J. Ocean Univ.

China (Oceanic and Coastal Sea Research)

DOI 10.1007/s11802-015-2601-5
ISSN 1672-5182, 2015 14 (4): 681-684
http://www.ouc.edu.cn/xbywb/
E-mail:xbywb@ouc.edu.cn

The Anti-allergic Activity of Polyphenol Extracted

from Five Marine Algae
CHEN Yu1), LIN Hong1), LI Zhenxing1), *, and MOU Quangui2)

1) Food Safety Laboratory, Ocean University of China, Qingdao 266003, P. R. China

2) National R&D Branch Center for Kelp Processing of China, Shandong Haizhibao Ocean Science and Technology,
Co. Ltd., Yantai 264300, P. R. China

(Received February 20, 2014; revised May 9, 2014; accepted June 6, 2015)
Ocean University of China, Science Press and Spring-Verlag Berlin Heidelberg 2015

Abstract Natural polyphenol has been widely believed to be effective in allergy remission. Currently, most of the natural poly-
phenol products come from terrestrial sources such as tea, grape seeds among others, and few polyphenols have been developed from
algae for their anti-allergic activity. The aim of the study was to screen some commercial seaweed for natural extracts with anti-al-
lergic activity. Five algae including Laminaria japonica, Porphyra sp., Spirulina platensis, Chlorella pyrenoidosa and Scytosiphon sp.
were extracted with ethanol, and the extracts were evaluated for total polyphenol contents and anti-allergic activity with the hyalu-
ronidase inhibition assay. Results showed that the total polyphenol contents in the ethanol extracts ranged from 1.67% to 8.47%,
while the highest was found in the extract from Scytosiphon sp. Hyaluronidase inhibition assay showed that the extracts from Scyto-
siphon sp. had the lowest IC50, 0.67 mg mL1, while Chlorella pyrenoidosa extract had the highest IC50, 15.07 mg mL1. The
anti-allergic activity of Scytosiphon sp. extract was even higher than the typical anti-allergic drug Disodium Cromoglycate (DSCG)
(IC50 = 1.13 mg mL1), and was similar with natural polyphenol from Epigallocatechin gallate (EGCG) (IC50 = 0.56 mg mL1). These
results indicated that the ethanol extract of Scytosiphon sp. contains a high concentration of polyphenol with high anti-allergic activ-
ity. Potentially Scytosiphon sp. can be developed to a natural anti-allergic compound for allergy remission.

Key words alga; polyphenol; hyaluronidase; anti-allergic activity

polyphenols (Kojima et al., 2000), polysaccharides (Tian

1 Introduction
With advancements in food processing technology and
the internationalization of the distribution and consump-
tion of food products, food allergy is becoming an in-
creasing concern and therefore gaining more attention of
the scientific community (Sicherer, 2011). Allergic or
hypersensitivity reactions can be divided into four general
categories based on the mechanism of immunological
response. Food allergy is considered as type I allergy,
with a clinical syndrome resulted from the release of al-
lergenic media by mast cells and basophils (Breiteneder
and Ebner, 2000). Allergic reactions can cause erythrism,
asthma, anaphylactic shock and a number of other ail-
ments (Helm and Burks, 2000).
Hyaluronidase (HAase, EC 3.2.1.35), a hyaluronic acid-
splitting enzyme, is a principal compound involved in
allergic reaction (Shibata et al., 2002). It is reported that
anti-allergic agent such as DSCG (disodium chromogly-
cate) has a strong inhibitory effect on the activation of
hyaluronidase. Several natural extracts from plants such as
* Corresponding author. Tel: 0086-532-82032389
E-mail: lizhenxing@ouc.edu.cn
et al., 2012), flavonoids (Shimosaki et al., 2011) etc. are
known to possess anti-allergic activity in addition to hav-
ing low side effects and high safety to human health. Re-
cently, marine source has attracted more and more atten-
tions for the development of active compounds. Currently,
some algal extracts such as ethanol extract or hydro ex-
tract are reported to possess immune-related (Katayama
et al., 2011), anti-inflammatory (Boonchum et al., 2011),
anti-tumor (Jiao et al., 2009), and bacteriostatic activities
(Kajiwara et al., 2006). However, few studies have been
reported on the anti-allergic activity from marine sources
(Samee et al., 2009).
A large number of algae species are abundantly distrib-
uted along the coast of China. Additionally, China pro-
duced 9900 thousand tons of algae and other aquatic
plants through aquaculture in 2008 alone (FAO, 2008).
Definitive information on the anti-allergic activities of
algae from China is lacking, and little is known about the
variations among different species of algae. In the present
study, we selected five algae belonging to four different
groups with the objectives of (a) measuring their anti-
allergic properties in terms of inhibition of hyaluronidase,
(b) selecting algae with high anti-allergic activity, and (c)
providing a sound basis for the development of anti-al-
682 CHEN et al. / J. Ocean Univ. China (Oceanic and Coastal Sea Research) 2015 14: 681-684
lergic compounds and functional foods from algae.
2 Materials and Methods
2.1 Algae
Laminaria japonica (Phaeophyta) (LJ) and Porphyra
sp. (Rhodophyta) (PP) were collected from the coastal
area of Qingdao, China, in March 2011. Spirulina platen-
sis (Cyanophyta) (SP), and Chlorella pyrenoidosa
(Chlorophyta) (CP) were purchased from Lvqi Biological
Engineering Corporation of Shandong, China in March
2011. Scytosiphon sp. (Phaeophyta) (SS) was collected
from the coastal area of Tuoji Island, China, in April
2011. Collected algae were washed three times with water
to remove epiphytes, sandand salt. Washing was followed
by air-drying in a shady area to avoid direct sunlight.
Dried algae were crushed and finely ground into a powder
and passed through a 40-mesh sieve and stored at room
temperature in air-tight containers.
2.2 Chemicals
Folin-Ciocalteus phenol reagent (2 mol L1), hyalu-
ronidase from bovine testes (500 U mL1), disodium cro-
moglycate (DSCG) (95%, purity), epigallocatechin gallate
(EGCG) from green tea and polyphenol from green tea
(TP) were purchased from Sigma-Aldrich, USA. Hyalu-
ronic acid sodium salt from Streptococcus equi was pur-
chased from Fluka, Germany. All other chemicals were at
AR grade. Milli-Q purified water was used to prepare the
reagents.
2.3 Extraction of Anti-allergic Compounds
Crude extracts of LJ, PP, SP, CP, and SS were pre-
pared using the method standardized in our laboratory
(Samee et al., 2009). Approximately, 10 g of dried sea-
weed samples were immersed in 100 mL 85% ethanol in a
conical flask and stirred for 24 h at 25. The extracts
were centrifuged and the supernatant was obtained. The
residues were subjected to extractions for two times. The
supernatant were pooled, filtered and then concentrated to
about 50 mL under vacuum by a rotary evaporator at 40
for each variety separately. Concentrated samples were
washed five times with one volume of chloroform to re-
move fats and pigments by precipitation. The upper layer
was mixed with 50 mL ethyl acetate thrice and the ethyl
acetate fraction was removed using a rotary evaporator at
40. Resulting residues were weighed and dissolved in
distilled water with a ratio 1:3.
2.4 Total Polyphenol Content
Total polyphenol content of the ethanol extracts were
determined using Folin-Ciocalteus method (Singleton,
1999) with some modifications. Briefly, the samples were
diluted considering the measurable range of the spectro-
photometer. About 0.1 mL of the diluted sample was
mixed in a test tube with 0.5 mL of 2 mol L1 Folin-Cio-
calteus reagent and 0.5 mL of water, followed by addi-
tion of 2.0 mL of 20% Na2CO3 after 3 min. Samples were
incubated in dark at room temperature for 45 min fol-
lowed by centrifugation at 9000 r min1 for 10 min. Ab-
sorbance of the supernatant were measured at 730 nm
using spectrophotometer (TU-1810, China). Phlorogluci-
nol was used as the standard. Total polyphenol content
was calculated using the standard curve.
2.5 Anti-hyaluronidase Activity Assay
Anti hyaluronidase activity was assayed using modi-
fied Morgan-Elson method (Muckenschnabel, et al.,
1998). Initially, hyaluronidase was dissolved in a buffer
solution (0.2 mol L1 sodium formate, 0.1 mol L1 NaCl,
and 0.2 mg mL1 bovine serum albumin (BSA) and ad-
justed to the pH 3.04.0 with formic acid) to get a final
concentration of 500 U mL1. A solution of 5 mg mL1
hyaluronic acid was prepared in triple distilled water and
stored at 4. About 17.3 g H3BO3 and 7.8 g KOH was
dissolved in 100 mL water to get borate solution to which
0.8 g mL1 K2CO3 solution was added at ratio of 10:1 be-
fore use. The streak reagent consisted of 20 g p-dime-
thylaminobenzaldehyde, 25 mL of concentrated hydro-
chloric acid and 75 mL of glacial acetic acid. The solution
was diluted for four times with glacial acetic acid before
use.
The anti-allergic activity assay was performed as de-
scribed below. Ahead of assay, 50 L of the hyaluronic
acid stock solution, 100 L of buffer and 250 L of water
were mixed and equilibrated at 37 for 10 min. Initially,
50 L of hyaluronidase and 100 L of sample each were
mixed and incubated at 37 for 20 min. The enzymatic
reaction was terminated by adding 110 L of borate solu-
tion and holding for 4.5 min in a boiling water bath, fol-
lowed by cooling the test tubes in ice-cold water for 20
min and adding 1.5 mL of streak reagent. The tubes were
then incubated at 37 for 20 min to maximize the color.
Finally, absorbance was measured on a TU-1810 spec-
trophotometer at 585 nm. Percentage inhibition was cal-
culated by the formula:
Inhibition (%) A B C D /( A B ) 100 ,
where A is the control absorbance, test sample of A was
replaced by the buffer solution; B is the control blank
absorbance, test samples and the hyaluronidase solution
were replaced by the buffer solution; C is the sample ab-
sorbance; and D is the absorbance of the blank sample in
which the hyaluronidase solution was replaced by the
buffer solution. IC50 was calculated using the mean of
three observations from each of the five concentrations
for all the samples including the positive controls. All
reactions were performed in triplicate including those for
the three positive controls.
2.6 Statistical Analysis
In the same experiments, the treatments were per-
formed at least in triplicate on the same turbot muscle
samples. Data were analyzed using SPSS 19.0 software