全 文 ：天然产物研究与开发 Nat Prod ResDev 2006 , 18:401-404
文章编号:1001-6880(2006)03-0401-04
Received September 21 , 2005;Accepted December 13 ,2005
＊Correspouding author Tel:86-29-88963253;E-mail:whm801@126.com
泡桐花体外抑菌作用及黄酮含量的测定
魏希颖＊ ,何 悦 ,蒋立锋 ,岳奇锋
(陕西师范大学生命科学学院 ,西安 710062)
摘 要:本文首次采用滤纸片法对泡桐花提取液的不同萃取部分进行了体外抑菌试验并用紫外分光光度法对
泡桐花的花冠和花萼中总黄酮含量进行了测定。结果表明不同萃取部分对金黄色葡萄球菌 , 大肠杆菌 , 枯草芽
孢杆菌均有不同程度的抑制作用 ,其中对金黄色葡萄球菌作用最强 , 而对黑曲霉 , 啤酒酵母 , 产黄青霉无明显抑
制作用。含量测定表明花萼中黄酮含量明显高于花冠。
关键词:泡桐花;体外抑菌;总黄酮
中图分类号:R282.71 文献标识码:A
The Study of the Antibacterial Activity in vitro and Determination of
Flavones of Flos Paulowniae
WEI Xi-ying＊ ,HE Yue , JIANG Li-Feng ,YUE Qi-feng
(College of life Sxienxes , Shaanxi Normal University , Xi an 710062 , China)
Abstract:In this paper , we adopt the filter paper to study antibiotic function of different component of Flos Paulowniae which is
extracted by ethanol and water.At the same time ,we also adopt an ultraviolet spectrophotometric method to measure the content
of flavones in corolla and calyces .The results show that different part extracted has strong antibiotic function on Staphylococcus
aureus 、Bacillus subtilis and Escherichia coli.The strongest effection is on Staphylococcus aureus , But has no effect on Saccha-
romyces carlsbergensis 、Aspergillus niger and Penicillium chrysogenum.The results of determination of flavones show that the con-
tent of flavones in calyces is higher than that in corolla.
Key words:Flos Paulowniae;flavones;antibiotic function
Paulownia tomentosa(Thub)Steud is also called , royal
paulownia , belonging to Scrophulariaceae , Paulownia.It
falls into deciduous tree , distributing in China s Gansu ,
Shaanxi , Liaoning , Hebei and Henan provinces[ 1] .The
medical value of paulownia has been recorded in Pen-ts
ao Kan-mu.Its flower , leaf , fruit ,wood , peel , root can all
be used as medicine.Flower can be used to cure upper
respiratory infection ,bronchitis , acute tonsillitis[ 2 ,3] bacil-
lary dysentery , acute enteritis , acute conjunctivitis and
parotitis ect.Clinical report that ointment made from Flos
Paulowniae can cure tinea pedis , burn[ 4 ,5] ,Folk use it to
cure dermatophytosis.Paulownia is quite resourceful but
unfortunately it has been left unnoticed for a long time ,
which leads to a big waste of resource.In order to make
better use of this resource ,we made a experiment to mea-
sure the antibiotic effect of different extraction part of Flos
Paulowniae and the total flavone content in it.
Experimental Material
Source of the sample
The flowers of Paulownia tomentosa(thunb)stend were
picked on the campus of ShaanXi Normal University in
April 2004.
Source of the bacterial
Bacillus subtilis;Staphylococcus aureus;Escherichia col-
i ;Saccharomyces carlsbergensis;Aspergillus niger;Peni-
cillium chrysogenum .
All of them are offered by microbiology laboratory in col-
lege of life science ,ShaanXi Normal University.
Apparatus and reagent
Rotary evaporimeter (RE-52AA), made in Shanghai
Yarong biochemistry apparatus factory ;722N visible
complementary filter ,made in Shanghai third analysis ap-
paratus factory;Excluding-water electro-heating standing-
temperature cultivator , PYX 50 ×65 , made in Shanghai
Yuejing medical apparatus factory;DL102 Electric drying
oven with forced convection ,made in Tianjin experimental
apparatus factory;Chrome-plated venire caliper ,(0 ～ 125
mm)×0.02 ,made in Xi an tool factory.
DOI :10.16333/j.1001-6880.2006.03.011
Mueller Hinton agar , made in Beijing AoBoXingSheng
biotechnology company limited by guarantee (batch num-
ber:20030306);Nutrient agar:made in Fujian Huian
chemical plant (batch number:921123);Tween 80:made
in Xi an chemical reagent factory.Ethanol , light
petroleum 、ethyl acetate 、aether are all analytically pure.
Experimentation
Antibacterial activity experiment
Sample preparation
Takes fresh pawlownia flowers each 150 gram , separately
uses the water backflow orcold soak them in 70% ethyl
alcohol.The extract fluid uses the light petroleum and the
ethyl acetate to extract separately , reduced pressure recy-
cling solvent , then with water hold the volume to 100mL ,
The samples of water distilled parts mark distinctly with
A1(light petroleum layer)A2 (ethyl acetate layer)A3
(water layer)and the samples of Alcohol distilled parts
mark distinctly with B1(light petroleum layer)B2(ethyl
acetate layer)the B3(water layer).
Methods
Preparation of the liquid of fungs[ 6 ,7]
Select the foregoing six kinds of bacterial to make individ-
ual inclined bacteria.Use aseptic water to elute them into
the aseptic test tube.After each tube is marked ,we use
careful counting method of the mirror to count the bacteri-
al.And then dilute them to make the concentration of
Bacillus subtilis between 2×109 ～ 5×109 per liter , and
Staphylococcus aureus between 2.5×1011 ～ 3.5×1011
per liter;Escherichia coli between 2×1010 ～ 4×1010 per
liter;Saccharomyces carlsbergensis 2×1010 ～ 5×1010 per
liter;Aspergillus niger and Penicillium chrysogenum 5×
109 ～ 1×1010 per liter respectively(filamentous fungi are
count by spores).
Preparation of culture medium
They are all prepared according to the references[ 8] M-H
agar medium;Czapek medium are sterilized at121 ℃,20
minutes for future spending.MacConkey medium is steril-
ized at 113 ℃,20 min for future spending.
Manipulating methods
Using holding type punching device ,we make the clean
filter paper into small round paperwith diameter of 6mm ,
which is then dry heat sterilized for future spending.We
use aseptic transfer pipet to imbibe 0.1mL liquid of bac-
terial and put it in the center of the culture dish which has
been prepared.Then coat it equally.With small round fil-
trate paper dipping in the foregoing samples ,we put asep-
tic forceps in the culture dish containing bacteria prepared
before.We put the bacterium s flat into the incubator of
constant temperature ,37 ℃ for 24 hours.And put the flat
of Saccharomyces carlsbergensis , Aspergillus niger and
Penicillium chrysogenum into incubator of constant tem-
perature ,28 ℃ for 48 hours.Measure the diameter of in-
hibition zone.Each sample is made into three flats , each
flat with 4 pieces of small round filtrate paper on it;we
use the mixture of tween 80 and aseptic water as antithe-
ses for sample A1&B1.And aseptic water as antitheses for
the rest samples.
Results
The effectiveness of bacteriostasis can be seen in table 1
and table 2.
Table 1 The diameter of inhibition zone of each bacterium in
sample A1;A2;A3(mm)
Bacterial A1 A2 A3
Bcillus subtilis 7.98 8.31 7.60
S taphylococcus aureus 8.52 9.29 9.14
Escherichia coli 8.32 8.98 9.43
Saccharomyces carlsbergensis 0 0 0
Aspergillus niger 0 0 0
Penici llium chrysogenum 0 0 0
Table 2 The diameter of inhibition zone of each bacterium in
sample B1;B2;B3(mm)
Bacterial A1 A2 A3
Baci llus subtilis 7.94 8.08 8.02
S taphylococcus aureus 8.64 10.20 9.24
Escherichia coli 9.04 8.30 9.06
Saccharomyces carlsbergensis 0 0 0
Aspergillus niger 0 0 0
Penici llium chrysogenum 0 0 0
402 Nat Prod ResDev Vol.18
Sterilizing effect to Staphylococcus aureus and Escherichia
coli (MBC)can be seen in table 3.
Table 3 Different extraction section s sterilizing effect to
Staphylococcusaureus and E.coli
Different extraction
section
Different concentration
(g/mL)
Bacterial 2 1 1/2 1/4 1/8 1/161/321/ 64
A1 + + + + + + + +
Escherichia coli A2 - + + + + + + +
A3 - + + + + + + +
Staphylococcuaureus B1 + + + + + + + +
B2 - + + + + + + +
B3 - + + + + + + +
“+” means positive(bacteria grows)“ -” means negative(no bacteria
grows)
Determination of Flavones
[ 9]
Preparation of the reference solution
Accurately measure 5 mg Quercetin as reference substance
and put it into the 50 mL methanol.
Preparation for the sample solutions
Take corolla and calyces each 10 g and put them into
70% ethanol , kept for 4 days.Retrieve the ethanol and
put the corolla and calyces in 80 ℃water bath , distilled
to dryness.Add methanol to the constant volume 50 mL.
Selection of maximum wavelength
Accurately extract 1 mL reference substance solution , 1
mL alchlor solution , 0.1 mL acetic acid-sodium acetate
buffer solution and put them into a fresh 5 mL volumetric
flask , adding methanol to the scale.Make up blank control
as the foregoing method dictates.Measure absorbance be-
tween 380 nm and 440 nm.The result is absorption maxi-
mum appears at 430 nm , so we choose 430 nm as the
standard wavelength in this experiment.
Review the linear relation
Separately and accurately measure reference substance so-
lution 0.2 mL、0.4 mL、0.6 mL 、0.8 mL 、1.0 mL、1.2
mL and put them into the six 5 mL volumetric flask.Sepa-
rately add 1mL alchlor solution ,0.1mL acetic acid-sodi-
um acetate buffer solution and add methanol to the scale.
Make up blank control using the same method.Measure
absorbance at 430 nm.Regarding density as ordinate and
absorbance as abscissa , draw regression curve and obtain
regression equation:Y =0.0153X - 0.0019 (R =
0.9933).The curve s linear relationship is good at 0.004
～ 0.024 mg/mL.
Precision experiment
Draw accurately 0.5 mL sample solution and put it into
the 5 mL volumetric flask , adding methanol to the scale.
Make up solution according to the method indicated in the
linear relationship , repeatedly measure absorbance for five
times at 430 nm , the result is as follows:
Table 4 Precision experiment measuring total flavones content
in Flos Paulowniae
Number of times of the experiment Absorbance A
1 0.830
2 0.833
3 0.833
4 0.833
5 0.829
Result:relative standard deviation:0.31%
Stability experiment
Draw sample solution to make solution using the same
method as precision experiment , measure absorbance at
430 nm every 15 min.The result is as follows:
Table 5 Stability experiment measuring total flavones content
in Flos Paulowniae
Time(min) Absorbance A
0 0.901
15 0.915
30 0.931
45 0.942
60 0.955
75 0.975
90 0.994
Relative standard deviation :3.17%
The content of the sample is determined
Draw accurately 0.5 mL sample solution and put it into
the 5 mL volumetric flask , adding methanol to the scale;
make up blank control using the same method as before.
Measure absorbance at 430 nm , the result is as follows:
Table 6 The total flavones content determining in Flos
Paulowniae
Sample Absorbance Content(mg/mL)Percentage(%)
Corolla 0.571 0.007 0.35
Calyces 0.850 0.011 0.55
Analysis and Discussion
(1)The result of antibiotic function experiment shows that
each part extracted of both the water distilled liquid and
alcohol distilled liquid of Flos Paulownia can inhibit
Staphylococcus aureus , Escherichia coli ;Bacillus subtilis
to a various degree.Staphylococcus aureus was inhibited
most , Escherichia coli take second place;Bacillus subtilis
was inhibited least.Aspergillus niger , Saccharomyces
carlsbergensis and Penicillium chrysogenum were not in-
hibited.
(2)In this paper UV ultraviolet spectrophotometer is used
to determine content of Flavone.From our study , the
means has preferable degree of precision and reliability
403Vol.18 WEI Xi-Ying et al:The Study of the Ant ibacterial Activity in Vitro And Determination of Flavones of Flos Paulowniae
(relative standard deviation:0.31%).Which has refer-
ence value to trace medicine components analysis as other
means.
(3)The results of total flavone content determining are:
corolla contains 0.35% flavone and calyces contains
0.55%.
(4)Flos Paulowniae is resourceful but the present utiliza-
tion ratio is relatively low , so it is a huge waste of re-
sources.This experiment result will offer a certain experi-
ment basis for the develop and utilize of Flos Paulowni-
ae.
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