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Identification of flavonoids in litchi (Litchi chinensisSonn.) leaf and evaluation of anticancer activities

1756-4646/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.jff.2013.11.022

* Corresponding author. Tel.: +86 20 37083042; fax: +86 20 37252960.E-mail address: yangbao@scbg.ac.cn (B. Yang).

Lingrong Wena,b, Dan Wua,c, Yueming Jianga, K. Nagendra Prasadd, Sen Lina, GuoxiangJianga, Jirui Hea,c, Mouming Zhaob, Wei Luob, Bao Yanga,*

aKey Laboratory of Plant Resource Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences,

Guangzhou 510650, ChinabCollege of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, ChinacUniversity of Chinese Academy of Sciences, Beijing 100049, ChinadSchool 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:

Received 10 October 2013

Received in revised form

21 November 2013

Accepted 22 November 2013

Available online 17 December 2013

Keywords:

Purification

Flavonoid

Cytotoxity

NMR

Litchi leaf

Six flavonoids, namely luteolin (1), epicatechin (2), kaempferol 3-O-b-glucoside (3),

kaempferol 3-O-a-rhamnoside (4), procyanidin A2 (5) and rutin (6) were purified from the

EtOAc-soluble extract of litchi leaf by column chromatography. Their structures were

elucidated by nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry

(MS) evidences. Luteolin and kaempferol 3-O-a-rhamnoside were found from this plant

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

activities against human lung cancer A549 and human breast cancer MCF-7 cells. Luteolin,

epicatechin, procyanidin A2 and rutin showed good antioxidant activities than butylated

hydroxytoluene (BHT). The antimicrobial activity assay indicated that luteolin possessed

the strongest antimicrobial activity against Staphylococcus aureus, Escherichia coli, Shigella

dysenteriae, Salmonella and Bacillus thuringiensis. Epicatechin, procyanidin A2 and rutin

showed relatively weak antimicrobial activities.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

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-

inflammatory, analgesic and antipyretic activities. The

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,

556 J O U R N A L O F F U N C T I O N A L F O O D S 6 ( 2 0 1 4 ) 5 5 5 – 5 6 3

& 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 (100–200 or

200–300 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-

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 (26–34 �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 CHCl3–MeOH solvent togain fractions E1-E20. Fractions

E6–E8 (5.318 g) was purified by silica gel again to obtain frac-

tions E6–1–E6–12, 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 MeOH–H2O, 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

J O U R N A L O F F U N C T I O N A L F O O D S 6 ( 2 0 1 4 ) 5 5 5 – 5 6 3 557

positive standard. The DPPH radical scavenging activity was

expressed as:

Scavenging activityð%Þ ¼ 1�As�AcA

� �� 100%

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 NaH2PO4–Na2HPO4 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 NaH2PO4–Na2HPO4 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 NaH2PO4–Na2HPO4 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 NaH2PO4–Na2HPO4 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:

ACU ¼Xn

i¼1

2f i � f0 � fn

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 ¼ 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

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

(1st–10th column). Then 100 lL of tested samples were added

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:

Cytotoxicityð%Þ ¼ 1�As�AcAc

� �� 100%

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 1–6) were isolated

558 J O U R N A L O F F U N C T I O N A L F O O D S 6 ( 2 0 1 4 ) 5 5 5 – 5 6 3

from the EtOAC extract with silica gel column, ODS column

and Sephadex LH-20 column. Their chemical structures were

identified on the basis of NMR and ESI-MS evidences and

comparison with literature. They were luteolin (1) (Ma et al.,

OOH

HO OOH

OH

2

345

67

89

10

1'2'

3'

4'5'

6'

OOH

HO O

OH

2

345

6

78

9

10

1'2'

3'

4'

5'

6'

1''

2'' 3'' 4''

5''

6''

O

O

OHHO

OH

O

OH

OH

OH

HO

OH

OH

2

345

6

7 8910

11

1213

14

15

2'3'

4'

5'

6'7'

8'9'

10'

11'

12'13'14'

15'

16

16'

O

HO

OH

OHHO

Fig. 1 – Chemical structur

2013), (�)–epicatechin (2) (Sun et al., 2006), kaempferol 3-O-

b-glucoside (3) (Lu & Yeap Foo, 1999), kaempferol 3-O-a-rham-

noside (4) (Cota et al., 2012), procyanidin A2 (5) (Kimiya,

Watanabe, Endang, Umar, & Satake, 2001) and rutin (6)

OH

OH

HO OOH

OH

2

345

6

7 89

10

1'2'

3'

4'5'

6'

OOH

HO O

OH

2

345

6

78

9

10

1'2'

3'

4'

5'

6'

1''

2''3''4''

5''6''

O

OOH

HO OOH

OH

7 2

3456

89

10

1112 13

14

15

16

1''

2''3''4''

HO

5''

1'

2'

3'

4'

5'6'

6''

es of compounds 1–6.

J O U R N A L O F F U N C T I O N A L F O O D S 6 ( 2 0 1 4 ) 5 5 5 – 5 6 3 559

(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

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 activityThe DPPH radical scavenging activities of compounds 1–6 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.

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-

ever, the scavenging activity was improved along with increas-

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

(2–4 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 capacityA 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

1–6 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.

Table 1 – Antioxidant activities of compounds 1–6.a

Flavonoids DPPH assay IC50 (lM) ORAC assay (lmol trolox equiv/lmol)

Luteolin (1) 9.98 ± 0.71A 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 3 – Cytotoxicities of procyanidin A2 against HepG2and Hela cells.a

Concentration (lg/mL) Cytotoxicity (%)

HepG2 HeLa

6.25 �23.43 ± 1.28D �12.14 ± 0.79D

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

100 78.16 ± 0.04A 80.91 ± 1.93A

200 81.57 ± 1.70A 82.77 ± 1.03A

a The values in a column having the same letters are in signifi-

cantly different (p > 0.05).

Table 2 – Antimicrobial activities of compounds 1–6.

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.

560 J O U R N A L O F F U N C T I O N A L F O O D S 6 ( 2 0 1 4 ) 5 5 5 – 5 6 3

3.4. Cytotoxicity of procyanidin A2

The in vitro cytotoxicities of procyanidin A2 were evaluated

against human hepatoma HepG2, human lung cancer A549,

human breast cancer MCF-7 and human cervical carcinoma

Hela cells. The effects of procyanidin A2 on the viability of

Hep-G2 and Hela cancer cells at different concentrations are

presented in Table 3. Procyanidin A2 showed high cytotoxic

activity at higher concentration, its activity against human

hepatoma HepG2 and human cervical carcinoma Hela

reached 81.57 ± 1.70 and 82.77 ± 1.03% at 200 lg/mL, respec-

tively. A concentration-dependent manner was observed in

the range of 25–200 lg/mL, and their EC50 values were

62.19 ± 5.05 and 66.07 ± 8.55 lg/mL, respectively. The

cytotoxicities against human lung cancer A549 and human

breast cancer MCF-7 were less than 50% at the highest

concentration (200 lg/mL) we used.

4. Discussion

4.1. Antioxidant activity

As described in our previous work, the assay of scavenging

the DPPH radicals is based on the reduction of DPPH

solution in the presence of a hydrogen or electron donating

antioxidants, leading to the formation of non-radical

form DPPH–H 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

responsible for the highest scavenging activity of procyani-

din A2.

In the ORAC assay, fluorescein was used as the fluores-

cent probe. According to the previous report, Ou, Hamp-

sch-Woodill, and Prior (2001) identified the fluorescein

oxidized products induced by peroxyl radical with LC/MS,

and found that the reaction mechanism was determined

to proceed as a classic hydrogen atom transfer mechanism

which can be elucidated based on FL oxidized products. As

described above, hydroxyl groups had the ability of donating

hydrogen atom to prevent the radical chain reaction, like

oxygen radical. Procyanidin A2 possessed the highest ORAC

value as its hydroxyl number was the maximum among

the test compounds.

Table 4 – The number of phenolic hydroxyl group and other important features of flavonoid compounds.

Sample Phenolic–OH 3 0,40-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

J O U R N A L O F F U N C T I O N A L F O O D S 6 ( 2 0 1 4 ) 5 5 5 – 5 6 3 561

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-Garcıa, Castillo, Marin, Ortuno, & Del Rıo,

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 J O U R N A L O F F U N C T I O N A L F O O D S 6 ( 2 0 1 4 ) 5 5 5 – 5 6 3

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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