87AsPac J. Mol. Biol. Biotechnol., Vol. 13 (2), 2005 Production of Saponin in Submerged Ginseng Root CultureAsia Pacific Journal of Molecular Biology and Biotechnology, 2005Vol. 13 (2) : 87-91

*Author for Correspondence.Mailing address: Hoon Il Oh, Dept. of Food Science and Technol-ogy, Sejong University, 98 Kunja-dong, Kwangjin-ku,Seoul 143-747, Korea. Tel: 822-3408-3229; Fax: 822-3408-356;Email: ohhi@kunja.sejong.ac.kr

Saponin Production in Submerged Adventitious Root Culture ofPanax ginseng as Affected by Culture Conditions and Elicitors

Jung Hea Kim, Eun Jung Chang and Hoon-Il Oh*

Department of Food Science and Technology, Sejong University98 Kunja-dong, Kwangjin-ku, Seoul, 143-747, Korea

Received 22 November 2005 / Accepted 30 December 2005

Abstract. Root growth and saponin production in the root culture of Panax ginseng C. A. Meyer were investigated undervarious pH values, concentrations of sucrose, nitrogen and phosphate and elicitors. The pH of a medium did not have asignificant effect on root growth, but apparently affected the saponin content. The maximum saponin content of 0.26 %was obtained at pH 6.0. The optimal concentrations of sucrose, nitrogen and phosphate on root growth and saponinproduction were 30 g/l, 382.7 mg/l and 40.40 mg/l, respectively. Saponin biosynthesis was stimulated by the addition ofpanaxatriol saponin (PTS) and total saponin (TS). However, fresh weight in PTS was so low that the theoretical totalsaponin in TS treatment was higher than that in PTS treatment. Thus, it appears that TS is a better elicitor than PTS in theginseng root culture. The saponin content in TS treated ginseng root was 0.38 % and approximately 1.5-fold higher than thecontrol. This work will be helpful for efficient large-scale bioprecessing of the ginseng root culture in a bioreactor.

Keywords. Panax ginseng C.A. Meyer, adventitious root, saponin, culture condition, elicitor

INTRODUCTION

Ginseng, the root of Panax ginseng C. A. Meyer, is avaluable Asian herb. It has been reported that ginseng hasmany benefical bioactive effects on human health, such asantitumor, antistress, antiaging and enhancing immunefunctions, although only a few of these have been clinicallyconfirmed (Akalezi et al., 1999). Until now, ginseng has beenreported to contain saponins, antioxidants, peptides,polysaccharides, alkaloids, lignans and polyacetylenes.Saponins, known as ginsenosides, are the principal bioactiveingredients (Jo et al., 1995; Sticher, 1998, Palazon et al., 2003).In recent years, ginseng has been increasingly used as a healthtonic formulated into a variety of commercial health products,including ginseng capsules, soups, drinks and cosmetics,which are marketed in the Asian as well as many othercountries around the world.

The study of ginseng culture started in the early 1960’s,and numerous studies were reported within the following 10years (Wu and Zhong, 1999). Most of these early studiesdealt with basic culture techniques, such as callus and cell lineinductions, and culture maintenance. They also dealt withthe chemical composition of the ginseng tissues and cellsobtained from in vitro cultures.

Bioprocess optimization and the scale-up of the planttissue and cell cultures require an understanding of nutrient

requirements for the production of cell mass and secondarymetabolites. Previous studies of ginseng culture have mainlyfocused on medium compositions (Akalezi et al., 1999; Zhangand Zhong, 1997; Zhang et al., 1996). There is a lack ofinformation of medium nutrients and elicitors on ginsengtissue culture for useful saponin production. In the presentwork, we studied the effects of initial pH, sucrose, nitrogenand phosphate concentrations of the medium and elicitorson the growth and saponin formation in root culture of P.ginseng for optimization of culture conditions.

MATERIALS AND METHODS

Plant material, medium and culture conditions. Panaxginseng C. A. Meyer seeds were obtained from the KoreaGinseng and Tobacco Research Institute Experimental Station(Suwon, Korea) and were surface-sterilized with 70 %(v/v)ethanol for 20 sec, immersed in 4 % sodium hypochlorite for15min, and finally rinsed five times with sterilized distilled

88 AsPac J. Mol. Biol. Biotechnol., Vol. 13 (2), 2005 Production of Saponin in Submerged Ginseng Root Culture

water. The initial culture was prepared by inoculating decoatedand dissected seeds onto a petri dish and incubated at 25°C inthe dark for three months. A solid Murashige and Skoog(MS) medium (Murashige and Skoog, 1962) withsupplemented 0.5 mg/l of 6-benzyl-aminopurine (BAP), 3.0mg/l of 1-naphthaleneacetic acid (NAA) and 30 g/l of sucrosewas used. Growth regulators used in this study were allpurchased from Sigma Chemical Co. (St. Louis, MO, USA).The pH of the medium was adjusted to 5.8 before autoclavingfor 15 min. at 121 °C. When the callus was induced, it wastransferred to the solid Schenk and Hildebrandt (SH) medium(Schenk and Hildebrandt, 1972) supplemented with 0.5 mg/l BAP, 3.0 mg/l NAA and 30 g/l sucrose for induction ofroot. The callus was incubated at 25 °C in the dark andsubcultured every four weeks. Adventitious roots from thecallus were obtained after three months. To induce mutipleadventitious roots from in vitro originated roots, each rootwas isolated from the callus tissue and cut into 1.0 cm inlength and inoculated into 100 ml Erlenmeyer flask containing40mL of the aforementioned liquid SH medium. The cultureswere maintained for more than three months in liquid SHmedium by shaking (60 rpm) under darkness at 25 °C beforeusing as a source for this study. Subculture was routinelyconducted at four weeks interval. For the study of the effects of the initial pH and nutrients,the medium pH was adjusted to pH 5.0, 5.5, 6.0, 6.5 and 7.0before autoclaved. Concentrations of sucrose, nitrogen andphosphate were ranged from 1~9 %, 127.6~1148 mg/l and26.9~242.1 mg/l, respectively. Six hundred mg of fresh rootsof 1.0 cm in length were transferred to a 100 ml Erlenmeyerflask containing 40 ml medium and cultured at 25 °C on ashaker at 60 rpm in the dark. Fresh weight and saponincontent were measured after 4 weeks.

Preparation and use of elicitors. Total saponin (TS),panaxadiol saponin (PDS) and panaxatriol saponin (PTS)were obtained from the Korea Ginseng and Tobacco ResearchInstitute (Taejeon, Korea) and water soluble chitosan was agift from Professor Jeon Dong Won at Ewha WomansUniversity (Seoul, Korea). To prepare the ginseng water extract,5-year-old fresh ginseng was purchased from a local retailstore, extracted with water at 75 °C for 24 hours and filter-sterilized before use. Each ginseng water extract, TS, PDSand PTS was added to the medium at the final concentrationof 0.1 %, and chitosan was added with a final concentrationof 0.0025 %.

Measurement of fresh weight and saponin content. Forthe measurement of the fresh weight of ginseng roots, theroots were filtered and washed with distilled water, thencollected and weighed. Ginseng saponin was extracted asdescribed by Shibata et al. (1966) and determined by vanillin-H2SO4 colorimetric method (Kim and Lee, 1978). Theoreticaltotal saponin is a value in that fresh weight is multiplied bythe saponin content.

Analyses of ginsenosides. Ginsenosides were extractedaccording to the procedure described by Shibata et al. (1966).The ginseng root was extracted with 80 % methanol for 3hours. Following the filtration, the extract was evaporated todryness and sequentially extracted with ethyl ether and butanolsaturated with H2O. The butanol extract was evaporated todryness and then dissolved in 1mL of methanol. The extractwas analyzed by HPLC as described by Han and Han (1995).The HPLC system consisted of Waters 600 pump and acarbohydrate column (Waters Assoc. Milford, MA, USA; 300× 7.8 mm, 10 µm). A gradient elution system of acetonitrile(A) and H2O (B) was used [20 % A (0 min); 20 % A (35 min);30 % A (36 min); 30 % A (80 min)]. The initial flow rate ofthe mobile phase was 1.5 ml/min from 0 to 35 min. Theflow rate was then changed to 2.0 ml/min between 35 and 36min and maintained at constant 2.0 ml/min, thereafter. Forthe analysis, 20 µl of the sample was injected into the systemand the ginsenoside peaks were detected by a UV detector at203 nm.

RESULT AND DISCUSSION

The morphology of the ginseng adventitious root. Theginseng callus was preferentially induced from the ginsengseed, and then the ginseng adventitious root was obtainedfrom the callus as described above. The morphology of theginseng adventitious root was thicker than the hairy rootinduced by Agrobacterium rhizogenes (Yoshikawa and Furuya,1987) and thinner than the cylinder-type root (Chi et al., 1989).Newly developing parts of the ginseng adventitious rootwere thin and yellowish, and became thick and brownish asthe incubation time increased.

Effect of the initial pH of the medium on saponinproduction. To investigate the effect of the initial pH of themedium on growth and saponin production in the ginsengroot culture, the roots were grown in media at different pHvalues, ranging from 5.0 ~ 7.0. Figure 1 shows that the pHof medium did not have a significant effect on fresh weightof the root, but apparently affected the saponin content. Themaximum saponin content reached to 0.26% at pH 6.0. Thesaponin contents of the ginseng root grown at a relativelyhigh pH of 6.0~7.0 was higher than those grown at pH 5.0and 5.5. This result was similar to that of Hwang et al. (1991)who reported that the optimal pH in culture of ginseng hairyroot was pH 5.5 ∼ 6.5.

Effect of sucrose concentration on saponin production.Sucrose is the common carbon source for the plant tissue andcell culture, serving as a principal energy source and acomponent for biosynthesis. The rate of biomass growth isusually directly correlated with sugar consumption (Wu andZhong, 1999). Therefore, we have tried to determine the

89AsPac J. Mol. Biol. Biotechnol., Vol. 13 (2), 2005 Production of Saponin in Submerged Ginseng Root Culture

optimum sucrose level of the medium for the optimal growthand saponin production of ginseng root. As shown in Figure2, an increase in sucrose level from 1 to 3 % remarkablyenhanced the root growth and saponin content, but furtherincrease in sucrose concentration up to 9 % repressed them.This can be attributed to the high osmotic pressure.Consequently, the most favorable sucrose concentration ofmedium was 3 %, and in this case, the fresh weight andsaponin content were 2.86 g and 0.25 %, respectively. A similarphenomenon was also reported in other ginseng cell cultures.Choi et al. (1994) found that the optimal concentration ofsucrose for cell growth was between 30 and 50 g/l. Insuspension cultures of Panax notoginseng cells, Zhang andZhong (1997) found that the constant or intermittent feedingof sucrose or other sugars was more effective than raising theinitial concentration to increase biomass and secondarymetabolites yield.

Effect of nitrogen concentration on saponin production.A nitrogen (N) source significantly affects the plant cell growthand metabolite formation. Ammonium (NH4

+) and nitrate(NO3

−) were mainly used as nitrogen sources in the plant celland tissue culture (Zhang et al., 1996). It is a general trendthat a lower NH4

+ to NO3− ratio is more favorable for the

plant tissue and cell growth (Wu and Zhong, 1999). Theeffect of nitrogen concentration on the tissue culture of P.ginseng was investigated by changing the nitrogen concentrationto be 127.6, 191.3, 382.7, 765.4 and 1148.0 mg/l respectivelyat constant NH4

+ to NO3− ratio of 1:9.5. The fresh weight,

saponin content and theoretical total saponin tended toincrease up to 382.7 mg/l of nitrogen concentration, butdecreased sharply thereafter (Figure 3). It appears that high

nitrogen concentration could cause adverse effects on the cellgrowth and saponin biosynthesis. Therefore, the mostsuitable concentration of nitrogen was 382.7 mg/l, in allaspects. Our result is different from that in P. notoginseng cellcultures, where low nitrate concentration had a positive effecton the saponin and polysaccharide contents (Zhang et al.,1996). This difference may be due to the different cell linesused.

Effect of phosphate concentration on saponinproduction. Phosphorus is another key nutrient for plantcell growth and secondary metabolite formation (Liu and

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Figure 1. Effects of initial pH of medium on the saponinformation.

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Figure 2. Effects of sucrose concentration on the saponinformation.

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Figure 3. Effects of nitrogen concentration on the saponinformation.

90 AsPac J. Mol. Biol. Biotechnol., Vol. 13 (2), 2005 Production of Saponin in Submerged Ginseng Root Culture

Zhong, 1998). As shown in Figure 4, phosphateconcentration did not have a significant effect on fresh weightof the root. However, the saponin content and theoreticaltotal saponin were significantly enhanced with an increase inphosphate concentration from 26.9 to 40.40 mg/l, and thendecreased markedly with an increase in phosphateconcentrations. In 40.40 mg/l of phosphate concentration,the saponin content and theoretical total saponin were 0.27% and 6.6 mg%, respectively. Similar results were reported inother papers. Zhang and Zhong (1997) found that an increasein initial phosphate concentration from 1.25 to 3.75 mMenhanced both cell growth and saponin yield in suspensioncultures of P. notoginseng cells. For the static callus culture,Choi et al. (1994) found that the saponin content was loweredwith an increase in phosphate concentration from 3.7 to 11mM. Liu and Zhong (1998) also reported that the crucialconcentrations of phosphate were 1.04 and 0.65 mM for thegrowth of P. ginseng and P. quinquefolium and 0.42 and 1.25mM for saponin production, respectively.

Effect of various elicitors on saponin production. Sincethe effects of elicitors on the ginseng tissue culture were notextensively studied, we explored the possibility of increasingthe production of ginseng saponin by adding elicitors to theculture medium. Among the various elicitors, TS and PTSshowed an eliciting effect on the ginseng root cultures (Figure5). The saponin contents in TS and PTS treated ginsengroots were 0.375 and 0.41 %, respectively. These values wereapproximately 1.5-fold higher than the control of 0.25 %.However, fresh weight in PTS was so low that the theoreticaltotal saponin in TS treatment was higher than that in PTStreatment. Thus, it appears that TS is a better elicitor thanPTS in the ginseng root culture. The elicitor-triggered increasein total saponin content was probably achieved by the

enhanced synthesis of different ginsenosides. Table 1 showsthe results of the ginsenoside analysis of ginseng roots grownin the medium with various elicitors added. In the control,the ginsenosede content of panaxadiol saponin (Rb1, Rb2,Rc, Rd) was higher than that of panaxatriol saponin(Re, Rf,Rg1). This result was contrary to that of Mallol et al. (2001)who analyzed ginseng hairy root. When PTS was used as anelicitor, Re, Rf and Rg1 contributed to the increase in thesaponin content, while the changes in the rest of ginsenosideswere insignificant. In contrast, when TS was used, the increasein the saponin content was mainly due to increase in the levelof Rc and Rf. These results suggest that these two elicitorshave different modes of action in the ginseng root culture.Some attempts have been made to increase the saponin yieldof the ginseng tissue culture through metabolic regulation,i.e. with the use of elicitors and precursors of saponinsynthesis. Linsefors et al. (1989) reported a significant increase(nearly 40 %) in the saponin content of ginseng tissue withthe addition of mevalonic acid to the medium. Moreover,

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Figure 4. Effects of phosphate concentration on the saponinformation.

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Figure 5. Effects of various elicitors on the saponin formation.GWE, Ginseng water extract; TS, Total saponin; PDS, Panaxadiolsaponin; PTS, Panaxatriol saponin.

Elicitor Contents of ginsenosides (% on dry basis)

Rb1 Rb2 Rc Rd Re Rf Rg1

Control 0.13 0.01 0.01 0.49 0.25 0.03 0.12GWEa 0.11 0.02 0.003 0.30 0.22 0.04 0.11TSb 0.14 0.02 0.05 0.45 0.27 0.15 0.13PDSc 0.02 0.05 - 0.13 0.03 0.04 0.02PTSd 0.14 0.009 0.05 0.43 0.70 0.52 0.50Chitosan 0.04 0.008 - - 0.11 0.14 0.05

Table 1. Contents of ginsenosides in roots of Panax ginseng liquidculture as affected by the addition of elicitors

aGinseng water extract, bTotal saponin, cPanaxadiol saponin,dPanaxatriol saponin

91AsPac J. Mol. Biol. Biotechnol., Vol. 13 (2), 2005 Production of Saponin in Submerged Ginseng Root Culture

Furuya et al. (1983) found that semicarbazide andthiosemicarbazide, which were assumed to be inhibitors ofthe side reactions of saponin synthesis, could significantlyenhance saponin production in the normal callus culture whenthey were indivisually used along with mevalonic acid.

ACKNOWLEGMENTS

This research was supported in part by a grant form the KoreanSociety of Ginseng, funded by Korea Ginseng and TobaccoCorp., Taejeon, Korea.

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