Vol.2, No.4, 117-122 (2013) Journal of Agricultural Chemistry and Environment
http://dx.doi.org/10.4236/jacen.2013.24017
Cultivation characteristics and flavonoid contents of
wormwood (Artemisia montana Pamp.)
Yong Joo Kim1, Jeong-Hoon Lee2*, Sun-Ju Kim1*
1Department of Bio-Environmental Chemistry, Chungnam National University, Daejeon, South Korea;
2Department of Herbal Crop Research NIHHS, RDA, Eumseong, South Korea
*Corresponding Authors:
Received 21 October 2013; revised 22 November 2013; accepted 30 November 2013
Copyright © 2013 Yong Joo Kim et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
The aim of this study was to establish the opti-
mum harvesting time and the content of flavon-
oids in the leaves, stems, and roots of Artemisia
montana Pamp. A. montana was monitored from
June to October in 2012. The yield of A. montana
at high density (30 × 10 cm) was higher than that
of A. montana at low density (30 × 20 and 30 cm).
Yield in terms of dry weight was increased with
an extended growth period and development
stage. High yield achieved at 2580 and 2757
kg·10 a−1 in September and October, respectively.
Among the leaves, stems, and underground plant
organs, jaceosidin and eupatilin were mainly
detected in the leaves, and the highest levels
were observed in June, at values of 66.6 and
158.2 mg·100 g−1, respectively. In contrast, api-
genin was the major compound detected in the
underground plant organs, with levels ranging
from 21.2 to 29.5 mg·100 g−1 until September.
Therefore, optimal harvest times were between
September and October, generating a high yield
and adding economic value although a higher
level of total flavonoids was observed in crops
harvested in June.
Keywords: Artemisia montana; Flavonoids; Harvest
Time; Plant Density
1. INTRODUCTION
Medicinal plants have long been recognized as natural
herbs that have minimal to negligible side effects. As the
culture of enhancing human well-being has gained
popularity, scientists have engaged in extensive studies
of medicinal plants, including studies on natural drugs,
herbal cosmetics, and natural pigments [1-3].
In Korea, wormwood has been used as an herb. Ar-
temisia spp. belongs to Compositae, and taxonomic es-
timates have indicated that 200 to 400 species exist
worldwide [4]. Approximately 40 species of Artemisia
are distributed in Korea, of which 26 species are re-
corded in the Color Illustrated Book about the Plants in
Korea [5]. In Korean traditional medicine, Artemisia spp.
is further classified as Chung-ho, Ae-yeop, In-jin, and
Am-ryeo. According to the herbal pharmacopoeia, the
Ae-yeop pertains to dried medicinal leaves and young
stems of Artemisia argyi Lev., A. princeps var. orientalis
(Pamp.) Hara., and A. montana Pamp. Ae-yeop has been
used as a medicinal herb [6]; it imparts warmth to the
body and controls blood circulation, body temperature,
bleeding, and pregnancy. It has also been used as a rem-
edy for abdominal pain due to complications, diarrhea,
chronic diarrhea, hematemesis, epistaxis, melena, and
amenorrhea [7]. Ae-yeop contains various compounds
such as flavonoids, steroids, phenylpropanoids, terpe-
noids, peptides, sesquiterpenoids, monoterpenoids, and
diterpenoids [8,9]. Among these, flavonoids are known
to possess excellent antioxidant activity that effectively
eliminates reactive oxygen species, as well as a variety
of other biological activities, including anti-cancer and
anti-inflammatory activities [10]. The major flavonoids
in Ae-yeop include eupatilin, jaceosidin, apigenin, and
eupafolin [9,11]. Its pharmacological activities include
anti-cancer, anti-inflammatory, anti-diabetic, and anti-
allergic activities [12-15]. Eupatilin is known to have
strong inhibitory effects on gastric ulcers and has been
used as the main raw material for the preparation of
natural drugs [16,17]. In addition, the size of A. montana
commonly used as Ae-yeop is larger than the size of
other wormwood species. It has also been proven to have
antioxidant and anti-diabetic effects because of compo-
nents such as caffeic acid, caffeoylquinic acid, catechol,
*These corresponding authors contributed equally to this work.

Y. J. Kim et al. / Journal of Agricultural Chemistry and Environment 2 (2013) 117-122
118
hyperoside, and protocatechuic acid making this herb a
potential drug resource [18,19]. However, agronomic
evaluation of this crop has not been conducted properly
because of difficulties in its morphological classification
among similar species. To establish “Good Agricultural
Practices” (GAP), plant growth characteristics and yields
of wormwood (Artemisia montana Pamp.) were evalu-
ated on the basis of plant density and harvest times.
Consequently, this study examined changes in the fla-
vonoid (apigenin, jaceosidin, and eupatilin) (Figure 1)
contents on the basis of cultivation characteristics and
harvest times of A. montana, to develop an economically
significant crop.
2. MATERIALS AND METHODS
2.1. Plant Materials
A. montana was collected from Gyeongsangbukdo
Ulleung-gun and planted in a test package at the De-
partment of Herbal Crop Research of the Korean Na-
tional Institute of Horticultural & Herbal Science on May
10, 2012. The plants were stored at KMRH under the
Voucher number: MPS0002514 (Figure 2). The plants
were grown in seedling trays containing 200 holes in a
greenhouse at the beginning of March 2012. After plant-
ing, the leaves were collected on the 10th day of each
month from July to October to investigate the crop char-
acteristics. Samples were harvested five times every
month from June to October for analysis of flavonoids.
The test package was prepared using 2000 kg·10 a−1 base
manure and covered with black plastic bags. A random-
ized complete block design was used in triplicate. For
planting density, spacing between furrows and rows was
90 and 30 cm, respectively. The planting intervals were
10, 20, and 30 cm. After planting, 20 specimens were
evaluated three times at 30-day intervals. For extraction,
block sampling was used. The quantity was converted to
the number per 10 a after harvesting in one m3 test envi-
ronment.
2.2. Seed Characteristics
To determine the seed characteristics of A. montana,
20 seeds were randomly selected in triplicate. The shape,
size, and color of the seeds were evaluated. For 1000-
seed weight, the average value of 10 measurements was
calculated. For germination, seeds with uniform size and
color, as well as devoid of pest contamination, were se-
lected using a caliper and microscope. The selected seeds
were placed in disposable petri dishes and maintained in
constant-temperature incubators set at 15, 20, 25, and
30˚C. The petri dishes were lined with filter paper soaked
with distilled water. Germination was defined as the
emergence of young leaves and roots of approximately 1
mm in length through the seed coat. The first germina-
tion time, bud burst period and germination rate were
monitored.
2.3. Equipment and Reagents
Flavonoid standards, jaceosidin, and eupatilin were
purchased from Chengdu Biopurify Phytochemicals Ltd.
(Chendu, Sichuan, China) and apigenin from Sigma-
Aldrich (St. Louis, MO, USA). Seed characteristics were
microscopically evaluated (Olympus SZ61; Olympus Co.
Tokyo, Japan). Seed germination was monitored in a
constant temperature incubator (Multi-Room Incubator,
Wisecube, Wonju-si, Korea). Flavonoid analysis based
on growth stage was performed using the Agilent 1100
series HPLC system (Agilent Technologies, CA, USA).
2.4. Extraction and Analysis of Flavonoids
Approximately 10 g of powder was extracted from
each plant organ of A. montana by using methanol
(MeOH), thus generating 2.4 g from the leaves, 1.7 g
from the stems, and 1.8 g from the roots. Each extract
(10 mg) was placed in a 2 mL Eppendorf tube and mixed
with 1 mL of MeOH. After 5 min of ultrasonic extraction,
the extracts were centrifuged at 3,000 rpm at 4˚C for 5
min. The supernatant was filtered using a 0.45 µm PTFE
hydrophilic syringe filter (i.d., 13 mm) and collected in a
vial for HPLC.
For the analysis of flavonoids, the Agilent 1100 Series
HPLC system (Agilent Technologies, CA, USA) equipped
with u-Bondapak TM C18 (10 µm, 3.9 × 300 mm, Wa-
ters, MA, USA) was used. Detection was conducted at a
wavelength of 354 nm, the flow rate was 1 mL·min−1,
and the column oven temperature was set at 30˚C. Ap-
proximately 20 µL of the sample was injected using an
auto sampler. The mobile phase solvents used were sol-
vent A [Water: H2PO4 (99.6: 0.4, v/v)] and solvent B
[acetonitrile]. The gradient program used as follows: 0 -
30 min, 30% → 70% solvent B; 30 - 40 min, 70% →
Figure 1. Chemical structure of flavonoids in A. montana. (a) apigenin; (b) jaceosidin; (c) eupatilin.

Y. J. Kim et al. / Journal of Agr icultural Chemistry and Environment 2 (2013) 117-122 119
Figure 2. Specimen of A. montana. Horti-
cultural traits: A. montana is a perennial
plant that belongs to Asteraceae and has
creeping roots and upright stems. The cau-
line leaves with hairs are alternately ar-
ranged. The size of A. montana is larger,
compared to the closely related taxa.
100% solvent B; 40 - 50 min, 100% → 30% solvent B;
50 - 55 min, keep 30% solvent B. A stock solution of
each flavonoid standards (apigenin, jaceosidin, and eu-
patilin) was made with 1 mL of MeOH and diluted with
MeOH to make 50, 100, 200, 250, and 500 µg·mL−1 for
standard solutions. After taking 20 μL of each standard
solution, HPLC chromatography was conducted to quan-
tify each component. A calibration curve was created
using the concentration of the standard solution as the
variable. The linear regression equation of the calibration
curve of each component was apigenin, y = 7.2412x +
8.8393; jaceosidin, y = 9.6193x + 8.8391; and eupatilin,
y = 8.5583x + 6.1677. The coefficient of determination
was R2 = 0.9999. By substituting the HPLC peak area
analyzed in each sample for the calibration curve regres-
sion equation, the amount of each compound (μg·mL−1)
was calculated. By calculating the yield, the extracts
were quantified (mg·100 g−1 of MeOH extracts).
2.5. Statistical Analysis
Data were analyzed by the application of the Duncan’s
multiple range test (DMRT, n = 3) at p ≤ 0.05 using the
SAS statistical program (SAS 9.3, SAS Institute Inc.,
Cary, NC, USA). The F-value is the ratio of the mean
square due to regression to the mean square due to error
and indicates the influence (significance) of each con-
trolled factor on the tested model.
3. RESULTS AND DISCUSSION
3.1. Seed Characteristics
A. montana seeds were oblong in shape, and the hair-
less achene was wrapped in a white fruit coat. The length,
width, and 1000-seed weight were 1.37 mm, 0.52 mm,
and 0.110 g, respectively (Figure 3). The first germina-
tion time was 2 days at 20˚C - 30˚C. However, the first
germination time was 3 days at 15˚C. The bud burst pe-
riod was observed at 20˚C - 30˚C and at 15˚C were 2
days and 5 days, respectively. This trend was similar to
that of A. capillaris, which is a closely related species
[20].
Germination rates at 15˚C, 20˚C, 25˚C, and 30˚C were
84.7%, 90.0%, 92.7%, and 87.3%, respectively, which
were slightly higher. The germination rate was the high-
est at 25˚C (Figure 3). The germination rate of A. mon-
tana increased up to a temperature of 30˚C, and, there-
after, decreased with higher temperature. Thus, 30˚C was
considered favorable for the initial germination of A.
montana. Our results were consistent with the findings of
Thompson [21]. Meanwhile, seed germination was close-
ly related to environmental conditions, such as genetic
differences, seed maturity, temperature, moisture, oxygen,
and sunlight [22]. The temperature has been reported to
have the greatest effect on germination rate [23,24]. Thus,
when considering the seed characteristics of A. montana
in this experiment, the optimum germination temperature
was 25˚C. This condition can influence the distribution
and seeding time of this species.
3.2. Growth Characteristics by Planting
Density
The growth characteristics and yields of A. montana
on the basis of planting density are shown in Table 1.
Plant height ranged from 168.3 to 176 cm. It appeared
that a higher density was often associated with a smaller
height. Such findings were contrary to the results of a
few studies [3,25,26], suggesting a higher planting den-
sity in Achyranthes japonica, Asparagus cochinchinensis,
and Ligusticum chuanxiong. Results indicated that the
heights of the plants were comparatively higher because
of the competition among the species and decrease in
light intensity. The results showed a similar tendency to
that of Song et al. [27], who reported that a higher plant-
ing density in P. sonchifolia and W. japonica led to a
lesser height because of competition between species [1].
Leaf dry weight ranged from 32 to 79.3 g. A lower plant
density was associated with a higher leaf dry weight. The
dry weight of the aerial plant organs per 10 a was the
highest in the 30 × 10 cm plots. However, no significant
differences were observed between the result and plant-
ing distance of 30 × 20 cm. A higher number of aerial
plant organs of A. montana were associated with a

Y. J. Kim et al. / Journal of Agricultural Chemistry and Environment 2 (2013) 117-122
120
(a) (b) (c)
Figure 3. Plant growth characteristics of A. montana according to different temperatures. (a) seed characteristics; (b) first
germination time and bud burst period (days); (c) germination rates (%).
Table 1. Plant growth characteristics and yields of A. montana according to plant density in September.
Yield (kg·10 a−1, dry weight)
Plant density
(cm)
No. of plant
(ea·10 a−1)
Plant height
(cm)
Stem diameter
(mm)
Leaf weight
(g, dry weight)
Dry weight
ratio (%) Aerial part
organa)
Underground part
organ
30 × 10 30,000 168.3 ± 4.10a 8.7 ± 1.40a 32.0 ± 1.50c 55.3a 2580 ± 224.0a 480.0 ± 91.24a
30 × 20 15,000 171.5 ± 6.17a 9.9 ± 1.87a 53.2 ± 8.14b 58.4a 2330 ± 91.65a 532.5 ± 68.74a
30 × 30 9,000 176.0 ± 6.42a 10.2 ± 170a 79.3 ± 4.25a 47.9b 1847 ± 69.18b 559.5 ± 53.31a
F-value 1.41NS 0.70NS 233.53*** 12.60** 19.75** 0.93NS
Within each column, values followed by the same letters in a column are not significantly different at p ≤ 0.05 (n = 3). Significance level about F-value is rep-
resented at *p < 0.05; **p < 0.01; ***p < 0.001; NSnot significant. a)Aerial plant organ indicated above-ground parts including stems and leaves.
greater planting density. These results were similar to
that of other medicinal plants such as A. japonica, A.
cochinchinensis, and L. chuanxiong [3,25,26].
3.3. Growth Characteristics by Harvest Time
Growth characteristics and yields of A. montana on the
basis of harvest time were investigated using 30 × 10 cm
plots (Table 2). The height sharply increased from
122.87 to 169.4 cm during the period of July-August,
without showing considerable differences after August.
The rainy season in July may have affected the growth of
A. montana because of the sufficient water and sunlight.
After the flowering period in August, growth stopped.
The stem diameter was the greatest in August when the
growth rate was the highest. Significant differences were
observed between August and other times. Leaf dry
weight was the highest in October. A longer growth pe-
riod was associated with a higher yield. Dry weight ratio
during the growth period decreased by 44.7% in Sep-
tember and by 71.9% in July. It may be possible that af-
ter the rainy season of July to August, the high moisture
content caused the higher dry weight ratio; in contrast, in
September the hot and dry weather caused the lower lev-
els of moisture content and dry weight ratio. Since Sep-
tember, the dry weight ratio has remained almost con-
stant. Apparently, the reason was that the change in cli-
mate after September was not significant. The dry weight
of the aerial plant organs by harvest time was 2757 kg·10
a−1, which was the highest in October. No significant
differences were observed between the results collected
in October, 2757 kg·10 a−1, and the dry weight of the
aerial plant organs harvested in September, 2580 kg·10
a−1. Thus, considering leaf dry weights and the yields, the
optimal harvesting time for A. montana was the period
between mid-September and early October.
3.4. Flavonoid Analysis
Jaceosidin and eupatilin were detected only in the
leaves, whereas apigenin was detected in the roots (Ta-
ble 3). Contents of jaceosidin and eupatilin with respect
to harvest time showed a similar pattern, and the contents
in the leaves harvested in June were the highest levels
(66.6 and 158.2 mg·100 g−1, respectively). The contents
of jaceosidin and eupatilin significantly decreased in July.
The contents of jaceosidin and eupatilin in the leaves of
A. princeps collected in May were the highest (38.6 and
211.4 mg 100 g−1, respectively) [28]. The levels of
monoterpene in A. princeps were documented the highest
level in May 8, and they decreased rapidly after mid-
May [29]. Our results were similar to those of previous
studies. The level of apigenin in the roots ranged from
21.2 to 29.5 mg 100 g−1. The content increased from June
to August and thereafter decreased. These results have
also been observed in other medicinal plants such as A.

Y. J. Kim et al. / Journal of Agr icultural Chemistry and Environment 2 (2013) 117-122 121
Table 2. Growth characteristics and yields of A. montana in different harvest times.
Harvest times Plant height
(cm)
Stem diameter
(mm)
Leaf weight
(g, dry weight)
Dry weight
ratio (%)
Ratio of leaf
weight (%)
Aerial part organa)
(kg·10 a−1, dry weight)
July 122.8 ± 0.23b 8.9 ± 0.59b 13.5 ± 0.68c 28.1c 43.3a 937.0 ± 15.10c
August 169.4 ± 0.42a 11.7 ± 0.47a 26.0 ± 2.53b 35.1b 31.7b 2,459 ± 62.45b
September 168.3 ± 0.10a 8.7 ± 1.40b 32.0 ± 1.50ab 55.3a 37.2b 2,580 ± 224.0ab
October 165.4 ± 1.27a 9.4 ± 1.04b 39.3 ± 7.66a 51.6a 42.8a 2,757 ± 137.2a
F-value 187.31*** 6.15* 21.92*** 109.72*** 10.46** 115.73**
Within each column, values followed by the same letters in a column are not significantly different at p ≤ 0.05 (n = 3). Significance level about F-value is rep-
resented at *p < 0.05; **p < 0.01; ***p < 0.001; NSnot significant. a)Aerial plant organ indicated above-ground parts including stems and leaves.
Table 3. Flavonoid contents (mg·100 g−1, n = 3) in MeOH extracts of A. montana harvested at different development stages from
June to October.
Harvest times Parts Apigenin Jaceosidin Eupatilin
Roots 21.2 ± 0.40 NDa) ND
Stems tr.b) ND ND
June
Leaves ND 66.6 ± 2.18 158.2 ± 15.5
Roots 24.8 ± 0.97 ND ND
Stems TR ND ND
July
Leaves ND 4.5 ± 0.32 14.6 ± 0.26
Roots 29.5 ± 0.24 ND ND
Stems TR ND ND
August
Leaves ND 17.6 ± 0.32 5.3 ± 0.11
Roots 25.7 ± 0.18 ND ND
Stems TR ND ND
September
Leaves ND 0.9 ± 0.05 8.8 ± 0.10
Roots 11.2 ± 0.12 ND ND
Stems TR ND ND
October
Leaves ND 2.1 ± 0.06 11.0 ± 0.10
a)ND, not detected. b)tr., trace amounts.
capillaris. Capillarisin content in A. capillaris increased
until flowering time and thereafter decreased [30]. After
August, which was the flowering time of A. montana,
certain ingredients in the underground plant organs de-
creased.
The levels of active ingredients are influenced by cli-
matic conditions, including precipitation and temperature.
For A. princeps, whose Ae-yeop was used as herbal drugs,
samples collected from April to May showed excellent
antioxidant effects. The samples collected from August
to September showed high antimicrobial activity [31].
Thus, according to the results of this study, A. montana
had the potential of serving as natural drugs, and farm-
ers may thus consider wormwood as an economically
beneficial crop.
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