Effects of Altitude on Polyphenols Contents in Cigar Leaves

LIU Bo-yuan ,CHEN Yong-wei ,JIN Ming-ke ,YANG Wei-li ,ZHAO Ming-qin *

1.College of Tobacco Science,Henan Agricultural University,Zhengzhou 450002,PRC;2.Dazhou Tobacco Company of Sichuan province,Dazhou 635000,PRC

Abstract Elevation affects the contents of polyphenols involved in the plants’resistance to the environment.In this study,polyphenolic contents,as well as the activity of polyphenol oxidase (PPO),were evaluated in mature cigar plants at different elevation levels.The results showed that:(1) The contents of chlorogenic acid,rutin,and scopolamine in cigar leaves increased gradually at all elevation levels,reaching a maximum 25 d after the mature stage.(2) With an increase in altitude,the polyphenolic compounds’ contents increased,having the lowest values at 350 m height and the highest at 1 090 m height.(3) As for the PPO activity in cigar leaves,the general trend showed an increase first followed by a decrease in activity during the mature period.(4) PPO activity in the leaves of cigar plants planted at 840 m height was substantially elevated compared to those growing at 600 m and 350 m height.Interestingly,the leaves planted at 1 090 m height had the lowest PPO activity.(5) There were significant positive correlations between altitude and the contents of chlorogenic acid,rutin,and scopolamine within the plants leaves.Altitude negatively correlated the proportion of chlorogenic acid and scopolamine whereas positively correlated rutin proportion within the total polyphenols.Temperature had negative correlations with the contents of chlorogenic acid,rutin,and scopolamine,while sunlight intensity had positive correlations on their contents.Conversely,there were no significant correlations between altitude,temperature,and light intensity on PPO activity.Therefore,this study can provide valuable insight and a theoretical basis for the production of premium cigars.

Key words Cigar;Elevation;Light intensity;Polyphenols;Polyphenol oxidase

1.Introduction

Tobacco is an agricultural product obtained by processing the leaves of the genusNicotiana tabacum[1].The cigar is a special tobacco product made by manually rolling the tobacco leaves.Thus,the tobacco leaves directly influence cigar quality.Polyphenols are an essential product of the plants' phenylpropane metabolic pathways and are widely expressed in plants that depend heavily on ecological adaptability[2-3].There are numerous kinds of polyphenols in tobacco,they mainly include tannins (chlorogenic acid),coumarins (scopoletin),flavonoids (rutin),as well as simple phenolic derivatives.Chlorogenic acid,scopoletin,and rutin account for more than 95%of the total polyphenols found within the plant[4-5].It is generally believed that polyphenols give tobacco a better aroma and taste.Although polyphenol compounds are low in volatility,they rarely enter the flue gas directly during combustion and inhalation.However,polyphenol compounds produce an acid reaction during the combustion and inhalation of tobacco,which can neutralize part of the alkalinity that makes tobacco’s taste mellow.Polyphenol compounds can also bind to proteins causing a browning reaction that is catalyzed by polyphenol oxidase (PPO),thus affecting the appearance,quality,and sensory quality of tobacco leaves[6-7].PPO is a Cu-binding metalloproteinase in plants,which are expressed in the thylakoids of normal photosynthetic cells and the plastids of non-photosynthetic cells[8-12].Polyphenols are easily oxidized to quinones by PPO in the tobacco plants,causing the quinones to turn brown after a series of reactions.The more polyphenols are oxidized,the worse the appearance and internal quality of the tobacco leaves become[13-17].

At present,studies have shown that with an increase in altitude,the contents of polyphenol compounds increased in tobacco[18-20].However,there are rarely any reported relationship between polyphenol content and altitude in the cigar.This study analyzed the changes of polyphenolic contents and PPO activity of tobacco plants within the mature stage at different elevations in Wanyuan,Sichuan,China.This research aims to provide a theoretical and evidencebased approach to producing high-quality cigars.

2.Materials and Methods

2.1.Altitude selection

The experiment was conducted in 2019 in Qinhe,Wanyuan,Sichuan Province (350 m above sea level),Hekou,Wanyuan,Sichuan Province (600 m and 840 m above sea level),and Zengjiaxiang,Wanyuan,Sichuan province (1 090 m above sea level).The experimental variety was Chuanxue 1.

2.2.Plant materials and growth condition

The selected cigar plant variety was transplanted on April 20th,2019,with a row spacing of 110 cm,a plant spacing of 45 cm,and a planting area of 6.8 hm2.A specialty fertilizer was compounded(N ∶P2O5∶K2O=1 ∶1 ∶2) and applied to the tobacco plants at a concentration of 820 kg/hm2.The base and topdressing ratio was 6 ∶4,and the application rate of the base and topdressing was 492,and 328 kg/hm2,respectively.For the application of the base fertilizer,it was added directly to the soil and covered in the transplanting line.The topdressing was prepared by dissolving it in water before application.After the topdressing was dissolved,a small hole was dug in between the tobacco plants with a depth of 15 cm;the topdressing fertilizer was then poured into the hole and covered with soil.Field management measured the intertillage and ridging,ensuring they were consistent with the local conventional level.During harvesting,cigar plants with the same growth and leaf size were selected,and the middle leaf (11th～12thleaf position) was used as the experimental material.

2.3.Determination items and methods

2.3.1.Measurement of field temperature

TESTO184T1 temperature and humidity recorder(Germany TESTO company) were used to record the field temperatures at 10:00～14:00 during the mature period (from June 20thto July 15th) .

2.3.2.Determination of field light intensity

Light intensity was measured using portable illuminators once every 5 d at 10:00 am starting from the beginning of plant maturity.A total of 4 measurements were taken during the course of this study.

2.3.3.Determination of enzyme activity

At each of the four elevations tobacco leaves were randomly selected every 5 d from the beginning of plant maturity.For each whole cigar leaf,the tip(occupying the top 1/3 of the leaf) and the leaf base(occupying the lower 1/3 of the leaf) were removed.The middle part of the leaf was kept to measure the activity of PPO using a colorimetric method.Each sample was tested in triplicate.

2.3.4.Determination of polyphenols

Cigar leaves at each of the four elevations were randomly selected every 5 d from the beginning of plant maturity.Cigar leaves were dried and grounded,then passed through 60 mesh screen.The content of chlorogenic acid,rutin,and scopolamine in the cigar leaves was determined using a 2695 high-performance liquid chromatograph.Each sample was tested in triplicate.

2.4.Data analysis and processing

Microsoft Excel 2016 software was used for data analysis and drawing,and SPSS 21.0 was used for significance and correlation analysis.

3.Results and Analysis

3.1.Field temperatures at different elevations

Field temperature was measured,as shown in Table 1.When the mature stage occurred,the field temperature at all elevations increased daily.The reason may be because of a strong correlation between the cigar plants maturity and the environmental temperature.On day 5,10,15,and 25,the temperature decreased with increasing altitude.There was a minor deviation on day 20,which may be due to a measurement error.

3.2.Field illumination intensity at different elevations

Field light intensity was measured as shown in Table 2.Proceeding plant maturity,the field light intensity increased at each altitude.The reason may be that with the increasing temperature,the field light intensity was also enhanced,and stronger light intensity promoted tobacco leaves’ ripening.On day 5,10,15,20,and 25,the illumination intensity was stronger with an increase in altitude.The lowest illumination was at an altitude of 350 m and the highest at 1 090 m.This observation was consistent with natural law.

3.3.Variation of polyphenol contents in cigars planted at different elevations

The variation of polyphenol contents in cigar plants was shown in Fig.1.The contents of chlorogenic acid in the cigar leaves under all elevations increased gradually,reaching a maximum at day 25.At altitudes of 1 090,840,600,and 350 m chlorogenic acid content in the cigar plants reached 15.26,14.37,13.15,and 12.08 mg/g,respectively 25 d after the mature stage.On day 5 and 25,the cigar plants chlorogenic acid content under all elevations reached statistically significant levels (P＜0.05).On day 10,15,and 20,the content of chlorogenic acid in the cigar plants planted at an altitude of 1 090 and 840 m did not reach a statistically significant level (P＞0.05).

The variation of rutin contents in cigarsis shown in Fig.2.The contents of rutin in cigars leaves under all elevations increased gradually,reaching the maximum at day 25.At the altitude of 1 090 m,the content of rutin in cigar plants reached 1.42 mg/g at day 25.At the altitude of 840,600,and 350 m,the content of rutin reached 1.21,0.99,and 0.89 mg/g,respectively.On day 5,10,20,and 25,the content of rutin in the cigar plants under all elevations reached statistically significant levels (P＜0.05).On day 15,the content of rutin in cigar plants that were planted at the altitude of 1 090 and 840 m did not reach a statistically significant level (P＞0.05).

Fig.2 Changes in rutin content of cigars planted at different elevations

The variation in scopolamine contents within the cigar plants was shown in Fig.3.The contents of scopolamine in cigars leaves under all elevations increased gradually,reaching the maximum at day 25.At the altitude of 1 090,840,600,and 350 m,the content of scopolamine in cigar plants reached 0.145,0.138,0.124,and 0.113 mg/g,respectively,25 days after maturity.On day 5,10,15,20,and 25,the content of scopolamine in cigar plants under all elevations reached statistically significant levels(P＜0.05).

Fig.3 Changes in scopolamine content of cigar plants planted at different elevations

3.4.Changes of PPO activity in cigars planted at different elevations

The changes in PPO activity within the cigar plants were shown in Fig.4.When the mature period occurred,the PPO activity in the cigar leaves had a general upward trend during the first 15 d at the altitude levels of 600 and 350 m,and an upward trend at the elevations of 1 090 and 840 m for the first 20 d,after which PPO activity declined at all altitudes.The maximum values reached at 1 090 and 840 m were 28.0 and 122.64 U/(g·min),respectively at day 20.The elevations of 600 and 350 m,the maximum values reached 112.76,and 87.24 U/(g·min),respectively at day 15.PPO activity in cigar plants planted at 840 m height was relatively higher,followed by those planted at 600 and 350 m height.PPO activity in the leaves planted at an altitude of 1 090 m was the lowest.The difference among treatments reached a statistically significant level (P＜0.05) on day 5 and 20.On day 10 and 25,the difference in PPO activity between the 840 and 600 m,and 600 and 350 m altitude levels was not statistically significant (P＞0.05) at day 10,and on day 15 the difference in PPO activity between the 840 and 600 m altitude levels was not statistically significant(P＞0.05).

Fig.4 Changes of PPO activity of cigars planted at different elevations

3.5.Correlation analysis between altitude and contents of polyphenols

The correlation analysis between altitude and polyphenol content was shown in Table 3.There was a significant positive correlation between altitude and chlorogenic acid,rutin,and scopolamine contents.There was no significant correlation between altitude and PPO activity indicating that altitude significantly affected polyphenol content.After the mature stage,the altitude had an extremely significant positive correlation with chlorogenic acid content except on day 10;rutin content also showed a significant positive correlation with altitude.On day 5 and 10,scopolamine content and altitude reached significant levels.On day 15～25,the scopolamine content was positively correlated with altitude.The correlation between PPO activity and altitude did not reached a statistically significant level.Altitude was negatively correlated with the proportion of chlorogenic acid and scopolamine in the total polyphenol content.However,altitude was positively correlated with the proportion of rutin within the total polyphenols.

Table 3 Correlation between altitude and polyphenol content

The correlation analysis between temperature and contents of polyphenol content was shown in Table 4.Temperature showed negative correlations with the contents of chlorogenic acid,rutin and scopolamine.There was no significant correlation between temperature and PPO activity.This indicated that temperature had a significant effect on polyphenol content.On day 5 and 25,chlorogenic acid and scopolamine contents were negatively correlated with the temperature.On day 10,the contents of rutin and scopolamine were negatively correlated with temperature.

Table 4 Correlation between temperature and polyphenol content

The correlation analysis between light intensity and polyphenol content was shown in Table 5.The light intensity showed positive correlations with the contents of chlorogenic acid,rutin and scopolamine.There was no significant correlation between illumination intensity and PPO activity.This indicated that illumination intensity had a significant effect on the content of polyphenols.After the mature stage,the contents of chlorogenic acid and scopolamine had a significant positive correlation with the illumination intensity.On day 10 and 20,the rutin contents were significantly positively correlated with the illumination intensity,and on day 15,rutin had the highest correlation with light intensity.

Table 5 Correlation between illumination intensity and polyphenol content

4.Discussion and Conclusion

The results showed that:1) With the increase in altitude,the field temperature decreased,and the light intensity increased.After the mature stage,the field temperature decreased,and the light intensity increased.In general,this was consistent with the laws of nature.2) As an essential chemical substance in tobacco,polyphenols content influenced tobacco quality[21].In this experiment,the contents of chlorogenic acid,rutin and scopolamine in cigar leaves under all elevations increased gradually,reaching the maximum concentration at day 25 with all compounds reaching a significant level.With an increase in altitude,polyphenol compounds’ contents increased,showing the lowest concentration at 350 m height and the highest at 1 090 m height.Previous studies have demonstrated that polyphenols content increased with maturity and elevation during tobacco leaves’ growth.The low temperature,ultraviolet light and other environmental stresses could induce polyphenols biosynthesis in plants[22-24].With an increase in altitude,the light intensity increased as the temperature decreased.Tobacco leaves had the ability to absorb and transform the intense ultraviolet radiation of sunlight into a less harmful wavelength of light.Therefore,they acted as an ultraviolet filter.It was believed that the reason for the high content of phenolic compounds in tobacco plant leaves that were exposed to high altitude,low temperature,and strong light intensity was to protect their DNA from degradation and harm[25-29].3) PPO is an essential enzyme in tobacco,found mainly in the chloroplast and is a direct mediator of browning enzymatic reaction.It can promote the deepening of tobacco leaves’ color,and an upregulation of PPO activity is conducive to the quality of tobacco[30-31].As for the PPO activity in cigar leaves,when mature period occurred,in the beginning of the mature period there is an upward general trend that slowly declines as time progresses.The PPO activity of cigars grown at 840 m above sea level was the highest,followed by 600 and 350 m,respectively,and finally the altitude at 1 090 m causing the lowest PPO activity.The reason for this phenomenon is that reducing light intensity increased PPO activity,whereas not having enough light intensity also adversely affects PPO activity.At the altitude of 1 090 m,the light intensity is too intense.Therefore,the PPO activity of cigars planted at this altitude is relatively low.At the altitudes of 350～840 m,the light intensity is weaker,hence the PPO activity is higher[32].4) This study analyzed the correlation of polyphenols and PPO activity to verify the relationship between altitude,temperature,and illumination intensity,on the content of polyphenols in cigars plants after maturity.The results showed a significant positive correlation between altitude and the contents of chlorogenic acid,rutin,and scopolamine.An extremely significant positive correlation was observed in some instances.After the mature stage,temperature was negatively correlated with the contents of chlorogenic acid,rutin,and scopolamine in the cigar plants.After the mature stage,illumination intensity had a significantly positive correlation with the contents of chlorogenic acid,rutin and scopolamine in cigars.There were no significant correlations between PPO activity altitude,temperature and light intensity.The experimental results were consistent with the research results of WANG Yet al.The content of polyphenols was positively correlated with the altitude,while the PPO activity was not significantly correlated with altitude.After the mature stage,the proportion of chlorogenic acid and scopolamine in total polyphenols was negatively correlated with altitude.The proportion of rutin in total polyphenols was positively correlated with altitude.The experimental results were consistent with the research results of FU Bet al[33].Their results showed that the polyphenols content of flue-cured tobacco was positively correlated with altitude.

In conclusion,with the increase in altitude,the field temperature decreased,and light intensity increased.After the mature stage,the contents of chlorogenic acid,rutin,and scopolamine within the cigar leaves under all elevations increased gradually,reaching the maximum at day 25.With the increase in altitude,polyphenol compounds content increased,showing the lowest levels at 350 m and the highest at 1 090 m.As for the PPO activity in cigar leaves,when the mature period occurred,an increase in enzyme activity was observed,and then decreased over time.The cigar plants’ PPO activity grown at 840 m above sea level was observed to be the highest,and the altitude of 1 090 m showed the lowest enzyme activity.There was a significant positive correlation between altitude and contents of chlorogenic acid,rutin and scopolamine.Altitude was negatively correlated with the proportion of chlorogenic acid and scopolamine in total polyphenols.However,it was positively correlated with the proportion of rutin within the total polyphenolic content.The temperature showed negative correlations with the contents of chlorogenic acid,rutin and scopolamine,while the light intensity had positive correlations.There was no significant correlation between PPO activity,altitude,temperature and light intensity.The results can provide a theoretical basis for the production of high quality cigars.