Choi SH et al. (2011), An edible gintonin preparation from ginseng.

XB-ART-47137

J Ginseng Res 2011 Nov 01;354:471-8. doi: 10.5142/jgr.2011.35.4.471.

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An edible gintonin preparation from ginseng.

Choi SH , Shin TJ , Lee BH , Hwang SH , Kang J , Kim HJ , Park CW , Nah SY .

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Ginseng, the root of Panax ginseng, is one of the oldest herbal medicines. It has a variety of physiological and pharmacological effects. Recently, we isolated a subset of glycolipoproteins that we designated gintonin, and demonstrated that it induced transient change in intracellular calcium concentration ([Ca(2+)]i) in cells via G-protein-coupled receptor signaling pathway(s). The previous method for gintonin isolation included multiple steps using methanol, butanol, and other organic solvents. In the present study, we developed a much simple method for the preparation of gintonin from ginseng root using 80% ethanol extraction. The extracted fraction was designated edible gintonin. This method produced a high yield of gintonin (0.20%). The chemical characteristics of gintonin such as molecular weight and the composition of the extract product were almost identical as the gintonin prepared using the previous extraction regimen involving various organic solvents. We also examined the physiological effects of edible gintonin on endogenous Ca(2+)-activated Cl(-) channel activity of Xenopus oocytes. The 50% effective dose was 1.03±0.3 μg/mL. Finally, since gintonin preparation through ethanol extraction is easily reproducible, gintonin could be commercially applied for ginseng-derived functional health food and/or drug following the confirmations of in vitro and in vivo physiological and pharmacological effects of gintonin.

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Species referenced: Xenopus
Genes referenced: clca1.3 gnas sult2a1

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	Fig. 1. Diagram for an edible gintonin (E-GT) preparation from Panax ginseng root. (A) A complete diagram for crude gintonin preparation from ginseng root. (B) The representative Ca2+-activated Cl- channel (CaCC) current traces are representative of one obtained from each preparation step. Treatment with ethanol extraction (EtOH ext) and the bound component obtained after elution with NaCl in Tris-HCl (pH 8.2) induced endogenous CaCC activation in Xenopus oocytes, whereas unbound component had no effect. The amount of each fraction used to test CaCC activity was 1 or 100 μg/mL. Inward currents were recorded at –80 mV holding potential.
	Fig. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of each gintonin prepared from ginseng root. SDS-PAGE of edible gintonin obtained from anion exchange chromatography as shown in Fig. 1. Coomassie Brilliant Blue staining was used to stain protein moieties of gintonin. Crude gintonin prepared from ginseng root in SDS-PAGE showed that apparent molecular weight of gintonin was about 13 kDa.
	Fig. 3. Elution patterns of gintonin prepared from ginseng root by gel chromatography. Gel filtration chromatograms on Superdex 75 column with phosphate buffered saline (pH 7.2) of edible gintonin prepared from ginseng root. Analysis through gel filtration chromatograms showed one main peak and other minor peaks. The main peak was active for the activation of Ca2+-activated Cl- channel.
	Fig. 4. Analysis of carbohydrate components of crude gintonin prepared from ginseng root by high performance anion exchange chromatography-pulsed ampherometric detection (HPAEC-PAD) chromatograms. HPAEC-PAD chromatograms revealed that gintonin to be mainly composed of three different kinds of neutral sugars: glucose, arabinose, galactose, and fucose and one amino sugar, glucosamine. (A) Std stands for standard carbohydrates used; 1. L-fucose, 2. L-rhamnose, 3. D-galactosamine, 4. D-arabinose, 5. D-glucosamine, 6. D-galactose, 7. D-glucose, 8. D-mannose, 9. D-xylose, 10. D-fructose. (B) HPAEC-PAD chromatograms of gintonin.
	Fig. 5. GC-MS spectral analysis of lipid components of edible gintonin prepared from ginseng root. Acid hydrolyzed gintonin were partitioned between distilled water and n-butanol. The n-butanol layer, after concentration, was further partitioned between distilled water and n-hexane. The n-hexane layer was subjected to gas chromatography-mass spectrometry with a DB5-MS capillary column. Several major peaks were present in hexane fraction of gintonin and were identified. The 20.94 peak was nonanedioic acid that is known to function to defense after infection in plants. The 22.04 peak of palmitic acid (C16:0) and 23.5 peak of linoleic acid (C18:2) were present as dominant fatty acids or their ester forms.
	Fig. 6. Edible gintonin prepared from ginseng root activates Ca2+-activated Cl- channel in a concentration-dependent manner in Xenopus oocytes. Experimental details are described in Materials and Methods. Data represent mean±SEM (n=7 or 8).

References [+] :

Berridge, Calcium signalling: dynamics, homeostasis and remodelling. 2003, Pubmed