REVIEW JURNAL

Establishment of cell suspension cultures of Withania somnifera for the production

of withanolide A

OLEH :
VINDHYA TRI WIDAYANTI, STP, MP
PS. DOKTOR ILMU PANGAN/ NIM. 197100100111003

1. Metode Regenerasi sel callus tanaman W. somnifera cv. Jawahar

MS (Murashige and Skoog’s) media

0,8% (b/v) agar Callus tanaman w. somnifera cv.Jawahar
30 g/L sukrosa
2,0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D)
0,5 mg/L Kinetin (KN)

Dibuat media agar dalam cawan

Diinokulasi

Kultur diinkubasi pada incubator dengan suhu 25o±1oC

dengan pencahayaan selama 16 jam (40µmol/m2s)
dengan lampu neon putih 40-W (Phillips, Kolkata, India)

Callus muda yang telah diregenerasi

2. Metode Perbanyakan Sel W. somnifera dengan media cair

MS (Murashige and Skoog’s) media cair Callus muda yang telah diregenerasi
2,0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D)
0,5 mg/L Kinetin (KN)

Diinokulasi

Media cair dalam

Erlenmeyer 250 ml

Kultur diinkubasi pada incubator dengan agitasi

110 rpm suhu 25o±1oC dengan pencahayaan
selama 16 jam (40µmol/m2s) dengan lampu neon
putih 40-W (Phillips, Kolkata, India)

Suspensi Sel Kultur

Disaring dengan kondisi steril menggunakan

saringan dengan ukuran 0,45µm

Media MS Cair

Sel Withania somnifera

3. Penentuan Biomassa Sel

Suspensi Sel Kultur

Disaring dengan kondisi steril menggunakan

saringan dengan ukuran 0,45µm

Media MS Cair

Sel Withania somnifera

Dikeringkan pada suhu 60oC hingga beratnya

konstan

ditimbang

Biomassa Sel Withania somnifera

4. Ekstraksi dan Analisis Senyawa WIthanolide A dengan metode HPLC

1 g Biomassa sel

3 ml MeOH

Sonikasi selama 10 menit, diulang sebanyak 3x

Disentrifuse 3000 rpm selama 5 menit Pelet

Supernatan Ditambah MeOh

hingga 10ml

Analisis senyawa Withanolide A menggunakan

HPLC
KONDISI SEL KULTUR UNTUK EKSPERIMEN
500 mg sel W. Somnifera cv. jauhar

Diinokulasikan pada 50ml media MS cair

VARIABEL BEBAS :
1. Media Growth Regulator
2. Lama inkubasi
3. Konsentrasi Inokulum
4. Jenis Media
5. Salt strength MS Medium
6. Jenis Sumber Karbon
7. Konsentrasi Sukrosa
8. pH

Kultur diinkubasi selama pada incubator dengan agitasi 110

rpm suhu 25o±1oC dengan pencahayaan selama 16 jam
(40µmol/m2s) dengan lampu neon putih 40-W (Phillips,
Kolkata, India)

PENGAMATAN/ RESPON:
1. Biomassa Sel Sel kultur W. somnifera
2. Senyawa Withanolid A
4. SET-UP EKSPERIMEN
4.1 PENGARUH MEDIA GROWTH REGULATOR PADA PERTUMBUHAN SEL W. Somnifera

TAHAP 1 - Untuk mengetahui Efek Media Growth Regulator terhadap Pertumbuhan Sel W.
Somnifera dan Produksi Senyawa Withanolide A

Perlakuan Respon
X1 (Jenis Media X2 (Konsentrasi (mg/L))
Growth Regulator)
2,4-D (2,4- 0,1 Y1 = Jumlah
Dichlorophenoxyacetic 0,5 Biomassa sel
acid) 1,0 Y2 = Senyawa
2,0 Withanolide A
5,0
NAA (Napthalene 0,1 Y1 = Jumlah
acetic acid) 0,5 Biomassa sel
1,0 Y2 = Senyawa
2,0 Withanolide A
5,0
IBA (Indole – 3 butyric 0,1 Y1 = Jumlah
acid) 0,5 Biomassa sel
1,0 Y2 = Senyawa
2,0 Withanolide A
5,0

Statistik Analisis : Menggunakan Rancangan Acak Lengkap, Uji Lanjut dengan DMRT
Dengan menggunakan software SPSS version 9.0

KESIMPULAN SET UP EKSPERIMEN TAHAP 1 :

PERLAKUAN TERBAIK PADA EKSPERIMEN TAHAP 1 ADALAH DENGAN MENGGUNAKAN

MEDIA GROWTH REGULATOR 2,4-D DENGAN KONSENTRASI 2.0 mg/L.
BIOMASSA SEL YANG DIHASILKAN SEBESAR 8,11 g/L (berat kering) DAN PRODUKSI
SENYAWA WITHANOLIDE A TERTINGGI SEBESAR 1,27 mg/g (berat kering) à LANJUT
EKSPERIMEN TAHAP 2
0.1, 0.5, 1.0 and withanolide A production were focused on. The type and con-
)] were as- centration of auxin or the auxin/cytokinin ratio alters dramatically
on cultures both the growth and the product formation in cultured plant cells
periment. A (Mantell and Smith, 1984). The biomass accumulation and withan-
thanolide A olide A content was differing with varied concentration of auxins.
, with sam- The maximum accumulation of biomass (8.11 g L!1 DW) and the dengan
TAHAP 2 - Untuk mengetahui Efek Kombinasi Media Growth Regulator
al inoculum highest production(BAP) of withanolide A (1.27 mg g Pertumbuhan
!1
DW) was ob-Sel W. Somnifera dan
Benzylaminopurine dan Kinetin (KN) terhadap
10.0, and served in the medium supplemented with 2.0 mg L !1
2,4-D. How-
Produksi Senyawa Withanolide A
other set of ever, the highest accumulation of biomass (10.79 g L!1 DW) and
e and Skoog withanolide KOMPOSISI
A (2.26 mg g !1
DW)GROWTH
has beenREGULATOR
observed with the cul-
Nitsch (NN, MEDIA RESPON (Y)
tures supplemented with 2.0 mg L!1 2,4-D + 0.5 mg L!1 KN (Ta-
and various VARIABEL PERLAKUAN
ble 1). These results showed that 2,4-D in combination with
d 2.0) were TERKONTROL X1 (JENIS X2 (KONSENTRASI
kinetin was suitable for withanolide A production when compared
A. The effect CYTOKININS) g/L)
with all other treatments. Similar to the above results the produc-
ctose, malt- 2,0
tionmg/L 2,4-D (2,4- A in Benzylaminopurine
of withanolide 0,1considerably (ca. Y1 = Jumlah
the shoot cultures varied
(1:1) and Dichlorophenoxyacetic
10-fold, 0.014–0.14 mg g(BAP)!1 0,5 to the phyto- Biomassa sel
fresh weight) according
ose concen- acid
hormone composition of the culture media as 1,0well as genotype Y2 = Senyawa
depending used as the explant source (Sangwan et al., 2007). 2,0 They have re- Withanolide A
er sterilized
Kinetin (KN)
ported that 1.0 ppm benzylaminopurine 0,1 0.5 ppm kinetin Y1 = Jumlah
plus
nto various was responsible for highest accumulation 0,5 of withanolide A Biomassa sel
5.8, 6.0 and (14.3 mg per 100 gm fresh weight and 238 mg
um by using
1,0 per 100 gm dry Y2 = Senyawa
weight, i.e., 0.24%) with the shoot cultures of W. 2,0somnifera. Withanolide A
acid (HCl)]
thanolide A
3.2. Growth
Statistik kinetics
Analisis of W. somnifera
: Menggunakan cell suspension
Rancangan culturesUji Lanjut dengan DMRT
Acak Lengkap,
Dengan menggunakan software SPSS version 9.0
Time course accumulation of biomass (dry wt) and production
of withanolide A are presented in Fig. 1. The maximum accumula-
HASIL PENELITIAN TAHAP 2
ssing them
eights were Table 1
nt weight is Withania somnifera cell suspension culture: effect of 2.0 mg L!1 2,4-dichlorophenoxy
acetic acid (2,4-D) in combination with different concentrations of cytokinins on
biomass accumulation and withanolide A production.a,b

Cytokinins Concentration Dry weight Withanolide A

(mg L!1) (g L!1) content (mg g!1 DW)
carried out Benzylaminopurine 0.1 5.48 ± 0.06d 1.36 ± 0.02d
thanolide A (BAP) 0.5 5.76 ± 0.18d 1.42 ± 0.06d
1.0 6.51 ± 0.09c 1.82 ± 0.03b
a Hills, CA,
2.0 6.69 ± 0.06c 1.63 ± 0.02c
Kinetin (KN) 0.1 7.17 ± 0.12b 1.78 ± 0.02b
0.5 10.79 ± 0.05a 2.26 ± 0.01a
1.0 6.74 ± 0.16c 1.81 ± 0.06b
2.0 6.66 ± 0.10c 1.78 ± 0.04b
domized de- a
Cells (0.5 g) were cultured in 50 ml of MS medium for 4 weeks.
d mean val- b
Data represents mean values ± SE of three replicates; each experiment was
using SPSS repeated twice. Mean separation within column by Duncan’s multiple range test at
P 6 0.05.

KESIMPULAN SET UP EKSPERIMEN TAHAP 1 :

PERLAKUAN TERBAIK PADA EKSPERIMEN TAHAP 2 ADALAH DENGAN MENGGUNAKAN
MEDIA GROWTH REGULATOR 2,4-D DENGAN KONSENTRASI 2.0 mg/L. YANG
DIKOMBINASIKAN DENGAN 0,5 mg/L KINENTIN (KN)
BIOMASSA SEL YANG DIHASILKAN SEBESAR 10,79 ± 0,05a g/L (berat kering) DAN
PRODUKSI SENYAWA WITHANOLIDE A TERTINGGI SEBESAR 2,26 ± 0,01a mg/g (berat
kering) à LANJUT EKSPERIMEN TAHAP 3
 yielded maximum biomass (10.88 g L DW) and highest produc-
tion of withanolide A (2.42 mg g!1 DW, Fig. 2). High (20 g L!1) 2
and low (2.5 and 5.0 g L!1) inoculums resulted in poor results. Sim-
ilar results were found in the cell culture of Taxus chinensis, where
1.5–3.0 g DW L!1 favored the total taxol productivity when com- 0
pared with the ranges of 1.5 to 6.0 g DW L!1 (Pan et al., 2000). As 2.5 5 10
-1
observed in the present study, it has been reported that an intracel- Inoculum density (g L
4.2 Kinetika Pertumbuhan
lular saponin accumulationSuspensi Sel W.insomnifera
was improved cell suspension cul-
Fig. 2. Withania somnifera cell suspension culture: effe
tures of Panax notoginseng under a moderately high inoculum
biomass accumulation and withanolide A production.
SET size (Zhang and Zhong, 1997).
UP EKSPERIMEN in 50 ml of MS medium supplemented with 2.0 mg L!1
4 weeks. Data represents mean values ± SE of three rep
PERLAKUAN Y (RESPON) repeated twice. Mean values with common letters are
3.4.XEffect
(LAMA of different
INKUBASI) media on biomass accumulation and P 6 0.05 according to Duncan’s multiple range test (D
withanolide A production
1 minggu Y1 = Jumlah Biomassa sel
2 minggu Y2 produced
Levels of secondary metabolites = SenyawabyWithanolide A
in vitro cultures used for the production of taxane diterp
3 minggu
can vary dramatically. Most reports focus on composition of med- (Pan et al., 2000) and ginsenoside producti
ium nutrients to achieve optimized accumulation of metabolites in
4 minggu (Wu and Zhong, 1999).
cultured cells (Rao and Ravishankar, 2002). In the present study
5 minggu
different media like MS, B5, NN and N6 were employed for cell sus-
pension culture and production of withanolide A and results re- 3.5. Effect of medium salt strength on biomas
Statistik
vealedAnalisis
that MS : Menggunakan Rancangan
medium was found Acak Lengkap,
to be superior comparedUji withanolide
toLanjut dengan ADMRT production
other media forDengan
both themenggunakan
biomass accumulation
softwareandSPSS
withanolide
version 9.0 The optimum nutrient concentration is a
A production (Fig. 3). Highest accumulation of biomass
(11.79 g L!1 DW) and withanolide A (2.39 mg g!1 DW) was ob- controlling the growth of the cells/organs an
HASIL PENELITIAN
served in the MS medium. Similar to our results MS medium was secondary metabolites (Rao and Ravishanka
study, we have tested the effect of 0.25, 0.5

12 2.5
a 14
DW DW
content Content
10 Withanolide A content (mg g -1 DW) 12
2
a a
b
10
Mean dry wt (g L-1)

8 b c
Mean dry wt (g L )
b
1.5 -1
8
6
c
b 1 6 a
b
4
d 4 b
d
c 0.5
2
d 2

0 0 0
1 2 3 4 5 MS B5 NN
Weeks Different media

Fig. 1. Time-profile of cell growth and withanolide A production in shake flask Fig. 3. Withania somnifera cell suspension culture: e
cultures of Withania somnifera. 500 mg of cells were cultured in 50 ml of MS biomass accumulation and withanolide A production.
medium supplemented with 2.0 mg L!1 2,4-D + 0.5 mg L!1 KN for 4 weeks. Data in 50 ml of medium supplemented with 2.0 mg L!1
represents mean values ± SE of three replicates; each experiment was repeated 4 weeks. Data represents mean values ± SE of three rep
twice. Mean values with common letters are not significantly different at P 6 0.05 repeated twice. Mean values with common letters are
according to Duncan’s multiple range test (DMRT). P 6 0.05 according to Duncan’s multiple range test (D

KESIMPULAN :
LAMA INKUBASI TERBAIK UNTUK PERTUMBUHAN BIOMASSA SEL W. somnifera DAN
PRODUKSI SENYAWA WITHANOLIDE A TERTINGGI ADALAH SELAMA 4 MINGGU.
PRODUKSI BIOMASSA PADA AKHIR MINGGU KEEMPAT ADALAH SEBESAR 11,02 g/L DAN
SENYAWA WITHANOLIDE A YANG DIHASILKAN SEBANYAK 2.03 mg/g.
 4.3 PENENTUAN JUMLAH INOKULUM W. somnifera YANG DITAMBAHKAN

SET UP EKSPERIMEN
PERLAKUAN Y (RESPON)
X (KONSENTRASI INOKULUM
YANG DITAMBAHKAN g/L)
2,5 Y1 = Jumlah Biomassa sel
5,0 Y2 = Senyawa Withanolide A
10,0
20,0

urthy / BioresourceStatistik Analisis

Technology : Menggunakan
101 (2010) 6735–6739 Rancangan Acak Lengkap, Uji Lanjut dengan
6737 DMRT
Dengan menggunakan software SPSS version 9.0
urth week 12 3
DW
n in the cul-
Content
m withano-
ourth week 10 a a 2.5

Withanolide A content (mg g DW)

a b
mass growth
on.

-1
8 2
Mean dry wt (g L )
-1

and
6 1.5
a
b b
, 10.0 and
4 c 1
s treatment
est produc-
h (20 g L!1) 2 0.5
esults. Sim-
ensis, where
when com- 0 0
l., 2000). As 2.5 5 10 20
-1
an intracel- Inoculum density (g L )
ension cul-
Fig. 2. Withania somnifera cell suspension culture: effects of inoculum densities on
h inoculum
biomass accumulation and withanolide A production. Cells (500 mg) were cultured
in 50 ml of MS medium supplemented with 2.0 mg L!1 2,4-D and 0.5 mg L!1 KN for
4 weeks. Data represents mean values ± SE of three replicates; each experiment was
repeated twice. Mean values with common letters are not significantly different at
nd P 6 0.05 according to Duncan’s multiple range test (DMRT).

KESIMPULAN :
tro cultures
KONSENTRASI INOKULUM YANG DITAMBAHKAN YANG TERBAIK UNTUK PERTUMBUHAN
used for the production of taxane diterpene from T. chinensis
BIOMASSA SEL W. somnifera DAN PRODUKSI SENYAWA WITHANOLIDE A TERTINGGI
ion of med- (Pan et al., 2000) and ginsenoside production from Panax ginseng
ADALAH 10 g/L. PRODUKSI BIOMASSA PADA KONDISI TERSEBUT ADALAH SEBESAR 10,88
etabolites in (Wu and Zhong, 1999).
g/L DAN SENYAWA WITHANOLIDE A YANG DIHASILKAN SEBANYAK 2.42 mg/g
esent study
for cell sus-
results re- 3.5. Effect of medium salt strength on biomass accumulation and
ompared to withanolide A production
withanolide
f biomass The optimum nutrient concentration is a critical determinant in
W) was ob- controlling the growth of the cells/organs and the accumulation of
 when com- 0 0
al., 2000). As 2.5 5 10 20
-1
an intracel- Inoculum density (g L )
pension cul-
Fig. 2. Withania somnifera cell suspension culture: effects of inoculum densities on
h inoculum
biomass accumulation and withanolide A production. Cells (500 mg) were cultured
in 50 ml of MS medium supplemented with 2.0 mg L!1 2,4-D and 0.5 mg L!1 KN for
4 weeks. Data represents mean values ± SE of three replicates; each experiment was
4.4 PENENTUAN JENIS MEDIA YANG TERBAIK UNTUK PRODUKSI BIOMASSA SEL W.
repeated twice. Mean values with common letters are not significantly different at
nd somnifera DAN SENYAWA
P 6 0.05 according WITHANOLIDE
to Duncan’s multiple A
range test (DMRT).

SET UP EKSPERIMEN
itro cultures used for the production of taxane diterpene from T. chinensis
PERLAKUAN Y (RESPON)
tion of med- (Pan et al., 2000) and ginsenoside production from Panax ginseng
etabolites in
X (JENIS MEDIA)
(Wu and Zhong, 1999).
resent study MS Y1 = Jumlah Biomassa sel
for cell sus- B5 Y2 = Senyawa Withanolide A
3.5. Effect of medium salt strength on biomass accumulation and
d results re- NN
ompared to withanolide A production
N6
withanolide
of biomass The optimum nutrient concentration is a critical determinant in
Statistik Analisis
controlling : Menggunakan
the growth Rancangan
of the cells/organs andAcak Lengkap, Uji of
the accumulation Lanjut dengan DMRT
W) was ob-
medium was secondary metabolites (Rao and Ravishankar, 2002). In the present9.0
Dengan menggunakan software SPSS version
study, we have tested the effect of 0.25, 0.5, 0.75, 1.0, 1.5 and 2.0
HASIL
2.5
14 3
DW
Content
Withanolide A content (mg g -1 DW)

12
2 2.5

Withanolide A content (mg g DW)

a
b
10 -1
c
Mean dry wt (g L )

c 2
-1

1.5
8
1.5
1 6 a

1
4 b
d c
0.5
0.5
2

0 0 0
MS B5 NN N6
Different media

in shake flask Fig. 3. Withania somnifera cell suspension culture: effects of different media on
n 50 ml of MS biomass accumulation and withanolide A production. Cells (500 mg) were cultured
4 weeks. Data in 50 ml of medium supplemented with 2.0 mg L!1 2,4-D and 0.5 mg L!1 KN for
was repeated 4 weeks. Data represents mean values ± SE of three replicates; each experiment was
rent at P 6 0.05 repeated twice. Mean values with common letters are not significantly different at
P 6 0.05 according to Duncan’s multiple range test (DMRT).

KESIMPULAN :
JENIS MEDIUM YANG TERBAIK UNTUK PERTUMBUHAN BIOMASSA SEL W. somnifera DAN
PRODUKSI SENYAWA WITHANOLIDE A TERTINGGI ADALAH MS. PRODUKSI BIOMASSA
PADA KONDISI TERSEBUT ADALAH SEBESAR 11,79 g/L DAN SENYAWA WITHANOLIDE A
YANG DIHASILKAN SEBANYAK 2.39 mg/g
 was found to be the ideal carbohydrate source for the biomass sion culture (Lian et
accumulation, which yielded the biomass of 10.47 g L!1 DW, fol- 2% sucrose favored
lowed by combination of sugars i.e., sucrose + glucose which accu- medium accumulate
mulated the biomass 9.32 g L!1 DW. The lowest accumulation of (Naik et al., 2010). I
biomass was recorded in the medium supplemented with maltose (1980) showed that
and fructose + sucrose which yielded about 7.06 g L
4.5 PENENTUAN MEDIUM SALT STRENGTH TERBAIK UNTUK PRODUKSI BIOMASSA SEL
!1
DW and W.
mulation in cell cult
7.73 g
somnifera DAN DW. The WITHANOLIDE
L!1SENYAWA highest production
A of withanolide A content of
2.95 mg g!1 DW was recorded in the medium supplemented with
3.8. Effect of initial m
SET UPsucrose followed by glucose, which produced 2.25 mg g!1 DW.
EKSPERIMEN withanolide A produc
The lowest
PERLAKUANproduction was recorded in the medium supplemented
Y (RESPON)
Xwith maltose,
(MS SALT which produced 1.70 mg g!1 DW of withanolide A
STRENGTH) The hydrogen ion
0,25content. These variations might be due
Y1 = Jumlah to differential
Biomassa sel signaling lev-
usually adjusted bet
0,5 els of sugar and sugar mixtures. However,
Y2 = Senyawa further studies
Withanolide A are essen-
centration of hydrog
0,75tial to assess the signal induced enzyme levels to prove this ture period. The
1 hypothesis. Systematic studies were carried out by Weathers et al. assimilation and inc
1,5 (2004) and Wang and Weathers (2007) and they verified the effect Jackman, 1989). The
2 of sugars on artemisinin production in the hairy roots as well as 5.8, 6.0 and 6.5) on b
seedling cultures of Artemisia annua. They showed maximum pro-
duction from cell sus
duction
Statistik of artemisinin
Analisis when
: Menggunakan hairy roots
Rancangan Acakwere grown
Lengkap, Ujiin the medium
Lanjut dengan DMRTest accumulation of
supplemented withmenggunakan
Dengan glucose, whereas the SPSS
software levelversion
of artemisinin
9.0 pro-
duced in the medium supplemented with fructose was twice that
HASIL 12
DW
14 3
DW Content
a 10
Content b
12

Withanolide A content (mg g DW)

2.5
c

-1

Mean dry wt (g L )
10 8

-1
Mean dry wt (g L )

a 2 d
-1

d
8
e e 6
b 1.5
6 c
d 1 4
d f
4

2 0.5 2
e

0 0 e
0
0.25 0.5 0.75 1 1.5 2 1
Medium strength Su

Fig. 4. Withania somnifera cell suspension culture: effects of medium strength on Fig. 5. Withania somnife
biomass accumulation and withanolide A production. Cells (500 mg) were cultured concentrations on biom
in 50 ml of MS medium supplemented with 2.0 mg L!1 2,4-D and 0.5 mg L!1 KN for (500 mg) were cultured in
4 weeks. Data represents mean values ± SE of three replicates; each experiment was D + 0.5 mg L!1 KN for 4 w
repeated twice. Mean values with common letters are not significantly different at each experiment was r
P 6 0.05 according to Duncan’s multiple range test (DMRT). significantly different at P

KESIMPULAN :
SALT STRENGTH MEDIUM MS YANG TERBAIK UNTUK PERTUMBUHAN BIOMASSA SEL W.
somnifera DAN PRODUKSI SENYAWA WITHANOLIDE A TERTINGGI ADALAH 1. PRODUKSI
BIOMASSA PADA KONDISI TERSEBUT ADALAH SEBESAR 12,13 g/L DAN SENYAWA
WITHANOLIDE A YANG DIHASILKAN SEBANYAK 2.35 mg/g
4.6 PENENTUAN JENIS SUMBER KARBON TERBAIK UNTUK PRODUKSI BIOMASSA SEL W.
somnifera DAN SENYAWA WITHANOLIDE A

SET UP EKSPERIMEN
PERLAKUAN RESPON
X (JENIS SUMBER KARBON) Y1 (BIOMASSA SEL) Y2 (WITHANOLIDE A)
g/L (berat kering) mg/g (berat kering)
Sukrosa 10,47 2,95
Glukosa 2,25
Fruktosa
Maltosa 7,06 1,70
Glukosa + Fruktosa (1:1)
Fruktosa + Sukrosa (1:1) 7,73
Sukrosa +Glukosa 9,32

Statistik Analisis : Menggunakan Rancangan Acak Lengkap, Uji Lanjut dengan DMRT
Dengan menggunakan software SPSS version 9.0

KESIMPULAN :
SUMBER KARBON YANG TERBAIK UNTUK PERTUMBUHAN BIOMASSA SEL W. somnifera
DAN PRODUKSI SENYAWA WITHANOLIDE A TERTINGGI ADALAH SUKROSA. PRODUKSI
BIOMASSA PADA KONDISI TERSEBUT ADALAH SEBESAR 10,47 g/L DAN SENYAWA
WITHANOLIDE A YANG DIHASILKAN SEBANYAK 2.95 mg/g
 sue and cell cultures, serving as the principal energy source and
a component for secondary metabolite biosynthesis. The rate of
act as energy biomass growth is usually directly correlated with sugar consump-
affect growth, tion (Rao and Ravishankar, 2002). Among the various concentra-
Rolland et al., tions of sucrose [1–8% (w/v)] tested in the present study, 4%
cose, fructose, sucrose found suitable for biomass accumulation (Fig. 5). However,
ucrose (1:1), accumulation of withanolide A was optimum with cultures supple-
e because su- 4.7 PENENTUAN
mented KONSENTRASI
with 3% sucrose SUKROSA
(Fig. 5). Similar to ourTERBAIK
results 3%UNTUK
sucrosePRODUKSI BIOMASSA SEL W.
tures. Sucrose somnifera
favored theDAN SENYAWA
ginsenoside WITHANOLIDE
production A
in Panax ginseng cell suspen-
r the biomass sion culture (Lian et al., 2002). In Bacopa monnieri shoot cultures,
g L!1 DW, fol- 2% sucrose favored biomass accumulation, whereas sucrose free
se which accu- SET UP EKSPERIMEN
medium accumulated maximum amount of bacoside A content
cumulation of PERLAKUAN
(Naik et al., Y (RESPON)
2010). In contrast to our results, Knobloch and Berlin
d with maltose X (KONSENTRASI
(1980) showed that 8% SUKROSA)
sucrose favored the indole alkaloid accu-
g L!1 DW and mulation in % (W/V)
cell cultures of Catharanthus roseus.
e A content of
emented with 1 Y1 = Jumlah Biomassa sel
3.8. Effect of initial medium pH on adventitious root growth and
25 mg g!1 DW. 2 Y2 = Senyawa Withanolide A
withanolide A production
supplemented 3
withanolide A
signaling lev-
4 The hydrogen ion concentration (pH) of the culture medium is
dies are essen- 6usually adjusted between 5 and 6 before autoclaving and the con-
centration of hydrogen ions in the medium changes during the cul-
to prove this 8ture period. The medium pH decreases during ammonia
Weathers et al.
assimilation and increases during nitrate uptake (McDonald and
ified the effect
ots as well as
Statistik
Jackman,Analisis : Menggunakan
1989). The effect of initial Rancangan Acak4.5,
medium pH (4.0, Lengkap,
5.0, 5.5, Uji Lanjut dengan DMRT
5.8, 6.0 and 6.5) onDengan
biomass menggunakan
accumulation andsoftware
withanolide
SPSS A pro-
version 9.0
maximum pro-
duction from cell suspension cultures are presented in Fig. 6. High-
in the medium HASIL :
est accumulation of biomass was observed when the medium pH
temisinin pro-
was twice that
12 a 3.5
DW b
3
Content
3
10
Withanolide A content (mg g DW)
Withanolide A content (mg g DW)

2.5 a
-1

2.5
-1

c
Mean dry wt (g L )

8
-1

2 d
2
e 6 b b c
1.5
1.5

1 4
f 1
c

0.5 2 0.5
e d

0 e
0 0
2 1 2 3 4 6 8
Sucrose concentration (%, w/v)

dium strength on Fig. 5. Withania somnifera cell suspension culture: effects of different sucrose
mg) were cultured concentrations on biomass accumulation and withanolide A production. Cells
0.5 mg L!1 KN for (500 mg) were cultured in 50 ml of MS medium supplemented with 2.0 mg L!1 2,4-
h experiment was D + 0.5 mg L!1 KN for 4 weeks. Data represents mean values ± SE of three replicates;
cantly different at each experiment was repeated twice. Means with common letters are not
significantly different at P 6 0.05 according to Duncan’s multiple range test (DMRT).

KESIMPULAN :
KONSENTRASI SUKROSA YANG TERBAIK UNTUK PERTUMBUHAN BIOMASSA SEL W.
somnifera DAN PRODUKSI SENYAWA WITHANOLIDE A TERTINGGI ADALAH 4%.
4.8 PENENTUAN pH TERBAIK UNTUK PRODUKSI BIOMASSA SEL W. somnifera DAN
SENYAWA WITHANOLIDE A

SET UP EKSPERIMEN
PERLAKUAN Y (RESPON)
X (pH)
4 Y1 = Jumlah Biomassa sel
4,5 Y2 = Senyawa Withanolide A
5
5,5
5,8
6
6,5

Statistik Analisis : Menggunakan Rancangan Acak Lengkap, Uji Lanjut dengan DMRT
Dengan menggunakan software SPSS version 9.0
P. Nagella, H.N. Murthy / Bioresource Technology 101 (2010) 6735–6739
HASIL
12 3 Chu, C.C., 1978. The N6 medium and its applications
crops. In: Proceedings of the Symposium Plant T
DW
a a Beijing, pp. 43–50.
Content Dhuley, J.N., 2000. Adaptogenic and cardioprotective a
10 2.5
Withanolide A content (mg g DW)
a and frogs. J. Ethanopharmacol. 70, 57–63.
b Furmanowa, M., Gajdzis-Kuls, D., Ruszkowska, J., C
b c Sadowska, A., Rani, R., Upadyay, S.N., 2001. In vi
-1

somnifera and isolation of withanolides with im

Mean dry wt (g L )

8 c 2
-1

c Planta Med. 67, 146–149.

d d d Gamborg, O.L., Miller, R.A., Ojima, K., 1968. Nutrient
e cultures of soybean root cells. Exp. Cell Res. 50, 15
6 1.5 Ganzera, M., Choudhary, M.I., Khan, I.A., 2003. Qu
withanolides in Withania somnifera. Fitoterapia 74
Gupta, G.L., Rana, A.C., 2007. Withania somnifera
Pharmacog. Rev. 1, 129–136.
4 1
Knobloch, K.H., Berlin, J., 1980. Influence of medium co
of secondary compounds in cell suspension culture
f Don. Z. Naturforsch. 35, 551–556.
2 0.5 Kuboyama, T., Tohda, C., Komatsu, K., 2005. Neuritic
g reconstruction induced by withanolide A. Br. J. Ph
Kulkarni, S.K., Dhir, A., 2008. Withania somnifera: an I
Psychopharmacol. Biol. Psychiatry 32, 1093–1105.
0 0 Lian, M.L., Chakrabarty, D., Paek, K.Y., 2002. Effect of
4 4.5 5 5.5 5.8 6 6.5 medium composition on cell growth and sapon
suspension culture of mountain ginseng (Panax gin
Hydrogen ion concentration (pH) 45, 201–206.
Mantell, S.H., Smith, H., 1984. Cultural factors that infl
Fig. 6. Withania somnifera cell suspension culture: effect of hydrogen ion concen- accumulation in plant cell and tissue cultures. In: M
tration (pH) on biomass accumulation and withanolide A production. Cells (500 mg) Plant Biotechnology. Cambridge, Cambridge Unive
were cultured in 50 ml of MS medium supplemented with 2.0 mg L!1 2,4-D and McDonald, K.A., Jackman, A.P., 1989. Bioreactor stud
0.5 mg L!1 KN for 4 weeks. Data represents mean values ± SE of three replicates; utilization in Alfalfa suspension cultures. Plant Cel
each experiment was repeated twice. Means with common letters are not Murashige, T., Skoog, F., 1962. A revised medium for
significantly different at P 6 0.05 according to Duncan’s multiple range test (DMRT). with tobacco tissue cultures. Physiol. Plant. 15, 47
Murthy, H.N., Dijkstra, C., Anthony, P., White, D.A., Da
KESIMPULAN : E.J., Paek, K.Y., 2008. Establishment of Withania som
was set at 5.8 and cells grown in this medium accumulated the production of withanolide A. J. Int. Plant Biol.
pH YANG TERBAIK UNTUK PERTUMBUHAN BIOMASSA SEL W. somnifera DAN PRODUKSI
10.27 g L!1 DW (Fig. 6). The maximum production of withanolide Naik, P.M., Manohar, S.H., Praveen, N., Murthy, H.N., 20
SENYAWA WITHANOLIDE
A content (2.51 mg g!1 A TERTINGGI
DW) ADALAH
was recorded when5,8.
the PRODUKSI
initial med- BIOMASSA PADA KONDISI
levels on in vitro shoot regeneration from leaf exp
accumulation of bacoside A in regenerated shoots.
TERSEBUT
ium pHADALAH
was 6.0 SEBESAR 10,27
(Fig. 6). High andg/L
lowDAN SENYAWA
medium WITHANOLIDE
pH did not favor A YANG 100, 235–239.
the biomass accumulation and withanolide A production. Whereas Nitsch, J.P., Nitsch, C., 1969. Haploid plants from pollen
DIHASILKAN SEBANYAK 2.51 mg/g Pan, Z.W., Wang, H.Q., Zhong, J.J., 2000. Scale-up stud
in Bacopa monnieri shoot cultures, initial medium pH set at 4.5 was Taxus chinensis cells for production of taxane dite
found suitable for biomass accumulation and bacoside A produc- 27, 714–723.
tion (Naik et al., 2010). Rao, R.S., Ravishankar, G.A., 2002. Plant cell cultu
secondary metabolites. Biotech. Adv. 20, 10–153.
Ray, S., Jha, S., 1999. Withanolide synthesis in cult
transformed with Agrobacterium tumefaciens. Plant
4. Conclusions Ray, S., Jha, S., 2001. Production of withaferin A in
somnifera Dunal. Planta Med. 67, 432–437.
In conclusion, in the present study we have successfully estab- Ray, S., Ghosh, B., Sen, S., Jha, S., 1996. Withanolide pr
Withania somnifera transformed with Agrobacteriu
lished cell suspension cultures of W. somnifera for the production 571–573.
of withanolide A. The productivity of withanolide A was found to Roja, G., Heble, M.R., 1991. Tissue cultures of Withan