Chemotaxonomic Importance of Sesquiterpenes and Flavonoids in ...

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for the presence of four drimane-type sesquiterpenes: polygodial (1), isopolygodial (2), ... erpene polygodial (1) could be also of diagnostic value for the.
Polygonum

Chemotaxonomic Importance of Sesquiterpenes and Flavonoids in Five Argentinian Species of Polygonum Genus M. Derita* and S. Zacchino Pharmacognosy Area, Faculty of Biochemical and Pharmaceutical Sciences, National University of Rosario, Rosario, Argentina Abstract Leaves of five species of Polygonum genus belonging to Persicaria section were collected from the northeast and central lowlands of Argentina and their dichloromethane extracts were analyzed by GC/EM. They were investigated for the presence of four drimane-type sesquiterpenes: polygodial (1), isopolygodial (2), drimenol (3) and confertifolin (4), previously isolated from P. acuminatum; and the presence of three flavonoids: pinostrobin (5), flavokawin B (6) and cardamonin (7), previously isolated from P. persicaria. Results showed that among the five species of Persicaria section studied, two species contained sesquiterpenes 1-4 but not flavonoids 5-7, other two species contained flavonoids 5-7 but not sesquiterpenes 1-4, and only one species contained compounds 1-7. These results add evidences to a previous proposal to sub-classify the Persicaria section of Polygonum genus from a chemotaxonomic point of view. [Editor’s note: This paper’s references are notated within the main body of the text in superscript, so as to avoid reader confusion with the discussed sesquiterpenes and flavonoids.] Key Word Index

Polygonum genus, Persicaria section, sesquiterpenes, flavonoids, chemotaxonomic importance.

Introduction The Polygonum genus (Polygonaceae) is well known for producing a variety of secondary metabolites including flavonoids, 1 triterpenoids,2 anthraquinones,3 coumarins,4 phenylpropanoids,5 lignans,6 stilbenoids,7 tannins8 and sesquiterpenoids.9 It is represented in Argentina by 21 species, which are divided into five sections: Echinocaulon, Amblygonum, Persicaria, Tiniaria and Polygonum.10 Polygonum punctatum Elliot, P. persicaria L., P. acuminatum Kunth., P. lapathifolium L. and P. hydropiperoides Michux var. hydropiperoides, are five out of the 11 perennial herbs, belonging to the Persicaria section, which grow in the northeast and central lowlands of Argentina. In a previous work, and considering that Gattuso11 and Cialdella10 suggested a delimitation of the Persicaria section to those species of Polygonum genus containing some kind of irritant valves, we suggested that the presence of the sesquiterpene polygodial (1) could be also of diagnostic value for the delimitation of the Persicaria section. Following this point of view, the inclusion of P. hydropiperoides var. hydropiperoides and P. lapathifolium (which do possess neither polygodial nor valvate glands) within the Persicaria section could be the

subject of a further revision.12 In this work we add evidence to that proposal, investigating the presence of three sesquiterpenes (in addition to polygodial) isolated from P. acuminatum [isopolygodial (2), drimenol (3) and confertifolin (4)], and three flavonoids isolated from P. persicaria [pinostrobin (5), flavokawin B (6) and cardamonin (7)], in DMC extracts of the species aforementioned. (Figure 1).

Experimental Plant material: Polygonum hydropiperoides Michux var. hydropiperoides was collected during the flowering season (March 2005) in San Luis province, Merlo district (32º35´S Lat., 65º03´O Long. and 850 m elevation), identified by Elisa Petenatti and deposited at the Herbarium of the National University of San Luis (UNSL # 9256). P. punctatum Elliot, P. persicaria L., P. acuminatum Kunth. and P. lapathifolium L. were harvested in March 2005 in Santa Fe province, Puerto Gaboto district (32º27´S Lat., 60º48´O Long. and 25 m elevation), identified by Susana Gattuso and deposited at the Herbarium of the National University of Rosario, Argentina (UNR Gattuso, S. 97, 108, 94, and 115, respectively).

*Address for correspondence: [email protected]

Rec: Jan 2011 Acc: April 2011

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Figure 1. A) Sesquiterpenes isolated from P. acuminatum: polygodial (1), isopolygodial (2), drimenol (3), confertifolin (4). B) Flavonoids isolated from P. persicaria: pinostrobin (5), flavokawin B (6), cardamonin (7).

Dried and powdered leaves of the species mentioned above (30 g each) were macerated with dichloromethane (DCM) for 24 h (3X). The solvent was evaporated under reduced pressure to yield 0.86, 1.14, 1.05, 0.80 and 0.50 g of DCM soluble extracts, respectively. Compounds isolation: Fractionation by column chromatography of P. acuminatum leaves DCM extract allowed us to isolate 70, 53, 35 and 27 mg of compounds 1-4 respectively. Fractionation by column chromatography of P. persicaria leaves DCM extract allowed us to isolate 40, 36 and 25 mg of compounds 5-7 respectively. Compounds 1-4 were previously isolated from Drymis spp.,13,14 P. punctatum15 and P. acuminatum,9 while compounds 5-7 were previously isolated from Boesenbergia pandurata, Myrica pensilvanica, P. ferrugineum and Piper spp.1,16,17 All spectrums of the isolated compounds in this work, were compared with the literature cited to assure the identity of each structure. Analysis of the DCM extracts: All the extracts were submitted to GC-MS using a Turbo Mass Perkin Elmer chromatograph, equipped with a fused silica column (SE-30 25 m x 0.22 mm ID) with He as a carrier gas, coupled to a mass selective detector, film 0.25 µm, ionization energy 70 eV with a temperature programme of 70-200ºC at 10ºC/min; total time 30 min. Compounds 1-7 were identified by comparison of their retention time and their MS spectrum with the authentic samples obtained from our previous works. Chromatograms are shown in Figure 2.

Figure 2: Gas chromatograms of dichloromethane extracts of P. acuminatum (A), P. persicaria (B), P. punctatum (C), P. lapathifolium (D) and P. hydropiperoides var. hydropiperoides (E). Peaks at 14.74/14.36 min in (A), (B) and (C) belong to drimenol (3). Peaks at 15.59/15.38 min in (A), (B) and (C) belong to confertifolin (4). Peaks at 16.16/16.38 min in (A), (B) and (C) belong to isopolygodial (2). Peaks at 17.41/17.40 min in (A), (B) and (C) belong to polygodial (1). Peaks at

20.81, 20.93, 20.91 min in (B), (D) and (E) belong to pinostrobin (5). Peaks at 22.69, 22.83, 22.80 min in (B), (D) and (E) belong to flavokawin B (6). Peaks at 23.51, 23.19, 23.14 min in (B), (D) and (E) belong to cardamonin (7). 12/Journal of Essential Oil Research

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Polygonum

Compound descriptions:

Polygodial (1) MP: 48ºC. [a]D: -27º (c 1.00, CHCl3). IR (KBr): 2927, 2850, 2726, 1722, 1680, 1642, cm-1. 1H NMR (300 MHz, CDCl3): 9.48 (1H, d, J= 4.2 Hz, H-11); 9.42 (1H, s, H-12); 7.11 (1H, m, H-7); 2.78 (1H, dddd, J= 6.2, 2.1, 2.1, 2.1 Hz, H-9); 2.55-2.40 (1H, m, H-6a); 2.35-2.20 (1H, m, H-6b); 1.82 (1H, m, H-1b); 1.54-1.43 (3H, m, H-2a, 2b, 3b); 1.34 (1H, td, J= 4.0 and 13.4 Hz, H-1a); 1.26-1.16 (2H, m, H-3a, H-5); 0.92; (3H, s, Me-15); 0.91 and 0.89 (6H, 2s, Me-14 and Me-15). 13 C NMR (75 MHz, CDCl3): 201.9 (HC=O); 193.2 (HC=O); 154.4 (=CH); 138.1 (=C); 60.2 (CH); 48,8 (CH); 41.7 (CH2); 39.5 (CH2); 36.8 (C); 33.0 (C); 33.0 (CH3); 25.1 (CH2); 21.9 (CH3); 17.9 (CH2); 15.2 (CH3). MS (EI, 70 eV): m/z (%) = 234 [M+], 216 [M+ - H2O], 206 [M+ - CO], 191 [206 - Me]. Isopolygodial (2) [a]D: +30º (c 1.00, CHCl3). IR (KBr): 2927, 2850, 2726, 1722, 1680, 1642, cm-1. 1H NMR (300 MHz, CDCl3): 9.86 (1H, d, J= 2.7 Hz, H-11); 9.41 (1H, s, H-12); 7.11 (1H, dd, J= 2.7 and 4.9 Hz, H-7); 3.26 (1H, bm, H-9); 2.57 (1H, dt, J= 5.0, 5.0, 20.6 Hz, H-6a); 2.22 (1H, dddd, J= 1.8, 2.6, 11.6 and 20.6 Hz, H-6b); 1.80 (1H, m, H-1b); 1.62-1.52 (5H, m, H-1a, 2a, 2b, 3b, 5); 1.19 (1H, ddd, J= 0.62, 4.1 and 12.0 Hz, H-3a); 0.97 (3H, s, Me-15); 0.94 and 0.92 (6H, 2s, Me-13 and Me-14). 13C NMR (75 MHz, CDCl3): 202.2 (HC=O); 192.8 (HC=O); 153.5 (=CH); 137.3 (=C); 58.5 (CH); 44.2 (CH); 42.0 (CH2); 37.6 (CH2); 37.1 (C); 32.9 (C); 32.7 (CH3); 25.5 (CH2); 21.9 (CH3); 21.5 (CH2); 18.4 (CH3). MS (EI, 70 eV): m/z (%) = 234 [M+], 216 [M+ - H2O], 206 [M+ - CO], 191 [206 - Me]. Drimenol (3) MP: 98ºC. [a]D: -15º (c 1.00, CHCl3). IR (KBr): 3405, 2922, 1620 cm-1. 1H NMR (300 MHz, CDCl3): 5.54 (1H, m, H-7); 3.85 (1H, dd, J= 3.3, 11.3 Hz, H-11B); 3.73 (1H, dd, J= 3.3, 11.3 Hz, H-11A); 1.97 (1H, m, H-6a); 1.95 (1H, m, H-1b); 1.92 (1H, m, H-6b); 1.86 (1H, m, H-9); 1.79 (3H, s, Me-12); 1.48 (2H, m, H-2a and 2b); 1.41 (1H, m, H-3b); 1.20 (2H, m, H-3a and H-5); 1.07 (1H, m, H-1a); 0.89, 0.87 and 0.85 (9H, 3s, Me-13, 14 and 15). 13C NMR (75 MHz, CDCl3): 132.9 (=C); 124.0 (=CH); 60.9 (H2C-OH); 57.3 (CH); 49.9 (CH); 42.1 (CH2); 39.8 (CH2); 36.0 (C); 33.3 (CH3); 32.9 (C); 23.6 (CH2); 22.0 (CH3); 21.9 (CH3); 21.9 (CH3); 18.8 (CH2); 14.9 (CH3). MS (EI, 70 eV): m/z (%) = 234 [M+], 216 [M+ - H2O], 206 [M+ - CO], 191 [206 - Me]. Confertifolin (4) MP: 153ºC. [a]D: +70º (c 1.00, CHCl3). IR (KBr): 1769, 1677 cm-1. 1H NMR (300 MHz, CDCl3): 4.72 (1H, ddd, J= 2.8, 2.8, 16.9 Hz, H-1a); 4.62 (1H, ddd, J= 1.7, 3.5, 16.9 Hz, H-1b); 2.54-2.05 (2H, m, H-4a and b); 1.92-1.30 (9H, m, H-5a and b, H-5a, H-7a and b, H-8a and b, H-9a and b); 1.15 (3H, s, Me-9a); 0.93 and 0.89 (6H, 2s, Me-6a and b). 13 C NMR (75 MHz, CDCl3): 174.5 (C=O); 170.8 (=C); 123.4 (=C); 68.2 (CH2); 51.2 (CH); 41.6 (CH2); 36.6 (CH2); 36.0 (C); 33.2 (C); 33.2 (CH3); 21.4 (CH2); 21.4 (CH3); 20.9 (CH3); 18.3 (CH2); 18.0 (CH2). MS (EI, 70 eV): m/z (%) = 234 [M+], 216 [M+ - H2O], 206 [M+ - CO], 191 [206 - Me]. Pinostrobin (5) MP: 87ºC. [a]D: -51º (c 1.00, CHCl3). IR (KBr): 3450, 2950, 1647, cm-1. 1H NMR (300 MHz, CDCl3): 12.04 (1H, s, OH); 7.49-7.40 (5H, m, H-2´-6´); 6.10 (1H, d, J= 2.3 Hz, H-8); 6.09 (1H, d, J= 2.3 Hz, H-6); 5.44 (1H, dd, J= 3.1 and 12.9 Hz, H-2); 3.83 (3H, s, O-Me); 3.11 (1H, dd, J= 12.9 and 17.2 Hz, H-3a); 2.84 (1H, dd, J= 3.1 and 17.2 Hz, H-3b). 13C NMR (75 MHz, CDCl3): 195.8 (C=O); 168.0 Vol. 23, September/October 2011

(=C); 164.1 (=C); 162.8 (=C); 138.4 (=C); 128.9 (=CH); 128.9 (=CH); 128.9 (=CH); 126.2 (=CH); 126.2 (=CH); 103.1 (=C); 95.1 (=CH); 94.2 (=CH); 79.2 (CH); 55.7 (OCH3); 43.4 (CH2). MS (EI, 70 eV): m/z (%) = 270 [M+]. Flavokawin B (6) MP: 95ºC. IR (KBr): 3450, 2940, 1630, cm-1. 1H NMR (300 MHz, CDCl3): 12.04 (1H, s, -OH); 7.92 (1H, d, J= 15.4 Hz, H-b); 7.79 (1H, d, J= 15.4 Hz, H-a); 7.647.28 (5H, m, H2-6); 6.12 (1H, d, J= 2.3 Hz, H-5´); 5.98 (1H, d, J= 2.3 Hz, H-3´); 3.93 and 3.85 (6H, 2s, 2 O-Me). 13C NMR (75 MHz, CDCl3): 192.6 (C=O); 168.4 (=C); 166.2 (=C); 162.5 (=C); 142.3 (=CH); 135.6 (=C); 130.0 (=CH); 128.9 (=CH); 128.9 (=CH); 128.4 (=CH); 128.4 (=CH); 127.5 (=CH); 106.3 (=C); 93.8 (=CH); 91.3 (=CH); 55.9 (O-CH3); 55.6 (O-CH3). MS (EI, 70 eV): m/z (%) = 284 [M+]. Cardamonin (7) MP: 98ºC. IR (KBr): 3400, 2924, 1638, cm-1. 1H NMR (300 MHz, DMSO-d6): 13.7 (1H, s, -OH); 7.83 (1H, d, J= 15.6 Hz, H-b); 7.73-7.70 (2H, m, H-2 and H-6); 7.68 (1H, d, J= 15.6 Hz, H-a); 7.46-7.44 (3H, m, H-3, 4 and 5); 6.01 (1H, d, J= 2.2 Hz, H-5´); 5.92 (1H, d, J= 2.2 Hz, H-3´); 3.88 (3H, s, O-Me). 13C NMR (75 MHz, DMSO-d6): 192.2 (C=O); 166.7 (=C); 165.6 (=C); 163.1 (=C); 142.3 (=CH); 135.4 (=C); 130.8 (=CH); 129.5 (=CH); 128.8 (=CH); 128.8 (=CH); 128.0 (=CH); 128.0 (=CH); 105.5 (=C); 96.3 (=CH); 92.2 (=CH); 55.5 (O-CH3). MS (EI, 70 eV): m/z (%) = 270 [M+].

Results and Discussion The analysis of the GC spectra clearly showed that P. hydropiperoides var. hydropiperoides and P. lapathifolium do not possess sesquiterpenes 1-4 but contain flavonoids 5-7; meanwhile P. punctatum and P. acuminatum do not possess flavonoids 5-7 but contain sesquiterpenes 1-4. Surprisingly, P. persicaria (the specie that gives the name to the whole section) possesses compounds 1-7. Hereby, taking into account our previous work12and these new results, we can propose that within the Persicaria section of Polygonum genus there are two kinds of species that could be sub-classified by a chemotaxonomic point of view: those that produce the sesquiterpenes (1-4) but not the flavonoids (5-7), and those that biosynthesize the flavonoids (5-7) but not the sesquiterpenes (1-4). To assure this proposal, we must go on studying other species that belong to the Persicaria section of Polygonum genus in terms of their content in sesquiterpenes and flavonoids. Acknowledgements

The authors wish to acknowledge CONICET, ANPCyT, ERASMUS MUNDUS lot 18 ARBOPEUE, UNR.

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2.

3.

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