Pharmaceutical Biology, 2009; 47(12): 1192–1197

R E S E A R C H A R T I C L E

The effect of sixteen medicinal plants used in the Brazilian pharmacopoeia on the expression and activity of glutathione S-transferase in hepatocytes and leukemia cells

Cássia Rosalina Príncipe, and Beny Spira

Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Brazil

Address for Correspondence: Beny Spira, Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Prof. Lineu Prestes, 1374, São Paulo-SP CEP:05508-900, Brazil. Tel: (5511) 30917347; Fax: (5511) 30917354; E-mail: benys@usp.br

(Received 22 July 2008; revised 01 September 2008; accepted 07 October 2008)

Introduction

Carcinogens and other xenobiotics are neutralized in mammalian cells by two groups of enzymes. Phase I enzymes, formed mainly by the cytochromes P450 introduce functional groups in the toxic compound, generally leading to non-electrophilic metabolites. However, in some instances the result is the production of highly reactive electrophiles which may end binding to nucleophilic centers on DNA, potentially enhancing carcinogenesis (van Bladeren, 2000). Phase II enzymes detoxify electrophilic compounds, including those pro-duced by phase I enzymes, through their conjugation with endogenous ligands (e.g., glutathione, glucuronic

acid) or by disabling functional groups (e.g., hydrolysis of epoxides to diols). Glutathione S-transferase (GST) is a family of phase II enzymes ubiquitous among all organisms hitherto studied. The primary function of GST is the conjugation of the nucleophilic sulfur atom of the tri-peptide glutathione (GSH) to the electrophilic center of the toxic compound. GSH binding reduces the toxicity and increases the solubility of the toxic com-pound enhancing its excretion from the cell (Jakoby, 1978). The GSTs are dimeric proteins formed by binary combinations of several subunits that are divided into two super families: cytosolic and microsomal. The cytosolic GSTs are further divided into seven classes: , , , , , and Ω (Hayes et al., 2005). They are widely

ISSN 1388-0209 print/ISSN 1744-5116 online © 2009 Informa UK LtdDOI: 10.3109/13880200903029340

AbstractGlutathione S-transferase (GST) is a family of enzymes involved in the detoxification of electrophilic com-pounds. Different classes of GST are expressed in various organs, such as liver, lungs, stomach and others. Expression of GST can be modulated by diet components and plant-derived compounds. The importance of controlling GST expression is twofold: increasing levels of GST are beneficial to prevent deleterious effects of toxic and carcinogenic compounds, while inhibition of GST in tumor cells may help overcoming tumor resistance to chemotherapy. A screening of 16 plants used in the Brazilian pharmacopoeia tested their effects on GST expression in hepatocytes and Jurkat (leukemia) T-cells. The methanol extracts of five plants inhibited GST expression in hepatocytes. Three plants significantly inhibited and four others induced GST expression in Jurkat cells. Among these, the extracts of Bauhinia forficata Link. (Leguminosae) and Cecropia pachystachya Tréc. (Urticaceae) inhibited GST expression at relatively low concentrations. With the excep-tion of B. forficata, all plants were cytotoxic when administered to Jurkat cells at high doses (1 mg/mL) and some extracts were considerably cytotoxic even at lower concentrations.

Keywords: Glutathione S-transferase; medicinal plants; Jurkat cells; hepatocytes; leukemia

http://www.informahealthcare.com/phb

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Effect of medicinal plants on GST 1193

distributed among all mammal tissues, while in the liver its concentration may account for 20% of the total hepatic proteins (Jakoby, 1978). Many vegetables or plant-derived compounds, known as chemopreven-tives induce GST expression (Williamson et al., 1997; Thornalley, 2002). Medicines or diet components that enhance GST expression might contribute to cancer prevention because a high and constant level of this enzyme helps mitigating the deleterious effects of carci-nogenic compounds (Wattenberg, 1985). On the other hand, overexpression of GST by tumor cells constitutes a serious drawback in cancer chemotherapy, due to the role of GST in the neutralization of chemotherapeutic drugs. GST over expression by tumor cells and its nega-tive effect on cancer treatment prognostic has been reported (Townsend & Tew, 2003; Ye & Song, 2005). Therefore, inhibitors of GST production would be valu-able as a complement for patients undergoing cancer chemotherapy.

Acute lymphoblastic leukemia (ALL) is the most com-mon malignancy in childhood, constituting about 30% of childhood cancers. Most studies support that GST plays a significant role in the susceptibility to ALL (Ye & Song, 2005). Also, the expression of GST π is associ-ated with a higher incidence of relapse in ALL patients, indicating that at least this GST class is involved in the development of tumor resistance to chemotherapy (Anderer et al., 2000; Gurbuxani et al., 2001; Tidefelt et al., 1992).

Epidemiological studies have shown that several vegetables or plants have a role in prevention of cancers (Steinmetz & Potter, 1996). A substantial part of this pro-tective effect is related to the induction of antioxidant and detoxifying enzymes such as GST. For instance,

isothiocyanates, widely found in cruciferous vegeta-bles are specific inducers of GST (Nho & Jeffery, 2001; Williamson et al., 1997). Emblica officinalis, an Indian medicinal plant and curcumin, an antioxidant isolated from turmeric (Curcuma longa) were shown to increase the level of GST in rodents (Piper et al., 1998; Sharma et al., 2000).

The use of medicinal plants (phytotherapy) is widely disseminated among Brazilians. To the best of our knowledge, the effect of Brazilian medicinal plants on GST or other phase II detoxifying enzymes has never been tested. However, the vast diversity found in Brazilian flora potentially holds many plants that might exert some kind of effect on GST activity. In the present study we evaluated the effect of 16 medicinal plants of the Brazilian folk medicine on the activity of GST in hepatocytes and leukemia T-cells.

Materials and methods

Plant collection and extraction

The plants used in this study are described in Table 1. Specimens were collected in the Parque Estadual da Ilha do Cardoso in June 2003 or in the district of Cananéia in October 2002, both in the southeast of the state of São Paulo, and were identified by Inês Cordeiro from the Instituto de Botânica of São Paulo. Voucher specimens are deposited in the herbarium of the Instituto de Botânica of São Paulo. The aerial parts of the plants were dried at room temperature or at 30°C in the shadow. The dried leaves were coarsely powdered in a blender and the dried powder extracted

Table 1. Plants collected for this study.

Scientific name Popular name Use in folk medicine Site of collection

Ageratum conyzoides L. (Asteraceae) Erva-de-São-João Analgesic, wound healing Cananéia

Baccharis trimera (Less.) DC. (Asteraceae) Carqueja Gastrointestinal diseases, anti-ulcerative, hangover

Ilha do Cardoso

Bauhinia forficata Link. (Leguminosae) Pata-de-vaca Diabetes Cananéia

Cecropia pachystachya Tréc. (Urticaceae) Embaúba branca Diuretic Ilha do Cardoso

Costus spiralis (Jacq.) Roscoe (Costaceae) Cana-do-brejo Diuretic, hemostatic Cananéia

Eugenia uniflora L. (Myrtaceae) Pitanga Rheumatism, diarrhea, coughing, bronchitis Ilha do Cardoso

Hydrocotyle bonariensis Lam. (Apiaceae) Chapéu-de-sapo Diuretic, liver and kidney problems Ilha do Cardoso

Jacaranda puberula Cham. (Bignoniaceae) Caroba Venereal diseases Cananéia

Kalanchoe pinnata (Lam.) Pers. (Crassulaceae) Fortuna Boils, coughing, gastritis Cananéia

Mimosa pudica L (Leguminosae) Dorme-dorme Sore throat, gastrointestinal diseases Cananéia

Pimenta pseudocaryophyllus Gomes Landrum var. (Myrtaceae)

Cataia Cold and respiratory diseases Ilha do Cardoso

Psidium cattleyanum Sabine (Myrtaceae) Araçá Diarrhea Ilha do Cardoso

Psychotria nuda (Cham. & Schltdl.) Wawra (Rubiaceae) Erva-de-anta Diabetes Ilha do Cardoso

Sambucus nigra L. (Caprifoliaceae) Sabugueira Respiratory diseases Cananéia

Schinus terebinthifolius Raddi (Anacardiaceae) Aroeira Respiratory and urinary diseases Ilha do Cardoso

Senna multijuga (Rich.) H.S. Irwin and Barneby (Leguminosae)

Caquera Purgative Cananéia

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1194 Cássia Rosalina Príncipe and Beny Spira

three times for 24 h with 100% methanol (20 g plant powder/200 mL methanol) under constant agitation. The three methanol extracts were pooled together and the solvent was evaporated by distillation under reduced pressure in a rotary evaporator. The extracts were then lyophilized and stored at −20°C until use. For the experiments with cell cultures the extracts were dissolved in dimethylsulfoxide (DMSO) at a stock concentration of 100 mg/mL.

Cell culture and treatments

L37 hepatocytes (Bulera et al., 1997) were grown in Dulbeco’s modified eagle medium supplemented with 10% fetal bovine serum. Jurkat cells were grown in RPMI medium supplemented with 10% fetal bovine serum. Plant extracts dissolved in 100% DMSO were added to semi-confluent cultures grown in 25 cm flasks at different final concentrations and incubated for 24 h. The final DMSO concentration in all assays was 1%, and that was also the concentration of DMSO in the control sam-ples. Following the treatment period, cells were washed twice with PBS and disrupted by incubating for 30 min at RT with 0.2 mL lysis buffer (1% Nonidet-P40; 0.05 M Tris-HCl pH 7.5; 0.15 M NaCl; 0.002 M EDTA) under mild agitation. The cell extract was then centrifuged for 15 min at 10,000 rpm. The supernatant was used as the cytosolic fraction.

GST assay

The assay was performed essentially as described (Spira & Raw, 2000). Cytosolic extracts were used as the enzyme source and 1-chloro-2,4-dinitrobenzene (CDNB) was the substrate whose conjugation to GSH was followed up by measuring the increase in product absorbance at 340 nm at 37°C. Results are given in enzyme units, which are defined as the amount of GST needed to conjugate 1 µmol of CDNB per minute per mg protein.

Measurement of protein concentration and cell viability

Protein concentration in the cell culture extracts was measured by the method of Sandermann and Strominger (1972). This is a modification of the Lowry method that allows to perform protein assays in the presence of nearly any detergent.

Cell viability was assessed by staining cell samples with trypan blue. Since only anchored cells (in the case of L-37 hepatocytes) and centrifuged Jurkat cells are disrupted and processed, the measurement of protein concentration also gives a reasonable estimate of cell viability and proliferation (Digel et al., 2007; Naim et al., 2005).

Results

The effect of 16 plant methanol extracts on the GST activity of L37 hepatocytes

Preliminary tests were performed to optimize the experi-mental conditions concerning plant extracts concentra-tion and solubility, treatment length, and method of cell disruption (data not shown). Eventually these were set as follows: the plant extracts were dissolved in DMSO and cells were treated with increasing log concentra-tions of each extract (from 1 µg/mL up to 1 mg/mL) for 24 h. To obtain the cytosolic extract, cells were disrupted by chemical means as described in Materials and meth-ods. Hepatocytes were chosen because of their ability to metabolize chemicals due to the presence of phase I and phase II enzymes. Even though it is an established cell culture, the L37 lineage retains many characteristics of hepatocytes (Bulera et al., 1997). Semi-confluent cells grown in DMEM were treated for 24 h with 1, 10, 100 and 1000 µg/mL of each extract. As a control, 1% DMSO was used, which is also the final concentration of the solvent in all treated cultures.

Table 2 shows the results of the GST assays with 16 different plant extracts. Most plants did not affect GST expression when compared to the control even at the highest tolerated concentration. Most extracts were toxic at 1000 µg/mL, as judged by Trypan blue exclusion assays (not shown) and by a sharp decrease in protein concentration. Methanol extracts of C. pachystachya Tréc. (Urticaceae) (10 and 100 µg/mL), E. uniflora L. (Myrtaceae) (100 µg/mL), H. bonarien-sis Lam. (Apiaceae) (1000 µg/mL), J. puberula Cham. (Bignoniaceae) (100 µg/mL), P. pseudocaryophylus Gomes Landrum var. (Myrtaceae) (10 and 100 µg/mL) and S. multijuga (Rich.) H.S. Irwin & Barneby (Leguminosae) (100 µg/mL) significantly inhibited the expression of GST. None of the tested extracts had a positive effect on GST, but it is worth noticing that although 1 mg/mL P. pseudocaryophylus displayed a high cytotoxicity, it also enhanced the level of GST activity by 2-fold (not shown), which may indicate a positive effect on GST at high concentrations, despite its strong cytotoxic effect.

The effect of plant extracts on the GST activity of Jurkat cells

Jurkat cells are T lymphoblasts derived from a patient with acute T-cell leukemia (Schneider et al., 1977). The rationale behind the use of Jurkat cells was the finding that these cells are resistant to some chemotherapeutic drugs such as arsenic trioxide, which is mainly due to the over-expression of GST π in the tumor cells (Zhou et al., 2005). Therefore, a plant extract with anti-GST activity might reduce the level of GST expression and

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consequently its resistance to chemotherapy. Semi-confluent Jurkat cells were treated with the 16 plant extracts as described above for the L-37 hepatocytes and assayed for GST activity (Table 3). With the exception of B. forficata, all plant extracts were toxic when admin-istered at the highest concentration (1000 µg/mL), and K. pinnata (Lam) Pers. displayed a strong cytotoxic effect even at 1 µg/mL. Five plants (C. pachystachya, J. puberula,

K. pinnata, S. nigra L. (Caprifoliaceae) and S. multi-juga) exhibited considerable cytotoxicity at 100 µg/mL. B. forficata (10, 100, and 100 µg/mL), C. pachystachya (10 µg/mL) and P. pseudocaryophylus (100 µg/mL) sig-nificantly inhibited GST expression, while E. uniflora (1 and 100 µg/mL), H. bonariensis (10 and 100 µg/mL), J. puberula (10 µg/mL) and M. pudica L. (Leguminosae)(100 µg/mL) induced GST in a significant manner.

Table 3. Effect of plant extracts on the GST activity of Jurkat cellsa.

Extract concentration (g/mL)

Plant 0 1 10 100 1000

Ageratum conyzoides 1 (± 0.07) 0.98 (± 0.18) 0.86 (± 0.1) 0.88 (± 0.21) TCb

Baccharis trimera 1 (± 0.09) 1.1 (± 0.24) 1.18 (± 0.18) 1.28 (± 0.3) TC

Bauhinia forficata 1 (± 0.09) 0.77 (± 0.12) 0.71 (± 0.11)* 0.63 (± 0.16)* 0.36 (± 0.04)*

Cecropia pachystachya 1 (± 0.09) 1.52 (± 0.46) 0.55 (± 0.25)* TC TC

Costus spiralis 1 (± 0.09) 0.82 (± 0.02) 0.84 (± 0.01) 0.97 (± 0.16) TC

Eugenia uniflora 1 (± 0.09) 1.73 (± 0.13)* 1.27 (± 0.28) 1.86 (± 0.15)* TC

Hydrocotyle bonariensis 1 (± 0.21) 1.11 (± 0.11) 1.58 (± 0.23)* 1.39 (± 0.31)* TC

Jacaranda puberula 1 (± 0.16) 1.18 (± 0.07) 1.82 (± 0.43)* TC TC

Kalanchoe pinnata 1 (± 0.14) TC TC TC TC

Mimosa pudica 1 (± 0.09) 0.69 (± 0.09) 1.21 (± 0.38) 1.8 (± 0.42)* TC

Pimenta pseudocaryophyllus 1 (± 0.22) 0.8 (± 0.06) 0.74 (± 0.08) 0.29 (± 0.04)* TC

Psidium cattleyanum 1 (± 0.22) 0.65 (± 0.05) 0.7 (± 0.07) 1.11 (± 0.24) TC

Psychotria nuda 1 (± 0.19) 0.92 (± 0.25) 1.17 (± 0.35) 0.88 (± 0.1) TC

Sambucus nigra 1 (± 0.1) 1.07 (± 0.16) 1.05 (± 0.26) TC TC

Schinus terebinthifolius 1 (± 0.1) 0.88 (± 0.09) 0.83 (± 0.13) 1.51 (± 0.29) TC

Senna multijuga 1 (± 0.06) 1.04 (0.04) 0.93 (0.11) TC TCaCells were treated for 24 h with 1, 10, 100 or 1000 g/mL of each extract. GST activity is expressed as enzyme units/mg protein. A value of 1 was assigned to the control. Values represent the mean ± SE of at least three independent experiments.bToxic concentration.*Values significantly different (p <0.05) from control by the Student’s t-test.

Table 2. Effect of plant extracts on the GST activity of L-37 cellsa.

Extract concentration (g/mL)

Plant 0 1 10 100 1000

Ageratum conyzoides 1 (± 0.15) 1.1 (± 0.25) 0.92 (± 0.13) 0.86 (± 0.07) TCb

Baccharis trimera 1 (± 0.04) 1.05 (± 0.03) 0.97 (± 0.16) 0.9 (± 0.12) TC

Bauhinia forficata 1 (± 0.17) 1 (± 0.03) 0.95 (± 0.23) 0.95 (± 0.09) 0.65 (± 0.09)

Cecropia pachystachya 1 (± 0.1) 0.83 (± 0.25) 0.5 (± 0.17)* 0.28 (± 0.06)* TC

Costus spiralis 1 (± 0.3) 1.13 (± 0.24) 1.41 (± 0.16) 1.14 (± 0.08) 0.85 (± 0.37)

Eugenia uniflora 1 (± 0.35) 0.77 (± 0.18) 0.64 (± 0.11) 0.59 (± 0.21)* TC

Hydrocotyle bonariensis 1 (± 0.21) 0.97 (± 0.11) 0.82 (± 0.16) 1.03 (± 0.23) 0.29 (± 0.05)*

Jacaranda puberula. 1 (± 0.3) 0.85 (± 0.12) 0.84 (± 0.3)* 0.65 (± 0.16)* TC

Kalanchoe pinnata 1 (± 0.2) 1.23 (± 0.33) 1.13 (± 0.26) 1.11 (± 0.49) 1.15 (± 0.21)

Mimosa pudica 1 (± 0.1) 0.98 (± 0.2) 1.09 (± 0.09) 0.93 (± 0.25) TC

Pimentapseudocaryophyllus 1 (± 0.15) 0.96 (± 0.24) 0.79 (± 0.26)* 0.55 (± 0.16)* TC

Psidium cattleyanum 1 (± 0.2) 1.05 (± 0.27) 0.97 (± 0.1) 0.98 (± 0.17) TC

Psychotria nuda 1 (± 0.12) 0.84 (± 0.2) 1.28 (± 0.09) 1.08 (± 0.18) 1.01 (± 0.17)

Sambucus nigra 1 (± 0.15) 1.18 (± 0.11) 1.09 (± 0.06) 1.03 (± 0.17) 1.0 (± 0.28)

Schinus terebinthifolius 1 (± 0.09) 1.44 (± 0.09) 1.69 (± 0.27) 1.38 (± 0.18) TC

Senna multijuga 1 (± 0.05) 0.91 (± 0.06) 0.95 (± 0.22) 0.51 (± 0.13)* TCaCells were treated for 24 h with 1, 10, 100 or 1000 g/mL of each extract. GST activity is expressed as enzyme units/mg protein. A value of 1 was assigned to the control. Values represent the mean ± SE of at least three independent experiments.bToxic concentration.*Values significantly different (p <0.05) from control by the Student’s t-test.

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1196 Cássia Rosalina Príncipe and Beny Spira

Table 4 summarizes the effects of the plant extracts on GST activity of both Jurkat T-cells and L-37 hepato-cytes. Only plants that displayed significant effects are shown.

Discussion

GST plays a central role in both prevention and treat-ment of cancer. Genetic polymorphisms in GST classes , , and are important markers for response to chemotherapy and patient survival (Lo & Ali-Osman, 2007). Likewise, development of drug resistance due to GST over-expression has been reported in various tumor types (Tew, 1994; Townsend & Tew, 2003). In particular, GST pi which is induced in response to a large number of chemical carcinogens is considered a marker for pre-neoplastic lesion in the liver and is present in high amounts in several tumor cell lines, including Jurkat T-cells (Sakai & Muramatsu, 2007; Zhou et al., 2005). The role of GST in the resistance of tumors is two-fold; by direct neutralization of the drug through conjuga-tion with GSH and by inhibition of the apoptosis-related proteins JNK1 and ASK1 (Adler et al., 1999; Cho et al., 2001). GST π and GST µ suppress via protein:protein interactions JNK1 and ASK1, respectively. The link with the apoptosis-related kinases explains how resistance to drugs that are not subject to conjugation by GSH is mediated by GST.

Expression of high levels of GST pi has been reported in several tumor types, including leukemia cells (Tew, 1994; Zhou et al., 2005). Jurkat cells, which are derived from an ALL patient, over-express GST π and are

resistant to treatment with As2O

3 (Zhou et al., 2005).

Therefore, a therapeutic adjuvant that would inhibit GST activity in ALL cells would contribute to the suc-cess of the treatment. Here, we show the effect of several medicinal plants used in the Brazilian pharmacopoeia on GST expression. Although most studies involving the search for GST inhibitors tested the direct influ-ence of the inhibitor/plant extract on GSH conjugation to an electrophile in vitro (Gyamfi et al., 2004; Merlos et al., 1991; Udenigwe et al., 2007; Zhang & Das, 1994), we opted to test the effect of the crude plant extracts on the expression of GST. This has the obvious advan-tage of testing the effect of the plants further upstream, probably at the level of GST transcription. Besides, the action of GST stimulators cannot be tested in such in vitro assays and do not take into account the likeli-ness of compound metabolism, once the effect of the compound or fraction is tested directly at the level of GST activity.

Most plant extracts tested in this study did not elicit any significant effect on GST expression, but most of the extracts that affected GST did so in a negative way. One exception was H. bonariensis that enhanced GST expression in Jurkat cells, pos-sibly due to the presence of isothiocyanates (Salgues, 1963). Jurkat cells were generally more sensitive than L-37 hepatocytes regarding the effect of the plant extracts on both GST activity and cell viability. The reason is probably related to the fact that unlike Jurkat T-cells, hepatocytes are able to metabolize and neu-tralize many of the compounds present in the extracts due to the presence of P450 cytochromes and phase II detoxifying enzymes.

The cytotoxic or anti-proliferative effects of the extracts was determined both by Trypan blue staining and by measurement of protein concentration in the cell extracts, as in other studies (Digel et al., 2007; Naim et al., 2005). Attempts to measure cell viability by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide] assay failed because the reagent reacted with several plant extracts resulting in the formation of for-mazan even in the absence of cells.

Some of the plant extracts, notably B. forficata (at 10, 100 and 1000 µg/mL) and C. pachystachya (at 10 µg/mL), inhibited GST expression in a significant manner. The former showed no sign of cytotoxicity even when administered at the highest dose (1 mg/mL), and pre-sented a dose-dependent GST inhibition. At this point, we can only speculate about the nature of the active chemical compounds in the crude plant extracts used in this study. A preliminary fractionation of B. forficata and C. pachystachya extracts suggests that polyphenols might be involved. Indeed, several polyphenols, such as tannic acid and ethacrynic acid have been shown to inhibit GST in vitro and B. forficata is reportedly rich in

Table 4. Summary of the effects of the plant extracts on the GST activity of L-37 and Jurkat cells.

Effect on GST activity

Plant extract L-37 Jurkat

Bauhinia forficata NSE -29% (10 µg/mL)a

-37% (100 µg/mL)

-74% (1000 µg/mL)

Cecropia pachystachya -50% (10 µg/mL) -45% (10 µg/mL)

-72% (100 µg/mL)

Eugenia uniflora -41% (10 µg/mL) +73% (1 µg/mL)

+86% (100 µg/mL)

Hydrocotyle bonariensis NSE +58% (10 µg/mL)

+39% (100 µg/mL)

Jacaranda puberula -16% (10 µg/mL) +82% (10 µg/mL)

-35% (100 µg/mL)

Mimosa pudica NSE +80% (10 µg/mL)

Pimenta pseudocaryophyllus

-21% (10 µg/mL) -71% (100 µg/mL)

-45% (100 µg/mL)

Senna multijuga -49% (10 µg/mL) NSEaPercentage of alteration in the level of GST relative to the control. The concentrations of the extracts are in parentheses.NSE, no significant effect.

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the flavonoid kaempferitrin (da Silva et al., 2000; Zhang & Das, 1994). Further fractionation of the crude extracts of these plants would hopefully lead to the identification of a novel GST inhibitor.

Acknowledgements

We thank Selmo Bernardo for helping with the harvest-ing of the plant specimens. This work was supported by the Bryan Gunn's Leukaemia Appeal (UK).

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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