POLYPHENOLS IN TOBACCO EXTRACTS OBTAINED BY MACROPOROUS RESIN

Transcription

1 Доклади на Българската академия на науките Comptes rendus de l Académie bulgare des Sciences Tome 68, No 2, 2015 POLYPHENOLS IN TOBACCO EXTRACTS OBTAINED BY MACROPOROUS RESIN CHIMIE Chimie organique Margarita Docheva, Soleya Dagnon (Submitted by Corresponding Member V. Bankova on September 9, 2014) Abstract The present study reports experimental data on the separation of polyphenols in extracts from Oriental and Virginia tobaccos obtained by using macroporous resin Amberlite XAD7. Additionally, separation conditions for phenolic acids and flavonoids were studied. The polyphenols were purified by adsorption/desorption process based on the different chemical and physical properties of the components. The content of polyphenols and nicotine in the tobaccos and in the obtained extracts was determined. Extract 1 was enriched in phenolic acids with a recovery of 76% from tobacco. Nicotine content in Extract 1 was between 6.48 mg/g and 21.0 mg/g. Extract 2 was enriched in flavonoids (92% recovery from tobacco) and showed a high radical scavenging activity determined by DPPH method. The IC 50 data varied from 9.20 ± 1.66 µg/ml to ± 7.03 µg/ml. The extracts enriched in flavonoids contained only minor amounts of nicotine (mean 0.4 mg/g). Key words: tobacco, polyphenols, macroporous resin, radical scavenging activity Introduction. Polyphenols are bioactive substances widely distributed in the plant kingdom. They are considered as bioactive non-nutritional compounds, due to their properties, including antioxidant functions. Polyphenol compounds are related to phenolic acids, flavonoids, tannins and stilbenoids. They possess a wide range of biological properties, such as antibacterial, antioxidant, antimicrobial, anticancer, antimutagenic and inhibitory of HIV-1 RT. The polyphenols are active against the human herpes simplex virus and adenoviruses [ 1 ]. As secondary metabolites the polyphenols are important components in tobacco. Tobacco studies have reported the identification of nearly 20 polyphenols. The major polyphenols in tobacco are the phenolic acids (chlorogenic acid, neochlorogenic acid and 4-O-Caffeoylquinic acid) and the flavonoids (rutin and 183

2 kaempferol-3 rutinoside) [ 2 5 ]. The content of rutin in Oriental tobacco (10 mg/g) is higher than the content of rutin determined in some traditional herbs such as Hypericum perforatum L. (8.4 mg/g), Tilia tomentosa L. (8.0 mg/g), Thymus vulgaris L. (4.5 mg/g), Satureja montana L. (3.8 mg/g), Chamomilla chamomila L. (2.8 mg/g), Melissa officinalis L. (0.3 mg/g) [ 2, 6 ]. The amount of chlorogenic acids in tobacco differs widely from 7.0 to 18 mg/g [ 7 ]. The current commercial sources of chlorogenic acids are the plants Lonicera japonica and Eucommia ulmoides. These sources are generally limited and therefore expensive [ 8 ]. In view of the high content of chlorogenic acid and rutin in tobacco, several attempts were made to obtain extracts, enriched in polyphenols [ 5, 9, 10 ]. The aim of this study was to obtain extracts enriched in phenolic acid or flavonoids from Oriental and Virginia tobaccos by using macroporous resin Amberlite XAD7. Additionally separation conditions for phenolic acids and flavonoids were studied. Materials and methods. Plant material. Dry leaves of Oriental and Virginia tobaccos Djebel Basma 1 cv. (Dj1), Krumovgrad 90 cv. (Kr90) and the variety Virginia 454 (Vir454) were used as a material. The cultivars were from the collection of the Institute of Tobacco and Tobacco Products, Plovdiv, Bulgaria. The tobacco Djebel Basma 1 cv. was developed by individual natural selection from local species. The cultivar Krumovgrad 90 cv. and the variety Virginia 454 were obtained by intervarietal hybridization. The plants were grown in the experimental fields of the Tobacco and Tobacco Products Institute, Plovdiv, Bulgaria, under agrotechnical conditions appropriate for Oriental and Virginia tobaccos. Reagents and adsorption medium. Macroporous resin Amberlite XAD7, Nicotine (CAS Number ), Chlorogenic acid (Cas Number ), Rutin (Cas Number ) and DPPH were purchased from Sigma Aldrich (Sigma-Aldrich Chemie GmbH, Buchs, SG, Switzerland); Methanol (MeOH) and Ethanol (EtOH) were HPLC grade (Merck, Germany). The resin was activated with EtOH. Six grams of the resin was shaken with 20 ml EtOH for 2 h on a mechanical shaker. The excess of solution was removed and the procedure was repeated once again. The resin was dried at 100 C for 2 h and stored under constant conditions. Adsorption of polyphenols from tobacco with macroporous resin. Dry tobacco powder (2 g) was extracted with 30 ml 60% (v/v) MeOH for 30 min on a mechanical shaker. The extract was filtered and diluted with 60% MeOH to 30 ml solution. The solution was added to 9 g macroporous resin Amberlite XAD7 weighed in a conical flask. The adsorption was carried out under static conditions for 2 h. After 2 h the excess of the solution was removed. Preparation of extract, enriched in phenolic acids (Extract 1). The resin with the adsorbed polyphenols was washed three times with distilled water. The washing water was removed every time. The phenolic acids were desorbed 184 M. Docheva, S. Dagnon

3 with 60 ml 30% MeOH (v/v) on a mechanical shaker for 2 h. The procedure was repeated three times and the liquid was removed every time. The washing water and the three desorbtion solutions were collected obtaining Extract 1. Preparation of extract, enriched in flavonoids (Extract 2). After obtaining Extract 1, the flavonoids were desorbed from the resin with 80 ml 100% MeOH on a mechanical shaker for 2 h. The procedure was repeated three times by removing the solution every time. The desorbtion solutions were collected obtaining Extract 2. Determination of polyphenols in tobacco and tobacco extracts. One tenth grams tobacco powder was sonicated for 30 min with 5 ml, 60% (v/v) MeOH. The extract was filtrated under vacuum. The polyphenols were purified by passing the solution through cartridge C18 according to the method described by Dagnon and Edreva in [ 2 ] and subjected to HPLC analysis. Aliquot of the obtained extracts (Extract 1 or Extract 2) was passed through a membrane filter 0.45 µm prior to HPLC analysis. The instrumentation used for HPLC analysis consisted of quaternary mixer Smartline Manager 5000, pump Smartline 1000 and PDA 2800 detector (Knauer, Germany). A Purospher (Merck, Germany) column (RP-18e 25 cm 4.6 mm i.d., 5 µm) was used. The mobile phase composition was: A CH 3 OH : 1% CH 3 COOH = 5:95 and B CH 3 OH : 1% CH 3 COOH = 85:15. The elution followed the gradient profile 100% A, for 20 min to 0% A (100%B). The detection wavelength was 320 nm (maximum adsorption for caffeoylquinic acids) and 352 nm (maximum adsorption for flavonoids). The flow rate was set at 1.0 ml/min and the injection volume was 20 µl. The confirmation of the identity of the chromatographic peaks was achieved by comparison of the retention times in the samples with those of the standard compounds chlorogenic acid and rutin. The content of the components was determined by an external method using a calibration curve established with five dilutions of each standard with correlation coefficients and 0.999, respectively. Determination of nicotine. The content of nicotine in the extracts was determined by using GC system according to the Coresta recommended method No 62, 2005 [ 11 ]. The Agilent 7890 GC-FID was equipped with HP-5, 0.32 mm i.d., 30 m, 0.25 µm column. The oven temperature was programmed from 110 C to 185 C at 10 C/min and from 185 C to 245 C at 6 C/min and the detector was set by 270 C. The operating temperature for the injector was 250 C, Split 5:1 and 1.0 µl injection volume. Identification and quantification of nicotine was accomplished by referring the peaks in the extracts to the peak of the standard nicotine using external calibration. Determination of radical scavenging activity of Extract 2 (DPPH assay). Previous studies have shown that the use of water or acetone as solvents 4 Compt. rend. Acad. bulg. Sci., 68, No 2,

4 in the reaction of phenolics with the stable radical DPPH results in low values of IC 50 [ 15 ]. Taking into account this recommendation we analyzed only Extract 2 for radical scavenging activity. The extract enriched in flavonoids (Extract 2) was evaporated to dryness and its weight was measured. The dry extract was solved in 5 ml MeOH. A solution of diphenylpicrylhydrazyl reagent (DPPH ) was prepared daily with concentration 0.12 mm ( g DPPH dissolved in 50 ml MeOH). Two ml of the DPPH solution was placed in a vessel and 2 ml of the extract was added in various concentrations (4, 8, 9, 18, 36, 48, 62, 72, 96, 144 µg/ml for Oriental tobaccos and 0.95, 1.9, 3.8, 7.6, 15, 30, 60, 120 µg/ml for Virginia tobacco). The mixtures were placed in the dark for 30 min at room temperature. The absorbance of the samples was measured at 515 nm with a spectrophotometer. All measurements were done under dim light. The percentage radical scavenging activity (RSA, %) of the samples was evaluated by comparing with a control (2 ml DPPH solution and 2 ml MeOH) and calculated by the following formula: RSA, % = [A 0 Ab/A 0 ] 100, where A 0 is the absorbance of the control blank and Ab is the absorbance of the samples. IC 50 (µg/ml) was defined as the concentration in the vessel of an extract that causes 50% loss of the colour. The higher the radical scavenging potential, the lower is the value of IC 50. Mean of the IC 50 value was calculated from three repetitions by interpolation of graphic based on minimum six concentration levels with correlation coefficient of To calibrate the DPPH assay, the radical scavenging potential of rutin and quercetin was determined with every set of samples. Statistics. All experimental procedures were done in triplicate. The quantitative data were expressed as mean ± standard deviation. Results and discussion. Polyphenols in tobacco and tobacco extracts. This investigation was conducted to obtain extracts from tobacco enriched in phenolic acids or enriched in flavonoids by using macroporous resin in static conditions. To achieve this goal, separation of the polyphenol components was undertaken on the basis of their different solubility in the desorption process from the macroporous resin. The solubility of chlorogenic acid in water is much higher than the solubility of rutin, which is better soluble in methanol [ 12, 13 ]. It is proven that the phenolic acids and flavonoids can be extracted from tobacco using 60 80% MeOH with high yield [ 5, 9 ]. The investigations published so far, discussing the adsorption of tobacco polyphenols by macroporous resin, have described the simultaneous purification of chlorogenic acid and rutin from aqueous-ethanolic extracts [ 5 ]. Moreover they have not reported the content of nicotine in the obtained extracts. Our special 186 M. Docheva, S. Dagnon

5 attention was directed to obtain extracts, which contained either flavonoids or phenolic acids and minor amount of nicotine. Figure 1 reveals the content of polyphenols in tobacco and in the obtained extracts (Extract 1 and Extract 2). The content of phenolic acids in the Oriental tobaccos Dj1 and Kr90 was equal (14 ± 1.04 mg/g) and it was higher than in Virginia tobacco Vir454 (10.02±0.75 mg/g). Accordingly, the amount of phenolic acids in Extract 1 from Oriental tobaccos (10.6 ± 0.86 mg/g) was higher than in Extract 1 from Virginia tobacco (7.7±0.63 mg/g). The recovery of phenolic acids from tobacco was mean 76%. It is evident that high-yield desorption of phenolic acids was achieved with 30% MeOH. The Oriental cultivar Dj1 was characterized by the highest flavonoid content 9.94±0.74 mg/g. The lowest amount of flavonoids was determined in V ±0.38 mg/g (Fig. 1). At the same time, the amount of flavonoids in the extracts (Extract 1) was decreased significantly and it was approximately equal mean 0.40 mg/g. Extract 2 obtained from Oriental tobaccos (Dj1 and Kr90) contained 8.48 ± 0.72 mg/g and 6.12 ± 0.53 mg/g flavonoids, respectively. The extract obtained from Virginia tobacco was characterized by smaller amount of flavonoids 5.18 ± 0.48 mg/g. The recovery of flavonoids from tobacco was mean 92%. In all tobacco extracts (Extract 2) the amount of phenolic acids was approximately 0.8 mg/g (6% recovery). Full desorbtion of flavonoids was achieved by using 100% MeOH. In this way, the polyphenols were separated in two different extracts one enriched in phenolic acids (Extract 1) and the other enriched in flavonoids (Extract 2). The content of the desorbed polyphenols in Extract 1 and in Extract 2 remained constant during the repetitions of the procedure (RSD = 6%) (Fig. 1). Nicotine in tobacco and tobacco extracts. The content of nicotine in tobacco and in the obtained tobacco extracts is presented in Table 1. Fig. 1. Content of polyphenols (mg/g) in Oriental tobaccos (Dj1 and Kr90), Virginia tobacco (Vir454) and in the obtained extracts (Extract 1 and Extract 2) Compt. rend. Acad. bulg. Sci., 68, No 2,

6 T a b l e 1 Content of nicotine (mg/g) in Oriental tobaccos (Dj1 and Kr90), Virginia tobacco (Vir454) and in the obtained extracts (Extract 1 and Extract 2) Tobacco Extract 1 Extract 2 Dj1 9.3 ± ± ± Kr ± ± ± Vir ± ± ± Nicotine in tobacco forms salt, which is extractable with acidic methanolwater mixture. In Extract 1 obtained by desorption with 30% MeOH, the amount of nicotine was higher in comparison with that of Extract 2. The recovery of nicotine in Extract 1 was mean 62%. Extract 2 obtained from Dj1, Kr90 and Vir454 showed only minor amount of nicotine 0.27 mg/g, 0.46 mg/g and 0.44 mg/g, respectively. The recovery of nicotine was less than 5%. Radical scavenging activity of Extract 2. The free radical scavenging activity of extracts enriched in flavonoids (Extract 2) is presented as IC 50 µg/ml in Table 2. Quercetin and rutin, well-known antioxidant compounds with a structure similar to those of the tobacco flavonoids, were employed as reference compounds with IC 50 = 1.36 ± 0.10 µg/ml and IC 50 = 3.06 ± 0.20 µg/ml, respectively. The data in Table 2 reveal the highest scavenging activity of Vir454 extract (IC 50 = 9.20 ± 1.66 µg/ml) in comparison with the data for Oriental tobaccos (Dj1 IC 50 = 31.95±5.75 µg/ml and Kr90 IC 50 = 39.05±7.03 µg/ml). The content of flavonoids in tobacco extract from Vir454 is nearly two times lower in comparison with that of Dj1 being the extract with a higher radical scavenging activity. Hence, the radical scavenging activity of the plant extracts depends on various T a b l e 2 Radical scavenging activity (IC 50) (µg/ml) of tobacco extracts, enriched in flavonoids (Extract 2), rutin and quercetin Sample IC 50 Dj ± 5.75 Kr ± 7.03 Vir ± 1.66 Rutin 3.06 ± 0.20 Quercetin 1.36 ± M. Docheva, S. Dagnon

7 biochemicals besides the flavonoid content. There might be some other chemical components, other than flavonoids, which influence DPPH radical scavenging activity of the extract. Comparing the data for the radical scavenging activity of Extract 2 from Oriental tobaccos obtained by macroporous resin with the previously reported data for extracts obtained by column chromatography, they appear very close [ 9 ]. The tobacco extracts, prepared according to the proposed procedure possess higher radical scavenging activity in comparison with the top five most active Bulgarian medicinal plants (Rumex crispus IC 50 =40.09 µg/ml, Rubus Occidentalis IC 50 =45.23 µg/ml, Rumex alpinus IC 50 =46.69 µg/ml, Euphorbia Helioscopia IC 50 =49.52 µg/ml and Rubus idaeus IC 50 =50.52 µg/ml [ 16 ]. Conclusion. By using macroporous resin Amberlite XAD7 for purification of tobacco extracts, separation of chlorogenic acid and rutin was achieved. The extracts, enriched in phenolic acids, were reduced by half in nicotine content, while the extracts, enriched in flavonoids contained only minor amounts of nicotine and possessed high radical scavenging activity. The obtained extracts may be regarded as a raw material for potential biopharmaceuticals. REFERENCES [ 1 ] Yordi E., E. Perez, M. Matos, E. Villares (2012) In: Nutrition, Well-Being and Health (ed. Dr. Jaouad Bouayed). InTech, 23 48, ISBN: [ 2 ] Dagnon S., A. Edreva (2003) Beitr. Tabakforsch. Int., 20, No 5, [ 3 ] Xie F., A. Yu, D. Hou, H. Liu, L. Ding, S. Zhang (2011) AJAC, 2, [ 4 ] Li F., W. Cai, X. Shao (2007) CJC, 25, No 4, [ 5 ] Wang H., M. Zhao, B. Yang, Y. Jiang, G. Rao (2008) Food Chemistry, 107, [ 6 ] Sofic E., A. Copra-Janicijelic, M. Salihovic, I. Tahirovic, G. Kroyer (2010) Medicinal Plants, 2, No 2, [ 7 ] Apostolova E., S. Dagnon, A. Edreva (2002) Beitr. Tabakforsch. Int., 20, 1, 1 6. [ 8 ] Chen Y., Q. Yu, X. Li, Y. Luo, H. Liu (2007) au, [ 9 ] Docheva M., S. Dagnon, S. Statkova-Abeghe (2014) NPR, 28, No 17, [ 10 ] Zhang J., S. Zhang, J. Wu, N. Li, J. Wang (2013) J. Med. Plants Res., 7, No 24, [ 11 ] Coresta Recommended Method No 62 (2005) Recommended_Methods/CRM_62.pdf [ 12 ] Pedriali C., A. Fernandes (2008) Quim. Nova, 31, No 8, [ 13 ] Xiang Z., Z. Ning (2008) LWT, 41, Compt. rend. Acad. bulg. Sci., 68, No 2,

8 [ 14 ] Buran T., A. Sandhu, Z. Li, C. Rock, W. Yang, L. Gu (2014) J. Food Engin., 128, [ 15 ] Molyneux P. (2004) Songklanakarin J. Sci. Technol., 26, No 2, [ 16 ] Nikolova M., L. Evstatieva, T. Nguyen (2011) Botanica Serbica, 35, No 1, Department of Tobacco Chemistry Tobacco and Tobacco Products Institute 4108 Markovo, Plovdiv, Bulgaria margarita 1980@abv.bg Department of Organic Chemistry Plovdiv University Paisii Hilendarski 24, Tsar Asen St 4000 Plovdiv, Bulgaria solbono@abv.bg 190 M. Docheva, S. Dagnon