Biochemical Systematics and Ecology 37 (2009) 2434

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Biochemical Systematics and Ecology

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Chemical variability of the invasive neophytes Polygonum cuspidatum Sieb. and Zucc. and Polygonum sachalinensis F. Schmidt ex Maxim
Peihong Fan a, b, Anne-Emmanuelle Hay a, Andrew Marston a, Hongxiang Lou b, Kurt Hostettmann a, *
a

Laboratory of Pharmacognosy and Phytochemistry, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Quai Ernest-Ansermet 30, CH-1211 Geneva 4, Switzerland b Department of Natural Products Chemistry, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China

a r t i c l e i n f o
Article history: Received 25 July 2008 Accepted 22 November 2008 Keywords: Polygonum cuspidatum Polygonum sachalinensis Polygonaceae HPLC MS ESI UV Coupled techniques

a b s t r a c t
Crude extracts of roots and stems of Polygonum cuspidatum Sieb. and Zucc. and Polygonum sachalinensis F. Schmidt ex Maxim from China and Switzerland were analyzed by online HPLC/UV/ESI-MS to ascertain the phytochemical differences between the original and invasive exotic varieties. A total of 36 constituents were identied by comparing their retention times, UV data, mass spectra with those of standards or with literature data. Certain constituents, such as avanol gallate dimers, were reported for the rst time from these species. The relative quantities of the major compounds (emodin glucoside, piceid, resveratroloside and piceatannol glucoside) differed in the samples of the Polygonum species from China and from Switzerland. Phenylpropanoid glucosides were the main constituents of the roots and stems of P. sachalinensis, while the roots of P. cuspidatum were characterized by the presence of stilbenes and anthraquinones. 2008 Elsevier Ltd. All rights reserved.

1. Introduction Polygonum cuspidatum Sieb. and Zucc. (syn: Fallopia japonica, Polygonum reynoutria, Reynoutria japonica, etc.) (Polygonaceae) originating from China, where it is commonly referred to as Hu Zhang, has migrated to Japan, where it is known as Kojo Kon. It is used in traditional Chinese medicine for the treatment of various inammatory diseases, hepatitis, tumors and diarrhea, and is ofcially listed in the Chinese Pharmacopoeia (China Pharmacopoeia Committee, 1999). Recently, antiviral (Chu et al., 2005) and bacterial DNA primase inhibiting activities (Hegde et al., 2004) were also reported. But it has gained much notoriety in Europe and North America (where it is known as Mexican Bamboo, Japanese Bamboo or Japanese Knotweed) as a pernicious weed, due to its virtually indestructible growing characteristics: a fast growing, robust perennial herb that emerges early in the spring and forms dense thickets up to nine feet in height. The thickets are so dense that they can reduce the diversity of plant species and signicantly alter natural habitats. Reproduction from rhizomes, even small fragments, enables the plant to be easily transferred to new sites, especially in riparian areas (Montpelier, 1998).

* Corresponding author. Tel.: 41 22 379 3401; fax: 41 22 379 3399. E-mail address: kurt.hostettmann@unige.ch (K. Hostettmann). 0305-1978/$ see front matter 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2008.11.018

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This plant may be confused with giant knotweed (Polygonum sachalinensis F. Schmidt ex Maxim, syn: Fallopia sachalinensis, Reynoutria sachalinensis), which has a similar growth form and is also a non-native species with its origins in Asia. Both of these two species are on the black list of invasive alien plants in Switzerland (http://www.cps-skew.ch/english/ black_list.htm). It is said that most invasive plants are phytochemically unique in their new habitats, and many of the phytochemicals from these plants have been reported to have multiple activities, including antiherbivore, antifungal, antimicrobial and allelopathic effects, which may provide the plants with several advantages in their new environments (Cappuccino and Arnason, 2006). As part of a study on the possible impact of their phytochemicals, different crude extracts of P. cuspidatum and P. sachalinensis were investigated. The aim of the work was to ascertain whether the plants growing in Switzerland differed in their chemical composition, since the presence of different metabolites might confer ecological advantages to the introduced varieties. This could be via alteration of soil microbial communities (Kourtev et al., 2002), effects on herbivores or interactions with native competitors. Another aspect is the possibility of alternative medical utilization since it is generally accepted that multiple constituents are responsible for the therapeutic effect of plants (Yi et al., 2007a). The technique of liquid chromatography simultaneously coupled to ultra-violet detection and electrospray ionization mass spectrometry (HPLC/UV/ESI-MS) was used since this is a reliable and sensitive method for the screening of plant secondary metabolites. By this means, the constituents of the different species and organs were analyzed. The chromatographic ngerprints of samples from China and Switzerland were run, to reveal the phytochemical differences between the original and invasive varieties.

Fig. 1. Structures of the main constituents of P. cuspidatum and P. sachalinensis.

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2. Materials and methods 2.1. Chemicals Analytical grade MeOH and DCM were used in the extraction. HPLC-grade CH3CN, Milli-Q water (Millipore Bedford, MA, USA) and analytical grade acetic acid were used for HPLC. Reference compounds piceid, emodin, catechin, quercetin3-O-rhamonoside and 2-caffeoyl glucoside were isolated and puried in-house and their identities were established by NMR and MS; resveratrol was purchased from SigmaAldrich (St. Louis, MO,USA).

2.2. Plant material and sample preparation Roots and stems of P. cuspidatum and P. sachalinensis in Switzerland were collected in Lausanne and Champex, respectively, in October 2007. Stems of P. cuspidatum in China were collected in Shandong in November 2007. The voucher samples of the plants are deposited at the herbarium of the Laboratory of Pharmacognosy and Phytochemistry (no. 2007012 for P. cuspidatum and no. 2007013 for P. sachalinensis). Three dried root samples of P. cuspidatum were purchased from local stores of three Provinces (Shandong, Shanxi, Yunnan) in China. The air-dried powdered materials were extracted at room temperature with CH2Cl2 (3 24 h) followed by MeOH (3 24 h). The extracts were stored at 4  C until use, after concentration under vacuum. Each extract (10 mg) was dissolved in 1 mL MeOH and then centrifuged before injection of 10 mL for HPLC/UV/ESI-MS analysis.

2.3. HPLC/MS analyses HPLC/ESI-MS analyses were performed with a Finnigan MAT (San Jose, CA, USA) model LCQ ion-trap mass spectrometer equipped with a Finnigan electrospray ionization (ESI) source and Xcalibur software. Extracts and reference compounds were analyzed on a Xterra C18 column (5 mm, 3.5 150 mm; Waters, Milford, MA, USA). The mobile phase consisted of 0.5% acetic acid in water (A) and acetonitrile (B). For root MeOH extracts, elution was performed using the following gradient prole: 1040% B in 40 min, then changing to 100% B in 20 min. The UV detector was set at 290 nm. For stem MeOH extracts, the gradient program was 1020% B in 10 min, hold 20% B for 10 min, then 2035% B in 10 min, 35 50% B in 20 min, 50100% B in 10 min. The UV detector was set at 320 nm. For DCM extracts, 1040% B in 10 min, 4080% B in 25 min, 80100% B in 10 min, then hold 100% B for 10 min, UV detector was set at 290 nm. The mobile phase ow rate was 1 mL/min. Before the source, the ow was split: 0.1 mL/min went to the MS and 0.9 mL/min went to the waste. The MS was operated mainly in the negative ion mode with a capillary voltage of 21 V, a source voltage of 4.0 kV, and a tube lens offset of 25 V, sheath gas ow of 60 arb, capillary temperature 240  C. To conrm some peaks, the positive ion mode with the same parameters was also used. Data were acquired in MS full scanning mode with different CID values (0 V, 20.0 V, 35.0 V separately).

mAU 700 600 500 400 300 200 100 0 200

mAU 500 400 300 200 100 250 300 350 400 450 nm 0 200 250 300 350 400 450 nm

Emodin (max 222, 254, 266, 288, 440 nm)

mAU 175 150 125 100 75 50 25 0 200 250 300 350

Piceid(max 222nm, 306, 319 nm)

mAU 2000 1750 1500 1250 1000 750 500 250 0 200

mAU 700 600 500 400 300 200 100 0 200

250

300

350

400

450

nm

400nm

250

300 nm

Quercetin-3-O-rhamnoside (max 256, 349 nm)

2-O-caffeoyl glucose(max 218, 245, 300(sh), 330 nm)

Catechin(max233, 280 nm)

Fig. 2. Characteristic UV spectra of representative compounds for each class studied.

Table 1 Retention times (see Fig. 4), MS data, and UV lmax values of the main constituents present in the root MeOH extracts of P. cuspidatum and P. sachalinensis. Compound 1 2 a 3 4 b 5 c d 6 7 8 9 10 e f g h i j k 11 l m 16
a b c d e

Retention time (min) 8.59 10.51 13.22 15.32 15.62 17.40 18.78 19.45 21.99 23.32 24.06 24.96 28.45 29.63 30.34 31.33 32.43 36.09 38.89 43.61 45.29 45.50 46.00 47.10 50.20

[M H] m/z 288.9 288.9 1152.8 404.8 389.0 728.8 388.7 440.7 880.8 540.8 540.9 406.8 430.9 227.0a 448.7 283.0b 516.7 486.6 778.9 1150.9 984.9 954.9 1133.1 1027.0 269.0

Other ions m/z 245.0 245.0 864.8 [M C15H13O6] 243 [M H-Glu] 227.0 [M H-Glu] 576.7 [M Galloyl] 227 288.8 [M Galloyl] 728.6 [M Galloyl] 226.9 [M H-GalloylGlu] 227.0 [M H-GalloylGlu] 245 [M H-Glu] 269 [M H-Glu] 269.1, 245.0 472.9 283.0 633.1 [M Coumaroyl] 955.0 838.8 [M Coumaroyl] 808.9 957.1 997.0 269.1

lmax (nm)
225, 225, 225, 220, 220, 225, 220, 225, 225, 225, 280 280 280 305, 320 305, 320 280 320 280 280 300

Class of compoundsc Flavanol Flavanol Flavanol Stilbene Stilbene Flavanol Stilbene Flavanol Flavanol Stilbene Stilbene Quinone Anthraquinone Stilbene Anthraquinone Anthraquinone Anthraquinone Anthraquinone Phenylpropanoid glucoside Phenylpropanoid glucoside Phenylpropanoid glucoside Phenylpropanoid glucoside Phenylpropanoid glucoside Phenylpropanoid glucoside Quinone

References or standards Catechin

Identication Catechin Epicatechin Flavanol tetramer Piceatannol glucoside Resveratroloside Flavanol gallate dimer P. Fan et al. / Biochemical Systematics and Ecology 37 (2009) 2434 Piceid Flavanol gallate Flavanol gallate dimer Piceid gallate Piceid gallate Torachrysone-8-Oglucoside Emodin-8-O-glucoside Resveratrol Undened Undened Undened Undened Undened Undened Undened

577.1 [M 2C15H13O6]

Vastano et al., 2000,d Vastano et al., 2000,d

288.9 [M GalloylC15H13O6]

Piceid

577.0 [M 2Galloyl]

Hegde et al., 2004,e Hegde et al., 2004,e Yi et al., 2007b,d Yi et al., 2007b Resveratrol
,d

225, 300 216, 280, 430 220, 280, 430 220, 305, 320 220, 280, 430 220, 280, 430 220, 280, 430 220, 280, 430 210, 300sh, 320 220, 300sh, 320 220, 300sh, 320 210, 300sh, 320 230, 300sh, 320 220, 300sh, 320 220, 280, 430

Kawai et al., 2006,e

Vanicoside B Undened Undened

Emodin

Emodin

In the negative mode, MS peaks are poorly dened, but they are clear in the positive mode ([M H] 229.1). The difference in retention time excludes the possibility of this being rhein, despite the identication from MS and UV data. The class of compound was determined by comparison with related standards. The reference substance proved the existence of the related compounds in the plant. The reference gave the prole information for the related compounds.

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3. Results and discussion 3.1. Standards and references P. cuspidatum and P. sachalinensis have been studied over many years. The main constituents of P. cuspidatum are anthraquinones, stilbenes, avonoids (Kimura et al., 1983), and other phenols such as gallic acid, benzoic acid, tryptophan, catechin, catechin glucoside, tachioside, isotachioside (Xiao et al., 2003). New compounds reported over the last few years include stilbene glycoside sulfates (Xiao et al., 2000), lignan sulfate (Xiao et al., 2002a), dimeric stilbene glycosides (Xiao et al., 2002b), stilbene glycoside gallates (Hegde et al., 2004) etc. In the Chinese pharmacopoeia, emodin and emodin-glucosides are chosen as marker compounds. Phenylpropanoid glycosides were reported as the main type of compounds in P. sachalinensis (Kumagai et al., 2005; Kawai et al., 2006). The main chemical structures are shown in Fig. 1. To acquire reasonable information and to facilitate the identication of all the analytes, representative references of each class of compounds were tested under the same conditions: piceid and resveratrol as representatives of stilbenes, emodin for anthraquinones, catechin for avonols, quercetin-3-O-rhamnoside for avonoids, 2-caffeoyl glucoside for phenylpropanoid glycosides. The classes of compounds can be recognized from their characteristic UV spectra (Fig. 2). 3.2. Optimization of HPLC/UV/MS conditions In order to construct a chromatographic ngerprint with good resolution of the constituents, the HPLC conditions were rst optimized. Both MeOH:H2O and CH3CN:H2O mobile phases with different gradients were tested in the initial part of the work. It was found that CH3CN:H2O mixtures gave better resolution. By comparing the HPLC chromatograms of extracts acquired at different wavelengths within the range 200500 nm, and the corresponding UV absorption maxima for each

Fig. 3. HPLC chromatograms of the MeOH extract (A) and DCM extract (B) of roots of P. cuspidatum from China with detection at 290 nm. (I) From Shandong Province, (II) from Shanxi Province, (III) from Yunnan Province. For conditions, see Materials and methods.

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standard compound, a wavelength of 290 nm was selected for the root MeOH extracts and DCM extracts, while 320 nm was selected for the stem MeOH extracts. Since the MS information can sometimes be ambiguous, it was optimized initially on the representative standards: piceid (stilbene), emodin (anthraquinone) and quercetin rhamnoside (avonoid). Both positive and negative ion modes were tried in order to gather the maximum amount of information about the structures. The results showed that the positive ion mode often produced adducts which interfered with the recognition of the molecular mass. The negative ion mode was chosen for most of the analyses, while the positive ion mode was used only to conrm some ambiguous peaks. The optimized conditions for piceid were chosen since the other types of compounds could be well ionized too. 3.3. Identication of constituents of root extracts of P. cuspidatum and P. sachalinensis by comparison with standards and literature data With HPLC/UV/MS, in many cases, direct identication of the peaks is possible based on a comparison with published data or standard compounds, and this has been a powerful tool for the rapid identication of the constituents in the plant extracts. The retention time, UV and MS data of the MeOH extracts of labeled peaks are listed in Table 1. Of the 25 labeled peaks,

Fig. 4. HPLC/UV (A) and ESI-MS (B) chromatograms of the MeOH extracts of root of P. cuspidatum from Switzerland (I) and Shandong, China (II). Key to peak identity as in Table 1 and Fig. 1. For conditions, see Materials and methods.

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numbers 1, 5, 10, 16 could be identied, respectively, as catechin, piceid, resveratrol and emodin by comparison with the corresponding reference compounds. Peaks 2, a, b, c, d with the same UV spectra (lmax 280 nm) as catechin were tentatively assigned to be avanols. MS data of their molecular ions [M H] and some fragments revealed that peak 2 corresponds to an isomer of catechin ([M H] at m/z 288.9); peak a to the avanol tetramer ([MH] m/z at 1152.8, with successive loss of two monomer units [M C15H13O6] at m/z 864.8, and [M 2C15H13O6] at m/z 577.1). For peaks b, c, d, loss of m/z 152 indicates the existence of a galloyl group. Peak b is a avanol gallate dimer ([M H] at m/z 728.8, [M Galloyl] at m/z 576.7, [M Galloyl-C15H13O6] at m/z 288.9), peak c is a avanol gallate ([M H] at m/z 440.7, [M Galloyl] at m/z 288.8), peak d is a avanol gallate dimer ([M H] at m/z 880.8, [M Galloyl] at m/z 728.6, [M 2Galloyl] at m/z 577.0). Peaks 3, 4, 6, 7 are stilbenes with the same UV spectra as piceid 5 and resveratrol 10 (lmax 220, 320 nm). Peaks 3, 4 correspond, respectively, to piceatannol glucoside ([M H] at m/z 404.8, [M H-Glu] at m/z 243.0) and resveratroloside ([M H] at m/z 389.0, [M H-Glu] at m/z 227.0), which were also present in Mexican Bamboo from South America and were unambiguously identied (Vastano et al., 2000). Peaks 6, 7 are thought to be two piceid gallates ([M H] at m/z 540.8/540.9, [M H-Galloyl-Glu] at m/z 226.9/227.0), which were isolated from P. cuspidatum (Hegde et al., 2004). UV spectra for peaks 8, 9, eh are typical of quinones (lmax 220, 280, 430 nm). Compounds 8 and 9 were tentatively identied as torachrysone-8-O-glucoside ([M H] at m/z 406.8, [M H-Glu] at m/z 245) and emodin-8-O-glucoside ([M H] at m/z 430.9, [M H-Glu] at m/z 269) according to their similar behavior on HPLC and MS data with those in the literature (Yi et al., 2007b). Peaks eh could not be identied with the present information. According to the UV proles, peaks im (Fig. 5) were assigned as phenylpropanoid glucosides. UV spectra for phenylpropanoid glucosides can be easily distinguished with those of stilbenes, since for the former, there is a shoulder at about 300 nm, while for the latter, from 300320 nm, the intensity of the absorbance is almost at the same level. Many compounds of this class have been isolated from the genus Polygonum (Brown et al., 1998; Sun et al., 2000; Takasaki et al., 2001a,b). Vanicoside B was reported as the main constituent in the root of P. sachalinensis (Kawai et al., 2006). With the molecular ion data, peak 11 was assigned as vanicoside B ([M H] at m/z 954.9). Peaks im cannot be completely identied with the present MS data. Compounds 24, 8 and 9 were isolated later and their online identication was conrmed by analysis of NMR and MS data. 3.4. HPLC/UV/MS chromatographic ngerprints of root extracts of P. cuspidatum and P. sachalinensis 3.4.1. Fingerprints of MeOH and DCM extracts of P. cuspidatum from China HPLC chromatographic ngerprints of MeOH (Fig. 3A) and DCM extracts (Fig. 3B) of three Chinese samples of P. cuspidatum (from Shandong, Shanxi, Yunnan Provinces, individually) were similar, and MS data also conrmed this fact. In the MeOH extracts of these three varieties, the predominant compounds were piceid (5, Rt 18.78, [M H] 388.7), emodin-8-O-b-Dglucoside (9, Rt 28.45, [M H] 430.9), emodin (16, Rt 50.20, [M H] 269.0), resveratrol (10, Rt 29.63, [M H] 227.0), torachrysone-8-O-b-D-glucoside (8, Rt 24.96, [M H] 406.8); the minor compounds observed were resveratroloside (4, Rt 15.62, [M H] 389.0), vanicoside B (11, Rt 45.50, [M H] 954.9), other unidentied anthraquinones (Rt 31.33, [M H] 283.0; Rt 32.43, [M H] 516.7; Rt 36.09, [M H] 486.6) and a phenylpropanoid glycoside (Rt 38.89, [M H] 778.9). More detailed information and identications are listed in Table 1. In the DCM extracts, emodin (16) and citreorosein (20, [M H] 285.1) were present. There were no obvious differences among these three varieties.

Fig. 5. HPLC chromatogram of MeOH extracts of roots of P. cuspidatum and P. sachalinensis from Switzerland. Key to peak identity as in Table 1 and Fig. 1. For conditions, see Materials and methods.

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Fig. 6. HPLC chromatogram of DCM extracts of roots of P. cuspidatum and P. sachalinensis. For conditions, see Materials and methods.

3.4.2. Fingerprints of MeOH extracts of P. cuspidatum from China and Switzerland To compare the Chinese and Swiss Polygonum samples, the one from Shandong province was chosen as a representative. HPLC/UV chromatograms of MeOH extracts of P. cuspidatum from Shandong, China (root) and Switzerland (root bark) are presented in Fig. 4A, together with their MS chromatograms (Fig. 4B). 3.4.3. Fingerprints of MeOH extracts of P. cuspidatum and P. sachalinensis from Switzerland Fig. 5 shows the comparison of the HPLC/UV chromatogram of MeOH extracts of root bark and root wood of P. cuspidatum (Switzerland) and roots of P. sachalinensis (Switzerland). 3.4.4. Fingerprints of DCM extracts of P. cuspidatum and P. sachalinensis from China and Switzerland The comparison of the DCM extracts of root of P. cuspidatum (China), root bark and wood of P. cuspidatum (Switzerland) and roots of P. sachalinensis (Switzerland) are presented in Fig. 6. For DCM extracts, the main constituents of P. cuspidatum were identied as emodin ([M H] at m/z 269, HPLC behaviour identical with standards); the presence of citreorosein was indicated by the data for [M H] at m/z 285.1, and lmax 430 nm (Kimura et al., 1983). But for the DCM extract of P. sachalinensis, there is no visible peak under the same conditions. 3.5. Comparisons of the extracts of P. cuspidatum and P. sachalinensis There are no signicant differences among the three Chinese samples from three provinces of China (Fig. 3), but by comparing P. cuspidatum from Switzerland and from China, it was found that stilbene glycosides such as piceatannol

Fig. 7. HPLC chromatograms of MeOH extracts of stems of P. cuspidatum and P. sachalinensis. For conditions, see Materials and methods.

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Table 2 Retention time, MS data, and UV (lmax) values of the main constituents present in the stem MeOH extracts of P. cuspidatum and P. sachalinensis. Compound 5 17 18 19 n 14 15 o p 13 11 q 12
a

Rt (min) 17.41 20.22 25.62 26.84 34.93 35.26 37.15 37.59 41.13 44.03 44.42 45.62 48.46

[M H] m/z 388.6 462.9 432.7 446.8 868.9 808.9 778.9 850.9 1150.9 984.9 954.9 1133 996.9

Other ions m/z 227.0 [M H-Glu] 301.0 [M H-Gla] 300.7 [M H-Ara] 301 [M H-Rha] 839.0[M CH3O], 809.0[M 2CH3O] 662.9 [M Coumaroyl] 632.9 [M Coumaroyl] 704.9 [M Coumaroyl] 954.9 [M C9H10O5] 838.8 [M Coumaroyl] 808.8 [M Coumaroyl] 1103 [M CH3O] 851.2 [M Coumaroyl]

lmax (nm)
230, 320 230,260, 350 230,260, 350 230,260, 350 230, 300sh, 320 230, 230, 230, 230, 230, 230, 230, 230, 300sh, 300sh, 300sh, 300sh, 300sh, 300sh, 300sh, 300sh, 320 320 320 320 320 320 320 320

Class of compounds Stilbene Flavone Flavone Flavone Phenylpropanoid glucoside Phenylpropanoid Phenylpropanoid Phenylpropanoid Phenylpropanoid Phenylpropanoid Phenylpropanoid Phenylpropanoid Phenylpropanoid glucoside glucoside glucoside glucoside glucoside glucoside glucoside glucoside

References or standards Piceid

Identication Piceid Quercetin-3-O-galactoside Avicularin Quercetin-3-O-rhamnoside Undened Lapathoside Ca C40H42O18 Hydropiperosidea C39H40O17 Undened Undened Lapathoside Aa C50H50O21 Vanicoside B Undened Vanicoside A

Quercetin-3-O-rhamnoside

692.8 [M Feruloyl]

662.8 [M 2Coumaroyl H] 955 [M Dihydrocoumaroyl] 704.9 [M Coumaroyl H]

Kawai et al., 2006 Kawai et al., 2006

Based on the corresponding molecules of the same class and MS data from the family Polygonaceae in Scinder database.

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glucoside (3) and resveratroloside (4) predominate in the invasive variety of Switzerland (Fig. 4A I), and proanthocyanidins such as catechin (1), epicatechin (2), avanol tetramer (a), avonol gallates (bd) are also obviously present. As observed in Fig. 4, stilbenes (peaks 3, 4, 5, 10) and anthraquinones (9, 16) are the main constituents of MeOH extracts of root of P. cuspidatum, while phenylpropanoid glycosides (11, im) are the main constituents of the MeOH extracts of root of P. sachalinensis (Fig. 5 I). There are no detectable stilbenes and anthraquinones in P. sachalinensis. In contrast, phenylpropanoid glycosides are present in P. cuspidatum (11 in Fig. 4A), especially in the wood (Fig. 5 II). In the DCM extracts, emodin and citreorosein are the main constituents of P. cuspidatum. There is no obvious difference between the samples from China and Switzerland. However, the two compounds are absent in the root of P. sachalinensis. 3.6. Identication of constituents and comparison of the stem extracts of P. cuspidatum and P. sachalinensis The stems of the two species have been less studied in the literature than the roots, so comparisons were performed in the present work. The three samples were stems of P. cuspidatum from Shandong (China) and from Switzerland, and of P. sachalinensis from Switzerland. Phenylpropanoid glycosides proved to be the main constituents in the MeOH extracts of these three samples. Only the relative quantities were different (Fig. 7). Peaks 11, 12, 13, 14, 15 were tentatively identied as vanicoside B, vanicoside A, lapathoside A, lapathoside C, hydropiperoside, respectively, based on the MS data and fragments, as well as from the fact that all these compounds have already been reported from the same genus Polygonum. The structures of peaks nq could not be deduced from MS and UV data alone (Table 2), so isolation work is being performed for their identication. Piceid and three avonoids, quercetin-3-O-galactoside (17, [M H] at m/z 462.9, [M Gal] at m/z 301.0, previously isolated from owers of P. sachalinensis (Zhang et al., 2005)), avicularin (18, [M H] at m/z 432.7, [M Ara] at m/ z 300.7, previously isolated from owers of P. sachalinensis (Zhang et al., 2005)) and quercetin-3-O-rhamnoside (19, [M H] at m/z 432.7, [M Rha] at m/z 301, identical with reference compound) were also found in the two varieties of P. cuspidatum. Piceid (5) was present in the stems of P. cuspidatum, but not in P. sachalinensis. Emodin and citreorosein were the main constituents in the DCM extracts of P. cuspidatum, but not in P. sachalinensis (Fig. 8). In summary, with the optimized HPLC/UV/ESI-MS analyses of the crude extracts of roots and stems of four samples of P. cuspidatum Sieb. and Zucc. and one sample of P. sachalinensis F. Schmidt ex Maxim from China or Switzerland, a total of 36 constituents were entirely or partially identied by comparing their retention times, UV data, mass spectra with those of standards or with literature. Flavanol gallate dimers (b and d) have not been previously cited in the literature. It was shown that while the constituents of the roots of the three samples of P. cuspidatum from China were almost identical, they were obviously different from the species collected in Switzerland (present as an invasive neophyte) with respect to piceatannol glucoside, resveratroloside and some proanthcyanidins. No piceatannol was detected in the roots of P. cuspidatum from China but could be found in P. cuspidatum from Switzerland. The main constituents of roots of P. sachalinensis were not comparable with those of P. cuspidatum, despite the similarity of the compounds in the stems of both species. Phenylpropanoid glucosides were the main constituents of the roots and stems of P. sachalinensis, and were present in the root wood and stems of P. cuspidatum, while stilbenes and anthraquinones were predominant constituents of the roots of P. cuspidatum.

mAU 300

*DAD1 C, Sig=290,4 Ref=550,4,P.sachalinensisstem (Switzerland) (I) *DAD1 C, Sig=290,4 Ref=550,4,P.cuspidatumstem (Switzerland) (II) *DAD1 C, Sig=290,4 Ref=550,4,P.cuspidatumstem (China) (III)

250

emodin [M-H]-m/z269.1 citreorosein [M-H]-m/z285.1

200

150

100 III 50 II I 0 0 10 20 30 40 50 min

Fig. 8. HPLC chromatogram of DCM extracts of stems of P. cuspidatum and P. sachalinensis. For conditions, see Materials and methods.

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P. Fan et al. / Biochemical Systematics and Ecology 37 (2009) 2434

The question is now whether the different proportions of constituents in the invasive P. cuspidatum and P. sachalinensis growing in Switzerland confer an ecological advantage to the plant. Are the stilbene glycosides piceatannol glucoside (3) and resveratroloside (4) important for the rapid spread of the introduced species? And what is their role? Stilbenes are a class of biologically active compounds which possess various medicinal activities (Goldberg et al., 1996). In view of the presence of stilbenes and the fast growing characteristics of P. cuspidatum, there may be a potential for this plant in the nutraceutical industry as a medicinal crop. On the other hand, it might also be that the different chemical compositions of the Swiss species preclude the use of P. cuspidatum as an herbal medicine. These aspects can be studied in further detail as the constituents of the Swiss and Chinese samples are now known. Acknowledgements This work was supported by the Swiss National Science Foundation (grant no. 200020-107775 to Prof. K. Hostettmann). The authors also gratefully acknowledge support by the China Scholarship Council. References
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