Journal of Natural Medicines

https://doi.org/10.1007/s11418-019-01367-8

ORIGINAL PAPER

Rosmarinic acid in Perilla frutescens and perilla herb analyzed by HPLC

Yuya Deguchi1 · Michiho Ito1

Received: 1 August 2019 / Accepted: 25 October 2019

© The Japanese Society of Pharmacognosy 2019

Abstract
High-quality perilla leaves are defined as those having purple upper and lower surfaces and a pleasant smell. The Japanese
Pharmacopoeia specifies the content of essential oils in perilla leaves but not the content of rosmarinic acid. Rosmarinic acid
is a common component of Labiatae plants such as shiso (Perilla frutescens Britton var. crispa W. Deane). Rosmarinic acid
has been reported to exhibit anti-inflammatory and anti-oxidant activity but the factors affecting the content of rosmarinic
acid in plants remain unknown. This study describes a simple and reproducible method for quantifying rosmarinic acid. We
elucidated the main causes for the different rosmarinic acid contents of plants by examining various samples of perilla using
the proposed method. Significant differences in rosmarinic acid content between varieties and cultivators were observed.
The rosmarinic acid content was higher in green perilla compared with red perilla, in wild species compared with cultivated
species, and in plants cultivated in outdoor nurseries compared with in indoor nurseries. The proposed quantitative method
was used to examine the rosmarinic acid content in a Kampo formula, Hangekobokuto, and was found to be higher in decoc-
tions prepared using the Kouge method compared with the typical preparation method. We examined the chlorophyll and
caffeic acid contents of several samples and their relationship with the rosmarinic acid content.

Keywords Perilla frutescens · Rosmarinic acid · HPLC analysis · Hangekobokuto

Introduction Rosmarinic acid is an ester of caffeic acid and 3, 4-dihy-

Perilla frutescens is an annual plant used as a fresh herb
in cooking. Purple perilla leaves are used pharmaceutically
to ease stomach disorders and induce sweating [1] and are
blended in several Japanese traditional medicinal formula-
tions such as Kososan and Hangekobokuto.
Rosmarinic acid (Fig. 1) is a common component of
Labiatae and Boraginaceae plants with reported anti-inflam-
matory and anti-oxidant activities [2, 3] and possible phar-
macological effects on hay fever and allergic diseases [4–6].
In addition, rosmarinic acid inhibits amyloid-β aggregation
and is thus expected to be effective in preventing Alzhei-
mer’s disease [7]. Furthermore, it inhibits the proliferation
of mesangial cells and suppresses mesangioproliferative
glomerulonephritis [8, 9].
droxyphenyllactic acid. The chemical synthesis of ros-
marinic acid using piperonal as a raw material involves many
reactions, is lengthy, and generates numerous byproducts
[10]. Gene cloning of enzymes from Coleus blumei involved
in rosmarinic acid biosynthesis has been reported and the
biosynthetic pathway of rosmarinic acid in plants is pro-
posed to be derived from the pathways for l-phenylalanine
and l-tyrosine [11, 12]. However, the environmental or
genetic factors affecting the content of rosmarinic acid in
plants have not been elucidated. Here, we describe a simple
and reproducible method for quantifying rosmarinic acid and
clarify the main causes for variation in the content of ros-
marinic acid in plants. In addition, we analyzed the content
of caffeic acid, a degradation product of rosmarinic acid, in
several samples.

* Michiho Ito
michihoi@pharm.kyoto‑u.ac.jp
1
Graduate School of Pharmaceutical Sciences, Kyoto
University, 46‑29 Yoshidashimoadachi‑cho, Sakyo‑ku,
Kyoto 606‑8501, Japan

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Fig. 1  Structural formula of rosmarinic acid
Materials and methods
Summary of the experiment
We determined the rosmarinic acid content of several
perilla groups and compared them according to the fol-
lowing characteristics.
(A) Investigation of the influence of growth environment,
harvest time, and perilla variety on rosmarinic acid
content, and correlation between chlorophyll a content
and rosmarinic acid content.
Numerous factors may affect the rosmarinic acid con-
tent of perilla leaves, including the growing environment,
harvest time, and the perilla variety. We examined 34
green perilla and 6 red perilla types. In addition, the chlo-
rophyll content of fresh leaves was examined and correla-
tions with rosmarinic acid content were investigated.
(B) Comparison of the rosmarinic acid content of different
perilla species.
Four Perilla species grow in Japan: the cultivated spe-
cies, P. frutescens Britton var. crispa (“Shiso”), and three
wild species, P. citriodora Nakai (“Lemon-egoma”), P.
hirtella Nakai (“Torano-o-jiso”), and P. setoyensis G.
Honda (“Seto-egoma”). P. frutescens Britton var. frute-
scens (“Egoma”) is a variety of P. frutescens Britton var.
crispa [13, 14]. We determined whether there is a differ-
ence in rosmarinic acid content per 1 g of dried leaves of
each of these Perilla species.
(C) Correlation between the extent of leaf growth and ros-
marinic acid content.
We examined the correlation between the extent of leaf
growth and the rosmarinic acid content of dried leaves. We
measured the degree of leaf growth by dividing the leaves
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Journal of Natural Medicines
depending on the length of the main vein in each leaf and
compared the rosmarinic acid content.
(D) Comparison of rosmarinic acid content in plants grown
in indoor nurseries (in a mechanical thermo-hygrostat
chamber for plants) and in outdoor nurseries.
We examined the difference in rosmarinic acid content
resulting from differences in the cultivation environment due
to indoor (mechanical thermo-hygrostat chamber for plants)
and outdoor cultivation.
(E) Changes in rosmarinic acid content due to differences
in the preparation of Hangekobokuto.
The 17th Japanese Pharmacopoeia states “Hangekobo-
kuto Extract contains not less than 4 mg (for preparation
prescribed 2 g of Perilla Herb) or not less than 6 mg (for
preparation prescribed 3 g of Perilla Herb) of rosmarinic
acid per extract prepared with the amount specified in the
Method of preparation.” in the section on Hangekobokuto
Extract. Rosmarinic acid is used in the confirmation test
and determination method. Here, we determined whether the
rosmarinic acid content in Hangekobokuto could be quanti-
fied by the method proposed below. Perilla herb is a crude
drug containing a large amount of essential oil and thus the
Kouge method, involving the addition of only perilla herb
immediately before the end of decoction, may be used in the
preparation of Hangekobokuto. The extract was prepared
using two methods (the typical method in which perilla herb
is added at the start of heating, and the Kouge method), then
we compared the weight of the extract, and the content of
rosmarinic acid and caffeic acid (a degradation product of
rosmarinic acid) in the extract.
Plant materials
1. Thirty-four green perilla and 6 red perilla plants were
grown in various environments, harvested in various
seasons. The plants were of various varieties and were
cultivated by specialist farmers (Table 1). Upper case
letters in the sample numbers indicate the cultivation
field, numbers indicate the harvest month, and lower
case letters indicate the variety.
2. P. frutescens Britton var. crispa (“Shiso”, Strain No.
25, 32), P. frutescens Britton var. frutescens (“Egoma”,
Strain No. 12, 5316, 5681), P. citriodora (“Lemon-
egoma”, Strain No. 5321, 5717), P. hirtella (“Torano-o-
jiso”, Strain No. 5042) and P. setoyensis (“Seto-egoma”,
Strain No. 5031) were grown in mechanical thermo-
hygrostat chambers for plants (Table 2) under the fol-
lowing conditions: temperature: 25 °C, humidity: 60%,
illumination hours: 5 AM to 10 PM.
Journal of Natural Medicines

3. Three plants each of P. frutescens Britton var. crispa harvested 132 days after sowing (Table 3). The growth
(Strain No. 5254) and P. frutescens Britton var. frutes- conditions were: temperature: 25 °C, humidity: 65%,
cens (Strain No. 12) were grown by soil cultivation in illumination hours: 5 AM to 10 PM. After collection,
a mechanical thermo-hygrostat chamber for plants and the harvested leaves of each plants were divided into 4

Table 1  Thirty-four green Sample numbers Harvest time Variety Leaf color Sample numbers Harvest time Variety Leaf color
perilla and 6 red perilla plants
were cultivated by specialist A-6-a 2017.6 a Red N-4-b 2017.4 b Green
farmers between April 2017 and
A-7-a 2017.7 a Red N-7-b 2017.7 b Green
January 2018
A-8-a 2017.8 a Red N-8-b 2017.8 b Green
B-6-a 2017.6 a Red O-4-b 2017.4 b Green
B-7-a 2017.7 a Red Q-10-b 2017.10 b Green
B-8-a 2017.8 a Red R-4-b 2017.4 b Green
C-4-b 2017.4 b Green R-6-b 2017.6 b Green
D-4-b 2017.4 b Green R-7-b 2017.7 b Green
D-6-b 2017.6 b Green R-8-b 2017.8 b Green
D-7-b 2017.7 b Green C-10-c 2017.10 c Green
E-6-b 2017.6 b Green D-13-c 2018.1 c Green
E-7-b 2017.7 b Green I-13-c 2018.1 c Green
E-8-b 2017.8 b Green J-4-c 2017.4 c Green
F-10-b 2017.10 b Green K-8-c 2017.8 c Green
G-4-b 2017.4 b Green K-10-c 2017.10 c Green
H-4-b 2017.4 b Green K-12-c 2017.12 c Green
H-6-b 2017.6 b Green M-4-c 2017.4 c Green
H-8-b 2017.8 b Green M-13-c 2018.1 c Green
H-13-b 2018.1 b Green N-6-c 2017.6 c Green
L-10-b 2017.10 b Green P-13-c 2018.1 c Green

Table 2  Laboratory-grown Strain No. Japanese name Species name Leaf color Essen-
perilla plants were used to tial oil
investigate differences in type
rosmarinic acid content between
strains 12 Egoma P. frutescens Britton var. frutescens Green PP
25 Shiso P. frutescens Britton var. crispa Green PP
32 Shiso P. frutescens Britton var. crispa Red PA
5031 Seto-egoma P. setoyensis G. Honda Green SF
5042 Torano-o-jiso P. hirtella Nakai Green PP
5316 Egoma P. frutescens Britton var. frutescens Green PP
5321 Lemon-egoma P. citriodora Nakai Green PP
5681 Egoma P. frutescens Britton var. frutescens Green SF
5717 Lemon-egoma P. citriodora Nakai Green C

Essential oil types: PA perillaldehyde, C citral, SF shisofuran, PP phenylpropanoid

Table 3  Laboratory-grown perilla plants were used to investigate the correlation between the degree of leaf growth and rosmarinic acid content,
and rosmarinic acid content in indoor- vs. outdoor-grown plants
Strain no. Japanese name Species name Leaf color Essen-
tial oil
type

12 Egoma P. frutescens Britton var. frutescens Green PP

5254 Shiso P. frutescens Britton var. crispa Red C

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 Journal of Natural Medicines

groups based on the main vein length of the leaf: < 3 cm,
3–6 cm, 6–9 cm, and 9–12 cm. The leaves were used as
experimental materials.
4. Three plants each of P. frutescens Britton var. crispa
(Strain No. 5254) and P. frutescens Britton var. frutes-
cens (Strain No. 12) were grown by soil cultivation in
outdoor nurseries and were harvested 132 days after
sowing. Strain No. 5254 leaves with the main vein length
of 3–6 cm (3 samples) and Strain No. 12 leaves with
main vein lengths of 3–6 cm (3 samples) and 6–9 cm (3
samples) were used as experimental materials.
5. Marketed products such as pinellia tuber, poria sclero-
tium, magnolia bark, and ginger were used as experi-
mental materials and purchased from Nakajima Herbal
Medicine Co. Thirteen Japanese-marketed perilla herb
products (I to XIII) were used and are described in
Table 4.
Reagents
Caffeic acid and rosmarinic acid were purchased from Wako
Pure Chemical Industries, Ltd. (Osaka, Japan). HPLC-grade
distilled water and acetonitrile, which were used in HPLC
analysis, and special-grade methanol were purchased from
Nacalai Tesque Inc. (Kyoto, Japan).
Experimental methods
Measurement of the caffeic acid and rosmarinic acid
content of 1 g of dried leaves
The perilla leaves were fully dried at room temperature,
frozen in liquid nitrogen and crushed, then the ground
materials were further dried using a desiccator. Dried leaf
powder (100 mg) was accurately weighed and extracted
by stirring with 5 mL or 10 mL of methanol for 24 h.
After separating the extract by suction filtration or cen-
trifugation, the extraction residue was subjected two more
times to the same extraction operation and the extracts
were combined and concentrated to dryness to completely
remove the methanol. The extract was dissolved in 1%
aqueous acetic anhydride solution, the insoluble matter
was removed by filtration, and the volume was increased
to 10 mL. An aliquot was passed through a syringe filter
(Cosmonice Filter W, 0.45 μm; Nacalai Tesque Inc.) and
used as an HPLC injection sample. Two sets of conditions
were used for the HPLC analysis. Isocratic mode was used
for experimental material 1 and gradient mode was used
for experimental materials 2, 3, 4, and 5 to determine the
caffeic acid content and to more accurately determine the
rosmarinic acid content.
The equipments were L-2130 pump, L-2300 column
oven, 2400 UV detector, D-2500 Chromato integrator
(Hitachi Ltd., Tokyo, Japan) and column was Cosmosil
Cholester packed column 4.6 mm ID × 150 mm (Nacalai
Tesque Inc.). Column temperature was 40 °C, detec-
tion wavelength was 330 nm and injection volume was
5 μL. Two eluents were used for mobile phase. Eluent A
was 20 mM, pH 2.5 potassium phosphate buffer, and elu-
ent B was acetonitrile. In isocratic mode eluent A/eluent
B was 82/18, and in gradient mode the program of eluent
B: 11% (0 min) → 11% (10 min) → 25% (50 min) → 45%
(51 min) → 45% (56 min) → 11% (57 min) → 11% (60 min).
Flow rate was 1.0 mL/min.
Solutions of caffeic acid and rosmarinic acid standards
were prepared in 1% acetic anhydride aqueous solution.
Calibration curves were prepared using dilutions of these
standard solutions and used for quantification.

Table 4  Japanese perilla herb Sample number Date of purchase Cultivation place Perillaldehyde
marketed products content (%)

I October 2017 Hebei Province (China) 0.48

II July 2016 Hebei Province (China) 0.39
III January 2016 Hebei Province (China) 0.53
IV January 2018 Guangdong Province (China) 0.01
V September 2015 Guangdong Province (China) 0.01
VI 2017 Kagawa Prefecture (Japan) 0.01
VII February 2018 Hebei Province (China) 0.64
VIII February 2018 Hebei Province (China) 0.66
IX February 2018 Hebei Province (China) 0.34
X 2018 China 0.44
XI January 2018 Hebei Province (China) 0.34
XII September 2012 Hebei Province (China) 0.22
XIII July 2016 Hebei Province (China) 0.38

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Journal of Natural Medicines
Simple quantification of chlorophyll a in fresh leaves
One fresh leaf was weighed, and 1.5 times this weight of
solid ­Na2HPO4 was added, then the mixture was finely pul-
verized together using liquid nitrogen. Exactly 500 mg of
the pulverized material was weighed, 8 mL of diethyl ether
was added, and extraction with stirring was performed for
24 h. After filtration, the volume was increased to 10 mL,
and the absorbance at 641.9 nm and 660.3 nm was meas-
ured with an absorbance meter. Chlorophyll a was quanti-
fied using the following equation based on references [15,
16].
Chlorophyll a(𝜇g/mL) = 10.02 × A−0.54 × B
(A absorbance at 660.3 nm, B absorbance at 641.9 nm).
Preparation of Hangekobokuto
An automatic decoction device (HMJ3-1000W; Hario Co.,
Ltd., Tokyo, Japan) was used.
(1) Typical preparation method (adding perilla herb at the
start of heating)
Pinellia tuber (6.00 g), poria sclerotium (5.00 g), magno-
lia bark (3.00 g), perilla herb (2.00 g) and ginger (1.00 g)
were accurately weighed, placed in an automatic decoction
device with 600 mL of tap water, and heated in automatic
mode for 1 h. Following suction filtration and washing,
the mixture was quickly cooled and freeze-dried to obtain
Hangekobokuto (1).
Fig. 2  The content of ros-
marinic acid per 1 g of dried
leaves in 34 green and 6 red
perilla samples. The experi-
ments were performed in trip-
licate for each sample and the
mean value and standard error
were calculated
(2) Kouge method (adding perilla herb after heating)
After heating the components except perilla herb in auto-
matic mode for 1 h as described in (1), perilla herb was
immediately added to the automatic decoction device, then
the device was covered and allowed to stand for 5 min with-
out heating to produce Hangekobokuto (2).
Freeze-dried Hangekobokuto (1) and (2) powders were
accurately weighed (10 mg) and dissolved in a 1% acetic
anhydride aqueous solution. Insoluble materials were fil-
tered off and the volume was increased to 10 mL. An aliquot
was passed through a syringe filter (Nacalai Tesque Inc.) and
used as an HPLC injection sample.
Measurement of perillaldehyde content
Perillaldehyde content in Japanese perilla herb marketed
products were measured by following the Japanese Phar-
macopoeia 17th edition [1].
Statistical analysis
Statistical analysis was performed by using R software. Sig-
nificant differences were calculated by t test. P value < 0.05
was considered as statistically significant.
Results and discussion
The content of rosmarinic acid in the perilla samples varied
and results are shown according to the conditions described
in “Materials and methods”.
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(A) Investigation of the influence of growth environment,
harvest time, and perilla variety on rosmarinic acid
content, and correlation between chlorophyll a content
and rosmarinic acid content.
The analysis was performed in triplicate for each of the
34 green and 6 red perilla samples shown in Table 1. The
mean value and standard deviation were calculated. The ros-
marinic acid content per 1 g of dried leaves varied between
0.177 mg (H-13-B) and 19.1 mg (H-8-B), and they differed
significantly (Fig. 2). No correlation was found with the
Fig. 3  Variation in rosmarinic acid content by harvest month for each field
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Journal of Natural Medicines
harvest month (Fig. 3) or with the content of chlorophyll a
per 1 g of fresh leaves (correlation coefficient r was 0.0818,
Fig. 4). We compared the rosmarinic acid content of the
three varieties of perilla plants cultivated in Aichi Prefecture
(Fig. 5). Rosmarinic acid tended to be more abundant in
green perilla than in red perilla leaves, although there was no
significant difference, consistent with the results reported by
Lu et al. [17]. Like anthocyanins [18], the biosynthetic path-
way for rosmarinic acid is believed to involve 4-coumaroyl-
CoA derived from phenylalanine [11, 12]. Red perilla is
believed to more actively biosynthesize anthocyanins than
Journal of Natural Medicines
Fig. 4  Correlation between
chlorophyll a content and
rosmarinic acid content. The
correlation coefficient r between
chlorophyll a content per 1 g
fresh leaves and rosmarinic acid
content per 1 g dried leaves was
0.0818, which indicates that
there is almost no correlation
green perilla, and 4-coumaroyl-CoA is consumed in the bio-
synthesis of anthocyanins. On the other hand, the biosynthe-
sis of anthocyanins may be suppressed in green perilla, so a
large amount of 4-coumaroyl-CoA, which is a substrate for
the biosynthesis of rosmarinic acid, is present and a large
amount of rosmarinic acid is thus biosynthesized.
(B) Comparison of the rosmarinic acid content of different
Perilla species.
Fig. 5  Comparison of perilla plant varieties. Circles: rosmarinic acid
content of each sample. Diamonds: the mean value of each vari-
ety. There was no significant difference between any two groups (P
value < 0.05). The mean value and the standard deviation were taken
from 6 samples of variety a, 23 samples of variety b, and 11 samples
of variety c
We conducted experiments using the laboratory-grown
perilla plants shown in Table 2. Comparison of the various
species showed that the rosmarinic acid content per 1 g of
dried leaves was significantly higher in P. citriodora than P.
frutescens Britton var. crispa and P. frutescens Britton var.
frutescens (P < 0.05, Fig. 6). In addition, comparison of cul-
tivated species and wild species indicated that the rosmarinic
acid content per 1 g of dried leaves was higher in wild spe-
cies than in cultivated species (P < 0.01, Fig. 7). The major
difference between cultivated and wild species is the number
of chromosomes: 2n = 40 in cultivated species and 2n = 20 in
wild species [13]. The cultivated species (2n = 40) are gener-
ated as double diploid species by crossing P. citriodora and
an unknown wild species (2n = 20) through hybridization
and chromosome doubling [19]. Further studies will be con-
ducted on the relationship between the genetic background
Perilla species and rosmarinic acid content.
(C) Correlation between the extent of leaf growth and ros-
marinic acid content.
Experiments were performed using the two strains of
perilla shown in Table 3. The rosmarinic acid content per 1 g
of dried leaves was higher in leaves with a main vein length
of < 3 cm or 9–12 cm (Fig. 8), suggesting that rosmarinic
acid is accumulated from the early stage of leaf growth.
Although accurate measurements are lacking, the leaves of
perilla plants are thought to grow rapidly between when the
main vein is from around 3 cm to around 9 cm long com-
pared with the preceding and subsequent growth periods.
Therefore, the rosmarinic acid content per 1 g of dried leaves
is less in leaves with a main vein length of 3–9 cm because
13

Fig. 6  Comparison of rosmarinic acid content between species. The
mean value and standard deviation were obtained for 2 strains of P.
frutescens Britton var. crispa, 3 strains of P. frutescens Britton var.
frutescens, and 2 strains of P. citriodora Nakai. The values for P. hir-
tella Nakai and P. setoyensis G. Honda were obtained from the analy-
sis of one strain each. There were significant differences between P.
frutescens Britton var. crispa and P. citriodora Nakai, and between
P. frutescens Britton var. frutescens and P. citriodora Nakai (P
value < 0.05)
Fig. 7  Comparison of cultivated and wild species. The mean value
and standard deviation were obtained from 5 strains of cultivated spe-
cies and 4 strains of wild species. There was a significant difference
between the 2 groups (**P value < 0.01)
the biosynthesis of rosmarinic acid lags behind the growth
rate of the leaves.
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Journal of Natural Medicines
(D) Comparison of rosmarinic acid content in plants grown
in indoor nurseries (in a mechanical thermo-hygrostat
chamber for plants) and in outdoor nurseries.
Further experiments were performed using the two
strains of perilla shown in Table 3. Comparison of plants
grown indoors (in a mechanical thermo-hygrostat chamber
for plants) and outdoors showed that the rosmarinic acid
content per 1 g of dried leaves was higher in Strain No. 12
and No. 5254 plants cultivated outdoors (Fig. 9). Whereas
plants cultivated in indoor nurseries are exposed to light
from fluorescent lamps and constant temperature, those cul-
tivated in outdoor nurseries are exposed to solar radiation
and variation in temperature, and these factors may increase
the rosmarinic acid content.
(E) Changes in rosmarinic acid content due to differences
in the preparation of Hangekobokuto.
Experiments were performed using the perilla herb Japa-
nese-marketed products shown in Table 4. Figure 10 shows
the rosmarinic acid content per 1 g of dried leaves of the
13 samples (I to XIII). The highest content was 8.83 mg
for sample II and the lowest content was 1.91 mg for sam-
ple XII. No correlation was found between the rosmarinic
acid content and the perillaldehyde content in the Japanese
perilla herb marketed products (correlation coefficient r was
0.00790, Fig. 11); perilla herb IV, V, VI were not removed as
outliers from the quartiles of box-whisker plot of perillalde-
hyde, however, when they were removed as outliers, a weak
correlation was found (correlation coefficient r was 0.381).
Hangekobokuto was prepared by methods (1) and (2)
using samples II and IV, respectively, and the total weight
of Hangekobokuto and the rosmarinic acid and caffeic acid
contents were measured. The total weight of Hangekobo-
kuto prepared using method (2) was lower for samples II
and IV compared with samples prepared using method (1)
(Fig. 12). The content of rosmarinic acid per 1 g of extract
and that in the recommended daily intake of Hangekobo-
kuto prepared using method (2) were higher (Figs. 13,
14) compared with samples prepared using method (1).
In addition, the content of caffeic acid, a decomposition
product of rosmarinic acid, was higher when perilla herb
was added at the start of heating [(1), Fig. 15]. These
results show that decoctions prepared using the Kouge
method have a higher content of rosmarinic acid than
when perilla herb is added at the start of heating. Yuki-
zaki et al. measured the content of rosmarinic acid in dried
lemon balm leaves and reported that the higher the tem-
perature at which the leaves are dried, the lower the con-
tent of rosmarinic acid in the leaves [20], suggesting that
heat reduces the content of rosmarinic acid. Variations of
the Kouge method involve heating for a short time after
Journal of Natural Medicines
Fig. 8  The correlation between
the length of the main vein
in leaves and rosmarinic acid
content. For leaves with veins
less than 3 cm long, the values
obtained from the analysis of
one plant sample are shown.
For the others, the mean value
and standard deviation obtained
from three plant samples are
shown (**P value < 0.01)
Fig. 9  Comparison of rosmarinic acid content of plants grown in
indoor and outdoor nurseries. Comparison of rosmarinic acid con-
tent of plants grown in indoor and outdoor nurseries. The mean value
and standard deviation for strain No. 12 were obtained using 6 sam-
adding perilla herb. There is no legally prescribed Kouge
method and the variation used in this experiment was
recommended by a physician who prescribes Traditional
Chinese Medicine at the Kyoto University Medical School
Hospital. The results of this experiment suggest that the
content of rosmarinic acid differs depending on the method
used to prepare Hangekobokuto decoction.
Conclusion
In this study, we elaborated a simple and highly repro-
ducible method to quantify the rosmarinic acid content in
perilla plants. Differences in the rosmarinic acid content
in perilla plants are largely related to differences among
ples (from 3 plants) whose main vein in the leaves was 3–6 cm and
6–9 cm long. The mean value and standard deviation for strain No.
5254 were obtained using 3 samples (from 3 plants) whose main vein
in the leaves was 3–6 cm long (**P value < 0.01, ***P value < 0.001)
species, and the rosmarinic acid content per 1 g of dried
leaves was significantly higher in wild species than in cul-
tivated species, suggesting that the ability to biosynthesize
rosmarinic acid may have changed during the hybridiza-
tion that resulted in the cultivated species. Comparison
of the rosmarinic acid content based on the length of the
main vein of the leaves suggests that the larger the leaf,
the higher the rosmarinic acid content. Furthermore, com-
parison of the rosmarinic acid content in indoor cultivated
(in a mechanical thermo-hygrostat chamber for plants) and
outdoor cultivated plants indicate that the rosmarinic acid
content is higher in outdoor cultivated plants. This study
showed that the rosmarinic acid content in perilla plants
significantly differs depending on the species, strain, and
growth environment.
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 Journal of Natural Medicines

Fig. 10  Rosmarinic acid content

per 1 g of dried leaves of 13
perilla herb marketed products
(mg). Analyses were performed
in triplicate for each of samples
I to VI and their mean value
and standard deviation were
calculated. The values obtained
from one analysis are shown for
all other samples

Fig. 11  Correlation between

perillaldehyde content and
rosmarinic acid content. The
correlation coefficient r between
perillaldehyde content and
rosmarinic acid content per 1 g
dried leaves was 0.00790

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Journal of Natural Medicines

Fig. 14  Rosmarinic acid content in a daily intake of Hangekobokuto

Fig. 12  Weight of Hangekobokuto decoction (g). The mean value and decoction (mg). The mean value and standard deviation of two analy-
standard deviation of two analyses using the same sample were calcu- ses using the same sample were calculated. There was no significant
lated. There was no significant difference between (1) and (2) difference between (1) and (2)

Fig. 13  Rosmarinic acid content per 1 g of Hangekobokuto decoction Fig. 15  Caffeic acid content per 1 g of Hangekobokuto decoction
(mg). The mean value and standard deviation of two analyses using (mg). The mean value and standard deviation of two analyses using
the same sample were calculated. There was no significant difference the same sample were calculated. There was no significant difference
between (1) and (2) between (1) and (2)

13

Acknowledgments We are very grateful to Mae Chu Co., Ltd., Nippon
Funmatsu Yakuhin Co., Ltd., Tochimoto Tenkaido Co., Ltd., Mikuni
& Co., Ltd., and Kotaro Pharmaceutical Co., Ltd. for providing perilla
herb marketed items. In addition, part of this research was conducted
as a joint research project with Science Create Co., Ltd.
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