2004 cytotoxic effects of mammea type coumarins from

www.elsevier.com/locate/lifescie

Life Sciences 75 (2004) 1635–1647

Cytotoxic effects of mammea type coumarins from

Calophyllum brasiliense

Ricardo Reyes-Chilpaa,*, Elizabet Estrada-Muniza, Teresa Ramırez Apana,Badia Amekrazb, Andre Aumelasb, Christopher K. Jankowskib, Mario Vazquez-Torresc

a Instituto de Quımica, Universidad Nacional Autonoma de Mexico, Ciudad Universitaria,

Delagacion Coyoacan Mexico D.F. 04510, MexicobDepartement de Chimie et Biochimie. Universite de Moncton, Moncton, Canada NBE1A 3E9

c Instituto de Investigaciones Biologicas. 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

0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.lfs.2004.03.017

* Corresponding author. Tel.: +52-56224430; fax: +52-56162203.

E-mail address: [email protected] (R. Reyes-Chilpa).

R. Reyes-Chilpa et al. / Life Sciences 75 (2004) 1635–16471636

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 Sarukhan, 1998). Numerous ethnomedical applications have been recorded for

this species throughout Latin America (Soto Nunez and Sousa, 1995; Garcıa Barriga, 1992; Mesıa-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

Vazquez Torres). A voucher specimen is deposited with the number 435 -Bojorquez et al.- in the

Herbarium of the Instituto de Investigaciones Biologicas, Universidad Veracruzana (CIB) at

Xalapa, Mexico.

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).


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


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-


methylbutyryl or 2-methylbutyryl) attached to C-8. Percentage of each isomer was determined from the1HNMR 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 Caceres 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 Quımica, 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


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.


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.

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.

Table 4

Effect of C. brasiliense coumarins* on enterobacteria

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.


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; Jimenez-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; Jimenez-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).

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 Muniz. We are grateful with Dr. Manuel Jimenez-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 Ramırez Munoz for laboratory assistance, with Beatriz Quiroz, Rocıo

Patino 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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