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Lee, Kim, and Kim: Maximization of Extracted Condition of Pro-angiogenic Components in Citrus unshiu Peels using Dimethyl Sulfoxide
Original Article

Natural Product Sciences 2016;22(4):287-292.

Published online: 20 January 2016

DOI: https://doi.org/10.20307/nps.2016.22.4.287

Maximization of Extracted Condition of Pro-angiogenic Components in Citrus unshiu Peels using Dimethyl Sulfoxide

Jungwhoi Lee1, ∗, Myungseung Kim1, Jae Hoon Kim1, 2, ∗

1College of Applied Life Science, SARI, Jeju National University, Jeju-do 690–756, Korea

2Subtropical/tropical Organism Gene Bank, Jeju National University, Jeju 63243, Republic of Korea

∗Author for correspondence Lee Jungwhoi, Department of Applied Life Science, Jeju National University, 102 Jejudaehak-ro, Jeju-si, Jeju-do 63243, Republic of Korea Tel: +82-64-729-8556; E-mail: sdjd1108@kaist.ac.kr Kim Jae Hoon, Department of Applied Life Science, Jeju National University, 102 Jejudaehak-ro, Jeju-si, Jeju-do 63243, Republic of Korea Tel: +82-64-729-8556; E-mail: kimjh@jejunu.ac.kr

Received 19 August 2016       Revised 26 September 2016       Accepted 27 September 2016

Copyright © 2016 The Korean Society of Pharmacognosy

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Aqueous extraction of Citrus unshiu peels (AECUP) is mainly comprised with pro-angiogenichesperidin and narirutin. In this study, we report approaches to increasing the yields of extracted hesperidin and narirutinfrom Citrus unshiu peels using proper solvents. Significantly improved yields of both compounds were obtained using methanol and dimethyl sulfoxide (DMSO) compared to acetonitrile, ethyl acetate, ethanol, and isopropyl alcohol. Especially, effect of DMSO was by far the better of the two solvents in extraction of hesperidin. In addition, the DMSO extracted hesperidin significantly induced the pro-angiogenic effects of human umbilical vein endothelial cells (HUVECs) and markedly up-regulated phosphorylation of the ERK1/2 signaling pathway. These results demonstrate that pro-angiogenic inducer; hesperidin and narirutin can be simply, easily, and effectively extracted from Citrus unshiu peels.

Keywords: Angiogenesis, Citrus unshiu peels, Hesperidin, Narirutin, DMSO, MeOH

References

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Fig. 1.
Chemical structures of hesperidin and narirutin.
nps-22-287f1.tif
Fig. 2.
Characterization of constituent elements using different solvents. Citrus Unshiu peel was extracted with sodium acetate buffer (A), MeOH (B), or DMSO (C). The extracts were dissolved in methanol and were injected into a HPLC system at a column flow rate of 1 mL/min and analyzed (two major peaks of narirutin and hesperidin were designated a and b, respectively).
nps-22-287f2.tif
Fig. 3.
Quantification of extracted narirutin and hesperidin.(A) LOD and LOQ values of standard hesperidin and narirutin were calculated using HPLC chromatograms, respectively. (B) Extracted hesperidin and narirutin were quantified using HPLC analysis (data represent the percentage ± SD and are representative of three individual experiments, ∗p < 0.05).
nps-22-287f3.tif
Fig. 4.
Purification of DMSO extracted hesperidin from Citrus Unshiu peel. (A) Extracted hesperidin was purified using a prep-LC system (two major peaks of narirutin and hesperidin were designated a and b, respectively). (B) Extracted hesperidin and standard hesperidin were dissolved in methanol and injected into the HPLC system at a column flow rate of 1 mL/min.
nps-22-287f4.tif
Fig. 5.
Pro-angiogenic effects of DMSO extracted hesperidin.(A) HUVECs were incubated with varying concentrations (0, 5, 10, and 20 µM) of DMSO extracted hesperidin for 72 h (data represent the percentage ± SD and are representative of three individual experiments, ∗p < 0.05, ∗∗p < 0.01). (B) HUVECs were incubated in the presence or absence of DMSO extracted hesperidin for 6 h using a Transwell migration assay (data represent the percentage ± SD and are representative of three individual experiments, ∗∗p < 0.01, ∗∗∗p <0.001). Representative image of a Transwell migration assay (scale bar=50µm). (C) Left, Capillary like structure (CLS) formation of HUVECs assayed after 16 h of incubation of cells in the presence or absence of DMSO extracted hesperidin(data represent the percentage ± SD and are representative of two individual experiments, ∗p < 0.05, ∗∗∗p < 0.001). Right, Representative image of tube-formation assay (scale bar = 50 µm).(D) Left, HUVECs were pre-incubated with different dose of RGD-peptide (0, 0.5, and 1 µM) for 30 min before DMSO extracted hesperidin (10 µM) were added for 60 min and cell lysates were subjected to immunoblot analysis using antibodies for p-ERK1/2 and ERK1. GAPDH was used as a loading control. Right, relative pixel intensities for p-ERK1/2 were measured using p-ERK1/2/GAPDH.
nps-22-287f5.tif
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