Journal of Applied Phycology 13: 489–492, 2001.

© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

489

Inhibitory effects of microalgae on the activation of hyaluronidase

Naoki Fujitani1, Setsuko Sakaki1 , Yuji Yamaguchi1 & Hiroyuki Takenaka1,2,∗

1 MAC Gifu Research Institute, 4-15 Akebono, Gifu (500-8148), Japan
2 MicroAlgae Corporation, 2-6-5 Ginza, Chuo-ku, Tokyo (104-0061), Japan

(∗ Author for correspondence; fax +81-582481820; e-mail takenaka@mac-bio.co.jp)

Received 6 May 2000; revised 18 March 2001; accepted 24 March 2001

Key words: anti-allergic effect, anti-hyaluronidase activity, microalgae, water extracts, ethanol-insoluble fraction,
Nostoc flagelliforme, Spirulina, Porphyridium, Dunaliella, Pleurochrysis

Abstract
The inhibitory effects of seven microalgae, Nostoc flagelliforme, Spirulina platensis, Porphyridium purpureum,
Rhodosorus marinus, Chlorella pyrenoidosa, Dunaliella salina and Pleurochrysis carterae on the activation of
hyaluronidase were evaluated. The inhibitory effect of the ethanol-insoluble fraction of each water extract from
microalgae was stronger than that of the ethanol-soluble fraction. The 50% inhibitory concentration (IC50 ) of the
ethanol-insoluble fraction of S. platensis, P. purpureum, R. marinus, C. pyrenoidosa, D. salina and P. carterae
was 0.15, 0.18, 0.26, 0.94, 0.15 and 0.41 mg mL−1 , respectively. The IC50 of N .flagelliforme was not calculated,
because there was no detectable inhibitory effect of this alga. The IC50 of disodium cromoglycate (DSCG) used as
the anti-allergic medicine was 0.14 mg mL−1 . The IC50 of S. platensis, P. purpureum and D. salina were almost the
same as that of DSCG. This suggests that the ethanol-insoluble fraction of S. platensis, P. purpureum and D. salina
might be an anti-allergic substance. The ethanol-insoluble fraction of S. platensis and D. salina was ultrafiltered
through a membrane having a molecular exclusion limit of 20 kDa. The IC50 of the residue was stronger than that
of the filtrate. These results suggest that the anti-allergic substance(s) of these microalgae may be polysaccharides.

Introduction as one of the target enzymes of influent calcium ion

in the mast cell and may directly control the mast cell
degranulation. Thus, hyaluronidase is involved in the
There are many patients with allergy, especially atopic
action that induces inflammation. It is reported that
dermatitis through out the world. Allergic responses
anti-allergic agents and anti-inflammation agents had a
can be divided into four general categories based
strong inhibitory effect on the activation of hyaluroni-
on the mechanism of immunological involvement
dase. Therefore, it may be expected that hyaluronidase
(Coombs & Gell, 1963). Atopic dermatitis is con-
is responsible for the processes causing inflammatory
sidered a type I allergy. The pathological mechanism
and allergic reactions. The inhibitory effects on hy-
of type I allergy has been explained as degranulation
aluronidase can be measured to evaluate anti-allergic
of mast cells and the release of chemical mediators
action (Maeda et al., 1990). Microalgae were the first
such as a histamine, leucotrienes and prostaglandins
plants to grow on earth (Takenaka, 1996) and there
from these cell (Ishizaka et al., 1975, 1977, 1978,
are estimated to be well over 30,000 different spe-
1979).
cies of microalgae. Because of their great species
Hyaluronidase is a hyaluronic acid-splitting en-
diversity, photosynthetic capability, high productivity
zyme and is involved in the permeability of the vascu-
and high product concentration, microalgae represent
lar system (Duran-Reynals, 1939; Chamber & Zwei-
a major resource for biotechnology and advanced ag-
fach, 1947), and inflammation (Rovelstad et al., 1958;
ricultural production. Not only are microalgae proving
Goggins et al., 1968). Hyaluronidase may be present
490
Table 1. Yield (%) of each fraction of microalga
to be valuable sources of novel biologically active mo-
lecules, but since many species can be cultured easily, Microalga Fraction
microalgae also have the advantage of being commer- Aqueous Ethanol- Ethanol-
cially viable, and have shown the potential to produce extract insoluble soluble
chemically complex molecules that are difficult to
synthesize. Nostoc flagelliforme 9.4 7.3 2.7
Spirulina platensis 15.2 4.7 9.0
The aim of the study reported here was to in-
Porphyridium purpureum 37.7 7.8 26.7
vestigate the inhibitory effects of some components
Rhodosorus marinus 20.3 5.0 14.3
extracted from seven microalgae on the activation of
Chlorella pyrenoidosa 9.0 5.0 2.7
hyaluronidase.
Dunaliella salina 7.3 5.0 2.6
Pleurochrysis carterae 19.4 14.4 5.8

Materials and methods

Algae Measurement of inhibition of activated hyaluronidase

Nostoc flagelliforme (Cyanophyta, harvested in
China), Spirulina platensis (Cyanophyta, produced
in Hawaii) and Chlorella pyrenoidosa (Chlorophyta,
produced in Taiwan) were obtained from MicroAl-
gae Pharmaceutical Corporation (Osaka, Japan).
Porphyridium purpureum (Rhodophyta, IAMR-1),
Rhodosorus marinus (Rhodophyta, collected from
Gero hot spa, Japan), Dunaliella salina (Chlorophyta,
collected from Great Salt Lake, USA) and Pleur-
ochrysis carterae (Haptophyta, obtained from Prof.
Okazaki of Tokyo Gakugei Univ.) were cultured in
flasks at our institute. The cells were harvested by
centrifugation and immediately dehydrated by freeze-
drying.
Reagents
Hyaluronidase (from bovine testes, type IV-S) and
compound 48/80 were purchased from Sigma Chem-
ical Co. (St Louis, Mo, USA). Hyaluronic acid po-
tassium salt (from rooster comb) was purchased from
Wako Pure Chemical Industries, Ltd (Osaka, Japan).
Disodium cromoglycate (DSCG) was purchased from
BIOMOL Research Laboratory, Inc., USA.
The test sample, hyaluronic acid potassium salt, hy-
aluronidase, compound 48/80, CaCl2 and NaCl were
all dissolved in 0.1 M acetate buffer (pH 4.0).
Hyaluronidase (400 unit mL−1 ), which had been
preincubated with the test sample at 37 ◦ C for 20
min in advance, was incubated with compound 48/80
(0.1 mg mL−1 ), CaCl2 (2.5 mM) and NaCl (0.15 M)
37 ◦ C for 20 min. After incubation, hyaluronic acid
potassium salt (0.4 mg mL−1 ) was added and the re-
action mixture was incubated at 37 ◦ C for 40 min,
and then the reaction was stopped by adding 0.4 N
NaOH and allowed to stand for 10 min in ice-water.
The inhibitory effect was determined by a modifica-
tion of the method described by Morgan-Elson (Maeda
et al., 1987). Test samples were replaced by the buf-
fer solution for the control, while the enzyme solution
was replaced by buffer solution for the blank. Percent
inhibition was calculated as follows:
Inhibition (%) = {(A-B)-(C-D)}/(A-B) × 100
A: control OD585
B: control blank OD585
C: sample OD585
D: sample blank OD585

Extraction
Three grams of N. flagelliforme and P .purpureum,
10 g S. platensis, C. pyrenoidosa, D. salina and P
.carterae and 8 g R. marinus were extracted with 100
mL of water at 90 ◦ C for 180 min, and then filtered
after cooling. These aqueous extracts were concen-
trated and freeze-dried. Each crude extract was frac-
tionated into ethanol-soluble and ethanol-insoluble
fraction. The yield of each fraction is shown in Table 1.
Results
The inhibitory effects of aqueous extract, ethanol-
insoluble fraction and ethanol-soluble fraction of
S.platensis on the activation of hyaluronidase are
shown in Figure1. The inhibitory effect of the ethanol-
insoluble fraction was the strongest among the three
fractions. This tendency was also found in another
microalgae (data not shown).

Figure 1. Inhibitory effects of aqueous extract, ethanol-insoluble
fraction and ethanol-soluble fraction of Spirulina platensis on the
activation of hyaluronidase.
aqueous extract;
ethanol-insoluble
fraction;  ethanol-soluble fraction.
Figure 2. The inhibitory effects of the ethanol-insoluble fraction
of microalgae. + Nostoc flagelliforme;
Spirulina platensis; 
Porphyridium purpureum;  Rhodosorus marinus;
Chlorella
pyrenoidosa;  Dunaliella salina;  Pleurochrysis carterae.
491
Figure 3. Inhibitory effects of the residue and filtrate of the eth-
anol-insoluble fraction of Spirulina platensis.
residue;
filtrate;
 ethanol-insoluble fraction.
Figure 2 shows the inhibitory effects of the
ethanol-insoluble fraction of microalgae and the 50%
inhibitory concentration (IC50 ) of each fraction of S.
platensis, P. purpureum, R.marinus, C. pyrenoidosa,
D. salina and P. carterae was 0.15, 0.18, 0.26, 0.94,
0.15 and 0.41 mg mL−1 , respectively. The IC50 of N.
flagelliforme was not detected. The IC50 of disodium
cromoglycate (DSCG) used as the anti-allergic medi-
cine was 0.14 mg mL−1 in this study. The IC50 of
S. platensis, P. purpureum and D. salina were almost
the same as that of DSCG. This result suggested that
the ethanol-insoluble fraction of S. platensis, P. pur-
pureum and D. salina might include an anti-allergic
substance.
The ethanol-insoluble fractions of S. platensis
and D. salina were ultrafiltered through a membrane
(USY-20, ADVANTEC, Tokyo, Japan) having a mo-
lecular exclusion limit of 20 kDa. Figure 3 shows
the inhibitory effects of the residue and filtrate of S.
platensis and the IC50 of the residue and filtrate was
0.08 and >2.00 mg mL−1 , respectively. The activ-
ity of the residue was stronger than that of DSCG.
The residue of D. salina indicated the same tendency.
These results suggested that the anti-allergic substance
of microalgae might be polysaccharides.
Discussion
Kanno et al. (1996) reported that the ethanol-insoluble
fraction of Chlorella included macromolecules such
as polysaccharides and the ethanol-soluble fraction
included low molecules such as alanine, glutamic
492
acid and oligopeptide. The polysaccharides contain
D-galacturonic acid from Gymnema sylvester and pec-
tic substances containing D-galacturonic acid from
Citrus reticulata (Ponkan mandarin) inhibited the ac-
tivation of hyaluronidase (Sawabe et al., 1990; Maeda
et al., 1991). It is suggested that one of substances in
microalgae that inhibits hyaluronidase may be poly-
saccharide.
Kakegawa et al. (1983, 1985) reported that there
was a good correlation between the inhibitory effect
on hyaluronidase and that of histamine release from
mast cell. It has been also reported that a hyalur-
onidase inhibitor is presumed to have an anti-allergic
activity on mast cell (Kakegawa et al., 1984). Yang et
al. (1977) reported that Spirulina decreased the serum
histamine level and inhibited compound 48/80 or
anti-dinitropheny IgE-induced histamine release from
peritoneal mast cell in rats. The ethanol-insoluble frac-
tion of S.platensis and other microalgae inhibited the
activation of hyaluronidase in this study. These find-
ings suggest that microalgae might have anti-allergic
activities that could be of potential clinical interest.
Acknowledgements
The authors thank Prof. Shepley Chen of University of
Illinois for pertinent advice. Thanks are also due to Mr
H. Tanaka and Miss H. Seki of MAC Gifu Research
Institute for their efforts in cultivating microalgae.
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