Confidential NITTO DENKO CORPORATION QUALITY CONTROL IN SUBCULTURING OF TISSUE-CULTURED GINSENG FOR...

NITTO DENKO CORPORATION

Confidential

QUALITY CONTROL IN SUBCULTURING OF TISSUE-CULTURED GINSENG FOR LONG PERIOD 　　　　　　　　

Yuko SAHASHI, Yoshiko TUJIKURA

Nitto Denko Co.

Cultivated ginsengCultivated ginseng

Tissue-cultured ginseng callus

Tissue-cultured ginseng callus

ExplantExplant

Regenerated ginsengRegenerated ginseng

Plant tissue culture technologyPlant tissue culture technology

Commercial production process of tissue-cultured ginseng

Commercial production process of tissue-cultured ginseng

Callus induction processCallus induction process

Selection of cell line, Examination of culture condition and Scale up processSelection of cell line, Examination of culture condition and Scale up process

TCG production systemTCG production system

Seed subculture (450ml/1L)

Seed culture (450ml/1L)

Jar fermentor culture by a cascade seeding system

TCG:Tissue-cultured ginseng 25 KL


Confidential

Quality evaluation of TCG seed

1. Ingredients analysis: Ginsenosides, Panaxans (P: rich fraction)

2. Growth (yield, Growth ratio, D/W ratio, YX/S, etc.)

3. Efficacy*

Effect on blood flow and stress-induced ulceration by extract

blood sugar level by P fraction

4. Safety*

Acute and chronic toxicological test

*:Animal test

Ingredients analysis is convenient to often check quality of subculture.Ingredients analysis is convenient to often check quality of subculture.

0

20

40

60

0 5 10

P fraction　 [mg/g-extract]G

inse

nos

ides

[mg/

g-ex

trac

t]O

O

OH

CH2 OH

CH2

OH

OH

CH2

OH

OHOH

OHOH

O

O

OH

O

CH2

O

OH

CH2

OH

OH

OO

O

O

OH

MM

M

L

K1 K2

Relation between Ginsenosides and P fraction in TCG different cell lines

Relation between Ginsenosides and P fraction in TCG different cell lines

Ginsenosides analysis is most suitable

for quality control of subculture.

Ginsenosides analysis is most suitable

for quality control of subculture.

P fraction:Panaxans-rich fraction K1,2, L, M, MM:TCG cell line derived from one plant


Confidential

Chromatogram of TCG Ginsenosides by HPLC

Rg1

Re

Rf

Rb1

RcRb2 Rd

Change of cell line for seed cultureChange of cell line for seed culture

Cell line K2nd line made from liquid subculture maintained for backup

Seed culture

3rd line which was a liquid culture made from solid subculture

Cell lines came from calli induced from one plant of ginseng about 25 years ago.

1985 1991 1996 1997

1st line selected for production

M

L

(450ml/1L)

1999

Change of TCG yield and Ginsenosides content of each cell line

Change of TCG yield and Ginsenosides content of each cell line


K: 1st line selected for production L:2nd line made from liquid subculture maintained for backup M:3rd line which was a liquid culture made from solid subculture

0

5

10

15

20

25

12/95 4 8 12 4 8 12 4

Date [month/year]

Dry

cel

l [g/

L]

cell line K L M

0

5

10

15

20

25

30

Date [month/year]

Gin

seno

side

con

tent

[mg/

g-D

.W.]

cell line K L M

450ml/1L

Master seed culture for quality control of seed cultureMaster seed culture for quality control of seed culture

Culture adding physical stress to TCG by a decrease of liquid medium Repression of a decrease of Ginsenosides production by adding stress　

Seed culture (450ml/1L)

Seed subculture (450ml/1L)

Master seed culture (300ml/1L)

Comparison between cell line M and MM on TCG yield and Ginsenosides content

Comparison between cell line M and MM on TCG yield and Ginsenosides content


M:3rd line which was subcultured in 450ml of medium MM:4th line which was made from 13th M subculture and subcultured in 300 ml of medium every 30 days.

0

5

10

15

1 6 11 16 21 26

Subculture No.

Dry

cel

l [g/

L]

cell line M

MM

0

5

10

15

20

25

30

1 6 11 16 21 26

Subculture No.

Gin

sen

osid

es c

onte

nt

[mg/

g-D

.W.]

cell line M

MM

450ml/1L

M

MM

300ml/1L


MM:4th line which was made from 13th M subculture and subcultured in 300 ml of medium every 30 days.

MM

300ml/1L

0

5

10

15

20

1 11 21 31 41 51 61 71 81 91

Subculture No.

Dry

cel

l [g/

L]

0

5

10

15

20

Gin

sen

osid

es[m

g/g-

D.W

.]

Dry cellGinsenosides

Effect of subculturing of cell line MM on TCG yield and Ginsenosides content

Effect of subculturing of cell line MM on TCG yield and Ginsenosides content

Culture method which gives TCG two different stress by turns; physical stress by a decrease of medium volume and chemical stress by a change of hormone concentration

[Physical stress]　Medium:300 ml Hormone:IBA 2ppm Kinetin 0.1ppm

[Chemical stress] Medium:450ml　 IBA:decrease Kinetin:increase

2 or 3 times by above condition

2 or 3 times2 or 3 times 　

[Physical stress]　Medium:300 ml IBA 2ppm Kinetin 0.1ppm

[Chemical stress] Medium:450ml　 IBA:decrease Kinetin:increase

Subculture:2or 3 times (culture condition of a master seed, MM)

Subculturing in different culture conditions by turnsSubculturing in different culture conditions by turns


MM:4th line which was made from 13th M subculture and subcultured in 300 ml of medium every 30 days.

Effect of subculturing in different culture conditions by turns on Ginsenosides content

Effect of subculturing in different culture conditions by turns on Ginsenosides content

0

4

8

12

16

76 81 86 91 96

Subculture No.

Dry

cel

l [g/

L]

MM(300)(450) ⇔　 (300)hormone 1(450) ⇔ (300)hormone 2(450) ⇔ (300)hormone 3(450) ⇔ (300)

0

4

8

12

16

76 81 86 91 96

Subculture No.

Gin

sen

osid

es c

onte

nt

[mg/

g-D

.W.]

MM(300)(450) ⇔　 (300)hormone 1(450) ⇔ (300)hormone 2(450) ⇔ (300)hormone 3(450) ⇔ (300)

hormone 1: 1/4 of IBA, 5-fold of K, 2:1/2 of IBA, 2-fold of K, 3: 1/2 of IBA, 2-fold of K


ConfidentialConclusion

1. We examined quality control of TCG in subculturing for long period. We made a master seed for quality control of seed. The master seed culture was more effective on quality control than the conventional seed culture.

2. After subculture was continued in 300ml of medium, Ginsenosides content was maintained for approximately 8 years.

3. After subculturing in combination of two culture conditions, some culture conditions with hormone change tended to show an increase of Ginsenosides content.

4. These results indicated that physical stress like shaking, or the combination of different stress was effective in quality control of TCG subculturing for long period.

　　　　 But we don’t know how long the cell line will keep stable condition. We will continue to examine quality control of TCG.


Confidential

Complementary data Complementary data

M 1 86 7 9 M3 4 52 M 1 86 7 9 M3 4 52OPC-5 OPC-20

2% agarose electropholysis M:100 base pair DNA ladder

1:White ginseng, 2: 山参 , 6:TCG MM, 7:TCG 3-A (new cell line)

1000

500

1000

500

(bp) (bp)

Gene analysis of cell line MM (RFLP)Gene analysis of cell line MM (RFLP)

TCG induced from Korean ginseng

Tissue-Cultured Ginseng Effects Tissue-Cultured Ginseng Effects

・ Effects on digestive organ

・ Improvement of blood circulation

・ Stimulation of energy

　 (stimulation of glucose metabolism)

0 5 10 15 20 25 30

＊＊

＊＊

Control

Tissue-cultured(400mg/kg)



Cultivated(400mg/kg)



Atropine

(Ulcer index)

(10mg/kg)

Cultivated 2

Tissue-cultured

Cultivated 1

Cultivated 3Cultivated 2

Tissue-cultured

Cultivated 1

Cultivated 3

Tissue-cultured

Cultivated 1

Cultivated 3

1 3 10 30[mg/kg]

**: Significantly different from control. P< 0.01

1 3 10 30[mg/kg]

**: Significantly different from control. P< 0.01

5

10

15

20

25

30**

0

5

10

15

20

25

30

5

10

15

20

25

30**

0

[%]

UDP-Glucose

Glucose-6-P

Fructose-6-P

Fluctose-1,6-diP

diOH-acetone-P

PFK

TCA cycleTCA cycle

G6PDHG6Pase

GK,HK

phosphorylase

glycogensynthetase

glycogensynthesisglycogensynthesissystem

20 9

100 129

100 241

100 70

100 172

Glucose-1-P

Glucose-6-P

UDP-Glucose

Glycogen

Fructose-6-P

Fructose-1,6-di-P

di-OH acetone-P

Glyceraldehyde-3-P

6-P-gluconateGlucose

AL-mice +Panaxan

PFK

G6PDH

100 71 71 83100 60100 46 46 89

60 94

*

* alloxane induced mice

Xylulose-5-P

Stimulation of glucolysis enzymes activities by hypoglycemic glycans, panaxans

Effect of ginsengs on blood flow on abdominal skin in rats

Effect of ginsengs on stress-induced ulceration in rats 　　　　　　 (Single administration)

Tissue-cultured ginseng has three main effects.

n=5, **: Significantly different from control. P< 0.01

Confidential NITTO DENKO CORPORATION QUALITY CONTROL IN SUBCULTURING OF TISSUE-CULTURED GINSENG FOR...

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