Environmental and Experimental Botany 56 (2006) 4453

Comparison of resveratrol, SOD activity, phenolic compounds

and free amino acids in Rehmannia glutinosa
under temperature and water stress
Ill Min Chung a,,1 , Jong Jin Kim b,1 , Jung Dae Lim c , Chang Yeon Yu c ,
Seung Hyun Kim d , Sang Joon Hahn a
a

Department of Applied Life Science, College of Life and Environment Science, Konkuk University,
GwangJinKu, HwaYangDong, Seoul 143-701, Korea
Department of Environmental Science, College of Life and Environment Science, Konkuk University, GwangJinKu,
HwaYangDong, Seoul 143-701, Korea
c College of Agriculture and Life Science, Kangwon National University, Chunchon 200-701, Korea
d Department of Agricultural Environmental Chemistry, University of California, Davis, CA 95616, USA
Received 30 September 2004; accepted 4 January 2005

Abstract
The objective of this study was to determine the contents of secondary metabolic substances such as resveratrol, SOD, phenolic compounds
and free amino acids in Rehmannia glutinosa under environmental stress. The content of resveratrol varied from 2.96 to 38.87 g g1 , while
SOD activity varied from 9.40 to 28.43%. Of the 16 individual phenolic compounds, myricetin showed the highest concentrations (34.30,
60.96, 35.70 g g1 ) in the water deficiency (1.18 MPa) and control (1.04 MPa) and low temperature (15 C) treatment. The 21 free
amino acids in R. glutinosa were only detected in small amounts and varied from 0.82 to 5.69 g g1 . Overall, these substances decreased at
high temperature and with a water deficiency. Other than for SOD activity, these substances were negatively correlated (r2 = 0.99** ) with
temperature and positively correlated with water stress (r2 = 0.99*** ). Also, other than for SOD activity under the water deficiency treatment,
resveratrol, SOD activity, phenolic compounds and free amino acids had over correlation coefficients (r2 = 0.99** ) in all treatments. Our study
suggests that it might be feasible to improve or develop R. glutinosa cultivation methods with high functional substances such as resveratrol,
even under poor environmental conditions for cultivation.
2005 Elsevier B.V. All rights reserved.
Keywords: Rehmannia glutinosa; Resveratrol; SOD activity; Phenolic compounds; Free amino acids; Temperature stress; Water stress

1. Introduction
Rehmannia glutinosa (Gaertn.) Libosch. is a perennial
medicinal plant that originates from China and belongs to
the family Scrophulariaceae. For centuries, many oriental
medical books in countries such as China, Japan and Korea
have listed this plants roots because of its medicinal purposes (Zhu et al., 2003). Although there are seven species
in the genus Rehmannia, only three are used for medici
1

Corresponding author. Tel.: +82 2 450 3730; fax: +82 2 446 7856.
E-mail address: imcim@konkuk.ac.kr (I.M. Chung).
These two authors contributed equally to this work.

0098-8472/$ see front matter 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.envexpbot.2005.01.001

nal purposes: R. glutinosa Libosch. in Korea, R. glutinosa

var. purpurea Makino in Japan and R. glutinosa var. hueichingensis (Chao and Schih) Hsiao in China. R. glutinosa
is generally cultivated in the southern provinces of Korea
due to its mild climate, where it is used in three ways: fresh,
dried and steamed. Each is known to have different medicinal effects (Hasegawa et al., 1982). The cultivated areas of
R. glutinosa in Korea has progressively decreased over the
last 20 years. In 2002, only 90 ha were planted, which was
just 6% of the total cultivated area of medicinal plants. This
trend is due to increasing imports of cheaper medicinal plants
from China, where over 2100 M/T are imported every year
(Ministry of Agriculture and Forestry, 2003).

I.M. Chung et al. / Environmental and Experimental Botany 56 (2006) 4453

R. glutinosa contains rehmaglutin A, B, C and D as an

iridoid and catalpol, aucubin, leonuride, melittoside, rehmanniosides, glucosides, amino acids and -stigmasterol as an
iridoid with glucosides, and has been reported to have various
medical effects such as reducing blood pressure in rabbits,
diuretic properties in mice with glycosuria and antibiotic
effects against bacteria. R. glutinosa also contains resveratrol, phenolic compounds, free amino acids and SOD, all of
which have been reported to have physiologically active substances that have anti-oxidative properties, aid the defence
mechanism, or possess chemicals related to resistance to environmental stresses in human beings or plants (Kubo et al.,
1994; Kim et al., 2000).
Resveratrol (trans-3,5,4 -trihydroxystilbene), a type of
stilbene, is found in peanuts, mulberries and grapes and accumulates in plants after exposure to UV light, aluminium chloride or infection by Plasmospora viticola or Botrytis cinerea
(Langcake, 1981; Sobolev and Cole, 1999). It is known to
have advantageous effects for diabetes, constipation, allergies and headaches. In addition, it can trigger apoptosis, has
antibacterial properties and can reduce inflammation (Kubo
et al., 1994). It also contains superoxide dismutase (SOD;
EC 1.15.1.1), which is present in most organisms, and was
first found by McCord and Fridovich (1969). There are three
types of SOD, Cu/Zn-SOD, Mn-SOD and Fe-SOD, all of
which have the following removal system for several in vivo
reactive oxygen species (ROS):
2O2

+ 2H H2 O2 + O2

The Cu/Zn-SOD type has generally been reported to occur

in the cytosol of most higher plants. Recently, there has been
an increase in interest in SOD as several research reports
related to human health have noted resistance mechanisms
against environmental stresses (Bryan et al., 2000). Free
amino acids and phenolic compounds composed of phenolic
acid (C6 C1 ), coumarins (C6 C3 ), flavonoids (C6 C3 C6 )
and condensed tannins are known to possess chemicals with
anticancer and anti-oxidative properties and can control physiological metabolites in humans and plants. The contents of
such chemicals vary with species, growth stage and cultivation method (Hong et al., 1989).
Since the industrial revolution in the 19th century, the
climate of the Earth has changed as a result of developing industry and science. This has led to abnormal climatic
conditions around the world. However, little is known about
variations in the aforementioned advantageous substances in
R. glutinosa to changing environmental conditions, such as
severe drought or elevated air temperatures. As a result, a
major focus has been placed on the analysis and evaluation of
medicinal chemicals in R. glutinosa and their effects against
several diseases.
The main purposes of this study were: (1) to examine
the content of resveratrol, phenolic compounds, free amino
acids and SOD activity in R. glutinosa cultivated under
water and temperature stress and; (2) to determine correlations between changes of these substances and environmental

45

factors. Such information in this study could help to both

develop more adaptable methods for cultivating R. glutinosa and in producing new cultivars with highly functional
substances.

2. Materials and methods

2.1. Plant material and growth conditions
R. glutinosa seedling which was grown in the seedling
culture box on 25 July 2003 and transplanted into plastic
pots (27 cm in diameter and 30 cm in height) filled with a
sandy loam soil on 11 September 2003. They were then cultivated in a greenhouse at Konkuk University. Fifty days
after transplanting (11 September), they were exposed to
two temperature and water conditions: low temperature (day:
20 C, night: 15 C, light intensity: 2050 mol m2 s1 ),
high temperature (day: 35 C, night: 30 C, light intensity:
2050 mol m2 s1 ), water deficiency (water potential in the
soil: 1.18 0.04 MPa), control (water potential in the soil:
1.04 0.07 MPa). Plants were harvested from each replicate and stored at low temperature (below 30 C) until
analysis. The experiment consisted of a completely randomized design with three replicates.
2.2. Measurement of superoxide dismutase (SOD)
activity
2.2.1. Enzyme extraction
For each sample, 0.2 g of dried root R. glutinosa was
ground, mixed with 0.4 g polyvinylpolypyrrol-idome (PVP)
and 2 mL extraction buffer with pH 7.0, 100 mM potassium phosphate, 10 mM sodium ascorbate and 5 mM EDTA.
After the homogenate was centrifuged at 15,000 rpm for
20 min, the upper solution was extracted using a PD-10
column of Sephadex G-25 and was used to estimate SOD
activity.
2.2.2. SOD activity test by the nitro blue tetrazolium
(NBT) reduction method
SOD activity of root R. glutinosa was measured by the
nitro blue tetrazolium (NBT) reduction method (Beyer and
Fridovich, 1987). Test tubes containing reaction solution
with 3 mL of assay buffer, 60 L of crude enzyme and
30 L of riboflavin were illuminated for 7 min in an aluminum foil lined box containing two 20-W Slyvania Groiux
Fluorescent lamps at 25 C. After reaction, the absorbance
of the blank solution and reaction solution was measured
with a spectrophotometer (Hitachi Ltd., Tokyo, Japan) at
560 nm. SOD activities were calculated as a following
equation:
SOD activity (%) = (1 A/B) 100
A: absorbance of sample; B: absorbance of blank.

46

I.M. Chung et al. / Environmental and Experimental Botany 56 (2006) 4453

2.3. Quantitative analysis of phenolic compounds and

resveratrol with HPLC
2.3.1. Preparation of R. glutinosa
Ground root parts of R. glutinosa (2 g) were mixed with
2 mL of 0.1 N HCl and 10 mL of acetonitrile (ACN) in a
125 mL screw-top flask and stirred for 2 h at room temperature. Following this, the solution was filtered through
a Whatman No. 42 filter paper. The filtrate was dried under
vacuum at a temperature below 30 C and then re-dissolved in
10 mL of 80% HPLC grade methyl alcohol in distilled water.
The re-dissolved sample was filtered through a 0.45 micro
filter unit (cameo 13N syringe-filter, nylon) and transferred
to a 1 mL vial.
2.3.2. Instrumental conditions for HPLC
Instrumentation for HPLC analysis was applied using the
method of Banwart et al. (1985). The mobile phase consisted
of solvent A and B. Solvent A contained 98% water and 2%
glacial acetic acid in 0.018 M ammonium acetate. Solvent
B was 70% solvent A and 30% organic solution, the latter
being composed of 82% methanol, 16% n-butanol and 2%
glacial acetic acid in 0.018 M ammonium acetate. Following
injection of 20 L of the sample, the flow rate of the mobile
phases was maintained at 1 mL min1 . A linear HPLC gradient was employed: (1) 0.01.0 min isocratic at 10% solvent
B; (2) 1.021.0 min linear gradient from 10 to 25% solvent
B; (3) 21.036.0 min linear gradient from 25 to 45% solvent
B; (4) 36.056.0 min linear gradient from 45 to 100% solvent
B; (5) 50.0050.15 min flow increased to 1.2 mL min1 ; (6)
82.0082.15 min linear gradient from 100 to 10% solvent
B; (7) 92.0092.15 min flow rate decreased to 1 mL min1 ;
and (8) after 99.0 min the sampled loop was rinsed and
the gradient repeated. The HPLC system consisted of a
Young-Lin M930 liquid chromatograph pump and an M720
detector (Young-Lin Instruments Co., Ltd). The column
for quantitative analysis was a YMC-Pack ODS-AM-303
(250 4.6 mm I.D.), and the UV absorption was measured
at 280 nm.
2.3.3. Making the calibration curves of resveratrol and
the 16 phenolic compounds
The 17 phenolic standards, including resveratrol, were
purchased from SigmaAldrich (USA) in 2002 and used for
the calibration curves. The purity of each standard was determined by HPLC chromatography, and the plotting standard
concentration was obtained at five concentrations (1, 25, 50,
75 and 100 ppm). A high linearity (r2 > 0.998) was obtained
for each curve. Resveratrol, gentisic acid, cathechin, hydroxybenzoic acid, chlorogenic acid, caffeic acid, syringic
acid, -coumaric acid, ferulic acid, hesperidin, narigin, salicylic acid, hyricetin, quercetin, t-cinnamic acid and naringenin were identified by comparing their retention times with
authentic standards. Their concentrations were calculated by
comparing peak areas of samples with those of the standards
(Table 1) and total phenolic compounds were summed by

Table 1
Calibration curves of 16 standard phenolic compounds and resveratrol
Standard chemicals

Retention
time (min)

Equations

Gentisic acid
p-Hydroxybenzoic acid
Catechin
Chlorogenic acid
p-Coumaric acid
Caffeic acid
Syringic acid
Ferulic acid
Hesperidin
Naringin
Salicylic acid
Myricetin
Quercetin
t-Cinnamic acid
Naringenin
Kaempferol
Resveratrol

17.577
19.030
19.505
21.160
24.040
24.212
26.307
39.413
49.675
50.357
52.292
55.753
65.077
66.845
67.800
76.122
57.148

y = 10.05x 3.96, r = 0.998***

y = 32.83x 3.42, r = 0.998***
y = 1.87x 0.18, r = 0.999***
y = 21.38x 7.85, r = 0.999***
y = 67.50x 5.55, r = 0.998***
y = 43.12x 7.43, r = 0.997***
y = 40.60x 0.03, r = 0.999***
y = 47.41x 14.45, r = 0.999***
y = 20.46x 4.47, r = 0.999***
y = 24.92x 3.50, r = 0.999***
y = 6.96x 5.78, r = 0.998***
y = 15.99x 36.52, r = 0.999***
y = 15.99x 36.52, r = 0.998***
y = 123.80x 19.08, r = 0.999***
y = 38.95x 18.70, r = 0.999***
y = 22.97x 32.75, r = 0.997***
y = 37.529x 8.72, r = 0.999***

the each identified 16 phenolic compound was analyzed by

HPLC, excluding resveratrol.
2.4. Quantitative analysis of free amino acids
2.4.1. Preparation of R. glutinosa
R. glutinosa root samples were dried under vacuum at a
temperature below 30 C and then ground. Powdered samples were passed through a 40 mesh screen and 0.1 g samples
made to derivatives for analyzing free amino acids using
phenylisothiocyanate (PITC, Aldrich). The PITC derivatives
were once again dried under vacuum at a temperature below
30 C and dissolved in 200 L solvent A buffer (pH 6.1).
The supernatant was analyzed after the homogenate was centrifuged at 30,000 rpm for 20 min and filtered through a 0.45
micro filter unit (Strydom and Cohen, 1988).
2.4.2. Instrumental conditions for HPLC
A linear HPLC gradient was employed. Solvent A contained 1.4 mM sodium acetate (NaHAc), 0.1% triethylamine
(TEA) and 6% acetonitrile (CH3 CN, pH 6.1), while solvent B was 60% CH3 CN. Following injection of 20 L
of the sample, solvent B was increased from 0 to 100%
over 30 min and the gradient repeated. The solvent flow
rate was 1 mL min1 . The HPLC system consisted of a
Hewlett Packard 1100 Series binary liquid chromatography
pump and detector (HP 1100 Series, USA). The reversedphased column for quantitative analysis was a Nova-Pak C18
(300 3.9 mm I.D. 4 m), the column oven temperature was
held at 46 C and UV absorption measured at 254 nm. The
concentrations of amino acids were calculated using a peak
area ratio of the control against the peak area ratio of the standard. When this value is multiplied by the concentration of
the amino acid standards, the concentration of specific amino
acids in R. glutinosa is obtained (Heinrikson and Meredith,
1984).

I.M. Chung et al. / Environmental and Experimental Botany 56 (2006) 4453

47

2.5. Statistical analysis

Analysis of variance for all data was undertaken using
the general linear model (GLM) procedure of the statistical analysis system (SAS) program developed by the SAS
Institute Inc. (2000). All of the aforementioned experiments
were replicated three times using a completely randomized
design. The pooled mean values were separated based on least
significant differences (LSD) at the 0.05 probability level.

3. Results and discussion

3.1. Quantication of resveratrol and evaluation of SOD
activity in R. glutinosa under temperature and water
stress
In general, the content of resveratrol in R. glutinosa
decreased more in treatments of temperature stress than water
deficiency (water stress). The water control treatment had the
highest resveratrol contents (38.87 g g1 ), while the high
temperature treatment had the lowest contents (2.96 g g1 ).
In particular, there were significant differences between the
water treatment control and the water deficiency treatment.
However, no significant differences were found in the temperature treatments (Fig. 1).
SOD activity in R. glutinosa differed from the results
found for resveratrol. The water deficiency treatment generally reduced inhibitory effects on various reactive oxygen species (ROS) to a greater extent than temperature
stress. There were significant differences among these treatments. The low temperature treatment (28.43%) had higher
inhibitory effects in removing ROS than the high temperature treatment (9.54%). In water treatments, although there
were no significant differences in the water stress treatments,
the control treatment (9.4%) had a lower inhibitory effect in
removing ROS than the water deficiency treatment (13.26%)
(Fig. 2).
In this study, the contents of resveratrol in R. glutinosa
was the highest in the water control treatment (Fig. 1), while

Fig. 1. Comparison of resveratrol concentrations in R. glutinosa under water

and temperature treatment.

Fig. 2. Comparison of SOD activity in R. glutinosa under water and temperature treatment.

SOD activity was the highest in the low temperature treatment (Fig. 2). Generally, stresses of water (deficiency) and
temperature are known to have side effects on growth and
development of the entire life cycle of plants. OToole and
Chang (1979) reported that water stress resulted in low individual grain weight due to a poor grain filling process. Boyer
and Bowen (1970) found a reduction in leaf area with water
stress, which would lead to a reduction in carbon assimilation
in addition to a reduction in plant growth. Stilbenes, such as
resveratrol, and non-flavonoid phytoalexins have been found
as major constituents in plants such as peanuts and grapes
following fungal infections or stress. Previous research on
R. glutinosa generally examined medical effects related to
human diseases such as diabetes and hypertension. However,
several recent research papers (Lim et al., 2004a,b) have
focused on other physiologically active chemicals and the
resistance of plants to environmental stress. For example,
Lim et al. (2004a) reported the potential of R. glutinosa as a
plant with high resistance to stress. As a result, the contents
of resveratrol were examined in it and several other medicinal plants. Lim et al. (2004b) carried out the transformation
of R. glutinosa from the RS3 gene of the peanut used by
Agrobacterium and reported that transformed plants might
have potential and useful effects on both plant and human
health under various environmental stresses, UV light.
SOD is generated from plant tissues when the plant
is under conditions of oxidative or environmental stress.
According to previous reports, SOD can easily convert superoxide anion radicals into H2 O2, and its activity is affected by
factors such as low temperature. In barley, mRNA amounts of
Cu/Zn-SOD tended to increase when placed under O3 stress,
and more was accumulated in the younger leaves (Chung et
al., 2000; Lee et al., 2002). SOD could also increase when
plants are under pathogenic attack or affected by environmental factors such as the accumulation of heavy metals and O3
(Raa, 1971; Chung et al., 2001). Lim et al. (2004c) recently
reported that SOD activity in R. glutinosa was higher than in
82 medicinal plants including Angelica dahurica. However,
little information on SOD activity in R. glutinosa is available.

I.M. Chung et al. / Environmental and Experimental Botany 56 (2006) 4453

48

Fig. 3. Comparison of total phenolic compounds in R. glutinosa under water

and temperature treatment.

As a result, further studies on resveratrol and SOD activity in

R. glutinosa are required in order to find correlations between
resveratrol, SOD activity and environmental stresses like UV
light.
3.2. Quantication of 16 phenolic compounds in R.
glutinosa under temperature and water stress
This study was conducted to examine differences in 16
phenolic compounds with both water and temperature stress.
Generally, the water deficiency treatment resulted in lower
total phenolic compounds than the temperature stress. The
low temperature treatment (98.63 g g1 ) had a higher total
phenolic compounds content than the high temperature treatment (16.86 g g1 ). These results were statistically significant (Fig. 3). In the water treatment, the control had
higher total phenolic compounds (113.49 g g1 ) than the
water deficiency treatment (62.27 g g1 ) (Fig. 3). These
results were also significant (Fig. 3). HPLC chromatogram
exhibited that the control extracts contained higher levels

of phenolic compounds than did those of the water deficiency (Fig. 4). Among the 16 phenolic compounds, gentisic
acid and kaempferol were not detected in any of the treatments, and 10 phenolic compounds, including gentisic acid,
were not detected in the high temperature and water deficiency treatments (Table 2). Myricetin in the water control
treatment (60.96 g g1 ) had the highest content of the 16
individual phenolic compounds in the four treatments. pHydroxybenzoic, caffeic and ferulic acids were only detected
in the water deficiency treatment and chlorogenic and caffeic
acids were detected in the high temperature. But, chlorogenic acid, there were no significant differences, increased
14.14 g g1 in the temperature treatments (Table 2).
Phenolic compounds are known to be secondary metabolic
substances with anti-oxidant and anticancer properties, as
well as conferring resistance to environmental disasters.
Lim et al. (2004c) reported that R. glutinosa had about
198.0 g g1 phenolic compounds, of which salicylic acid
comprised 110.0 g g1 (about 55%). Our results agreed with
those of Lim et al. (2004c), where R. glutinosa was found
to have higher contents of salicylic acid in all four treatments. This study might also suggest that growth at high
temperature or under drought conditions could decrease the
content of phenolic compounds such as hesperidin and salicylic acid. Kim et al. (2004) reported that the content of
phenolic compounds was correlated with climatic characteristics in the cropping year, in particular the temperature in
July (r2 = 0.25* ). Other research reported that drought conditions in the cropping year could lead to noticeably decreased
contents of cinnamic and benzoic acid derivatives in wheat
leaves (Tsai and Todd, 1972). It is thought that the decrease
in phenolic compounds might result from a decline in the
activity of key enzymes related to the biosynthesis of phenolic compounds. The content of phenolic compounds also
increased under conditions of water stress in the cucum-

Table 2
Distribution of 16 phenolic compounds in R. glutinosa with treatments of water and temperature stress
Compounds

Gentisic acid
Catechin
p-Hydroxybenzoic acid
Chlorogenic acid
Caffeic acid
Syringic acid
p-Coumaric acid
Ferulic acid
Hesperidin
Naringin
Salicylic acid
Myricetin
Quercetin
t-Cinnamic acid
Naringenin
Kaempferol
Tr: Trace.

Water treatment

LSD(0.05)

Deficiency (1.18 MPa)

(g g1 )

Control (1.04 MPa)

(g g1 )

Tr
Tr
Tr
8.73
Tr
0.05
Tr
Tr
4.2
0.91
14.08
34.30
Tr
Tr
Tr
Tr

Tr
Tr
0.28
10.56
1.62
0.12
Tr
3.21
1.92
1.18
33.64
60.96
Tr
Tr
Tr
Tr

Temperature treatment
(15 C)

0
0
0.06
1.04
0.02
0.06
0
0.28
1.90
0.40
4.24
7.33
0
0
0
0

LSD(0.05)
(35 C)

Low
(g g1 )

High
(g g1 )

Tr
0.35
Tr
2.83
3.25
2.07
0.86
4.01
10.75
5.21
10.97
35.70
20.92
0.43
1.28
Tr

Tr
Tr
Tr
14.14
2.72
Tr
Tr
Tr
Tr
Tr
Tr
Tr
Tr
Tr
Tr
Tr

0
0.01
0
2.78
1.03
0.89
0.01
1.33
8.33
3.72
4.53
9.56
4.51
0.15
0.49
0

I.M. Chung et al. / Environmental and Experimental Botany 56 (2006) 4453

49

Fig. 4. HPLC chromatogram of phenolic compounds in R. glutinosa under water treatment. (A) Standard phenolic compounds; (B) control (1.18 MPa);
(C) deficiency (1.04 MPa)

ber (Cucumis sativus), although no effect was found for the

radish (Raphanus sativus) under drought conditions (Tevini
et al., 1983). There were no clear relationship between water
stress and the level of phenolic compounds in the R. glutinosa
although a few studies have been reported in the correlation
between water stress and the level phenolic compounds in the

cotton (Gossypium hirsutum) (Guinn and Eidenbock, 1982),

Heteromeles arbutifolia (Dement and Mooney, 1974), plum
(Prunus domestica) (Hillis and Swain, 1959). In the future,
we will need to study the variation of phenolic compounds
from more varieties in order to determine more correctly the
distribution pattern of phenolic compounds in R. glutinosa.

I.M. Chung et al. / Environmental and Experimental Botany 56 (2006) 4453

50

3.3. Quantication of 21 free amino acids in R.

glutinosa under temperature and water stress
In general, the 21 free amino acids in R. glutinosa were
detected in lower amounts than the phenolic compounds.
However, they were detected in all four treatments. In general, there was a higher content of total free amino acids in
the water stress treatment than in the temperature stress treatment. Overall, the low temperature treatment (3.84 g g1 )
and the water stress control (5.69 g g1 ) had the highest
contents, and these results were significant. Tryptophan was
found in larger amounts than the other free amino acids
in all treatments except for the low temperature treatment.
Generally, the high temperature and water deficiency treatments significantly tended to reduce the amount of free amino
acids, although cysteine was not detected in both treatments
(Table 3, Figs. 5 and 6).
In this study, the total content of the 21 free amino acids
(0.825.69 g g1 ) were found at lower levels than in other
research papers (Hong et al., 1989; Borress et al., 1976;
Hansan and Nelson, 1978). Hong et al. (1989) reported that
the total content of 18 free amino acids varied from 17.60 to
32.05 g g1 in three kinds of mushrooms, and the content
varied with the plant part. The report of Borress et al. (1976)
indicated that free proline would accumulate in the plant as a
result of synthesis de novo under water stress, while Hansan
and Nelson (1978) found that betaine, synthesized from serine, would vary as a result of de novo synthesis from one- and
two-carbon precursors under similar conditions. These studies have showed the higher contents of the free amino acid in

Fig. 5. Comparison of total free amino acids in R. glutinosa under water

and temperature treatment. (A) Water treatmentcontrol (1.18 MPa);
(B) water treatmentdeficiency (1.04 MPa); (C) temperature treatment
low temperature (15 C); (D) temperature treatmenthigh temperature
(35 C).

the water stress treatment with aerial part, leaves, stems and
seeds not root part analysis. Ordinarily, elevated temperature
during growth periods would not only lead to a deterioration
of plant tissues because of a lack of life supporting energy
from a reduced photosynthesis, but would also alter proteins
and enzymes (Maynard and David, 1987). Also, there were
no clear relationship between water stress and free amino
acid such as proline and serine content has been established
in R. glutinosa. The cause of the fluctuations in free amino
acids concentrations between water deficiency (1.18 MPa)

Table 3
Distribution of 21 free amino acids in R. glutinosa with treatments of water and temperature stress
Compounds

Cysteine
Aspartic acid
Glutamic acid
Asparagine
Serine
Glutamine
Glycine
Histidine
Arginine
Threonine
Alanine
Proline
Tyrosine
Valine
Methionine
Cystin
Isoleucine
Leucine
Phenylalanine
Tryptophan
Lysine
Tr: Trace.

Water treatment

LSD(0.05)

Deficiency (1.18 MPa)

(g g1 )

Control (1.04 MPa)

(g g1 )

Tr
0.012
0.020
0.014
0.012
0.002
0.001
Tr
0.016
0.001
0.018
0.017
Tr
0.019
0.002
0.015
0.031
0.055
0.052
0.418
0.110

Tr
0.262
0.206
0.105
0.067
0.061
0.045
0.086
0.561
0.052
0.169
2.273
0.089
0.133
0.073
0.178
0.186
0.139
Tr
1.002
Tr

Temperature treatment
(15 C)

0
0.024
0.029
0.078
0.020
0.024
0.023
0.017
0.029
0.015
0.024
0.051
0.061
0.025
0.046
0.017
0.020
0.017
0.017
0.015
0.013

LSD(0.05)
(35 C)

Low
(g g1 )

High
(g g1 )

Tr
0.863
0.296
0.060
0.040
0.261
0.058
0.153
0.779
0.029
0.156
0.190
0.188
0.150
0.116
Tr
0.097
0.200
0.055
0.102
0.050

Tr
0.027
0.036
0.047
0.030
0.014
0.010
Tr
0.017
Tr
0.081
0.016
0.012
0.039
0.034
0.031
0.032
0.017
Tr
0.505
Tr

0
0.047
0.053
0.034
0.020
0.124
0.011
0.025
0.159
0.013
0.034
0.017
0.102
0.025
0.076
0.015
0.028
0.102
0.016
0.002
0.032

I.M. Chung et al. / Environmental and Experimental Botany 56 (2006) 4453

51

Fig. 6. Chromatogram of free amino acids in R. glutinosa under water and temperature treatment (a: cysteine; b: aspartic acid; c: glutamic acid; d: asparagine,
e: serine; f: glutamine; g: glycine; h: histidine; i: arginine; j: threonine; k: alanine; l: proline; m: tyrosine; n: valine; o: methionine; p: cystin; q: isoleucine; r:
leucine; s: phenylalanine; t: tryptophan; u: lysine).

52

I.M. Chung et al. / Environmental and Experimental Botany 56 (2006) 4453

and control (1.04 MPa) treatments exhibited in this study

could not be concluded because R. glutinosa medicinal plant
characteristics is growing well under dry soil conditions and
has strong tolerance in the water stress in the field. Thus
the analysis of free amino acid requires considerable further
study.
Results from this study indicated that temperature
stress was negatively correlated with resveratrol (r2 =
0.99** ), SOD activity (r2 = 0.99** ), phenolic compounds
(r2 = 0.99*** ) and free amino acid (r2 = 0.99*** ), while
water stress was positively correlated with resveratrol
(r2 = 0.99*** ), phenolic compounds (r2 = 0.99*** ) and free
amino acid (r2 = 0.99*** ). SOD activity was not found to be
correlated. That is, the values of the above parameters tended
to decrease with elevated temperature or the imposition of a
water deficiency. Hence, other than SOD activity, the contents
of these substances are thought to be related with environmental factors such as temperature or water in the cropping
year.
Thus, we found that resveratrol, SOD activity, phenolic
compounds and free amino acids could change as a result
of different cropping environments. In the future, we will
conduct further studies on correlations between the contents
of resveratrol, SOD activity, phenolic compounds and free
amino acids and other cropping factors such as the management of fertilizers or planting density.

Acknowledgement
This work was supported as part of a grant from the Rural
Development Administration, Korea (Biogreen 21 project) in
2003.

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