Anchoring 20(R)-Ginsenoside Rg3 onto Cellulose Nanocrystals To

Research Article pubs.acs.org/journal/ascecg

Anchoring 20(R)-Ginsenoside Rg3 onto Cellulose Nanocrystals To Increase the Hydroxyl Radical Scavenging Activity Chunxia Tang,†,‡ Yulong Wang,*,† Yunduo Long,‡ Xingye An,‡,∥ Jing Shen,§ and Yonghao Ni*,‡ †

College of Chemical and Biological Engineering, Changsha University of Science and Technology, No. 960, Wanjiali South Road, Changsha 410004, China ‡ Department of Chemical Engineering, University of New Brunswick, Fredericton, New Brunswick E3B 5A3, Canada § Key Laboratory of Bio-based Material Science and Technology of Ministry of Education, Northeast Forestry University, Harbin 150040, China ∥ Tianjin Key Laboratory of Pulp and Paper, Tianjin University of Science & Technology, Tianjin 300457, China ABSTRACT: Cellulose nanocrystals (CNCs) exhibit attractive properties as carriers for bioactive molecules, including their large surface area, charge characteristics, and abundant surface hydroxyl groups, among others. In this study, we used CNCs as carriers for a hydrophobic drug, 20(R)-ginsenoside Rg3 (here abbreviated as 20(R)-Rg3), to improve its bioavailability, dispersity, and antioxidation activity in an aqueous system. The CNC/20(R)-Rg3 nanocomposites were fabricated and then characterized by transmission electron microscopy, Fourier transform IR spectroscopy, and X-ray diffraction. The particle size of 20(R)-Rg3 was in the range of 8−22 nm. In vitro hydroxyl radical scavenging by the 2deoxyribose oxidation method and the salicylic acid hydroxylation method showed that the CNC/20(R)-Rg3 nanocomposites had much higher antioxidation activity (33.58% and 48.62%) than CNC-free 20(R)-Rg3, water-dispersed 20(R)-Rg3, and CNCs. KEYWORDS: Cellulose nanocrystals, Carrier, 20(R)-Ginsenoside Rg3, Bioavailability, Dispersity, Antioxidation activity

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INTRODUCTION Ginseng has a long history in traditional Chinese medicine.1 This drug has gained popularity because of the public’s demand for natural/organic products, and its promising applications as an anticancer, antiaging, antidiabetes, and anti-inflammation agent, and the enhancement of immune function and cognition.2−5 Ginsenosides, the main therapeutic constituents of ginseng, are composed of hydrophobic triterpenes or steroid aglycones and hydrophilic sugar side chains.6 Ginsenoside Rg3 family, a group of typical ginsenoside biomacromolecules, have relatively high anticancer and immune activities compared with the majority of ginsenosides both in vivo and in vitro.7−11 However, the main challenges of using ginsenosides Rg3 in pharmacy are their insolubility in water and poor oral bioavailability due to their inherent hydrophobic properties, which can hamper clinical applications.10 Nanonization and amorphization represent an effective strategy in terms of improving the bioavailability of water-insoluble drugs in aqueous systems.12,13 Solvent/antisolvent precipitation is an easily scalable approach to obtain nanosized amorphous drug particles.14 For example, Han et al.15 fabricated amorphous 20(S)-protopanaxadiol nanoparticles with bovine serum albumin as a carrier to improve its bioavailability and activity for hepatic cancer treatment. Cellulose nanocrystals (CNCs) are advantageous as nanoscale carriers for bioactive molecules.16−19 The abundant surface OH groups on CNCs provide sites for surface modification, and © 2017 American Chemical Society

CNCs have been studied as the supporting materials for hydrophobic drugs.20,21 Jackson et al.22 used cetyltrimethylammonium bromide (CTAB)-modified CNCs as carriers for hydrophobic anticancer drugs (docetaxel, paclitaxel, and etoposide) and found that CTAB-coated CNCs had good capacity to bind nonionized hydrophobic drugs, thereby enhancing their bioavailability and antioxidant ability. In another case, Mohanta et al.23 used CNCs as the support for curcumin, a hydrophobic drug, to prepare porous thin films through the layer-by-layer method and found that the CNCs played an important role in improving the bioavailability and dispersity of curcumin as a result of hydrogen-bond formation and van der Waals interactions between CNCs and curcumin. In this study, 20(R)-ginsenoside Rg3 (20(R)-Rg3), a drug belonging to the ginsenoside Rg3 family, was loaded onto CNCs to enhance its hydroxyl radical (OH·) scavenging activity. The resultant CNC/20(R)-Rg3 nanocomposites were characterized by transmission electron microscopy (TEM), Fourier tranform IR spectroscopy (FTIR), and X-ray diffraction (XRD), and the in vitro OH· scavenging activity was investigated.

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MATERIALS AND METHODS

Materials. CNCs were supplied by Cellulose Lab Inc. (Canada); they were produced by dissolving pulp via sulfuric acid (64 wt Received: December 9, 2016 Revised: June 12, 2017 Published: July 31, 2017 7507

DOI: 10.1021/acssuschemeng.6b02996 ACS Sustainable Chem. Eng. 2017, 5, 7507−7513

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ACS Sustainable Chemistry & Engineering

Figure 1. Schematic illustration of the formation of CNC/20(R)-Rg3 nanocomposites. containing 0.2 mL of FeSO4·7H2O (10 mM), EDTA (10 mM), and 2deoxyribose (10 mM) was mixed with 0.2 mL of sample solution (with a 20(R)-Rg3 concentration of 0−15 μg/mL). Subsequently, 0.1 M phosphate buffer (pH 7.4) was added to the mixture to reach a volume of 1.8 mL. Finally, 0.2 mL of H2O2 (10 mM) was added to the system to start the reaction, and the mixture was incubated at 37 °C for 4 h. After the incubation, 1 mL of 2.8% trichloroacetic acid (TCA) and 1.0% thiobarbituric acid (TBA) were added. The mixture was then placed in boiling water for 10 min and cooled in an ice−water bath. The absorbance was measured against a blank at 532 nm using a UV−vis spectrophotometer (TU-1801PC, Persee). The scavenging rate of OH· was calculated using eq 1:

%)-catalyzed hydrolysis and sodium hydroxide treatment.24 20(R)-Rg3 (≥99%, HPLC) was purchased from Fusheng Pharmaceuticals Inc. (Dalian, China). All of the other chemicals were purchased from SigmaAldrich (Canada) and used as received. Deionized water was used throughout this study. Fabrication of CNC/20(R)-Rg3 Nanocomposites. The procedure for the fabrication of CNC/20(R)-Rg3 nanocomposites was similar to that in the previously published report related to the use of CNCs as carriers for spirooxazine dyes.25 20(R)-Rg3 powder was first dissolved in anhydrous ethanol at a concentration of 100 μg/mL, followed by ultrasonic treatment (20 min). The 20(R)-Rg3 solution (10.60 mL) was added dropwise to a CNC dispersion (60.00 mL, 0.2 wt %) under vigorous stirring for 10 min at 0 °C. The temperature was maintained using a water/ice bath. For comparison, 20(R)-Rg3 ethanol solution (10.60 mL) was added dropwise to water (60.00 mL) under the same conditions to prepare CNC-free 20(R)-Rg3, and 20(R)-Rg3 powder was well-dispersed in 60.00 mL of water at a concentration of 15.00 μg/mL by ultrasonic treatment to form the water-dispersed 20(R)-Rg3 sample. TEM Observations. The CNC/20(R)-Rg3 dispersion was diluted with deionized water to 0.01 wt %, and a drop of the diluted dispersion was transferred to a carbon-coated copper grid using a pipet. The grid was then air-dried overnight at room temperature. TEM observations were conducted using a JEOL 2010 STEM instrument (Japan) operated at an accelerating voltage of 200 keV. FTIR Analyses. The CNC/20(R)-Rg3 dispersion was passed through a stirred ultrafiltration cell with filters having a pore size of 50 nm to remove the unloaded 20(R)-Rg3 according to the procedure reported by Akhlaghi et al.26 The filtered nanocomposites and control samples (CNC and 20(R)-Rg3) were dried in a vacuum oven at 40 °C. FTIR absorption spectra were recorded with a Thermo Scientific Nicolet 6700 FTIR spectrometer (USA) from KBr pellets. Specifically, 1 mg of sample was thoroughly ground with 100 mg of anhydrous KBr, and the resultant powder was made into a pellet by pressing at 7 MPa for 1 min. XRD Analyses. XRD patterns of air-dried samples were obtained on a Bruker D8 Advance powder X-ray diffractometer (Germany) operated at an acceleration voltage of 40 kV and using Cu Kα radiation (λ = 1.54 Å). The data were collected with a 2θ scanning range of 10°−80° and a speed of 6 deg/min.27 In Vitro Hydroxyl Radical Scavenging Activity. Two methods were used to generate and determine hydroxyl radical (OH·), the 2deoxyribose oxidation method and the salicylic acid hydroxylation method. These assays of OH· scavenging activity are described below. 2-Deoxyribose Oxidation Method. This is a widely accepted method as described by Heo et al.28 and Chung et al.29 Briefly, a mixture

OH· scavenging (%) = (Ac − A s) × 100/ Ac

(1)

where Ac is the absorbance of the control (deionized water instead of sample solution) and As is the absorbance of the solution in the presence of the sample. Salicylic Acid Hydroxylation Method. In this system, OH· was generated by the Fenton reaction and trapped by salicylic acid, in accordance with the study of Smirnoff and Cumbes.30 The mixture had a total volume of 3 mL and contained 150 mM phosphate buffer (pH 7.4), 0.15 mM Fe3+-EDTA, 2 mM salicylic acid, 0.26 mM ascorbic acid, sample solution (with a 20(R)-Rg3 concentration of 0−15 μg/mL), and 0.6 mM H2O2. The mixture was incubated at 37 °C for 100 min, and the absorbance of the solution at 530 nm was measured against a blank using a UV−vis spectrophotometer (TU-1801PC, Persee). The scavenging rate of OH· was calculated according to eq 1, where Ac is the absorbance of the control (deionized water instead of sample solution), representing the total amount of OH·, and As is the absorbance of the sample. Reaction of Hydrogen Peroxide with 20(R)-Rg3. The lab study was carried out following the procedure of Ruch et al.31 Specifically, a solution of 43 mM H2O2 was prepared in 0.1 mM phosphate buffer (pH 7.4); the concentration of H2O2 was determined from the expression (absorbance at 230 nm) = 0.038[H2O2] + 0.4397. Then 3.4 mL of sample solution (with a 20(R)-Rg3 concentration of 0−15 μg/mL) was added to 0.6 mL of H2O2 solution, and the absorbance at 230 nm was measured after 10 min. The scavenging rate of hydrogen peroxide was calculated using eq 2:

H 2O2 scavenging (%) = (Ac − A s) × 100/Ac

(2)

where Ac is the absorbance of the control (deionized water instead of sample solution), which is correlated to the total amount of hydrogen peroxide, and As is the absorbance of the solution in the presence of the sample, which represents the residual hydrogen peroxide. 7508


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Figure 2. TEM images of (a) water-dispersed 20(R)-Rg3, (b) CNC-free 20(R)-Rg3, (c) CNCs, and (d, e) CNC/20(R)-Rg3 nanocomposites. Scale bars are (a−d) 0.5 μm and (e) 0.2 μm.

Figure 3. 20(R)-Rg3 particle size distributions of (a) CNC/20(R)-Rg3 nanocomposites and (b) CNC-free 20(R)-Rg3. The particle size distribution data were estimated from the TEM images using ImageJ software. Fe2+ Chelating Ability. The Fe2+ chelating activities of the investigated samples were measured using the method of Dairam et al.32 Briefly, 2 mL of sample solution (with a 20(R)-Rg3 concentration from 0 to 15 μg/mL) was added to deionized water to reach a volume of 3.7 mL, followed by the addition of 0.1 mL of FeCl2 (2 mM) to the mixture. The reaction was initiated by the addition of 0.2 mL of ferrozine (5 mM), and then the mixture was shaken vigorously and allowed to react for 20 min. The absorbance of the solution was then measured spectrophotometrically at 562 nm. EDTA was used as a positive control. The percentage of inhibition of ferrozine−Fe2+ complex formation was calculated using eq 3:

Fe2 + chelating activity (%) = (Ac − A s) × 100/Ac

where Ac is the absorbance of the control (deionized water instead of sample solution) and As is the absorbance of the solution in the presence of the sample.

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RESULTS AND DISCUSSION

CNCs as Carriers for 20(R)-Rg3. The concept of preparing CNC/20(R)-Rg3 nanocomposites via antisolvent precipitation33 is shown in Figure 1. After addition of 20(R)-Rg3 ethanol solution to a CNC water suspension, 20(R)-Rg3 is at a supersaturated concentration in the antisolvent (water), which is the main driving force for nucleation. Then nuclei consisting of several molecules are formed. CNCs can provide sites for these nuclei.25 The nuclei then grow into nanoparticles by the addition

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ACS Sustainable Chemistry & Engineering of more 20(R)-Rg3 ethanol solution and finally reach equilibrium.34,35 Morphology and Size Distribution of CNC/20(R)-Rg3 Nanocomposites. Figure 2 shows TEM images of (a) waterdispersed 20(R)-Rg3, (b) CNC-free 20(R)-Rg3, (c) CNCs, and (d, e) CNC/20(R)-Rg3 nanocomposites. It can be observed that the water-dispersed 20(R)-Rg3 sample is in a large aggregate particle (cluster of 20(R)-Rg3 crystals) because of its hydrophobic nature, and its size is as large as several micrometers (Figure 2a). The well-dispersed, rodlike CNCs with a length of about 60−200 nm and a width of about 10−20 nm can be clearly observed in Figure 2c. As shown in Figure 2d,e, the CNC/20(R)Rg3 nanocomposites are the same size as the CNCs, while their 20(R)-Rg3 nanoparticles are 15 ± 7 nm in size, indicating that the CNCs were effective in dispersing hydrophobic 20(R)-Rg3 in the aqueous system because of the high specific surface are. In contrast, Figure 2b shows that the size of CNC-free 20(R)-Rg3 particles is in the range of 20−80 nm. Nanosized drug particles have advantages in terms of bioavailability and exposure, but they are prone to agglomerate.34,36 In this study, CNCs acted as carriers and stabilizers for 20(R)-Rg3 in the aqueous system. As shown in Figure 3, 20(R)Rg3 particles loaded on the surface of CNCs (Figure 3a) had a smaller size than CNC-free 20(R)-Rg3 particles (Figure 3b); in addition, they were more uniform. These results support the conclusion that CNCs are effective carriers for hydrophobic compounds like 20(R)-Rg3. FTIR Analysis of CNC/20(R)-Rg3 Nanocomposites. The FTIR spectrum of CNC/20(R)-Rg3 nanocomposites is shown in Figure 4. It can be seen that the absorptions at 3370, 2945,

coating/encapsulation. The absorption peak related to C−H stretching of 20(R)-Rg3 at 2945 cm−1 disappeared and new peaks pertaining to C−C skeletal stretching and C−H/O−H bending vibrations appeared at 1340−1700 cm−1, indicating that the molecular skeleton of 20(R)-Rg3 was anchored to the CNC.38 XRD Analyses of CNC/20(R)-Rg3 Nanocomposites. As shown in the Figure 5 inset, 20(R)-Rg3 used in this study was a

Figure 5. XRD patterns of 20(R)-Rg3, CNCs, and CNC/20(R)-Rg3 nanocomposites.

crystalline drug with sharp and intense peaks.39 The peaks for CNC were rather evident, with those at 2θ ≈ 15°, 16.5°, 20.5°, and 22.5° being characteristic of cellulose I crystals and those at 2θ ≈ 12.5° and 20° being characteristic of cellulose II crystals.40 However, in the CNC/20(R)-Rg3 nanocomposites, the characteristic peaks of 20(R)-Rg3 at 2θ = 15.7°, 17.5°, and 19° were not detected. These results revealed that the crystallinity of 20(R)-Rg3 nanoparticles was significantly decreased during composite formation. Amorphous 20(R)-Rg3 nanoparticles were formed after the precipitation process. Han et al.15 prepared 20(S)-protopanaxadiol nanoparticles with bovine serum albumin as the carrier via antisolvent precipitation and found that these nanoparticles had an amorphous nature. In another study, Khan and Rathod14 reported that curcumin nanoparticles showed decreased crystallinity and were formed in an amorphous state after the antisolvent-induced precipitation process. In Vitro Hydroxyl Radical Scavenging Activity. Oxidative stress, a state caused by reactive oxygen species (ROS) that exist in all aerobic organisms, is significantly responsible for a variety of inflammatory diseases, including cardiovascular disease, cancer, diabetes, and aging.41 ROS are highly reactive, and hydroxyl radical (OH·) is a typical ROS with a very short in vivo half-life of about 10−9 s and a reaction rate of about 109−1010 M−1·s−1.42 For these reasons, it would be reasonable to use OH· to evaluate the reactivity of antioxidants. Moreover, previous studies on ginseng have made it clear that its medical efficacy is closely related to its protective effects against free radical attack and its function as an antioxidant.43−45 In this work, we used two widely accepted methods, the 2deoxyribose oxidation method and the salicylic acid hydroxylation method, to generate and determine hydroxyl radical (OH·). In the first system, 2-deoxyribose is oxidized by OH·

Figure 4. FTIR spectra of 20(R)-Rg3, CNCs, and CNC/20(R)-Rg3 nanocomposites.

1635, 1390, and 1080 cm−1 are all typical peaks of 20(R)-Rg3, representing O−H stretching, saturated C−H stretching, CO stretching, C−C skeletal vibration, and C−H bending vibrations, respectively, in agreement with those reported by Kim et al.37 For CNCs, the absorption peaks at 3300−3500 (O−H stretching), 2904 (C−H stretching), 1635 (CO stretching), 1058 (C−O stretching), and 900 cm−1 (C−H ring stretching) are rather evident. In the case of the composites, the hydroxyl band (attributed to CNCs) at 3446 cm−1 was decreased and slightly shifted, probably as a result of hydrogen-bond formation between CNCs and 20(R)-Rg3 as well as the impact of surface 7510


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the CNC-free 20(R)-Rg3 and water-dispersed 20(R)-Rg3 samples. For example, under the conditions studied and at a 20(R)-Rg3 concentration of 15 μg/mL, the OH· scavenging was 33.58% for the CNC/20(R)-Rg3 nanocomposites, while it was 11.23% and 2.53% for the CNC-free 20(R)-Rg3 and waterdispersed 20(R)-Rg3 samples, respectively. The OH· scavenging efficiency measured by the salicylic acid hydroxylation method is shown in Figure 7. Similar to the results from the 2-deoxyribose oxidation method, the CNC/20(R)-Rg3 nanocomposite samples showed the strongest OH· scavenging ability among these studied samples. At concentrations ranging from 0 to 15 μg/mL, the hydroxyl radical scavenging activity of CNC/20(R)-Rg3 increased markedly with increasing dosage, reaching 48.62% at 15 μg/mL. Both methods showed that the best OH· scavenging ability was exhibited by the CNC/20(R)-Rg3 samples. Furthermore, our experimental results showed that CNC nanoparticles themselves have very weak hydroxyl radical scavenging activity. At the same concentrations of CNCs as of the CNC/20(R)-Rg3 nanocomposite samples in Figures 6 and 7, the hydroxyl radical scavenging was just 1.25% and 1.56%, respectively (not shown in Figures 6 and 7). This is consistent with the results reported by Kang et al.44 that the hydroxyl radical scavenging activity of ginsenosides is directly related to their steroid aglycone group, while the hydrophilic sugar moiety has a very weak influence on the scavenging activity. Moreover, it seems that water-dispersed 20(R)-Rg3 is not effective in scavenging hydroxyl radicals (a very high concentration of 2 mM was used).44 It was proposed that the mechanism for scavenging of hydroxyl radicals by ginsenoside includes (1) hydrogen atom abstraction and (2) hydroxylation of unsaturated structures, as noted by Lü et al.47 in their study using ginsenoside Rb1. The above results support the notion that the hydroxyl radical scavenging ability of 20(R)-Rg3 is directly correlated to how well it is dispersed: among the samples examined in this study, the CNC/20(R)-Rg3 nanocomposite samples have the best dispersion, as supported by the TEM results and the particle size distribution data (Figures 2 and 3); the CNC-free 20(R)Rg3 sample (Figure 3b) has larger-sized particles than the CNC/ 20(R)-Rg3 sample (Figure 3a); and the particle size for the water-dispersed 20(R)-Rg3 sample is in the range of several micrometers, thus having the worst dispersity and consequently the poorest hydroxyl radical scavenging results (Figures 6 and 7). Therefore, it can be concluded that (1) CNCs are effective carriers/supports for 20(R)-Rg3 to form CNC/20(R)-Rg3 nanocomposites so that hydrophobic 20(R)-Rg3 can be welldispersed in aqueous systems (Figures 2 and 3) and (2) the hydrophilic CNCs will render the CNC/20(R)-Rg3 nanocomposites compatible with the aqueous system, thus increasing the 20(R)-Rg3 bioavailability. The OH· scavenging activities of the samples in the present Fenton system may also be affected by (1) possible reaction of radical scavengers with H2O2 and (2) potential chelation with Fe(II). Therefore, these two aspects were further investigated, and the results are shown in Figures 8 and 9, respectively. As can be seen in Figure 8, all of the test samples showed very weak H2O2 reactivity. For example, at a 20(R)-Rg3 concentration of 15 μg/mL, the H2O2 consumption was 1.25%, 0.42%, and 0% for CNC/20(R)-Rg3, CNC-free 20(R)-Rg3, and water-dispersed 20(R)-Rg3, respectively. Also, compared with that of EDTA, the Fe2+ chelation ability of different forms of Rg3 (CNC/20(R)Rg3 nanocomposites, CNC-free 20(R)-Rg3, and water-dispersed 20(R)-Rg3) was negligible or absent (Figure 9).

generated from the Fenton reaction and degraded to malondialdehyde (MDA). The MDA can react with thiobarbituric acid (TBA) to form a pink chromogen under a low pH condition. The absorbance of the pink chromogen is lower when a lesser amount of OH· is present in the system (e.g., if a OH· scavenger is present).29 In the second system, OH· is generated using a mixture of ascorbate, hydrogen peroxide, and iron. The OH· is effectively trapped by salicylic acid, producing stable hydroxylated salicylic acid derivatives, namely, 2,3- and 2,5dihydroxybenzoic acid and catechol. 46 The amount of hydroxylated salicylic acid formed, which can be quantitatively determined by the UV−vis spectrophotometric method, is decreased in the presence of antioxidants. The results on the OH· radical scavenging rate (with various 20(R)-Rg3 concentrations from 0 to 15 μg/mL) determined by the 2-deoxyribose oxidation method and the salicylic acid hydroxylation method are shown in Figures 6 and 7, respectively.

Figure 6. Hydroxyl radical scavenging activities of CNC/20(R)-Rg3 nanocomposites, CNC-free 20(R)-Rg3, and water-dispersed 20(R)-Rg3 measured by the 2-deoxyribose oxidation method. Experiments were carried out in triplicate, and the averages are reported.

Figure 7. Hydroxyl radical scavenging activities of CNC/20(R)-Rg3 nanocomposites, CNC-free 20(R)-Rg3, and water-dispersed 20(R)-Rg3 measured by the salicylic acid hydroxylation method. Experiments were carried out in triplicate, and the averages are reported.

Samples included CNC/20(R)-Rg3 nanocomposites, CNC-free 20(R)-Rg3, and water-dispersed 20(R)-Rg3. For comparison, the CNC system alone was also studied. As shown in Figure 6, the CNC/20(R)-Rg3 nanocomposite samples exhibited consistently higher OH· scavenging efficiencies in comparison with 7511


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hydrophobic compounds like 20(R)-Rg3, making them compatible with aqueous systems and thus improving their antioxidation capability.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: +1 506 452 6084. Fax: +1 506 453 4767. *E-mail: [email protected]. Tel: +86 15116156013. ORCID

Chunxia Tang: 0000-0001-9396-7189 Yonghao Ni: 0000-0001-6107-6672 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the Canada Research Chairs Program as well as the Natural Science Foundation of Hunan Province (Grant 2015JJ2008) for financial support.

Figure 8. Investigation of H2O2 with different forms of 20(R)-Rg3 (CNC/20(R)-Rg3 nanocomposites, CNC-free 20(R)-Rg3, and waterdispersed 20(R)-Rg3). Experiments were carried out in triplicate, and the averages are reported.

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REFERENCES

(1) Wong, A. S.; Che, C. M.; Leung, K. W. Recent advances in ginseng as cancer therapeutics: a functional and mechanistic overview. Nat. Prod. Rep. 2015, 32 (2), 256−72. (2) Li, K.-K.; Gong, X.-J. A review on the medicinal potential of Panax ginseng saponins in diabetes mellitus. RSC Adv. 2015, 5 (59), 47353− 47366. (3) Ganesan, P.; Ko, H.-M.; Kim, I.-S.; Choi, D.-K. Recent trends of nano bioactive compounds from ginseng for its possible preventive role in chronic disease models. RSC Adv. 2015, 5 (119), 98634−98642. (4) Ru, W.; Wang, D.; Xu, Y.; He, X.; Sun, Y. E.; Qian, L.; Zhou, X.; Qin, Y. Chemical constituents and bioactivities of Panax ginseng (C. A. Mey.). Drug Discoveries Ther. 2015, 9 (1), 23−32. (5) Vermaak, I.; Viljoen, A. M.; Hamman, J. H. Natural products in anti-obesity therapy. Nat. Prod. Rep. 2011, 28 (9), 1493−533. (6) Dai, L.; Liu, K.; Si, C.; Wang, L.; Liu, J.; He, J.; Lei, J. Ginsenoside nanoparticle: a new green drug delivery system. J. Mater. Chem. B 2016, 4 (3), 529−538. (7) Qian, T.; Cai, Z.; Wong, R. N.; Mak, N. K.; Jiang, Z. H. In vivo rat metabolism and pharmacokinetic studies of ginsenoside Rg3. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2005, 816 (1−2), 223− 32. (8) Xu, T. M.; Cui, M. H.; Xin, Y.; Gu, L. P.; Jiang, X.; Su, M. M.; Wang, D. D.; Wang, W. J. Inhibitory effect of ginsenoside Rg3 on ovarian cancer metastasis. Chin. Med. J. 2008, 121 (15), 1394−1397. (9) Xu, T. M.; Xin, Y.; Cui, M. H.; Jiang, X.; Gu, L. P. Inhibitory effect of ginsenoside Rg3 combined with cyclophosphamide on growth and angiogenesis ovarian cancer. Chin. Med. J. 2007, 120 (7), 584−588. (10) Yu, H.; Teng, L.; Meng, Q.; Li, Y.; Sun, X.; Lu, J.; Lee, R. J.; Teng, L. Development of liposomal Ginsenoside Rg3: formulation optimization and evaluation of its anticancer effects. Int. J. Pharm. 2013, 450 (1− 2), 250−2588. (11) Zhang, Q.; Kang, X.; Yang, B.; Wang, J.; Yang, F. Antiangiogenic effect of capecitabine combined with ginsenoside Rg3 on breast cancer in mice. Cancer Biother.Radiopharm. 2008, 23 (5), 647−653. (12) Cheow, W. S.; Kiew, T. Y.; Yang, Y.; Hadinoto, K. Amorphization strategy affects the stability and supersaturation profile of amorphous drug nanoparticles. Mol. Pharmaceutics 2014, 11 (5), 1611−1620. (13) Zhang, Z.-B.; Xie, M.-L.; Kuang, Y.-Y.; Wang, J. X.; Le, Y.; Zeng, X. F.; Chen, J. F. Preparation of amorphous drug nanoparticles by highgravity reactive precipitation technique. Chem. Eng. Process. 2016, 104, 253−261. (14) Khan, W. H.; Rathod, V. K. Process intensification approach for preparation of curcumin nanoparticles via solvent-nonsolvent nanoprecipitation using spinning disc reactor. Chem. Eng. Process. 2014, 80, 1−10.

Figure 9. Comparison of Fe2+ chelation activities of EDTA, CNC/ 20(R)-Rg3 nanocomposites, CNC-free 20(R)-Rg3, and water-dispersed 20(R)-Rg3. Experiments were carried out in triplicate, and the averages are reported.

Overall, these results further demonstrated that the CNC/ 20(R)-Rg3 sample exhibited strong OH· scavenging activity under current conditions, which mainly contributed to the effective carrier/support of CNCs to render 20(R)-Rg3 with good dispersity and compatibility in aqueous systems.

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CONCLUSIONS In this study, we have presented a simple method to improve the compatibility/effectiveness of hydrophobic drug (20(R)-Rg3) in an aqueous system, which was achieved by using cellulose nanocrystals as a carrier. TEM and FTIR results showed that the 20(R)-Rg3 nanoparticles were anchored onto CNCs with a diameter of 8−22 nm. XRD results suggested that 20(R)-Rg3 nanoparticles were in an amorphous state in the nanocomposites. The CNC/20(R)-Rg3 nanocomposites showed a high hydroxyl radical (OH·) scavenging activity of 33.58% and 48.62% (as determined by the 2-deoxyribose oxidation method and the salicylic acid hydroxylation method, respectively), while the CNC-free 20(R)-Rg3, water-dispersed 20(R)-Rg3, and CNC control samples exhibited a gradually decreased OH· scavenging activity under otherwise the same conditions. These results support the conclusion that CNCs can be effective carriers for 7512


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Anchoring 20(R)-Ginsenoside Rg3 onto Cellulose Nanocrystals To

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