Alkaloid Accumulation in Catharanthus roseus
Increases with Addition of Seawater Salts to the
Nutrient Solution1
5
Wang Jing-Yan, Liu Zhao-Pu2
College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095
(China)
ABSTRACT
10
A sand culture experiment was conducted to determine the effects of different seawater (5 and 10%)
treatments on plant growth, inorganic ions, indole alkaloid concentrations and yield in Catharanthus
roseus, in an effort to increase the alkaloid yield by artificial cultivation. The total fresh and dry weight
and tissue K+ concentration of C. roseus decreased, and increased Na+ concentrations in roots, stems
15
and leaves in seawater stressed plants as compared to the control. The concentrations and yield of
vindoline, catharanthine, vinblastine and vincristine increased under seawater stress. The
concentrations and yield of these alkaloids were higher in 5% seawater-stressed plants than that of the
10%. Considering the industrial production, 5% seawater treatments could reduce the cost of
producing of alkaloid. In the control plants, the highest alkaloid concentrations reached a peak on 100
20
days after planting (DAP); suggesting that plant harvest must be optimized in terms of growth
duration.
Key words: alkaloids contents, C. roseus, growth, inorganic ions
25
INTRODUCTION
Salt stress induces various biochemical and physiological responses in plants and affects almost all
plant processes (Nemoto and Sasakuma, 2002). Salt damage to plants can be due to a combination of
30
several causes, like osmotic and specific ion toxicity (Daniela et al., 2004) that affect a wide spectrum
of physiological and metabolic processes in plants (Silveira et al., 2001). Salinity not only induces
high Na+ concentrations in plant tissues but also reduces the uptake of essential nutrients such as K+
and Ca2+ (Parida and Das, 2005). Most agricultural plants use K+, rather than Na+, as an important
component of osmotic adjustment and as an essential macronutrient (Schachtman and Liu, 1999).
1
Project supported by the National High Technology Research and Development Program (2007AA091702) and Open
Foundation of Key Laboratory of Jiangsu Province (K04009).
2 Corresponding author. E-mail: [email protected]
1
Consequently, plants growing in saline soils may suffer both Na+ toxicity and K+ deficiency.
Salinity also modulates the levels of secondary metabolites that have an important physiological
influence, particularly on plant stress tolerance (Liu et al., 1997; Dutta et al., 2005). Some of these
metabolites affect light absorptive properties, harvesting light for photosynthesis and protecting the
5
cells from damage effects caused by high energy radiation. Others promote defensive action against
herbivores and pathogens (Harborne and Williams, 2000), and some of them, like vinbalstine and
vincristine in Catharanthus roseus are very useful drugs against cancer and Parkinson’s disease. It seems
practically important to study the relation between salt stress influences on the increasing content of
human health important secondary metabolites of medicinal plants.
10
The exploitation of medicinal plants was recently increased (Tan et al., 20 06). Catharanthus roseus
(L.) G. Don is a perennial important medicinal and evergreen herb of the dogbane family
(Apocynaceae), produces more than 100 monoterpenoid indole alkaloids (TIAs) in it’s different
organs. The terpenoid indole alkaloids such as catharanthine, vinblastine and vincristine in plants are
very important secondary metabolites owing to their wide pharmaceutical applications for human
15
health (Tan et al., 2006; Sreevalli et al., 2004). Vindoline and catharanthine are the precursors of
vinblastine and vincristine. However, the contents of these alkaloids are usually too low to meet the
health market requirements. Despite a number of reports on the medicinal aspects (Filippini et al.,
2003), growth-regulator effects (El-Sayed and Verpoorte, 2005; Jaleel et al., 2006; 2007a), and
water-stress responses (Jaleel et al., 2007b; 2007c) of C. roseus plants, only a few attempts to explain
20
the physiological basis of salt tolerance and secondary metabolites’ accumulation were found. The
objectives of this study were to determine the effects of partial concentrations of seawater salts in the
irrigation water on growth and indoline alkaloid content of C. roseus, and on enhancing the indole
alkaloid accumulation by changing the culture condition.
25
MATERIALS AND METHODS
The seeds of C. roseus were collected from Seashore Technology Institute of Nanjing Agricultural
University, Hainan, China. The seeds were surface sterilized in an aqueous solution of 0.1% (w/v)
HgCl2 for 1 min to prevent fungal contamination, and then thoroughly rinsed in several changes of
30
sterile water. Seeds were sown in plastic containers (30×20 cm) containing moist vermiculite. After
germination, the seedlings were selected for uniformity and grown in pots (a radius of 25 cm and depth
of 40cm) filled with quartz sand in the glasshouse, and watered with half-strength Hoagland nutrient
solution every two days. Forty days after planting (DAP), the plants were cultured with full-strength
Hoagland nutrient solution containing ( in g·L-1, KNO3 5.05×10-1, Ca(NO3)2 8.20×10-1, KH2PO4
35
13.60×10-2, MgSO4 2.4×10-1, FeSO4·7H2O 5.57×10-3, H3BO3 2.86×10-3, MnCl2·7H2O 1.81×10-3,
ZnSO4·7H2O 2.2×10-4, CuSO4·5H2O 0.8×10-4, (NH4)2MoO4 0.9×10-4) supplemented with dried
seawater salt. The crude salt was produced by evaporation from seawater in Laizhou bay (laying at
2
latitude 36°59′-37°28′ N and longitude 119°33′-120°18′ E). The major ions in 100% seawater were (in
g·L-1): HCO3- 0.13, Cl- 17.52, SO42- 3.87, Ca2+ 0.79, Mg2+ 1.03, K+ 0.60, Na+ 9.48. Three treatments
including control (no added seawater), 5% (w/v) and 10% (w/v) seawater were established with twelve
replicates each and irrigated very two days.
5
Plants samples were taken at random on 60, 80, 100 and 120 DAP and were divided into two sub
samples: one for biomass determination and inorganic ions analysis and the another for alkaloid
extraction. Samples for biomass and inorganic ions analysis were immediately oven-dried in paper
bags at 110 °C for 5 min and then at 70 °C to a constant weight (DW). The samples for alkaloid
extraction were oven-dried in paper bags at 70 °C (Luo et al., 2005).
10
A 50 mg sample was ashed in a muffle stove. The ash was dissolved in concentrated nitric acid and
diluted to 100 mL with distilled water. The contents of Na+ and K+ were determined by an atomic
absorption spectrometer (Hitachi Z-800, Japan)
Vindoline, catharanthine, vinblastine and vincristine extraction from C.roseus was carried out
according to Luo (2005) with slight changes. Alkaloids were extracted with hot methanol (100%) from
15
plant dry powder of C. roseus. The alkaloid extract was filtered and was analyzed by HPLC system.
The detailed method was as follows: these four kinds of alkaloid were separated on a Diamonsil (C18
column 250 mm×4. 6 mm), using 380 ml solution A [water: diethylamide = 986:14 (v/v),pH 7. 2 ]
combine with 620 ml solution B [methanol: acetonitrile = 4:1] as the mobile phase. The detection was
performed at a flow rate of 1.5 mL·min-1, and detected at 220 nm within 40 min.
20
Statistical analysis was performed using Excel and SPSS 13.0 followed by Tukey’s multiple range
tests. The values are means ± S.D. for at list four samples in each group. P values≦0.05 were
considered as significant. The figures were drawn by Originpro8.0 (soft ware) and Excel.
RESULTS
25
Seawater salts addition effects on of C.roseus growth and composition
Addition of seawater salts to the Hoagland nutrition solution significantly decreased the dry weights
of roots, stems and leaves of C. roseus when compared to control (Table ). In the treatment with 5%
30
seawater, the decrease of dry weight ranged from 32% of the control after 60 days to 21% after 120
days. Comparable numbers for the 10% seawater treatment were 40 and 48% of control after 60 and
120 days respectively.
35
40
3
TABLE 
Seawater salt stress effects on the dry weight yields of C.roseus plant parts
Seawater
salts
concentration
(%)
0
5
10
Tissue
Duration of treatments (Days After Planting)
60
80
100
120
(gplant-1)
Roots
0.47 a
0.30 b
0.23 c
0.76 a
0.67 b
0.40 c
1.27 a
0.96 b
0.64 c
2.49 a
1.41 b
0.99 c
0
5
10
Stems
0.54 a
0.45 b
0.40 b
2.35 a
1.42 b
0.82 c
3.28 a
2.29 b
1.45 c
7.79 a
6.56 b
4.28 c
0
5
10
Leaves
1.41 a
0.89 b
0.83 b
2.11 a
1.70 b
0.99 c
2.81 a
2.40 b
1.46 c
2.94 a
2.58 b
1.60 c
5
Notes: Values in the same row not sharing a common letter differ significantly at P< 0.05. Composed
of Hoagland’s nutrient solution with the addition of 5 or 10% dried seawater salts. The same as
follows.
10
Effects of seawater salts addition on the tissue concentrations of K+ and Na+ in C. roseus plant tissues
The K+ concentration in root and stem decreased slightly with duration of treatments in the control
plants, but increased in the leaves. With seawater irrigation, the concentrations of K+ decreased with
the duration of the treatment. The K+ concentration in the root was greater in the seawater-treated
15
plants than in those of the control treatment, possibly because seawater had higher concentration of K+
than that in Hoagland nutrient solution (see M&M). Plants treated with 10% seawater had lower K+
concentration than those with 5% treatment (Fig.1a).
Fig.1b showed the Na+ content of C. roseus organs in the presence and absence of seawater stress.
Control plants kept low concentrations of Na+. Seawater-treated plants showed a significant increase in
20
Na+ contents compared to the control.
Fig.1
Fig.1 Effects of various durations of seawater salts additon on tissue concentrations of K+ (a) and Na+
(b) in C.roseus. The vertical bars represent ± SD when greater than the symbol size. (DAP=days after
25
planting)
Seawater salts additiont effects on the alkaloid contents in C. roseus
The vindoline contents in roots, stems and leaves increased significantly under seawater stress in
30
three organs when compared with the control, and increased with the duration of seawater stress.
However, there was no significant difference in the vindoline contents in roots and stems between 5%
4
and 10% seawater treated plants. The vindoline contents in leaves was increased with increasing
seawater concentration except in the 60 DAP sampling (Table Ⅱ).
5
TABLE Ⅱ
Duration of seawater salts addition treatment effects on tissue contents of vindoline in C.roseus
Seawater
salts
concentration
(%)
0
5
10
Tissue
60
Vindoline contents (mg·g-1DW)
Days After Planting
80
100
120
Roots
0.746 b
0.930 a
0.899 a
0.680 b
0.988 a
0.903 a
0.877 b
1.09 a
1.07 a
0.809 b
1.13 a
1.33 a
0
5
10
Stems
0.311 b
0.402 a
0.477 a
0.342 b
0.456 a
0.482 a
0.352 b
0.461 a
0.474 a
0.243 b
0.555 a
0.544 a
0
5
10
Leaves
1.85 b
3.18 a
3.89 a
1.86 c
3.42 b
4.83 a
2.59 c
3.30 b
4.20 a
2.15 c
5.05 b
6.04 a
The catharanthine content changed in different treatments in a similar way to the contents of
10
vindoline (Table Ⅲ). However, catharanthine concentrations in roots and leaves were higher than that
of vindoline.
TABLE Ⅲ
Duration of seawater salts addition treatment effects on tissue contents of catharanthine in C.roseus
15
Seawater
salts
concentration
(%)
0
5
10
Tissue
60
Catharanthine contents (mg·g-1DW)
Days After Planting
80
100
120
Roots
0.792 b
2.48 a
2.74 a
0.821 b
2.90 a
3.16 a
1.34 b
2.85 a
3.02 a
0.786 b
3.00 a
3.14 a
0
5
10
Stems
0.248 b
0.522 a
0.623 a
0.172 b
0.650 a
0.680 a
0.336 c
0.615 b
0.707 a
0.259 b
0.658 a
0.695 a
0
5
10
Leaves
2.45 b
4.63 a
4.81 a
2.19 c
4.55 b
5.83 a
4.25 c
5.42 b
7.65 a
1.38 c
6.02 b
7.67 a
In roots, no significant difference of the vinblastine content was found between 5% seawater
irrigated plants and that in the control plants except for 80 DAP. However, 5% seawater increased
20
vinblastine contents in stems and leaves. With 10% seawater irrigation, the vinblastine contents
5
increased significantly when compared with that found in fresh water irrigated plants (Table Ⅳ).
TABLE Ⅳ
Duration of seawater salts addition treatment effects on tissue contents of vinblastine in C.roseus
Seawater
salts
concentration
(%)
0
5
10
0
5
10
0
5
10
Tissue
60
Vinblastine contents (mg·g-1DW)
Days After Planting
80
100
120
Roots
0.235 b
0.264 b
0.355 a
0.221b
0.302 a
0.346 a
0.263 b
0.322 ab
0.365 a
0.259 bc
0.284 b
0.389 a
Stems
0.154 b
0.258 a
0.254 a
0.148 b
0.242 a
0.266 a
0.185 b
0.249 a
0.337 a
0.164 b
0.246 a
0.276 a
Leaves
0.266 b
0.302 a
0.301 a
0.238 b
0.311 a
0.319 a
0.263 b
0.311 a
0.322 a
0.259 b
0.313 a
0.319 a
5
Vincristine content in C. roseus increased notably when seawater salts were present in the nutrient
solution as compared with that without salts. There was no significant difference in the vincristine
contents in roots and stems between 5% and 10% seawater treatments. However, the vincristine
10
content in leaves of 10% seawater-treated plants increased significantly when compared to the 5%
seawater treatment (Table Ⅴ).
TABLE Ⅴ
Duration of seawater salts addition treatment effects on tissue contents of vincristine in C.roseus
15
Seawater
salts
concentration
(%)
0
5
10
0
5
10
0
5
10
Tissue
60
Vincristine contents (mg·g-1DW)
Day after planting
80
100
120
Roots
0.049 b
0.112 a
0.113 a
0.097 b
0.166 a
0.183 a
0.130 b
0.180 a
0.168 a
0.065 b
0.185 a
0.178 a
Stems
0.015 b
0.041 a
0.058 a
0.015 b
0.074 a
0.074 a
0.083 a
0.097 a
0.086 a
0.015 b
0.078 a
0.079 a
Leaves
0.155 c
0.355 b
0.430 a
0.151 c
0.296 a
0.389 a
0.177 c
0.304 b
0.437 a
0.128 c
0.390 b
0.514 a
Alkaloid yield in C. roseus due to seawater salts addition treatment
20
With the duration of the seawater added salt irrigation, the yield of four kinds of alkaloids increased
6
gradually from 60 DAP to 120DAP. At 60 DAP, there was no significant difference in alkaloid yield
between seawater-treated plants and the control. The alkaloid yield per plant was higher in 5%
seawater salts addition treated plants than in the 10% treated plants. From 80 to 120 DAP, the yield of
vindoline, catharanthine and vincristine in 5% treated plants increased significantly when compared
5
with the fresh water irrigated plants. However, there was no significant change in vinblastine yield at
5% seawater-salt addition as compared with to the control (Fig.2).
Under control conditions, the vindoline, catharanthine and vincristine reached the highest yield on
100 DAP and then decreased (Fig.2).
10
Fig.2
Fig.2 Effects of various duration (DAP=days after planting) of seawater salts addition treatment on
alkaloid yield in C.roseus.
15
DISCUSSION
Salinity can inhibit root growth, decrease external water potential and cause ion imbalance and ionic
toxicity (Greenway and Munns, 1980; Hasegawa et al., 2000). The reduction in plant growth may be
an adaptive response to salt stress (Jaleel et al., 2007d). A growth decrease under unfavorable
20
conditions allows the conservation of energy, thereby launching the appropriate defense response and
also reducing the risk of heritable damage as reported in Calendula officinalis plants under salinity
(Chaparzadeh et al., 2004). In the present study with seawater stress, salinity decreased plant dry
weight to a large extent when compared to the control (Table ). The differences in dry weights
between 5% seawater-treated plants and that of the control decreased with the duration of the seawater
25
stress, probably because of acclimation to the relatively low salinity level of 5% seawater-salt
additions.
High Na+ concentration competes with the uptake of other nutrient ions, especially K+, leading to
their deficiency in the plant. Increased NaCl concentration induced an increase in Na+ and Cl- and a
decrease in K+ tissue concentrations in a number of plant species (Khan et al., 1999; 2000; 2001), as
30
well as in present study (Fig.1). Seawater salts used in the nutrient solution of the present study
contained 9.48 g·L-1 Na+. Seawater salts stress caused an increase in the Na+ and Cl- tissue
concentration of C.roseus, with the highest ion accumulation in the leaves (Fig.1). There was a
negative relationship between Na+ and K+ concentrations in plant organs (Fig.1). The 5% and 10%
seawater additional salts used here also contained 0.03 and 0.06 g·L-1 K+ respectively, so the K+
35
concentration in roots of seawater-treated plants was slightly higher than that of control (Fig.1b).
The alkaloid concentration in C. roseus was influenced by factors like age of plant, salinity stress,
and nitrogen fertilization (Misra and Guptab, 2006), as well as on the treatment with plant-growth
regulator triadimefon (Jaleel et al., 2006), MeJA (El-Sayed and Verpoorte, 2005), GA3 (Jaleel et al.,
7
2007a). Recently, it has also been reported that indole alkaloid production in C. roseus can be
increased by abiotic stresses, like drought (Jaleel et al., 2007b; 2007c) and wounding (Flota et al.,
2004). In the present study, another abiotic stress (seawater salts addtition) increased the
concentrations of vindoline and catharanthine, especially in C. roseus leaves.
5
The metabolism and regulation of C. roseus alkaloids have been investigated under multiplicate
regulats.
Alkaloids
accumulation
are
controlled
by
organ-,
development
stage
and
environment-specific (De Luca and Thomas，1999). Previous studies with immature and mature
seedlings suggested that alkaloid biosynthesis and accumulation were associated with plant
development stage (De Luca et al., 1986; Rafael et al., 2001).
10
In the present study, the alkaloid contents in the plants were highest in the leaves when compared
with that in roots and stems. The vindoline, catharanthine and vinristine yield increased with
increasing duration of plant growth in sand culture until 100 DAP and then decreased. These results
suggest that plant alkaloid metabolism is under organ- and development- specific control.
15
CONCLUSIONS
Dry weight of the C.roseus was increased by partial seawater stress (5 and 10%). The content of
vindoline, cantharanthine, vinblastine and vincristine was also increased. It is suggested that careful
use of seawater salt additions to a nutrient solution can be used for irrigation of the C.roseus plants, for
20
industrial alkaloid production, and by that could reduce the cost of alkaloid production. The peak in
alkaloid concentrations were obtained at 100 days after planting, and decreased after that; suggesting
that plant harvest time must be optimized to obtain maximized alkaloid yields. Further studies are
needed to clarify the relationships between the secondary plant products and salt stress.
25
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25
30
35
10
1.2
a
Leaves
-1
K Concentrations (mmolg DW)
1.0
0.8
0.6
Roots
+
Stems
0.4
1.0
0.9
0.8
0.7
0.6
0.5
0.4
1.4
1.2
1.0
0.8
0.6
0.4
Leaves
Stems
-1
Na Concentrations (mmolg DW)
60
1.5
1.2
0.9
0.6
0.3
0.0
80
DAP
control
5%
100
10%
120
80
100
120
b
1.0
0.8
0.6
0.4
0.2
0.0
Roots
+
0.8
0.6
0.4
0.2
0.0
60
control
5
DAP
5%
10%
Fig.1 Effects of various durations of seawater salts additon on tissue concentrations of K+ (a) and Na+
(b) in C.roseus. The vertical bars represent ± SD when greater than the symbol size. (DAP=days after
planting)
10
11
Catharanthine
Cont
20
5%
Cont
10%
Alkaloid yield (mg·plant DW)
16
-1
-1
Alkaloid yield (mg·plant DW)
Vindoline
12
8
4
0
60
80
100
Days after planting (DAP)
20
15
10
5
0
60
120
10%
-1
2.5
2
1.5
1
0.5
0
80
100
Cont
2.5
120
5%
10%
2
1.5
1
0.5
0
60
120
80
100
120
Days after planting (DAP)
Day after planting (DAP)
5
100
Vincristine
3
60
80
Days after planting (DAP)
Alkaloid yield (mg·plant DW)
-1
Alkaloid yield (mg·plant DW)
5%
10%
25
Vinblastine
Cont
5%
30
Fig.2 Effects of various duration (DAP=days after planting) of seawater salts addition treatment on
alkaloid yield in C.roseus.
12