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J Ginseng Res
2011 Jun 01;352:209-18. doi: 10.5142/jgr.2011.35.2.209.
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A simple method for the preparation of crude gintonin from ginseng root, stem, and leaf.
Pyo MK
,
Choi SH
,
Shin TJ
,
Hwang SH
,
Lee BH
,
Kang J
,
Kim HJ
,
Lee SH
,
Nah SY
.
Abstract
Ginseng has been used as a general tonic agent to invigorate the human body as an adaptogenic agent. In a previous report, we have shown that ginseng contains a novel glycolipoprotein called gintonin. The main function of gintonin is to transiently enhance intracellular free Ca(2+) [Ca(2+)]i levels in animal cells. The previous method for gintonin isolation included multiple steps using organic solvents. In the present report, we developed a simple method for the preparation of crude gintonin from ginseng root as well as stem and leaf, which produced a higher yield of gintonin than the previous one. The yield of gintonin was 0.20%, 0.29%, and 0.81% from ginseng root, stem, and leaf, respectively. The apparent molecular weight of gintonin isolated from stem and leaf through sodium dodecyl sulfate polyacrylamide gel electrophoresis was almost same as that from root but the compositions of amino acids, carbohydrates or lipids differed slightly between them. We also examined the effects of crude gintonin from ginseng root, stem, and leaf on endogenous Ca(2+)-activated Cl- channel (CaCC) activity of Xenopus oocytes through mobilization of [Ca(2+)]i. We found that the order of potency for the activation of CaCC was ginseng root > stem > leaf. The ED50 was 1.4±1.4, 4.5±5.9, and 3.9±1.1 μg/mL for root, stem and leaf, respectively. In the present study, we demonstrated for the first time that in addition to ginseng root, ginseng stem and leaf also contain gintonin. Gintonin can be prepared from a simple method with higher yield of gintonin from ginseng root, stem, and leaf. Finally, these results demonstrate the possibility that ginseng stem and leaf could also be utilized for ginstonin preparation after a simple procedure, rather than being discarded.
Fig. 1. Diagram for simple preparation of crude gintonin 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 butanol fraction and the bound component obtained after elution with NaCl in Tris-HCl (pH 8.2) induced endogenous CaCC activation in Xenopus oocytes, whereas H2O layer and unbound component had no effect. The amount of each fraction used to test CaCC activity was 10 μg/mL. Inward currents were recorded at â80 mV holding potential. CGSF, crude ginseng total saponin fraction; fr., fraction; GT, gintonin.
Fig. 2. Diagram for preparation of gintonin fraction from Panax ginseng stem. (A) A complete diagram for crude gintonin preparation from ginseng stem. (B) The representative Ca2+-activated Cl- channel (CaCC) current traces are representative of one obtained from each crude gintonin preparation step. Treatment with butanol fraction and the bound component obtained after elution with NaCl in Tris-HCl (pH 8.2) induced endogenous CaCC activation in Xenopus oocytes, whereas H2O layer and the unbound component had no effect. The amount of each fraction used to test CaCC activity was 10 μg/mL. Inward currents were recorded at â80 mV holding potential. CGSF, crude ginseng total saponin fraction; fr., fraction; GT, gintonin.
Fig. 3. Diagram for preparation of gintonin fraction from Panax ginseng leaf. (A) A complete diagram for crude gintonin preparation from ginseng leaf. (B) The representative Ca2+-activated Cl- channel (CaCC) current traces are representative of one obtained from each gintonin preparation step. Treatment with butanol fraction and the bound component obtained after elution with NaCl in Tris-HCl (pH 8.2) induced endogenous CaCC activation in Xenopus oocytes, whereas H2O layer and the unbound component had no effect. The amount of each fraction used to test CaCC activity was 10 μg/mL. Inward currents were recorded at â80 mV holding potential. CGSF, crude ginseng total saponin fraction; fr., fraction; GT, gintonin.
Fig. 4. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of each gintonin prepared from ginseng root, stem, and leaf. (A) SDS-PAGE of the crude gintonins obtained from anion exchange chromatography. Coomassie Brilliant blue staining was used to stain protein moieties of gintonins. Crude gintonin prepared from ginseng root (R), stem (S), and leaf (L) in SDSâPAGE showed that the apparent molecular weight of gintonin is about 13 kDa. (B) Periodic acid-Schiff staining demonstrated that crude gintonins contain carbohydrate moiety.
Fig. 5. Elution patterns of gintonin prepared from ginseng root, stem, or leaf by gel chromatography. Gel filtration chromatograms on Superdex 75 column with phosphate buffer saline (pH 7.2) of crude gintonin prepared from ginseng root (A), stem (B) or leaf (C). Analysis through gel filtration chromatograms showed that the main peaks of crude gintonin prepared from root, stem, or leaf are almost same but ginseng stem and leaf showed an additional peak after the main peak. We found that the main peak was active for the activation of Ca2+-activated Cl- channel.
Fig. 6. Analysis of carbohydrate components of crude gintonin prepared from ginseng root, stem, or leaf by HPAEC-PAD chromatograms. (A-C) HPAEC-PAD chromatograms show that gintonins are composed of two different kinds of neutral sugars:glucose and galactose, one amino sugar, galactosamine, and one acid sugar, glucuronic acid. (A) Std stands for standard carbohydrates used; 1. L-fucose, 2. L-rhamnose, 3. D-galactosamine, 4. D-glucosamine, 5. D-galactose, 6. D-glucose, 7. D-mannose, 8, D-xylose. (B) Acid sugar; 1. Galacturonic acid, 2. Glucuronic acid.
Fig. 7. GC-MS spectral analysis of lipid components of crude gintonin prepared from ginseng root, stem, or leaf. Acid hydrolyzed gintonin was partitioned between distilled water and n-butanol (n-BuOH). The n-BuOH layer, after concentration, was further partitioned between distilled water and n-hexane. The n-hexane layer was subjected to GC-MS with a DB5-MS capillary column. Several peaks were present in the hexane fraction of gintonin and were identified as palmitic acid, stearic acid, oleic acid, linoleic acid ester or free form. The 22.11 peak of palmitic acid (C16:0) and 23.59 peak of linoleic acid (C18:2) were dominant fatty acid in gintonins.
Fig. 8. Crude gintonin prepared from ginseng root, stem, or leaf activates Ca2+-activated Cl- channel (CaCC) with concentration-dependent manner. Crude gintonin prepared from ginseng root, stem, and leaf increased inward CaCC currents in a concentration-dependent manner. The order of gintonin potency for CaCC activation was root > stem > leaf. Data represent means±SEM (n=7-8).
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