全 文 ：第 21卷　第 4期 　　　　　　　　　　　　　　云南农业大学学报　　　　　　　 Vo l. 21　No.4
2006年　8月 　　　　　　　　　　 Journa l o fYunnanA griculturalUniversity　　　　　 Aug. 2006
　收稿日期:2006 - 03 - 01
*基金项目:云南省自然科学基金资助项目(2004C0034M)　　　　**通讯作者　　　　
　作者简介:彭春秀(1977 -), 女 ,重庆人 , 硕士 ,讲师 , 主要从事植物生物技术研究。
Production ofArbutin through B iotransformation
ofExogenousHydroquinone byDatura stramonium
Cell Suspension Cultures
*
PENG Chun-xiu1 , GONG Jia-shun2**
(1. Facu lty o fModern Agricultural Enginee ring, Kunm ing University of Science and Techno logy, Kunm ing 650224, Ch ina;
2. Faculty o f Food Techno logy, Yunnan Ag ricu ltu ra lUniversity, Kunm ing 650201, China)
Abstract:To investigate the b iotransforma tion o f hydroquinone by ce ll suspension cu ltures ofDatura
stramonium. Cultured ce lls derived from stems ofDa tura stramonium were m ain tained in Linsma iher
and Skoog (LS) so lidmedium. Datura stramonium ce lls in suspension cultu res did not accumu late ar-
butin (4-hydroxypheny l-β-D-g lucopy ranoside) but w ere able to specifica lly o-g lucosyla te exogenous
hydroquinone at position 1. In particu lar, Da tura cu ltures g lucosyla ted ca 93.4% of hydroquinone
(240μmol /100mL cultu res)w ithin 8 days after hydroquinone adm in istered. The arbutin ob tained w as
extrac ted from the cu ltures and further pu rified by silicon G el co lumn chromatography. The exogenous
hydroquinone and arbutin w ere ana lyzed by HPLC.
Key words:g lucosyla tion;hydroquinone;arbu tin;Da tura stramon ium;biotransform ation
CLCN:S 567. 239.01　　Docum ent code:A　　A rtic le ID:1004 - 390X(2006)04 - 0429 - 06
白花曼陀罗细胞悬浮培养生物转化外源氢醌合成熊果苷的研究
彭春秀 1 , 龚加顺 2
(1.昆明理工大学现代农业工程学院 ,云南 昆明 650224;
2.云南农业大学食品科学技术学院 ,云南 昆明 650201)
摘要:研究了白花曼陀罗细胞悬浮培养对外源氢醌的糖基化。转化细胞来自白花曼陀罗嫩茎在 LS固体培养基
上诱导产生的愈伤组织。白花曼陀罗悬浮培养细胞不能分泌熊果苷 ,但能糖基化外源氢醌合成熊果苷。当氢醌
添加量达 240μm o l /100 mL培养物时 ,约有 93. 4%的氢醌转化形成了熊果苷 , 并应用多种色谱技术进行分离纯
化 , 进行了 HPLC分析和结构鉴定。
关键词:糖基化;氢醌;熊果苷;白花曼陀罗;生物转化
1　 Introduction
Plant ce ll cultures are conside red to be use fu l for
transfo rm ing cheap and plentifu l substances into rare
and expensive substances by using the cell cu lture as
a bioreac to r
[ 1]
. T issue o f intact plan ts can ab le to con-
vert exogenous pheno lic compounds in to their co rre-
sponding g lucoside s
[ 2]
. S im ilarly, many w orkers have
repo rted tha t plant ce ll cultu res are a lso capable of
g lucosy la ting such substra te as phenols, ste ro ids, te r-
peno ids, and ca rdeno lides[ 1] . P ILGRIM (1970) has
suggested that the callus cultu res o f three p lant spe-
cies w ere ab le to conve rt some simple pheno ls inclu-
ding hyd roquinone in to the corresponding mono g luco-
sides. Recen tly, MAMORU et a.l (1988) have con-
firmed tha t suspension cultures ofDatura innoxia is
capab le of g lucosy lating m-hydroxybenzoic acid, um-
belliferone, escu letin, daphne tin, and liquiritige-
nin
[ 2]
.
A rbutin(4-hyd roxyphenyl-β-D-g lucopy ranoside)
is a g lycosyla ted hydroquinone, wh ich consists o f a
phenolmo lecule w ith a g lucose moie ty in the parapo-
sition. It is we ll know n as a substrate of the enzyme
β-g lucosidase and also for its d iure tic and urinary an-
ti-infective properties [ 3] . The A rbu tin has sligh t an ti-
b iotic ac tiv ity based on the re lease o f the ag ly cone hy-
droquinone during its intestinal metabolism. It a lso
inh ib its the me lanin biosynthe tic pathw ay and is
therefore app lied as a skin lightener in specific cos-
me tics
[ 4]
. N ow the a rbutin is w ide ly app lied in cos-
me tic, bu t the price of arbutin in the marke t is ve ry
expensive than hydroquinone, so it is an impo rtant
value to conve rt hydroqu inone into arbutin.
How ever, the successfu l g lucosy la tion of an ex-
ogenous substra te by ce ll suspension culture s o fDatu-
ra stramonium is no t repo rted now. The objective o f
th is study w as to dea l w ith the production of arbutin
th rough bio transfo rmation o f exogenous hydroqu inone
byDatura stramonium ce lls suspension cu ltures.
2　Materials and methods
2. 1　P lants
P lant materia ls o fDatura stramon ium were ob-
tained from p lant Garden of Kunm ing, and identified
by Pro fessor ZHUWei-M ing.
2. 2　Tissue and cell cultures
S tems o fDa tura stramonium were d isinfected by
immersing in 70% ethano l fo r 60 s, fo llow ed by
0.1% HgC l2 fo r 10m in. Then plan t materials we re
w ashed five times w ith ste rilized w ate r. Fo r initiation
of ca llus cultures, disinfected stems w ere cut into
sma ll pieces ( length 0.5 cm) and asep tically trans-
ferred to B5 medium supp lemented w ith 1μmo l /L 2,
4-D and 1μmo l /L KT and 8.5 g /L agar. The pH
value of themedium was adjusted to 5.8 and then au-
toclaved a t 121℃ fo r 20m in. The ca llus cu ltures
we re maintained on themed ium w ith the same compo-
sition at(25±1) ℃ in darkness by subcu lturing ev-
ery two w eeks.
Suspension cultu res we re initiated by transfe rring
pieces of callus to the Linsmaier and Skoog (LS)me-
dium w ithout agar. C ells we re g row n at 25℃ on a gy-
ratory shaker (110 r /m in) in na tura l ligh.t The cu l-
tures w ere transferred at w eekly intervals, w ith a
packed-vo lume dilu tion o f ca 3 fo ld. One w eek o ld,
m id-exponential ce ll suspensions we re used.
2. 3　Exogenous substrate
Hydroquinone w as supplied by S igma Co. ,
LTD. Itw as disso lved in E tOH and d ilu te to 10mg /
mL befo re use.
2. 4　B io transfo rmation
Datura stramonium cell suspensions (ca 40 ～ 60
mL packed volume) were transferred into 250 mL
flasks each con taining 120μmol hydroqu inone in 100
mL basa l med ium , and cu ltured as above. A fter 8
days incuba tion, the cultures we re assayed for arbutin
by HPLC method. The cells and the supernan tmedi-
um were boiled for 10m in and then centrifuged
(4 000 r /m in , 1 h). The supernan t w as passed
through silica gel G (200 to 300 mesh) co lumn
(10 cm ×30 cm ) and w ashed w ith CHC l3 ∶MeOH
(19∶1). The fractions con tained arbu tin w ere freeze-
dried and ana ly zed by NMR and E IM S.
2. 5　Th in layer chrom atog raphy
The mobile phase w as ch loroform:me thanol (7
∶3) and detected by spray ing w ith 70% H2SO4 , fo l-
low ed by hea ting a t90 ～ 100℃. The TLC chromatog-
raphy showed that the co lo r of do tw as yellow.
2. 6　Assay o fHPLC
Conditions for reversed-phase HPLC were as fo l-
low s, co lumn:TSK GEL ODS120A , 10μm , 1504. 6
mm , flow rate∶1 mL /m in, pressu re:20 kg /cm2 , de-
tection:abso rp tion at 220 nm for arbutin, so lvent sys-
tem:CH3CN - H2O (90∶10) fo r arbutin, reten tion
time:3.42m in.
430 云南农业大学学报 　　　　　　　　　　　　　　第 21卷
2. 7　NMR and E IM S
1
H and
13
C NMR spectra we re obtained at 400
MH z and 100MHz, and reco rded in CD3OD at room
temperature, respective ly. EIM S spectrum was meas-
u red on anAE IMS -50mass spectrome ter.
3　Results
3. 1　Fo rmaton ofmono-β-D-g lucoside r arbutin
The suspension cu ltures ofDa tura stramon ium at
the la te exponential g row th phase (5 day s after inocu-
lation)we re fed aseptica lly w ith a hydroqu inone and
subsequen tly inocu lated fo r 8 day s be fore harves.t The
g lucosy la ted hyd roquinone iso lated from the E tOH ex-
tracts o f the harve sted ce llsw ere cha racterized by TLC
analyses and HPLC measuremen ts (Fig. 1). In a ll
cases, the mono-β-D-g lucosider produced w as found
mostly in the cu lture medium. The b iotransforma tion
pa thw ay o f hyd roquinone by Datura stramonium ce ll
suspension cu ltures w as shown in F ig. 2.
3. 2　E ffect o f ce ll inoculums
The amoun t o f cells con tained in cu ltures had an
importan t e ffec t on the biotransform ation of hydroqui-
none (Fig. 3). The production o f arbu tin w as en-
hanced w ith quantities of ce lls increasing. W hen the
ce ll inocu lums we re added to 5 g /100mL cultu res,
the production of arbutin w as the h ighest and subse-
quen tly kep t stationary phase.
3. 3　E ffect o f hydroqu inone concentration
The effect o f substra te concentration added to the
cu lturemedium on the g lucoside forma tion w as exam-
431第 4期　　　彭春秀 , 等:白花曼陀罗细胞悬浮培养生物转化外源氢醌合成熊果苷的研究
ined. Hydroqu inone w as adm inistered to the medium
for 5 days after cells inoculation and the cells we re
cu ltured for an additional 8 days. The amount o f arbu-
tin produced w as decreased w ith the amount o f hydro-
quinone increasing as shown in F ig. 4.
The cu ltured ce lls converted the exogenous hy-
droquinone into arbu tin by 93.4%, when the hydro-
quinonew as added at the concentra tion o f 240μmo l /
100mL culture s. The production o f arbutin w as inhib-
ited by 31.5%, when the hydroquinone w as added at
the concen tration o f 485μmo l /100mL cu ltures. To a-
vo id the production inhibition o f arbu tin caused by a
high concen tra tion of hydroquinone, an effo rt to sup-
ply the substrate in four 120 μmol /f lask doses at 2-
day in terva l is now underw ay.
3. 4　E ffect o f sucrose concentra tion
In orde r to exam ine the e ffec t of sucrose concen-
tration on the g luco sy la tion o f hydroquinone, cells
w ere grown in the cultu re med ia containing diffe rent
amoun ts of sucro se. Hydroquinone was supplied 5
day s after inocu lation o f the ce lls and the culture w as
continued fo r an additional 8 days. A s show n in Fig.
5, the glucoside y ield per flask increased w ith the
concentration o f sucrose increasing.
3. 5　E ffect o f initia l pH va lue
To exam ine the e ffec t o f various in itia l pH value
in the medium , suspension cells, which had been
subcu ltured in the medium containing 2, 4-D and K i-
netin, we re transferred to new medium ad justed for
diffe rent pH value and cu ltured fo r 5 days. The cells
w ere incubated fo r an add itiona l8day s after hydroqui-
none adm inistered. As shown in Fig. 6, the produc-
tion of arbu tin w as the h ighest in the med ium at pH
value 6.0, while the production w as the low est at pH
value 8.0. In otherw ords, the high pH value forme-
dium inhibited the biotransform ation of hydroqu inone.
3. 6　E ffect of various ca rbon sources
E ffects o f various carbohydra tes on the bio trans-
forma tion of hydroquinone w ere revealed. V arious
carbon source s(30 g /L) inc lud ing fructose, glucose,
sorbitol, mannito l, sucro se /g lucose (2∶1), suc rose
/ fructose (2∶1) instead of sucrose w as supplied to
the medium , respectively. The ce lls, which had been
subcultu red in the medium containing 2, 4-D and K i-
ne tin, were transferred to the new medium conta ining
above carbohydrates in the presence o f kine tin (1
μmo l /L) and 2, 4-D(10μmol /L) and cu ltured fo r 5
days be fore hyd roquinone m inistered. A fter the ce lls
cu ltured fo r 5 days, the amoun t of hydroquinone (240
μmo l) was adm iniste red to the medium for inocula-
tion 8 days. A s show n in Fig. 7 , using sucrose and its
m ix tu res w ith g lucose and fructose as carbon sou rces
432 云南农业大学学报 　　　　　　　　　　　　　　第 21卷
in the mediaw ere benefited to the biotransform ation o f
hydroqu inone, the production of arbutin ob tained in
these med iaw as higher than o thers.
3. 7　E ffect o f culturedmedium
The medium has lo ts o f componen ts for cell
grow th, and it affec ts the metabolism of cell in sus-
pension cu ltures. The e ffects of various kinds ofmedi-
um such as LS, B5 , MS and N6 [ 5] on the conve rsion
of hyd roquinone in ce lls suspension culture ofDatura
stramon ium was studied. The results suggested that
the kind of medium had a little e ffec t on the produc-
tion of arbu tin through biotransforma tion o f hydroqui-
none (F ig. 8).
3. 8　E ffect o f exogenous pheny lalanine
In orde r to improve the efficiency o f bio transfo r-
ma tion o f hydroqu inone by ce ll suspension cultu res,
the induc tive e ffec t o f pheny lalanine w as exam ined.
The cells w ere transferred to media contain ing various
amoun t of pheny lalanine and cu ltured for 5 day s, and
the exogenous hydroquinone (364μmo l /L)was sup-
plied to the medium for culturing 8 day s before har-
ves.t A s show n in Fig. 9, pheny lalan ine adm inistered
to the medium improved the g lucosy lation activ ity,
when the pheny lalanine w as added at the concen tra-
tion of 40μmol /100mL cu ltures, and then g radually
decreased w ith the concen tra tion of pheny lalanine in-
creased.
3. 9　Chem ical structu re
The
1
H and
13
C NMR spectra w ere ob tained at
400MH z and 100MHz, and recorded in CD3OD at
room temperature (shown in tab. 1), respec tive ly.
The resu lts show ed that the compound obta ined from
fermentation o f cell cu lturesw as iden tified as arbutin.
E IM S m /z(%):272(1.2), 162(17.2), 110(100);
85(18), 73(20.2), 57(15).
Tab. 1　 13C NMR and 1H NMR spectral data fo r a rbu tin
13C NMR 1H NMR
C -1 152.3 7. 06(2H , d, J=9.7, H - 2, H -6)
C - 2, 6 119.3 6. 70(2H , d, J=9.7, H - 3, H -5)
C - 3, 5 116.6 4. 77(1H , d, J=7. 6, H - 1′)
C -4 153.6 3. 87(dd, H - 6′A, J=1.7, 12.4)
C - 1′ 103.4 3.70(dd, H -6′B, J=4. 3, 12. 4)
C - 2′ 74.8 3.28 ～ 3.45 (m, H - 2′～ H - 5′)
C - 3′ 77.8
C - 4′ 71.2
C - 5′ 77.8
C - 6′ 62.4
4　Discussion
G lycosy lated hydroquinone arbutin (4-hydroxy-
pheny l-β-D-glucopy ranoside) is abundant in certain
re surrection p lants, which can survive a lmost com-
plete dehydration for pro longed pe riods
[ 6] .
433第 4期　　　彭春秀 , 等:白花曼陀罗细胞悬浮培养生物转化外源氢醌合成熊果苷的研究
PRIDHAM(1964)was first to confirm that hydroqui-
none adm iniste red to the leaves and seedling s of vari-
ous higher p lants w ere rapidly conve rted to the co rre-
sponding g lucosides. It has been reported the reafter
that cultu red ce lls o f higher p lants, such asDatura
innoxia
[ 7] , D. ferox, Agrostemma githago, D igitalis
purpurea , Gardenia jasm inoides, C itrus P aradis,
Rauvolfia serpentine a lso converted hydroquinone to
themonoglucoside arbu tin
[ 1, 4, 8 ～ 10]
.
The presen t experiments have shown that exoge-
nous hydroquinone when introduced in to the cultu red
ce lls ofDa tura stramonium are readily converted to
the co rresponding mono-β-D-g lucosides (arbu tin). It
suggested that the usefulness o f cu lturedDatura stra-
monium ce lls as biocatalysts for the production o f val-
uab le g lucosides. M any factors, such as cell inocu-
lum s, hydroquinone concentra tion, initia l pH va lue o f
media, sucrose concen tration, carbon sou rces, k ind
of cu lture medium , induce r (pheny la lanine) e tc. all
in fluenced the conversion efficiency ofDa tura stramo-
nium cel.l
E fforts to de tect sim ilar transg lucosy lase activ ity
in the cu ltured ce lls o fDatura stramon ium are now
unde rway.
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434 云南农业大学学报 　　　　　　　　　　　　　　第 21卷