全 文 ：外植体与不同理化因子对虎杖愈伤组织诱导及
次生代谢产物形成的影响?
刘晓琴1 ,2 , 张 卫1
??
, 金美芳1 , 虞星炬1
(1 中国科学院大连化学物理研究所海洋生物产品工程组 , 辽宁 大连 116023; 2 中国科学院研究生院 , 北京 100039)
摘要 : 考察了外植体、培养基及光照条件对中药植物虎杖愈伤组织形成及次生代谢产物生产的影响。总的
来看 , 所有愈伤组织中总酚和总黄酮含量比原植物的含量高 2～3 倍 ; 而蒽醌的含量比原植物中含量低。
外植体对愈伤组织形成及次生代谢产物生产的影响很大 , 所考察的 3 种外植体中 , 叶外植的愈伤组织诱导
率最高而源于根外植体的愈伤组织具有最好的次生代谢能力。所考察的 6 种培养基中 , MS+ 0.5 mg?L 2 ,4 -D
+ 1.0 mg?L 6-BA 和 N6 + 0.5 mg?L 2 ,4 -D + 1.0 mg?L 6-BA 无论对于愈伤组织的产生还是次生代谢产物的累积都
有较优表现。光照对愈伤组织诱导及次生代谢产物产生有明显影响 , 但二者无规律性联系。
关键词 : 虎杖 ; 愈伤组织 ; 外植体 ; 培养基 ; 蓼科 ; 总酚 ; 黄酮 ; 蒽醌
中图分类号 : Q 945 文献标识码 : A 文章编号 : 0253 - 2700(2006)04 - 403 - 07
Effects of Explants, Medium Formulations and Light on Callus
Induction and Secondary Metabolites Accumulated in the
Calli of Polygonum cuspidatum (Polygonaceae)
LIU Xiao-Qin1 , 2 , ZHANG Wei1 * * , J IN Mei-Fang1 , YU Xing-Ju1
(1 Marine Bioproducts Engineering Group, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023 , China;
2 Graduate School of The ChineseAcademy of Sciences, Beijing 100039 , China)
Abstract: Callus initiation of aChinese traditional medical plant, PolygonumcuspidatumSieb . et Zucc (Polygonaceae)
wasstudied, with afocus onthefactors that could influencethecallus inductionability and secondary metabolites accumu-
lation . In comparison with the parent explants, the levels of total phenolics and flavoneswere 2 to 3-fold higher and the
levels of anthraquinones (AQs) were lower in all calli induced . Different explants exhibited great variability in callus in-
duction rateand secondarymetaboliteaccumulation . Among thethree explants tested, leaf explantswerewith thebest ca-
pacity in callus induction, while root explants accumulated the highest levels in secondary metabolites . Two media of six
tested, MS and N6 respectively plus 0 .5 mg?L 2 , 4-dichlorophenoxy acetic acid and 1 .0 mg?L 6-benzylaminopurine,
showed the best performances in bothcallus growth and secondary metabolite accumulation . Illumination shower a remark-
able effects on both callus induction and secondary metabolite accumulation, but no regular pattern was observed .
Key words: Polygonum cuspidatum; Polygonaceae; Callus culture; Explant; Medium formulation; Phenolics; Fla-
vones; Anthraquinones (AQs)
Introduction
PolygonumcuspidatumSieb . et Zucc, a Chi- nese traditional medical plant named“Hu Zhang”, be-
云 南 植 物 研 究 2006 , 28 (4) : 403～409
Acta Botanica Yunnanica

?
?? ?Author for correspondence .
Received date: 2005 - 11 - 21 , Accepted date: 2006 - 03 - 10
作者简介 : 刘晓琴 , 女 , 硕士 , 主要从事药用植物细胞培养体系的建立及代谢调控。 ?
Foundation item: This research was financially supported by the National Natural Science Foundation of China ( No . 20176058)
longs to the family of Polygonaceae ( Xiao et al .,
2002) . It is also known as Mexican Bamboo, Japa-
neseBamboo, or Japanese Knotweed (Vastano et al .,
2000) . The dried root of P . cuspidatum is a well-
known traditional medicine in China and Japan ( Xiao
et al ., 2002 ) . It is used as analgesic, antipyretic,
diuretic, expectorant, and antitussive; and for the
treatments of arthralgia, jaundice, amenorrhea, and
chronic bronchitis ( Chu et al ., 2004 ) . P . cus-
pidatum has recently attracted great attention for its
bioactivity of anti-bacterial ( Hegde et al ., 2004; Ki-
ma et al ., 2005 ) , anticancer ( Kimura et al .,
2001) , and benefit for cardiovascular system ( Brada-
mante et al ., 2003 ) . Various chemical compounds
have been isolated from this plant, including anthra-
quinones, stilbenes, flavonoids, and other phenolics
(Yang et al ., 2001; Chen et al ., 2001; Jayatilake
et al ., 1993) . Someof thesechemicals with interest-
ed bioactivities, such as polyphenol , anthraquinones
derivatives in this plant have been widely reported
( Park et al ., 2004; Matsuda et al ., 2001;
Savouret et al ., 2002) .
Plant tissue and cell culture has great potential as
an alternative source to traditional agriculture or wild
plant harvest in the industrial production of bioactive
plantmetabolites ( Rao et al ., 2000 ) . It offers the
advantages of producing a large amount of useful sec-
ondary metabolites under controlled conditions . How-
ever, the in vitro cell and tissue culture of P . cus-
pidatum for the production of secondary metabolites has
not been reported .
The aimof this paper is therefore to establish the
conditions for callus induction of P . cuspidatumfor the
production of secondary metabolites . Effects of differ-
ent factors, explants, basal medium, phytohormones
and light were investigated on the callus induction
ability and the accumulationof total phenolics, anthra-
quinones and flavonoids, which were deemed as the
overall secondary metabolic activity . To facilitate fur-
ther work of the cell line selection for theproductionof
these bioactivemetabolites, wereported the factors af-
fecting callus induction of P . cuspidatum for the pro-
ductionof secondary metabolites during the initial stag-
es of establishing a new cell line .
Materials and Methods
Plant materials
The explants, lateral roots, stems and leaves of P . cus-
pidatumwere collected from the herbs transplanted from Lushan
Arboretumto theCampus of Dalian Institute of Chemical Physics
4 years ago, which have been growing under natural conditions .
Callus culture
The explants were washed thoroughly with tap water and
surface sterilized in a laminar airflow hood following the proce-
dures listed below: First, the explantswere immersed into70%
aqueous ethanol for 20 seconds followed by rinsing them with
sterile deionizedwater for threetimes; secondly, theroot, stem
and leaf explantswere soaked in 0.1 % mercury dichloride solu-
tion for 8 , 22 and 16 minutes respectively according to the re-
sults of a preliminary experiment on the surface sterilization time
(data not shown) . Thirdly, theexplantswere washed with ade-
quate sterile double distilled water ( dd water) for five times,
and excised into proper sizes (stems and lateral roots, segments
approximately 8 mmin length; leaves, 5 mm×5 mmsections) .
Every four explants were scattered in 100 ml flasks contained 40
ml culturemediumsolidifiedwith 7g?L agar . Six different media
containing changes both in basal medium (MS, B5 or N6 ) and
phytohormones (0 . 5 mg?L 2 ,4 -D + 1.0 mg?L 6-BA , or 0 .5 mg?
L NAA + 1 .0 mg?L KT ) weretested . ThepH valueof themedia
was adjusted to5 .8 before autoclaving at 115℃ for 15 minutes .
The flasks with explants were sealed with aluminum foil under
aseptic condition and incubated ina culture chamber at 25±2℃
under light and darkness, respectively . In light induction, sev-
eral Philip40W fluorescent tubeswere used toprovidewhite light
of 300μEm- 1 S- 1 for a photoperiod of 14 h . Subculturewas done
at 40 days interval by transferring the calli to freshmedium con-
tainingthe same ingredients as those used for callus initiation .
Determination of callus induction rate and its growth (DW
and DFR)
Both callus induction ability ( % ) and callus growth were
used to illustrate the effect of different factors on callus culture
establishment . In addition, another parameter, the weight ratio
of dry to fresh callus (DFR) , was also used to indicate the cal-
lus growth status .
25 days after inoculation, theability of the explants to de-
velop callus under various conditions was recorded as the callus
induction ability ( % ) which is defined as:
404 云 南 植 物 研 究 28 卷
callus induction ability ( % ) = Total mumber of explants producing callus
Total mumber of explants cultured
×100 %
After 3 subcultures, FW (fresh cell weight) of callus was
measured byweighing1 - 4 pieces of calli . DW (dry cell weight)
was determined bydryingthecallus at 80℃ in an ovenfor acon-
stant weight (48 h) . DFR is defined as (Naill et al ., 2005) .
DFR ( % ) = Dry callusweight
Fresh callus weight
×100%
In general , a lower DFR value indicates higher water cont-
ent and consequently more friable callus .
Analytical procedures for total phenolics
A modified Folin-Ciocalteu technique (Singleton and Rossi,
1965) was used for determining the total phenolics accumulated
in the calli, with a detailed protocol shown in ( Fig . 1 : A ) .
The phenolic content in calli was calculated using a standard
curve of gallic acid as shown in Table3 .
Fig . 1 Flow chart for the quantification of total phenolics and flavones contents in callus cultures
of P . cuspidatum A : phenolics; B: flavones .
Analytical procedures for total flavones
Theconcentration of total flavones in extracts was measured
by UV-Visspectrophotometry, based on a colorimetric oxidation?
reduction reaction with the analytical procedures shown in ( Fig .
1 : B ) . Flavones concentration was calculated from a standard
curve of Rutin . (Chen et al ., 2002) .
5044 期 LIU Xiao-Qin et al : Effects of Explants, Medium Formulations and Light on Callus Induction . . .
Analytical procedures for AQs
0 .05 g freshcalluswas extractedwith hot methanol ( kept in
80℃ water bath before use) at roomtemperature for 30 minutes
for AQs quantification . The samples were centrifuged at 4000 r?
min for 5 minutes and0 .5 ml supernatant or standard solutionwas
transferred to a10 ml glass test tube . The solvent was evaporated
in 80℃ water bath to dryness in a draught cupboard before add-
ing 10 ml 1% Mg ( CH3 COO)2 -methanol solution, a specific
chromogenic reagent for AQs . Absorbance atλ507 nmwasmea-
sured and total AQs concentration was calculated from a standard
curve of emodin, a most common ingredient of anthraquinone
structure in wild-grown P . cuspidatumherb .
Results and Discussion
Callus induction and growth status
The callus induction abilitywas evaluated by three
parameters: induction rate ( % ) , callus growth and
DFR . Induction rate ( % ) represents the percentageof
explants that could form callus without considering the
quantity and growth status of the calli . Callus growth
and DFR serve to quantitatively evaluate the callus
growth status . As shown in Table 1 .
Leaf explants were most capable for callus induc-
tion under all conditions . Induction rates of 100%
were achieved for leave explants initiated inMS and N6
media, except for the useof N6 basal mediumstrength-
ened by phytohormone combinationB (N6 -B) . B5 gave
thepoorest induction rate ranging from 16 .7 - 41 .7%
for leaf explants . No callus induction was observed for
root explants under B5 -B medium in light, and for
stem explants under three conditions of B5 -A in the
dark, B5 -B in the light as well as MS-B in the dark .
When root explants were used, N6 basal medium was
much better than the rest two in most conditions, ex-
cept with phytohormone combination B, in dark . For
the stem explants, MS basal medium appeared to be
the best with the highest induction rate of 50% achie-
ved, with hormone combination A under light .
Quantitative analysis of callus growth status in
terms of callus growth and DFR for 3 subcultures was
shown in Table2 and Table 3 , respectively . Compared
with Table 1 , many induced calli failed to grow up
when continuously subcultured in the same conditions
as the callus initiation . It was possibly related to the
quantity of the calli when originally induced, as well
as the potential for continuous growthof the calli .
Table 1 Callus induction rate ( % ) of P . cuspidatum
Expl-
ants
Light
condition
MS basal
medium
A a Bb
B5 ?basal
medium
A B
N6 basal
medium
A B
Root lightc 33 ?. 3 16 .7 8 A. 3 0 \66 .7 83 .3
darkd 25 ?. 0 41 .7 33 T. 3 16 J. 7 50 .0 16 .7
Leaf light 100 M100 C25 T. 0 41 J. 7 100 ?100
dark 100 M100 C33 T. 3 16 J. 7 100 ?66 .7
Stem light 50 ?. 0 8 .3 25 T. 0 0 \41 .7 8 u. 3
dark 33 ?. 3 0 0 f8 7. 3 27 .3 16 .7
a: phytohormone combination A : 0 . 5 mg?L 2 , 4 -D and 1 .0 mg?L 6-BA ;
b: phytohormone combination B: 0 . 5 mg?L NAA and 1 .0 mg?L KT ; c:
cultured under white light at light intensity of 300μEm- 1 S - 1 for aphotope-
riod of 14 h; d: cultured in dark at light intensity less than 1μEm- 1S - 1 .
Table 2 Qualitative analysis of callus growth of P . cuspidatum
Expl-
ants
Light
condition
MS basal
medium
A B
B5 ?basal
medium
A B
N6 basal
medium
A B
Root light + + + + — + + — + + + +
dark + + — — — + + + +
Leaf light + + + + + — — + + + + +
dark + + + + — — — + + —
Stem light + + + — + — + + —
dark + + — — — — +
+ + + + : represents growth status of the calli , the higher the better ;
— : no growth
Table 3 DFR value ( % ) of the calli of P . cuspidatum
Explants
Light
condition
DFR of
explants
MS basal medium
A B
B5 ?basal medium
A B
N6 \basal medium
A B
light 12 _. 56 — 17 ?. 95 — 20 ?. 52 14 .32
Root 64 J. 30
dark 15 _. 39 — — — 17 ?. 28 12 .97
light 13 _. 19 20 K. 77 — — 9 ?. 45 22 .96
Leaf 51 J. 76
dark 11 _. 54 — — — 10 ?. 58 —
light 9 M. 92 — 9 ?. 04 — 6 .40 —
Stem 46 J. 82
dark 7 M. 54 — — — — 9 ?. 01
604 云 南 植 物 研 究 28 卷
However the callus induced from the leaf explants
maintained a strong growth in MS and B5 -based media
containing phytohormone combination A . Calli derived
from roots grew better over those fromstems . In view
of the medium ingredient, MS-A was most suitable for
callus growth, followed by N6 -A . While with phyto-
hormone combination B, only a few calli continued to
give poor growth .
DFR value of the calli indicates the water content
in calli and therefore, to some extent, the rigidity of
the calli . Generally, looser callus with lower DFR
value was more desirable for establishment of suspen-
sion cultures . Calli from stem explants were generally
low in DFR (Table 3) .
Total phenolics production
Phenolic compounds are secondary metabolites
synthesized by plants both during normal development,
and in response to stress conditions such as infection,
wounding, and UV radiation ( Naczk et al ., 2004 ) .
In plants, it may act as phytoalexins, antifeedants,
and attractants for pollinators, and contributors to the
plant co-pigments, antioxidants, and protective agents
against UV light (Naczk et al ., 2004) . The synthe-
sis of phenolic compounds strongly dependson environ-
mental conditions ( Lus-Endrich et al ., 2000) . Cal-
lus cultures of P . cuspidatumcan produce a significant
amount of phenolic substances ( Table 4 ) under various
conditions .
Table 4 Analysis of total phenolics content in callus cultures of P . cuspidatum ( phenolics content in mg per 1 g DW callus or plant)
Explants
Light
condition
Content in
explants
MS basal medium
A B
B5 ?basal medium
A B
N6 ?basal medium
A B
light 47 ?. 8±0 E. 25 — 34 .2±0 .97 — 31 *. 2±1 .48 37 h. 5±1 ). 01
Root 9 !. 43±0 ?. 72
dark 40 ?. 5±1 E. 56 — — — 35 *. 4±0 .10 44 h. 7±0 ). 39
light 29 ?. 4±1 E. 29 23 .0±1 Z. 07 — — 36 *. 5±1 .66 21 h. 2±0 ). 31
Leaf 8 3. 1±0 ?. 48
dark 23 ?. 5±2 E. 01 — — — 40 *. 1±8 .87 —
light 35 ?. 1±2 E. 01 — 4 .5±0 . 08 — 23 *. 2±1 .29 —
Stem 1 !. 57±0 ?. 07
dark 42 ?. 5±1 E. 62 — — — — 25 h. 4±3 ). 46
All cultures tested produced phenolics contents
higher than that of parent explants, with amaximumof
3 - 5 folds increases . The remarkable increase indi-
cates the active construction of carbon-based defense
compounds in the callus of P . cuspidatum ( Lus-En-
drich et al ., 2000) . Thehighest phenolics contentof
47 .8 mg?gDW was obtained in root-derived calli ,
which was 5 folds higher than the parent root explants
(9 . 43 mg?gDW) . Only one callus derived from leaf
and stem each has a phenolics content over 40 mg?
gDW . No clear correlation could be concluded for the
phenolics content between the calli and their parent ex-
plants . A phenolic content of 42 .5 mg?gDW was
achieved in callus fromstemexplants that has the low-
est phenolics content among all the explants tested .
Total Flavones production
Depending on the degree of oxidation, flavones
always co-occur with other forms of flavonoid-related
compounds . There is an increasing interest in the bio-
logical effects of flavone compounds that showing abil-
ities in reducing the risk of cardiovascular disease,
quenching active oxygen species and reducing throm-
botic tendency, as well as anti-inflammation, anti-
carcinogenic ( Skerget et al ., 2005; Karakaya et
al ., 1999 ) . In this study, similar results werefound
in flavones accumulation as that of the phenolics . As
shown in Table 5 , all calli had much higher flavones
content, when compared with their parent explants,
except one stem-derived callus on B5 -A medium in
light . The highest flavones content of 27 .6 mg?gDW
was obtained fromthe root-derived callus in MS-B me-
dium under darkness, however no clear correlation
could be concluded for flavones content between the
calli and their parent explants .
AQs production
As shown in Table 6 , in contrast with the results
of phenolics and flavones discussed above, AQs accu-
mulation was predominantly in root-derived callus . The
7044 期 LIU Xiao-Qin et al : Effects of Explants, Medium Formulations and Light on Callus Induction . . .
Table 5 Analysis of flavones content in callus cultures of P . cuspidatum ( flavones content in mg per 1 g DW callus or plant)
Explants
Light
condition
Content in
explants
MS basal medium
A B
B5 ?basal medium
A B
N6 ?basal medium
A B
light 17 ?. 3±0 E. 38 — 26 .7±0 .54 — 24 *. 1±2 .48 15 h. 2±0 ). 42
Root 12 F. 6±0 ?. 49
dark 27 ?. 6±0 E. 87 — — — 13 *. 5±0 .54 19 h. 5±0 ). 39
light 10 ?. 9±0 E. 57 22 .4±0 Z. 52 — — 8 ?. 8±0 .23 16 h. 7±0 ). 83
Leaf 3 !. 29±0 ?. 35
dark 9 q. 7±0 2. 27 — — — 15 *. 6±0 ?. 64 —
light 10 ?. 6±0 E. 64 — 0 .4±0 . 43 — 10 *. 2±2 .19 —
Stem 1 !. 11±0 ?. 11
dark 21 ?. 5±2 E. 67 — — — — 8 U. 4±0 ?. 45
total AQs content accumulated in all thecalli was much
lower than that of their parent explants . The highest
AQ content of 2 .9 mg?gDW, obtained in root derived
callus on N6 -B mediumin light, was about 47% of the
AQs content of its parent explants ( 6 . 1 mg?gDW ) .
This result may be discouraging but similar with some
results reported . In the case of Rheum palmatum,
Farzami et al . (2002) reported that total AQs content
was much lower in cultures ( 0 . 3% ) than that in the
parent plant (2 - 5% ) . WhileHan et al . (2003) re-
ported that cell cultures of M. cittrifolia accumulated
very small amounts of AQs in the presence of 2 , 4 -D
before a fungal elicitor, Paph, was added to the cell
culture at day 4 . In addition, it was observed a step-
wise decrease in the color of the calli whichwas mainly
correlated with the AQs accumulation, starting with
bright yellow, then yellow, and finally a steadily
straw yellow after 3 subcultures .
Table 6 Analysis of AQs content in callus cultures of P . cuspidatum ( AQs content in mg per 1 g DW callus or plant)
Explants
Light
condition
Content in
explants
MS basal medium
A B
B5 ?basal medium
A B
N6 ?basal medium
A B
light 1 q. 6±0 2. 19 — 2 ?. 1±0 . 09 — 2 ?. 9±0 .21 0 U. 9±0 .08
Root 6 !. 12±0 ?. 39
dark 1 q. 3±0 2. 04 — — — 1 ?. 5±0 .08 1 U. 3±0 .43
light 1 q. 1±0 2. 15 0 ?. 3±0 G. 07 — — 0 ?. 1±0 .02 0 U. 3±0 .09
Leaf 2 !. 31±0 ?. 27
dark 0 q. 2±0 2. 13 — — — 0 ?. 3±0 .14 —
light 0 q. 2±0 2. 07 — 0 ?. 0±0 . 02 — 0 ?. 7±0 .03 —
Stem 0 !. 47±0 ?. 02
dark 0 q. 3±0 2. 08 — — — — 0 U. 3±0 ?. 18
Conclusion
This study has successfully initiated the callus
cultures from different explants ( roots, leaves and
stems) of P . cuspidatum . The callus cultures could
produceboth total phenolics and flavones metabolites 2
- 3-fold higher than their parent explants . AQs accumu-
lation in all callus lines was much lower than their par-
ent explants . However no clear correlationwas found for
theproductionof all three classes of secondary metabo-
lites between theparent explants and their derived calli .
Leaf explants exhibited the best performance in callus
induction and growth, especially in MS-A medium,
however calli-derived from roots produced the highest
level of secondary metabolites . B5 basal medium was
generally proved to be least effective in both callus in-
duction and secondary metabolite accumulation . Given
the important medicinal uses of P . cuspidatum, the
successful establishment of in vitro callus culture in this
study might lead to an alternative source for controlled
production of potential bioactive metabolites . Further
work will be focusedon the identification of themetab-
olite structures among the phenolics and flavones pro-
duced by callus culture, and the establishment and
manipulation of suspension cell culture for hyper-pro-
duction of identified bioactivemetabolites .
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