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Quality assessment of Japanese knotweed (Fallopia japonica) grownon Prince Edward Island as a source of resveratrol
Chen, Huaguo; Tuck, Tina; Ji, Xiuhong; Zhou, Xin; Kelly, Glen; Cuerrier,
Alain; Zhang, JunzengThis publication could be one of several versions: author’s original, accepted manuscript or the publisher’s version. /La version de cette publication peut être l’une des suivantes : la version prépublication de l’auteur, la version
acceptée du manuscrit ou la version de l’éditeur.
For the publisher’s version, please access the DOI link below./ Pour consulter la version de l’éditeur, utilisez le lien
DOI ci-dessous.

Publisher’s version / Version de l'éditeur:
https://doi.org/10.1021/jf4019239

Journal of Agricultural and Food Chemistry, 61, 26, pp. 6383-6392, 2013-06-06

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Articlepubs.acs.org/JAFC

Quality Assessment of Japanese Knotweed (Fallopia japonica) Grown
on Prince Edward Island as a Source of Resveratrol
Huaguo Chen,†,§ Tina Tuck,† Xiuhong Ji,† Xin Zhou,§ Glen Kelly,# Alain Cuerrier,⊥and Junzeng Zhang*,††

Aquatic and Crop Resource Development, National Research Council Canada, 550 University Avenue, Charlottetown, PE C1A 4P3,
Canada§The Research Center for Quality Control of Natural Medicine, Guizhou Normal University, 116 Baoshan North Road, Guiyang,
Guizhou 550001, People’s Republic of China#
Northeast Biological Processors Ltd., R.R. 3, Charlottetown, PE C1A 7J7, Canada⊥
Jardin botanique de Montréal, Institut de recherche en biologie végétale, Université de Montréal, 4101 rue Sherbrooke Est,
Montréal, Québec H1X 2B2, Canada
ABSTRACT: Japanese knotweed (Fallopia japonica, also known as Polygonum cuspidatum) is a common invasive plant species
on Prince Edward Island (PEI), Canada, whereas it has been used in Chinese medicine and more recently as a raw material for
extracting resveratrol. This paper reports on the quantiﬁcation of resveratrol, polydatin, emodin, and physcion in roots, stems,
and leaves of Japanese knotweed samples from PEI and British Columbia (BC), Canada, and nine provinces of China, by
ultraperformance liquid chromatography (UPLC). The results showed that the root contains a much higher level of resveratrol
than the stem and leaf, and it is accumulated in its highest level in October. PEI-grown knotweed contains similar levels of
resveratrol and polydatin compared to Chinese samples collected in the month of October, but the contents of the otheranthraquinones (emodin and physcion) are diﬀerent. As such, Japanese knotweed grown in PEI could be a commercially viable
source of raw material for resveratrol production; however, caution has to be taken in harvesting the right plant species.
KEYWORDS: Japanese knotweed, Fallopia japonica, UPLC, resveratrol, quality assessment
commercially viable natural sources of resveratrol for the growing
nutraceutical market. The rhizome or root of F. japonica, also
known as Japanese knotweed, is currently a major source for thenatural resveratrol ingredients sold in dietary supplement
markets worldwide.22,23 On Prince Edward Island (PEI), andother places in Canada, Japanese knotweed is a common invasiveplant species. However, until now no information was available
on the level of resveratrol in knotweed grown in PEI.
As the demand for natural resveratrol is expected to growrapidly in the future with increasing applications in the
nutraceutical, therapeutic, and cosmeceutical markets, assessing
PEI-grown knotweed as a commercial source could present newmarket opportunities for agriculture and bioresource sectors in
the region. In this study, more than 50 knotweed samples
collected from 4 diﬀerent sites from PEI over various seasons
have been analyzed for their levels of resveratrol and othercomponents. Knotweed samples from China, which have beenused as raw material for resveratrol production, were also studiedin parallel.

INTRODUCTION
Fallopia japonica (Houtt.) Ronse Decraene (previously known as
Polygonum cuspidatum Sieb. & Zucc.), a well-known Chinese
herb and oﬃcially listed in the Chinese Pharmacopoeia, has been
traditionally used for treatment of various inﬂammatory diseases,
hepatitis, tumors, and diarrhea in Eastern Asian countries such as
China, Korea, and Japan.1−6 In North America, the young stemof F. japonica (Japanese knotweed) is edible and was consumed
as a vegetable in the past.7 Japanese knotweed is listed as one of
the “World’s Worst” invaders, and the farming and landscaping
sectors have to control its growth and invasive nature.8
Over the past decades, the chemical composition9−11 andbiological activity4,5,12,13 as well as quality control2,9 of F. japonica
have been widely reported, and resveratrol, polydatin, emodin,
physcion, chrysophanol, rhein, and emodin-8-O-β-D-glucosidewere revealed as some of the main constituents.
Resveratrol, or trans-3,5,4′-trihydroxystilbene, is a naturally
occurring antioxidant compound typically associated with grapes
and red wine14,15 and is also present in F. japonica.16 Extensive
research on resveratrol over the past decade has demonstrated
signiﬁcant beneﬁcial eﬀects including the promotion of heart
health, neuroprotection, anti-inﬂammation, and anticancer andantiaging eﬀects, as well as diabetes and obesity prevention.17−20
These documented health beneﬁts have raised the proﬁle andinterest in resveratrol as an active ingredient in the growing
nutraceutical and cosmeceutical industries.
Due to the relatively low level of resveratrol present in grapeskin,14,21 other plants have been investigated as potential and■© 2013 American Chemical Society■

MATERIALS AND METHODS

Instrumentation and Reagents. UPLC analysis was carried out onan Agilent 1200 liquid chromatography system, equipped with a vacuum
Received: May 2, 2013
Revised: June 3, 2013
Accepted: June 6, 2013
Published: June 6, 2013
6383

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Journal of Agricultural and Food Chemistry

ArticleTable 1. Fallopia japonica and Other Knotweed Samples Analyzed in This Study
sampleNRC-INH code

Latin nameorigin/collection siteacquisition/collection time

plant part

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61

INH-OS-114
INH-OS-115
INH-OS-116
INH-OS-117
INH-OS-118
INH-OS-119
INH-OS-120
INH-OS-121
INH-OS-122
INH-OS-123
INH-OS-124
INH-OS-125
INH-OS-126
INH-OS-127
INH-OS-135
INH-OS-136
INH-OS-137
INH-OS-138
INH-OS-139
INH-OS-140
INH-OS-141
INH-OS-142
INH-OS-143
INH-OS-144
INH-OS-145
INH-OS-146
INH-OS-147
INH-OS-148
INH-OS-149
INH-OS-150
INH-OS-151
INH-OS-152
INH-OS-153
INH-OS-154
INH-OS-155
INH-OS-156
INH-OS-157
INH-OS-158
INH-OS-159
INH-OS-160
INH-OS-161
INH-OS-162
INH-OS-163
INH-OS-164
INH-OS-165
INH-OS-166
INH-OS-167
INH-OS-168
INH-OS-169
INH-OS-170
INH-368
INH-369
INH-367
INH-366
INH-365
INH-373
INH-372
INH-371
INH-370
INH-382
INH-381

Fallopia japonica
Fallopia japonica
Fallopia japonica
Fallopia japonica
Fallopia japonica
Fallopia japonica
Fallopia japonica
Fallopia japonica
Fallopia japonica
Fallopia japonica
Fallopia japonica
Fallopia japonica
Fallopia japonica
Fallopia japonica
Fallopia × bohemica
Fallopia japonica
Fallopia japonica
Fallopia × bohemica
Fallopia japonica
Fallopia japonica
Fallopia × bohemica
Fallopia × bohemica
Fallopia × bohemica
Fallopia × bohemica
Fallopia japonica
Fallopia × bohemica
Fallopia japonica
Fallopia × bohemica
Fallopia japonica
Fallopia × bohemica
Fallopia × bohemica
Fallopia × bohemica
Fallopia × bohemica
Fallopia × bohemica
Fallopia × bohemica
Fallopia japonica
Fallopia japonica
Fallopia japonica
Fallopia × bohemica
Fallopia × bohemica
Fallopia × bohemica
Fallopia × bohemica
Fallopia japonica
Fallopia japonica
Fallopia japonica
Fallopia × bohemica
Fallopia × bohemica
Fallopia × bohemica
Fallopia × bohemica
Fallopia × bohemica
Fallopia japonica
Fallopia japonica
Fallopia japonica
Fallopia japonica
Fallopia japonica
Fallopia × bohemica
Fallopia × bohemica
Fallopia × bohemica
Fallopia × bohemica
Fallopia × bohemica
Fallopia × bohemica

Zunyi, Guizhou Province, China
Bijie, Guizhou Province, China
Leishan, Guizhou Province, China
Dafang, Guizhou Province, China
Anshun, Guizhou Province, China
Guiyang, Guizhou Province, China
Zhejiang Province, China
Fujian Province, China
Sichuan Province, China
Yunnan Province, China
Henan Province, China
Jiangxi Province, China
Jiangsu Province, China
Hubei Province, China
St. Margaret’s, PEI, Canada
Dunstaffnage, PEI, Canada
Dunstaffnage, PEI, Canada
St. Margaret’s, PEI, Canada
Dunstaffnage, PEI, Canada
Dunstaffnage, PEI, Canada
St. Margaret’s, PEI, Canadasite 1, Summerside, PEI, Canadasite 1, Summerside, PEI, Canada
St. Margaret’s, PEI, Canada
Dunstaffnage, PEI, Canada
St. Margaret’s, PEI, Canada
Dunstaffnage, PEI, Canada
St. Margaret’s, PEI, Canada
Dunstaffnage, PEI, Canadasite 1, Summerside, PEI, Canadasite 1, Summerside, PEI, Canadasite 1, Summerside, PEI, Canada
St. Margaret’s, PEI, Canada
St. Margaret’s, PEI, Canada
St. Margaret’s, PEI, Canada
Dunstaffnage, PEI, Canada
Dunstaffnage, PEI, Canada
Dunstaffnage, PEI, Canadasite 1, Summerside, PEI, Canadasite 1, Summerside, PEI, Canadasite 1, Summerside, PEI, Canadasite 1, Summerside, PEI, Canada
Dunstaffnage, PEI, Canada
Dunstaffnage, PEI, Canada
Dunstaffnage, PEI, Canadasite 1, Summerside, PEI, Canadasite 1, Summerside, PEI, Canada
St. Margaret’s, PEI, Canada
St. Margaret’s, PEI, Canada
St. Margaret’s, PEI, Canada
Dunstaffnage, PEI, Canada
Dunstaffnage, PEI, Canada
Dunstaffnage, PEI, Canada
Dunstaffnage, PEI, Canada
Dunstaffnage, PEI, Canada
St. Margaret’s, PEI, Canada
St. Margaret’s, PEI, Canada
St. Margaret’s, PEI, Canada
St. Margaret’s, PEI, Canadasite 2, Summerside, PEI, Canadasite 2, Summerside, PEI, Canada

Jan 2009
March 2009
June 2009
Aug 2009
Jan 2009
April 2009
Nov 2010
Nov 2010
Nov 2010
Nov 2010
Nov 2010
Nov 2010
Nov 2010
Nov 2010
May 14, 2010
May 5, 2010
May 5, 2010
May 20, 2010
May 20, 2010
May 20, 2010
May 20, 2010
May 20, 2010
May 20, 2010
July 13, 2010
July 13, 2010
July 13, 2010
July 13, 2010
July 13, 2010
July 13, 2010
July 13, 2010
July 13, 2010
July 13, 2010
Aug 28, 2010
Aug 28, 2010
Aug 28, 2010
Aug 28, 2010
Aug 28, 2010
Aug 28, 2010
Aug 28, 2010
Aug 28, 2010
Aug 28, 2010
Oct 14, 2010
Oct 14, 2010
Oct 14, 2010
Oct 14, 2010
Oct 14, 2010
Oct 14, 2010
Oct 14, 2010
Oct 14, 2010
Oct 14, 2010
Aug 30, 2011
Aug 30, 2011
Aug 30, 2011
Aug 30, 2011
Aug 30, 2011
Aug 30, 2011
Aug 30, 2011
Aug 30, 2011
Aug 30, 2011
Aug 30, 2011
Aug 30, 2011

root
root
root
root
root
root
root
root
root
root
root
root
root
root
root
root
stem and leaf
root
root
stem and leaf
stem and leafroot
stem and leaf
stem
leaf
leaf
root
root
stem
stem
root
leaf
stem
leaf
root
stem
leaf
root
stem
leaf
root
stem
stem
leaf
root
leaf
root
stem
leaf
root
root (old)root (young)
stem
leaf
flower
root
stem
leaf
flowerroot (old)root (young)6384

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Journal of Agricultural and Food Chemistry

ArticleTable 1. continued
sampleNRC-INH code

Latin nameorigin/collection siteacquisition/collection time

62
63
64
65
66
67
68
69
70
71
72
73
74
75
76

INH-380
INH-379
INH-378
INH-374
INH-375
INH-376
INH-377
INH-OS-242
INH-OS-243
INH-OS-238
INH-OS-241
INH-OS-248
INH-OS-247
INH-OS-240
INH-OS-239

Fallopia × bohemica
Fallopia × bohemica
Fallopia × bohemica
Fallopia × bohemica
Fallopia × bohemica
Fallopia × bohemica
Fallopia × bohemica
Fallopia japonica
Fallopia × bohemica
Fallopia × bohemica
Fallopia japonica
Polygonum polystachyum
Polygonum polystachyum
Fallopia × bohemica
Fallopia × bohemica

site 2, Summerside, PEI, Canadasite 2, Summerside, PEI, Canadasite 2, Summerside, PEI, Canadasite 1, Summerside, PEI, Canadasite 1, Summerside, PEI, Canadasite 1, Summerside, PEI, Canadasite 1, Summerside, PEI, Canada
Fraser Valley, BC, Canada
Fraser Valley, BC, Canada
Fraser Valley, BC, Canada
Fraser Valley, BC, Canada
Fraser Valley, BC, Canada
Fraser Valley, BC, Canada
Fraser Valley, BC, Canada
Fraser Valley, BC, Canada

Aug 30, 2011
Aug 30, 2011
Aug 30, 2011
Aug 30, 2011
Aug 30, 2011
Aug 30, 2011
Aug 30, 2011
Aug 31, 2011
Aug 31, 2011
Aug 31, 2011
Aug 31, 2011
Aug 31, 2011
Aug 31, 2011
Aug 31, 2011
Aug 31, 2011

plant part
stem
leaf
flower
root
stem
leaf
flower
leaf
stem
leaf
root
root
stem
stem
root

Figure 1. Eﬀect of diﬀerent extraction methods (A) and solvents (B) on contents of targeted compounds. UE, ultrasonic extraction; SEX, Soxhlet
extraction; SE, soak extraction.
degasser, a quaternary pump, an autosampler, and a diode array detector(DAD), connected to a reversed-phase column (ZORBAX RRHD SBC18 2.1 × 100 mm, 1.8 μm, Agilent USA). Data collection was
performed using ChemStation (Agilent). An ultrasonic cleaner was used
for sample extraction (VMR International, West Chester, PA, USA).
The water used for all of the solutions and dilutions was prepared with aMillipore FONA84400 water puriﬁcation system (USA). Methanol is of
HPLC grade (Caledon Laboratories Ltd., Canada). Resveratrol,
polydatin, emodin, and physcion (>98%) were purchased from
Sigma-Aldrich (USA).
Knotweed Samples. Fourteen rhizome and root samples of F.
japonica acquired commercially from 9 provinces in China, 54 samplesof F. japonica and a related knotweed species from PEI, Canada, and 8 F.
japonica and other knotweed samples (F. × bohemica and F. polystachya)from British Columbia (BC), Canada, were investigated (Table 1).
Voucher specimens for knotweed samples collected in PEI have been
kept at the National Research Council Canada.
Following closer taxonomical investigations, we were able to reveal
two distinct taxa among the PEI samples, Fallopia japonica (Houtt.)Ronse Decraene and the hybrid F. × bohemica (Chrtek & Chrtková) J. P.
Bailey. On the basis of previous systematic studies,24−26 we identiﬁedsome samples and came across numerous populations of F. × bohemica.
Description of the voucher specimens can be helpful for researchers
involved in natural health products and members of industries who
might want to address harvesting, cultivation, and processing of speciﬁc
taxa/samples.
F. japonica (Houtt.) Ronse Decraene var. japonica (NRC voucher
specimen code INH-365-369) is a rhizomatous shrub bearing rather
6385

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Journal of Agricultural and Food Chemistry

ArticleTable 2. Orthogonal Design and Statistical Analysis for Optimization of Extraction Conditions(A) Factors and Levels of Orthogonal Design
factorsolvent/sample ratio (mL/g) (A)

1
2
3

25:1
30:1
35:1

extraction time (min) (B)methanol/water ratio (v/v) (C)factorsatimes of extraction (D)

10
50:50
20
60:40
30
70:30
(B) Arrangement and Results of L9 (34) Orthogonal Test

1
2
3

content (mg/g)

no.

A

B

C

1
2
3
4
5
6
7
8
9
K1
K2
K3
R

25:1
25:1
25:1
30:1
30:1
30:1
35:1
35:1
35:1
57.66
48.95
47.31
10.35

10
20
30
10
20
30
10
20
30
51.11
51.17
51.64
0.53

50:50
60:40
70:30
60:40
70:30
50:50
70:30
50:50
60:40
54.05
49.50
50.37
4.55

D

resveratrol

polydatin

1
5.66
4.28
2
5.11
4.05
3
4.88
4.36
3
4.29
2.99
1
4.52
3.84
2
4.12
3.8
2
3.7
3.33
3
3.38
3.52
3
3.57
4.06
53.17
51.06
49.69
3.48
(C) Analysis of Variance (ANOVA) Results

emodinphyscion

total

5.63
5.51
5.06
3.81
4.79
5.19
4.55
4.33
4.01

5.12
3.82
4.18
4.02
3.43
4.15
3.73
4.87
4.26

20.69
18.49
18.48
15.11
16.58
17.26
15.31
16.10
15.90

sourcesum of squares

degrees of freedom

F value

P value

Aa
B
Ca
Da
total

20.63
0.056
3.88
2.04
26.61

2
2
2
2
8

367.38
1.00
69.26
36.48

<0.05
>0.05
<0.05
<0.05

Signiﬁcant (P < 0.05), F0.05(3, 2) = 19.16419.

Figure 2. UPLC chromatograms of standard compounds (A, peak 1, polydatin; 2, resveratrol; 3, emodin; and 4, physcion), PEI-grown Japaneseknotweed extract (B), and extract of F. japonica from China (C).

6386

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Journal of Agricultural and Food Chemistry

ArticleTable 3. Contents of Resveratrol, Polydatin, Emodin, and Physcion from Diﬀerent Samples (n = 3)
samplepolydatin(mg/g)

resveratrol(mg/g)

emodin(mg/g)

physcion(mg/g)

samplepolydatin(mg/g)

resveratrol(mg/g)

emodin(mg/g)

physcion(mg/g)

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38

6.57 ± 0.12
6.53 ± 0.11
12.55 ± 0.19
12.33 ± 0.20
8.87 ± 0.15
5.72 ± 0.11
8.77 ± 0.13
10.45 ± 0.16
8.82 ± 0.11
9.20 ± 0.14
8.57 ± 0.12
12.01 ± 0.07
7.61 ± 0.11
11.73 ± 0.19
0.74 ± 0.02
11.40 ± 0.25
0.30 ± 0.05
0.56 ± 0.07
10.34 ± 0.21
0.16 ± 0.03
0.28 ± 0.05
7.18 ± 0.20
0.20 ± 0.02
0.04 ± 0.04
0.13 ± 0.02
0.06 ± 0.01
12.16 ± 0.27
0.67 ± 0.05
0.08 ± 0.01
0.09 ± 0.01
11.88 ± 0.25
0.13 ± 0.01
0.01 ± 0.01
0.13 ± 0.02
0.62 ± 0.01
0.08 ± 0.01
0.16 ± 0.02
13.38 ± 0.24

4.72 ± 0.09
2.77 ± 0.07
2.92 ± 0.06
2.89 ± 0.07
3.41 ± 0.07
2.88 ± 0.09
6.39 ± 0.07
3.94 ± 0.06
5.50 ± 0.07
2.06 ± 0.03
2.76 ± 0.04
12.07 ± 0.13
3.71 ± 0.05
4.11 ± 0.04
0.11 ± 0.02
0.83 ± 0.05
0.15 ± 0.02
0.14 ± 0.02
0.85 ± 0.04
0.03 ± 0.01
0.03 ± 0.01
0.56 ± 0.02
0.05 ± 0.01
000.04 ± 0.01
1.06 ± 0.09
0.07 ± 0.04
00.01 ± 0.01
1.12 ± 0.05
0000.08 ± 0.01
002.13 ± 0.05

6.59 ± 008
4.48 ± 0.06
7.23 ± 0.10
9.02 ± 0.6
5.94 ± 0.10
4.37 ± 0.07
4.08 ± 0.06
6.15 ± 0.09
7.79 ± 0.08
6.45 ± 0.09
6.05 ± 0.07
8.39 ± 0.06
8.25 ± 0.07
9.23 ± 0.10
0.54 ± 0.05
3.59 ± 0.11
0.41 ± 0.03
0.34 ± 0.03
4.58 ± 0.15
0.06 ± 0.01
0.11 ± 0.01
4.60 ± 0.09
0.12 ± 0.01
0.03 ± 0.01
0.05 ± 0.01
0.05 ± 0.01
6.51 ± 0.13
0.54 ± 0.06
0.04 ± 0.01
0.04 ± 0.01
5.42 ± 0.11
0.05 ± 0.01
00.01 ± 0.01
0.64 ± 0.07
0.04 ± 0.01
0.03 ± 0.01
2.65 ± 0.10

5.55 ± 0.08
2.59 ± 0.05
9.13 ± 0.11
15.27 ± 0.23
12.66 ± 0.21
6.29 ± 0.18
3.73 ± 0.08
10.23 ± 0.10
12.37 ± 0.18
15.72 ± 0.28
12.86 ± 0.21
12.84 ± 0.20
11.30 ± 0.17
13.84 ± 0.21
1.52 ± 0.09
15.54 ± 0.30
0.77 ± 0.03
1.04 ± 0.08
15.42 ± 0.28
0.16 ± 0.01
0.39 ± 0.04
9.71 ± 0.20
0.24 ± 0.02
0.07 ± 0.01
0.13 ± 0.02
0.06 ± 0.01
14.54 ± 0.21
1.43 ± 0.10
0.04 ± 0.01
07.20 ± 0.14
0.06 ± 0.01
001.17 ± 0.08
0.08 ± 0.01
0.89 ± 0.03
9.49 ± 0.19

39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76

0.10 ± 0.01
0.13 ± 0.01
13.47 ± 0.30
0.07 ± 0.01
0.10 ± 0.02
0.25 ± 0.04
8.28 ± 0.18
0.41 ± 0.03
9.34 ± 0.17
0.03 ± 0.01
0.06 ± 0.01
0.43 ± 0.03
12.18 ± 0.22
3.11 ± 0.02
0.11 ± 0.01
0.15 ± 0.02
012.95 ± 0.19
0.08 ± 0.01
0.05 ± 0.01
013.26 ± 0.21
13.68 ± 0.27
0.09 ± 0.01
0.17 ± 0.01
01.13 ± 0.03
0.11 ± 0.01
0.06 ± 0.01
00.07 ± 0.01
0.07 ± 0.01
0.06 ± 0.01
6.33 ± 0.13
8.25 ± 0.14
0.09 ± 0.01
0.07 ± 0.01
6.72 ± 0.12

002.01 ± 0.06
000
6.27 ± 0.13
02.74 ± 0.07
00.01 ± 0.01
0.05 ± 0.01
4.31 ± 0.03
1.36 ± 0.04
0002.17 ± 0.03
0002.35 ± 0.03
2.28 ± 0.07
0000.18 ± 0.01
0
0
0
0
0
0
1.28 ± 0.02
1.97 ± 0.05
001.39 ± 0.03

001.41 ± 0.03
0.02 ± 0.01
00.03 ± 0.01
6.85 ± 0.06
00.27 ± 0.02
0002.74 ± 0.05
0.55 ± 0.02
0.06 ± 0.01
001.48 ± 0.06
0.05 ± 0.01
001.79 ± 0.03
1.32 ± 0.03
0.05 ± 0.01
000.54 ± 0.02
0.08 ± 0.01
0000.09 ± 0.01
00.67 ± 0.02
1.21 ± 0.05
00.05 ± 0.01
0.78 ± 0.03

002.30 ± 0.08
00010.89 ± 0.16
01.15 ± 0.06
000
8.98 ± 0.29
3.97 ± 0.06
0.08 ± 0.01
0.33 ± 0.01
02.33 ± 0.03
0002.48 ± 0.03
2.69 ± 0.05
0001.25 ± 0.05
0
0
0
0
0
0
0.82 ± 0.03
0.67 ± 0.02
0.03 ± 0.01
00.85 ± 0.03

small, acuminate, entire leaves with blades showing truncate bases.
Limbs are slightly longer than wide, no longer than 11 cm on thevoucher specimen. Veins on the abaxial surface are devoid of hairs ortrichomes; they show irregular knobs as shown in Zika and Jacobson.26
The presence or absence of hairs or knobs is the best character that
diﬀerentiates Fallopia sachalinensis and F. × bohemica from F. japonica
var. japonica. The inﬂorescences are loose and longer than thesubtending leaf.
All of the other specimens were identiﬁed as Fallopia × bohemica(Chrtek & Chrtková) J. P. Bailey (NRC voucher specimen codes INH370-373, INH-374-377, INH-378-382), although they showed diﬀerences in relation to the size of blades and the degree of compactness of
inﬂorescences. The specimens bearing the accession number INH-370373 as well as INH-374-377 possess blades of 15 cm or longer withtruncate bases (or very slightly cordate); the apex is acuminate.
Inﬂorescences are compact and short (much shorter than thesubtending leaf). On the abaxial surface, short hairs are conspicuousalong the veins, which is characteristic of the hybrid. The last specimen(INH-378-382) shows characters from the species and the hybrid.
Leaves are shorter than the latter specimens and oscillate around 12 cm
long. Bases are again truncate (or slightly cordate), and blades terminate
with an acuminate apex. The presence of short hairs instead of knobs andthe fact that the inﬂorescences are slightly shorter than the subtending

leaf indicate that we have the hybrid. Although shorter, inﬂorescences
are less compact than the other F. × bohemica specimens.
Sample Preparation. Fresh samples were cleaned with water andthen milled into powder by a versatile plant grinder after being air-dried
and then oven-dried (50−60 °C) right before grinding. Before weighingout, all samples were further dried on a freeze-dryer to remove moisture.
Extraction Method. A 0.4 g aliquot of dry sample was accurately
weighed out, and 10 mL of methanol/water (50:50, v/v) was added; thesample was then placed into an ultrasonic cleaner. The mixture was
extracted for 30 min, and then the extraction solution was centrifugally
separated and ﬁltered through 0.45 μm Millipore nylon membrane, andan aliquot of the ﬁltrate was used for UPLC analysis.
Preparation of Reference Substance Solutions. Resveratrol,
polydatin, emodin, and physcion are the common components
reportedly present in F. japonica, so they were commercially sourced
and used as the reference substances in this study. Stock solutions ofresveratrol (0.20 mg/mL), polydatin (0.60 mg/mL), emodin (0.25 mg/mL), and physcion (0.10 mg/mL) were prepared by dissolving the purecompounds in methanol. After ﬁltering through a 0.45 μm Millipore
membrane, the reference substance solution was directly used for UPLC
analysis.
UPLC Analysis. A reverse phase SB-C18 column (2.1 × 100 mm, 1.8μm) from Agilent was used with the mobile phase consisting of
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Journal of Agricultural and Food Chemistry

ArticleFigure 3. Contents of four targeted compounds in diﬀerent F. japonica samples collected from Dunstaﬀnage, PEI, Canada.

Figure 4. Levels of four targeted compounds in roots of F. japonica collected at diﬀerent times in PEI, Canada.
methanol (A) and water (B). The optimized gradient elution was
performed using the following linear gradient: 0 min, 15% A; 3 min, 20%
A; 5 min, 25% A; 8 min, 40% A; 11 min, 80% A; 15 min, 90% A; 17 min,
90% A. The column compartment was kept at a temperature of 50 °C,
detection wavelength was set at 303 nm, and the volume of sample
injected was 2 μL.
Multivariate Data Analysis. The data set from UPLC analysis wasimported to SIMCA-P+ (version 12.0, Umetrics, Umea, Sweden) for
conducting partial least-squares discriminant analysis (PLS-DA).
Quantiﬁed compound concentrations were all mean-centered andPareto-scaled prior to the analysis.

Table 4. Average Contents of Four Targeted Compounds in F.
japonica Root Samples from China and PEI, CanadaacompoundChinese samples(mg/g)PEI samples(mg/g)PEI samples (Oct)(mg/g)

polydatin
rresveratrol
emodinphyscion

9.27 ± 1.02
4.3 ± 0.16
6.72 ± 0.25
10.31 ± 0.68

11.04 ± 1.35
2.68 ± 0.18*a
5.15 ± 0.21*a12.58 ± 0.71*a

10.69 ± 1.17
4.25 ± 0.14
5.13 ± 0.22*a
12.67 ± 0.58*a*, signiﬁcant (P < 0.05).

Analysis. Column temperatures of 45, 50, and 55 °C and ﬂow
rates of 0.4, 0.5, 0.8, and 1.0 mL/min were also investigated.
Considering system back pressure and running time, the ﬂow
rate was set at 0.5 mL/min when the column temperature was
kept at 50 °C. Also, the detection wavelength was examined.
When the wavelength was at 303 nm, most components showedstrong responses; thus, the DAD detection was performed at 303
nm.
Under these optimal conditions, resveratrol, polydatin,
emodin, physcion, and other minor components in the extractsof F. japonica samples were well separated. All method validation

RESULTS AND DISCUSSION
Optimization of UPLC-DAD Method. To obtain optimal
conditions for separation and quantiﬁcation, the mobile phaseand its ﬂow rate, conditions for elution, column temperature, anddetection wavelength were investigated in this study.
The chromatographic conditions were optimized on the basisof conditions given in the literature.27,28 In the gradient
optimization, gradient time, shape, and initial composition of
the mobile phase were taken into consideration, and the
optimized gradient elution was presented above under UPLC

■

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Journal of Agricultural and Food Chemistry

ArticleFigure 5. Contents of four targeted compounds in F. japonica root samples from China and PEI, Canada.

tests below were carried out on F. japonica extracts prepared as
described above. The injection precision was determined byrepeating UPLC injection of the same sample solution six times
per day. The sample stability was determined with measurementsfrom a single sample solution stored at room temperature for 2, 4,
6, 8, and 12 h. The repeatability was assessed by analyzing six
separate samples.
The relative peak area (RPA) of each characteristic peak was
calculated for the estimation of injection precision, stability, andrepeatability and the results were as follows: for injection
precision, the relative standard deviations (RSD) of the RPA <0.56%; for sample stability, RSD < 1.68%; for repeatability, RSD< 1.71% for six independent samples. Thus, the results indicatedthat the UPLC-DAD method was suitable for this quantitative
analysis work.
Optimization of Extraction Conditions. To exhaustively
extract resveratrol, polydatin, emodin, and physcion from F.
japonica, three extraction approaches were compared: ultrasonicextraction (UE), conventional Soxhlet extraction (SXE), andsoak extraction (SE). The extraction eﬃciencies of resveratrol,
polydatin, emodin, and physcion using the three proposed
extraction methods were measured, as milligrams per gram of raw
sample, to be 4.72, 6.57, 6.59, and 5.55 mg/g; 4.22, 5.18, 5.48,
4.92 mg/g; and 4.09, 5.17, 5.56, and 4.91 mg/g, respectively(Figure 1A). Obviously, ultrasonic extraction had the highest
extraction eﬃciency, which was then chosen as the extraction
approach for the following experiments.
Then, the extraction solvent, a key factor for UE, wasevaluated. Three solvent mixtures were tested, methanol−
ethanol, methanol−water, and acetonitrile−methanol, in a ratioof 80:20 (v/v) to evaluate their eﬃciency for extraction of
resveratrol, polydatin, emodin, and physcion from F. japonica.
After ultrasonic extraction, the levels of resveratrol, polydatin,
emodin, and physcion by using the three solvent mixtures were
found to be 4.43, 6.24, 6.47, and 5.52 mg/g; 4.72, 6.57, 6.59, and5.55 mg/g; and 4.36, 6.15, 6.07, and 5.31 mg/g, respectively(Figure 1B). This showed that the extraction of resveratrol,
polydatin, emodin, and physcion with methanol−water (80:20)

was the best. For dry plant materials, solvents such as methanol−
water with higher polarity could penetrate plant cell membranes
and enhance the extraction of bioactive components. Therefore,
the methanol−water (80:20) was chosen as the solvent for
further investigation.
Subsequently, the eﬀects of diﬀerent factors on ultrasonicextraction of resveratrol, polydatin, emodin, and physcion were
investigated, and orthogonal experiment design was used todetermine the signiﬁcance of four environmental factors
including solvent/sample ratio (mL/g), methanol/water ratio(v/v), extraction time (min), and times of extraction (number oftimes).
The experimental factors with corresponding levels arepresented in Table 2A, and the results of orthogonal design L9(34) are presented in Table 2B. The intuitional analysis from R
value in Table 2B indicates that the inﬂuence order of eachvariable on the extraction eﬃciency is A > C > D > B. It also
showed that K1 had the highest K value in factor A, which meansthat compared with other levels of factor A, level 1 has the highest
extraction eﬃciency and is the most suitable level for extraction.
On the basis of the same principle, level 3 of factor B, level 1 offactor C, and level 1 of factor D were chosen for extraction. To
verify the data analysis of orthogonal experiment design, analysis
of variance (ANOVA) was used to further determine which
factor was signiﬁcant in aﬀecting the extraction yield ofresveratrol, polydatin, emodin, and physcion. As shown in
Table 2C, factors A, C, and D are clearly the most signiﬁcant ones
(P < 0.05) aﬀecting extraction.
The orthogonal experiment design combined with statisticalanalysis had been applied to optimize the conditions of extraction
for improving the yield for resveratrol, polydatin, emodin, andphyscion in F. japonica, and the optimal extraction condition wasused as described above.
Results of Analysis. Chromatograms of Reference
Compounds and F. japonica Extract. F. japonica contains
emodin, emodin-6-ether, emodin-8-monomethyl ether, chrysophanol, rhein, emodin-8-β-D-glucoside, physcion, resveratrol,
and polydatin, with resveratrol, polydatin, emodin, and physcion
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ArticleFigure 6. PLS-DA score plot (A) and loading plot (B) showing clusters based on contents of polydatin, resveratrol, emodin, and physcion in root
samples of F. japonica (Fj), F. × bohemica (Fxb), and Polygonum polystachyum (Pp) from China and Canada.

emodin, and physcion (Figure 2A) are compared with theextracts of PEI-grown F. japonica (Figure 2B) and F. japonica
from China (Figure 2C). On the basis of UPLC analysis, PEI-

as the main and biologically active components. To identify themain components of knotweed species grown in PEI, the UPLC
proﬁles of the standard compounds resveratrol, polydatin,
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Journal of Agricultural and Food Chemistry

Article20 samples collected at three diﬀerent locations (4 sites) in PEI,
Canada, 3 samples acquired from BC, Canada (as shown in Table1), and 14 samples from China. The results indicated that thecontents of resveratrol and polydatin were higher in Japaneseknotweed, F. japonica, grown in PEI or from China. In
comparison, we observed much lower levels of these compounds
in other related species, F. × bohemica and P. polystachyum, with adiﬀerence of >10 times in some samples.
This diﬀerence of key metabolite abundances was further
demonstrated by using a multivariate data analysis tool, whichshowed a fairly clear separation of F. japonica from other related
species, based on levels of the four compounds analyzed in root
samples. As shown in Figure 6, the score and loading plots (A and
B) of the partial least-squares discriminant analysis (PLS-DA)
demonstrated that F. japonica contains distinctly higher levels of
resveratrol, polydatin, emodin, and physcion. One sample (no.
72), acquired from BC, Canada, as F. japonica, seems to containlower levels of resveratrol and other compounds, indicating theeﬀect of environmental factors.
In summary, this study showed that the content of resveratrolin Japanese knotweed (F. japonica) grown in PEI, Canada, varies
in diﬀerent parts of the plant and that the root contains a higher
level of resveratrol than stem and leaf. The level of resveratrol inroot samples of F. japonica from PEI is comparable to that ofChinese samples, whereas a signiﬁcant diﬀerence was foundbetween the common Japanese knotweed (F. japonica) and its
closely related species F. × bohemica. Japanese knotweed
contains >10 times the level of resveratrol as F. × bohemica.
Also, time of collection was found to have a great inﬂuence on thecontents of resveratrol; resveratrol level is at its highest in
October. Additionally, PEI-grown knotweed contains a similarlevels of resveratrol and polydatin as the Chinese samples
collected in the month of October, but the contents of emodin
and physcion are statistically diﬀerent (lower for emodin andhigher for physcion). As such, we conclude that Japaneseknotweed grown in PEI, Canada, is a commercially viable sourceof raw material for resveratrol production.

grown knotweed shows a similar composition proﬁle incomparison with the Chinese samples except that it contains
an additional, more polar analogue of resveratrol, identiﬁed as
resveratroloside29 (based on NMR and MS analysis of purecompounds obtained through preparative HPLC, data notshown).
Contents of Resveratrol, Polydatin, Emodin and Physcionin Diﬀerent Plant Parts of Knotweed Grown in PEI, Canada. It
is well-known that diﬀerent parts of a raw herb may havesomewhat diﬀerent composition proﬁles and thus may be used
for diﬀerent therapeutic purposes. In this study, the contents ofresveratrol, polydatin, emodin, and physcion of various parts of F.
japonica grown in PEI were measured and compared. Root, stem,
ﬂower, and leaf samples of F. japonica collected from PEI wereextracted and analyzed on UPLC. The contents of resveratrol,
polydatin, emodin, and physcion are shown in Table 3. The
contents of resveratrol, polydatin, emodin, and physcion were
found to vary considerably in various parts of the plant andsamples collected during diﬀerent seasons (Figure 3).
Contents of Resveratrol, Polydatin, Emodin, and Physcionin the Root of F. japonica Harvested at Diﬀerent Times in PEI,
Canada. As shown above, diﬀerent plant parts contain various
levels of resveratrol, polydatin, and emodin, and the root was
found to contain higher levels of these compounds. Therefore,
we studied the eﬀect of harvesting time on the levels of
resveratrol, polydatin, emodin, and physcion various in root
samples of F. japonica, and the results are shown in Figure 4. The
results indicated that polydatin in F. japonica increases from May
to July, reaches its highest in August, and then begins to decrease.
Resveratrol, on the other hand, gradually increased from May to
October, and the level was 2.5 times higher in October than inMay. However, emodin in F. japonica appeared not to have aparticular trend of accumulation during the year, whereas
physcion seemed to decrease gradually from May to October.
Comparison on Contents of Polydatin, Resveratrol,
Emodin, and Physcion among Root Samples of F. japonica
from China and PEI, Canada. It is well-known that diﬀerent
growth environments have an enormous impact on the content
of active ingredients in plants.30 China is in eastern Asia andCanada is located in North America, so there is a great diﬀerenceof climate and environmental factors related to plant growth. It isthus very interesting to compare the levels of these key
components among the F. japonica root samples.
As shown in Table 4 and Figure 5, polydatin level was found to
be within a similar range in both Chinese and PEI samples; theaverage content of polydatin in the root of F. japonica grown in
PEI is about 11.04 mg/g, slightly higher than the average ofChinese samples (9.27 mg/g). The level of resveratrol in the
Chinese and PEI samples diﬀers considerably, with the averagecontent in Chinese samples being 4.30 mg/g, 1.6 times higher
than that of the PEI samples. However, if we choose only the
samples harvested in October for comparison, the contents ofresveratrol appeared to be at similar levels. Chinese samples also
contain a higher level (1.3 times) of emodin and less physcion
than the PEI samples (6.72 mg/g emodin and 10.31 mg/g
physcion in Chinese samples; and 5.15 and 12.58 mg/g in PEI
samples).
Comparison of Contents of Polydatin, Resveratrol, Emodin,
and Physcion among Root Samples of F. japonica, F. ×bohemica, and Polygonum polystachyum Collected at
Diﬀerent Sites from PEI, and Acquired from BC, Canada,
and China. In this study, we compared the contents of polydatin,
resveratrol, emodin, and physcion in all root samples, including■

AUTHOR INFORMATION

Corresponding Author*(J.Z.) Present address: Aquatic and Crop Resource Development, National Research Council Canada, 1411 Oxford Street,
Halifax, NS B3H 3Z1, Canada. Phone: +1-902-426-7408. Fax:
+1-902-426-9413. E-mail: junzeng.zhang@nrc.gc.ca.
Funding

Financial support for the present work was received from the
National Research Council Canada − Industrial Research
Assistance Program (NRC-IRAP) and Guiyang Science andTechnology Plan projects (No. [2012]7024, No. [2012101]3-4,
No. ZY[2011]3013, and No. [2011]4008).
Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS
Stacey Barker and Brad Alexander of Environmental Services,
Fraser Valley Regional District, BC, Canada, are appreciated forcollecting and processing plant samples.

■■

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