Development of catechin production process by means of teacell culture.Naomi Shibasaki-Kitakawa, Junna Takeishi and Toshikuni Yonemoto

Department of Chemical Engineering, Tohoku UniversityAoba-yama 07, Sendai 980-8579, JAPAN

SummaryThe suspension cultures of tea callus cells, C.sinensis ev. Yabukita, were performed

using the liquid Gamborg B5 medium, supplemented with 0.1 mglL kinetin, 0.5 mglL 2,4­dichlorophenoxyacetic acid (2,4-D) and 20 giL glucose, in the dark at 28 ac. The effects of

culture conditions, such as culture period, phytohormone addition and light irradiation, oncell growth and catechin production were investigated. The production of flavonoid (catechin+ proanthocyanidin) was promoted by inoculating the cells to the fresh medium at the timegiving the maximum flavonoid content in the cell. By repeating the cultivation using themedium with naphthaleneacetic acid (NAA), the differentiation from the callus cells intothe adventitious roots was observed. The flavonoid production was accelerated but the cellgrowth was suppressed. By repeating the cultivation under the continuous fluorescent lightirradiation, the flavonoid production was significantly increased without inhibiting the cellgrowth. The cultivation was repeated several times under the appropriate condition and themaximum flavonoid production, about 1.5 giL-medium, and the maximum content, about150 mg/gDCW, were obtained. The later value was larger than that in the leaves of teaplant.

KeywordsTea callus cells, suspension culture, catechin production,

1. IntroductionTea plant synthesizes a lot of secondary metabolites such as caffeine, theanine and cat­

echin. Recently, catechin has received much attention because it has antioxidative, an­tibacterial and antiallergic activities. However, only few attempts have been made on thecatechin -production by means of the tea cell culture. The research group of Zaprometovet al. 1- 3) studied on the cultivation of cells derived from stems and leaves of tea plant,

Camellia sinensis, using solid medium, and reported that both cells retained the ability tosynthesize flavonoid, catechin and proanthocyanidin. They also investigated the effects ofadding kinetin, one of phytohormone, and of light irradiation on the flavonoid production.In their cultures using the solid medium, the cell growth rate was significantly small andthe maximum flavonoid content in the cells was lower than that in the intact tea plant.

In this research, we performed the suspension cultures of tea callus cells using the liquidmedium, which permitted the large-scale cultivation and a higher catechin productivity.Culture period, phytohormone addition and light irradiation were selected as operatingconditions and the effects of the culture conditions on cell growth and catechin productionwere investigated. The appropriate culture condition was proposed.

2. Materials and MethodsThe callus cells were derived from seeds of tea plant, Camellia sinensis cv. Yabukita,

kindly provided by National Institute of Vegetable and Tea Science, Japan. The GamborgB5 medium used for cultivation was supplemented with phytohormones (O.lx10-3 g/dm3

-5- Session n

DDO

('1)-15 ......- .....- ......--......- .......-_.--...E (a)dry cell conc.

;!2;:g 10C)

kinetin and 0.5x10-3 gjdm3 2,4-D), vitamins (1.0x10-2 gjdm3 nicotinic acid, 1.0x10-3

gjdm3 thiamine Hel, 0.1 gjdm3 myoinositol, 1.0x10-2 g/dm3 ascorbic acid and 0.25 gjdm3

glutamine) and 20 gjdm3 glucose. Cell suspension was subcultured every 28 days in 0.5­

dm3 Erlenmeyer flasks containing 0.2 dm3 of the medium in the dark on a rotary shaker

(100 rpm) at 28°C over one year. The amount of the cells for inoculation was 5.0 g on the

basis of wet weight.

In order to discuss the effect of the culture period, the two-stage cultivation was per­

formed. In the first stage, the subcultured cells were inoculated to the medium and then

- cultivated for 7, 14, 21 or 28 days in the dark. In the second stage, the 1.0 g (wet weight)

cells cultured in the first stage were inoculated to many 0.1-dm3 Erlenmeyer flasks contain­

ing 0.04 drr13 of the medium, respectively, and then simultaneously cultivated to obtain the

data for a different culture period. The first stage with the culture period of 28 days was

set to be the control culture -and it was identical to the subculture condition. On the other

hand, to investigate the effect of the phytohormone addition, the subcultured cells were

inoculated to the medium with 0.5x10-3 gjdm3 NAA instead of 2,4-D and then cultivated

similarly to the second stage of the two-stage cultivation. In addition, the subcultured cells

were also inoculated to the medium with 2,4-D and then cultivated under the continuous

fluorescent light irradiation (3000 lux). All experiments were repeated at least two times

under the same condition to confirm the reproducibility of the data.

At specified time intervals, one flask was taken out and then the cells and the medium

were separated by centrifugation. The cells were lyophilized until there was no change of the

weight and the dry cell concentration was determined. The flavonoid were extracted fromthe powdered cells with 1 % (vjv) HCI--':methanol solution. The total flavonoid content in

the cells was determined by the colorimetric method using spectrophotometer. There are

many kinds of catechin and proanthocyanidin in the flavonoid extracted from the tea cells,

so that the catechin compositi~n in the flavonoid was determined using HPLC system with

an Inertsil ODS column and a diode array detector.

3. Results and Discussion3.1 Effect of 'culture period

The effect of the culture period of the first

stage on cell growth and flavonoid production in

the second stage of two-stage cultiv~tion is shown

in Fig.l. Under any condition, the dry cell con- g8centration increased until about 20 days and then 5

gradually decreased. The cell growth in the second ~ PQ

u period [days]stage was hardly affected by the culture period of :. 0 <> 7

the first stage. The flavonoid content in the cells ~ 60 (b)f1avonoid content ~ ~~cultured for 21 days in the first stage was the high- ~ • 28(controQ

est and the content in the cells cultured for 7 days, J=»40othe lowest. The maximum flavonoid content in the Xsecond stage increased as the initial content was 1:: 20 0

higher. The time course of the flavonoid content § 0 ~ D.D .tIe 8in the first stage was considered to be the same as "C <> Q 6. e <>that in the control culture and the content gave a .~ 0 0~-0111111115~-1~O~-1~5~~2~O-~2~5"""

minimum at about 7 days and a maximum at about ~ time [day]20 days. Thus, the cells cultured for 21 days in the Fig. 1 Effect of culture period

Session II -6-

first stage had a high content. By inoculating the cells at the time giving the maximum

flavonoid content, the content obtained in the next generation increased.

30

0.21619.8

5

(b) 2,4-0 (control)'f'"'

~ '. ;~

(a)dry cell conc.

0 ~

~ ~ ~<>~

0 <>0

0 repeated time

t\ 0 o 1

l~ 0 02/':;.3

(b)f1avonoid content• dark

/':;.

~<>

~

/':;. <)~

<> <>/':;.0> <> 0

) ~ ~ 0 • •10 15 20 25

time [day]Fig. 3 Effect of culture period

10.9

10.7 35.9 0.3855.1 45.6 0.234

78.1

~

Fig. 2 Photographs of cells

cell conc. flavonoid content productivity[gDCWjdm3 ] [xI0-3gjgDCW] [gjdm3 ]

Growth and flavonoid production at 20 daysTable 1

NAA(lst)NAA(2nd)NAA(3rd)2,4-D (control)

generation

3.2 Effect of phytohormone additionThe cultivation using the medium with NAA addition was repeated three times every

28 days in the dark. The cultures repeated one, two and three times are termed the first,

second and third generations. The photograph of the cells in the third generation with NAA

is shown in Fig.2, compared with that in the control culture with 2,4-D. The cells in the

control culture were loose white calluses. In the third generation, projections were observed

in the cell aggregates and the differentiation from the callus cells into the adventitious roots

occurred. Table 1 shows the growth and the flavonoid production at 20 days in each culture.

The cell concentration de- (a) NAA (3rd)creased significantly in the

second generation and was

not measured in the third

generation due to the dif­

ferentiation into the adven­

titious roots. The flavonoid

content increased by repeat­

ing the cultivation with NAA

addition. The productivity

was the highest in the first

generation without inhibiting

cell growth.

3.3 Effect of light irradi­ation

The cultivation under the

continuous fluorescent light

irradiation was repeated three

times every 28 days using the medium with 2,4-D. (")~15E

The results for the repeated cultures are shown in ~

Fig.3, compared with that in the control culture in gthe dark. The cell concentration in the first gener- 0)

10

oation increased more slowly than that in the dark. §

(.)

The concentrations in the second and third gener- 5~

ations increased more rapidly than that in the first ~-0

generation and overlapped with that in the dark. 0

By repeating the cultivation under the light irra- ~150o

diation, the delay of the cell growth observed in .g>0)

the first generation disappeared. Thus, the delay ~100

of the cell growth seemed to arise from the op- xtical stress induced by transferring the cells from ~ 50

the dark place to the bright one. On the other c::8

hand, the flavonoid content in the first generation "0'0

was much higher than that in the dark after 15 ~ 00

days. The initial flavonoid content in the second ~

generation was much larger than that in the first

-7- Session IT

1st

,,"DO

fit!o

repeated time4,5th 0 1hrd <>2

~~d L..-_~----=-~....,..-J<>

<><>~100

E <

"* 508

~ 0[:-_--=-D_---:"'="D_--:'::-D_-='=~__='':_____:=''='~ 0 5 10 15 20 25 30~ time [day]

Time course of flavonoid contentunder appropriate condition

Fig. 5

~200 f-oC)

-;;'150..,o

generation and the maXImumvalue was attained at about 25

days. The contents in the sec­

ond and third generations weresimilar. By repeating the cul­

tivation under the light irradia­

tion, the flavonoid accumulation

in the cells was significantly pro-

moted without inhibiting the cell Fig. 4growth. The microscopic photograph of the cells

obtained in the third generation is shown inFig.4, compared with that in the control culture

in the dark. A lot of chloroplasts were present in

the cells under the light irradiation, whereas the

cells in the dark were colorless. The differentia­

tion from the callus cells into the leaves occurred

by repeating the cultivation under the light irra­diation. The chloroplasts were known to be one

of the important sites of flavonoid synthesis2).

3.4 Cultured results under appropriatecondition

For the improvement of the flavonoid productivity, it was very effective to inoculate

the cells at the time giving maximum flavonoid content, to prevent the differentiation into

adventitious roots by 2,4-D addition and to repeat the cultivation under the light irradiation.Thus, the cultivation was repeated several times under the appropriated condition combined

with the above-mentioned results. The cell growth in the repeated cultures was the same

as that in the control culture (not shown). The time course of the flavonoid content in each

generation is shown in Fig.5. The maximum content in the first generation was attained

at 30 days and that in the second generation was given at 22 days. The time giving the

maximum value became shorter by repeating the cultivation. This was because the cells

having a higher activity for flavonoid synthesis were inoculated. The flavonoid contents in

the third, fourth and fifth generations were almost similar and the flavonoid productionseemed to be stabilized. As a result, we obtained the maximum flavonoid production, about

1.5 giL-medium, and the maximum content, about 150 mg/gDCW. The later value waslarger than that in the leaves of tea plant. The ratio of total catechin to flavonoid was larger

than 90 %.

4. AcknowledgementThis research was supported in part by the O-CHA (tea) Pioneer Academic Research

Grant from the Organizing Committee of 2001 International Conference on O-CHA (tea)Culture and Science.

5. References1) T.F.Koretskaya, M.N.Zaprometov, Soviet Plant.Physiol.,22,825-829(1975).2) M.N.Zaprometov, N.V.Zagoskina, Soviet Plant.Physiol.,34,136-143(1987).

3) N.V.Zagoskina, T.U.Usik, M.N.Zaprometov, Soviet Plant.Physiol.,37,829-834(1990).

Session II -8-