Chemical Constituents Isolated from the Bark of Guatteria blepharophylla (Annonaceae) and their Antiproliferative and Antimicrobial Activities

Departamento de Química, Universidade Federal do Paraná, CP 19081, 81531-990 Curitiba-PR, Brazil Departamento de Química, Universidade Federal de Sergipe, 49100-000 São Cristovão-SE, Brazil Departamento de Química, Universidade Federal do Amazonas, 69077-000 Manaus-AM, Brazil Instituto de Química, Universidade Estadual de Campinas, CP 6154, 13083-970 Campinas-SP, Brazil Divisão de Microbiologia and Divisão de Farmacologia e Toxicologia, CPQBA, Universidade Estadual de Campinas, CP 6171, 13083-970 Campinas-SP, Brazil


Introduction
The genus Guatteria Ruiz & Pav.contains close to 290 species and is the largest genus within the Annonaceae family. 1 Species of Guatteria are frequent constituents of Neotropical (lowland) forests, and it is widely distributed throughout Mesoamerica, the Caribbean and tropical South America. 1 Plants of this family are known for their edible fruits and medicinal properties of many species. 2Previous chemical and pharmacological investigations on some species of this family, including Guatteria, have indicated the presence of important bioactive compounds, exhibiting many pharmacological activities, such as, cytotoxicity against human tumor cell lines, [3][4][5] antimicrobial, 5,[6][7][8][9] and antiparasitic properties, particularly against Leishmania sp., 3,7,[10][11][12] Plasmodium falciparum 5,12,13 and Trypanosoma sp. 3,12,14espite the importance of annonaceous members in folk medicine, the number of species that have been chemically investigated is still very small.One of them is Guatteria blepharophylla Mart in Mart, a small tropical tree that occurs in the Amazonian region (Brazil, Peru, Guyana, Ecuador and Venezuela). 1 In Brazil this species is common in Amazonas and Pará states, where it is known as "envireira". 15Previous phytochemical studies on this species described the isolation and identification of essential oils and isoquinoline alkaloids. 8,15,16n continuation of our research on bioactive compounds from Amazonian annonaceous plants, we report herein the phytochemical study of the bark of G. blepharophylla and the evaluation of antiproliferative and antimicrobial properties of the main isolated compounds.

General experimental procedures
Melting points were determined on a Quimis Q-340S23 micromelting point apparatus.UV-Vis spectra were obtained in CH 3 OH on a Hewlett-Packard HP 8452A diode array spectrophotometer.IR spectra were acquired on a BIORAD FTS-3500 GX spectrophotometer.Mass spectra were recorded on a Varian Saturn 2000 spectrometer operating at 70 eV.NMR data, 1D and 2D, were recorded at 293 K in CDCl 3 or CDCl 3 + CD 3 OD or CD 3 OD on a Bruker Avance DRX 400 and Brucker ARX 200 spectrometers.The spectrometers were equipped with a 5 mm multinuclear direct detection probe with z-gradient.One-bond and longrange 1 H- 13 C correlation (HSQC, (heteronuclear single quantum correlation) and HMBC (heteronuclear multiple bond coherence), respectively) experiments were optimized for an average coupling constants 1 J (C,H) and LR J (C,H) of 140 and 8 Hz, respectively.All 1 H and 13 C NMR chemical shifts (d) are given in ppm related to the TMS (tetramethylsilane) signal at 0.00 ppm as internal reference and the coupling constants (J) in Hz.Silica gel 60 (70-230 mesh) was used for column chromatography, while silica gel 60 F 254 were used for analytical (0.25 mm), and preparative (1.00 mm) TLC.Compounds were visualized by exposure under UV 254/366 light, spraying p-anisaldeyde reagent followed by heating on a hot plate, or spraying with Dragendorff's reagent.

Plant material
The bark of G. blepharophylla was collected in January 2005 on the campus of the Universidade Federal do Amazonas (UFAM), Manaus-AM, Brazil, and identified by the taxonomist Prof. Dr. A. C. Webber from UFAM.A voucher specimen (number 7340) has been deposited at the Herbarium of the UFAM.After identification, G. blepharophylla bark was dried at room temperature and finely powdered.

Extraction and isolation of the chemical constituents
The dried powdered bark (1500 g) of G. blepharophylla was successively extracted with n-hexane followed by MeOH to yield n-hexane (28.18 g) and MeOH (212.50 g) extracts.
Penicillin:streptomycin (1000 μg mL -1 :1000 UI mL -1 , 1 mL L -1 ) was added to experimental cultures.Cells in 96 well plates (100 μL cells well-1) were exposed to sample concentrations in DMSO/RPMI (0.25, 2.5, 25 and 250 μg mL -1 ) (DMSO, dimethyl sulfoxide) at 37 °C, 5% of CO 2 in air for 48 h.Final DMSO concentration did not affect cell viability.Afterwards, cells were fixed with 50% trichloroacetic acid and cell proliferation determined by spectrophotometric quantification (540 nm) of cellular protein content using sulforhodamine B assay.Using the concentration-response curve for each cell line, TGI (concentration that produces total growth inhibition or cytostatic effect) 17 was determined through non-linear regression analysis (Table 1) using software ORIGIN 7.5 (OriginLab Corporation).

In vitro antimicrobial activity
The growth inhibitory activity of the crude extracts, fractions and isolated compounds was tested against  The bacteria strains were cultured overnight at 36 o C in Nutrient Agar (Merck), while C. albicans was cultured in Saboraud Dextrose Agar.Inoculum for the assays was prepared by diluting a scraped cell mass in 0.85% NaCl solution, adjusted to McFarland scale 0.5 and confirmed by spectrophotometer reading at 580 nm.Cell suspensions were finally diluted to 10 4 CFU mL -1 for using in the activity assays.MIC (minimal inhibitory concentration) tests were carried out according to Eloff, 18 using Müller-Hinton broth on a tissue-culture test plate (96 wells).The stock solution crude extracts, fractions and isolated compounds were diluted and transferred into the first well, and serial dilutions were made so that concentrations in the range of 1.0-0.015mg mL -1 were obtained.Chloramphenicol and nystatin (Merck) were used as the reference antibiotic control in the range of 0.25-0.002mg mL -1 .The inoculum was added to all wells, and the plates were incubated at 36 o C for 48 h.Each concentration was screened in triplicate.Antimicrobial activity was detected by adding 20 μL of 0.5% TTC (triphenyl tetrazolium chloride, Merck) aqueous solution.MIC was defined as the lowest concentration of the sample that inhibited visible growth, as indicated by TCC staining (dead cells are not stained by TTC).

Results and Discussion
Once n-hexane and MeOH crude extracts were found to have antiproliferative activity (Table 1), these extracts were subjected to successive chromatographic fractionations as described in the Experimental section leading to the isolation of the chemical constituents 1-12 (Figure 1).
Compounds 1-10 were identified by comparison of their spectroscopic data with those reported in the literature as caryophyllene oxide 19 (1), lichexanthone 20 (2), spathulenol 19 (3), a mixture of b-sitosterol 21 (4), and stigmasterol 21 (5), O-methylmoschatoline 22,23  (6), lysicamine 22,23  (7), nornuciferine 22  (8), liriodenine 6,23  (9), and isocoreximine 15 (10).Compounds 7-10 were recently found on the stem of this species, 15 while compounds 1-6, 11 and 12 were reported for the first time in this specie.Compound 2 is related for the first time in the genus Guatteria, and it is the second report in the Annonaceae, previously isolated from the roots of Rollinia leptopetala. 24ompound 11 was obtained as an orange amorphous powder which was positive to the Dragendorff's test.Its UV-vis spectrum showed absorption peaks at 204, 238,  2).The hydrogen H-8 at d 7.82 showed strong long-range 1 H- 13 C correlation with the carbon C-7 at d 183.1, which confirmed the oxoaporphine skeleton.According with 1 H and 13 C NMR 1D/2D this compound was identified as the oxoaporphine alkaloid known subsessiline.This compound has been found only in two species of Annonaceae Guatteria ouregou 25 and G. subsessilis, 26 but the physical data are incomplete.The 13 C NMR data are reported for the first time.The complete physical data for this compound are described in this work.
Compound 12 was obtained as a blue amorphous powder.Its UV-Vis spectrum showed absorption peaks at 204, 238, 282, 304, 468 and 595 nm, which are typical of molecules with an oxoaporphine skeleton.The IR spectrum showed absorption bands at 3325 and 1660 cm -1 , characteristic of phenolic hydroxyl and carbonyl groups.The 1 H and 13 C NMR spectra of 12 were very similar to those of 11, except for the absence of a methoxyl signal, which was replaced by a hydroxyl group, and absence of substitution in the D ring, in structure 12 according to the IR spectrum and NMR data (Table 2).The absence of The experiments were obtained at 293 K and TMS as internal reference (0.00 ppm) in a CDCl 3 + drops of CD 3 OD or b CD 3 OD.c Long-range 1 H- 13 C HMBC correlations, optimized for 8 Hz, is from hydrogens stated for the indicated carbon.
substitution in the D ring was confirmed by the presence of four adjacent aromatic hydrogens at d 9.08 (1H, ddd, J 8.6, 1.0 and 0.5 Hz), d 8.41 (1H, ddd, J 8.0, 1.6 and 0.5 Hz), d 7.68 (1H, ddd, J 8.6, 7.0 and 1.6 Hz), and d 7.33 (1H, ddd, J 8.0, 7,0 and 1.0 Hz) which were attributed to H-11, H-8, H-10, and H-9, respectively (Table 2).The position of the hydroxyl group at C-3 was established on the basis of the long-range 1 H- 13 C correlation of the hydrogen H-4 at d 8.60 with the carbon C-3 at d 166.0, which showed no correlation with the methoxyl groups.According with 1 H and 13 C NMR 1D/2D, this compound was identified as the oxoaporphine alkaloid known isomoschatoline.As well as observed for 11, the physical data published for this compound are incomplete and presented in this work.The 13 C NMR data is reported for the first time.This compound has been found only in three species of Annonaceae Uvaria mocoli, 27 Guatteria melosma and Cleistopholis patens. 28he isolated compounds 1-2 and 6-12 were evaluated for in vitro antiproliferative activity against nine human tumour cell lines (Table 1), while compounds 6, 7, 10 and 12 were also tested for antimicrobial activity.Compound 9 showed the higher antiproliferative activity for breast (MCF-7) with a TGI value of 37.67 mmol L -1 , more active than positive control doxorubicin (TGI value of 46.04 mmol L -1 ).Compound 7 presented significant antiproliferative activity for lung, non-small cells (NCI-H460) with a TGI value of 49.52 mmol L -1 (doxorubicin, TGI value of 14.27 mmol L -1 ), while compound 12 showed significant activity for breast (MCF-7) with TGI value of 73.97 mmol L -1 .Compound 10 showed selective activity for ovarian expressing phenotype for multiple drug resistance (NCI-ADR/RES) with a TGI value of 131.50 mmol L -1 , but was less active than doxorubicin (TGI value of 14.80 mmol L -1 (Table 1).Compounds 9 and 12 also showed significant antiproliferative activity against different tumor cell lines with TGI values below to 100 mmol L -1 (Table 1).
It is important to notice that compounds 6, 7, 9, 11 and 12 shared the same basic skeleton with different substitution patterns.This way, our results suggested that a methoxylated substitute at R 3 reduces antiproliferative activity (compound 11) or even results in an inactive compounds as 6, but a hydroxyl group at R 3 favored the activity (compound 12).The best results were obtained for methylenedioxy group (compound 9) or methoxy groups (compounds 7 and 12) at R 1 and R 2 .
No significant antibacterial activity was observed for the tested compounds.The only significant antimicrobial result was observed for compound 12 that showed antifungal activity against C. albicans (MIC value of 50.81 mmol L -1 ) similar to the positive control nystatin (MIC value of 54.00 mmol L -1 ).

Conclusion
The chemical investigation of the bark of G. blepharophylla has resulted in the isolation of several compounds common in the taxon Guatteria, such as, 1, 3, 6, 7 and 9 that could be considered chemotaxonomic markers of this genus.Compounds 1-6, 11 and 12 are being reported for the first time in this species, and are important for the chemotaxonomy of Annonaceae family.The significant in vitro antiproliferative results obtained against several human tumour cell lines and antimicrobial activities demonstrated by the major compounds indicate that this species is a natural source of biologically active compounds.Compound 12 showed significant antifungal and antiproliferative activities against C. albicans and human tumor cell lines of breast (MCF-7).Therefore, studies involving mechanisms of action are necessary to fully understand its biological significance.

Figure S7. 1 H
Figure S7. 1 H NMR spectrum of the mixture of compounds 4 and 5 in CDCl 3 at 200 MHz.

Figure S22. 1
Figure S22. 1 H-13 C one-bond correlation map from HSQC NMR experiment of compound 11 in CDCl 3 + drops of CD 3 OD at 400 and 100 MHz.

Figure S23. 1 H
Figure S23. 1 H-13 C long-range correlation map from HMBC NMR experiment of compound 11 in CDCl 3 + drops of CD 3 OD at 400 and 100 MHz.

Figure S26. 1
Figure S26. 1 H-13 C one-bond correlation map from HSQC NMR experiment of compound 12 in CD 3 OD at 400 and 100 MHz.

Table 1 .
13tiproliferative activity of extracts, fractions and major compounds against cancer cell lines The signals at d 4.05, d 4.11 and d 4.17 (each 3H, s) were assigned to three methoxyl groups located in the A ring, according to long-range 1 H-13C correlation in the HMBC NMR experiment (Table2).The presence of a hydroxyl group in the molecule located in the D ring at C-9 was established on the basis of the long-range 1 H-13C correlation of the hydrogen H-11 at d 8.96 with the carbon C-9 at d 157.3, which showed no correlation with the methoxyl groups.The13C NMR experiment along with HSQC and HMBC experiments allowed to verify the presence of 19 carbons, including a carbonyl group at d 183.1, 15 aromatic carbons between d 157.3-113.4,and three methoxyl groups at d 61.9, d 61.4, and d 60.9, which was in agreement with structure 11 (Table a Positive control; NT (Not tested); UACC-62 (melanoma), MCF-7 (breast), NCI-H460 (lung, non-small cells), OVCAR-03 (ovarian), PC-3 (prostate), HT-29 (colon), 786-0 (renal), K562 (leukemia), and NCI-ADR/RES (ovarian expressing phenotype multiple drugs resistance); TGI (Total Growth Inhibition).Figure 1.Chemical constituents isolated from the bark of Guatteria blepharophylla.