全 文 ：Ｒeceived:March 22，2013 Accepted:September 5，2013
* Corresponding author Tel:86-20-85217701;E-mail:d_zhm@ 163． com
天然产物研究与开发 Nat Prod Ｒes Dev 2014，26:392-397
文章编号:1001-6880(2014)3-0392-06
肾茶提取物抗氧化及保护线粒体作用研究
陈地灵1，龙贺明2，张鹤鸣1* ，李 宁1，刘颂豪1
1华南师范大学药物研究院，广州 510631;2 赣南医学院第一附属医院肿瘤科，赣州 341000
摘 要:肾茶水提液依次用石油醚、氯仿、乙酸乙酯萃取，与水层得到 4 个不同极性部位。以清除 DPPH·、超氧
阴离子和羟自由基的能力，络合 Fe2 +能力，体外抗活性氧能力以及对 FeSO4 和 H2O2 诱导小鼠肾脏组织匀浆脂
质过氧化产物 MDA和 GSH-Px水平的影响，综合评价其抗氧化活性;以抑制 Fe2 + -L-Cys诱导肾脏线粒体肿胀能
力，初步评价其保护肾脏线粒体的作用。结果显示肾茶水提液 4 个不同极性部位均具有不同程度的清除 DPPH
·、O-·2 、·OH和络合 Fe
2 +能力;其中乙酸乙酯和水提部位具有显著体外抗 ＲOS 能力，降低 MDA 和升高 GSH-
Px水平，以及抑制肾脏线粒体肿胀的能力，且其作用效果均与剂量呈正相关。本实验结果可为肾茶用于治疗肾
脏疾病的药效物质基础研究提供依据。
关键词:肾茶;抗氧化活性;金属络合能力;活性氧;线粒体肿胀
中图分类号:Ｒ285. 5 文献标识码:A
In vitro Antioxidant and Mitochondria Protective Activities
of Clerodendranthus spicatus Extracts
CHEN Di-ling1，LONG He-ming2，ZHANG He-ming1* ，LI Ning1，LIU Song-hao1
1Southern Institute of Pharmaceutical Ｒesearch，South China Normal University，Guangzhou Guangdong 510631，China;
2 Department of Oncology，The First Affiliated Hospital of Gannan Medical University，Jiangxi Ganzhou 341000，China
Abstract:Water extract of Clerodendranthus spicatus was successively fractionated using 3 different solvents to provide
petroleum ether extract (PE) ，chloroform extract (CE) ，ethyl acetate extract (EAE)and water extract (WE)． Different
antioxidant assays including OH·，O-·2 ，DPPH· scavenging activity，reducing power，metal chelating activity，anti-reac-
tive oxygen species (anti-ＲOS)activity and effects on FeSO4 and H2O2 induced MDA and GSH-Px production increase
were employed to evaluate the antioxidant activities of four extracts． The inhibition rate of mitochondrial swelling induced
by Fe2 + -L-Cys in vitro was employed to evaluate the protective effects on mitochondrion． The results showed that all the
4 different extracts had the ability of scavenging DPPH·，O-·2 ，·OH and chelating Fe
2 +，anti-ＲOS in vitro，significantly
lower the levels of MDA，rised the levels of GSH-Px in renal homogenate and inhibited the renal mitochondria swelling，
in concentration dependent manner． The results provided a basis for C． spicatus to be used in the treatment of renal dis-
ease．
Key words:Clerodendranthus spicatus;antioxidant activity;metal chelating activity;reactive oxygen species;mitochondri-
al swelling
Introduction
Clerodendranthus spicatus Thunb．，scattered in Fujian，
Taiwan，Hainan，Guangdong and Yunnan provinces of
China［1］，is normally used as folk medicine to treat a-
cute or chronic nephritis，urocystitis，urolithiasis，gall-
stone，rheumatic arthritis，etc［2］． More than 200 chemi-
cal constituents were isolated from C． spicatus，inclu-
ding flavonoids (5-hydroxy-6，7，4-trimethoxyflavone
and 5-hydroxy-6，7，3，4-tetramethoxyflavone) ，phenol-
ic acids (orthosiphol，siphonol，oleanolic acid，ursolic
acid，rosmarinic acid and glucuronic acid) ，saponins
and internal compensation inositol［3］． The pharmaco-
logical researches showed that the extracts of C． spica-
tus had antihypertensive effect，inhibitory effect on
Staphylococcus aureus，Pseudomonas aeruginosa and
Candida albicans［4］．
Oxidative stress，defined as a disturbance in the bal-
ance between the production of reactive oxygen species
(ＲOS) and antioxidant defense systems． Excessive
ＲOS production is known to cause oxidative damage to
major macromolecules in cells，including DNA，lipids
and proteins，thereby disrupting cellular functions and
integrity，which will cause cell death and tissue dam-
age． The role of ＲOS has been implicated in several
diseases，including cancer，diabetes and cardiovascular
diseases，aging，etc． Antioxidants are vital substances
which possess the ability to reduce oxidative damage
caused by ＲOS． As the possible carcinogenic effects of
synthetic antioxidants，there is a growing demand for
replacing synthetic antioxidants by natural antioxidants
from herbs． Hence，the development of natural antioxi-
dant has gained more attentions．
To the best of the author s knowledge，no report about
the antioxidant properties of C． spicatus has been pub-
lished until now． The aim of this study is to evaluate
the antioxidant properties of different extracts of C． spi-
catus，to investigate the relationship between concentra-
tion and antioxidant properties．
Materials and Methods
Chemicals
Trichloroacetic acid (TCA) ，2-deoxy-D-ribose，ascor-
bic acid，linoleic acid，Tween-40，3-(2-pyridyl)-5，6bis
(4-phenylsulfonic acid)-1，2，4-triazine (ferrozine) ，
ethylenediamine trtra acetic acid (EDTA) ，2，2-di-
phenyl-1-picrylhydrazyl(DPPH) ，phenazine methosul-
phate (PMS) ，potassium ferricyanide，thiobarbituric
acids (TBA) ，nitroblue tetrazolium (NBT) ，β-nicotin-
amide adenine dinucleotide reduced (β-NADH)and
ammonium thiocyanate were obtained from Sigma
Aldrich Chemical Co．(USA)． All other chemicals and
solvents were of analytical or high performance liquid
chromatography grade．
Animals and tissue homogenate preparation
Adult Kunming mice，18-25 g，were obtained from Cen-
ter of Laboratory Animal of Guangzhou University of
Chinese Medicine，Lot No．:SCXK (Yue)2008-0020，
SYXK (Yue)2008-0085． The mice were pair-housed
in plastic cages in a temperature-controlled (25 ℃)
colony room with a 12 /12 h light /dark cycle． Food and
water were available ad libitum． All experimental proto-
cols were approved by the Center of Laboratory Animal
of Guangzhou University of Chinese Medicine． All ef-
forts were made to minimize the number of animals
used． The mice were executed by cervical vertebra dis-
location and the kidney were dissected，then grinded
into 10% tissue homogenate using glass pestle with 4
℃ physiological saline in ice-bath and stored at 4 ℃
before used．
Plant material
The whole plant was collected on August 20th，2012
from Medicinal Plants Mountain in Guangzhou Univer-
sity of Chinese Medicine and was identified by Prof．
LIN Li，a Chinese medicine resources specialist． The
fresh plant was dried at room temperature，and then
ground into fine powder． The voucher specimen (No．
20120820-SC)was deposited in botanic specimen cen-
ter of college of Chinese Materia Medica，Guangzhou U-
niversity of Chinese Medicine．
Preparation of extracts
10 g of the dried powder of C． spicatus was extracted
with 3 × 200 mL water at 100 ℃ for 1． 5 h each time．
The water solutions were combined and evaporated to
dryness under reduced pressure． The residue was dis-
solved in 200 mL of 30% aqueous ethanol (V /V)
completely． The resulting solution was kept in a refrig-
erator at 4 ℃ overnight，and was centrifuged at 5000 r /
min for 10 min to eliminate chlorophyll． The superna-
tant solution was collected and evaporated to dryness．
The residue was added with 100 mL distilled water，and
shaken vigorously to form a suspension，followed by
fractionation using different solvents in order of:petro-
leum ether，chloroform and ethyl acetate． Three extracts
and the water phase were evaporated to dryness under
reduced pressure by rotary evaporator to provide petro-
leum ether extract (PE) ，chloroform extract (CE) ，
ethyl acetate extract (EAE) ，and water extract (WE) ，
respectively． All of these extracts were stored at 4 ℃
until used．
The stock solution of these extracts were weekly pre-
pared at a concentration of crude drug 100 mg /mL in
50% aqueous methanol (V /V)and stored at 4 ℃ ． Di-
393Vol. 26 CHEN Di-ling，et al:In vitro Antioxidant and Mitochondria Protective Activities of Clerodendranthus spicatus Extracts
luted extracts were prepared daily by diluting the stock
solutions with 50% aqueous methanol (V /V)．
DPPH radical scavenging activity
The free-radical scavenging activity was measured by
using 1，1-diphenyl-2-picrylhydrazyl (DPPH·) as-
say［5］． The testing of each sample was performed in
triplicate． Percent inhibition was measured according to
following formula and IC50 value was calculated by
graph pad prism software． The radical-scavenging ca-
pacity (ＲSC)was calculated using the equation:
ＲSC = ［1-(A1-A0)/A2］ × 100%
Where A1 is the absorbance of probe，at a given sample
concentration level (average of three probes) ;A0 is the
correction or the absorbance of the extract alone (with-
out reagents) ，and A2 is the absorbance of the DPPH
radical reagent without extracts．
Superoxide radical scavenging activity
Superoxide radical scavenging activity (SＲSA)was
determined by the nitroblue tetrazolium reduction meth-
od described by Zhang et al［6］． All measurements were
made in triplicate． The abilities to scavenge the super-
oxide radical were calculated using the following equa-
tion:
SＲSA = (1-A1 /A0) × 100%
Where A1 is the absorbance of sample，A0 is the absor-
bance of control．
Hydroxyl radical scavenging activity
Hydroxyl radical scavenging assay was carried out using
a method described by Zhang et al［6］ with slight modifi-
cations． The absorbance of supernatant was measured at
536 nm． The hydroxyl radical-scavenging activity was
expressed as:
Scavenging percentage = ［(A1-A0)/ (A-A0) ］×
100%
Where A1 is the absorbance of reaction mixture with
sample，A0 is the absorbance of reaction mixture with
sample replaced by equivalent volume of deionized wa-
ter，and A is the absorbance of the reaction mixture
with sample and H2O2 replaced by equivalent volume
of deionized water． Vitamin C was used as a positive
control． The IC50 value was defined as the concentration
of sample (mg /mL)require to scavenging 50% of hy-
droxyl radical and calculated by graph pad prism soft-
ware．
Phosphomolybdenum (P-Mo)inhibition assay
The antioxidant activity was evaluated by phosphomo-
lybdenum method according to the reported procedure
［5］． Percent inhibition was calculated by the following
formula．
Inhibition ratio = (1-absorbance of sample /absorbance
of control) × 100%
Fe2 + chelating activity assay
1 mL of different concentrations of sample solution and
3． 7 mL of 50% aqueous methanol (V /V)and 0． 1 mL
of 2 mmol /L FeSO4(7H2O solution were mixed and re-
acted for 30 s，0． 1 mL of 5 mmol /L ferroustriazine so-
lution，mixed and measured the absorbance A1 at the
wavelength of 562 nm after reaction at room tempera-
ture for 10 min． The sample s solvent was used as
blank A0 ． Percent chelating was measured according to
following formula and IC50 value was calculated by
graph pad prism software．
Chelating ability = (A1-A0)/ A0 × 100%
Effects on ＲOS in chemical modified systems
The levels of anti-reactive oxygen species (anti-ＲOS)
were measured strictly following the instructions of de-
termination kit purchased from Nanjing Jiancheng Bio-
engineering Institute． (China)． The anti-ＲOS unit was
calculated as 1 mL of sample solution lowering 1
mmol /(L·min)of H2O2 in the chemical modified
systems at 37 oC．
Ｒenal mitochondrial swelling degree assay
10 μL of different concentrations of sample solution and
40 μL of 0． 01 mol /L L-Cys and 200 μL of 5 mmol /L
FeSO4 solution and 50 μL of 0． 5 mg /mL prot renal mi-
tochondria suspension liquid，then added to 3 mL with
0． 1 mol /L PBS (pH7． 4)mixed and measured the ab-
sorbance at the wavelength of 520 nm after reacting 0，
10，15，30 min． The absorbance without sample solution
was used as control and the renal mitochondria suspen-
sion liquid was used as blank．
Effects on MDA in renal tissue homogenate
The levels of MDA were measured strictly following the
instructions of determination kit purchased from Nan-
jing Jiancheng Bioengineering Institute (China)． 0． 2
mL of different concentrations of sample solution were
493 Nat Prod Ｒes Dev Vol. 26
kept in 37℃ for 1h，then added 1 mL of 15% trichlo-
roacetic acid solution and 1 mL of 0． 67% TBA，vortex
and developed in boiling water bath for 15 min，cooled
and then centrifuged at 3000 rpm for 10 min，and
measured the absorbance of supernatant fluid at 532
nm，and the normal，FeSO4 and H2O2 induced renal tis-
sue homogenate were measured，respectively． Percent
inhibition was calculated by the following formula．
Inhibition ratio = (A0-A1)/A0) × 100%
Where A1 is the absorbance of reaction mixture with
sample，A0 is the absorbance of reaction mixture with
sample replaced by equivalent volume of deionized wa-
ter．
Effects on GSH-Px in renal tissue homogenate
280 μL of 10% renal tissue homogenate and 40 μL of
different concentrations of sample solution were kept in
the 37 ℃ for 1 h，ice-bath for 10 min and centrifuged
at 3000 rpm for 10 min，measured the absorbance of
supernatant fluid strictly following the instructions of
determination kit purchased from Nanjing Jiancheng
Bioengineering Institute (China)． The control group
tube was the absorbance of 5 mg /mL Vc solution and
normal group was the absorbance of the PBS (pH7．
4)．
Statistical analysis
All assays were carried out in triplicates，and results
were expressed as mean ± s． ANOVA was used to ana-
lyze the differences among IC50 of various samples for
different antioxidant assays，with least significance
difference (LSD)P ＜ 0． 01 as a level of significance u-
sing SPSS 17． 0 (SPSS，Abaus Concepts，Berkeley，
CA)and graph pad prism (Graph Phad，San Diego，
USA)software．
Ｒesults and Discussion
Antioxidant activity
The results of Duncan s multiple Ｒange Tests showed
that there were no significant differences (P ＜ 0． 05)
among IC50(concentration of crude drug，similarly here-
inafter)values of WE and EAE except on DPPH，while
there were significant different from those of PE and
CE，as shown in Table 1．
Table 1 The IC50 values of four extracts of C． spicatus (mean ± s，n = 3)
Group DPPH· O-·2 ·OH P-Mo Fe2 +
PE 460． 22 ± 21． 48 330． 54 ± 12． 46 38． 55 ± 2． 61 1213． 39 ± 35． 46 302． 67 ± 10． 91
CE 721． 42 ± 15． 17 314． 72 ± 8． 42 29． 75 ± 2． 45 1071． 14 ± 40． 13 263． 24 ± 8． 26
EAE 2． 05 ± 0． 24 5． 09 ± 0． 72 3． 52 ± 0． 37 50． 69 ± 2． 72 53． 03 ± 2． 27
WE 1． 22 ± 0． 07 3． 84 ± 0． 46 2． 89 ± 0． 21 50． 21 ± 3． 84 47． 17 ± 1． 84
Because the IC50 values of PE and CE on DPPH·，
O-·2 ，·OH，P-Mo and Fe
2 + were lower than that of
EAE and WE (as shown in Table 1) ，only WE and
EAE were used for the anti-ＲOS，inhibition MDA and
GSH-Px and renal mitochondrial swelling assays．
As shown in Table 2，the WE and EAE showed signifi-
cant activity on anti-ＲOS，the A520 of control tube was
significant different from the sample tube (P ＜ 0． 01)．
The anti-ＲOS activity assay showed significant dose-
effect relationship at the concentrations 0． 25-2． 00 mg /
mL． At the same concentration，the inhibition rate (IＲ)
of WE was higher than that of EAE，but the difference
was not significant (P ＞ 0． 05)．
The Fe2 + and H2O2 can induced the rising of levels of
MDA in kidney tissue，as shown in Table 3，compared
Table 2 Effects of WE and EAE on ＲOS in chemical mod-
ified systems (mean ± s，n = 3)
Sample Dosage
(mg /mL)
A520
Inhibition rate
(U /mg(prot．)
Control － 0． 827 ± 0． 012 －
EAE 0． 25 0． 517 ± 0． 007＊＊ 37． 61
1． 00 0． 207 ± 0． 001＊＊ 74． 97
2． 00 0． 089 ± 0． 003＊＊ 82． 10
WE 0． 25 0． 429 ± 0． 016＊＊ 48． 13
1． 00 0． 187 ± 0． 009＊＊ 77． 63
2． 00 0． 024 ± 0． 002＊＊ 85． 01
＊＊P ＜ 0． 01 vs control tube
with the control tube，the levels of MDA of the Fe2 +，
H2O2 induced tube and the spontaneous tube were sig-
nificant different (P ＜ 0． 01) ，respectively． It showed a
593Vol. 26 CHEN Di-ling，et al:In vitro Antioxidant and Mitochondria Protective Activities of Clerodendranthus spicatus Extracts
significant dose-effect relationship，and the effect of
WE was better than that of EAE． The effect of extracts
on H2O2-induced tube was the best，followed by the
spontaneous tube and Fe2 + -induced tube． It suggested
that the extracts of C． spicatus have a strong antioxidant
activity． The same effects on levels of GAH-Px in renal
homogenate were showed as in Table 4，the extracts of
C． spicatus can significantly improve the antioxidant ac-
tivity of kidney．
Table 3 Effects of EAE and WE on MDA in renal homogenate (mean ± s，n = 3)
Sample Dosage
(mg /mL)
MDA content /(nmol /mg)
Normal IＲ /% FeSO4-induced IＲ /% 3% H2O2-induced IＲ /%
Blank － 2． 515 ± 0． 147 － 4． 530 ± 0． 461 － 4． 048 ± 0． 261 －
Control － 13． 911 ± 1． 571△ － 21． 460 ± 1． 647△ － 19． 529 ± 1． 671△ －
EAE 0． 25 8． 507 ± 0． 543＊＊ 38． 85 17． 829 ± 1． 173* 16． 92 12． 338 ± 1． 438＊＊ 36． 82
1． 00 5． 988 ± 0． 416＊＊ 56． 96 15． 392 ± 1． 249＊＊ 28． 28 9． 924 ± 1． 084＊＊ 49． 18
2． 00 4． 745 ± 0． 278＊＊ 65． 89 12． 283 ± 1． 073＊＊ 42． 76 5． 695 ± 0． 671＊＊ 70． 84
WE 0． 25 8． 142 ± 0． 671＊＊ 41． 47 17． 464 ± 1． 548* 18． 62 11． 974 ± 1． 004＊＊ 38． 69
1． 00 5． 623 ± 0． 346＊＊ 59． 58 15． 027 ± 1． 230＊＊ 29． 98 9． 560 ± 0． 917＊＊ 51． 05
2． 00 4． 380 ± 0． 315＊＊ 68． 51 11． 918 ± 0． 973＊＊ 44． 46 5． 330 ± 0． 536＊＊ 72． 71
△P ＜ 0． 01 vs blank tube;* P ＜ 0． 05，＊＊P ＜ 0． 01 vs control tube．
Table 4 Effects of EAE and WE on GSH-Px and renal mitochondrial swelling (mean ± s，n = 3)
Sample Dosage
(mg /mL)
GSH-Px
content
(nmol /mg)
Mitochondrial swelling A520
0 min 5 min 15 min 30 min
Blank － 48． 162 ± 1． 273 0． 760 ± 0． 021 0． 751 ± 0． 003 0． 738 ± 0． 005 0． 728 ± 0． 003
Control － 84． 511 ± 1． 489△ 0． 749 ± 0． 014 0． 722 ± 0． 005 0． 687 ± 0． 010 0． 673 ± 0． 004
EAE 0． 25 69． 614 ± 1． 181 0． 857 ± 0． 018 0． 839 ± 0． 007 0． 836 ± 0． 009 0． 833 ± 0． 007
1． 00 95． 894 ± 2． 374* 0． 876 ± 0． 016 0． 868 ± 0． 006 0． 866 ± 0． 004 0． 865 ± 0． 009
2． 00 106． 829 ± 2． 813＊＊ 1． 189 ± 0． 024 1． 184 ± 0． 005 1． 184 ± 0． 012 1． 183 ± 0． 011
WE 0． 25 81． 278 ± 2． 107 0． 862 ± 0． 017 0． 859 ± 0． 002 0． 856 ± 0． 005 0． 854 ± 0． 009
1． 00 101． 453 ± 3． 491＊＊ 0． 891 ± 0． 020 0． 889 ± 0． 001 0． 887 ± 0． 007 0． 886 ± 0． 007
2． 00 113． 896 ± 3． 617＊＊ 1． 214 ± 0． 024 1． 213 ± 0． 008 1． 213 ± 0． 013 1． 212 ± 0． 005
△P ＜ 0． 01 vs blank tube;* P ＜ 0． 05，＊＊P ＜ 0． 01 vs control tube．
Effects on renal mitochondrial swelling degree
The reaction of unsaturated fatty acid with free radicals
induced by Fe2 + -L-Cys would produce much lipid per-
oxide and then the mitochondria would be swelling，and
the absorbance at 520 nm would taper off with the ex-
tended response time． It meant that the mitochondria
swelling will be more and more seriously． As shown in
Table 4 and Fig． 1，the reduction rate of control tube
was 12． 69% and higher than the blank tube of
4. 22%，the difference was significant (P ＜ 0． 01) ，
while the sample tube of WE and EAE were lower，
which showed that the extracts of C． spicatus can signif-
icantly inhibit the mice renal mitochondria swelling and
positively dependent on the concentration．
14
12
10
8
6
4
2
0
0% 5 15% 30
Reaction%time(min)
Cu
m
ul
at
iv
e%r
ed
uc
tio
n%
ra
te
(%
) Blank
Control
0.25鄄EAE
1.00鄄EAE
2.00鄄EAE
0.25鄄WE
1.00鄄WE
2.00鄄WE
Fig. 1 The cumulative reduction rate of WE and EAE
on absorbance of renal mitochondrial swelling
Conclusion
Ｒecent studies showed that more than 30 flavonoids
693 Nat Prod Ｒes Dev Vol. 26
were isolated from C． spicatus［7，8］which were tested to
have strong antioxidant activities． More than 30 phenol
and polyphenol acid were isolated which were tested to
be the active ingredient of anti-inflammatory，antibiosis
and diuresis［9］． Based on the results obtained in the
present study，it was concluded that the C． spicatus had
significant antioxidant activities and the extracts of C．
spicatus can be used as an antioxidant for adjuvant
therapy on renal diseases．
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