Research Article Vitislabrusca Extract(HP01 ...

Research ArticleVitis labrusca Extract (HP01) Improves Blood Circulation andLipid Metabolism in Hyperlipidemic Rats

Bo Yoon Chang 1 Dae Sung Kim2 and Sung Yeon Kim 1

1Institute of Pharmaceutical Research and Development College of Pharmacy Wonkwang University Iksan Jeonbuk 54538Republic of Korea2HanpoongPharmCo Ltd 333-24 1st Palbok-dong Deokjin-gu Jeonju-si Jeonbuk 561-841 Republic of Korea

Correspondence should be addressed to Sung Yeon Kim sungykimwonkwangackr

Received 23 July 2020 Revised 9 November 2020 Accepted 1 December 2020 Published 28 December 2020

Academic Editor Shin Takayama

Copyright copy 2020 Bo Yoon Chang et al is is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Excessive intake of high-lipid foods and lifestyle changes can easily cause hyperlipidemia Hyperlipidemia is clinically considered amajor risk factor for cardiovascular disease which is the second leading cause of death worldwide In this study the effects of aVitis labrusca extract (HP01) on coagulation platelet aggregation and lipid metabolism were investigated in hyperlipidemic ratsA rat model of high-fat diet- (HFD-) induced hyperlipidemia was used Hemostatic parameters and lipid levels were investigatedafter HP01 treatment of hyperlipidemic rats Different doses of HP01 (200mgkgday and 400mgkgday po) were administeredfor 3 weeks and prothrombin time (PT) activated partial thromboplastin time (aPTT) and platelet aggregation and bleed time(BT) were determined e levels of thromboxane B(2) (TXB(2)) and serotonin were measured using enzyme-linked immu-nosorbent assay kits Simultaneously hepatic function and blood fat indexes including aspartate aminotransferase (AST) alanineaminotransferase (ALT) triglyceride (TG) malondialdehyde (MDA) and glutathione (GSH) superoxide dismutase (SOD)catalase (CAT) and glutathione peroxidase (GPx) were also measured In comparison with the data obtained for rats in theuntreated HFD group HP01 (200mgkg) treatment prolonged PT but did not affect aPTT HP01 treatment did not alter plasmaTXB(2) PGI2 or serotonin levels However HP01 showed some effects in improving liver function by reducing the levels ofhepatic lipids ALT MDA and hepatic TG levels significantly decreased whereas GSH GPx CAT and SOD levels significantlyincreasedese results confirm the HP01 extract will improve thromboplastic and the liver metabolic disorders in hyperlipidemiaby oxidative stress response

1 Introduction

e incidence of heart and vascular diseases is increasingeveryday because of the recent improvement in livingstandards westernized dietary habits and changes inliving standards Cardiovascular disease (CVD) whichdescribes all conditions affecting the heart and bloodvessels is currently the leading cause of death worldwideere are several risk factors associated with the devel-opment of CVD such as obesity hypertension diabetesand hyperlipidemia Among them hyperlipidemia playsan important role in inducing various types of CVD [1ndash3]Hyperlipidemia plays an important role in the

pathogenesis of CVDs such as hypercoagulabilityhypofibrinolysis and atherosclerosis [4]

To prevent poor quality of life and increased medicalexpenses because of CVD efforts have been made to preventthe occurrence of CVD risk factors early on Hyperlipidemiais a major risk factor for CVD Serum low-density lipo-protein (LDL) cholesterol levels significantly affect themorbidity and mortality in CVD [5ndash7] In addition bloodcirculation disorder is a state in which the homeostasis ofblood flow is disturbed where vascular hemostasis andinhibitory action are balanced and normal blood flow ismaintained [8 9] However if the blood coagulation factor isexcessively activated or platelet aggregation is promoted

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2020 Article ID 6180310 12 pageshttpsdoiorg10115520206180310

blood flow homeostasis is disrupted and blood circulation isaffected resulting in thrombosis and embolism

rombus which is generated during the coagulationprocess gets attached to the blood vessel wall and obstructsthe flow of blood increases the viscosity of blood inhibitsblood circulation and interferes with the inflow of oxygen ornutrients into the tissue [10 11] rombosis and embolismdue to hematogenous disorders cause serious CVDs such asmyocardial infarction and stroke us the application ofdietary therapy has been shown to improve hyperlipidemiaand blood circulation disorders [12 13]

is indirectly suggests that dietary changes will play animportant role as one of the lifestyle adjustments Manyprevious studies have focused on foods and nutrients thatare expected to lower lipid levels and improve blood cir-culation and examined their potential in lowering lipid levelsand alleviating blood circulation disorders after ingestingfunctional foods containing them Eating habits and lifestylehyperlipidemia hypertension and diabetes are known asrisk factors for blood circulation disorders [14ndash16]

Drugs used as anticoagulants include heparin injectablessuch as warfarin and apixaban and ingestible drugs such asedoxaban and rivaroxaban [17ndash19] However hemostaticagents traditionally used in medical practice have not alwaysbeen effective in reducing the dangerous loss of blood becauseof hemorrhagic shock dilute coagulation or complications ofthrombosis It is especially important to develop new he-mostatic agents with high efficacy and fewer side effects [20]In anticoagulant research the advantageous low toxicity ofnatural substances has attracted attention Currently far morestudies are focusing on natural products and have discoveredsome natural products such as the ginkgo biloba leaf extractthat can coagulate blood effectively [21ndash25]

Apart from its stronger antioxidation ability [26ndash28]Vitis labrusca shows other effects such as hepatoprotection[29] cardioprotection renal protection and neuro-protection [30 31] e grape leaf extract (V labrusca ex-tract VLE) was recently reported to improve the antiplateletactivity by inhibiting TXB(2) and serotonin when it wasadministered to SD rats for one week under normal con-ditions [32] However antiplatelet activity under normalconditions may imply an increased risk for both nonmajorand major bleeding erefore it is essential to confirm theefficacy of antiplatelet activity in an experimental model inwhich blood coagulation is delayed such as dyslipidemiaand arteriosclerosis e ability of HP01 to improvethrombotic and hyperlipidemia through the antioxidantactivity on the hyperlipidemia model was investigated

2 Materials and Methods

21 Chemicals 2 2-Diphenyl-1-picrylhydrazyl (DPPH) vita-min C (50μM) Trolox thiobarbituric acid glutathione gluta-thione reductase (GR) 5 5-dithio-bis-(2-nitrobenzoic acid(DTNB) and sodium dodecyl sulfate (SDS) were purchasedfromSigma (St LouisMO) CaCl2 prothrombin time (PT) andactivated partial thromboplastin time (aPTT) were purchasedfrom Siemens (USA) Ammonium dihydrogen phosphate(ADP) was purchased from Chrono-log (Pennsylvania USA)

22 Extract of HP01 Sample extraction was conductedaccording to the method described by Kwon et al [32] withminormodificationseVitis labrusca leaves were collectedfrom Baekgu-myeon Gimje-si Jeollabuk-do and Hwa-seong-si Gyeonggi-do Republic of Korea in April 2019ebotanical identification was authenticated by Dr Hyoung-Kwon Cho of the Hanpoong Pharm CO Ltd where thevoucher specimen (HP-01) was deposited V labruscaleaves (1 g) were extracted with 20mL of acidified hydro-alcoholic solvent (ethanol water acetic acid 50 47 3 v v v) for 3 h at 80ndash90degC After filtration the residue was re-extracted as described above two additional times ecombined filterates were the was centrifuged at 1500 rpm atroom temperature and then evaporated and lyophilized

23 HPLC Estimation of Quercetin-3-O-glucuronideQuercetin-3-O-glucuronide in HP01 was quantified byHPLC (Shimadzu LC-3030C3D Kyoto Japan) using aWaters Xbridge shield RP18 column 46mm IDtimes150mm35 μm (Waters Co Milford MA USA) In the HPLCquantitative analysis the mobile phase was a mixture of 01formic acid (A) and acetonitrile (B) with a gradient elutionmethod 0-40 min a linear gradient from 15 B to 25 B40ndash60 min held at 15 B Flow rate was 06 mLmin in-jection volume was set to 10 μL and the peaks were detectedat 330 nm Equipment control data acquisition and inte-gration were performed with Shimabzu software epresence of quercetin-3-O-glucuronide was confirmed byretention time that was the same as that of the appropriatestandards (EXTRASYNTHESE France)

24 Antioxidant Activities of HP01 To confirm the antiox-idant activity of HP01 DPPH radical-scavenging assay wasperformed using the method described by Klouwen [33]e extracts were added to 02mMDPPH and incubated for30min Absorbance was measured at 520 nme inhibitionrate was calculated as follows

Inhibition rate 1 minusA

B1113874 1113875 times 100 (1)

where A is the absorbance of the sample and B is the ab-sorbance of the control

Superoxide radical scavenging assay was also performedusing the SOD assay kitmdashWST (Dojindo Tokyo Japan)according to the manufacturerrsquos instructions Optical den-sity was measured at 450 nm Vitamin C (50 μM) or Trolox(500 μgmL) was used as the reference

25 Animal Care e animals used in this study were 5-week-old male SpraguendashDawley (SD) rats (initial weights2271plusmn 379 g) that were purchased from Orient Bio Inc(Kwangju Republic of e platelet count was adjustedKorea) Rats were housed in specific pathogen-free (SPF)conditions at 21degCminus24degC and between 40 and 60 relativehumidity with a 12 h light-dark cycle All animals wereacclimatized for at least 1 week prior to the start of ex-periments All studies were performed in accordance with

2 Evidence-Based Complementary and Alternative Medicine

the guidelines for animal experimentation by WonkwangUniversity and were approved (No WK19-048) e ratswere randomly selected and assigned to 5 groups (6 ratsgroup) One group served as normal (NOR) and was fed aNOR diet (5053 Pico Lab Rodent Diet Purina LabDiet CAUSA) e other 4 groups were fed a high-fat diet (HFD)(D12492 New Brunswick NJ USA) containing 60 kcalfrom fat for 8 weeks without HP01 e HFD groups wereadditionally treated by gavage with HP01 (dissolved indistilled water) at 200mgkg and 400mgkg bodyweight for4 weeks after hyperlipidemia induced Gingko extract (GK50mgkg po) was used as the positive control During the12 weeks experimental period mice had free access to foodand water

26 Preparation of Platelets Blood was collected from theabdominal aortas using sodium citrate tubes (BD Biosci-ences USA) e collected blood was centrifuged to obtainthe platelet-rich plasma (PRP) and platelet-poor plasma(PPP) e platelet count was adjusted to 3times108 plateletsmL for the platelet aggregation and anticoagulant assay

27 Platelet Aggregation Aggregation was measured tur-bidimetrically with slight modifications Briefly PRP waspreincubated for 5min at 37degC Platelet aggregation wasinduced by adding ADP (25molmL Chrono-log Haver-town Pennsylvania USA) e amount of light transmittedthrough the sample was observed for 10min at 37degC using anaggregometer (Model 700-2 Chrono-Log HavertownPennsylvania USA)

28 Anticoagulant Assay aPTT (Pathrombin SL SiemensHealthcare Diagnostics Inc Marburg Germany) and PT(romborel S Siemens Healthcare Diagnostics IncMarburg Germany) were determined according to themanufacturerrsquos protocols For the PT assay 25 μL of PRPwas incubated for 15min at 37degC e assay reaction wasstarted by adding 50 μL of PT reagent and was observed for3min

298ickness of theCarotidArtery e carotid artery tissueswere fixed in 10 formalin buffer solution and then rou-tinely processed for paraffin embedding e 4 μm sectionsof each tissue were stained with hematoxylin and eosin andobserved under an optical microscope (Zeiss AxioscopeJena Germany) at 200x magnification

210Determination ofTGandTC e levels of TG and totalcholesterol in the blood and liver were determined usingBioVision assay kits (Audit Diagnostics Cork Ireland) andAsan cholesterol enzymatic kit (Asan Seoul Korea)according to themanufacturerrsquos instructionse lipids wereextracted from the liver samples following the modifiedmethod described by Folch et al Briefly total lipids wereextracted from the liver samples in the homogenizing buffer(0154M KCl 50mM Tris-HCl and 1mM EDTA)

211 Determination of Hepatic Damage and AntioxidantActivity Serum isolated from the collected blood sampleswas used for determining the ASTand ALT levels accordingto themethod described by Reitman and Frankel [34] MDAthe end product of lipid peroxidation was measured using aslightly modified thiobarbituric acid reactive substance assay[35] e total GSH was measured according to the methoddescribed by Griffith et al [36] Antioxidant enzyme activitywas analyzed using specific enzyme assay kits (CaymanChemical Michigan USA) in hepatic cytosol Superoxidedismutase (SOD) activity (CuZn Mn and FeSOD) wasmeasured by diluting samples to 1 1000 Catalase (CAT)activity was measured by 4-amino-3-hydrazino-5-mercapto-1 2 4-triazole (purpald) in a sample diluted to 1 1000Glutathione peroxidase (GPx) assay was performed in thepresence of glutathione (GSH) and oxidized glutathione(GSSG)

212 Statistical Analysis All data are expressed asmeanplusmn SD of at least three independent experiments Sig-nificant differences were compared using repeated measuresANOVA followed by the NewmanndashKeuls multiple rangetest Statistical significance was defined as Plt 005 Allstatistical analyses were performed using GraphPad Soft-ware Inc (San Diego CA)

3 Results

31 Proximate Composition of HP01 Proximate analysisinvolves the determination of major components of anextract including carbohydrate crude fat crude proteinmoisture ash and sodium e proximate composition ofHP01 accessions is presented in Table 1

32 Chromatographic Analysis of the Quercetin-3-O-glucu-ronide Profile in HP01 Quercetin-3-O-glucuronide contentin HP01 as determined by HPLC was 1413plusmn 283mggPeak identification was based on the UV spectrum and theretention times of the quercein-3-O-glucuronide standardused in our laboratory HPLC analysis at 330 nm revealedpeaks at 2654 minutes and 2656 minutes for the standardand HP01 respectively (Figure 1)

33 Effects of HP01 on Radical Scavenging Activity DPPHradical scavenging activity was determined to confirm theantioxidant effects of HP01 HP01 increased DPPH radicalscavenging activity in a concentration-dependent mannerand exhibited up to 943 scavenging activity at 100 μgmLand 500 μgml Ascorbic acid (50 μM) was used as thepositive control SOD-like activity measurements revealedthat the positive control Trolox (500 μgmL) showed ascavenging activity of 562 HP01 showed a significantdifference in scavenging activity compared with the controland a radical scavenging activity of 519 was confirmed at500 μgmLese data suggest a dose-dependent antioxidanteffect of HP01 (Figure 2)

Evidence-Based Complementary and Alternative Medicine 3

34 Effects of HP01 on Bodyweight Food Intake and TissueWeights e average bodyweight of the rats fed a HFD for 8weeks was 6650plusmn 218 (g) and the groups were reposi-tioned and the candidates were administered for 3 weeks

No significant differences in bodyweight were observedamong groups during the experimental period e weightgain in rats of the GK (621plusmn 98 g) and 200mgkg HP01(739plusmn 98 g) groups compared to that of the HFD group(938plusmn 107 g) showed a tendency to decrease by 338 and212 respectively

e HFD group consumed 1013plusmn 163 kcal (212plusmn 31 g)of diet (HFD 51 kcal 1 g) on an average No change infood intake because of HP01 was observed

After 4 weeks of administration the experimental ani-mals were anesthetized and then the pDEXA equipmentwas used to measure the bone density of the thigh body fatmass of the whole body and muscle mass of the calf musclepart ere was no increase in bone density and calf muscleweight because of HP01 administration However HP01(400mgkg) significantly attenuated the HFD-induced in-crease in fat mass (Plt 005) (Table 2) ere was no changein the weight of the liver spleen kidney testis heart andlung because of HP01 administration Epididymal fat weightshowed a 372 decrease in the GK group and HP01 causeda decrease of 331 and 239 at 200mgkg and 400mgkgrespectively compared to that of the control group (Table 3)

35 Effects of HP01 on 8ickness of the Carotid Arterye carotid blood vessels of the SD rats were isolated toobserve changes in wall thickness of the blood vessels fol-lowing HFD intake e GK (240plusmn 25 μm) and HP01400mgkg (284plusmn 34mm) groups showed significant de-creases in wall thickness of 342 and 223 respectivelycompared to that of the HFD group (365plusmn 80 μm)(Figure 3)

PTwas shortened by HFD intake and HP01 treatment at200mgkg and 400mgkg improved PT by 55 and 54seconds respectively aPTT was significantly delayed by109 s by GK and did not show an increase with HP01treatment e effect of blood coagulation on HP01 wasconfirmed by the impedance method and platelet aggre-gation was confirmed by the optical method e test wasperformed on PRP and ADP was used as an agonist Plateletaggregation which was increased by HFD intake decreasedby 259 in the GK group and a significant decrease wasobserved in the HP01 200mgkg group Serum TXA2 andserotonin levels were measured but no significant increase

or decrease was observed in the HFD fed groups comparedto those in the control group (Figure 4)

36 Effects ofHP01 on Lipid Profile To examine the effects ofHP01 on the lipid profile we determined the levels of TGand TC in the liver and serum Administration of HFD wasfound to result in significantly increased hepatic TG and TClevels in the HFD group e HP01 group treated with SBSshowed markedly lower levels of TG and TC in the liver Inaddition after HFD intake for 12 weeks hepatic totalcholesterol levels increased compared to those of the NORgroup However hepatic total cholesterol levels in the HP01group decreased by approximately 362 compared to thelevels in rats on a HFD However no significant change inblood TG levels was observedese results can be attributedto the ability of HP01 to effectively suppress the accumu-lation of hepatic TG and TC in HFD-fed rats (Table 3)

37 Effects of HP01 on Hepatic Antioxidant Activity andDamage MDA an indicator of oxidative stress increasedwith HFD intake However 200mgkg and 400mgkg ofHP01 decreased oxidative stress by 237 and 74 re-spectively and a significant difference was observed withHP01 200mgkg (Figure 5(a)) When HP01 was adminis-tered at 200mgkg and 400mgkg GSH levels decreasedbecause of a significant increase in oxidative stress by 216and 109 respectively Similarly the HFD group exhibitedsignificantly declined activity of hepatic GPx (Figure 5(c))SOD (Figure 5(d)) and CAT (Figure 5(e)) when comparedwith the control group 400mgkg of HP01 administrationsignificantly improved the activity of GPx SOD and CAT by429 316 and 324 respectively in the liver of theHFD group Increased ALT levels due to HFD intake de-creased by 693 in the GK group and significantly de-creased by 702 and 684 in the 200mgkg and 400mgkgHP01 groups respectively However no decrease in ASTwasobserved with HP01 treatment (Figure 6)

4 Discussion

Grapes are rich in organic acids such as tartaric acid malicacid citric acid and vitaminsA B andC In particular it hasbeen reported that grapes contain various polyphenoliccomponents including flavonoids such as anthocyaninscatechin and resveratrol [37 38] It is known that poly-phenolic components in grapes are mainly present in thegrape skin and seeds and are distributed not only in theedible parts but also in the leaves ese polyphenoliccomponents are known to be effective in preventing CVDcancer arteriosclerosis aging and thrombosis and havebeen reported to be involved in exerting antioxidant effectsthrough antioxidation of serum LDL When problems suchas dietary habits and lifestyles act in combination to increaseoxidative stress in the body the risk of developing chroniccarcinoma vascular disease and metabolic syndrome in-creases ese phytochemicals protect the body from oxi-dative damage inflicted by reactive oxygen species (ROS)through their antioxidant effects and free radical scavenging

Table 1 Proximate composition of HP01

Composition (unit) AmountGross energy (kcal100 g) 34310plusmn 51Carbohydrate () 7287plusmn 24Crude fat () 274plusmn 07Crude protein () 674plusmn 18Moisture () 891plusmn 17Ash () 874plusmn 08Sodium (mg100 g) 7996plusmn 51


400

300

200

100

00 5 10 15 20 25 30 35 40 45 50 55 60 (min)

Retention time

STD Quercetin-3-O-glucuronide

265

47

OH

OH

OH

OH

OH

O

OO

O

OHO

HO

HO

(a)

400

300

200

100

00 5 10 15 20 25 30 35 40 45 50 55 60 (min)

Retention time

Sample HP01

265

40

(b)

Figure 1 High-performance liquid chromatography analysis of quercein-3-O-glucironide in HP01 e marker compound of HP01 wasanalyzed using high-performance liquid chromatography

HP01 (μgkg)CON Vit C 100 300 1000

DPP

H ra

dica

l sc

aven

ging

activ

ity (

)

A

B

CD E

0

20

40

60

80

100

120

(a)

CON 100 300 1000HP01 (μgkg)

Trolox

Supe

roxi

de ra

dica

l sc

aven

ging

activ

ity (

)

A

B

C

DE

0

20

40

60

80

100

120

(b)

Figure 2 Effects of HP01 on antioxidant activities (a) Hydroxyl and (b) superoxide radical scavenging activities of HP01 Data are shown asmeanplusmn SD of changes in the DPPH and superoxide radical scavenging activity e results are presented as the meanplusmn SD Values withdifferent letters (A B C D and E) are significantly different one from another (one-way ANOVA followed by the NewmanndashKeuls multiplerange test Plt 005)


ese phytochemicals possess antioxidant effects and freeradical scavenging that protects the body against oxidativedamage produced by ROS [39ndash42] In the present studyHP01 showed a high antioxidant effect

Polyphenolsflavonoids which contain two or morearomatic rings are contained in various fruits and legumesaccording to the studies by Ahmad et al [43] 5000 fla-vonoids have been found so far Flavonoids provide colorand flavor to fruits and vegetables [43 44]

In case of natural products the components of the ex-tract may vary depending on the harvest time and placethus a marker compound was selected to ensure the qualityof the HP01 extract According to Singh and Zhao [45]markers are chemically defined constituents of a herbal drugthat are of interest for the quality control purposes inde-pendent of whether they have any therapeutic activity or notWe have selected quercetin-3-O-glucuronide as the markercompound of HP01

Table 2 Effects of HP01 on bodyweight food consumption fat mass and relative epididymal fat in hyperlipidemic rats

Groupsfactors

BodyweightFood intake

(kcal)

FAT

Initial Final Gain Mass ()Relative

epididymalfat ()

NOR 5452plusmn 638 a 6176plusmn 614 a 780plusmn 201 a 875plusmn 09 a 451plusmn 121 a 21plusmn 05 a

HFD

CON 6685plusmn 273 b 7623plusmn 255 b 938plusmn 107 a 1013plusmn 163 a 628plusmn 40 b 46plusmn 12 bGK 6647plusmn 236 b 7058plusmn 239 b 621plusmn 152 b 881plusmn 185 a 494plusmn 67 a 29plusmn 03 a

HP01 (mgkg) 200 6627plusmn 236 b 7360plusmn 234 b 739plusmn 152 a 914plusmn 185 a 508plusmn 68 b 31plusmn 03 a400 6637plusmn 212 b 7730plusmn 173 b 868plusmn 72 a 927plusmn 101 a 476plusmn 25 b 35plusmn 05 b

HP01 was administered orally once a day for 4 weeks in hyperlipidemic rats Gingko extract (GK 50mgkg po) was used as the positive control e resultsare presented as the meanplusmn SD Values with different letters (a and b) are significantly different one from another (one-way ANOVA followed by theNewmanndashKeuls multiple range test Plt 005)

Table 3 Effects of HP01 on lipid profile in hyperlipidemic rats

Group Hepatic (mgg liver) Serum (mgdL)TG Total cholesterol TG Total cholesterol

NOR 28plusmn 11 a 22plusmn 03 a 3518plusmn 1682 a 947plusmn 293 a

HFD

CON 160plusmn 29 b 134plusmn 74 b 3689plusmn 1662 a 1275plusmn 369 aGK 122plusmn 37 a 76plusmn 31 a 5125plusmn 1142 a 1026plusmn 209 a

HP01 (mgkg) 200 147plusmn 29 a 86plusmn 10 a 5933plusmn 2668 a 1387plusmn 312 a400 108plusmn 16 c 125plusmn 68 a 5872plusmn 1480 a 1307plusmn 300 a

HP01 was administered orally once a day for 4 weeks in hyperlipidemic rats Gingko extract (GK 50mgkg po) was used as the positive control Values arereported as meanplusmn standard deviation Values with different letters (a b and c) are significantly different one from another (one-way ANOVA followed bythe NewmanndashKeuls multiple range test Plt 005)

HFD ndash + + + +

HP01 (mgkg)

CON GK 200 400NOR

(a)

Caro

tid ar

tery

thic

knes

s (μm

)

A

B

AA

AB

0

10

20

30

40

50

NOR HFD ndashGK ndash ndash + ndash ndash

+ + + +

HP01 ndash ndash ndash 200 400 (mgkg)

(b)

Figure 3 Effects of HP01 on carotid artery thickness in hyperlipidemic rats HP01 was administered orally once a day for 4 weeks inhyperlipidemic rats Gingko extract (GK 50mgkg po) was used as the positive control (a) Hematoxylin and eosin-stained sections ofcarotid artery tissues (original magnification times200) (b)Measurement of carotid artery thickness Values are reported as the meanplusmn standarddeviation Values with different letters (A and B) are significantly different one from another (one-way ANOVA followed by the New-manndashKeuls multiple range test Plt 005)


In this study we demonstrated for the first time thatHP01 can improve blood circulation by suppressing coag-ulation and platelet activation and reducing lipid levels in SDrats fed a HFD for 8 weeks Hyperlipidemia refers to theconcentration of one or several components exceedingnormal levels in the hepatic and blood lipid profiles such ascholesterol triglyceride phospholipid and free fatty acid[46] As these variations are frequently observed in diseasessuch as obesity insulin resistance and diabetes they can beused as biomarkers of the diseases

Ageno et al reported that mean triglyceride levels werehigher and HDL-C levels were lower in venous thrombosispatients than in control patients [47] Morelli et al havepreviously shown that lipid-lowering drugs (statins mostnotably rosuvastatin) are associated with a decreased risk ofvenous thrombosis which might indicate a possible role oflipids in the pathophysiology of venous thrombosis [48]Zajtchuk and Zajtchuk had reported the data revealing thatpatients with high triglyceride levels also had a high inci-dence of low antithrombin activity and increased plateletaggregation [49] It is likely that hyperlipidemic patients aremore prone to thrombosis of diseased coronary arteries

erefore we selected the increase in TG and totalcholesterol as the biomarkers for thrombosis and conducted astudy on the blood circulation improving function of HP01

We administered a 60 HFD to induce hyperlipidemiabefore HP01 administration and measured TG levels at 2

week intervals No change was observed in blood TG andtotal cholesterol levels of SD rats fed a HFD by week 8However hepatic TG and total cholesterol levels showed asignificant increase of 337-fold and 57-fold respectivelycompared to that of the normal groups ese results weresimilar to the results obtained from previous study by Chiuet al where the hepatic TG and total cholesterol weremeasured after 8 weeks of ingesting HFD [50] HP01 wasadministered from 8 weeks and the TG and total cholesterolconcentration showed a significant difference compared tothat of the normal group

In the present study high-fat feeding induced a sig-nificant increase in fat weight which represents adipocytehypertrophy Our results confirm the suppressive effect of-HP01 on HFD-induced adiposity No anatomical abnor-malities were found between the NOR group and the HFDgroup ese results showed that the color and pattern ofeach organ other than the liver were not different from thoseof the normal group indicating that hyperlipidemia andother diseases were not induced

In our experiments there was no change in the amountof lipids in serum but an increase in the amount of the livertissue lipids was observed In animals that induced hyper-lipidemia by HFD during a period of less than 12 weeksblood did not show any changes in the blood lipid profile butonly differences in lipids in the liver tissue [16 51ndash54]According to the previous studies by Kim et al HFD must

PT (s

ec)

AB

A A A

05

101520253035

NOR HFD ndash

GK ndash ndash + ndash ndash

+ + + +


(a)

aPTT

(sec

)

A B B B B

NOR HFD ndash


+ + + +


05

101520253035

(b)

Agg

rega

tion

() A B

A AB

NOR HFD ndash


+ + + +


0

20

40

60

80

(c)

TXA

2 (ng

mL)

A AA A A

NOR HFD ndash


+ + + +


0

2080

100

(d)

Sero

toni

ne (n

gm

L) AB

A B B

NOR HFD ndash


+ + + +


0

2080

100

120

140

(e)

Figure 4 Effects of HP01 on platelet activity in hyperlipidemic rats HP01 was administered orally once a day for 4 weeks in hyperlipidemicrats Gingko extract (GK 50mgkg po) was used as the positive control e platelet activity shown as (a) PT (b) aPTT and (c) plateletaggregation Serum levels of (d) TXA2 and (e) serotonin Values are reported as meanplusmn standard deviation Values with different letters (Aand B) are significantly different one from another (one-way ANOVA followed by the NewmanndashKeuls multiple range test Plt 005)


ALT

(uni

tmL)

NOR HFD ndash


+ + + +


A

B

C C C

0

50

100

150

200

250

300

(a)

AST

(uni

tmL)

NOR HFD ndash


+ + + +


A

B

BB

B

0

50

100

150

200

250

300

(b)

Figure 6 Effects of HP01 on the hepatic protection in hyperlipidemic rats HP01 was administered orally once a day for 4 weeks inhyperlipidemic rats Gingko extract (GK 50mgkg po) was used as the positive control e serum levels of (a) ALTand (b) AST Valuesare reported as the meanplusmn standard deviation Values with different letters (A B and C) are significantly different one from another (one-way ANOVA followed by the NewmanndashKeuls multiple range test Plt 005)

MD

A (μ

Mg

live

r)

A

A

B BB

NOR HFD ndash


+ + + +


020406080

100120140

(a)

AA

BA A

GSH

(μM

g li

ver)

NOR HFD ndash


+ + + +


02468

101214

(b)

GPx

activ

ity

(nm

olm

inm

g pr

otei

n) A

BA

B

A

NOR HFD ndash


+ + + +


02468

10121416

(c)

SOD

activ

ity (U

mg

prot

ein)

AA

BB

B

NOR HFD ndash


+ + + +


0

200

400

600

800

1000

1200

(d)

Cata

lase

activ

ity(n

mol

min

mg

prot

ein) A

B

AB AB A

NOR HFD ndash


+ + + +


0

20

40

60

80

100

120

(e)

Figure 5 Effects of HP01 on the hepatic protection in hyperlipidemic rats HP01 was administered orally once a day for 4 weeks inhyperlipidemic rats Gingko extract (GK 50mgkg po) was used as the positive control e hepatic levels of (a) MDA (b) GSH (c) GPx(d) SOD and (e) catalase Values are reported as the meanplusmn standard deviation Values with different letters (A B and C) are significantlydifferent one from another (one-way ANOVA followed by the NewmanndashKeuls multiple range test Plt 005)


be taken for at least 15 weeks for changes in blood lipids tooccur [55] e changes in lipid in the hepatic tissue fol-lowing the induction of hyperlipidemia were decreased bythe administration of HP01 [56]

Increased vascular endocardial thickness is an importantbiomarker for diagnosing CVD e arterial plaque withhypertrophy was significantly increased by lipid accumu-lation in HFD-induced hyperlipidemia Our results also havedemonstrated HFD-induced hypertrophic changes in theaortic wall A significant improvement effect was confirmedin the HP01 group

In hyperlipidemia altered lipid levels disrupt the balanceof the procoagulant and anticoagulant pathways In addi-tion it induces the activation of procoagulation enzymaticcomplexes (factor Xa and factor XaVa-mediated factorVII) Our results show significantly shorter PT and aPTT inhyperlipidemic rats than in normal rats

Blood clotting occurs through a series of proteolyticreactions including the stepwise activation of coagulationfactors IndashXII Some of these factors are activated by twopathways the extrinsic pathway and the intrinsic pathwayIn general aPTTis used to determine the overall efficiency ofthe extrinsic clotting pathway and PT is an intrinsic clottingpathway screening test Our results showed that HP01prolonged PT in hyperlipidemic rats However it did notaffect aPTT High blood lipid levels are associated withincreased clotting factors [57] Kim et al have reported asignificant correlation between total cholesterol and tri-glyceride levels and procoagulant factors II VII IX and Xexcept fibrinogen implying that the elevation of procoa-gulant factors may contribute to shorter PT values in sub-jects with high blood lipids [58] Because there was noassociation of factor VIII with blood lipid levels in our studysubjects with HP01 also did not show a delayed aPTT valuecompared to those with hyperlipidemia ese findingssuggest that HP01 contributes to improving the blood flowby regulating the extrinsic clotting pathway

Serotonin and TXA2 are known as the most typicalvascular regulators that mediate vasoconstriction becauseof platelet aggregation TXA2 is a metabolite of arachidonicacid and is known to regulate platelet aggregation andvasomotor responses along with nitric oxide In the studyby Kim et al study the GBx administration reduced theHFD-induced TXA2 and serotonin levels [59] Howeverthe results of our experiments did not show a significantdecrease in increased TXA2 and serotonin by HP01 eseresults imply that HP01 is not associated with vasomotorresponses

Hyperlipidemia induces oxidative stress because of lipidaccumulation Monteiro et al reported that metabolic ab-normalities as a consequence of HFD in rats cause platelethyperaggregability involving enhanced intraplatelet ROSproduction and decreased NO bioavailability that are ac-companied by potential defects in the prosthetic heme groupof sGC [60] Our data showed that ADP-induced plateletaggregation was significantly higher in the HFD groupHP01 significantly downregulated the aggregation comparedto that of the HFD group ese results can be attributed tothe antioxidant effect of HP01

Matsuzawa-Nagata et al showed that pathways involvedin the mitochondrial respiratory chain were downregulatedin both the liver and adipose tissue of mice fed HFD [61]High mitochondrial β-oxidation rates may help metabolizeexcess free fatty acid (FFA) but a large number of electronsentering the respiratory chain can cause abnormal oxygendepletion e production of mitochondrial ROS throughwhich electrons flow can be increased is suggests that therespiratory dysfunction can increase the production ofmitochondrial ROS Hyperlipidemia results in unbalancebetween oxidation and antioxidation and produces a largenumber of oxygen free radicals in vivo Furthermore ox-ygen-free radicals translate to MDA Oxygen-free radicalsand MDA result in the injury of vascular endothelial cell andpromote the formation and development of atherosclerosise activities of GSH-Px SOD and CATdirectly reflect theability to scavenging oxygen-free radicals Santos et al re-ported that V vinifera L grape skin extract (ACH09) pre-vented the increase inMDA and protein carbonylation levelsand reduced the antioxidant activity of SOD CAT and GPxsuggesting an important protective effect against oxidativestress that may contribute toward the improvement ofmitochondrial function and reduction of lipid accumulationin the liver [53] Also our research showed that HP01 ad-ministration significantly decreased hepatic MDA levels andincreased the total GSH SOD CAT and GPx activitycompared to those of the HFD group ese results indicatethe strong antioxidant capacity of HP01 Aminotransferaseactivity is altered during the course of lipid metabolismbecause of fat accumulation Measurement of the liver damagebecause of interstitial fat accumulation is important for thediagnosis of NAFLD erefore AST and ALT levels weremeasured as indicators of liver damage In the current studyAST and ALT levels showed a more pronounced increase inrats fed HFD than in rats fed ND However HP01 supple-mentation significantly reduced ALTandAST levels indicatinga strong hepatoprotective effect in HFD-induced liver injuryTaken together all of the above results show that HP01 exhibitsremarkable free radical scavenging activity both in vitro and invivo as well as hypolipidemic and hepatoprotective activities invivo Despite these findings the precise mechanism by whichthe HP01 exhibited its antioxidant and hypolipidemic effectscannot be concluded from these data which is a limitation ofthis study Hence further studies using the HP01 as well asconstituents onmolecular pathways such as lipid synthesis andantioxidant regulator are highly required

5 Conclusions

Administration of HP01 improved blood circulation andameliorated lipid-related disorders in the hyperlipidemiamodel ese effects are considered that potent antioxidantactivity of HP01 may be directly or indirectly responsiblefor its hypolipidemic properties e HP01 might beconsidered as an alternative functional food or pharma-ceutical in the prevention and treatment of hyperlipid-emia As a powerful antioxidant HP01 needs furtherresearch as a therapeutic agent for the treatment ofhyperlipidemia


Abbreviations

HFD High-fat dietPT Prothrombin timeaPTT Activated partial thromboplastin timeTXB romboxane BAST Aspartate aminotransferaseALT Alanine aminotransferaseTG TriglycerideMDA MalondialdehydeGSH GlutathioneCVD Cardiovascular diseaseLDL Low-density lipoproteinPGI2 Prostaglandin I2DMEM Dulbeccorsquos modified eagle mediumSD SpraguendashDawleySPF Specific pathogen-freeNOR NormalHPLC High-performance liquid chromatographyDPPH 22-Diphenyl-1-picrylhydrazylSOD Superoxide dismutasePRP Platelet-rich plasmaPPP Platelet-poor plasmaADP Ammonium dihydrogen phosphateHCl Hydrogen chlorideUV UltravioletGK Ginkgo extractDEXA DexamethasoneROS Reactive oxygen speciesVLE Vitis labrusca extractGBx Ginsenoside berryNO Nitric oxideFFA Free fatty acid

Data Availability

e datasets used andor analyzed during the current studyare available from the corresponding author upon request

Conflicts of Interest

e authors declare that they have no conflicts of interest

Authorsrsquo Contributions

All authors have read and approved the manuscript BYCDSK and SYK designed the study conducted the researchanalyzed the data contributed reagentsmaterialsanalysistools and wrote the article

Acknowledgments

is work was supported by Korea Institute of Planning andEvaluation for Technology in Food Agriculture and Forestry(IPET) through Agri-Bio Industry Technology DevelopmentProgram funded by MAFRA (118041-3)

References

[1] A S Go D Mozaffarian V L Roger et al ldquoExecutivesummary heart disease and stroke statistics-2013 update areport from the American Heart Associationrdquo Circulationvol 127 no 1 pp 143ndash152 2013

[2] G A Mensah and D W Brown ldquoAn overview of cardio-vascular disease burden in the United Statesrdquo Health Affairsvol 26 no 1 pp 38ndash48 2007

[3] India State-Level Disease Burden Initiative CVD Collabora-tors ldquoe changing patterns of cardiovascular diseases andtheir risk factors in the states of India the global burden ofdisease study 1990ndash2016rdquo Lancet Glob Health vol 6 no 12pp e1339ndashe1351 2018

[4] S N Randeria and G J A omson ldquoInflammatory cyto-kines in type 2 diabetes mellitus as facilitators of hyper-coagulation and abnormal clot formationrdquo CardiovascularDiabetology vol 18 no 1 p 72 2019

[5] R Ariyanti and B Besral ldquoDyslipidemia associated withhypertension increases the risks for coronary heart disease acase-control study in Harapan Kita Hospital National Car-diovascular Center Jakartardquo Journal of Lipids vol 2019Article ID 2517013 6 pages 2019

[6] S S Martin ldquoCalculating LDL cholesterol in familial com-bined hyperlipidemia out with the old in with the newrdquoAtherosclerosis vol 277 pp 172ndash174 2018

[7] AH Wu ldquoBiomarkers for cholesterol absorption and syn-thesis in hyperlipidemic patients role for therapeutic selec-tionrdquo Clinics in Laboratory Medicine vol 34 no 1pp 157ndash166 2014

[8] L Lind and H Lithell ldquoDecreased peripheral blood flow in thepathogenesis of the metabolic syndrome comprising hyper-tension hyperlipidemia and hyperinsulinemiardquo AmericanHeart Journal vol 125 no 5 pp 1494ndash1497 1993

[9] X Ye and C Zhang ldquoEffects of hyperlipidemia and cardio-vascular diseases on proliferation differentiation and homingof mesenchymal stem cellsrdquo Current Stem Cell Research and8erapy vol 12 no 5 pp 377ndash387 2017

[10] F F Mussa J D Horton R Moridzadeh J NicholsonS Trimarchi and K A Eagle ldquoAcute aortic dissection andintramural hematoma a systematic reviewrdquo Journal of theAmerican Medical Association vol 316 no 7 pp 754ndash7632016

[11] P C Choy Y L Siow D Mymin and O Karmin ldquoLipids andatherosclerosisrdquo Biochemistry and Cell Biology vol 82 no 1pp 212ndash224 2004

[12] S N Doshi I F McDowell S J Moat et al ldquoFolic acidimproves endothelial function in coronary artery disease viamechanisms largely independent of homocysteine loweringrdquoCirculation vol 105 no 1 pp 22ndash26 2002

[13] K ompson W Pederick and A B Santhakumar ldquoAn-thocyanins in obesity-associated thrombogenesis a review ofthe potential mechanism of actionrdquo Food amp Function vol 7no 5 pp 2169ndash2178 2016

[14] D R Harder K R Rarick D Gebremedhin and S S CohenldquoRegulation of cerebral blood flow response to cytochromeP450 lipid metabolitesrdquo Comprehensive Physiology vol 8no 2 pp 801ndash821 2018

[15] X Cheng and R J Lee ldquoe role of helper lipids in lipidnanoparticles (LNPs) designed for oligonucleotide deliveryrdquoAdvanced Drug Delivery Reviews vol 99 pp 129ndash137 2016


[16] R Ryu H J Kim B Moon et al ldquoEthanol extract of per-simmon tree leaves improves blood circulation and lipidmetabolism in rats fed a high-fat dietrdquo Journal of MedicinalFood vol 18 no 7 pp 715ndash723 2015

[17] S Khalid and H Daw ldquoe role of apixaban in the treatmentof heparin-induced thrombocytopeniardquo Cureus vol 9 no 7p e1428 2017

[18] P Dhakal R Pathak S Giri G S Murthy and V R BhattldquoNew oral anticoagulants for the management of heparininduced thrombocytopenia a focused literature reviewrdquoCardiovascular and Hematological Agents in MedicinalChemistry vol 13 no 2 pp 87ndash91 2015

[19] M A Miyares and K A Davis ldquoDirect-acting oral antico-agulants as emerging treatment options for heparin-inducedthrombocytopeniardquo Annals of Pharmacotherapy vol 49no 6 pp 735ndash739 2015

[20] J W Skelley J A Kyle and R A Roberts ldquoNovel oral an-ticoagulants for heparin-induced thrombocytopeniardquo Journalof 8rombosis and 8rombolysis vol 42 no 2 pp 172ndash1782016

[21] S Choi D S Oh and U M Jerng ldquoA systematic review of thepharmacokinetic and pharmacodynamic interactions ofherbal medicine with warfarinrdquo PLoS One vol 12 no 8Article ID e0182794 2017

[22] G Y Yeh R B Davis and R S Phillips ldquoUse of comple-mentary therapies in patients with cardiovascular diseaserdquoAmerican Journal of Cardiology vol 98 no 5 pp 673ndash6802006

[23] T Q Rui L Zhang H Z Qiao et al ldquoPreparation andphysicochemical and pharmacokinetic characterization ofginkgo lactone nanosuspensions for antiplatelet aggregationrdquoJournal of Pharmaceutical Sciences vol 105 no 1 pp 242ndash249 2016

[24] K H Ryu H Y Han S Y Lee et al ldquoGinkgo biloba extractenhances antiplatelet and antithrombotic effects of cilostazolwithout prolongation of bleeding timerdquo8rombosis Researchvol 124 no 3 pp 328ndash334 2009

[25] A J Lau D F Toh T K Chua Y K Pang S O Woo andH L Koh ldquoAntiplatelet and anticoagulant effects of Panaxnotoginseng comparison of raw and steamed Panax noto-ginseng with Panax ginseng and Panax quinquefoliumrdquoJournal of Ethnopharmacology vol 125 no 3 pp 380ndash3862009

[26] E S Kovaleski L K Gonccedilalves G Bortolato et al ldquoEffects ofthe ingestion of different kinds of white grape juice (Vitislabrusca) during adolescence on body weight biochemicalparameters and oxidative stress in liver of adult Wistar ratsrdquoFood Chemistry vol 291 pp 110ndash116 2019

[27] R Guo H Qiao J Zhao et al ldquoe grape VlWRKY3 genepromotes abiotic and biotic stress tolerance in transgenicArabidopsis thalianardquo Frontiers in Plant Science vol 9 p 5452018

[28] C L Dalla Corte N R de Carvalho G P Amaral et alldquoAntioxidant effect of organic purple grape juice on ex-haustive exerciserdquo Applied Physiology Nutrition and Meta-bolism vol 38 no 5 pp 558ndash565 2013

[29] L S Oliboni C Dani C Funchal J A Henriques andM Salvador ldquoHepatoprotective cardioprotective and renal-protective effects of organic and conventional grapevine leafextracts on Wistar rat tissuesrdquo Anais da Academia Brasileirade Ciencias vol 83 no 4 pp 1403ndash1411 2011

[30] G Scola V L Laliberte H K Kim et al ldquoVitis labruscaextract effects on cellular dynamics and redox modulations in

a SH-SY5Y neuronal cell model a similar role to lithiumrdquoNeurochemistry International vol 79 pp 12ndash19 2014

[31] C Dani L S Oliboni F Agostini et al ldquoPhenolic content ofgrapevine leaves (Vitis labrusca var Bordo) and its neuro-protective effect against peroxide damagerdquo Toxicology inVitro vol 24 no 1 pp 148ndash153 2010

[32] S U Kwon H Y Lee M Xin et al ldquoAntithrombotic activityof Vitis labrusca extract on rat platelet aggregationrdquo BloodCoagulation and Fibrinolysis vol 27 no 2 pp 141ndash146 2016

[33] H M Klouwen ldquoDetermination of the sulfhydryl content ofthymus and liver using DPPHrdquo Archives of Biochemistry andBiophysics vol 99 no 1 pp 116ndash120 1962

[34] S Reitman and S Frankel ldquoA colorimetric method for thedetermination of serum glutamic oxalacetic and glutamicpyruvic transaminasesrdquo American Journal of Clinical Pa-thology vol 28 no 1 pp 56ndash63 1957

[35] C G Fraga B E Leibovitz and A L Tappel ldquoLipid per-oxidation measured as thiobarbituric acid-reactive substancesin tissue slices characterization and comparison with ho-mogenates and microsomesrdquo Free Radical Biology andMedicine vol 4 no 3 pp 155ndash161 1988

[36] O W Griffith ldquoDetermination of glutathione and glutathionedisulfide using glutathione reductase and 2-vinylpyridinerdquoAnalytical Biochemistry vol 106 no 1 pp 207ndash212 1980

[37] A Rauf M Imran M S Butt M Nadeem D G Peters andM S Mubarak ldquoResveratrol as an anti-cancer agent a re-viewrdquo Critical Reviews in Food Science and Nutrition vol 58no 9 pp 1428ndash1447 2018

[38] J Tabeshpour S Mehri F Shaebani Behbahani andH Hosseinzadeh ldquoProtective effects of Vitis vinifera (grapes)and one of its biologically active constituents resveratrolagainst natural and chemical toxicities a comprehensivereviewrdquo Phytotherapy Research vol 32 no 11 pp 2164ndash21902018

[39] K B Pandey N Mishra and S I Rizvi ldquoProtective role ofmyricetin on markers of oxidative stress in human erythro-cytes subjected to oxidative stressrdquo Natural Product Com-munications vol 4 no 2 pp 221ndash226 2009

[40] H S Demrow P R Slane and J D Folts ldquoAdministration ofwine and grape juice inhibits in vivo platelet activity andthrombosis in stenosed canine coronary arteriesrdquo Circulationvol 91 no 4 pp 1182ndash1188 1995

[41] I Belinha M A Amorim P Rodrigues et al ldquoQuercetinincreases oxidative stress resistance and longevity in Sac-charomyces cerevisiaerdquo Journal of Agricultural and FoodChemistry vol 55 no 6 pp 2446ndash2451 2007

[42] S I Rizvi and P K Maurya ldquoAlterations in antioxidantenzymes during aging in humansrdquo Molecular Biotechnologyvol 37 no 1 pp 58ndash61 2007

[43] A Ahmad R M Khan and K M Alkharfy ldquoEffects of se-lected bioactive natural products on the vascular endothe-liumrdquo Journal of Cardiovascular Pharmacology vol 62 no 2pp 111ndash121 2013

[44] K B Pandey and S I Rizvi ldquoPlant polyphenols as dietaryantioxidants in human health and diseaserdquo Oxidative Med-icine and Cellular Longevity vol 2 no 5 pp 270ndash278 2009

[45] A Singh and K Zhao ldquoHerb-drug interactions of commonlyused Chinese medicinal herbsrdquo International Review ofNeurobiology Neurobiology of Chinese Herb Medicinevol 135 pp 197ndash232 2017

[46] L Williams Y Seki P M Vuguin and M J CharronldquoAnimal models of in utero exposure to a high fat diet areviewrdquo Biochimica et Biophysica Acta vol 1842 no 3pp 507ndash519 2014


[47] W Ageno C Becattini T Brighton R Selby andP W Kamphuisen ldquoCardiovascular risk factors and venousthromboembolism a meta-analysisrdquo Circulation vol 117no 1 pp 93ndash102 2008

[48] V M Morelli W M Lijfering M H A Bos F R Rosendaaland S C Cannegieter ldquoLipid levels and risk of venousthrombosis results from theMEGA-studyrdquo European Journalof Epidemiology vol 32 no 8 pp 669ndash681 2017

[49] R Zajtchuk and J Zajtchuk ldquoRelationship of triglyceridelevels to thrombosis in patients with coronary artery diseaserdquo8e Annals of 8oracic Surgery vol 35 no 3 pp 274ndash2761983

[50] C Y Chiu T E Yen and S H Liu ldquoComparative effects andmechanisms of chitosan and its derivatives on hypercholes-terolemia in high-fat diet-fed ratsrdquo International Journal ofMolecular Sciences vol 21 no 1 p 92 2019

[51] A Holmes L J Coppey E P Davidson and M A YorekldquoRat models of diet-induced obesity and high fatlow dosestreptozotocin type 2 diabetes effect of reversal of high fat dietcompared to treatment with enalapril or menhaden oil onglucose utilization and neuropathic endpointsrdquo Journal ofDiabetes Research vol 2015 Article ID 307285 8 pages 2015

[52] P Chan B Tomlinsoin C W Tsai W H Pan and Y S Leeldquorombophilia in patients with hypercholesterolemiardquoMetabolism vol 45 no 8 pp 966ndash969 1996

[53] I B Santos G F de Bem V S C Cordeiro et al ldquoSup-plementation with Vitis vinifera L skin extract improvesinsulin resistance and prevents hepatic lipid accumulationand steatosis in high-fat diet-fed micerdquo Nutrition Researchvol 43 pp 69ndash81 2017

[54] C Y Chiu L P Wang and S H Liu ldquoFish oil supple-mentation alleviates the altered lipid homeostasis in bloodliver and adipose tissues in high-fat diet-fed ratsrdquo Journal ofAgricultural and Food Chemistry vol 66 no 16 pp 4118ndash4128 2018

[55] J Kim H Lee J Lim J Oh S S Shin and M Yoon ldquoeangiogenesis inhibitor ALS-l1023 from lemon-balm leavesattenuates high-fat diet-induced nonalcoholic fatty liverdisease through regulating the visceral adipose-tissue func-tionrdquo International Journal of Molecular Sciences vol 18no 4 p 846 2017

[56] C Marques M Meireles S Norberto et al ldquoHigh-fat diet-induced obesity rat model a comparison between wistar andsprague-dawley ratrdquo Adipocyte vol 5 no 1 pp 11ndash21 2016

[57] A Branchi A Rovellini D Sommariva A G Gugliandoloand A Fasoli ldquoEffect of three fibrate derivatives and of twoHMG-CoA reductase inhibitors on plasma fibrinogen level inpatients with primary hypercholesterolemiardquo8rombosis andHaemostasis vol 70 no 2 pp 241ndash243 1993

[58] J A Kim J E Kim S H Song and H K Kim ldquoInfluence ofblood lipids on global coagulation test resultsrdquo Annals ofLaboratory Medicine vol 35 no 1 pp 15ndash21 2015

[59] M H Kim J Lee S Jung J W Kim J H Shin and H J Leeldquoe involvement of ginseng berry extract in blood flow viaregulation of blood coagulation in rats fed a high-fat dietrdquoJournal of Ginseng Research vol 41 no 2 pp 120ndash126 2017

[60] P F Monteiro R P Morganti M A Delbin et al ldquoPlatelethyperaggregability in high-fat fed rats a role for intraplateletreactive-oxygen species productionrdquo Cardiovascular Dia-betology vol 11 no 1 p 5 2012

[61] N Matsuzawa-Nagata T Takamura H Ando et al ldquoIn-creased oxidative stress precedes the onset of high-fat diet-induced insulin resistance and obesityrdquo Metabolism vol 57no 8 pp 1071ndash1077 2008


blood flow homeostasis is disrupted and blood circulation isaffected resulting in thrombosis and embolism

rombus which is generated during the coagulationprocess gets attached to the blood vessel wall and obstructsthe flow of blood increases the viscosity of blood inhibitsblood circulation and interferes with the inflow of oxygen ornutrients into the tissue [10 11] rombosis and embolismdue to hematogenous disorders cause serious CVDs such asmyocardial infarction and stroke us the application ofdietary therapy has been shown to improve hyperlipidemiaand blood circulation disorders [12 13]

is indirectly suggests that dietary changes will play animportant role as one of the lifestyle adjustments Manyprevious studies have focused on foods and nutrients thatare expected to lower lipid levels and improve blood cir-culation and examined their potential in lowering lipid levelsand alleviating blood circulation disorders after ingestingfunctional foods containing them Eating habits and lifestylehyperlipidemia hypertension and diabetes are known asrisk factors for blood circulation disorders [14ndash16]

Drugs used as anticoagulants include heparin injectablessuch as warfarin and apixaban and ingestible drugs such asedoxaban and rivaroxaban [17ndash19] However hemostaticagents traditionally used in medical practice have not alwaysbeen effective in reducing the dangerous loss of blood becauseof hemorrhagic shock dilute coagulation or complications ofthrombosis It is especially important to develop new he-mostatic agents with high efficacy and fewer side effects [20]In anticoagulant research the advantageous low toxicity ofnatural substances has attracted attention Currently far morestudies are focusing on natural products and have discoveredsome natural products such as the ginkgo biloba leaf extractthat can coagulate blood effectively [21ndash25]

Apart from its stronger antioxidation ability [26ndash28]Vitis labrusca shows other effects such as hepatoprotection[29] cardioprotection renal protection and neuro-protection [30 31] e grape leaf extract (V labrusca ex-tract VLE) was recently reported to improve the antiplateletactivity by inhibiting TXB(2) and serotonin when it wasadministered to SD rats for one week under normal con-ditions [32] However antiplatelet activity under normalconditions may imply an increased risk for both nonmajorand major bleeding erefore it is essential to confirm theefficacy of antiplatelet activity in an experimental model inwhich blood coagulation is delayed such as dyslipidemiaand arteriosclerosis e ability of HP01 to improvethrombotic and hyperlipidemia through the antioxidantactivity on the hyperlipidemia model was investigated

2 Materials and Methods

21 Chemicals 2 2-Diphenyl-1-picrylhydrazyl (DPPH) vita-min C (50μM) Trolox thiobarbituric acid glutathione gluta-thione reductase (GR) 5 5-dithio-bis-(2-nitrobenzoic acid(DTNB) and sodium dodecyl sulfate (SDS) were purchasedfromSigma (St LouisMO) CaCl2 prothrombin time (PT) andactivated partial thromboplastin time (aPTT) were purchasedfrom Siemens (USA) Ammonium dihydrogen phosphate(ADP) was purchased from Chrono-log (Pennsylvania USA)

22 Extract of HP01 Sample extraction was conductedaccording to the method described by Kwon et al [32] withminormodificationseVitis labrusca leaves were collectedfrom Baekgu-myeon Gimje-si Jeollabuk-do and Hwa-seong-si Gyeonggi-do Republic of Korea in April 2019ebotanical identification was authenticated by Dr Hyoung-Kwon Cho of the Hanpoong Pharm CO Ltd where thevoucher specimen (HP-01) was deposited V labruscaleaves (1 g) were extracted with 20mL of acidified hydro-alcoholic solvent (ethanol water acetic acid 50 47 3 v v v) for 3 h at 80ndash90degC After filtration the residue was re-extracted as described above two additional times ecombined filterates were the was centrifuged at 1500 rpm atroom temperature and then evaporated and lyophilized

23 HPLC Estimation of Quercetin-3-O-glucuronideQuercetin-3-O-glucuronide in HP01 was quantified byHPLC (Shimadzu LC-3030C3D Kyoto Japan) using aWaters Xbridge shield RP18 column 46mm IDtimes150mm35 μm (Waters Co Milford MA USA) In the HPLCquantitative analysis the mobile phase was a mixture of 01formic acid (A) and acetonitrile (B) with a gradient elutionmethod 0-40 min a linear gradient from 15 B to 25 B40ndash60 min held at 15 B Flow rate was 06 mLmin in-jection volume was set to 10 μL and the peaks were detectedat 330 nm Equipment control data acquisition and inte-gration were performed with Shimabzu software epresence of quercetin-3-O-glucuronide was confirmed byretention time that was the same as that of the appropriatestandards (EXTRASYNTHESE France)

24 Antioxidant Activities of HP01 To confirm the antiox-idant activity of HP01 DPPH radical-scavenging assay wasperformed using the method described by Klouwen [33]e extracts were added to 02mMDPPH and incubated for30min Absorbance was measured at 520 nme inhibitionrate was calculated as follows

Inhibition rate 1 minusA

B1113874 1113875 times 100 (1)

where A is the absorbance of the sample and B is the ab-sorbance of the control

Superoxide radical scavenging assay was also performedusing the SOD assay kitmdashWST (Dojindo Tokyo Japan)according to the manufacturerrsquos instructions Optical den-sity was measured at 450 nm Vitamin C (50 μM) or Trolox(500 μgmL) was used as the reference

25 Animal Care e animals used in this study were 5-week-old male SpraguendashDawley (SD) rats (initial weights2271plusmn 379 g) that were purchased from Orient Bio Inc(Kwangju Republic of e platelet count was adjustedKorea) Rats were housed in specific pathogen-free (SPF)conditions at 21degCminus24degC and between 40 and 60 relativehumidity with a 12 h light-dark cycle All animals wereacclimatized for at least 1 week prior to the start of ex-periments All studies were performed in accordance with










3 Results














4 Discussion





400

300

200

100

00 5 10 15 20 25 30 35 40 45 50 55 60 (min)

Retention time


265

47

OH

OH

OH

OH

OH

O

OO

O

OHO

HO

HO

(a)

400

300

200

100

00 5 10 15 20 25 30 35 40 45 50 55 60 (min)

Retention time

Sample HP01

265

40

(b)


HP01 (μgkg)CON Vit C 100 300 1000

DPP

H ra

dica

l sc

aven

ging

activ

ity (

)

A

B

CD E

0

20

40

60

80

100

120

(a)

CON 100 300 1000HP01 (μgkg)

Trolox

Supe

roxi

de ra

dica

l sc

aven

ging

activ

ity (

)

A

B

C

DE

0

20

40

60

80

100

120

(b)







Groupsfactors


(kcal)

FAT


epididymalfat ()


HFD







HFD




HFD ndash + + + +

HP01 (mgkg)

CON GK 200 400NOR

(a)

Caro

tid ar

tery

thic

knes

s (μm

)

A

B

AA

AB

0

10

20

30

40

50


+ + + +


(b)










PT (s

ec)

AB

A A A

05

101520253035

NOR HFD ndash


+ + + +


(a)

aPTT

(sec

)

A B B B B

NOR HFD ndash


+ + + +


05

101520253035

(b)

Agg

rega

tion

() A B

A AB

NOR HFD ndash


+ + + +


0

20

40

60

80

(c)

TXA

2 (ng

mL)

A AA A A

NOR HFD ndash


+ + + +


0

2080

100

(d)

Sero

toni

ne (n

gm

L) AB

A B B

NOR HFD ndash


+ + + +


0

2080

100

120

140

(e)



ALT

(uni

tmL)

NOR HFD ndash


+ + + +


A

B

C C C

0

50

100

150

200

250

300

(a)

AST

(uni

tmL)

NOR HFD ndash


+ + + +


A

B

BB

B

0

50

100

150

200

250

300

(b)


MD

A (μ

Mg

live

r)

A

A

B BB

NOR HFD ndash


+ + + +


020406080

100120140

(a)

AA

BA A

GSH

(μM

g li

ver)

NOR HFD ndash


+ + + +


02468

101214

(b)

GPx

activ

ity

(nm

olm

inm

g pr

otei

n) A

BA

B

A

NOR HFD ndash


+ + + +


02468

10121416

(c)

SOD

activ

ity (U

mg

prot

ein)

AA

BB

B

NOR HFD ndash


+ + + +


0

200

400

600

800

1000

1200

(d)

Cata

lase

activ

ity(n

mol

min

mg

prot

ein) A

B

AB AB A

NOR HFD ndash


+ + + +


0

20

40

60

80

100

120

(e)










5 Conclusions



Abbreviations


Data Availability






Acknowledgments


References










































































3 Results














4 Discussion





400

300

200

100

00 5 10 15 20 25 30 35 40 45 50 55 60 (min)

Retention time


265

47

OH

OH

OH

OH

OH

O

OO

O

OHO

HO

HO

(a)

400

300

200

100

00 5 10 15 20 25 30 35 40 45 50 55 60 (min)

Retention time

Sample HP01

265

40

(b)


HP01 (μgkg)CON Vit C 100 300 1000

DPP

H ra

dica

l sc

aven

ging

activ

ity (

)

A

B

CD E

0

20

40

60

80

100

120

(a)

CON 100 300 1000HP01 (μgkg)

Trolox

Supe

roxi

de ra

dica

l sc

aven

ging

activ

ity (

)

A

B

C

DE

0

20

40

60

80

100

120

(b)







Groupsfactors


(kcal)

FAT


epididymalfat ()


HFD







HFD




HFD ndash + + + +

HP01 (mgkg)

CON GK 200 400NOR

(a)

Caro

tid ar

tery

thic

knes

s (μm

)

A

B

AA

AB

0

10

20

30

40

50


+ + + +


(b)










PT (s

ec)

AB

A A A

05

101520253035

NOR HFD ndash


+ + + +


(a)

aPTT

(sec

)

A B B B B

NOR HFD ndash


+ + + +


05

101520253035

(b)

Agg

rega

tion

() A B

A AB

NOR HFD ndash


+ + + +


0

20

40

60

80

(c)

TXA

2 (ng

mL)

A AA A A

NOR HFD ndash


+ + + +


0

2080

100

(d)

Sero

toni

ne (n

gm

L) AB

A B B

NOR HFD ndash


+ + + +


0

2080

100

120

140

(e)



ALT

(uni

tmL)

NOR HFD ndash


+ + + +


A

B

C C C

0

50

100

150

200

250

300

(a)

AST

(uni

tmL)

NOR HFD ndash


+ + + +


A

B

BB

B

0

50

100

150

200

250

300

(b)


MD

A (μ

Mg

live

r)

A

A

B BB

NOR HFD ndash


+ + + +


020406080

100120140

(a)

AA

BA A

GSH

(μM

g li

ver)

NOR HFD ndash


+ + + +


02468

101214

(b)

GPx

activ

ity

(nm

olm

inm

g pr

otei

n) A

BA

B

A

NOR HFD ndash


+ + + +


02468

10121416

(c)

SOD

activ

ity (U

mg

prot

ein)

AA

BB

B

NOR HFD ndash


+ + + +


0

200

400

600

800

1000

1200

(d)

Cata

lase

activ

ity(n

mol

min

mg

prot

ein) A

B

AB AB A

NOR HFD ndash


+ + + +


0

20

40

60

80

100

120

(e)










5 Conclusions



Abbreviations


Data Availability






Acknowledgments


References











































































4 Discussion





400

300

200

100

00 5 10 15 20 25 30 35 40 45 50 55 60 (min)

Retention time


265

47

OH

OH

OH

OH

OH

O

OO

O

OHO

HO

HO

(a)

400

300

200

100

00 5 10 15 20 25 30 35 40 45 50 55 60 (min)

Retention time

Sample HP01

265

40

(b)


HP01 (μgkg)CON Vit C 100 300 1000

DPP

H ra

dica

l sc

aven

ging

activ

ity (

)

A

B

CD E

0

20

40

60

80

100

120

(a)

CON 100 300 1000HP01 (μgkg)

Trolox

Supe

roxi

de ra

dica

l sc

aven

ging

activ

ity (

)

A

B

C

DE

0

20

40

60

80

100

120

(b)







Groupsfactors


(kcal)

FAT


epididymalfat ()


HFD







HFD




HFD ndash + + + +

HP01 (mgkg)

CON GK 200 400NOR

(a)

Caro

tid ar

tery

thic

knes

s (μm

)

A

B

AA

AB

0

10

20

30

40

50


+ + + +


(b)










PT (s

ec)

AB

A A A

05

101520253035

NOR HFD ndash


+ + + +


(a)

aPTT

(sec

)

A B B B B

NOR HFD ndash


+ + + +


05

101520253035

(b)

Agg

rega

tion

() A B

A AB

NOR HFD ndash


+ + + +


0

20

40

60

80

(c)

TXA

2 (ng

mL)

A AA A A

NOR HFD ndash


+ + + +


0

2080

100

(d)

Sero

toni

ne (n

gm

L) AB

A B B

NOR HFD ndash


+ + + +


0

2080

100

120

140

(e)



ALT

(uni

tmL)

NOR HFD ndash


+ + + +


A

B

C C C

0

50

100

150

200

250

300

(a)

AST

(uni

tmL)

NOR HFD ndash


+ + + +


A

B

BB

B

0

50

100

150

200

250

300

(b)


MD

A (μ

Mg

live

r)

A

A

B BB

NOR HFD ndash


+ + + +


020406080

100120140

(a)

AA

BA A

GSH

(μM

g li

ver)

NOR HFD ndash


+ + + +


02468

101214

(b)

GPx

activ

ity

(nm

olm

inm

g pr

otei

n) A

BA

B

A

NOR HFD ndash


+ + + +


02468

10121416

(c)

SOD

activ

ity (U

mg

prot

ein)

AA

BB

B

NOR HFD ndash


+ + + +


0

200

400

600

800

1000

1200

(d)

Cata

lase

activ

ity(n

mol

min

mg

prot

ein) A

B

AB AB A

NOR HFD ndash


+ + + +


0

20

40

60

80

100

120

(e)










5 Conclusions



Abbreviations


Data Availability






Acknowledgments


References


































































400

300

200

100

00 5 10 15 20 25 30 35 40 45 50 55 60 (min)

Retention time


265

47

OH

OH

OH

OH

OH

O

OO

O

OHO

HO

HO

(a)

400

300

200

100

00 5 10 15 20 25 30 35 40 45 50 55 60 (min)

Retention time

Sample HP01

265

40

(b)


HP01 (μgkg)CON Vit C 100 300 1000

DPP

H ra

dica

l sc

aven

ging

activ

ity (

)

A

B

CD E

0

20

40

60

80

100

120

(a)

CON 100 300 1000HP01 (μgkg)

Trolox

Supe

roxi

de ra

dica

l sc

aven

ging

activ

ity (

)

A

B

C

DE

0

20

40

60

80

100

120

(b)







Groupsfactors


(kcal)

FAT


epididymalfat ()


HFD







HFD




HFD ndash + + + +

HP01 (mgkg)

CON GK 200 400NOR

(a)

Caro

tid ar

tery

thic

knes

s (μm

)

A

B

AA

AB

0

10

20

30

40

50


+ + + +


(b)










PT (s

ec)

AB

A A A

05

101520253035

NOR HFD ndash


+ + + +


(a)

aPTT

(sec

)

A B B B B

NOR HFD ndash


+ + + +


05

101520253035

(b)

Agg

rega

tion

() A B

A AB

NOR HFD ndash


+ + + +


0

20

40

60

80

(c)

TXA

2 (ng

mL)

A AA A A

NOR HFD ndash


+ + + +


0

2080

100

(d)

Sero

toni

ne (n

gm

L) AB

A B B

NOR HFD ndash


+ + + +


0

2080

100

120

140

(e)



ALT

(uni

tmL)

NOR HFD ndash


+ + + +


A

B

C C C

0

50

100

150

200

250

300

(a)

AST

(uni

tmL)

NOR HFD ndash


+ + + +


A

B

BB

B

0

50

100

150

200

250

300

(b)


MD

A (μ

Mg

live

r)

A

A

B BB

NOR HFD ndash


+ + + +


020406080

100120140

(a)

AA

BA A

GSH

(μM

g li

ver)

NOR HFD ndash


+ + + +


02468

101214

(b)

GPx

activ

ity

(nm

olm

inm

g pr

otei

n) A

BA

B

A

NOR HFD ndash


+ + + +


02468

10121416

(c)

SOD

activ

ity (U

mg

prot

ein)

AA

BB

B

NOR HFD ndash


+ + + +


0

200

400

600

800

1000

1200

(d)

Cata

lase

activ

ity(n

mol

min

mg

prot

ein) A

B

AB AB A

NOR HFD ndash


+ + + +


0

20

40

60

80

100

120

(e)










5 Conclusions



Abbreviations


Data Availability






Acknowledgments


References






































































Groupsfactors


(kcal)

FAT


epididymalfat ()


HFD







HFD




HFD ndash + + + +

HP01 (mgkg)

CON GK 200 400NOR

(a)

Caro

tid ar

tery

thic

knes

s (μm

)

A

B

AA

AB

0

10

20

30

40

50


+ + + +


(b)










PT (s

ec)

AB

A A A

05

101520253035

NOR HFD ndash


+ + + +


(a)

aPTT

(sec

)

A B B B B

NOR HFD ndash


+ + + +


05

101520253035

(b)

Agg

rega

tion

() A B

A AB

NOR HFD ndash


+ + + +


0

20

40

60

80

(c)

TXA

2 (ng

mL)

A AA A A

NOR HFD ndash


+ + + +


0

2080

100

(d)

Sero

toni

ne (n

gm

L) AB

A B B

NOR HFD ndash


+ + + +


0

2080

100

120

140

(e)



ALT

(uni

tmL)

NOR HFD ndash


+ + + +


A

B

C C C

0

50

100

150

200

250

300

(a)

AST

(uni

tmL)

NOR HFD ndash


+ + + +


A

B

BB

B

0

50

100

150

200

250

300

(b)


MD

A (μ

Mg

live

r)

A

A

B BB

NOR HFD ndash


+ + + +


020406080

100120140

(a)

AA

BA A

GSH

(μM

g li

ver)

NOR HFD ndash


+ + + +


02468

101214

(b)

GPx

activ

ity

(nm

olm

inm

g pr

otei

n) A

BA

B

A

NOR HFD ndash


+ + + +


02468

10121416

(c)

SOD

activ

ity (U

mg

prot

ein)

AA

BB

B

NOR HFD ndash


+ + + +


0

200

400

600

800

1000

1200

(d)

Cata

lase

activ

ity(n

mol

min

mg

prot

ein) A

B

AB AB A

NOR HFD ndash


+ + + +


0

20

40

60

80

100

120

(e)










5 Conclusions



Abbreviations


Data Availability






Acknowledgments


References









































































PT (s

ec)

AB

A A A

05

101520253035

NOR HFD ndash


+ + + +


(a)

aPTT

(sec

)

A B B B B

NOR HFD ndash


+ + + +


05

101520253035

(b)

Agg

rega

tion

() A B

A AB

NOR HFD ndash


+ + + +


0

20

40

60

80

(c)

TXA

2 (ng

mL)

A AA A A

NOR HFD ndash


+ + + +


0

2080

100

(d)

Sero

toni

ne (n

gm

L) AB

A B B

NOR HFD ndash


+ + + +


0

2080

100

120

140

(e)



ALT

(uni

tmL)

NOR HFD ndash


+ + + +


A

B

C C C

0

50

100

150

200

250

300

(a)

AST

(uni

tmL)

NOR HFD ndash


+ + + +


A

B

BB

B

0

50

100

150

200

250

300

(b)


MD

A (μ

Mg

live

r)

A

A

B BB

NOR HFD ndash


+ + + +


020406080

100120140

(a)

AA

BA A

GSH

(μM

g li

ver)

NOR HFD ndash


+ + + +


02468

101214

(b)

GPx

activ

ity

(nm

olm

inm

g pr

otei

n) A

BA

B

A

NOR HFD ndash


+ + + +


02468

10121416

(c)

SOD

activ

ity (U

mg

prot

ein)

AA

BB

B

NOR HFD ndash


+ + + +


0

200

400

600

800

1000

1200

(d)

Cata

lase

activ

ity(n

mol

min

mg

prot

ein) A

B

AB AB A

NOR HFD ndash


+ + + +


0

20

40

60

80

100

120

(e)










5 Conclusions



Abbreviations


Data Availability






Acknowledgments


References


































































ALT

(uni

tmL)

NOR HFD ndash


+ + + +


A

B

C C C

0

50

100

150

200

250

300

(a)

AST

(uni

tmL)

NOR HFD ndash


+ + + +


A

B

BB

B

0

50

100

150

200

250

300

(b)


MD

A (μ

Mg

live

r)

A

A

B BB

NOR HFD ndash


+ + + +


020406080

100120140

(a)

AA

BA A

GSH

(μM

g li

ver)

NOR HFD ndash


+ + + +


02468

101214

(b)

GPx

activ

ity

(nm

olm

inm

g pr

otei

n) A

BA

B

A

NOR HFD ndash


+ + + +


02468

10121416

(c)

SOD

activ

ity (U

mg

prot

ein)

AA

BB

B

NOR HFD ndash


+ + + +


0

200

400

600

800

1000

1200

(d)

Cata

lase

activ

ity(n

mol

min

mg

prot

ein) A

B

AB AB A

NOR HFD ndash


+ + + +


0

20

40

60

80

100

120

(e)










5 Conclusions



Abbreviations


Data Availability






Acknowledgments


References









































































5 Conclusions



Abbreviations


Data Availability






Acknowledgments


References


































































Abbreviations


Data Availability






Acknowledgments


References


































































Research Article Vitislabrusca Extract(HP01 ...

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