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International Journal of Nanomedicine Volume 10, 2015 - Issue
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Original Research

Aqueous extract of Rabdosia rubescens leaves: forming nanoparticles, targeting P-selectin, and inhibiting thrombosis

Yuji Wang1 Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, Engineering Research Center of Endogenous Prophylactic of Ministry of Education of China, Beijing Laboratory of Biomedical Materials, College of Pharmaceutical Sciences, Capital Medical University, Beijing, People’s Republic of China
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Jingcheng Tang1 Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, Engineering Research Center of Endogenous Prophylactic of Ministry of Education of China, Beijing Laboratory of Biomedical Materials, College of Pharmaceutical Sciences, Capital Medical University, Beijing, People’s Republic of China
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Haimei Zhu1 Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, Engineering Research Center of Endogenous Prophylactic of Ministry of Education of China, Beijing Laboratory of Biomedical Materials, College of Pharmaceutical Sciences, Capital Medical University, Beijing, People’s Republic of China
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Xueyun Jiang1 Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, Engineering Research Center of Endogenous Prophylactic of Ministry of Education of China, Beijing Laboratory of Biomedical Materials, College of Pharmaceutical Sciences, Capital Medical University, Beijing, People’s Republic of China
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Jiawang Liu1 Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, Engineering Research Center of Endogenous Prophylactic of Ministry of Education of China, Beijing Laboratory of Biomedical Materials, College of Pharmaceutical Sciences, Capital Medical University, Beijing, People’s Republic of China
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Wenyun Xu1 Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, Engineering Research Center of Endogenous Prophylactic of Ministry of Education of China, Beijing Laboratory of Biomedical Materials, College of Pharmaceutical Sciences, Capital Medical University, Beijing, People’s Republic of China
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Haiping Ma1 Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, Engineering Research Center of Endogenous Prophylactic of Ministry of Education of China, Beijing Laboratory of Biomedical Materials, College of Pharmaceutical Sciences, Capital Medical University, Beijing, People’s Republic of China
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Qiqi Feng1 Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, Engineering Research Center of Endogenous Prophylactic of Ministry of Education of China, Beijing Laboratory of Biomedical Materials, College of Pharmaceutical Sciences, Capital Medical University, Beijing, People’s Republic of China
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Jianhui Wu1 Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, Engineering Research Center of Endogenous Prophylactic of Ministry of Education of China, Beijing Laboratory of Biomedical Materials, College of Pharmaceutical Sciences, Capital Medical University, Beijing, People’s Republic of China
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Ming Zhao1 Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, Engineering Research Center of Endogenous Prophylactic of Ministry of Education of China, Beijing Laboratory of Biomedical Materials, College of Pharmaceutical Sciences, Capital Medical University, Beijing, People’s Republic of China;2 Faculty of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung, TaiwanCorrespondence[email protected] [email protected]
&
Shiqi Peng1 Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, Engineering Research Center of Endogenous Prophylactic of Ministry of Education of China, Beijing Laboratory of Biomedical Materials, College of Pharmaceutical Sciences, Capital Medical University, Beijing, People’s Republic of ChinaCorrespondence[email protected] [email protected]
 show all
Pages 6905-6918 | Published online: 04 Nov 2015

Figures & data

Figure 1 IC50s of AERL inhibiting platelet aggregation induced by PAF, ADP, AA, and TH.

Notes: Aspirin was the positive control. n=6.

Abbreviations: AA, arachidonic acid; ADP, adenosine diphosphate; AERL, aqueous extract of Rabdosia rubescens leaves; PAF, platelet-activating factor; SD, standard deviation; TH, thrombin; IC50, half maximal inhibitory concentration.

Figure 1 IC50s of AERL inhibiting platelet aggregation induced by PAF, ADP, AA, and TH.Notes: Aspirin was the positive control. n=6.Abbreviations: AA, arachidonic acid; ADP, adenosine diphosphate; AERL, aqueous extract of Rabdosia rubescens leaves; PAF, platelet-activating factor; SD, standard deviation; TH, thrombin; IC50, half maximal inhibitory concentration.

Figure 2 Thrombus weights of rats orally receiving AERL and rosmarinic acid.

Notes: (A) Thrombus weights of rats orally receiving AERL. (B) Thrombus weights of rats orally receiving rosmarinic acid. Aspirin was the positive control. n=10.

Abbreviations: AERL, aqueous extract of Rabdosia rubescens leaves; NS, normal saline; SD, standard deviation.

Figure 2 Thrombus weights of rats orally receiving AERL and rosmarinic acid.Notes: (A) Thrombus weights of rats orally receiving AERL. (B) Thrombus weights of rats orally receiving rosmarinic acid. Aspirin was the positive control. n=10.Abbreviations: AERL, aqueous extract of Rabdosia rubescens leaves; NS, normal saline; SD, standard deviation.

Figure 3 Effect of AERL on the in vitro release of sP-selectin from platelets.

Notes: IQCA was the reference compound. n=4.

Abbreviations: AERL, aqueous extract of Rabdosia rubescens leaves; IQCA, 3S-tetrahydroisoquinoline-3-carboxylic acid; NS, normal saline; SD, standard deviation; sP-selectin, soluble P-selectin.

Figure 3 Effect of AERL on the in vitro release of sP-selectin from platelets.Notes: IQCA was the reference compound. n=4.Abbreviations: AERL, aqueous extract of Rabdosia rubescens leaves; IQCA, 3S-tetrahydroisoquinoline-3-carboxylic acid; NS, normal saline; SD, standard deviation; sP-selectin, soluble P-selectin.

Figure 4 TEM and AFM images showing the nanostructure of the lyophilized solids of AERL in water and rat plasma.

Notes: (A) TEM image of nanoparticles of AERL in water, 4.5–63.6 nm in diameter (concentration, 1.0 mg/mL). (B) TEM image of nanoparticles of AERL in water, 32.9–180.8 nm in diameter (concentration, 0.5 mg/mL). (C) TEM image of nanoparticles of AERL in water, 38.9–227.8 nm in diameter (concentration, 0.25 mg/mL). (D) TEM image of nanoparticles of AERL in water, 45.7–234.3 nm in diameter (concentration, 0.12 mg/mL). (E) AFM image of nanoparticles of AERL in rat plasma, 53–159 nm in diameter (concentration, 0.5 mg/mL). Two red arrowheads indicate the diameter of the nanoparticle. (F) AFM image of nanostructure of rat plasma alone.

Abbreviations: AERL, aqueous extract of Rabdosia rubescens leaves; AFM, atomic force microscopy; TEM, transmission electron microscopy.

Figure 4 TEM and AFM images showing the nanostructure of the lyophilized solids of AERL in water and rat plasma.Notes: (A) TEM image of nanoparticles of AERL in water, 4.5–63.6 nm in diameter (concentration, 1.0 mg/mL). (B) TEM image of nanoparticles of AERL in water, 32.9–180.8 nm in diameter (concentration, 0.5 mg/mL). (C) TEM image of nanoparticles of AERL in water, 38.9–227.8 nm in diameter (concentration, 0.25 mg/mL). (D) TEM image of nanoparticles of AERL in water, 45.7–234.3 nm in diameter (concentration, 0.12 mg/mL). (E) AFM image of nanoparticles of AERL in rat plasma, 53–159 nm in diameter (concentration, 0.5 mg/mL). Two red arrowheads indicate the diameter of the nanoparticle. (F) AFM image of nanostructure of rat plasma alone.Abbreviations: AERL, aqueous extract of Rabdosia rubescens leaves; AFM, atomic force microscopy; TEM, transmission electron microscopy.

Figure 5 HPLC–PDA 3D chromatogram of AERL and docking feature of rosmarinic acid in the active site of P-selectin.

Notes: (A) Docking feature of rosmarinic acid in the active site of P-selectin. (B) HPLC–PDA 3D chromatogram of AERL (20.34% in whole extract, calculated from HPLC analysis, 254 nm).

Abbreviations: AERL, aqueous extract of Rabdosia rubescens leaves; AU, absorbance unit; HPLC, high-performance liquid chromatography; PDA, photodiode array detector; min, minutes.

Figure 5 HPLC–PDA 3D chromatogram of AERL and docking feature of rosmarinic acid in the active site of P-selectin.Notes: (A) Docking feature of rosmarinic acid in the active site of P-selectin. (B) HPLC–PDA 3D chromatogram of AERL (20.34% in whole extract, calculated from HPLC analysis, 254 nm).Abbreviations: AERL, aqueous extract of Rabdosia rubescens leaves; AU, absorbance unit; HPLC, high-performance liquid chromatography; PDA, photodiode array detector; min, minutes.

Figure 6 Depression of P-selectin expression by rosmarinic acid.

Notes: (A) Unlabeled platelets (background). (B) PE-anti-CD62P-labeled platelets. (C) PE-anti-CD62P-labeled and AA-activated platelets without rosmarinic acid. (D) PE-anti-CD62P-labeled and AA-activated platelets with 1 μM of rosmarinic acid. P2, a high fluorescence intensity area in which there is no fluorescence in NS group.

Abbreviations: AA, arachidonic acid; NS, normal saline; PE, phycoerythrin.

Figure 6 Depression of P-selectin expression by rosmarinic acid.Notes: (A) Unlabeled platelets (background). (B) PE-anti-CD62P-labeled platelets. (C) PE-anti-CD62P-labeled and AA-activated platelets without rosmarinic acid. (D) PE-anti-CD62P-labeled and AA-activated platelets with 1 μM of rosmarinic acid. P2, a high fluorescence intensity area in which there is no fluorescence in NS group.Abbreviations: AA, arachidonic acid; NS, normal saline; PE, phycoerythrin.

Figure S1 1H NMR of rosmarinic acid.

Notes: (A) 1H NMR of rosmarinic acid; (B) enlarged image of 1H NMR of rosmarinic acid. 1H NMR (500 MHz, MeOD): δ/ppm =7.54 (d, J=15.5 Hz, 1H), 7.03 (d, J=2 Hz, 1H), 6.94 (dd, J=8.0 Hz, 2.0 Hz, 1H), 6.77 (d, J=8.0 Hz, 1H), 6.74 (d, J=2.0 Hz, 1H), 6.69 (d, J=8.5 Hz, 1H), 6.60 (dd, J=8.0 Hz, 2.0 Hz, 1H), 6.26 (d, J=15.5 Hz, 1H), 5.18 (d, J=8.5 Hz, 4.5 Hz, 1H), 3.09 (dd, J=14.5 Hz, 4.5 Hz, 1H), 3.00 (dd, J=14.5 Hz, 8.5 Hz, 1H).

Abbreviations: NMR, nuclear magnetic resonace; H, hydrogen.

Figure S1 1H NMR of rosmarinic acid.Notes: (A) 1H NMR of rosmarinic acid; (B) enlarged image of 1H NMR of rosmarinic acid. 1H NMR (500 MHz, MeOD): δ/ppm =7.54 (d, J=15.5 Hz, 1H), 7.03 (d, J=2 Hz, 1H), 6.94 (dd, J=8.0 Hz, 2.0 Hz, 1H), 6.77 (d, J=8.0 Hz, 1H), 6.74 (d, J=2.0 Hz, 1H), 6.69 (d, J=8.5 Hz, 1H), 6.60 (dd, J=8.0 Hz, 2.0 Hz, 1H), 6.26 (d, J=15.5 Hz, 1H), 5.18 (d, J=8.5 Hz, 4.5 Hz, 1H), 3.09 (dd, J=14.5 Hz, 4.5 Hz, 1H), 3.00 (dd, J=14.5 Hz, 8.5 Hz, 1H).Abbreviations: NMR, nuclear magnetic resonace; H, hydrogen.

Figure S2 13C NMR of rosmarinic acid.

Notes: 13C NMR (125 MHz, MeOD): δ/ppm =172.04 (C11), 167.05 (C9), 148.33 (C3), 146.33 (C2), 145.41 (C17), 144.77 (C7), 143.89 (C16), 127.87 (C13), 126.28 (C6), 121.74 (C5), 120.41 (C14), 116.19 (C4), 115.11 (C18), 114.91 (C8), 113.85 (C15), 113.03 (C1), 73.19 (C10), 36.52 (C12).

Abbreviations: NMR, nuclear magnetic resonace; C, carbon.

Figure S2 13C NMR of rosmarinic acid.Notes: 13C NMR (125 MHz, MeOD): δ/ppm =172.04 (C11), 167.05 (C9), 148.33 (C3), 146.33 (C2), 145.41 (C17), 144.77 (C7), 143.89 (C16), 127.87 (C13), 126.28 (C6), 121.74 (C5), 120.41 (C14), 116.19 (C4), 115.11 (C18), 114.91 (C8), 113.85 (C15), 113.03 (C1), 73.19 (C10), 36.52 (C12).Abbreviations: NMR, nuclear magnetic resonace; C, carbon.

Figure S3 Faraday–Tyndall effect, zeta potential, and size of AERL in pH 7.0 and pH 2.0 water.

Notes: (A) 0.5 mg/mL of AERL. (B) 15 mg/mL of AERL. (C) Distilled water. (D) Size (a) and zeta potential (b) of AERL in pH 7.0 water (0.5 mg/mL). (E) Size (a) and zeta potential (b) of AERL in pH 2.0 water (0.5 mg/mL).

Abbreviations: AERL, aqueous extract of Rabdosia rubescens leaves; SD, standard deviation.

Figure S3 Faraday–Tyndall effect, zeta potential, and size of AERL in pH 7.0 and pH 2.0 water.Notes: (A) 0.5 mg/mL of AERL. (B) 15 mg/mL of AERL. (C) Distilled water. (D) Size (a) and zeta potential (b) of AERL in pH 7.0 water (0.5 mg/mL). (E) Size (a) and zeta potential (b) of AERL in pH 2.0 water (0.5 mg/mL).Abbreviations: AERL, aqueous extract of Rabdosia rubescens leaves; SD, standard deviation.

Table S1 Thirty peaks and (−)ESI-MS/MS spectrum assigned structures

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