(Nexia)

Study on the Safety and Antitumor Activity

of Rhus Verniciflua Extract (Nexia)

2006 8

(Nexia)

Study on the Safety and Antitumor Activity
of Rhus Verniciflua Extract (Nexia)

2006 8

 (Nexia)

Study on the Safety and Antitumor Activity
of Rhus Verniciflua Extract (Nexia)

2006 8

2006 8

. 1

. 3
1. (Nexia) 3
1.1. Nexia 3
1.2. Nexia 3
1.3. , 4

2. Nexia 4
2.1. Nexia 13 4 4
2.2. Nexia 5
2.3. Nexia 5

3. Nexia 6
3.1. 6
3.2. 7
3.3. 7
3.4. 8

4 Nexia 9
4.1. 9

4.2. 9
4.3. Nexia 10

. 11
1. Nexia 11
1.1. Nexia 11
1.1.1. 11
1.1.2. 12
1.1.3. 13
1.1.4. 14
1.2. Nexia 14
1.3. , 17

2. Nexia 19
2.1. Nexia 13 4 19
2.2. Nexia 26
2.3. Nexia 30

3. Nexia 33
3.1. Nexia HUVEC 33
3.2. Nexia 34
3.2.1. Nexia VEGF HUVEC 34
3.2.2. Nexia VFGF HUVEC 35
3.3. Nexia A549 36

4. Nexia 43
4.1. 1 43
4.2. 2 44
4.3. 3 45
4.4. 4 46

. 52

. 60

62
ABSTRACT 67

.

,
.

1)

. ,

.
, .
,
, ,
2)

3)

. , , ,

4)

5)

6)

, ,

, , , , , ,
7)

65.4% ,

8-9)

.
()
, ,

10)

, .

,
11)

urushiol T

.
(Rhus verniciflua STOKES)

- 1 -

 (Nexia;
0394089 /0504160) . Nexia
Nexia ,
, 4

- 2 -

1. (Nexia)
1.1. Nexia

10 15
0504160 Nexia . Nexia

.
.
, , , , ,
.

1.2. Nexia

(Nexia)
,
, ,
. Nexia High
Performance Liquid Chromatography (HPLC)
.

- 3 -

1.3. ,

(Merk Co., 70-230 mesh)

Sephadex LH-20 . NMR
VARIAN 400 (Unity Inova 400 STNMR Spectrometer, USA)
CD3OD .

2. Nexia
2.1. Nexia 13 4

Nexia 13
, 4
. GLP
( 2000-63)
( 1999-61) .
(n=10), (n=6), (n=6), (n=10)
Nexia (mg/kg) 0, 50, 150, 450 13
5 mL/kg. 450 mg/kg 4
. 50:50
.
, , , ,
, , , , ,
.

- 4 -

Levene's

test

data

transformation transformed data Levene's test

one way ANOVA test ,
Dunnett's t-test
.

2.2. Nexia

Nexia
(), , , ,

. 200400800
1,600 mg/kg 4 , 1
, . 5
mL/kg 2, 2 .

2.3. Nexia (
)

Nexia
(Active Systemic Anaphylaxis, ASA)


(Passive Cutaneous Anaphylaxis, PCA)
.
Nexia

- 5 -

, (45 mg/kg), (200 mg/kg), +FCA (200

mg/kg) (OVA 5mg/kg+FCA) 5
. 6-7 5 3
, 9
3 5
.
12
1 2
10 5120 . 4
.
15
30 .

3. Nexia
3.1.

2,3-bis [2-methoxy-4-nitro-5-sulfo]-2H-tetrazolium-5carboxanilide (XTT) .

4

(HUVEC) 0.1% gelatin 96-well 110 . 24

5% heat-inactivated fetal bovine serum(FBS) M199
100 24 . 1 XTT
(1 / in PBS) stock solution 10 PME (1.53 / in PBS) 50
well microplate reader (Molecular Device Co.) 450
nm .

- 6 -

3.2.

(HUVEC) vascular endothelial growth

factor(VEGF) XTT .
3

0.1% gelatin 96-well plate 510 . 24

5% heat-inactivated FBS M199 6
starvation. 5% heat-inactivated FBS, 10 ng/ VEGF 5 units/ heparin
M199 Nexia 100 48
. 1 XTT (1 / in PBS) stock solution 10 PME (1.53
/ in PBS) 50 well microplate reader
(Molecular Device Co.) 450 nm
.

3.3.

wound healing assay .

5

0.1% gelatin 6-well plate 310

. 24 6-well yellow tip . 5%
heat-inactivated FBS, 10 ng/ VEGF 5 units/ heparin M199
Nexia 100 17 . 17
PBS washing . Fixing solution
Diff-quick yellow tip
.

- 7 -

3.4.

A549

5x10

Immnunodeficient mice(male), CanN. Cg-Foxn 1nu/CrljBgi ) ,

, , ,

(gemcitabine) , 7
. Nexia 50 mg/kg, 150 mg/kg, 300 mg/kg 24
, 100 mg/kg 1 8 15
. 1 2 caliper
, 27
.
ANOVA Student's t-test
5% .

: V(mean tumor volume, mm )=AB /2

- 8 -

(A=, B=)

4. Nexia
4.1.

1997 3 2001 5

(complete remission) 4
.

, , , ,

4.2.

(Korea Clean Cancer Association) . Lost follow up
,

(Kwanghyewon
oriental medicine hospital call back system).
2005 3 2
(current status)
2006 3 22 .

- 9 -

4.3. (Nexia)

Nexia 1 3
. Nexia .
Nexia 1 150mg 2
(KHW) 300 1 450mg
1(DX) 300 .
.
Nexia , ,
, , , , , ,
.

- 10 -

.
1. Nexia
1.1. Nexia

1.1.1.

scale-up
.
.
. (chip shape)
(sawdust shape) .
2% 8.1% 4
.
90~95 , 10 , 6
.

.
.

2
. 1 80%, 2
20% . 3
. 2

- 11 -

1.1.2.

. 40, 60, 80, 100

. Fig. 1
80~100
4 .

1.8
1.6

Abs(500nm)

1.4
1.2
1
0.8
0.6

40
60
80
100

0.4
0.2
0
0

10

Extraction Time(hrs)

Fig 1. Extraction profile at different extraction temperature.

- 12 -

1.1.3.

.

80%(v/v) . Fig. 2
40% 60%
60% . 100%

.

20%EtOH
40%EtOH
60%EtOH
80%EtOH
100%EtOH
80%MeOH

4
3.5

Abs(500nm)

3
2.5
2
1.5
1
0.5
0
0

10

Extraction Time(hrs)

Fig. 2. Extraction profile at different ratio of BuOH

- 13 -

12

1.1.4.

810
.

.
.
8.1%(w/w) 60%
9.8%(w/w) . 90~95, 10, 2,
1, 6,
60% .

1.2. Nexia

Nexia Fig. 3 (A),

CHCl3 (B), Ethylacetate (C), n-butanol (D), (E)
.
37.2% .

HPLC Fig. 4 ,

fustin
fisetin, sulfuretin, butein . HPLC

.
.
Nexia

urushiol, fustin, fisetin, sulfuretin butein

- 14 -

 HPLC .

HPLC Shimadzu LC-10A w/ two pumps and autoinjector

, YMC-pack ODS-A, S-5, 12nm, 250x4.6mm I.D. column

,

20

40

urushiol

mobile

phase

methanol:water=85:15, flow rate 1.2ml/min , flavonoid mobile

phase phosphate buffer20mM(pH=3):methanol:THF=5:3:2methanol:
water=85:15, flow rate 1.0ml/min .

Fig. 3. Schematic diagram of fractionation of Nexia.

- 15 -

a) HPLC chromatogram of A.

b) HPLC chromatogram of B.

c) HPLC chromatogram of ethylacetate fraction, C.

- 16 -

d) HPLC chromatogram of n-butanol fraction, D.

e) HPLC chromatogram of water fraction, E.

Fig. 4. HPLC pattern of each slovent fraction of Nexia

1.3. ,

Fig. 5 RVC-4-1 RVC-2-2-3-1 1H, 13C, NMR

RVC-4-1 2,4-dihydroxybenzaldehyde, RVC-2-2-3-1 sinapyl
aldehyde . ethylacetate
fustin, fisten, sulfuretin .
. HPLC
95%

- 17 -

Fig. 5. Isolation scheme of effcetive compounds from CHCl3 fraction of Nexia

- 18 -

2. Nexia
2.1. Nexia 13 4

Nexia 13
, 4
. GLP
( 2000-63)
( 1999-61) .
(n=10), (n=6), (n=6), (n=10)
(mg/kg) 0, 50, 150, 450 13 .
450 mg/kg 4 .
50:50 .
86 450 mg/kg 1

. .

, 50 150 mg/kg
,
150 mg/kg 450 mg/kg
.
. 450 mg/kg 1
.
, ,

, leukocyte ,

- 19 -

 ,
,
.
ALT, Glu, T-Chol, Na, GGT
APTT ,
.
50 mg/kg 1
, 50 150 mg/kg

, 1
,

. 50 mg/kg
, (Table
1).
450 mg/kg 3
2
. ,
(transit time)
, .

. 4 450 mg/kg
2 1 50%
, .
, , , , ,
(background

- 20 -

lesion) (Table 2).

, Nexia 13 450
mg/kg ,
, 150 450 mg/kg
, (NOAEL) 150 mg/kg,
50 mg/kg (MTD)
450 mg/kg . 4 , 1
,
.

- 21 -

Table 1. Necropsy findings(main test)

Organs

Abdominal
cavity
Adrenals

Aorta

Brain

Esophagus

Eyes

Heart

Kidneys
Large
Intestine
Liver with
gall bladder
Lungs

Sex
Group
Dose(mg/kg)
Number of
animals
No. of examined
Not Remarkable
Remarkable
No. of examined
Not Remarkable
Remarkable
No. of examined
Not Remarkable
Remarkable
No. of examined
Not Remarkable
Remarkable
No. of examined
Not Remarkable
Remarkable
No. of examined
Not Remarkable
Remarkable
No. of examined
Not Remarkable
Remarkable
No. of examined
Not Remarkable
Remarkable
No. of examined
Not Remarkable
Remarkable
No. of examined
Not Remarkable
Remarkable
No. of examined
Not Remarkable

G1
0

Male
G2
G3
50
100

G4
450

G1
0

Female
G2
G3
50
100

G4
450

3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3

3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3

3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3

3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
2

3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3

3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3

3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3

3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3

- 22 -

Remarkable
-Dark-red,
diffuse, severe
No. of examined
Pancreas
Not Remarkable
Remarkable
No. of examined
Skin
Not Remarkable
Remarkable
No. of examined
Small intestine Not Remarkable
Remarkable
No. of examined
Spinal cord
Not Remarkable
Remarkable
No. of examined
Spleen
Not Remarkable
Remarkable
No. of examined
Stomach
Not Remarkable
Remarkable
No. of examined
Not Remarkable
Testis/Ovaries
Remarkable
-Small, bilateral

3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
0

3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
2
1
1

3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
0

3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
0

3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
0

3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
0

3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
0

3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
3
3
0
0

- 23 -

Table 2. Necropsy findings(recovery test)

Organs

Sex
Group
Dose(mg/kg)
Number of

animals
No.
of
examined
Abdominal
Not Remarkable
cavity
Remarkable
No. of examined
Adrenals
Not Remarkable
Remarkable
No. of examined
Aorta
Not Remarkable
Remarkable
No. of examined
Brain
Not Remarkable
Remarkable
No. of examined
Esophagus
Not Remarkable
Remarkable
No. of examined
Eyes
Not Remarkable
Remarkable
No. of examined
Heart
Not Remarkable
Remarkable
No. of examined
Kidneys
Not Remarkable
Remarkable
No. of examined
Large Intestine Not Remarkable
Remarkable
No. of examined
Liver with
Not Remarkable
gall bladder
Remarkable
No. of examined
Lungs
Not Remarkable

Male

Female

G1
0

G4
450

G1
0

G4
450

2
2
0
2
2
0
2
2
0
2
2
0
2
2
0
2
2
0
2
2
0
2
2
0
2
2
0
2
2
0
2
2

2
2
0
2
2
0
2
2
0
2
2
0
2
2
0
2
2
0
2
2
0
2
2
0
2
2
0
2
2
0
2
1

2
2
0
2
2
0
2
2
0
2
2
0
2
2
0
2
2
0
2
2
0
2
2
0
2
2
0
2
2
0
2
2

2
2
0
2
2
0
2
2
0
2
2
0
2
2
0
2
2
0
2
2
0
2
2
0
2
2
0
2
2
0
2
2

- 24 -

Remarkable
-Dark-red,

Pancreas

Skin

Small intestine

Spinal cord

Spleen

Stomach

Testis/Ovaries

diffuse, severe
No. of examined
Not Remarkable
Remarkable
No. of examined
Not Remarkable
Remarkable
No. of examined
Not Remarkable
Remarkable
No. of examined
Not Remarkable
Remarkable
No. of examined
Not Remarkable
Remarkable
No. of examined
Not Remarkable
Remarkable
No. of examined
Not Remarkable
Remarkable

2
2
0
2
2
0
2
2
0
2
2
0
2
2
0
2
2
0
2
2
0

2
2
0
2
2
0
2
2
0
2
2
0
2
2
0
2
2
0
2
2
0

2
2
0
2
2
0
2
2
0
2
2
0
2
2
0
2
2
0
2
2
0

2
2
0
2
2
0
2
2
0
2
2
0
2
2
0
2
2
0
2
2
0

- 25 -

2.2. Nexia

Nexia
4
2004008001,600 mg/kg 4
, 1 ,
, , , , , ,
.

(Table 3)
1,600 mg/kg 1
1 .

(Table 4, 5) ,
.
, 1

.


.
, Nexia
1,600 mg/kg

1,600 mg/kg 800 mg/kg .

- 26 -

Table 3. Mortality
Group
G1
G2

Dose(mg/kg)

Sex

No. of animals No. of death

Mortality

Male

(dead/total)
0%(0/2)

Female

0%(0/2)

200400

Male

0%(0/2)

8001600

Female

0%(0/2)

Control

- 27 -

Table 4. Changes of body weight

Sex : male
Group/

(kg)
Animal

Dose(m
g/kg)
G1
00
00

G2
200
400
800
1,600

ID
1M01
1M02
Mean
S.D.
N
2M03
2M04
Mean
S.D.
N

Treatment period (day)

0

12

16

22

9.54
10.37
9.96
0.59
2
10.37
9.72
10.05
0.46
2

9.93
10.96
10.45
0.73
2
10.56
10.09
10.33
0.33
2

10.02
10.65
10.34
0.45
2
10.52
9.97
10.25
0.39
2

10.10
10.52
10.31
0.30
2
10.87
10.32
10.60
0.39
2

10.33
10.94
10.64
0.43
2
10.60
10.26
10.43
0.24
2

10.34
11.35
10.85
0.71
2
10.68
10.34
10.51
0.24
2

Sex : female
Group/Dose
(mg/kg)
G1
00
00

G2
200
400
800
1,600

(kg)
Animal
ID
1F01
1F02
Mean
S.D.
N
2F03
2F04
Mean
S.D.
N

0
9.61
9.54
9.58
0.05
2
9.35
9.77
9.56
0.30
2

Treatment period (day)

4
8
12
9.70
9.85
9.95
9.62
9.94
10.14
9.66
9.90
10.05
0.06
0.60
0.13
2
2
2
9.60
9.63
9.88
9.95
9.95
10.12
9.78
9.79
10.00
0.25
0.23
0.17
2
2
2

- 28 -

16
10.20
10.30
10.25
0.07
2
9.75
10.20
9.98
0.32
2

22
10.32
11.56
10.44
0.17
2
9.74
10.25
10.00
0.36
2

Table 5. Food consumption

Sex : male
Group/Dose
(mg/kg)
G1
Control

G2
200400
8001,600

(g/day)
Animal
ID
1M01
1M02
Mean
S.D.
N
2M03
2M04
Mean
S.D.
N

0
300
300
300
0
2
300
300
300
0
2

4
300
300
300
0
2
300
300
300
0
2

Treatment period (day)

8
12
16
300
300
300
300
300
300
300
300
300
0
0
0
2
2
2
300
300
300
300
300
300
300
300
300
0
0
0
2
2
2

Sex : female
Group/Dose
(mg/kg)
G1
Control

G2
200400
8001,600

20
300
300
300
0
2
300
300
300
0
2

21
300
300
300
0
2
300
300
300
0
2

(g/day)
Animal
ID
1M01
1M02
Mean
S.D.
N
2M03
2M04
Mean
S.D.
N

0
300
300
300
0
2
300
300
300
0
2

4
300
300
300
0
2
300
300
300
0
2

- 29 -

Treatment period (day)

8
12
16
300
300
300
300
300
300
300
300
300
0
0
0
2
2
2
300
300
300
300
300
300
300
300
300
0
0
0
2
2
2

20
300
300
300
0
2
300
300
300
0
2

21
300
300
300
0
2
300
300
300
0
2

2.3. Nexia (
)

, (45mg/kg)
(200mg/kg) 3 9 , (200mg/kg)
(5mg/kg) 3 3 ,
, 15 30
.
12
,

.

15 . ,
,
.
3
(Table 6).
,
,
2560 (Table 7).
,
1 .
.
, Nexia
, ,

- 30 -

Table 6. Active systemic anaphylactic(ASA) symptoms

G1
Saline

G2
Nexia

G3
Nexia

G4
Nexia

G5
OVA

45

200

200

10

Asymptomatic

Restlessness

Piloerection

Tremor
Rubbing or

Symptom

Group
Test item
Challenge dose
(mg/kg)
Number of animal

licking nose
Sneezing

Coughing

Hyperpnea

Urination

Evaculation

Lacrimation

Dyspnea

Rhonchus

Cyanosis

Staggering gait

Jumping
Gasping

and Writhing
Side position
Chene-Stokes
respiration

- 31 -

Death
FCA : Freund's Complete Adjuvant

OVA : Ovalbumin

Table 7. Passive cutaneous anaphylaxis(PCA) test

Group/

Dilution factor of antiserum

Test

Sensitization Challenge item

Dose
Dose
(mg/kg)

(mg/kg)

G1

G6

G2

G7

45

200

G3

G8

200

G4

G9

200+FCA

200

G5

G10

5+FCA

10

Number
of animal
0 5120 2560 1280 640 320 160 80 40 20 10

Saline

Nexia

Nexia

Nexia

OVA

FCA : Freund's Complete Adjuvant

OVA : Ovalbumin

- 32 -

3. Nexia
3.1. Nexia HUVEC

In vitro Nexia 50 %
(concentration of 50 % inhibition ; IC50) A549 68.8 /ml, K562 81.0
/ml, KATO 77.3 /ml, SK-OV-3 165.2 /ml ,
HUVEC 200 /ml
(Fig. 6).

140
Nexia
IC50

120

% of viability

100
80
60
40
20
0

100

200

300

400

500

600

Concentration (ug/ml)

Fig. 6. Effect of Nexia on the cytotoxicity against HUVEC

- 33 -

3.2. Nexia

3.2.1. Nexia VEGF HUVEC

HUVEC VEGF

66%

Nexia 120/ml
(Fig. 7).

150

% of viability

125

###

100

***

75

50

25

VEGF
Nexia(g/ml)

+
-

+
15

+
30

+
60

+
120

Fig. 7. Effect of Nexia on the proliferation of HUVEC stimulated by VEGF

### p<0.001 compared with negative control, *** p<0.001 compared with VEGF.

- 34 -

3.2.2. Nexia VFGF HUVEC (migration )

HUVEC VEGF window scrape . Fig. 8

window scrape VEGF
Nexia 50/ml

100/ml HUVEC

120

C e ll m ig r a t io n
( % o f c o n t r o l)

100

80

60

40

20

VEGF
Nexia (g/ml)

+
-

(A)

+
50

+
100

(B)

Fig. 8. Effect of Nexia on the migratory activity of VEGF stimulated HUVEC

(A) Photographs of migrated cells in window scraped field; Left upper (Negative
control),

Right

upper(VEGF

Lower(VEGF+100/ml Nexia)

alone),

Left

Lower(VEGF+50/ml

Nexia),

Right

(B) Graphs of effect of Nexia on the migratory

activity. # p<0.001 compared with negative control, * p<0.001 compared with VEGF.

- 35 -

3.3. Nexia A549

Nexia
(A549) (xenograft model) ,
.
(non-small lung cancer cell)
A549 ,
.
(Table 8, 10).

, , ,

(gemcitabine) , 7
. 50 mg/kg, 150 mg/kg, 300 mg/kg 24
, 100 mg/kg 1 8
15 . 27 613.98mm
3

536.53mm , 570.80mm , 364.18mm 449.58 mm

12.6%, 7.0%, 40.7% 26.8 % (Fig. 9, Table
9, 11).
(p>0.05)
.
gemcitabine
.
1, 8 15 25
.

. Nexia
(300 mg/kg) .

- 36 -

1200

1000

mm3

800

600

400

200

0
Control

NEX 50

NEX150

NEX300

GEM100

Fig. 9. Inhibitory effect of Nexia on the growth of A549 cells in athymic nude
mice

- 37 -

- 38 -

Table 8. Change of body weight

(g)

Day after administration

Group/
Dose(mg/kg)

11

14

18

21

25

28

G1

Mean 26.07

26.09

25.85

26.44

26.58

26.71

26.74

26.87

26.80

S.D.

1.97

2.47

2.45

2.24

2.20

2.09

2.16

1.89

1.86

G2

Mean 24.80

24.74

24.78

25.16

25.86

25.85

26.13

26.54

26.47

50

S.D.

1.32

1.35

1.31

1.54

1.36

1.42

1.26

1.21

1.13

G3

Mean 25.52

25.47

25.08

25.13

25.51

26.07

26.37

26.72

26.76

150

S.D.

1.53

1.61

2.56

3.03

2.55

1.95

1.77

1.91

1.65

G4

Mean 25.85

25.87

25.44

26.05

26.19

26.00

26.54

26.72

27.12

300

S.D.

1.00

1.05

1.17

1.19

1.04

0.95

0.97

1.16

1.11

25.55

25.94

26.61

26.79

26.57

27.27

27.62

27.94

1.54

1.59

1.30

1.39

0.95

1.27

1.29

1.33

1.29

G5

Mean 26.11

100
S.D.
(Gemci
N
tabine)

N : Number of animal

- 39 -

Table 9. Response of tumor volume to treatment with test item and gemcitabine
3

(mm )

Day after administration

Group/
Dose(mg/kg)

11

14

18

21

25

28

G1

Mean 78.14

150.73 199.18 285.33 342.41 444.06 484.97 531.39 588.68

S.D.

42.32

105.98 114.25 187.64 225.58 289.29 281.26 299.96 341.22

G2

Mean 77.76

143.94 194.54 233.89 283.41 403.71 415.54 488.65 527.43

50

S.D.

42.89

83.83

121.81 143.70 166.54 247.99 282.30 315.51 321.86

G3

Mean 78.00

133.65 222.90 256.59 313.70 479.90 427.68 616.81 671.48

150

S.D.

42.10

55.84

90.84

109.61 134.21 209.68 87.34

227.34 215.90

G4

Mean 74.67

108.86 179.92 227.40 242.34 350.94 343.15 431.78 509.45

300

S.D.

44.41

82.28

129.26 144.20 165.78 180.44 180.00 262.52 282.78

G5

Mean 77.47

100
S.D.
(Gemci
N
tabine)

100.40 164.37 209.33 264.11 328.01 367.82 526.88 639.84

42.26

75.93

98.30

146.49 174.12 162.03 181.94 307.61 350.43

N: Number of animal

- 40 -

Table 10. Tumor weight of sacrificed mice

Group/
Dose (mg/kg)
G1
Mean
0
S.D.
N
G2
Mean
50
S.D.
N
G3
Mean
150
S.D.
N
G4
Mean
300
S.D.
N
G5
Mean
100
S.D.
(Gemcitabine) N

Tumor weight
0.398
0.216
7
0.394
0.238
7
0.450
0.145
7
0.342
0.160
7
0.380
0.147
7

(g)

% Inhibitiona)
0.0

1.0

-13.1

14.1

4.5

N: Number of animal
a) % Inhibition : (1-mean tumor weight of test item group/mean tumor weight of
vehicle group)100

- 41 -

Table 11. Mean tumor volume of sacrificed mice

Group/
Dose (mg/kg)
G1
Mean
0
S.D.
N
G2
Mean
50
S.D.
N
G3
Mean
150
S.D.
N
G4
Mean
300
S.D.
N
G5
Mean
100
S.D.
(Gemcitabine) N

Tumor volume
613.98
358.99
7
536.53
318.04
7
570.80
197.43
7
364.18
162.24
7
449.58
187.67
7

(mm )

% Inhibitiona)
0.0

12.6

7.0

40.7

26.8

N: Number of animal
a) % Inhibition : (1-mean tumor volume of test item group/mean tumor volume of
vehicle group)100

- 42 -

4. Nexia
4.1. 1

59 1998 2 1 S
type pre B-cell.
. 98 2 1 S 98 10
15 8 , , (PBSC)
. 98 2 16 98 4 8
idarubicin, VPL
, , steroid induced DM, . 98 3
26 98 4 8 (Prophylactic Cranial
Irradiation) 8 . 98 3 28
98 4 6 BUN/CR (40/1.3 mg/dl)
. 98 5 16 5 20 1 98 6 5
(PBSC) 1 98 7 22 10 15
3 . 98 7 21 20% CR, 98 10
5 30% CR .
99 4 24
. Nexia 99 5 14 2000
5 24 1 10 50mg/ 1 3 . ,
, ,
.
. 2004 8 10 WBC
3

6.48(10 /) RBC 3.32(10 /) Hb 10.7(g/dL) Hct 33(%) PLT 157(10 /)

. 1

- 43 -

 9 2 .

4.2. 2

9 1999 5 3 P
L1 type . , ,
.

99

vincristine, MTX, mercaptopurine, asparaginase

99 6 1 1 . 99 5 10
urine culture 99 5 21
(pancytopenia) (neutropenia) 99 7
99 7 5
.
mercaptopurine steroid

.
99 7 5 2000 2 Nexia 150mg/ 1 3

2000

daunorubicin,

vincristine, MTX, asparaginase, steroid 2000

3 30 2 . vincristine, MTX,
mercaptopurine 2004 4 Nexia
.
. Nexia ,
, .
3

2000
6

2 7 S WBC 12.7(10 /) RBC 4.17(10 /)

3

Hb 11.7(g/dL) Hct 35(%) PLT 139(10 /) 20004 8 18

- 44 -

WBC 8.18(10 /) RBC 4.09(10 /) Hb 11.5(g/dL) Hct

3

35.1(%) PLT 272(10 /) .

2 5 1
.

4.3. 3

15 96 11 13 S
L1, biphenotype .
. 96 11 97 3 5 ,
. 96 11 13 WBC
3

3.4(10 /) ANC 11 Hb 7.9(g/dL) PLT 177(10 /) 96 11 16

96 12 14 vincristine, daunorubicin, prednisolone, MTX
96 12 11 . 96
12 20 97 1 3 MTX 4 CNS prophylaxis
2 180cGy 1800cGY . 97 1
8

97

12

vincristine,

asparaginase,

cyclophosphamide,

dexamethason, prednisolone
. 97 3 20

WBC 0.9 Hb 11.2

PLT 73 97
3 31 .
Nexia 97 3 31 2000 5 10 3 1 10 150mg/
1 3 .
2002 7 15

WBC

6.6(10 /)

RBC

4.7(10 /)

Hb

309(10 /) .

- 45 -

13.3(g/dL)

Hct

40.2(%)

PLT

 1 8 3
.

4.4. 4

9 99 1 18 S ,
L2 type . 98 12 SG
. 99 1
99 10 10 , .
99 1 20 cyclophosphamide, daunorubicin, dexamethasone, MTX
99 2 23 .
99 3 17 99 10 14 99 5
27 99 6 9 (Prophylactic Cranial Irradiation) 10
. 99 3 6 S WBC
3

0.19(10 /) Hb 12.0(g/dL) PLT 109(10 /) AST 64(IU/L) ALT 128(IU/L)

.
99 3 23
, ,
. Nexia 99 3 23
2000 5 24 1 2 50 mg/ 1 3 .
99 10 28
Nexia . , ,
, . 2004 8 18
3

WBC 10.3(10 /) RBC 5.21(10 /) Hb 13.7(g/dL) Hct

3

40.6(%) PLT 408(10 /) .

1 6

- 46 -

- 47 -

Table 12. Clinical features and therapy history of the acute leukemia
No. Name

Sex
Age

Dx.

Type

First Dx.

First Dx.
hospital

Chemotherapy

Radiation Stem cell

PCI

PBSC

8 months

None

2 months

PCI

None

5 months

CCR

PCI

None

10 months

CCR

PYJ

F/59 ALL

Pre-B 98/2/1

S. Hospital

IDA, VPL, MTZ, ARA,

VM21, MTX

SKH

M/9

ALL

L1

99/5/3

P. Hospital

VCR, MTX, 6-MP, ARA,

None
ASP, LON

KKB

F/15 ALL

L1

VCR, DX, ARA, MTX,

96/11/13 S. Hospital ASP, LON, CPV,
C-VMA, AR10

KGH

M/9

L2

99/1/18

S. Hospital

CPV, CRV, ARA, DX,

VCR, MTX, ASP, AR10

Relapse

transplantation treatment*

ALL

Western medicine

therapy

CCR
Relapse,
2nd CR

UK=Unknown; AML=Acute Myeloid Leukemia; ALL=Acute Lymphoblastic Leukemia; ARA=daunorubicin; 6-MP=6-mercaptopurine;

MTX=methotrexate; VCR=vincristine; ASP=asparaginase; DX=dexamethasone; CPV=cyclophosphamide; PCI=prophylactic cranial
irradiation; IDA=idarubicin; LON=prednisolone; VPA=vepesid; AR10=cytarabine; PBSC=peripheral blood stem cell; CR=complete
remission; CCR=continuous complete remission.
* : Western medicine therapies including remission induction chemotherapy, consolidation chemotherapy, intensification
chemotherapy, prophylaxis cranial irradiation and stem cell transplantation.

- 48 -

Table 13. Immunophenotypic markers and cytogenetic analysis of the acute leukemia
No. Name

Sex
Age

Dx.

Subtype

Immunophenotypic markers

Cytogenetics
46 XX t(1:19)(1p19q:1q19p)[10]

PYJ

SKH

F/59 ALL

M/9

ALL

KKB

F/15 ALL

KGH

M/9

ALL

Pre-B cell

L1

CD10(+) CD19(+) CD22(-) CD13(-) HLA-DR(+)

CD45(+) CD34(+) CD10(+) CD19(+) HLA-DR(+)

sIg(-)

46 XX t(1:19)(1p19q:1q19p)
t(7:21)(p22:q22)[7]/46XX[12]
hyperdiploid
46XY
56XY +X +4 +6 +9 +10 +14 +17
+18 +21 +21

L1,

CD2(+) CD3(-) CD10(+) CD15(-) CD19(+) CD20(-)

biphenotype

CD5(-)

L2

CD2(-) CD10(+) CD19(+) CD20(-) CD24(-)

UK=Unknown; CD=cluster of differentiation; HLA-DR=human leukocyte antigen D-related

- 49 -

UK

UK

Table 14. Side effects of chemotherapy in the acute leukemia

No. Name

Sex/
Age

Dx.

Treatment
ChemoTx

PYJ

F/59

ALL

PCI
PBSC

SKH

M/9

ALL

KKB

F/15

ALL

KGH

M/9

ALL

ChemoTx
ChemoTx
PCI
ChemoTx
PCI

Side effects

Changed treatment plan

pulmonary edema, ARF, steroid induced DM, oral

candidasis
glomerulonephritis, hepatitis, pancytopenia,
neutropenia, UTI(pseudomonas aeruginosa)

UK

refuse treatment

UK

refuse treatment

UK

refuse treatment

UK=Unknown; PBSC=peripheral blood stem cell; PCI=prophylactic cranial irradiation; ChemoTx=Chemotherapy; BMT=bone
marrow transplantation; UTI=urinary tract infection; DIC=disseminated intravascular coagulation

- 50 -

Table 15. Nexia administration and survival times of the acute leukemia
No. Name

First visit Visit KHW

Purpose

KHW

to first Dx.

to visit

PYJ

99/4/24

14 months

SKH

99/7/5

2 months

KKB

97/3/31

5 months

KGH

99/3/23

2 months

Combined
Treatment
Refuse
treatment
Refuse
treatment
Combined
Treatment

Nexia adm. Nexia dose

Combined

Survival* after Disease Free

Treatment Nexia adm.

12 months 150 mg/day None

Survival*

71 months

85 months

15 months 450 mg/day 15 months 68 months

61 months

37 months 450 mg/day UK

96 months

99 months

14 months 150 mg/day 7 months

72 months

72 months

UK=Unknown; adm=administration; KHW=Kwanghyewon oriental medicine hospital

* : Survival Time was recorded at March 2, 2005 according to Korea Clean Cancer Association.
: Current status was checked at March 22, 2006 according to KHW call back system.

- 51 -

Current status
Alive, in CR, 110
months after ALL Dx.
Alive, in CR, 83
months after ALL Dx.
Alive, in CR, 97
months after ALL Dx.
Alive, in CR, 86
months after ALL Dx.

.
,
.
DNA ras,
myc

(Proto-oncogen) Rb, P53

(Tumor suppressor gene)
13)

14-15)

,
16)

.
, , , , , , ,
, , . ,
.
Laacca Sinica Exsiccata , , ,
.


. urushiol laccase
renin .

, , , ,
10)

52

 (, Rhus Verniciflua)
, , ,
, , ( )
10-12)

. Nexia

.
Nexia 1996 (), ,
17)

KAIST .
, ''
1996 ,
( 0394089) .
(urushiol) (cortex)
.
T lymphocyte monocyte
cytokine, chemotactic factor adhesion molecule
18)

.
cytokine, chemotactic
factor adhesion molecule ,
.
19-20)

.
Hydroquinone, K1, K2, K3 Mitomycin C
DNA ,
DNA mitosis .
DNA

53

 - free radical
free radical
18,21)

(free radical transfer reaction)

hydroquinone aromatic di-alcohol

ROS(Reactive Oxygen Species)
K1

22-23)

810
.
80100 4
.
40% 60%
60%
.
(A), CHCl3 (B), Ethylacetate (C), n-butanol
(D), (E)

HPLC Chromatogram

fustin fisetin,
sulfuretin, butein .
Nexia Nexia
. (KFDA GLP)
(Blokhin Cancer Research Center) LG
LS .
Nexia 13 4
Nexia 13 450 mg/kg ,
, 150 450

54

mg/kg ,
(NOAEL) 150 mg/kg, 50 mg/kg
(MTD) 450 mg/kg
. 4 , 1
,
.
Nexia
1,600 mg/kg
1,600 mg/kg
800 mg/kg .
Nexia Nexia
,

.
Nexia A549

.
40.7 %
26.8 % . 0.342g
14.1 %
. (Nexia) A549 68.8 /ml, K562
81.0 /ml, KATO 77.3 /ml, SK-OV-3 165.2 /ml,

HUVEC

200 /ml IC50 ,

. Nexia Nexia VEGF HUVEC

. Nexia

55

 (A549), (K562), (KATO III), (SK-OV-3)

DNA fragmentation
.
3%
24)

.
70%
24)

30% ,
65
25)

.
,
1
5 45-50%
27-31)

80%

26)

. 2
27-33)

20-30%

10 25%
34)

.
90%

35-37)

90% 50~75%

20%
36~67%

38-40)

1) , 2)
, 3) B-

56

41)

 1) , 2) 3) {t(9;22), t(4;11), t(8;14)}, 4)

42)

, 5) .
30,000/
. 23%
15% . (Ph1)
.

43-44)

CALGB

50~60% .
8090%
. 8090%
3040% 1530%
.
. L2 myeloid
.
.
Ph1 23% 15% . Ph1
.
Methotrexate(MTX) polyglutamination
45)

.
.
.

.
1997 3 2001 5

57

 (Complete Remission) 4
.
4 (case 1) 1 3
(case 2,3,4) 15 . Case 1 pre-B type case 2, 3, 4 L1,
L1, L2 type. 3 (case 1,3,4)
case 1 . Case 2
2 1
(Table 12).
Case 1 , , steroid induced DM,
case 2 (pancytopenia),
(neutropenia), , . 3
(Table 14).

14, 2, 5, 2 case 1
. Case1, 4

, case 2, 3

. case 2, 4 15
, 7 . Nexia 12, 15, 37, 14
150, 450, 450, 150 mg/day . Nexia
2005 3 2 71, 68, 96, 72
85, 61, 99, 72 . 2006 3 22
110, 83, 97, 86
(Table 15).
,

26)

80%

2
27-33)

20-30%

58

 10
19)

25% . GIMEMA Group

778 2.2
9 27% ,
2.4 (CCR)
46)

9 33% .
4
Nexia
7 9 (CCR)
Nexia
.
.

59

.
(Nexia) , ,
.

1. Nexia 90~95, 10,

1 , 6 , , 60%
, urushiol
fusetin, fisetin, sulfuretin, butein HPLC .

2. Nexia 13 4 ,
,
.

3. Nexia

300 mg/kg A549

40.7% .

4. Nexia A549 68.8 /ml, K562 81.0 /ml, KATO 77.3 /ml,
SK-OV-3 165.2 /ml, HUVEC 200 /ml IC50 .

5. Nexia VEGF HUVEC .

6. 4 Nexia
110, 83, 97, 86
.

60


Nexia ,
.

61


1. . . : ; 989-9992, 2002.
2. . . , 1995:1-24, 65-74.
3. . . , 1995:1107.
4. , .
. 1991;12(2):1-10.
5. , .
. 1991;1(1):153-60.
6. , , .
. 1999;14(1):37-44.
7.

1995:15.
8. Lee HJ, Lee EO, Rhee YH, Ahn KS, Li GX, Jiang C, Lu J, Kim SH.,
, , , , ,
,, 2004 ; 18(4) : 1056-1060.
9.

An

oriental

herbal

cocktail,

ka-mi-kae-kyuk-tang,

exerts

anti-cancer

activities by targeting angiogenesis, apoptosis and metastasis.Carcinogenesis.

2006 Jun 15; [Epub ahead of print]
10.

2002;15(1):60-76.
11. , .
. . 1998;7(1):700.
12. Hong DH, Han SB, Lee CW et al. Cytotoxicity of urushiols isolated from
sap of Korean lacquer tree (Rhus vernicifera Stokes). Arch Pharm Res. 1999
Dec;22(6):638-41.

62

13. Herman J. G., Baylin S. B. Mechanisms of Disease: Gene Silencing in

Cancer in Association with Promoter Hypermethylation. N Engl J Med 2003;
349:2042-2054, Nov 20, 2003
14. Chao EC, Lipkin SM. Molecular models for the tissue specificity of DNA
mismatch

repair-deficient

carcinogenesis.

Nucleic

Acids

Res.

2006

Feb

6;34(3):840-52.
15. Huang J, Li X, Hilf R, Bambara RA, Muyan M. Molecular basis of
therapeutic strategies for breast cancer. Curr Drug Targets Immune Endocr
Metabol Disord. 2005 Dec;5(4):379-96.
16. Katsutoshi Terasawa. Evidence-based Reconstruction of Kampo Medicine:
Part IIThe Concept of Sho. Evid. Based Complement. Altern. Med., Sep
2004;1:119 - 123.
17. 8. (NexiaQ)
. 1053:12(11 ICOM )
18. Schmidt RJ, Khan L, Chung LY. Are free radicals and not quinones the
haptenic species derived from urushiols and other contact allergenic mono- and
dihydric alkylbenzenes? The significance of NADH, glutathione, and redox
cycling in the skin.Arch Dermatol Res. 1990;282(1):56-64.
19.

SH

Kaufman.

Apoptosis:

Pharmacological

Implication

and

Therapeutic

Opportunities, Academic Press, San Diego, CA, USA. 1997.

20. Reynolds NJ, Yi JY, Fisher GJ, et al. Down-regulation of Langerhans cell
protein kinase C-beta isoenzyme expression in inflammatory and hyperplastic
dermatoses. Br J Dermatol. 1995 Aug;133(2):157-67.
21. R.A. Floyd. Free Radical and Cancer, Marcel Dekker, New York, USA,
Chap.1, 1982.
22. Bolton JL, Pisha E, Shen L, et al. The reactivity of o-quinones which do

63

not isomerize to quinone methides correlates with alkylcatechol-induced toxicity

in human melanoma cells. Chem Biol Interact. 1997 Sep 12;106(2):133-48.
23. Wu FY, Liao WC, Chang HM. Comparison of antitumor activity of vitamins
K1, K2 and K3 on human tumor cells by two (MTT and SRB) cell viability
assays. Life Sci. 1993;52(22):1797-804.
24. , , . . , 2003:786.
25. Kasper et al. Harrison's principal of internal medicine 16th edition Vol.1.
McGrawHill, 2005:631-2.
26. Griffin P. Bethesda Handbook of clinical hematology. Lippincott Williams &
Wilkins, 2005:136, 141-2, 153.
27. Cliff RA, Buckner CD, Thomas ED, et al. The treatment of acute
nonlymphoblastic leukemia by allogeneic marrow transplantation. Bone Marrow
Transplant. 1987;2:243-58.
28. Zittoun RA, Mandelli F, Willemze R, et al. Autologous or allogeneic bone
marrow

transplantation

compared

with

intensive

chemotherapy

in

acute

myelogenous leukemia. N Engl J Med. 1995;332(4):217-23.

29. Reiffers J, Stoppa AM, Attal M, et al. Allogeneic vs autologous stem cell
transplantation vs chemotherapy in patients with acute myeloid leukemia in first
remission: the BGMT 87 study. Leukemia. 1996;10(12):1874-82.
30. Harousseau JL, Cahn JY, Pignon B, et al. Comparison of autologous bone
marrow transplantation and intensive chemotherapy as postremission therapy in
adult acute myeloid leukemia. Blood. 1997;90(8):2978-86.
31. Cassileth PA, Harrington DP, Appelbaum FR, et al. Chemotherapy compared
with autologous or allogeneic bone marrow transplantation in the management
of

acute

myeloid

leukemia

in

first

1998;339(23):1649-56.

64

remission.

Engl

Med.

32. Keating S, Suciu S, de Witte T, et al. Prognostic factors of patients with

acute myeloid leukemia (AML) allografted in first complete remission: an
analysis of the EORTC-GIMEMA AML 8A trial. Bone Marrow Transplant.
1996;17(6):993-1001.
33. Mehta J, Powles R, Treleaven J, et al. Long-term follow-up of patients
undergoing allogeneic bone marrow transplantation for acute myeloid leukemia
in first complete remission after cyclophosphamide-total body irradiation and
cyclosporine. Bone Marrow Transplant. 1996;18(4):741-6.
34. Mandelli F, Annino L, Rotoli B. The GIMEMA ALL 0183 trial: analysis of
10-year follow-up. Br J Haematol. 1996 Mar;92(3):665-72.
35. Nesbit ME, Sather H, Robison LL. et al. Sanctuary therapy: a randomized
trial of 724 children with previously untreated acute lymphoblastic leukemia: A
Report from Children's Cancer Study Group. Cancer Res. 1982 Feb;42(2):674-80.
36.

Riehm

H.

Gadner

H,

Henze

G,

et

al.

The

Berlin

childhood

acute

lymphoblastic leukemia therapy study. Am J Pediatr Hematol Oncol.. 1980:2299.

37. Sallan SE, Hitchcock-bryan S, Gelber R. Influence of intensive asparaginase
in the treatment of childhood non-T-cell acute lymphoblastic leukemia. Cancer
Res. 1983;43:5601.
38. Gee TS, Hanhbin M, Dowing MD, et al. Acute lymphoblastic leukemia in
adults and children. Cancer. 1979;37:1256.
39. Jacob AD, Gale RP. Recent advances in the biology and treatment of acute
lymphoblastic leukemia in adults. N Engl J Med. 1984;311:1219.
40. Chessells JM. Acute lymphoblatic leukemia. Semin Hematol. 1982;19:155.
41. Copelen EA, McGuire EA. The biology and treatment of acute lymphoblastic
leukemia in adults. Blood. 1995;85:1151.
42. Hoelzer D, Thiel E, Loeffler H, et al. Intensified therapy in acute

65

lymphoblastic and acute undifferentiated leukemia in adults. Blood. 1984;64:38.

43. Boucheix C, David B, Sebban C, et al. For the French Group on Therapy
for Adult Acute Lymphoblastic Leukemia Immunophenotype of a prospective
trial including 562 tested patients (LALA87). Blood. 1994;84:1603.
44. Larson RA, Dodge RK, Burns CP, et al. A five drug remission induction
regimen

with

intensive

consolidation

for

adults

with

acute

lymphoblastic

leukemia : Cancer and Leukemia Group B study 8811. Blood. 1995;85:2025.

45. Goker E, Lin JT, Trippett T, et al. Decreased poluglutamylation of
methotrexate in acute lymphoblastic leukemia blasts in adults compared to
children with this disease. Leukemia. 1993;7:1000.
46. Annino L, Vegna ML, Camera A, et al. Treatment of adult acute
lymphoblastic leukemia (ALL): long-term follow-up of the GIMEMA ALL 0288
randomized study. Blood. 2002 Feb 1;99(3):863-71

66

ABSTRACT
Study on the Safety and Antitumor Activity of Rhus
Verniciflua Extract (Nexia)
Won-Cheol Choi
Department of East and West Medical Science,
Graduate School of East and West Medicine
(Supervised by Sung-Hoon Kim, OMD, Ph.D.)

This study was undertaken to evaluate safety and antitumor activity of Nexia,
a processed extract of Rhus verniciflua STOKES by heating according to
Korean patent 0504160 through the manufacture processing, constituent analysis
for standardization, toxicology, cancer research and clinical application.
The best processing conditions were obtained at 90~95 for 6 hours under one
air pressure, using 10 times of water or 60% ethanol. Urushiol, fusetin, fisetin,
sulfuretin and butein were confirmd in Nexia by high performance liquid
chromatography(HPLC). Nexia did not exhibit any toxicological symptoms
through

13

week

continuous

treatment,

dosage

accumulation

study

and

anaphylaxis response. Nexia exerted cytotoxicity against A549, K562, KATO ,

SK-OV-3 and HUVEC with IC50 of

68.8 /ml,

81.0 /ml, 77.3 /ml, 165.2

/ml and 200 /ml, respectively.

In addition, Nexia significantly inhibited the proliferation and

migratory

activity in vascular endothelial growth factor(VEGF) treated human umbilical

67

vein endothelial cells (HUVEC). Furthermore, Nexia suppressed the growth of

A549 cells innoculated sc into the back of athymic nude mice, CanN. Cg-Foxn
1nu/CrljBgi.
The lifespan of four patients with acute lymphoblastic leukemia under complete
remission

were

110,

83,

97,

and

86

months

after

treatment

of

Nexia,

respectively.
These data suggest that Nexia, a processed extract by removing allergen from
Rhus verniciflua STOKES, may have safety and antitumor activity and also still
need continuous study on its mechanism and clinical trial.

68