horticulturae

Article
Improvement of Phenylpropanoid Production with
Elicitor Treatments in Pimpinella brachycarpa Nakai
Nam Su Kim 1 , Dae Hui Jung 2 , Chung Ryul Jung 2 , Kwon Seok Jeon 2 , Hong Woo Park 2, *
and Sang Un Park 1,3, *
1 Department of Smart Agriculture Systems, Chungnam National University, 99 Daehak-ro, Yuseong-gu,
Daejeon 34134, Korea; kns917555@cnu.ac.kr
2 Forest Medicinal Resources Research Center, National Institute of Forest Science, Yeongju 36040, Korea;
najdhda@korea.kr (D.H.J.); crjung92@korea.kr (C.R.J.); jks2029@korea.kr (K.S.J.)
3 Department of Crop Science, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 34134, Korea
* Correspondence: redrain39@korea.kr (H.W.P.); supark@cnu.ac.kr (S.U.P.);
Tel.: +82-54-630-5649 (H.W.P.); +82-42-821-5730 (S.U.P.); Fax: +82-42-822-2631 (S.U.P.)




Received: 4 November 2020; Accepted: 15 December 2020; Published: 17 December 2020


Abstract: Pimpinella brachycarpa Nakai, known as cham-na-mul in Korea, is a popular edible herb
and vegetable. Phenolic compounds are recognized as a vital group of plant secondary metabolites
that provide innumerable, valuable therapeutic properties. Elicitors are biofactors or chemicals from
diverse sources that can trigger morphological and physiological responses in the target organism.
This study examined the effect of methyl jasmonate (MeJA), salicylic acid (SA), and chitosan treatment
on the accretion of phenolic compounds in P. brachycarpa Nakai. This plant was harvested under
different concentration of elicitor treatment for time course. Eight phenolic compounds including
were detected in response to elicitor using HPLC. While the untreated controls showed the lowest
phenolic content, treatment with 0.3% chitosan, 0.1 mM SA, and 0.1 mM MeJA resulted in 1.43-,
1.39-, and 1.35-fold increase in the phenolic content, respectively. The highest content of most of
the individual phenolic compounds followed a similar trend according to treatment type, with
chitosan treatment showing the highest content, followed by SA and then MeJA treatments. Thus, we
demonstrate that the treatment with optimal concentrations of these elicitors for an optimal period of
time increases the production of phenolic compounds in P. brachycarpa Nakai.

Keywords: Pimpinella brachycarpa nakai; elicitors; phenolic compounds; methyl jasmonate; salicylic
acid; chitosan

1. Introduction
Pimpinella brachycarpa Nakai, which belongs to the Apiaceae family, is widely distributed in
Asia, Africa, and Europe [1]. This plant, known as cham-na-mul in Korea, is a popular edible herb
and vegetable [2]. In addition, this plant has been used as folk medicine in Korea for the treatment
of persistent cough, bronchial asthma, gastrointestinal disturbances, and insomnia [3]. In previous
phytochemical studies, essential oil components, terpenes, and flavonoids were isolated from P.
brachycarpa Nakai [3–5]. An EtOH extract of P. brachycarpa Nakai exhibited a positive effect on the lipid
metabolism of rats with high cholesterol and chronic hepatotoxicity in rat liver induced by ethanol [3,6].
Moreover, a MeOH extract of P. brachycarpa Nakai showed positive effects on antioxidant, antifungal,
antithrombotic, and antibacterial activities [7,8]. In addition, many previous studies have reported the
content of inorganic components, tannin, fiber, general components, and vitamins in the stem and
leaves of P. brachycarpa Nakai [7–10].
Phenolic compounds, which are commonly found in the plant kingdom, are secondary metabolites
arising biogenetically from either the shikimate or the phenylpropanoid pathway [11]. They play a

Horticulturae 2020, 6, 108; doi:10.3390/horticulturae6040108 www.mdpi.com/journal/horticulturae

Horticulturae 2020, 6, 108 2 of 16

key role as defense compounds against biotic and abiotic stresses, such as pathogen infection, insects,
low temperature, and high light, and perform many physiological functions such as the mediation of
plant-pollinator interactions [12,13]. Previous studies have reported that phenolic compounds affect
antioxidant [14], anticarcinogenic [15], anti-inflammatory [16], anticancer, [17] and cardioprotective [18]
activities. Phenolic compounds are classified into different groups according to their basic skeleton: C6
(simple phenol, benzoquinones), C6—C1 (phenolic acid), C6—C2 (acetophenone, phenylacetic acid),
C6—C3 (hydroxycinnamic acid, coumarin, phenylpropanes, chromones), C6—C4 (naphthoquinones),
C6—C1—C6 (xanthones), C6—C2—C6 (stilbenes, anthraquinones), and C6—C3—C6 (flavonoids,
isoflavonoids, neoflavonoids) [11]. The biosynthesis of phenylpropanoids and flavonoids as well as
the various catalyzing enzymes involved in the process are shown in Figure 1 [19].

Figure 1. Schematic representation of phenylpropanoid and flavonoid biosynthetic pathways. Red

bold color indicates the phenylpropanoids and flavonoids measured in this study. PAL, phenylalanine
ammonia lyase; TAL, tyrosine ammonia lyase; C4H, cinnamate 4-hydroxylase; C3H, coumarate
3-hydroxylase; COMT, caffeic acid 3-O-methyltransferase; 4CL, 4-coumaroyl CoA ligase; HCT,
hydroxycinnamoyl-coA shilimate/quinate hydroxycinnamoil transferase; C30 H, p-coumaroyl ester
30 -hydroxilase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F30 H,
flavonoid 30 -hydroxylase; FLS, flavonol synthase; DFR, dihydroflavonol-4 reductase; 3GT, flavonoid
3-O-glucosyltransferase [19].

Elicitors are biofactors or chemicals from diverse sources that can trigger morphological and
physiological responses in the target organism [20]. Elicitors are classified as either biotic or abiotic.
Biotic elicitors may include viruses, bacteria, fungi, or herbivores, and abiotic elicitors may include
inorganic compounds and metal ions. In addition, they can include chemicals that are generated for
defense against herbivore and pathogen attack [20]. The production of secondary metabolites in plants
can be activated by signal molecules, abiotic stresses, and elicitors. In particular, the accumulation of
secondary metabolites can be stimulated by elicitors such as jasmonic acid (JA), salicylic acid (SA),
chitosan, and ultraviolet-A or B (UV-A or B) radiation [21]. Previous studies have reported that JA from
the cells of Hypericum perforatum L. (St. John’s wort) cause an increase in the total phenolic content [22].
Furthermore, the phenolic content of chitosan-elicited Cocos nucifera (coconut) cells was shown to be
Horticulturae 2020, 6, 108 3 of 16

increased [23]. In tomato seedlings, UV-A treatment stimulated phenylalanine ammonia lyase (PAL),
which is an important enzyme in the phenylpropanoid biosynthesis and increases the accumulation of
anthocyanin in the hypocotyls and fruits [24].
Likewise, the production of hypericin and hyperforin in H. perforatum was shown to be increased
upon exposure to UV-B [25].
Studies on the phenolic content of P. brachycarpa Nakai are insufficient and the effect of elicitor
treatment has not been investigated. Therefore, this study examined the production of phenolic
compounds in P. brachycarpa Nakai upon treatment with the elicitor methyl jasmonate (MeJA), SA,
and chitosan.

2. Materials and Methods

2.1. Plant Materials and Elicitor Treatment

P. brachycarpa Nakai seedlings were planted at intervals of 15 to 20 cm in a shading-shielded
greenhouse and cultivated for 60 days. Then, 0.1, 0.3, or 0.5 mM MeJA (Sigma-Aldrich Co., St. Louis,
MO, USA) in aqueous solution containing 0.05% Tween 20 was sprayed on the leaves and stems of P.
brachycarpa Nakai. Treated samples were harvested on days 3, 6, and 9 post-treatment for comparison
of their phenylpropanoid content. SA (Sigma-Aldrich Co., St. Louis, MO, USA) and chitosan (Ja
Kwang Co. Ltd., Anseong, Korea) treatments were performed using the same method. Treatment
concentrations of SA were 0.1, 0.3, and 0.5 mM. The treatment concentrations of chitosan were 0.1, 0.3,
and 0.5%. For each elicitor treatment, three plants were harvested. As a control, 0.05% Tween 20 in
distilled water was used.

2.2. Extraction Procedure of Phenolic Compounds

For phenolic compound extraction, the harvested P. brachycarpa Nakai samples were deep-frozen
in liquid nitrogen and freeze-dried at −70 ◦ C for 72 h. Then, the samples were pulverized to fine
powder using a pestle until 100 mg of each sample was obtained. For phenylpropanoid extraction,
2 mL of 80% methanol was added to the samples, followed by vortexing for 2 min and sonication
at 37 ◦ C for 1 h. Then, the sonicated samples were centrifuged at 12,000 rpm for 12 min at 4 ◦ C and
the supernatants were filtered through 0.45 µm filter papers. The solution was then transferred to
vials for high-performance liquid chromatography (HPLC) analysis. The content of each phenolic
compound was calculated based on the calibration curve and the peak area of the standard compounds
for each sample.

2.3. HPLC Analysis of Phenolic Compounds

The HPLC conditions are presented in Table 1. For peak identification and quantification,
spike tests were performed, and the content of phenolic compounds was quantified using standard
calibration curves. The calibration curves of the standard compounds were as follows: catechin hydrate,
y = 28025.39 x − 2223.87 (R2 = 0.9997); chlorogenic acid, y = 52135.70 x − 57351.97 (R2 = 0.9991); caffeic
acid, y = 101427.93 x − 19999.04 (R2 = 0.9990); p-coumaric acid, y = 172376.91 x − 2288.50 (R2 = 0.9991);
ferulic acid, y = 115029.24 x − 16713.48 (R2 = 0.9995); benzoic acid, y = 16424.67 x + 1917.49 (R2 = 0.9996);
rutin, y = 25662.41 x − 3352.89 (R2 = 0.9990); quercetin, y = 40520.94 x + 12547.82 (R2 = 0.9994). All
standards used in this work were purchased from Sigma-Aldrich (USA).
Horticulturae 2020, 6, 108 4 of 16

Table 1. The HPLC conditions for phenylpropanoid analysis.

Futecs HPLC system NS-4000

HPLC
(Futecs Co. Ltd., Daejeon, Korea)
OptimaPak C18-51002546
Column (250 mm × 4.6 mm I.d., particle size 5 µm)
(RStech Co. Ltd., Daejeon, Korea)
Solvent A (0.15% Acetic acid water)
Mobile phase
Solvent B (Methanol)
Flow rate 1.0 mL/min
Column temperature 30 ◦ C
Injection Volume 10 µL
UV wavelength 280 nm
Time (min) Solvent A (%) Solvent B (%)
0–1 95 5
1–9 85 15

Gradient elution 9–24 80 20

24–54 70 30
54–65 55 45
65–75 44 56
75–80 40 60
80–90 20 80
90–105 95 5

2.4. Statistical Analysis

The content of the different phenylpropanoid compounds is presented as the mean ± standard
deviation of three replicates. The data were analyzed using IBM SPSS statistics 26 software (IBM Corp.,
Armonk, NY, USA). Statistical significance was evaluated with analysis of variance (ANOVA) using
Duncan’s multiple range test (DMRT) with a significance level of p ≤ 0.05.

3. Results

3.1. Effect of MeJA Treatment on The Phenolic Content (mg/g dry wt.) of P. brachycarpa Nakai Samples
HPLC analysis was performed to examine and compare the phenolic content of P. brachycarpa
Nakai sprouts taken at different harvest times (3, 6, and 9 days) after MeJA treatment. Eight phenolic
compounds (catechin hydrate, chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, benzoic
acid, rutin, and quercetin) were detected in different amounts at different sampling times (Figure 2).
MeJA treatment significantly influenced the production of all phenolic compounds except quercetin.
The total phenolic content ranged between 10.20 and 13.78 mg/g dry wt., depending on the harvesting
day. The highest total phenolic content (13.78 mg/g dry wt.) was detected 9 days after treatment with
0.1 mM MeJA, which was 1.35 times higher than the lowest total phenolic content detected in a control
sample. The second highest total phenolic content (13.71 mg/g dry wt.) was detected 6 days after
treatment with 0.1 mM MeJA. The content of catechin hydrate MeJA-treated samples ranged between
0.19 and 0.37 mg/g dry wt., depending on the harvesting time, with the highest content (0.37 mg/g dry
wt.) detected 3 days after treatment with 0.3 mM MeJA, followed by that found 6 days after treatment
with 0.1 mM MeJA (0.37 mg/g dry wt.). After 6 days from MeJA application, the catechin hydrate
content tended to decline. It is noteworthy that the catechin hydrate content in any of the MeJA-treated
samples was higher than that in any of the control samples. Among all detected phenolic compounds,
Horticulturae 2020, 6, 108 5 of 16

chlorogenic acid showed the highest content, ranging between 8.14. and 11.38 mg/g dry wt. The
highest chlorogenic acid content (11.38 mg/g dry wt.) was detected 9 days after exposure to 0.1 mM
MeJA, which was 1.4 times higher than the lowest chlorogenic acid content found in a control sample.
The second highest chlorogenic acid content was observed 6 days after exposure, to 0.3 mM MeJA
(11.29 mg/g dry wt.), followed by that found 3 days after exposure to 0.1 mM MeJA (10.92 mg/g dry
wt.). Remarkably, the chlorogenic acid content found in any of the MeJA-treated samples was higher
than that found in any of the control samples. The levels of caffeic acid, p-coumaric acid, and ferulic
acid were lower and their values were almost similar irrespective of the treatment used. The range of
caffeic acid content in MeJA-treated samples at different harvesting times was 0.09–0.16 mg/g dry wt.
The highest content of caffeic acid at any harvesting time was detected after treatment with 0.1 mM
MeJA, with the highest amount (0.16 mg/g dry wt.) found 9 days after treatment, which was 1.78 times
higher than the lowest content found in a control sample. The range of p-coumaric acid content in
MeJA-treated samples at different harvesting times was 0.04–0.09 mg/g dry wt. The highest content
of p-coumaric acid at any harvesting time was detected after treatment with 0.3 mM MeJA, with the
highest amount (0.09 mg/g dry wt.) found 3 days after treatment, which was 2.25 times higher than the
lowest content found in a control sample. The range of ferulic acid content in MeJA-treated samples at
different harvesting times was 0.04–0.05 mg/g dry wt., showing hardly any variation between samples.
The trend in caffeic acid, benzoic acid, rutin, and total phenolic content was the same, with the highest
content of these compounds at any harvesting time detected after treatment with 0.1 mM MeJA. The
range of benzoic acid content in MeJA-treated samples at different harvesting times was 0.49–0.71
mg/g dry wt. The highest benzoic acid content (0.71 mg/g dry wt.) was found 9 days after treatment
with 0.1 mM MeJA, which was 1.45 times higher than that found 9 days after exposure to 0.5 mM
MeJA, corresponding to the lowest benzoic acid content found. The rutin content in MeJA-treated
samples at different harvesting times ranged between 0.60. and 0.93 mg/g dry wt. The highest rutin
content (0.93 mg/g dry wt.) was found 3 days after treatment with 0.1 mM, which was 1.55 times higher
than that found 9 days after exposure to 0.5 mM MeJA, corresponding to the lowest rutin content
found. The quercetin content in MeJA-treated samples at different harvesting times ranged between
0.28 and 0.51 mg/g dry wt., and the highest quercetin content was observed in the control samples.
This indicated that MeJA treatment did not influence quercetin content.
Horticulturae 2020, 6, 108 6 of 16

Figure 2. The content of phenolic compounds (mg/g dry wt.) in P. brachycarpa Nakai samples after
treatment with different concentrations of methyl jasmonate (MeJA). The values marked above the
bars with different letters (a–i) indicate a statistically significant difference between treatments, as
determined using DMRT (p < 0.05).
Horticulturae 2020, 6, 108 7 of 16

3.2. Effect of SA Treatment on The Phenolic Content (mg/g dry wt.) of P. brachycarpa Nakai Samples
The levels of phenolic compounds in P. brachycarpa Nakai sprouts were investigated at different
harvesting times (3, 6, and 9 days) after treatment with different concentrations of SA using HPLC. Eight
different phenolic compounds were observed in different amounts at different sampling times (Figure 3).
Treatment with SA increased the content of most phenolic compounds, except for benzoic acid, rutin,
and quercetin. Here, the content of benzoic acid, rutin, and quercetin was lower in SA-treated samples
than in the control samples. The content of the different phenolic compounds followed different trends
depending on the SA concentration used and the harvesting time. SA treatments had a significant
influence on the total phenolic content, ranging between 10.20 and 4.14 mg/g dry wt. at different
harvesting times. The highest total phenolic content (14.14 mg/g dry wt.) was detected 9 days after
exposure to 0.1 mM SA, which was 1.39 times higher than the lowest content found in a control sample.
The second highest total phenolic content (13.47 mg/g dry wt.) was detected 6 days after treatment
with 0.1 mM SA, followed by that found 3 days after exposure to 0.3 mM SA (13.5 mg/g dry wt.). The
content of chlorogenic acid was much higher than that of any other phenolic compound, irrespective
of the treatment type. The chlorogenic acid content in SA-treated samples ranged between 8.14. and
12.34 mg/g dry wt. The highest content (12.34 mg/g dry wt.) was detected 9 days after treatment
with 0.1 mM SA, which was 1.52 times higher than the lowest content found in a control sample. The
second highest chlorogenic acid content was observed 6 days after treatment with 0.1 mM SA (11.61
mg/g dry wt.), followed by that found 3 days after exposure to 0.3 mM SA (11.60 mg/g dry wt.). The
chlorogenic acid content was much higher in any of the SA-treated samples than in any control sample.
The content of catechin hydrate in SA-treated samples ranged between 0.19. and 0.32 mg/g dry wt.
The highest content (0.32 mg/g dry wt.) was detected 3 days after treatment with 0.1 mM SA. The
catechin hydrate content of the samples treated with either 0.1 mM or 0.3 mM SA was very similar at
all sampling dates. The catechin hydrate content was higher in any of the SA-treated samples than that
in the control samples. In comparison, the content of p-coumaric acid, caffeic acid, and ferulic acid
was much lower irrespective of the treatment type. The caffeic acid content in SA-treated samples
ranged between 0.09 and 0.12 mg/g dry wt. The highest caffeic acid content was detected 3 days after
treatment with 0.1 mM SA (0.12 mg/g dry wt.). On the other hand, the content of p-coumaric acid in
SA-treated samples ranged between 0.04 and 0.06 mg/g dry wt., and was almost the same in all treated
samples, indicating that SA-treatment did not influence the content of p-coumaric acid. The content of
ferulic acid in SA-treated samples ranged between 0.05. and 0.08 mg/g dry wt., and the values were
almost the same among the different treated samples.
Horticulturae 2020, 6, 108 8 of 16

Figure 3. The content of phenolic compounds (mg/g dry wt.) in P. brachycarpa Nakai samples after
treatment with different concentrations of salicylic acid (SA). The values marked above the bars with
different letters (a–f) indicate a statistically significant difference between treatments, as determined
using DMRT (p < 0.05).
Horticulturae 2020, 6, 108 9 of 16

3.3. Effect of Chitosan Treatment on The Phenolic Content (mg/g dry wt.) of P. brachycarpa Nakai Samples
P. brachycarpa Nakai samples treated with different concentrations of chitosan were harvested on
days 3, 6, and 9 post-treatment to examine the content of different phenolic compounds using HPLC
analysis. Eight different phenolic compounds were detected at varying amounts on different sampling
dates (Figure 4). Chitosan treatment increased the content of all detected phenolic compounds except
for rutin. Here, the content of rutin was lower in the chitosan-treated samples than in the control
samples. The different phenolic compounds followed different trends depending on the chitosan
concentration and the harvesting times. The total phenolic content of chitosan-treated samples ranged
between 10.20 and 14.63 mg/g dry wt. The highest total phenolic content (14.63 mg/g dry wt.) was
detected 9 days after treatment with 0.3% chitosan, which was 1.43 times higher than the lowest total
phenolic content of a control sample. The second highest total phenolic content (14.47 mg/g dry wt.)
was detected 6 days after treatment with 0.5% chitosan. The content of chlorogenic acid was much
higher than that of any other phenolic compounds irrespective of the treatment type. The range of
chlorogenic acid content in chitosan-treated samples was 8.14–11.87 mg/g dry wt., with the highest
content (11.874 mg/g dry wt.) detected 9 days after treatment with 0.3% chitosan. The second highest
chlorogenic acid content (11.74 mg/g dry wt.) was found 6 days after treatment with 0.5% chitosan.
The catechin hydrate content in chitosan-treated samples ranged between 0.19 and 0.47 mg/g dry
wt. The highest catechin hydrate content (0.47 mg/g dry wt.) was detected 9 days after treatment
with 0.5% chitosan. The levels of p-coumaric acid, caffeic acid, and ferulic acid were much lower,
and the values were almost identical irrespective of the treatment type. The caffeic acid content in
chitosan-treated samples ranged between 0.09 and 0.13 mg/g dry wt. The highest caffeic acid content at
any harvesting time was detected after treatment with the highest concentration of chitosan (0.5%). The
range of p-coumaric acid content in chitosan-treated samples was 0.04–0.12 mg/g dry wt. The highest
p-coumaric acid content (0.12 mg/g dry wt.) was detected 9 days after treatment with 0.5% chitosan.
The ferulic acid content in chitosan-treated samples ranged between 0.05 and 0.11 mg/g dry wt. The
highest ferulic acid content (0.11 mg/g dry wt.) was detected 6 and 9 days after treatment with 0.5%
chitosan. The benzoic acid content in chitosan-treated samples was 0.51–0.79 mg/g dry wt. The highest
benzoic acid content (0.79 mg/g dry wt.) was found 3 days after treatment with 0.5% chitosan, which
was 1.55 times higher than the lowest content found in a control sample. The content of quercetin in
chitosan-treated samples ranged between 0.48 and 0.73 mg/g dry wt. The highest quercetin content
(0.73 mg/g dry wt.) was found 6 days after treatment with 0.5% chitosan, which was 1.52 times higher
than the lowest content detected in a control sample.
Horticulturae 2020, 6, 108 10 of 16

Figure 4. The content of phenolic compounds (mg/g dry wt.) in P. brachycarpa Nakai samples taken at
different harvesting days after treatment with different chitosan concentrations. The values marked
above the bars with different letters (a–f) indicate statistically significant differences between treatments,
as determined using DMRT (p < 0.05).
Horticulturae 2020, 6, 108 11 of 16

3.4. Comparison of The Effect of MeJA, SA, and Chitosan Treatments on The Phenolic Content (mg/g dry wt.) of
P. brachycarpa Nakai Samples
The highest total phenolic content (14.63 mg/g dry wt.) was detected 9 days after treatment with
0.3% chitosan, followed by that found 9 days after treatment with 0.1 mM SA (14.14 mg/g dry wt.)
and then by that found 9 days after treatment with 0.1 mM MeJA (13.78 mg/g dry wt.) (Table 2). The
phenolic content 9 days after treatment with 0.3% chitosan, 0.1 mM SA, or 0.1 mM MeJA was 1.43,
1.39, and 1.35 times higher than the lowest content found in a control sample (10.20 mg/g dry wt.),
respectively. The highest chlorogenic acid content (12.34 mg/g dry wt.) was detected 9 days after
treatment with 0.1 mM SA, followed by that found 9 days after treatment with 0.3% chitosan (11.87
mg/g dry wt.) and then by that found 9 days after treatment with 0.1 mM MeJA (11.38 mg/g dry wt.).

Table 2. The content of total phenolic compounds (mg/g dry wt.) in P. brachycarpa Nakai under different
elicitor treatment.

Treatment Total Treatment Total Treatment Total

0 Days Control 10.20 ± 0.20 g 1 Control 10.20 ± 0.20 f Control 10.20 ± 0.20 i
Control 10.41 ± 0.30 fg Control 10.41 ± 0.30 ef Control 10.41 ± 0.30 hi
MeJA 0.1 13.26 ± 0.18 bc SA 0.1 11.47 ± 0.01 c Chitosan 0.1% 11.58 ± 0.34 de
3 Days
MeJA 0.3 13.06 ± 0.28 c SA 0.3 13.50 ± 0.15 b Chitosan 0.3% 11.47 ± 0.33 ef
MeJA 0.5 10.63 ± 0.12 fg SA 0.5 11.14 ± 0.57 cd Chitosan 0.5% 13.48 ± 0.30 b
Control 10.86 ± 0.42 e Control 10.86 ± 0.42 cdef Control 10.86 ± 0.42 g
MeJA 0.1 13.71 ± 0.14 ab SA 0.1 13.47 ± 0.07 b Chitosan 0.1% 12.07 ± 0.23 cd
6 Days
MeJA 0.3 13.62 ± 0.10 ab SA 0.3 11.42 ± 0.33 c Chitosan 0.3% 13.51 ± 0.60 b
MeJA 0.5 11.96 ± 0.13 d SA 0.5 10.92 ± 0.13 cde Chitosan 0.5% 14.47 ± 0.23 a
Control 10.59 ± 0.30 fg Control 10.59 ± 0.30 def Control 10.59 ± 0.30 hi
MeJA 0.1 13.78 ± 0.27 a SA 0.1 14.14 ± 0.44 a Chitosan 0.1% 12.48 ± 0.34 c
9 Days
MeJA 0.3 10.63 ± 0.02 fg SA 0.3 10.71 ± 0.86 def Chitosan 0.3% 14.63 ± 0.33 a
MeJA 0.5 10.50 ± 0.47 fg SA 0.5 10.68 ± 0.16 def Chitosan 0.5% 10.92 ± 0.31 fg
1 Different letters (a–i) with in a column indicate significant difference between areas for that parameter using DMRT

(Duncan’s Multiple Range Test) at 5% level (p < 0.05).

The chlorogenic acid content 9 days after treatment with 0.1 mM SA, 0.3% chitosan, or 0.1 mM
MeJA was 1.52, 1.46, and 1.40 times higher than the lowest content found in a control sample (8.14
mg/g dry wt.), respectively. The highest catechin hydrate content (0.47 mg/g dry wt.) was detected 9
days after treatment with 0.5% chitosan, followed by that found 3 days after 0.3 mM MeJA treatment
(0.37 mg/g dry wt.), and then by that found 3 days after 0.1 mM SA treatment (0.32 mg/g dry wt.). The
catechin hydrate content in these three samples was 2.47, 1.95, and 1.68 times higher than the lowest
catechin hydrate content found in a control sample (0.19 mg/g dry wt.), respectively. The highest caffeic
acid content (0.16 mg/g dry wt.) was detected 9 days after treatment with 0.1 mM MeJA, followed
by that found 3 days after treatment with 0.5% chitosan (0.13 mg/g dry wt.), and then that found 3
days after treatment with 0.1 mM SA (0.12 mg/g dry wt.). These treatments resulted in 1.78, 1.44, and
1.33 times higher caffeic acid content than the lowest content found in a control sample (0.09 mg/g
dry wt.), respectively. The highest p-coumaric acid content (0.12 mg/g dry wt.) was detected 9 days
after treatment with 0.5% chitosan, followed by those found 3 days after treatment with 0.3 mM MeJA
(0.0.9 mg/g dry wt.) and 0.1 mM SA (0.06 mg/g dry wt.). These three treatments resulted in 3.0, 2.25,
and 1.5 times higher p-coumaric acid content than the lowest content found in a control sample (0.09
mg/g dry wt.), respectively. The highest ferulic acid content (0.11 mg/g dry wt.) was detected 9 days
after treatment with 0.5% chitosan, followed by that found 3 days after treatment with 0.5 mM SA (0.0.9
mg/g dry wt.). These treatments resulted in 2.2 and 1.6 times higher ferulic acid content, respectively,
than the lowest content found in a control sample (0.05 mg/g dry wt.). The highest benzoic acid content
(0.79 mg/g dry wt.) was detected 9 days after treatment with 0.3% chitosan, followed by that found 9
days after treatment with 0.1 mM MeJA (0.71 mg/g dry wt.). These treatments resulted in 1.55 and
1.39 times higher benzoic acid content, respectively, than the lowest content found in a control sample
(0.51 mg/g dry wt.). A slightly higher rutin content was only observed after 0.1 mM MeJA treatment.
SA and chitosan treatments did not influence the rutin content. Moreover, the quercetin content was
Horticulturae 2020, 6, 108 12 of 16

increased slightly after treatment with 0.5% chitosan, whereas MeJA and SA treatments did not show
any effect on quercetin content.

4. Discussion
Elicitation is recognized as one of the most important techniques for the stimulation of secondary
metabolites. The accretion of any metabolites from either part of parent or transformed plants is largely
reliant on the source; however, it may be influenced by treatments as well as environmental factors.
Elicitors trigger an increase in the content of phenolic compounds, pigments, flavones, phytoalexins,
and other defense-related compounds when in contact with the cells of higher plants [26–32].
The levels of phenolic compounds in P. brachycarpa Nakai were investigated in response to
different elicitor (MeJA, SA, and chitosan) treatments at different harvesting times (3, 6, and 9 days)
post-treatment. In this study, eight phenolic compounds (catechin hydrate, chlorogenic acid, caffeic
acid, p-coumaric acid, ferulic acid, benzoic acid, rutin, and quercetin) were detected in different
amounts at different sampling times. All the elicitors significantly increased the content of most of the
phenolic compounds compared to the untreated controls. These results are consistent with previous
studies reporting the identification of these types of compounds in common buckwheat [33,34], tartary
buckwheat [35], hairy root cultures of tartary buckwheat [36], Agastache rugosa [37], sorghum [38],
Scutellaria baicalensis [39], and Kohlrabi [40].
Our time course analysis revealed that MeJA, SA, and chitosan treatment progressively enhanced
the production of phenolic compounds in the studied species. This might be due to an increase in the
expression levels of phenylpropanoid-related genes following treatment with MeJA, SA, or chitosan,
since previous studies proclaimed that different types of elicitors increase the expression levels of
phenlypropanoid-related genes and enhance the accumulation of phenolic compounds. Some of these
findings are relevant to our study and are discussed here. MeJA treatment was shown to increase
the expression levels of phenlypropanoid-related genes and enhance the accumulation of phenolic
compounds in radish sprouts [41] and A. rugosa Kuntze [37]. Light treatment with 100 µM methyl
jasmonate of radish seedlings increased the anthocyanin content in radish [42]. The rosmarinic acid
content in A. rugosa Kuntze was 2.1-, 4.7-, and 3.9-fold higher after exposure to 10, 50, and 100 mM
MeJA, respectively, than that in untreated controls [37]. The content of phenolic acids was increased
in Exacum affine shoot cultures by adding the precursor (L-phenylalanine), the elicitor (MeJA), and
by increasing the sucrose concentration [43]. At least a 6–8-fold increase in PAL and CHI activities
was observed in JA-elicited cells, confirming a strong activation of the phenylpropanoid pathway.
JA elicitation increased the production of phenylpropanoids and naphtodianthrones in a Hypericum
perforatum cell suspension without differentiation of dark glands under a 16 h photoperiod [22]. Most
of the above-mentioned findings coincide with our study, in which MeJA treatment significantly
increased the content of all detected phenolic compounds, except quercetin.
The rutin content has been shown to increase following treatment with SA in tartary buckwheat [44].
After 72 h from the application of SA, the levels of 4-hydroxybenzoic acid, 4-coumaric acid, and
ferulic acid were shown to be 3.5-, 2.9-, and 3.8-fold higher than those in the control samples,
respectively, in eggplant roots [45]. At 50 µM, SA enhanced the production of hypericin (7.98-fold) and
pseudohypericin (13.58-fold) in H. hirsutum, and at 200 µM, enhanced the production of hypericin
(2.2-fold) and pseudohypericin (3.94-fold) in H. maculatum [46]. Thus, our results are consistent with
those from others, as SA treatment increased the content of most phenolic compounds, except benzoic
acid, rutin, and quercetin.
Eight different phenolic compounds were detected in different amounts at different sampling times
in response to different concentrations of chitosan in P. brachycarpa Nakai. Treatment with chitosan
increased the content of all detected phenolic compounds except rutin. Our results on phenolic content
upon chitosan treatment are supported by previous studies. In soybean leaves, the total phenolic
content increased following chitosan and chitin oligomer treatments [47]. Treatment with 0.1% chitosan
and 150 µM JA resulted in the highest levels of phenolic compounds compared to those of the control,
Horticulturae 2020, 6, 108 13 of 16

whereas SA treatment did not affect the production of phenolic compounds in sprouted buckwheat.
A sample of buckwheat taken 72 h after 0.1% chitosan treatment showed higher levels of phenolic
compounds than those in all control samples [48]. The treatment of Cocos nucifera (coconut) endosperm
cell suspension cultures with chitosan led to the rapid formation of phenylpropanoid derivatives [23].
Moreover, 48 h post-elicitation with chitosan, the content of 4-hydroxybenzoic acid, 4-coumaric acid,
and ferulic acid in the cell wall of eggplants was 2.6-, 2.8-, and 3.0-fold higher than that in the control
sample, respectively [45].
Qi et al. [49] revealed that exogenous MeJA treatment up-regulated many photosynthesis and
defense-related proteins, and down-regulated a number of ribosomal proteins. They also discovered
that MeJA treatment can maintain a better reactive oxygen species (ROS) scavenging capability,
by decreasing Hydrogen peroxide, peroxidase. And they suggested that up-regulated proteins
stress-related, such as DHAR1, ATCOR47, PDE334, ATHM1 can affect antioxidant activity. The
previous study suggested that salicylic acid was synthesized in cells can move freely in and out of the
cells, tissues, and organs, and this movement is finely regulated by ROS and Ca2+ [50]. The treatment
with a moderate concentration of SA increased the activity of the enzyme, finally increased growth and
bio-productivity. However, the treatment with higher concentrations of SA decreased the activity of
the enzyme by reducing the photosynthesis-related enzyme activity such as ribulose-1,5-biphosphate
carboxylase/oxygenase (RuBPCO) [50]. In a previous study revealed that chitosan-treated plants
up-regulated PAL, peroxidase, glucanase, polyphenol oxidase, and defense-responsive enzymes [51].
Farag et al. [52] investigated elicitor-specific biosynthesis of isoflavonoids in response to yeast elicitor
(YE) and MeJA in Medicago truncatula Cell Cultures. The response to YE increased isoflavonoid
biosynthesis, including the shikimic acid pathway and flavonoid biosynthesis. MeJA treatment
resulted in contrast to that of YE, increased in afromosin and medicarpin and, but many pathway
precursors were not changed. Based on the abundant precursor pathways, an increased medicarpine
of isoflavonoid for YE is achieved from de novo biosynthesis; in contrast, MeJA-induced medicarpine
accumulation occurs through remigration of the vacuole pool of formononetin glucoside and hydrolysis.
Kubes et al. [53] investigated the elicitation effect of two vanadium compounds, which could make a
positive effect on the isoflavones production in Trifolium pratense. The content of isoflavones in T. pratense
suspension culture was increased after 24 h by vanadium compounds, NH4 VO3 . However, increased
isoflavones since been suppressed by transport-mechanisms inhibitors. This suppress process was
mostly facilitated by vesicle transport and ATP-binding cassette protein. These findings suggest that
elicitors can have a positive effect on plants through various mechanisms and physiological activities.
A previous study revealed that studies on the biological activities of rare phenylpropanoids of the
genus Pimpinella, such as anoltiglate, isoeugenolisobutyrate, epoxy-pseudoisoeugenoltiglate [54].
Delazar et al. [55] investigated a chemical profile of the essential oils of Pimpinella
aurea that was significantly different from the previously published reports, β-Bisabolene
(39.559%) and geranyl 2-methylbutanoate (25.334%) were the two most abundant components.
Additionally, both phenylpropanoid derivatives, erythro-10 -(4-methoxyphenyl)-propan-10 ,20 -diol,
and erythro-10 -[4-(sec-butyl)-phenyl]-propan-10 ,20 -diol, were identified by using vacuum liquid
chromatography (VLC) and preparative thin-layer chromatography (PTLC). And these were exhibited
low levels of antioxidant activity in the DPPH assay. In this study, we investigate the accumulation of
phenolic compounds in P. brachycarpa Nakai upon treatment with the elicitor MeJA, SA, and chitosan.
The samples were taken 9 days after treatment with 0.3% chitosan, 0.1 mM SA, and 0.1 mM MeJA
showed 1.43-, 1.39-, and 1.35-fold increases in total phenolic content, respectively, compared to the
lowest total phenolic content found in a control sample. Therefore, chitosan, SA, and MeJA treatment
augment the production of phenolic compounds in P. brachycarpa Nakai.

5. Conclusions
This study shows that chitosan, SA, and MeJA enhance the production of different phenolic
compounds. Eight different phenolic compounds, including chlorogenic acid, catechin hydrate, caffeic
Horticulturae 2020, 6, 108 14 of 16

acid, p-coumaric acid, ferulic acid, benzoic acid, rutin, and quercetin were detected in P. brachycarpa
Nakai. All the elicitors tested increased the content of most of the detected phenolic compounds.
Particularly, 1.43-, 1.39-, and 1.35-fold increases in total phenolic content were achieved 9 days after
treatment with 0.3% chitosan, 0.1 mM SA, and 0.1 mM MeJA, respectively, compared to the lowest
total phenolic content detected in a control sample. These results might help to create robust strategies
for the improvement of phenolic production by P. brachycarpa Nakai, which will positively impact its
use as an edible herb and folk medicine in Korea and elsewhere.

Author Contributions: H.W.P. and S.U.P. designed the experiments and analyzed the data. N.S.K., D.H.J., C.R.J.
and K.S.J. performed the experiments and analyzed the data. N.S.K., H.W.P. and S.U.P. wrote the manuscript. All
authors have read and agreed to the published version of the manuscript.
Funding: This research was supported by the National Institute of Forest Science, Korea Forest Service, Korea
(Project No. FG0502-2017-01).
Conflicts of Interest: The authors declare no conflict of interest.

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