Coumarins of Matricaria chamomilla L.: Aglycones and glycosides

Food Chemistry 141 (2013) 54–59

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Coumarins of Matricaria chamomilla L.: Aglycones and glycosides Veronika Petrul’ová-Poracká a, Miroslav Repcˇák a,⇑, Mária Vilková b, Ján Imrich b a b

Institute of Biology and Ecology, Faculty of Science, P. J. Šafárik University, Mánesova 23, SK-04001 Košice, Slovak Republic Institute of Chemistry, Faculty of Science, P. J. Šafárik University, Moyzesova 11, SK-040 01 Košice, Slovak Republic

a r t i c l e

i n f o

Article history: Received 26 September 2012 Received in revised form 14 January 2013 Accepted 4 March 2013 Available online 14 March 2013 Keywords: Matricaria chamomilla L. Asteraceae Coumarins Daphnin Skimmin

a b s t r a c t The identity and quantity of coumarin-like compounds in leaves and anthodia of Matricaria chamomilla L. were studied by LC-DAD and NMR. So far, two monosubstituted coumarins, herniarin and umbelliferone, and two herniarin precursors were identified therein. In this paper, two other coumarin glycosides and one aglycone were confirmed. Skimmin (umbelliferone-7-O-b-D-glucoside), daphnin (daphnetin-7-O-bD-glucoside) and daphnetin (7,8-dihydroxycoumarin) were found for the first time in diploid and tetraploid leaves and anthodia of M. chamomilla L. Daphnetin is known as a strong sensitizer, so this compound and its glycosidic derivative can contribute to the allergic potential of chamomile. Commercial chamomile preparations were tested for their presence. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction The coumarins (1,2-benzopyrans), lactones of O-hydroxycinnamic acid derivatives, are widely distributed in many plant families, such as Apiaceae, Asteraceae, Rosaceae, Rutaceae, Rubiaceae, Solanaceae, and Lamiaceae (Smyth, Ramachandran, & Smyth, 2009). They are found in all plant organs and are usually accumulated in seeds, roots, reproductive organs and epidermal cells. Coumarins exist in plant cells in two forms – free or conjugated by a glycosidic bond with other molecules, e.g. glucose. As the free form is more effective than the conjugated one, this bond is broken in physiological processes by a specific glucosyltransferase (Chong, Baltz, Fritig, & Saindrenam, 1999). Matricaria chamomilla L. is a medical herb, well-known for its various therapeutical effects, which are influenced by synthesis and accumulation of specific secondary metabolites. Main active constituents present in the plant are flavonoids (apigenin and its derivatives), constituents of essential oil, mainly chamazulene, farnesene, ()-a-bisabolol, dicycloethers, and coumarins (Power & Browning, 1914). In existing papers that deal with the content of chamomile coumarin compounds, seven coumarins (herniarin, umbelliferone, coumarin, isoscopoletine, scopoletine, esculetin, and fraxidin) were described (Iverson, Zahid, Shoqafi, Ata, & Samarasekera, 2010; Kotov, Khvorost, & Komissarenko, 1991). Under normal physiological Abbreviation: GMCA, b-D-glucopyranosyloxy-4-methoxycinnamic acid.

⇑ Corresponding author. Tel.: +421 55 234 2310.

E-mail address: [email protected] (M. Repcˇák). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.03.004

conditions, plants accumulate high amounts of herniarin and its precursors, (Z)- and (E)-b-D-glucopyranosyloxy-4-methoxycinnamic acids (Z- and E-GMCA), but only traces of umbelliferone (Hofer, Szabó, & Greger, 1986; Repcˇák, Eliašová, & Rušcˇancˇinová, 1998). Literature indicates that umbelliferone is formed in plants by lactonisation of the (Z)-form of dihydroxycinnamic acid and acts as the precursor of other coumarins (herniarin, esculetin, daphnetin, skimmin) (Bourgaud et al., 2006; Dewick, 2002). However, in chamomile, neither its precursors, nor any derivatives, have yet been found. Skimmin (umbelliferone-7-O-b-D-glucoside) has been reported from Matricaria matricarioides, another plant species of the same plant genus (Ma, Winsor, & Daneshtalab, 2007). The presence of other identified coumarins mentioned above can result from two biosynthetic pathways (Bourgaud et al., 2006), either from lactonisation of cinnamic acid derivatives or from derivatization reactions of umbelliferone or herniarin. Neither of these mentioned pathways were confirmed in chamomile. So, the aim of our work was the isolation and identification of minor coumarin compounds of M. chamomilla L., that could follow the coumarin biosynthesis. Chamomile drug is very popular, as an infusion in traditional medicine, because of its antioxidant and therapeuthic effects (Horzˇic´ et al., 2009; McKay & Blumberg, 2006). Extracts from this plant are used in pharmaceutical, food and cosmetic industries. Despite the positive effects of chamomile, people who are sensitive to some Asteraceae species, can be sensitive to chamomile. Though overall allergenic potential of the chamomile drug is considered to be low, some chemical constituents, such as sesquiterpene alcohols, flavonoids and coumarins, can evoke the allergic reaction

V. Petrul’ová-Poracká et al. / Food Chemistry 141 (2013) 54–59

called skin contact allergy. Herniarin and umbelliferone, when tested, expressed only weak effects (Masamoto, 2001; Paulsen, 2002; Paulsen, Otkjær, & Andersen, 2010), but the presence of other coumarins with sensitizer character can contribute to the allergic potential of this plant. Therefore, tests of minor coumarins in commercial samples of the chamomile plant purchased in pharmacies were performed. 2. Material and methods 2.1. Chemicals Solvents for HPLC were acetonitrile (J.T. Barker, Deventer, Holland), methanol (Sigma–Aldrich) and deionised water (Millipore Direct-Q 3UV with Pump). Skimmin (P99%) was purchased from Apin Chemicals LTD, daphnetin (P99%) and herniarin (P98%) from Extrasynthése, and umbelliferone (P99%) from Sigma Aldrich. (Z)- and (E)-GMCA and daphnin were isolated from a chamomile leaf extract and their purity was assessed by HPLC-DAD and NMR as P99%. 2.2. Plant material Plants of M. chamomilla L. (Asteraceae) diploid cv. ‘Bona’ and tetraploid cv. ‘Lutea’ (Oravec et al., 2005) were grown in field conditions in the Botanic Garden of the Pavol Jozef Šafárik University in Košice. Leaves and anthodia were harvested and dried at room temperature. Anthodia were harvested in the phase when white ligulate flowers were flowering and half of the total number tubular flowers were in bloom. For the evaluation of coumarins in chamomile teas available in the Slovak pharmacies, ten commercial drugs were purchased. Three samples contained only anthodia, one was an ethanolic extract, and 7 preparations were drugs in tea bags:  Herbex s.r.o., Slovakia, ‘‘Rumancˇek pravy´‘‘, loose-flower, 50 g,  Juvamed s.r.o., Slovensko, ‘‘Rumancˇek kamilkovy´‘‘, loose-flower, 40 g,  Agrokarpaty Plavnica s.r.o., Slovakia,‘‘Rumancˇek kamilkovy´‘‘, loose-flower, 40 g,  Baliarne obchodu Poprad a.s., Slovakia, ‘‘Rumancˇek pravy´’’, 20 bags, 1.5 g per bag,  Herbex s.r.o., Slovakia, ‘‘Rumancˇek pravy´’’, 20 bags, 2.5 g per bag,  Lipton, Unilever Slovensko, s.r.o., Camomile, 20 bags, 0.9 g per bag,  Lord Nelson, Germany, Kamille, 25 bags, 2.25 g per bag,  Agrokarpaty Plavnica s.r.o., Slovakia, ‘‘Rumancˇek kamilkovy´‘‘, 20 bags, 1.5 g per bag,  Klember a spol. s.r.o., Slovakia, ‘‘Rumancˇek pravy´’’, 20 bags, 1.5 g per bag,  IVAX Pharmaceuticals s.r.o., Chamomilla-IVAX, Matricariae extractum fluidum, 25 ml. 2.3. Extraction and isolation of minor coumarins An ethanolic extract of leaves (46.4 g) was fractionated and re-chromatographed by silicagel column chromatography (70–140 lm, deactivated by 11% of water), and eluted with CHCl3 containing from 5 to 20% of methanol. Semipreparative HPLC and DAD detection were used for separation and purification of individual compounds: column 8  250 mm, SGX C18, 7 lm, Tessek Ltd., Prague, mobile phase A (10% acetonitrile) and mobile phase B (80% acetonitrile), UV detection was at 320 nm, injection volume 100 ll, flow 4 ml/1 min.

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2.4. Quantification Dry anthodia and leaf rosettes of diploid and tetraploid chamomile were homogenised and extracted by 75% MeOH (100 mg DW/ 5 ml MeOH) at room temperature. Extracts were centrifuged at 3000 rpm for 5 min and analysed by using HPLC-DAD. The HPLC system consisted of an Agilent Technologies 1260 Infinity device, made of a degasser, autosampler, binary solvent pump, and diode array detector (DAD). LC separation was performed on a reversed phase column ECOM 250  4.6 mm, Kromasil, C18 100–7 lm. Mobile phase A {water with 1% (v/v) trifluoroacetic acid} and mobile phase B {70% acetonitrile}were used in a gradient programme with a flow of 1.1 ml/min: 0–20 min 40% B; 20–30 min 100% B; 30–37 min 100% B, 37–40 min 0% B. The volume of the single injection was 20 ll. After each injection, the column was equilibrated for 5 min. For quantitative analysis, a wavelength of 320 nm was used. Furthermore, UV/Vis spectra between 210 and 400 nm were recorded to verify the peak identity of coumarins and the compounds’ purity. Quantitative data were collected using Chemstation software rev. B.04.03 [16]. 2.5. Calibration curves Six-point calibration curves were constructed by analysing various amounts of coumarins (2; 1; 0.5; 0.4; 0.25; 0.18 mg) and their glucosides dissolved in 5 ml of methanol. Every point of the curve is the result of three-injection analyses. They showed good linearity and the whole range of tested concentrations with a regression coeficient (R2) better than 0.999. The limits of detection (LOD) and quantification (LOQ) for the minor coumarin-like compounds analysed by HPLC/DAD were the following: LODskimmin = 1.1 ng/ml, LOQskimmin = 1.65 ng/ml; LODumbelliferone = 0.35 ng/ml, LOQumbelliferone = 0.7 ng/ml; LODdaphnetin = 0.55 ng/ml, LOQdaphnetin = 1.1 ng/ml. Because of high accumulation of herniarin and its precursors in plants, LODs and LOQs for these compounds were not determined. 2.6. NMR The structure of compounds were determined by 1D (1H, 13C, selective NOESY, TOCSY) and 2D (gCOSY, NOESY, gHSQCAD, gHMBCAD) NMR spectroscopy. The NMR spectra were recorded on a 600 MHz Varian VNMRS NMR spectrometer equipped with a 5 mm OneNMR probe at 25 °C. The residue was dissolved in deuterated methanol (CD3OD) and chemical shifts were expressed relative to an internal standard, tetramethylsilane (TMS). Assignments of newly isolated compounds 1 and 2 were achieved as shown in Supplementary data. 2.6.1. Skimmin (1) 1 H NMR (CD3OD, 600 MHz) d 7.90 (1H, d, J = 9.6 Hz, H-4), 7.56 (1H, d, J = 9.0 Hz, H-5), 7.08 (1H, m, H-6), 7.07 (1H, m, H-8), 6.28 (1H, d, J = 9.6 Hz, H-3), 5.03 (1H, m, H-10 ), 3.91 (1H, dd, J = 11.7, 2.4 Hz, H-60 a), 3.70 (1H, dd, J = 11.7, 6.0 Hz, H-60 b), 3.48–3.52 (3H, m, H-20 ,30 ,50 ), 3.40 (1H, dd, J = 9.0, 5.4 Hz, H-40 ); 13C NMR (CD3OD, 150.837 MHz) d 161.7 (C, C-2), 160.8 (C, C-7), 155.3 (C, C-9), 144.1 (CH, C-4), 129.0 (CH, C-5), 113.9 (CH, C-6), 112.9 (CH, C-3), 103.6 (CH, C-8), 100.5 (CH, C-10 ), 77.0, 76.4, 73.3 (3  CH, C-20 , C30 , C-50 ), 69.8 (CH, C-40 ), 61.0 (CH2, C-60 ). 2.6.2. Daphnin (2) 1 H NMR (CD3OD, 600 MHz) d 7.86 (1H, d, J = 9.6 Hz, H-4), 7.22 (1H, d, J = 7.8 Hz, H-6), 7.08 (1H, d, J = 7.8 Hz, H-5), 6.29 (1H, d, J = 9.6 Hz, H-3), 4.94 (1H, d, J = 7.8 Hz, H-10 ), 3.90 (1H, m, H-60 a), 3.69 (1H, dd, J = 11.4, 5.4 Hz, H-60 b), 3.56 (1H, dd, J = 9.0, 7.8 Hz,

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V. Petrul’ová-Poracká et al. / Food Chemistry 141 (2013) 54–59

OH

Glucose

O

O

O

Glucose

1

H3C

OH O

HO

3

H3C

O

2 Glucose O COOH

O

O

O

O

4 Glucose O

O

O

HO

O

COOH

5

6 H3C

O

O

O

7

Fig. 1. Structures of chamomile mono- and disubstituted coumarins and their precursors.

H-20 ), 3.48 (2H, m, H-30 ,50 ), 3.43 (1H, m, H-40 ); 13C NMR (CD3OD, 150.837 MHz) d 162.8 (CH, C-2), 149.7 (C, C-7), 146.1 (CH, C-4), 144.4 (C, C-9), 135.9 (C, C-8), 119.6 (CH, C-5), 116.5 (C, C-10), 114.6 (CH, C-3), 114.4 (CH, C-6), 103.6 (CH, C-10 ), 78.5 (CH, C-50 ), 77.5 (CH, C-30 ), 74.8 (CH, C-20 ), 71.2 (CH, C-40 ), 62.4 (CH2, C-60 ). The quantification experiments were performed using a quick and easy qNMR method (Crouch & Russell, 2011) which does not require any internal chemical standard or external signal injection, using Agilent VnmrJ 3.2 software. All data were collected with active sample temperature regulation at 25 °C, employing stored calibration data. 3. Results and discussion In chamomile, two monosubstituted and four disubstituted coumarins have so far been confirmed. Combination column liquid chromatography, HPLC-DAD, and NMR analyses allowed confirmation of three other coumarins, one mono- and two disubstituted ones. Structures of analysed compounds are summarized in Fig. 1. Skimmin (umbelliferone-7-O-b-D-glucoside, 1) was identified from 5.3 mg of a white powder and analysis of 1.792 mg of an amorphous white-yellow material confirmed the daphnin structure (daphnetin-7-O-b-D-glucoside, 2). Daphnetin (7,8-dihydroxycoumarin, 4) was identified in trace amounts in leaves and anthodia using a daphnetin standard. These compounds are shown in Fig. 2 which present HPLC-DAD chromatograms of methanol extracts of chamomile anthodia (A) and leaves (B).

Fig. 2. HPLC-DAD chromatograms of methanol extracts of chamomile anthodia (A) and leaves (B) and UV spectra of isolated compounds.

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V. Petrul’ová-Poracká et al. / Food Chemistry 141 (2013) 54–59 7.07

OH OH

103.6

5.03160.8 113.8

H OH H

OH

7.08

OH

O

O

O

155.3

161.7

113.9

112.9 144.1

129.0

OH OH

OH

H

O100.5 O

H

H

7.56

7.90

H

OH

7.22

H OH H

O H

OH OH

O

O 162.8

116.5

114.6

H

146.1

H

H

7.08

7.86

6.29

OH O

O

H OH H

O H

H

1 Fig. 3. Important 1H and

O 144.4

119.6

OH O

H

135.9

4.94149.7 114.4

H OH H

OH

6.28

H O

OH

O103.6 O

H

H

2 13

C NMR chemical shifts and key HMBC correlations.

The structure determination of newly isolated compounds 1 and 2 that involved unequivocal localization of the glucose unit on the aromatic skeleton was accomplished using gHMBC spectra (Fig. 3 and 4). NMR data corresponded to the literature (Guinot et al., 2009; Ozgen et al., 2012). Quantified levels of coumarins in diploid and tetraploid leaves and anthodia of M. chamomilla L. are shown in Table 1. From comparison of the minor coumarin glycosides in leaves of both cultivars and tetraploid anthodia, levels of daphnin were found to be

higher than those of skimmin. The content of the main chamomile glycosides, (Z)- and (E)-GMCA, was higher in leaves, too. In flowers and leaves, the levels of aglycones were determined to be several times lower than those of glycosides. In general, chamomile leaves had higher levels of coumarins (glycosides and aglycones) than had anthodia. This is in agreement with the assumption that coumarin synthesis is localised in chloroplasts (Bourgaud et al., 2006). Accumulation of glycosides or precursors of coumarins corroborates the fact that free forms of phenolic compounds are more reactive and subject to oxidation and, so, for their storage and transport, some of their active groups (HO–, CH3O–) have to be inactivated by a linkage with glucose (Chong et al., 2002). In comparison with other aglycones, anthodia and leaves of both cultivars have very low concentrations of daphnetin. This may indicate that its production results from derivatization of other aglycones present in chamomile. In the report of Bourgaud et al. (2006), hydroxylation of umbelliferone to dihydroxycoumarins esculetin or daphnetin was suggested. However, this process has not been confirmed in plants, but only in vitro experiments. According to results of this paper, an important role of umbelliferone as a precursor for production of other coumarins in chamomile can be suggested. In this way, the low concentration of umbelliferone under plant physiological conditions can be explained. Comparison of diploid and tetraploid coumarins (Fig. 5) showed no significant differences in levels of skimmin, daphnin, and

Fig. 4. HMBC spectra of 1 and 2.

Table 1 Identification and quantification of coumarin-like compounds in diploid and tetraploid anthodia and leaves of Matricaria chamomilla L. (Asteraceae) with HPLC-DAD. Peak

Compound

UV k max (nm)

1

Skimmin (umbelliferone 7-O-b-D-glucoside)

258, 311

2

Daphnin (daphnetin-7-O-b-D-glucoside)

252, 325

3

Z-GMCA

220, 280, 302

4

Daphnetin

209, 262, 327

5

E-GMCA

216, 236, 294, 320

6

Umbelliferone

210, 216 (sh), 322

7

Herniarin

216 (sh), 320

x – Average values [mg g1 DW]; sx – standard deviations, n = 3.

Diploid

Tetraploid

Leaves x sx

Anthodia x sx

t-stat P

0.130 0.100 0.168 0.026 10.3 2.80 0.007 0.002 15.9 9.15 0.061 0.017 1.67 0.651

0.183 0.088 0.142 0.049 5.84 3.68 0.021 0.019 9.82 6.47 0.053 0.014 0.607 0.115

0.360 0.731 1.95 0.100 3.44 0.014 7.36 <0.001 0.296 0.777 3.39 0.015 0.765 0.473

Leaves x sx

Anthodia x sx

t-stat P

0.165 0.097 0.207 0.020 16.1 0.511 tr.

0.230 0.108 0.257 0.037 8.60 1.62 0.018 0.012 11.6 4.50 0.067 0.024 0.413 0.017

0.591 0.586 2.68 0.055 7.65 0.002 2.55 0.064 1.22 0.289 2.96 0.042 3.55 0.024

17.8 7.62 0.025 0.005 2.06 0.804

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Fig. 5. Levels of minor (A and C) and major (B and D) coumarin compounds (mg g1 DW) in anthodia and leaves of diploid and tetraploid cultivars. Error bars represent standard deviations.

Table 2 Levels of coumarin glycosides and aglycones in commercial available chamomile drugs. Compound

Skimmin x sx

Daphnin x sx

Z-GMCA x sx

Daphnetin x sx

E-GMCA x sx

Umbelliferone x sx

Herniarin x sx

Juvameda

0.140 0.018 0.176 0.016 0.284 0.092 0.015 0.001 0.148 0.015 0.253 0.051 0.181 0.013 0.295 0.051 0.220 0.031 0.179 0.053

0.054 0.028 0.082 0.011 0.047 0.008 0.005 0.004 0.075 0.022 0.071 0.062 0.094 0.011 0.159 0.039 0.125 0.065 0.075 0.014

5.45 1.56 5.76 0.236 4.90 0.857 0.866 0.037 2.07 0.341 2.84 0.148 3.33 0.322 4.56 0.521 3.87 0.434 2.69 0.488

0.031 0.012 0.007 0.007 0.000 0.000 0.018 0.004 0.022 0.001 0.012 0.011 0.032 0.001 0.025 0.003 0.024 0.006 0.007 0.012

8.47 2.18 9.55 0.426 9.76 1.817 1.52 0.034 2.31 0.577 2.34 0.262 4.60 2.14 5.20 0.727 8.25 0.945 1.61 0.318

0.043 0.014 0.016 0.028 0.018 0.009 0.042 0.006 0.091 0.012 0.075 0.031 0.102 0.007 0.126 0.030 0.007 0.013 0.027 0.025

0.719 0.159 0.348 0.037 0.241 0.041 0.236 0.021 0.578 0.026 0.390 0.021 0.386 0.010 0.450 0.025 0.398 0.047 0.406 0.031

Herbex

a

Agrokarpaty Plavnicaa IVAXb Lord Nelsonc Popradskyc Liptonc Herbexc Agrokarpaty Plavnicac Klemberc

x – Average values [mg g1 DW]; for IVAX, x means mg/1 mL of the extract. sx – Standard deviation (n = 3). a Loose flower tea. b Chamomile alcohol extract. c Tea bag.

(E)-GMCA in leaves or anthodia. Significant differences were recorded in leaf levels of (Z)-GMCA and daphnetin. Tetraploid leaves and anthodia contained more of all analysed compounds than did diploids, in agreement with the results previously published (Repcˇák, Imrich, & Franeková, 2001; Repcˇák et al., 1998; Repcˇák, Švehlíková, Imrich, & Pihlaja, 1999). The presence of other disubstituted coumarins in our samples was not confirmed, probably due to inadequate detection limits (LOD and LOQ) of our HPLC method with DAD detection. In the

paper where authors isolated these compounds, methods with MS detection have been used. Moreover, plants of the same kind cultivated under different climate conditions may not show the same metabolite pattern (McKay & Blumberg, 2006; Paulsen, 2002). Use of the chamomile drug is sometimes accompanied by skin allergic reactions that may be the result of coumarin effects. In previous reports (Masamoto, 2001; Paulsen, 2002; Paulsen et al., 2010), herniarin and umbelliferone are described as weak and

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daphnetin as a strong sensitizer. The molecule of the latter contains two hydroxyl groups, unlike umbelliferone, possessing only one and herniarin none. Lin et al. (2008) demonstrated that coumarins with higher numbers of hydroxyl groups were more reactive. So this compound and its glycoside might contribute to the allergic potential of the plant more than umbelliferone or herniarin themselves. To conclude, ten commercial drugs available in pharmacies were tested for the presence of coumarins (Table 2). All tested drugs contained these compounds and their precursors (1–7). Levels of glycosides (1, 2, 3, 5) found in teas were lower than those in diploid and tetraploid anthodia cultivated in experimental fields. Contents of coumarin aglycones in tested preparations were not too different from those grown in the field. The Ivax-extract had the lowest content of glycosides, but contents of aglycones were comparable to those in tea bags. Acknowledgement This work was supported by the Grant Agency, VEGA, of the Slovak Ministry of Education, grants No. 1/0122/09, 1/0672/11, the State NMR Program, grant no. 2003SP2002802031/2544/08, and Structural Funds of EU under the contract No007/20092.1/OPVaV. We thank Mrs. Anna Michalcˇová and Mrs. Margita Buzinkaiová for their valuable technical assistance. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2013. 03.004. References Bourgaud, F., Hehn, A., Larbat, R., Doerper, S., Gontier, E., Kellner, S., et al. (2006). Biosynthesis of coumarins in plants: A major pathway still to be unravelled for cytochrome P450 enzymes. Phytochemistry Reviews, 5, 293–308. Chong, J. Ch., Baltz, R., Fritig, B., & Saindrenam, P. (1999). An early salicylic acid-, pathogen- and elicitor-inducible tobacco glucosyltransferase: Role in compartmentalization of phenolics and H2O2 metabolism. FEBS Letters, 458, 204–208. Chong, J., Baltz, R., Schmitt, C., Beffa, R., Fritig, B., & Saindrenam, P. (2002). Downregulation of a pathogen-responsive Tobacco UDP-Glc: Phenylpropanoid glucosyltransferase reduces scopoletin glucoside accumulation, enhances oxidative stress and weakens virus resistance. The Plant Cell, 14, 1093–1107.

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