Natural Product Communications 2009 Vol 4 No to

1371 - 1376 Ellagic Acid Derivatives from Syzygium cumini Stem Bark Investigation of their Antiplasmodial Activity

bClaudia A Simoes-Piresa Sandra Vargasa Andrew MarstonS Jean-Robert Iosets

bull

Maral Q Paulo C An Matheeussend and Louis Maesd

Laboratory ojPharmacognosy and Phytochemistry Ecole de Pharmacie Geneve-Lausanne University ojGeneva University ojLausanne CH-1211 Geneve 4 Switzerland

bDrugsJor Neglected Diseases initiative (DNDi) 15 Chemin Louis Dunant 1202 Geneva Switzerland

cLaboratory oJNatural Product Chemistry Department oJChemistry Federal University oJParaiba 58059-900 Jofio Pessoa Brazil

dLaboratory ojMicrobiology Parasitology and Hygiene (LMPH) University ojAntwerp B-2020 Wilrijk Antwerp

Claudiapiresunigech

Received June 2nd 2009 Accepted September loth 2009

Bioguided fractionation of Syzygium cumini (Myrtaceae) bark decoction for antiplasmodial activity was performed leading to the isolation of three known ellagic acid derivatives (ellagic acid ellagic acid 4-0-a-L-2-acetylrhamnopyranoside 3-0-methylellagic acid 3-O-a-L-rhamnopyranoside) as well as the new derivative 3-0-methylellagic acid 3-O-f)-Dshyglucopyranoside Activity investigation was based on the reduction of P Jalciparum (PjKl) parasitaemia in vitro and the inhibition of f)-hematin formation a known mechanism of action of some antimalarial drugs Among the investigated ellagic acid derivatives only ellagic acid was able to reduce P Jalciparum parasitaemia in vitro and inhibit f)-hematin formation suggesting that free hydroxyl groups are necessary for activity within this class ofcompounds

Keywords Syzygium cumini Myrtaceae ellagic acid derivatives antiplasmodial f)-hematin

Syzygium cumini (L) Skeels (Myrtaceae) is found in Thailand Philippines and Madagascar as well as in tropical and subtropical regions of America Some of the vernacular names of the species are Jam Jaman (in India) Luk wa (in Thailand) Madan (in Japan) Rotra (in Madagascar) Jambo and Jambolao (in Brazil) [I] The plant has been used for years in traditional medicine especially for the treatment of diabetes but also as a digestive astringent anthelmintic and in the treatment of bronchitis dysentery and ulcers [2] A few compounds have been previously identified in the bark of the plant mostly ellagic and gallic acid derivatives and flavonoids However a series of ellagitannins has not been structurally elucidated [34]

We herein present the bioguided fractionation of S cumini bark decoction for antiplasmodial activity and the isolation of active and non-active ellagitannins one of them elucidated as a new natural

product Activity investigation was based on the reduction of P Jalciparum (PjKI) parasitaemia in vitro and the inhibition of ~-hematin formation a known mechanism of action of some antimalarial drugs [5]

High performance liquid chromatography coupled with UV detection and electrospray mass spectrometry (HPLCruVESI-MS) in the negative mode was conducted for the crude extract showing the presence of ellagic acid with a pseudomolecular ion at mlz 30 I [M-Hr Other compounds presented molecular pseudomolecular ions corresponding to known ellagitannins However the exact structure of these compounds could not be determined on the basis of on-line data Thus a fractionation procedure was established in order to isolate compounds for complete structure elucidation and activity determination

1372 Natural Product Communications Vol 4 (10) 2009 Simoes-Pires et al

I~CH~ I H

HO h 0 0 ~ OH O 0

HO 1 3~OHH

OH

H

HOH$ - OJ HO ~ ~O 4 HaC

6

As a preliminary actlVlty investigation an HPLC micro fractionation was conducted and 40 fractions of I mL were collected in a 96-well plate Fractions were tested for the inhibition of ~-hematin fonnation in an in-house assay particularly developed for the screening of plant extracts and fractions By this means two peaks could be linked to significant activity one at 68 min and the other at 232 min corresponding to gallic and ellagic acids respectively

Four major fractions (A-D) were obtained by centrifugal partition chromatography (CPC) All fractions were tested against P jalciparum in vitro Fraction C was considered highly active with an ICso value lower than 025 IlglmL while fractions Band D were considered moderately active with IC50

values of 271 and 198 llgmL respectively

Gallic acid (1 22 mg) was obtained as yellow crystals by filtration of fraction B Fraction C was further fractionated by medium pressure liquid chromatography (MPLC) followed by semishypreparative HPLC isolation to give ellagic acid (2 16 mg) and the new ellagitannin 3-0-methylellagic acid 3-O-J3-D-glucopyranoside (3 6 mg) Semishypreparative HPLC of fraction D provided ellagic acid 4-0-a-L-2-acetylrhamnopyranoside (4 3 mg) and 3-0-methylellagic acid 3 -O-a-L-rhamnopyranoside (53 mg)

Known compounds (1 2 4 and 5) had their structures confinned by the analysis of their spectral data which were compared with those in the literature Compound 3 was elucidated by the careful analysis of spectral data including high resolution mass spectrometry H 3C homo- and heteronuclear NMR experiments (67]

The high-resolution electrospray mass spectrum (HR-ESI-MS) of compound 3 showed a pseudomolecular ion at mlz 4770633 (M-Hr calculated for C2I H I703 (4770669) corresponding to the molecular fonnula C21HIS013 the same as that of several methyleUagic acid glucosides previously published [8-10] The H NMR spectrum of3 showed a singlet at OH 405 indicating the presence of one O-methyl substituent Enzymatic hydrolysis with ~-D-glucosidase was successfully achieved confinning a ~-D-glucopyranoside moiety and the connectivity between protons of the sugar moiety was established by analysis of the COSY correlations The attachment of the sugar at C-3 could be detennined by the HMBC correlation between the anomeric proton (OH 506) and C-3 (oc 1416) together with the upfield shift of H-5 (OH 771 instead of 780) compared with a glucoside substitution at position 4 [11] The methoxyl substitution at C-3 was detennined by the I3C shift of ca 64 ppm instead of the typical value of 57 ppm of an O-methyl substitution at C-4 [8] According to previous work [12] 4-0-methylellagic acid derivatives present a NOESY correlation between the methoxyl signal and that of H-5 This correlation was not observed in the NOESY spectrum of 3 It is also important to notice that both compounds 3 and 5 having a sugar substitution at C-3 presented l3C NMR shifts of C-l at Oc 1156 and 1144 respectively rather than 1122 as in ellagic acid (2) and 1126 in its derivative with the sugar moiety at C-4(4) Therefore this shift difference is suggested as a useful tool in the differentiation of sugar substitution patterns at C-3 and C-4

Isolated compounds were tested against P jalciparum (PjKl) and for their cytotoxicity on human fibroblasts ICso values are presented in Table 1 According to these results none of the compounds were considered cytotoxic and only ellagic acid was able to reduce in vitro parasitaemia Interestingly ellagic acid derivatives were not active

The inhibition of J3-hernatin fonnation has previously been raised as one of the mechanisms of action for the in vitro antiplasmodial activity of ellagic acid [13] When the activity on the inhibition of J3-hernatin fonnation was assessed for isolated compounds only 1 and 2 presented a significant inhibition (Figure I)

Throughout the validation process of our in-house ~shyhematin assay gallic acid (1) was actually considered as an interfering compound with no reduction of

--

Antiplasmodial ellagie aeid derivatives from Syzygium cumini

Table 1 Antiplasmodial and cytotoxic activities of compounds isolated from S cumini stem bark

Compounds PfKl Cytotoxicity

ICso (11M) I gt3200 2941 2 278 gt3200

3 gt3200 gt3200 4 gt3200 gt3200 5 gt3200 gt3200

Tested on human fibroblasts

14

12

08 ~ ~

~ 06

~ 04

02

0

)2 2 3 4 5

Figure 1 [nhibition of p-hematin formation by isolated compounds 1-5 Significant inhibition is denoted by positive values for IAnalys

in vitro parasitaemia On the other hand the inhibition of ~-hematin formation by ellagic acid (5) has previously been investigated and two possible explanations have been raised 1) its flat molecular structure with a high electronic density would be able to form TC-TC interaction complexes with hematin monomers and 2) the aryl hydroxyl groups of ellagic acid would be able to form coordination bonds with the Fe(lII) of hematin [14] In the case of compounds 3-5 the obtained results are in accordance with the second explanation since they are OH-substituted derivatives presenting no significant inhibition on ~-hematin formation

Experimental

Plant material and extraction Syzygium cumini (L) Skeels (Myrtaceae) stem bark was collected in the city of Joao Pessoa state of Paraiba Brazil in July 2004 The bark was dried at 60degC ground and 400 g was extracted by decoction in 2 L distilled water for 15 min The resulting extract was filtered and freezeshydried to give 80 g ofpowdered extract

HPLCuVIESI-MS Analyses were performed on a HP-IIOO system (Hewlett Packard Palo Alto CA USA) equipped with a binary pump a DAD and an autosampler MS analyses were made on a Finnigan MAT (San Jose CA USA) LCQ ion trap mass

Natural Product Communications Vol 4 (10) 20091373

spectrometer using an electrospray ionization (ESI) interface Samples were chromatographed on a CIS

Zorbax Extendt column (250 x 40 mm id 5 lm) (Agilent Germany) with a 10 mLlmin flow rate The mobile phase was MeOH (+01 formic acid) water (+01 formic acid) in gradient mode UV spectra were recorded at 210 254 and 366 nm MS were obtained in negative mode ESI conditions capillary temperature 200degC source voltage 5 kV source current 80 lA corona needle current 5 lA collision sheath gas pressure 80 psi collision energy 15 eV

HPLC microfractionation The same HPLC conditions described above were used Samples were collected every 1 min (I mL) in a 96-weU plate using a Gilson model FC204 collector (Middleton USA) Fractions were evaporated to dryness on a Speedvac system (model ReT 90 Jouan Ec1epens Switzerland) The content of each fraction was suspended in test solution for the ~-hematin

inhibition assay

Centrifugal partition chromatography (CPC) Fractionations were conducted on a counter-current chromatograph CCC-IOOO (Pharma-Tech Research Corp) equipped with dynamic coils of 650 mL total volume The rotation speed was set at 1000 rpm Two LC-300 pumps (Scientific Systems Inc) were used to pump either upper or lower phase into the coils at a flow rate of 30 mLimin for each phase Samples were diluted in 30 mL of a mixture of upper and lower phases (I I) and introduced through the injection loop Crude extract (20 g) was chromatographed using the solvent system tert-butylshymethyl-ether - n-butanol acetonitrile (22 I 5) + 005 TF A The lower phase was used as mobile phase followed by phase inversion after 13 h

Medium pressure liquid chromatography (MPLC) The equipment used consisted of a BUchi B-681 pump a Knauer K 200 I UV detector a Pharmacia LKB Rec I recorder and an automatic BUchi B-684 fraction collector Lichroprepreg CIS stationary phase (15-25 lm Merck) was packed in a pressure-resistant column A stepwise gradient of MeOH-water was employed as mobile phase starting at 595 and gradually increasing to 1000 according to the onshyline UV chromatographic profile Fraction C (500 mg) was placed in an introduction cartridge after having been mixed with 3 times its weight of stationary phase The fractionation provided 4 major fractions (C I-C4)

1374 Natural Product Communications Vol 4 (10) 2009 Simoes-Pires et al

Semi-preparative high performance liquid chromatography A Shimadzu LC-SA binary pump equipped with a SPD-IOA VP Shimadzu UV -Vis detector (Kyoto Japan) was employed at a flow rate of 10 mLimin A Symmetry PrepTM CI8 column (150 x 19 mm id 7 ~m) was used The solvent system was A) water + 005 TFA and B) MeOH + 005 TFA in isocratic mode Fraction C3 (32 mg) and D (28 mg) were chromatographed using 30 and 35 B as mobile phase respectively

Antiplasmodial activity assay A chloroquineshyresistant strain of P Jalciparum (PjKl) was used The strain was maintained in RPMI -1640 medium supplemented with 037 mM hypoxanthine 25 mM Hepes 25 mM NaHC03 and 05 Albumaxreg with 2-4 washed human erythrocytes All cultures and assays were conducted at 37degC Extractcompound stock solutions were prepared in 100 DMSO at 20 mgmL or mM Samples were serially pre-diluted (2shyfold or 4-fold) in DMSO followed by a further dilution in demineralized water to assure a final inshytest DMSO concentration of lt1 Assays were performed in 96-well microtiter plates each well containing 10 ilL of the watery compound dilutions together with 190 ilL of the malaria parasite inoculum (1 parasitaemia 2 HCT) Parasite growth was compared with untreated-infected (100 growth) and uninfected-controls (0 growth) After 72 h incubation at 37degC plates were frozen at -20dege After thawing 20 ilL of each well was transferred onto another plate together with 100 ~L Malstattrade reagent and 20 ilL of a 1 1 mixture of PES (phenazine ethosulfate 01 mgmL) and NBT (Nitro Blue Tetrazolium Grade III 2 mgmL) The plates were kept out of light for 2 h and change in color was measured spectrophotometrically at 655 nm The results were expressed as reduction in parasitaemia compared with control wells and IC50 values were calculated Compoundsextracts were tested at 5

concentrations (64 164 1 and 025 11M or IlgmL) Chloroquine was used as reference compound (ICso=035 ~M)

Cytotoxicity assay Human fibroblast MRC-5 SV2 cells were cultured in MEM + Earls salts-medium supplemented with L-glutamine NaHC03 and 5 inactivated fetal calf serum All cultures and assays were conducted at 37degC under an atmosphere of 5 CO2 Compoundextract stock solutions were prepared in 96-well microtiter plates as described for antiplasmodial activity Each well contained 10 ilL of the watery compound dilutions together with 190 ~L of MRC-5 SV2 inoculum (3xl04 cellsmL) Cell growth was compared with untreated-control wells (100 cell growth) and medium-control wells (0 cell growth) After 3 days incubation cell viability was assessed fluorimetrically after addition of 50 ilL resazurin per well After 4 h at 37degC fluorescence was measured (Aex 550 nm Aem 590 nm) Results were expressed as reduction in cell growthviability compared with control wells and ICso values were determined Compoundsextracts were tested at 5 concentrations (64 16 4 1 and 025 11M or IlgmL) using tamoxifen as cytotoxic reference compound (lC5o = 1 06 ~M)

~Hematin formation inhibition assay Solutions (50 mM) of pure compounds were prepared in 10 M HCl-MeOH-DMSO (532) Compoundsfractions were distributed in a 96-well plate A previously established Phi~-assay [15] was used to develop the in-house inhibition assay based on ~-hematin

photocolorimetric determination Colorimetric measurements were performed at 405 nm in an ELshy808 apparatus (Bio-Tek Instruments Inc) and qualitative results were statistically obtained by IAnalysis

Table 2 H NMR shiftsmiddot of ellagic acid derivatives (2-5) isolated from S cumini stem bark (500 MHz)

C 2 4

5 747 (IH s) 3-0CH

5 747 (lH s) I 2 3 4 5 6 8

749 (IH s) 405 (3H s) 771 (1H s) 506 (IH brs) 407 (1H brs) 352 (lH d 98) 348 (1H brs) 373 (1H brs) 351

789 (lH s)

755 (IH s) 560 (IH brs) 539 (1H brs) 417 (IH d 63) 349 (1H t 97) 379 (1H dd 63 93) 129 (3H d 59) 216 (3H s)

751 (1H s) 404 (3H s) 776 (lH s) 546 (1H brs) 400 (1H brs) 385 (I H dd 33 93) 334 (1H brs) 354 (I H dd 62 95) 114 (3H d 60)

middotChemical shift values are reported in ppm relative to TMS number of protons signal multiplicities and coupling constants (Hz) are shown in parentheses OMSO-d6 - 0 20 (I I) at 35degC bOMSO-d6 at 40degC OMSO-d6 at 35degC dCOOO at 27degC eOveriapped with solventH20 signal obtained from the HSQC spectrum

1374 Natural Product Communications Vol 4 (10) 2009 Simoes-Pires et al

Semi-preparative high performance liquid chromatography A Shimadzu LC-SA binary pump equipped with a SPD-IOA VP Shimadzu UV -Vis detector (Kyoto Japan) was employed at a flow rate of 10 mLimin A Symmetry PrepTM CI8 column (150 x 19 mm id 7 ~m) was used The solvent system was A) water + 005 TFA and B) MeOH + 005 TFA in isocratic mode Fraction C3 (32 mg) and D (28 mg) were chromatographed using 30 and 35 B as mobile phase respectively

Antiplasmodial activity assay A chloroquineshyresistant strain of P Jalciparum (PjKl) was used The strain was maintained in RPMI -1640 medium supplemented with 037 mM hypoxanthine 25 mM Hepes 25 mM NaHC03 and 05 Albumaxreg with 2-4 washed human erythrocytes All cultures and assays were conducted at 37degC Extractcompound stock solutions were prepared in 100 DMSO at 20 mgmL or mM Samples were serially pre-diluted (2shyfold or 4-fold) in DMSO followed by a further dilution in demineralized water to assure a final inshytest DMSO concentration of lt1 Assays were performed in 96-well microtiter plates each well containing 10 ilL of the watery compound dilutions together with 190 ilL of the malaria parasite inoculum (1 parasitaemia 2 HCT) Parasite growth was compared with untreated-infected (100 growth) and uninfected-controls (0 growth) After 72 h incubation at 37degC plates were frozen at -20dege After thawing 20 ilL of each well was transferred onto another plate together with 100 ~L Malstattrade reagent and 20 ilL of a 1 1 mixture of PES (phenazine ethosulfate 01 mgmL) and NBT (Nitro Blue Tetrazolium Grade III 2 mgmL) The plates were kept out of light for 2 h and change in color was measured spectrophotometrically at 655 nm The results were expressed as reduction in parasitaemia compared with control wells and IC50 values were calculated Compoundsextracts were tested at 5

concentrations (64 164 1 and 025 11M or IlgmL) Chloroquine was used as reference compound (ICso=035 ~M)

Cytotoxicity assay Human fibroblast MRC-5 SV2 cells were cultured in MEM + Earls salts-medium supplemented with L-glutamine NaHC03 and 5 inactivated fetal calf serum All cultures and assays were conducted at 37degC under an atmosphere of 5 CO2 Compoundextract stock solutions were prepared in 96-well microtiter plates as described for antiplasmodial activity Each well contained 10 ilL of the watery compound dilutions together with 190 ~L of MRC-5 SV2 inoculum (3xl04 cellsmL) Cell growth was compared with untreated-control wells (100 cell growth) and medium-control wells (0 cell growth) After 3 days incubation cell viability was assessed fluorimetrically after addition of 50 ilL resazurin per well After 4 h at 37degC fluorescence was measured (Aex 550 nm Aem 590 nm) Results were expressed as reduction in cell growthviability compared with control wells and ICso values were determined Compoundsextracts were tested at 5 concentrations (64 16 4 1 and 025 11M or IlgmL) using tamoxifen as cytotoxic reference compound (lC5o = 1 06 ~M)

~Hematin formation inhibition assay Solutions (50 mM) of pure compounds were prepared in 10 M HCl-MeOH-DMSO (532) Compoundsfractions were distributed in a 96-well plate A previously established Phi~-assay [15] was used to develop the in-house inhibition assay based on ~-hematin

photocolorimetric determination Colorimetric measurements were performed at 405 nm in an ELshy808 apparatus (Bio-Tek Instruments Inc) and qualitative results were statistically obtained by IAnalysis

Table 2 H NMR shiftsmiddot of ellagic acid derivatives (2-5) isolated from S cumini stem bark (500 MHz)

C 2 4

5 747 (IH s) 3-0CH

5 747 (lH s) I 2 3 4 5 6 8

749 (IH s) 405 (3H s) 771 (1H s) 506 (IH brs) 407 (1H brs) 352 (lH d 98) 348 (1H brs) 373 (1H brs) 351

789 (lH s)

755 (IH s) 560 (IH brs) 539 (1H brs) 417 (IH d 63) 349 (1H t 97) 379 (1H dd 63 93) 129 (3H d 59) 216 (3H s)

751 (1H s) 404 (3H s) 776 (lH s) 546 (1H brs) 400 (1H brs) 385 (I H dd 33 93) 334 (1H brs) 354 (I H dd 62 95) 114 (3H d 60)

middotChemical shift values are reported in ppm relative to TMS number of protons signal multiplicities and coupling constants (Hz) are shown in parentheses OMSO-d6 - 0 20 (I I) at 35degC bOMSO-d6 at 40degC OMSO-d6 at 35degC dCOOO at 27degC eOveriapped with solventH20 signal obtained from the HSQC spectrum

Antiplasmodial ellagic acid derivatives from Syzygium cumini Natural Product Communications Vol 4 (10) 20091375

Table 3 llC NMR shiftsmiddot of eUagic acid derivatives isolated from S NMR analyses IH_ and 13C_NMR spectra were cumini stem bark (500 MHz)

recorded in a Varian (Palo Alto CA USA) Unity C 2 36 4 sJ

Inova 500 spectrometer at 500 and 125 MHz1 1122 1125 1086 1114 2 1363 nd nd nd respectively Chemical shifts were recorded in ppm 3 1394 1402 1420 1401 as 8 relative to tetramethylsilane (TMS) as internal 4 1480 1493 1467 1527 5 1102 1111 1128 1114 standard 6 1495 nd nd 1130 7 1590 1592 1601 1586

3-0CH 645 698 HR-ESI-MS High resolution MS were obtained on a I 1122 1156 1126 1144 Micromass-LCT Premier Time of Flight (TOF) mass 2 1363 nd nd nd 3 1394 1416 1400 1465 spectrometer (Waters MA USA) with an 4 1480 1475 1501 1412 electro spray interface coupled with an Acquity5 1102 1122 1111 1112

UPLC system (Waters MA USA) ESI conditions 6 1495 nd nd 1072 7 1590 1595 1608 1586 capillary voltage 2800 V cone voltage 40 V MCP I 1023 978 1001

detector voltage 2650 V source temperature 120degC 2 751 722 699 3 773 691 700 desolvation temperature 250degC cone gas flow 10 4 742 728 717 5 LIh desolvation gas flow of 550 Lih Detection was 776 705 698 6 6949 1615 178 performed in positive and negative ion modes in the 7 1728 mlz range 100-1000 with a scan time of 025 s in 8 198

middotChemical shift values are reported in ppm relative to TMS OMSO-d6- centroid mode 0 20 (I I) at 35degC bOMSO-d6 at 40degC OMSO-d6 at 35degC dC0300 at 27degC nd not detected 3-0-MethyJellagic acid 3-O-j3-D-gJucopyranoside

(3)Enzymatic hydrolysis Compound 3 was treated with

H NMR Table 2j3-D-glucosidase (from almonds Sigma St Louis 3C NMR Table 3MO USA) in 1 mL NaOAc buffer (pH 50) for one HR-ESI-MS mlz 4770633 [M-Hr calculated forday at 37degC The aglycone was extracted by partition C2 H 170 I34770669)with n-BuOH and submitted to LCfUV -DAD

analysis in order to confirm hydrolysis

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[12] Elkhateeb A Subeki Takahashi K Matsuura H Yamasaki M Yamato 0 Maede Y Katakura K Yoshihara T Nabeta K (2005) Anti-babesial ellagic acid rhamnosides from the bark of Elaeocarpus parvifolius Phytochemistry 66 2577-2580

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[13J Banzouzi JT Prado R Menan H Valentin A Roumestan C Mallie M Pelissier Y Blache Y (2002) In vitro antiplasmodial activity of extracts of Alchornea cordialia and identification of an active constituent ellagic acid Journal ofEthnopharmacology 81399-401

[14] DellAgli M Parapini S Basilico N Verotta L Taramelli D Berry C B05i510 E (2003) In vilro studies on the mechanism of action of two compounds with antiplasmodial activity EUagic acid and 345-trimethoxyphenyl (6-O-galloyl)-beta -D-glucopyranoside Pianta Medica 69162-164

[15] Ncokazi KK Egan TJ (2005) A colorimetric high-throughput beta-hematin inhibition screening assay for use in the search for antimalarial compounds Analytical Biochemistry 338306-319

1376 Natural Product Communications Vol 4 (10) 2009 Simoes-Pires et al

[13J Banzouzi JT Prado R Menan H Valentin A Roumestan C Mallie M Pelissier Y Blache Y (2002) In vitro antiplasmodial activity of extracts of Alchornea cordialia and identification of an active constituent ellagic acid Journal ofEthnopharmacology 81399-401

[14] DellAgli M Parapini S Basilico N Verotta L Taramelli D Berry C B05i510 E (2003) In vilro studies on the mechanism of action of two compounds with antiplasmodial activity EUagic acid and 345-trimethoxyphenyl (6-O-galloyl)-beta -D-glucopyranoside Pianta Medica 69162-164

[15] Ncokazi KK Egan TJ (2005) A colorimetric high-throughput beta-hematin inhibition screening assay for use in the search for antimalarial compounds Analytical Biochemistry 338306-319