35 CHAPTER 2 DEVELOPMENT AND ELICITATION OF VINCA ROSEA HAIRY ROOT CULTURES 2.1 INTRODUCTION Two anti cancer compounds, bisindole alkaloids, vinblastine and vincristine, accumulated in low amounts by Vinca rosea plants, are of great interest to the pharmaceutical industry. Vinblastine and vincristine at present, are produced by extraction of the plant material or by semi-synthesis using the monomeric precursors, Vindoline and catharanthine. 208 Vindoline is accumulated at a relatively higher level in the aerial parts, whereas catharanthine is present at a much lower level. 209, 210 Cell and tissue cultures of Vinca rosea have been extensively studied as alternative sources of anticancer and other alkaloids. Vinblastine and vincristine are not produced in undifferentiated cell suspension cultures, the main reason being the absence of vindoline production. Moreover, cell suspension cultures produce low levels of alkaloids and are unstable. Root cultures inculcated by Agrobacterium rhizogenes mediated transformation have become of much interest in recent times as sources of useful compounds due to their rapid growth and high productivity. 211,212 Hairy root cultures also possess a higher level of differentiation and an inherent genetic stability, resulting in a stable production of secondary metabolites when compared to cell suspension cultures. Potential of the hairy root cultures of Vinca rosea has been investigated by some research groups with respect to the production of indole alkaloids. 210, 213-216 Transformed root cultures have been found to be productive by at least one order of magnitude more than the cell suspension cultures. 217 Parr et al., 218 reported an interesting result, the detection by immunoassay of a small level of vinblastine in Vinca rosea hairy root cultures.The presence of bisindole alkaloids has
53
Embed
CHAPTER 2 DEVELOPMENT AND ELICITATION OF VINCA ROSEA …shodhganga.inflibnet.ac.in/bitstream/10603/14131/7/07_chapter 2.pdf · 40 and Cefotaxime were added, filter sterilized (Milex
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
35
CHAPTER 2
DEVELOPMENT AND ELICITATION OF VINCA ROSEA
HAIRY ROOT CULTURES
2.1 INTRODUCTION
Two anti cancer compounds, bisindole alkaloids, vinblastine and vincristine,
accumulated in low amounts by Vinca rosea plants, are of great interest to the
pharmaceutical industry. Vinblastine and vincristine at present, are produced by
extraction of the plant material or by semi-synthesis using the monomeric precursors,
Vindoline and catharanthine.208 Vindoline is accumulated at a relatively higher level
in the aerial parts, whereas catharanthine is present at a much lower level.209, 210Cell
and tissue cultures of Vinca rosea have been extensively studied as alternative sources
of anticancer and other alkaloids. Vinblastine and vincristine are not produced in
undifferentiated cell suspension cultures, the main reason being the absence of
vindoline production. Moreover, cell suspension cultures produce low levels of
alkaloids and are unstable.
Root cultures inculcated by Agrobacterium rhizogenes mediated
transformation have become of much interest in recent times as sources of useful
compounds due to their rapid growth and high productivity.211,212 Hairy root cultures
also possess a higher level of differentiation and an inherent genetic stability,
resulting in a stable production of secondary metabolites when compared to cell
suspension cultures. Potential of the hairy root cultures of Vinca rosea has been
investigated by some research groups with respect to the production of indole
alkaloids.210, 213-216 Transformed root cultures have been found to be productive by at
least one order of magnitude more than the cell suspension cultures.217 Parr et al., 218
reported an interesting result, the detection by immunoassay of a small level of
vinblastine in Vinca rosea hairy root cultures.The presence of bisindole alkaloids has
36
not been confirmed in any other hairy root lines produced since parr et al., (1988)218.
Another important result with transformed root cultures of Vinca rosea was that of
palazon et al., (1998)219 which demonstrated the ability of root lines to synthesize
vindoline. These findings in the literature promoted us to undertake studies on hairy
root cultures of Vinca rosea for exploring their potential as sources of therapeutically
interesting indole alkaloids.
2.2. MATERIALS AND METHODS
Table 5. Culture vessels, equipment and glassware
Round bottom flask (100 and 250ml)
Beakers (50,100,250,500 and 1000ml)
Pipettes (1,2,5 and 10ml)
Volumetric flasks (10,50,100 and 250ml)
Culture conical flasks (50 and 100 ml)
Screw cap tubes (5 and 10ml)
Conical flasks (250,500 and 1000ml)
Measuring cylinders (10,25,50,100,250,500 and 1000ml)
Separating funnels (125 and 250ml)
Culture tubes (50ml)
Petri dishes (90mm diameter)
Culture vessels and glassware were purchased from Borosil glass works limited,
Mumbai (Borosil) and Schott Duran.
37
The following are some of the major equipment used in thisstudy. ( Table 6)
Table 6. Major equipment used in the study
Bath sonicator (Loba 3.5 L100, Loba Chemie, Hyderabad)
Laminar flow clean air work station (Steri Vertical YSI 189, Yorko Sales Pvt. Ltd., New Delhi ) Vertical autoclave (Life Steriware, Yorko Sales Pvt. Ltd., New Delhi)
Electronic balance (Shimadzu ELB-300 And EIB 200G ,Shimadzu Philippines)
Ph meter (Elico LI 127, Elico Ltd .,Hyderabad)
Water purification system (Millipore Elix 3,Millipore India Ltd., Bangalore)
linoleic acid (350 µg/ml) were prepared by dissolving in double distilled water. Stock
62
solution of salicylic acid was prepared in double distilled water by using a few drops
of 0.1M sodium hydroxide. Arachidonic acid was dissolved in ethanol to get a stock
solution of 2.5 mg/l and the Ph of the solutions was adjusted to 5.5 wherever required
and filter sterilised.
Elicitation
Hairy root cultures were subjected to treatment with abiotic and biotic elicitors
on day 21. The abiotic elicitors were tested at three concentrations each. Copper
sulphate (100,500 and 1000µM), cadmium acetate (100,500 and 1000µM), aluminium
chloride (10, 50, and 100 mM) and silver nitrate (10,100, and 1000 µM) were tested
in one experiment. Sodium chloride (50,100, and 250Mm) were added in another
experiment. Salicylic acid (100 and 1000µM), arachidonic acid (1 and 5 mg/l) and
linoleic acid (50 and 250µM) were tested in a third separate experiment. The
incubation conditions were same as described above. The roots were harvested on day
23 in each study.
Alkaloid Analysis
After harvesting, hairy roots were washed once with double distilled water,
blotted and fresh weights recorded. Hairy roots were extracted and analyzed for
alkaloids by TLC and HPLC as described in Chapter 2.
Statistical Analysis
Analysis of variance (ANOVA) and Tukey’s test were carried out using prism
5 (GraphPad Software Inc., USA) for determining the significance of treatment
effects. A p value of <0.05 was considered significant.
63
2.8. RESULTS AND CONCLUSIONS
Hairy root cultures of Vinca rosea were treated with abiotic and biotic (signal
and signal transduction molecules) elicitors with an aim of increasing the production
of indole alkaloids. The accumulation of ajmalicine and catharanthine takes place
parallel to growth while the production of serpentine is non-growth phase were
exposed for elicitors and roots were harvested 48 hours after treatment for extraction
and analysis of alkaloids.
Elicitation with Metal Ions
Heavy metal ions especially at the highest concentration were generally found to be
detrimental to growth. Significant reduction of the root biomass occurred with copper
(340 mg, 23% of FW of control culture), cadmium (300mg, 21% of FW of control
culture) and silver (250mg, 17% of FW of control culture) treatments. This may be
due to the death of some cells because of stress condition as indicated by a change in
colour from cream to light brown to light black compared to the control roots where
the colour remained unchanged. The effects of elicitors on indole alkaloid production
are shown in table 10-12.
64
Table-10 Effect of Metal ions on Ajmalicine production in Hairy root cultures of Vinca rosea
Elicitor Concentration Ajmalicine Contenta(mg/1) CR CM CT Copper Sulphate
1000 µm 0.78±0.08 4.24±0.15 5.03±0.23*
500 µm 3.94±0.12 2.41±0.11 6.35±0.23*
100 µm 4.41±0.11 0.36±0.04 4.78±0.14*
Cadmium Acetate
1000 µm 0.65±0.04 0.45±0.05 1.09±0.09
500 µm
1.45±0.09
0.34±0.03
1.8±0.12
100 µm
3.20±0.16
ND
3.20±0.16*
Aluminium Chloride
100 mM
0.55±0.05 ND 0.55±0.05
50 mM
0.81±0.07
1.48±0.1
2.29±0.17*
10 mM 0.34±0.04 0.92±0.08 1.26±0.11
Silver nitrate
1000 µm 1.74±0.15 5.16±0.14 6.90±0.29*
100 µm
6.31±0.13
ND
6.31±0.13*
10 µm
5.98±0.17
ND
5.98±0.17*
Control
Water 1.73±0.14 ND 1.73±0.14
a n=3, values are mean ±, standard deviation; * significantly different from
control (p<0.05); AR- Ajmalicine in root ; AM- Ajmalicine in medium; AT -total
Ajmalicine; ND-not detected.
65
Table-11 Effect of Metal ions on Serpentine production in Hairy root cultures of Vinca rosea
Elicitor Concentration Serpentine Contenta(mg/1) S R SM ST Copper Sulphate
1000 µm 1.94±0.05 2.12±0.12 4.06±0.09*
500 µm 3.72±0.13 1.99±0.1 5.72±0.23*
100 µm 4.18±0.12 ND 4.18±0.12*
Cadmium Acetate
1000 µm 1.77±0.15 0.75±0.07 2.52±0.22*
500 µm 1.81±0.12 0.69±0.07 2.5±0.19*
100 µm 2.34±0.11 ND 2.34±0.11*
Aluminium Chloride
100 mM
3.02±0.12 ND 3.02±0.12*
50 mM
1.95±0.13
0.75±0.09
2.70±0.22*
10 mM 2.49±0.11 0.22±0.03 2.72±0.14*
Silver nitrate
1000 µm 1.40±0.09 ND 1.40±0.09
100 µm
3.34±0.13
2.58±0.09
5.93±0.22*
10 µm
3.53±0.13
ND
3.53±0.13*
Control
Water 1.42±0.17 ND 1.42±0.17
a n=3, values are mean ±, standard deviation; * significantly different from
control (p<0.05); SR- Serpentine in root ; SM- Serpentine in medium; ST-total
Serpentine; ND-not detected.
66
Table-12 Effect of Metal ions on Catharanthine production in Hairy root cultures of Vinca rosea
Elicitor Concentration Catharanthine Contenta(mg/1) CR CM CT Copper Sulphate
1000 µm 0.57±0.06 2.65±0.12 3.21±0.18
500 µm 1.98±0.1 1.45±0.07 3.43±0.17
100 µm 3.5±0.12 ND 3.50±0.12
Cadmium Acetate
1000 µm 0.53±0.06 ND 0.53±0.06
500 µm 1.1±0.11 ND 1.1±0.11
100 µm 2.3±0.13 ND 2.3±0.13*
Aluminium Chloride
100 mM
0.59±0.06 ND 0.59±0.06
50 mM
0.37±0.05
1.21±0.11
1.58±0.16
10 mM 0.29±0.04 2.06±0.08 2.34±0.12*
Silver nitrate
1000 µm 0.49±0.06 ND 0.49±0.06
100 µm
1.46±0.13 ND 1.46±0.13
10 µm
3.73±0.19 ND 3.73±0.19*
Control
Water 1.22±0.12 ND 1.22±0.12
a n=3, values are mean ±, standard deviation; * significantly different from
control (p<0.05); CR-Catharanthine in root ; CM-Catharanthine in medium;
CT-total Catharanthine; ND-not detected
67
Copper ions were found to stimulate alkaloid production as well as their
release into the medium (table 10-12). The highest concentration of cu²+ (1000 µM)
produced more release than the lower concentrations (500 and 100 µM). At 1000 µM
Cu, the amount of ajmalicine, serpentine and catharanthine found in the media were
4.24, 2.12 and 2.65 mg/l, respectively which were not detected in the media of control
cultures. Maximal production of alkaloid contents (root and media) was favoured with
lower concentration of the elicitor (Fig.12). Accumulation of ajmalicine (6.35 mg/l)
and serpentine (5.72mg/l) were improved with 500 µM Cu²+ by about 3.7 and 4 times,
respectively over the control cultures (1.73 and 1.42 mg/l, respectively). There was
nearly 3 times improvement in the production of catharanthine (3.5 mg/l) with the
lowest concentration of elicitor (100µM) when compared to control cultures
(1.22mg/l).
Elicitation with Cd²+ ions did not result in significant improvement in either
production or release of alkaloids, except for treatment with the lowest concentration
(100 µM). There were nearly 2 times improvements in the production of ajmalicine
(3.2mg/l), serpentine (2.52mg/l) and catharanthine (2.3mg/l) when compared to
control cultures (Fig. 13)
Treatment with aluminium chloride (100 mM) enhanced significantly the
production of serpentine (3.02 mg/l) by about 2 times while the accumulation of
ajmalicine and increases in the levels of TDC transcript and cellular tryptamine
(Zheng and Wu, 2004)249 Moreno-Valenzuela et al., (2003)252 found that cadmium
chloride which blocks ca2+ flux across the plasma membrane increased the total
alkaloid content of transformed root cultures of Vinca rosea by 25% and their release,
10 times. However, copper treatment decrease the total and individual valepotriate
contents of transformed roots of Valeriana locusta.255 Copper and cadmium were
found to affect the distribution of tropane alkaloids between hairy roots of
D.stramonium and the media, the effect being dependent on the concentration and
nature of the ion used for elicitation.255
68
1000 500 100 control0
1
2
3
4
5
6
7
AjmalicineSerpentineCatharanthine
*
*
*
**
*
**
*
Concentration (micromoles)
Alk
aloi
d co
nte
nt (
mg
/l)
Fig. 12 Effect of Copper Sulphate on Alkaloid Production in Vinca rosea hairy root cultures. Data with * are significantly different from control (p<0.05)
69
1000 500 100 control0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
AjmalicineSerpentineCatharanthine*
**
*
*
Concentration (micromoles)
Alk
alo
id C
on
ten
t (m
g/l)
Fig. 13 Effect of Cadmium Acetate on Alkaloid Production in Vinca rosea hairy root cultures. Data with * are significantly different from control (p<0.05)
70
100 50 10 control0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5Ajmalicine
Serpentine
Catharanthine
**
**
*
Concentration (milli moles)
Alk
alo
id C
on
ten
t (m
g/l)
Fig. 14 Effect of Aluminium chloride on Alkaloid Production in Vinca rosea hairy root cultures. Data with * are significantly different from control (p<0.05)
71
1000 100 10 control0
1
2
3
4
5
6
7
8Ajmalicine
Serpentine
Catharanthine*
**
*
* *
Concentration (micro moles)
Alk
aloi
d c
ont
ent
(mg
/l)
Fig. 15 Effect of Silver Nitrate on Alkaloid Production in Vinca rosea hairy root cultures. Data with * are significantly different from control (p<0.05)
72
Aluminim chloride was found to increase the accumulation of tropane
alkaloids (43-83%) in hairy root cultures of Brugmansia candiada with significant
release of scopolamine into the medium (Spollansky et al., 2000),240 Hamel et al.,
(1998)256 suggested that aluminium may act as an elicitor as most of the genes up
regulated by it shared homologies with pathogenesis-related ones. Ag+ has been found
to be an effective inducer of secondary metabolite production in several studies. It
improved theaccumulation of tropane alkaloids by 5- to 8 times in transformed root
cultures of B.candida and increased significantly the release of scopolamine.239 The
addition of Ag+ resulted in more than 2 times improvement in the yield of tanshinones
in Salvia miltiorrhiza hairy root cultures.257
From the data it is clear that there is stimulation in the accumulation of
alkaloids in hairy root cultures of Vinca rosea in the case of copper and silver
treatment. However, alkaloid production was not affected substantially by treatment
with aluminium and cadmium, which may be due to toxic effects overwhelming any
possible elicitation. The toxic effects of elicitors may be mechanisms of action. The
cellular damage caused by copper and other metal ions especially to membranes could
release endogenous elicitors that increase the production of secondary metabolites as
a stress response to the damage.258 Moreover, the positive effects of silver nitrate on
indole alkaloid production may probably be associated with the stimulation of the
activity of enzymes involved in the formation of terpenoid moiety donor,
secologanine. Ag+ was shown to stimulate the activity of 3-hydroxy-3-methylglutaryl
CoA reductase (HMGR) and 1-deoxy-D-xylulose 5-phosphate synthase(DXS) in
hairy root cultures of S. miltiorrhiza, thereby enhancing the production of diterpenoid,
tanshinone.259 Thus, elicitation of hairy rootcultures of Vinca rosea with metal ions
appears to be an effective strategy for improving the production of indole alkaloids.
73
Treatment with Sodium Chloride and Mannitol
Salt stress by sodium chloride and osmotic shock by mannitol did not affect
adversely the growth of hairy roots of Vinca rosea. Sodium chloride addition
stimulated the production and release of alkaloids (Table-13). The maximum amount
of ajmalicine, serpentine and catharanthine found in the media were 1.39, 1.43 and
0.44 mg/l, respectively. The highest ajmalicine and serpentine yield (4.21 and 4.08
mg/l, respectively) were obtained with 250 mM concentration (Fig. 16).The yields
were about 2.5 times higher than the control values (1.83 and 1.52 mg/l for ajmalicine
and serpentine, respectively).However catharanthine yield (2.36 mg/l) was improved
by 1.8 times over the control cultures (1.25 mg/l).
Treatment with mannitol improved the production of catharanthine and
serpentine with significant amounts being retained within the root tissues (Table-
14).With regard to catharanthine production (4.54 mg/l), 3.6 times improvement was
seen with 200 mM of mannitol (Fig. 17).Serpentine production (4.16 mg/l) was
increased by 2.7 times while ajmalicine contents (3 mg/l) were improved by 1.6 times
over the control.
74
Table-13 Effect of Sodium Chloride on Alkaloid production in Hairy root cultures of Vinca rosea
Elicitor Concentration Alkaloid Contenta(mg/1) Of NaCl Root Medium Total
Ajmalicine 250 mM 2.83±0.13 1.39±0.09 4.21±0.22*
100 mM 2.25±0.13 0.59±0.09 2.84±0.2*
50 mM 1.54±0.14 ND 1.54±0.14
Control
1.83±0.12
ND
1.83±0.12
Serpentine 250 mM 2.65±0.14 1.43±0.11 4.08±0.25*
100 mM 1.27±0.12 ND 1.27±0.12
50 mM 1.1±0.09 ND 1.1±0.09
Control
1.52±0.07
ND
1.52±0.07
Catharanthine
250 mM 1.92±0.08 0.44±0.05
2.36±0.13*
100 mM 2.18±0.17 0.13±0.02
2.31±0.19*
50 mM 1.22±0.1 ND 1.22±0.1
Control
1.25±0.08
ND
1.25±0.08
a n=3, values are mean ±, standard deviation; * significantly different from
control (p<0.05); ND-not detected
75
250 100 50 control0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
AjmalicineSerpentineCatharanthine
*
*
*
*
Concentration (milli moles)
Alk
alo
id c
on
ten
t (m
g/l)
Fig. 16 Effect of Sodium chloride on Alkaloid Production in Vinca rosea hairy root cultures. Data with * are significantly different from control (p<0.05)
76
Table-14 Effect of Mannitol on Alkaloid production in Hairy root cultures of Vinca rosea
Elicitor Concentration Alkaloid Contenta (mg/1) of Mannitol Root Medium Total
Ajmalicine 400 mM 1.75±0.12 1.25±0.12 3.0±0.24*
200 mM 2.51±0.19 0.23±0.03 2.75±0.21*
100 mM 2.35±0.14 0.18±0.03 2.52±0.17
Control
1.83±0.12
ND
1.83±0.12
Serpentine 400 mM 3.26±0.08 ND 3.26±0.08*
200 mM 4.16±0.13 ND 4.16±0.13*
100 mM 2.91±0.09 ND 2.91±0.09*
Control
1.52±0.07
ND
1.52±0.07
Catharanthine 400 mM 4.13±0.14 ND
4.13±0.14*
200 mM 4.43±0.17 0.37±0.03
4.5±0.17*
100 mM 3.50±0.17 0.1±.02 4.54±0.19*
Control
1.25±0.08
ND
1.25±0.08
a n=3, values are mean ±, standard deviation; * significantly different from control (p<0.05); ND-not detected
77
400 200 100 control0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
Ajmalicine
Serpentine
Catharanthine* *
*
*
**
**
*
Concentration (milli moles)
Alk
aloi
d co
nten
t (m
g/l)
Fig. 17 Effect of Mannitol on Alkaloid Production in Vinca rosea hairy root cultures. Data with * are significantly different from control (p<0.05)
78
In Vinca rosea plants, drought has been found to promote alkaloid
accumulation.260,261,262 On the other hand salt stress and osmotic shock have been
used for increasing the alkaloid accumulation in cell suspensioncultures.90 Higher
levels of ajmalicine and catharanthine were found to be accumulated in a Vinca rosea
salt tolerant line.216 Zhao et al., (2000b)263 reported that osmotic shock was more
effective than salt stress in improving indole alkaloid yields. A combination treatment
of fungal elicitation and mannitol effectively improved alkaloid production in cell
cultures of Vinca rosea.264 The results of our study support the positive effects of salt
and osmotic stress in improving secondary metabolite production in plant cell
cultures.
Effects of signal and signal Transduction Molecules
Salicylic, arachidonic and linoleic acids were added to 21-day-old hairy root
cultures of Vinca rosea to study their effect on alkaloid production. Growth of the
cultures was not affected significantly with these treatments. But hairy roots turned
light brown with the addition of arachidonic acid. This is presumably a manifestation
of the hyper sensitive response and browning has also been observed with cell
cultures of Taxus species elicitation with arachidonic acid.238 The results pertaining
to the effect of these agents on alkaloid yields are presented in Table 15-17.
Treatment with salicylic acid did not improve the accumulation of ajmalicine
and serpentine (Fig. 18). However, the contents of catharanthine (2.04 mg/l) were
found to be increased by 1.6 times with the lowest concentration (100 µM). The lower
levels seen with salicylic acid treatment may be due to the inhibitory effect of the
elicitor on alkaloid biosynthesis especially at the highest concentration.
79
Significant improvements in ajmalicine and serpentine production were
observed with arachidonic and linoleic acids. Concentration of the elicitor seemed to
be important as positive effects were seen at higher doses (Fig. 19 and 20). Also, these
treatments induced the release of ajmalicine and serpentine but the site of
accumulation of catharanthine was not affected (Table 15-17). Arachidonic acid
(5 mg/l) enhanced the yield of ajmalicine (4.3 mg/l) by 2.5 times when compared to
the control (1.73 mg/l) (fig. 19). A greater improvement was obtained for serpentine
production (4.87 mg/l) which was 3.4 fold more than the control (1.45 mg/l).
However, there was no significant effect on catharanthine accumulation (1.61 mg/l)
when compared to control cultures (1.36 mg/l).
80
Table-15 Effect of signal and signal transduction molecules on Ajmalicine Production in Vinca rosea hairy root cultures
Substance Concentration Ajmalicine Contenta(mg/1) AR Am At
Salicylic acid 1000µM 1.36±0.12 ND 1.36±0.12
100 µM 1.74±0.16 ND 1.74±0.16
Arachidonic acid
5mg/1 3.5±0.16 0.8±0.04 4.3±0.19*
1mg/1 1.73±0.1 ND 1.73±0.1
Linoleic acid 250 µM 2.74±0.03 ND
2.74±0.13*
50 µM 1.54±0.12 ND
1.54±0.12
Control
Water Ethanol(50%)
1.65±0.16 1.73±0.06
ND ND
1.65±0.16 1.73±0.06
a n=3, values are mean ±, standard deviation; * significantly different from
control (p<0.05); Ar-ajmalicine in root; Am-ajmalicine in medium; At-total
Ajmalicine;ND-not detected
81
Table-16 Effect of signal and signal transduction molecules on Serpentine Production in Vinca rosea hairy root cultures
Substance Concentration Serpentine Contenta(mg/1) SR SM ST
a n=3, values are mean ±, standard deviation; * significantly different from control (p<0.05); SR-Serpentine in root;Sm- Serpentine in medium;St-total Serpentine ; ND-not detected
82
Table-17 Effect of signal and signal transduction molecules on Catharanthine Production in Vinca rosea hairy root cultures
Substance Concentration Catharanthine Contenta(mg/1) CR CM CT
Salicylic acid 1000µM 1.22±0.12 ND 1.22±0.12
100 µM 2.04±0.17 ND 2.04±0.17
Arachidonic acid
5mg/1 1.61±0.12 ND 1.61±0.12
1mg/1 1.2±0.07 ND 1.2±0.07
Linoleic acid 250 µM 1.35±0.13 ND 1.35±0.13
50 µM 1.38±0.1 ND
1.38±0.1
Control
Water Ethanol(50%)
1.24±0.1 1.36±0.09
ND ND
1.24±0.1 1.36±0.09
a n=3, values are mean ±, standard deviation; * significantly different from control (p<0.05); CR- Catharanthine in root;Cm- Catharanthine in medium; Ct-total Catharanthine; ND-not detected
83
1000 100 control0.0
0.5
1.0
1.5
2.0
2.5 Ajmalicine
Serpentine
Catharanthine
*
Concentration (micromoles)
Alk
alo
id C
on
ten
t (m
g/l)
Fig. 18 Effect of Salicylic acid on Alkaloid Production in Vinca rosea hairy root cultures. Data with * are significantly different from control (p<0.05)
84
5 1 control (ethanol)0
1
2
3
4
5
6AjmalicineSerpentineCatharanthine*
*
Concentration (mg/l)
Alk
alo
id c
on
ten
t (m
g/l)
Fig. 19 Effect of Arachidonic acid on Alkaloid Production in Vinca rosea hairy root cultures. Data with * are significantly different from control (p<0.05)
85
250 50 control0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5 Ajmalicine
Serpentine
Catharanthine
*
*
*
Concentration (micromoles)
Alk
aloi
d c
on
ten
t (m
g/l)
Fig. 20 Effect of Linoleic acid on Alkaloid Production in Vinca rosea hairy root cultures. Data with * are significantly different from control (p<0.05)
86
Treatment with linoleic acid increased significantly the accumulation of
serpentine (3.65 mg/l), which was 2.5 times more than the control (Fig. 20).
Ajmalicine yield (2.74 mg/l) was improved by 1.7 times but catharanthine production
was not affected.
Salicylic acid is a signaling molecule that plays an essential role in many
plant defense reactions and up regulates the expression of various defense related
genes.265 It also inhibits the biosynthesis of ethylene, a phytohormone that is also
active in plant defense mechanisms. Treatment with salicylic acid increased the
production and release of hyoscyamine and scopolamine in hairy roots of
B.candida.239 But salicylic acid did not increase the total yield of tropane alkaloids
but induced their release from transformed root cultures of A.belladona.266 Salicylic
acid has also been found to be ineffective in increasing the alkaloid contents in cell
cultures of Vinca rosea.244,267,246 Moreover, Yahia et al., (1998)268 demonstrated that
there was no relation between the accumulation of ajmalicine and the evolution of
ethylene in cell cultures of Vinca rosea.
Arachidonic acid is a fatty acid present in the lipids of plant pathogenic
Oomycete fungi and a potent elicitor of sesquiterpenoid phytoalexins and suppressor
of glycol alkaloid synthesis in Solanaceous Species.269 Arachidonic acid has been
used to promote the production of taxanes in cell cultures of Taxus species in several
studies.270,271,272 Moreover, arachidonic acid has been found to alter the expression of
HMGR isogenes in potato and tomato.269,273 Also, it has been reported that there
exists a cross talk between mevalonate and non-mevalonate pathways in the synthesis
of alkaloids during stationary growth phase of hairy root cultures of Vinca rosea.274
The positive effects of arachidonic acid in the present study may be due to
stimulation of the mevalonate pathway, which supplies terpenoid precursors towards
87
alkaloid biosynthesis. Moreover, the polyunsaturated fatty acids arachidonic and
linoleic acids were found to induce extremely rapid generation of hydrogen peroxide
in the cultured potato plant cells.275 Such a stimulation of free–radical generation may
also be associated with hairy roots of Vinca rosea upon treatment with arachidonic
and linoleic acids, which may result in the enhancement of alkaloid yields.