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Cytotoxic effects of mammea type coumarins from 
Calophyllum brasiliense 
Ricardo Reyes-Chilpaa,*, Elizabet Estrada-Mun˜iza, Teresa Ramı´rez Apana, 
Badia Amekrazb, Andre Aumelasb, Christopher K. Jankowskib, Mario Va´zquez-Torresc 
a Instituto de Quı´mica, Universidad Nacional Auto´noma de Me´xico, Ciudad Universitaria, 
Delagacio´n Coyoacan Me´xico D.F. 04510, Mexico 
bDe´partement de Chimie et Biochimie. Universite de Moncton, Moncton, Canada NBE1A 3E9 
c Instituto de Investigaciones Biolo´gicas. Universidad Veracruzana, Apartado Postal 294, Xalapa, Veracruz, 91000, Mexico 
Received 24 September 2003; accepted 29 March 2004 
Abstract 
Calophyllum brasiliense (Clusiaceae) is a big tree from the Tropical Rain Forests of the American continent. 
The organic extracts from the leaves yielded coumarins of the mammea type: mammea A/BA, A/BB, B/BA, B/BB, 
C/OA, C/OB, B/BA cyclo F, B/BB cyclo F, and isomammeigin. The triterpenoids friedelin and canophyllol, as 
well as the biflavonoid amentoflavone, protocatechuic and shikimic acids, were also obtained. Most of the isolated 
compounds were tested in vitro against K562, U251, and PC3 human tumor cell lines. The coumarins were 
cytotoxic against the three cell lines, the highest activity was shown by mammea A/BA (IC50 = 0.04 to 0.59 AM). 
The mixtures of mammea A/BA + A/BB, mammea B/BA + B/BB and mammea C/OA + C/OB were also highly 
active (IC50 < 4.05 AM). Friedelin was cytotoxic only against PC3, and U251 lines. Inhibition of HIV-1 reverse 
transcriptase was also assayed in vitro; however, none of the tested compounds (250 AM) prevented the activity of 
this enzyme. Most of the isolated compounds were also inactive against fourteen bacterial strains; however 
mammea A/BA + A/BB, and mammea C/OA + C/OB inhibited the growth of Staphylococcus aureus, S. 
epidermidis and Bacillus subtilis. 
D 2004 Elsevier Inc. All rights reserved. 
Keywords: Calophyllum brasiliense; Clusiaceae; Mammea; Coumarins; Triterpenoids; Biflavonoids; Cytotoxic activity; Tumor 
cell lines; HIV; Reverse transcriptase; Enterobacteria 
* Corresponding author. Tel.: +52-56224430; fax: +52-56162203. 
E-mail address: chilpa@servidor.unam.mx (R. Reyes-Chilpa). 
0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved. 
doi:10.1016/j.lfs.2004.03.017 
www.elsevier.com/locate/lifescie 
Life Sciences 75 (2004) 1635–1647
1636 R. Reyes-Chilpa et al. / Life Sciences 75 (2004) 1635–1647 
Introduction 
The Calophyllum genus (Clusiaceae) is composed by 180–200 tree species, most of them thrive 
within the Indo-Pacific zone, particularly from the Malay Peninsula to New Guinea (Stevens, 1980). 
Calophyllum species have recently received considerable attention from a pharmacological point of 
view, since some of them produce potent inhibitors of reverse transcriptase of human immunode-ficiency 
virus type 1 (HIV-1 RT) (Kashman et al., 1992; Patil et al., 1993; McKee et al., 1996; 
Dharmaratne et al., 2002). These compounds known as calanolides, inophyllums and cordatolides 
are tetracyclic dipyranocoumarins with a propyl, phenyl or methyl substituent attached to C-4, 
respectively (Fig. 1; Ishikawa, 2000). Since HIV-1 is characterized by its cytopathic effects, 
specially on CD-4 lymphocytes, it is necessary that RT inhibitors also show low cytotoxicity 
(Weislow et al., 1989). Under these criteria, the most promising tetracyclic dipyranocoumarin 
suitable to be developed as a drug is (+)-calanolide A, which is currently investigated in phase II/III 
clinical trials. This compound was isolated in low yields from the leaves of C. lanigerum var. 
austrocoriaceum, but now it is also available synthetically (Flavin et al., 1996). Besides tetracyclic 
coumarins, tricyclic pyranocoumarins have been isolated from Calophyllum species (Ishikawa, 
2000). In addition, it was recently reported that C. dispar contains coumarins of the mammea 
type, such as mammea A/BA cyclo F (Fig. 1), several of them resulted highly cytotoxic against 
human epidermoid carcinoma cells (KB) (Guilet et al., 2001a,b). Mammea coumarins are 
characterized by a simple 5,7-dioxygenated coumarin skeleton bearing a phenyl, or an alkyl chain 
on C-4, acyl and prenyl (free or cyclized) substituents on either C-6 or C-8 (Crombie and Games, 
1966; Crombie and Games, 1967; Crombie et al., 1972). They are common constituents of Mammea 
and Mesua species (Clusiaceae), but have been only found once in a Calophyllum species (Guilet et 
al., 2001a,b). To our best knowledge, the anti-HIV-1 properties of this class of compounds have not 
been investigated yet. 
Calophyllum brasiliense is the widest distributed species among the eight Calophyllum species 
found in the American Continent, it grows in the Tropical Rain Forests from Brazil to Mexico (Stevens, 
1980; Pennington and Sarukha´n, 1998). Numerous ethnomedical applications have been recorded for 
this species throughout Latin America (Soto Nun˜ez and Sousa, 1995; Garcı´a Barriga, 1992; Mesı´a-Vela 
Fig. 1. Tetracyclic dipyranocoumarins and tricyclic dehydrofuranocoumarin.
R. Reyes-Chilpa et al. / Life Sciences 75 (2004) 1635–1647 1637 
et al., 2001), but apparently none is related to AIDS or cancer treatment. Previous chemical analysis of 
C. brasiliense bark have reported the presence of xanthones with cancer chemopreventive properties 
(Ito et al., 2002). Other xanthones with antifungal activity have been isolated from the heartwood 
(Reyes-Chilpa et al., 1997). Chromanone carboxylic acids were obtained from the bark latex (Stout et 
al., 1968) and seeds (Plattner et al., 1974). The polar extracts of the leaves contain hyperin 
(hyperoside), amentoflavone, quercetin, gallic acid, and protocatechuic acid; some of these phenolic 
compounds exhibited analgesic activity (da Silva et al., 2001). We are now reporting the cytotoxic 
effects against three human tumor cell lines of mammea type coumarins isolated from the leaves of C. 
brasiliense. These compounds, along with several phenolic acids, triterpenoids, and a biflavonoid 
obtained from the same source, were also tested against HIV-1 reverse transcriptase, and fourteen 
enteropathogenic bacteria. 
Material and methods 
Plant material 
Calophyllum brasiliense Cambess. (Clusiaceae) was collected at Ejido Benigno Mendoza, Sierra 
de Santa Marta, State of Veracruz, Mexico. Authentication was done by one of us (Mario 
Va´zquez Torres). A voucher specimen is deposited with the number 435 -Bojo´rquez et al.- in the 
Herbarium of the Instituto de Investigaciones Biolo´gicas, Universidad Veracruzana (CIB) at 
Xalapa, Me´xico. 
Extraction and isolation of compounds 
The dried leaves (2075 g) were extracted at room temperature over a period of one week with hexane, 
acetone, and methanol, successively. Compounds were isolated after spontaneous crystallization or by 
column chromatography on Silica Gel-60 (CC), and identified by their spectroscopic (1HNMR, 
13CNMR, IR, UV), EIMS, and CIMS, and comparison with published data. 
Hexane extract (70.3 g). While in solution, the extract suffered spontaneous precipitation obtaining 
a white powder (19.8 g). Part of this material (5.1 g) was treated with CH2Cl2, the insoluble part was 
a mixture (583 mg) of friedelin (1) and canophyllol (2), while the soluble part afforded a mixture (4 
g) of mammea A/BA (3) and mammea A/BB (4). The extract was then concentrated in vacuo (35.3 
g), and 9.4 g were subjected to CC (200 g). Elution with hexane yielded first friedelin (45.9 mg), 
afterwards a mixture (5 mg) of mammea B/BA (5) and B/BB (6), and finally a mixture of mammea 
A/BA and mammea A/BB (720 mg). Elution with hexane-EtOAc (9:1) yielded initially canophyllol 
(20 mg, m.p. 180–182jC), then a mixture (50 mg) of mammea C/OA (7) and mammea C/OB (8), 
final fractions afforded a mixture (30 mg) of mammea B/BA cyclo F (9), and mammea B/BB cyclo 
F (10). 
Mammea B/BA (5) & mammea B/BB (6) 
1HNMR(200 MHz, CDCl3/TMS): 6.86 s, 1H, (OH-5); 6.03 s, 1H, (H-3); 2.93 t, J = 7.5 Hz, 2H, 
(CH2-CH2 -CH3); 1.38 m, 2H, (CH2-CH2 -CH3); 0.91 t, J = 6.4 Hz, 3H, (CH2-CH2 -CH3).
1638 R. Reyes-Chilpa et al. / Life Sciences 75 (2004) 1635–1647 
Isoprenyl on C-6: 5.24 t, J = 7.2 Hz, 1H (CH); 3.49 d, J = 7.2 Hz, 2H (CH2); 1.82 and 1.87, both s & 
3H, 2 CH3. 
(5): 14.61 s, 1H (OH-7). R = 3-methylbutyryl: 3.16 d, J = 6.6 Hz, 2H, (CH2); 2.27 m, J = 6.6 Hz, 1H 
(CH); 1.03 d, J = 6.7 Hz, 6H, 2(CH3). 
(6): 14.68 s, 1H (OH-7). R = 2-methylbutyryl: 3.93 m, J = 6.5 Hz, 1H (CH); 1.25 d, J = 6.6 Hz, 3H 
(CH3); 1.93 m, 2H, (CH2); 0.97 t, J = 6.4 Hz, 3H, (CH3). 
IR r max (KBr): 3325(OH); 2960, 2931, 2858 (C-H); 1722(C = O); 1602(C = C); 1560 (C = C); 1462(C 
= C); 1329(C-O); 1116 (C-O). 
Mammea C/OA (7) & mammea C/OB (8) 
1HNMR (200 MHz, CDCl3/TMS): 6.04 s, 1H (H-3); 7.10 s wide, 1H (OH-5); 6.25 s, 1 H (H-6); 6.04 s, 
1H (H-3), 2.95 t, J = 7.5 Hz, 2H (CH2-(CH2)3-CH3); 1.64 m, 2H (CH2-CH2-(CH2)2-CH3); 1.37 m, 2H 
(CH2)3-CH2-CH3); 0.90 t, J = 6.8 Hz, 3H (CH2-(CH2)4-CH3). 
(7): 14.16 s, 1H (OH-7). R = 3-methylbutyryl: 3.14 d, J = 6.6 Hz, 2H (CH2); 2.26 m, J = 6.6 Hz, 1H 
(CH); 1.01 d, J = 6.6 Hz, 6H, 2(CH3). 
(8): 14.12 s, 1 H (OH-7). R = 2-methylbutyryl: 3.90 m, 1H (CH); 1.24 d, J = 6.6 Hz, 3H (CH3), 1.88 m, 
2H (CH2); 0.94 t, J = 5.9 Hz, 3H (CH3). 
EMIE: 70 ev, m/z (%): 332 M+ (36.6%) [C19H24O5]+, 317 (21.10%) [M+-CH3], 299 (7.0%) [M+-CH3- 
H2O], 276 (66.1%) [M+-C4H8], 275 (100%) [M+-C4H9], 234(13.3%), 219 (12.6%). IR r max 
(KBr): 3164 (OH); 2961, 2932, 2872 (C-H); 1726 (C = O); 1622 (C = C); 1595 (C = C); 1390 
(C-O); 1270 (C-O); 1117 (C-O). 
Mammea B/BA cyclo F (9) & mammea B/BB cyclo F (10) 
1HNMR(200 MHz, CDCl3/TMS): 14.18 s, 1H (OH-7); 5.95 s, 1H (H-3); 2.82 m, J = 7.2 Hz, 2H 
(CH2- CH2- CH3); 1.64 m, J = 7.4 Hz, 2H (CH2-CH2-CH3); 1.03 t, J = 6.4 Hz, 3H (CH2-CH2-CH3); 
4.85 t, J = 9.0 Hz, 2H, (CH-cyclo F), 3.17 d, J = 6.6 Hz, 2H, (CH2-cyclo F), 2.00 s wide, 1H (OH cyclo 
F); 1.41 and 1.28 both s & 3H, 2(CH3 cyclo F). R = 3-methylbutyryl (B/BA cyclo F): 3.08 d, J = 6.7 
Hz, 2H (CH2); 2.24 m, J = 6.6 Hz, 1H (CH); 1.03 d, J = 6.1 Hz 6H, 2(CH3). R = 2-methylbutyryl (B/BB 
cyclo F): 3.85 m, 1H (CH); 1.23 d, J = 8 Hz, 3H (CH3); 1.92 and 1.5 both m  1H (CH2); 0.99 t, J = 5.6 
Hz, 3H (CH3). 
EIME: 70 ev, m/z (%): 388 M+ (54.9%) [C22H28O6]+; 373(14.0%)[M+-CH3]; 355 (11.2%); 332(22.5%); 
331(100%); 301(7.0%); 287(4.9%); 259(14.7%). IR r max (KBr): 2964(C-H); 1728(C = O); 
1631(C = C); 1606(C = C); 1429(C = C); 1393(C-O); 1300(C-O); 1150(C-O); 1121(C-O). 
Acetone extract 
Concentration in vacuo yielded a total of 101 g. A sample (19 g) was macerated with EtOAc 
obtaining an insoluble brown colored powder (6 g), which after redisolving in methanol yielded 
amentoflavone (11) (yellow crystals, mp 300 jC, 430 mg). The soluble part of the extract was treated
R. Reyes-Chilpa et al. / Life Sciences 75 (2004) 1635–1647 1639 
with activated carbon to remove chlorophylls, filtered on celite, and concentrated to 50 ml. From this 
solution a mixture (561 mg) of friedelin (1) and canophyllol (2) precipitated. The remaining material 
(5.3 g) was subjected to CC. Fractions eluted with hexane-EtOAc (9:1) afforded (41.8 mg) isomam-meigin 
(mammea A/BA cyclo D) (12), and finally friedelin (1). Elution with hexane-EtOAc (8.5:1.5) 
yielded a mixture (174 mg) of mammea A/BA (3) and A/BB (4). Elution with the same solvent mixture 
yielded canophyllol (2), and finally a mixture (102 mg) of mammea B/BA cyclo F (9), and mammea B/ 
BB cyclo F (10). Fractions obtained with hexane-EtOAc (6.5:3.5) afforded protocatechuic acid (13) 
(100 mg). Purification of compound 3 (mp 122–124 jC) was achieved by HPLC using ODS-column 
eluting with 90% MeOH, meanwhile compound 9 (mp 125–126 jC) was obtained pure after 
recristalyzation from CHCl3-hexane. 
Mammea A/BA (3)  mammea A/BB (4) 
1HNMR(200 MHz, CDCl3/TMS): 7.56 m, 3H (Ar); 7.41 m, 2H (Ar); 6.0 s, 1H (H-3); 5.95 s, 1H 
(OH-5). Isoprenyl on C-6: 5.09 tm, J = 6.9 Hz, 1H (CH); 3.29 d, J = 6.9 Hz, 2H (CH2); 1.65 and 1.70, 
both s  3H, (2 CH3). 
(3): 14.61 s, 1H (OH-7). R = 3-methylbutyryl: 3.19 d, J = 6.7 Hz, 2H (CH2);2.32 m, J = 6.7 Hz, 
1H (CH);1.06 d, J = 6.6 Hz, 6H, (2 CH3). 
(4): 14.57 s, 1H (OH-7). R = 2-methylbutyryl (A/BB): 3.95 m, J = 6.6 Hz, 1H (CH-CH3); 1.29 d, J = 6.7 
Hz, 3H (CH-CH3), 1.94 m, 2H (CH2); 1.01 t, J = 7.2 Hz, 3H (CH3). 
EMIE: 70 ev m/z (%): 406 M+ (96.4%)[C25H26O5]+; 363(55.6%); 351(76.0%); 293 (100 %). IR r max 
(KBr) 3985(OH); 2964, 2932, 2872(C-H); 1729(C = O); 1724(C = O); 1614 (C = C);1556 
(C = C); 1390(C-O). 
Isomammeigin (mammea A/BA cyclo D) (12). Yellow crystals, mp 162–164 jC 
1HNMR(200 MHz, CDCl3/TMS): 14.78 s, 1H (OH-7); 7.39 m, 3H (Ar); 7.23 m, 2H (Ar); 6.0 s, 1H 
(H-3); 5.38 d, J = 10.0 Hz, 1H (H-3V); 6.62 d, J = 10.0 Hz, 1H (H-4V); 0.95 s, 6H, 2(CH3-chromen). 
R = 3-methylbutyryl: 3.19 d, J = 6.7 Hz, 2H (CH2); 2.32 sept., J = 6.7 Hz, 1H (CH); 1.06 d, J = 6.7 Hz, 
6H, 2(CH3). 
Methanol Extract 
Concentration in vacuo yielded a total of 181 g. A sample (60 g) was macerated with EtOAc (800 ml) 
obtaining an insoluble brown colored powder (34 g). The soluble part was treated for removing 
chlorophylls as previously indicated, concentrated in vacuo, and after adding warm hexane, canophyllol 
(2) precipitated. The remaining solution was concentrated and dissolved with acetone obtaining crystals 
of shikimic acid (14) (663 mg). 
Bioasays 
Triterpenes, phenolic acids, and isommameigin (12) were tested as pure compounds. The coumarins 
were generally tested as mixtures of two isomers which differ only by the kind of acyl substituent (3-
1640 R. Reyes-Chilpa et al. / Life Sciences 75 (2004) 1635–1647 
methylbutyryl or 2-methylbutyryl) attached to C-8. Percentage of each isomer was determined from the 
1HNMR spectrum, and in some cases by GC-MS of sylilated mixtures. Coumarin mixtures were: 
mammea A/BA + mammea A/BB (3  4, 70:30%), mammea B/BA + mammea B/BB (5  6, 45:55%), 
mammea C/OA + mammea C/OB (7  8, 70:30%), and mammea B/BA cyclo F + mammea B/BB cyclo 
F (9  10, 70:30%). 
Citotoxicity assay and estimation of IC50 
Assays were performed by the method of sulforradamine B (SRB) as previously described (Skehan et 
al., 1990) with 3 human tumor cell lines K562 (lymphoma), U251 (central nervous system), and PC3 
(prostata). Briefly, this method is based on the bonding of anionic dye SRB to proteins of cells fixed with 
10% trichloroacetic acid. The complex protein-SRB is solubilized with a Tris buffer, which is read at 515 
nm in a microtiter plate reader. Preliminary screening was carried out at 31 AM; all compounds were 
dissolved in DMSO with a maximum concentration at 0.5%. DMSO at this concentration was 
completely innocuous. Reported values are means of 3 experiments with 3 replicates each one. Only 
those compounds that inhibited cellular growth in more than 50% of at least two tumor lines followed to 
determination of IC50 values. In this case the coumarins mammea A/BA (3) and mammea B/BA cyclo F 
(9) were tested as pure compounds. Positive control was adriamycin. 
Screening of HIV-1 RT inhibition 
Possible inhibition by isolated compounds of reverse transcriptase of human inmunodeficiency 
virus -type 1-, was tested with the Lenti RT kit (Cavidi Tech, Uppsala, Sweden). The principle, 
performance, and composition of this nonradioactive microtiter plate RT assay have been described 
previously (Ekstrand et al., 1996; Shao et al., 1997). Screening was carried out at 2.5, 25 and 250 
AM, compounds were dissolved in DMSO with a maximum final concentration at 10%. At this 
concentration DMSO inhibited in less than 10% HIV-1 RT activity. Reported values were corrected 
for solvent activity, and are means of two experiments with 3 replicates each one. As positive 
control, nevirapine, a non nucleoside HIV reverse transcriptase inhibitor, was employed. This 
compound was extracted with CH2Cl2 from commercial tablets, its identity and purity was confirmed 
by 1HNMR. 
Screening of antibiotic activity 
Assays were performed following the method described by Ca´ceres et al. (1990). Eleven bacterial 
strains from the collection of the Department of Public Health, School of Medicine, National University 
of Mexico –UNAM- (Cravioto et al., 1996) were tested: Escherichia coli enteropatogenic (EPEC) 
(029358), E. coli enteropatogenic (ETEC) (050933), E. coli enteroinvasive (EIEC) (28918), E. coli 
(ATCC 25922), E. coli agregative (AGG) (049766), Salmonella typhi (clinical isolation RRE) (095426), 
Salmonella typhi (ATCC 6539), Salmonella typhimurium (074289), Shigella dysenteriae (Dys 3), 
Staphylococcus aureus, Pseudomonas aeruginosa (ATCC 27853). In addition 3 bacterial strains from 
the collection of Instituto de Quı´mica, UNAM, were tested: Staphylococcus aureus, Bacillus subtilis and 
S. epidermidis. Substances were dissolved in acetone or water, impregnated in filter paper disks 
(Whatmann 3, 6 mm of diameter) and tested at 500 Ag/disc with plates (Muller-Hington agar) previously
R. Reyes-Chilpa et al. / Life Sciences 75 (2004) 1635–1647 1641 
inoculated, and incubated (37j, 24 h). Reported values are means of inhibition halus in milimeters of 
three replicates. Chloramphenicol (0.25 Ag/disc) was used as positive control. 
Results 
The hexane extract of Calophyllum brasiliense leaves afforded the triterpenoids, friedelin (1), and 
canophyllol (2), and eight coumarins (3–10) belonging to the mammea type (Fig. 2). The acetone 
extract yielded several of the above mentioned compounds (1, 2, 3  4, 9  10), but in addition the 
Fig. 2. Compounds isolated from C. brasiliense leaves.
1642 R. Reyes-Chilpa et al. / Life Sciences 75 (2004) 1635–1647 
biflavonoid amentoflavone (11), isomammeigin (12), and protocatechuic acid (13). The methanol 
extract afforded friedelin (1), and shikimic acid (14). Coumarins and triterpenoids were abundant 
secondary metabolites, since they represented approximately 1.5% and 0.5% of the dry weight of the 
leaves, respectively. Predominant compounds were mammea A/BA (3), mammea A/BB (4), friedelin 
(1), and canophyllol (2). 
All the mammea coumarins tested showed citotoxicity at 31 AM against the three human tumor cell 
lines (Table 1). The most active were the mixtures of 3  4, and 7  8, which showed inhibition values 
of 88 to 100%. The mixture 9  10, as well as isomammeigin (12), only inhibited the growth of tumor 
cells in 38 to 69% (Table 1). The triterpene friedelin (1) inhibited in 61.9% the PC3 line, while U251 was 
less affected, and was harmless to K562. The other triterpene canophyllol (2), as well as, shikimic and 
protocatechuic Acids (14 and 13) were inactive (Table 1). Structurally friedelin (1) differs from 
canophyllol (2) just for lacking one hydroxyl on C-28, therefore presence this functional group seems 
to decrease activity. 
IC50 values (Table 2) were calculated for compounds that showed inhibition values higher than 50% 
with at least 2 cell lines. The highest activity was recorded for mammea A/BA (3) (IC50 = 0.04 to 0.59 
AM), followed by the mixtures of mammea A/BA + A/BB (3  4), mammea B/BA + B/BB (5  6) and 
mammea C/OA + C/OB (7  8) also with high activity (IC50 V 4.05 AM). In contrast mammea B/BA 
cyclo F pure (9) or in mixture with mammea B/BB cyclo F (10) were less potent with at IC50 5.0–63 
AM. The above data suggests that a propyl, pentyl or phenyl on C-4 (3–8) is relevant for high cytoxicity 
activity. On the other hand, a 6-prenyl chain (3–8) increases cytotoxicity, but this effect diminish if this 
substituent is cyclized to a dihydrofuran or a pyran ring (9–10). 
Regarding to HIV-1 reverse transcriptase, the C. brasiliense compounds tested did not inhibited its 
polymerase activity (Table 3). For instance, the best of all compounds, isomammeigin (12) only showed 
8.6 % inhibition; meanwhile the positive control, nevirapine, at the same concentration (250 AM) 
inhibited in 74.1% the enzyme. 
Table 1 
Inhibition of human tumor cell lines by C. brasiliense compounds* 
% of growth inhibition 
PC3 K562 U251 
Triterpenes 
Friedelin (1) 61.9 0 25.8 
Canophyllol (2) 7.0 0 13.7 
Coumarins 
Mammea A/BA + A/BB (3  4) 96.4 88.6 93.1 
Mammea C/OA + C/OB (7  8) 100 89.1 100 
Mammea B/BA cyclo F + B/BB cyclo F (9  10) 48.4 69.5 57.8 
Isomammeigin (12) 63.4 46.8 38.9 
Acids 
Protocatechuic acid (13) 0 0 0 
Shikimic acid (14) 2.32 0 5.26 
*31 AM; mean of 3 experiments with 3 replicates each one.
R. Reyes-Chilpa et al. / Life Sciences 75 (2004) 1635–1647 1643 
Table 2 
IC50 of C. brasiliense compounds* on human tumor cell lines 
IC50 (AM) 
PC3 K562 U251 
Coumarins 
Mammea A/BA (3) 0.31 F 0.01 0.04 F 0.02 0.59 F 0.10 
Mammea B/BA cyclo F (9) 8.31 F 0.45 10.79 F 1.14 25 
Mammea A/BA + A/BB (3  4) 0.34 F 0.10 4.05 F 0.99 1.92 F 0.17 
Mammea B/BA + B/BB (5  6) 1.02 F 0.01 0.65 F 0.09 1.47 F 0.10 
Mammea C/OA + C/OB (7  8) 1.91 F 1.18 1.59 F 0.41 1.85 F 0.30 
Mammea B/BA cyclo F + B/BB cyclo F (9  10) 63.02 F 0.58 5.00 F 1.07 9.4 F 1.03 
Positive Control 
Adriamycin 0.12 F 0.07 1.04 F 0.53 0.054 F 0.02 
*Means F S.E. of 3 independent experiments with 3 replicates each one. 
Many of the compounds isolated from C. brasiliense failed to inhibit (500 Ag/disc) the growth of 
fourteen bacterial strains. These were mammea A/BA + A/BB (3  4), mammea C/OA + C/OB (7  8), 
mammea B/BA cyclo F + B/BB cyclo F (9  10), friedelin (1), canophyllol (2), protocatechuic acid (13), 
and shikimic acid (14). However, mammea A/BA + A/BB (3  4), and mammea C/OA + C/OB (7  
Table 3 
Effect of C. brasiliense compounds* on HIV-1 RT activity 
Inhibition (%) 
Triterpenes 
Friedelin (1) 6.1 
Canophyllol (2) 0.0 
Coumarins 
Mammea A/BA + A/BB (3  4) 0.0 
Mammea C/OA + C/OB (7  8) 0.0 
Mammea B/BA cyclo F + B/BB cyclo F (9  10) 4.7 
Biflavonoid 
Amentoflavone (11) 0.0 
Coumarins 
Isomammeigin (12) 8.6 
Acids 
Protocatechuic acid (13) 2.0 
Shikimic acid (14) 2.7 
Positive control 
Nevirapine 74.1 
*250 AM. Means of two experiments with 3 replicates each one.
1644 R. Reyes-Chilpa et al. / Life Sciences 75 (2004) 1635–1647 
Table 4 
Effect of C. brasiliense coumarins* on enterobacteria 
8), reduced the growth of Staphylococcus aureus, S. epidermidis, and Bacillus subtilis (Table 4). Mean 
inhibition halus were smaller than that induced by the antibiotic chloramphenicol. 
Discussion 
Previous investigations have demonstrated that coumarins can be cytotoxic in vitro to several 
human tumor cells. Although the type and number of cell lines differ among these studies, it is 
tempting to do some rough comparisons with the most active compounds here described. The 
coumarins mammea A/BA (3), and the mixtures of mammea A/BA + A/BB (3  4), mammea B/BA + 
B/BB (5  6) and mammea C/OA + C/OB (7  8) showed an IC50 V 4.05 AM (Table 2). Their 
potency is clearly superior if compared with coumarin itself, and a series of monoxygenated coumarins 
which exhibited IC50 values 400 AM (Kawaii et al., 2001; Jime´nez-Orozco et al., 1999). Less contrast 
can be observed when compared with the most active of a number of dihydroxylated coumarins, 
esculetin (6,7-dihydroxycoumarin) and nordalbergin (4-phenyl-6,7-dihydroxycoumarin), which showed 
an IC50 = 17–30 AM (Kawaii et al., 2001). The structurally related coumarins, mammea A/BA cyclo F 
(Fig. 1), and mammea A/BB cyclo F, tested against KB cells showed IC50 values of 9 and 15 Ag/ml 
(21 and 35 AM), respectively (Guilet et al., 2001b). Several preliminary conclusions can be depicted 
from the above data. As indicated by Kawaii et al. (2001), a dihydroxylated coumarin moiety is 
important for cytotoxic activity. However, regarding to mammea type coumarins it seems that a 5,7 
dioxygenated pattern is highly effective. Other features are: substituents at C-4 can be either phenyl or 
alkyl, a 6 or 8-acyl substituent ortho to an hydroxyl, a 6 or 8-prenyl chain ortho to an hydroxyl 
increases activity while it diminishes if cyclized to a dihydrofuran or a pyran ring. 
Mechanism of action of cytotoxic coumarins has not been investigated in detail; however, it is known 
that esculetin, and umbelliferone (7-hydroxycoumarin), can arrest cell cycle, and induce apoptosis (Chu 
et al., 2001; Jime´nez-Orozco et al., 2001; Wang et al., 2002). In the case of esculetin, arrest occurs in G1 
phase; it is caused by hypophosphorilation of Rb protein, downregulation of CDK4 kinase and cyclin 
D1, as well as upregulation of p27 inhibitor (Wang et al., 2002). Apoptosis induced by esculetin involves 
chromatin condensation, DNA fragmentation, and formation of apoptotic bodies, liberation of mito-chondrial 
cytocrome C, activation of caspases 9 and 3, and downregulation of antiapoptotic Bcl-2 
protein (Chu et al., 2001). 
Inhibition (mm) 
S. aureus B. subtilis S. epidermidis 
Coumarins 
Mammea A/BA + A/BB (3  4) 15.2 14.5 14.3 
Mammea C/OA + C/OB (7  8) 13.3 12.0 12.5 
Positive control 
Chloramphenicol* 19.0 15.0 15.3 
**concentration: 0.25 Ag/disc. 
*500 Mg/disc, means of 3 repetitions, mm = milimeters.
R. Reyes-Chilpa et al. / Life Sciences 75 (2004) 1635–1647 1645 
Fig. 3. Mammea A/BB as an hypothetical precursor of Inophyllum B. 
None of the compounds here isolated was able to inhibit HIV-1 RT. In the case of mammea type 
coumarins, their inactivity could be explained by the lack of ring D (2,3-dimethylcroman-4-ol ring) 
attached to C-7 and C-8, and present in the calanolides, inophyllums, and cordatolides. The 
importance of ring D for HIV-1 RT inhibitory properties has been thoroughly demonstrated (McKee 
et al., 1996; Ishikawa et al., 1997; Dharmaratne et al., 2002). Mammea A/BB, or a related 
molecule with a 2-methylbut-2-enoyl substituent on C-8, could be a hypothetical precursor of 
inophyllum B. Rings C and D may arise from cyclizations of hydroxyl on C-5 with C-6 prenyl 
chain, and C-7 hydroxyl with C-8 acyl substituent (followed by C-12 carbonyl reduction), 
respectively (Fig. 3). 
The eight coumarins isolated from C. brasiliense leaves (Fig. 2) have been previously reported 
from Mammea americana, and M. africana seeds (Crombie and Games, 1966; Crombie et al., 1985; 
Carpenter et al., 1970), as well as Mesua racemosa (Morel et al., 1999). Nevertheless, this class of 
compounds have only been described previously from one species within the Calophyllum genus. 
Mammea coumarins with a saturated acyl substituent attached to C-6 or C-8 are chemotaxonomically 
important since they link a small group of Calophyllum species, composed by C. dispar, and now C. 
brasiliense, with Mammea and Mesua genus, all of them included in the Calophylloideae subfamily of 
the Clusiaceae (Guilet et al., 2001a). While performing botanical collects, we have detected that C. 
brasiliense in Mexico may have two different populations differing in their leaves chemistry. One 
chemotype contains mainly mammea type coumarins in the leaves, object of this report. In a next 
paper we will provide evidences of the existence of another chemotype, its chemistry and 
pharmacological properties. 
Acknowledgements 
This research was supported by grant IN207301 from DGAPA-UNAM, and a scholarship from 
CONACyT to Elizabet Estrada Mun˜ iz. We are grateful with Dr. Manuel Jime´nez-Estrada, and Dr. Carlos 
Eslava from the National University of Mexico –UNAM-, for their kind support and technical advice. 
We are also indebted with Raquel Ramı´rez Mun˜oz for laboratory assistance, with Beatriz Quiroz, Rocı´o 
Patin˜o Maya, Luis Velasco, and Javier Perez for recording some of the spectra, and Laura Cortes Zarraga 
for assisting with botanical bibliography.
1646 R. Reyes-Chilpa et al. / Life Sciences 75 (2004) 1635–1647 
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2004 cytotoxic effects of mammea type coumarins from

  • 1. Cytotoxic effects of mammea type coumarins from Calophyllum brasiliense Ricardo Reyes-Chilpaa,*, Elizabet Estrada-Mun˜iza, Teresa Ramı´rez Apana, Badia Amekrazb, Andre Aumelasb, Christopher K. Jankowskib, Mario Va´zquez-Torresc a Instituto de Quı´mica, Universidad Nacional Auto´noma de Me´xico, Ciudad Universitaria, Delagacio´n Coyoacan Me´xico D.F. 04510, Mexico bDe´partement de Chimie et Biochimie. Universite de Moncton, Moncton, Canada NBE1A 3E9 c Instituto de Investigaciones Biolo´gicas. Universidad Veracruzana, Apartado Postal 294, Xalapa, Veracruz, 91000, Mexico Received 24 September 2003; accepted 29 March 2004 Abstract Calophyllum brasiliense (Clusiaceae) is a big tree from the Tropical Rain Forests of the American continent. The organic extracts from the leaves yielded coumarins of the mammea type: mammea A/BA, A/BB, B/BA, B/BB, C/OA, C/OB, B/BA cyclo F, B/BB cyclo F, and isomammeigin. The triterpenoids friedelin and canophyllol, as well as the biflavonoid amentoflavone, protocatechuic and shikimic acids, were also obtained. Most of the isolated compounds were tested in vitro against K562, U251, and PC3 human tumor cell lines. The coumarins were cytotoxic against the three cell lines, the highest activity was shown by mammea A/BA (IC50 = 0.04 to 0.59 AM). The mixtures of mammea A/BA + A/BB, mammea B/BA + B/BB and mammea C/OA + C/OB were also highly active (IC50 < 4.05 AM). Friedelin was cytotoxic only against PC3, and U251 lines. Inhibition of HIV-1 reverse transcriptase was also assayed in vitro; however, none of the tested compounds (250 AM) prevented the activity of this enzyme. Most of the isolated compounds were also inactive against fourteen bacterial strains; however mammea A/BA + A/BB, and mammea C/OA + C/OB inhibited the growth of Staphylococcus aureus, S. epidermidis and Bacillus subtilis. D 2004 Elsevier Inc. All rights reserved. Keywords: Calophyllum brasiliense; Clusiaceae; Mammea; Coumarins; Triterpenoids; Biflavonoids; Cytotoxic activity; Tumor cell lines; HIV; Reverse transcriptase; Enterobacteria * Corresponding author. Tel.: +52-56224430; fax: +52-56162203. E-mail address: chilpa@servidor.unam.mx (R. Reyes-Chilpa). 0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.03.017 www.elsevier.com/locate/lifescie Life Sciences 75 (2004) 1635–1647
  • 2. 1636 R. Reyes-Chilpa et al. / Life Sciences 75 (2004) 1635–1647 Introduction The Calophyllum genus (Clusiaceae) is composed by 180–200 tree species, most of them thrive within the Indo-Pacific zone, particularly from the Malay Peninsula to New Guinea (Stevens, 1980). Calophyllum species have recently received considerable attention from a pharmacological point of view, since some of them produce potent inhibitors of reverse transcriptase of human immunode-ficiency virus type 1 (HIV-1 RT) (Kashman et al., 1992; Patil et al., 1993; McKee et al., 1996; Dharmaratne et al., 2002). These compounds known as calanolides, inophyllums and cordatolides are tetracyclic dipyranocoumarins with a propyl, phenyl or methyl substituent attached to C-4, respectively (Fig. 1; Ishikawa, 2000). Since HIV-1 is characterized by its cytopathic effects, specially on CD-4 lymphocytes, it is necessary that RT inhibitors also show low cytotoxicity (Weislow et al., 1989). Under these criteria, the most promising tetracyclic dipyranocoumarin suitable to be developed as a drug is (+)-calanolide A, which is currently investigated in phase II/III clinical trials. This compound was isolated in low yields from the leaves of C. lanigerum var. austrocoriaceum, but now it is also available synthetically (Flavin et al., 1996). Besides tetracyclic coumarins, tricyclic pyranocoumarins have been isolated from Calophyllum species (Ishikawa, 2000). In addition, it was recently reported that C. dispar contains coumarins of the mammea type, such as mammea A/BA cyclo F (Fig. 1), several of them resulted highly cytotoxic against human epidermoid carcinoma cells (KB) (Guilet et al., 2001a,b). Mammea coumarins are characterized by a simple 5,7-dioxygenated coumarin skeleton bearing a phenyl, or an alkyl chain on C-4, acyl and prenyl (free or cyclized) substituents on either C-6 or C-8 (Crombie and Games, 1966; Crombie and Games, 1967; Crombie et al., 1972). They are common constituents of Mammea and Mesua species (Clusiaceae), but have been only found once in a Calophyllum species (Guilet et al., 2001a,b). To our best knowledge, the anti-HIV-1 properties of this class of compounds have not been investigated yet. Calophyllum brasiliense is the widest distributed species among the eight Calophyllum species found in the American Continent, it grows in the Tropical Rain Forests from Brazil to Mexico (Stevens, 1980; Pennington and Sarukha´n, 1998). Numerous ethnomedical applications have been recorded for this species throughout Latin America (Soto Nun˜ez and Sousa, 1995; Garcı´a Barriga, 1992; Mesı´a-Vela Fig. 1. Tetracyclic dipyranocoumarins and tricyclic dehydrofuranocoumarin.
  • 3. R. Reyes-Chilpa et al. / Life Sciences 75 (2004) 1635–1647 1637 et al., 2001), but apparently none is related to AIDS or cancer treatment. Previous chemical analysis of C. brasiliense bark have reported the presence of xanthones with cancer chemopreventive properties (Ito et al., 2002). Other xanthones with antifungal activity have been isolated from the heartwood (Reyes-Chilpa et al., 1997). Chromanone carboxylic acids were obtained from the bark latex (Stout et al., 1968) and seeds (Plattner et al., 1974). The polar extracts of the leaves contain hyperin (hyperoside), amentoflavone, quercetin, gallic acid, and protocatechuic acid; some of these phenolic compounds exhibited analgesic activity (da Silva et al., 2001). We are now reporting the cytotoxic effects against three human tumor cell lines of mammea type coumarins isolated from the leaves of C. brasiliense. These compounds, along with several phenolic acids, triterpenoids, and a biflavonoid obtained from the same source, were also tested against HIV-1 reverse transcriptase, and fourteen enteropathogenic bacteria. Material and methods Plant material Calophyllum brasiliense Cambess. (Clusiaceae) was collected at Ejido Benigno Mendoza, Sierra de Santa Marta, State of Veracruz, Mexico. Authentication was done by one of us (Mario Va´zquez Torres). A voucher specimen is deposited with the number 435 -Bojo´rquez et al.- in the Herbarium of the Instituto de Investigaciones Biolo´gicas, Universidad Veracruzana (CIB) at Xalapa, Me´xico. Extraction and isolation of compounds The dried leaves (2075 g) were extracted at room temperature over a period of one week with hexane, acetone, and methanol, successively. Compounds were isolated after spontaneous crystallization or by column chromatography on Silica Gel-60 (CC), and identified by their spectroscopic (1HNMR, 13CNMR, IR, UV), EIMS, and CIMS, and comparison with published data. Hexane extract (70.3 g). While in solution, the extract suffered spontaneous precipitation obtaining a white powder (19.8 g). Part of this material (5.1 g) was treated with CH2Cl2, the insoluble part was a mixture (583 mg) of friedelin (1) and canophyllol (2), while the soluble part afforded a mixture (4 g) of mammea A/BA (3) and mammea A/BB (4). The extract was then concentrated in vacuo (35.3 g), and 9.4 g were subjected to CC (200 g). Elution with hexane yielded first friedelin (45.9 mg), afterwards a mixture (5 mg) of mammea B/BA (5) and B/BB (6), and finally a mixture of mammea A/BA and mammea A/BB (720 mg). Elution with hexane-EtOAc (9:1) yielded initially canophyllol (20 mg, m.p. 180–182jC), then a mixture (50 mg) of mammea C/OA (7) and mammea C/OB (8), final fractions afforded a mixture (30 mg) of mammea B/BA cyclo F (9), and mammea B/BB cyclo F (10). Mammea B/BA (5) & mammea B/BB (6) 1HNMR(200 MHz, CDCl3/TMS): 6.86 s, 1H, (OH-5); 6.03 s, 1H, (H-3); 2.93 t, J = 7.5 Hz, 2H, (CH2-CH2 -CH3); 1.38 m, 2H, (CH2-CH2 -CH3); 0.91 t, J = 6.4 Hz, 3H, (CH2-CH2 -CH3).
  • 4. 1638 R. Reyes-Chilpa et al. / Life Sciences 75 (2004) 1635–1647 Isoprenyl on C-6: 5.24 t, J = 7.2 Hz, 1H (CH); 3.49 d, J = 7.2 Hz, 2H (CH2); 1.82 and 1.87, both s & 3H, 2 CH3. (5): 14.61 s, 1H (OH-7). R = 3-methylbutyryl: 3.16 d, J = 6.6 Hz, 2H, (CH2); 2.27 m, J = 6.6 Hz, 1H (CH); 1.03 d, J = 6.7 Hz, 6H, 2(CH3). (6): 14.68 s, 1H (OH-7). R = 2-methylbutyryl: 3.93 m, J = 6.5 Hz, 1H (CH); 1.25 d, J = 6.6 Hz, 3H (CH3); 1.93 m, 2H, (CH2); 0.97 t, J = 6.4 Hz, 3H, (CH3). IR r max (KBr): 3325(OH); 2960, 2931, 2858 (C-H); 1722(C = O); 1602(C = C); 1560 (C = C); 1462(C = C); 1329(C-O); 1116 (C-O). Mammea C/OA (7) & mammea C/OB (8) 1HNMR (200 MHz, CDCl3/TMS): 6.04 s, 1H (H-3); 7.10 s wide, 1H (OH-5); 6.25 s, 1 H (H-6); 6.04 s, 1H (H-3), 2.95 t, J = 7.5 Hz, 2H (CH2-(CH2)3-CH3); 1.64 m, 2H (CH2-CH2-(CH2)2-CH3); 1.37 m, 2H (CH2)3-CH2-CH3); 0.90 t, J = 6.8 Hz, 3H (CH2-(CH2)4-CH3). (7): 14.16 s, 1H (OH-7). R = 3-methylbutyryl: 3.14 d, J = 6.6 Hz, 2H (CH2); 2.26 m, J = 6.6 Hz, 1H (CH); 1.01 d, J = 6.6 Hz, 6H, 2(CH3). (8): 14.12 s, 1 H (OH-7). R = 2-methylbutyryl: 3.90 m, 1H (CH); 1.24 d, J = 6.6 Hz, 3H (CH3), 1.88 m, 2H (CH2); 0.94 t, J = 5.9 Hz, 3H (CH3). EMIE: 70 ev, m/z (%): 332 M+ (36.6%) [C19H24O5]+, 317 (21.10%) [M+-CH3], 299 (7.0%) [M+-CH3- H2O], 276 (66.1%) [M+-C4H8], 275 (100%) [M+-C4H9], 234(13.3%), 219 (12.6%). IR r max (KBr): 3164 (OH); 2961, 2932, 2872 (C-H); 1726 (C = O); 1622 (C = C); 1595 (C = C); 1390 (C-O); 1270 (C-O); 1117 (C-O). Mammea B/BA cyclo F (9) & mammea B/BB cyclo F (10) 1HNMR(200 MHz, CDCl3/TMS): 14.18 s, 1H (OH-7); 5.95 s, 1H (H-3); 2.82 m, J = 7.2 Hz, 2H (CH2- CH2- CH3); 1.64 m, J = 7.4 Hz, 2H (CH2-CH2-CH3); 1.03 t, J = 6.4 Hz, 3H (CH2-CH2-CH3); 4.85 t, J = 9.0 Hz, 2H, (CH-cyclo F), 3.17 d, J = 6.6 Hz, 2H, (CH2-cyclo F), 2.00 s wide, 1H (OH cyclo F); 1.41 and 1.28 both s & 3H, 2(CH3 cyclo F). R = 3-methylbutyryl (B/BA cyclo F): 3.08 d, J = 6.7 Hz, 2H (CH2); 2.24 m, J = 6.6 Hz, 1H (CH); 1.03 d, J = 6.1 Hz 6H, 2(CH3). R = 2-methylbutyryl (B/BB cyclo F): 3.85 m, 1H (CH); 1.23 d, J = 8 Hz, 3H (CH3); 1.92 and 1.5 both m 1H (CH2); 0.99 t, J = 5.6 Hz, 3H (CH3). EIME: 70 ev, m/z (%): 388 M+ (54.9%) [C22H28O6]+; 373(14.0%)[M+-CH3]; 355 (11.2%); 332(22.5%); 331(100%); 301(7.0%); 287(4.9%); 259(14.7%). IR r max (KBr): 2964(C-H); 1728(C = O); 1631(C = C); 1606(C = C); 1429(C = C); 1393(C-O); 1300(C-O); 1150(C-O); 1121(C-O). Acetone extract Concentration in vacuo yielded a total of 101 g. A sample (19 g) was macerated with EtOAc obtaining an insoluble brown colored powder (6 g), which after redisolving in methanol yielded amentoflavone (11) (yellow crystals, mp 300 jC, 430 mg). The soluble part of the extract was treated
  • 5. R. Reyes-Chilpa et al. / Life Sciences 75 (2004) 1635–1647 1639 with activated carbon to remove chlorophylls, filtered on celite, and concentrated to 50 ml. From this solution a mixture (561 mg) of friedelin (1) and canophyllol (2) precipitated. The remaining material (5.3 g) was subjected to CC. Fractions eluted with hexane-EtOAc (9:1) afforded (41.8 mg) isomam-meigin (mammea A/BA cyclo D) (12), and finally friedelin (1). Elution with hexane-EtOAc (8.5:1.5) yielded a mixture (174 mg) of mammea A/BA (3) and A/BB (4). Elution with the same solvent mixture yielded canophyllol (2), and finally a mixture (102 mg) of mammea B/BA cyclo F (9), and mammea B/ BB cyclo F (10). Fractions obtained with hexane-EtOAc (6.5:3.5) afforded protocatechuic acid (13) (100 mg). Purification of compound 3 (mp 122–124 jC) was achieved by HPLC using ODS-column eluting with 90% MeOH, meanwhile compound 9 (mp 125–126 jC) was obtained pure after recristalyzation from CHCl3-hexane. Mammea A/BA (3) mammea A/BB (4) 1HNMR(200 MHz, CDCl3/TMS): 7.56 m, 3H (Ar); 7.41 m, 2H (Ar); 6.0 s, 1H (H-3); 5.95 s, 1H (OH-5). Isoprenyl on C-6: 5.09 tm, J = 6.9 Hz, 1H (CH); 3.29 d, J = 6.9 Hz, 2H (CH2); 1.65 and 1.70, both s 3H, (2 CH3). (3): 14.61 s, 1H (OH-7). R = 3-methylbutyryl: 3.19 d, J = 6.7 Hz, 2H (CH2);2.32 m, J = 6.7 Hz, 1H (CH);1.06 d, J = 6.6 Hz, 6H, (2 CH3). (4): 14.57 s, 1H (OH-7). R = 2-methylbutyryl (A/BB): 3.95 m, J = 6.6 Hz, 1H (CH-CH3); 1.29 d, J = 6.7 Hz, 3H (CH-CH3), 1.94 m, 2H (CH2); 1.01 t, J = 7.2 Hz, 3H (CH3). EMIE: 70 ev m/z (%): 406 M+ (96.4%)[C25H26O5]+; 363(55.6%); 351(76.0%); 293 (100 %). IR r max (KBr) 3985(OH); 2964, 2932, 2872(C-H); 1729(C = O); 1724(C = O); 1614 (C = C);1556 (C = C); 1390(C-O). Isomammeigin (mammea A/BA cyclo D) (12). Yellow crystals, mp 162–164 jC 1HNMR(200 MHz, CDCl3/TMS): 14.78 s, 1H (OH-7); 7.39 m, 3H (Ar); 7.23 m, 2H (Ar); 6.0 s, 1H (H-3); 5.38 d, J = 10.0 Hz, 1H (H-3V); 6.62 d, J = 10.0 Hz, 1H (H-4V); 0.95 s, 6H, 2(CH3-chromen). R = 3-methylbutyryl: 3.19 d, J = 6.7 Hz, 2H (CH2); 2.32 sept., J = 6.7 Hz, 1H (CH); 1.06 d, J = 6.7 Hz, 6H, 2(CH3). Methanol Extract Concentration in vacuo yielded a total of 181 g. A sample (60 g) was macerated with EtOAc (800 ml) obtaining an insoluble brown colored powder (34 g). The soluble part was treated for removing chlorophylls as previously indicated, concentrated in vacuo, and after adding warm hexane, canophyllol (2) precipitated. The remaining solution was concentrated and dissolved with acetone obtaining crystals of shikimic acid (14) (663 mg). Bioasays Triterpenes, phenolic acids, and isommameigin (12) were tested as pure compounds. The coumarins were generally tested as mixtures of two isomers which differ only by the kind of acyl substituent (3-
  • 6. 1640 R. Reyes-Chilpa et al. / Life Sciences 75 (2004) 1635–1647 methylbutyryl or 2-methylbutyryl) attached to C-8. Percentage of each isomer was determined from the 1HNMR spectrum, and in some cases by GC-MS of sylilated mixtures. Coumarin mixtures were: mammea A/BA + mammea A/BB (3 4, 70:30%), mammea B/BA + mammea B/BB (5 6, 45:55%), mammea C/OA + mammea C/OB (7 8, 70:30%), and mammea B/BA cyclo F + mammea B/BB cyclo F (9 10, 70:30%). Citotoxicity assay and estimation of IC50 Assays were performed by the method of sulforradamine B (SRB) as previously described (Skehan et al., 1990) with 3 human tumor cell lines K562 (lymphoma), U251 (central nervous system), and PC3 (prostata). Briefly, this method is based on the bonding of anionic dye SRB to proteins of cells fixed with 10% trichloroacetic acid. The complex protein-SRB is solubilized with a Tris buffer, which is read at 515 nm in a microtiter plate reader. Preliminary screening was carried out at 31 AM; all compounds were dissolved in DMSO with a maximum concentration at 0.5%. DMSO at this concentration was completely innocuous. Reported values are means of 3 experiments with 3 replicates each one. Only those compounds that inhibited cellular growth in more than 50% of at least two tumor lines followed to determination of IC50 values. In this case the coumarins mammea A/BA (3) and mammea B/BA cyclo F (9) were tested as pure compounds. Positive control was adriamycin. Screening of HIV-1 RT inhibition Possible inhibition by isolated compounds of reverse transcriptase of human inmunodeficiency virus -type 1-, was tested with the Lenti RT kit (Cavidi Tech, Uppsala, Sweden). The principle, performance, and composition of this nonradioactive microtiter plate RT assay have been described previously (Ekstrand et al., 1996; Shao et al., 1997). Screening was carried out at 2.5, 25 and 250 AM, compounds were dissolved in DMSO with a maximum final concentration at 10%. At this concentration DMSO inhibited in less than 10% HIV-1 RT activity. Reported values were corrected for solvent activity, and are means of two experiments with 3 replicates each one. As positive control, nevirapine, a non nucleoside HIV reverse transcriptase inhibitor, was employed. This compound was extracted with CH2Cl2 from commercial tablets, its identity and purity was confirmed by 1HNMR. Screening of antibiotic activity Assays were performed following the method described by Ca´ceres et al. (1990). Eleven bacterial strains from the collection of the Department of Public Health, School of Medicine, National University of Mexico –UNAM- (Cravioto et al., 1996) were tested: Escherichia coli enteropatogenic (EPEC) (029358), E. coli enteropatogenic (ETEC) (050933), E. coli enteroinvasive (EIEC) (28918), E. coli (ATCC 25922), E. coli agregative (AGG) (049766), Salmonella typhi (clinical isolation RRE) (095426), Salmonella typhi (ATCC 6539), Salmonella typhimurium (074289), Shigella dysenteriae (Dys 3), Staphylococcus aureus, Pseudomonas aeruginosa (ATCC 27853). In addition 3 bacterial strains from the collection of Instituto de Quı´mica, UNAM, were tested: Staphylococcus aureus, Bacillus subtilis and S. epidermidis. Substances were dissolved in acetone or water, impregnated in filter paper disks (Whatmann 3, 6 mm of diameter) and tested at 500 Ag/disc with plates (Muller-Hington agar) previously
  • 7. R. Reyes-Chilpa et al. / Life Sciences 75 (2004) 1635–1647 1641 inoculated, and incubated (37j, 24 h). Reported values are means of inhibition halus in milimeters of three replicates. Chloramphenicol (0.25 Ag/disc) was used as positive control. Results The hexane extract of Calophyllum brasiliense leaves afforded the triterpenoids, friedelin (1), and canophyllol (2), and eight coumarins (3–10) belonging to the mammea type (Fig. 2). The acetone extract yielded several of the above mentioned compounds (1, 2, 3 4, 9 10), but in addition the Fig. 2. Compounds isolated from C. brasiliense leaves.
  • 8. 1642 R. Reyes-Chilpa et al. / Life Sciences 75 (2004) 1635–1647 biflavonoid amentoflavone (11), isomammeigin (12), and protocatechuic acid (13). The methanol extract afforded friedelin (1), and shikimic acid (14). Coumarins and triterpenoids were abundant secondary metabolites, since they represented approximately 1.5% and 0.5% of the dry weight of the leaves, respectively. Predominant compounds were mammea A/BA (3), mammea A/BB (4), friedelin (1), and canophyllol (2). All the mammea coumarins tested showed citotoxicity at 31 AM against the three human tumor cell lines (Table 1). The most active were the mixtures of 3 4, and 7 8, which showed inhibition values of 88 to 100%. The mixture 9 10, as well as isomammeigin (12), only inhibited the growth of tumor cells in 38 to 69% (Table 1). The triterpene friedelin (1) inhibited in 61.9% the PC3 line, while U251 was less affected, and was harmless to K562. The other triterpene canophyllol (2), as well as, shikimic and protocatechuic Acids (14 and 13) were inactive (Table 1). Structurally friedelin (1) differs from canophyllol (2) just for lacking one hydroxyl on C-28, therefore presence this functional group seems to decrease activity. IC50 values (Table 2) were calculated for compounds that showed inhibition values higher than 50% with at least 2 cell lines. The highest activity was recorded for mammea A/BA (3) (IC50 = 0.04 to 0.59 AM), followed by the mixtures of mammea A/BA + A/BB (3 4), mammea B/BA + B/BB (5 6) and mammea C/OA + C/OB (7 8) also with high activity (IC50 V 4.05 AM). In contrast mammea B/BA cyclo F pure (9) or in mixture with mammea B/BB cyclo F (10) were less potent with at IC50 5.0–63 AM. The above data suggests that a propyl, pentyl or phenyl on C-4 (3–8) is relevant for high cytoxicity activity. On the other hand, a 6-prenyl chain (3–8) increases cytotoxicity, but this effect diminish if this substituent is cyclized to a dihydrofuran or a pyran ring (9–10). Regarding to HIV-1 reverse transcriptase, the C. brasiliense compounds tested did not inhibited its polymerase activity (Table 3). For instance, the best of all compounds, isomammeigin (12) only showed 8.6 % inhibition; meanwhile the positive control, nevirapine, at the same concentration (250 AM) inhibited in 74.1% the enzyme. Table 1 Inhibition of human tumor cell lines by C. brasiliense compounds* % of growth inhibition PC3 K562 U251 Triterpenes Friedelin (1) 61.9 0 25.8 Canophyllol (2) 7.0 0 13.7 Coumarins Mammea A/BA + A/BB (3 4) 96.4 88.6 93.1 Mammea C/OA + C/OB (7 8) 100 89.1 100 Mammea B/BA cyclo F + B/BB cyclo F (9 10) 48.4 69.5 57.8 Isomammeigin (12) 63.4 46.8 38.9 Acids Protocatechuic acid (13) 0 0 0 Shikimic acid (14) 2.32 0 5.26 *31 AM; mean of 3 experiments with 3 replicates each one.
  • 9. R. Reyes-Chilpa et al. / Life Sciences 75 (2004) 1635–1647 1643 Table 2 IC50 of C. brasiliense compounds* on human tumor cell lines IC50 (AM) PC3 K562 U251 Coumarins Mammea A/BA (3) 0.31 F 0.01 0.04 F 0.02 0.59 F 0.10 Mammea B/BA cyclo F (9) 8.31 F 0.45 10.79 F 1.14 25 Mammea A/BA + A/BB (3 4) 0.34 F 0.10 4.05 F 0.99 1.92 F 0.17 Mammea B/BA + B/BB (5 6) 1.02 F 0.01 0.65 F 0.09 1.47 F 0.10 Mammea C/OA + C/OB (7 8) 1.91 F 1.18 1.59 F 0.41 1.85 F 0.30 Mammea B/BA cyclo F + B/BB cyclo F (9 10) 63.02 F 0.58 5.00 F 1.07 9.4 F 1.03 Positive Control Adriamycin 0.12 F 0.07 1.04 F 0.53 0.054 F 0.02 *Means F S.E. of 3 independent experiments with 3 replicates each one. Many of the compounds isolated from C. brasiliense failed to inhibit (500 Ag/disc) the growth of fourteen bacterial strains. These were mammea A/BA + A/BB (3 4), mammea C/OA + C/OB (7 8), mammea B/BA cyclo F + B/BB cyclo F (9 10), friedelin (1), canophyllol (2), protocatechuic acid (13), and shikimic acid (14). However, mammea A/BA + A/BB (3 4), and mammea C/OA + C/OB (7 Table 3 Effect of C. brasiliense compounds* on HIV-1 RT activity Inhibition (%) Triterpenes Friedelin (1) 6.1 Canophyllol (2) 0.0 Coumarins Mammea A/BA + A/BB (3 4) 0.0 Mammea C/OA + C/OB (7 8) 0.0 Mammea B/BA cyclo F + B/BB cyclo F (9 10) 4.7 Biflavonoid Amentoflavone (11) 0.0 Coumarins Isomammeigin (12) 8.6 Acids Protocatechuic acid (13) 2.0 Shikimic acid (14) 2.7 Positive control Nevirapine 74.1 *250 AM. Means of two experiments with 3 replicates each one.
  • 10. 1644 R. Reyes-Chilpa et al. / Life Sciences 75 (2004) 1635–1647 Table 4 Effect of C. brasiliense coumarins* on enterobacteria 8), reduced the growth of Staphylococcus aureus, S. epidermidis, and Bacillus subtilis (Table 4). Mean inhibition halus were smaller than that induced by the antibiotic chloramphenicol. Discussion Previous investigations have demonstrated that coumarins can be cytotoxic in vitro to several human tumor cells. Although the type and number of cell lines differ among these studies, it is tempting to do some rough comparisons with the most active compounds here described. The coumarins mammea A/BA (3), and the mixtures of mammea A/BA + A/BB (3 4), mammea B/BA + B/BB (5 6) and mammea C/OA + C/OB (7 8) showed an IC50 V 4.05 AM (Table 2). Their potency is clearly superior if compared with coumarin itself, and a series of monoxygenated coumarins which exhibited IC50 values 400 AM (Kawaii et al., 2001; Jime´nez-Orozco et al., 1999). Less contrast can be observed when compared with the most active of a number of dihydroxylated coumarins, esculetin (6,7-dihydroxycoumarin) and nordalbergin (4-phenyl-6,7-dihydroxycoumarin), which showed an IC50 = 17–30 AM (Kawaii et al., 2001). The structurally related coumarins, mammea A/BA cyclo F (Fig. 1), and mammea A/BB cyclo F, tested against KB cells showed IC50 values of 9 and 15 Ag/ml (21 and 35 AM), respectively (Guilet et al., 2001b). Several preliminary conclusions can be depicted from the above data. As indicated by Kawaii et al. (2001), a dihydroxylated coumarin moiety is important for cytotoxic activity. However, regarding to mammea type coumarins it seems that a 5,7 dioxygenated pattern is highly effective. Other features are: substituents at C-4 can be either phenyl or alkyl, a 6 or 8-acyl substituent ortho to an hydroxyl, a 6 or 8-prenyl chain ortho to an hydroxyl increases activity while it diminishes if cyclized to a dihydrofuran or a pyran ring. Mechanism of action of cytotoxic coumarins has not been investigated in detail; however, it is known that esculetin, and umbelliferone (7-hydroxycoumarin), can arrest cell cycle, and induce apoptosis (Chu et al., 2001; Jime´nez-Orozco et al., 2001; Wang et al., 2002). In the case of esculetin, arrest occurs in G1 phase; it is caused by hypophosphorilation of Rb protein, downregulation of CDK4 kinase and cyclin D1, as well as upregulation of p27 inhibitor (Wang et al., 2002). Apoptosis induced by esculetin involves chromatin condensation, DNA fragmentation, and formation of apoptotic bodies, liberation of mito-chondrial cytocrome C, activation of caspases 9 and 3, and downregulation of antiapoptotic Bcl-2 protein (Chu et al., 2001). Inhibition (mm) S. aureus B. subtilis S. epidermidis Coumarins Mammea A/BA + A/BB (3 4) 15.2 14.5 14.3 Mammea C/OA + C/OB (7 8) 13.3 12.0 12.5 Positive control Chloramphenicol* 19.0 15.0 15.3 **concentration: 0.25 Ag/disc. *500 Mg/disc, means of 3 repetitions, mm = milimeters.
  • 11. R. Reyes-Chilpa et al. / Life Sciences 75 (2004) 1635–1647 1645 Fig. 3. Mammea A/BB as an hypothetical precursor of Inophyllum B. None of the compounds here isolated was able to inhibit HIV-1 RT. In the case of mammea type coumarins, their inactivity could be explained by the lack of ring D (2,3-dimethylcroman-4-ol ring) attached to C-7 and C-8, and present in the calanolides, inophyllums, and cordatolides. The importance of ring D for HIV-1 RT inhibitory properties has been thoroughly demonstrated (McKee et al., 1996; Ishikawa et al., 1997; Dharmaratne et al., 2002). Mammea A/BB, or a related molecule with a 2-methylbut-2-enoyl substituent on C-8, could be a hypothetical precursor of inophyllum B. Rings C and D may arise from cyclizations of hydroxyl on C-5 with C-6 prenyl chain, and C-7 hydroxyl with C-8 acyl substituent (followed by C-12 carbonyl reduction), respectively (Fig. 3). The eight coumarins isolated from C. brasiliense leaves (Fig. 2) have been previously reported from Mammea americana, and M. africana seeds (Crombie and Games, 1966; Crombie et al., 1985; Carpenter et al., 1970), as well as Mesua racemosa (Morel et al., 1999). Nevertheless, this class of compounds have only been described previously from one species within the Calophyllum genus. Mammea coumarins with a saturated acyl substituent attached to C-6 or C-8 are chemotaxonomically important since they link a small group of Calophyllum species, composed by C. dispar, and now C. brasiliense, with Mammea and Mesua genus, all of them included in the Calophylloideae subfamily of the Clusiaceae (Guilet et al., 2001a). While performing botanical collects, we have detected that C. brasiliense in Mexico may have two different populations differing in their leaves chemistry. One chemotype contains mainly mammea type coumarins in the leaves, object of this report. In a next paper we will provide evidences of the existence of another chemotype, its chemistry and pharmacological properties. Acknowledgements This research was supported by grant IN207301 from DGAPA-UNAM, and a scholarship from CONACyT to Elizabet Estrada Mun˜ iz. We are grateful with Dr. Manuel Jime´nez-Estrada, and Dr. Carlos Eslava from the National University of Mexico –UNAM-, for their kind support and technical advice. We are also indebted with Raquel Ramı´rez Mun˜oz for laboratory assistance, with Beatriz Quiroz, Rocı´o Patin˜o Maya, Luis Velasco, and Javier Perez for recording some of the spectra, and Laura Cortes Zarraga for assisting with botanical bibliography.
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