marine drugs

Article Pyrogallol--6,6’-Bieckol from Ecklonia cava Improved Blood Circulation in Diet-Induced Obese and Diet-Induced Hypertension Mouse Models

1,2, 1, 1 3 3 4 Myeongjoo Son † , Seyeon Oh †, Hye Sun Lee , BoMi Ryu , Yunfei Jiang , Ji Tae Jang , You-Jin Jeon 3,* and Kyunghee Byun 1,2,*

1 Functional Cellular Networks Laboratory, Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Incheon 21999, Korea; [email protected] (M.S.); [email protected] (S.O.); [email protected] (H.S.L.) 2 Department of Anatomy & Cell Biology, Graduate School of Medicine, Gachon University, Incheon 21936, Korea 3 Department of Marine Life Science, School of Marine Biomedical Sciences, Jeju National University, 1 Ara 1-dong, Jejudaehak-ro, Jeju 63243, Korea; [email protected] (B.R.); [email protected] (Y.J.) 4 Aqua Green Technology Co., Ltd., Smart Bldg., Jeju Science Park, Cheomdan-ro, Jeju 63243, Korea; [email protected] * Correspondence: [email protected] (Y.-J.J.); [email protected] (K.B.); Tel.: +82-64-754-3475 (Y.-J.J.); +82-32-899-6511 (K.B.) These authors contributed equally to this work. †  Received: 1 April 2019; Accepted: 5 May 2019; Published: 8 May 2019 

Abstract: Blood circulation disorders, such as hyperlipidemia and arteriosclerosis, are not easily cured by dietary supplements, but they can be mitigated. Although Ecklonia cava extract (ECE), as dietary supplements, are associated with improving the conditions, there are not many studies verifying the same. In this study, the beneficial effect of ECE and leaf of Ginkgo biloba extract (GBE), which is a well-known dietary supplement, were first confirmed in a diet induced-obese model. Afterwards, 4 were isolated from ECE, and their inhibitory effects on vascular cell dysfunction were validated. Pyrogallol-phloroglucinol-6,6-bieckol (PPB) was selected to be orally administered in two mice models: the diet induced obese model and diet induced hypertension model. After four weeks of administration, the blood pressure of all mice was measured, after which they were subsequently sacrificed. PPB was found to significantly improve blood circulation, including a reduction of adhesion molecule expression, endothelial cell (EC) death, excessive vascular smooth muscle cell (VSMC) proliferation and migration, blood pressure, and lipoprotein and cholesterol levels. Based on the excellent efficacy in diet-induced mouse models of obese and hypertension, our results demonstrate that PPB is a valuable active compound from among the phlorotannins that were isolated and it has the potential to be used in functional foods for improving the blood circulation.

Keywords: insufficient blood flow; Ecklonia cava; Pyrogallol-phloroglucinol-6,6’-bieckol; vascular dysfunction

1. Introduction The blood circulatory system is responsible for supplying the body with oxygenated blood, and it promotes the delivery of hormones, nutrients, and medication to different organs. However, insufficient blood flow results in restricted delivery to some parts of the body, particularly the extremities [1]. Although not considered to be a disease per se, inadequate blood circulation causes health problems, such as atherosclerosis, coronary artery disease, and hypertension.

Mar. Drugs 2019, 17, 272; doi:10.3390/md17050272 www.mdpi.com/journal/marinedrugs Mar. Drugs 2019, 17, 272 2 of 14

The endothelium forms the active luminal surface of blood vessels and it constitutes an interface between circulating blood in lumen and vessel walls; the luminal surface is composed of a continuous single layer of endothelial cells (ECs) and it is supported by connective tissue. ECs control the vascular tone and contractile tension in walls, by releasing diverse relaxing and contracting factors, which include acetylcholine, ATP and ADP, substance P, bradykinin, histamine, thrombin, serotonin, reactive oxygen species (ROS), nitric oxide (NO), arachidonic acid and its metabolites, and vasoactive peptides [2]. Tunica media is distinguished from tunica intima by a transverse arrangement of its elastin and collagen fibers. The media layer is mainly composed of vascular smooth muscle cells (VSMCs) and its thickness is related to blood vessel type. VSMCs maintain homeostasis in the vascular system by actively contracting and relaxing, and the sympathetic nervous system, ROS, the renin-angiotensin-aldosterone system, and immune activation influence these functions [3]. Furthermore, EC and VSMC dysfunctions can dysregulate blood circulation and they are characterized by cell death, the over-expressions of cell adhesion molecules (e.g., E-selectin, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1)) and Von Willebrand factor (VWF), platelet activation, excessive smooth muscle cell proliferation and migration, smooth muscle cell phenotypic modulation, and vascular permeability [4–6]. Recent times have seen a considerable interest in the use of functional foods to improve blood circulation, such as green tea, nuts, cayenne, Ginkgo biloba (GB), and Ecklonia cava (E. cava). GB is used as a herbal medicinal product to help in relieving peripheral arterial occlusive disease [7], and experimental evidence has shown that the leaf extract of GB (GBE) has numerous effects that are of medical interest, which include increasing the cerebral, peripheral, and microvascular blood flow and decreasing capillary permeability [8–10]. In addition, GBE is shown to protect against P-selectin-mediated immune cell adhesion and vascular inflammation [11]. The effects of GBE are attributed to the presence of two major fractions: flavonoids (under 24% of GBE) and ginkgolides (under 6% of GBE). It has been demonstrated that the ginkgolides reduce platelet aggregation and activation, and they act as platelet-activating factor antagonists, thereby encompassing the potential to improve blood circulation [12]. Of the various brown algal species, which include E. cava, Sargassum coreanum, Sargassum thunbergii, Scytosiphon lomentaria, Ishige okamurae, Sargassum fulvellum, and Sargassum horneri, it has been shown that E. cava has the potential to improve blood circulation, and its extracts are currently available as dietary supplements [13]. Coagulation studies have demonstrated that E. cava effectively prolonged thrombin time (TT), activated partial thromboplastin time (aPTT), and prothrombin time (PT), among brown algal [12]. In a Goldblatt mouse model of hypertension, increased systolic blood pressure and reduced blood flow in the renal arteries were significantly alleviated when compared to the values that were observed in control hypertension mice (15% reduction at 50 mg/kg Ecklonia cava extract (ECE)). In addition, the angiotensin converting enzyme (ACE) inhibitory effect, which is important for blood pressure control, was found to be most effective in the mice that were orally administrated 200 mg/kg ECE. The antihypertensive efficacy of ECE was confirmed by ACE inhibition [13]. Despite the well-known effect of ECE on blood circulation, the effects of the phlorotannins that comprise the ECE are not well known. Phlorotannins appear to be unique to marine algae and they are known to neutralize ROS activity; for example, they reduce the oxidizing capacity of oxidized low-density lipoprotein (LDL) and peroxynitrite, and it scavenges the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical [14]. Four active phlorotannins have been identified in E. cava, namely, (DK), 2,7-phloroglucinol- 6,6-bieckol (PHB), pyrogallol-phloroglucinol-6,6-bieckol (PPB), and (PFFA) [15]. PPB treatment significantly modulates the monocyte migration by reducing the inflammatory factor levels and macrophage differentiation in vitro. In addition, PPB treatment markedly reduced monocyte-induced endothelial cell death and the related caspase levels, and reduces monocyte-induced vascular smooth muscle cell excessive proliferation and migration in vitro. Although PPB shown beneficial effects in vitro, there are not many studies regarding improving blood circulation in vivo. Mar. Drugs 2018, 16, x FOR PEER REVIEW 3 of 14

levels, and reduces monocyte-induced vascular smooth muscle cell excessive proliferation and

Mar.migration Drugs 2019 in, 17vitro., 272 Although PPB shown beneficial effects in vitro, there are not many studies3 of 14 regarding improving blood circulation in vivo. However, although PPB is shown to have beneficial effects in vitro, few studies have examined its effect on blood circulation in vivo. Therefore, the However,present study although was undertaken PPB is shown to toexamine have beneficial the effects e ffofects PPBin on vitro vascular, few studies dysfunction have examinedin obese and its ehypertensionffect on blood mouse circulation models.in vivo . Therefore, the present study was undertaken to examine the effects of PPB on vascular dysfunction in obese and hypertension mouse models. 2. Results and Discussion 2. Results and Discussion 2.1. ECE and GBE Treatments Improved ECs and VSMCs Functions In Vitro and In Vivo 2.1. ECE and GBE Treatments Improved ECs and VSMCs Functions In Vitro and In Vivo Palmitate-acid (PA) conjugated bovine serum albumin (BSA) treated cell assay was performed Palmitate-acid (PA) conjugated bovine serum albumin (BSA) treated cell assay was performed to to confirm the efficacies of GBE and ECE. The EC survival assay result illustrated reduced EC confirm the efficacies of GBE and ECE. The EC survival assay result illustrated reduced EC survival survival ratios in the PA treated cells, which were dose-dependently higher in the PA plus ECE ratios in the PA treated cells, which were dose-dependently higher in the PA plus ECE (PA/ECE) or (PA/ECE) or GBE (PA/GBE) treated cells (Figure 1A). On the other hand, the VSMC proliferation GBE (PA/GBE) treated cells (Figure1A). On the other hand, the VSMC proliferation and migration and migration was inhibited after exposure to PA/ECE or PA/GBE as compared with PA treated was inhibited after exposure to PA/ECE or PA/GBE as compared with PA treated cells (Figure1B,C). cells (Figure 1B,C). The percentage of TUNEL positive cells were higher in diet induced obese mice The percentage of TUNEL positive cells were higher in diet induced obese mice that were orally that were orally administrated saline (DIO/saline) than in chow diet fed mice (Sham); these levels administrated saline (DIO/saline) than in chow diet fed mice (Sham); these levels were significantly were significantly lower in ECE orally administrated obese mice (Obese/ECE) than in GBE orally lower in ECE orally administrated obese mice (Obese/ECE) than in GBE orally administrated obese administrated obese mice (Obese/GBE) (Figure 1D). In addition, the proliferating cell nuclear mice (Obese/GBE) (Figure1D). In addition, the proliferating cell nuclear antigen (PCNA) positivity was antigen (PCNA) positivity was less in ECE administrated obese mice when compared to saline less in ECE administrated obese mice when compared to saline administrated obese mice (Figure1E) administrated obese mice (Figure 1E) and, consequently, the media layers of blood vessels in and, consequently, the media layers of blood vessels in hematoxylin and eosin stained aortas were hematoxylin and eosin stained aortas were thinner in ECE administrated mice than in the saline thinner in ECE administrated mice than in the saline administrated mice (Figure S1). Furthermore, administrated mice (Figure S1). Furthermore, the levels of systolic, diastolic, and mean artery the levels of systolic, diastolic, and mean artery pressures (Figure1F) were significantly lower in pressures (Figure 1F) were significantly lower in Obese/ECE mice than in Obese/Saline mice. These Obese/ECE mice than in Obese/Saline mice. These results indicate that, in the diet induced-obese results indicate that, in the diet induced-obese mouse models, ECE and GBE were similarly effective mouse models, ECE and GBE were similarly effective in terms of improving the blood circulation. in terms of improving the blood circulation.

FigureFigure 1. 1.E ffEffectsects of Ginkgoof Ginkgo biloba bilobaextract extract (GBE) and(GBE)Ecklonia and cavaEckloniaextract cava (ECE) extract on vascular (ECE) dysfunctionon vascular indysfunction vitro and in in vivo vitro(A and) endothelial in vivo (A cells) endothelial (ECs) (SVEC4-10; cells (ECs) mouse (SVEC4-10; endothelial mouse cells)endothelial were exposed cells) were to Palmitateexposed /Phosphate-buto Palmitate/Phosphate-bufferedffered saline (PA/PBS), Palmitatesaline /(PA/PBS),ECE (PA/ECE) Palmitate/ECE or Palmitate /GBE(PA/ECE) (PA/GBE). or ThePalmitate/GBE survival ratios (PA/GBE). of ECs were The measured survival ratios using aof cell ECs survival were measured assay. (B,C using) PA/ PBS,a cell PA survival/ECE, or assay. PA/GBE (B, wereC) PA/PBS, administered PA/ECE, to vascular or PA/GBE smooth were muscle administered cells (VSMCs) to (MOVAS,vascular mousesmooth vascular muscle smoothcells (VSMCs) muscle cells),(MOVAS, and proliferatingmouse vascular to migrating smooth muscle VSMC ratioscells), wereand pr measuredoliferating by to respective migrating assays. VSMC (D ratios) Confocal were microscopicmeasured by images respective showing assays. apoptotic (D) Confocal cells as microscopic TUNEL positive images cells showing (red, arrows) apoptotic and cells DAPI as stainedTUNEL nucleipositive (blue). cells (red, Scale arrows) bar = 50 andµm DAPI (E) Lightstained microscopic nuclei (blue). images Scale showing bar = 50 proliferatingμm (E) Light cellmicroscopic nuclear antigenimages (PCNA)showing positive proliferating cells (brown, cell nuclear arrow) inantigen obese mice.(PCNA) Scale positive bar = 10 cellsµm (brown, (F) Blood arrow) pressure in plotsobese mice. Scale bar = 10 μm (F) Blood pressure plots of systolic, diastolic, and mean artery blood pressure in obese mice. *, P < 0.05 and ***, P < 0.001 vs. the PBS (Sham) group; $, P < 0.05, $$, P < 0.01 and $$$, P

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Mar.of Drugs systolic, 2018, diastolic,16, x FOR PEER and mean REVIEW artery blood pressure in obese mice. *, P < 0.05 and ***, P < 0.001 vs. 4 of 14 the PBS (Sham) group; $, P < 0.05, $$, P < 0.01 and $$$, P < 0.001 vs. PA (or DIO/Saline) group; NS, not< 0.001 significant. vs. PA (or Results DIO/Saline) are presented group; NS, as meansnot significant.SD. ECE, ResultsE. cava areextract; presented GBE, as leafmeans of GB± SD. extract; ECE, ± PCNA,E. cava extract; Proliferating GBE, cellleafnuclear of GB ex antigen;tract; PCNA, TUNEL, Proliferating Terminal deoxynucleotidyl cell nuclear antigen; transferase TUNEL, dUTP Terminal nick enddeoxynucleotidyl labeling. transferase dUTP nick end labeling.

2.2. PPB Effectively Reduced ECs and VSMCs Dysfunction In Vitro 2.2. PPB Effectively Reduced ECs and VSMCs Dysfunction In Vitro In order to identify the active component of the ECE, four phlorotannins that were isolated fromIn ECE order were to identify compared, the active as previously component describe of the ECE,d [16]. four The phlorotannins four isolated that phlorotannins were isolated fromwere ECEconfirmed were compared, to be DK, as PHB, previously PPB, describedand PFFA. [16 ].To The investigate four isolated the phlorotannins protective effects were confirmedof adhesion to bemolecule DK, PHB, expression PPB, and on PFFA. ECs Toas investigatemouse endothelial the protective cells, etheffects ECs of were adhesion exposed molecule to either expression PBS, 0.25 on ECsmM asof mousePA, or endothelial0.25 mM of cells, PA plus the ECsfour were phlorotannins exposed to for either 48 h PBS, (Figure 0.25 2A). mM The of PA, expression or 0.25 mM levels of PA of plusadhesion four phlorotanninsmolecules, such for as 48 E-selectin, h (Figure 2ICAM-1,A). The expressionVCAM-1, and levels vWF of adhesionin PA/PBS molecules, treated SVEC4-10 such as E-selectin,were significantly ICAM-1, higher VCAM-1, than andthose vWF in the in PAPBS/PBS trea treatedted ECs, SVEC4-10 but the level were in significantly PA/PPB co-treated higher thancells thosewas lower in the than PBS treatedthose in ECs, PA/PBS but the treated level in ECs PA/ PPB(Figures co-treated 2B–E). cells Furthermore, was lower thansurvival those ratios in PA were/PBS treatedsignificantly ECs (Figure lower2 inB–E). the Furthermore,PA/PBS treated survival ECs than ratios in werethe PBS significantly treated ECs lower (Figure in the 2F). PA The/PBS survival treated ECsratios than were in thesignificantly PBS treated lower ECs (Figurein PA/PBS2F). Thetreated survival SVEC4-10 ratios cells were than significantly those in lower the PBS in PA treated/PBS treatedSVEC4-10 SVEC4-10 cells. cells than those in the PBS treated SVEC4-10 cells.

FigureFigure 2.2.Comparison Comparison of theof the four four phlorotannins phlorotannins isolated isol fromated ECE from on ECECE adhesion on EC moleculeadhesion expression molecule andexpression cell survival and cellin vitro survival.(A) Illustrationin vitro. (A explains) Illustration palmitate explains induced palmitate EC dysfunction induced EC model dysfunctionin vitro. (modelB–E) mRNA in vitro. expression (B–E) mRNA levels expression of the adhesion levels molecules, of the adhesi includingon molecules, E-selsectin, including ICAM-1, E-selsectin, VCAM-1 andICAM-1, vWF inVCAM-1 ECs as determinedand vWF in byECs qRT-PCR. as determined (F) The by graph qRT-PCR. shows PA(F) withThe graph or without shows PA with or (dieckolwithout (DK),phlorotannin 2,7-phloroglucinol-6,6-bieckol (dieckol (PHB),(DK), Pyrogallol-Phloroglucinol-6,6-Bieckol 2,7-phloroglucinol-6,6-bieckol (PPB),(PHB), and phlorofucofuroeckolPyrogallol-Phloroglucinol-6,6-Bieckol A (PFFA)) treated (PPB), EC survival and phlorofucofuroeckol ratio. *, P < 0.05 and A **, (PFFA)) P < 0.01 treated vs. the EC PBS survival group; $,ratio. P < 0.05*, P < and 0.05 $$, and P < **,0.01 P < vs.0.01 PA vs./PBS the group;PBS group; #, P < $,0.05 P < vs.0.05 PA and/PPB. $$, PA, P < palmitate0.01 vs. PA/PBS conjugation group; to #, BSA; P < DK,0.05 dieckol;vs. PA/PPB. PHB, PA, 2,7-phloroglucinol-6,6-bieckol; palmitate conjugation to BSA; PPB, DK, pyrogallol-phloroglucinol-6,6-bieckol;dieckol; PHB, 2,7-phloroglucinol-6,6-bieckol; PFFA, phlorofucofuroeckolPPB, pyrogallol-phloroglucinol-6,6-bieckol; A. PFFA, phlorofucofuroeckol A.

Apart from mouse endothelial cells, the exposure to PA/PBS also increased the excessive proliferation and migration of VSMCs. The treatment of VSMCs with PA resulted in significantly higher cell proliferating ratios than PBS treated cells; also, the proliferating ratios of PA/PPB co-treated MOVAS were significantly lower than PA/PBS treated cells (Figure 3A,B). Similarly, the

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Apart from mouse endothelial cells, the exposure to PA/PBS also increased the excessive proliferation and migration of VSMCs. The treatment of VSMCs with PA resulted in significantly higher cell proliferating ratios than PBS treated cells; also, the proliferating ratios of PA/PPB co-treated Mar. Drugs 2018, 16, x FOR PEER REVIEW 5 of 14 MOVAS were significantly lower than PA/PBS treated cells (Figure3A,B). Similarly, the migrating ratiosmigrating were ratios higher were in PA higher/PBS treatedin PA/PBS VSMCs treated than VS PBSMCs treated than PBS cells. treated The proliferating cells. The proliferating and migrating and cellmigrating ratios of cell PA ratios/PPB co-treatedof PA/PPB VSMCs co-treated were VSMCs also lower were than also those lower of than other those phlorotannin of other phlorotannin treated cells (Figuretreated3 C).cells (Figure 3C).

FigureFigure 3.3. ComparisonComparison ofof thethe fourfour phlorotanninsphlorotannins isolatedisolated fromfrom ECEECE ononexcessive excessive proliferationproliferation andand migrationmigration ofof VSMCsVSMCsin in vitro vitro..( A(A)) IllustrationIllustration explainsexplains palmitatepalmitate conjugationconjugation toto BSABSA (PA)(PA) induced induced VSMCVSMC dysfunction dysfunction model modelin vitroin vitro..(B) PA(B) with PA orwith without or without phlorotannin phlorotannin treated VSMCs treated were VSMCs measured were usingmeasured a proliferation using a proliferation assay, and (C )assay, trans-well and migration(C) trans-well assay. migration *, P < 0.05 assay. vs. the *, PBS P < group; 0.05 vs. $, Pthe< 0.05PBS vs.group; PA group; $, P < and,0.05 #,vs. P

2.3. PPB Helps Rescue Vascular Dysfunction in the Diet-Induced Mouse Models 2.3. PPB Helps Rescue Vascular Dysfunction in the Diet-Induced Mouse Models WhenWhen consideringconsidering thatthat PPBPPB waswas moremore activeactive thanthan thethe otherother threethree activeactive compoundscompounds inin vitrovitro,, wewe examined examined thethe e effectsffects of of PPB PPB in in diet-induced diet-induced mouse mouse models models of of obese obese and and hypertension. hypertension. ProteinProtein levelslevels of of the the adhesion adhesion molecules, molecules, such such asas E-selectin, E-selectin, ICAM-1,ICAM-1, VCAM-1,VCAM-1, andand vWF,vWF, were were lower lower in in the the ObeseObese/PPB/PPB mice mice group group than than in thein the Obese Obese/Saline/Saline group. group. The adhesionThe adhesion molecule molecule levels werelevels also were higher also inhigher the diet-induced in the diet-induced hypertension hypertension mice model mice mode (Hypertensionl (Hypertension/Saline)/Saline) than inthan the in Sham the Sham group, group, but thebut expressions the expressions in Hypertension in Hypertension/PPB/PPB mice mice group group were were statistically statistically significantly significantly lower lower than than in the in Hypertensionthe Hypertension/Saline/Saline group group (Figure (Figure4A–F). 4A–F).

Mar. Drugs 2018, 16, x FOR PEER REVIEW 5 of 14 migrating ratios were higher in PA/PBS treated VSMCs than PBS treated cells. The proliferating and migrating cell ratios of PA/PPB co-treated VSMCs were also lower than those of other phlorotannin treated cells (Figure 3C).

Figure 3. Comparison of the four phlorotannins isolated from ECE on excessive proliferation and migration of VSMCs in vitro. (A) Illustration explains palmitate conjugation to BSA (PA) induced VSMC dysfunction model in vitro. (B) PA with or without phlorotannin treated VSMCs were measured using a proliferation assay, and (C) trans-well migration assay. *, P < 0.05 vs. the PBS group; $, P < 0.05 vs. PA group; and, #, P < 0.05 vs. the PA/PPB group. NS, not significant. PA, palmitate conjugation to BSA; DK, dieckol; PHB, 2,7-phloroglucinol-6,6-bieckol; PPB, pyrogallol-phloroglucinol-6,6-bieckol; PFFA, phlorofucofuroeckol A.

2.3. PPB Helps Rescue Vascular Dysfunction in the Diet-Induced Mouse Models When considering that PPB was more active than the other three active compounds in vitro, we examined the effects of PPB in diet-induced mouse models of obese and hypertension. Protein levels of the adhesion molecules, such as E-selectin, ICAM-1, VCAM-1, and vWF, were lower in the Obese/PPB mice group than in the Obese/Saline group. The adhesion molecule levels were also higher in the diet-induced hypertension mice model (Hypertension/Saline) than in the Sham group,

Mar.but Drugsthe expressions2019, 17, 272 in Hypertension/PPB mice group were statistically significantly lower than6 of 14in the Hypertension/Saline group (Figure 4A–F).

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FigureFigure 4.4. InhibitoryInhibitory eeffectsffects ofof thethe PPBPPB isolatedisolated fromfrom ECEECE onon ECEC adhesionadhesion moleculemolecule expressionexpression inin diet-induceddiet-induced mousemouse modelsmodels ofof obeseobese andand hypertension.hypertension. ((AA,,BB)) ArrowsArrows inin confocalconfocal microscopicmicroscopic imagesimages showshow adhesionadhesion moleculemolecule expressionsexpressions (green,(green, arrow;arrow; E-selectin,E-selectin, ICAM-1,ICAM-1, VCAM-1,VCAM-1, vWF)vWF) oror nucleinuclei (DAPI,(DAPI, blue)blue) inin obeseobese andand hypertensionhypertension mousemouse models.models. ((CC––FF)) QuantitativeQuantitative graphsgraphs showshow fluorescencefluorescence intensityintensity fromfrom representative representative images images (dark (dark gray gray bar, bar, obese obese mouse mouse model; model; gray bar,gray hypertension bar, hypertension mouse model).mouse model). Scale bar Scale= 50 barµm = *,50 P μ

TheThe expressionsexpressions ofof ECEC survivalsurvival relatedrelated molecules,molecules, suchsuch asas PI3K-pAKTPI3K-pAKT andand pAMPK,pAMPK, inin thethe ObeseObese/PPB/PPB micemice group group and and Hypertension Hypertension/PPB/PPB mice mice group group were were significantly significantly higher higher than than those those in the in Obesethe Obese/Saline/Saline mice mice group group and Hypertension and Hypertension/S/Saline micealine groups mice groups (Figure 5(FigureA–D). The5A–D). TUNEL The positiveTUNEL cellspositive were cells also were markedly also markedly lower in the lower Obese in /thePPB Obese/PPB mice and Hypertension mice and Hypertension/PPB/PPB mice groups mice than groups in the Obesethan in/Saline the Obese/Saline and Hypertension and Hypert/Salineension/Saline groups (Figure groups5E,F). These(Figure results 5E,F). indicate These thatresults PPB indicate modulates that thePPB expressions modulates ofthe adhesion expressions molecules of adhesion and cell molecules survival and in vivo. cell survival in vivo.

Figure 5. Inhibitory effects of the PPB isolated from ECE on EC dysfunction in diet-induced mouse modelsFigure 5. of Inhibitory obese and effects hypertension. of the PPB (A )isolated Light microscopic from ECE on images EC dysfunction showing the in ECdiet-induced survival related mouse molecules,models of suchobese as and PI3K, hypertension. pAKT and pAMPK (A) Light (brown, microscopic arrow). (imagesB–D) Quantitative showing the graphs EC survival show intensity related frommolecules, representative such as imagesPI3K, (darkpAKT gray and bar, pAMPK obese mouse(brown, model; arrow). gray (B bar,–D) hypertensionQuantitative mouse graphs model). show Scaleintensity bar =from10 µ m.representative (E,F) TUNEL images positive (dark cells gray (red, bar, arrow) obese indicate mouse apoptotic model; cells,gray andbar, nucleihypertension stained DAPImouse (blue). model). Quantitative Scale bar graphs= 10 μm. show (E,F percentage) TUNEL positive of TUNEL cells positive (red, cellsarrow) from indicate representative apoptotic images. cells, Scaleand nuclei bar = stained50 µm. DAPI *, P < (blue).0.05 and Qu ***,antitative P < 0.001 graphs vs. the show sham percentage group; $, of P TUNEL< 0.05 vs. positive Obese /cellsSaline from or Hypertensionrepresentative/Saline images. group. Scale PPB, bar = pyrogallol-phloroglucinol-6,6-bieckol. 50 μm. *, P < 0.05 and ***, P < 0.001 vs. the sham group; $, P < 0.05 vs. Obese/Saline or Hyertension/Saline group. PPB, pyrogallol-phloroglucinol-6,6-bieckol.

In addition, the inhibitory effects of PPB on the excessive proliferation and migration of VSMCs were validated in the two animal models. The expression levels of pERK1/2 and pAKT in VSMCs were the highest in the Obese/Saline and Hypertension/Saline mice groups (Figure 6), which indicates the involvement in the observed reductions in numbers of proliferative VSMCs and in wall thicknesses.

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In addition, the inhibitory effects of PPB on the excessive proliferation and migration of VSMCs were validated in the two animal models. The expression levels of pERK1/2 and pAKT in VSMCs were the highest in the Obese/Saline and Hypertension/Saline mice groups (Figure6), which indicates the involvementMar. Drugs 2018 in, 16 the, x observedFOR PEER REVIEW reductions in numbers of proliferative VSMCs and in wall thicknesses. 7 of 14

FigureFigure 6.6. Inhibitory effects effects of of the the PPB PPB isolated isolated from from ECE ECE on on VSMC VSMC dysfunction dysfunction related related molecules molecules in indiet-induced diet-induced mouse mouse models models of of obese obese and and hypertension. hypertension. ( (AA)) Light Light microscopic imagesimages showingshowing abnormallyabnormally highhigh expressionsexpressions ofof proliferationproliferation andand migrationmigration relatedrelated moleculesmolecules (pERK1(pERK1/2/2 andand pAKT)pAKT) inin VSMCsVSMCs inin thethe ObeseObese andand inin HypertensionHypertension models.models. ((BB,,CC)) QuantitativeQuantitative graphsgraphs showshow intensityintensity fromfrom representativerepresentative imagesimages (dark(dark graygray bar,bar, ObeseObese mousemouse model;model; graygray bar,bar, HypertensionHypertension mousemouse model).model). ScaleScale barbar= = 1010 µμm.m. *,*, PP< < 0.050.05 vs.vs. thethe shamsham group; group; $, $, P P< <0.05 0.05 vs. vs. Obese Obese/Saline/Saline or or Hypertension Hyertension/Saline/Saline group.group. PPB,PPB, pyrogallol-phloroglucinol-6,6-bieckol.pyrogallol-phloroglucinol-6,6-bieckol.

TheThe PCNAPCNA positivepositive cellscells werewere higherhigher inin thethe ObeseObese/Saline/Saline andand HypertensionHypertension/Saline/Saline micemice groupsgroups thanthan inin thethe ShamSham group,group, butbut aa reducedreduced numbernumber ofof positivepositive cellscells werewere observedobserved inin thethe ObeseObese/PPB/PPB andand HypertensionHypertension/PPB/PPB micemice groupsgroups thanthan inin thethe ObeseObese/Saline/Saline andand HypertensionHypertension/Saline/Saline mice groups. Finally,Finally, aortaaorta wallwall thicknessthickness waswas alsoalso significantlysignificantly thinnerthinner inin thethe ObeseObese/PPB/PPB andand HypertensionHypertension/PPB/PPB micemice groupsgroups thanthan inin thethe ObeseObese/Saline/Saline andand HypertensionHypertension/Saline/Saline groupsgroups (Figure(Figure7 7A–D).A–D). These These results results indicateindicate thatthat PPB PPB inhibits inhibits excessive excessive VSMC VSMC proliferation proliferation and and migration, migration, and itand probably it probably reduces reduces blood vesselblood wallvessel thickening wall thickening in diet-induced in diet-induced mouse models mouse of obesemodels and of hypertension. obese and hypertension. Some experiments Some furtherexperiments validated further whether validated the uncontrolled-blood whether the uncontro pressurelled-blood and lipoproteins pressure and in lipoproteins these mouse in models these weremouse improved models were after exposureimproved toafter PPB. exposure Systolic, to diastolic, PPB. Systolic, and mean diastolic, blood and pressures mean blood were higherpressures in thewere Obese higher/Saline in the and Obese/Saline Hypertension and/Saline Hypertension/S mice groupsaline than mice in sham groups group, than butin sham the blood group, pressures but the wereblood significantly pressures were lower significantly in the Obese lower/PPB in and the Hypertension Obese/PPB and/PPB Hypertension/PPB groups than in Obese groups/Saline than and in HypertensionObese/Saline and/Saline Hypertension/Saline mice groups (Figure mice7E). groups In addition, (Figure serum 7E). In total addition, cholesterol, serum triglyceride, total cholesterol, and LDLtriglyceride,/HDL ratios and were LDL/HDL significantly ratios were lower significan in the Obesetly lower/PPB and in the Hypertension Obese/PPB/ PPBand Hypertension/PPB groups than in the Obesegroups/Saline than in and the Hypertension Obese/Saline/Saline and Hy groupspertension/Saline (Figure S2). groups (Figure S2).

Mar. Drugs 2019, 17, 272 8 of 14

Mar. Drugs 2018, 16, x FOR PEER REVIEW 8 of 14

FigureFigure 7. 7.Eff ectsEffects of PPBof isolatedPPB isolated from ECEfrom on ECE vascular on dysfunctionvascular dysfunction and blood pressureand blood in diet-induced pressure in mousediet-induced models ofmouse obese models and hypertension of obese and (A,C )hypertension Light microscopic (A,C) images Light showingmicroscopic proliferation images showing marker (PCNA)proliferation and H&E marker stained (PCNA) blood and vessels. H&E stained (B,D) Plotsblood showing vessels. ( numbersB,D) Plots of showing PCNA positivenumbers cells of PCNA and intima-mediapositive cells ratios and intima-media obtained using ra representativetios obtained using H&E imagesrepresentati (darkve gray H&E bar, images Obese (dark mouse gray model; bar, garyObese bar, mouse Hypertension model; mousegary bar, model). Hypertension (E) Blood mouse pressures, model). such as(E) systolic, Blood pressures, diastolic, and such mean as systolic, artery pressurediastolic, in and Obese mean and artery Hypertension pressure mousein Obese models. and Hypertension Scale bar = 10 mouseµm (PCNA) models. and Scale 200 barµm = (H&E). 10 μm *,(PCNA) P < 0.05 vs.and Sham 200 group;μm (H&E). $, P < 0.05*, P vs.< 0.05 Obese vs./Saline Sham or Hypertensiongroup; $, P /

E.E. cava cavais ais brown a brown seaweed seaweed that that is used is used in food in productsfood products and it isand the it most is the common most common in the seas in aroundthe seas Jejuaround Island Jeju [17 Island]. Several [17]. studies Several have studies been have conducted been conducted on the association on the association between Ecklonia between cava Eckloniaand cardiovascularcava and cardiovascular health [13– 15health,17]. Some[13–15,17]. studies Some on phlorotannins studies on phlorotannins that were isolated that were from isolated ECE or ECEfrom itselfECE report or ECE potent itself anti-oxidant report potent properties anti-oxidant against toxinproperties or stress against induced toxin ROS or induction stress induced [18,19]. ECEROS isinduction also reported [18,19]. to suppress ECE is also lipopolysaccharide reported to suppress (LPS)-induced lipopolysacch inflammatoryaride (LPS)-induced responses in inflammatory endothelial cells,responses and decreases in endothelial the expressions cells, and of decreases adhesion factorsthe expressions (E-selectin, of VCAM-1, adhesion ICAM-1, factors and(E-selectin, vWF) andVCAM-1, immune ICAM-1, cell adhesion and vWF)in vitro and. immune Additionally, cell adhesion ECE is reportedin vitro. Additionally, to have anti-inflammatory ECE is reported and to anti-oxidanthave anti-inflammatory effects in endothelial and anti-oxidant cells [20]. effects in endothelial cells [20]. AnAn interesting interesting result result of of the the present present study study is that is that the etheffect effect of GBE of isGBE not is significantly not significantly different different from thatfrom of that GB, whichof GB, iswhich currently is currently one of theone 10 of most the 10 popular most popular supplements supplements in the western in the western world [ 7world–11]. In[7–11]. rats, GBE In rats, supplementation GBE supplementation for four weeksfor four reduces weeks the reduces systolic, the but systolic, not diastolic but not blood diastolic pressure, blood inpressure, a dose and in a time dose dependent and time mannerdependent [21 ,manner22]. Interestingly, [21,22]. Interestingly, systolic and systolic diastolic and blood diastolic pressures blood werepressures also lower were inalso Obese lower/ECE in Obese/ECE and Obese/ GBEand Obese/ mice gropesGBE mice than gropes those inthan the those Obese in/Saline the Obese/Saline and there wasand no there significant was no di ffsignificanterence in betweendifference Obese in /betweenGBE and Obese/GBE Obese/ECE miceand group.Obese/ECE However, mice meangroup. arteryHowever, blood mean pressure artery and blood blood vesselpressure thicknesses and blood were vessel significantly thicknesses lower were in Obesesignificantly/ECE than lower those in inObese/ECE Obese/Saline than (Figure those1 inF). Obese/Saline (Figure 1F). E.E. cavacava isis currentlycurrently beingbeing exploredexplored forfor itsits health-promotinghealth-promoting propertiesproperties duedue toto itsits highhigh phlorotanninphlorotannin contentscontents (~18%(~18% onon aa dry dry weight weight basis). basis). ItIt isis knownknown toto containcontain numerousnumerous phlorotannins,phlorotannins, which which includes includes dieckol, dieckol, PFFA, PHB,PFFA, PPB, PHB, phlorofucofuroeckol, PPB, phlorofucofuroeckol, 6,60-bieckol, 8,86,6’-bieckol,0-bieckol, 2-8,8’-bieckol,O-(2,4,6-trihydroxyphenyl)-6,6 2-O-(2,4,6-trihydroxyphenyl)-6,60-bieckol, eckstolonol,′ 2-phloroeckol,-bieckol, eckstolonol, 1-(30,50-dihydroxyphenoxy)- 2-phloroeckol, 7-(2”,4”,6-trihydroxyphenoxy)-2,4,9-trihydroxydibenzo-1,4-dioxin,1-(3’,5’-dihydroxyphenoxy)-7-(2’’,4’’,6-trihydroxyphenoxy)-2,4,9-trihydroxydibenzo-1,4-dioxin, , 7-phloroeckol, phlorotannin A,eckol, and phloroglucinol7-phloroeckol, [23phlorotannin–28]. The phlorotannins A, and phlo haveroglucinol a resilient [23–28]. dibenzo-1,4-obesityxin The phlorotannins backbone, have a whichresilient presumably dibenzo-1,4-obesityxin aids molecular backbone, interactions which with variouspresumably biomolecules aids molecular [29,30]. Interestingly,interactions with the various biomolecules [29,30]. Interestingly, the anti-oxidant effects of these phlorotannins are related to the number of hydroxyl groups that are present. According to a study by Li et al., dieckol

Mar. Drugs 2019, 17, 272 9 of 14 anti-oxidant effects of these phlorotannins are related to the number of hydroxyl groups that are present. According to a study by Li et al., dieckol and 6,60-bieckol, which both containing more than 10 hydroxyl groups, have a higher anti-oxidant efficacy than phloroglucinol or eckol (less than 10 hydroxyl groups) [26]. Thus, since PPB possesses 15 hydroxyl groups, it might also be expected to improve blood circulation effects, such as monocyte migration and macrophage polarization. We isolated four phlorotannins from ECE and observed that all of the reduced adhesion molecules expression and increased EC survival. In addition, PPB effectively increases the EC survival and decreases the adhesion molecule expression in diet-induced mouse models of obese and hypertension (Figure4). Previous studies have shown that adhesion molecule expression and pAKT-PI3K negatively affect EC survival [15,31]. The effects of PPB on VSMCs, which play important roles in blood circulation-related diseases, were also examined. Our results show that exposure to PA causes VSMC dysfunction, and PPB treatment helps to reduce the excessive proliferation in MOVAS and in these two mouse models. High fat and high cholesterol diets induce the de-differentiation of contractile vascular smooth muscle cells into synthetic smooth muscles cells, and these phenotype changes play important roles in vascular diseases, like atherosclerosis [32]. Synthetic cells exhibit excessive proliferation, migration, and neo-intima formation. Proliferative cell (marked PCNA) and intima-media ratio were found to be significantly lower in the Obese/PPB and Hypertension/PPB group than those in the Obese/Saline and Hypertension/Saline group (Figure5). Interestingly, PPB as an active phlorotannin from ECE significantly reduces the levels of lipoprotein, cholesterols, and blood pressure (Figure7 and Figure S2). This potency is presumably related to the anti-oxidant efficacy of PPB and the number of hydroxyl groups that are attached to its ring system [27,33].

3. Materials and Methods

3.1. Preparations of ECE and GBE and Isolation of Four Phlorotannins ECE was obtained from Aqua Green Technology Co. Ltd. (Jeju, Korea). Briefly, Ecklonia cava were washed, air-dried for 48 h at room temperature, ground, and extracted while using 50% aqueous ethanol for 12 h at 85 ◦C. The mixture was then filtered, concentrated, sterilized by heating for 60 min. at 85 ◦C, and spray-dried. The four phlorotannins that were used in this study were isolated from ECE using a previously described method [16]. Briefly, centrifugal partition chromatography (CPC) was performed while using a two-phase solvent system comprised of water/ethyl acetate/methanol/n-hexane (7:7:3:2, v/v/v/v). For separation, the stationary phase was an organic solution that was filled in the CPC column; the mobile phase was pumped into the column in a descending mode at the flow rate 2 mL/min. The positive control GBE was purchased ready to use from Hanzhong TRG Biotech Co. Ltd. (Hanzhong, China).

3.2. Endothelial Cell Survival Assay SVEC4-10 cells (104) were purchased from the Korean cell line bank (Seoul, Korea). The cells were seeded in wells of 96-well plates and, after 24 h incubation, the attached cells were rinsed twice with PBS. The cells were then treated with BSA plus PBS (PBS), PA plus PBS (PA/PBS), or PA plus phlorotannin (PA/DK, PA/PHB, PA/PPB, or PA/PFFA) for 48 h. The treated cells were rinsed twice with PBS and cell survival was determined using the Transdetect cell counting kit (Transgen Biotech Co., Ltd., Beijing, China). Absorbance was measured at 450 nm using a microplate reader (Spectra max plus, Molecular Devices, San Jose, CA, USA).

3.3. VSMC Proliferation Assay MOVAS cells (104) were purchased from the Korean cell line bank (Seoul, Korea). The cells were seeded in 96-well plates; after 24 h incubation, the attached cells were rinsed twice with PBS and subsequently treated with BSA plus PBS (PBS), PA plus PBS (PA/PBS), or PA plus phlorotannin Mar. Drugs 2019, 17, 272 10 of 14

(PA/DK, PA/PHB, PA/PPB, or PA/PFFA) for 4 h, rinsed twice with PBS, and cell proliferation assay was determined while using the Transdetect cell counting kit. Absorbance was measured at 450 nm using a microplate reader (Spectra max plus, Molecular Devices).

3.4. VSMC Trans-Well Migration Assay MOVAS cells (5 105) were seeded in the upper compartments of trans-well chambers and × they were treated with BSA plus PBS (PBS), PA plus PBS (PA/PBS), or PA plus phlorotannin (PA/DK, PA/PHB, PA/PPB, or PA/PFFA) for 24 h. The attached cells were rinsed twice with PBS, and cells remaining in the upper compartments of the trans-well chamber were thoroughly removed with cotton swabs. The migrating cells were detected using the Transdetect cell counting kit to quantify cell migration to lower compartments; absorbance was measured at 450 nm using a micro plate reader (Spectra max plus).

3.5. Quantitative Real Time Polymerase Chain Reaction (qRT-PCR) Cellular RNA was isolated using RNAiso Plus (TAKARA, Kusatsu, Japan), according to the manufacturer’s instructions. Briefly, the cell pellets were resuspended in 1 mL of RNAiso Plus by pipetting, mixed with 0.1 mL chloroform (Amresco, Cleveland, OH, USA), and centrifuged at 12,000 g × for 15 min at 4 ◦C. The supernatants were mixed with 0.25 mL 100% isopropanol, and isolated RNA pellets were washed with 70% ethanol and centrifuged at 7500 g for 5 min. Dried pellets were × dissolved in 30 µL diethyl pyrocarbonate (DEPC) water, and RNA was quantified while using a Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was prepared from RNA using a cDNA synthesis kit (PrimeScript™, TAKARA). qRT-PCR was performed to determine the RNA levels. The primer was mixed with distilled water and then placed in 384-wells. Template (cDNA) and SYBR green (TAKARA) were subsequently added, and then validated using PCR machine (Bio-Rad, Hercules, CA, USA). Table S1 lists the genes of interest.

3.6. Diet Induced Mice Model C57BL/6N male mice (seven weeks old) were obtained from Orient Bio (Sungnam, Korea) and maintained under controlled conditions (room temperature 23 ◦C and 50% humidity under a 12 h dark/light cycle). High fat diet-induced obese mice model (obese) was used in this study. Briefly, the mice were randomly assigned to five groups: (group 1, Sham) the mice were fed a regular chow diet (PicoLab Rodent diet, Fort Worth, TX, USA) for four weeks and then orally administrated 0.9% normal saline for four weeks; (group 2, Obese/Saline) mice were fed a 45% high fat diet (HFD; Research Diet Inc., New Brunswick, NJ, USA) for four weeks to procure diet-induced obese mice, and then orally administrated saline for four weeks; (group 3, Obese/ECE) mice were fed a 45% HFD for four weeks and then orally administrated ECE (70 mg/kg/day) for four weeks; (group 4, Obese/GBE) mice were fed a 45% HFD for four weeks and then orally administrated GBE (500 mg/kg/day) for four weeks [34]. To confirm the effects of Pyrogallol-Phloroglucinol-6,6-Bieckol (PPB) as a single phlorotannin from ECE, (group 5, Obese/PPB) the mice were fed HFD for four weeks and then orally administrated PPB (2.5 mg/kg/day (ECE contains 3.57% PPB)) for four weeks. Hypertension mice model (Hypertension) was used in this study. Briefly, the mice were randomly assigned to two groups: (group 1, Hypertension/Saline) mice were fed with high cholesterol diet (Harlan Laboratories, Indianapolis, IN, USA) with salt (0.03 mg/kg/day, Hanju Corp., Ulsan, Korea) for four weeks and then orally administrated 0.9% saline for four weeks; (group 2, Hypertension/PPB) mice were fed high cholesterol diet with salt for four weeks and then orally administrated PPB (2.5 mg/kg/day) for four weeks. At eight weeks after treatment commencement, all of the mice were sacrificed in accordance with the ethical principles issued by the Institutional Animal Care and Use Committee of Gachon University (approval number LCDI-2017-0034). Table S2 and Figure S3 contain the schemes for the entire constructed animal model. Mar. Drugs 2019, 17, 272 11 of 14

3.7. DAB Immunohistochemistry

Aorta paraffin block tissues were sectioned at 7 µm, placed on slides, and dried at 37 ◦C for 24 h. The slides with the paraffin embedded sections were incubated in 0.3% H2O2 (Sigma-Aldrich, saint louis, MO, USA) for 30 min, rinsed three times with PBS, incubated in animal serum to block antibody binding, incubated with primary antibodies (listed in Table S1), and then rinsed three times with PBS. The probed slides were then exposed to biotinylated secondary antibodies from the ABC kit (Vector Laboratories, Burlingame, CA, USA), incubated for 1 h in blocking solution, and rinsed three times with PBS. Thr slides were developed with DAB (3,3-diaminobenzidine) substrate for 5 to 15 min, mounted with cover slips and DPX mounting solution (Sigma-Aldrich), and visualized by light microscopy (Olympus Optical Co., Tokyo, Japan). Table S3 lists the antibodies used this study.

3.8. Immunofluorescence (IF) Paraffin block sections (7 µm) were de-paraffinized, incubated in animal serum to block antibody binding, incubated with primary antibodies (listed in Table S1) for two days at 4 ◦C, rinsed tree times with PBS, incubated for 1 h with fluorescence conjugated secondary antibody, and then rinsed three times with PBS. The rinsed sections were then incubated for 5 min. in DAPI solution, rinsed three times with PBS, and mounted with cover slip using vector shield solution (Vector Laboratories). Fluorescence was detected using a confocal microscope (LSM 710, Carl Zeiss, Oberkochen, Germany). Table S3 lists the antibodies used this study.

3.9. Blood Pressure Measurements Blood Pressure is measured using by a noninvasive tail-cuff CODA system (Kent Scientific Corp., Torrington, CT, USA), as previously described in mice [35]. The mice sublimation was conducted for 5 min. for two days, 10 min. for two days, and 15 min. for two days in each group (total six days), and the last seven days we measured blood pressure.

3.10. Data Analysis The results are expressed as the means standard deviations (S.D.). Kruskal–Wallis tests were ± used to determine the differences between groups, and applying the Mann–Whitney U test in SPSS ver. 22 software completed post-hoc comparisons. Significant differences are indicated, as follows: asterisk (*) versus PBS and Sham; $ versus PA/PBS, Obese/Saline and Hypertension/Saline; and, # versus PA/PPB, Obese/PPB, and Hypertension/PPB.

4. Conclusions The efficacy of ECE for improving blood circulation was confirmed by administration to a high fat diet-induced mouse model for four weeks. ECE contains phlorotannins, such as dieckol (DK), 2,7-phloroglucinol-6,6-bieckol (PHB), pyrogallol-phloroglucinol-6,6-bieckol (PPB), and phlorofucofuroeckol A (PFFA). Especially, PPB markedly reduces adhesion molecule expression, EC death and excessive migration, and the proliferation of VSMCs in vitro and in the obese and hypertension mouse models. Additionally, PPB, as a dietary supplement, remarkably reduces the blood pressure and the serum lipoprotein levels in vivo. As expected, PPB plays an important role as the active substance in ECE for improving blood circulation. Currently, it is anticipated that functional foods, such as substances that are effective in alleviating various blood circulation related problems, will be effective in improving blood circulation. It is believed that such substances have the potential for future applications in the clinical setting. Mar. Drugs 2019, 17, 272 12 of 14

Supplementary Materials: The following are available online at http://www.mdpi.com/1660-3397/17/5/272/s1, Table S1: List of primer for quantitative real time polymerase chain reaction (qRT-PCR); Table S2: Composition of experimental diets; Table S3: List of antibodies for Immunohistochemistry, Immunofluorescence and immunoblotting (Western blot); Figure S1: Comparative analysis of GBE and ECE on blood vessel wall thickness; Figure S2: Effects of PPB isolated from ECE on serum lipoprotein, cholesterols in obese and hypertension; Figure S3: Scheme of experimental animal models Author Contributions: K.B. and Y.-J.J. were responsible for study conceptualization and methodology. Formal analysis and data collection were implemented by M.S. and S.O.; Data analysis and interpretation by H.S.L., B.R. and Y.J.; Provide the substance by J.T.J.; Draft preparation by M.S. and S.O.; and supervision and provision of funding acquisition K.B. and Y.-J.J. Funding: This research was a part of a project entitled ‘Development of functional food products with natural materials derived from marine resources’ (no. 20170285), funded by the Ministry of Oceans and Fisheries, Republic of Korea. Acknowledgments: E. cava extract was obtained from Aqua Green Technology Co. Ltd. (Jeju, Korea). Conflicts of Interest: The authors have no conflict of interest to declare.

Abbreviation

PA palmitate conjugation to BSA DK dieckol PHB 2,7-phloroglucinol-6,6-bieckol PPB pyrogallol-phloroglucinol-6,6-bieckol PFFA phlorofucofuroeckol A α-SMA α-smooth muscle actin PCNA Proliferating cell nuclear antigen H&E Hematoxylin and eosin

References

1. Norgren, L.; Hiatt, W.R.; Dormandy, J.A.; Nehler, M.R.; Harris, K.A.; Fowkes, F.G.; Rutherford, R.B. Inter-society consensus for the management of peripheral arterial disease (TASC II). J. Vasc. Surg. 2007, 45, 5–67. [CrossRef][PubMed] 2. Vanhoutte, P.M.; Zhao, Y.; Xu, A.; Leung, S.W. Thirty years of saying NO: Sources, fate, actions, and misfortunes of the endothelium-derived vasodilator mediator. Circulation Res. 2016, 2, 375–396. [CrossRef] 3. Touyz, R.M.; Alves-Lopes, R.; Rios, F.J.; Camargo, L.L.; Anaqnostopoulou, A.; Amer, A.; Montezano, A.C. Vascular smooth muscle contraction in hypertension. Cardiovasc. Res. 2018, 114, 529–539. [CrossRef] 4. Vanhoutte, P.M.; Shimokawa, H.; Tang, E.H.; Feletou, M. Endothelial dysfunction and vascular disease. Acta Physiol. 2009, 196, 193–222. [CrossRef][PubMed] 5. Bennett, M.R.; Sinha, S.; Owens, G.K. Vascular smooth muscle cells in atherosclerosis. Circulation Res. 2016, 4, 692–702. [CrossRef][PubMed] 6. Iba, T.; Levy, J.H. Inflammation and thrombosis: Roles of neutrophils, platelets and endothelial cells and their interactions in thrombus formation during sepsis. J. Thromb. Haemost. 2018, 2, 231–241. [CrossRef] 7. DeKosky, S.T.; Williamson, J.D.; Fitzpatrick, A.L.; Kronmal, R.A.; Ives, D.G.; Saxton, J.A.; Lopez, O.L.; Burke, G.; Carlson, M.C.; Fried, L.P.; et al. Ginkgo biloba for prevention of dementia: A randomized controlled trial. JAMA 2008, 19, 2253–2262. [CrossRef] 8. Nash, K.M.; Shah, Z.A. Current perspectives on the beneficial role of Ginkgo biloba in neurological and cerebrovascular disorders. Integr. Med. Insights 2015, 10, 1–9. [CrossRef] 9. Zuo, W.; Yan, F.; Zhang, B.; Li, J.; Mei, D. Advances in the studies of Ginkgo biloba leaves extract on aging-related diseases. Aging Dis. 2017, 6, 812. [CrossRef] 10. Tian, J.; Liu, Y.; Chen, K. Ginkgo biloba extract in vascular protection: Molecular mechanisms and clinical applications. Curr. Vasc. Pharmacol. 2017, 15, 532–548. [CrossRef][PubMed] 11. Fei, R.; Fei, Y.; Zheng, S.; Gao, Y.G.; Sun, H.X.; Zeng, X.L. Purified polysaccharide from Ginkgo biloba leaves inhibits P-selectin-mediated leucocyte adhesion and inflammation. Acta Pharmacol. Sin. 2008, 29, 499–506. [CrossRef] Mar. Drugs 2019, 17, 272 13 of 14

12. Yang, Y.; Li, Y.; Wang, J.; Sun, K.; Tao, W.; Wang, Z.; Xiao, W.; Pan, Y.; Zhang, S.; Wang, Y. Systematic Investigation of Ginkgo Biloba Leaves for Treating Cardio-cerebrovascular Diseases in an Animal Model. ACS Chem. Biol. 2017, 5, 1363–1372. [CrossRef] 13. Seca, A.M.L.; Pinto, D.C.G.A. Overview on the Antihypertensive and Anti-Obesity Effects of Secondary Metabolites from Seaweeds. Mar. Drugs 2018, 7, 237. [CrossRef] 14. Venkatesan, J.; Kim, S.K.; Shim, M. Antimicrobial, antioxidant, and anticancer activities of biosynthesized silver nanoparticles using marine algae Ecklonia cava. Nanomaterials 2016, 12, 235. [CrossRef] 15. Oh, S.; Son, M.; Lee, H.S.; Kim, H.S.; Jeon, Y.J.; Byun, K. Protective Effect of Pyrogallol-Phloroglucinol- 6,6-Bieckol from Ecklonia cava on Monocyte-Associated Vascular Dysfunction. Mar. Drugs 2018, 16, 441. [CrossRef] 16. Lee, J.H.; Ko, J.Y.; Oh, J.Y.; Kim, C.Y.; Lee, H.J.; Kim, J.; Jeon, Y.J. Preparative isolation and purification of phlorotannins from Ecklonia cava using centrifugal partition chromatography by one-step. Food Chem. 2014, 158, 433–437. [CrossRef] 17. Kim, T.H.; Bae, J.S. Ecklonia cava extracts inhibit lipopolysaccharide induced inflammatory responses in human endothelial cells. Food Chem. Toxicol. 2010, 48, 1682–1687. [CrossRef] 18. Takahashi, M.; Satake, N.; Yamashita, H.; Tamura, A.; Sasaki, M.; Matsui-Yuasa, I.; Tabuchi, M.; Akahoshi, Y.; Terada, M.; Kojima-Yuasa, A. Ecklonia cava protects the liver against ethanol-induced injury in rats. Biochim. Biophys. Acta 2012, 1820, 978–988. [CrossRef] 19. Jang, J.; Ye, B.R.; Heo, S.J.; Oh, C.; Kang, D.H.; Kim, J.H.; Affan, A.; Yoon, K.T.; Choi, Y.U.; Park, S.C.; et al. Photo-oxidative stress by ultraviolet-B radiation and antioxidative defense of eckstolonol in human keratinocytes. Environ. Toxicol. Pharmacol. 2012, 34, 926–934. [CrossRef] 20. Lee, S.H.; Heo, S.J.; Hwang, J.Y.; Han, J.S.; Jeon, Y.J. Protective effects of enzymatic digest from Ecklonia cava against high glucose-induced oxidative stress in human umbilical vein endothelial cells. J. Sci. Food Agric. 2010, 90, 349–356. [CrossRef] 21. Beck, S.M.; Ruge, H.; Schindler, C.; Burkart, M.; Miller, R.; Kirschbaum, C.; Goschke, T. Effects of Ginkgo biloba extract EGb 761® on cognitive control functions, mental activity of the prefrontal cortex and stress reactivity in elderly adults with subjective memory impairment–a randomized double-blind placebo-controlled trial. Hum. Psychopharmacol. Clin. Exp. 2016, 3, 227–242. [CrossRef] 22. Mansour, S.M.; Bahgat, A.K.; El-Khatib, A.S.; Khayyal, M.T. Ginkgo biloba extract (EGb 761) normalizes hypertension in 2K, 1C hypertensive rats: Role of antioxidant mechanisms, ACE inhibiting activity and improvement of endothelial dysfunction. Phytomedicine 2011, 18, 641–647. [CrossRef] 23. Ahn, G.; Park, E.; Lee, W.W.; Hyun, J.W.; Lee, K.W.; Shin, T.; Jeon, Y.J.; Jee, Y. Enzymatic extract from Ecklonia cava induces the activation of lymphocytes by IL-2 production through the classical NF-κB pathway. Mar. Biotechnol. 2011, 13, 66–73. [CrossRef] 24. Lee, D.H.; Park, M.Y.; Shim, B.J.; Youn, H.J.; Hwang, H.J.; Shin, H.C.; Jeon, H.K. Effects of Ecklonia cava polyphenol in individuals with hypercholesterolemia: A pilot study. J. Med. Food 2012, 15, 1038–1044. [CrossRef] 25. Yoon, N.Y.; Eom, T.K.; Kim, M.M.; Kim, S.K. Inhibitory effect of phlorotannins isolated from Ecklonia cava on mushroom tyrosinase activity and melanin formation in mouse B16F10 melanoma cells. J. Agric. Food Chem. 2009, 57, 4124–4129. [CrossRef] 26. Li, Y.; Qian, Z.J.; Ryu, B.; Lee, S.H.; Kim, M.M.; Kim, S.K. Chemical components and its antioxidant properties in vitro: An edible marine brown alga, Ecklonia cava. Bioorg. Med. Chem. 2009, 17, 1963–1973. [CrossRef] [PubMed] 27. Kang, S.M.; Lee, S.H.; Heo, S.J.; Kim, K.N.; Jeon, Y.J. Evaluation of antioxidant properties of a new compound, pyrogallol-phloroglucinol-6,6’-bieckol isolated from brown algae, Ecklonia cava. Nutr. Res. Pract. 2011, 5, 495–502. [CrossRef] 28. Kim, M.J.; Kim, H.K. Insulinotrophic and hypolipidemic effects of Ecklonia cava in streptozotocin–induced diabetic mice. Asian Pac. J. Trop. Med. 2012, 5, 374–379. [CrossRef]

29. Kong, C.S.; Kim, J.A.; Ahn, B.N.; Vo, T.S.; Yoon, N.T.; Kim, S.K. 1-(30, 50-dihydroxyphenoxy)-7-(2”, 4”, 6-trihydroxyphenoxy)-2, 4, 9-trihydroxydibenzo-1, 4-dioxin Inhibits Adipocyte Differentiation of 3T3-L1 Fibroblasts. Mar. Biotechnol. 2010, 12, 299–307. [CrossRef] Mar. Drugs 2019, 17, 272 14 of 14

30. Shin, H.C.; Kim, S.H.; Park, Y.; Lee, B.H.; Hwang, H.J. Effects of 12-week oral supplementation of Ecklonia cava on anthropometric and blood lipid parameters in overweight Korean individuals: A double-blind randomized clinical trial. Phytother. Res. 2012, 26, 363–368. [CrossRef] 31. Manning, B.D.; Toker, A. AKT/PKB Signaling: Navigating the Network. Cell 2017, 169, 381–405. [CrossRef] 32. Cai, Y.; Knight, W.E.; Guo, S.; Li, J.D.; Knight, P.A.; Yan, C. Vinpocetine suppresses pathological vascular remodeling by inhibiting vascular smooth muscle cell proliferation and migration. J. Pharmacol. Exp. Ther. 2012, 343, 479–488. [CrossRef] 33. Zou, Y.; Qian, Z.J.; Li, Y.; Kim, M.M.; Lee, S.H.; Kim, S.K. Antioxidant effects of phlorotannins isolated from Ishige okamurae in free radical mediated oxidative systems. J. Agric. Food Chem. 2008, 56, 7001–7009. [CrossRef][PubMed] 34. Banin, R.M.; de Andrade, I.S.; Cerutti, S.M.; Oyama, L.M.; Telles, M.M.; Ribeiro, E.B. Ginkgo biloba Extract (GbE) Stimulates the Hypothalamic Serotonergic System and Attenuates Obesity in Ovariectomized Rats. Front. Pharmacol. 2017, 8, 605. [CrossRef] 35. Wang, Y.; Thatcher, S.E.; Cassis, L.A. Measuring blood pressure using a noninvasive tail cuff method in mice. Methods Mol. Biol. 2017, 1614, 69–72.

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