Chemical Modification of Ginsenoside on Cell Viability and Cytokine Secretion

Abstract

Background: Rb1 is a ginsenoside steroid glycoside found exclusively in the plant Panax ginseng. In an earlier report, we showed that Rb1 increased cell proliferation and reduced VEGF (vascular endothelial growth factor) secretion by human retinal pigment epithelial (ARPE19) cells.

Objective: In the present study, we hypothesized that chemical modification of Rb1 changes the level of VEGF secretion by ARPE19 cells.

Method: Three derivatives of Rb1 were chemically synthesized by hydrogenation (Rb1-H2), acetylation (Rb1-Acyl), and epoxidation (Rb1-Epoxy). Structural modifications were confirmed by 1H Nuclear Magnetic Resonance (NMR) spectra and Mass Spectrometry (MS). To test the biological activity, chemically modified compounds were added to cell culture media and incubated for 72 hours at a concentration of 250 nM at 37°C. Conditioned media were collected and cells were harvested/ counted after treatment. Viable cell numbers were determined by the trypan blue dye exclusion method and VEGF levels by Enzyme-Linked Immunosorbent Assays (ELISA).

Results: Consistent with the prior report, results of the present study show Rb1 increased cell proliferation and decreased VEGF secretion. Similar to Rb1's effect on cell proliferation, treatment with Rb1-H2, Rb1-Acyl and Rb1-Epoxy resulted in an increase in cell numbers. In contrast to Rb1- induced decrease in VEGF secretion, treatment with Rb1-H2, Rb-Acyl and Rb1-Epoxy resulted in increased VEGF levels.

Conclusion: Chemical modifications of the ginsenoside Rb1 significantly affect the biological activity of VEGF secretion by ARPE19 cells. Additional SAR (Structure Activity Relationship) experiments will be conducted to study the detailed mechanisms by which how specific modifications of Rb1 functional groups alter biological activities.

Fig. (1)

Structure of Ginsenoside, Rb1 showing functional groups with chemical modification.

Fig. (2)

NMR Spectra of Rb1 and chemically modified Rb1. A) Rb1 without chemical modification. B) Rb1-H₂. C) Rb1-Acyl. D) Rb1-Epoxy. See “Result” section for a description on changes in ¹H NMR signals corresponding to hydrogenation, acylation and epoxidation of Rb1.

Fig. (2)

NMR Spectra of Rb1 and chemically modified Rb1. A) Rb1 without chemical modification. B) Rb1-H₂. C) Rb1-Acyl. D) Rb1-Epoxy. See “Result” section for a description on changes in ¹H NMR signals corresponding to hydrogenation, acylation and epoxidation of Rb1.

Fig. (3)

Photomicrographs of ARPE-19 cells in culture. A) confluent cells a Passage 9 after 5 days in culture in a P75 flask, 100× magnification B) Cells at Passage 10 were seeded in a 1 ml well at a seeding density of 20,000 cells per well and incubated for 24 hr in 37C, 5% CO, 200× magnification.

Fig. (4)

Increase in cell proliferation after with Rb1 and Rb1-modified compounds. Data in this figure show the increase in viable cell number between vehicle control (0-Rb1) group and group of cell with Rb1 (or Rb1 modified compounds) treatment at 250 nM for 72 hr. (n = 3 for all data points; ±SEM).

Fig. (5)

hVEGF secretion by ARPE 19 cells is inhibited by Rb1 and enhanced by Rb1H2, Epoxide, or Rb1 Acylated Treatments. A) After 72 hrs, hVEGF secretion from cells treated with Rb1 at 250 nM decreased to 382 pg/mL (compared to 600 pg/mL in vehicle control of 0-Rb1) whereas cells treated with Rb1-H₂ at 250 nM increased to 659 pg/mL. Similarly, cell treated with Rb1-Acyl increased to 1157 pg/mL and Rb1-Epoxy, increased to 657 pg/mL. B) Compared to 0.05 pg/cell/mL in vehicle control of 0-Rb1, hVEGF decreased to 0.02 pg/cell in respond to Rb1. In contrast, Rb1-H₂ treatment increased hVEGF secretion to 0.21 pg/cell/mL, Rb1-H₂, to 0.33 pg/cell by Rb1-Acyl, and to 0.09 pg/cell by Rb1-Epoxy treatment (n = 3 for all data points; ±SEM).