Effect of NP Supply on Herb Yield, Hypericin Content and Cadmium
Accumulation of St. John’s Wort (Hypericum perforatum L.)
M. Azizi and R. Omidbaigi
Tarbiat Modarres University
Department of Horticulture,
Tehran, Iran
Keywords: fertilisation, herb quality, number of flowers
Abstract
The experiment was done on `Topas` cultivar in the Research Station of
Tarbiat Modarres University located near Tehran and was conducted through two
successive seasons of 1998-1999. We have studied the effect of three levels of
nitrogen fertiliser (0, 150, 250 kg ha-1) and three levels of phosphorous fertiliser (0,
100, 200 kg ha-1) on herb yield, hypericin content and cadmium accumulation of the
plant. Results show that fertiliser treatments can affect herb quality and yield. The
highest herb yield (1053.9 g m-2) was produced in the plots that received 250 kg N
and 100 kg P fertiliser as compared to control plot(745.8 g m-2). According to the
results NP-supply increased the number of flowering stems per plant and hypericin
content. There is a significant correlation between the number of flowering stems
and the hypericin content of the St. John’s wort herb. All fertiliser treatments
increased cadmium accumulation of the herb as compared to the control treatment.
The highest cadmium content (0.269 mg kg-1 dry weight basis) was measured in the
plot that received 250 kg N and 200 kg P fertiliser, the lowest cadmium content was
measured (0.091 mg kg-1 dry weight basis) in the control plot.
INTRODUCTION
St. John’s wort (Hypericum perforatum L.) is an important medicinal plant that
has been used since ancient for pharmaceutical effects such as wound healing and
antidepressive properties (Hungarian Pharmacopoeia, 1984, Bombardelli and Morazzoni,
1995, Cellarova et al., 1995). Hypericin, a polycyclic aromatic dione, is one of the most
important secondary metabolites. It shows antiviral and antiretroviral activities (ESCOP,
1996, Cellarova et al., 1995).
The formation and accumulation of active substances in medicinal plants are
affected by environmental condition (Franz 1983, Palevitch, 1987. Edaphic factors
especially chemical properties of the soil also have particular importance in the
development of active substances (Franz, 1983). Hypericum perforatum is a cadmium
accumulator (Marquard and Schneide, 1996, Buter et al., 1996), nitrogen and phosphorus
fertilisers affect cadmium accumulation in plants (Grant et al., 1998), therefore we studied
the effects of NP-supply on active substances and cadmium accumulation in the plant.
MATERIALS AND METHODS
Field Experiment
Seeds of Hypericum perforatum L. cultivar `Topas` were soaked in water
overnight, dried, and were sown at a depth of 5 mm in spring in the open field nursery
and irrigated afterwards regularly. When the seedlings reached the 25 cm height, they
were transplanted into the field for fertiliser treatments. Each plot was 125×160 cm in size
and 40 cm x 25 cm plant spacing was applied. Three levels of nitrogen (0, 150, 250 kg
ha-1) and three levels of phosphorus (0, 100, 200 kg ha-1) were provided. Sources of N and
P fertilisers were urea and super phosphate. The P fertiliser contained an average of 26
mg Cd kg-1.
Proc. Int. Conf. on MAP
Eds. J. Bernáth et al.
Acta Hort. 576, ISHS 2002
267
Hypericin Analysis
One gram of air-dried and homogenised aerial part of Hypericum perforatum was
extracted in a Soxhlet with chloroform (200 ml) until chlorophyll ceased extracting (4 h).
During this period the temperature of water bath was 70 ºC. After removal of chloroform
the thimble was reextracted with acetone (200 ml) until the red pigment, hypericin, ceased
extracting (6 h). The water bath temperature was 65 ºC. The filtrate allowed to evaporate
in a rotary evaporator and the water bath temperature was set at 35 ºC. This was followed
by addition of methanol to make up 25 ml. The mixture was then mixed and filtered,
supernatant was then discarded and 5 ml of the mixture were diluteded with 20 ml of
methanol. The methanolic extract was then analysed by spectrophotometer (Unicam 8700
series U.V. visible) at 590 nm (Upton, 1997), and the hypericin content was calculated by
the following equation (Anounymus, 1991, Upton, 1997):
A × h × 1000
H = -----------------------------B × 718 × (100 – a)
H : Hypericin content (µg g-1); A : Absorbance at 590 nm; h : Dilution parameter
(125) ; B : Diameter of Cuvette (1 cm); 718 : Specific absorbance of hypericin; a: Sample
moisture content.
Cadmium Analysis
The cadmium content of the samples was determined according to the LUFAmethod introduced for the analysis of animal food products (Anonymous, 1988). The
dried samples were ashed at 550 ºC by dissolving the tissue in 20 % HNO3. Cadmium
concentration was measured using Varian 300/400 atomic absorption spectrophotometer
at a wavelength of 228.8 nm using a graphite furnace with deuterium correction.
RESULTS
Herb Yield
Results show that fertiliser treatments increased the dry herb yield of St. John’s
wort as compared to control (Fig. 1). According to the results of two years, control plots
produced the lowest herb yield and the highest herb yield was harvested from the plots
that received 250 kg N and 100 kg P fertiliser (Fig. 1).
Hypericin Content
The results indicated that NP supplies increased the hypericin content of St.John’s
wort significantly (Fig. 2). The highest hypericin contents in 1998 and 1999 (1052.03 and
882.63 µg g-1 dry weight) were extracted from the plot that was treated with N250, P100
and N250, P200 kg ha-1 respectively. The lowest hypericin contents in 1998 and 1999
(434.26 and 224.43 µg g -1 dry weight) were obtained from the control plots (Fig. 2).
Comparisons between the number of flowering stems/plant (Fig. 3) and hypericin content
show that there is a positive correlation between these two characteristics (R2 = 0.81).
Cadmium Content
Cadmium analysis of the samples shows that Hypericum perforatum accumulates
cadmium, which is promoted by fertiliser treatments. On the basis of our results (Fig. 4)
the highest cadmium accumulation occurred in the plants that were treated with high level
of N and P fertilisers, the lowest cadmium accumulation was observed in the control
plants.
DISCUSSION
The yield and quality of St. John’s wort are affected by chemical fertilisers as
other agricultural crops but because of the pharmaceutical use of the plant these effects
268
have a special importance on modification of hypericin and cadmium content. Therefore
such a fertiliser treatment which has a positive effect on active substance should be
recommended. Because the hypericin content in the flowers is characteristically high
(Martonfi and Repcak, 1994, Repcak and Martonfi, 1997, Pluhar and Zelnik, 1994) the
positive correlation between the flower number and fertilisation is rather important.
However, the cadmium content was increased by application of fertilisers, especially by
phosphorus, in harmony with results of Grant et al. (1998) and Eriksson (1990) the
cadmium content of the soil before cultivation must also be controlled.
Literature Cited
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19884.
Anonymous, 1988. LUFA-Methodenbuch Band 111, Die chemische Untersuchung der
Futtermittel, 2 .Erganzung, VDLUFA-Verlag, Darmstadt, Abschnitt A 17(2):2.
Bombardelli, E. and Morazzoni, P. 1995. Hypericum perforatum. Fitoterapia. 66(1):4368.
Buter, B., Soldati, A., Schaffner, W., Berger, K. and Pank, F. 1996. Site-specific shoot
dry matter and concentrations of hypericin, biflavons and cadmium in different
Hypericum spp. accessions (first year results). Proceedings. International Symposium
on Breeding Research on Medicinal and Aromatic Plants, Quedlinburg, Germany, 30
June-4 July. 2:84-87.
Cellarova, E., Kimakova, K., Daxnerova, Z. and Martonfi, P. 1995. Hypericum
perforatum (St Johns wort ): In vitro culture and the production of hypericin and other
secondary metabolites. In : Bajaj, P.S.(ed): Biotechnology in Agriculture and Forestry.
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261-275.
Eriksson, J. 1990. Factors influencing adsorption and plant uptake of cadmium from
agricultural soil. Department of Soil Sciences Reports and Dissertations, 4. Swedish
University of Agricultural Science, Upssala, Sweden 29.
ESCOP, 1996. Monographs on the medicinal use of plants drugs, Fasicule 1, Hyperici
herba.
Franz, Ch. 1983. Nutrient and water management for medicinal and aromatic plant. Acta
Hort. 132:203-215.
Grant, C.A., Buckley, W.T., Bailey, L.D. and Selles, F. 1998. Cadmium accumulation in
crops. Can. J. Plant Sci. 78:1-17.
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Budapest
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Martonfi, P and Repcak, M. 1994. Secondary metabolites during flower ontogenesis of
Hypericum perforatum L. Zahradnictvi. 21(1):37-44.
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208:29-34.
Pluhar, Z. and Zelnik, K. 1994. Introduction of Hypericum perforatum cultivar `Topas.`
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Hypericum perforatum flower. Biologia-Bratislava. 52(1):91-94.
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269
Dry herb yield ( g/m2)
1200
1000
a
800
bc
c
a
ab
a
a
bc
c
600
400
200
0
two year mean
N0P0
N0P100
N0P200
N150P0
N150P100
N150P200
N250P0
N250P100
N250P200
745.883
829.817
789.25
984.567
832.133
961.483
932.883
1053.9
964.85
tw
o
ye
ar
m
NP-Supply
Fig.1- Effect of NPsupply on dry herb yield of St.John's wort
Hypericin content( micg/g)
1200
1000
ab
800
bc
c
600
400
a
bc
a
abc abc
bc
c
c
c
ab
bc
d
d
de
200
e
N 150
P 100
N150
P 200
4 3 4 .3 6 9 8 .8 6 8 7 .6 8 0 5 .5 8 3 2 .4 7 1 7 .6
N250
P0
N250
P 100
N250
P200
941
1052
8 2 1 .1
N 0P 0
2 2 4 .4
N 0P 1 N0P 2
00
00
445
N 150
P0
N 150
P 100
N150
P 200
N250
P 100
N250
P200
3 7 3 .2 6 2 2 .2 7 6 5 .2 7 8 0 .2 6 8 1 .2 7 9 0 .9 8 8 2 .6
N P -S u p p ly
F ig . 2 - E ffe c t o f N P s u p p ly o n h y p e ric in c o n te n t o f S t.J o h n 's w o rt
270
N250
P0
98
1999
N 150
P0
19
1998
N0P1 N0P2
00
00
9
N 0P 0
9
19
0
ea
n
20
18
ab
No of stem / plant
16
ab
14
12
ab
cd
cd
d
ab
bc
c
10
8
b c
b c
a
ab
b c
a
e
e
d
6
4
2
N 15
0P 0
N 15
0P 1
00
N 15
0P 2
00
N 25
0P 0
N 25
0P 1
00
N 25
0P 2
00
8 .8 9
1 3 .4
1 1 .6
1 3 .8
1 4 .3
1 5 .5
1 3 .2
1 5 .1
1 6 .6
1999
N 0P
0
N 0P
100
N 0P
200
N 15
0P 0
N 15
0P 1
00
N 15
0P 2
00
N 25
0P 0
N 25
0P 1
00
N 25
0P 2
00
1 0 .5
1 1 .2
1 4 .1
1 6 .1
1 7 .5
1 4 .8
1 5 .1
1 7 .4
1 8 .1
8
N 0P
200
99
N 0P
100
9
19
1998
N 0P
0
19
0
N P -S u p p ly
F ig . 3 -
E ff e c t o f N P s u p p ly o n n u m b e r o f s t e m /p la n t o f S t .J o h n 's w o r t
0 .3
Cadmium content(mg/kg)
0 .2 5
a
a
a
a
0 .2
ab
ab
0 .1 5
ab
ab
ab
abc
ab
bc
ab
0 .1
bc
bc
bc
b
c
0 .0 5
0
1998
1999
N0P0
N0P100
N0P200
N150P 0
N150P100
N150P200
N250P0
N250P100
N250P200
0 .0 9 2
0 .1 4 9
0 .1 8 9
0 .1 4 7
0 .1 6 8
0 .1 7 1
0 .1 1 8
0 .2 5 9
0 .2 6 9
N0P0
N0P100
N0P200
N150P0
N150P100
N150P200
N250P0
N250P100
N250P200
0 .0 5 9
0 .0 9 6
0 .1 4 7
0 .1 2 1
0 .1 1 4
0 .1 5 2
0 .1 0 4
0 .2 3 1
0 .2 2 1
19
98
19
99
F i g . 4 - E f f e c t o f N P s u p p l y o n c a d m i u m a c c u m u l a t io n in S t .J o h n ' s w o r t
271