Characterization, purification of Poncirin from edible citrus Ougan (Citrus reticulate cv. Suavissima) and its growth inhibitory effect on human gastric cancer cells SGC-7901

Abstract

Poncirin is a bitter flavanone glycoside with various biological activities. Poncirin was isolated from four different tissues (flavedo, albedo, segment membrane, and juice sac) of Ougan fruit (Citrus reticulate cv. Suavissima). The highest content of poncirin was found in the albedo of Ougan fruit (1.37 mg/g DW). High speed counter-current chromatography (HSCCC) combined with D101 resin chromatography was utilized for the separation and purification of poncirin from the albedo of Ougan fruit. After this two-step purification, poncirin purity increased from 0.14% to 96.56%. The chemical structure of the purified poncirin was identified by both HPLC-PDA and LC-MS. Poncirin showed a significant in vitro inhibitory effect on the growth of the human gastric cancer cells, SGC-7901, in a dose-dependent manner. Thus, poncirin from Ougan fruit, may be beneficial for gastric cancer prevention. The purification method demonstrated here will be useful for further studies on the pharmacological mechanism of poncirin activity, as well as for guiding the consumption of Ougan fruit.

Figure 1

Molecular structure of poncirin.

Figure 2

Dynamic leakage curves (A), dynamic desorption curves of poncirin by gradient elution (B), and isocratic elution (C) on column packed with D101 resin. Initial poncirin concentration in feed solution: 0.05 mg/mL; isocratic elution solution: 60% (v/v) ethanol; both adsorption and desorption flow rate: 1.0 mL/min.

Figure 3

HSCCC separation chromatogram of poncirin. Solvent system: chloroform–methanol–n-butanol–water (4:3:0.5:2, v/v/v/v); flow rate (mobile phase): 2.0 mL/min; revolution speed: 850 rpm; sample size: 60 mg; detection wavelength: 280 nm. Fraction I was collected from tube # 8-27.

Figure 4

HPLC chromatogram and UV absorption spectrum of poncirin from crude extract before (A) and after (B) treatment with D101 resin and the purified product (C) from fraction I obtained by HSCCC. Peak I represents poncirin. (D) and (E), LC-MS chromatogram of HSCCC purified poncirin product.

Figure 4

HPLC chromatogram and UV absorption spectrum of poncirin from crude extract before (A) and after (B) treatment with D101 resin and the purified product (C) from fraction I obtained by HSCCC. Peak I represents poncirin. (D) and (E), LC-MS chromatogram of HSCCC purified poncirin product.

Figure 5

Inhibitory activity of purified poncirin on the growth of gastric cancer SGC-7901 cells. 5-Fu (positive control), 5-fluorouracil; Results are presented as mean ± S.D. (n = 9).