Sacrolide A, a new antimicrobial and cytotoxic oxylipin macrolide from the edible cyanobacterium Aphanothece sacrum

Article abstract of DOI:10.3762/bjoc.10.190

Macroscopic gelatinous colonies of freshwater cyanobacterium Aphanothece sacrum, a luxury ingredient for Japanese cuisine, were found to contain a new oxylipin-derived macrolide, sacrolide A (1), as an antimicrobial component. The configuration of two chi

Full text of DOI:10.3762/bjoc.10.190

Sacrolide A, a new antimicrobial and cytotoxic oxylipin
macrolide from the edible cyanobacterium
Aphanothece sacrum
Naoya Oku1, Miyako Matsumoto1, Kohsuke Yonejima1, Keijiroh Tansei2
and Yasuhiro Igarashi*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2014, 10, 1808–1816.
1Biotechnology Research Center and Department of Biotechnology,
Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama
939-0398, Japan and 2Suizenjinori-Hompo Tanseidoh, 5-13-3 Tsuboi,
Chuo-ku, Kumamoto, Kumamoto 860-0863, Japan
doi:10.3762/bjoc.10.190
Received: 21 April 2014
Accepted: 14 July 2014
Published: 07 August 2014
Email:
Yasuhiro Igarashi* - yas@pu-toyama.ac.jp
This article is part of the Thematic Series "Natural products in synthesis
and biosynthesis".
* Corresponding author
Guest Editor: J. S. Dickschat
Keywords:
Aphanothece sacrum; cyanobacterium; food intoxication; natural
products; sacrolide A; suizenji-nori
© 2014 Oku et al; licensee Beilstein-Institut.
License and terms: see end of document.
Abstract
Macroscopic gelatinous colonies of freshwater cyanobacterium Aphanothece sacrum, a luxury ingredient for Japanese cuisine, were
found to contain a new oxylipin-derived macrolide, sacrolide A (1), as an antimicrobial component. The configuration of two chiral
centers in 1 was determined by a combination of chiral anisotropy analysis and conformational analysis of different ring-opened
derivatives. Compound 1 inhibited the growth of some species of Gram-positive bacteria, yeast Saccharomyces cerevisiae and
fungus Penicillium chrysogenum, and was also cytotoxic to 3Y1 rat fibroblasts. Concern about potential food intoxication caused
by accidental massive ingestion of A. sacrum was dispelled by the absence of 1 in commercial products. A manual procedure for
degrading 1 in raw colonies was also developed, enabling a convenient on-site detoxification at restaurants or for personal
consumption.
Introduction
Cyanobacteria continue to be core sources for bioactive second- As a part of our program to exploit untapped microbes for drug
ary metabolites [1,2], and their significance in drug discovery discovery, we focus on cyanobacteria that are eaten in Japan,
has increased for the past two decades [3]. Some cyanobacteria and previously reported the isolation of an unusual ω-1 fatty
form macroscopic colonies and are harvested for human acid (9Z,12Z)-9,12,15-hexadecatrienoic acid from a freshwater
consumption in many parts of the world [4].
periphytic cyanobacterium Nostoc verrucosum [4].
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Beilstein J. Org. Chem. 2014, 10, 1808–1816.
Aphanothece sacrum is also an edible cyanobacterium, which is (Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus,
an endemic species in the Aso water system in the Kyushu and Streptomyces lividans), one Gram-negative bacterium
District, Japan. It inhabits oligotrophic but mineral-rich fresh- (Escherichia coli), two yeasts (Candida albicans and Saccha-
water such as spring-fed ponds with a narrow temperature range romyces cerevisiae), and two fungi (Aspergillus oryzae and
of 18 °C–20 °C [5] and forms thin crispate gelatinous floating Penicillum chrysogenum) revealed that all these layers varied in
colonies. The first record of this alga being consumed as food the extent of activity against certain test organism(s). Espe-
appeared 250 years ago. Various laver sheets, such as suizenji- cially intriguing was the antifungal activity exhibited by the
nori, akizuki-nori, jusentai, or shikintai, were developed and n-hexane-soluble fraction, which, in most cases, comprises
designated as regional specialty goods by the local lords [6]. lipids. To identify the responsible constituent, the fraction was
These products have also been presented as a tribute to the successively purified by silica gel chromatography, gel filtra-
shogun family, and because of its economic value, the feudal tion on Sephadex LH-20, and finally by ODS HPLC to yield 1
domains protected habitats from overharvesting and water (1.5 mg, 3.0 × 10−4% w/w). In addition, the antifungal prin-
pollution. In 1924, the government designated the habitat in ciple in the aqueous MeOH fraction was isolated and identified
Kamiezuko Pond as a national treasure. However, with the as the same substance (6.0 mg, 1.2 × 10−3% w/w).
increasing urbanization in this region, the supply of under-
ground water has decreased significantly, which further The molecular formula C18H28O4 was assigned to 1 based on a
narrowed the originally limited distribution of this species [7]. molecular ion m/z 331.1889 [M + Na]+ observed by HRMS
The only remaining natural habitat is reportedly in the Kogane- (ESI) (calcd for C18H28NaO4, 331.1880). Of the five degrees of
gawa Stream, a 2 km-long shallow stream where two compa- unsaturation calculated from the molecular formula, four were
nies engage in aquaculture [8]. A. sacrum is officially cata- accounted for shielded carbonyl (δC 196.7, Table 1) and
logued as a Class IA endangered species by the Ministry of carboxyl (δC 172.9) groups, and two double bonds (δC 149.3,
Environment [9].
135.6, 124.5, and 121.9) as observed in the 13C NMR spectrum.
Thus, the remaining one degree of unsaturation is attributed to a
The aquaculture industry has supported chemical investigations cyclic structure. Because a resonance for a formyl proton (δH
of this rare alga. However, to date, only two substances have 9–10) was not observed in the 1H NMR spectrum, the carbonyl
been reported: pseudovitamin B12 [10] and sulfated polysaccha- group must be part of a conjugated ketone. Other structural
ride sacran [11].
pieces were deduced from an HSQC spectrum to be two oxyme-
thines (δH/δC 5.23/77.3 and 4.50/71.1), nine methylenes (δH/δC
We evaluated the antimicrobial activity of this alga and found 1.74/33.81; 2.36, 2.50/33.82; 2.61, 2.56/28.7; 1.50/26.5; 1.35/
that a lipophilic fraction of an ethanolic extract showed 26.4; 1.34/25.8; 1.68/24.3; 1.28/21.7; 2.06/20.8), and a methyl
inhibitory activity against several microbes. Activity-guided group (δH/δC 0.97/14.1), which indicated that a lactone linkage
fractionation resulted in the discovery of a new macrolactonic is the only possible cyclization topology. The analysis of the
oxylipin, sacrolide A (1, Figure 1).
COSY spectrum (Figure 2) helped trace six-carbon and ten-
carbon spin systems, both of which include an oxymethine (H9:
δH 4.50, H13: δH 5.23) and a disubstitued olefin. The
geometry of the olefins was determined as E for Δ10 and Z for
Δ15 based on the coupling constants JH10,H11 ~ 15.8 Hz and
JH15,H16 ~ 10 Hz, respectively. The former spin system
(O-H13-H214-H15=H16-H217-H318) had a methyl group at
one end and was therefore deemed to constitute a side chain
group, whereas the latter (-H22-H23-H24-H25-H26-H27-H28-
H9(-O-)-H10=H11-) were bidirectionally opened by a meth-
ylene and a double bond at the termini and should constitute a
body of the macrocycle core. The connectivity of the two frag-
ments was verified by HMBC correlations from H10 (δH 6.93),
H11 (δH 6.56), H13 (δH 5.23), and H214 (δH 2.61, 2.56) to C12
(δC 196.7), forming an α,β-unsaturated α'-acyloxy ketone func-
Figure 1: Structure of sacrolide A (1).
Results and Discussion
A. sacrum (500 g) was extracted with EtOH, and the combined tionality, and from H22 (δH 2.50, 2.36), H3 (δH 1.68), and H13
extract was fractionated by solvent partitioning into n-hexane-, to C1 (δC 173.1), bridging a lactone linkage. The structure
10% aqueous MeOH-, 1-BuOH-, and water-soluble fractions. assembled to this point used up C18H27O4 of the molecular
Antimicrobial testing against four Gram-positive bacteria formula C18H28O4 and a remaining proton, not observed in the
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Beilstein J. Org. Chem. 2014, 10, 1808–1816.
1H spectrum, was assigned to a hydroxy proton (9-OH). Thus,
the planar structure of 1 was determined to be a new
14-membered macrolide.
Table 1: 1H (500 MHz) and 13C (125 MHz) NMR data for sacrolide A
(1) in CDCl3 (δ in ppm).
Position δC
δH, mult. (J in Hz),
HMBC
integr.
(1H to 13C)
Figure 2: Selected COSY (bold lines) and HMBC (arrows) correla-
tions for sacrolide A (1).
1
2
172.9
33.81
2.36, m, 1H
2.50, m, 1H
1.68, m, 2H
1.34, m, 2H
1.35, m, 2H
1.50, m, 2H
1.28, m, 2H
1.74, m, 2H
4.50, dt (4.7, 4.5), 1H
1
1
The stereogenic center C13 constitutes an α-acyloxy ketone
moiety and deacylation catalyzed by a base may result in the
loss of chirality because of epimerization [12]. To circumvent
this problem, we initially attempted to protect the ketone group
prior to chemical conversion (Scheme 1). Compound 1 was
acetylated (Supporting Information File 1) and subjected to
protection as 1,3-dithiane [13] or 1,4-dinitrophenylhydrazone
[14]; however, neither of them resulted in the desired products.
Then a strategy without protection was explored (Scheme 2).
Reduction with L-selectride (lithium tri-sec-butylborohydride)
gave the desired alcohol 3 and its ester-exchanged isomer 2
with a ratio of 2:1. To facilitate chiral anisotropy analysis at C9
and C12, the more abundant 3 was condensed either with (S)- or
(R)-α-methoxyphenylacetic acid (MPA) [15] to give bis-MPA
esters 3a and 3b. The δΔ values for the 2-en-1,4-diol moiety do
not hold a diagnostic value because this region is under the
influence of overlapping anisotropic effects by the flanking of
two MPA residues. However, the consistent distribution of posi-
tive and negative values at the right half and on the side chain
3
24.3
25.8
26.4
26.5
21.7
33.82
71.1
149.3
124.5
196.7
77.3
28.7
1, 2, 4, 5
2, 5
4
5
6
6
4, 5
7
4, 5, 6, 8, 9
5, 7, 9, 10
7, 8, 11
8
9
10
11
12
13
14
6.93, dd (4.7, 15.8), 1H 8, 9, 11, 12
6.56, d (15.8), 1H
9, 12
5.23, dd (6.0, 7.6), 1H
2.61, m, 2H
1, 12, 14, 15
12, 13, 15,
16
2.56, m, 1H
12, 13, 15,
16
15
16
17
18
121.9
135.6
20.6
5.30, td (7.9, 9.8), 1H
5.54, td (7.6, 10.0), 1H
2.06, qd (7.6, 7.4), 2H
0.97, t (7.6), 3H
15, 16, 18
16, 17
14.1
Scheme 1: Initial derivatization strategy for the stereochemical analysis of sacrolide A (1).
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Beilstein J. Org. Chem. 2014, 10, 1808–1816.
Scheme 2: Determination of the stereochemistry of sacrolide A (1).
of the molecule was most reasonably explained by R-configura- dation at the α'-position, and macrolactonization of the resulting
tions for both the chiral centers. Thus, the (9R,12R)-configur- α',γ-ketodiol would yield 1. Similar, but rather simpler metabo-
ation was established for 3.
lites were found from corn [17,18]. However, they comprise a
pair of enantiomers and thus should be generated non-enzymati-
cally from α-linoleic acid-derived allene oxide. In contrast, 1 is
optically active and is more likely to be produced by a sequence
of enzymatic reactions.
To correlate the R-configuration of C12 to the chirality of C13,
3 was treated with NaOMe in methanol, and the obtained
product triol 4 was converted to acetonide 5. The conformation
of the 2,2-dimethyl-1,3-dioxolane ring in 5 was analyzed by
NOESY and HSQC experiments, both of which confirmed a Sacrolide A (1) inhibited the growth of Gram-positive bacteria
trans relationship of H12 and H13 as evidenced by one-to-one (Micrococcus luteus, Streptomyces lividans, Staphylococcus
NOE correlations between the acetonide methyl groups and aureus, Bacillus subtilis), a yeast (Saccharomyces cerevisiae)
oxymethine protons. Therefore, it was concluded that C13 has and a fungus (Penicillium chrysogenum) (Table 2). Moreover, 1
an R-configuration.
was cytotoxic to 3Y1 rat fibroblasts (GI50 4.5 μM). Under
microscopic observation, cells exposed to 13 μM of 1 rapidly
Sacrolide A (1) is apparently a member of the oxylipins, lipid lost anchorage to the substratum. Blebs developed on the
oxidation products reported from all organisms, and its biosyn- cellular surface (Figure 3B) and the cells eventually detached
thesis should be routed through α-linolenic acid-derived allene from the substratum and died (Figure 3C). Because this series
oxide (Scheme 3) [16]. The addition of H2O to this intermedi- of changes occurred within a time frame which was much
ate gives α,β-unsaturated-γ-ketol, which accepts further oxi- shorter than the doubling time of the cells, 1 is is not expected
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Beilstein J. Org. Chem. 2014, 10, 1808–1816.
Scheme 3: Plausible biosynthesis of sacrolide A (1).
to target cell cycle-associated events, such as the synthesis of
proteins, DNA, or RNA. Despite their structural diversity, many
oxylipins induce a common stress response in plants and animal
cells, and this activity is attributed to their ability to modify the
cysteinyl residues in proteins, thereby affecting redox-
controlled signal transduction pathways [19]. The induction of
membrane blebbing has been reported for lipophilic inhibitors
of protein phosphatase 2A, which is a cysteine-dependent phos-
phatase [20,21]. However, 1 did not inhibit this enzyme (data
not shown).
Table 2: Antimicrobial activity of sacrolide A (1).
MICa value
(μg/mL)
microbe
Gram-positive
Bacillus subtilis
>8.0
>8.0
0.5
Micrococcus luteus
Staphylococcus aureus
Streptomyces lividans
1.0
Gram-negative
yeast
Escherichia coli
>8.0
Candida albicans
8.0
Saccharomyces
cerevisiae
>8.0
In Japanese cuisine, A. sacrum is used as an ingredient in soups
and marinades or as a garnish for sashimi (thin sliced raw fish).
It is sold dry or brined for nationwide distribution or as a fresh
food item directly to local restaurants. Although a massive
ingestion is rare to happen, the cytotoxicity of 1 raised concerns
about potential food intoxication. To evaluate the presence of 1
fungus
Penicillium chrysogenum 1.0
aMinimum inhibitory concentration.
in these products, extracts were prepared and examined by anti- antimicrobial screening. The aqueous 90% MeOH fraction of
fungal testing and LC–MS analysis. The products were soft- the raw sample exhibited significant antifungal activity (12 mm/
ened or desalted by soaking in water and then subjected to the 6 mm Φ disk) against Penicillium chrysogenum at 200 μg/disk
same extraction and fractionation scheme used at the initial and an intense molecular ion of 1 at m/z 307 [M – H]− in nega-
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Beilstein J. Org. Chem. 2014, 10, 1808–1816.
Figure 3: The effect of sacrolide A (1) on 3Y1 rat fibroblastic cells. (a) Control. (b) 45 min after exposure to 13 μM of 1. (c) 20 h after exposure to
13 μM of 1. Arrowheads indicate blebs on the cell surface. The magnification is identical in (a), (b) and (c), the bar represents 100 μm.
tive mode LC–MS analysis (Figure 4). In contrast, the same and a peak for 1 in the LC–MS analysis completely disap-
fractions from the commercial products were both inactive and peared (Figure 4). Although the exact degradation pathway of 1
devoid of a peak corresponding to 1, which dispelled the was not identified during this treatment, a facile deactivation of
concern about food intoxication for processed A. sacrum. 1 in the raw alga was achieved in a fast, simple, and inexpen-
sive manner, which offers a useful option when consumers wish
To eliminate the uncertainty with regard to the safety of eating to enjoy the crispy texture of fresh alga, but feel uneasy about
substantial amounts of raw alga, a precooking treatment to the possibility of food intoxication.
manually deactivate 1 was desired. We hypothesized that the
modification of the reactive enone functionality or the degrad- Conclusion
ation of the cyclic structure may eliminate the bioactivity of 1, We explored a bioactive secondary metabolite from the very
and envisioned that the treatment with an alkaline solution may rare but edible cyanobacterium A. sacrum for the first time. A.
serve this purpose. In fact, by boiling the alga in 3% aqueous sacrum is endemic to the Aso water system in Japan. As a
NaHCO3 for 1 min prior to extraction, the antifungal activity result, we discovered a new oxylipin-derived macrolide 1 with a
Figure 4: Extracted ion chromatograms for sacrolide A (1) molecular ion at m/z 307 [M – H]− in the LC–MS analysis of aqueous 90% MeOH fractions
from the ethanolic extracts of (a) raw alga, (b) dry product, (c) brined product, and (d) raw alga boiled in 3% NaHCO3 for 1 min. Chromatogram (e)
shows the elution of purified sacrolide A (1) at tR 23.6 min.
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Beilstein J. Org. Chem. 2014, 10, 1808–1816.
broad antimicrobial spectrum and cytotoxicity. Although the chloroform/MeOH 99:1, 97:3, 95:5, 9:1, and chloroform/
safety of this alga as food is secured by 250 years of consump- MeOH/H2O 6:4:1 to give ten fractions. The second fraction, the
tion as a premium delicacy and the lack of a single episode of one with the highest activity, was gel-filtered on Sephadex
food intoxication, safety was reconfirmed by checking the LH-20 (chloroform/MeOH 1:1) followed by silica gel HPLC
absence of 1 in the commercial products. Moreover, a conveni- (Cosmosil 5SL-II 1 × 25 cm) eluted with n-hexane/Et2O (2:1)
ent precooking treatment was developed to deactivate 1 in the to yield 1 (6.0 mg). Starting with the second most active
raw alga, which prevents food intoxication caused by a high- n-hexane layer (380.1 mg) and following the same procedure
dose intake of 1 and allows for the consumption of a substantial gave 1.5 mg of 1.
amount of unprocessed alga.
Sacrolide A (1): yellow solid; soluble in MeOH, CHCl3, MeCN,
In conclusion, the present study illustrates the importance of EtOAc, and Et2O, and insoluble in H2O. [α]D24.5 +41.4 (c
exploring untapped biological resources for the discovery of 0.115, MeCN); UV (MeCN) λmax, nm (ε) 232 (8000), 283
new bioactive molecules, and also encourages the careful (320), 331 (160); HRMS–ESI (m/z): [M + Na]+ calcd for
conservation of a variety of habitats to maximize the availabil- C18H28NaO4, 331.1880; found, 331.1889.
ity of genetic resources.
Paper disk agar diffusion method
The antimicrobial potency of the chromatographic fractions was
Experimental
General methods
evaluated by a paper-disk agar diffusion method. Fractions at
Cosmosil 75C18-PREP (Nacalai Tesque Inc., 75 µm) was used each stage of the purification were diluted to the same concen-
for ODS flash chromatography. NMR spectra were obtained by tration with MeOH, and 10 μL aliquots were loaded on paper
a Bruker AVANCE 500 spectrometer at 500 MHz for 1H. disks with 6 mm diameter, which were left until completely
Residual solvent peaks at δH/δC 7.27/77.0 ppm in CDCl3 were dried. A loop of the test organism, suspended in a small amount
used as chemical shift reference signals. HRMS–ESI–TOF and water, was mixed with liquefied agar medium precooled to
LC–MS analyses were carried out by an Agilent 1200 HPLC- nearly body temperature, and the inoculated medium was
DAD system coupled with a Bruker micrOTOF mass spectrom- quickly poured into a sterile plastic dish. The medium was
eter. Optical rotation and UV spectra were recorded on a composed of 0.5% yeast extract, 1.0% tryptone, 1.0% NaCl,
JASCO DIP-3000 polarimeter and a Hitachi U-3210 spec- 0.5% glucose, and 1.5% agar. After the agar solidified, the
trophotometer, respectively.
drug-loaded disks were placed on the plate, and the test cultures
were incubated at 32 °C for a day or two until the diameters of
inhibitory haloes turned measurable.
Biological material
A. sacrum is commercially cultured by one of the authors
(K. T.) in a groundwater-irrigated aquaculture pond located in Microculture antimicrobial testing
Kumamoto, Kyushu District. The algal mass collected in a sieve 100 μL of Mueller–Hinton broth supplemented with
basket was immediately frozen at −20 °C and shipped to the CaCl2·2H2O (5% w/v) and MgCl2·6H2O (2.5% w/v) were
laboratory in October, 2012. Both the dry and brined A. sacrum transferred to the wells of a sterilized 96 well microtiter plate.
products were purchased from Kisendo Ltd. (Fukuoka, Japan) The test samples were dissolved in DMSO and diluted 20 times
in January 2014.
with the same broth medium. For a two-fold serial dilution
along the columns each 100 μL aliquot of the test solution was
added to the top row, mixed with the pre-dispensed medium,
Extraction and isolation
500 g of a water-thawed specimen of the alga was ground with and the same amount was then transferred to the second row.
an equal amount of Celite in EtOH (1 L). The resulting slurry The same operation was repeated until reaching the bottom row,
was paper-filtered to separate an ethanolic extract and an algal where the last transfer was discarded to equalize the volume of
cake, and the latter was extracted three more times with the the medium. The indication strains were grown on agar media
same amount of alcohol. The combined extract (1.5 g) was (Staphylococcus aureus FDA209P JC-1, Micrococcus luteus
partitioned between aqueous 60% MeOH (400 mL) and ATCC9341, Escherichia coli NIHJ JC-2, Saccharomyces cere-
dichloromethane (400 mL × 3), the former of which was further visiae S100, and Candida albicans A9540) or in Sabouraud
separated between aqueous 90% MeOH and n-hexane, whereas broth (Penicillum chrysogenum NBRC4626, Streptomyces livi-
the latter was further separated between H2O (200 mL) and dans TK23) overnight and then diluted with sterile saline or the
n-BuOH (200 mL × 3). The most active aqueous MeOH layer same broth to 0.5 McFarland (~108 cfu/mL). Two μL of these
(422.9 mg) was subjected to silica gel chromatography with a suspensions were immediately inoculated to the microcultures,
stepwise elution by n-hexane/EtOAc 4:1, 2:1, 1:1, 1:2, 1:4, and the plates were incubated at 32 °C for 18–24 h. The concen-
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Beilstein J. Org. Chem. 2014, 10, 1808–1816.
tration required to completely inhibit the growth of the
microbes was defined as the minimum inhibitory concentration
(MIC).
Supporting Information
Supporting Information File 1
Procedures for chemical conversion/derivatization, NMR
assignments, copies of NMR, MS and UV spectra for 1,
NMR spectra for 2, 3, 3a and 3b.
Cytotoxicity testing and cell morphology
observation
The cytotoxicity against 3Y1-B clone 1-6 rat fibroblasts and the
action of sacrolide A (1) on the morphology of the same cell
line were evaluated by the procedures described in [22].
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-10-190-S1.pdf]
Method to precook raw alga for the elimination of sacrolide
A (1): A tablespoon of NaHCO3 (15 g) was added to a pot
containing 500 mL of water, and the solution was boiled at high
temperature. Then 40 g of raw A. sacrum was added and boiled
with occasional stirring for 1 min. The boiled alga was drained
in a colander, washed three times with cold water, and then
subjected to the extraction and fractionation procedure
described in the next section.
Acknowledgements
This work was supported in part by the President’s Fund Initia-
tive at the Toyama Prefectural University. 3Y1-B clone 1-6 rat
fibroblastic cells (resource number JCRB0734) were obtained
from the Health Science Research Resources Bank.
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commercial products and precooked sample of A. sacrum:
Dry or brined alga was soaked in water for 30 min and weighed
prior to extraction. A 20 g portion from each product,
precooked or raw alga, was extracted with EtOH, and the
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commercial products or precooked sample exhibited an activity
against Penicillium chrysogenum, whereas the aqueous 90%
MeOH fraction from raw alga exhibited an inhibition circle
with a diameter of 12.0 mm, which indicates the presence of 1.
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column (Nacalai Tesque, 2.0 mm × 150 mm) under the elution
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