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Extract of Ginkgo biloba induces glutathione-S-transferase subunit-P1 in vitro.


The extract of Ginkgo biloba (EGb), containing 24% flavone glycosides and 6% terpenoids, is widely used to treat early-stage Alzheimer's disease, vascular dementia, peripheral claudication and vascular tinnitus. Its remarkable antioxidant activity has recently been demonstrated in both cell lines and animals. Glutathione-S-transferases (GSTs) are a class of important detoxification enzymes in the antioxidant system and GST-P1 is the major GST isoform highly expressed in human tissues. Over expression of GST-P1 protected prostate cells from cytotoxicity and DNA damage by the heterocyclic amine carcinogen, while inhibition of expression of GST-P1 by transfecting GST-PI antisense cDNA or targeted deletion of GST-P1 has been found to sensitize ceils to cytotoxic chemicals. It is obvious that induction of GST-P1 expression should be a promising alternative for chemoprevention. The present study aimed to investigate the induction effect of EGb on GST-P1 in HepG2 and Heplclc7 cell lines and found that GST-P1 was increased both at the expression and enzyme activity levels.

[C] 2008 Elsevier GmbH. All rights reserved.

Keywords: Extract of Ginkgo biloba; Glutathione-S-transferase-subunit-P1 (GST-PI); Herb


Ginkgo is a gymnosperm tree that exists since the last glacial period and is considered as a living fossil (Zhou and Zheng, 2003). Ginkgo biloba, a member of the Ginkgoaceae family, has been cultivated in China for hundreds of years. Extract from the leaves of Ginkgo biloba (EGb) has been used as a traditional Chinese medicine for centuries in the treatment of asthma and bronchitis (Kleijnen and Knipschild, 1992). Currently, it is used widely to treat cerebrovascular and peripheral vascular insufficiency, neurosensory problems, and disturbances in vigilance, short-term memory and other cognitive functions that are associated with dementias, ageing and senility (DeFeudis et al., 2003). In recent decades, EGb has been reported to possess antioxidant activity and to fight against oxidative processes in both cell lines and animals (Droy-Lefaix et al., 1995; Rong et al., 1996; Gohil et al., 2000; Bridi et al., 2001; Chen et al., 2001; Ahmad et al, 2005).

In the antioxidant system, glutathione-S-transferases (GSTs) are a class of detoxification (or antioxidant) enzymes that catalyse the conjugation of reduced glutathione to electrophilic compounds, including endogenous or exogenous sources of reactive oxygen species (ROS) and environmental carcinogenic compounds (Hayes and Pulford, 1995). On the basis of their amino acid sequence eight classes of mammalian GSTs (namely GST alpha, mu, pi, theta, sigma, zeta, kappa, and omega) have been identified whose expr ession is both tissue- and species-specific (Ketterer, 2001). GST-PI is the major GST isoform in mouse liver and is reported to be highly expressed in both human liver and skin (Raza et a., 1991; Desmots et al., 2001). Over expression of GST-PI protected prostate cells from cytotoxicity and DNA damage by the heterocyclic amine carcinogen PhIP (Nelson et al., 2001), while inhibition of expression of GST-P1 by transfecting GST-P1 antisense cDNA has been found to sensitize cells to cytotoxic chemicals (Ban et al., 1996). Targeted deletion of GST-Pi significantly enhanced the sensitivity of mice to polycyclic hydrocarbon-induced skin carcinogenesis (Henderson et al., 1998; Long et al., 2000). Since GST-P1 exerts important detoxification or antioxidant functions in body, induction of its expression should be a promising alternative for chemoprevention. This study aimed to investigate the induction effect of EGb on GST-P1 in vitro.

Materials and methods

Cell culture

Human hepatoma cell line HepG2 and mouse hepatoma cell line Hepalclc7, from cells storage in the cell lab of Pharmacology and Therapeutics Department of Liverpool University, were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2mM glutamine, 10% fetal calf serum, 100 U/ml penicillin and 100 [mu]g/ml streptomycin and incubated at 37 [degrees]C in a 5% [CO.sub.2] atmosphere.


EGb containing 24% ginkgo-flavone glycosides and 6% terpenoids was provided by The Research Department of Hunan Warrant Pharmaceutical Co. (1) and was dissolved in DMSO. The final concentration of DMSO in the cell culture medium was controlled to <0.1%. Other reagents were purchased from Sigma (Poole, UK) unless otherwise stated.

RNA isolation and real-time PCR

Hepalclc7 cells were seeded in 6-well plates at 2 x [10.sup.6]/well and cultured for 24h. Cells were then washed in Hank's balanced salt solution (HBSS) and incubated in complete DMEM containing EGb at the indicated concentrations for 4h. Total RNA was isolated using TRIzol reagent and 1 [mu]g RNA was reverse transcribed using TaqMan reagents (N808-0234) according to the manufacturer's instructions. GST-P1 and GAPDH (housekeeping gene) mRNA levels were quantified by real-time quantitative PCR, using pre-developed TaqMan assay reagents and the AB 7000 sequence detection system.

Western blot for GST-P1

Cells were seeded at 50,000/well in 96-well plates and cultured for 24 h. Cells were then washed in Hank's balanced salt solution (HBSS) and incubated in complete DMEM containing EGb at the indicated concentrations for 24 h. Cells were washed twice in HBSS before being lysed in 50 [mu]l lysis buffer (0.2% Triton X-100, 5mM NaCl, 10mM HEPES, (sub.pH 8), 0.5 mM sucrose, 1 mM EDTA, 0.5 mM spermidine, 0.15mM spermine) and centrifuged at 3500g for 3min. Supernatants were retained as cytosolic extracts and total protein concentrations were determined (Bradford, 1976). Equal amounts of proteins were mixed with loading buffer (70% LDS, 30% reducing agent), and heated at 70 [degrees]C for 5min. The samples were then loaded onto a NuPAGE 4-12% SDSpolyacrylamine gel (Invitrogen, Paisley, UK) and separated at 170 V for 1 h. The proteins were transferred onto a Hybond ECL membrane at 80 V for 1 h. The membrane was blocked in 10% non-fat milk in tris-buffered saline containing 0.1% Tween 20 (T-TBS) for 15min, then probed with a primary antibody in 2% non-fat milk (GST-PI and actin 1:4000) for 1 h. Membranes were washed repeatedly in 0.1% T-TBS before being probed with secondary antibody (1:4000) for 1 h. Following repeated washes, visualization of the protein antibody conjugate was performed using enhanced chemiluminescence and the band volumes were quantified by UVISoft [TM] software (UVITech, Cambridge, UK).

Assay of GST activity

Cells were split to 200,000/well in 24-well plates and cultured for 24 h. Cells were then washed in Hank's balanced salt solution (HBSS) and incubated in complete DMEM containing EGb at the indicated concentrations for 24 h. GST activity was determined (Habig and Jakoby, 1981). Cells were washed twice in HBSS before being lysed in 200 [mu]l lysis buffer and centrifuged at 5000 rpm for 3 min. Ten microliters supernatant was placed in a 96-well plate along with 300 [mu]l phosphate buffer containing 1.5 mM l-chloro2,4-dinitrobenzene (DNCB) and 1.5mM GSH. The formation of the DNCB-GSH conjugate was determined from the increase in absorbanee at 340 nm. Cytosolic total protein was also quantified by the Bradford method.

Statistical analysis

Data were expressed as mean [+ or -] S.E.M and analyzed by SPSS, with a p value of less than 0.05 considered statistically significant.


To verify the induction effect of EGb on GST-P1, we detect it at gene transcription, protein and enzyme activity levels. At the gene transcription level, we demonstrated the up-regulation effect of EGb with real-time PCR, which was embodied in our last report (liu et al., 2007).

At the protein level, the cytosolic GST-PI was elevated by EGb dose-dependently (p<0.01) (Fig. 1). The strongest induction (1.68 folds) was found at the concentration of 62.5 [mu]g/ml in the Hepalclc7 cells.


We further tested the induction effect of GST-P1 with the measurement of enzyme activity. As shown in Fig. 2, EGb increased the GST activity significantly. In the Hepa1clc7 cells, all four concentrations of EGb increased GST activity (p<0.005) and the peak was found at the concentration of 62.5 [mu]g/ml (p<0.01, compared with the control) (Fig. 2A). In the HepG2 cells, the GST activity was also increased by EGb significantly (p<0.005) and the strongest induction was found at the concentration of 125 [mu]g/ml compared with control (p<0.01) (Fig. 2B). The data from the two cell lines both demonstrated the induction effect of EGb from the enzyme activity level.



The present research demonstrated that EGb induced GST-P1 and elevated the cellular GST activity both in the mice and human cell lines. It strongly supports the notion that EGb possess the antioxidant effect through induction GST-P1, besides induction of other antioxidant enzymes or scavenge of the reactive oxygen species (ROS) directly (DeFeudis et al., 2003). But these effects were all tested in vitro, the next verification of the induction of EGb on GST-P1 in vivo should be investigated.

EGb is an extract mainly containing flavonoids (ginkgo-flavone glycosides) and terpenoids (ginkgolides and bilobalides). The flavonoids were reported to play antioxidant roles in this extract and the terpenoids are known to antagonize platelet-activating factor in the processes of platelet aggregation and arterial thrombosis, acute inflammation, allergic reactions and cardiovascular insufficiency (DeFeudis, 1998; Chen et al., 2005). With regards to this induction effect on GST-P1, whether flavonoids or other constituent(s) or the total extract (EGb) is the most active ingredient requires delineation.

The GST-P1 was reported to be the most a bundant isoenzyme in the GST family and present in almost all human tissues, but it is also over-expressed in various malignancy tissues (Coles and Ketterer, 1990; Tsuchida and Sato, 1992; Mohammadzadeh et al., 2003). Consequently, GST-P1 expression has been considered as a diagnostic indicator of chemical carcinogenesis (Tidefelt et al., 1992; Grignon et al., 1994; Hamada et al., 1994). Furthermore, a number of studies have shown a correlation between over expression of GST-P1 and the development of resistance towards various anti-cancer drugs (O'Brien et al., 2000; Townsend and Tew, 2003). It is obvious that GST-P1 not only protects the normal cells but also the cancer cells against various attacks including chemotherapy. In this regard, induction of GST-P1 may not favorable for cancer chemotherapy. But many researches demonstrated that EGb possesses the anti-cancer effects on different models (Griffiths et al., 1999; Mutoh et al., 2000; Papadopoulos et al., 2000). It is possible that EGb may exert anti-cancer effect through other mechanisms or has different influence on GST-P1 expression or other antioxidant enzymes in tumor cells.

In our last report, EGb was found to induce typical phase 2 genes, including GST-P1, through the Keap1-Nrf2-ARE signaling pathway. ARE, antioxidant response element, is a cis-acting motif located in the 5'-flanking promoter region of nearly all phase 2 genes and mediates the transcriptional up-regulation of phase 2 genes caused by different classes of chemical compounds, including Michael reaction acceptors, diphenols, quinones, isothiocyanates, peroxides, mercaptans, trivalent arsenicals, heavy metals, and dithio-lethiones (Prestera et al., 1993). The inducer signal is transmitted to the ARE by two important proteins--Nrf2, a transcription factor homologous to Drosophila cap 'n' collar proteins, and Keap1, a cytosolic protein homologous to the Drosophila actin-binding protein Kelch, which plays an active role in Nrf2 regulation, by directing it for proteosomal degradation (Itoh et al., 1997, 1999; McMahon et al., 2003). How does EGb interact with the Keapl-Nrf2-ARE system or whether it activates other signaling pathways will be investigated further.


This work was supported by China-UK Science Networks, NW Cancer Research Fund and Grant from Anhui Province, PR China (2004KJ340). The Wellcome Trust provided materials for works carried out in the UK.

(1) In the %-content of the main compounds comparable with that of the special Ginkgo extract EGb761, Firma Schwabe (Germany).


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Abbreviations: EGb, extract of Ginkgo biloba; GST-PI, glutathioneS-transferase subunit-pl: ARE, antioxidant response element: Nrf2, nuclear factor erythroid 2-related factor 2; Keapl, Kelch-like ECH-associated protein 1.

Xiao-Ping Liu (a), *, Christopher E.P. Goldring (b), Hai-Yi Wang (b), Ian M. Copple (b), Neil R. Kitteringham (b), B. Kevin Park (b)

(a) Department of Pharmacology, Wannan Medical College. Wuhu, Anhui, PR china

(b) Department of Pharmacology and Therapeutics, The University of Liverpool, Liverpool, Merseyside, UK

* Corresponding author. Tel./fax: + 86 553 3932619.

E-mail address: (X.-P. Liu).

0944-7113/$ - see front matter [C] 2008 Elsevier GmbH. All rights reserved.

doi: 10.1016/j.phymed.2008.11.001
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Author:Liu, Xiao-Ping; Goldring, Christopher E.P.; Wang, Hai-Yi; Copple, Ian M.; Kitteringham, Neil R.; Par
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:May 1, 2009
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