LWT - Food Science and Technology 42 (2009) 131136

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LWT - Food Science and Technology

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TLC bioautography-guided isolation of antioxidants from fruit

of Perilla frutescens var. acuta
Lihua Gu a, b, Tao Wu a, b, *, Zhengtao Wang a, b, **
a
Key Laboratory of Standardization of Chinese Medicines, Ministry of Education, Institute of Chinese Materia Medica,
Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai 201203, PR China
b
Shanghai R&D Centre for Standardization of Chinese Medicines, Shanghai 201210, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Guided isolation through bioautography on TLC using 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH ) as
Received 3 January 2008 a detection reagent led to the isolation of four antioxidant compounds from fruit of Perilla frutescens var.
Received in revised form 15 April 2008 acuta. These compounds were identied as rosmarinic acid (1), luteolin (2), apigenin (3), and chrysoeriol
Accepted 16 April 2008
(4), by means of UV, NMR, and ESI MS. All the compounds were isolated for the rst time from the fruit of

the plant. Compounds 1 and 2 showed signicant DPPH scavenging capacities, with IC50 values of 8.61
Keywords: and 7.50 mM, respectively. Further quantitative HPLC analysis conrmed that compounds 14 are the
TLC bioautography
predominant contributors to the free radical scavenging activity of the extract of P. frutescens var. acuta.
Perilla frutescens var. acuta
DPPH
2008 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.
Antioxidant

1. Introduction
A growing amount of evidence has shown that free radical
mediated damage plays an important role in the aetiology of
several human diseases. For example, oxidative stress has been
widely postulated to be involved in the development and
progression of some chronic diseases such as cardiovascular
disease, neuronal disease, cataracts, and several types of cancer
(Thomas & Kalyanaraman, 1997). Recent epidemiological studies
indicated that a high intake of antioxidants was positively associ-
ated with the reduced risk of coronary heart diseases and ageing-
related diseases (Flora, 2007; Valko et al., 2007). However, some
synthetic antioxidants such as BHA and BHT, have been revealed to
be potentially toxic and carcinogenic (Thamavit et al., 1985;
Williams, Shimada, McQueen, Tong, & Ved Brat, 1984). Conse-
quently, there is an increasing interest to search for antioxidants
naturally present in vegetables, fruits and functional herbs. Due to
the complicated compositions of natural materials, it is challenging
to rapidly screen the active antioxidants. A number of screening
assays were developed and used to search potential antioxidants,
* Corresponding author. Key Laboratory of Standardization of Chinese Medicines,
Ministry of Education, Institute of Chinese Materia Medica, Shanghai University of
Traditional Chinese Medicine, 1200 Cailun Road, Shanghai 201203, PR China. Tel.:
86 21 51 322 513; fax: 86 21 51 322 519.
** Corresponding author.
E-mail addresses: laurawu2000@hotmail.com (T. Wu), wangzht@hotmail.com
(Z. Wang).
which include, but are not limited to, high-throughput relative
DPPH radical scavenging capacity (RDSC) assay (Cheng, Moore, &
Yu, 2006), HO radical scavenging capacity (HOSC) assay (Moore,
Yin, & Yu, 2006), and thin layer chromatography (TLC)
bioautography assay (Cimpoiu, 2006; Jayasinghe, Puvanendran,
Hara, & Fujimoto, 2004). Compared to other methods, the TLC
bioautography method can quickly detect and separate the active
components in a complicated plant extract, and has additional
advantages such as convenience, being simple to run, and requiring
no specialized equipment. Herein, we describe an activity-guided
isolation of natural antioxidants from fruit of Perilla frutescens (L.)
Britt. var. acuta by TLC bioautography method.
P. frutescens var. acuta (Lamiaceae) has been used as an edible
biologic medicine in Eastern Asia for more than a thousand years.
The leaves, stems, and fruit of this plant are used individually to
treat a variety of diseases (Chinese Pharmacopoeia Commission,
2005). Folium Perillae is used to induce perspiration and dispel
chills, and to regulate stomach function. Caulis Perillae is tradi-
tionally used as an analgesic and anti-abortive agent, while Fructus
Perillae is employed for dyspnea and cough relief, phlegm elimi-
nation, and the bowel relaxation (Chinese Pharmacopoeia
Commission, 2005). In addition, edible Perillae oil made from the
seeds has a high iodine content, is rich in polyunsaturated fatty
acids, and is used as a preservative.
Compared to Folium Perillae and Caulis Perillae (Banno et al.,
2004; Liu, Steigel, Reininger, & Bauer, 2000; Nakanishi et al., 1990;
Nakazawa & Ohsawa, 2000; Takeda, Tsuji, Inazu, Egashira, &
Matsumiya, 2002), little is known about the active phytochemicals

0023-6438/$34.00 2008 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.lwt.2008.04.006
132 L. Gu et al. / LWT - Food Science and Technology 42 (2009) 131136

of Fructus Perillae. A few studies have shown that fatty acids and
polyhydric phenols in the fruit may have anti-anaphylactic,
anti-inammatory, and antimicrobial properties, and may also
inhibit arachidonate lipoxygenase (Wang et al., 2001; Yamamoto &
four compounds were obtained including 1 (4.5 mg), 2 (8 mg), 3 (3
mg), and 4 (8 mg) based on their elution order.
2.4. The conventional spectrophotometric DPPH scavenging


Ogawa, 2002; Yamamoto, Sakakibara, Nagatsu, & Sekiya, 1998).
In the present study, an activity-guided purication was
conducted to isolate the free radical scavenging components from
fruit of P. frutescens var. acuta. In addition, semi-quantication of
the isolates was carried out by reverse phase high performance
liquid chromatography (HPLC).
2. Materials and methods
2.1. General
UV spectra were recorded on a Beckman DU 640 spectrometer
(Beckman Instruments Inc., Fullerton, CA, USA). 1H and 13C NMR
spectra were recorded on Bruker AM-500 spectrometer using
DMSO-d6 or D2O as solvents. Electrospray ionization (ESI) mass
spectra were obtained with a Bruker-daltonics APES-III 7.0 JESLA
FTMS spectrometer (Bruker, USA). HPLC analysis was performed on
an Agilent 1100 series HPLC apparatus (Agilent, USA), using
a Polaris C18 column (Metachem, Switzerland) (250  4.6 mm i.d.,
5 mm), and a gradient elution with the mobile phase comprising
water and methanol. Silica gel 60 F254 TLC plates (Merck, Germany)
were used for TLC bioautography analysis.

1,1-Diphenyl-2-picrylhydrazyl radical (DPPH ) and butylated
hydroxyanisole (BHA) were purchased from Sigma-Aldrich (Stein-
heim, Germany). All solvents used for chromatography and
extraction were of HPLC grade and obtained from Fisher Scientic
(Fair Lawn, NJ, USA). All other chemicals were of analytical grade
without further purication.
2.2. Plant material
capacity assay

The conventional spectrophotometric DPPH scavenging capacity
assay was conducted as described by Blois (1958) with minor
modication. Briey, an aliquot of 500 mL of different concentrations
of PES (in acetone), AMS or individual pure compounds in methanol

was added to 500 mL of 0.205 mM DPPH methanol solution. After
gentle mixing and 40 min of standing at room temperature,

the DPPH level was spectrophotometrically determined at 517 nm.
The free radical scavenging capacity was expressed as percent of

DPPH scavenged that was calculated as [(A0  A1/A0)]  100 (where
A0 was the absorbance of the reagent blank, and A1 was the absor-
bance with antioxidants). In addition, the IC50 value of each sample

was obtained by plotting the percent DPPH scavenging of each
concentration of an antioxidant sample against the sample con-
centrations. The IC50 value is the concentration of an antioxidant to

quench 50% of DPPH in the reaction mixture under the assay con-
dition. BHA was used as a positive control. Duplicate reactions were
carried out for each concentration of each individual sample.
2.5. TLC bioautography analysis
An aliquot of AMS methanol solution (1 mg/mL, 3 mL) or
individual pure isolate methanol solutions (1.0 mg/mL, 2 mL) was
directly deposited (as spots or bands) onto the TLC plates. TLC
plates were developed in a presaturated solvent chamber with n-
hexanetolueneethyl acetateformic acid (2:5:2.5:0.5) as
developing reagents until the solvent front reached 1 cm from the
top of plates. The developed TLC plates were then removed from

Fruit of P. frutescens var. acuta was collected from Anhui

province, China, in October 2006, and authenticated by Prof. Mian
Zhang, the Department of Pharmacognosy, China Pharmaceutical
University, Nanjing, China. A voucher specimen (No. zsz-20030820-
hubei-ahbz) is deposited at Shanghai R&D Centre for Standardiza-
tion of Traditional Chinese Medicines, Shanghai, China.

2.3. Extraction and isolation of antioxidant compounds

The air-dried, powdered fruit (620 g) of P. frutescens var. acuta

were extracted thrice using 1250 mL of petroleum ether
(6090  C) under reux for 2 h. The supernatant was separated
from the solid residue by paper ltration (Whatman No. 1 lter,
Whatman Paper Ltd., Maidstone, UK). Petroleum ether in the
supernatant was removed by evaporation at 50  C under reduced
pressure to give a residue, named as PES. The defatted fruit was
re-extracted twice with 2000 mL of 80% aqueous methanol under
reux for 2.5 h. The extract was combined, and methanol was
evaporated at 50  C under reduced pressure. The resulting sus-
pension was then lyophilized to yield 24 g powder. This dry frac-
tion was named AMS.
PES and AMS were dissolved in acetone and methanol,

respectively, and tested against DPPH using the conventional

spectrophotometric assay. The DPPH -active AMS was subjected to
column chromatography on a C18 column using an analytical HPLC
system equipped with a DAD detector set at 360 nm and a fraction
collector. A 40-min linear gradient was used, consisting of 80/20


MeOH/H2O to 50/50 MeOH/H2O with a ow rate of 1.0 mL/min.
The individual eluted components from one HPLC run were
monitored by TLC bioautography, while the active ones were
separately combined and subjected to lyophilization. As a result,
Fig. 1. TLC plates stained with 2.54 mM DPPH solution in methanol, and visualized (A)
under visible light, (B) under UV 254 nm, and (C) under UV 366 nm. Three microlitres
of the 80% methanol extract (AMS, 1 mg/mL) of Perilla frutescens var. acuta was applied
as dots on TLC layer. The spots marked with a, b, c, d, and e indicate compounds with

DPPH scavenging activities.
 L. Gu et al. / LWT - Food Science and Technology 42 (2009) 131136 133

OCH3

OH
2 7 OH
HO HO O
3 1 1'
8 2'
9' HO O
9 6'
6 OH
4 8' 7' 3'
HO O O
5
5' 4'

OH O

1 Rosmarinic acid 3 Chrysoeriol

OH

3' OH
4' OH
2'

8 5'
HO O HO O
7 9 2 1'
6'

6 3
10 4
5

OH O OH O

2 Luteolin 4 Apigenin

Fig. 2. Chemical structures of compounds (14) isolated from fruit of Perilla frutescens var. acuta.

the chamber, and allowed to air-dry for 30 min, followed by Shanghai, China) prior to HPLC analysis. An aliquot of the ltrate

spraying with a 2.54 mM DPPH methanol solution for derivatization. (10 mL) was injected into a Polaris HPLC C18 column (250 

Bands with the DPPH scavenging activity were observed as white 4.6 mm i.d., 5 mm) and eluted with a linear gradient with
yellow bands on a purple background. Each TLC plate was also a mobile phase containing solvent A (methanol) and solvent B
monitored under UV light at 254 and 366 nm. (water). The solvent gradient was programmed from 20% to 50%
B in 40 min with a ow rate of 1.0 mL/min. The separation was
2.6. HPLC quantitative analysis carried out at room temperature and monitored at 360 nm. The
semi-quantitative analysis of four active components was ach-
AMS methanol solution (1.0 mg/mL) was ltered through ieved by peak area normalization measurements, expressed as g
a cellulose acetate membrane lter (0.45 mm, Anpu Co., per 100 g.

Table 1
NMR data of compounds 14 (DMSO)
13 1
Carbon C NMR H NMR

1a 2 3 4 1a 2 3 4
1 129.60
2 116.75 167.36 163.56 164.03 7.19 (s)
3 146.44 101.94 103.60 102.73 6.56 (s) 6.62 (s) 6.78 (s)
4 149.82 181.01 181.70 181.66
5 119.93 161.27 157.20 161.35 7.12 (d, 7.6)
6 125.42 99.47 98.70 98.73 6.96 (d, 7.9) 6.33 (d, 1.8) 6.18 (br. s) 6.49 (d, 1.8)
7 149.17 163.49 164.10 163.62 7.57 (d, 16.1)
8 118.91 94.17 94.00 93.86 6.78 (d, 16.0) 6.08 (d, 1.8) 6.87 (br. s) 6.20 (d, 1.5)
9 171.38 157.40 161.40 161.07
10 102.31 103.10 103.59
10 132.06 120.21 120.30 121.06 6.91 (s)
20 146.44 112.48 110.10 128.39 7.37 (d, 2.2) 7.55 (br. s) 6.93 (d, 8.6)
30 149.17 146.21 150.70 115.85 7.92 (d, 8.6)
40 118.84 151.30 147.90 157.20 6.90 (d, 9.5)
50 117.84 115.79 115.70 115.85 6.63 (d, 7.7) 7.36 (d, 7.9) 6.93 (d, 7.9) 7.92 (d, 8.3)
60 124.49 118.75 121.40 128.39 6.82 (d, 8.1) 7.55 (br. s) 6.93 (d, 8.6)
70 39.03 3.29 (m)
80 75.92 5.23 (m)
90 180.08
C30 OCH3 55.89 3.89 (s)
a
NMR measurements were performed in D2O. Values in parentheses are coupling constants (Hz).
134 L. Gu et al. / LWT - Food Science and Technology 42 (2009) 131136
3. Results and discussion
3.1. Isolation of antioxidant compounds

The conventional spectrophotometric DPPH capacity assay was
rst used to screen the potential antioxidant fractions of P.

frutescens var. acuta. The DPPH was signicantly scavenged by AMS
in a dose-dependent manner, with an IC50 value of 69.0  4.63 mM,
and PES with an IC50 value of 35.0  4.25 mM. It is interesting that
a higher free radical scavenging activity was observed in PES,
compared with AMS. This is probably due to the high essential oil
components such as fatty acids present in P. frutescens var. acuta
(Chen, Zhang, & Guo, 2004; Choi, 2004). In addition, it is
noteworthy that a considerable amount of a-linolenic acid was
reported present in Perilla oil (Hilditch, 1956; Lee, Ryu, & Kwak,
2002). The high content of a-linolenic acid might partly contribute
to the antioxidant activity of PES and other health benets of Perilla
oil (Simopoulos, 2004). In the present study, the 80% methanol
extract was selected for further purication, since less is known
about the polar antioxidants in the fruit of this plant. This active
AMS was then monitored by a TLC bioautography method to guide
the separation because this method gives quick access for detection
and localization of the active compounds in a complicated plant
extract (Tasdemir, Donmez, Calis, & Ruedi, 2004). In the method,

the DPPH scavenging activity was observed visually as white
yellow spots on a purple background. Fig. 1A shows a prole of the
antioxidant components in the 80% methanol extract of P. frutescens
var. acuta under visible light. At least eight spots (such as spots ae)

in the chromatograms were observed to have DPPH scavenging
activities. In addition, the same stained TLC plate was also inspected
under UV 254 and 366 nm (Fig. 1B and C). Note that the antioxidant
spots shown in Fig. 1A were also observed in those of Fig. 1B and C.
However, it needs to be pointed out that the different detection
sensitivities observed among Fig. 1AC were due to the diverse
nature of the antioxidative compounds. For example, spot e was
easily better visualized under 254 and 366 nm than that under
visible light.
The active AMS was directly subjected to a HPLC C18 column
eluted with a gradient of water and methanol. Finally, four pure
compounds were isolated and identied, the structures of which
are presented in Fig. 2.
3.2. Structure determinations of isolated compounds
All the compounds were identied by UV, 1H, 13C NMR, ESI MS
spectra and by comparison with the literature data.
Compound 1 was obtained as a yellow powder, with UV (MeOH)
lmax, 290, 327 nm. Detailed analysis of its 1H and 13C NMR spectra
(Table 1) established the structure of 1 as rosmarinic acid, which

Fig. 3. TLC plates stained with 2.54 mM DPPH solution in methanol, and visualized (A) under visible light, (B) under UV 254 nm, and (C) under UV 366 nm. Three microlitres of the
80% extract (AMS, 1 mg/mL) of Perilla frutescens var. acuta, and 2 mL of 1.0 mg/mL of pure isolates were applied as bands on TLC layers. Bands 14, and y stand for rosmaniric acid,
luteolin, chrysoeriol, apigenin, and the 80% methanol extract of extract of Perilla frutescens var. acuta, respectively.
agreed well with the reported data (Zheng, Li, & Feng, 2004). The
identication was further conrmed by a negative ESI MS analysis
(m/z 359, [M  H]). This is the rst report for the isolation of
rosmarinic acid from fruit of P. frutescens var. acuta, though it was
previously isolated as a main component from leaves of this plant
(Takeda et al., 2002).
Compound 2 was also obtained as a yellow powder, and gave
a positive reaction with AlC13 reagent, probably indicating a avo-
noid nature. Its UV spectrum was consistent with that of a avonoid
with maxima at 252, 265, and 347 nm (Gonzalez-Guevara et al.,
2006). A direct comparison of 1H, 13C NMR data (Table 1) with the
reported data (Li, Luo, He, & Zhang, 2007; Shen, Liang, Peng, & Ding,
2004) led to identication of 2 as luteolin, which was further
conrmed by a negative ESI MS analysis (m/z 285 [M  H]). This is
the rst report on the isolation of luteolin from this plant.
Compound 3 was obtained as a yellow powder, and gave
a positive reaction with A1C13 reagent. Combined with the analysis
of its UV (MeOH, lmax 265, 340 nm), ESI MS (m/z 299 [M  H]), 1H
and 13C NMR (Table 1), compound 3 was identied as chrysoeriol,
which agreed well with the data reported (He, Zhu, Wang, Xu, & Hu,
2004; Shen et al., 2004). Again, this is the rst time to report the
isolation of chrysoeriol from this plant.
Similar to compounds 2 and 3, compound 4 was obtained as
a yellow powder, and shows characteristic avonoid reaction with
AlC13 reagent. Its detailed UV (MeOH, lmax 265, 335 nm), 1H and 13C
NMR data (Table 1), negative ESI MS data (m/z 269 [M  H]),
agreed well with the data reported (Li et al., 2007; Shen et al.,
2004). Therefore, this compound was identied as apigenin, and
isolated from genus Perilla for the rst time.
Further TLC bioautographic analysis conrmed that these four

isolated compounds (14) were DPPH -active components that
corresponded to the active bands of the 80% methanol extract, as

shown in Fig. 3. This indicated that the DPPH -active components of
P. frutescens var. acuta can be obtained with the guidance of TLC
bioautography method.

3.3. DPPH scavenging activity of the isolates

The DPPH scavenging activities of all the isolated compounds

were estimated using the conventional spectrophotometric DPPH
scavenging capacity assay, except compound 3 due to limited
amounts available. Fig. 4 shows the doseresponse curves for the

DPPH scavenging activities of compounds 1, 2, 4, and the positive
control, BHA. Rosmarinic acid (1) and luteolin (2) showed signi-

cant DPPH scavenging activities with IC50 values of 8.61 1.08 and
7.50  2.05 mM, respectively, which were comparable to that of BHA
(IC50 30.2  4.04 mM). However, no 50% of inhibition was
achieved for apigenin with concentrations up to 5.0 mM. The
 L. Gu et al. / LWT - Food Science and Technology 42 (2009) 131136 135

A 100
BHA
% DPPHscavenged

75

50

25

0
0 100 200 300 400 500
Concentration ( M)

B 100 Rosmarinic acid

% DPPH scavenged

75
Fig. 5. HPLC chromatogram at 360 nm of the 80% methanol extract (AMS) of fruit of
Perilla frutescens var. acuta: (1) rosmaniric acid, (2) luteolin, (3) chrysoeriol, and (4)
50
apigenin. The inset showed online UV spectra of isolates 14, and unknown peaks
58.
25

0 41.7, 2.2, and 16.3 g/100 g, respectively, as determined by the area

0 30 60 90 120 150
normalization method. In addition to peaks 14, peaks 58 were
Concentration ( M)
observed as well as their online UV spectra. By comparing the UV
spectra of peak 6 with peaks 24, and peaks 5, 7 and 8 with peak 1,
C 100
Luteolin it was deduced that peaks 5, 7 and 8, and peak 6 were derivatives of
% DPPHscavenged

rosmarinic acid and avonoids, respectively. This nding indicated

75
their potential in contributing to the antioxidant activity of the
plant. Further studies are required to isolate and identify these
50 unknown compounds.
Based on the above HPLC quantication analysis, together with
25 antioxidant activity data, we can conclude that the isolated
compounds including rosmarinic acid, luteolin, and apigenin are
0 the predominate contributors to the overall antioxidant activity of
0 20 40 60 80 100
the fruit of the plant.
Concentration ( M)

D 100
Apigenin 4. Conclusions
% DPPH scavenged

75 The TLC bioautography-guided strategy was used to separate

the antioxidant compounds from plant extracts. Four antioxidant
50 compounds were isolated from fruit of P. frutescens var. acuta for
the rst time. The isolated rosmarinic acid and luteolin demon-
25 strated signicant free radical scavenging activities. Quantitative
HPLC analysis conrmed that these isolated compounds are
predominate components of fruit of the plant, indicating their
0
0 1 2 3 4 5 6 signicant contribution to the overall antioxidant activity.
Concentration (mM)

Fig. 4. Dose effects of the pure isolates-DPPH reactions (A) BHA, (B) rosmarinic acid, Acknowledgements

(C) luteolin, and (D) apigein. The nial concentration of DPPH in all reactions was
102.5 mM. The absorbance at 517 nm of the reactions was measured at minute 40 of the This study was nancially supported in part by a grant from the
reaction. All the tests were conducted in duplicate, and the means were used.
Shanghai Rising Star Project of Shanghai Science and Technology
Committee (No. 06QA14047 awarded to Dr. T. Wu), and by a grant
from the Shanghai Leading Academic Discipline Project (No.
antioxidant activity decreased as follows: rosmarinic acid > Y0301). The author would like to thank Miss Jessica Blackford in
luteolin > BHA > apigenin. USA for the modication of this manuscript.

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