRL of 0.6 μg g−1 for tea. With the exception of a few samples, nicotine contents obtained in this study were lower than this value, which suggests that tea has no toxic effect when consumed as a beverage. In light of our data, we recommend future revision of this MRL, with careful consideration given to samples exceeding this value to avoid the risk of contamination.