Concentrated Phosphatidic Acid in Cereal Brans as Potential

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Concentrated phosphatidic acid in cereal brans as potential protective agents against indomethacin-induced stomach ulcer Sheuli Afroz, Teru Ikoma, Ayano Yagi, Kentaro Kogure, Akira Tokumura, and Tamotsu Tanaka J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02884 • Publication Date (Web): 26 Aug 2016 Downloaded from http://pubs.acs.org on August 27, 2016

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Journal of Agricultural and Food Chemistry

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Title:

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Concentrated phosphatidic acid in cereal brans as potential protective

3

agents against indomethacin-induced stomach ulcer

4 5

Authors:

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Sheuli Afroz†, Teru Ikoma†, Ayano Yagi†, Kentaro Kogure†, Akira Tokumura‡ and

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Tamotsu Tanaka*†

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Affiliations:

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†

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770-8505, Japan

12

‡

13

University, Hiroshima 731-0351, Japan

Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima

Department of Pharmaceutical Life Sciences, Faculty of Pharmacy, Yasuda Women’s

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*To whom correspondence should be addressed.

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Tel: +81-88-633-7249; Fax: +81-88-633-9572

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E-mail: [email protected]

18 19

Running title: Concentrated phosphatidic acid in cereal brans

1 ACS Paragon Plus Environment


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ABSTRACT

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One of complications associated with long-term use of non-steroidal

22

anti-inflammatory drugs (NSAIDs) is peptic ulcer. Recently, we found that orally

23

administered phosphatidic acid (PA) ameliorated aspirin-induced stomach lesions in

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mice. In this study, we identified PA-rich food sources, and examined the effects of the

25

food materials on indomethacin-induced stomach ulcer. Among examined, buckwheat

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(Fagopyrum esculentum) bran contained the highest level of PA (188 mg/100 g). PA

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was the richest phospholipid (25%) in the lipid fraction of the buckwheat bran.

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Administration of the lipid extracts of buckwheat bran significantly ameliorated

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indomethacin-induced stomach lesions in mice. In contrast, wheat (Triticum durum)

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bran lipids (PA, 4%) and soybean (Glycine max) lipids (PA, 3%) were not associated

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with ameliorative effects. These results indicated that PA-rich lipids can be used as

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effective supplement for prevention of NSAIDs-induced stomach ulcer.

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KEYWORDS: phosphatidic acid, cereal brans, stomach ulcer, phospholipase A2,

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lysophosphatidic acid


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INTRODUCTION

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NSAIDs are widely used for reduction of inflammation and risk of deep vein

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thrombosis. A recent medical survey revealed that as many as 25% of chronic NSAIDs

38

users may develop peptic ulcer disease, of which bleeding may occur in 2−4% cases.1

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Proton pump inhibitors (PPIs) are the first-line drugs for gastric ulcer treatment.

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However, long-term use of PPIs has been associated with development of

41

iron-deficiency anemia,2 owing to impaired mineral absorption. Gastro-protective

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agents that are easy to take, inexpensive, and have fewer side effects can resolve this

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NSAIDs-associated problem.

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Phosphatidic acid (PA) is a natural phospholipid, that is predominantly present

45

in plant foodstuffs.3 Recently, we found that oral administration of synthetic PA and

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lysophosphatidic acid (LPA) ameliorated aspirin-induced stomach mucosal injury in

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mice and PA was converted to LPA in the stomach by phospholipase A2 (PLA2).4 LPA

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is a bioactive glycerophospholipid with diverse biological properties. LPA elicits

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various cellular responses, including cell proliferation, migration, and protection

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against apoptotic cell death in numerous types of cells through its specific G

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protein-coupled receptors (LPA1−6). Recent studies have revealed the roles of LPA

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signaling in maintaining the epithelial integrity of mammals gastrointestinal (GI)

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tract.5-9 Previously, we demonstrated that orally administered LPA prevented

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water-immersion stress-induced gastric ulcer in rats.10 Our recent study found that

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LPA2 was localized on the apical side of gastric surface mucous cells and enhanced

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production of prostaglandin (PG) E2, a cytoprotective PG that augments the mucous

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gel barrier in the GI tract.11 These results suggest the possibility that gastric epithelia

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respond to luminal LPA formed from ingested PA and that PA-rich foods contribute to

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epithelial integrity of the stomach by augmentation of the cytoprotective function.

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However, it remains unknown whether a PA-rich food actually protects against peptic

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ulcer induced by NSAIDs. 3 ACS Paragon Plus Environment


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Previously, we examined PA content in various foods and found that cruciferous

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vegetables, such as cabbage and radish, are PA-rich foodstuffs.3 In this study, we

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focused on cereals because they are staple foods that are often eaten daily in large

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amounts. Several investigators reported lipids compositions of cereals.12-15 However,

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there are few reports on the PA content of cereals. We also examined the effect of

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materials prepared from PA-rich foods on the acute NSAIDs-induced stomach lesions

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in mice.

69 70 71

MATERIALS AND METHODS

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Food sources. Flour of common buckwheat endosperm (Fagopyrum esculentum)

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grown in Hokkaido Japan was obtained from a mail-order grocery. Seeds of buckwheat

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grown in Tokushima Japan were a gift from a local farmer. The buckwheat seeds were

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from the same species but of different strains. In fact, seeds from Hokkaido were

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bigger than those from Tokushima. The milling company, Tani Syokuryou, Co.

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(Tokushima Japan) kindly produced the powder from different parts of the buckwheat

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seeds grown in Tokushima. Flour of tartary buckwheat endosperm (Fagopyrum

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tartaricum) grown in Hokkaido was purchased from a mail-order grocery. Flours from

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wheat (Triticum durum) grown in Ontario (Canada), Kumamoto (Japan), and Hokkaido

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(Japan) were purchased from a mail-order grocery and the names of the strains are

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Supernnove, Minaminomegumi, and Haruyutaka, respectively. All flours were from

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durum wheat species. Flours of whole wheat and wheat bran were prepared from the

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Haruyutaka strain grown in Hokkaido Japan. Flour of rye (Secale cereale) grown in

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Hokkaido Japan was obtained from a mail-order grocery. The name of the strain is

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unknown. Brown rice (Oryza sativa) and its polished form (white rice) were purchased


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from a local market. Flours of the brown and white rice were prepared by milling in a

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mill mixer. Flour of rice bran was obtained from the brown rice by using a rice mill.

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The first milling flour was collected and used as rice bran. Cornmeal (Zea mays),

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soybean flour (Glycine max) and peanuts (Arachis hypogaea) were purchased from a

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local market. Flours of these nuts were prepared by milling in a mill mixer. Cabbage

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(Brassica oleracea), radish (Raphanus sativus) and carrot (Daucus carota) were

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obtained from a local market.

94 95

Reagents.

1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphate

(16:0/18:2

PA),

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1-palmitoyl-2-lyso-sn-glycero-3-phosphate

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1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (16:0/18:2 PC), egg yolk PA

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were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Egg yolk lecithin was

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purchased from Tokyo Chemical Industry Co. (Tokyo, Japan). Phos-tag was obtained

100

from Wako Pure Chemical Industries (Osaka, Japan). Carboxymethyl cellulose (CMC)

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and indomethacin were obtained from Kanto Chemical Co. (Tokyo, Japan) and

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NACALAI TESQUE Inc. (Kyoto, Japan), respectively.

(16:0

LPA),

103 104

Extraction of lipids and isolation of PA from cereal lipids. Lipids were

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extracted from vegetables and cereal flours by using the Bligh and Dyer method,16 as

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described previously.17 In brief, 2 g of cereal flours or minced vegetables were added to

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an equal amount of water and homogenized. The homogenates were heated in hot

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water for 3 min to increase the efficacy of lipid extraction and inactivate lipolytic

109

enzymes. After heating, the homogenates were added to an appropriate volume of

110

chloroform,

111

chloroform/methanol/water (7.5:15:6, v/v/v). Then, the suspension was homogenized

methanol

and

water

to

yield

a

mixture


consisting

of


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again and centrifuged. After collecting the supernatant, the pellet was added to 11.4 mL

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of solvent consisting of chloroform/methanol/water (3:6:2.4, v/v/v), mixed vigorously,

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and centrifuged. The supernatant was combined and added to an appropriate volume of

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chloroform and water to make chloroform/methanol/water (21:21:18.9, v/v/v). After

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acidification with 0.2 mL of 5 N HCl, the suspension was centrifuged. Lipids were

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obtained from the chloroform phase (lower phase). Phospholipids in the extracted

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lipids were quantified on the basis of phosphomolybudenum-malachite green

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formation.18 PA in lipid extracts was isolated by TLC. The mobile phase used in

120

chromatography was chloroform/methanol/28% aqueous ammonia (60:35:8, v/v/v).

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After development, the plate was sprayed with primuline for visualization of lipids

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under UV light. PA was extracted from the silica gel by using the Bligh and Dyer

123

method16 and quantified.18 Mole percentages of PA in total phospholipids were

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calculated from the amounts of PA and total phospholipids. Because the amounts of

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phospholipids were determined on the basis of lipid phosphorus, calculations were

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required for conversion of the weight of lipid phosphorus (mg Pi/g) to the weight of

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phospholipids (mg/g). In this calculation, 25 was used as the conversion factor for total

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phospholipids. In the case of PA, 21.8 was used as the conversion factor. The factors

129

used were in accordance with the official method recommended by the American Oil

130

Chemists' Society (AOCS).13

131 132

Analysis of PA and LPA molecular species. PA and LPA molecular species were

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determined by matrix-assisted laser desorption ionization time-of-flight mass

134

spectrometry (MALDI-TOF MS) with phos-tag, as described previously.19 In brief,

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aliquots of PA and LPA were dissolved in 100 µL of methanol containing 0.1%

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aqueous ammonia. This solution (10 µL) was mixed with 5 µL of 0.1 mM

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phos-tag solution, and a small portion (0.5 µL) of this mixture was spotted on a sample


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Zn

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plate. Immediately, 0.5 µL of 2,4,6-trihydroxyacetophenone (THAP) solution was

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layered on the mixture as a matrix solution and subjected to MALDI-TOF MS.

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MALDI-TOF mass spectra were acquired by using a Voyager DE STR mass

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spectrometer (Applied Biosystems, Framingham, MA, USA) in positive-ion detection

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mode. The wavelength of the nitrogen-emitting laser, pressure in the ion chamber, and

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accelerating voltage were 337 nm, 3.7×10−7 Torr, and 20 kV, respectively. To enhance

144

the reproducibility, 300 laser shots were averaged for each mass spectrum.

145 146

Indomethacin-induced stomach lesions. Indomethacin was suspended in 3%

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(w/v) CMC and then sonicated for 30 sec. Synthetic PA, LPA, phosphatidylcholine

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(PC), or natural lipids extracted from cereal bran were mixed with 3% CMC, shaken

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vigorously, and sonicated for 1 min. Five-week-old male ICR mice (35 g body weight)

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fasted overnight were administered 0.2 mL of the lipids suspension intragastrically.

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After 30 min, 0.2 mL of the indomethacin suspension (22.9 mg/kg body weight) was

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administered intragastrically. The mice were anesthetized with diethyl ether and

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sacrificed 5 h after the indomethacin administration. Ulcer severity was evaluated on

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the basis of the total lesion lengths, as described previously.4 Briefly, the isolated

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stomach was immersed in 2% formalin for 15 min. Then, the stomach was cut along

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the greater curvature, and the lengths of lesion on the stomach wall were measured by

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using a mm scale and a magnifying glass. The total lengths of lesion were expressed as

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a lesion index. The care and handling of mice were in accordance with the National

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Institution of Health guidelines. All experimental procedures described above were

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approved by the Tokushima University Animal Care and Use Committee.

161 162

Formation of LPA from buckwheat bran lipids in mouse stomach. Formation

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of LPA from buckwheat bran lipids in an isolated mouse stomach was examined ex

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vivo. The buckwheat bran lipids (600 nmol phospholipids) suspension was injected 7 ACS Paragon Plus Environment


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into an isolated mouse stomach in which both ends of the stomach were ligated and

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then incubated at 37 oC with continuous aeration. After incubation, the lipids were

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washed and then extracted by using the method of Bligh and Dyer, with acidification

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of the aqueous phase.17 The lipids were separated by two dimensional TLC. The

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mobile

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chloroform/methanol/28%

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chloroform/methanol/acetone/acetic acid/water (50:10:20:13:5, v/v/v/v/v), respectively.

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The resulting LPA was isolated and quantified, as described above. The molecular

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species of the PA and LPA were determined by MALDI-TOF MS, as phos-tag

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complexes.

phases

for

the

first

and

ammonia

second (60:35:8,

chromatography v/v/v)

were and

175 176 177

Statistical Analysis. Statistical analyses of the differences between two means were performed by using Student’s t-test.

178 179 180

RESULTS

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Phosphatidic acid in cereals. We quantified amounts of total phospholipids and

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PA, focusing on cereals. Percentages of PA in total phospholipids were calculated from

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these values (Figure 1). Wheat, rye, and rice contained phospholipids at levels of

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approximately 140 to 340 mg/100 g. PA accounted only for 2 to 4% of the total

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phospholipids in these flours. Amounts of PA in these cereal flours (2−7 mg/100 g)

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were low relative to those in other foodstuffs examined. There were no significant

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differences in the amounts of phospholipids or PA among the different strains of wheat.

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Common buckwheat (Fagopyrum esculentum) and tartary buckwheat (Fagopyrum

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tartaricum) are different species belonging to the same genus. Although the amounts of

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phospholipids were similar between these two species of buckwheat flours, the PA

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content in tartary buckwheat flour (41 mg/100 g) was 2.4 to 3.7 times that of common 8 ACS Paragon Plus Environment

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buckwheat flours (11−17 mg/100 g). The PA level of the tartary buckwheat flour was

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comparable to that of cabbage (53 mg/100 g wet weight), which was identified as

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PA-rich foodstuff in our previous study.3 The percentages of PA in the total

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phospholipids of cornmeal (15%) was considerably higher than those of wheat and rice.

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As a result, PA was much higher in cornmeal (20 mg/100 g) than in wheat and rice.

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Peanut flour and soybean flour characteristically contained abundant phospholipids;

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therefore, the amounts of PA in these nuts flours were relatively high (21−45 mg/100

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g). However, the percentages of PA in the total phospholipids in peanut and soybean

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flours were not so high (3%).

201 202

PA content in different parts of cereal seeds. We found that rice bran contained

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50 times as much PA (104 mg/100 g) as that of rice endosperm (2.1 mg/100 g).

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Similarly, the PA content in buckwheat bran (188 mg/100 g) was 10 times that of

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buckwheat endosperm (19 mg/100 g). The percentages of PA in total phospholipids

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were also high in rice bran (12%) and buckwheat bran (25%). A similar tendency was

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observed in wheat seeds. However, the absolute amounts of PA and percentages of PA

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in total phospholipids in wheat bran were considerably lower than those of rice bran

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and buckwheat bran (Figure 2). The ameliorative effect of these bran lipids on

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indomethacin-induced stomach lesions was examined, as described below.

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PA content in boiled and non-boiled cereals. Previously, we showed that cutting

213

or mashing of raw cabbage leaves induced PA formation in the leaves,20 which was

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caused by hydrolysis of endogenous phospholipids, including PC by activated

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phospholipase D (PLD) during tissue destruction. Consistent with our previous

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findings, levels of PA prepared from boiled cabbage leaves, radish roots, and carrot

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roots were low relative to those prepared from raw ones. By contrast, levels of PA

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prepared from boiled brans of buckwheat and rice were similar to those of PA prepared 9 ACS Paragon Plus Environment


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from non-boiled brans (Figure 3), which indicated the absence of active PLD enzyme

220

and presence of abundant PA in buckwheat and rice brans.

221 222

Anti-ulcer effect of synthetic PA and LPA on indomethacin-induced stomach

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lesions. Indomethacin is a widely used NSAID that causes gastric ulceration as a side

224

effect. Previously, we found that pre-administration of synthetic PA and synthetic LPA

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significantly reduced aspirin-induced stomach lesions.4 To determine whether PA and

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LPA ameliorate stomach lesions induced by NSAIDs other than aspirin, we established

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an indomethacin-induced ulcer mouse model. Oral administration of 22.9 mg/kg

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indomethacin produced 8 to 10 linear mucosal lesions extended from the fundic to

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pyloric area of stomach wall (Figure 4A). We found that indomethacin-induced lesions

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were sustained for a longer period of time than those induced by aspirin. This feature

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of the indomethacin-induced ulcer model was advantageous for suppressing

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experimental errors in the ulcer index. Pre-administration of 0.2 mL of 1 mM synthetic

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PA and synthetic LPA significantly reduced indomethacin-induced ulcer lesions. PA

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reduced lesion formation more effectively than LPA at low concentrations (Figure 4C).

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PC is the most abundant phospholipid both in animal and plant foods. We found that

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synthetic PC showed marginal ameliorative effects at 1 mM. Egg yolk PA significantly

237

reduced lesion formation at 0.3 mM and 1 mM. On the other hand, egg yolk lecithin

238

did not reduce lesion formation in the same range of concentrations (Figure 4D).

239 240

Effects of cereal brans lipid extracts on indomethacin-induced stomach lesions

241

in mice. We prepared lipid extracts from cereal brans and soybean, and examined their

242

anti-stomach ulcer activity. The percentages of PA in the lipid extracts from buckwheat

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bran, rice bran, wheat bran, and soybean were 25%, 12%, 4%, and 3%, respectively.

244

The amounts of lipids administered were determined on the basis of their

245

phospholipids content. For example, administration of 0.2 mL of CMC suspension of

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buckwheat bran lipids at 1 mM means administration of the lipid extracts containing

247

0.2 µmol of phospholipids in 0.2 mL. As shown in Figure 5A, administration of

248

buckwheat bran lipids and rice bran lipids at 1 mM significantly reduced


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indomethacin-induced ulcer lesions. Neither wheat bran lipids nor soybean lipids at the

250

same concentration significantly reduced ulcer lesions. The maximum protection rate

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(49%) was observed at 1 mM of buckwheat bran lipids. A higher concentration (3 mM)

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of buckwheat bran lipids did not reduce ulcer lesions (Figure 5B). Similarly, a higher

253

concentration of synthetic PA did not show an ameliorative effect (Figure 5C). As an

254

extended study, we prepared a suspension of buckwheat bran powder and administered

255

it orally to mice. We found that buckwheat bran suspension which containing

256

phospholipids at 0.2 µmol significantly reduced indomethacin-induced ulcer lesions.

257

However, the ameliorative effect of the powder was relatively weak. Its protection rate

258

did not exceed 30% (data not shown).

259 260

Formation of LPA from buckwheat bran lipids in mouse stomach. To

261

determine whether LPA was actually formed from PA during digestion of buckwheat

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bran lipids in mouse stomach, we conducted an ex vivo experiment by using isolated

263

mouse stomach. Before digestion, LPA spot was scarcely detected in buckwheat bran

264

lipids (Figure 6A). After incubation of buckwheat bran lipids for 30 min in an isolated

265

stomach, LPA was detected as an apparent spot on TLC (Figure 6B). Quantification of

266

LPA and PA before and after incubation revealed that about 16 nmol LPA was formed

267

with concomitant decrease of the amount of PA in an isolated stomach. The elevated

268

level of LPA remained unchanged up to 1 h of incubation (Figure 6C). The sum of the

269

amounts of LPA and PA at the end of incubation was only 30% of the PA injected as

270

buckwheat bran lipids. The lower recovery of PA plus LPA may have been due to

271

incorporation or decomposition of PA and LPA during incubation in the stomach. The

272

formation of LPA was confirmed by MALDI-TOF MS. The major molecular species

273

of LPA that formed in stomach from buckwheat bran lipids were found to be 16:0,

274

18:2, and 18:1 LPA (Figure 6E). This is consistent with the fact that major molecular

275

species of buckwheat bran PA were 16:0/18:2, 16:0/18:1, 18:2/18:2, 18:1/18:2, and

276

18:1/18:1 PA (Figure 6D). We also treated buckwheat bran lipids with stomach lavage

277

for confirmation of phospholipase A2 activity. Treatment of buckwheat bran lipids

278

containing 17 nmol of PA with stomach lavage for 30 min, approximately 8 nmol LPA

279

was formed (data not shown). These results are consistent with our previous results 11 ACS Paragon Plus Environment


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showing that stomach PLA2 can hydrolyze PA to form LPA in stomach without

281

sodium deoxycholate.4

282 283 284

DISCUSSION

285

Previously, we reported that orally administrated PA ameliorate aspirin-induced

286

stomach lesions in mice.4 Here, we found that PA is concentrated in the bran parts of

287

rice and buckwheat seeds, and that lipid extracts from these PA-rich cereal brans have

288

ameliorative activity against indomethacin-induced stomach lesions over a limited

289

range of concentrations.

290

Weihrauch and Son have compiled data on the total lipid and phospholipid contents

291

of 140 foods, including cereals.13 The phospholipid contents of cereals obtained in this

292

study (wheat, rye, rice, and corn flours) were in a similar range as those of the data

293

reported earlier. To our knowledge, PA contents of wheat, rye, and rice flours are

294

unknown. Here, we found that the PA contents of these cereals were 2−3, 7, and 2.1

295

mg/100 g, respectively.

296

Buckwheat seeds are good sources of valuable nutrients, and their consumption has

297

been associated with numerous beneficial effects on health.21 The amounts of

298

phospholipids in buckwheat flour have been reported by Mazza to be 0.3% on a dry

299

weight basis (300 mg/100 g).22 Consistent with this data, we found that the

300

phospholipid contents in common buckwheat and tartary buckwheat flours were 417 to

301

486 mg/100 g and 421 mg/100 g, respectively. In addition, we found that tartary

302

buckwheat flour contained a higher level of PA (41 mg/100 g) than common

303

buckwheat flours (11−17 mg/100 g).

304

Enrichment of phospholipids in rice bran has been reported by Choudhury.23

305

Enrichment of fats in buckwheat bran has been reported by Bonafaccia.24 In this study,

306

PA contents in rice bran and buckwheat bran were determined to be 104 mg/100 g and

307

188 mg/100 g, respectively. We also found that PA accounted for relatively higher

308

percentages in the total phospholipids (12% and 25% in rice bran and buckwheat bran,

309

respectively) in these brans. To our knowledge, this is the first study to show that PA is

310

concentrated in the bran parts of rice and buckwheat seeds. The physiological 12 ACS Paragon Plus Environment

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significance of bran parts of seeds is protection of embryos from disease, pests,

312

mechanical injury, and unlimited water penetration. It has been reported that PA plays

313

a vital role in plant defense response against various environmental stresses.25-27

314

Abundant PA in the bran parts of cereal seeds may have a role in protection of internal

315

seed parts from such stresses.

316

Indomethacin is a widely used NSAID to relieve pain, swelling, and symptoms of

317

arthritis. Like aspirin, indomethacin causes gastrointestinal adverse events, including

318

bleeding and ulceration in the stomach. Previously, we demonstrated that

319

pre-administration of synthetic PA and LPA ameliorated aspirin-induced acute stomach

320

lesions in mice and that free fatty acids, lysophosphatidylcholine (LPC), tri- and

321

diacylglycerol did not reduce ulcer lesions over the same range of concentrations.4

322

Consistent with our previous study with aspirin, both synthetic PA and LPA exerted

323

ameliorative activity against indomethacin-induced stomach lesions. We showed that

324

egg yolk PA but not egg lecithin had an ameliorative effect on indomethacin-induced

325

stomach lesions. These results indicated that pre-administration of purified PA

326

effectively suppressed tissue damage caused by NSAIDs in stomach.

327

In the present study, we demonstrated that lipids containing high percentages of

328

PA, such as buckwheat bran lipids significantly ameliorated indomethacin-induced

329

stomach lesions. By contrast, lipids containing low percentages of PA, such as wheat

330

bran and soybean lipids did not significantly reduce ulcer lesions over the same range

331

of concentrations. We also observed that the ameliorative effect was more potent for

332

synthetic PA than for lipid extracts of buckwheat bran, especially at low concentrations.

333

These results indicated that anti-ulcer potency of PA was affected by co-existing lipids.

334

Lipid components other than PA present in the extract may interfere with the effects of

335

PA and LPA. PC, lysoPC, free fatty acids, and other surface-active lipids in the extract

336

may trap and hide PA as inclusion micelles or liposomes. The fact that buckwheat bran

337

powder showed a lower ameliorative effect than those of buckwheat bran lipids and

338

synthetic PA may be explained by this interference of the co-existing components.

339

Defensive strategies of gastric mucosa include suppression of gastric acid

340

secretion, formation of a mucus gel layer of mucin, secretion of bicarbonate and

341

enhancement of mucosal blood flow. PGE2 is considered to play pivotal roles in these 13 ACS Paragon Plus Environment


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342

mucosal protections. Previously, we found that LPA2 was localized on the apical side

343

of gastric surface mucous cells and that LPA increase PGE2 level by upregulation of

344

COX-2 mRNA in human stomach cells.11 Pre-administration of LPA is considered to

345

strengthen a barrier function and keep the mucous cells vigorous. In addition,

346

anti-apoptosis via LPA28 and enhancement of cell migration17 have been shown as LPA

347

action in gastrointestinal cells. The protective effect of pre-administered LPA against

348

NSAIDs-induced

349

LPA-receptor-mediated actions in the stomach. In fact, we demonstrated LPA

350

formation from buckwheat bran lipids in isolated mouse stomach. In this experiment,

351

amount of LPA formed was determined to be 16 nmol from buckwheat bran lipids

352

containing 100 nmol PA (Figure 6C). Based on this result, 6 nmol LPA is considered to

353

be formed from effective dose of buckwheat bran lipids. Previously, we showed that

354

gastric PLA2 effectively hydrolyzes PA to LPA only at neutral pH.4 Considering that

355

neutral space in stomach cavity is mucous gel layer, formation of LPA is likely to

356

proceed in the gel layer. LPA formed in such a limited space (estimated to

357

approximately 0.4 mL/mouse stomach) may effectively interact with LPA2 (Figure 7).

358

The elevated concentration of LPA in stomach gel layer would be retained up to 1 h,

359

when PA-rich lipid was administered. Formation of LPA at the gel layer may explain

360

why administration of small amount of PA is effective than that of much amount of

361

LPA.

stomach

ulcer

can

be

partially

explained

by

these

362

PA has been shown to modulate cellular function. It has been reported that

363

exogenous PA enhanced calcium influx and calcium release from intracellular

364

stores.28-30 Schepp et al. have shown that increase of intracellular calcium

365

concentration enhances PGE2 production in human gastric mucous cells.31 It has been

366

reported that mammalian target of rapamycin (mTOR) is a critical target for cell

367

survival and that both intracellular and extracellular PA activated mTOR signaling.32,

368

33

369

cell membrane through dephosphorylation and phosphorylation reactions.34 These

370

biological actions of PA may involve in the ameliorative effect of PA-rich foods.

In this regard, Pagano et al. have shown that exogenous PA can diffuse across the

371

Synthetic PA significantly reduced the total length of lesions at concentration from

372

0.1 to 1 mM, which corresponds to 0.4 to 4 mg/kg body weight. However, PA at 3 mM 14 ACS Paragon Plus Environment

Page 15 of 31


373

(12 mg/kg body weight) was found to have no effect. Ineffectiveness at high

374

concentrations was also observed in an experiment with synthetic LPA (data not

375

shown). At present, the exact reasons for this phenomenon are unknown. A possible

376

explanation is desensitization of the LPA receptor by supraphysiological concentration

377

of LPA. Considering that LPA concentration in saliva has been reported to be

378

approximately 0.9 µM,35 the LPA receptor expressed on the apical side of the stomach

379

epithelia may respond to LPA at similar concentration. LPA administration at high

380

concentrations may induce internalization of the LPA receptor and hamper

381

LPA-receptor-mediated signaling.

382

In conclusion, we characterized buckwheat bran and rice bran as PA-rich

383

foodstuffs. We also demonstrated that natural PA, such as egg yolk PA, and PA-rich

384

lipids, such as buckwheat bran lipids, significantly reduced indomethacin-induced

385

stomach lesions over a limited range of concentrations. Our results suggest that the

386

purified PA or lipid extracts from PA-rich foodstuffs described here may be effective

387

as anti-ulcer supplements.

388 389 390

ABBREVIATIONS USED

391

COX, cyclooxygenase; GI, gastrointestinal; LPA, lysophosphatidic acid; mTOR,

392

mammalian target of rapamycin; PA, phosphatidic acid; PC, phosphatidylcholine; TLC,

393

thin-layer chromatography; MALDI-TOF MS, matrix-assisted laser desorption

394

ionization time-of-flight mass spectrometry; CMC, carboxymethyl cellulose; PLA2,

395

phospholipase A2; PLD, phospholipase D; PGE2, prostaglandin E2; NSAIDs,

396

non-steroidal anti-inflammatory drugs.

397 398 399

ACKNOWLEDGEMENT

400

We thank Fumihiro Okamoto and Toshio Minami for donating of buckwheat seeds.

401



402 403

SUPPORTING INFORMATION

404

This work was partly supported by grants-in-aid from the Ministry of Education,

405

Science, Sports, and Culture of Japan (15K07430), the research program for

406

development of an intelligent Tokushima artificial exosome (iTEX), and the Sugiyama

407

Sangyou Kagaku Research Foundation.

408


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409

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521

FIGURE LEGENDS

522

Figure 1. Amounts of phospholipid and PA and percentages of PA in cereal flours.

523

Lipids were extracted from cereal flours and plant foodstuffs. PA was isolated by TLC

524

from the extracts. The amounts of PA and total phospholipids were determined by

525

measurement of phosphorus of the lipids. The percentages of PA were calculated from

526

the amounts of total phospholipids and PA. Names of strains or producing areas are

527

indicated in parenthesis. Haru, Haruyutaka strain; Mina, Minaminomegumi strain;

528

Super, Supernova strain; Hkd, Hokkaido area; Tks, Tokushima area.

529 530

Figure 2. Amounts of phospholipid and PA and percentages of PA in whole seed,

531

endosperm, and bran of cereal seeds.

532

Lipids were extracted from each part of cereal seeds. PA was isolated by TLC from the

533

lipid extracts. Haru, Haruyutaka strain; Tks, Tokushima area

534 535

Figure 3. Amounts of PA in non-boiled and boiled foodstuffs.

536

Lipids were extracted from boiled or non-boiled vegetables or cereal brans. PA was

537

isolated by TLC from the lipid extracts. Significantly different from values of

538

non-boiled foodstuffs, ***P

Concentrated Phosphatidic Acid in Cereal Brans as Potential

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