JOURNAL OF FERMENTATION AND BIOENGINEBRINO Vol. 83, No. 2, 206-208. 1997

Effect of Chitosan on Indirubin Production from Suspension Culture of Polygonurn tinctorium

JU HWAN KIM,’ JOONG HAN SHIN,’ HO JAE LEE,2 IN SIK CHUNG,3 AND HYONG JO0 LEE’*

Department of Food Science and Technology, Seoul National University, Suwon 441-744,l Department of Food Technology, Dongeui Technical Junior College, Pusan 614-715,2 and Department of Genetic

Engineering, Kyung Hee University, Suwon, 449-701,3 Korea

Received 17 June 1996/Accepted 15 October 1996

The effect of cbitosan on indirubin production by Polygonum tinctorium cells cultured in shake flasks was investigated. The optimum concentration of cbitosan for indirubin production was found to be 200 mg/l, at which the specific indirubin concentration (indirubin concentration per unit weight of cells) was 5.17 mg/g dry cell weight (DCW). The optimum period of elicitation was 5 d in the presence of chitosan. Indirubin production was enhanced by 72% when the cells were cultured in the presence of 200 mg/Z of cbitosan for 5 d using SH medium supplemented with 2.72 mM calcium chloride and 5 mM indole.

[Key words: chitosan, indirubin, Polygonum tinctorium, elicitation]

Indirubin and indigo-related compounds are of sig- nificant interest as natural colorants and for the treat- ment of chronic granulocytic leukemia (l-3). Production of indirubin and indigo-related compounds by plant cell culture is important because intact plants such as Zn- digofera spp., Zsatis tinctoria, Polygonum tinctorium, and Lonchocarpus cyanexens produce the compounds in only small amounts over a l-2 year growth period (4). Maier et al. and Kim et al. studied biotransformation pathways for indirubin and indigo production from in- dole precursors using cultures of P. tinctorium cells (5, 6). Recently Chung et al. (7) reported that the maximum yield of indirubin in indole-supplemented suspension culture of P. tinctorium cells was only 7% of the theo- retical yield, significantly lower than that of P-methyl- digoxin in cell culture of Digitalis lanata.

A wide variety of elicitors have been employed to alter cell metabolism in order to enhance the production of secondary metabolites in plant cell cultures (8-13). Among these elicitors, chitosan (B-1 ,Clinked glucosa- mine) proved to be very effective in suspension culture of Mentha piperita, Vanilla plantfolia and Glycine max cells (9-11). This suggests that indirubin production can be enhanced if such an elicitor is used to suspension cul- ture of P. tinctorium cells. Therefore, in this study, we examined the effect of elicitors on indirubin production by cultured P. tinctorium cells.

Calli of a Korean cultivar of P. tinctorium were in- duced in B5 medium (14) containing 2 mg/l 2,4-D, 1 g/l yeast extract, and 0.8% agar as described previously (15). Suspension cells of P. tinctorium were established and maintained in the same B5 medium without agar. SH medium (16) with 0.2 mg 2,4-D and 1 g yeast extract was used for suspension cultures. The initial pH of these media was adjusted to 5.7 before autoclaving. Indole dissolved in ethanol was used as a stock solution. In all experiments, indole was added to the SH medium to the final concentration of 5 mM.

Nigeran, pectin, chitosan and pectinase were pur- chased from Sigma Chemical Co. The chitosan was purified by a method described elsewhere (9). Briefly, it

* Corresponding author.

was dissolved in 90 ml of 0.1 N acetic acid, and the solu- tion was centrifuged for 30min. Then, the insoluble fractions were discarded. This procedure was performed four times. After centrifugation, the supernatant was precipitated by adjustment of its pH to 8.0 with 5 N NaOH. The precipitates were washed extensively with distilled water and then freeze-dried.

The purified chitosan was dissolved in 0.1 N acetic acid (1 g chitosan/90 ml acetic acid) and the pH of the solution was adjusted to 5.0. Chitosan, nigeran or pectin was added to the SH medium to an appropriate concen- tration before autoclaving. Pectinase was filter-sterilized before addition to the SH medium. The amounts of the elicitors used were 50mg/l for nigeran, lOOmg/f for pectin, 50-400 mg/l for chitosan, and 10 U/I for pectinase. Cultivation was performed at an agitation rate of 100 rpm and 27°C in 500ml round-bottomed shake flasks with a working volume of 200 ml.

The suspension cells were collected by filtration, washed twice with distilled water, and dried at 70°C for 24 h for dry cell weight (DCW) determination. For in- dirubin analysis, the cells were collected by vacuum-filtra- tion (Whatman no. 41), dried, and immersed in CHC13 solution for 24 h. The red-colored extract was filtered (polyvinylidene fluoride, 0.2 pm pore size, German Science Inc.). The filtrate was concentrated under vacu- um using a rotary evaporator, and the concentrate was dried at 80°C for 30 min. The absorbance of indirubin at 535 nm was measured (Pye Unicam PU8600 UV/VIS spectrophotometer, Philips) and converted to indirubin concentration.

A set of experiments was conducted to determine the effects of various elicitors on the level of indirubin production in suspension cultures of P. tinctorium cells. We determined the appropriate elicitor concentrations based on the data obtained from the literature (8-12). The cells were cultured in the presence of chitosan (100 mg/f), pectinase (lOU/t), nigeran (50mg/f), or pectin (lOOmg/Z) for 30 d. As shown in Fig. 1, pectin does not induce an increase in the rate of accumulation of indirubin in suspension culture of P. tinctorium cells. We found that chitosan was the best among the elicitors tested in terms of indirubin concentration. On the other

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VOL. 83, 1997 NOTES 207

con chi pet ptn nig

Ekitor

FIG. 1. Effects of various elicitors on indirubin production in suspension cultures of P. tinctoriwm cells. Cultivation time: q , 10 d; q , 20 d; q , 30 d. con, Control: chi, chitosan; pet, pectinase; ptn, pectin; nig, nigeran.

hand, pectinase and nigeran were suboptimal, although they are effective for increasing the yields of secondary metabolites in cultures of Lithospermum erythrorhizon and Solanum meiongena cells (17, 18). Our results are in good agreement with those obtained by Chang and Lee for chitosan effects on secondary metabolite production by Mentha piperita cells (9).

Several runs were performed to determine the effect of chitosan at various concentrations on indirubin produc- tion in suspension cultures of P. tinctorium cells. The specific indirubin concentration (indirubin concentration per unit weight of cells) at 30 d increased as the chitosan concentration was increased from 50mg/l to 2OOmg/l, whereas that in the case of a chitosan concentration of 400 mg/l was lower than that of 2OOmg/2 (Fig. 2), sug- gesting that 2OOmg/l is the optimum concentration of chitosan for indirubin production by P. tinctorium cells. At this chitosan concentration the specific indirubin concentration was 5.17 mg/g DCW. Furthermore, our results show that the dose-dependent response curve for chitosan treatment during the cell culture was a satura- tion curve. This tendency is similar to that reported for induction of enzymes involved in phenylpropanoid metabolism by chitosan in Vanilla planifolia (10).

For determination of the effect of changes in elicita-

0 50 100 200 400 Chitosan concentration (me//)

FIG. 2. Effect of chitosan at various concentrations on indirubin production in suspension cultures of P. tinctorium cells. Cultivation time: @, 10d; 0, 20d; q , 30d.

1 3 5 30 Duration of cultivation (d)

FIG. 3. Effect of changes in duration of cultivation in the presence of chitosan on indirubin production in suspension cultures of P. tinctorium cells. Total cultivation time: q , 10 d; q , 20 d; q , 30 d.

tion period on indirubin production by P. tinctorium cells, 2OOmg/f of chitosan was added to the medium at the start of culture for 1, 3, 5 or 30 d, after which the cells were collected by filtration, washed, and transferred into flasks containing conditioned medium without chito- San. The conditioned medium was the supernatant ob- tained from centrifugation of cells grown identically in medium lacking chitosan. Cultivation was then con- tinued until total cultivation time of 30 d. Figure 3 shows that the amount of indirubin produced varied with the duration of incubation in the presence of chito- San. The specific indirubin concentration in the cells cul- tured in the presence of chitosan for 5 d was 3.90mg/g DCW, higher than that for any of the other durations of cultivation in the presence of chitosan. This reveals that the duration of cultivation in the presence of chitosan was rather important with respect to indirubin produc- tion.

Experiments were carried out to determine the optimal concentrations of CaC12, MgS04, and NaH2P04 in SH medium for indirubin production in the presence of chitosan by P. tinctorium cells. The data in Table 1 show that concentrations of MgS04 and NaH2P04 as in SH medium (1X) are optimal for indirubin production. In case of CaC&, two-fold concentration (addition of calcium chloride to SH medium to a final concentration of 2.72mM) resulted in a 16% increase in the level of indirubin production, compared to the level when the SH medium (1X) was used.

For determination of the effect of intermittent addi- tion of chitosan to the SH medium on indirubin produc- tion by P. tinctorium cells, 50mg/l of chitosan was added to the flasks four times, on days 0, 5, 10, and 15 while a control was prepared by adding 2OOmg/l of chitosan to a flask at the start of the culture. Figure 4 shows that the specific indirubin concentration in the case of the intermittent addition was slightly less than that in the case of the control, indicating that addition of 2OOmg/l of chitosan at one time is a better method for indirubin production. It should be pointed out that the specific indirubin concentration at the late stage of the experiment performed under the optimal conditions (Fig. 4) was somewhat lower than that at the initial stage of the experiment performed under the nonoptimal con- ditions (Fig. 1). This might have been due to differences in cell viability for specific concentration determination

208 KIM ET AL. J. FERMENT. BIOENG.,

TABLE 1. Effects of inorganic salts at various concentrations on indirubin production in suspension cultures of P. tinctorium cells

Salt strength Indirubin (mg/g DCW)

0 1/2x 1x 2x

CaC& 3.077 2.623 3.202 3.699 MgSG4 1.205 2.449 2.533 2.475 NaH,POI 1.104 1.374 1.508 1.438

Treatments: 0, l/2 x , 1 x , and 2 x of original SH medium.

or to cell line variability during the course of the experi- ment.

Another experiment was performed to investigate the chitosan effects on indirubin production under optimal conditions. The cells were cultured in the presence of 200 mg/f of chitosan for 5 d in SH medium supplement- ed with calcium chloride to a concentration twice that in SH medium (1X). The specific indirubin concentration at this condition was 72% higher than that for the cul- tured cells in the SH medium (1X) without chitosan (non- elicited cells). This value (72% increase) is lower than that (496% increase) for hernandulcin production in chitosan-treated Lippia dulcis hairy root culture (19). However, our result appears to be promising, consider- ing that chitosan is not effective for inducing benzo- phenanthridine synthesis in Eschschoftzia cell culture (20). The overall conclusion from the results of this study is that an chitosan-elicited P. tinctorium cell cul- ture produces much higher levels of indirubin than does a non-elicited P. tinctorium cell culture.

This work was supported by Grants from the Korea Science and Engineering Foundation through the Research Center for New Bio- Materials in Agriculture.

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