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Identification of flavonoids in litchi (Litchi

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anticancer activities

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DOI: 10.1016/j.jff.2013.11.022

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Identification of flavonoids in litchi (Litchi chinensis

Sonn.) leaf and evaluation of anticancer activities

Lingrong Wena,b, Dan Wua,c, Yueming Jianga, K. Nagendra Prasadd, Sen Lina, Guoxiang
Jianga, Jirui Hea,c, Mouming Zhaob, Wei Luob, Bao Yanga,*
a
Key Laboratory of Plant Resource Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences,
Guangzhou 510650, China
b
College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, China
c
University of Chinese Academy of Sciences, Beijing 100049, China
d
School of Engineering, Monash University, Selangor 46150, Malaysia

A R T I C L E I N F O A B S T R A C T

Article history: Six flavonoids, namely luteolin (1), epicatechin (2), kaempferol 3-O-b-glucoside (3),
Received 10 October 2013 kaempferol 3-O-a-rhamnoside (4), procyanidin A2 (5) and rutin (6) were purified from the
Received in revised form EtOAc-soluble extract of litchi leaf by column chromatography. Their structures were
21 November 2013 elucidated by nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry
Accepted 22 November 2013 (MS) evidences. Luteolin and kaempferol 3-O-a-rhamnoside were found from this plant
Available online 17 December 2013 for the first time. Procyanidin A2 exhibited high anticancer activities against human hepa-
toma HepG2 and human cervical carcinoma Hela cells. However, it had poor anticancer
Keywords: activities against human lung cancer A549 and human breast cancer MCF-7 cells. Luteolin,
Purification epicatechin, procyanidin A2 and rutin showed good antioxidant activities than butylated
Flavonoid hydroxytoluene (BHT). The antimicrobial activity assay indicated that luteolin possessed
Cytotoxity
the strongest antimicrobial activity against Staphylococcus aureus, Escherichia coli, Shigella
NMR
dysenteriae, Salmonella and Bacillus thuringiensis. Epicatechin, procyanidin A2 and rutin
Litchi leaf
showed relatively weak antimicrobial activities.
 2013 Elsevier Ltd. All rights reserved.

1. Introduction inflammatory, analgesic and antipyretic activities. The

Litchi (Litchi chinensis Sonn.) is a non-climacteric subtropical-
fruit originated from Southeast Asia, belonging to the Sapind-
aceae family (Hwang et al., 2013; Jiang et al., 2013). Due to the
good taste and abundant nutrition, litchi fruit is widely
accepted by consumers over the world as other subtropical
fruits, like longan (Yang, Jiang, Shi, Chen, & Ashraf, 2011).
As a tissue of litchi plant, litchi leaf has been used in tradi-
tional Chinese medicine for the treatment of heartstroke, flat-
ulence and detoxication. Pharmacological studies conducted
by Besra, Sharma and Gomesl (1996) indicated that the
petroleum ether extract of litchi leaf had significant anti-
extract could inhibit the cyclooxygenase pathway of arachi-
donic acid metabolism rather than inhibiting arachidonic
acid-induced inflammation.
Flavonoids, including anthocyanins, flavones, flavonols,
flavanols, chalcones, dihydrochalcones, dihydroflavonols
and isoflavonoids, are important secondary metabolites of
plants, which are beneficial for the plant as physiologically
active compounds or stress-resistant agents (Treutter, 2006).
In addition, flavonoids possess diverse biological activities,
such as antioxidant, anti-inflammatory, immunomodulatory,
anticancer and antimicrobial effects, which have attracted
much attention in recent years (Costa, Garcia-Diaz, Jimenez,

* Corresponding author. Tel.: +86 20 37083042; fax: +86 20 37252960.

E-mail address: yangbao@scbg.ac.cn (B. Yang).
1756-4646/$ - see front matter  2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jff.2013.11.022
556 JOURNAL OF FUNCTIONAL FOODS
& Silva, 2013; Yang et al., 2012). A significant amount of flavo-
noids were found in litchi seed and pericarp, which are
byproducts of litchi processing and are usually discarded. Pre-
vious reports demonstrated that the major flavonoids in litchi
pericarp were epicatechin, epicatechin gallate, rutin, querce-
tin 3-O-glucoside, procyanidin B4 and B2, while epicatechin,
procyanidin A1 and A2, rutin, phlorizin, tamarixetin 3-O-ruti-
noside and litchioside D were the primary flavonoids in litchi
seeds (Li & Jiang, 2007; Xu, Xie, Hao, Jiang, & Wei, 2011; Xu,
Xie, Wang, & Wei, 2010; Zhao, Yang, Wang, Li, & Jiang, 2006).
As various tissues of a plant should have similar phyto-
chemical composition, it implies that litchi leaf should be a
good source of flavonoids. Moreover, litchi leaf is much easier
to collect than litchi pericarp and seed. However, the informa-
tion on flavonoids of litchi leaf is still limited. In order to re-
veal the flavonoid composition in litchi leaf, ethanolic
extract of litchi leaf was fractionated and further purified.
Six flavonoids were identified, and their antioxidant and anti-
microbial activities were evaluated. Moreover, in vitro antican-
cer activities of purified flavonoids against human hepatoma
Hep-G2 and human cervical carcinoma Hela, human lung
cancer A549 and human breast cancer MCF-7 were measured.
2. Materials and methods
2.1. Plant material
Fresh litchi leaves were collected from an orchard in Guangz-
hou, China on July, 2010, and were carefully washed with
distilled water, then sun-dried and ground into fine powder
witha laboratory mill (FW100, Taisite Instrument Co., Ltd,
Tianjin, China). The materials were stored at room tempera-
ture in a desiccator till use.
2.2. General methods
Nuclear magnetic resonance (1H NMR (400 MHz) and 13C NMR
(100 MHz)) spectra were recorded on a Bruker DRX-400 instru-
ment (Bruker BioSpin Gmbh, Rheinstetten, Germany) in deu-
terated solvent with different solvent residual peaks
(CD3OD: dH 3.31 and dC 49.30 ppm, DMSO: dH 2.49 and dC
39.70 ppm) as references. Electrospray ionization mass spec-
trometry (ESI-MS) data were acquired on a MDS SCIEX API
2000 LC/MS apparatus (MDS Sciex, Ontario, Canada). Column
chromatography was performed over silica gel (100200 or
200300 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao,
China), Sephadex LH-20 (GE Healthcare, Shanghai, China) and
Develosil ODS (S-75 lm, Nomura Chemical Co., Ltd., Seto, Ja-
pan), respectively. Thin layer chromatography was performed
on precoated silica gel HSGF254 plates. High performance li-
quid chromatography (HPLC) was carried out using a Shima-
dzu LC-20AT liquid chromatography (Shimadzu Corp., Kyoto,
Japan) equipped with a Shimadzu UV detector, and Shima-
dzu-Pack ODS-A columns (250 4.6 mm and 250 20 mm)
were used for the analysis and preparation, respectively.
Bacterialstrains: Staphylococcus aureus, Escherichia coli, Shigella
dysenteriae, Salmonella and Bacillus thuringiensis were
generously offered by Bioorganic Chemistry Research group,
South China Botanical Garden. The strains were cultivated
in LuriaBertani broth. In addition, Human hepatoma Hep-
6 ( 2 0 1 4 ) 5 5 5 5 6 3
G2, human lung cancer A549, human breast cancer MCF-7
and human cervical carcinoma Hela cells were provided by
Guangzhou Jinan Biomedicine Research and Development
Center, Guangzhou, China. The cells were maintained in
RPMI-1640 medium plus 10% heat-inactivated fetal bovine
serum in a humidified atmosphere with 5% CO2 at 37 C.
2,20-Azobis(2-methylpropionamidine)dihydrochloride (AAPH),
fluorescein sodium salt, Trolox, resazurin sodium salt, 1,1-di-
phenyl-2-picryldydrazyl (DPPH), 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) and quercetin were
purchased from Sigma Chemical Co. (St. Louis, MO, USA).
Methnol and trifluoroacetic acid used for HPLC analysis was
got from CNW Technologies Gmbh (Dusseldorf, Germany).
Other reagents were obtained from Guangzhou Reagent Co.
(Guangzhou, China).
2.3. Extraction and isolation
Dried leaf powders (8500 g) were extracted with 95% EtOH
(20 L 3) at room temperature (2634 C) for 4 days each time.
Solvent removal and concentration was achieved using a ro-
tary evaporator (N-1001, EYELA Co., Tokyo, Japan) under re-
duced pressure at 45 C which gave a dark green solid
(1956 g, 23.01%), most of which (1500 g) were suspended in
water and then fractionated successively by petroleum ether,
ethyl acetate (EtOAc), and n-butanol (n-BuOH). The petroleum
ether extract (559.95 g, 37.33%), EtOAc extract (701.25 g,
46.75%), and n-BuOH extract (68.25 g, 4.55%) fractions after
drying in vacuo were obtained. The EtOAc-soluble extract
(200 g) were subjected to purification by silica gel column
using CHCl3MeOH solvent togain fractions E1-E20. Fractions
E6E8 (5.318 g) was purified by silica gel again to obtain frac-
tions E61E612, and fraction E6-5 (1.13 g) was further puri-
fied by ODS column eluted with MeOH-H2O, the 45% MeOH
eluate was submitted to Sephadex LH-20 column eluted with
MeOH to yield compound 1 (8.4 mg). Fraction E10 (9.56 g) was
further purified by silica gel and recrystallized to yield com-
pound 2 (2.36 g). Fraction E11 (3.75 g) was further purified by
ODS column eluted with MeOH-H2O, the 30% and 35% MeOH
eluates were recrystallized and loaded to Sephadex LH-20 col-
umn eluted with MeOH to yield compound 3 (800 mg) and
compound 4 (11 mg), respectively. Fraction E15 (13.683 g)
was purified by silica gel and recrystallized to yield compound
5 (2.32 g), and fraction E17 (4.47 g) was further purified by ODS
column eluted with MeOHH2O, the 20% MeOH eluate was
submitted to Sephadex LH-20 column eluted with MeOH to
yield compound 6 (56 mg).
2.4. Assay of DPPH radical scavenging activity
The DPPH radical scavenging activity was measured by the
method of Wen, Yang, Cui, You, and Zhao (2012). Chemicals
were accurately weighed and dissolved in methanol to obtain
a final concentration of 4 mM. The diluted sample (0.1 mL)
was added to 2.9 mL of 0.1 mM DPPH in methanol. After vor-
tex, the fluid was kept in the dark at room temperature for
30 min. The absorbance was measured at 517 nm.The control
was carried out with methanol instead of sample solution,
while methanol was used as the blank, BHT was used as
 JOURNAL OF FUNCTIONAL FOODS
positive standard. The DPPH radical scavenging activity was
expressed as:
 
As  Ac
Scavenging activity% 1   100% (1st10th column). Then 100 lL of tested samples were added
A
where As is the absorbance of the reaction solution, Ac is the
absorbance of the solution including 0.1 mL of sample and
2.9 mL of methanol, and A is the absorbance of the solution
including 2.9 mL of DPPH and 0.1 mL of methanol.
2.5. Assay of oxygen radical absorption capacity
The oxygen radical absorption capacity (ORAC) was deter-
mined as previously described by Lin, Zhao, Dong, Yang and
Zhao (2012) with some modifications. The final reaction mix-
ture was 200 lL. Trolox was accurately weighed and dissolved
in ethanol to give a concentration of 2 mM, and diluted with
75 mM NaH2PO4Na2HPO4 buffer (pH 7.4) to a series of con-
centrations (1, 2, 4, 6, 8 and 10 lM). The fluorescein sodium
salt and AAPH were made in 75 mM NaH2PO4Na2HPO4 buffer
(pH 7.4) to give a final concentration of 70 and 12 mM in the
final reaction mixture, respectively. All the reagents were
made before use. The tested chemicals were dissolved in
DMSO and diluted with 75 mM NaH2PO4Na2HPO4 buffer (pH
7.4). At first, 20 lL of sample (or Trolox) were added in a well
of 96-well microtitre plate, and then 120 lL of fluorescein so-
dium salt were added. After the mixture was incubated for
15 min at 37 C, 60 lL of AAPH were added before analysis.
Additionally, 75 mM NaH2PO4Na2HPO4 buffer (pH 7.4) was
used as blank. The fluorescence was measured every 2 min
for 120 min. The reaction was carried out at a constant tem-
perature of 37 C. All the measurements were performed on
a Varioskan Flash spectral scan multimode plate reader (Ther-
mo Fisher Scientific, Thermo Electon Co., Waltham, MA, USA)
with an excitation wavelength of 485 nm and an emmission
wavelength of 520 nm.
Finally, ORAC values were calculated according to the
regression equation between Trolox concentration and the
net area under curve (AUC) and were expressed as lmol Trol-
ox equivalents per lmol sample (lmol Trolox equiv/lmol).
The AUC is calculated as:
X
n
ACU 2f i  f0  fn
i1
where f0 is the initial fluorescence reading at 0 min; fi is the
fluorescence reading at time 2 i min; and fn is the final fluo-
rescence reading at 120 min. The net AUC was obtained by sub-
tracting the AUC of the blank from that of the sample, that is:
Net AUC AUCsample  AUCblank
2.6. Assay of antimicrobial activity
According to the method of Rahman and Gray (2005), the anti-
microbial activity was determined by a microdilution titre
technique using 96-well plate with the advantage of determin-
ing the minimum inhibitory concentration (MIC) conveniently
and efficiently. In brief, 100 lL of resazurin sodium salt (100 lg/
mL, indicator solution) were placed into the sterile control
wells (11th column) on the 96-well plate. After the indicator
6 ( 20 1 4) 5 5 556 3 557
solution (100 lg/mL, 7.5 mL) were mixed with tested organism
(108 cfu/mL, 5 mL), 100 lL of the mixture were transfered into
the growth control wells (12th column) and all the tested wells
into the 1st clolumn wells on the plate. Once all solutions were
mixed together, half of the content (100 lL) from 1st column
wells were then transferred to the 2nd column of wells. And
the following well was treated accordingly (double dilution)
up to the 10th column, followed by discarding last 100 lL ali-
quot. Finally, the plates were incubated at37 C for about 5
6 h, until the growth control wells change colour from blue to
pink. In a plate, up to six samples could be applied leaving
two for positive and negative controls (kanamycin and metha-
nol, respectively). The lowest concentration at which colour
change occurred was considered as the MIC of a test sample,
and the antimicrobial activity was determined by the compar-
ison of the sample and controls.
2.7. Cytotoxicity assay
The cytotoxicity assay was performed by MTT staining meth-
od using 96-well flat-bottom microtiter plate (Zhu et al., 2013).
A preliminary assay was conducted for six purified flavonoids
at 200 lg/mL. Only procyanidin A2 showed significant cytotox-
icity. Therefore, it was chosen for determination of dose-
dependent effect. Procyanidin A2 was dissolved in DMSO to
different concentrations. An aliquot of 5 lL were added to a
well, including 195 lL of cancer cells (5 104 cell/mL) culture
medium. The final concentrations of procyanidin A2 were
6.25, 12.5, 25, 50, 100 and 200 lg/mL, respectively. After the
plate was incubated at 37 C in a humidified atmosphere with
5% CO2 for 72 h, 10 lL of MTT solution (5 mg/mL) were added
to each well and incubated for 4 h. The supernatant was care-
fully removed before DMSO (200 lL) were added to each well
and shaken for 15 min to dissolve formazan crystals. The
absorbance of the above DMSO solution was measured on a
Bio-Rad model 550 microplate reader (Bio-Rad Laboratories,
Hercules, CA, USA) at 570 nm. MTT solution with DMSO (with-
out both cells and medium) was used as blank control, and
the cytotoxicities of procyanidin A2 to HepG2, Hela, A549
and MCF-7 cells were calculated as:
 
As  Ac
Cytotoxicity% 1   100%
Ac
where As is the absorbance of the solution with tested com-
pound, Ac is the absorbance of blank control.
2.8. Statistical analysis
All data were expressed as the means of three replicated deter-
minations. Statistical calculations were conducted to calculate
the correlation. P-values <0.05 were regarded as significant.
3. Results
3.1. Structural identification
The EtOH extract of litchi leaf was initially partitioned
sequentially by using petroleum ether, EtOAC and n-BuOH
sequentially. Six flavonoids (compounds 16) were isolated
558 JOURNAL OF FUNCTIONAL FOODS 6 ( 2 0 1 4 ) 5 5 5 5 6 3

from the EtOAC extract with silica gel column, ODS column 2013), ()epicatechin (2) (Sun et al., 2006), kaempferol 3-O-
and Sephadex LH-20 column. Their chemical structures were b-glucoside (3) (Lu & Yeap Foo, 1999), kaempferol 3-O-a-rham-
identified on the basis of NMR and ESI-MS evidences and noside (4) (Cota et al., 2012), procyanidin A2 (5) (Kimiya,
comparison with literature. They were luteolin (1) (Ma et al., Watanabe, Endang, Umar, & Satake, 2001) and rutin (6)

5'
OH 5'
6' 4' OH
4'
6'
3'
HO O
1' HO O 3'
8 OH
7 9 2 2' 7 8 1'
2 OH
9 2'
6
10 3
5 4 6 10 3
5 4
OH
OH O
OH

5'
OH
5' 6'
OH 4'
6'
4'
HO O 3'
8 1'
HO O 3' 9 2
8 1' 7 2'
7 9 2 2' 6 10 3
6 HO 5 4
10 3 OH
5 4
3'' 4''
2''
OH O
O
OH O OH
1'' 6''
5'' 5'' 1''

6'' 3'' 2''

HO 4''

OH
15
13 OH
OH
12 16
14
14
11 HO O
HO O 16 8 12
15 13
2
7 8 9 2
7 9 11 OH
6 10 3 HO
5 4 O
OH 6 5 10 4 3 O 2'
7' 4'
OH 6' 3'
8' OH O
9' 5' 1' 6'
5'
O OH
10'
16' 2' 4' 6''
11' 3' 5''
15' 1''

14' 13' OH 3''

12' 4'' 2''
HO

OH

Fig. 1 Chemical structures of compounds 16.

 JOURNAL OF FUNCTIONAL FOODS
(Wan, Yu, Zhou, Tian, & Cao, 2011), respectively. Their chem-
ical structures are shown in Fig. 1.
The contents of luteolin, epicatechin, kaempferol 3-b-
glucoside, kaempferol 3-a-rhamnoside, procyanidin A2 and
rutin were determined by HPLC to be 0.04 0.01, 2.18 0.02,
1.05 0.08, 0.22 0.01, 8.06 0.10 and 2.34 0.02 mg/g on a ever, the scavenging activity was improved along with increas-
fresh weight basis, respectively. The flavonoids compositions
of litchi fruit and flower are similar to litchi leaf (Chang et al.,
2013). However, the individual flavonoid contents in litchi flesh
are much lower than leaf, as epicatechin and rutin levels are
1.04 and 1.13 lg/g on fresh weight basis (Zhang et al., 2013).
Litchi pericarp is a good source of above the flavonoids with
the levels of epicatechin and procyanidin A2 being 3.31 and
1.21 mg/g on fresh weight basis, respectively (Li et al., 2012).
3.2. Antioxidant activity
3.2.1. DPPH radical scavenging activity
The DPPH radical scavenging activities of compounds 16 at
different concentrations are displayed in Fig. 2. As shown in
the figure, most of the tested compounds possessed remark-
Fig. 2 DPPH scavenging activities of compounds 1-6 (a
means P < 0.01). A, DPPH scavenging activities of
procyanidin A2, epicatechin, luteolin and rutin. B, DPPH
scavenging activities of kaempferol 3-O-b-glucoside and
kaempferol 3-O-a-rhamnoside.
6 ( 20 1 4) 5 5 556 3 559
able scavenging activity against DPPH radicals in a concentra-
tion-dependent manner except for compounds 3 and 4,
whose scavenging activity was much lower than those of the
other four compounds. The possible reason was the fewer
number of hydroxyl group and occurrence of glycoside. How-
ing the concentration of samples to different degrees. The
DPPH radical scavenging activities of epicatechin and procy-
anidin A2 had no significant differences at low concentrations
(24 lM), while the difference increased with the increased
concentration, and the scavenging activity of procyanidin A2
was significantly higher than that of epicatechin at high con-
centration (>5 lM). In addtion, luteolin had a significantly
weaker DPPH scavenging activity than epicatechin, procyani-
din A2 and rutin at the same concentration, while that of rutin
was lower than those of epicatechin and procyanidin A2.
As shown in Table 1, all the six flavonoids exhibited DPPH
radical scavenging activity with IC50 values ranging from 5.08
to 113.79 lM. The IC50 values of scavenging activity were in
the increasing order of procyanidin A2 = epicatechin = querce-
tin < rutin < luteolin < BHT < kaempferol 3-O-a-rhamno-
side < kaempferol 3-O-b-glucoside. Amongst them,
procyanidin A2 and epicatechin showed the highest DPPH
radical scavenging activities with IC50 values of 5.08 0.37
and 5.54 0.28 lM, respectively.
3.2.2. Oxygen radical absorption capacity
A higher ORAC value indicates astronger oxygen radical
absorbance capacity of the sample. As displayed in Table 1,
all the tested compounds showed stronger oxygen radical
absorbance capacity than Trolox with their ORAC values
ranging from 11.91 to 30.41 lmol Trolox equiv/lmol. The
ORAC values were in the decreasing order of procyanidin
A2 = quercetin > epicatechin > rutin > kaempferol 3-O-b-glu-
coside = kaempferol 3-O-a-rhamnoside = luteolin. Similar to
the DPPH radical scavenging activity, procyanidin A2 and
epicatechin were found to be the most potent antioxdants
of the six flavonoids with ORAC values of 30.41 0.87 and
26.85 0.99 lmol Trolox equiv/lmol, while kaempferol 3-O-
b-glucoside and kaempferol 3-O-a-rhamnoside possessed
the same absorption capacity against peroxyl radical as
luteolin.
3.3. Antimicrobial activity
The minimum inhibitory concentration (MIC) of compounds
16 against S. aureus, E. coli, S. dysenteriae, Salmonella and B.
thuringiensis are listed in Table 2. Amongst the tested flavo-
noids, luteolin showed the strongest antimicrobial activity
with the minimum MIC value of 14.06 lg/mL, which was low-
er than that of kanamycin (15.63 lg/mL) against S. dysenteriae,
but higher than that of kanamycin (7.81 lg/mL) against S. aur-
eus, E. coli, Salmonella and B. thuringiensis. Other compunds like
epicatechin, procyanidin A2 and rutin had relatively weak
antimicrobial activity with minimum MIC values of 62.5 lg/
mL, while kaempferol 3-O-b-glucoside and kaempferol 3-O-
a-rhamnoside showed no antimicrobial activity in the tested
concentration range.
560 JOURNAL OF FUNCTIONAL FOODS 6 ( 2 0 1 4 ) 5 5 5 5 6 3

Table 1 Antioxidant activities of compounds 16.a

Flavonoids DPPH assay IC50 (lM) ORAC assay (lmol trolox equiv/lmol)
A
Luteolin (1) 9.98 0.71 11.91 1.24A
Epicatechin (2) 5.54 0.28B 26.85 0.99B
Kaempferol 3-O-b-glucoside (3) 113.79 1.06C 14.28 0.63A
Kaempferol 3-O-a-rhamnoside (4) 78.71 0.76D 14.20 0.54A
Procyanidin A2 (5) 5.08 0.37B 30.41 0.87C
Rutin (6) 7.40 0.12E 23.24 0.83D
Quercetin 5.52 0.51B 29.79 0.33C
BHT 38.66 1.29F b
a
The values having the same letters are insignificantly different (p > 0.05).
b
Undetermined.

Table 2 Antimicrobial activities of compounds 16.

Flavonoids MIC (lg/mL)
Staphylococcus aureus Escherichia coli Shigella dysenteriae Salmonella Bacillus thuringiensis

Luteolin 14.06 14.06 14.06 14.06 14.06

Epicatechin 62.50 62.50 62.50 62.50 62.50
Kaempferol 3-O-b-Glucoside
Kaempferol 3-O-a-Rhamnoside
Procyanidin A2 62.50 62.50 62.50 62.50 62.50
Rutin 62.50 62.50 62.50 62.50 62.50
Kanamycin 7.81 7.81 15.63 7.81 7.81
() Not detected in the tested concentrations.

cytotoxicities against human lung cancer A549 and human

breast cancer MCF-7 were less than 50% at the highest
Table 3 Cytotoxicities of procyanidin A2 against HepG2
concentration (200 lg/mL) we used.
and Hela cells.a
Concentration (lg/mL) Cytotoxicity (%)
HepG2 HeLa 4. Discussion
D D
6.25 23.43 1.28 12.14 0.79
4.1. Antioxidant activity
12.5 31.64 4.86D 19.99 3.33D
25 5.67 2.59C 11.10 2.10A
50 46.53 5.58B 37.77 5.34A As described in our previous work, the assay of scavenging
100 78.16 0.04A 80.91 1.93A the DPPH radicals is based on the reduction of DPPH
200 81.57 1.70A 82.77 1.03A solution in the presence of a hydrogen or electron donating
a
The values in a column having the same letters are in signifi- antioxidants, leading to the formation of non-radical
cantly different (p > 0.05). form DPPHH and colour change from purple to yellow.
The DPPH radical scavenging activity of flavonoids is deter-
mined by the presence of hydroxyl groups which are able
to provide hydrogen. The number of hydroxyl could be
3.4. Cytotoxicity of procyanidin A2 responsible for the highest scavenging activity of procyani-
din A2.
The in vitro cytotoxicities of procyanidin A2 were evaluated In the ORAC assay, fluorescein was used as the fluores-
against human hepatoma HepG2, human lung cancer A549, cent probe. According to the previous report, Ou, Hamp-
human breast cancer MCF-7 and human cervical carcinoma sch-Woodill, and Prior (2001) identified the fluorescein
Hela cells. The effects of procyanidin A2 on the viability of oxidized products induced by peroxyl radical with LC/MS,
Hep-G2 and Hela cancer cells at different concentrations are and found that the reaction mechanism was determined
presented in Table 3. Procyanidin A2 showed high cytotoxic to proceed as a classic hydrogen atom transfer mechanism
activity at higher concentration, its activity against human which can be elucidated based on FL oxidized products. As
hepatoma HepG2 and human cervical carcinoma Hela described above, hydroxyl groups had the ability of donating
reached 81.57 1.70 and 82.77 1.03% at 200 lg/mL, respec- hydrogen atom to prevent the radical chain reaction, like
tively. A concentration-dependent manner was observed in oxygen radical. Procyanidin A2 possessed the highest ORAC
the range of 25200 lg/mL, and their EC50 values were value as its hydroxyl number was the maximum among
62.19 5.05 and 66.07 8.55 lg/mL, respectively. The the test compounds.
 JOURNAL OF FUNCTIONAL FOODS 6 ( 20 1 4) 5 5 556 3 561

Table 4 The number of phenolic hydroxyl group and other important features of flavonoid compounds.
Sample PhenolicOH 3 0 ,4 0 -OH C2 = C3 C4 = O 3,5-OH

Luteolin (1) 4 1 1 0
Epicatechin (2) 4 1 0 1
Kaempferol 3-O-b-glucoside (3) 3 0 1 0
Kaempferol 3-O-a-rhamnoside (4) 3 0 1 0
Procyanidin A2 (5) 7 2 0 2
Rutin (6) 4 1 1 0
Quercetin 4 1 1 1

Flavonoids, a group of phenolic compounds widely present
in plants have been reported to possess strong antioxidant
activity. Previous reports indicated that antioxidant activity
of phenolics would be determined by the number and posi-
tions of hydroxyl group, glycosylation, and ortho-dihydroxy
groups were the most important structural features for the
antioxidant activity of phenolics (Cai, Mei, Jie, Luo, & Corke,
2006). The antioxidant activity of any flavonoid depended on
the presence of the o-dihydroxy structure in the B-ring (3 0 ,
4 0 -OH), the 2,3-double bond in the combination with a 4-oxo
group and the existence of both hydroxyl groups in positions
3 and 5 (Benavente-Garca, Castillo, Marin, Ortuno, & Del Ro,
1997). The test compounds had different antioxidant activi-
ties due to their structural variations. As shown in Table 4,
there are seven phenolic hydroxyl groups, two o-dihydroxy
groups and two hydroxyl groups in the position 3 and 5 (3,
5-OH) in procyanidin A2, much more than that of epicatechin,
which possess weaker antioxidant activity than procyanidin
A2. Quercetin has an identical number of hydroxyl groups at
the same positions as epicatechin but also contains a 2,3-dou-
ble bond in the combination with a 4-oxo group in the C ring,
this structure provides an enhancement of antioxidant activ-
ity as the results demonstrated. The antioxidant activity of
quercetin was much higher than luteolin, which lacked 3-
OH group. In addition, rutin, glycosylation at 3-OH group by
rutinoside, had also a weaker antioxidant activity than quer-
cetin. All these results confirmed the importance of 3-OH
group in the C ring, as Rice-Evans, Miller, and Paganga
(1996) reported. They found that 4-keto group was only func-
tional in combination with the 2,3 double bond, o-dihydroxy
groups, especially the orthodiphenolic structure in the B ring
was important to the antioxidant activity.
4.2. Antimicrobial activity
A number of flavonoids such as flavones, flavonols, flava-
nones and isoflavones, as well as their acylated derivatives,
show good antimicrobial activities (Harborne & Williams,
2000). Previous reports have indicated that antimicrobial
activities against Gram-postive and Gram-negative bacteria
were related to its inhibition of DNA and (or) protein syn-
thesis. It was important to possess at least one hydroxyl
group in ring A or B at C-3,5,7 for flavonoid, and flavonoids
without hydroxyl groups in ring B or where the hydroxyl
was replaced with other groups would turn out to be less
active or even inactive (Bylka, Matlawska, & Pilewski,
2004). Glycosides in general had less antimicrobial activity,
and that of rutin was much weaker than that of luteolin
in the present work.
4.3. Cytotoxicity
Doxorubicin, a wide-spectrum antitumor antibiotics, is effec-
tive in the treatment of human cancer, such as human breast
cancer, lung cancer (Arcamone, 1981). Xu et al. (2010) found
that the EC50 values of doxorubicin on HepG2 and Hela cancer
cells were 79.50 and 22.64 lM (43.21 and 12.31 lg/mL), respec-
tively, which were lower than that of procyanidin A2. It is wor-
thy noting that epicatechin, luteolin and rutin have
cytotoxicities against human cancer according to literatures.
Epicatechin showed weak cytotoxicity to human breast can-
cer cell MCF-7 (Zhao et al., 2007). Luteolin was demonstrated
to be cytotoxic against human oesophageal adenocarcinoma
and human hepatoma HepG2, SK-Hep-1, PLC/PRF/5, Hep3B,
and HA22T/VGH (Chang et al., 2005; Yoo et al., 2009). Rutin
had a significant stimulatory effect on human leukemia LH-
60, OCM-1 melanoma, carcinoma of human squamous cells
and colon cancer lines (Benavente-Garcia & Castillo, 2008;
Lin et al., 2012).
5. Conclusions
Luteolin, epicatechin, kaempferol 3-O-b-glucoside, kaempfer-
ol 3-O-a-rhamnoside, procyanidin A2 and rutin were purified
from litchi leaf and identified. Epicatechin and procyanidin
A2 showed good antioxidant activities. Luteolin possessed
the strongest antimicrobial activities against S. aureus,
E. coli, S. dysenteriae, Salmonella and B. thuringiensis. Epicate-
chin, procyanidin A2 and rutin also showed a weaker antimi-
crobial activity. Furthermore, procyanidin A2 was found to
exhibit good cytotoxicities against human hepatoma HepG2
and human cervical carcinoma Hela. Further studies on
determining other chemical compounds and bioactivity of
litchi leaf are worthwhile in the future for better understand-
ing and exploiting this plant.
Acknowledgements
We are grateful to the financial support from Guangdong Nat-
ural Science Funds for Distinguished Young Scholar (No.
S2013050014131), Youth Innovation Promotion Association of
Chinese Academy of Sciences, Pearl River Science and
562 JOURNAL OF FUNCTIONAL FOODS
Technology New Star Fund of Guangzhou, International
Foundation for Science (No. F/4451-2), Guangdong Natural
Science Foundation (No. S2011020001156), Science and
Technology Planning Project of Guangdong Province (No.
2011A020102006), Special Fund for Agro-Scientific Research
in the Public Interest (No. 201303073), and National Natural
Science Foundation of China (No. 3110122).
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