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Journal of Ethnopharmacology 129 (2010) 208–213

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Journal of Ethnopharmacology

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Chemopreventive and anti-cancer properties of the aqueous extractof flowers of Butea monosperma

Tenzin Choedon1, Surendra Kumar Shukla1, Vijay Kumar ∗

Virology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India

a r t i c l e i n f o

Article history:Received 29 October 2009Received in revised form 9 February 2010Accepted 13 March 2010Available online 20 March 2010

Keywords:Butea monospermaHCCHBx-transgenic micePlant extractTUNEL assayMAP kinase

a b s t r a c t

Ethnopharmacological relevance: Butea monosperma (Lam.) (Fabaceae) popularly known as ‘flame of theforest’ has been widely used in the traditional Indian medical system of ‘Ayurveda’ for the treatment ofa variety of ailments including liver disorders.Aim of the study: To evaluate the antioxidative, anti-inflammatory, hepatoprotective and anti-canceractivities of the aqueous extract of Butea monosperma flowers.Materials and methods: Dried flowers of Butea monosperma were extracted with water. The extract wastested for its anti-proliferative, pro-apoptotic and anti-carcinogenic effects in hepatoma cell lines. Thechemopreventive and anti-angiogenic effects of the extract were evaluated by its daily oral administrationin a HBV-related X15-myc mouse model of hepatocellular carcinoma (HCC).Results: Treatment with the aqueous extract inhibited cell proliferation and accumulation of cells in G1phase. This was accompanied by a marked reduction in the levels of activated Erk1/2 and SAPK/JNKand induction of apoptotic cell death. Oral administration of the extract in transgenic mice conferredhepatoprotection as is evident from normal serum ALT levels and improved liver histopathology andlowered serum VEGF level.Conclusions: The ability of aqueous extract of Butea monosperma flowers to impose growth arrest andtrigger pro-apoptotic death in cell culture strongly correlated with its strong chemopreventive effect invivo when given orally.

© 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Butea monosperma (Lam.) (family: Fabaceae) also known asflame of the forest or Palasa in Sanskrit, is a medium sized treefound in most parts of India. In the traditional system of medicineknown as ‘Ayurveda’, Butea monosperma has been used in the treat-ment of a variety of ailments including liver disorders (Burlia andKhadeb, 2007). Nearly every part of Butea monosperma has beenused as tonic, astringent, aphrodisiac and diuretic. About 45 medic-inal uses are associated with Butea monosperma and out of theseclaims almost half of the number of claims have been reported

Abbreviations: AEBM, aqueous extract of Butea monosperma flowers; ALT, ala-nine aminotransferase; DAPI, 4′-6-diamidino-2-phenylindole; DMEM, Dulbecco’smodified Eagle’s medium; ERK, extracellular signal-regulated kinases; FBS, fetalbovine serum; HCC, hepatocellular carcinoma; JNK, Jun-amino-terminal kinase;LFT, liver function tests; MAPK, mitogen activated protein kinase; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; PBS, phosphate-bufferedsaline; SAPK, stress-activated protein kinase; TUNEL, Terminal deoxynucleotidyltransferase dUTP nick end labeling; VEGF, vascular endothelial growth factor.

∗ Corresponding author at: Virology Group, International Centre for Genetic Engi-neering and Biotechnology (ICGEB), P.O. Box 10504, Aruna Asaf Ali Marg, New Delhi110067, India. Tel.: +91 11 26741680; fax: +91 11 26742316.

E-mail address: vijay@icgeb.res.in (V. Kumar).1 Equal first authors.

to be associated with flowers of the plant (Burlia and Khadeb,2007). The main constituent of the flower is butrin (1.5%) besidesbutein (0.37%) and butin (0.04%) (Chokchaisiri et al., 2009). Fla-vanoids butrin (7,3′,4′-trihydroxyflavanone-7,3′-diglucoside) andisobutrin (3,4,2′,4′-tetrahydroxychalcone-3,4′-diglucoside) havebeen shown to be hepatoprotective (Wagner et al., 1986). Its gumextract has shown significant antimicrobial and antifungal activity(Gurav et al., 2008). Antioxidative, anti-inflammatory, hepatopro-tective and chemopreventive properties of different constituents ofButea monosperma have been also reported (Sehrawat and Sultana,2006). In the present study, we have evaluated the anti-cancerpotential of the aqueous extract of Butea monosperma flowers inhepatic cell lines in culture and its chemopreventive efficacy in vivoin a transgenic mouse model of hepatocellular carcinoma (HCC) andshow that the extract has a strong ameliorative effect on cancerboth in vitro and in vivo.

2. Material and methods

2.1. Plant material and aqueous extract

Flowers of Butea monosperma were purchased from thelocal market and were identified by expert botanists, National

0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.jep.2010.03.011

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Institute of Science Communication and Information Resources(NISCAIR), New Delhi. A voucher specimen is deposited atthe Raw Materials Herbarium and Museum of NISCAIR (Ref.No. NISCAIR/RHMD/Consult/2009-10/1226/30). The dry powderedflowers (500 g) were soaked in 3L distilled water and heated onsteam bath for 4 h. The aqueous extract of Butea monosperma flow-ers (AEBM) was filtered and concentrated using a rotary evaporator.Finally, it was then freeze-dried to get dried powder and stored ina desiccator at +4 ◦C until used.

2.2. Chemicals

Caspase 3 and caspase 9 antibodies were purchased fromSanta Cruz, CA. Propidium iodide, Silibinin, DMSO, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)and Methanol reagent were purchased from Sigma Chemical Co.(St Louis, MO). Dulbecco’s modified Eagle’s medium (DMEM), fetalbovine serum (FBS), Streptomycin and Penicillin were purchasedfrom Gibco BRL. VEGF quantikine ELISA kit was procured fromR&D system (Minneapolis, USA), TUNEL assay kit from PromegaCorporation, Madison, USA.

2.3. Cell lines

Human hepatoma cells Huh7 (Nakabayashi et al., 1982), HepG2cells (HB-8065) and AML12 cells (immortalized mouse hepato-cytes, CRL 2254) were purchased from ATCC. All cultures weremaintained in DMEM supplemented with 10% FBS, Penicillin(100 �g/ml) and Streptomycin (100 �g/ml) incubated at 37 ◦C ina humidified chamber, 5% CO2 atmosphere.

2.4. Cell viability assay

MTT assay was performed according to manufacturer’s protocol.Cells were seeded at a density of 0.6 × 106 cells in 60 mm dish for24 h and then treated with AEBM or reference drug Silibinin (posi-tive control). After 48 h, cells were incubated with MTT for 45 minat 37 ◦C in dark. Formazan crystals were solubilized in dimethyl sul-foxide and the absorbance was recorded at 560 nm. Untreated cellswere used as control of viability (100%). The mean absorbance val-ues of three experiments were expressed as percentage of viabilityin relative to control.

2.5. Flow cytometric analysis of cell cycle distribution

Cell cycle analysis was performed by the method described pre-viously (Mukherji et al., 2007). Briefly, cells were washed in PBS,fixed in 70% ethanol and stained with propidium iodide (50 �g/ml).Cell suspension was analyzed with a FACS Calibur BD Bioscience,San Jose, CA. The percentages of cell cycle distribution were deter-mined by using FlowJo software.

2.6. Western blot analysis

Aliquots of samples with 70 �g of protein (determined by Brad-ford method) were fractionated in 12% SDS-PAGE and transferredto 0.45 �m nitrocellulose by wet transfer. The membranes wereblocked with 5% fat free milk in PBS and then probed with primaryantibody followed by secondary antibody. After washing 3 times inPBST, the membranes were visualized using enhanced chemilumi-nescence Western blotting detection system (Santa Cruz, CA).

2.7. Animal tumor model

Development of the HBV-related transgenic mouse model (X15-myc) of HCC has been described earlier (Lakhtakia et al., 2003).

The transgene-positive mice was identified by polymerase chainreaction using tail DNA biopsies and the 4–6 weeks old pupswere further used for screening of different fractions of drug forits anti-cancer property. Mice were housed in humidity (30–70%)and temperature (23 ± 2 ◦C). Necessary ethical clearance was takenfrom the Institutional Animal Ethics Committee for doing animalexperiments. Animals were divided into two groups – Group I(400 mg/kg) and Group II (100 mg/kg) and treated daily (five daysper week) by oral administration of AEBM as described in Choedonet al. (2006). Silibinin – a well known hepatoprotective agent (Ranaet al., 2005) – was used as a positive control at two doses: Group I– 100 mg/kg and Group II – 25 mg/kg and also administered orallyas mentioned above.

2.8. Assessment of liver functions and histopathology

For liver function assessment blood was collected from exper-imental mice of all groups by retro orbital bleeding. Serum wasseparated by centrifugation at 3000 rpm for 10 min. The activity ofthe enzyme alanine aminotransferase (ALT) – a well known serumbiomarker of hepatotoxicity (Ozer et al., 2008) was measured byautoanalyzer. Histopathology of liver samples was assessed, usinghematoxylin and eosin staining of paraffin-embedded liver tissuesections. Tissues were fixed in 10% buffered formalin for 24 h andwere processed for paraffin embedding and sectioning (5 �m thick-ness), then processed for hematoxylin and eosin (H/E) staining.

2.9. TUNEL assay

Apoptosis was detected in paraffin-embedded liver sections byTUNEL staining using DeadEndTM Fluorometric TUNEL kit. Paraffin-embedded sectioned tissue was processed for TUNEL labeling.Assay was done as per manufacturer’s protocol. Each tissue sec-tion was fixed in PBS containing 4% paraformaldehyde for 15 minat 25 ◦C, washed with PBS and then processed for TUNEL labeling.The images were obtained using confocal microscopy (Nikon A1R).Colors were assigned as fluorescein UTP (green) and DAPI (blue).

2.10. VEGF estimation

Levels of the angiogenic cytokine VEGF were measured in theserum samples of experimental mice as per manufacturer’s proto-col (R& D system, Minneapolis, USA).

2.11. Statistical analysis

Results were expressed as Means ± S.D. and all statistical com-parisons were made by Student’s ‘t’ test and p values less than orequal to 0.05 were considered significant.

3. Results

3.1. Effect of AEBM on cell proliferation

The growth inhibition property of AEBM was evaluated in threedifferent cell lines, viz. HepG2, Huh7 and AML12. Cells were treatedwith increasing concentration (0.1–1 mg/ml) of AEBM and cell via-bility was measured after 48 h. As shown in Fig. 1, AEBM treatmentled to a significant decrease in cell viability (p < 0.001) and 50%inhibition was observed at 0.1 mg/ml in both Huh7 and HepG2cells. Interestingly, under these conditions, little or no change inthe viability of AML12 cells was observed.

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Fig. 1. Effect of AEBM on viability of hepatoma cells. Huh7, HepG2 and AML12 cellswere treated with increasing concentrations (0.1, 0.3 and 1 mg/ml) of AEBM for 48 hand cell viability was measured by MTT assay. Per cent survival was determined ascompared to untreated cells. The difference in cell viability between untreated andAEBM-treated Huh7 and HepG2 cells were found to be highly significant (p < 0.001).

3.2. Effect on cell cycle

Since AEBM exhibited a strong anti-proliferative activity, wenext analyzed the regulation of cell cycle in the presence of AEBMby flow cytometry. A dose-dependent growth inhibition of Huh7cells was observed with majority of cells (∼80%) arrested in G1phase at 0.9 mg/ml (Fig. 2A). Silibinin was found to be relativelyless effective.

3.3. Effect on apoptotic pathway

To understand the molecular mechanism of AEBM-inducedgrowth inhibition, we measured the intracellular levels of pro-caspases. There was a marked increase in the activation of bothcaspase 3 and caspase 9 (∼2 fold) as evident from decrease in thelevels of respective pro-caspases (Fig. 2B and C) suggesting that cas-pase dependent apoptotic death could be another mechanism forthe beneficial effects of Butea monosperma. We further evaluatedthe pro-apoptotic effects of AEBM in the liver of treated X15-mycmice by TUNEL assay. The liver of these mice showed a dramaticincrease in the labeling (∼3 fold) as compared to untreated trans-genic mice or non-transgenic littermates (Fig. 2D). Silibinin alsoexhibited a strong pro-apoptotic effect (Fig. 2D).

3.4. Effect on activated MAPK and SAPK/JNK

Since MAPK and SAPK/JNK pathways are involved in cell pro-liferation and tumor development, we now measure the levels of

Fig. 2. Effect of AEBM on cell cycle progression and apoptotic cell death. (A) Huh7 cells were grown in the presence of AEBM (0.3, 0.6, 0.9 mg/ml) or Silibinin (0.05, 0.1,0.15 mg/ml) for 48 h. After staining with propidium iodide, the cell cycle analysis was done by flow cytometry; (B) Huh7 cells were treated with AEBM for 48 h and the levelsof pro-caspase 3 and pro-caspase 9 were determined by Western blot; (C) quantitative data of panel B. The level of significance (p < 0.005) is shown as *; (D) the X15-myctransgenic (Tg) mice were treated orally with AEBM or Silibinin for 12 months and the liver sections were analyzed for apoptosis by TUNEL assay. The data is presented asbar graph. Levels of significance (as against Tg control): *, p < 0.005; and **, p < 0.01.

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Fig. 3. Effect of AEBM on signaling kinases. (A) Cells were treated with AEBM (1 mg/ml) for 24 h and the levels of total and phosphorylated ERK and JNK were measured byWestern blot analysis; (B) bar graph showing levels of pJNK and pERK in the control and AEBM-treated cells (n = 3). The levels of significance are indicated.

activated kinases post-AEBM treatment. As shown in Fig. 3, treat-ment of Huh7 cells with AEBM led to a dramatic (∼3 fold) decreasein the levels of both phosphorylated ERK and JNK and further sub-stantiated its anti-cancerous property.

3.5. In vivo effect on liver histopathology and liver function

Next we examined the effect of AEBM treatment on thehistopathological changes in the liver of X15-myc mice. As shown inFig. 4A, there was a dramatic reversal in tissue architecture by AEBMjust as with Silibinin control. In contrast, the untreated control miceshowed more vascularized liver with distorted architecture.

In the assessment of individual hepatic function in cancerpatient, liver function tests (LFT) represent a broad range of normalfunctions performed by the liver. LFT are usually abnormal in HCCcases with elevated blood levels of ALT due to excessive liver dam-age. Analysis of ALT levels in both treatment groups of mice showeda dramatic reduction in its levels (p < 0.001) indicating ameliorativeeffects of Butea monosperma against HCC and restoration of normalliver physiology (Fig. 4B).

3.6. Anti-angiogenic effect of AEBM

Since VEGF is considered as an important marker of angiogen-esis and tumor proliferation, we now measure the serum levels ofVEGF in the AEBM-treated X15-myc mice. As shown in Table 1, asignificant reduction in VEGF levels (∼2 fold) was observed in bothtreatment groups of mice across all age groups suggesting a stronganti-angiogenic property of AEBM as well as Silibinin.

Table 1Serum VEGF levels in the AEBM-treated X-15-myc mice.

Treatment group/age Control AEBM Silibinin

Group I6 months 1004.3 ± 265.9 561.7 ± 201.7b 478.9 ± 129.7b

9 months 984.8 ± 155.2 490.7 ± 144.8b 260.0 ± 123.0a

12 months 945.5 ± 82.6 455.3 ± 96.7a 261.9 ± 12.9a

Group II6 months 1004.3 ± 265.9 513.4 ± 275.1b 413.5 ± 154.3b

9 months 984.8 ± 155.2 476.0 ± 195.0b 525.3 ± 142.1b

12 months 945.5 ± 82.6 482.9 ± 41.8b 473.7 ± 46.5b

Values (pg/ml) are expressed as mean ± SEM of three independent observations.Level of significance — a: p < 0.001; b: p = 0.01.

4. Discussion

In the Indian traditional system of medicine, a number ofmedicinal uses are known to be associated with flowers of Buteamonosperma (Burlia and Khadeb, 2007). Here we have studied theanti-cancer properties of the aqueous extract of Butea monospermaflowers on growth inhibition and induction of apoptotic death ofhepatoma cells and looked for its ameliorative effects in a trans-genic model of liver cancer.

Induction of apoptosis is considered as one of the key mech-anisms for the targeted therapy of various cancers (Constantiniet al., 2000). Working on AEBM, we found that while it dramati-cally reduced the viability of hepatoma cells, the non-transformedhepatocytes remained unaffected (Fig. 1) thus, scoring an impor-tant point in favor of developing Butea monosperma as a drug ofchoice. Analysis of the AEBM-induced growth inhibition and apop-totic death of hepatoma cells suggested their arrest in G1 phase(Fig. 2A) as reported previously for Silibinin (Rana et al., 2005). Itmay be argued that inhibition of deregulated cell cycle progres-sion in cancer cells could be an effective strategy to control tumorgrowth (Carnero, 2002).

It is now well established that activation of caspases lead todegradation of cellular proteins, cell shrinkage, DNA fragmenta-tion, loss of plasma membrane potential and membrane blebbing(Nicholson, 1999). The pro-apoptotic effect of AEBM (Fig. 2B andC) was evident not only in cell culture but also in vivo. Accord-ingly, we observed a significant increase in tumor cell death in liverwhen AEBM was administered orally in transgenic mice (Fig. 2D).This imposition of growth arrest and apoptotic cell death alsocorrelated with down-regulation of MAP kinase and SAPK/JNKsignaling pathways (Fig. 3) which are considered important forthe management of cancer and other chronic diseases (Kaminska,2005).

Our in vivo studies in the X15-myc mice suggested that AEBMcarries a strong hepatoprotective and anti-cancer activity similar to“Silymarin” – a well known herbal drug (Pradhan and Girish, 2006).We observed a marked improvement in the tissue architecture andnormalization of liver vasculature, and lowering of serum ALT levelsof these mice (Fig. 4). Reportedly, anti-inflammatory drugs that areknown to regulate ALT levels can prolong the recurrence intervals ofdisease in HCC patients (Tarao et al., 2000). Further, normalizationof serum VEGF levels in the AEBM-treated mice (Table 1) also cor-roborated its potential as an anti-cancer agent (Finn and Zhu, 2009;Kaseb et al., 2009). Thus, the hepatoprotective and anti-angiogenicproperties of the Butea monosperma extract are likely to have signif-

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Fig. 4. Hepatoprotective effect of AEBM in the X15-myc transgenic mice. (A) Groups of four to six-week-old mice (n = 6) were treated orally with AEBM or Silibinin for 12months and the liver samples were examined for histopathological changes in H/E stained micro-slides (magnification, 200×); (B) blood samples of the treated mice werecollected at 6, 9 and 12 months of age and the serum levels of ALT was measured using an autoanalyzer. Silibinin-treated and -untreated transgenic mice were used ascontrols. Values are presented as mean ± SEM (n = 5).

icant implications in cancer therapy similar to Silymarin (Pradhanand Girish, 2006).

5. Conclusion

Our present findings suggest that aqueous extract of Buteamonosperma flowers exhibited a strong anti-cancer activity(growth inhibition, cell cycle arrest, pro-apoptotic activity andinterference with mitogenic signaling) in hepatoma cells and showsminimal cytotoxic effect on non-transformed AML12 hepatocytes.These results warranted an in vivo study with the extract ina genetic mouse model of liver cancer which showed a clearhepatoprotective and anti-angiogenic effects. Thus, the benefi-cial effects of the aqueous extract of Butea minosperma flowersmakes a strong case for developing novel therapeutics againstcancer.

Acknowledgements

The authors acknowledge the Council of Scientific and IndustrialResearch (CSIR), New Delhi, India for fellowship of SK Shukla. Thiswork was supported by the core grant of International Centre forGenetic Engineering and Biotechnology, New Delhi.

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