Confidential NITTO DENKO CORPORATION QUALITY CONTROL IN SUBCULTURING OF TISSUE-CULTURED GINSENG FOR LONG PERIOD Yuko SAHASHI, Yoshiko TUJIKURA Nitto Denko Co.

Cultivated ginseng Tissue-cultured ginseng callus Explant Regenerated ginseng Plant tissue culture technology

Commercial production process of tissue-cultured ginseng Callus induction process Selection of cell line, Examination of culture condition and Scale up process

TCG 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 NITTO DENKO CORPORATION Quality evaluation of TCG seed 1.Ingredients analysis: Ginsenosides, Panaxans (P: rich fraction) 2.Growth (yield, Growth ratio, D/W ratio, Y X/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.

MM M L K1 K2 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 fractionK1,2, L, M, MM:TCG cell line derived from one plant

Confidential NITTO DENKO CORPORATION Chromatogram of TCG Ginsenosides by HPLC Rg1 Re Rf Rb1 Rc Rb2Rd

Change of cell line for seed culture Cell line K 2 nd line made from liquid subculture maintained for backup Seed culture 3 rd 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 st line selected for production M L (450ml/1L) 1999

Change of TCG yield and Ginsenosides content of each cell line Cell lines came from calli induced from one plant of ginseng about 25 years ago. K: 1 st line selected for production L:2 nd line made from liquid subculture maintained for backup M:3 rd line which was a liquid culture made from solid subculture 450ml/1L

Master 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 Cell lines came from calli induced from one plant of ginseng about 25 years ago. M:3 rd line which was subcultured in 450ml of medium MM:4 th line which was made from 13 th M subculture and subcultured in 300 ml of medium every 30 days. 450ml/1L M MM 300ml/1L

Cell lines came from calli induced from one plant of ginseng about 25 years ago. MM:4 th line which was made from 13 th M subculture and subcultured in 300 ml of medium every 30 days. MM 300ml/1L 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 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 turns

Cell lines came from calli induced from one plant of ginseng about 25 years ago. MM:4 th line which was made from 13 th M subculture and subcultured in 300 ml of medium every 30 days. Effect of subculturing in different culture conditions by turns on Ginsenosides content 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

Confidential NITTO DENKO CORPORATION Conclusion 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 NITTO DENKO CORPORATION Complementary data

M18679M3452M18679M3452 OPC-5OPC-20 2% agarose electropholysisM:100 base pair DNA ladder 1:White ginseng, 2: 山参, 6:TCG MM, 7:TCG 3-A (new cell line) (bp) Gene analysis of cell line MM (RFLP) TCG induced from Korean ginseng

Tissue-Cultured Ginseng Effects ・ Effects on digestive organ ・ Improvement of blood circulation ・ Stimulation of energy (stimulation of glucose metabolism) 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