Anticancer Plants Used in Bangladesh

Journal of Ethnopharmacology 99 (2005) 21–30

Studies of the anticancer potential of plants used inBangladeshi folk medicine

Letıcia Veras Costa-Lotufoa,∗, Mahmud Tareq Hassan Khanb,1, Arjumand Atherc,Diego Veras Wilkea, Paula Christine Jimeneza, Claudia Pessoaa,

Maria Elisabete Amaral de Moraesa, Manoel Odorico de Moraesa

a Department of Physiology and Pharmacology, School of Medicine, Federal University of Cear´a, Fortaleza Rua Cel Nunes deMelo 1127, Caixa Postal-3157, 60430-270 Fortaleza Ce, Brazil

b Pharmacology Research Laboratory, Faculty of Pharmaceutical Sciences, University of Science and Technology Chittagong,Foy’ lake, Chittagong, Bangladesh

c Plant Biotechnology Division, HEJ Research Institute of Chemistry, University of Karachi, Karachi, Pakistan

Received 1 November 2003; received in revised form 10 January 2005; accepted 19 January 2005Available online 11 March 2005

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bstract

The present study evaluated the anticancer potential of 11 plants used in Bangladeshi folk medicine. The extracts were tested forsing the brine shrimp lethality assay, sea urchin eggs assay, hemolysis assay and MTT assay using tumor cell lines. The extractOroxylumndicumshowed the highest toxicity on all tumor cell lines tested, with an IC50 of 19.6�g/ml for CEM, 14.2�g/ml for HL-60, 17.2�g/ml for-16 and 32.5�g/ml for HCT-8. On the sea urchin eggs, it inhibited the progression of cell cycle since the frist cleavage (IC50 = 13.5�g/ml).he extract ofAegle marmelosexhibited toxicity on all used assays, but in a lower potency thanOroxylum indicum. In conclusion, amonll tested extracts, only the extracts ofOroxylum indicum, Moringa oleiferaandAegles marmeloscould be considered as potential sourf anticancer compounds. Further studies are necessary for chemical characterization of the active principles and more extensivvaluations.2005 Elsevier Ireland Ltd. All rights reserved.

eywords: Oroxylum indicum; Bangladeshi folk plants; Cytotoxicity

. Introduction

The use of natural products as anticancer agents has aong history that began with folk medicine and throughhe years has been incorporated into traditional and allo-athic medicine. Several drugs currently used in chemother-py were isolated from plant species or derived from aatural prototype. They include theVinca alkaloids, vin-lastine and vincristine, isolated fromCatharanthus roseus,

∗ Corresponding author. Tel.: +55 85 3288 8255; fax: +55 85 3288 8333.E-mail addresses:[email protected] (L.V. Costa-Lotufo),

[email protected] (M.T.H. Khan).1 Present address: Department of Biochemistry and Molecular Biology,enter for Biotechnology, University of Ferrara, Ferrara 44100, Italy.

etoposide and teniposide, the semisynthetic derivativeepipodophyllotoxin, isolated from species of the gePodophyllum, the naturally derived taxanes isolated frspecies of the genusTaxus, the semisynthetic derivativescamptothecin, irinotecan and topotecan, isolated fromCamp-totheca acuminata, and several others (Cragg et al., 19931994; Wang, 1998). According toCragg and Newman (2000,over 50 % of the drugs in clinical trials for anticancertivity were isolated from natural sources or are relatethem.

Most of the research performed today focuses on thvelopment of new drugs to treat cancer, as well as viramicrobial infections. The present study aims to provideon the cytotoxic potential of 11 extracts from Bangladeplants belonging to different families (Table 1) on developing

378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2005.01.041

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Table 1List of plants species used in this study

Plant species (family) voucher no. Time ofcollection

Area of collection Dry weight(in kg)

Part used, extraction process andyield (%)

Some reported pharmacologicalactivies

References

Aegle marmelos(L.) Correa.(Rutaceae) EPL-106

May 1996 Chittagong Hilltract, Bangladesh

31.0 Stem bark; 100% ethanol (cold)partition: ether (9.9)

Antibacterial Rani and Khullar (2004)

Anti-enteric potential Rani and Khullar (2004)NO scavenging activity Jagetia and Baliga (2004)Antioxidant Kamalakkannan and Prince

(2003)Antidiabetic Sabu and Kuttan (2004), Saxena

and Vikram (2004)Radioprotective Jagetia et al. (2004)Antiviral activity Badam et al. (2002)Antiproliferative Khan et al. (2002), Lambertini et

al. (2004), Lampronti et al.(2003)

Inhibits estrogen receptor Lambertini et al. (2004)Antidiarrheal Shoba and Thomas (2001)Antiulcer activity Goel et al. (1997)Antifungal activity Rana et al. (1997)

Aphanamixis polystachya(Wall.)R. Parker (Meliaceae)GDK-8913

July 1996 Tangail,Bangladesh

200.0 Stem bark; 80% ethanol:water(cold) (11.8)

Antihepatotoxic Gole and Dasgupta (2002)

Oil (2.5) and solid (8.9) Antiproliferative Khan et al. (2002)Cuscuta reflexaRoxb.

(Convolvulaceae) BC-2411August1996

Chittagong,Bangladesh

3.5 Whole plant; Absolute ethanol(cold) (10.3)

Anti-steroidogenic Gupta et al. (2003)

Analgesic Pal et al. (2003)Antiproliferative Khan et al. (2002)

9(2005)21–30

Antiviral Awasthi (1981)Emblica officinalisGaertn.

(Euphorbiaceae) BC-3823May 1996 Chittagong Hill

tract, Bangladesh25.0 Fruit; 100% ethanol (cold) (15.0) Antiproliferative Khan et al. (2002), Lambertini et

al. (2004),Nemmani et al. (2002)Antipyretic and analgesic Perianayagam et al. (2004)Antiepileptic Achliya et al. (2004a)Hepatoprotective Achliya et al. (2004b)Against oxidative stress Rajak et al. (2004), Bhattacharya

et al. (2002)Esterogen receptor inhibitor Lambertini et al. (2004)Immunomodulator Ganju et al. (2003)Antitussive activity Nosal’ova et al. (2003)Apoptocic on cancer cells Rajeshkumar et al. (2003)Anticarcinogenic Rajeshkumar et al. (2003)Cytoprotective Sai Ram et al. (2003), Sai Ram et

al. (2002a)Antimutagenic Jena et al. (2003), Kaur et al.

(2002)

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Table 1 (Continued)





References

Antioxidant Bhattacharya et al. (2000),Chaudhuri (2002), Sabu andKuttan (2002)

Gastroprotective Al-Rehaily et al. (2002)Antiulcerogenic Sai Ram et al. (2002b)Antidiabetic Sabu and Kuttan (2002)Antihyperlipidemic Augusti et al. (2001)Against ophthalmic disorders Biswas et al. (2001)

Hemidesmus inidicus(L.) Br. exSchult. (Asclepiadaceae)BC-1083

February1996

Dhaka, Bangladesh 2.0 Root; 80% ethanol:water (cold)(8.9)

Antidiarrheal activity Das et al. (2003)

Inhibit hepatocarcinogenesis Iddamaldeniya et al. (2003)Antiatherogenic Mary et al. (2003b)Antioxidant and antithrombotic Mary et al. (2003a)Anti-ulcerogenic Anoop and Jegadeesan (2003)Antiinflammatory Jain and Basal (2003)

Moringa oleiferaLam.(Moringaceae) BCS-4575

November1996


25.0 Root; 100% ethanol (cold) (14.5),partition: hexane (7.4),chloroform (5.9), methanol (9.5),water (9.5)

Inhibits central neurotransmitters Ray et al. (2003)

Anti-HSV-1 Lipipun et al. (2003)Antioxidant Kumar and Pari (2003),

Siddhuraju and Becker (2003)Chemomodulator Bharali et al. (2003)Hypocholesterolaemic Mehta et al. (2003)Hypoglycaemic Kar et al. (2003)Hepatoprotective Pari and Kumar (2002)
(2
005)21–30

23

Antimicrobial activities Suarez et al. (2003)Antiplasmodial activities Kohler et al. (2002)Radioprotective effect Rao et al. (2001)

Nigella sativaL. (Ranunculaceae)SDK-84

January1996


12.0 Seeds; ethanol 100%(hot-soxhlet) (25.4) partition:ether (7.7)

Triggers apoptosis in cancer cells Gali-Muhtasib et al. (2004)

Lipid-lowering Le et al. (2004)Insulin-sensitizing Le et al. (2004)Antidiabetic Kanter et al. (2004), Fararh et al.

(2004)Immunopotentiating Fararh et al. (2004)Immunosuppressive Islam et al. (2004)Cytotoxic Islam et al. (2004)Protects nephrotoxicity Ali (2004)Analgesic Hajhashemi et al. (2004)

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Table 1 (Continued)





References

Anti-inflammatory Hajhashemi et al. (2004)Tracheal relaxant Boskabady et al. (2004)Lipid metabolic Morikawa et al. (2004a),

Morikawa et al. (2004b)Anticonvulsant Hosseinzadeh and Parvardeh

(2004)Liver protectant Al-Ghamdi (2003)Anti-allergic Kalus et al. (2003)

Oroxylum indicum(L.) Kurz(Bignoniaceae) EPL-0978

February1996


10.0 Stem bark; 100% ethanol(hot-soxhlet) (15.2)

Antiproliferative Khan et al. (2002), Tepsuwan etal. (1992)

Against breast cancer Lambertini et al. (2004)Estrogen receptor inhibitor Lambertini et al. (2004)Antioxidant Jiwajinda et al. (2002)Anti-arthritic Laupattarakasem et al. (2003)Antimutagenic Nakahara et al. (2002), Nakahara

et al. (2001), Tepsuwan et al.(1992)

Paederia foetidaL. (Rubiaceae)BNH-25372

May 1996 Chittagong,Bangladesh

8.0 Whole plant; ethanol 100%(cold) (11.9)

Anti-inflammatory De et al. (1994)

Anti-shigellosis Haider et al. (1991)Anthelmintic Roychoudhury et al. (1970)

Polyalthia longifolia(Sonn.)Thwaites (Annonaceae)GFS-738

February1996

Chittagong,Bangladesh 2.1 Stem bark; 80% ethanol:water(6.9)

Cytotoxic activity Wu et al. (1990), Zhao et al.(1991), Chen et al. (2000)

Antimicrobial activity Faizi et al. (2003a), Faizi et al.

9(2005)21–30

(2003b)Tribulus terrestris L.

(Zygophyllaceae) EPL-012August1996

Dhaka, Bangladesh 4.0 Fruit; 100% ethanol (hot-soxhlet)(17.9)

Antihypertensive Sharifi et al. (2003)

Sexual effects Gauthaman et al. (2003)Inhibition of breast cancer Sun et al. (2003)Diuretic activity Al-Ali et al. (2003)Anthelmintic Deepak et al. (2002)Hypoglycemic Li et al. (2002)COX-2 inhibitor Hong et al. (2002)Anticancer Bedir et al. (2002), Ali et al.

(2001)Antifungal Bedir et al. (2002)Aphrodiasic Gauthaman et al. (2002)Antibacterial Ali et al. (2001)

All voucher specimens have been deposited in the following herbaria: BCSIR Laboratory; Jahangirnagar University at Dhaka; and Bangladesh NationalHerbarium.

L.V. Costa-Lotufo et al. / Journal of Ethnopharmacology 99 (2005) 21–30 25

embryos of sea urchins, on tumor cell lines, erythrocytes andbrine shrimp nauplii.

2. Materials and methods

2.1. Preparation of crude extract

The roots ofHemidesmus indicusand the stem barks ofPolyalthia longifoliaandAphanamixis polystachyawere ex-tracted with 80% ethanol:water in cold extraction process.The first plant yielded 8.9% extract, the second, 6.87%, andthe third, which resulted in two extracts, an oily phase and asolid mass, yielded 2.54 and 8.91%, respectively. The stembark ofOroxylum indicumthe, fruits ofTribulus terrestrisandthe seeds ofNigella sativawere extracted in a soxhlet appa-ratus using absolute ethanol as solvent. The first two speciesyielded 15.24 and 17.94% extracts, respectively. The ex-tract ofNigella sativawas further partitioned in diethyletherand yielded 7.65%. The other plants,Cuscuta reflexa(wholeplant), Paederia foetida(whole plant),Emblica officinalis(fruits),Moringa oleifera(roots), andAegle marmelos(stembark), were extracted with absolute ethanol in a cold extrac-tion process.Cuscuta reflexa, Paederia foetidaandEmblicaofficinalisyielded 10.32, 11.92 and 15.0% extracts, respec-tively. The crude extract ofMoringa oleifera, which yielded1 ),c %),ai sw e re-c d asn

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urchins (Lytechinus variegatus) were collected at Pecembeach, on the northeastern coast of Brazil. The gamete elimi-nation was induced by injecting 3.0 ml of 0.5 M KCl into theurchins coelomic cavity via the periostomial membrane. Theeggs were washed twice using filtered seawater to removethe jelly coat surrounding the cells. Concentrated sperm wascollected with a Pasteur pipette and maintained under lowtemperatures until the moment of fertilization. For fertiliza-tion, 1 ml of a sperm suspension (0.05 ml of concentratedsperm in 2.45 ml of filtered seawater) was added to every50 ml of egg solution. Each well received 1 ml of fertilizedegg suspension. The extracts were added immediately afterfertilization (within 2 min) to get concentrations of 10, 30,100, 300 and 1000�g/ml in a final volume of 2 ml. Doxoru-bicin (0.058–58.0�g/ml) was used as positive control. Theplates were then shaken on a constant temperature water bathat 26± 2◦C. At appropriate intervals, aliquots of 200�l werefixed in the same volume of 10% formaldehyde to obtain firstand third cleavages, and blastulae. One hundred eggs or em-bryos were counted for each concentration of test substanceto obtain the percentage of normal cells.

2.4. MTT assay

The cytotoxicity of the extracts was tested against B-1 a),C n’sM ul-t etalc d1 ,c -eoi for3 nt ofD ec bil-i -2-t abi reshm rsl lvedi plater effectw ce ofr

2

them lr MC nedo the

4.54%, was further partitioned inn-hexane (7.39% yieldhloroform (5.94%), methanol (9.46%) and water (9.48nd the extract derived fromAegle marmeloswas partitioned

n diethylether (9.87% yield) (Khan et al., 2002). The extractere dried in a rotary vacuum evaporator and the residuonstituted in DMSO before testing. The vehicle was useegative control.

All of these plants were collected from various locies, including Chittagong, Chittagong Hill Tract, Tangail ahaka, of Bangladesh, in different months of 1996.Table 1hows species used in this study and the location of thosit of their respective voucher specimens.

.2. Brine shrimp assay

Brine shrimp (Artemia salinaLeach) eggs were hatchn a becker filled with seawater under constant aerafter 48 h the nauplii were collected by pipette and wounted macroscopically in the stem of the pipette aglighted background. Ten nauplii were transferred toell of 24-multiwell plates containing the samples. Txtract concentration ranged from 10 to 1000�g/ml. Thelates were maintained under illumination. Survivors wounted after 24 h of incubation and the percentageaths at each dose and control (seawater plus vehicleetermined (Meyer et al., 1982).

.3. Assay on sea urchins

The test was performed in 24-well plates followingethod described byCosta-Lotufo et al. (2002). Adult sea

6 (murine melanoma), HCT-8 (human colon carcinomEM and HL-60 (leukemia) tumor cell lines (Childreercy Hospital, Kansas City, MO, USA). Cells were c

ured in RPMI-1640 medium, supplemented with 10% falf serum, 2 mM glutamine, 100�g/ml streptomycin an00 U/ml penicillin at 37◦C with 5% CO2. For experimentsells were plated in 96-well plates (105 cells/well for adhernt cells or 0.3× 106 cells/well for suspended cells in 100�lf medium). After 24 h, the extracts (2–125�g/ml) dissolved

n DMSO (1%) was added to each well and incubateddays (72 h). Control groups received the same amouMSO. Doxorubicin (0.01–0.58�g/ml) was used as positivontrol. Growth of tumoral cells was quantitated by the a

ty of living cells to reduce the yellow dye 3-(4,5-dimethylhiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) tolue formazan product (Mosmann, 1983). At the end of 72 h

ncubation, the medium in each well was replaced by fedium (200�l) containing 0.5 mg/ml of MTT. Three hou

ater, the formazan product of MTT reduction was disson DMSO, and absorbance was measured using a multi-eader (Spectra Count, Packard, Ont., Canada). Drugas quantified as the percentage of control absorban

educed dye at 550 nm.

.5. Hemolytic assay

The test was performed in 96-well plates followingethod described byCosta-Lotufo et al. (2002). Each wel

eceived 100�l of 0.85% NaCl solution containing 10 maCl2. The first well was the negative control that containly the vehicle (distilled water or DMSO 10%), and, in

26 L.V. Costa-Lotufo et al. / Journal of Ethnopharmacology 99 (2005) 21–30

second well, 100�l of test substance that was diluted in halfwas added. The extracts were tested at concentrations rang-ing from 10 to 2500�g/ml. The serial dilution continued untilthe 11th well. The last well received 20�l of 0.1% Triton X-100 (in 0.85% saline) to obtain 100% hemolysis (positivecontrol). Then, each well received 100�l of a 2% suspen-sion of mouse erythrocytes in 0.85% saline containing 10 mMCaCl2. After incubation at room temperature for 30 min andcentrifugation, the supernatant was removed and the liberatedhemoglobin was measured spectroscopically as absorbanceat 540 nM.

2.6. Statistical analysis

Data are presented as mean± S.E.M. The IC50 or EC50values and their 95% confidence intervals (CI 95%) were ob-tained by nonlinear regression using the GRAPHPAD pro-gram (Intuitive Software for Science, San Diego, CA).

LC50 on brine shrimp was obtained from the 24 h countsusing the probit analysis method described byFinney (1971).

3. Results

3.1. Brine shrimp assay

ts1

3

as-s s theo nI hind e ex-

tract obtained fromAegle marmeloswas also strongly active,inhibiting the first cleavage with an IC50 of 50.5�g/ml, thethird cleavage with an IC50 of 22.7�g/ml, and the blastu-lae with an IC50 of 13.2�g/ml. As it can be observed fromthe IC50 values, its efficacy increased with the time of con-tact with the embryos. The extracts obtained fromMoringaoleifera,Emblica officinalis,Aphanamixis polystachya(solidresidue from the ethanolic extract),Cuscuta reflexa, TribulusterrestrisandNigella sativaalso possessed inhibitory activ-ity on the sea urchin egg development, but with a moderateefficacy. The other tested extracts were inactive in this assay.

3.3. MTT assay

The cytotoxicity on the tumor cell lines was evaluated us-ing the MTT assay and the results are presented inTable 3.The extract obtained fromOroxylum indicumwas the mostactive in this assay, exhibiting lower IC50 values againstall tested cell lines, 14.2�g/ml for HL-60, 19.6�g/ml forCEM, 17.2�g/ml for B-16 and 32.5�g/ml for HCT.8. Theextracts obtained fromAegle marmelos, Moringa oleifera,Aphanamixis polystachya(solid residue from the ethanolicextract),Cuscuta reflexa, Emblica officinalis, Tribulus ter-restrisandNigella sativaalso possessed cytotoxic activity,mainly against drug-sensitive lines like the leukemias (HL-6 theyw tede

3

re-l d fort ultsoT -t -

TI sea urcLy s

S (�g/ml

DA )AA )C 56.3)E )HM )N 4.5)O )PPT 56.3)

T s 95 % n. ND:n nsidere

The extract ofAegle marmeloswas the only one thahowed a positive result in this assay, presenting a LD50 of7.5± 2.0�g/mL.

.2. Assay on sea urchin embryos

Table 2shows the results for the sea urchin embryoay using the plant extracts. The most active extract wane obtained fromOroxylum indicum, which presented a

C50 around 10�g/ml in all tested phases of the sea urcevelopment, first and third cleavages, and blastulae. Th

able 2nhibition of the cell cycle by the plant extracts on the embryos of the

pecies Embryonic stage IC50

First cleavage

oxorubicin 6.3 (4.3–9.1)egle marmelos 50.5 (41.0–62.3)phanamixis polystachyaoil >1000phanamixis polystachyasolid 413.0 (156.4–1096)uscuta reflexa 386.5 (168.7–885.4)mblica officinalis 400.1 (ND)emidesmus indicus >1000oringa oleifera 169.4 (155.2–184.9)igella sativa 425.3 (ND)roxylum indicum 13.5 (12.4–14.7)aederia foetida >1000olyalthia longifolia >1000ribulus terrestris 404.8 (155.6–1056)

he extract was added 2 min after fertilization of the eggs. The IC50 and itot determined. Extracts with an IC50 value lower than 100�g/ml were co

0 and CEM) and the melanoma. However, generally,ere not able to block HCT-8 proliferation. The other tesxtracts were inactive in this assay.

.4. Hemolytic assay

In order to verify whether the observed cytotoxicity isated to membrane disruption, plant extracts were testeheir ability to induce lysis of mouse erythrocytes. The resbtained from the hemolytic assay are presented inTable 4.he extract obtained fromNigella sativawas the most ac

ive in this assay (EC50 = 116.8�g/ml), followed by the ex

hintechinus variegatuson the first and third cleavage and blastulae stage

)

Third cleavage Blastulae

0.3 (0.2–0.7) 0.5 (0.3–1.1)22.7 (20.1–25.6) 13.2 (11.1–15.6

>1000 >100078.9 (50.5–91.9) 40.1 (24.7–65.1400.3 (230.3–695.7) 406.0 (268.5–6

125.9 (ND) 123.4 (32.5–468.3>1000 >1000

119.5 (74.7–190.9) 52.5 (41.0–67.3243.2 (193.5–305.8) 363.1 (303.4–4013.0 (11.8–14.4) 11.5 (10.4–12.7

>1000 >1000>1000 >1000195.6 (169.8–225.3) 393.9 (236.4–6

confidence interval (CI 95%) were obtained by non-linear regressiod active.


Table 3In vitro cytotoxicity of the plants extracts on four tumor cell lines measured by the MTT assay

Species Cell line IC50 (�g/ml)

HL-60 CEM HCT-8 B-16

Doxorubicin 0.02 (0.01–0.02) 0.02 (0.01–0.02) 0.04 (0.03–0.05) 0.03 (0.02–0.04)Aegle marmelos 46.4 (42.5–50.8) 28.6 (23.2–35.3) >125 23.8 (20.3–28.0)Aphanamixis polystachyaoil >125 >125 >125 >125Aphanamixis polystachyasolid 87.2 (70.2–108.3) 73.7 (65.9–82.3) >125 78.6 (55.4–111.5)Cuscuta reflexa 55.0 (48.0–63.1) 51.5 (44.8–59.3) 79.4 (52.3–120.7) >125Emblica officinalis 70.4 (55.6–89.2) 33.2 (26.9–40.9) 66.5 (50.7–87.2) 63.3 (55.3–72.5)Hemidesmus indicus >125 >125 >125 >125Moringa oleifera 60.0 (59.2–61.4) 12.7 (8.4–19.4) 113.8 (ND) 28.8 (ND)Nigella sativa 68.7 (ND) 61.0 (ND) 112.8 (ND) 81.1 (32.0–205)Oroxylum indicum 14.2 (10.9–18.5) 19.6 (17.8–21.6) 32.5 (ND) 17.2 (15.3–19.3)Paederia foetida >125 >125 >125 >125Polyalthia longifolia >125 >125 >125 >125Tribulus terrestris 39.8 (28.7–55.2) 52.4 (44.4–61.8) >125 89.9 (52.7–152.8)

The IC50 and its 95 % confidence interval (CI 95%) were obtained by non-linear regression. ND: not determined. Extracts with an IC50 value lower than30�g/ml were considered active.

Table 4Hemolytic activity of the plant extracts on mouse erythrocyte (2%)

Species EC50 (�g/ml) CI 95%

Aegle marmelos 395.8 (344.8–454.4)Aphanamixis polystachyaoil >2500Aphanamixis polystachyasolid >2500Cuscuta reflexa 846.9 (732.2–979.7)Emblica officinalis >2500Hemidesmus indicus >2500Moringa oleifera >2500Nigella sativa 116.8 (95.7–142.6)Oroxylum indicum >2500Paederia Foetida >2500Polyalthia longifolia >2500Tribulus terrestris 622.7 (558.8–693.9)

The total hemolysis was obtained with 50�l of Triton X-100 1% and 1hincubation. The EC50 and 95 % confidence interval (CI 95%) were obtainedby non-linear regression. Extracts with an EC50 value lower than 200�g/mlwere considered active.

tracts fromAegle marmelos, Tribulus terrestris, andCuscutareflexa, which presented EC50 values of 395.8, 622.7, and846.9�g/ml, respectively. The other tested extracts were in-active in this assay.

4. Discussion and conclusions

In this study, the antimitotic potential of 11 medicinal plantextracts from Bangladesh was investigated. The in vitro an-timitotic potential was estimated as the ability of these ex-tracts to inhibit tumor cell line growth and sea urchin eggdevelopment. The toxicity toArtemia salinanauplii and thehemolytic activity on mouse erythrocytes were also evalu-ated.

The extract ofOroxylum indicumshowed the highest ac-tivity in the MTT assay using tumor cell lines and on seaurchin egg development, indicating the presence of cytotoxic

compounds in this extract. IC50 values for the four tested celllines and for different stages of embryo development werearound 15�g/ml, indicating that this extract did not presentany selectivity.Khan et al. (2002)also demonstrated thatthe extract of the stem bark ofOroxylum indicumpresentedstrong in vitro antiproliferative activity on chronic myeloge-nous leukemia K562. These authors compared the effect often plant species on cell proliferation of K562 cells, and foundthat the extract ofEmblica officinalisfruits was the mostactive followed byOroxylum indicum. Interestingly, in thepresent study, the extract ofEmblica officinalisfruits wastwo to three times less active than the one obtained from thestem bark ofOroxylum indicum.

Despite this cytotoxic activity, theOroxylum indicumex-tract did not possess any activity againstArtemia salinanau-plii or against mouse erythrocytes. These data suggest thatthe cytotoxic activity was not related to the lytic propertiesor membrane instability induced by the extract.

The inhibitory effect ofOroxylum indicumon sea urchinegg development could be observed since the first cleavage,and the cells presented a homogeneous cytoplasm, indicat-ing that the activity was related to DNA or protein synthesis(Fusetani, 1987).

The extract obtained fromAegle marmelosshowed activ-ity in all assays, indicating the presence of cytotoxic sub-s on ob-tt

Ts -s e al-r ty insr al.,2 ic

tances. The present data suggest that the ether fractiained from the ethanol extract ofAegle marmelosmight beoxic to tumor and normal cells.

The extracts ofMoringa oleifera, Emblica officinalis,ribulus terrestris, Cuscuta reflexa, Nigella sativaand theolid fraction ofAphanamixis polystachyaextract also preented moderate cytotoxic activity. Previous studies haveady demonstrated the occurrence of cytotoxic activiome of these species, such asEmblica officinalis, CuscutaeflexaandNigella sativa(Swamy and Tan, 2000; Jose et001; Khan et al., 2002). On the other hand, no cytotox


activity was observed inPolyalthia longifolia, HemidesmusindicusandPaederia foetidaextracts.

Finally, this study revealed that 8 among 11 tested speciesof plants used in Bangladeshi folk medicine presented someactivity in the used assays. However according to the crite-ria of the American National Cancer Institute, the IC50 limitto consider a crude extract promising for further purifica-tion is lower than 30�g/mL (Suffness and Pezzuto, 1990).Thus, only the extracts ofOroxylum indicum(three cell lines),Moringa oleifera(two cell lines) andAegles marmelos(twocell lines) could be considered as potential sources of anti-cancer compounds. Further, studies are necessary for chem-ical characterization of the active principles and more exten-sive biological evaluations.

Acknowledgements

We wish to thank the Brazilian National Research Council(CNPq), Claude Bernard Institute and the Research SupportFoundation of Ceara (FUNCAP) for financial support in theform of grants and fellowship awards. MTHK gratefully ac-knowledges the travel support of the Third World Academyof Sciences (TWAS), Italy, for his visit to Pakistan through a“South–South Fellowship”. The technical assistance of Sil-v ed.

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Anticancer Plants Used in Bangladesh