Bioactive flavonoids and triterpenes from Terminalia fagifolia (Combretaceae)

Two 1,3-diarylpropanes, 1-(4'-hydroxy-2'-methoxyphenyl)-3-(3"-methoxy-4" -hydroxyphenyl)-propane and 1-(2'-hydroxy-4',6'-dimethoxyphenyl)-3-(3"-methoxy-4" -hydroxyphenyl)-propane, seven flavanones, naringenin, naringenin-4',7-dimethyl-ether, sakuranetin, isosakuranetin, liquiritigenin-4',7-dimethyl-ether, liquiritigenin-7-methyl-ether and liquiritigenin-4'-methyl-ether, two chalcones, isoliquiritigenin-4-methyl-ether and isoliquiritigenin-4'-methyl-ether, one flavan, 7,4'-dihydroxy-3'-methoxyflavan, nine triterpenes, arjunic acid, arjunetin, arjungenin, arjunglucoside I, arjunolic acid, arjunglucoside II, 23-galloylarjunglucoside II (isolated as its mono-, di- and tri-O-methyl derivatives after methylation with diazomethane), betulinic acid and ursolic acid acetate, along with gallic acid and sitosterol were isolated from the heartwood and trunk bark of of Terminalia fagifolia. The flavanones and chalcones obtained in the present work are new in the Combretaceae and this is the first report of the occurrence of 1,3-diarylpropanes in this family. The isolated compounds were evaluated for their in vitro cytotoxic activity against two human cancer cell lines (Hep2 larynx carcinoma and H292 lung mucoepidermoid carcinoma) and antioxidant properties. Isoliquiritigenin-4-methyl-ether, isoliquiritigenin-4'-methyl-ether, 1-(2'-hydroxy-4',6'-dimethoxyphenyl)-3-(3"-methoxy -4"-hydroxyphenyl)-propane and the di- and tri-O-methyl derivatives of 23-galloylarjunglucoside II were the most active in the cytotoxic assay.

In a continuation of our study on constituents of plants of the Terminalia genus which occur in the central-western region of Brazil, 2,3 we have examined the composition of the ethanol extracts from the heartwood and the trunk bark of T. fagifolia. Herein we describe the isolation and structural identification of twelve flavonoids, comprising seven flavanones (1-7), one flavan (8), two chalcones (9 and 10), two diarylpropanes (11 and 12) and nine pentacyclic triterpenes (13)(14)(15)(16)(17)(18)(19)(20)(21), in addition to gallic acid and sitosterol. The flavanones and chalcones are new in the Combretaceae. To date, diarylpropanes were mostly reported in members of the Myristicaceae and this is the first report for their occurrence in the Combretaceae family.
The structural elucidation of these isolates was established on the basis of 1D and 2D NMR spectroscopic techniques.
The evaluation of the in vitro cytotoxic activities of sixteen compounds against two human cancer cell lines (Hep 2 and H 292 ), as well as the antioxidative properties of eighteen isolated compounds are also reported.
Compounds 13-18 and 20, known pentacyclic triterpenes whose occurrence is not uncommon in the genus Terminalia, were identified as arjunic acid, arjunetin, arjungenin, arjunglucoside I, arjunolic acid, arjunglucoside II and betulinic acid, respectively, and their corresponding NMR spectral data were in full agreement with those reported in the literature. 15,16 23-Galloylarjunglucoside II (19) has been formerly obtained solely from T. macroptera 17 and in the present work it was isolated and characterized after methylation with diazomethane as its mono-, di-and tri-O-methyl derivatives 23-(4"-O-methyl)-galloylarjunglucoside II (19a), 23-(3",4"-di-O-methyl)-galloylarjun- glucoside II (19b) and 23-(3",4",5"-tri-O-methyl)galloylarjunglucoside II (19c). The carbon signals of 19a-19c were very similar to those of 19, except for the signals attributable to the galloyl moiety, which were consistent with the presence of methoxy groups at C-4" in 19a, C-3" and C-4" in 19b and C-3", C-4" and C-5" in 19c, as shown in Table 2. Compound 21 was identified as ursolic acid acetate 15 and in spite of the wide distribution of this triterpene in other plant genera, no record is available for its presence in Terminalia.
Compounds 2, 3, 6, 8-16, 19a-19c and 22 were assayed in vitro against the Hep 2 (larynx carcinoma) and H 292 (lung mucoepidermoid carcinoma) human cell lines. As depicted in Table 3, compounds 9, 10, 12, 19b and 19c displayed significant cytotoxic activity on both cell lines (IC 50 values in the range of 9.7-23.2 μg mL -1 ), while 11 and 13 exhibited weak cytotoxicity in this assay (IC 50 values in the range of 30.7 -41.4 μg mL -1 ). The cytotoxic antitumor drug cisplatin was taken as positive control (IC 50 5.1 and 7.8 μg mL -1 ). The detection of cytotoxic compounds in Terminalia fagifolia associated to its use in the Brazilian folk medicine for the treatment of tumors requires further investigations.
In the antioxidant assay, compounds 8, 11-13 and 19a-19c strongly inhibited bleaching of β-carotene on TLC plates, while 14-16 and 22 showed moderate activity. On the other hand, compounds 2-6 and 9-10 were inactive. Although the antioxidative properties of a number of ortho-dioxygenated flavonoid derivatives are well known and several oxygenated pentacyclic triterpenes have also been reported as antioxidant compounds, 18,19 there is no report for the antioxidant activity of diarylpropanes 11 and 12 and triterpenes 19a-19c. Recently arjungenin (15) and its glucoside (16) were found to show a moderate free radical scavenging activity in the DPPH model, while arjunic acid (13) and arjunetin (14) exhibited no significant activity in the same assay. 19 However, in the present work 13 and 14 showed strong and moderate activities, respectively, in the autography assay towards β-carotene.  Although a large number of cytotoxic constituents from different flavonoid classes has been described, to the best of our knowledge this is the first report on the cytotoxicity of 1,3-diarylpropanes. In addition, the isolation of this class of compounds from a member of the Combretaceae adds new phytochemical data, which might have chemotaxonomical importance.

Extraction and isolation of chemical constituents
Air-dried and powdered heartwood (2684 g) was extracted at room temperature with EtOH. After concentration in vacuo, the residue was partitioned between hexane/CH 3  Air-dried and powdered trunk bark (1500 g) was extracted at room temperature with EtOH. The residue obtained from the EtOH extract was subsequently partitioned between MeOH/H 2 O (9:1) and hexane; and MeOH/H 2 O (1:1) and CH 2 Cl 2 . The CH 2 Cl 2 phase (22.2 g) was subjected to CC on silica gel (70-230 mesh) eluted with hexane, CH 2 Cl 2 , EtOAc and EtOAc/MeOH 20% to 1227Garcez et al. Vol. 17, No. 7, 2006

In vitro cytotoxic assay
In vitro cytotoxic activities were measured against Hep 2 , a human larynx carcinoma cell line and H 292 , a human lung mucoepidermoid carcinoma cell line, obtained from Instituto Adolfo Lutz (São Paulo, SP, Brazil). Cells were cultivated in DMEM medium supplemented with 10% foetal calf serum, 100 μg mL -1 streptomycin, 100 U mL -1 penicillin and 0.25 μg mL -1 anfotericin B, at 37 o C in a humidified incubator with 5% CO 2 . Cellular viability was assessed by formazan production from methylthiazolyldiphenyltetrazolium bromide (MTT colorimetric assay) as described previously. 20

Bleaching experiments on β-carotene
The test was carried out on TLC plates, using a solution of β-carotene as a spraying reagent and α-tocopherol as the reference compound. 21