molecules
Article
Effects of Light Intensity on the Growth,
Photosynthetic Characteristics, and Flavonoid
Content of Epimedium pseudowushanense B.L.Guo
Junqian Pan and Baolin Guo *
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Peking Union Medical College,
Beijing 100193, China; [email protected]
* Correspondence: [email protected]; Tel.: +86-139-1146-9895; Fax: +86-105-783-3172
Academic Editor: Derek J. McPhee
Received: 7 October 2016; Accepted: 2 November 2016; Published: 4 November 2016
Abstract: Epimedium pseudowushanense B.L.Guo is used in traditional medicine as an aphrodisiac
and to strengthen muscles and bones. Several recent reports have shown that flavonoids from
Epimedium also significantly affect the treatment of breast cancer, liver cancer, and leukemia.
However, few studies have examined the medicinal-ingredient yield of Epimedium, a light-demanding
shade herb, under different light intensities. To investigate the effects of light intensity on
medicinal-ingredient yields, Epimedium was exposed to five levels of light intensity until harvest time.
Leaf dry biomass under L4 was the highest among different light treatments. L4 was also associated
with the highest net photosynthetic rate. Quantification of epimedin A, epimedin B, epimedin C,
and icariin showed that L3 produced the highest amount of epimedin C, and that flavonoid content
responded to light levels differently. Results indicated that L3 and L4 were the optimal light levels
for medicinal-ingredient yield.
Keywords: Epimedium pseudowushanense B.L.Guo; light intensity growth; photosynthetic
characteristics; flavonoid content; medicinal-ingredient yield
1. Introduction
Epimedii Folium, commonly called Yinyanghuo, is an important perennial shade herb in traditional
Chinese medicine. Epimedium belongs to the Berberidaceae family and is widely used as an aphrodisiac
and to strengthen muscles and bones. Epimedium leaves contain high amounts of flavonoid glycosides,
such as epimedin A, epimedin B, epimedin C, and icariin [1,2]. Recent reports have shown
that flavonoids from Epimedium significantly affect the treatment of breast cancer, liver cancer,
and leukemia [3–5]. The recognition of the health benefits of Epimedium has increased its market
demand. However, its resource recycling rate is low and environmentally dependent. Furthermore,
its natural sources are endangered, further increasing prices. Commercial culture can address resource
constraints of Epimedium. E. pseudowushanense B.L.Guo is an important resource of Epimedii Folium and
is the first Epimedium species introduced and cultivated in Guizhou Province, China.
Light is an important environmental factor influencing plant growth, development, and secondary
metabolism [6]. Under high light conditions, certain plant species, such as bayberry trees, do not
grow because light irradiance decreases photosynthetic rates [7]. Therefore, all plants have their
own optimal light intensity ranges for growth. Light intensity that is too high or too low impacts
morphology, photosynthetic physiology, and secondary metabolite production. These characteristics
are closely related to medicinal plant productivity. For example, Ma and others [8] reported that
Camptotheca acuminata grown under 75% irradiance had significantly greater height, net photosynthetic
rate (Pn), stomatal conductance (gs), total aboveground biomass, and chlorophyll fluorescence than
plants grown under 100% (1500 ± 30 µmol·m−2 ·s−1 ), 50%, and 25% irradiance.
Molecules 2016, 21, 1475; doi:10.3390/molecules21111475
www.mdpi.com/journal/molecules
Molecules 2016, 21, 1475
2 of 11
Molecules
21, 1475of different flavonoids varies according to the structure and specific flavonoids
2 ofare
12
The2016,
function
involved for instance in limiting exogenous microbial growth, helping generate new fruits or leaves,
providing
light protection,
andflavonoids
enhancingvaries
antioxidant
defense
[9,10].
Qualitative
and quantitative
The function
of different
according
to the
structure
and specific
flavonoids
composition
of
flavonoids
varies
under
different
environments
[9].
Changes
in
light
intensity
may
are involved for instance in limiting exogenous microbial growth, helping generate
new fruits
influence
because the
flavonoid
hydroxyl
groups
on the[9,10].
A and Qualitative
B rings varyand
in
or leaves, flavonoid
providingcontent
light protection,
and
enhancing
antioxidant
defense
number
and
position.
Several
studies
have
shown
that
high
light
irradiance
promotes
the
quantitative composition of flavonoids varies under different environments [9]. Changes in light
biosynthesis
flavonoids,
such as
dihydroxy
B-ring-substituted
flavonoids
(luteolin
and
intensity mayofinfluence
flavonoid
content
because
the flavonoid hydroxyl
groups
on the7-OA and
B
quercetin
3-O-glycosides)
but
does
not
influence
the
biosynthesis
of
monohydroxy
B-ring-substituted
rings vary in number and position. Several studies have shown that high light irradiance promotes
flavonoids
(pigenin
7-O- and kaempferol
3-O-glycosides)
[11–14]. Pacheco
and others
[15] 7-Oreported
the biosynthesis
of flavonoids,
such as dihydroxy
B-ring-substituted
flavonoids
(luteolin
and
that
Piper
aduncum
grown
under
50%
natural
light
irradiance
had
higher
total
flavonoid
concentration
quercetin 3-O-glycosides) but does not influence the biosynthesis of monohydroxy B-ring-substituted
than
those (pigenin
grown under
100%
natural irradiance.
Based [11–14].
on thesePacheco
reports and
and others
the harvest
time of
flavonoids
7-O- and
kaempferol
3-O-glycosides)
[15] reported
E.
pseudowushanense,
the
present
study
exposed
Epimedium
to
five
different
light
intensities
for
that Piper aduncum grown under 50% natural light irradiance had higher total flavonoid concentration
60
days.
than
those grown under 100% natural irradiance. Based on these reports and the harvest time of
In the natural environment,
E. pseudowushanense
grows
in woodlands,
scrub,
and forest
E. pseudowushanense,
the present study
exposed Epimedium
to five
different light
intensities
for 60edges.
days.
In commercial
culture,
E.
pseudowushanense
seedlings
are
usually
shade-grown
by
a
simulated
wild
In the natural environment, E. pseudowushanense grows in woodlands, scrub, and forest edges.
cultivation
method.
However,
our understanding
of are
the usually
photosynthesis
and flavonoid
content
of
In commercial
culture,
E. pseudowushanense
seedlings
shade-grown
by a simulated
wild
E.
pseudowushanense
under
shade
management
is
limited.
This
study
aims
to
determine
the
optimal
cultivation method. However, our understanding of the photosynthesis and flavonoid content of
light
conditions for commercial
Epimedium
production.
This
study
alsoaims
aimstotodetermine
investigate
flavonoid
E. pseudowushanense
under shade
management
is limited.
This
study
the
optimal
accumulation
different
light intensities
improve
propagation
and
light conditionsinforEpimedium
commercialunder
Epimedium
production.
This studytoalso
aims to its
investigate
flavonoid
cultivation.
The
results
of
the
present
study
may
contribute
to
the
scientific
culture
and
management
accumulation in Epimedium under different light intensities to improve its propagation and cultivation.
of
as present
well asstudy
provide
reference to
forthe
other
Epimedium
We anticipate
that our
TheEpimedium,
results of the
maya contribute
scientific
culturespecies.
and management
of Epimedium,
results
will
improve
our
understanding
of
the
changes
in
photosynthetic
parameters
and
as well as provide a reference for other Epimedium species. We anticipate that our results will improve
concentrations
of
secondary
metabolites
in
Epimedium
under
different
light
treatments.
Our
results
our understanding of the changes in photosynthetic parameters and concentrations of secondary
will
providein aEpimedium
sound theoretical
foundation
for the standardized
cultivation
this theoretical
important
metabolites
under different
light treatments.
Our results will
provide aofsound
medicinal
plant.
foundation for the standardized cultivation of this important medicinal plant.
2.
2. Results
Results
2.1. Effects of Light Intensity on Plant Growth
Clear external differences were observed among plants grown under 60 days of different light
intensities. Compared
Compared with
with high
high light
light intensities
intensities (L3,
(L3, L4,
L4, and
and L5),
L5), leaf areas were higher under low
light intensities
intensities (L1
(L1and
andL2).
L2).L2
L2resulted
resultedininthe
thehighest
highestleaf
leaf
area.
Light
intensity
had
different
effects
area.
Light
intensity
had
different
effects
on
onpseudowushanense
E. pseudowushanense
growth:
L3,and
L4,L5
and
L5 resulted
in number
higher number
of branches.
On the
E.
growth:
L3, L4,
resulted
in higher
of branches.
On the contrary,
contrary,
and L2in
resulted
higher
SPAD
water The
content.
The of
number
of branches
under
L3,
L1 and L2L1
resulted
higher in
SPAD
and
waterand
content.
number
branches
under L3,
L4, and
L4,
and L5
were(p156.3%
(p187.5%
< 0.05),(p187.5%
< 0.05),
and(p203.1%
< 0.05)respectively,
higher, respectively,
than
L5 were
156.3%
< 0.05),
< 0.05),(pand
203.1%
< 0.05)(phigher,
than of plants
of
plants
grown
SPADunder
values
L1were
and L2
were
< 0.05)
and(p54.3%
(p higher,
< 0.05)
grown
under
L1.under
SPADL1.
values
L1under
and L2
53.1%
(p53.1%
< 0.05)(pand
54.3%
< 0.05)
higher,
respectively,
than ingrown
plants under
grownL5.
under
L5.content
Water content
responded
similarly.
The highest
respectively,
than in plants
Water
responded
similarly.
The highest
values
valuesobserved
were observed
inunder
plantsL1
under
the were
lowest
inunder
plantsL5
under
L5 (Figure
1).
were
in plants
and L1
theand
lowest
inwere
plants
(Figure
1).
Figure 1. Cont.
Molecules 2016, 21, 1475
Molecules 2016, 21, 1475
3 of 12
3 of 11
Molecules 2016, 21, 1475
3 of 11
Figure 1.1.Morphological
Morphological
parameters
E. pseudowushanense
light intensities.
Figure
parameters
of E.ofpseudowushanense
under under
differentdifferent
light intensities.
The data
The
data
are
expressed
as
mean
±
SD.
Different
letters
indicate
significant
differences
between
light
1. Morphological
of E.indicate
pseudowushanense
different
lightlight
intensities.
areFigure
expressed
as mean ± SD.parameters
Different letters
significant under
differences
between
intensity
intensity
treatments
n = ±30.
The data
are< expressed
as mean
SD. Different letters indicate significant differences between light
treatments
(p
0.05);(p
n <= 0.05);
30.
intensity treatments (p < 0.05); n = 30.
2.2. Effects
of Light
Light Intensity
Intensity on
on Plant
Plant Photosynthetic
Photosynthetic Parameters
Parameters
2.2.
Effects of
2.2. Effects of Light Intensity on Plant Photosynthetic Parameters
Figure 22 presents
presents the
the effects
effects of
of different
different light
light intensities
intensities on
on leaf
leaf photosynthetic
photosynthetic parameters.
Figure
parameters.
Figure
2 presents
the
effects
of different(Pn),
lighttranspiration
intensities onrate
leaf(T),
photosynthetic
parameters.
Compared
with
L1,
leaf
net
photosynthesis
and water
use
efficiency
Compared with L1, leaf net photosynthesis (Pn), transpiration rate (T), and water
use efficiency
(WUE)
Compared
withasL1,
leafintensity
net photosynthesis
(Pn),
transpiration
rate
(T), and
water
use efficiency
(WUE)
increased
light
increased,
but
not
under
L5.
L4
resulted
in
the
highest
Pn,
and
increased as light intensity increased, but not under L5. L4 resulted in the highest Pn, T, and T,
WUE,
(WUE)
increased
as
light
intensity
increased,
but
not
under
L5.
L4
resulted
in
the
highest
Pn,
T,
and
WUE, whereas
L1 resulted
in the measurements.
lowest measurements.
Compared
with
L1, L4 increased
Pn,
T, and
whereas
resulted
the lowest
Compared
with L1,with
L4 increased
Pn, T, and
WUE
WUE, L1
whereas
L1 in
resulted
in the lowest measurements.
Compared
L1, L4 increased
Pn, T,
andby
WUE
by
457.9%
(p
<
0.05),
107.6%
(p
<
0.05),
and
173.7%
(p
<
0.05),
respectively.
The
highest
gs
values
457.9%
(p
<
0.05),
107.6%
(p
<
0.05),
and
173.7%
(p
<
0.05),
respectively.
The
highest
gs
values
were
WUE by 457.9% (p < 0.05), 107.6% (p < 0.05), and 173.7% (p < 0.05), respectively. The highest gs values
were
observed
under
L4.
No
significant
differences
were
observed
between
other
treatments
(p
>
0.05).
observed
under L4.
No
differences
were
observed
treatments(p(p> 0.05).
> 0.05).
were observed
under
L4.significant
No significant
differences
were
observedbetween
between other
other treatments
Compared
with
L1,
intercellular
CO
2 concentration (Ci) decreased by 33.1% (p < 0.05), 34.3%
Compared
with
L1, L1,
intercellular
CO2CO
concentration
(Ci) decreased
by 33.1%
(p < 0.05),
< 0.05),
Compared
with
intercellular
2 concentration
(Ci) decreased
by 33.1%
(p <34.3%
0.05),(p34.3%
(p < 0.05),
and
36.2%
(p
< 0.05)
under
L3, L5,
L4, and
L5, respectively.
and
36.2%
(p
<
0.05)
under
L3,
L4,
and
respectively.
(p < 0.05), and 36.2% (p < 0.05) under L3, L4, and L5, respectively.
L1
L1
L2
L2
L3
L4
L5
L3
L4
L5
10
10
L4L4 L5L5
b
a a a a
b
b
b
b
6
b
a a
8
8
aa
4
c
L3L3
4
b
L2
L2
b
b
b
b
2
b
aa
L1
L1
L1
L1
L2
L2
L3
L4
L3
L4
L5
L5
0
b
L5
L5
2
b
0.10
c
b
L4
L4
bb
0
0.20
b
L3
L3
-1
CiCi
(mmol·mol
(mmol·mol)-1)
0 0 100
200
300
100 200 300 400
400 500
500
L2L2
aa
0.00
0.00
T (mmol·m-2·s-1)
-2 -1
T (mmol·m0.20
·s )
0.10
0.30
0.30
L1L1
a
6
0.0
0.0
0.000
0.000
dd
bb
WUE (%)
WUE (%)
cc
bb
0.010
0.010
bb
a
b
b
0.005
0.005
Pn (μmol·m-2·s-1)
bb
gs
gs (mmol·m
(mmol·m-2-2·s·s-1-1) )
Pn (μmol·m-2·s-1)
0.5
1.0
1.5
0.5
1.0
1.5
2.0
0.015
0.015
2.0
a
a
L1
L1
L2
L2
L3
L4
L3
L5
L4
Figure
Net
photosyntheticrate
rate(Pn),
(Pn),stomatal
stomatal conductance
conductance (gs),
2 concentration
(Ci),
Figure
2. 2.
Net
photosynthetic
(gs),intercellular
intercellularCO
CO
2 concentration (Ci),
Figure
2. Net photosynthetic
rate (Pn),
stomatal conductance
(gs), intercellular CO2 concentration
(Ci),
transpiration
rate
(T),and
andwater
water
useefficiency
efficiency
(WUE) of
of E.
different
transpiration
rate
(T),
use
(WUE)
E. pseudowushanense
pseudowushanenseleaves
leavesunder
under
different
transpiration
rate
(T),
and
water
use
efficiency
(WUE)
of
E.
pseudowushanense
leaves
under
different
light
intensities.The
Thedata
dataare
areexpressed
expressedas
asmean
mean ±
± SD;
light
intensities.
SD; nn == 9.
9. Different
Differentletters
lettersindicate
indicatesignificant
significant
light
intensities.
The data
are
expressed
as mean
± SD; n = 9. Different letters indicate significant
differences
between
light
intensity
treatments
(p
<
0.05).
differences between light intensity treatments (p < 0.05).
differences between light intensity treatments (p < 0.05).
L5
Molecules 2016, 21, 1475
Molecules 2016, 21, 1475
4 of 12
4 of 11
2.3.
2.3.Effects
EffectsofofLight
Light Intensity
Intensity on
on Chloroplast
Chloroplast Ultrastructure
Ultrastructure
Light
Lightintensity
intensitymarkedly
markedlyinfluenced
influencedthe
theshape
shapeand
andnumber
numberofofstarch
starchgrains
grainsand
andgrana
granalamellae
lamellaein
Epimedium
(Figure
3).
L4
resulted
in
more
grana
lamellae
than
other
treatments,
with
approximately
in Epimedium (Figure 3). L4 resulted in more grana lamellae than other treatments, with
11
to 14 grana lamellae
grana.
Plantsper
under
L5 had
more
starch
per chloroplast.
approximately
11 to 14per
grana
lamellae
grana.
Plants
under
L5 grains
had more
starch grainsStarch
per
grains
seldom
appeared
under
L1.
Interestingly,
plants
under
L1
and
L2
had
narrower
chloroplasts
chloroplast. Starch grains seldom appeared under L1. Interestingly, plants under L1 and L2 had
than
plantschloroplasts
under L3, L4,
and
L5. under L3, L4, and L5.
narrower
than
plants
Figure3.3.Chloroplast
Chloroplastultrastructure
ultrastructureofofE.E.pseudowushanense
pseudowushanensemesophyll
mesophyll
cells
under
(A);
Figure
cells
under
L1L1
(A);
L2L2
(B);(B);
L3 L3
(C);
(C);
L4
(D);
and
L5
(E)
treatment
at
60
days.
Abbreviation:
SG,
starch
grains;
GL,
grana
lamellae.
L4 (D); and L5 (E) treatment at 60 days. Abbreviation: SG, starch grains; GL, grana lamellae.
2.4.Effect
EffectofofLight
Light Intensity
Intensity on
on Flavonoid
Flavonoid Content
Content in
in Epimedium
2.4.
Epimedium
Table 1 shows that the method of determining the flavonoid glycosides is highly accurate.
Table 1 shows that the method of determining the flavonoid glycosides is highly accurate. Figure 4
Figure 4 shows the changes in the contents of four different flavonoid glycosides in
shows the changes in the contents of four different flavonoid glycosides in E. pseudowushanense under
E. pseudowushanense under different light intensities. Epimedin A and epimedin B amounts showed
different light intensities. Epimedin A and epimedin B amounts showed similar changes. Epimedin
similar changes. Epimedin A and epimedin B increased when light intensity increased from L1 to L4,
A and epimedin B increased when light intensity increased from L1 to L4, whereas both decreased
whereas both decreased under L5. The highest epimedin A and epimedin B contents were observed
under L5. The highest epimedin A and epimedin B contents were observed under L4. Epimedin
under L4. Epimedin A and epimedin B contents were 191.4% (p < 0.05) and 95.8% (p < 0.05) higher,
A and epimedin B contents were 191.4% (p < 0.05) and 95.8% (p < 0.05) higher, respectively, than
respectively, than under L1. Furthermore, epimedin C content increased as light intensity increased
under L1. Furthermore, epimedin C content increased as light intensity increased from L1 to L3,
from L1 to L3, whereas epimedin C content decreased as light intensity increased from L3 to L5. Thus,
whereas epimedin C content decreased as light intensity increased from L3 to L5. Thus, L3 increased
L3 increased epimedin C by 483.6% (p < 0.05) compared with L1. Interestingly, icariin content was
epimedin C by 483.6% (p < 0.05) compared with L1. Interestingly, icariin content was unaffected by
unaffected by light treatment. Icariin contents in plants under L4 and L1 were significantly higher
light treatment. Icariin contents in plants under L4 and L1 were significantly higher than in plants
than in plants under L5. Icariin contents under L2 and L3 were intermediate. Epimedin C was the
under L5. Icariin contents under L2 and L3 were intermediate. Epimedin C was the major flavonoid
major flavonoid in Epimedium.
in Epimedium.
1475
Molecules 2016, 21, 1475
Analyte a
a
Analyte
1
2
1
3
2
43
Linear Range
(μg·mL−1)
Linear Range
3.1~32.2
(µg
·mL−1 )
3.1~32.2
3.1~32.2
20.5~520.6
3.1~32.2
3.1~32.2
20.5~520.6
11
5 of 12
Table 1. Calibration data of four analytes.
Table 1. Calibration data of four analytes.
Intra-Day RSD (%) (n = 6)
LOD
LOQ
r2
Calibration Equation b
−1)
−1)
(μg·mL
(μg·mL
L1
Intra-Day
RSD L3
(%) (n = 6)L5
LOD
LOQ
Calibration
2
r0.9992
−
1
−
1
Y = Equation
7701.546xb + 9983
0.26
0.81
2.46
1.91
1.36
(µg·mL )
(µg·mL )
L1
L3
L5
Y = 5548.525x + 15,762
0.9995
0.29
0.95
3.27
2.38
1.57
Y = 7701.546x + 9983
0.9992
0.26
0.81
2.46
1.91
1.36
Y = 7171.274x + 31,544
0.9993
0.03
0.11
3.36
1.50
2.02
Y = 5548.525x + 15,762
0.9995
0.29
0.95
3.27
2.38
1.57
Y
=
9108.011x
+
6141
0.9996
0.11
0.37
2.50
1.66
1.46
Y = 7171.274x + 31,544
0.9993
0.03
0.11
3.36
1.50
2.02
Inter-Day RSD (%) (n = 6) Recovery and RSD
(%) (Mean, n = 6)
L1 RSDL3
Inter-Day
(%) (n = 6)L5 Recovery
and RSD (%)
1.06
0.98
1.39
105.54,
3.32
(Mean,
n = 6)
L1
L3
L5
1.62
1.78
1.20
104.06, 2.96
1.06
0.98
1.39
105.54, 3.32
1.83
1.19
1.14
102.95, 3.37
1.62
1.78
1.20
104.06, 2.96
1.79
1.54
1.44
104.92,
1.83
1.19
1.14
102.95, 3.372.70
4
3.1~32.2
9108.011x
+ 6141
0.11 A , epimedin
0.37 B, epimedin
2.50 C, and
1.66 icariin,1.46
1.79 b Y: peak
1.54 area; X:
1.44
2.70
The
compound
codes 1, 2,Y3,=and
4 of each
analyte0.9996
refers to epimedin
respectively;
concentration104.92,
of compound
a
b
−1).
The compound
codes 1, 2, 3, and 4 of each analyte refers to epimedin A, epimedin B, epimedin C, and icariin, respectively; Y: peak area; X: concentration of compound (µg·mL−1 ).
(μg·mL
a
4. Epimedin A, epimedin
epimedin B, epimedin
epimedin C,
C, and
and icariin
icariin content
content of
of E.
E. pseudowushanense
pseudowushanense under
under different
different light
light intensities.
intensities. The data
data are
are expressed
expressed as
as mean
mean ±
± SD.
Figure 4.
SD.
Different letters
letters indicate
indicate significant
significant differences
differencesbetween
betweenlight
lightintensity
intensitytreatments
treatments(p
(p<< 0.05);
0.05);nn== 30.
30.
Different
Molecules 2016, 21, 1475
Molecules 2016, 21, 1475
Molecules
21, Intensity
1475
2.5. 2.5.
Effect
of2016,
Light
ononLeaf
YieldofofEpimedium
Epimedium
Effect
of Light
Intensity
LeafDry
DryBiomass
Biomass and
and Medicinal-Ingredient
Medicinal-Ingredient Yield
6 of 12
6 of 11
6 of 11
Different
light
intensities
were
associated
with
differences
inYield
leafofbiomass
dry
biomass
2.5.
Effect
of Light
Intensity
on Leaf
Dryassociated
Biomass and
Medicinal-Ingredient
Epimedium
Different
light
intensities
were
with
differences
in leaf
dry
(Figure (Figure
5). The 5).
Thehighest
highest
leaf
dry
biomass
was
observed
in
plants
under
L4.
Leaf
dry
biomass
gradually
increased
leaf drylight
biomass
was observed
in plantswith
under
L4. Leaf dry
biomass
gradually
increased
as
Different
intensities
were associated
differences
in leaf
dry biomass
(Figure
5). The
as light
intensity
increased
from
L1
to
L4.
L4
increased
leaf
dry
biomass
by
926.3%
(p
<
0.05)
compared
light
intensity
increased
from
L1
to
L4.
L4
increased
leaf
dry
biomass
by
926.3%
(p
<
0.05)
compared
highest leaf dry biomass was observed in plants under L4. Leaf dry biomass gradually increased as
Interestingly,
leaf
dry
biomass
decreased
as light
lightdry
intensity
increased
from
toto
L5.
withwith
L1. L1.
Interestingly,
leaf
dry
biomass
as
intensity
increased
from
L5.
light
intensity
increased
from
L1
to L4.decreased
L4
increased
leaf
biomass
by 926.3%
(p L4
< L4
0.05)
compared
with L1. Interestingly, leaf dry biomass decreased as light intensity increased from L4 to L5.
Figure
Leafdry
dry biomass
E. E.
pseudowushanense
underunder
different
light intensities.
The data areThe
expressed
Figure
5. 5.Leaf
biomassofof
pseudowushanense
different
light intensities.
data are
as
mean
±
SD.
Different
letters
indicate
significant
differences
between
light
intensity
treatments
Figure 5.asLeaf
dry biomass
of E. pseudowushanense
undersignificant
different light
intensities. between
The data are
expressed
expressed
mean
± SD. Different
letters indicate
differences
light
intensity
(p
0.05); ±n SD.
= 30.Different letters indicate significant differences between light intensity treatments
as <mean
treatments
(p < 0.05);
n = 30.
(p < 0.05); n = 30.
Given that the total of epimedin A, epimedin B, epimedin C, and icariin content in flavonoid
Given that
the total
of 90%
epimedin
A, epimedin
epimedin
C, and
icariin
content
in flavonoid
glycosides
than
in any treatment
(dataB,
shown), C,
these
components
represented
Givenwas
thatmore
the total
of epimedin
A, epimedin
B,not
epimedin
andfour
icariin
content in
flavonoid
glycosides
was
more
than
90%
in
any
treatment
(data
not
shown),
these
four
components
represented
flavonoid
in Epimedium.
yield these
was calculated
as the represented
product
of
glycosidesglycosides
was more than
90% in any Medicinal-ingredient
treatment (data not shown),
four components
flavonoid
glycosides
in
Epimedium.
Medicinal-ingredient
yield
was
calculated
as
the
product
flavonoid
glycoside
content
and
leaf
dry
biomass
per
plant.
Figure
6
shows
that
plants
under
60
days
flavonoid glycosides in Epimedium. Medicinal-ingredient yield was calculated as the product of of
flavonoid
glycoside
leafdry
dry
biomass
per
plant.
Figure
6that
shows
that
plants
under
of
L3 and
L4 hadcontent
higher and
medicinal-ingredient
yields
than
under
other
treatments
(p
<600.05).
flavonoid
glycoside
content
and
leaf
biomass
per
plant.
Figure
6 shows
plants
under
days
Medicinal-ingredient
yield
gradually
increased
as
light
intensity
increased
from
L1
to
L3,
whereas
it
60 days
L3 and
L4 had
higher
medicinal-ingredient
yields
under
other
treatments
< 0.05).
of L3ofand
L4 had
higher
medicinal-ingredient
yields
thanthan
under
other
treatments
(p <(p0.05).
decreased
by
40.3%
(p
<
0.05)
as
light
intensity
increased
from
L3
to
L5.
Medicinal-ingredient
yield
gradually
intensityincreased
increasedfrom
from
whereas
Medicinal-ingredient
yield
graduallyincreased
increased as
as light intensity
L1L1
to to
L3,L3,
whereas
it it
decreased
by 40.3%
(p (p
< 0.05)
fromL3
L3totoL5.
L5.
decreased
by 40.3%
< 0.05)asaslight
lightintensity
intensity increased
increased from
Figure 6. Medicinal-ingredient yields of E. pseudowushanense under different light intensities.
The
data6.are
expressed as mean ±yields
SD. Different
letters indicate significant
differences
between
light
Figure
Medicinal-ingredient
E. pseudowushanense
different
light
intensities.
Figure
6. Medicinal-ingredient
yields of E.of
pseudowushanense
underunder
different
light intensities.
The data
intensity
treatments
(p
<
0.05);
n
=
30.
The data are expressed as mean ± SD. Different letters indicate significant differences between light
are expressed as mean ± SD. Different letters indicate significant differences between light intensity
intensity treatments (p < 0.05); n = 30.
treatments
(p < 0.05); n = 30.
Molecules 2016, 21, 1475
7 of 12
3. Discussion
Although Epimedium is a shade-tolerant species, light intensity plays an important role in its
survival, early growth, development of photosynthetic apparatus, and production of secondary
metabolites [16–18]. Four major flavonoids including icariin, epimedin A, epimedin B, and epimedin
C are quality indicators of Epimedii Folium [2]. Our study showed that medicinal-ingredient yields of
individual plants were higher under 60 days of L3 and L4 treatment (Figure 6). This result suggests
that the L3 and L4 are the optimal conditions for the medicinal ingredient production of Epimedium.
Meanwhile, content of epimedin C, the major Epimedium flavonoid, was lower under low and high
light intensity treatments, therefore reducing the medicinal-ingredient yield of individual plants under
L1, L2, and L5 treatments (Figure 4). Interestingly, the production of different flavonoids was induced
by different light ranges. A large number of other flavones were probably stimulated by lower light in
addition to the four major components. In addition, epimedin A and epimedin B contents increased
significantly as light intensity increased from L1 to L4, but decreased as light intensity increased
from L4 to L5. Epimedin C content increased significantly as light intensity increased from L1 to L3,
whereas it decreased as light intensity increased from L3 to L5. Furthermore, icariin content changed
differently compared with other flavonoids. These results signify the complex relationships between
light intensity and epimedin A, epimedin B, epimedin C, and icariin biosynthesis. Further studies
are necessary to better understand the factors regulating the synthetic balance of the four medicinal
flavones in the flavonoid pathway.
Leaf dry biomass influences the medicinal-ingredient yield of Epimedium. Leaf dry biomass is
influenced by light intensity. Various plant characteristics, such as leaf area, number of branches,
water content, and SPAD are influenced by light intensity [19]. Differences in plant morphology are
documented in different species adapted to various light environments [20,21]. In the present study,
E. pseudowushanense seedlings grown under relatively low light intensities (L1–L2) had larger leaves
compared with those grown under high light intensities (L3–L5) (Figure 1). These findings are similar
to the results reported by Tang and others [22]. Our study showed that E. pseudowushanense seedlings
grown under L3–L5 treatments had significantly higher number of branches and lower water content
(Figure 1). In E. pseudowushanense, the number of branches was directly associated with leaf biomass.
More branches signify more leaves and more leaf dry biomass. Lower water content influenced leaf dry
biomass similarly. Additionally, the highest Pn, gs, and T were observed in plants under L4 (Figure 2).
Decreased Pn was associated with reduced T, gs, and Ci as light intensity increased from L4 to L5. This
result indicates that stomatal limitation occurred and is presumably consistent with E. pseudowushanense
stomatal traits. SPAD is another important factor determining photosynthetic rate and plant growth.
In this study, we observed that SPAD decreased as light intensity increased from L3 to L5 (Figure 1),
suggesting that excessive light intensity induced pigment damage [23]. This finding is consistent with
the response of T. hemsleyanum [19]. Interestingly, higher SPAD and lower Pn were observed under L1
and L2. Thus, these results might be considered as responses to different light conditions [24].
Chloroplasts are the only organelles in which photosynthesis occurs. Photoreactions are localized
in the internal chloroplast membrane (i.e., the thylakoid). Thylakoid structure and integrity are
critical for effective photosynthesis [25]. In this study, leaves grown under L4 had better-developed
grana and more thylakoids than those under other treatments (Figure 3), consistent with the highest
photosynthetic rates and leaf dry biomass under L4. E. pseudowushanense chloroplasts were damaged
under high light intensities (L5), thus decreasing flavonoid glycoside content because chloroplasts
synthesize flavonoids in the plant cell [26,27]. Similar results were reported for Camptotheca acuminata
seedlings [8].
E. pseudowushanense is most commonly utilized in the treatment of osteoporosis, liver cancer,
and leukemia because of its high flavonoid content [4,28,29]. Based on previous studies on Epimedium
species [30], the most suitable intensity for flavonoid accumulation in E. sagittatum ranged from
40–160 µmol·m−2 ·s−1 . We set the L1 to L5 light regimes from 9.1–127.3 µmol·m−2 ·s−1 . We conclude
that L3 (54.6 ± 2.5 µmol·m−2 ·s−1 ) and L4 (90.9 ± 2.5 µmol·m−2 ·s−1 ) treatments achieved higher
Molecules 2016, 21, 1475
8 of 12
medicinal-ingredient yields after 60 days, and are therefore recommended for Epimedium cultivation.
The present
study
provides a better understanding of the responses of growth, photosynthesis,
Molecules 2016,
21, 1475
8 of 11 and
secondary metabolite accumulation in E. pseudowushanense seedlings exposed to various light intensities.
presentcould
studyserve
provides
better understanding
the responsescultivation
of growth,ofphotosynthesis,
and
Our results
as a atheoretical
basis for theofstandardized
E. pseudowushanense.
secondary metabolite accumulation in E. pseudowushanense seedlings exposed to various light
intensities.
Our
results could serve as a theoretical basis for the standardized cultivation of
4. Materials and
Methods
E. pseudowushanense.
4.1. Plant Materials and Growth Conditions
4. Materials and Methods
E. pseudowushanense buds germinate in February. Flowering occurs from March to April and
4.1. Plant
Materials
and[31].
GrowthE.Conditions
fruiting
occurs
in May
pseudowushanense is cultivated under 10% to 30% irradiance in
greenhouses.
Its
rhizome
buds
develop
during
winter. Flowering
New leaves
and from
stems
with to
flowers
develop
E. pseudowushanense buds germinate
in February.
occurs
March
April and
in thefruiting
following
early
ForE.this
study, healthy,ishomogenous
two-year-old
E. pseudowushanense
occurs
in spring.
May [31].
pseudowushanense
cultivated under
10% to 30%
irradiance in
◦ N,New
seedlings
were collected
from
Leidevelop
Shan County
108◦leaves
E) in Guizhou
Each
stem had
greenhouses.
Its rhizome
buds
during (16
winter.
and stems Province.
with flowers
develop
in the following
early
spring.
For this study,
healthy,
two-year-old
E. pseudowushanense
two compound
leaves.
Each
compound
leaf had
three homogenous
leaflets (Figure
7). Seedlings
were transferred to
seedlings
werediameter:
collected from
Lei height:
Shan County
(16°
N, 108°
E) in Guizhou
Province.
Each
stem
had
plastic
pots (inner
10 cm,
10 cm)
with
drainage
holes. One
seedling
was
transferred
two
compound
leaves.
Each
compound
leaf
had
three
leaflets
(Figure
7).
Seedlings
were
transferred
per pot. All pots contained a substrate mixture of 75% peat and 25% vermiculite. On 26 December
to plastic pots (inner diameter: 10 cm, height: 10 cm) with drainage holes. One seedling was transferred
2013, the transplanted seedlings were transferred to the greenhouse of the Institute of Medicinal Plant
per pot. All pots contained a substrate mixture of 75% peat and 25% vermiculite. On 26 December
Development. Seedlings were then grown under shade. Each seedling was disinfected with 800-fold
2013, the transplanted seedlings were transferred to the greenhouse of the Institute of Medicinal Plant
carbendazim
solution.
A completely
randomized
design
with
threewas
replications
treatment
Development.
Seedlings
were then grown
under shade.
Each
seedling
disinfectedper
with
800-fold and
10 seedlings
per replicate
set up onrandomized
1 February design
2014, or
approximately
one month
after transferring.
carbendazim
solution.was
A completely
with
three replications
per treatment
and 10
Inflorescences
were
removed
promote
growth of the
utilized
in medicinal
seedlings per
replicate
was upon
set up blossoming
on 1 Februaryto
2014,
or approximately
one leaves
month after
transferring.
Inflorescences
were removed
growth
of the cultivation
leaves utilized
in medicinal
treatments.
Temperature
rangeupon
was blossoming
set as 20–21to◦promote
C during
the entire
period.
Humidity
treatments. Temperature
range
set intensities
as 20–21 °Cwere
during
the entireby
cultivation
period. lamps
Humidity
was maintained
at 60%. The
fivewas
light
supplied
T5-fluorescent
at 16-h
−2 ·s−1 ),
was maintained
60%.
TheLight
five treatment
light intensities
were
supplied
by T5-fluorescent
lamps
at·m
16-h
irradiation
durationsatper
day.
groups
were
as follows:
L1 (9.1 ± 2.5
µmol
−2 −1
irradiation
durations
Light
treatment
groups
2.5 μmol·m
1 ), L3
2 ·s−as
1 ),follows:
L2 (18.2
± 2.5 µmol
·m−2per
·s−day.
(54.6
± 2.5 µmol
·m−were
L4 (90.9L1±(9.1
2.5±µmol
·m−2 ·s·s−1 ),), L2
and L5
−2
−1
−2
−1
−2
−1
(18.2 ± 2.5 μmol·m−2·s −),1 L3 (54.6 ± 2.5 μmol·m ·s ), L4 (90.9 ± 2.5 μmol·m ·s ), and L5 (127.3 ± 2.5
·
(127.3 ± 2.5 −2µmol
·
m
s
).
Light
intensities
were
measured
by
LI-6400
external
quantum
sensor
μmol·m ·s−1). Light intensities were measured by LI-6400 external quantum sensor (LI-COR, Lincoln,
(LI-COR, Lincoln, NE, USA) system. Sufficient water was provided for plants once every four days
NE, USA) system. Sufficient water was provided for plants once every four days until the end of
until experimentation.
the end of experimentation.
Figure 7. E. pseudowushanense flowers and leaves. There are two compound leaves on each stem and
Figure 7. E. pseudowushanense flowers and leaves. There are two compound leaves on each stem and
each compound leaf consisted of three leaflets.
each compound leaf consisted of three leaflets.
Molecules 2016, 21, 1475
9 of 12
4.2. Plant Growth, Chlorophyll Content, and Leaf Dry Biomass
The number of newly germinated branches was recorded after 60 days of light treatment. Leaf
areas of three leaflets were measured by LI-3000C (LI-COR, Lincoln, NE, USA). Fresh leaf weights
were measured by an electronic balance. Leaf dry weights were measured after oven drying leaves
at 60 ◦ C to constant weight. Water content was calculated as: water content = 1 − dry weight/wet
weight. Chlorophyll content was measured by a chlorophyll meter SPAD-502 (Konica Minolta Inc.,
Tokyo, Japan). Measurement light wavelengths were 650 nm and 940 nm. At the end of each treatment,
all leaves of intact plants were collected to calculate leaf dry biomass.
4.3. Photosynthetic Parameters
Photosynthetic measurements were taken between 9:00 AM and 11:00 AM. Healthy,
fully-developed, topmost leaflets of fronds were taken from three randomly selected plants per
replicate treatment. Photosynthesis (Pn) was measured by a LI-6400 portable photosynthesis system
(Li-Cor, Inc., Lincoln, NE, USA) with a standard leaf chamber equipped with a 6400-02B LED light
source. Data were recorded under 21% O2 , 500 µmol (CO2 )·m−2 ·s−1 air concentration, 60% relative
humidity, 200 µmol (CO2 )·s−1 air flow, and 20 ± 0.5 ◦ C temperature. Vapor pressure deficit (VPD)
was calculated with leaf and air temperatures and relative humidity. VPD was similar among the five
experimental treatments.
4.4. Assessment of Chloroplast Ultrastructure
To observe the chloroplast ultrastructure of mesophyll cells in E. pseudowushanense seedlings
subjected to different light intensities for 60 days, a fully expanded leaflet from a randomly selected
plant from each replicate per treatment was collected. Fresh leaves were cut into 4–6 slices (1 mm2 )
at their adaxial surfaces for transmission electron microscopy. Sections were placed immediately in
2.5% (v/v) glutaraldehyde (0.1 M phosphate buffer, pH 7.2), then de-gassed and fixed for at least 4 h.
Samples were then post-fixed with 1% (v/v) osmium acid. After fixation, samples were washed three
times with the same buffer for 15 min. Specimens were dehydrated by a graded ethanol series (50%,
70%, 90%, 95%, and 100% at 30 min each) then embedded in epoxy resin, from which ultrathin sections
were prepared. The ultrathin sections were sequentially stained with uranyl acetate and lead citrate
and then examined with a transmission electron microscope (JEM-1400, Hitachi, Tokyo, Japan).
4.5. Flavonoid Content and Medicinal-Ingredient Yield
One ternate leaf was collected per plant after 30, 60, 90, and 120 days of exposure to different light
intensity treatments. Fresh leaves of 10 seedlings per replicate were fixed for 3 min in a microwave,
then dried to a constant weight at 60 ◦ C. Sample pretreatment was performed according to the
Chinese Pharmacopoeia Commission (2010). Samples were crushed and sifted through a No. 3
pharmacopoeia sieve. Approximately 200 mg of sample was added to 50 mL 70% ethanol then extracted
by ultrasonication for 30 min. The extract was then filtered with a 0.45 µm microfiltration membrane
for HPLC analysis. Eluent A contained double distilled water and eluent B contained acetonitrile.
The gradient elution program was as follows: 0–17 min (25%–26% B), 17–26 min (26%–100% B).
The column was washed with 100% eluent B between every two samples for 15 min and then
re-equilibrated with 25% eluent B for 10 min. Elution conditions were as follows: 1.0 mL·min−1
flow rate, 25 ◦ C column temperature, 270 nm detection wavelength, and 20 µL injection volume.
Six gradient concentrations of epimedin A (3.1–32.3 µg·mL−1 ), epimedin B (3.1–32.3 µg·mL−1 ),
epimedin C (20.5–520.6 µg·mL−1 ), and icariin (3.1–32.3 µg·mL−1 ) were prepared. A Zorbax SB-C18
column (Agilent Technologies, Palo Alto, CA, USA) was utilized as the chromatographic column in
chromatographic analysis (250 mm × 4.6 mm I.D., 5 µm). HPLC analysis data were processed by
PerkinElmer ChemStation software (version 6.3.1, PerkinElmer, Waltham, MA, USA).
Molecules 2016, 21, 1475
10 of 12
Medicinal ingredient yield was calculated as: medicinal-ingredient yield = (epimedin A
content + epimedin B content + epimedin C content + icariin content) × leaf dry biomass.
4.6. Data Analysis
Statistical analysis was conducted with one-way ANOVA software (R language version 3.1.1,
https://www.r-project.org/about.html). Differences between means were detected with Tukey HSD
test. p value was set at 0.05 and 0.01 for ANOVA and Tukey HSD tests, respectively.
Acknowledgments: The National Natural Science Foundation of China (81473302) supported this study.
We would like to thank Zhang Yali for his insightful suggestions regarding the manuscript and Ding Wanlong for
taking pictures. We are very grateful for the experiment support of GUOYAOJITUAN TONGJITANG (GUIZHOU)
PHARMACEUTICAL CO.LTD, and some personal support from Xiangbo Yang, Zhihai Jiang, and Li Li. Funding:
The National Natural Science Foundation of China (81473302).
Author Contributions: B.G. and J.P. conceived and designed the experiments; J.P. performed the experiments;
J.P. analyzed the data; B.G. contributed reagents/materials/analysis tools; J.P. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
Pn
gs
SPAD
T
WUE
Ci
SG
GL
VPD
Net photosynthetic rate
Stomatal conductance
Soil and Plant Analyzer Development (the relative content of chlorophyll)
Transpiration rate
Water use efficiency
Intercellular CO2 concentration
Starch grains
Grana lamellae
Vapor pressure deficit
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Wu, H.; Lien, E.J.; Lien, L.L. Chemical and pharmacological investigations of Epimedium species: A survey.
In Progress in Drug Research; Jucker, E., Ed.; Birkhäuser Basel: Basel, Switzerland, 2003; pp. 1–57.
Liu, J.J.; Li, S.P.; Wang, Y.T. Optimization for quantitative determination of four flavonoids in Epimedium
by capillary zone electrophoresis coupled with diode array detection using central composite design.
J. Chromatogr. A 2006, 1103, 344–349. [CrossRef] [PubMed]
Kim, B.; Park, B. Baohuoside I suppresses invasion of cervical and breast cancer cells through the
downregulation of CXCR4 chemokine receptor expression. Biochemistry 2014, 53, 7562–7569. [CrossRef]
[PubMed]
Lin, C.C.; Ng, L.T.; Hsu, F.F.; Shieh, D.-E.; Chiang, L.C. Cytotoxic effects of coptis chinensis and Epimedium
sagittatum extracts and their major constituents (berberine, coptisine and icariin) on hepatoma and leukaemia
cell growth. Clin. Exp. Pharmacol. Physiol. 2004, 31, 65–69. [CrossRef] [PubMed]
Kang, S.H.; Jeong, S.J.; Kim, S.H.; Kim, J.H.; Jung, J.H.; Koh, W.; Kim, J.H.; Kim, D.K.; Chen, C.Y.; Kim, S.H.
Icariside ii induces apoptosis in U937 acute myeloid leukemia cells: Role of inactivation of STAT3-related
signaling. PLoS ONE 2012, 7, e28706.
Zoratti, L.; Karppinen, K.; Luengo Escobar, A.; Häggman, H.; Jaakola, L. Light-controlled flavonoid
biosynthesis in fruits. Front. Plant Sci. 2014, 5, 534. [CrossRef] [PubMed]
Guo, Y.-P.; Guo, D.-P.; Zhou, H.-F.; Hu, M.-J.; Shen, Y.-G. Photoinhibition and xanthophyll cycle activity in
bayberry (myrica rubra) leaves induced by high irradiance. Photosynthetica 2006, 44, 439–446. [CrossRef]
Ma, X.; Song, L.; Yu, W.; Hu, Y.; Liu, Y.; Wu, J.; Ying, Y. Growth, physiological, and biochemical responses of
Camptotheca acuminata seedlings to different light environments. Front. Plant Sci. 2015, 6, 321. [CrossRef]
[PubMed]
Jaakola, L.; Hohtola, A. Effect of latitude on flavonoid biosynthesis in plants. Plant Cell Environ. 2010, 33,
1239–1247. [CrossRef] [PubMed]
Guo, J.; Han, W.; Wang, M.-H. Ultraviolet and environmental stresses involved in the induction and
regulation of anthocyanin biosynthesis: A review. Afr. J. Biotechnol. 2008, 7, 4966–4972.
Molecules 2016, 21, 1475
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
11 of 12
Agati, G.; Stefano, G.; Biricolti, S.; Tattini, M. Mesophyll distribution of ‘’antioxidant“ flavonoid glycosides
in ligustrum vulgare leaves under contrasting sunlight irradiance. Ann. Bot. 2009, 104, 853–861. [CrossRef]
[PubMed]
Agati, G.; Biricolti, S.; Guidi, L.; Ferrini, F.; Fini, A.; Tattini, M. The biosynthesis of flavonoids is enhanced
similarly by UV radiation and root zone salinity in L. Vulgare leaves. J. Plant Physiol. 2011, 168, 204–212.
[CrossRef] [PubMed]
Koyama, K.; Ikeda, H.; Poudel, P.R.; Goto-Yamamoto, N. Light quality affects flavonoid biosynthesis in
young berries of cabernet sauvignon grape. Phytochemistry 2012, 78, 54–64. [CrossRef] [PubMed]
Agati, G.; Brunetti, C.; di Ferdinando, M.; Ferrini, F.; Pollastri, S.; Tattini, M. Functional roles of flavonoids in
photoprotection: New evidence, lessons from the past. Plant Physiol. Biochem. 2013, 72, 35–45. [CrossRef]
[PubMed]
Pacheco, F.V.; Alvarenga, I.C.A.; Ribeiro Junior, P.M.; Pinto, J.E.B.P.; Avelar, R.D.P.; Alvarenga, A.A. Growth
and production of secondary compounds in monkey-pepper (Piper aduncum L.) leaves cultivated under
altered ambient light. Aust. J. Crop Sci. 2014, 8, 1510–1516.
Gottschalk, K.W. Shade, leaf growth and crown development of Quercus rubra, Quercus velutina,
Prunus serotina and Acer rubrum seedlings. Tree Physiol. 1994, 14, 735–749. [CrossRef] [PubMed]
Müller, V.; Albert, A.; Barbro Winkler, J.; Lankes, C.; Noga, G.; Hunsche, M. Ecologically relevant UV-B dose
combined with high PAR intensity distinctly affect plant growth and accumulation of secondary metabolites
in leaves of Centella asiatica L. Urban. J. Photochem. Photobiol. B Biol. 2013, 127, 161–169. [CrossRef] [PubMed]
Wang, M.L.; Jiang, Y.S.; Wei, J.Q.; Wei, X.; Qi, X.X.; Jiang, S.Y.; Wang, Z.M. Effects of irradiance on growth,
photosynthetic characteristics, and artemisinin content of Artemisia annua L. Photosynthetica 2008, 46, 17–20.
[CrossRef]
Dai, Y.; Shen, Z.; Liu, Y.; Wang, L.; Hannaway, D.; Lu, H. Effects of shade treatments on the photosynthetic
capacity, chlorophyll fluorescence, and chlorophyll content of Tetrastigma hemsleyanum Diels et Gilg.
Environ. Exp. Bot. 2009, 65, 177–182. [CrossRef]
Valladares, F.; Chico, J.; Aranda, I.; Balaguer, L.; Dizengremel, P.; Manrique, E.; Dreyer, E. The greater seedling
high-light tolerance of Quercus robur over Fagus sylvatica is linked to a greater physiological plasticity. Trees
2002, 16, 395–403.
Aleric, K.M.; Katherine Kirkman, L. Growth and photosynthetic responses of the federally endangered
shrub, Lindera melissifolia (Lauraceae), to varied light environments. Am. J. Bot. 2005, 92, 682–689. [CrossRef]
[PubMed]
Tang, H.; Hu, Y.-Y.; Yu, W.-W.; Song, L.-L.; Wu, J.-S. Growth, photosynthetic and physiological responses of
Torreya grandis seedlings to varied light environments. Trees 2015, 29, 1011–1022. [CrossRef]
Shao, Q.; Wang, H.; Guo, H.; Zhou, A.; Huang, Y.; Sun, Y.; Li, M. Effects of shade treatments on photosynthetic
characteristics, chloroplast ultrastructure, and physiology of Anoectochilus roxburghii. PLoS ONE 2014, 9,
e85996. [CrossRef] [PubMed]
Murchie, E.H.; Horton, P. Acclimation of photosynthesis to irradiance and spectral quality in british plant
species: Chlorophyll content, photosynthetic capacity and habitat preference. Plant Cell Environ. 1997, 20,
438–448. [CrossRef]
Liu, Y.; Li, X.; Liu, M.; Cao, B.; Tan, H.; Wang, J.; Li, X. Responses of three different ecotypes of reed
(Phragmites communis trin.) to their natural habitats: Leaf surface micro-morphology, anatomy, chloroplast
ultrastructure and physio-chemical characteristics. Plant Physiol. Biochem. 2012, 51, 159–167. [CrossRef]
[PubMed]
Saunders, J.A.; McClure, J.W. The distribution of flavonoids in chloroplasts of twenty five species of vascular
plants. Phytochemistry 1976, 15, 809–810. [CrossRef]
Agati, G.; Matteini, P.; Goti, A.; Tattini, M. Chloroplast-located flavonoids can scavenge singlet oxygen.
New Phytol. 2007, 174, 77–89. [CrossRef] [PubMed]
Zhai, Y.-K.; Guo, X.; Pan, Y.-L.; Niu, Y.-B.; Li, C.-R.; Wu, X.-L.; Mei, Q.-B. A systematic review of the efficacy
and pharmacological profile of Herba epimedii in osteoporosis therapy. Pharmazie 2013, 68, 713–722. [PubMed]
Li, C.; Li, Q.; Mei, Q.; Lu, T. Pharmacological effects and pharmacokinetic properties of icariin, the major
bioactive component in Herba epimedii. Life Sci. 2015, 126, 57–68. [CrossRef] [PubMed]
Molecules 2016, 21, 1475
30.
31.
12 of 12
Liang, Q.; Wei, G.; Chen, J.; Wang, Y.; Huang, H. Variation of medicinal components in a unique geographical
accession of horny goat weed Epimedium sagittatum maxim. (Berberidaceae). Molecules 2012, 17, 13345–13356.
[CrossRef] [PubMed]
Guo, B.-L.; He, S.-Z.; Zhong, G.-Y.; Xiao, P.-G. Two new species of Epimedium (Berberidaceae) from China.
Acta Phytotaxon. Sin. 2007, 45, 813–821. [CrossRef]
Sample Availability: Samples of the compounds are available from the authors.
© 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).