VNU Joumal of Science, Natural Sciences and Technology 23 (2007) 269-274

Effecting of medium composition on biomass and ginsenoside

production in cell suspension culture of Panax vietnamensis
Ha et Grushv.

Nguyen Trung Thanh1’* , Nguyen Van Ket2, Paek Kee Yoeup3

'Department o f Bioỉogy, Colỉege o f Science, VNU, 334 Nguy en Trai, Hanoi, Vietnam
1Department o f Agroforestry, Dalat University
^Department o/Horỉiculíure, Chungbuk National ưniversity, Cheongịu, South Korea

Received 15 August 2007

Abstract. We established cell suspension culture on Panax vietnamensis and some attempts have
been made to increase ginsenoside yield of ginseng cell culture through manipulation various
culture íactors and process variable. Half and fiill strength MS medium were found to be equally
suitable for both biomass as well as ginsenoside production. The biomass production and
ginsenoside yield vvere obtained 9.8 g/L DW and 6.81 mg/g DW, respectively. The eíĩect of initial
sucrose concentrations were also investigated in suspension cultures of p. vietnamensis for
biomass and production of ginseng saponin (secondary metabolite). The íĩnal dry ccll weight was
increased from 5.4 to 10.3 g/L with an increase of initial sucrose concentration from 20 to 50 g/L,
but an even higher sucrose concentration of 60 g/L seemed to repress the ccll growth, íiirther
increase of sucrose concenừation upto 70 g/L led to a decrease in ginsenoside accumulation and
biomass production. The maximum growth and ginsenoside production was obtained for nitrogen
concentration of 30 mM.
Kev\vords: MS strength, sucrose, nitrogen, auxin, and cytokinin.

1. Introduction treatment of many serious diseases and for

enhancing body strength in long joumeys in
Vietnamese ginseng was found at highland high mountains.
o f Central Vietnam in 1973, and was regarded The demand for ginseng has increased
as a new species as Panax vietnamensis Ha et dramatically worldwide and ginseng becomes
Grushv. (1985). This is the most Southern very expensive because o f its long-term
distribution o f Panax genus (Araliaceae). It is a conventional (5-7 years) and troublesome
secret medicine o f the Sedang ethnic group as a production cycles. The annual tumover of
miraculous, life-saving plant drug used for the ginseng in the United States was $98 million
with a growth rate o f 26% [1]. Thereíore, plant
• Tác giả liên hệ. ĐT: 84-4-8582178. cell and tissue culture methods have been
E-mail: thanhntsh@ gm ail.com
269
270 N .T . T h a n h e t al. / V N U Ị o u m a l o f S á e n c e , N a tu r a l S c ie n c e s a n d T e c h n o lo g y 23 (2 0 0 7 ) 2 6 9 -2 7 4

explored as potentially more efficient 2. M ate rials and m ethods

altematives for the mass production o f ginseng
and its active components. Research into Induction o f callus
ginseng cell and tissue culture started in the
Fresh mountain ginseng roots were
early 1960s and commercial application has
collected from Ngoe Linh mountain, Quang
undenvay since the late 1980s. The powder and
Nam province. Selected root were washed with
extracts from ginseng cell culture were used to
a detergent solution for 5-10 min and then
make health foods, drinks and cosmetics. The
rinsed with running tap water for 5-10 min.
ginseng culture has continued to attract
They were rinsed with sterilized water after
considerable research and development effort in
being soaked in 70% aqueous EtOH for 0.5-3
recent years and scientists seek to understand min under reduced pressure, íurther sterilized
and optimize the culture conditions [2]. with 1% sodium hypochloride for 10-30 min,
As earlier reported [3] that p. ginseng callus and then rinsed repeatedly with sterile distilled
produces almost the same pharmacologically water. The sterilized roots were cut into
active saponins, ginsenosides as that of sections o f 2-10 mm and then were inoculated
cultivated ginseng root. In a 30-liter jar into MS solid medium (Murashige and Skoog,
fermentor culture, the increase o f the grovvth 1962) containing 30 g/L sucrose, 1 mg/L 2,4-D,
ratio and dry weight were not accompanied by and 0.1 mg/L kinetin. After 1 month callus
an increase o f the saponin content. Using MS were induced. The callus were subcultured into
medium minus NH 4NO 3 and plus 0.5% glucose above medium after every 20 days for
and 2% sucrose and 2% sucrose added after 2 proliferation o f callus. After 5 times of
weeks o f culture resulted in a higher growth subculture into the solid medium the callus
ratio and higher dry weight than using regular were inoculated into liquid medium (same with
MS medium containing 3% sucrose. above).

Effects o f application sole nitrate (NOj ) Stock cell culture and culture condỉtion
and in combination with ammonium (N H /) on
Suspended cells o f p. vietnamensừ were
production of ginscng saponin and
initiated through callus induction from the
polysaccharides by suspension cultures of
cultivated plant root [5]. The cell line was
Panax ginseng were observed by [4]. The
maintained in MS liquid medium supplemented
results indicated that the specific production
with 3 mg/L indole-3-butyric acid (IBA), 0.1
(content) o f ginseng ỊK)lysaccharide was not
mg/L o f kinetin and 30 g/L sucrose. The pH
signiíicantly affected by alteration o f the N
was adjusted to 5.8 before autoclaving.
source and the saponin production was
Cells were cultivated in 300 ml conical
relatively higher within the initial N
ílasks w ith a working volume 100 ml on a
concentration o f 5 mM with nitrate alone or a
rotary shaker in darkness at a rotation speed of
(N 0 3)/(NH 4+) ratio o f 2:1.
105 rpm and a culture temperature o f 25°c.
In this paper, we established cell suspension Cells cultivated for 15 days were used in the
culture o f ginseng ccll and some attempts have experiment and the inoculum size 6 g/flask
been made to increase biomass and ginsenoside (fresh weight). The other cultural conditions
yield o f Ngoe Linh ginseng cell culture. were done as described by [6].
 N .T . T h a n h e t al. / V N U ịo u r n a l o f S cien c e, N a tu r a l S c ie n c e s a n d T e c h n o lo g y 2 3 (2 0 0 7 ) 2 6 9 -2 7 4 271

Determination and analyses Table 1 shows the effects of different

Extraction and determination o f ginsenoside strength o f MS medium on biomass and
production were determined as reported ginsenoside production. H alf and full strength
previously [5,6]. MS medium were found to be equally suitable
Experimental design and data analysis for both biomass as well as ginsenoside
All experiment were repeated three times production. The highest biomass production
with 3 replicates. Data were subjected to and ginsenoside yield were obtained 9.8 g/L
Duncan’s multiple range test using SAS DW and 6.81 mg/g DW, respectively. High salt
program (Version 6.12, SAS Institute Inc., strength (2.0) inhibited a cell growth and
Caiỹ, USA). ginsenoside production accumulation. Such a
phenomenon was also described in provious
3. R esults and dỉscussion cultures o f Panax ginseng adventitìous roots [7].

1. Effects different strength o f MS medium

on biom ass and ginsenoside production

Table 1. EíTect of different strength of MS međium on biomass and ginscnosidc production

MS medium Fresh wt. Dry wt. Ginsenoside (mg/g DW)

concentration (g/L) (g/L) (g/L)
Rg Rb Total
0.5 153 az 9.5 a 2.39 4.42 6.81
1.0 162 a 9.8 a 2.27 4.39 6.66
1.5 120 b 7.3 b 1.95 3.88 5.83
2.0 89 c 5.4 c 1.52 2.92 4.42
2Mean separation by Duncan’s multiple range test at p < 0.05

2. Effect o f different sucrose concentrations relatively high sucrose level was beniíĩcial to
on cell growth and ginsenoside production secondary metabolite synthesis [8]. For
The effect o f initial sucrose concentration example, [9] reported that the triacylglycerol
(0, 20, 30, 50, 60 and 70 g/L) was also content o f the cells o f oil seed rape could be
investigated in suspension cultures o f p. increase about 8-fold on a fresh weight basis
vietnamensis for biomass and production of when sucrose concentration in the growth
ginseng saponin (secondary metabolite). The medium was raise from 2 to 22% (w/v). [10,
íĩnal dry cell weight was increased from 5.4 to 11] found that the optimal concentration of
10.3 g/L with an increase o f initial sucrose sucrose for cell growth was between 30 and 50
concentration from 20 to 50 g/L, but an even g/L and upto 70 g/L sucrose inhibited cell
higher sucrose concentration o f 60 g/L seemed growth, while the ginsenoside content shovved a
to repress the cell grovvth. Further increase of steady increase with sucrose concentration of
sucrose concentration upto 70 g/L led to a upto 60 g/L. Based on our results it can be
decrease in ginsenoside accumulation and concluded that high sucrose level and secondary
biomass production (Table 2). On the contrary metablite production is not a general
of our results, several authors suggested that a phenomenon and depends on plant species.
272 N .T . T h a n h e t a i / V N U Ị o u m a l o f S cien c e, N a tu r a l S à e rtc e s a n d T e c h n o lo g y 2 3 ( 2 0 0 7 ) 2 6 9 -2 7 4

Table 2. EÍTect of diíĩerent sucrose concentrations on ccll growth and ginsenoside production

Sucrose Fresh wt. Dry wt. Ginsenoside (mg/g DW)

concentr. (g/L) (g/L) (g/L)
Rg Rb Total

N
O n
0 00 5.4c 1.52 2.92 4.42
20 u
158a 9.6a 2.32 4.31 6.63
30 165a 9.9a 2.95 4.01 6.96
50 171 a 10.3a 2.13 4.69 6.82
60 134b 8.1b 1.49 3.42 4.91
70 93c 5.6c 1.25 2.81 4.06
zMean separation by Duncan’s multiple range test at p < 0.05

3. Effect o f đifferent nitrogen concentration weight) in various cultures is shown in (Table

on cell growth and ginsenoside production 3). It is apparent that growth was inhibited at a
The effect o f the initial nitrogen high initial N concentration. The highest dry
concentration in the medium for cell growth weight reached 10.2 g/L at an initial nitrogen
and metabolite production was studied in p. concentration o f 30 mM. The highest
vietnamensis cell cultures. The initial nitrogen ginsenoside production was (7.35 mg/g DW) at
level was adjusted to 0, 10, 30, 60, 90 and 120 initial medium nitrogen concenừation of 30
mM. The kinetics o f growth (based on dry mM after 25 days o f culture.

Table 3. EíTect of diíĩerent niứogen concentration on cell growth and ginsenoside production

Nitrogen Fresh wt. Dry wt. Ginsenoside (mg/g DW)

conccnt. (mM) (g/L)
Rg Rb Total
0 79cz 5.2c 1.47 2.81 4.28
10 122b 8.1b 2.25 4.33 6.58
30 176a 10.2a 2.81 4.54 7.35
60 156a 10.la 2.52 4.59 7.11
90 119b 7.9b 1.95 4.02 5.97
120 86c 5.4c 1.21 3.34 4.55
zMean separation by Duncan’s multiple range test at p < 0.05
In cell cultures o f p. quinquefolium, [12] maximum production o f ginseng saponin and
reported that the íinal dry cell weight was polysaccharide obtained (1.5 g/L and 2.19 g/L)
relatively low with the low nitrogen at the initial niừogen concentration o f 40 mM
concentration. Maximum cell dry weight [12]. In the simultaneous production of ginseng
obtained (15 g/L) at a total initial nitrogen saponin and polysaccharide by suspension
concentration o f 40 mM and the cell growth cultures o f p. ginseng, [4] reported that
was inhibited at a high initial nitrogen production of ginseng saponin was related with
concentratìon o f 80 mM. Similarly, the the total nitrogen concentration. The result
accumulation of total saponin and suggested that a low nitrogen concentration was
polysaccharide were also iníluenced by initial beneficial for the stimulation o f total saponin
nitrogen concentration in the medium. The production.
 N .T . T h a n h e t al. Ị V N U Ị o u r n a l o f S cie n c e , N a tu r a l S c ie n c e s a n d T e c h n o lo g y 23 ( 2 0 0 7 ) 2 6 9 -2 7 4 273

Acknovvledgments Science, Natural Sciences and Technology 23,

No. 1S (2007) 167.

This work was supported by grants from the [6] N.T. Thanh, L .v . Can, K.Y. Paek, The
Department o f Science and Technology, adventitious root cultures o f Ngoe Linh ginseng
(Panax vietnamensis Ha et Grushv), Proceeding
Vietnam National University Hanoi
of N ational Conference on Life Sciences,
(QG.06.14), and Basic Research Program in
Vietnam, 2007, pp 828-831.
Life Sciences, Ministry o f Science and
[7] K .w . Yu, Production o f the useful metabolites
Technology (6.090.06) to Hanoi University of
through bioreactor culture o f Korean ginseng (p .
Science, Faculty o f Biology. The authors are
ginseng c . A. Meyer). Ph.D. thesis, Chungbuk
also gratefưl to Dr. Niranjana H. Murthy for
National University, South Korca, 2000.
reading English manuscript.
[8] c .o . Akalezi, s. Liu, Q.s. Li, J.T. Yu, J.J.
Z hong> Com bined effects o f initial sucrose
concentration and inocuỉum size on ccll grovvth
R eíerences and ginseng production by suspension cultures
o f p . ginseng. J. Pro Biochem. 34 (1999) 639.
[1] B.K. Voglcr, M.H. Pittler, E. Em st, The cfficacy [9] R.J. W cselake., S.D. Bycrs, J.M. Davoren, A.
o f ginscng: a system atic review o f random ised Larochc, D.M. Hodges, M.K. Pomcroy and T.L.
clinica) trials, European J. of Cỉinical Furukaw a-Stoffer, Triacylglyccrol biosynthesis
Pharm acoỉogy 55 (1999) 567. and genc cxpression in m icrosporc derived cell
[2] J. W u, J.J. Zhong, Production o f ginscng and its suspension cultures o f oilseed rape, J. Exp. Bot
bioactive com ponents in plant ccll culturc: 4 9 (1 9 9 8 )3 3 .
currcnt technoìogical and applicd aspccts, [10] K.T. Choi, C.H. Lee, 1.0. Ahn, J.H. Lee, J.c.
J. Biotechnology 6 8 (1 9 9 8 ) 89. Park, Characteristics of the growth and
[3] T. Furuya, T. Yoshikavva, Y. Orihara, Oda ginscnosidcs in the suspension culturc cells o f
Hirohiko, Studies o f Ihe culturc conditions for Korcan ginseng (P. gìnseng C.A. Meyer). In
Panax ginseng cells in ja rs ĩerm entors, W .G. Bailey, c . W hitehead, J.T.A. Proctor, J.T.
J. N atural Products 47 (1984) 70. Kyle. (eds), Proce Int G inseng Con., Vancouver,
[4] s. Liu, J.J. Zhongt Sim ultaneous production o f 1994, pp. 259-268.
ginscng saponin and polysaccharide by [11] K.T. Choi, 1.0. A hn, J .c . Park, Production o f
suspension cultures o f Panax g in sen g : Nitrogen ginseng saponin in tissue culturc o f ginscng (P.
effccts, J. Enzym e and M icrobial Technology 21 ginseng c . A. M eyer), Russ. J. Plant Physioỉ. 40
(1 9 9 7 )5 1 8 . (1994)784.
[5] N.T. Thanh, L.T. Son, K.Y. Paek, Induction and [12] J.J. Zhong, S.J. W ang, E íĩccts o f nitrogcn source
proliferation o f callus o f N goe Linh ginseng on the production o f ginseng saponin and
(P anax vieínamensis H a et G rushv): E íĩects o f polysaccharide by cell cultures of p.
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274 N.T. T h a n h t i aỉ. / V N U Ị o u m a l o f S c ie n c e, N a tu r a l S c ie n c e s a n d T e c h n o lo g y 2 3 ( 2 0 0 7 ) 2 6 9 -2 7 4

Ảnh hưởng mồi trường nuôi cấy đến sự tăng trưởng sinh khối
và sự tích lũy sản phẩm ginsenoside trong nuôi cấy tế bào lỏng
của Sâm Ngọc Linh (Panax vietnam ensỉs Ha et Grushv.)

Nguyễn Trung Thành1, Nguyễn Văn Kết2, Paek Kee Yoeup3

^Khoa Sinh học, Trường Đại học Khoa học Tự nhiên, ĐHQGHN, 334 Nguyễn Trãi, Hà Nội, Việt Nam
2Khoa Nông lăm, Trường Đại học Đà Lạt
*Khoa Cây trồng, Đại học Quốc gia Chungbuk, Cheongịu, Hàn Quốc

Để sản xuất sinh khối và sản phẩm trao đổi chất thứ cấp ginsenoside, các thí nghiệm nuôi cấy tế
bào lỏng cùa Sâm Ngọc Linh (Panax vietnamensis Ha et G nishv.) đã được tiến hành với các thành
phần khác nhau cùa môi trường nuôi cấy. Đổi với nồng độ môi trường MS cho thấy với tỷ ]ệ 50 hoặc
100% là thích hợp cho sự tích luỹ sinh khối tế bào và sự tổng hợp sản phẩm thứ cấp ginsenoside.
Nồng độ đường trong môi trường nuôi cấy cũng được thay đổi, kết quà cho thấy 30 g/L là thích hợp
cho sự tích luỹ sinh khối tế bào và sự tổng hợp sản phẩm ginsenoside. Sinh khối khô tăng từ 5.4 đến
10.3 g/L khi tăng nồng độ đường từ 0 đến 50 g/L. Tiếp tục tăng nồng độ đường sẽ kìm hãm sự sinh
trường tế bào cũng như sự tổng hợp ginsenoside. Tương tự, ờ nồng độ 30 mM nitrogen là tối ưu cho
sự sinh trưởng tế bào và sự tích luỹ sản phẩm trao đổi chất thứ cấp ginsenoside.
Từ khóa: Nồng độ môi trường MS, đường, nitơ, auxin và cytokinin.