Against Cadmium Induced Cytotoxicity(VII) The Inhibitory Effects of Quercitrin from Houttuynia Cordata Quercitrin (VIII)   

Journal of the Korean Chemical Society 2003, Vol. 47, No. 2

Printed in the Republic of Korea

175



Quercitrin     (VIII)

^†
^†
^† ^‡ ^#*

  

 

†
  
3

‡
  

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(2003. 1. 28 )

The Inhibitory Effects of Quercitrin from Houttuynia Cordata Against Cadmium Induced Cytotoxicity(VII)

Jeongho Lee^†, Kinam Lee^†, Chunwoo Lee^†, Hyunja Chun^‡, Ilsoo You^#, Jina Lim, and Seunghwa Baek*

Department of Herbal Resources, Professional Graduate School of Oriental Medicine, Wonkwang University, Iksan 570-749, Korea

†Department of Third Medicine, Professional Graduate School of Oriental Medicine, Wonkwang University, Iksan 570-749, Korea

‡Division of Natural Science, Wonkwang University, Iksan 570-749, Korea

#Department of Environmental Engineering & Chemical Technology, Iksan College, Iksan 570-110, Korea (Received January 28, 200
)

: , !", quercitrin, 3T3 #$%&'(

Keywords: Houttuynia Cordata, Cadmium, Quercitrin, NIH 3T3 Fibroblasts

 

)*+ ,-
. ./01 23 45 /06 7, 8. cadmium1 *96 :;< =>? @A> B CDE FEG, H6 I4J> KL MNOPQ AR

> SOT7, ;UO, VO WXY Z[ \]O, $

O, ;O ^_ `Pa bc de3 $ f.^1-4

? g-I hi, 6j, kl, mn, ophi, gqhi, grhi SpI, st, uv-, w5, x2, ylI, z{I, |We, }~, d, €je, ‚ƒ, . /0 m2hi, uE-, „-, …-, †‡, ˆ-, ‰ n, Šc, nU, ‹LU&, c, Œe, , Ž, g

‘> ’V6 “P7,  ”. quercitrin1 ‚u

Shi, gqhi k• 6jhi6 “P7, decanoylacetaldehyde1 –— g; ’4 “?

˜PQ ™š› “f.^5-8

Flavonoid? œQ o€A(glycoside) \]Q žA

> ŸU ”(T “P7, *91 1` 1 g  ¡ ¢

£D? ¤žQ¥ g¦6§¨, g‚©ª, «„-

¬, ‚ƒ, €j RW ­ˆ, 6j, %'‚u®

 ¬, g, g-, 5, ‚ƒkD, ¯°’ ‘

> ±6 “? ˜PQ ™š› “f.⁹

 

. _ methanolQ ²:D³ n-hexane, chloroform, ethyl acetate, n-butanol, žQ ´}”µ ˜ . ethyl acetate ”µž3 silica gel3 6iD³ chloroform

 methanolQ 2+ ”U ¶ methacrylic acid polymer 4 °5· recycling prep-HPLC(SUNIL JAI)_ 6iD

Journal of the Korean Chemical Society 176   

³ ¸ ¤ž3 ”UD¹f.

Compound. Yellow needle, TLC: Rf= 0.725(CHCl3: MeOH=5:5), UV λmax(MeOH nm) 256, 350, IR max (KBr cm⁻¹) 3424(br, -OH), 1655(α β-unsaturated, C=O), 1607, 1498(aromatic C=C), 1088(C-O), ¹H-NMR(DMSO- d6, 500 MHz): δ12.64(brs, C5-OH), 7.26(1H, d, J=2.3 Hz, H-2'), 7.22(1H, dd, J=2.3, 8.3 Hz, H-6'), 6.82(1H, d, J=8.3 Hz, H-5'), 6.21(1H, s, H-8), 6.04(1H, d, J=0.7 Hz, H-6), 5.23(1H, d, J=1.0 Hz, H-1"), 3.95(1H, dd, J=1.4, 3.0 Hz, H-2"), 3.49(1H, dd, J=3.2, 9.2 Hz, H-3"), 3.22(1H, m, H-5"), 3.15(1H, t, J=9.7 Hz, H-4"), 0.81 (3H, d, J=5.4 Hz, H-6"), ¹³C-NMR(DMSO-d6, 125 MHz) 156.71(C-2), 133.78(C-3), 176.97(C-4), 160.94(C-5), 99.61(C-6), 161.10(C-7), 94.08(C-8), 156.43(C-9), 102.60(C-10), 120.39(C-1'), 115.20(C-2'), 145.35(C-3'), 148.95(C-4'), 115.31(C-5'), 120.98(C-6'), 101.71(C-1"), 70.02(C-2"), 70.45(C-3"), 71.18(C-4"), 70.30(C-5"), 17.49(C-6").

   . 0.2 M NH³-0.2 M NH4Cl(pH 8.0) i©PQ pH p¡_ $Eºa, 17»20^oC

¼¡>¥ ½ºD¹f. ¾ ¨¿ÀL Á Â…? 190

»700 nm …>¥ Á D¹P7, ¾”O ˆÃ

>  ĤA ŤÆ? ÇÆÃ3 *iD¹f.⁶⁴ Ç

¾´? Beer Ã, A= εb cPQ ´D¹f.^10-12

  

. UV/VIS spectrophotometer>¥ 256 nm, 350 nm>¥ ¾M6 ÈÉÊP7, IR spectrophotometer

>¥? 3424 cm⁻¹>¥ sugar OH4 broadDË ÈÉ ÊP7, 1655 cm⁻¹>¥ α,β-unsaturated C=O4 ÈÉÊ

, 1607 cm⁻¹, 1498 cm⁻¹>¥ aromatic C=C4 Á TÌ

, 1088 cm⁻¹>¥ glycosidic C-O4 Á TÌf.^13,14

1H-NMR spectrum>¥ 0.81(J=5.4 Hz) ppm>¥ rhamnose CH3 proton singnal6 doubletPQ ÈÉÊP7, 5.23 ppm

>¥ rhamnose anomeric proton signal6 J=1.0 HzQ doubletQ ÈÉʁ, 3.15»3.95 ppm> rhamnose> 

@D? \O@ proton signal6 uÍTÌf. AU>

¥ ÈÉÎ signal6 6.04 ppm(d, J=0.7 Hz) 6.21 ppm

>¥ bb H-6, H-8 proton signal6 H-6' proton ortho- couplingD “Ï3 ™  “f. H-8 proton6 6.21 ppm>¥ singletQ ÈÉʁ, AU oxygenated pattern 4ÐÑ¥ 4W Ò6 ÈÉÈ? 5,7-dihydroxy group>¥

6Ó proton6 8Ó proton Ôf high field>¥ uÍTÌ f. 7.26 ppm(J=2.3 Hz)>¥ proton signal6 doubletP Q H-2'4 ÈÉÊP7, BU 5', 6' proton signal6 6.82 ppm(d, J=8.3 Hz) 7.22 ppm(dd, J=2.3, 8.5 Hz)

>¥ ÈÉÈ A UÔf low field>¥ uÍTÌf.

12.64 ppm>¥ 5-OH proton signal6 ÈÉÈ? ˜3 ™

 “Ìf.^13,1413C-NMR spectrum>¥ C-36 133.8 ppm, C-2, 156.7 ppm, C-4, 177.0 ppm, C-3' 145.4 ppm, C-4', 148.9 ppm, 177.0 ppm>¥ C-4 carbonylY 101.7 ppm>¥ rhamnose anomeric carbon signal, 17.5 ppm

>¥ rhamnose C-6"4 ÈÉÊf.^15,166Õ ÅQ quercitrin(Quercetin-3-O-α-L-rhamnopyranoside)@ ˜ 3 Ö@D¹f.¹⁷

   . 0.2 M NH3-0.2 M NH4Cl(pH 8.0) i©>¥ cadmium quercitrin UV

¾ ¨¿À×1 ¾¡4 274 nm 6¶>¥ mole ratio 4 I4J> KL ¾¡? hyperchromic effect )Õ3 ÈÉØP7, ¾¡4 251 nm, 354 nm, 370 nm>¥

isosbestic point4 ÈÉÊf. ¾¡4 267 nm>¥

cadmium quercitrin6 1:1Q complexT? ˜PQ È ÉÊP7, ٞ p¡Y Ǿ´Q 6= p¡4 53.52µMŒ3 ™  “ÌP7, λ1=370 nm, λ2=270 nm 6¶>¥ ÚQÐ ¾4 ÈÉÊf (Fig. 1). Quercitrin

> cadmium3 p¡ÛQ Ü4DÝ spectra4 ^D? ˜ 3 ™  “́, 6§ spectra ^? cadmium

quercitrin„ Þ  ٞ\>  ˜PQ ;b·

Fig. 1. Absorption spectra of quercitrin (50µM) at 0.2 M NH₃-0.2 M NH₄Cl (pH 8.0) in the presence of various con- centrations of cadmium. R=[quercitrin]/[cadmium]; R=0.0(A), 0.6(B), 0.8(C), and 1.0(D).

Quercitrin    !" 177

2003, Vol. 47, No. 2

f. Cadmium quercitrin mole ratio4 1.06 TÝ

¾¡ ^4 ß4 T, mole ratio4 àáÈ â PÝ ¾¡4 âË ÈÉÊf. Mole ratio4 1.0>¥ ” ãäO ٞ3 \D? ˜PQ ;bT7, Cadmium 6¼ BU diol@ å U U„!(bidentate ligand) 4 U_ \D³ Þ  cadmium Ù¤ž3 \

D? ˜PQ æç·f(Fig. 2).^12,18-20

NIH 3T3 fibroblasts   . Mosmann

 ˆÃPQ NIH 3T3 #$%'(> !"3 èU

¶, >¥ ”U quercitrin> m NIH 3T3 #

$%'( !" 2>  Á 3 D¹f. äé

Fig. 2. The proposed structure of 1:1 complex of cadmium and quercitrin.

Table 1. The cytotoxicity of quercitrin by MTT assay against NIH 3T3 fibroblasts cell lines

Concentration (µM)

A540

Mean±S.D.^a (% of control)

Control 0.734±0.05 100.00

25.0µM 0.755±0.04 102.86

50.0µM 0.763±0.05 103.95

100.0µM 0.702±0.06 195.64

150.0µM 0.689±0.04 1 93.87

Fig. 3. The MTT absorbance of quercitrin on 3T3 fibroblasts treated with cadmium (MTT50). Cells were incubated for 48 hrs. The cells were harvested with trypsin-EDTA. aThe val- ues represent the mean±standard deviations for triplicate experiments. Significantly different from the control value;

*p<0.05, **p<0.01(Student’s t-test).

Fig. 4. (A) Inverted photomicrograph of NIH 3T3 fibroblasts treated with MTT for additional 3hrs after incubation unmodified medium (control) for 2 days ×200. Most cells had abundant cytoplasm and formed round shape, (B) Inverted photomicrograph of NIH 3T3 fibroblasts after incu- bation in the Cd₅₀ concentration for 2 days ×200. Most cells were formed round type and number of cells were decreased, (C) Inverted photomicrograph of NIH 3T3 fibroblasts after incubation in the medium containing Cd50 concentration plus 53.52µM quercitrin for 2 days ×200. Most cells were showed regenerative and number of cells were increased.

Journal of the Korean Chemical Society 178   

 ¾¡_ 100%Q D³ ê6ëQÇp¡>  ¾

¡_ ÆìOPQ Á D¹P7, IC50@ MTT501 33.04 µM3 ÈÉØf.^12,21 MTT ¾¡? !" p¡>

BD³ íŠD¹P7, !"G èUé>¥? 29.9%

 ¾¡_ Ô¹P7, 10µM quercitrin3 èU p¡>

? 51.8%Q }´O $(p<0.05)3 Ô¹P7, 25.0µ M quercitrin p¡>¥? 67.1%, 53.52µM>¥? 76.6%

 m2í’_ Ô¹, 100µM quercitrin3 èU

 p¡>¥? 74.9%Q !" m2í’4 íŠ

D? ˜PQ ÈÉØf. 6§ )Õ1 quercitrin> 

2> @· ˜PQ ;bT7(Table 1), !"

quercitrin ٞ\(1:1) p¡(53.52µM)>¥ 4W à1 m2í’ ÈÉÊf(Fig. 3).^21,22

   !". '( )îO u Í>¥? äé3 24º„ oïDÝ well ¦ð6 ñò

 ó3 ô? ˆ²\PQ çõ3 6ö7, NIH 3T3 #

$%'(÷6 ÙT “́(Fig. 4A), IC50(MTT50)

 !"3 èU é>¥? '(4 íŠD¹, ' ( \]4
\PQø ^\T? ïÕ3 ù  “Ì f(Fig. 4B). IC50p¡ !" quercitrin 53.52µM 3 èU é>¥? IC⁵⁰é> ÆD³ '(4 I4D

 C;)Õ6 ñúD¹f(Fig. 4C). û ½üÅ>  DÝ, !">  '(23 quercitrin6 ¬D?

2í’4 “? ˜PQ *ý·f.^21,22

 

(Houttuynia cordata THUNB)>¥ ”U quercitrin 3 UV-spectrophotometer_ 6iD³ 0.2 M NH3-0.2 M NH4Cl pH 8.0 i©>¥ ºýY !"„ 1:1 ٞ

\ p¡? 53.52µM p¡>¥ T? ˜PQ ÈÉ Êf. NIH 3T3 #$%'( !" 2>  C;

’ 53.52µM quercitrin p¡>¥ MTT ¾¡4 }

´OPQ $ “? m2í’_ ÈÉØP7, 

)îO Šþ>¥¡ '( C;6 ñúD¹P7,

¸ quercitrin6 !"> D³ ÿÕ· NIH 3T3

#$%'( C;’4 “? ˜PQ ÈÉÊf.

û ã? BK21* ãÆ E
PQ 
TÌP 7, 6> í* !f.

  !

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