Skip to main content
NCBI home page
ACS Omega logoLink to ACS Omega
. 2021 Sep 20;6(39):25361–25371. doi: 10.1021/acsomega.1c03275

Screening of Polyphenols in Tobacco (Nicotiana tabacum) and Determination of Their Antioxidant Activity in Different Tobacco Varieties

Xinda Zou †, Amrit BK †, Abdur Rauf ‡,*, Muhammad Saeed §, Yahya S Al-Awthan ∥,⊥, Mohammed A Al-Duais #,∇, Omar Bahattab ∥, Muhammad Hamayoon Khan ‡, Hafiz A R Suleria †,*
PMCID: PMC8495694  PMID: 34632194

Abstract

graphic file with name ao1c03275_0004.jpg

Tobacco (Nicotiana tabacum) is an herbaceous plant originating from South America and processed into cigarettes for consumption. Polyphenols are considered vital components of tobacco in view of their contribution to antioxidant properties. This study aimed to determine the phenolic compounds in different tobacco varieties by applying cold extraction with methanol and distilled water. The extracts were screened for phenolic compound diversity and distribution as well as their antioxidant potential in different tobacco varieties. The results showed that the methanolic extract of tobacco SP-28 exhibited the highest value in the total phenolic content (24.82 ± 0.07 mg GAE/gd.w.) and total flavonoid content (4.42 ± 0.01 mg QE/gd.w.), while the water extract of tobacco SN-2 exhibited the highest value in the total condensed tannin (1.12 ± 0.03 mg CE/gd.w.). The radical scavenging capacities of tobacco SP-28 were relatively high in DPPH (18.20 ± 0.01 mg AAE/gd.w.) and FRAP (3.02 ± 0.10 mg AAE/gd.w.), whereas the ABTS value was the highest in tobacco SN-2 (37.25 ± 0.03 mg AAE/gd.w.), and the total antioxidant capacity was the highest in tobacco SN-1 (7.43 ± 0.18 mg AAE/gd.w.). LC-ESI-QTOF-MS/MS identified a total of 49 phenolic compounds, including phenolic acids (14), flavonoids (30), and other polyphenols (5) in four different tobacco varieties. Tobacco SP-28 showed the highest number of phenolic compounds, especially enriched in flavones. Our study highlights the antioxidant potential of tobacco extracts and reveals the phenolic distribution among different tobacco varieties that could support tobacco utilization in different pharmaceutical industries.

1. Introduction

Tobacco (Nicotiana tabacum) is one of the annual or limited perennial herbaceous plants in the Solanaceae family, which originated from South America.1 There are more than 60 known tobacco plants in the genus of Nicotiana, but only two of them, Nicotiana rustica and N. tabacum, are known to be made into cigarettes.2 Currently, tobacco becomes a very popular commercial plant because it is able to grow on relatively infertile land and is extremely profitable.3 China has been the largest tobacco grower in the world, which achieved above 1 million hectares of cultivated area in 2017.4 In recent years, the extraction of bioactive compounds from different plant and marine sources has become a popular trend.5−8 Tobacco (N. tabacum) has been proven to contain a large number of biologically active ingredients, such as alkaloids and polyphenols,9 which contain anti-oxidation, anti-inflammatory, and anti-fungal functions. The bioactive components in tobacco (N. tabacum) are mainly phenolics, flavonoids, terpenoids, alkaloids, and polysaccharides, which contribute to the functions of tobacco extracts.10 Meanwhile, it also contains a lot of aromatic compounds such as limonene, indole, pyridine, and phytosterols.9 The chemical composition of tobacco leaves is influenced by factors such as ripening, drying, fermentation, treatment processing, and storage. Polyphenols are important flavoring substances in tobacco, accounting for approximately 7% of dry weight, and their concentrations are determined by maturity, variety,11 and the temperature of the air-curing process.12

Polyphenols are the most common antioxidants in the daily diet,13 which can scavenge free radicals produced during cellular respiration and normal metabolism.14 There are three main mechanisms of antioxidant action: regulation of the activities of antioxidant enzymes to reduce the production of oxygen radicals,15 combination with free radicals to form phenolic oxygen radicals to stop the chain reaction,16 and reduction of the Fenton reaction by chelating with metal ions.17 The chlorogenic acid of tannin, scopolamine, hyoscyamine of coumarin, and rutin, flavone, and rhamnose of flavonoids are the main polyphenols in tobacco, among which chlorogenic acid, rutin, and scopolamine account for over 80% of the total content of polyphenols and are the most abundant polyphenols in tobacco leaves.18 Polyphenols are not only influencing the growth of tobacco, but the phenolic compounds and their metabolites are also aroma substances of cigarettes.12 Therefore, the content of polyphenols in tobacco determines the quality and flavor of cigarette products. The accumulation of scopolamine in tobacco plants may be a reaction of tobacco plants to adverse factors such as bacteria, mold, and chemical and mechanical damage.18 The known pathways for the synthesis of polyphenols in tobacco can be generally divided into three: the shikimic acid pathway, the acetic acid–malonic acid pathway, and the acetic acid–mevalonate pathway.19 A previous study shows that the content of polyphenols in different parts of tobacco also varies,12 and the main trend is the concentrate of middle leaf > lower leaf > upper leaf.

In this study, the phenolic compounds were estimated by the total phenolic content (TPC) assay, total flavonoid content (TFC) assay, and total condensed tannin (TCT) assay. Also, different antioxidant methods were applied to determine the antioxidant of these tobacco sample powders such as total antioxidant capacity (TAC) assay, ferric reducing antioxidant power (FRAP) determination, and 2,2′-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis-3- ethylbenzothiazoline-6-sulfonic acid (ABTS) free radical scavenging evaluation. Further, phenolic compounds were characterized by the LC-ESI-QTOF-MS/MS analysis and reported in several groups and subgroups. The Venn diagrams were sketched to illustrate the distribution of phenolic compounds in different tobacco varieties and Pearson’s correlation coefficient and the principal component analysis (PCA) were applied to explain the differences between antioxidant assays and phenolic composition. This work reveals the differences of phenolics among four different tobacco varieties and provides comprehensive information about their phenolic composition and distribution to support the utilization of tobaccos in different industries.

2. Results and Discussion

2.1. Polyphenol Estimation (TPC, TFC, and TCT)

The phenolic compounds were extracted using cold extraction of methanol (MK-399, MSN-1, MSN-2, and MP-28) and distilled water (WK-399, WSN-1, WSN-2, and WSP-28). The phenolic estimation was carried out by TPC, TFC, and TCT assays. Tobacco has been shown to contain a variety of phenolic compounds.20 These plant secondary metabolites are synthesized mainly through the shikimic acid and malonic acid pathways21 and provide protection against abiotic stress and pathogen infection.22 The main polyphenols in tobacco are chlorogenic acid and rutin (quercetin-3-rhamnosyl glucoside), which could be extracted from tobacco and tobacco waste.23 The data in Table 1 showed that the TPC values of SP-28 were higher than the other four varieties. These results also tally with the fact that SP-28 tobacco is more resistant to stress and has higher bacterial tolerance. A higher concentration of total phenols (15.80 mg GAE/g) from the Oriental tobacco sample has been reported in a previous study,24 which further proved the great potential of tobacco as a raw material of polyphenol products.

Table 1. Polyphenol Content Estimation of Four Tobacco Varietiesa.

sample name TPC (mg GAE/g) TFC (mg QE/g) TCT (mg CE/g)
WK-399 5.87 ± 0.05d 0.25 ± 0.00d 1.05 ± 0.02c
WSN-1 6.91 ± 0.15d 0.05 ± 0.00d 1.19 ± 0.04b
WSN-2 1.78 ± 0.23e 0.12 ± 0.00d 1.12 ± 0.03b
WSP-28 12.93 ± 0.29b 1.22 ± 0.00c 2.98 ± 0.04a
MK-399 11.15 ± 0.19b 3.01 ± 0.01b 1.14 ± 0.02b
MSN-1 4.85 ± 0.08d 1.13 ± 0.00c 0.73 ± 0.00d
MSN-2 8.53 ± 0.24c 4.04 ± 0.01a 0.80 ± 0.01c
MP-28 24.82 ± 0.07a 4.42 ± 0.01a 0.95 ± 0.01b,c
Open in a new tab
a

GAE, gallic acid equivalents; QE, quercetin equivalents; CE, catechin equivalents; WK-399, WSN-1, WSN-2, and WSP-28 are distilled water extractions, while MK-399, MSN-1, MSN-2, and MSP-2 are methanol extractions. Superscripts a, b, and c reveal a significant difference between different samples in a column, which was analyzed by one-way ANOVA Tukey’s HSD test (P < 0.05).

Flavonoids are formed from phenylalanine, tyrosine, and malonic acid and are commonly found in plants as glycosylated derivatives, such as rutin, which is abundant in tobacco.25 The main structures of flavonoids consisted of a C6–C3–C6 carbon skeleton.26 It was found that the flavonoids were extracted significantly higher in methanol than water as an extraction solvent. Generally, chalcone synthase is the key to the synthesis of flavonoids and is regulated by five genes.27 The expression intensity of these genes determines the difference in the concentration of flavonoids in different tobacco varieties.20 Meanwhile, the tobacco flavonoid content is also affected by disease, temperature, light intensity, and other factors.25 These reasons could illustrate why the value of the SN-1 tobacco total flavonoid content was merely 0.05 mg QE/g in water extraction and 1.13 mg QE/g in methanolic extraction. For total condensed tannins, the values of water extracts were significantly higher than methanolic extracts, which were 1.05 ± 0.02 mg CE/g in WK-399, 1.19 ± 0.04 mg CE/g in WSN-1, 1.12 ± 0.03 mg CE/g in WSN-2, and 2.98 ± 0.04 mg CE/g in WSP-28. A previous study has shown that this ingredient has inhibitory activity on the tobacco Mosaic virus.28 Therefore, the higher tannins can be extracted from the highly antiviral tobacco varieties. This result is consistent with the characteristics of SP-28 tobacco.

2.2. Antioxidant Activities (TAC, DPPH, FRAP, and ABTS)

Four different methods (TAC, DPPH, FRAP, and ABTS) were applied to identify the antioxidant activity of tobacco water and methanolic extract. DPPH and ABTS are stable free radicals that could determine the free radical scavenging capacity of antioxidants.29 The DPPH radical scavenging result of this study is from 8.89 ± 0.08 mg AAE/g (MSN-1) to 18.20 ± 0.01 mg AAE/g (WSP-28) (Table 2). Other research also has determined that the tobacco extract is an excellent antioxidant that contains a strong ability to scavenge DPPH free radicals.30 The scavenging effect is positively correlated with the concentration of the extract and is significantly superior to the vitamin C antioxidants commonly used in the food industry.31 Moreover, the highest value exists in tobacco SP-28 again, which means that the samples with the highest total phenol content have the strongest free radical scavenging ability. A similar trend appeared in the ABTS test, which has shown that antioxidant activities of SP-28 extraction were 37.01 ± 0.13 mg AAE/g. Therefore, tobacco SP-28 extraction has greater potential as an antioxidant in the food industry.

Table 2. Determination of Antioxidant Activities by the Free Radical Capture Capacitya.

sample DPPH (mg AAE/g) FRAP (mg AAE/g) ABTS (mg AAE/g) TAC (mg AAE/g)
WK-399 10.24 ± 0.05c 2.39 ± 0.10b 33.71 ± 0.26b 5.89 ± 0.31a,b
WSN-1 14.62 ± 0.06b 2.25 ± 0.06b 33.30 ± 0.03b 7.43 ± 0.18a
WSN-2 11.25 ± 0.04c 1.45 ± 0.07c 37.25 ± 0.03a 6.12 ± 0.24a
WSP-28 18.20 ± 0.01a 3.02 ± 0.10a 36.94 ± 0.14a 3.29 ± 0.27c
MK-399 10.39 ± 0.06c 1.67 ± 0.05b,c 25.00 ± 0.30c 5.95 ± 0.19a,b
MSN-1 8.89 ± 0.08d 1.31 ± 0.07c 26.25 ± 0.39c 4.32 ± 0.11c
MSN-2 12.17 ± 0.23b,c 2.87 ± 0.17a 26.79 ± 0.14c 3.79 ± 0.23c
MP-28 14.28 ± 0.09b 1.75 ± 0.01b,c 37.01 ± 0.13a 6.58 ± 0.14a
Open in a new tab
a

AAE, ascorbic acid equivalents; WK-399, WSN-1, WSN-2, and WSP-28 are distilled water extractions, while MK-399, MSN-1, MSN-2, and MSP-2 are methanol extractions. Superscripts a, b, and c reveal a significant difference between different samples in a column, which was analyzed by one-way ANOVA Tukey’s HSD test (P < 0.05).

The FRAP method can measure the antioxidant and reduction abilities of plant samples according to their ability to reduce Fe3+ to Fe2+.32 In FRAP, all values were significantly lower than the other assays, which showed that the highest value was merely 3.02 ± 0.10 mg AAE/g. This indicates that the tobacco extract has only a moderate reduction ability to trivalent iron. A study of tobacco and tobacco waste extract confirmed this view and showed that the tobacco extract was less effective at reducing Fe3+ ions than ascorbic acid.30 This cements the point that the antioxidant activity of tobacco polyphenols is not completely realized by electron transfer. Some researchers reported that the total phenol content in tobacco leaves has a strong positive correlation with FRAP results.33 In spite of this, such a kind of correlation was not found in this study; there was no consistency between the differences in iron reduction capacity and total phenol content among different tobacco varieties. In addition, tobacco SN-1 (6.12 ± 0.24 mg AAE/g) revealed a stronger antioxidant capacity than SP-28 in aqueous extraction. Thus, the antioxidant capacity is hard to predict from a result of one essay to another. According to a series of research results, different antioxidant activity determination methods give similar results in the detection of spices and seaweed extracts,34 but there are great differences in the detection of fruit and vegetable samples.35

Although the total phenolic content of the methanol extract was much higher than that of water extracts, it did not show the same trend in the antioxidant activity test. This might be due to the different phenolic components and concentrations in different tobacco varieties. Thus, LC-ESI-QTOF-MS/MS was applied to characterize untargeted phenolic compounds present in these different tobacco varieties.

2.3. LC-ESI-QTOF-MS/MS Characterization of Tobacco Extraction

The extractions of four tobacco samples were analyzed by LC-ESI-QTOF-MS/MS and several chemicals were identified. In this study, negative ([M – H]−) and positive ([M + H]+) ionization modes are processed (see the Supporting Information). The polyphenols were tentatively characterized in Agilent LC/MS mass hunter qualitative software, which was based on the differences in m/z ratio in the MS spectra, and Personal Compounds Database and Library (PCDL) was also applied. Further analysis sorted out 49 compounds, which had over an 80 PCDL library score and the mass error < 5 ppm.

Table 3 shows that 49 polyphenols are present in solvent mixtures of aqueous and methanolic extracts in a ratio of 1:1 (v/v) of four different tobacco species (K-399, SN-1, SN-2, and SP-28). Phenolic compounds were classified into phenolic acids (14), flavonoids (30), and other polyphenols (5). Most of the phenolic compounds were included in flavonoids and phenolic acids and the flavonoids were more abundant. The variation in polyphenol compounds in the four tobacco species led to different free radical scavenging abilities between four kinds of tobacco varieties.

Table 3. Polyphenols in Different Tobacco Samples Using LC-ESI-QTOF-MS/MSa.

proposed compound molecular formula RT (min) ionization (ESI+/ESI–) molecular weight theoretical (m/z) observed (m/z) error (ppm) MS/MS product ion sample
phenolic acids                  
hydroxybenzoic acids                  
1. gallic acid C7H6O5 11.133 [M – H]− 170.0215 169.0142 169.0135 –4.1 125 SN-2
2. protocatechuic acid 4-O-glucoside C13H16O9 12.325 [M – H]− 316.0794 315.0721 315.0709 –3.8 153 SN-1
3. 2,3-dihydroxybenzoic acid C7H6O4 15.181 [M – H]− 154.0266 153.0193 153.0191 –1.3 109 *K-399, SN-2
4. 2-hydroxybenzoic acid C7H6O3 20.014 **[M – H]− 138.0317 137.0244 137.0243 –0.7 93 *SP-28, K-399, SN-2
hydroxycinnamic acids                  
5. verbascoside C29H36O15 4.228 [M – H]− 624.2054 623.1981 623.1989 1.3 477, 461, 315, 135 SN-1
6. 3-feruloylquinic acid C17H20O9 11.461 [M – H]− 368.1107 367.1034 367.1032 –0.5 298, 288, 192, 191 SN-1
7. caffeoyl glucose C15H18O9 12.666 [M – H]− 342.0951 341.0878 341.0891 3.8 179, 161 SN-1
8. caffeic acid C9H8O4 15.871 **[M – H]− 180.0423 179.0350 179.0341 –5.0 143, 133 *SP-28, SN-2
9. caffeic acid 3-O-glucuronide C15H16O10 19.297 [M – H]− 356.0743 355.0670 355.0653 –4.8 179 *SP-28, SN-2
10. ferulic acid 4-O-glucuronide C16H18O10 20.530 [M – H]− 370.0900 369.0827 369.0826 –0.3 193 SP-28
11. sinapic acid C11H12O5 22.639 **[M – H]− 224.0685 223.0612 223.0605 –3.1 205, 163 *SP-28, K-399
12. m-coumaric acid C9H8O3 28.689 **[M – H]− 164.0473 163.0400 163.0397 –1.8 119 *SP-28, K-399
hydroxyphenylpropanoic acids                  
13. dihydrocaffeic acid 3-O-glucuronide C15H18O10 19.003 [M – H]− 358.0900 357.0827 357.0817 –2.8 181 SP-28
14. dihydroferulic acid 4-O-glucuronide C16H20O10 23.973 [M – H]− 372.1056 371.0983 371.0977 –1.6 195 SP-28
flavonoids                  
dihydrochalcones                  
15. 3-hydroxyphloretin 2′-O-glucoside C21H24O11 17.087 **[M – H]− 452.1319 451.1246 451.1226 –4.4 289, 273 *SP-28, K-399, SN-2
16. phloridzin C21H24O10 31.058 [M – H]− 436.1369 435.1296 435.1278 –4.1 273 SN-1
dihydroflavonols                  
17. dihydromyricetin 3-O-rhamnoside C21H22O12 19.604 **[M – H]− 466.1111 465.1038 465.1021 –3.7 301 *SP-28, K-399
18. dihydroquercetin 3-O-rhamnoside C21H22O11 31.493 [M – H]− 450.1162 449.1089 449.1075 –3.1 303 *SN-1, SP-28
flavanols                  
19. 4′-O-methylepigallocatechin C16H16O7 10.052 [M + H]+ 320.0896 321.0969 321.0963 –1.9 302 SP-28
20. (+)-catechin C15H14O6 13.897 **[M – H]− 290.0790 289.0717 289.0707 –3.5 245, 205, 179 *SN-2, SP-28, K-399
21. procyanidin dimer B1 C30H26O12 16.775 **[M – H]− 578.1424 577.1351 577.1359 1.4 451 *K-399, SP-28
22. procyanidin trimer C1 C45H38O18 19.173 **[M – H]− 866.2058 865.1985 865.1990 0.6 739, 713, 695 *K-399, SP-28
23. (+)-gallocatechin C15H14O7 20.183 **[M – H]− 306.0740 305.0667 305.0666 –0.3 261, 219 *SN-2, K-399
flavanones                  
24. neoeriocitrin C27H32O15 22.819 [M – H]− 596.1741 595.1668 595.1669 0.2 431, 287 SP-28
25. narirutin C27H32O14 30.646 [M – H]− 580.1792 579.1719 579.1721 0.3 271 SN-1
flavones                  
26. 6-hydroxyluteolin 7-O-rhamnoside C21H20O11 25.270 [M – H]− 448.1006 447.0933 447.0935 0.4 301 *SP-28, SN-1
27. apigenin 6,8-di-C-glucoside C27H30O15 26.952 [M – H]− 594.1585 593.1512 593.1524 2.0 503, 473 SP-28
28. rhoifolin C27H30O14 27.010 **[M – H]− 578.1636 577.1563 577.1573 1.7 413, 269 *SP-28, SN-1
29. apigenin 6-C-glucoside C21H20O10 27.933 **[M – H]− 432.1056 431.0983 431.0968 –3.5 413, 341, 311 *SN-1, SN-2, SP-28
30. diosmin C28H32O15 29.100 **[M + H]+ 608.1741 609.1814 609.1787 –4.4 301, 286 *SP-28, SN-1
31. chrysoeriol 7-O-glucoside C22H22O11 30.050 **[M + H]+ 462.1162 463.1235 463.1217 –3.9 445, 427, 409, 381 SN-1, *SP-28
flavonols                  
32. myricetin 3-O-galactoside C21H20O13 16.784 [M – H]− 480.0904 479.0831 479.0818 –2.7 317 SP-28
33. kaempferol 3-O-glucosyl-rhamnosyl-galactoside C33H40O20 24.110 [M – H]− 756.2113 755.2040 755.2040 0.0 285 SP-28
34. kaempferol 3-O-(2″-rhamnosyl-galactoside) 7-O-rhamnoside C33H40O19 26.474 [M – H]− 740.2164 739.2091 739.2124 4.5 593, 447, 285 SP-28
35. kaempferol 3,7-O-diglucoside C27H30O16 28.515 **[M-H]− 610.1534 609.1461 609.1451 –1.6 447, 285 *SP-28, SN-1, SN-2
36. myricetin 3-O-rhamnoside C21H20O12 29.834 **[M-H]− 464.0955 463.0882 463.0863 –4.1 317 *SP-28, SN-1, SN-2
37. quercetin 3-O-(6″-malonyl-glucoside) C24H22O15 31.680 [M + H]+ 550.0959 551.1032 551.1008 –4.4 303 SN-2
isoflavonoids                  
38. 6″-O-acetylglycitin C24H24O11 9.159 **[M + H]+ 488.1319 489.1392 489.1391 –0.2 285,270 SP-28
39. 3′-hydroxygenistein C15H10O6 17.278 [M + H]+ 286.0477 287.0550 287.0539 –3.8 269,259 *K-399, SP-28
40. 3′-hydroxydaidzein C15H10O5 22.627 [M + H]+ 270.0528 271.0601 271.0588 –4.8 253, 241, 225 K-399
41. 5,6,7,3′,4′-pentahydroxyisoflavone C15H10O7 29.837 **[M + H]+ 302.0427 303.0500 303.0490 –3.3 285, 257 K-399, *SP-28, SN-2
42. violanone C17H16O6 31.058 [M – H]− 316.0947 315.0874 315.0862 –3.8 300, 285, 135 SN-1
43. 6″-O-malonylgenistin C24H22O13 31.447 **[M + H]+ 518.1060 519.1133 519.1114 –3.7 271 SN-1, *SP-28
44. glycitin C22H22O10 35.318 [M + H]+ 446.1213 447.1286 447.1274 –2.7 285 SP-28
other polyphenols                  
hydroxybenzaldehydes                  
45. 4-hydroxybenzaldehyde C7H6O2 31.363 **[M – H]− 122.0368 121.0295 121.0292 –2.5 77 *SN-1, SP-28, K-399, SN-2
hydroxycoumarins                  
46. coumarin C9H6O2 9.554 [M + H]+ 146.0368 147.0441 147.0445 2.7 103, 91 *SP-28, SN-2
tyrosols                  
47. hydroxytyrosol 4-O-glucoside C14H20O8 24.382 [M – H]− 316.1158 315.1085 315.1078 –2.2 153, 123 SP-28
lignans                  
48. schisandrol B C23H28O7 5.936 [M + H]+ 416.1835 417.1908 417.1926 4.3 224, 193, 165 *SN-2, SP-28
stilbenes                  
49. 3′-hydroxy-3,4,5,4′-tetramethoxystilbene C17H18O5 30.612 [M + H]+ 302.1154 303.1227 303.1217 –3.3 229, 201, 187, 175 SP-28
Open in a new tab
a

Single asterisk (*): compounds are characterized in more than one sample, but data presented in the table belong to the asterisk sample. Double asterisk (**): compounds were detected in both positive ionization mode [M + H]+ and negative ionization mode [M – H]−, whereas the data were presented in single mode.

2.3.1. Phenolic Acids

Generally, most of the phenolic acid compound ionization was presented in negative mode; this was due to the fact that ESI– mode was more sensitive to the characterization of phenolic acids.36 The hydrogen atom donation ability provides phenolic acids radical scavenging activity, which makes these compounds be able to act as natural antioxidants.37 Among, 14 kinds of phenolic acids were identified in the water and methanol extractions of four tobacco species. They were further classified as hydroxybenzoic (4), hydroxycinnamic (8), and hydroxyphenylpropanoic acids (2).

2.3.1.1. Hydroxybenzoic Acids and Hydroxyphenylpropanoic Acids

Gallic acid, 2,3-dihydroxybenzoic acid, and 2-hydroxybenzoic acid were detected in tobacco SN-2 at m/z 169.0135, m/z 153.0191, and m/z 137.0243. Gallic acid was also reported in mango by-products38 and ginger.37 Compound 2, which was extracted from tobacco SN-1, was tentatively identified as protocatechuic acid 4-O-glucoside, which generated a [M – H]− ion at m/z 315.0709. Protocatechuic acids were abundant in fruits of fishtail palm and jelly palm.39 A previous study has shown that gallic acid and 2,3-dihydroxybenzoic acid exist in strawberry hops and juniper berries.40 The product ions of 2,3-dihydroxybenzoic acid in MS/MS analysis indicated the loss of CO2 (44 Da) from precursor ions.41 Hydroxyphenylpropanoic acid components were only found in tobacco SP-28, which were dihydrocaffeic acid 3-O-glucuronide (RT = 19.003 min with m/z 357.0817) and dihydroferulic acid 4-O-glucuronide (RT = 23.973 min with m/z 371.0977). These ingredients are naturally versatile antioxidants with a wide range of potential medical and industrial applications.42

2.3.1.2. Hydroxycinnamic Acids

Ferulic acid was commonly found in foods including rice, oats, pineapple, coffee, and peanuts,43 but it was rarely found in tobacco. In this study, two ferulic acid derivatives were detected in tobacco SN-1 and SP-28, which were 3-feruloylquinic acid (RT = 11.461 min with m/z 367.1032) and ferulic acid 4-O-glucuronide (RT = 20.530 min with m/z 20.530). Caffeic acid and its derivatives, which normally are glycosides formed primarily with other sugars, are common in tobacco and its smoke.44 In our study, the precursor ions found at m/z 341.0891 (compound 7), m/z 179.0341 (compound 8), and m/z 355.0653 (compound 9) represented the existence of caffeoyl glucose, caffeic acid, and caffeic acid 3-O-glucuronide. The MS/MS product ions at m/z 143 and m/z 133 were formed by caffeic acid losing 2H2O and HCOOH.40 The derivatives of caffeic, sinapic, and ferulic acids were also detected in edible parts of palm fruits,39 black spices,45 garlic, and cherry.46

2.3.2. Flavonoids

Flavonoids are the largest phenolic compound group in this study; 30 identified flavonoids were divided into flavanols (5), flavanones (2), flavones (6), flavonols (6), isoflavonoids (7), dihydrochalcones (2), and dihydroflavonols (2). It is worth noting that tobacco SP-28 contains almost all kinds of flavonoids that have been isolated. There is evidence that dietary intake of isoflavones and flavones is inversely associated with cancer risk.47 Therefore, tobacco SP-28 extraction has the potential as a functional food additive.

2.3.2.1. Dihydrochalcones

In the present work, only two dihydrochalcones were present in tobacco samples. Compound 15 with precursor ions found at m/z 451.1226 in both positive and negative modes was identified as 3-hydroxyphloretin 2′-O-glucoside. This substance was also reported in black spices,45 fruit peels,48 and juniper berries.49 The other dihydrochalcone compound was phloridzin (RT = 31.058 min with m/z 435.1278), which contained peak fragmentation at m/z 273 caused by the consecutive loss of glucoside.40

2.3.2.2. Flavanols and Flavanones

A total of five flavanols and two flavanones were divided from tobacco extractions. The procyanidin dimer B1 (RT = 16.775 min with m/z 577.1359) and procyanidin trimer C1 (RT = 19.173 min with m/z 865.1990) were only detected in tobacco K-399 and SP-28. More cyanidins were reported in previous studies, such as cyanidin 3-O-rutinoside, acylated cyanidin 3-O-(coumaroyl) rutinoside,50 and cyanidin 3-O-rutinoside chloride.25 The catechin and their derivatives were the most abundant components in group flavanols, among which 4′-O-methylepigallocatechin was identified with the precursor ion [M + H]+ at m/z 321.0963. This compound has also been reported in Elaeodendron transvaalense, a kind of medicine plant located in southern African countries.51

2.3.2.3. Flavones, Flavonols, and Dihydroflavonols

There were six flavanols and six flavanones detected in tobacco samples except K-399. Several flavone and flavonol components identified were in the form of glycosides, most of which were combined with rhamnoside and glucoside. Compounds 33, 34, and 35, which had the precursor ion [M – H]− at m/z 755.2040, 739.2124, and 609.1451, were tentatively characterized as kaempferol 3-O-glucosyl-rhamnosyl-galactoside, kaempferol 3-O-(2″-rhamnosyl-galactoside) 7-O-rhamnoside, and kaempferol 3,7-O-diglucoside. In addition, many other kaempferol derivatives have been reported, such as astragalin and nicotiflorin.25 The precursor ion [M – H]− at m/z 285 was generated by losing a neutral hexose [M-C5H10O5]− of the ion [M – H]− at m/z 285. The other groups of flavonols detected in this study were myricetin 3-O-galactoside (RT = 16.784 min with m/z 479.0818) and myricetin 3-O-rhamnoside (RT = 29.834 min with m/z 463.0863) in negative mode. Apigenin derivatives have also been found in air-cured tobacco in a previous study.52 In tobacco SN-1, SN-2, and SP-28, compound 29 was identified as apigenin 6-C-glucoside, which had the precursor ion [M – H]− at m/z 431.0968.

2.3.2.4. Isoflavonoids

Compounds 38, 40, and 44 with precursor ions [M + H]+ at m/z 489.1391, m/z 271.0588, and m/z 447.1274 were tentatively characterized as 6″-O-acetylglycitin, 3′-hydroxydaidzein, and glycitin. These three isoflavonoids were rarely found in tobacco samples but frequently observed in legume plants, such as soybean53 and black bean.54 Moreover, compound 42 was identified as violanone, which detected precursor ions at m/z 315.0862 in negative ionization mode. This substance was found in tobacco samples for the first time.

2.3.3. Other Polyphenols

Five other polyphenol compounds were detected in these four kinds of tobaccos, which were divided into hydroxybenzaldehydes, hydroxycoumarins, tyrosols, lignans, and stilbenes. The product ions of coumarin (RT = 9.554 min with m/z 147.0445), which were located at m/z 103 and m/z 91, were generated by losing CO2 and two CO.55 4-Hydroxyfenzaldehyde was the only detected hydroxybenzaldehyde (RT = 31.363 min with m/z 121.0292), which exists in all four tobacco varieties. This compound was found in both negative and positive ionization modes, which contained peak fragmentation at m/z 77. In this group, tobacco SP-28 contained all species of polyphenols including hydroxytyrosol 4-O-glucoside, schisandrol B, and 3′-hydroxy-3,4,5,4′-tetramethoxystilbene.

2.4. Phenolic Content Distribution in Tobacco

Various polyphenols exist in tobacco samples that have conjugated structures in forms, and there are differences in their distribution in different tobaccos. Therefore, analyzing the variability of these polyphenols species in different tobacco samples at the same time would be a complex task. The Venn diagrams (Figure 1) were sketched in this study to offer a synopsis of different phenolic compound distributions, which were labeled with different colors in tobacco SN-1 (yellow), SN-2 (green), SP-28 (red), and K-399 (blue).

Figure 1.

Figure 1

Open in a new tab

Venn diagrams of polyphenol components determined in different tobacco samples. (A) Total phenolic compound distribution in different tobacco species and (B) relations of phenolic acids present in different tobacco samples. (C) Flavonoid distribution in tobacco samples and (D) other phenolic distribution situation in all four different varieties of tobaccos.

The Venn diagrams showed that SP-28 contained 29 unique compounds, which account for 16% of the total phenolic compound. Meanwhile, tobacco SN-1, SN-2, and K-399 contain 17 (9.4%), 15 (8.3%), and 11 (6.3%), respectively. The maximum value of overlapping total phenolic compounds that were distributed in tobacco SP-28 and SN-1 was 30 (16.6%), among which 24 (27.3%) of them belonged to flavonoids. The minimum value of overlapping total phenols present in tobacco SN-1, SN-2, and K-399 was 2 (1.1%). Moreover, there were 15 total phenolic compounds commonly existing in four different tobacco species. Previous researchers found that the composition of polyphenols could be applied as a parameter to characterize the class of tobaccos.56 This indicates that tobacco SP-28 is the most abundant polyphenol tobacco species. It has been shown in the literature that phenols are responsible for tobacco resistance to pathogens and can influence aromatic properties.24 As a result, tobacco with high stress resistance and a superior aroma would contain higher phenolics.

In other studies, flue-cured tobacco leaves contain more flavonoids than phenolic acids,57 and this pattern is also reflected in the present study. The Venn diagram shows that all tobacco species presented more phenol species in most overlapping phenols and all unique phenols. The maximum unique phenolic acids and flavonoids were still located in tobacco SP-28, which were 8 (19%) and 14 (15.9%), respectively. One obvious difference is that there was no common overlapping phenolic acid among tobacco SP-28 and SN-1, while this area had the highest similarity of flavonoids, which was 24 (27.3%). The other trend was that seven phenols were commonly found in four tobaccos, which were distributed in one phenolic acid, five flavonoids, and other phenolics.

2.5. Correlation between the Phenolic Content and Antioxidant Activities

The correlation of the phenolic content (TPC, TFC, and TCT) and antioxidant activities (DPPH, FRAP, ABTS, and TAC) was evaluated by Pearson’s correlation test, and the correlation coefficients are presented in Table 4. Additionally, the similarities and differences between methods applied to estimate antioxidant activity and measure the phenolic content were investigated by principal component analysis (PCA), and these are summarized in Figure 2.

Table 4. Correlation between the Phenolic Content (TPC, TFC, and TCT) and Antioxidant Activities (DPPH, FRAP, and ABTS) Performed as Pearson’s Correlation Coefficients (r).

variable TPC TFC TCT DPPH FRAP ABTS
TFC 0.724a          
TCT 0.165 –0.216        
DPPH 0.484 0.063 0.790b      
FRAP 0.113 0.082 0.563 0.629a    
ABTS 0.246 –0.302 0.458 0.601 0.153  
TAC 0.079 –0.188 –0.422 –0.141 –0.457 0.253
Open in a new tab
a

Significant level, P ≤ 0.05 of correlation.

b

Significant level, P ≤ 0.01 of correlation.

Figure 2.

Figure 2

Open in a new tab

Principal component analysis (PCA) of the phenolic content (TPC, TFC, and TCT) and antioxidant determination assays (DPPH, ABTS, FRAP, and TAC) of four tobacco species.

Only two antioxidant assays showed a positive significant correlation, which were DPPH and FRAP with the Pearson’s correlation coefficient r = 0.629 (P < 0.05). A previous study has proved a significant positive correlation existing between FRAP and other antioxidant assays.58 Furthermore, a highly significant positive correlation has been found between TCT and DPPH with Pearson’s correlation coefficient r = 0.79 (P < 0.01), which revealed the same trend in the studies of Li et al.59 and Wang et al.60 The reason is that the phenolic compounds in tobacco are able to offer H to DPPH free radicals to form DPPH-H.59 The TPC assay was detected having a significant positive correlation with antioxidant activity including ABTS, DPPH, and FRAP.61 However, these correlations did not reach a significant level in this study. Other antioxidant assays were also strongly correlated with each other in a previous report62 but not presented in this study. This was due to the finding that phenols in extracts of tobacco samples have different scavenging abilities to DPPH, ABTS free radicals, and reducing Fe3+-TPTZ. Apart from the above, the TPC has shown a significant positive correlation with the TFC; this supported the finding in phenolic content distribution in tobacco that flavonoids occupy a dominant position in tobacco polyphenols.

Figure 2 indicates that there was 66.14% of total variability present in the initial data, which was kept by the first two factors F1 and F2. The distance between two assays presents the proximity level of them; the closer the two vectors are, the more significant the correlation is. For instance, the distances of assays FRAP and DPPH and assays DPPH and TCT were very close, so it contained significant positive correlations, which are shown in Pearson’s correlation coefficient table (Table 4). In short, this study highlights the antioxidant potential of tobacco extraction and reveals the polyphenol diversity among different tobacco species, which could support tobacco by-products utilized as additives in different industries for bioactive product development.

3. Methodology

3.1. Chemical and Reagents

Several chemicals of analytical grade that were used for extraction and characterization were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia) including 2,2′-azino-bis (3 ethylbenzothiazoline-6-sulfonic acid) (ABTS), Folin and Ciocalteu’s phenol reagent, 2,2′-diphenyl-1-picrylhydrazyl (DPPH), l-ascorbic acid, vanillin, hexahydrate aluminum chloride, ferric chloride, gallic acid, quercetin, and 2,4,6-tripyridyl-s-triazine (TPTZ). The supplier of hydrochloric acid, glacial acetic acid, hydrated sodium acetate, methanol, and anhydrous sodium acetate was Thermo Fisher Scientific Inc. (Waltham, MA, USA). Sodium carbonate (anhydrous) was supplied by Chem-Supply Pty Ltd. (Adelaide, SA, Australia), while sulfuric acid (98%) was purchased from RCI Labscan (Rongmuang, Thailand). Deionized water (resistivity, 18.2 MΩ/cm) was prepared by a Millipore Milli-Q Gradient Water Purification System (Darmstadt, Germany) and the filtration was processed by a 0.22 μm type Millipak Express 20 filter (Milli-Q, Darmstadt, Germany).

3.2. Sample Preparation and Extraction

Four kinds of tobacco samples (K-399, SN-1, SN-2, and SP-28) of N. tabacum were collected from various regions of Swabi, KPK, Pakistan. The plant specimen was identified by a botanist in the Department of Botany, University of Swabi KP, Pakistan. The drying procedure was processed under shade at room temperature, ground into uniform powder, and stored at −20 °C. Then, the powder samples were cold-extracted with organic solvents methanol (MK-399, MSN-1, MSN-2, and MSP-28) and distilled water (WK-399, WSN-1, WSN-2, and WSP-28). The methanolic extracts were concentrated by a rotary evaporator at low temperature (50–55 °C) and the water extracts were concentrated by a water bath. Finally, the aqueous and methanol extracts were filtrated by a syringe filter (0.45 μm, Thermo Fisher Scientific Inc., Waltham, MA, USA) and the supernatant was used for further analysis, conducted at the Department of Agriculture and Food Systems, The University of Melbourne in Australia.

3.3. Antioxidant Activity Determination

3.3.1. Determination of the Total Phenolic Content (TPC)

The total phenolic content of tobacco extracts was determined by following the protocol of the Folin–Ciocalteu method63 with some modifications. Twenty-five microliters of extract and 25 μL of Folin–Ciocalteu’s reagent (1:3 diluted with water) were mixed in a 96-well plate (Corning Inc., Corning, NY, USA), followed by 5 min of incubation at 25 °C. Then, 200 μL of water and 25 μL of 10% (w/w) sodium carbonate were added to dilute and another 1 h of incubation was required. Finally, the absorbance at 725 nm was measured in a microplate reader, and gallic acid (0–200 μg/mL) in ethanolic solution was added for standard curve generation. The result was presented in mg gallic acid equivalents/gd.w..

3.3.2. Determination of the Total Flavonoid Content (TFC)

The total flavonoid content was measured by the AlCl3 colorimetry-based method.38 The tobacco extract (80 μL) was mixed with 80 μL of 2% aluminum chloride and 120 μL of 50 g/L sodium acetate (water solution) in a 96-well plate, followed by 2.5 h of incubation at 25 °C. The absorbance at 440 nm was measured in a microplate reader, and quercetin methanolic solution (0–50 μg/mL) was added for standard curve generation. Each sample was processed in triplicate, and the result was presented in mg quercetin equivalents.

3.3.3. Determination of the Total Condensed Tannin (TCT)

The total tannin content measurement was based on a previously reported method.60 Twenty-five microliters of tobacco extract was mixed with 150 μL of 4% vanillin solution and 25 μL of 32% sulfuric acid in a 96-well plate and incubated for 15 min at 25 °C. Finally, the absorbance at 500 nm was measured in a microplate reader, and catechin (0–1000 μg/mL) in methanolic solution was added for standard curve generation. The measurements were repeated three times, and the result was presented in mg catechin equivalents.

3.3.4. 2,2′-Diphenyl-1-picrylhydrazyl (DPPH) Antioxidant Assays

The process was based on the published protocol of Zhu et al.64 DPPH solution was diluted with analytical grade methanol to 0.1 M. Forty microliters of the extraction was added into 260 μL of DPPH radical methanol solution in a 96-well plate and incubated for 30 min at 25 °C. The standard curve was generated by ascorbic acid solution with 0–30 μg/mL. For accuracy, each sample was measured in triplicate, and results were expressed in mg ascorbic acid equivalents.

3.3.5. Ferric Reducing Antioxidant Power (FRAP) Assay

The antioxidant power was also determined by the ferric reducing capability assay, which was based on the reported method of Hong et al.65 The FRAP reagent was composed of 300 mM acetate buffer, 10 mM TPTZ, and 20 mM ferric chloride in a volume ratio of 10:1:1. Twenty microliters of tobacco extract was mixed with 280 μL of FRAP reagent in a 96-well plate, followed by 10 min of incubation at 37 °C. Absorbances of samples were measured by a microplate reader at 593 nm, and ascorbic acid (0–50 μg/mL) solution was added as a reference for standard curve generation. Measurements done three times were expressed in mg AAE (ascorbic acid equivalents).

3.3.6. 2,2′-Azino-bis-3-ethylbenzothiazoline-6-sulfonic Acid (ABTS) Radical Scavenging Assay

The ABTS+ radical cation decolorization assay was applied to measure the ABTS antioxidant activity of tobacco extracts.66 The ABTS+ stock solution was prepared by mixing 7 mM ABTS and 140 mM potassium persulfate solutions, followed by 16 h of incubation in the dark environment. Then, the ABTS+ solution was diluted with ethanol until it achieved the absorbance of 0.70 ± 0.02 at 734 nm. Finally, the absorbance of samples, which included 10 μL of sample extract and 290 μL of prepared ABTS+ solution, followed by another 6 min of incubation, was measured at 500 nm in a microplate reader. The ascorbic acid aqueous solution with the concentration of 0–200 μg/mL was applied for standard curve generation. The measurements were repeated three times, and the result was presented in mg AAE.

3.3.7. Total Antioxidant Capacity (TAC) Assay

The total antioxidant capacity measurement method was based on the published protocol of Prieto et al.67 The tobacco extracts were pipetted (40 μL) and added into 260 μL of phosphomolybdate reagent, which was prepared by mixing 0.6 M sulfuric acid, 0.028 M sodium phosphate, and 0.004 M ammonium molybdate. The following procedure was incubation of 300 μL samples at 95 °C for 10 min and then cooling to room temperature. Finally, the absorbance of sample solution at 695 nm was measured and compared with the standard curve, which was structured by ascorbic acid with a predetermined gradient concentration.

3.4. Polyphenol Identification by LC-ESI-QTOF-MS/MS Analysis

The identification of the polyphenol content was carried out according to the previously published method of Suleria et al.48 A liquid chromatograph (Agilent Technologies, Santa Clara, CA, USA) was connected with a mass Q-TOF liquid chromatograph and continued to connect to a double mass spectrometer by an electrospray ionization (ESI) source. Separation was processed in a Synergi Hydro-RP 80 Å, LC Column 250 mm × 4.6 nm, 4 μm (Phenomenex, Torrance, CA, USA) with a stable column temperature at 25 °C and a sample temperature at 10 °C. Mobile phase A was 2% acetic acid in 98% water, and mobile phase B was a mixture of acetonitrile/water/acetic acid (100:99:1, v/v/v). The whole gradient program was set to 85 min in length, with a mobile phase flow of 0.8 mL/min, and the volume of sample injection was 5 μL. Mass spectrum parameters were executed as follows: nebulizer gas pressure, 45 psi; 250 °C sheath gas with a flow rate of 11 L/min; 300 °C N2 with a flow rate of 5 L/min. The software of Agilent (Agilent Technologies, Santa Clara, CA, USA) was specialized for data acquisition and analysis. The peak identification was executed in both negative ([M – H]−) and positive ([M + H]+) ionization modes. The working voltages were 3.5 kV (capillary) and 500 V (nozzle), and the mass spectra ranged from 50 amu to 1300 amu. Agilent LC-ESI-QTOF-MS/MS Mass Hunter workstation software (Qualitative Analysis, version B.03.01, Agilent) was applied for data acquisition and analysis performance.

3.5. Statistical Analysis

The chemical composition of each sample will be represented as the mean ± standard deviation of the three independent repetitions. Data differences between four different tobacco species were analyzed by one-way ANOVA Tukey’s HSD test, and the statistically significant level was set as P < 0.05. The group differences between the aqueous extract and the methanolic extract were also tested. The correlation between antioxidant assays and phenolic compound estimation methods was evaluated by Pearson’s correlation coefficient at P < 0.05 and the principal component analysis (PCA) was executed by XLSTAT-2019.1.3 (Addinsoft Inc., New York, NY, USA).

4. Conclusions

In conclusion, all tobacco samples, SN-1, SP-28, K-399, and SN-2, contain a wide variety of polyphenols and are able to scavenge free radicals efficiently, showing strong antioxidant potential. According to the result of LC-ESI-QTOF-MS/MS, 49 phenolic compounds were characterized; some variations in polyphenols existed in different tobacco species. The polyphenols of tobacco SP-28 were the most abundant, which might be the reason that the ability to scavenge free radicals is better than the other tobacco samples. These identified polyphenols revealed the potential value of tobacco by-products. This project would promote tobacco by-product recycling and offer new raw materials for food industries and pharmaceuticals. Further studies about tobacco extraction toxicological, bioavailability, and animal studies are required for developing tobacco by-products as commercial ingredients.

Acknowledgments

The authors are thankful to the Higher Education Commission of Pakistan for funding this work under grant number 7343/KPK/NRPU/R&D/HEC/2017. We would like to thank Michael Leeming, Nicholas Williamson, and Shuai Nie from the Mass Spectrometry and Proteomics Facility, Bio21 Molecular Science and Biotechnology Institute, the University of Melbourne, VIC, Australia, for providing access and support for the use of HPLC-PDA and LC-ESI-QTOF-MS/MS and data analysis.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c03275.

  • LC-ESI-QTOF-MS/MS basic peak chromatography for the characterization of phenolic compounds of tobaccos (PDF)

Author Contributions

X.Z. and H.A.R.S. conceptualized the study. X.Z., A.R., A.B., and M.S. provided the methodology. Y.S.A., F.A.A., and H.A.R.S. validated the data. O.B., X.Z., and A.R. conducted the formal analysis. M.H.M. and H.A.R.S. performed the investigation. X.Z and H.A.R.S. provided the resources, curated the data, and prepared the original draft of the manuscript. All authors read the paper and agreed to the process for publications.

The authors declare no competing financial interest.

Supplementary Material

ao1c03275_si_001.pdf (318KB, pdf)

References

  1. Briški F.; Horgas N.; Vuković M.; Gomzi Z. Aerobic composting of tobacco industry solid waste simulation of the process. Clean Technol. Environ. Policy 2003, 5, 295–301. 10.1007/s10098-003-0218-7. [DOI] [Google Scholar]
  2. Tayoub G.; Sulaiman H.; Alorfi M. Determination of nicotine levels in the leaves of some Nicotiana tabacum varieties cultivated in Syria. Herba Pol. 2015, 61, 23–30. 10.1515/hepo-2015-0028. [DOI] [Google Scholar]
  3. Zhang J.; Zhang J.; Wang M.; Wu S.; Wang H.; Niazi N. K.; Man Y. B.; Christie P.; Shan S.; Wong M. H. Effect of tobacco stem-derived biochar on soil metal immobilization and the cultivation of tobacco plant. J. Soils Sediments 2019, 19, 2313. 10.1007/s11368-018-02226-x. [DOI] [Google Scholar]
  4. Yang X.; Lu K.; McGrouther K.; Che L.; Hu G.; Wang Q.; Liu X.; Shen L.; Huang H.; Ye Z.; Wang H. Bioavailability of Cd and Zn in soils treated with biochars derived from tobacco stalk and dead pigs. J. Soils Sediments 2017, 17, 751. 10.1007/s11368-015-1326-9. [DOI] [Google Scholar]
  5. Suleria H. A. R.; Gobe G.; Masci P.; Osborne S. A. Marine bioactive compounds and health promoting perspectives; innovation pathways for drug discovery. Trends Food Sci. Technol. 2016, 50, 44–55. 10.1016/j.tifs.2016.01.019. [DOI] [Google Scholar]
  6. Akhtar S.; Khalid N.; Ahmed I.; Shahzad A.; Suleria H. A. R. Physicochemical Characteristics, Functional Properties, and Nutritional Benefits of Peanut Oil: A Review. Crit. Rev. Food Sci. Nutr. 2014, 54, 1562–1575. 10.1080/10408398.2011.644353. [DOI] [PubMed] [Google Scholar]
  7. Nigam M.; Suleria H. A. R.; Farzaei M. H.; Mishra A. P. Marine anticancer drugs and their relevant targets: a treasure from the ocean. DARU J. Pharm. Sci. 2019, 27, 491–515. 10.1007/s40199-019-00273-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Butt M. S.; Imran A.; Sharif M. K.; Ahmad R. S.; Xiao H.; Imran M.; Rsool H. A. Black Tea Polyphenols: A Mechanistic Treatise. Crit. Rev. Food Sci. Nutr. 2014, 54, 1002–1011. 10.1080/10408398.2011.623198. [DOI] [PubMed] [Google Scholar]
  9. Banožić M.; Babić J.; Jokić S. Recent advances in extraction of bioactive compounds from tobacco industrial waste-a review. Ind. Crops. Prod. 2020, 144, 112009. 10.1016/j.indcrop.2019.112009. [DOI] [Google Scholar]
  10. Shen J.; Shao X. Determination of tobacco alkaloids by gas chromatography–mass spectrometry using cloud point extraction as a preconcentration step. Anal. Chim. Acta 2006, 561, 83–87. 10.1016/j.aca.2006.01.002. [DOI] [Google Scholar]
  11. Huabing L.; Xiaoyin S.; Jianguo L.; Changhe C.; Xiaobin Z.; Shen X.; Yonggang Z.; Huiqing J.; Shixiao X.; Tiezhao Y.; Guangjin Z. Effects of maturity, variety and their interaction on polyphenols in flue-cured tobacco. Chinese Tobacco Sci. 2019, 4, 29. [Google Scholar]
  12. Shaohao L.; Jianwen Z.; Zhe Z.; Qiu Z.; Ruina Z.; Mingqin Z. Effect of temperature on polyphenols metabolism of cigar tobacco leaf during air-curing process. Journal of Northwest A & F University(Natural Science Edition) 2021, 43, 1–9. [Google Scholar]
  13. Septembre-Malaterre A.; Stanislas G.; Douraguia E.; Gonthier M. P. Evaluation of nutritional and antioxidant properties of the tropical fruits banana, litchi, mango, papaya, passion fruit and pineapple cultivated in Reunion French Island. Food Chem. 2016, 225–233. 10.1016/j.foodchem.2016.05.147. [DOI] [PubMed] [Google Scholar]
  14. Yan Z.; Zhong Y.; Duan Y.; Chen Q.; Li F. Antioxidant mechanism of tea polyphenols and its impact on health benefits. Anim. Nutr. 2020, 6, 115–123. 10.1016/j.aninu.2020.01.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Xuezhi Z.; Chong T.; Nana Z.; Weiye R.; Yi M.; Xin J.; Ying Z.; Shibin D.; Chenjiang Y.; Xiaolei Y. Tea polyphenols alleviate high fat and high glucose-induced endothelial hyperpermeability by attenuating ROS production via NADPH oxidase pathway. BMC Res. Notes. 2014, 7, 1–17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Rong T.; Hongyan L. Antioxidant properties in vitro and in vivo: realistic assessments of efficacy of plant extracts. Plant Sci. Reviews 2012, 7, 11–17. [Google Scholar]
  17. Yiannakopoulou E. C. Targeting oxidative stress response by green tea polyphenols: clinical implications. Free Radical Res. 2013, 47, 667–671. 10.3109/10715762.2013.819975. [DOI] [PubMed] [Google Scholar]
  18. Meiling X.; Xiaohui H.; Yuchuan S.; Youyin F.; Yongpin L. Correlation analysis of routine chemical components,polyphenol and sensory quality of cigar germplasm resources. J. Agric. Sci. Technol. 2019, 21, 124–134. [Google Scholar]
  19. Wenjun H.; Jia H.; Zhihao S.; Tiezhao Y. Research progress on factors affecting metabolism of polyphenols in flue-cured tobacco leaves. Guizhou Agri. Sci. 2019, 47, 4–7. [Google Scholar]
  20. Tsaballa A.; Sarrou E.; Xanthopoulou A.; Tsaliki E.; Kissoudis C.; Karagiannis E.; Michailidis M.; Martens S.; Sperdouli E.; Hilioti Z.; Fotopoulos V.; Nianiou-Obeidat I.; Tsaftaris A.; Madesis P.; Kalivas A.; Ganopoulos I. Comprehensive approaches reveal key transcripts and metabolites highlighting metabolic diversity among three oriental tobacco varieties. Ind. Crops. Prod. 2020, 143, 111933. 10.1016/j.indcrop.2019.111933. [DOI] [Google Scholar]
  21. Azmir J.; Zaidul I. S. M.; Rahman M. M.; Sharif K. M.; Mohamed A.; Sahena F.; Jahurul M. H. A.; Ghafoor K.; Norulaini N. A. N.; Omar A. K. M. Techniques for extraction of bioactive compounds from plant materials: a review. J. Food Eng. 2013, 426–436. 10.1016/j.jfoodeng.2013.01.014. [DOI] [Google Scholar]
  22. Deng Y.; Shanfa L. Biosynthesis and Regulation of Phenylpropanoids in Plants. Crit. Rev. Plant Sci. 2017, 36, 257–290. 10.1080/07352689.2017.1402852. [DOI] [Google Scholar]
  23. Shifflett J. R.; Watson L.; McNally D. J.; Bezabeh D. J. Analysis of the Polyphenols of Tobacco Using Pressurized Liquid Extraction (PLE) and Ultra Performance Liquid Chromatography With Electrospray Ionization-Tandem Mass Spectometric Detection (UPLC-ESI-MS/MS). Beitr. Tab. Int. 2017, 27, 195–207. [Google Scholar]
  24. Popova V.; Gochev V.; Girova T.; Iliev I.; Ivanova T.; Stoyanova A. Extraction products from tobacco–aroma and bioactive compounds and activities. Curr. Bioact. Compd. 2015, 11, 31–37. 10.2174/157340721101150804150016. [DOI] [Google Scholar]
  25. Fu B.; Ji X.; Zhao M.; He F.; Wang X.; Wang Y.; Liu P.; Niu L. The influence of light quality on the accumulation of flavonoids in tobacco (Nicotiana tabacum L.) leaves. J. Photochem. Photobiol., B 2016, 162, 544–549. 10.1016/j.jphotobiol.2016.07.016. [DOI] [PubMed] [Google Scholar]
  26. Ververidis F.; Trantas E.; Douglas C.; Vollmer G.; Kretzschmar G.; Panopoulos N. Biotechnology of flavonoids and other phenylpropanoid-derived natural products. Part II: Reconstruction of multienzyme pathways in plants and microbes. Biotechnol. J. 2007, 2, 1235–1249. 10.1002/biot.200700184. [DOI] [PubMed] [Google Scholar]
  27. Chen S.; Pan X.; Li Y.; Cui L.; Zhang Y.; Zhang Z.; Pan G.; Yang J.; Cao P.; Yang A. Identification and Characterization of Chalcone Synthase Gene Family Members in Nicotiana tabacum. J. Plant Growth Regul. 2017, 36, 374. 10.1007/s00344-016-9646-6. [DOI] [Google Scholar]
  28. Liu G.; Zujian W. The inhibition of infectivity of tobacco mosaic virus by tannins extracted from plants. J. Fujian Agric. For. University (Nat. Sci. Ed.) (China) 2003, 32, 292–295. [Google Scholar]
  29. Molnar M.; Jerković I.; Suknović D.; Bilić Rajs B.; Aladić K.; Šubarić D.; Jokić S. Screening of Six Medicinal Plant Extracts Obtained by Two Conventional Methods and Supercritical CO2 Extraction Targeted on Coumarin Content, 2,2-Diphenyl-1-picrylhydrazyl Radical Scavenging Capacity and Total Phenols Content. Molecules 2017, 22, 348. 10.3390/molecules22030348. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Jing Y.; Gao Y.; Wang W.; Cheng Y.; Lu P.; Ma C.; Zhang Y. Optimization of the extraction of polysaccharides from tobacco waste and their biological activities. Int. J. Biol. macromol. 2016, 91, 188–197. 10.1016/j.ijbiomac.2016.05.069. [DOI] [PubMed] [Google Scholar]
  31. Haiyan W.; Mouming Z.; Bao Y.; Yueming J.; Guohua R. Identification of polyphenols in tobacco leaf and their antioxidant and antimicrobial activities. Food Chem. 2008, 1399–1406. 10.1016/j.foodchem.2007.09.068. [DOI] [Google Scholar]
  32. Paiva L.; Lima E.; Motta M.; Marcone M.; Baptista J. Variability of antioxidant properties, catechins, caffeine, L-theanine and other amino acids in different plant parts of Azorean Camellia sinensis. Curr. Res. Food Sci. 2020, 3, 227–234. 10.1016/j.crfs.2020.07.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Majer P.; Stoyanova S.; Hideg É. Do leaf total antioxidant capacities (TAC) reflect specific antioxidant potentials? – A comparison of TAC and reactive oxygen scavenging in tobacco leaf extracts. J. Photochem. Photobiol., B. 2010, 100, 38–43. 10.1016/j.jphotobiol.2010.04.007. [DOI] [PubMed] [Google Scholar]
  34. Matanjun P.; Mohamed S.; Mustapha N. M.; Muhammad K.; Ming C. H. Antioxidant activities and phenolics content of eight species of seaweeds from north Borneo. J. Appl. Phycol. 2008, 20, 367–373. 10.1007/s10811-007-9264-6. [DOI] [Google Scholar]
  35. Nilsson J.; Pillai D.; Onning G.; Persson C.; Nilsson A.; Akesson B. Comparison of the 2,2′-azinobis-3-ethylbenzotiazo-line-6-sulfonic acid (ABTS) and ferric reducing anti-oxidant power (FRAP) methods to asses the total antioxidant capacity in extracts of fruit and vegetables. Mol. Nutr. Food Res. 2005, 49, 239–246. 10.1002/mnfr.200400083. [DOI] [PubMed] [Google Scholar]
  36. Spínola V.; Pinto J.; Castilho P. C. Identification and quantification of phenolic compounds of selected fruits from Madeira Island by HPLC-DAD–ESI-MSn and screening for their antioxidant activity. Food Chem. 2015, 173, 14–30. 10.1016/j.foodchem.2014.09.163. [DOI] [PubMed] [Google Scholar]
  37. Yang D.; Dunshea F. R.; Suleria H. A. R. LC-ESI-QTOF/MS characterization of Australian herb and spices (garlic, ginger, and onion) and potential antioxidant activity. J. Food Process. Preserv. 2020, 44, e14497 10.1111/jfpp.14497. [DOI] [Google Scholar]
  38. Peng D.; Zahid H. F.; Ajlouni S.; Dunshea F. R.; Suleria H. A. R. LC-ESI-QTOF/MS Profiling of Australian Mango Peel By-Product Polyphenols and Their Potential Antioxidant Activities. Processes. 2019, 7, 764. 10.3390/pr7100764. [DOI] [Google Scholar]
  39. Ma C.; Dunshea F. R.; Suleria H. A. R. LC-ESI-QTOF/MS Characterization of Phenolic Compounds in Palm Fruits (Jelly and Fishtail Palm) and Their Potential Antioxidant Activities. Antioxidants 2019, 8, 483. 10.3390/antiox8100483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Subbiah V.; Zhong B.; Nawaz M. A.; Barrow C. J.; Dunshea F. R.; Suleria H. A. R. Screening of Phenolic Compounds in Australian Grown Berries by LC-ESI-QTOF-MS/MS and Determination of Their Antioxidant Potential. Antioxidants. 2021, 10, 26. 10.3390/antiox10010026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Escobar-Avello D.; Lozano-Castellón J.; Mardones C.; Pérez A. J.; Saéz V.; Riquelme S.; von Baer D.; Vallverdú-Queralt A. Phenolic Profile of Grape Canes: Novel Compounds Identified by LC-ESI-LTQ-Orbitrap-MS. Molecules 2019, 24, 3763. 10.3390/molecules24203763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Abu-Reidah I. M.; Gil-Izquierdo A.; Medina S.; Ferreres F. Phenolic composition profiling of different edible parts and by-products of date palm (Phoenix dactylifera L.) by using HPLC-DAD-ESI/MSn. Food Res. Int. 2017, 100, 494–500. 10.1016/j.foodres.2016.10.018. [DOI] [PubMed] [Google Scholar]
  43. Mukherjee C.; Chakraborty S. Study of dietary Polyphenols from natural herbal sources for providing protection against Human Degenerative Disorders. Biocatal. Agric. Biotechnol. 2021, 101956. 10.1016/j.bcab.2021.101956. [DOI] [Google Scholar]
  44. Rodgman A.; Perfetti T. A., The chemical components of tobacco and tobacco smoke; Taylor & Francis: 2009. [Google Scholar]
  45. Feng Y.; Dunshea F. R.; Suleria H. A. R. LC-ESI-QTOF/MS characterization of bioactive compounds from black spices and their potential antioxidant activities. J. Food Sci. Technol. 2020, 57, 4671–4687. 10.1007/s13197-020-04504-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Gu C.; Howell K.; Dunshea F. R.; Suleria H. A. R. LC-ESI-QTOF/MS Characterisation of Phenolic Acids and Flavonoids in Polyphenol-Rich Fruits and Vegetables and Their Potential Antioxidant Activities. Antioxidants 2019, 8, 405. 10.3390/antiox8090405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Rossi M.; Strikoudi P.; Spei M. E.; Parpinel M.; Serraino D.; Montella M.; Libra M.; Vecchia C. I.; Rosato V. Flavonoids and bladder cancer risk. Cancer Causes Control 2019, 30, 527–535. 10.1007/s10552-019-01158-2. [DOI] [PubMed] [Google Scholar]
  48. Suleria H. A. R.; Barrow C. J.; Dunshea F. R. Screening and Characterization of Phenolic Compounds and Their Antioxidant Capacity in Different Fruit Peels. Foods. 2020, 9, 1206. 10.3390/foods9091206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Tang J.; Dunshea F. R.; Suleria H. A. R. LC-ESI-QTOF/MS characterization of phenolic compounds from medicinal plants (hops and juniper berries) and their antioxidant activity. Foods. 2020, 9, 7–7. 10.3390/foods9010007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Appelhagen I.; Wulff-Vester A. K.; Wendell M.; Hvoslef-Eide A.-K.; Russell J.; Oertel A.; Martens S.; Mock H.-P.; Martin C.; Matros A. Colour bio-factories: Towards scale-up production of anthocyanins in plant cell cultures. Metab. Eng. 2018, 48, 218–232. 10.1016/j.ymben.2018.06.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Khumalo G. P.; Sadgrove N. J.; Van Vuuren S. F.; Van Wyk B. E. Antimicrobial lupenol triterpenes and a polyphenol from Elaeodendron transvaalense, a popular southern African medicinal bark. S. Afr. J. Bot. 2019, 122, 518–521. 10.1016/j.sajb.2018.07.020. [DOI] [Google Scholar]
  52. Miao M.-M.; Li L.; Shen Q.-P.; Liu C.-B.; Li Y.-K.; Zhang T.; Zhang F.-M.; He P.; Wang K.-M.; Zhu R.-Z.; Chen Y.-K.; Yang G.-Y. Anti-TMV activity flavones from the leaves of Yunnan local air cured tobacco. Fitoterapia 2015, 103, 260–264. 10.1016/j.fitote.2015.04.014. [DOI] [PubMed] [Google Scholar]
  53. Chen L. J.; Zhao X.; Plummer S.; Tang J.; Games D. E. Quantitative determination and structural characterization of isoflavones in nutrition supplements by liquid chromatography–mass spectrometry. J. Chromatogr. A. 2005, 1082, 60–70. 10.1016/j.chroma.2005.03.066. [DOI] [PubMed] [Google Scholar]
  54. Kim J.; Lee H.-I.; Lim Y. J.; Park Y. J.; Kim W.; Kim D.-O.; Kim B.-Y.; Eom S. H.; Baik M.-Y. Antioxidant and phytoestrogenic activities of puffed black soybeans (Glycine max). LWT. 2020, 118, 108780. 10.1016/j.lwt.2019.108780. [DOI] [Google Scholar]
  55. Zeng Y.; Lu Y.; Chen Z.; Tan J.; Bai J.; Li P.; Wang Z.; Du S. Rapid Characterization of Components in Bolbostemma paniculatum by UPLC/LTQ-Orbitrap MSn Analysis and Multivariate Statistical Analysis for Herb Discrimination. Molecules 2018, 23, 1155. 10.3390/molecules23051155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Xiang G.; Yang H.; Yang L.; Zhang X.; Cao Q.; Miao M. Multivariate statistical analysis of tobacco of different origin, grade and variety according to polyphenols and organic acids. Microchem. J. 2010, 95, 198–206. 10.1016/j.microc.2009.12.001. [DOI] [Google Scholar]
  57. Sifola M. I.; Carrino L.; Cozzolino E.; del Piano L.; Graziani G.; Ritieni A. Potential of Pre-Harvest Wastes of Tobacco (Nicotiana tabacum L.) Crops, Grown for Smoke Products, as Source of Bioactive Compounds (Phenols and Flavonoids). Sustainability 2021, 13, 2087–2087. 10.3390/su13042087. [DOI] [Google Scholar]
  58. Pulido R.; Bravo L.; Saura-Calixto F. Antioxidant activity of dietary polyphenols as determined by a modified ferric reducing/antioxidant power assay. J. Agric. Food Chem. 2000, 48, 3396–3402. 10.1021/jf9913458. [DOI] [PubMed] [Google Scholar]
  59. Li X.; Wang T.; Zhou B.; Gao W.; Cao J.; Huang L. Chemical composition and antioxidant and anti-inflammatory potential of peels and flesh from 10 different pear varieties (Pyrus spp.). Food Chem. 2014, 152, 531–538. 10.1016/j.foodchem.2013.12.010. [DOI] [PubMed] [Google Scholar]
  60. Wang Z.; Barrow C. J.; Dunshea F. R.; Suleria H. A. R. A Comparative Investigation on Phenolic Composition, Characterization and Antioxidant Potentials of Five Different Australian Grown Pear Varieties. Antioxidants. 2021, 10, 151–151. 10.3390/antiox10020151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Zhong B.; Robinson N. A.; Warner R. D.; Barrow C. J.; Dunshea F. R.; Suleria H. A. R. LC-ESI-QTOF-MS/MS Characterization of Seaweed Phenolics and Their Antioxidant Potential. Mar. Drugs. 2020, 18, 331. 10.3390/md18060331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Floegel A.; Kim D. O.; Chung S. J.; Koo S. I.; Chun O. K. Comparison of ABTS/DPPH assays to measure antioxidant capacity in popular antioxidant-rich US foods. J. Food Compos. Anal. 2011, 24, 1043–1048. 10.1016/j.jfca.2011.01.008. [DOI] [Google Scholar]
  63. Singleton V. L.; Orthofer R.; Lamuela-Raventós R. M. [14] Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. Methods Enzymol. 1999, 299, 152–178. 10.1016/S0076-6879(99)99017-1. [DOI] [Google Scholar]
  64. Zhu C.; Chou O.; Lee F. Y.; Wang Z.; Barrow C. J.; Dunshea F. R.; Suleria H. A. R. Characterization of Phenolics in Rejected Kiwifruit and Their Antioxidant Potential. Processes. 2021, 9, 781. 10.3390/pr9050781. [DOI] [Google Scholar]
  65. Hong Y.; Wang Z.; Barrow C. J.; Dunshea F. R.; Suleria H. A. R. High-Throughput Screening and Characterization of Phenolic Compounds in Stone Fruits Waste by LC-ESI-QTOF-MS/MS and Their Potential Antioxidant Activities. Antioxidants. 2021, 10, 234. 10.3390/antiox10020234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Du J.; Zhong B.; Subbiah V.; Barrow C. J.; Dunshea F. R.; Suleria H. A. R. LC-ESI-QTOF-MS/MS Profiling and Antioxidant Activity of Phenolics from Custard Apple Fruit and By-Products. Separations 2021, 8, 62. 10.3390/separations8050062. [DOI] [Google Scholar]
  67. Prieto P.; Pineda M.; Aguilar M. Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of vitamin E. Anal. Biochem. 1999, 269, 337–341. 10.1006/abio.1999.4019. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ao1c03275_si_001.pdf (318KB, pdf)

Articles from ACS Omega are provided here courtesy of American Chemical Society

RESOURCES