Food Additives & Contaminants
ISSN: 0265-203X (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/tfac19
Cytoprotective effects of Gypsophila saponins towards isolated rat hepatocytes F. Braut‐Boucher , S. Achard‐Ellouk , D. Pauthe‐Dayde , M. Henry & H. Hoellinger To cite this article: F. Braut‐Boucher , S. Achard‐Ellouk , D. Pauthe‐Dayde , M. Henry & H. Hoellinger (1990) Cytoprotective effects of Gypsophila saponins towards isolated rat hepatocytes, Food Additives & Contaminants, 7:S1, S127-S130, DOI: 10.1080/02652039009373863 To link to this article: http://dx.doi.org/10.1080/02652039009373863
Published online: 10 Jan 2009.
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Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tfac19 Download by: [University of Florida]
Date: 06 November 2015, At: 01:58
FOOD ADDITIVES AND CONTAMINANTS, 1990, VOL. 7, SUPPLEMENT NO. 1, S127-S130
Cytoprotective effects of Gypsophila saponins towards isolated rat hepatocytes F. BRAUT-BOUCHER, S. ACHARD-ELLOUK, D. PAUTHE-DAYDE†, M. HENRY† and H. HOELLINGER
Downloaded by [University of Florida] at 01:58 06 November 2015
UDC CNRS-INSERM (UA 400), Faculté de Médecine, Université René Descartes, Paris, France, and † Laboratoire de Botanique, Faculté de Pharmacie, Toulouse, France Saponins are glycosides widely distributed in the plant kingdom and are found in many foods. The hepatoprotective potential of glucuronogypsogenin (GG) and gypsoside (GY) towards isolated rat hepatocytes treated by three toxic models used at sub-lethal doses: galactosamine (5 x 10-3 M), CCl4 (5 x 10-4 M) and erythromycin (5 x 10-4 M) was investigated. Two schedules were carried out corresponding to curative or preventive treatment. No protection was observed on hepatocytes treated with GY before or after addition of the toxicants. In contrast, a protective action was detected when hepatocytes were pretreated with GG (5 X 10-5 M) as probe, by the normalisation of LDH leakage and ATP content. It depends on the toxicant: the cytoprotective spectrum is 5 x 10-5 to 5 x 10-7 M with galactosamine; 5 x 10-5 to 5 x 10-6 M with CCU; and around 5 X 10-5 M with erythromycin. Taking into account the importance of LDH as an indicator of membrane damages, GG was assumed to interact with membrane hepatocyte. Keywords: triterpenic-saponins; isolated rat hepatocytes; galactosamine; CCl4; erythromycin; cytoprotection; hepatoprotection
Introduction
Recently, Oakenfull (1981) revealed the potential of diet saponins to lower blood cholesterol and Kiso et al. (1984) demonstrated the anti-hepatotoxic action of triterpenic saponins. Our purpose was to investigate the properties of two new saponins: gypsoside (GY) and glucuronogypsogenin (GG), as potential hepatoprotective molecules. Suspensions of freshly isolated rat liver parenchymal cells treated with sub-lethal doses of three toxicants (CCU, erythromycin and galactosamine) were used as an in vitro model. To evaluate the cytoprotective effect of the molecules the lactate dehydrogenase leakage (LDH) and the ATP content were measured. In this paper we report the in vitro cellular responses of isolated hepatocytes exposed to saponins prior to intoxication.
Material and methods
Chemicals GY and GG were obtained by purification of pure white saponin (Merck, Darmstadt) (Henry et al. 1989) (figure 1). Preparation of hepatocytes Hepatocytes were isolated from Sprague-Dawley male rats' liver (Berry and 0265-203X/90 $3.00 © 1990 Taylor & Francis Ltd.
F. Braut-Boucher et al.
S128
Downloaded by [University of Florida] at 01:58 06 November 2015
Gjpioild (GY)
.lCH3
Glucurono-gypioglnln (GG)
OOOH OOOH
CH3 CH3
CHO
Figure 1. Structure of glucuronogypsogenin (GG) and gypsoside (GY).
Friend 1979). The cells were suspended at 3 X 106 cellsml"1 in MEM Eagle medium. Treatment schedules and biochemical analysis Hepatocytes were pre-incubated with various concentrations of saponin for 1 h prior to addition of the toxicant. The total incubation period was 3 h. LDH was assayed in the cell free medium and ATP in the cellular pellet as previously described by Braut-Boucher et al. (1988). Three separate experiments were realized in duplicate. Results Effects of gypsoside in intoxicated rat hepatocytes No protective action was observed towards the three toxicants used when hepatocytes were pretreated by GY. Effects of glucuronogypsogenin in intoxicated hepatocytes With GG (figure 2) used in the concentration range from 5 x 10~7 to 5 x 10~5 M a cytotprotective effect was registered towards all the toxicants tested. The LDH leakage in the supernatants and the ATP cellular level were brought back to normal values compared to untreated rat hepatocyte data. The cytoprotective effect was more pronounced when hepatocytes were treated with galactosamine. The range of protective effect was estimated to be from 5 X 10~6 to 5 x 10"5 M with CCU, and around 5 x 10"5 M towards erythromycin.
Cytoprotective effects of saponins % of control
ftof control 5.10-3M
170.
170.
S129
tluaircc»gypjoj£nin+ rartcn ttrKUccidc • 5.1O-4M
150 ^
150.
130.
130 110.
LDH
90.
ATP
LDH
110. 90.
70.
70.
SO.
50.
ATP
30.
30.
5.10-*
5.10-8
5.10-4
5.10-6
5.10-6
3.10-4
CoocentntiaD (M) %of control
Downloaded by [University of Florida] at 01:58 06 November 2015
170^
glucurooo-gyp»og£iiin+ eryihromycii u 5.10-4M
13V 130-
LDH
110-
90ATP
70^ 50. 301 5.10-8
5.10-6
5.10-4
Concentnticn (M)
Figure 2. Cytoprotective effect of glucuronogypsogenin towards intoxicated rat hepatocytes.
Discussion The main result of this study was that the monodesmoside GG, isolated from Gypsophila pacifica K was less toxic than the bidesmoside GY on hepatocytes (Braut-Boucher et al. 1988). Moreover, this latter compound did not present any cytoprotective effect. Hikino et al. (1984) demonstrated the highest hepatoprotective properties of the aglycone against carbon tetrachloride- or galactosamineinduced hepatotoxicity. In this work, we were able to compare the activity of two molecules differing by the number of sugars connected to the genin. We could define the broader protection range of monodesmoside GG towards galactosamine injuries comparatively to those registered with CCU or erythromycin. In conclusion, further studies have been developed to approach the mechanism of action of GG on membrane hepatocytes and to explain the cytoprotective properties of this pure saponin. References BERRY, M. N., and FRIEND, D. S., 1969, High yield preparation of isolated rat liver parenchymal cells. A biochemical and fine structural study. Journal of Cell Biology, 43, 29-43. BRAUT-BOUCHER, F., ACHARD-ELLOUK, S., HOELLINGER, H., PAUTHE-DAYDE, D., and HENRY,
M.,
1988, Isolated and cultured rat hepatocytes as models for studying the cytotoxicity of two triterpenoid saponins. Liver Cells and Drugs, edited by A. Guillouzo (Colloque INSERM: John Libbey Eurotext), 164, pp. 379-383. HENRY, M., PAUTHE-DAYDE, D., and ROCHD, M., 1990, Extraction and quantitative HPLC determination of gypsogenin-3-glucuronide. Journal of Chromatography (in press). HIKINO, H., OSAWA, T., KISO, Y., and OSHIWA, Y., 1984, Analgesic and antihepatotoxic actions of
dianosides, triterpenoid saponins of Dianthus superbus var. longicalicynus herbs, Planta Medica, 353-355.
Si30
F. Braut-Boucher et al.
KISO, Y., TOHKIN, M., HIKINO, H., HATTORI, M., SAKAMOTO, T., and NAMBA, T., 1984, Antihepato-
Downloaded by [University of Florida] at 01:58 06 November 2015
toxic activity of glycyrrhizin 1. Effects of free radical generation and lipid peroxidation. Planta Medica, 298-302. OAKENFULL, D., 1981, Saponins in food, a review. Food and Chemical Toxicology, 6, 19-40.
ISSN: 0265-203X (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/tfac19
Cytoprotective effects of Gypsophila saponins towards isolated rat hepatocytes F. Braut‐Boucher , S. Achard‐Ellouk , D. Pauthe‐Dayde , M. Henry & H. Hoellinger To cite this article: F. Braut‐Boucher , S. Achard‐Ellouk , D. Pauthe‐Dayde , M. Henry & H. Hoellinger (1990) Cytoprotective effects of Gypsophila saponins towards isolated rat hepatocytes, Food Additives & Contaminants, 7:S1, S127-S130, DOI: 10.1080/02652039009373863 To link to this article: http://dx.doi.org/10.1080/02652039009373863
Published online: 10 Jan 2009.
Submit your article to this journal
Article views: 6
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tfac19 Download by: [University of Florida]
Date: 06 November 2015, At: 01:58
FOOD ADDITIVES AND CONTAMINANTS, 1990, VOL. 7, SUPPLEMENT NO. 1, S127-S130
Cytoprotective effects of Gypsophila saponins towards isolated rat hepatocytes F. BRAUT-BOUCHER, S. ACHARD-ELLOUK, D. PAUTHE-DAYDE†, M. HENRY† and H. HOELLINGER
Downloaded by [University of Florida] at 01:58 06 November 2015
UDC CNRS-INSERM (UA 400), Faculté de Médecine, Université René Descartes, Paris, France, and † Laboratoire de Botanique, Faculté de Pharmacie, Toulouse, France Saponins are glycosides widely distributed in the plant kingdom and are found in many foods. The hepatoprotective potential of glucuronogypsogenin (GG) and gypsoside (GY) towards isolated rat hepatocytes treated by three toxic models used at sub-lethal doses: galactosamine (5 x 10-3 M), CCl4 (5 x 10-4 M) and erythromycin (5 x 10-4 M) was investigated. Two schedules were carried out corresponding to curative or preventive treatment. No protection was observed on hepatocytes treated with GY before or after addition of the toxicants. In contrast, a protective action was detected when hepatocytes were pretreated with GG (5 X 10-5 M) as probe, by the normalisation of LDH leakage and ATP content. It depends on the toxicant: the cytoprotective spectrum is 5 x 10-5 to 5 x 10-7 M with galactosamine; 5 x 10-5 to 5 x 10-6 M with CCU; and around 5 X 10-5 M with erythromycin. Taking into account the importance of LDH as an indicator of membrane damages, GG was assumed to interact with membrane hepatocyte. Keywords: triterpenic-saponins; isolated rat hepatocytes; galactosamine; CCl4; erythromycin; cytoprotection; hepatoprotection
Introduction
Recently, Oakenfull (1981) revealed the potential of diet saponins to lower blood cholesterol and Kiso et al. (1984) demonstrated the anti-hepatotoxic action of triterpenic saponins. Our purpose was to investigate the properties of two new saponins: gypsoside (GY) and glucuronogypsogenin (GG), as potential hepatoprotective molecules. Suspensions of freshly isolated rat liver parenchymal cells treated with sub-lethal doses of three toxicants (CCU, erythromycin and galactosamine) were used as an in vitro model. To evaluate the cytoprotective effect of the molecules the lactate dehydrogenase leakage (LDH) and the ATP content were measured. In this paper we report the in vitro cellular responses of isolated hepatocytes exposed to saponins prior to intoxication.
Material and methods
Chemicals GY and GG were obtained by purification of pure white saponin (Merck, Darmstadt) (Henry et al. 1989) (figure 1). Preparation of hepatocytes Hepatocytes were isolated from Sprague-Dawley male rats' liver (Berry and 0265-203X/90 $3.00 © 1990 Taylor & Francis Ltd.
F. Braut-Boucher et al.
S128
Downloaded by [University of Florida] at 01:58 06 November 2015
Gjpioild (GY)
.lCH3
Glucurono-gypioglnln (GG)
OOOH OOOH
CH3 CH3
CHO
Figure 1. Structure of glucuronogypsogenin (GG) and gypsoside (GY).
Friend 1979). The cells were suspended at 3 X 106 cellsml"1 in MEM Eagle medium. Treatment schedules and biochemical analysis Hepatocytes were pre-incubated with various concentrations of saponin for 1 h prior to addition of the toxicant. The total incubation period was 3 h. LDH was assayed in the cell free medium and ATP in the cellular pellet as previously described by Braut-Boucher et al. (1988). Three separate experiments were realized in duplicate. Results Effects of gypsoside in intoxicated rat hepatocytes No protective action was observed towards the three toxicants used when hepatocytes were pretreated by GY. Effects of glucuronogypsogenin in intoxicated hepatocytes With GG (figure 2) used in the concentration range from 5 x 10~7 to 5 x 10~5 M a cytotprotective effect was registered towards all the toxicants tested. The LDH leakage in the supernatants and the ATP cellular level were brought back to normal values compared to untreated rat hepatocyte data. The cytoprotective effect was more pronounced when hepatocytes were treated with galactosamine. The range of protective effect was estimated to be from 5 X 10~6 to 5 x 10"5 M with CCU, and around 5 x 10"5 M towards erythromycin.
Cytoprotective effects of saponins % of control
ftof control 5.10-3M
170.
170.
S129
tluaircc»gypjoj£nin+ rartcn ttrKUccidc • 5.1O-4M
150 ^
150.
130.
130 110.
LDH
90.
ATP
LDH
110. 90.
70.
70.
SO.
50.
ATP
30.
30.
5.10-*
5.10-8
5.10-4
5.10-6
5.10-6
3.10-4
CoocentntiaD (M) %of control
Downloaded by [University of Florida] at 01:58 06 November 2015
170^
glucurooo-gyp»og£iiin+ eryihromycii u 5.10-4M
13V 130-
LDH
110-
90ATP
70^ 50. 301 5.10-8
5.10-6
5.10-4
Concentnticn (M)
Figure 2. Cytoprotective effect of glucuronogypsogenin towards intoxicated rat hepatocytes.
Discussion The main result of this study was that the monodesmoside GG, isolated from Gypsophila pacifica K was less toxic than the bidesmoside GY on hepatocytes (Braut-Boucher et al. 1988). Moreover, this latter compound did not present any cytoprotective effect. Hikino et al. (1984) demonstrated the highest hepatoprotective properties of the aglycone against carbon tetrachloride- or galactosamineinduced hepatotoxicity. In this work, we were able to compare the activity of two molecules differing by the number of sugars connected to the genin. We could define the broader protection range of monodesmoside GG towards galactosamine injuries comparatively to those registered with CCU or erythromycin. In conclusion, further studies have been developed to approach the mechanism of action of GG on membrane hepatocytes and to explain the cytoprotective properties of this pure saponin. References BERRY, M. N., and FRIEND, D. S., 1969, High yield preparation of isolated rat liver parenchymal cells. A biochemical and fine structural study. Journal of Cell Biology, 43, 29-43. BRAUT-BOUCHER, F., ACHARD-ELLOUK, S., HOELLINGER, H., PAUTHE-DAYDE, D., and HENRY,
M.,
1988, Isolated and cultured rat hepatocytes as models for studying the cytotoxicity of two triterpenoid saponins. Liver Cells and Drugs, edited by A. Guillouzo (Colloque INSERM: John Libbey Eurotext), 164, pp. 379-383. HENRY, M., PAUTHE-DAYDE, D., and ROCHD, M., 1990, Extraction and quantitative HPLC determination of gypsogenin-3-glucuronide. Journal of Chromatography (in press). HIKINO, H., OSAWA, T., KISO, Y., and OSHIWA, Y., 1984, Analgesic and antihepatotoxic actions of
dianosides, triterpenoid saponins of Dianthus superbus var. longicalicynus herbs, Planta Medica, 353-355.
Si30
F. Braut-Boucher et al.
KISO, Y., TOHKIN, M., HIKINO, H., HATTORI, M., SAKAMOTO, T., and NAMBA, T., 1984, Antihepato-
Downloaded by [University of Florida] at 01:58 06 November 2015
toxic activity of glycyrrhizin 1. Effects of free radical generation and lipid peroxidation. Planta Medica, 298-302. OAKENFULL, D., 1981, Saponins in food, a review. Food and Chemical Toxicology, 6, 19-40.
