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Acetylsalicylic acid application decreased tobacco-specific nitrosamines and its precursors but maintained quality of air-cured burley tobacco (Nicotiana tabacum L.)
Industrial Crops & Products 104 (2017) 221–228
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Acetylsalicylic acid application decreased tobacco-specific nitrosamines and its precursors but maintained quality of air-cured burley tobacco (Nicotiana tabacum L.)
MARK
⁎
Xiang Chena, Li Liub, Yanmin Zhangb, Xiaofeng Zhoub, Tao Linb, Youhong Songa, Jincai Lia, , ⁎ Xinsheng Chengb, a b
School of Agronomy, Anhui Agricultural University, Hefei 230036, China Research Center of Tobacco and Health, University of Science and Technology of China, Hefei 230051, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Nicotiana tabacum L. Tobacco-specific nitrosamines (TSNA) Nitrosamine precursor Sensory quality Acetylsalicylic acid
Burley tobacco (Nicotiana tabacum L.) is an important raw material in the production of Chinese-style blended cigarettes. However, the high levels of tobacco-specific nitrosamines (TSNA) have devalued the industrial use of burley tobacco and limited its sustainable use. The objective of this paper was to investigate the effects of acetylsalicylic acid (ASA) application on accumulation of TSNA, its precursors and quality of air-cured burley tobacco (Nicotiana tabacum L. cv. DaBai 1). Field experiments were conducted to determine the contents of TSNA and the precursors and the sensory quality of burley tobacco under 0, 0.1, 0.3 mM of ASA in 2014 and 2015 at Dazhou, Sichuan Province, China. The results showed that TSNA, nitrate, nitrite and alkaloids levels were significantly reduced by the application of ASA, of which the total TSNA were significantly lower by 10.40–24.75%, and two of the strong animal carcinogens in tobacco products, i.e. N-nitrosonornicotine (NNN) and 4-(methylnitrosamino) -1- (3- pyridyl) -1- butanone (NNK) were reduced by 9.01–24.23% and 11.43–26.86%, respectively. Year had significant effects on TSNA and the TSNA contents in 2014 were higher than in 2015. Interestingly, the sensory quality of burley tobacco remained or was even better after application of ASA. In conclusion, this study revealed that ASA application was able to reduce the levels of TSNA and its precursors in sustaining burley tobacco industry potentially.
1. Introduction Sichuan Province, located in southwest China, has a unique climate and geographical environment. Burley tobacco (Nicotiana tabacum L.) is a local major industrial crop and plays important roles in local economy and agricultural production. High-quality burley tobacco produced in Sichuan Province has been widely used in the production of Chinesestyle blended cigarettes because of a unique structure, physical properties and signature flavor. It is known that tobacco and its products contain a certain number of carcinogenic substances, and the carcinogenicity of tobacco products is primarily attributed to the presence of tobacco-specific nitrosamines (TSNA) (Hecht, 1998, 2008). With increasing concerns on smoking related health problems, the high levels of TSNA have affected the industrial development of burley tobacco and limited its sustainable use. It is generally thought that TSNA are undetectable or at a very low
level in fresh leaves prior to harvesting, but can be readily measured in air-cured leaves after curing. This is because TSNA are generated through the nitrosation of tobacco alkaloids in processes predominantly taking place during the curing stage of tobacco leaves, although additional formation can occur in the subsequent storage and processing of cured leaves, as well as via pyrosynthesis during combustion in some circumstances (Bush et al., 2001; Shi et al., 2013). The major four kinds of TSNA found in tobacco and its products are N-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), Nnitrosoanabasine (NAB) and N-nitrosoanatabine (NAT). Tobacco alkaloids and nitrite are important precursors of TSNA. Alkaloids can react with nitrite to form relevant TSNA i.e. NNN, NNK, NAB and NAT derived from nornicotine, nicotine, anabasine and anatabine, respectively (Fig. 1) (Bush et al., 2001; Hecht et al., 1983). NNN and NNK have been classified as Group I carcinogens (the highest designation) by International Agency for Research on Cancer (2007). Additionally, as
Abbreviations: ASA, acetylsalicylic acid; NAB, N-nitrosoanabasine; NAT, N-nitrosoanatabine; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NNN, N-nitrosonornicotine; TSNA, tobacco-specific nitrosamines ⁎ Corresponding authors. E-mail addresses: [email protected] (X. Chen), [email protected] (J. Li), [email protected] (X. Cheng). http://dx.doi.org/10.1016/j.indcrop.2017.04.031 Received 3 January 2017; Received in revised form 21 March 2017; Accepted 19 April 2017 0926-6690/ © 2017 Elsevier B.V. All rights reserved.
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Fig. 1. TSNA formed by nitrosation of tobacco alkaloids.
objective of this study was to investigate the effects of ASA application on TSNA accumulation and quality of air-cured burley tobacco leaves in the production of burley tobacco.
the NNN and NNK are the most prevalent strong carcinogens in tobacco and its products, they may ultimately be regulated in United States by U.S. Food and Drug Administration (FDA) (2011). Therefore, in recent years it has been a focus on reducing TSNA formation and accumulation in burley tobacco leaves and their products. To reduce TSNA in cured tobacco leaves, in addition to through the processing techniques, breeding new varieties and rational agronomic managements are proved to be effective. The levels of nicotine and TSNA were lower in tobacco products processed by the leaves from low nicotine varieties bred with genetic engineering techniques (Conkling, 2005). In recent years, the United States tobacco industry and other main tobacco producing countries have established a standard with a maximum limit of nornicotine content in burley tobacco leaves, and application of low nicotine conversion varieties to reduce TSNA contents in the production of burley tobacco (Jack et al., 2007; Shi, 2013). Burley tobacco requires a large amount of nitrogen fertilizer to produce high yield of cured leaf in ensuring profitable yields. However, excessive nitrogen fertilizer application produces air-cured leaves with undesirable levels of NO3-N and alkaloid (MacKown et al., 1984, 2000). The appropriate management of nitrogen fertilization, according to the local practice, is an effective measure to reduce TSNA levels in air-cured leaves (Chamberlain and Chortyk, 1992; Wahlberg et al., 1999). Cui et al. (1994) reported that the levels of TSNA in air-cured burley tobacco with maleic hydrazide application were reduced by 30–50% compared with the hand-suckered control. Furthermore, the addition of antioxidants such as ascorbic acid (Vitamin C) and tocopherol (Vitamin E) to tobacco prior to the harvesting were capable to reduce TSNA levels by scavenging free radicals and inhibiting the nitrosation reaction of nitrite and alkaloids (Krauss et al., 2003; Lathia et al., 1988; Lathia and Blum, 1989; Li et al., 2006; Rundlöf et al., 2000). It has been reported that 2-(acetyloxy) benzoic acid (acetylsalicylic acid, ASA) can increase yield, quality and the resistance to biotic and abiotic stresses in several plant species, for instance, it enhanced the resistance to tobacco mosaic virus in tobacco (Bokshi et al., 2003; Gimenez et al., 2014; Hashmi et al., 2012; Heller et al., 2013; White, 1979). As an important agronomic management to regulate the nutrition and quality of tobacco, topping (removal of the developing inflorescence) changed the sink–source relationships, hormonal balance and root development. Additionally, the wound caused by topping could induce the biosynthesis of many secondary metabolites e.g. nicotine (Baldwin, 1989; Baldwin et al., 1994; Fragoso et al., 2014; Qi et al., 2012). Recently, ASA was found to be applied to wounds at the main stalk of burley tobacco after topping, resulting in lower levels of alkaloids, nitrate and nitrite (Hua et al., 2010; Huang et al., 2010). The functional mechanism of ASA action in tobacco is unclear, though a hypothesis has been proposed (Zhang et al., 2011). Given ASA application is able to reduce the levels of alkaloids, nitrate and nitrite, it may have some effects on TSNA accumulation. Therefore the
2. Materials and methods 2.1. Experimental site The field experiments were conducted on a private farm located in Xuanhan, Dazhou, Sichuan Province, China (Latitude 31°16' N, Longitude 107°42' E; 545 m altitude) from April to October respectively in 2014 and 2015. The soil type at the field site is a typical brown soil with a medium loam texture. The 0- to 20-cm soil depth had the following characteristics: available N of 131.3 mg kg−1, Olsen P of 25.1 mg kg−1, available K of 106.9 mg kg−1, organic matter of 36 g kg−1, and pH 6.4. Monthly mean temperature and rainfall data during the growing season are shown in Fig. 2. Mean monthly temperatures from April to October in both experimental years closed to the 30-yr average temperature (Fig. 2a). The total rainfall from April to October was 1494 mm in 2014 and 1021 mm in 2015, respectively, which was different from 30-yr average (Fig. 2b). From August to September, the total rainfall in 2014 was higher by 190% than in 2015, indicating that burley tobacco air-curing processes in 2014 was exposed to a wetter condition. 2.2. Experimental design A randomized complete block design of three treatments was used, each treatment having three replicates. Treatment A was applied with 0.1 mM ASA (0.25 mL plant−1); treatment B was applied with 0.3 mM ASA (0.25 mL plant−1); treatment C (Ck) was applied with water only (0.25 mL plant−1) as the control treatment. The selection of two different concentrations in application of ASA was on the basis of previous studies (Hua et al., 2010; Huang et al., 2010). ASA was obtained from Sigma Chemical Company (St Louis, MO, USA) and freshly dissolved in water about 1-h before smearing. A commercial Burley tobacco (Nicotiana tabacum L. cv. DaBai 1) was used and grown in a seedbed with a growing medium consisting of 70% (v/v) peat culture substrate and 30% (v/v) perlite in both years. Pelleted seeds were sown on 24 February 2014 and 1 March 2015, and grown in a naturally illuminated glasshouse. Transplanting tobacco seedlings into the field was done on 25 and 29 April in 2014 and 2015 according to standard practices of plant arrangement with a row spacing of 1.20 m and a plant spacing of 0.45 m. The plant density was 16, 500 plants ha−1. The experiment consisted of 9 subplots in both years. Each subplot size was 6.0 m wide by 8.4 m long. Plots within a block were separated by a 1.2 m bare space and independent block were separated by a 2.5 m bare space. The 4 outside edges of the whole planting area 222
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Fig. 2. Mean monthly temperature (°C) (a) and rainfall (mm) (b) in 2014 (dark column) and 2015 (blank column) during the experiments (April-October) and 30-yr average (dark line) temperature (°C) (a) and rainfall (mm) (b) at Dazhou, Sichuan, China.
laboratory for sensory quality evaluation and chemical analysis. The sensory quality was determined by China Tobacco Anhui Industrial Co., Ltd. All the samples were freeze-dried, ground to pass through a 0.3mm screen and subsequently stored at −20 °C until analysis. NNN, NNK, NAB, NAT, nitrate-N, nitrite-N, nornicotine, nicotine, anatabine and anabasine were quantitatively analyzed with the methods presented below.
were bordered by two rows of extra guarding plants. Subsequent irrigation, depending on rainfall, was applied to maintain soil moisture around the field capacity level and ensure optimal burley tobacco plant water supply. No other pesticides were applied during the experiment. 210 kg N ha−1 in total were used in the burley tobacco plots in each year. P and K were also applied to the plots. The overall ratio of N: P2O5:K2O was 1:1:2 in both 2014 and 2015. The plots received 70% of N rate, 100% of P rate, and 60% of K rate with a compound fertilizer (N:P:K = 10:10:28) prior to burley tobacco transplanting. The rest of N and K were applied at 26-d after transplanting. The weeds in the field were removed manually. The cultural practices followed as recommendation for commercial tobacco cultivation by Sichuan Tobacco Company in both growing seasons. Burley tobacco plants were topped when plants in the field had produced 22–24 leaves on 5 and 12 July in 2014 and 2015, respectively. Subsequently, different concentrations of ASA were applied with a medium goat-hair smear to wounds at burley tobacco main stalk within 1–2 h after topping. Burley tobacco leaves were harvested manually from 12 July to 20 August in 2014 and 2015, and air cured by the recommended practices of the region.
2.3.1. Tobacco-specific nitrosamines analysis Quantitative determinations of NNK, NNN, NAT and NAB were performed according to China Tobacco Standard (YQT 29-2013). In brief, 1 g of ground sample was accurately weight into a conical flask and combined with 30 mL of 0.1 M ammonium acetate solution. The mixture was extracted in the ultrasonic generator for 40 min and 2 mL of extraction solution was centrifuged at 10,000 rpm for 5 min. Then 1 mL of supernatant was filtered through a 0.22 μm membrane and collected for analysis. TSNA were quantified by liquid chromatography tandem mass spectrometry (LC–MS/MS) coupled with electrospray ionization (ESI) (LTQ-Orbitrap XL, Thermo Fisher Scientific, USA). The analysis was performed on a 100 × 2.1 mm Hypersil Gold C18 column with 3 μm particle size (Thermo Fisher Scientific, USA) and the column temperature was set as 40 °C with an injection volume of 5 μL. The mobile phases were methanol solution (A) and 0.2% acetic acid water solution (B) with a flow rate 0.4 mL/min. The gradient elution conditions were programmed as follows: 0.0–2.0 min, 10–100% A; 2.0–4.5 min, 100–10% A; 4.5–6 min, 10% A. Mass spectrometer condi-
2.3. Sampling and measurements Fifty burley tobacco plants were randomly selected from each plot (excluding the edge row) and their middle leaves (from bottom to top: 11th-13th) were labeled to assist to mark the number for samples. Aircured burley tobacco leaf samples were collected and brought to the 223
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tions were controlled as follows: ESI ion source, positive ion mode, multiple reaction monitoring (MRM) mode, 3400 V spray voltage, 120 °C ion source temperature, 700 L/h nebulizer gas flow rate, 70 L/ h curtain gas flow rate, 100 ms scan time. Total TSNA were calculated as the sum of NNN, NNK, NAB and NAT. The method has the limits of quantification (LOQ) for NNK, NNN, NAT and NAB were 0.89 ng/g, 2.38 ng/g, 1.19 ng/g and 1.78 ng/g, respectively, and the limits of detection (LOD) were 0.27 ng/g, 0.72 ng/g, 0.36 ng/g and 0.54 ng/g, respectively.
Table 1 Analysis of variance for treatment (T) and year (Y) effects on NNK, NNN, NAT, NAB and total TSNA contents of air-cured burley tobacco. F-test values are shown. Source
df
NNK
NNN
NAT
NAB
Total TSNA
T Y T×Y
2 1 2
83.01*** 846.98*** 12.97**
11.26** 960.13*** 1.16ns
27.96*** 1194.49*** 8.89**
42.21*** 1458.11*** 14.56**
28.67*** 2011.83*** 3.99ns
ns, not significant; *, ** and *** indicate significance at P = 0.05, 0.01 and 0.001 levels, respectively.
2.3.2. Nitrate-N and nitrite-N analysis Nitrate-N and Nitrite-N contents in air-cured burley tobacco samples were quantified with UV–vis Spectrophotometer (Model 756P, Spectrum Instrument Company, Shanghai, China) according to the methodology of Hua et al. (2010). Sodium nitrate and sodium nitrite were served as the standard substance for nitrate and nitrite, respectively. For determination of nitrate, 0.2 mL of ground tobacco sample solution and 0.8 mL of 5% salicylic acid solution were mixed and added into 19 mL of 8% sodium hydroxide solution. The reaction mixture was stood for 25 min and determined for its absorbance at 410 nm. For determination of nitrite, 2 mL of ground tobacco sample solution, 4 mL of 1% sulfanilamide solution and 4 mL of 0.02% α-naphthylamine solution were mixed in the glass test tube and maintained in 25 °C thermostat water bath for 30 min. The absorbance of the sample was determined at 540 nm.
NAT, NAB and total TSNA contents in treatment B were significantly reduced by 24.98, 17.58, 26.74, 29.53 and 19.40%, respectively (Table 3). The year also had significant effects on the NNK, NNN, NAT, NAB and total TSNA contents (Table 1). The mean NNK and NNN contents were higher in 2014 than in 2015, and the same trend exhibited by NAT, NAB and total TSNA. Moreover, significant year × treatment interactions were observed for NNK, NAT and NAB (Table 1). As a consequence, the results indicated that the application of ASA was able to effectively reduce TSNA content, particularly reduce NNK and NNN contents. These suggested that the reduction of TSNA in aircured burley tobacco leaves by the application of ASA might reduce health risk associated with the use of tobacco and its products. 3.2. Effects of ASA application on Nitrate-N and Nitrite-N contents of aircured burley tobacco
2.3.3. Alkaloid analysis Nicotine, nornicotine, anatabine and anabasine contents in aircured leaf samples were quantitatively analyzed by gas chromatography-mass spectrometry (GC–MS) (Model HP6890/5975, Agilent Technologies Inc., USA) according to the China Tobacco Standard (YCT 383-2010). Trichloromethane was used as the extraction solvent, and 2-methylquinoline and 2, 4′-Bipyridine were served as the internal standards. For each sample, 0.3 g of ground tobacco was accurately weight into a conical flask, and 2 mL of 5% aqueous sodium hydroxide was added to moisten the burley tobacco sample and stood for 15 min. Then 20 mL of extraction solution was added into the tube and extracted in the ultrasonic generator for 15 min. The mixture was centrifuged for 5 min. After the sample and extraction solvent were separated, water was removed using anhydrous sodium sulfate and 2 mL of solvent was collected into chromatography bottle. Subsequently, extracts were injected into the gas chromatography for alkaloid separation and quantification. Total alkaloids were calculated as the sum of nicotine, nornicotine, anatabine and anabasine.
Significant differences were observed among three treatments for nitrate-N and nitrite-N (Table 2). Both nitrate-N and nitrite-N contents of ASA-treated burley tobacco were reduced in 2014 and 2015 (Table 4). Compared with control, the 2-yr mean nitrate-N content in treatment A and B were significantly reduced by 10 and 36.05%, respectively, and the 2-yr mean nitrite-N content in treatment A and B were also significantly reduced by 8.92 and 10.60%, respectively. The analysis of variance results also showed that the nitrate-N and nitrite-N contents were significantly affected by year (Table 2). The mean nitrate-N and mean nitrite-N contents were both higher in 2014 than in 2015, which was probably related with the difference in the precipitation dispersal during the two growing seasons as shown in Fig. 2b. Moreover, the interaction of the year with the treatment was significant for nitrate-N (Table 2). Overall, the results of the present study demonstrated that the application of ASA was capable of reducing the nitrate-N and nitrite-N contents of air-cured burley tobacco leaves.
2.4. Statistical analysis
3.3. Effects of ASA application on alkaloid contents of air-cured burley tobacco
All statistical analyses were conducted using Statistical Product and Service Solutions (SPSS) version 17.0 software (SPSS Inc., Chicago IL). Analysis of variance (ANOVA) was conducted on all data combined across years. For nitrate-N, nitrite-N, alkaloids and TSNA, analysis of variance was also conducted for each individual year. Treatment means for all above analysis were separated with Fisher’s protected LSD test at a 0.05 significant level.
Alkaloid contents of air-cured burley tobacco were significantly affected by different treatments (Table 2). It has to be noted that the four individual alkaloids and total alkaloids contents were reduced by application of ASA compared with the control in the 2-yr of the study (Table 5). Moreover, the 2-yr mean alkaloid contents in treatment B were lower than those in treatment A and control, of which nicotine, nornicotine, anatabine, anabasine and total alkaloids contents were significantly reduced by 14.44, 16.95, 7.31, 10.53 and 14.48%, respectively. It has also noticeable that four individual alkaloids and total alkaloids contents were significant differences between years, and no significant year × treatment interactions were observed for alkaloids (Table 2). The mean nicotine, nornicotine, anatabine, anabasine and total alkaloids contents in 2015 were lower than 2014 (Table 5). The results indicated that in the production of burley tobacco, the application of ASA was able to reduce alkaloid contents, particularly reduce nornicotine content. Since the nornicotine is the major metabolite of nicotine and precursor of NNN, these data also suggested that the reduction in nornicotine content by the application of ASA might be
3. Results 3.1. Effects of ASA application on TSNA contents of air-cured burley tobacco Analysis of variance of our data revealed that NNK, NNN, NAT, NAB and total TSNA contents were significantly different across treatments (Table 1). It is noticeable that the application of ASA reduced the NNK, NNN, NAT, NAB and total TSNA contents of air-cured burley tobacco in 2014 and 2015 (Table 3). And the 2-year mean TSNA contents of treatment B were lowest among three treatments, of which NNK, NNN, 224
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Table 2 Analysis of variance for treatment (T) and year (Y) effects on nitrate-N, nitrite-N, nicotine, nornicotine, anatabine, anabasine and total alkaloids contents of air-cured burley tobacco. Ftest values are shown. Source
df
Nitrate-N
Nitrite-N
Nicotine
Nornicotine
Anatabine
Anabasine
Total alkaloids
T Y T×Y
2 1 2
37.54*** 1123.87*** 29.86***
16.85*** 3322.82*** 0.09ns
11.14** 719.05*** 0.08ns
14.88** 11.44** 3.04ns
12.59** 961.69*** 0.06ns
5.98* 672.77*** 1.68ns
24.76*** 963.46*** 0.19ns
ns, not significant; *, ** and *** indicate significance at P = 0.05, 0.01 and 0.001 levels, respectively.
conducive to reducing NNN content and its undesirable effects of tobacco leaves.
Table 4 Mean effects of treatments on nitrate-N and nitrite-N contents of air-cured burley tobacco.
3.4. Effects of ASA application on sensory quality of air-cured burley tobacco The internal quality of burley tobacco leaves was reflected by the sensory quality. The sensory quality score indicators include style, aroma quality, aroma amount, concentration, undesirable smell, strength and residue taste (Table 6). Significant differences were observed among three treatments for all determinations except concentration, strength and residue taste (Table 6). The 2-yr mean total score of sensory quality of treatment B was highest among three treatments. The style, aroma quality and aroma amount of this treatment were higher than those in other treatments. The 2-yr mean total score of control treatment was lowest, indicating that the application of ASA have no negative effects on sensory quality of burley tobacco leaves. Overall, the application of ASA remained or improved the sensory quality of burley tobacco, especially improved aroma quality and aroma amount, and reduced undesirable smell. The year was also a significant factor for all determinations except residue taste (Table 6), which suggested that the sensory quality of burley tobacco was affected by different weather conditions in this study. The interaction of the year with the treatment was not significant for sensory quality parameters. Overall, the sensory quality of burley tobacco was better in 2015 than in 2014. These results were in accordance with the findings in TSNA contents of air-cured burley tobacco leaves in this study.
Treatment1
Year
Nitrate-N (μg g−1)
Nitrite-N (μg g−1)
Ck A B
Mean Mean Mean Level of 2014 2015 Level of 2014 2014 2014 Level of 2015 2015 2015 Level of
2563.89a2 2307.71b 1639.65c *** 3677.82a 663.01b *** 4382.50a 3988.91a 2662.05b ** 745.27 626.51 617.24 ns
39.44a 35.92b 35.26b *** 50.07a 18.68b *** 57.61a 54.28b 53.31b * 21.23a 17.55b 17.22b *
Ck A B Ck A B
significance
significance
significance
significance
ns, not significant; *, ** and *** indicate significance at P = 0.05, 0.01 and 0.001 levels, respectively. 1 Three concentrations of acetylsalicylic acid (ASA) were applied to wounds at burley tobacco main stalk within 1–2 h after topping. Treatment A, 0.1 mM ASA; Treatment B, 0.3 mM ASA; and the control treatment (Ck), smear water only. 2 Means in column within the treatments of each year, 2-yr mean or within the years averaged over the treatments followed by the same letter are not significantly different from each other at the P = 0.05 level of significance.
associated with the nasal cavities cancer, while NNK may be a predominantly determinant of lung cancers (Hecht, 2003, 2008), the tobacco industry is concerned with methodologies that could have effects on reducing TSNA levels. As ASA has been previously shown to reduce the levels of nitrate, nitrite and alkaloids (Hua et al., 2010; Huang et al., 2010), we investigated the accumulation of TSNA as affected by pre-harvest application of ASA. Interestingly, as shown in this study, the 2-yr mean total TSNA contents of air-cured burley
4. Discussion Since TSNA are by far the most prevalent strong carcinogens in tobacco and its products, of which NNN has been reported to be
Table 3 Mean effects of treatments on NNK, NNN, NAT, NAB and total TSNA contents of air-cured burley tobacco. Treatment1
Year
NNK (ng g−1)
NNN (ng g−1)
NAT (ng g−1)
NAB (ng g−1)
Total TSNA (ng g−1)
Ck A B
Mean Mean Mean Level of 2014 2015 Level of 2014 2014 2014 Level of 2015 2015 2015 Level of
61.27a2 51.99b 45.97c *** 67.29a 38.86b *** 78.88a 65.30b 57.69c ** 43.66a 38.67b 34.25c **
1616.22a 1420.00b 1332.01b ** 2231.78a 680.39b *** 2429.43 2210.70 2055.46 ns 803.21a 629.39b 608.56b ***
338.91a 284.03b 248.27c *** 462.69a 118.11b *** 538.55a 452.73b 396.80c ** 139.27a 115.33ab 99.73b *
9.38a 8.76a 6.61b *** 13.17a 3.32b *** 14.66a 14.29a 10.56b ** 4.09a 3.22b 2.66c **
2025.78a 1764.79b 1632.86b *** 2774.93a 840.68b *** 3061.32a 2742.97b 2520.51b * 990.23a 786.61b 745.20b ***
Ck A B Ck A B
significance
significance
significance
significance
ns, not significant; *, ** and *** indicate significance at P = 0.05, 0.01 and 0.001 levels, respectively. 1 Three concentrations of acetylsalicylic acid (ASA) were applied to wounds at burley tobacco main stalk within 1–2 h after topping. Treatment A, 0.1 mM ASA; Treatment B, 0.3 mM ASA; and the control treatment (Ck), smear water only. 2 Means in column within the treatments of each year, 2-yr mean or within the years averaged over the treatments followed by the same letter are not significantly different from each other at the P = 0.05 level of significance.
225
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Table 5 Mean effects of treatments on nicotine, nornicotine, anatabine, anabasine and total alkaloids contents of air-cured burley tobacco. Treatment1
Year
Nicotine (mg g−1)
Nornicotine (mg g−1)
Anatabine (mg g−1)
Anabasine (mg g−1)
Total alkaloids (mg g−1)
Ck A B
Mean Mean Mean Level of 2014 2015 Level of 2014 2014 2014 Level of 2015 2015 2015 Level of
24.80a2 22.81b 21.22b ** 31.26a 14.62b *** 33.22 30.95 29.61 ns 16.37a 14.66b 12.82c ***
5.84a 5.42b 4.85c ** 5.12b 5.62a ** 5.69a 5.32a 4.34b ** 5.98 5.52 5.36 ns
1.64a 1.64a 1.52b ** 1.94a 1.25b *** 1.99 1.98 1.86 ns 1.29a 1.29a 1.18b *
0.19a 0.18b 0.17b * 0.24a 0.12b *** 0.26 0.24 0.23 ns 0.13 0.12 0.11 ns
32.46a 30.04b 27.76c *** 38.56a 21.61b *** 41.15a 38.48ab 36.04b * 23.77a 21.59b 19.47c ***
Ck A B Ck A B
significance
significance
significance
significance
ns, not significant; *, ** and *** indicate significance at P = 0.05, 0.01 and 0.001 levels, respectively. 1 Three concentrations of acetylsalicylic acid (ASA) were applied to wounds at burley tobacco main stalk within 1–2 h after topping. Treatment A, 0.1 mM ASA; Treatment B, 0.3 mM ASA; and the control treatment (Ck), smear water only. 2 Means in column within the treatments of each year, 2-yr mean or within the years averaged over the treatments followed by the same letter are not significantly different from each other at the P = 0.05 level of significance. Table 6 Mean effects of treatments on the sensory quality of air-cured burley tobacco leaves. Treatment
Year
Style1
Aroma quality
Aroma amount
Concentration
Undesirable smell
Strength
Residue taste
Total score
Ck A B
Mean Mean Mean Level of significance 2014 2015 Level of significance
6.30b2 6.30b 6.35a * 6.20b 6.43a ***
5.27c 5.45b 5.60a *** 5.20b 5.68a ***
6.10c 6.23b 6.38a *** 6.17b 6.31a **
5.77 5.77 5.75 ns 5.50b 6.02a ***
5.53b 5.77a 5.80a *** 5.64b 5.76a *
6.27 6.27 6.28 ns 6.03b 6.51a ***
4.98 5.00 5.03 ns 5.00 5.01 ns
40.22c 40.78b 41.20a *** 39.74b 41.72a ***
ns, not significant; *, ** and *** indicate significance at P = 0.05, 0.01 and 0.001 levels, respectively. 1 The perfect total score is 70, composed of style 10, aroma quality 10, aroma amount 10, concentration 10, undesirable smell 10, strength 10, residue taste 10. 2 Means in column within the years averaged over the treatment followed by the same letter are not significantly different from each other at the P = 0.05 level of significance.
conducive to microorganism activity and the reduction of nitrate to nitrite. On the other hand, it is known that chemical, visual and physical characteristics are important traits of cured tobacco leaves, of which traits such as alkaloid contents and their composition ratio significantly influence the quality and safety of burley tobacco (Bilalis et al., 2015; Shi, 2013). The results of our study showed that the 2-yr mean alkaloid contents were reduced by the application of ASA, particularly the nornicotine content (Table 5). These observations were consistent with previous report of the application of ASA in tobacco (Huang et al., 2010). In addition to being the precursor of NNN, nornicotine may have harmful effects on human by inducing aberrant protein glycation and covalent binding to commonly used prednisone drugs (Dickerson and Janda, 2002). Therefore, the reduction in nornicotine content contributes to reducing tobacco-related health risks and improving the industrial availability of burley tobacco. In addition, although TSNA formation predominantly take place during the curing stage of tobacco leaves (Burton et al., 1989; Bush et al., 2001), nitrite and alkaloid in cured leaves continue to generate substantial TSNA during storage after curing. Recently, Shi et al. (2013) found that the total TSNA content was significantly increased by 215% after 12 months storage under the natural environment. It is also noteworthy that the levels of nitrite and alkaloids were reduced by the application of ASA. These observations also suggested that the lower levels of nitrite and alkaloids by the application of ASA might have a potential in reducing TSNA formation and accumulation in cured leaves during storage. Tobacco industry have made considerable efforts to produce tobacco leaves with lower TSNA levels, however, the challenge is to
tobacco leaves were significantly reduced by the application of 0.3 mM ASA (Table 3). It is also noted that 2-yr mean NNK and NNK contents in ASA treatments were significantly reduced, which has a positive effect to reduce the undesirable effects on the users of tobacco products. Moreover, the mean TSNA contents in 2014 were higher than those in 2015, which was probably related to the fact that the total rainfall from August to September (air-curing period) was higher in 2014 than that in 2015. This can be explain with the finding that curing tobacco at a higher humidity increased the accumulation of TSNA (Burton et al., 1989). Furthermore, this study also investigated the precursors of TSNA i.e. nitrite and alkaloid as affected by the application of ASA after topping. In the production of burley tobacco, the changes of the nitrite and alkaloid levels caused by topping would be reflected on TSNA contents of burley tobacco leaves. Therefore, if the levels of TSNA precursors i.e. nitrite and alkaloid could be reduced, the formation and accumulation of TSNA might be reduced as well. In this study, the nitrate-N and nitrite-N contents of air-cured burley tobacco leaves were significantly reduced by the application of ASA (Table 4). Our study was in agreement with those reports in Hua et al. (2010). High level of nitrate was common in burley tobacco due to high demanding of nitrogen fertilizer. Nitrite, the direct precursor and key limiting factor of TSNA formation, is mainly produced via leaf microbe-mediated reduction of nitrate, and the reduction in nitrate levels of tobacco leaves contributed to lowering nitrite levels and subsequently reducing TSNA formation during senescence and curing (Burton et al., 1992, 1994; Bush et al., 2001; Lu et al., 2016; Wei et al., 2014). Moreover, the higher mean nitrite-N content in 2014 may be related to wetter air-curing condition from August to September (Burton et al., 1992, 1994), which is 226
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find the best balance between having good quality and lower TSNA. It is of interest to note that the sensory quality of air-cured burley tobacco leaves remained or was even better by the application of ASA (Table 6). The aroma became more pure, undesirable smell was reduced, and the style of burley tobacco was maintaining, which is probably related the lower nornicotine levels by the application of ASA (Shi et al., 2011). These observations also suggested the value of ASA application in tobacco industry. In addition to being the raw material in manufacturing of cigarettes, the dried tobacco leaves are also an important source of various aroma products such as essential oil, concrete and absolute (Nedeltcheva-Antonova et al., 2016). Hence, the lower levels of TSNA in air-cured burley tobacco leaves by the application of ASA might be potentially used in various natural cosmetic and perfume formulations, and add additional economic value of burley tobacco. However, little is known about the effect of ASA application on TSNA of different tobacco types and varieties. Thus, further studies are necessary. Meanwhile, the detailed functional mechanism of ASA for reducing TSNA in air-cured burley leaves will be further investigated in a future study. These will contribute to the application of ASA in a feasible agronomic practice to lower TSNA formation and accumulation in the production of burley tobacco and expand the industrial availability of burley tobacco.
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5. Conclusions This study showed positive effects of ASA application on TSNA accumulation and quality of burley tobacco. The application of ASA reduced levels of TSNA, particularly the levels of NNK and NNN. Also, the application of ASA reduced the levels of nitrate-N, nitrite-N and alkaloid. Surprisingly after the application of ASA the sensory quality of air-cured burley tobacco leaves remained or was even better. Therefore, ASA could be considered as a promising strategy in balancing the levels of TSNA with other considerations such as flavor and smokeability. In particular, under strict regulation of TSNA levels in tobacco products, the application of ASA may be of significant value in sustaining tobacco industry. Conflict of interest The authors declare that they have no conflicts of interest. Acknowledgments This work was financially supported by a grant from the China National Tobacco Corporation (No. 2013JH04), and the Sichuan Province Corporation of China National Tobacco Corporation (No. 201304). We are very grateful to Dazhou City Corporation of Sichuan Province Tobacco Corporation for providing support during the field experiments. Also, we would like to thank the staff at the Anhui Tobacco Industrial Co., Ltd., Anhui, China for their technical assistance. References Baldwin, I.T., Schmelz, E.A., Ohnmeiss, T.E., 1994. Wound-induced changes in root and shoot jasmonic acid pools correlate with induced nicotine synthesis in Nicotiana sylvestris spegazzini and comes. J. Chem. Ecol. 20, 2139–2157. http://dx.doi.org/10. 1007/BF02066250. Baldwin, I.T., 1989. Mechanism of damage-induced alkaloid production in wild tobacco. J. Chem. Ecol. 15, 1661–1680. http://dx.doi.org/10.1007/BF01012392. Bilalis, D.J., Travlos, I.S., Portugal, J., Tsioros, S., Papastylianou, Y., Papatheohari, Y., Avgoulas, C., Tabaxi, I., Alexopoulou, E., Kanatas, P.J., 2015. Narrow row spacing increased yield and decreased nicotine content in sun-cured tobacco (Nicotiana tabacum L.). Ind. Crops Prod. 75, 212–217. http://dx.doi.org/10.1016/j.indcrop. 2015.05.057. Bokshi, A.I., Morris, S.C., Deverall, B.J., 2003. Effects of benzothiadiazole and acetylsalicylic acid on beta-1, 3-glucanase activity and disease resistance in potato. Plant Pathol. 52, 22–27. http://dx.doi.org/10.1046/j.1365-3059.2003.00792. x. Burton, H.R., Childs, G.H., Andersen, R.A., Fleming, P.D., 1989. Changes in chemical composition of burley tobacco during senescence and curing. 3. Tobacco-specific nitrosamines. J. Agric. Food Chem. 37, 426–430. http://dx.doi.org/10.1021/ jf00086a034.
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Acetylsalicylic acid application decreased tobacco-specific nitrosamines and its precursors but maintained quality of air-cured burley tobacco (Nicotiana tabacum L.)
MARK
⁎
Xiang Chena, Li Liub, Yanmin Zhangb, Xiaofeng Zhoub, Tao Linb, Youhong Songa, Jincai Lia, , ⁎ Xinsheng Chengb, a b
School of Agronomy, Anhui Agricultural University, Hefei 230036, China Research Center of Tobacco and Health, University of Science and Technology of China, Hefei 230051, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Nicotiana tabacum L. Tobacco-specific nitrosamines (TSNA) Nitrosamine precursor Sensory quality Acetylsalicylic acid
Burley tobacco (Nicotiana tabacum L.) is an important raw material in the production of Chinese-style blended cigarettes. However, the high levels of tobacco-specific nitrosamines (TSNA) have devalued the industrial use of burley tobacco and limited its sustainable use. The objective of this paper was to investigate the effects of acetylsalicylic acid (ASA) application on accumulation of TSNA, its precursors and quality of air-cured burley tobacco (Nicotiana tabacum L. cv. DaBai 1). Field experiments were conducted to determine the contents of TSNA and the precursors and the sensory quality of burley tobacco under 0, 0.1, 0.3 mM of ASA in 2014 and 2015 at Dazhou, Sichuan Province, China. The results showed that TSNA, nitrate, nitrite and alkaloids levels were significantly reduced by the application of ASA, of which the total TSNA were significantly lower by 10.40–24.75%, and two of the strong animal carcinogens in tobacco products, i.e. N-nitrosonornicotine (NNN) and 4-(methylnitrosamino) -1- (3- pyridyl) -1- butanone (NNK) were reduced by 9.01–24.23% and 11.43–26.86%, respectively. Year had significant effects on TSNA and the TSNA contents in 2014 were higher than in 2015. Interestingly, the sensory quality of burley tobacco remained or was even better after application of ASA. In conclusion, this study revealed that ASA application was able to reduce the levels of TSNA and its precursors in sustaining burley tobacco industry potentially.
1. Introduction Sichuan Province, located in southwest China, has a unique climate and geographical environment. Burley tobacco (Nicotiana tabacum L.) is a local major industrial crop and plays important roles in local economy and agricultural production. High-quality burley tobacco produced in Sichuan Province has been widely used in the production of Chinesestyle blended cigarettes because of a unique structure, physical properties and signature flavor. It is known that tobacco and its products contain a certain number of carcinogenic substances, and the carcinogenicity of tobacco products is primarily attributed to the presence of tobacco-specific nitrosamines (TSNA) (Hecht, 1998, 2008). With increasing concerns on smoking related health problems, the high levels of TSNA have affected the industrial development of burley tobacco and limited its sustainable use. It is generally thought that TSNA are undetectable or at a very low
level in fresh leaves prior to harvesting, but can be readily measured in air-cured leaves after curing. This is because TSNA are generated through the nitrosation of tobacco alkaloids in processes predominantly taking place during the curing stage of tobacco leaves, although additional formation can occur in the subsequent storage and processing of cured leaves, as well as via pyrosynthesis during combustion in some circumstances (Bush et al., 2001; Shi et al., 2013). The major four kinds of TSNA found in tobacco and its products are N-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), Nnitrosoanabasine (NAB) and N-nitrosoanatabine (NAT). Tobacco alkaloids and nitrite are important precursors of TSNA. Alkaloids can react with nitrite to form relevant TSNA i.e. NNN, NNK, NAB and NAT derived from nornicotine, nicotine, anabasine and anatabine, respectively (Fig. 1) (Bush et al., 2001; Hecht et al., 1983). NNN and NNK have been classified as Group I carcinogens (the highest designation) by International Agency for Research on Cancer (2007). Additionally, as
Abbreviations: ASA, acetylsalicylic acid; NAB, N-nitrosoanabasine; NAT, N-nitrosoanatabine; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NNN, N-nitrosonornicotine; TSNA, tobacco-specific nitrosamines ⁎ Corresponding authors. E-mail addresses: [email protected] (X. Chen), [email protected] (J. Li), [email protected] (X. Cheng). http://dx.doi.org/10.1016/j.indcrop.2017.04.031 Received 3 January 2017; Received in revised form 21 March 2017; Accepted 19 April 2017 0926-6690/ © 2017 Elsevier B.V. All rights reserved.
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Fig. 1. TSNA formed by nitrosation of tobacco alkaloids.
objective of this study was to investigate the effects of ASA application on TSNA accumulation and quality of air-cured burley tobacco leaves in the production of burley tobacco.
the NNN and NNK are the most prevalent strong carcinogens in tobacco and its products, they may ultimately be regulated in United States by U.S. Food and Drug Administration (FDA) (2011). Therefore, in recent years it has been a focus on reducing TSNA formation and accumulation in burley tobacco leaves and their products. To reduce TSNA in cured tobacco leaves, in addition to through the processing techniques, breeding new varieties and rational agronomic managements are proved to be effective. The levels of nicotine and TSNA were lower in tobacco products processed by the leaves from low nicotine varieties bred with genetic engineering techniques (Conkling, 2005). In recent years, the United States tobacco industry and other main tobacco producing countries have established a standard with a maximum limit of nornicotine content in burley tobacco leaves, and application of low nicotine conversion varieties to reduce TSNA contents in the production of burley tobacco (Jack et al., 2007; Shi, 2013). Burley tobacco requires a large amount of nitrogen fertilizer to produce high yield of cured leaf in ensuring profitable yields. However, excessive nitrogen fertilizer application produces air-cured leaves with undesirable levels of NO3-N and alkaloid (MacKown et al., 1984, 2000). The appropriate management of nitrogen fertilization, according to the local practice, is an effective measure to reduce TSNA levels in air-cured leaves (Chamberlain and Chortyk, 1992; Wahlberg et al., 1999). Cui et al. (1994) reported that the levels of TSNA in air-cured burley tobacco with maleic hydrazide application were reduced by 30–50% compared with the hand-suckered control. Furthermore, the addition of antioxidants such as ascorbic acid (Vitamin C) and tocopherol (Vitamin E) to tobacco prior to the harvesting were capable to reduce TSNA levels by scavenging free radicals and inhibiting the nitrosation reaction of nitrite and alkaloids (Krauss et al., 2003; Lathia et al., 1988; Lathia and Blum, 1989; Li et al., 2006; Rundlöf et al., 2000). It has been reported that 2-(acetyloxy) benzoic acid (acetylsalicylic acid, ASA) can increase yield, quality and the resistance to biotic and abiotic stresses in several plant species, for instance, it enhanced the resistance to tobacco mosaic virus in tobacco (Bokshi et al., 2003; Gimenez et al., 2014; Hashmi et al., 2012; Heller et al., 2013; White, 1979). As an important agronomic management to regulate the nutrition and quality of tobacco, topping (removal of the developing inflorescence) changed the sink–source relationships, hormonal balance and root development. Additionally, the wound caused by topping could induce the biosynthesis of many secondary metabolites e.g. nicotine (Baldwin, 1989; Baldwin et al., 1994; Fragoso et al., 2014; Qi et al., 2012). Recently, ASA was found to be applied to wounds at the main stalk of burley tobacco after topping, resulting in lower levels of alkaloids, nitrate and nitrite (Hua et al., 2010; Huang et al., 2010). The functional mechanism of ASA action in tobacco is unclear, though a hypothesis has been proposed (Zhang et al., 2011). Given ASA application is able to reduce the levels of alkaloids, nitrate and nitrite, it may have some effects on TSNA accumulation. Therefore the
2. Materials and methods 2.1. Experimental site The field experiments were conducted on a private farm located in Xuanhan, Dazhou, Sichuan Province, China (Latitude 31°16' N, Longitude 107°42' E; 545 m altitude) from April to October respectively in 2014 and 2015. The soil type at the field site is a typical brown soil with a medium loam texture. The 0- to 20-cm soil depth had the following characteristics: available N of 131.3 mg kg−1, Olsen P of 25.1 mg kg−1, available K of 106.9 mg kg−1, organic matter of 36 g kg−1, and pH 6.4. Monthly mean temperature and rainfall data during the growing season are shown in Fig. 2. Mean monthly temperatures from April to October in both experimental years closed to the 30-yr average temperature (Fig. 2a). The total rainfall from April to October was 1494 mm in 2014 and 1021 mm in 2015, respectively, which was different from 30-yr average (Fig. 2b). From August to September, the total rainfall in 2014 was higher by 190% than in 2015, indicating that burley tobacco air-curing processes in 2014 was exposed to a wetter condition. 2.2. Experimental design A randomized complete block design of three treatments was used, each treatment having three replicates. Treatment A was applied with 0.1 mM ASA (0.25 mL plant−1); treatment B was applied with 0.3 mM ASA (0.25 mL plant−1); treatment C (Ck) was applied with water only (0.25 mL plant−1) as the control treatment. The selection of two different concentrations in application of ASA was on the basis of previous studies (Hua et al., 2010; Huang et al., 2010). ASA was obtained from Sigma Chemical Company (St Louis, MO, USA) and freshly dissolved in water about 1-h before smearing. A commercial Burley tobacco (Nicotiana tabacum L. cv. DaBai 1) was used and grown in a seedbed with a growing medium consisting of 70% (v/v) peat culture substrate and 30% (v/v) perlite in both years. Pelleted seeds were sown on 24 February 2014 and 1 March 2015, and grown in a naturally illuminated glasshouse. Transplanting tobacco seedlings into the field was done on 25 and 29 April in 2014 and 2015 according to standard practices of plant arrangement with a row spacing of 1.20 m and a plant spacing of 0.45 m. The plant density was 16, 500 plants ha−1. The experiment consisted of 9 subplots in both years. Each subplot size was 6.0 m wide by 8.4 m long. Plots within a block were separated by a 1.2 m bare space and independent block were separated by a 2.5 m bare space. The 4 outside edges of the whole planting area 222
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Fig. 2. Mean monthly temperature (°C) (a) and rainfall (mm) (b) in 2014 (dark column) and 2015 (blank column) during the experiments (April-October) and 30-yr average (dark line) temperature (°C) (a) and rainfall (mm) (b) at Dazhou, Sichuan, China.
laboratory for sensory quality evaluation and chemical analysis. The sensory quality was determined by China Tobacco Anhui Industrial Co., Ltd. All the samples were freeze-dried, ground to pass through a 0.3mm screen and subsequently stored at −20 °C until analysis. NNN, NNK, NAB, NAT, nitrate-N, nitrite-N, nornicotine, nicotine, anatabine and anabasine were quantitatively analyzed with the methods presented below.
were bordered by two rows of extra guarding plants. Subsequent irrigation, depending on rainfall, was applied to maintain soil moisture around the field capacity level and ensure optimal burley tobacco plant water supply. No other pesticides were applied during the experiment. 210 kg N ha−1 in total were used in the burley tobacco plots in each year. P and K were also applied to the plots. The overall ratio of N: P2O5:K2O was 1:1:2 in both 2014 and 2015. The plots received 70% of N rate, 100% of P rate, and 60% of K rate with a compound fertilizer (N:P:K = 10:10:28) prior to burley tobacco transplanting. The rest of N and K were applied at 26-d after transplanting. The weeds in the field were removed manually. The cultural practices followed as recommendation for commercial tobacco cultivation by Sichuan Tobacco Company in both growing seasons. Burley tobacco plants were topped when plants in the field had produced 22–24 leaves on 5 and 12 July in 2014 and 2015, respectively. Subsequently, different concentrations of ASA were applied with a medium goat-hair smear to wounds at burley tobacco main stalk within 1–2 h after topping. Burley tobacco leaves were harvested manually from 12 July to 20 August in 2014 and 2015, and air cured by the recommended practices of the region.
2.3.1. Tobacco-specific nitrosamines analysis Quantitative determinations of NNK, NNN, NAT and NAB were performed according to China Tobacco Standard (YQT 29-2013). In brief, 1 g of ground sample was accurately weight into a conical flask and combined with 30 mL of 0.1 M ammonium acetate solution. The mixture was extracted in the ultrasonic generator for 40 min and 2 mL of extraction solution was centrifuged at 10,000 rpm for 5 min. Then 1 mL of supernatant was filtered through a 0.22 μm membrane and collected for analysis. TSNA were quantified by liquid chromatography tandem mass spectrometry (LC–MS/MS) coupled with electrospray ionization (ESI) (LTQ-Orbitrap XL, Thermo Fisher Scientific, USA). The analysis was performed on a 100 × 2.1 mm Hypersil Gold C18 column with 3 μm particle size (Thermo Fisher Scientific, USA) and the column temperature was set as 40 °C with an injection volume of 5 μL. The mobile phases were methanol solution (A) and 0.2% acetic acid water solution (B) with a flow rate 0.4 mL/min. The gradient elution conditions were programmed as follows: 0.0–2.0 min, 10–100% A; 2.0–4.5 min, 100–10% A; 4.5–6 min, 10% A. Mass spectrometer condi-
2.3. Sampling and measurements Fifty burley tobacco plants were randomly selected from each plot (excluding the edge row) and their middle leaves (from bottom to top: 11th-13th) were labeled to assist to mark the number for samples. Aircured burley tobacco leaf samples were collected and brought to the 223
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tions were controlled as follows: ESI ion source, positive ion mode, multiple reaction monitoring (MRM) mode, 3400 V spray voltage, 120 °C ion source temperature, 700 L/h nebulizer gas flow rate, 70 L/ h curtain gas flow rate, 100 ms scan time. Total TSNA were calculated as the sum of NNN, NNK, NAB and NAT. The method has the limits of quantification (LOQ) for NNK, NNN, NAT and NAB were 0.89 ng/g, 2.38 ng/g, 1.19 ng/g and 1.78 ng/g, respectively, and the limits of detection (LOD) were 0.27 ng/g, 0.72 ng/g, 0.36 ng/g and 0.54 ng/g, respectively.
Table 1 Analysis of variance for treatment (T) and year (Y) effects on NNK, NNN, NAT, NAB and total TSNA contents of air-cured burley tobacco. F-test values are shown. Source
df
NNK
NNN
NAT
NAB
Total TSNA
T Y T×Y
2 1 2
83.01*** 846.98*** 12.97**
11.26** 960.13*** 1.16ns
27.96*** 1194.49*** 8.89**
42.21*** 1458.11*** 14.56**
28.67*** 2011.83*** 3.99ns
ns, not significant; *, ** and *** indicate significance at P = 0.05, 0.01 and 0.001 levels, respectively.
2.3.2. Nitrate-N and nitrite-N analysis Nitrate-N and Nitrite-N contents in air-cured burley tobacco samples were quantified with UV–vis Spectrophotometer (Model 756P, Spectrum Instrument Company, Shanghai, China) according to the methodology of Hua et al. (2010). Sodium nitrate and sodium nitrite were served as the standard substance for nitrate and nitrite, respectively. For determination of nitrate, 0.2 mL of ground tobacco sample solution and 0.8 mL of 5% salicylic acid solution were mixed and added into 19 mL of 8% sodium hydroxide solution. The reaction mixture was stood for 25 min and determined for its absorbance at 410 nm. For determination of nitrite, 2 mL of ground tobacco sample solution, 4 mL of 1% sulfanilamide solution and 4 mL of 0.02% α-naphthylamine solution were mixed in the glass test tube and maintained in 25 °C thermostat water bath for 30 min. The absorbance of the sample was determined at 540 nm.
NAT, NAB and total TSNA contents in treatment B were significantly reduced by 24.98, 17.58, 26.74, 29.53 and 19.40%, respectively (Table 3). The year also had significant effects on the NNK, NNN, NAT, NAB and total TSNA contents (Table 1). The mean NNK and NNN contents were higher in 2014 than in 2015, and the same trend exhibited by NAT, NAB and total TSNA. Moreover, significant year × treatment interactions were observed for NNK, NAT and NAB (Table 1). As a consequence, the results indicated that the application of ASA was able to effectively reduce TSNA content, particularly reduce NNK and NNN contents. These suggested that the reduction of TSNA in aircured burley tobacco leaves by the application of ASA might reduce health risk associated with the use of tobacco and its products. 3.2. Effects of ASA application on Nitrate-N and Nitrite-N contents of aircured burley tobacco
2.3.3. Alkaloid analysis Nicotine, nornicotine, anatabine and anabasine contents in aircured leaf samples were quantitatively analyzed by gas chromatography-mass spectrometry (GC–MS) (Model HP6890/5975, Agilent Technologies Inc., USA) according to the China Tobacco Standard (YCT 383-2010). Trichloromethane was used as the extraction solvent, and 2-methylquinoline and 2, 4′-Bipyridine were served as the internal standards. For each sample, 0.3 g of ground tobacco was accurately weight into a conical flask, and 2 mL of 5% aqueous sodium hydroxide was added to moisten the burley tobacco sample and stood for 15 min. Then 20 mL of extraction solution was added into the tube and extracted in the ultrasonic generator for 15 min. The mixture was centrifuged for 5 min. After the sample and extraction solvent were separated, water was removed using anhydrous sodium sulfate and 2 mL of solvent was collected into chromatography bottle. Subsequently, extracts were injected into the gas chromatography for alkaloid separation and quantification. Total alkaloids were calculated as the sum of nicotine, nornicotine, anatabine and anabasine.
Significant differences were observed among three treatments for nitrate-N and nitrite-N (Table 2). Both nitrate-N and nitrite-N contents of ASA-treated burley tobacco were reduced in 2014 and 2015 (Table 4). Compared with control, the 2-yr mean nitrate-N content in treatment A and B were significantly reduced by 10 and 36.05%, respectively, and the 2-yr mean nitrite-N content in treatment A and B were also significantly reduced by 8.92 and 10.60%, respectively. The analysis of variance results also showed that the nitrate-N and nitrite-N contents were significantly affected by year (Table 2). The mean nitrate-N and mean nitrite-N contents were both higher in 2014 than in 2015, which was probably related with the difference in the precipitation dispersal during the two growing seasons as shown in Fig. 2b. Moreover, the interaction of the year with the treatment was significant for nitrate-N (Table 2). Overall, the results of the present study demonstrated that the application of ASA was capable of reducing the nitrate-N and nitrite-N contents of air-cured burley tobacco leaves.
2.4. Statistical analysis
3.3. Effects of ASA application on alkaloid contents of air-cured burley tobacco
All statistical analyses were conducted using Statistical Product and Service Solutions (SPSS) version 17.0 software (SPSS Inc., Chicago IL). Analysis of variance (ANOVA) was conducted on all data combined across years. For nitrate-N, nitrite-N, alkaloids and TSNA, analysis of variance was also conducted for each individual year. Treatment means for all above analysis were separated with Fisher’s protected LSD test at a 0.05 significant level.
Alkaloid contents of air-cured burley tobacco were significantly affected by different treatments (Table 2). It has to be noted that the four individual alkaloids and total alkaloids contents were reduced by application of ASA compared with the control in the 2-yr of the study (Table 5). Moreover, the 2-yr mean alkaloid contents in treatment B were lower than those in treatment A and control, of which nicotine, nornicotine, anatabine, anabasine and total alkaloids contents were significantly reduced by 14.44, 16.95, 7.31, 10.53 and 14.48%, respectively. It has also noticeable that four individual alkaloids and total alkaloids contents were significant differences between years, and no significant year × treatment interactions were observed for alkaloids (Table 2). The mean nicotine, nornicotine, anatabine, anabasine and total alkaloids contents in 2015 were lower than 2014 (Table 5). The results indicated that in the production of burley tobacco, the application of ASA was able to reduce alkaloid contents, particularly reduce nornicotine content. Since the nornicotine is the major metabolite of nicotine and precursor of NNN, these data also suggested that the reduction in nornicotine content by the application of ASA might be
3. Results 3.1. Effects of ASA application on TSNA contents of air-cured burley tobacco Analysis of variance of our data revealed that NNK, NNN, NAT, NAB and total TSNA contents were significantly different across treatments (Table 1). It is noticeable that the application of ASA reduced the NNK, NNN, NAT, NAB and total TSNA contents of air-cured burley tobacco in 2014 and 2015 (Table 3). And the 2-year mean TSNA contents of treatment B were lowest among three treatments, of which NNK, NNN, 224
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Table 2 Analysis of variance for treatment (T) and year (Y) effects on nitrate-N, nitrite-N, nicotine, nornicotine, anatabine, anabasine and total alkaloids contents of air-cured burley tobacco. Ftest values are shown. Source
df
Nitrate-N
Nitrite-N
Nicotine
Nornicotine
Anatabine
Anabasine
Total alkaloids
T Y T×Y
2 1 2
37.54*** 1123.87*** 29.86***
16.85*** 3322.82*** 0.09ns
11.14** 719.05*** 0.08ns
14.88** 11.44** 3.04ns
12.59** 961.69*** 0.06ns
5.98* 672.77*** 1.68ns
24.76*** 963.46*** 0.19ns
ns, not significant; *, ** and *** indicate significance at P = 0.05, 0.01 and 0.001 levels, respectively.
conducive to reducing NNN content and its undesirable effects of tobacco leaves.
Table 4 Mean effects of treatments on nitrate-N and nitrite-N contents of air-cured burley tobacco.
3.4. Effects of ASA application on sensory quality of air-cured burley tobacco The internal quality of burley tobacco leaves was reflected by the sensory quality. The sensory quality score indicators include style, aroma quality, aroma amount, concentration, undesirable smell, strength and residue taste (Table 6). Significant differences were observed among three treatments for all determinations except concentration, strength and residue taste (Table 6). The 2-yr mean total score of sensory quality of treatment B was highest among three treatments. The style, aroma quality and aroma amount of this treatment were higher than those in other treatments. The 2-yr mean total score of control treatment was lowest, indicating that the application of ASA have no negative effects on sensory quality of burley tobacco leaves. Overall, the application of ASA remained or improved the sensory quality of burley tobacco, especially improved aroma quality and aroma amount, and reduced undesirable smell. The year was also a significant factor for all determinations except residue taste (Table 6), which suggested that the sensory quality of burley tobacco was affected by different weather conditions in this study. The interaction of the year with the treatment was not significant for sensory quality parameters. Overall, the sensory quality of burley tobacco was better in 2015 than in 2014. These results were in accordance with the findings in TSNA contents of air-cured burley tobacco leaves in this study.
Treatment1
Year
Nitrate-N (μg g−1)
Nitrite-N (μg g−1)
Ck A B
Mean Mean Mean Level of 2014 2015 Level of 2014 2014 2014 Level of 2015 2015 2015 Level of
2563.89a2 2307.71b 1639.65c *** 3677.82a 663.01b *** 4382.50a 3988.91a 2662.05b ** 745.27 626.51 617.24 ns
39.44a 35.92b 35.26b *** 50.07a 18.68b *** 57.61a 54.28b 53.31b * 21.23a 17.55b 17.22b *
Ck A B Ck A B
significance
significance
significance
significance
ns, not significant; *, ** and *** indicate significance at P = 0.05, 0.01 and 0.001 levels, respectively. 1 Three concentrations of acetylsalicylic acid (ASA) were applied to wounds at burley tobacco main stalk within 1–2 h after topping. Treatment A, 0.1 mM ASA; Treatment B, 0.3 mM ASA; and the control treatment (Ck), smear water only. 2 Means in column within the treatments of each year, 2-yr mean or within the years averaged over the treatments followed by the same letter are not significantly different from each other at the P = 0.05 level of significance.
associated with the nasal cavities cancer, while NNK may be a predominantly determinant of lung cancers (Hecht, 2003, 2008), the tobacco industry is concerned with methodologies that could have effects on reducing TSNA levels. As ASA has been previously shown to reduce the levels of nitrate, nitrite and alkaloids (Hua et al., 2010; Huang et al., 2010), we investigated the accumulation of TSNA as affected by pre-harvest application of ASA. Interestingly, as shown in this study, the 2-yr mean total TSNA contents of air-cured burley
4. Discussion Since TSNA are by far the most prevalent strong carcinogens in tobacco and its products, of which NNN has been reported to be
Table 3 Mean effects of treatments on NNK, NNN, NAT, NAB and total TSNA contents of air-cured burley tobacco. Treatment1
Year
NNK (ng g−1)
NNN (ng g−1)
NAT (ng g−1)
NAB (ng g−1)
Total TSNA (ng g−1)
Ck A B
Mean Mean Mean Level of 2014 2015 Level of 2014 2014 2014 Level of 2015 2015 2015 Level of
61.27a2 51.99b 45.97c *** 67.29a 38.86b *** 78.88a 65.30b 57.69c ** 43.66a 38.67b 34.25c **
1616.22a 1420.00b 1332.01b ** 2231.78a 680.39b *** 2429.43 2210.70 2055.46 ns 803.21a 629.39b 608.56b ***
338.91a 284.03b 248.27c *** 462.69a 118.11b *** 538.55a 452.73b 396.80c ** 139.27a 115.33ab 99.73b *
9.38a 8.76a 6.61b *** 13.17a 3.32b *** 14.66a 14.29a 10.56b ** 4.09a 3.22b 2.66c **
2025.78a 1764.79b 1632.86b *** 2774.93a 840.68b *** 3061.32a 2742.97b 2520.51b * 990.23a 786.61b 745.20b ***
Ck A B Ck A B
significance
significance
significance
significance
ns, not significant; *, ** and *** indicate significance at P = 0.05, 0.01 and 0.001 levels, respectively. 1 Three concentrations of acetylsalicylic acid (ASA) were applied to wounds at burley tobacco main stalk within 1–2 h after topping. Treatment A, 0.1 mM ASA; Treatment B, 0.3 mM ASA; and the control treatment (Ck), smear water only. 2 Means in column within the treatments of each year, 2-yr mean or within the years averaged over the treatments followed by the same letter are not significantly different from each other at the P = 0.05 level of significance.
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Table 5 Mean effects of treatments on nicotine, nornicotine, anatabine, anabasine and total alkaloids contents of air-cured burley tobacco. Treatment1
Year
Nicotine (mg g−1)
Nornicotine (mg g−1)
Anatabine (mg g−1)
Anabasine (mg g−1)
Total alkaloids (mg g−1)
Ck A B
Mean Mean Mean Level of 2014 2015 Level of 2014 2014 2014 Level of 2015 2015 2015 Level of
24.80a2 22.81b 21.22b ** 31.26a 14.62b *** 33.22 30.95 29.61 ns 16.37a 14.66b 12.82c ***
5.84a 5.42b 4.85c ** 5.12b 5.62a ** 5.69a 5.32a 4.34b ** 5.98 5.52 5.36 ns
1.64a 1.64a 1.52b ** 1.94a 1.25b *** 1.99 1.98 1.86 ns 1.29a 1.29a 1.18b *
0.19a 0.18b 0.17b * 0.24a 0.12b *** 0.26 0.24 0.23 ns 0.13 0.12 0.11 ns
32.46a 30.04b 27.76c *** 38.56a 21.61b *** 41.15a 38.48ab 36.04b * 23.77a 21.59b 19.47c ***
Ck A B Ck A B
significance
significance
significance
significance
ns, not significant; *, ** and *** indicate significance at P = 0.05, 0.01 and 0.001 levels, respectively. 1 Three concentrations of acetylsalicylic acid (ASA) were applied to wounds at burley tobacco main stalk within 1–2 h after topping. Treatment A, 0.1 mM ASA; Treatment B, 0.3 mM ASA; and the control treatment (Ck), smear water only. 2 Means in column within the treatments of each year, 2-yr mean or within the years averaged over the treatments followed by the same letter are not significantly different from each other at the P = 0.05 level of significance. Table 6 Mean effects of treatments on the sensory quality of air-cured burley tobacco leaves. Treatment
Year
Style1
Aroma quality
Aroma amount
Concentration
Undesirable smell
Strength
Residue taste
Total score
Ck A B
Mean Mean Mean Level of significance 2014 2015 Level of significance
6.30b2 6.30b 6.35a * 6.20b 6.43a ***
5.27c 5.45b 5.60a *** 5.20b 5.68a ***
6.10c 6.23b 6.38a *** 6.17b 6.31a **
5.77 5.77 5.75 ns 5.50b 6.02a ***
5.53b 5.77a 5.80a *** 5.64b 5.76a *
6.27 6.27 6.28 ns 6.03b 6.51a ***
4.98 5.00 5.03 ns 5.00 5.01 ns
40.22c 40.78b 41.20a *** 39.74b 41.72a ***
ns, not significant; *, ** and *** indicate significance at P = 0.05, 0.01 and 0.001 levels, respectively. 1 The perfect total score is 70, composed of style 10, aroma quality 10, aroma amount 10, concentration 10, undesirable smell 10, strength 10, residue taste 10. 2 Means in column within the years averaged over the treatment followed by the same letter are not significantly different from each other at the P = 0.05 level of significance.
conducive to microorganism activity and the reduction of nitrate to nitrite. On the other hand, it is known that chemical, visual and physical characteristics are important traits of cured tobacco leaves, of which traits such as alkaloid contents and their composition ratio significantly influence the quality and safety of burley tobacco (Bilalis et al., 2015; Shi, 2013). The results of our study showed that the 2-yr mean alkaloid contents were reduced by the application of ASA, particularly the nornicotine content (Table 5). These observations were consistent with previous report of the application of ASA in tobacco (Huang et al., 2010). In addition to being the precursor of NNN, nornicotine may have harmful effects on human by inducing aberrant protein glycation and covalent binding to commonly used prednisone drugs (Dickerson and Janda, 2002). Therefore, the reduction in nornicotine content contributes to reducing tobacco-related health risks and improving the industrial availability of burley tobacco. In addition, although TSNA formation predominantly take place during the curing stage of tobacco leaves (Burton et al., 1989; Bush et al., 2001), nitrite and alkaloid in cured leaves continue to generate substantial TSNA during storage after curing. Recently, Shi et al. (2013) found that the total TSNA content was significantly increased by 215% after 12 months storage under the natural environment. It is also noteworthy that the levels of nitrite and alkaloids were reduced by the application of ASA. These observations also suggested that the lower levels of nitrite and alkaloids by the application of ASA might have a potential in reducing TSNA formation and accumulation in cured leaves during storage. Tobacco industry have made considerable efforts to produce tobacco leaves with lower TSNA levels, however, the challenge is to
tobacco leaves were significantly reduced by the application of 0.3 mM ASA (Table 3). It is also noted that 2-yr mean NNK and NNK contents in ASA treatments were significantly reduced, which has a positive effect to reduce the undesirable effects on the users of tobacco products. Moreover, the mean TSNA contents in 2014 were higher than those in 2015, which was probably related to the fact that the total rainfall from August to September (air-curing period) was higher in 2014 than that in 2015. This can be explain with the finding that curing tobacco at a higher humidity increased the accumulation of TSNA (Burton et al., 1989). Furthermore, this study also investigated the precursors of TSNA i.e. nitrite and alkaloid as affected by the application of ASA after topping. In the production of burley tobacco, the changes of the nitrite and alkaloid levels caused by topping would be reflected on TSNA contents of burley tobacco leaves. Therefore, if the levels of TSNA precursors i.e. nitrite and alkaloid could be reduced, the formation and accumulation of TSNA might be reduced as well. In this study, the nitrate-N and nitrite-N contents of air-cured burley tobacco leaves were significantly reduced by the application of ASA (Table 4). Our study was in agreement with those reports in Hua et al. (2010). High level of nitrate was common in burley tobacco due to high demanding of nitrogen fertilizer. Nitrite, the direct precursor and key limiting factor of TSNA formation, is mainly produced via leaf microbe-mediated reduction of nitrate, and the reduction in nitrate levels of tobacco leaves contributed to lowering nitrite levels and subsequently reducing TSNA formation during senescence and curing (Burton et al., 1992, 1994; Bush et al., 2001; Lu et al., 2016; Wei et al., 2014). Moreover, the higher mean nitrite-N content in 2014 may be related to wetter air-curing condition from August to September (Burton et al., 1992, 1994), which is 226
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find the best balance between having good quality and lower TSNA. It is of interest to note that the sensory quality of air-cured burley tobacco leaves remained or was even better by the application of ASA (Table 6). The aroma became more pure, undesirable smell was reduced, and the style of burley tobacco was maintaining, which is probably related the lower nornicotine levels by the application of ASA (Shi et al., 2011). These observations also suggested the value of ASA application in tobacco industry. In addition to being the raw material in manufacturing of cigarettes, the dried tobacco leaves are also an important source of various aroma products such as essential oil, concrete and absolute (Nedeltcheva-Antonova et al., 2016). Hence, the lower levels of TSNA in air-cured burley tobacco leaves by the application of ASA might be potentially used in various natural cosmetic and perfume formulations, and add additional economic value of burley tobacco. However, little is known about the effect of ASA application on TSNA of different tobacco types and varieties. Thus, further studies are necessary. Meanwhile, the detailed functional mechanism of ASA for reducing TSNA in air-cured burley leaves will be further investigated in a future study. These will contribute to the application of ASA in a feasible agronomic practice to lower TSNA formation and accumulation in the production of burley tobacco and expand the industrial availability of burley tobacco.
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