Journal of Bioscience and Bioengineering
VOL. 111 No. 6, 706 – 710, 2011
www.elsevier.com/locate/jbiosc
Enhanced plumbagin production in elicited Plumbago indica hairy root cultures
Moumita Gangopadhyay,1,⁎ Saikat Dewanjee,2 and Sabita Bhattacharya1
Medicinal Plant Laboratory, Department of Botany, Bose Institute, 93/1 APC Road, Kolkata 700009, India 1 and Advanced Pharmacognosy Research Laboratory,
Department of Pharmaceutical Technology, Jadavpur University, Raja SC Mallik Road, Kolkata 700032, India 2
Received 3 August 2010; accepted 3 February 2011
Available online 5 March 2011
Elicitation of Plumbago indica hairy roots with yeast carbohydrate fraction, chitosan, manganese chloride, copper chloride
and methyl jasmonate exhibited significant elevation (~ 1.2 to 2 fold) of plumbagin production in shake flask culture as
compared with control. Chitosan and methyl jasmonate elicitation also caused simultaneous plumbagin leaching into culture
media. Three days’ exposure of chitosan (200 mg l−1) and methyl jasmonate (80 μM) together synergized total plumbagin
yield to its maximum 11.96 ± 0.76 mg g−l DW in shake flask culture. In bioreactor cultivation, a significant raise in fresh root
biomass was recorded on day 20 as compared with control shake flask culture. Three days’ exposure of chitosan (200 mg l−1)
and methyl jasmonate (80 μM) with 20 days old bioreactor-culture significantly improved total plumbagin production to
13.16 ± 1.72 mg g−l DW with simultaneous plumbagin leaching into bioreactor media.
© 2011, The Society for Biotechnology, Japan. All rights reserved.
[Key words: Plumbago indica; Hairy roots; Elicitors; Bioreactor; Chitosan; Methyl jasmonate]
Plants are considered as chemical factories for biosynthesis of a
huge array of structurally diverse bioactive secondary metabolites. The
isolation of bioactive compounds from whole plants creates pressure
on natural germplasm. Therefore, the biotechnological production of
valuable secondary metabolites in plant cell culture is a striking choice.
It offers attractive alternatives to classical technologies for the
production of plant-based metabolites without hampering natural
flora. Moreover, the production of plant metabolites in plant cell
culture is independent of seasonal and geographical variations. In vitro
culture systems provide various ways to boost the yields of desired
metabolites conveniently and cost-effectively. Elicitation is one of the
most effective approaches for increasing the production of secondary
metabolites in plant cell culture (1,2). Elicitors are signals compounds
triggering the formation of secondary metabolites by activating the
pathways in response to exogenous stresses (3). Though plant cell
cultures could be a potential source of wide varieties of valuable
metabolites, but the recovery of metabolites from culture medium and
down-stream processing is very much challenging. Low hydrophilicity
of most of secondary metabolites restricts their release into the
medium from in vitro maintained cell culture. Thus, it is necessary to
remove the water insoluble products from the culture medium
without disturbing the cell metabolic activities. In this connection,
the extracellular leaching is also important.
Plumbago indica (family Plumbaginaceae), a dicotyledonous plant,
is well known for its ethnomedicinal values. P. indica is a rich source of
therapeutically active, root specific, natural napthoquinone plumbagin. Plumbagin has been reported to possess filaricidal (4) anticancer
(5), cardiotonic (6), antimalarial (7), antimicrobial (8) and anti⁎ Corresponding author. Tel.: + 91 9830854626; fax: + 91 33 23500595.
E-mail address: moumita_gangopadhyay@yahoo.co.uk (M. Gangopadhyay).
fertility (9) activities. Among six different species of Plumbago, P. indica is the richest source of plumbagin (10). The annual requirement
for plumbagin in Indian subcontinent is about 7 metric tonnes (11).
Increasing demand of plumbagin in both domestic and international
markets has led to the overexploitation of P. indica from natural
habitat. Presently, this plant has become a rare categorized (12).
Traditional agricultural methods take several years to achieve desired
level of plumbagin in the roots of this seedless plant (11). On the other
hand, synthetic approach of plumbagin production is not commercially promising (13,14). In this situation, intervention of modern
biotechnological approaches to enhance plumbagin production
through plant cell culture is the only way to fulfill market demands,
as well to save this plant from becoming extinct. During past few
decades, several attempts were made to improve plumbagin
production through suspension (15) and adventitious root culture
(16), but the product yield was small in industrial aspect. Since the
synthesis of plumbagin is linked to root differentiation, undifferentiated cell cultures did not produce this metabolite efficiently. In this
respect, hairy or transformed root cultures have several advantages
over normal cell and organ cultures (17). Hairy root culture of P.
indica has been successfully established in the Medicinal Plant
Laboratory, Bose Institute, Kolkata, India (18). P. indica hairy roots
were able to grow faster than non-transformed roots in hormone-free
media and produced plumbagin at comparatively higher levels (18)
than non-transformed roots. Since the increasing market demand
necessitates further improvement of plumbagin production for viable
commercial exploitation, the present study was undertaken to
enhance plumbagin production in P. indica hairy roots under the
influences of different biotic and abiotic elicitors. Since the plumbagin
production is intracellular and growth associated, it was further
aimed to culture hairy roots in a bioreactor (19) to obtain significant
1389-1723/$ - see front matter © 2011, The Society for Biotechnology, Japan. All rights reserved.
doi:10.1016/j.jbiosc.2011.02.003
VOL. 111, 2011
PLUMBAGIN PRODUCTION IN ELICITED P. INDICA HAIRY ROOTS
higher root biomass. Conventional bioreactors for cell suspension
cultures cannot be used for hairy roots because the later form root
clumps with intertwined and self-immobilized morphologies, which
resist the percolation of oxygen into the hairy roots, leading to poor
growth and metabolite production (20). In this study, P. indica hairy
roots were cultured in a special bioreactor with continuous air supply.
After a significant raise in fresh root biomass, hairy roots were
exposed to selected and optimized elicitors for enhanced plumbagin
production.
MATERIALS AND METHODS
Plant material
A fast growing hairy root clone (H13) of P. indica established in
Medicinal Plant Laboratory, Bose Institute, India (18) was used as source material. The
root clone was obtained by infecting the midribs of leaf explants of in vitro grown P.
indica with Agrobacterium rhizogenes strain ATCC 15834 (18). Hairy roots (1.5 g l−1)
were sub-cultured in 50 ml of hormone-free liquid MS medium (21) with 3% sucrose at
25 ± 2°C in dark on an orbital shaker at 70 rpm for 20 d.
Preparation of elicitors
The fungal cultures used for the elicitation were
Fusarium solani, Aspergillus niger and Rhizopus oryzae (22). The fungi were maintained
in potato-dextrose agar slant at 30 ± 2°C. The cultures were transferred into 250 ml
Erlenmeyer flasks containing 50 ml of potato-dextrose broth at 30 ± 2°C on an orbital
shaker at 70 rpm. After one week, cultures were harvested, filtered and dried at 60°C
for 24 h. The dry cell powders were dissolved separately in double distilled water
(10 g l-1) and autoclaved for 15 min at 121°C.
The carbohydrate fraction isolated from yeast (Saccharomyces cerevisiae) extract
was prepared by ethanol precipitation method (23). Briefly, 50 g of the yeast extract
was dissolved in 250 ml double distilled water. Ethanol was added to 80% (v/v). After
incubation at 6°C for 4 d, the precipitate was collected. The process was repeated thrice
and the precipitate was dissolved in 200 ml double distilled water, yielding the crude
preparation that was used without further purification.
Chitosan (Hi media, India) was purified by the method described Kim et al. (24) with
little modification. Briefly, 1 g chitosan was dissolved in 90 ml, 0.1 N acetic acid and the
solution was centrifuged for 20 min at 6700× g. After centrifugation, the supernatant was
precipitated by adjusting its pH to 8.0 with 5 N NaOH. The precipitate was washed
repeated with double distilled water and lyophilized. One gram lyophilized chitosan was
dissolved in 100 ml, 0.1 N acetic acid and the pH of the solution was adjusted to 5.0.
Methyl jasmonate (95% pure) in 96% ethanol was purchased from Sigma Aldrich,
USA and filtered through a syringe filter (25 μm, Gelman Sciences, Ann Arbor, MI, USA).
Chloride salts of manganese, zinc, lead, cobalt, nickel, silver, copper and calcium
were used for the elicitation. Stock solutions were prepared separately by dissolving 1 g
of salt in 100 ml double distilled water. The pH of individual salt solutions was adjusted
to 5.5 (25). The solutions were autoclaved for 15 min at 121°C.
Selection and optimization of elicitors
Elicitation studies were carried out
with selected fungal biomass (1, 2 and 3 mg l−1), yeast carbohydrate fraction (1, 2 and
−1
−1
3 mg l ), chitosan (100, 200 and 300 mg l ), inorganic salts (100, 200 and
300 mg l−1) and methyl jasmonate (20, 40, 80 and 100 μM). Twenty days’ old hairy
roots (0.075 g on fresh weight basis) were transferred to fresh liquid MS medium
containing selected concentrations of elicitors. One set of shake flask without elicitor
served as control. Intracellular plumbagin content and plumbagin leaching was
estimated on days 1, 3 and 7.
To study the synergistic effect of elicitors, P. indica hairy roots were cultured with
the exposure of selected elicitors (based on the effect of individual elicitors on
plumbagin accumulation in shake flask cultures) in combination, namely chitosan
(200 mg l−1) + methyl jasmonate (80 μM), yeast carbohydrate fraction (1 mg l−1) +
chitosan (200 mg l−1) + methyl jasmonate (80 μM) and yeast carbohydrate fraction
(1 mg l−1) + chitosan (200 mg l−1) + Manganese chloride (200 mg l−1) + Copper
chloride (100 mg l−1) + methyl jasmonate (80 μM). Plumbagin content and extracellular plumbagin leaching was estimated on days 1, 3 and 7.
Bioreactor cultivation of P. indica hairy roots with elicitors
After selection
and optimization of the elicitors in shake flasks, 3 g l−1 of P. indica hairy roots were
cultivated in a 3 l bioreactor (length 22 cm and diameter 14 cm) with a working
volume of 1.75 l. The reactor was provided with openings for air inlet, air outlet,
inoculation port and sampling port. The air was supplied through a glass sparger,
molded into a circular shape with pores of size 1 mm at the bottom of the reactor. The
reactor was provided with an autoclavable perforated plastic basket, which was open
on the top. The basket was placed at a height of 7 cm from the bottom of the reactor
vessel on a stainless steel stand. The distance between the sparger and the basket was
4 cm. Air was sparged at a rate of 30.4 cm3 s−1. The bioreactor was maintained in dark at
25 ± 2°C. The reactor containing 1.75 l phytohormone-free liquid MS medium with 3%
sucrose was inoculated with hairy roots (5.25 g on fresh weight basis) of P. indica. After
20 d, the fresh root biomass, dry root biomass, plumbagin content and total plumbagin
yield were determined. Then the culture within the bioreactor was treated with
elicitors i.e. chitosan (200 mg l−1) + methyl jasmonate (80 μM) which were selected
from shake flask studies. One set without elicitor served as control. The cultivation was
continued for 3 d (based on the results of shake flask culture) and the results were
recorded.
707
Estimation of plumbagin by HPLC
The plumbagin content was estimated by
HPLC (LC-20 AT Liquid Chromatogram, Shimadzu, Japan) employing isocratic linear
solvent system of water and acetonitrile (20:80, v/v) as per the method of
Gangopadhyay et al. (18). Plumbagin leaching into culture media was also estimated
for each set of experiment. Briefly, the 20 ml medium was collected and extracted with
20 ml ethyl acetate. The methanol soluble fraction of ethyl acetate extract was
subjected to HPLC analysis to estimate plumbagin leaching. Plumbagin content was
calculated as mg g–1 DW.
Data analysis
Three replicates were made for each experimental set. Data were
statistically calculated by utilizing one way ANOVA and expressed as mean ± standard
deviation followed by Turkey–Kramer's t-test using computerized GraphPad InStat
version 3.05, GraphPad Software, La Jolla, CA, USA. The values were considered
significant when p b 0.05.
RESULTS AND DISCUSSION
Effect of fungal biomass The effect of different fungi viz.
F. solani, A. niger and R. oryzae on plumbagin production is shown in
Table 1. The elicitation with F. solani and A. niger in hairy root culture
of P. indica caused a slight, statistically insignificant improvement of
intracellular plumbagin content in hairy roots at the dose of 1 mg l−1
up to 3 d. One day exposure of F. solani and A. niger at a dose of
1 mg l−1 showed the product yield of 5.92 ± 0.40 and 5.62 ±
0.32 mg g−l DW respectively. On other hand, intracellular plumbagin
content was adversely affected with R. oryzae. The metabolite
production gradually decreased with increasing dose and exposure
time with all the selected fungi. The media leaching of plumbagin was
not observed in any of fungal elicitation. F. solani, A. niger and R. oryzae
are commonly employed biotic elicitors for enhancing secondary
metabolites in hairy root culture of different plant species (20).
However, fungal elicitors produce very species-specific action of
elicitation (26). In present study, insignificant alteration of intracellular plumbagin content in P. indica hairy roots by elicitation with the
fungi suggested that the plant species is not specific toward selected
fungi.
Effect of yeast carbohydrate fraction
Yeast carbohydrate
fraction has been employed as biotic elicitor for enhancing secondary
metabolites in hairy root cultures of various species (2). Addition of
yeast carbohydrate fraction exhibited positive effect on plumbagin
production in P. indica hairy roots without altering root biomass
(Table 1). Maximum increase in plumbagin accumulation (6.13 ±
0.38 mg g−l DW, p b 0.05, ~ 1.2 fold higher than control) was obtained
at the dose of 1 mg l−1 on day 3. The intracellular plumbagin content
TABLE 1. Effect of different fungal elicitors and yeast carbohydrate fraction on
plumbagin accumulation in P. indica hairy roots.
Elicitors
Concentrations
(mg l−1)
Control
F. solani
–
1
2
3
1
2
3
1
2
3
1
2
3
A. niger
R. oryzae
Yeast carbohydrate fraction
Plumbagin
(mg g−1 DW)
Day 1
Day 3
Day 7
5.33 ± 0.21
5.92 ± 0.40
5.41 ± 0.17
4.02 ± 0.22
5.62 ± 0.32
4.91 ± 0.18
4.10 ± 0.24
4.92 ± 0.14
4.33 ± 0.21
4.25 ± 0.15
5.46 ± 0.31
5.39 ± 0.23
5.31 ± 0.24
5.31 ± 0.35
5.66 ± 0.47
5.39 ± 0.21
3.88 ± 0.25
5.42 ± 0.28
4.22 ± 0.21
4.01 ± 0.30
4.87 ± 0.09
4.12 ± 0.26
4.03 ± 0.14
6.13 ± 0.38*
6.01 ± 0.32
5.88 ± 0.26
5.29 ± 0.25
5.39 ± 0.35
5.03 ± 0.26
3.04 ± 0.28
5.01 ± 0.30
4.09 ± 0.19
3.90 ± 0.21
4.66 ± 0.12
4.01 ± 0.19
3.74 ± 0.18
6.04 ± 0.42
5.83 ± 0.29
5.07 ± 0.14
Values presented as mean ± SD. Data marked with an asterisk are significantly different
(elevated) with respect to the corresponding control according to Tukey's test
(p b 0.05). The final root biomass was nearly the same (0.077 ± 0.003 g on fresh weight
basis) in all cases and no extracellular plumbagin leaching was observed.
5.29 ± 0.25
7.97 ± 0.14*
8.73 ± 0.40*
8.06 ± 0.51*
7.19 ± 0.75*
9.18 ± 0.35*
10.19 ± 0.67*
10.37 ± 0.22*
(mM)
Methyl Jasmonate
Values presented as mean ± SD. Data marked with an asterisk are significantly different (elevated) with respect to the corresponding control according to Tukey's test (p b 0.05).
The final root biomass was nearly the same (0.076 ± 0.002 g on fresh weight basis) in all cases.
–
2.26 ± 0.08
3.37 ± 0.14
3.08 ± 0.23
2.09 ± 0.33
4.08 ± 0.25
4.98 ± 0.32
5.48 ± 0.35
5.29 ± 0.25
5.70 ± 0.09
5.36 ± 0.35
4.98 ± 0.28
5.09 ± 0.45
5.10 ± 0.30
5.21 ± 0.43
4.89 ± 0.37
5.31 ± 0.35
8.87 ± 0.13*
8.96 ± 0.84*
8.13 ± 0.26*
8.23 ± 0.56*
8.87 ± 0.16*
10.46 ± 0.90*
8.98 ± 0.74*
–
2.04 ± 0.14
3.12 ± 0.33
3.01 ± 0.15
1.89 ± 0.22
2.04 ± 0.23
3.21 ± 0.42
3.09 ± 0.32
5.31 ± 0.35
6.83 ± 0.16*
5.83 ± 0.55
5.12 ± 0.23
6.13 ± 0.31
6.83 ± 0.36*
7.25 ± 0.49*
5.89 ± 0.42
5.33 ± 0.21
7.71 ± 0.11*
8.38 ± 0.68*
6.91 ± 0.12*
6.79 ± 0.51*
7.29 ± 0.34*
9.20 ± 0.43*
7.59 ± 0.64*
–
1.99 ± 0.12
2.71 ± 0.32
1.62 ± 0.17
0.12 ± 0.05
0.22 ± 0.09
1.09 ± 0.10
0.89 ± 0.22
5.33 ± 0.21
5.72 ± 0.11
5.67 ± 0.47
5.28 ± 0.24
6.67 ± 0.51*
7.07 ± 0.25*
8.12 ± 0.33*
6.70 ± 0.44*
100
200
300
20
40
80
100
Control
Chitosan
(mg l− 1)
Day 7
Day 3
Intracellular plumbagin Plumbagin leaching Total plumbagin Intracellular plumbagin Plumbagin leaching Total plumbagin Intracellular plumbagin Plumbagin leaching Total plumbagin
(mg g−1 DW)
(mg g−1 DW)
(mg g−1 DW)
(mg g− 1 DW)
(mg g− 1 DW)
(mg g− 1 DW)
(mg g− 1 DW)
(mg g− 1 DW)
(mg g− 1 DW)
Day 1
Concentrations
was found to be inversely related to the increasing dose of yeast
extract. The media leaching was not observed with yeast elicitation.
Effect of chitosan Chitosan is a deacetylated derivative of
chitin found in the cell walls of fungi, crustacean exoskeletons,
cuticles of insects and some algae (27). Chitosan acts as an exogenous
elicitor of response mechanisms and has been demonstrated to induce
plant defenses. Application of chitosan and chitin oligomers increased
the activities of phenylalanine ammonia-lyase (PAL) and tyrosine
ammonia-lyase (TAL) (28). The products of PAL and TAL are modified
via the phenylpropanoid pathways to produce precursors of secondary metabolites which play an important role in plant–pathogen
interactions (28). In this study, the addition of chitosan significantly
increased plumbagin production (p b 0.05, ~ 1.3–1.7 fold higher than
control) in P. indica hairy roots with simultaneous leaching of
plumbagin into culture media (Table 2). The concentration of
plumbagin in culture media gradually increased with time and
reached its maximum on day 7. The total plumbagin yield was
found to be maximum (8.96 ± 0.84 mg g−l DW, p b 0.05, ~ 1.7 fold
higher than control) at the dose of 200 mg l−1 on day 3. These results
have interesting biotechnological implication, since the use of
chitosan not only augmented the production of plumbagin but also
stimulated its release into culture medium within 24 h of exposure.
Effect of methyl jasmonate Amongst various elicitors, exogenously applied methyl jasmonate has been confirmed as effective for
the induction of secondary metabolites in plant cell cultures (29).
Methyl jasmonate are considered to be involved in a part of the signal
transduction pathway that induces particular enzymes to catalyze
biochemical reactions to form low molecular weight secondary
metabolites as defense compounds (30). In this study, plumbagin
accumulation was significantly improved with methyl jasmonate
elicitation with simultaneous leaching of plumbagin into culture
media (Table 2). The intracellular plumbagin content gradually
decreased with time due to continuous leaching of plumbagin into
culture media. The concentration of plumbagin in culture media
gradually increased with time and reached its maximum on day 7.
The total plumbagin yield was found to be maximum (10.46 ±
0.90 mg g−l DW, p b 0.05, ~2.0 fold higher than control) at the dose of
80 μM on day 3. The leaching of plumbagin into culture media would
serve as an additional advantage for industrial purpose.
Effect of inorganic salts The effects of various inorganic salts
on plumbagin production in P. indica hairy roots were recorded in this
study (Table 3). Manganese chloride elicitation caused significant
increase in intracellular plumbagin content (7.26 ± 0.36 mg g−l DW,
p b 0.05, ~1.4 fold higher than control) on day 3 at the dose of
200 mg l−1 without significant alteration of root biomass. Addition of
copper chloride caused a dose-independent effect on plumbagin
production which was increased up to 6.99 ± 0.33 mg g−l DW
(p b 0.05, ~ 1.3 fold higher than control) at the dose of 300 mg l−1 on
day 1. A slight elevation of intracellular plumbagin content (6.02 ±
0.52 mg g−l DW, p N 0.05) was observed with calcium chloride
elicitation at the dose of 300 mg l−1 on day 1. Plumbagin content
remained almost unaltered in other concentrations of calcium
chloride throughout the duration of the experiment. The intracellular
plumbagin content was negatively affected by zinc chloride, lead
chloride, cobalt chloride and nickel chloride, while no significant
change in intracellular plumbagin content was observed with silver
chloride. The leaching of plumbagin was not observed with any of
these inorganic salts.
Combined effect of the elicitors
Based on the effect of
different biotic and abiotic elicitors, P. indica hairy roots were exposed
to a combination of selected elicitors. The combined effects of the
elicitors on plumbagin production in P. indica hairy roots are shown in
Table 4. Significant elevation of total plumbagin was observed with all
the selected combinations of elicitors within 24 h. Amongst various
combinations, Chitosan (200 mg l−1) + methyl jasmonate (80 μM)
J. BIOSCI. BIOENG.,
TABLE 2. Effect of chitosan and methyl jasmonate in different concentrations and exposure times on plumbagin accumulation in P. indica hairy roots and leaching of plumbagin into culture media.
GANGOPADHYAY ET AL.
Elicitors
708
VOL. 111, 2011
PLUMBAGIN PRODUCTION IN ELICITED P. INDICA HAIRY ROOTS
TABLE 3. Effect of different inorganic salts on plumbagin accumulation in P. indica hairy
roots.
Elicitors
Control
Manganese chloride
Zinc chloride
Lead chloride
Cobalt chloride
Nickel chloride
Silver chloride
Copper chloride
Calcium chloride
Concentrations
(mg l− 1)
–
100
200
300
100
200
300
100
200
300
100
200
300
100
200
300
100
200
300
100
200
300
100
200
300
Plumbagin
(mg g− 1 DW)
Day 1
Day 3
Day 7
5.33 ± 0.21
5.67 ± 0.25
6.33 ± 0.42
5.31 ± 0.31
2.47 ± 0.33
2.01 ± 0.18
1.86 ± 0.15
3.12 ± 0.18
3.67 ± 0.15
3.86 ± 0.15
3.87 ± 0.18
3.04 ± 0.33
2.88 ± 0.15
4.21 ± 0.13
4.07 ± 0.28
5.31 ± 0.32
5.25 ± 0.32
5.41 ± 0.44
5.24 ± 0.18
6.21 ± 0.43
6.09 ± 0.38
6.99 ± 0.33*
5.67 ± 0.45
5.67 ± 0.24
6.02 ± 0.52
5.31 ± 0.35
6.12 ± 0.33
7.26 ± 0.36*
6.33 ± 0.45
1.95 ± 0.27
1.89 ± 0.32
1.5 ± 0.18
3.03 ± 0.23
3.05 ± 0.13
3.01 ± 0.09
3.78 ± 0.15
3.12 ± 0.19
2.75 ± 0.17
3.91 ± 0.17
3.82 ± 0.16
6.31 ± 0.36
5.45 ± 0.24
5.82 ± 0.38
5.49 ± 0.27
6.76 ± 0.34*
6.15 ± 0.42
5.89 ± 0.28
5.45 ± 0.33
5.42 ± 0.34
5.80 ± 0.42
5.29 ± 0.25
5.22 ± 0.31
5.89 ± 0.33
4.89 ± 0.38
1.67 ± 0.22
1.37 ± 0.24
1.22 ± 0.21
2.88 ± 0.12
2.97 ± 0.22
2.78 ± 0.12
3.01 ± 0.22
3.09 ± 0.24
2.67 ± 0.21
3.78 ± 0.25
3.67 ± 0.26
4.89 ± 0.28
4.83 ± 0.33
5.32 ± 0.29
4.98 ± 0.24
6.09 ± 0.29
5.88 ± 0.40
5.68 ± 0.37
5.02 ± 0.40
5.32 ± 0.28
5.49 ± 0.45
Values presented as mean ± SD. Data marked with an asterisk are significantly different
(elevated) with respect to the corresponding control according to Tukey's test
(p b 0.05). The final root biomass was nearly the same (0.076 ± 0.003 g on fresh weight
basis) in all cases and no plumbagin leaching was observed.
elicitation caused the highest production of total plumbagin (11.96 ±
0.76 mg g−l DW, p b 0.05, ~2.3 fold higher than control) on day 3 with
subsequent plumbagin leaching (4.68 ± 0.25 mg. g−l DW) which
reached its maximum on day 7. Based on total plumbagin production
and leaching behavior, 3 days’ exposure of chitosan (200 mg l−1) +
methyl jasmonate (80 μM) was used for bioreactor cultivation.
Production of plumbagin in a bioreactor In present study,
P. indica hairy roots were cultured in a bioreactor with continuous air
supply. Since different amounts of hairy roots were cultured in shake
flask and in bioreactor, the elevation of fresh root biomass and
plumbagin production was compared with respect to initial fresh root
biomass taken for respective studies. The fresh root biomass and
intracellular plumbagin content were estimated after harvesting on
709
day 20. Almost ~ 1.8 fold increase in fresh root biomass without
substantial change in intracellular plumbagin content (mg. g−l DW)
was noticed after day 20. Since plumbagin production was growth
associated, the plumbagin production was increased (~2.5 fold) as
compared to shake flask culture. Chitosan (200 mg l−1) + methyl
jasmonate (80 μM) were added on day 20 of cultivation in the
bioreactor when sufficient biomass had accumulated. The culture
within bioreactor was exposed to the optimized concentration of the
selected elicitors for 3 d. One set without elicitor served as control.
The root biomass remained almost unaltered within 3 days exposure
of elicitors as compared to the control culture without elicitor.
Bioreactor culture with 3 days’ exposure of chitosan (200 mg l−1) +
methyl jasmonate (80 μM) improved significantly plumbagin synthesis (28.56 ± 3.33 mg g−l DW, p b 0.05, ~2.2 fold higher than control
culture. The exposure of chitosan + methyl jasmonate for 3 days also
improved plumbagin leaching into the culture media as compared to
the shake flask culture. Agitation of media through aerator might have
facilitated the metabolite leaching into media.
To conclude, combined exposure of chitosan and methyl jasmonate elicitors caused a substantial improvement of plumbagin
production with continuous leaching into the media, which may
serve as an alternate process for its production by Pharmaceutical
industries without affecting natural germplasm of this rare categorized medicinal plant.
ACKNOWLEDGEMENTS
The authors acknowledge the Department of Agricultural and Food
Engineering, Indian Institute of Technology, Kharagpur, India for their
cordial support.
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TABLE 4. Combined effect of elicitors on plumbagin accumulation in P. indica hairy roots.
Elicitors
Day 1
Intracellular
plumbagin
(mg g− 1 DW)
Control
Chitosan (200 mg l−1)+
methyl jasmonate (80 μM)
Yeast carbohydrate
fraction (1 mg l−1)+
chitosan (200 mg l−1)+
methyl jasmonate (80 μM)
Yeast carbohydrate
fraction (1 mg l−1)+
chitosan (200 mg l−1)+
manganese chloride (200 mg l−1)+
copper chloride (100 mg l−1)+
methyl jasmonate (80 μM)
Day 3
Plumbagin
leaching
(mg g− 1 DW)
Total
plumbagin
(mg g− 1 DW)
Intracellular
plumbagin
(mg g− 1 DW)
5.33 ± 0.21
8.65 ± 0.44*
–
2.79 ± 0.37
5.33 ± 0.21
11.45 ± 0.77*
7.32 ± 0.41*
1.10 ± 0.21
6.54 ± 0.38*
0.57 ± 0.18
Day 7
Plumbagin
leaching
(mg g− 1 DW)
Total
plumbagin
(mg g− 1 DW)
Intracellular
plumbagin
(mg g− 1 DW)
Plumbagin
leaching
(mg g− 1 DW)
Total
plumbagin
(mg g− 1 DW)
5.31 ± 0.35
8.14 ± 0.43*
–
3.82 ± 0.42
5.31 ± 0.35
11.96 ± 0.76*
5.29 ± 0.25
7.12 ± 0.43*
–
4.68 ± 0.25
5.29 ± 0.25
11.80 ± 0.46*
8.42 ± 0.63*
7.24 ± 0.36*
1.44 ± 0.27
8.68 ± 0.42*
6.92 ± 0.31*
2.16 ± 0.28
9.08 ± 0.33*
7.11 ± 0.30*
6.42 ± 0.28*
0.80 ± 0.17
7.22 ± 0.26*
6.40 ± 0.21*
1.16 ± 0.26
7.57 ± 0.47*
Values presented as mean ± SD. Data marked with an asterisk are significantly different (elevated) with respect to the corresponding control according to Tukey's test (p b 0.05).
The final root biomass was nearly the same (0.077 ± 0.002 g on fresh weight basis) in all cases.
710
GANGOPADHYAY ET AL.
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