Anti-cancer and cytotoxic constituents from some Bangladeshi medicinal plants

Abstract

This thesis describes the isolation and structure elucidation of secondary metabolites from three plants namely Polyalthia simiarum Hook. F. and Thom. ( Family- Annonaceae ) , Glochidion multiloculare ( Roxb. ex Willd.) Muell.-Arg. and Glochidion lanceolarium ( Roxb.) Voigt ( Family- Euphorbiaceae ) as well as biological studies ( anti-cancer, antioxidant, cytotoxic, antimicrobial, analgesic and antiinflamatory ) of the extractives from these plants. A total of twelve compounds were isolated of which one ( PSB-001, 162 ) has been reported as a new compound by us ( Kabir et al., 2010) . The structures of the isolated compounds were elucidated mainly by high field NMR and mass spectroscopic techniques. The petroleum ether extract of the stem bark of P. simiarum afforded four compounds viz. PSB-001 ( 2-Oxo-14,15-bisnor-3,11E-kolavadien-13-one, 162 ) , PSB-007 ( Kolavenic acid, 163 ) , PSB-004 ( 16β-Hydroxycleroda-3,13( 14 ) Z-dien-15,16-olide, 164 ) and PSB-008 ( 16Oxocleroda-3,13( 14) E-dien-15-oic acid, 165 ) . These four compounds are the first report of their occurance from this respective plant. Among these compounds PSB-001 ( 2-Oxo-14,15bisnor-3,11E-kolavadien-13-one, 162 ) represents the second report of the isolation of any 18carbon containing bisnor-clerodane diterpenoid from nature and it appears to be new. The petroleum ether soluble fraction of the methanol extract of the stem bark of Glochidion multiloculare provided five comounds namely, GM-022 ( 3-Epi-lupeol, 166 ) , GM-023 ( Lupeol, 167 ) , GM-029 ( Glochidone, 168 ) , GM-032 ( Glochidonol, 169 ) and GM-035 ( Glochidiol, 170 ) . On the other hand, chemical investigation of carbon tetrachloride soluble fraction of the methanol extract of the stem bark of Glochidion lanceolarium yielded three compounds GL-022 ( Epilupeol, 171 ) , GL-023 ( Glochidonol, 172 ) and GL-035 ( Glochidone, 173 ) . The extractives of P. simiarum, G. multiloculare and G. lanceolarium were subjected to assay for the anti-cancer activity against EAC tumor bearing mice. The antitumor activity of the ethyl acetate ( EA ) extract of P. simiarum was determined against Ehrlich ascites carcinoma ( EAC ) in mice at 25 mg/kg and 50 mg/kg body weight intraperitoneally. Significant ( p<0.001 ) increase of survival time by ( 25 ± 0.57 and 27 ± 0.40 days) by the EA extract treated tumor bearing mice was confirmed with respect to the control group ( 22 ± 0.12 days ) . The antitumor activity of the extract/fractions ( MEGM, PEFGM, CTFGM, CHFGM)of stem bark of G. multiloculare and also the extracts ( MEGL, PEFGL, CTFGL ) of G. lanceolarium was determined against Ehrlich ascites carcinoma ( EAC ) in mice at 20 mg/kg body weight intraperitoneally. Significant ( p<0.001) increase of survival time by 24 ± 0.12, 26 ± 0.40, 21 ± 0.12 and 27±0.42 days was observed by the extracts MEGM, PEFGM, CTFGM and CHFGM treated tumor bearing mice with respect to the control group ( 20 ± 0.12 days ) , whereas the increase of survival time by ( 25 ± 0.32, 26 ± 0.10 and 23 ± 0.19 days) by the extracts MEGL, PEFGL and CTFGL treated tumor bearing mice was also observed with respect to the control group ( 20 ± 0.12 days) . Treatment with different extracts of the above mentioned plants decreased the intraperitonial tumor burden, thereby reducing the tumor volume, tumor weight, viable tumor cell count and increased the life span of the tumor bearing mice. Hematological studies revealed that the heamoglobin ( Hb) content was decreased in EAC treated mice whereas restoration to close to normal levels was observed in extract/fractions treated animals. There was a significant ( p<0.001) decrease in RBC and increase in WBC counts in extract/fractions treated animals when compared to EAC affected animals. All the extractives and purified compounds of P. simiarum, G. multiloculare, and G. lanceolarium were studied for free radical scavenging activity where tert-butyl-1hydroxytoluene ( TBHT ) was used as reference standard. In this study, the extracts MEGM, MEGL, PEFGM, PEFGL and PEFPSB of the above mentioned plants showed highest free radical scavenging activity with IC50 values 16.40, 18.32, 19.85, 20.29 and 21.5 μg/ml, respectively. The carbon tetrachloride soluble material ( CTFGL ) of G. lanceolarium exhibited promising antioxidant activity having IC50 value of 22.24 μg/ml. Compound PSB-004 ( 164 ) and ethyl acetate fraction ( EAFPSB ) of P. simiarum revealed potential antioxidant capacity with IC50 values of 23.5 and 24.5 μg/ml, respectively. In this investigation, the CTFGM, CFFGM and AQFGM of P. simiarum showed moderate free radical scavenging activity with IC50 values of 27.41, 32.30, and 38.17 μg/ml, respectively. The crude extracts/fractions of the plants P. simiarum, G. multiloculare and G. lanceolarium and two pure compounds ( PSB-004 and PSB-007) were subjected to brine shrimp lethality bioassay for probable cytotoxic activity. The PEFPSB of P. simiarum exhibited highest cytotoxicity with LC50 value of 1.91 μg/ml whereas EAFPSB of P. simiarum showed promising activity with LC50 value of 3.65 μg/ml. The degree of lethality was directly proportional to the concentration of extract ranging from the lowest concentration ( 0.78125μg/ml ) to the highest concentration ( 400 μg/ml ) . The positive control drug, vincristine sulfate showed LC50 at 0.32 μg/ml. The LC50 values of PEFGM, CTFGM, CFFGM, MeEGM and AQFGM of G. multiloculare were found to be 3.11, 4.96, 7.56, 9.23 and 16.32 μg/ml ) , respectively. The PEFGM exhibited significant mortality whereas CTFGM and CFFGM showed moderate activity. Besides these, the LC50 values PEFGL, CTFGL, CFFGL, MeEGL and AQFGL of G. lanceolarium were found to be 3.09, 6.70, 9.18, 14.24 and 17.68 μg/ml, respectively. In this experiment, compound PSB-004 ( 164 ) isolated from of P. simiarum demonstrated strong cytotoxic activity with LC50 value of 2.29 μg/ml, whereas compound PSB-007 ( 163 ) of the same plant showed moderate activity with LC50 value of 8.84 μg/ml. The extractives of the experimental three plants and two purified compounds ( PSB-004 and PSB-007 isolated from P. simiarum ) were screened for their antimicrobial activity against a number of test organism ( both Gram-positive, Gram-negative and fungi ) by the standardized disc diffusion method. The results obtained were compared with that produced by the standard antibiotic, Kanamycin. The petroleum ether ( PEFPSB ) and ethyl acetate ( EAFPSB ) soluble fractions of P. simiarum showed promising antibacterial activity with the average zone of inhibition of 20-28 mm and 21-28 mm, respectively at 400 μg/disc. The pet-ether soluble fraction revealed the highest activity against the growth of B. megaterium having the zone of inhibition of 28 mm. In this study, the zones of inhibition produced by the extracts CFFGM, PEFGM and CTFGM of G. multiloculare ranged from 9-12 mm, 8-10 mm and 7-9 mm, respectively. The carbon tetrachloride fraction ( CTFGL ) of G. lanceolarium was screened against 12 test bacteria and 3 fungi. The zone of inhibition was 07-10 mm. The fraction exhibited moderate activity against the test bacteria Salmonella typhi and Vibrio parahemolyticus and Candida albicans, Aspergillus niger whereas, the petroleum ether soluble fraction ( PEFGL) showed poor activity against the test organism. In case of pure compounds, PSB-004 ( 164 ) obtained from P. simiarum demonstrated moderate antimicrobial activity against 12 test bacteria and 3 fungi, having the zone of inhibition 10-14 mm whereas, PSB-007 ( 163 ) showed poor activity against test bacteria and fungi. The extracts/fractions of investigated plants were screened for probable analgesic activity using Swiss albino mice as experimental animal. The EA and PE extracts of P. simiarum showed analgesic activity in both tail flick and acetic acid induced methods at 50 and 100 mg/kg body weight. Among the extracts, the EA extract revealed a dose dependent analgesic activity in the methods. Both the extractives of G. multiloculare ( MEGM, PEFGM, CTFGMand CHFGM) and G. lanceolarium ( MEGL, PEFGL and CTFGL ) at 100 mg/kg b.w., produced significant increase in pain threshold in tail flick method whereas significantly reduced the writhing caused by acetic acid. All the tested extracts/fractions of investigated plants were capable to exhibit moderate analgesic activity. The extractives of P. simiarum, G. multiloculare and G. lanceolarium were subjected to assay for the anti-inflamatory activity using Male Wister rats and Swiss albino mice as experimental animal. The EA and PE extracts of P. simiarum, showed anti-inflammatory activities at 50- and 100 mg/kg body weight. The EA extract reduced the paw edema considerably ( 27.5% and 39.10% inhibition after 4h) . In addition the extractives of G. multiloculare ( MEGM, PEFGM, CTFGM and CHFGM) and G. lanceolarium ( MEGL, PEFGL and CTFGL ) at the dose 100 mg/kg body weight showed anti-inflammatory activity. The CTFGL of G. lanceolarium reduced the paw edema ( 0.85± 0.07, after 4h) , when compared to carrageenan induced control mice. Therefore, the extractives of the above mentioned plants were found to exhibit moderate anti-inflammatory activity. For the statistical validity of the results, all the tests were performed in triplicate or more and the data have been presented as mean ± standard deviation.