全 文 ：　　　　　　 天然产物研究与开发　 　　　　　
2005　Vol.17　No.5 NATURAL PRODUCT RESEARCH AND DEVELOPMENT　 　　　　　　　　
　
　
　
　
　　Received Febrary 22 , 2005;Accepted March 8 , 2005
　　Fundation Item:This project was supported by agricultural research foun-
dation of Guangdong province(No.2003A203507)
　＊Corresponding author　E-mail:yangbao@china.com.cn
Identification of the Major Flavanols in Litchi Pericarp
YANG Bao＊ ,ZHAO Mou-ming ,LIU Yang ,LI Bao-zhen
(College of Light Industry and Food Science , South of China University of Technology , Guangzhou 510640 , China)
Abstract:Litchi pericarp contains significant amounts of polyphenolic compounds.Solvent classification of crude flavonoids ex-
tracts from litchi pericarp was used in this research.The results showed ethyl acetate fraction gathered the most flavonoids.The
major flavonoids(P1 , P2 and P3)were isolated by using reverse phase liquid chromatography.Ultraviolet and visible light scan-
ning analysis showed they were flavanols.Nuclear magnetic resonance(NMR)spectra and mass spectra(MS)analysis further in-
dicated P1 , P2 and P3 were proanthocyanidin B4 , proanthocyanidin B2 and epicatechin , respectively.
Key words:litchi pericarp;epicatechin;proanthocyanidin B4;proanthocyanidin B2;NMR;MS
荔枝壳主要黄烷醇类物质分析
杨　宝＊ ,赵谋明 ,刘　洋 ,李宝珍
(华南理工大学轻工与食品学院 , 广州 510640)
摘　要:本研究采用乙醇溶液提取荔枝壳中的黄酮 ,并利用不同有机溶剂进行分级。通过反相液相色谱对主要
单体成分 P1 , P2 和 P3分离纯化。经紫外可见光扫描分析表明为黄烷醇类物质。通过核磁共振波谱和质谱分析
证明 P1 , P2和 P3分别为表儿茶素 , 原花青素 B2 和原花青素 B4。
关键词:荔枝壳;表儿茶素;原花青素 B2;原花青素 B4;核磁共振波谱;质谱
中图分类号:TS202.3;Q946.91　
The litchi(Litchi chinensis Sonn.)is an exotic fruit in
southeast Asia , especially in China.In the last 30 years ,
other semitropical regions began to plant litchi tree[ 1] .It
has a delicious taste and lovely shape , and is desired by
many people.In China , annual litchi production is about
1.5million tons.Litchi pericarp accounts for approximate-
ly 15 percent of the total weight of fresh fruit.It contains
significant amounts of flavonoids and can be taken as an
important bioactive substances resource to produce func-
tional food.
At present , most papers of litchi pericarp reported the
browning phenomena of litchi pericarp and how to inhibit
it[ 2-4] .Very few reports were combined with identification
of the polyphenols in litchi pericarp.A systematic study of
litchi pericarp compositions will be helpful to know the
mechanism of browning and find more effective way to re-
solve this problem.This research isolated the major
flavonoids by using reverse phase liquid chromatography ,
and determined its molecular weight and chemical struc-
ture by using MS and NMR , respectively.
Results and Discussion
Comparison of percentage of total flavonoids content
in four extracts
Litchi pericarp comprised flavnonoids with different polar-
ity.So they could be partitioned by hexane , ethyl acetate ,
butanol and water.Hayder et al.[ 5] have used hexane ,
chloroform and ethyl acetate to partition extracts of
flavonoids.Ethyl acetate extract gathered most of the
flavonoids in extracts , accounting for 83.1% of total
falvonoids.The hexane fraction ,butanol fraction and water
fraction accounted for 0.2%,13.6% and 3.1%, respec-
tively.
Isolation and identification of P1 ,P2 and P3 fraction
Reverse phase HPLC gave a successful resolution of major
phenol compounds[ 6 , 7] .The profile of reverse phase HPLC
showed three sharp peaks(P1 ,P2 and P3)(Fig.1).The
peak of P3 was substantially higher than the other two.
This meant P3 fraction was the major flavonoid in litchi
pericarp.These three fractions were collected for further i-
dentification.
577
Fig.1　Elution profiles of ethyl acetate extract separated by
reverse phase HPLC
Fig.2　Molecular formula of epicatechin, proanthocyanidin
B2 and proanthocyanidin B4
Gerardo
[ 8]
have reported that Flavanol has maximum ab-
sorbance at about 280 nm.Flavonol has maximum ab-
sorbance at about 350 nm.Anthocyanin has maximum ab-
sorbance at about 280 nm and 520 nm.The UV-visible
scanning results showed these three compounds had maxi-
mum absorbance around 280 nm.This meant that they
were flavanols.The results of UV-visible scanning , NMR
andMS of P1 ,P2 and P3 were as follows.
P1:UVλmax at 279 nm;[M-H] - peak at m/z 577.1;13
C NMR:29.6 , 38.9 , 67.7 , 73.7 , 79.9 , 83.8 , 83.9 ,
96.3 ,97.5 , 97.7 , 99.5 , 108.2 , 114.2 ～ 115.9 , 119.2 ,
120.3 ,131.2 ～ 132.6 ,145.6 ～ 146.5 ,155.4 ～ 158.7.It
was proved to be proanthocyanidin B4(Fig.2).
P2:UVλmax at 279.5 nm;[M-H] -peak at m/z 577.3;
13C NMR:29.3 , 36.9 , 66.4 , 72.5 , 76.3 , 79.2 , 95.8 ,
96.3 , 97.2 , 100.5 , 102.1 , 107.4 , 114.9 ～ 115.8 ,
119.2 , 119.3 , 131.4 , 131.6 , 145.0 ～ 146.1 , 155.1 ～
157.9.It was proved to be proanthocyanidin B2(Fig.2).
P3:UVλmax at 278 nm;[M-H] - peak at m/z 289.1;
13C NMR:28.9(C-4),66.9(C-3), 79.4(C-2),95.5(C-
8), 96.2(C-6), 99.7(C-4a), 115.3(C-2′), 115.6(C-
5′),119.3(C-6′), 132.1(C-1′), 145.3(C-3′), 145.5
(C-4′),157.0(C-5), 157.5(C-7),157.6(C-8a).It was
proved to be epicatechin(Fig.2).
Experimental
Plant material
Fresh litchi fruit(Litchi chinensis Sonn.)cv.Huaizhi at
the commercially mature stage were picked up from a
commercial orchard in Guangzhou ,China.Fruits were se-
lected for uniformity of shape and colour , then stored at 4
℃ in refrigerator.
Extraction of polyphenolic compounds
Phenolic compounds preparation and partition were done
according to the method reported by Lee and Wicker
[ 9]
and Argolo et al.[ 10] with some modifications.Five grams
of lyophilized pericarp ground in liquid nitrogen were ex-
tracted three times with 100 mL of ethanol/1.5M HCl
(85:15 , v/v)for 2 hours at 4 ℃.The three extracts were
pooled and filtered on filters before concentration under
vacuum at 40 ℃.The dried extract was dissolved in water
(100 mL)and partitioned successively with hexane , ethyl
acetate and butanol , yielding the hexane , ethyl acetate and
butanol extracts , respectively.The total flavonoids content
in each extract was determined by aluminum nitrate
method
[ 11] .
Purification of the major polyphenolic compounds
The ethyl acetate extract was evaporated to dryness under
vacuum at 40 ℃ and dissolved in ethanol.It was submit-
ted to semipreparative scale HPLC procedure on a reverse
phase polystyrene/divinyl column (100×6.4 mm i.d.)
(Pharmacia ,Swiss).Elution conditions were as follows:2
mL/min flow rate;solvent A , water/ formic acid (98:2 ,
v/v);solvent B , acetonitrile/water/ formic acid (80:19:
1 , v/v/v);isocratic for 4 min with 3%B , from 3 to 50%
B in 14 min.The compounds were detected at 280 nm.
The elution volume of three main peaks was collected , e-
vaporated under vacuum , and freeze-dried to obtain com-
pounds(P1 ,P2 and P3)in a pure dried state.
UV-visible spectophotometric analysis
P1 ,P2 and P3 were prepared in ethanol.The spectra was
measured on UV-2102 PC UV-visible spectrophotometer
578 天然产物研究与开发　　　　　　　　　　　　　 　　　2005　Vol.17　No.5
(Unico ,China)and recorded from 200 nm to 580 nm.
Molecular weight estimation by ESI-MS
MS system (LCQDECA , Finigan company , USA)equipped
with a Hewlett-Packard 9000 computer system was used to
detect the molecular weight.Mass spectroscopy was
recorded with a heat capillary voltage of 4.5 kV , a heat
capillary temperature of 270 ℃, sheath gas flow rate of 70
units , and auxiliary gas flow rate of 10 units.The scan
range of m/z was 200 ～ 1200.
NMR spectroscopy
13C NMR spectra was measured at 30 ℃with a using 3
mm tubes with MeOH-d4 as the solvent.13 C chemical
shifts was given in ppm relative to TMS as an internal
standard.
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5792005　Vol.17　No.5 杨　宝等:荔枝壳主要黄烷醇类物质分析