Nanoencapsulation of Red Ginseng Extracts Using Chitosan with

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Nanoencapsulation of red ginseng extracts using chitosan with polyglutamic acid or fucoidan for improving antithrombotic activities Eun Suh Kim, Ji-Soo Lee, and Hyeon Gyu Lee J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00911 • Publication Date (Web): 16 May 2016 Downloaded from http://pubs.acs.org on May 16, 2016

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Journal of Agricultural and Food Chemistry

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(To be submitted to Journal of Agricultural and Food Chemistry)

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Nanoencapsulation of red ginseng extracts using chitosan with

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polyglutamic acid or fucoidan for improving antithrombotic activities

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Eun Suh Kim†, Ji-Soo Lee†, Hyeon Gyu Lee†, *

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Department of Food and Nutrition, Hanyang University,

222, Wangsimni-ro, Seongdong-gu, Seoul 04763, Republic of Korea

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Running title: Nanoencapsulation of red ginseng extracts

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*Corresponding author.

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Tel: +82-2-2220-1202; Fax: +82-2-2281-8285;

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E-mail address: [email protected] (H.G. Lee).

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ACS Paragon Plus Environment


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Abstract

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The potential of nanoencapsulation using bioactive coating materials for

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improving antithrombotic activities of red ginseng extract (RG) was examined. RG-

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loaded chitosan (CS) nanoparticles were prepared using antithrombotic materials,

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polyglutamic acid (PGA) or fucoidan (Fu). Both CS-PGA (P-NPs, 360 ± 67 nm) and

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CS-Fu nanoparticles (F-NPs, 440 ± 44 nm) showed sustained ginsenoside release in an

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acidic environment and improved ginsenoside solubility by approximately 122.8%.

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Both in vitro rabbit and ex vivo rat platelet aggregation of RG (22.3% and 41.5%) were

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significantly (p < 0.05) decreased within P-NPs (14.4% and 30.0%) and F-NPs (12.3%

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and 30.3%), respectively. Though RG exhibited no effect on in vivo carrageenan-

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induced mouse tail thrombosis, P-NPs and F-NPs demonstrated significant effects,

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likely the anticoagulation activity of PGA and Fu. Moreover, in in vivo rat arteriovenous

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shunt model, P-NPs (156 ± 6.8 mg) and F-NPs (160 ± 3.2 mg) groups showed

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significantly lower thrombus formation than RG (190 ± 5.5 mg). Therefore,

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nanoencapsulation using CS, PGA, and Fu is a potential for improving the

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antithrombotic activity of RG.

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Keywords: red ginseng extracts; chitosan nanoparticles; ionic gelation; platelet

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aggregation; antithrombotic activity

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Introduction Platelet activation and aggregation resulting in thrombus formation are essential

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events for hemostasis to cure vascular injury.1,

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endogenous agonists such as collagen, adenosine diphosphate (ADP), and thrombin are

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generated at the site of injury.3, 4 These agonists activate and aggregate platelets and

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support the platelet adhesion to the site of vascular injury.5, 6. However, over-activation

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of platelet can cause venous and arterial thrombosis that interrupt blood stream and

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vascular diseases such as edema, inflammation, myocardial infarction, and cerebral

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apoplexy.7,

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thrombopoiesis and regulating cardiovascular health.9, 10

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8

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When blood vessels are damaged,

Thus, antiplatelet management is important for preventing excessive

Red ginseng, a traditional Korean functional food, has been known to get

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pharmacological effects on hypertension, diabetes, immunity, and fatigue.11,

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ginseng extracts (RG) can be isolated into ginsenoside derivatives, the main active

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constituents of RG that have unique inherent bioactivities.13, 14 Among the derivatives,

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ginsenoside Rb1 and Rg1 have significant effects on antithrombosis and antiplatelet

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which refers to the general inhibition of blood clotting and platelet aggregation,

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respectively.15, 16 However, low stability and permeability in the gastrointestinal (GI)

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tract have been identified as impediments of oral administration of the ginsenosides.17, 18

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Encapsulation can be defined as a process to entrap one substance (bioactive

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compounds) within another substance (wall materials) in order to prevent degradation,

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control release, and modify physical characteristics.19 Recently, nanoencapsulation has

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been widely investigated for drug delivery because of its higher surface areas arising

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from nano-sized particles, which results in extent of absorption and uptake efficiency.20 ３


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Red


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Polymer nanoparticles prepared by nontoxic, biodegradable, and biocompatible natural

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polysaccharides are suitable for food applications. Chitosan (CS), a cationic

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polysaccharide, is commonly used in oral medications due to its high membrane

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permeability and biodegradability by lysozyme in serum.21 Polyglutamic acid (PGA),

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consists of glutamic acid units connected by amide linkages22, 23 and fucoidan (Fu), an

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anionic sulfated polysaccharide found in seaweed, can produce nanoparticles by ionic

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gelation with CS.24 Furthermore, PGA and Fu are known to have anticoagulation

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activity.25, 26 Therefore, nanoencapsulation using PGA and Fu is expected to contribute

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to enhance the antithrombotic activities of RG by an additive effect of the coating

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materials as well as overcome the poor oral bioavailability of ginsenoside by

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encapsulation.

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The aim of this study was to investigate the effects of nanoencapsulation using

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CS, PGA, and Fu on the physicochemical properties and antithrombotic activities of RG.

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Physicochemical properties of nanoparticles, including particle size, morphological

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characteristics, encapsulation efficiency (EE), solubility, and in vitro release behavior

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were assessed. Moreover, inhibitory activities on in vitro and ex vivo platelet activity,

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and in vivo carrageenan-induced tail thrombosis and arteriovenous (AV) shunt

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thrombosis were also evaluated.

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Materials and Methods

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Materials CS (M.W. 1,000-3,000), PGA (M.W. 50 kDa), and Fu from

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Laminaria japonica were purchased from Kitto life Co. (Seoul, Korea), Bio leaders

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Corp. (Daejeon, Korea), and Haewon Biotech Inc. (Seoul, Korea), respectively. RG was

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supplied from CheonJiYang Co., Ltd. (Seoul, Korea). Citrate dextrose solution (ACD), ４


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Hepes buffer, bovine serum albumin (BSA), and carboxymethyl cellulose (CMC) were

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purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Additionally,

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collagen was purchased from Chrono-Log Co. (Havertown, PA, USA). All other

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chemicals were reagent grade and used without further purification.

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RG nanoencapsulation Two types of RG-loaded nanoparticles were prepared

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based on ionic gelation of CS with PGA (P-NPs) or Fu (F-NPs).23, 24 Briefly, CS and RG

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were dissolved in distilled water (DW) and completely mixed at 2 and 10 mg/mL of

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final concentration, respectively. Under continuous stirring at 1,000 rpm (MS-MP8,

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Daihan Co., Seoul, Korea), the PGA or Fu solution, varied from 0.6 to 1.6 mg/mL and

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from 2.5 to 250 µg/mL were added to the mixture, respectively. Further stirring of the

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mixture led to the immediate formation of nanoparticles.

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Physicochemical properties of nanoparticles Particle size, zeta potential (ZP),

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polydispersed index (PDI), and derived count rate (DCR) of RG-loaded nanoparticles

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were investigated by dynamic light scattering (DLS) using a Zetasizer Nano ZS

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(Malvern Instruments Ltd., Worcestershire, UK). The morphological characteristics of

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the nanoparticles were analyzed using a high resolution transmission electron

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microscope (TEM, JEM 2100F, JEOL, Tokyo, Japan). Nanosuspension was prepared by

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placing one drop onto 200 mesh copper grid and air-dried at 37˚C. Samples were

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stained with 2% phosphotungstic acid solution for 30 min and subjected to TEM

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analysis.

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Ginsenoside determination Ginsenoside was quantified using a high

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performance liquid chromatography (HPLC) system consisting of two 515 HPLC

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pumps, a 486 tunable absorbance detector, a manual injector with a 50 µL loop (all from ５



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Waters Corp., Milford, MA, USA), and a degasser (ERC-3215α, ERC Inc., Kawaguchi,

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Japan). Separation was carried out on a YMC-Pack Pro C18 column (4.6×250 mm, 5 µm,

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YMC Co., Ltd., Kyoto, Japan) at 40˚C using a column heater (CH-500, Eppendorf,

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Hamburg, Germany). The binary gradient system consisted of DW (A) and acetonitrile

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(B). The elution conditions were as follows: 0 min, 80% A; 5 min, 80% A; 13 min, 75%

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A; 85 min 55% A; 90 min, 10% A; 95 min, 55% A; 98 min, 80% A; re-equilibration

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from 98 to 100 min with 80% A. Flow rate was set to 1.6 mL/min and detected at UV

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wavelength of 203 nm. The solubility of ginsenosides were evaluated after filtering the

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suspension of samples throughout 0.45 µm-pore-size nylon membrane filter to remove

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the insoluble RG.27 Completely dissolved ginsenosides Rg1 and Rb1 were determined

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by analogy with HPLC peaks of commercial standards.

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Encapsulation efficiency The encapsulation efficiency (EE) was indirectly

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determined using the amount of free ginsenoside Rg1 and Rb1 in the supernatant

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following nanoparticle ultracentrifugation at 15,000×g for 30 min (Optima TL

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Ultracentrifuge, Beckman, Fullerton, CA, USA).28 EE was calculated by Eq. (1):

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EE % = × 100

(1)

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In vitro release properties The in vitro ginsenoside release from nanoparticles

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was examined under simulated gastric (SGF) and intestinal fluid (SIF) by adjusting the

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nanoparticle suspension which had a pH of approximately 7.38 to pH 1.2 with 0.1 M

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HCl and adjusting pH back to 6.8 with 0.1 M NaOH.29 RG-loaded nanoparticles were

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successively exposed to SGF for 2 h and SIF for 4 h at 37˚C collected following SGF

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and SIF exposure. Nanoparticle suspensions were centrifuged at 15,000×g for 30 min,

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and the ginsenoside in the supernatant was quantified using HPLC as described above.

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Ginsenoside release rate was calculated by Eq. (2):

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Release % =

"

× 100

(2)

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Animals New Zealand white rabbits were purchased from Koatech Co.

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(Pyongtaek, Korea). All rabbits were housed at 24 ± 1 ˚C with a relative humidity of 55

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± 5% and provided food pellet and tap water. All animals and procedures were approved

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by the Institutional Animal Care and Use Committee (IACUC) guidelines for the care

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and use of laboratory animals of Hanyang University. Five-week-old male ICR mice (25

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± 2 g) and male Sprague–Dawley (SD) rats (200–250 g) were purchased from Orient

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Bio. Inc. (Seongnam, Korea). The animals were placed in controlled environments at 22

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± 2 ˚C and a relative humidity of 55±5% with a 12 h light-dark cycle, and acclimatized

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with standard chow for at least one week prior to use. All animal experiments were

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performed according to the IACUC guidelines of Daejeon University.

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In vitro platelet aggregation assay In vitro platelet aggregation assay was

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performed using washed platelets (WP) isolated from rabbit blood. Whole blood was

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collected directly from the ear artery of rabbits into an ACD solution and centrifuged at

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230×g for 10 min (Combi 408, Hanil Co., Seoul, Korea) to obtain platelet rich plasma

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(PRP). Platelets were collected as precipitate after centrifugation of PRP at 800×g for 15

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min. Platelet were washed with Hepes buffer (137 mM NaCl, 2.7 mM KCl, 1 mM

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MgCl2, 5.6 mM glucose, and 3.8 mM Hepes, pH 6.5) containing 0.35% BSA and 0.4

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mM EGTA (ethylene glycol-bis (13-aminoethyl ether) N,N,N'N'-tetraacetic acid). WP

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was dispersed in Hepes buffer (pH 7.4) and adjusted to 4×108 cells/mL using

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hematology analyzer (Vet ABC, Horiba, Kyoto, Japan). WP was incubated at the

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aggregometer (Chrono-log 490, Chrono-log Co., Havertown, PA, USA) with 1,000 rpm

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at 37°C and treated with samples for 3 min. Platelet aggregation was induced using 5

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µg/mL of collagen and the aggregation rate was evaluated by turbidimetry method.15

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Ex vivo platelet aggregation assay The rats were divided into 4 groups of 4

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animals each for administration of freeze-dried RG, P-NPs, F-NPs, and control. The

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samples in 0.25% (w/v) CMC solution were orally administered to the rats at the dose

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of 300 mg/ kg body weight for three consecutive days at the same time of day. PRP was

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obtained from whole blood by centrifuging at 230×g for 10 min and platelet poor

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plasma (PPP) was obtained from PRP by centrifuging at 800×g for 15 min. PRP was

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collected 1 h after sample treatment, and adjusted to 3×108 platelets/mL with PPP.

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Platelet aggregation was induced by 10 µg/mL of collagen and the rate of aggregation

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was determined by the turbidimetry method.15

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In vivo carrageenan-induced mouse tail thrombosis model The mice were

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divided into 4 groups of 6 animals each for administration of freeze-dried RG, P-NPs,

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F-NPs, and control. The samples in 0.25% (w/v) CMC solution were orally

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administered to mice at the dose of 300 mg/kg. After administration of samples for 1 h,

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40 µL of 1% (w/v) sterile carrageenan (Type I) dissolved in physiological saline was

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injected in the right hind paw. Mice were observed for the thrombus in the tail and

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thrombus lengths were measured at 24, 48, and 72 h.30

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In vivo AV shunt model The rats were divided into 4 groups of 5 animals each

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for administration of freeze-dried RG, P-NPs, F-NPs, and control. The samples in 0.25%

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(w/v) CMC solution were orally administered to the rats at the dose of 300 mg/kg at the ８


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same time on each three consecutive days. In vivo AV shunt thrombosis model was

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tested after 2 h of the last administration. After the rats were under anesthesia with

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pentobarbital sodium salt (60 mg/kg, i.p.), polyethylene tube was inserted between the

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left jugular vein and the right carotid artery. The saline-filled shunt which was

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assembled by cotton thread (diameter, 0.25 mm) with tygon tube (internal diameter, 2

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mm) was connected with two cannulas. After extracorporeal circulation for 15 min,

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associated thrombus on the cotton thread was weighed by subtracting the weight of the

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dry cotton thread.31

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Statistical analysis All experiments were performed in triplicate. All data were

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expressed as the mean value ± standard deviation. Significant differences (p < 0.05)

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among corresponding mean values were identified using one-way analysis of variance

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(ANOVA) followed by Duncan’s multiple comparison test (SPSS 12.0.1, SPSS Inc.,

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Chicago, IL, USA).

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Results and discussion

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Physicochemical properties of RG-loaded nanoparticles At the identical

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concentration of 2 mg/mL CS, particle size of P-NPs significantly decreased with an

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increase in PGA from 0.6 to 1.2 mg/mL, followed by dramatic increase with an increase

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in PGA above 1.2 mg/mL (Table 1). The smallest and largest particle sizes of 331 ± 6

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and 805 ± 49 nm were obtained at 1.2 and 0.8 mg/mL PGA, respectively. The excess of

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PGA over 1.6 mg/mL resulted in aggregation and precipitation. Nanoparticles formed

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by ionic gelation between multiple charged materials are closely affected by the

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proportions of the materials and possibly prepared at the particular proportion.32 PGA

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concentration is critical in determining particle size of P-NPs, since the PGA have the ９



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negatively charged carboxylic acid (-COO-) which resulted in electrostatic interaction

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with the positively charged amino groups (-NH3+) on CS. In the concentration range

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from 0.6 to 1.2 mg/mL of PGA concentration, the increase of PGA concentration

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increased the cross-linking properties between CS and PGA resulting in denser structure

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of P-NPs which had smaller particle size. On the other hand, in presence of higher PGA

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above the proper proportion, superfluous PGA led to more CS involved in formation of

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single nanoparticles, resulting in formation of larger nanoparticles.33 ZP, an electrostatic

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potential at the interface of fluid and particle surfaces in solutions or colloidal

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suspensions, is an important factor in determining the stability of nanoparticles.34

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Absolute ZP values of P-NPs were significantly increased with an increase in PGA

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concentrations. It can be explained that stability of P-NPs were increased due to the

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increase of reaction proportion between CS and PGA. PDI, a measure of particle size

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distribution, is a critical factor for size uniformity of the resultant nanoparticles. PDI

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value lower than 0.3 is regarded as evidence of homogeneous particle size and adequate

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dispersion, while values higher than 0.7 indicate poor uniformity and dispersity.35 At all

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PGA conditions, P-NPs demonstrated PDI values lower than 0.3. Therefore, PGA from

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0.6 to 1.6 mg/mL is considered a suitable condition for preparation of P-NPs. DCR, the

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scattering intensity of the particles in solution per second, is roughly proportional to the

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concentration of nanoparticles in the colloidal suspensions.36 The results of DCR

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showed opposite trend compared with particle size that DCR increased with an increase

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in PGA from 0.6 to 1.2 mg/mL, and then decreased dramatically in PGA above 1.2

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mg/mL. Moreover, preparation of P-NPs at 1.2 mg/mL PGA seemed to produce the

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largest amount of P-NPs in the suspensions due to its the highest DCR values.37

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Therefore, PGA concentration of 1.2 mg/mL was considered to be proper preparation １０


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condition of P-NPs which produced the smallest nanoparticles with the satisfactory

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stability and dispersion.

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F-NPs were also prepared by ionic gelation of CS and various concentration of

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Fu (Table 2). At a Fu range from 2.5 to 100 µg/mL, F-NPs sizes varied from 440 to 496

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nm (no significant differences). However, particle size significantly increased with an

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increase in Fu above 150 µg/mL. Moreover, particles on the micro-scale over 1,000 nm

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were prepared at 250 µg/mL Fu. As mentioned above, ionic gelation rate is highly

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dependent on charge balance between oppositely charged materials. Therefore, when Fu

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exceeded critical concentrations for balanced interaction with CS, aggregation was

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occurred due to increased electrostatic attraction between CS and Fu. PDI of the F-NPs

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prepared with Fu ranged from 2.5 to 150 µg/mL was stable (< 0.3) and regarded as high

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dispersion of nanoparticles. However, PDI significantly increased above 200 µg/mL Fu,

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and F-NPs prepared by 250 µg/mL Fu exhibited poor PDI (> 0.7). For ZP, F-NPs

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demonstrated lower absolute ZP values compared with P-NPs. Again, DCR exhibited

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opposite increasing and decreasing trends compared with those of particle size. The

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effects of nanoencapsulation are increased with an increase of surface area to volume

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ratios which derived from smaller particle size.38 Therefore, the smallest P-NPs by 1.2

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mg/mL PGA, and F-NPs by 10 µg/mL Fu were analyzed in further studies.

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Morphological

observations

P-NPs

demonstrated

uniform

spherical

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morphologies and F-NPs demonstrated irregular surface morphologies with partial

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aggregation (Figure 1). Both P-NPs and F-NPs were arranged layer-by-layer from the

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core to the edge, indicating encapsulation of RG within coating materials. Particle sizes

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of P-NPs by 1.2 mg/mL PGA and F-NPs by 10 µg/mL Fu which yielded the smallest

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size in DLS analysis were varied approximately from 300 to 400 nm, and from 350 to

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600 nm, respectively. The size differences between TEM and DLS analysis can be

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attributed to hydration and the three-dimensional considerations.

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Ginsenoside solubility and EE As shown in Table 3, solubility of ginsenoside

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Rg1 and Rb1 were significantly increased within both nanoparticles approximately from

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48.43 to 59.07 mg/g and from 61.94 to 76.71 mg/g, respectively. Moreover, EE of Rg1

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and Rb1 were 21.64% and 38.04% in P-NPs, and 22.63% and 40.13% in F-NPs,

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respectively. However, significant differences of solubility and EE of each ginsenosides

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between the types of nanoparticles were not observed.

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In vitro release properties Release rate of ginsenoside Rg1 and Rb1 from the

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nanoparticles after 2 h of exposure to SGF were varied from 37.63 to 45.56%, and from

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23.97 to 30.76%, respectively (Figure 2). Moreover, after consecutive 4 h exposure to

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SIF, release rate of ginsenoside Rg1 and Rb1 were varied from 74.98 to 76.52%, and

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from 56.71 to 57.53%, respectively. Therefore, both P-NPs and F-NPs can be expected

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to increase the stability of ginsenosides in GI tract due to its sustained release of

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ginsenosides under gastric environment where ginsenoside Rg1 and Rb1 were instable.

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Compared with the types of nanoparticles, P-NPs showed significantly lower release

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rates of ginsenoside than F-NPs in SGF. Aggregation of nanoparticles would lead to

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release of core materials from the particles into the dissolution media.39 Moreover,

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colloidal instability which can lead to aggregation is mainly attributed by decrease of

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electrostatic repulsion between the particles. Therefore, lower stability of F-NPs than

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that of P-NPs which was indicated from the results of ZP might promote the release of

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ginsenosides. However, after continuous exposure to SIF, both nanoparticles gave

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similar release rates of ginsenoside Rg1 and Rb1. Moreover, release of ginsenoside Rg1

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from the nanoparticles was significantly higher than that of Rb1 during the constant

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release period in both P-NPs and F-NPs. Regarding the molecular weight of ginsenoside

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Rg1 and Rb1 reported to be 801.02 and 1,109.28, respectively, it can be assumed that

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release of smaller molecule of Rg1 were faster than the release of Rb1. 40, 41

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In vitro platelet aggregation Blood coagulation mechanisms mainly consist of

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primary and secondary events, and the test of antiplatelet activity belongs to the former

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event. Platelet is activated via multiple pathways which are triggered by several agonists

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such as collagen, ADP, and thrombin. While each agonist is involved in different

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pathways, collagen contributes to the all steps of platelet activation including ADP

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release, thromboxane A2 formation, and platelet activation.16 Therefore, a collagen-

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induced platelet aggregation test can be used to evaluate the interference activities on

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the general mechanism of platelet aggregation.

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To evaluated the protective effects of nanoencapsulation on antithrombotic

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activities of RG, in vitro antiplatelet activities of free RG, P-NPs, and F-NPs were

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analyzed in pH 2 where RG is instable (Figure 3). RG significantly inhibited collagen-

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induced platelet aggregation (22 ± 1%) compared with control (37 ± 8%), and the

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activity of RG was significantly elevated within both P-NPs (14 ± 3%) and F-NPs (12 ±

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3%). Primarily, encapsulated ginsenoside Rg1 and Rb1 encapsulated in nanoparticles

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exhibited more stable antiplatelet activity compared with free RG, due to reduced

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degradation induced by sustained release in the acidic environment. Furthermore,

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increased solubility of ginsenoside resulted in higher concentration of dissolved

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ginsenoside Rg1 and Rb1, and these active forms seem to contribute to the increased

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antiplatelet activity. However, the effects of types of nanoparticles on antiplatelet

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activities showed no significant difference.

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Ex vivo platelet aggregation Platelet aggregation of free RG group was 41.5 ±

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3.5%, while that of P-NPs and F-NPs groups were 30.0 ± 5.7% and 30.3 ± 4.5%,

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respectively (Figure 3). The results confirmed the findings of in vitro study, in that RG

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had significant inhibitory effects on platelet aggregation and that the activity was

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significantly increased by nanoencapsulation. In a previous study, heparin, a

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representative anticoagulant, was encapsulated within polymeric nanoparticles for oral

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administration. Evaluation factors for anticoagulation activity were significantly

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improved due to an increase in solubility and controlled release by nanoencapsulation.42

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The results of ex vivo antiplatelet activity study also can be explained by the increased

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stability in gastric environments during digestion and the solubility of therapeutically

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active ginsenoside forms, contributing to an increase in ginsenoside bioavailability and

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activity.

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In vivo carrageenan-induced mouse tail thrombosis The secondary blood

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coagulation events could have been divided into intrinsic and extrinsic pathways and the

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two pathways were integrated into the platelet activation step. Carrageenan most likely

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causes thrombosis by altering the Hageman factor of the intrinsic coagulation pathway

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not by platelet activation.43 After subcutaneous injection, carrageenan induces

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inflammation at local blood vessel and leads to the release of inflammation factors.

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These factors then damage the endothelial cell and interrupt hemagglutination and

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fibrinolysis likely resulting in thrombus formation.

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The thrombus at the end of the tails appeared after 48 h of injection, as red wine

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colored region which progressed to dry necrosis (Figure 4). The mean thrombus length

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of the each groups after 48 h showed no significant differences (Figure 5). However,

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after 72 h, reduce of thrombus lengths were observed in the nanoparticles groups.

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Particularly, thrombus lengths of P-NPs (3.86 ± 0.94 cm) were significantly reduced by

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42.73 and 34.46% compared with control (6.74 ± 0.75 cm) and free RG (5.89 ± 1.83

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cm), respectively. In previous researches, RG had no effect on the in vitro activated

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partial thromboplastin time (aPTT) study which represents the intrinsic coagulation

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pathways16 while PGA and Fu showed significant effects.25, 26 Therefore, the inhibitory

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effects of P-NPs and F-NPs on thrombosis followed by the intrinsic pathways can be

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explained by the presence of PGA and Fu while RG showed no effect.

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In vivo AV shunt thrombosis As shown in Figure 6, RG showed significant

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inhibitory activity on thrombus formation in in vivo AV shunt model (190 ± 5.5 mg)

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compared with control (276 ± 10.3 mg). Moreover, antithrombotic activities of RG

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within P-NPs (156 ± 6.8 mg) and F-NPs (160 ± 3.2 mg) were significantly increased by

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approximately 17.89 and 15.79%, respectively. However, significant differences

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between the mean thrombus weight of P-NPs and F-NPs groups was not observed.

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AV shunt model is considered an integrated method for the evaluation of

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antithrombotic activity, as thrombus in the AV shunt model is influenced by both

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intrinsic and extrinsic coagulation pathways.44 Therefore, the results convinced the

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antithrombotic activity of ginsenoside and increased activity within nanoencapsulation.

333

Increase in inhibitory activity of thrombosis in AV shunt model can be caused not only

334

by nanoencapsulation (via increasing stability and solubility of ginsenoside), but also by

１５



335

the complex inhibitory effects of the coating materials. Therefore, though both P-NPs

336

and F-NPs could not exhibit satisfying encapsulation efficiency, RG-loaded CS

337

nanoparticles prepared with PGA or Fu can be regarded as the promising delivery

338

systems for improving the antithrombotic activities of RG, and beneficial for

339

cardiovascular diseases prevention. However, further study for improving the EE and

340

ZP is necessary to increase the stability and effectiveness of nanoparticles. Moreover, it

341

is also interesting to investigate the complex use of PGA, Fu, and other bioactive

342

coating materials in nanoencapsulation of RG.

343

Acknowledgments

344

This work was supported by the Technological Innovation R&D Program

345

(S2042340) funded by the Small and Medium Business Administration (SMBA, Korea).

346

References

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480

Figure captions

481

Figure 1. TEM image of P-NPs with 1.2 mg/mL PGA (a), (b) and F-NPs with 10 µg/mL

482

Fu (c), (d) at different magnifications.

483

Figure 2. In vitro ginsenoside Rg1 and Rb1 release from P-NPs (a) and F-NPs (b) in

484

SGF (0-2 h) and SIF (2-6 h).

485

Figure 3. In vitro and ex vivo antiplatelet aggregation activities of RG, P-NPs, and F-

486

NPs. All data are presented as mean ± standard deviation and different lowercase letters

487

indicate significant differences (p < 0.05).

488

Figure 4. Representative images of carrageenan-induced mice tail thrombus of RG

489

groups after 48 h (a) and 72 h (b), P-NPs groups after 48 h (c) and 72 h (b), and F-NPs

490

groups after 48 h (e), and 72 h (f), respectively.

491

Figure 5. Inhibitory activities of in vivo carrageenan induced mouse tail thrombus of

492

RG, P-NPs, and F-NPs. All data are presented as mean ± standard deviation and

493

different lowercase letters indicate significant differences (p < 0.05).

494

Figure 6. Inhibitory activities of in vivo AV shunt thrombus of RG, P-NPs, and F-NPs.

495

All data are presented as mean ± standard deviation and different lowercase letters

496

indicate significant differences (p < 0.05).

497

２３



Tables Table 1. Preparation conditions and physicochemical properties of P-NPsa Chitosanb (mg/mL)

PGA (mg/mL)

Particle size (nm)

Zeta potential (mV)

2

0.6

798 ± 57 a

0.8

Polydispersed Index

Derived count rate (kcps)

8.6 ± 1.4 e

0.205 ± 0.038 b

40,247 ± 11,350 d

805 ± 49 a

13.9 ± 1.2 d

0.118 ± 0.045 d

41,415 ± 14,585 d

1.0

360 ± 67 c

18.6 ± 1.0 c

0.136 ± 0.041 cd

182,103 ± 52,229 c

1.2

331 ± 6 d

19.5 ± 1.1 c

0.163 ± 0.029 c

274,421 ± 50,064 a

1.4

424 ± 10 c

21.0 ± 2.1 b

0.210 ± 0.020 b

219,406 ± 10,918 b

1.6

526 ± 16 b

23.8 ± 1.4 a

0.267 ± 0.034 a

194,126 ± 17,085 bc

a

All data are presented as mean ± standard deviation. The red ginseng extracts concentration was fixed at 10 mg/mL. a-d Different letters in the same column indicate significant differences (p < 0.05). b

２４


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Table 2. Preparation conditions and physicochemical properties of F-NPsa Chitosanb (mg/mL)

Fuoidan (µg/mL)

Particle size (nm)

Zeta potential (mV)

2

2.5

474 ± 87 c

5.0

Polydispersed index

Derived count rate (kcps)

2.7 ± 0.3 d

0.239 ± 0.062 c

35,786 ± 6,541 e

467 ± 66 c

3.0 ± 0.3 d

0.267 ± 0.089 c

54,733 ± 5,040 d

7.5

449 ± 40 c

2.7 ± 0.3 d

0.255 ± 0.088 c

59,989 ± 4,316 cd

10

440 ± 44 c

2.8 ± 0.2 d

0.243 ± 0.064 c

62,169 ± 4,137 cd

100

496 ± 69 c

3.6 ± 0.4 c

0.208 ± 0.033 c

86,692 ± 4,481 a

150

648 ± 84 b

4.1 ± 0.4 b

0.233 ± 0.050 c

74,759 ± 1,217 b

200

686 ± 96 b

4.4 ± 0.7 b

0.385 ± 0.079 b

65,749 ± 4,709 c

250

1,020 ± 348 a

4.9 ± 0.4 a

0.824 ± 0.124 a

56,670 ± 13,616 d

a

All data are presented as mean ± standard deviation. The red ginseng extracts concentration was fixed at 10 mg/mL. a-e Different letters in the same column indicate significant differences (p < 0.05). b

２５



Table 3. Ginsenoside solubility and encapsulation efficiency of RG, P-NPs, and F-NPsa Aqueous solubility (mg/g)

Encapsulation efficiency (%)

Rg1

Rb1

Rg1

Rb1

Free RG

48.43 ± 0.95 b

61.94 ± 2.10 b

P-NPsb

58.75 ± 0.63 a

76.71 ± 0.61 a

21.64 ± 1.03

38.04 ± 1.06

F-NPsc

59.07 ± 0.39 a

74.41 ± 0.52 a

22.63 ± 0.84

40.13 ± 0.75

a

All data are presented as mean ± standard deviation. P-NPs was prepared by 2 mg/mL CS, 1.2 mg/mL PGA and 10 mg/mL RG. c F-NPs was prepared by 2 mg/mL CS, 10 µg/mL Fu and 10 mg/mL RG. a-b Different letters in the same column indicate significant differences (p < 0.05). b

２６


Page 26 of 33

Page 27 of 33


Figures

Figure 1. TEM image of P-NPs with 1.2 mg/mL PGA (a), (b) and F-NPs with 10 µg/mL Fu (c), (d) at different magnifications.

２７



Figure 2. In vitro ginsenoside Rg1 and Rb1 release from P-NPs (a) and F-NPs (b) in SGF (0-2 h) and SIF (2-6 h).

２８


Page 28 of 33

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Figure 3. In vitro and ex vivo antiplatelet aggregation activities of RG, P-NPs, and F-NPs. All data are presented as mean ± standard deviation and different lowercase letters indicate significant differences (p < 0.05).

２９



Figure 4. Representative images of carrageenan-induced mice tail thrombus of RG groups after 48 h (a) and 72 h (b), P-NPs groups after 48 h (c) and 72 h (b), and F-NPs groups after 48 h (e), and 72 h (f), respectively.

３０


Page 30 of 33

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Figure 5. Inhibitory activities of in vivo carrageenan induced mouse tail thrombus of RG, P-NPs, and F-NPs. All data are presented as mean ± standard deviation and different lowercase letters indicate significant differences (p < 0.05).

３１



Figure 6. Inhibitory activities of in vivo AV shunt thrombus of RG, P-NPs, and F-NPs. All data are presented as mean ± standard deviation and different lowercase letters indicate significant differences (p < 0.05).

３２


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Graphic for table of contents


Nanoencapsulation of Red Ginseng Extracts Using Chitosan with

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