Cytotoxicity on Cancer cells of the Extract of Sophora flavescens Ait.

     

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1 

Cytotoxicity on Cancer cells of the Extract of Sophora flavescens Ait.

Hyun-Ok Lee^†, Ju-Yeon Chun, Ji-Youn Lee and Chang-Hee Kim¹

Department of Dental Hygiene, Wonkwang Health Science College, Iksan-City 570-750, Korea

1Department of Dental Hygiene, Shin-Sung College, Choongnam 343-861, Korea


 In this study, we investigated the cytotoxicity of ethyl acetate subfraction of Sophora flavescens Ait.(EASS) on cancer cells using MTT quantitative analysis. The EASS was cytotoxicity from the concentration of 6.25 g/ml to KB, B16, HeLa, and MCF-7 cancer cells and the cytotoxicity was significant, (p < 005) increased as the concentrations of EASS were increased, (12.5, 25, 50, 100 g/ml). The IC for KB, B16, HeLa, and MCF-7 were 56.58, 65.43, 83.95, and 106.65 g/ml, respectively. Conclusively, the EASS inhabited the growth of cancer cells and the order of potency of cytotoxicity was KB > B16 > HeLa > MCF-7.

   Cytotoxicity, Sophora flavescens Ait., Subfraction, MTT assay



         

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&‡ "¦ 'y;§¨©h 3.0 g 10 ml ¯¥ °Wx±

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KB´5 ±¿ÀAWÁT 4UV J  ÃÄ2. ;D J  MeOH:CHCI3, MeOH ;ÅP ![GÄi,

;ÅP ÆÇ; 1:10, 3:10, MeOH:CHCI3 <B¼ E (275 mg) KL" ;<%É2(Fig. 1).

†Corresponding author Tel: 063-840-1265 Fax: 063-840-1269

E-mail: [email protected]

 Cytotoxicity on Cancer cells of the Extract of Sophora flavescens Ait.

2.  

Æ+6 _* Ê_ 4^oC ˄&" †5%É&, Ì< (4"

10% DMSO" Í­%f KL" Ì<%É2.

3.    

Z[” Β e› Ì<6
 pÏ !8PÁ

(KB, ATCC OCL-17), 9Ђ\
(HeLa, KCLB 1002), 8{
(MCF-7, KCLB 30022)7 mouse Ñ«U

(B16, KCLB 8006): Ì<%É2.

4.   

ÒE" Ì<¼ Ò L-glutamine; Ó¼ DMEM (Dulbecco's Modified Eagle's Medium, Sigma, USA) RPMI 1640(Gibco, USA)" NaHCO3 ÔÕ6 ³, 3l V"

kp 2Ö membrane filter(0.2µm) f6 ³, f×" 56

oC"b 30 š inactivation _» J Q' ØÙ FBS: 4Ï E 1% G#Ú ÔÕ6 2Ö, 1N NaOH7 1N HCl Ì

<%f pH 7.2 G#Ú %É2.

5.  

s[ Β% e%f pÏ HeLa , MCF-7 

7 mouse B16  trypsin-EDTA(0.05% trypsin, 0.53

mM EDTA, Gibco, USA) <× ÛB%f FBS(Fetal Bovine Serum, Gibco, USA) Ó¼ DMEM"b ÒE%

Éi, KB  RPMI 1640" 10% FBS7 Penicillin G(25 unit/ml) Ü%f ÒE%É2. Ý  ÒEÞ#

37^oC, ß# 95%, 5% CO2 ÒE"b ÒE%É2.

6. MTT 

 ÒE¼ Ý well" MTT(3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide, Sigma, USA) Ó¼ ÒE× well à 1 ml á ² 4_š Åâ CO2 ÒE"b ÒE ³ ãä ’_å e%f ÒE× Ŭ ãä’

×(Isopropanol <›6 0.04 N HCl) ²& MTT formazan

 <›6 ³ ELISA(Spectra Max 250, Molecula Devices)

" 6 æç„ 570 nm"b æè#: Β%f éÆê

ëì ­%É2.

7. 

KLí ŸNÛB Ý " é6 _* î#ï l;

: ð% e%f ñ ò ­(one-way ANOVA) _ó%

É2. 86 l; 1  Duncan 2Èôe ª’ Ì

³ª’ %Éi, 8# õ 0.05 %É&, ŸN ­

SPSS Win Ì<%É2. IC50" 56 ŸNÛB Student's


 Extraction and fractionation of S. flavescens Ait.

a) ethyl acetate, ^b) hexane

t-test: ;<%É&, p-value 0.05 ;% ‚ƒ 86 ö

 ÷’%É2.

 

1.   ! "#$ %& '( )* +,-.

/ (KB) 0 

&‡ "¦ 'y;§ J Â KB " é6 Z[

 çø% e6 MTT ’¬ ­T" 6 KLí Table 1 ù2. KL í, úû î#"b ŸNa 86 l;

 ]ü2(p<0.001). î#ï ̳ ª’6 í, &‡ "¦

'y;§ J Â î# 6.25µg/ml \ý KB  [

„ …+%Éi, 100µg/ml î#þ _* î#  ÿÚ KB  [„…+I dF ]ü&, ŸNa

86 l; 1Ä2(p<0.05). _* î# 100µg/ml"b

 KB " é6 Z[; „ dF(76.1%) ]ü2.

_*: 0.0, 6.25, 12.5, 25, 50 > 100µg/ml î# ÛB 6 í KB " é6 3„
 éÆê ëìÿ  ëì

 ÝÝ 100.0, 86.5, 75.2, 71.4, 49.7 > 23.9% ]

ü2. Ê, &‡ "¦ 'y;§ J Â KB " é6

[„ …+
 _*î# 6.25, 12.5, 25, 50 > 100µg/

ml"b ÝÝ 13.5, 24.8, 28.6, 50.3 > 76.1% ]ü2.

&‡ "¦ 'y;§ J Â úû î#"b KB " é

%f 86 Z[I ]üi, î# ÿÚ pÏ !8PÁ
U " é6 Z[ dF ]ü&, IC₅₀ 56.58µg/ml î# 5®GÄ2.

2.   ! "#$ %& 123 -456/

(B16) 0 

&‡ "¦ 'y;§ J Â B16 " é6 Z[

 çø% e6 MTT ’¬ ­T" 6 KLí Table 27 ù2. KLí, úû î#"b ŸNa 86 l;

]ü2(p<0.001). î#ï ̳ª’6 í, &‡ "¦ '

y;§ J Â î# 6.25µg/ml \ý B16  [„

 …+%Éi, 100µg/ml î#þ _* î# ÿ

Ú B16  [„…+I ]ü&, 86 l; 1 Ä2(p<0.05). _* î# 100µg/ml"b B16 "

é6 =
s[(69.5%); „ dF ]üH, KB "

é6 Z[(76.1%)†2 F ]ü2. _*: 0.0, 6.25, 12.5, 25, 50 > 100µg/ml: ÛB6 í B16 " é 6 [„
 éÆê ëìÿ , ëì
 ÝÝ 100.0, 95.4, 74.4, 61.0, 40.9 > 30.5% ]ü2. Ê, &‡ "¦ '

y;§ J Â B16 " é6 Z[ _* î# 6.25, 12.5, 25, 50 > 100µg/ml"b ÝÝ 4.7, 25.6, 39.0, 59.1

> 69.48% ¿ƒx B16  [„ …+%É2. &‡

"¦ 'y;§ J Â úû î#"b B16 " é6 86 Z[; ]üi, î# ÿÚ Z[

dF ]ü&, IC50 65.43µg /ml î# 5®GÄ2.

3.   ! "#$ %& '( 7894.

(HeLa) 0 

&‡ "¦ 'y;§ J Â HeLa " é6 Z [ çø% e6 MTT ’¬ ­T" 6 KLí

Table 3 ù2. KLí, úû î#"b ŸNa 86 l; ]ü2(p<0.001). î#ï ̳ª’6 í, &‡

"¦'y;§ J Â î# 6.25µg/ml \ý HeLa 

 [„ …+%Éi, 100µg/ml î#þ _* î#

ÿÚ  [„…+I ]ü2. _* î#

100µg/ml"b HeLa " é6 Z[(50.4%); „ dF ]üH, KB " é6 Z[(76.1%); ¿ƒ

x B16  Z[(69.5%)†2 F ]ü2.

_*: 0.0, 6.25, 12.5, 25, 50 > 100µg /ml î#

ÛB6 í HeLa " é6 [„
 éÆê ëìÿ , ëì
 ÝÝ 100.0, 88.9, 86.6, 72.6, 56.1 > 49.6%

]ü2. Ê, &‡ "¦ 'y;§ J Â HeLa "

é6 [„ …+
 _* î# 6.25, 12.5, 25, 50 >

100µg/ml Ü_ ÝÝ 11.1, 13.4, 27.4, 43.9 > 50.4%

 
 The Cytotoxicity of the ethyl acetate soluble subfractions of Sophora flavescens Ait. by the MTT assay on human KB cells

Cell KB Cells

Concentration (µg / ml) Mean
S.D.¹ % of control

Control 0.318
0.034 100.0

6.25 0.275
0.022^a 86.5

12.5 0.239
0.011^b 75.2

25 0.227
0.015^b 71.4

50 0.158
0.006^c 49.7

100 0.076
0.006^d 23.9

F 65.081

p 0.000

IC₅₀ 56.59

Cells were incubated for 48 hrs. The cells were harvested with trypsin- EDTA. ¹The values represent the mean
standard deviations for quad- riplicates experiments. Means followed by the same letters are not sig- nificantly different at the 5% level by Duncan's multiple range test.

F : F ratio, p : probability

   The Cytotoxicity of the ethyl acetate soluble subfractions of Sophora flavescens Ait. by the MTT assay on mouse B16 cells

Cell B16 Cells

Concentration (µg/ml) Mean
S.D.¹ % of control

Control 0.403
0.012 100.0

6.25 0.384
0.007^a 95.3

12.5 0.300
0.016^b 74.4

25 0.246
0.007^c 61.0

50 0.165
0.021^d 40.9

100 0.124
0.008^e 30.5

F 200.870

p 0.000

IC50 65.43

Cells were incubated for 48 hrs. The cells were harvested with trypsin- EDTA. ¹The values represent the mean
standard deviations for quad- riplicates experiments. Means followed by the same letters are not sig- nificantly different at the 5% level by Duncan's multiple range test.

F : F ratio, p : probability

Cytotoxicity on Cancer cells of the Extract of Sophora flavescens Ait.

 ]ü2. &‡ "¦ 'y;§ J Â úû î#"b HeLa " é6 86 Z[; ]üi, î#  ÿÚ Z[ dF ]ü&, IC50 83.95µg/ml î

# 5®GÄ2.

4.   ! "#$ %& '( +:.

(MCF-7) 0 

&‡ "¦ 'y;§ J Â MCF-7 " é6 

Z[ çø% e6 MTT ’¬ ­T" 6 KLí

Table 47 ù2. KLí, úû î#"b ŸNa 86 l; ]ü2(p<0.05). î#ï ̳ª’6 í, &‡ "

¦ 'y;§ J Â î# 6.25µg/ml \ý MCF-7 

 [„ …+%Éi, 100µg /ml þ î# ÿ

Ú  [„…+I ]ü2. _* î# 100µg/

ml"b MCF-7 " é6 Z[(41.3%); „ dF

]üH, pÏ KB " é6 Z[(76.1%), ¿ƒx B16  Z[(69.5%), pÏ HeLa " é6 

Z[(50.4%) †2 F ]ü2. _*: 6.25, 12.5, 25, 50

> 100µg/ml î# ÛB6 í MCF-7 " é6 [

„
 éÆê ëìÿ , ëì
 ÝÝ 93.1, 89.0, 83.9, 64.2 > 58.7% ]ü2. Ê, &‡ "¦ 'y;§ J Â

 MCF-7" é6 [„ …+
 î# 6.25, 12.5, 25, 50 > 100µg/ml"b ÝÝ 6.9, 11.0, 16.1, 35.8 > 41.3%

 MCF-7  [„ …+%É&, &‡ "¦ 'y;§

J Â úû î#"b MCF-7 " é6 86 Z[

; ]üi, î# ÿÚ Z[ dF ]ü&, IC₅₀ 106.6µg/ml î# 5®GÄ2.



&‡ ¨©h
" é6 Z[ çø% e%f

&‡ "¦ 'y;§ J Â MTT ’¬ ­ K_%

É2. &‡ "¦ 'y;§ J Â
p KB , B16 , HeLa 7 MCF-7 " é%f 6.25µg/ml î#\ý Z[; ]üi, 12.5, 25, 50 > 100µg/ml þ ÝÝ î#ï Z[ %É&, ŸNa 8

[; p’GÄ2(p<0.05). KB " é6 IC50 56.58µg/

ml, B16 " é6 IC50 65.43µg/ml, HeLa " é 6 IC50 83.95µg/ml, MCF-7 " é6 IC50 106.65 µg/ml ]ü2. &‡ "¦ 'y;§ J Â


 [„ …+%É&, Z[ # B16 , HeLa

, MCF-7 , KB  b dF ]ü2.

 

! 2002Š# ñè†ÇéC !ë ñ" %f  ó¼ í ;" ·Ì2.



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   The Cytotoxicity of the ethyl acetate soluble subfractions of Sophora flavescens Ait. by the MTT assay on HeLa cells

Cell HeLa Cells

Concentration (µg/ml) Mean
S.D.¹'' % of control

Control 0.871
0.034 100.0

6.25 0.774
0.017^a 88.9

12.5 0.754
0.035^a 86.6

25 0.632
0.012^b 72.6

50 0.489
0.015^c 56.1

100 0.432
0.053^d 49.6

F 36.641

p 0.000

IC₅₀ 83.95

Cells were incubated for 48 hrs. The cells were harvested with trypsin- EDTA. ¹The values represent the mean
standard deviations for quad- riplicates experiments. Means followed by the same letters are not sig- nificantly different at the 5% level by Duncan's multiple range test.

F : F ratio, p : probability

   The Cytotoxicity of the ethyl acetate soluble subfractions of Sophora flavescens Ait. by the MTT assay on MCF-7 cells

Cell MCF-7 cells

Concentration (µg/ml) Mean
S.D.¹ % of control

Control 0.848
0.004 100.0

6.25 0.790
0.005^a 93.5

12.5 0.755
0.011^b 89.0

25 0.712
0.010^c 83.9

50 0.545
0.011^d 64.2

100 0.498
0.009^e 58.7

F 440.120

p 0.000

IC₅₀ 106.6

Cells were incubated for 48 hrs. The cells were harvested with trypsin- EDTA. ¹The values represent the mean
standard deviations for quad- riplicates experiments. Means followed by the same letters are not sig- nificantly different at the 5% level by Duncan's multiple range test.

F : F ratio, p : probability

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11. , , , , :   !" #

$%&'() *+&, -.. / 01 45(6): 587-590, 2001.

12. 23, 456, 789: 6;: $%& *+<=(I). / 01 43(4): 419-422, 1999.

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539, 1994.

(Received May 13, 2002; Accepted June 24, 2002)