Bioactive Azafluorenone Alkaloids from Polyalthia debilis (Pierre) Finet & Gagnep

This study investigated bioactive extracts of Polyalthia debilis (Annonaceae) with antimicrobial, antimalarial and cytotoxic activities. Extensive chromatographic isolations provided azafluorenone alkaloids; onychine (1) and 7-methoxyonychine (2) together with a mixture of β–sitosterol and stigmasterol. The two alkaloids were isolated from the P. debilis for the first time. Isolated fractions containing a mixture of triterpenoids (C7, C8 and C9) exhibited the most potent antimicrobial activity against many bacterial strains with minimum inhibitory concentration of 64 μg/mL. Fractions with antimalarial and cytotoxic activities were also observed. The findings suggest the potential use of P. debilis in medicinal applications.


Antimicrobial activity
This activity was tested against 27 strains of microorganisms using the agar dilution method [14]. Results (Table 1) showed that except for fractions C6 and E5 that exhibited partial growth inhibition, the tested extracts and fractions displayed complete antigrowth activity against many microorganisms with MIC values ranging from 64-256 g/mL. It is interesting to note that fractions C7, C8 and C9 from the chloroform extract (PC) displayed the highest antimicrobial activity against Corynebacterium diphtheriae NCTC 10356, Bacillus subtilis ATCC 6633 and Bacillus cereus with MICs of 64 g/mL, while the extracts (PC and PE) and fraction E4 from PE showed antigrowth activity against such organisms with MICs of 256 g/mL. The aforementioned organisms were also inhibited by fraction C5, with MICs of 128-256 g/mL. In addition, the fractions containing a mixture of triterpenoids (C7, C8 and C9) also inhibited the growth of Branhamella catarrhalis with a MIC of 64 g/mL. At the MIC of 256 g/mL fraction C2 selectively showed antigrowth activity agains B. subtilis ATCC 6633, while E3.6 selectively inhibited the growth of C. diphtheriae NCTC 10356. Fraction C3 (against C. diphtheriae NCTC 10356), C7 (against Plesiomonas shigelloides) and C8 (against Micrococcus lutens ATCC 10240, P. shigelloides and Streptococcus pyogenes) exerted potent antimicrobials with MIC of 64 g/mL. Complete inhibition of Staphylococcus epidermidis ATCC 12228 and Enterococcus faecalis ATCC 29212 were observed from fractions C8 and C9 with MIC of 256 g/mL. Staphylococcus aureus ATCC 25923 was inhibited by the fraction C8 with MIC of 256 g/mL. Moreover, C8 was the only component that showed antifungal activity against Candida albicans (75% inhibition) at 256 g/mL. Unfortunately, the isolates 1 and 2 were not evaluated for antimicrobials, due to the limited quantity of the compounds available. Onychine (1) was shown to be active against C. albicans B311 with MIC of 3.12 g/mL [15]. In addition, compound 1 also exhibited antimicrobial activity against many organisms e.g. S. aureus NCTC 8530, B. subtilis IFO 3007, Escherichia coli IFO 3545 and Saccharomyces cerevisiae IFO 0203 with MIC range 50 to >100 g/mL [16]. To date, the antimicrobial activity of P. debilis was not reported in the literature. Previously, a mixture of 6-and 7-methoxyonychines was reported to show a weak DNA-damaging potential [9].

Antimalarial activity
Antimalarial activity of the extracts and fractions was tested [17] against chloroquine resistant Plasmodium falciparum (T9.94). It was revealed ( Table 2) that most of the tested extracts (PC and PE) and fractions (C2-C9, E4 and E5) displayed fair activity with IC 50 values of 100-1000 g/mL. Only the fraction E3.6 showed good activity, with an IC 50 of 10-100 g/mL. Previously, the dichloromethane extract and isolates; dimeric aporphine alkaloids of P. debilis were reported to be antimalarials [2]. The alkaloids 1 and 2, including chloroform and ethyl acetate extracts, as well as the isolated fractions of P. debilis were not tested for antimalarial action.

Cytotoxic activity
Cytotoxic assay was evaluated [18] against three cell lines. The results (Table 3) showed that the extract (PC) and many fractions (C3-C9, E3.6 and E5) exhibited cytotoxic activity against all the tested cell lines. However, ethyl acetate extract and fractions (C2 and E4) were shown to be inactive cytotoxic agents (IC 50 > 50 g/mL). Among the cytotoxic agents, fractions C3 and C9 exhibited the highest activity against A549 cells with comparable IC 50 of 6.0 ± 2.8 and 6.75 ± 0.4 g/mL, respectively. Fraction C5 displayed the most potent activity against HepG2 cells with IC 50 of 7.0 ± 2.5 g/mL. In addition,the C5 also showed the highest cytotoxic activity against HCC-S102 cells (IC 50 8.5 ± 0.7 g/mL). So far cytotoxic activity of the P.debilis was not reported elsewhere.

Conclusions
Bioactive extracts (chloroform and ethyl acetate) of P. debilis were investigated and found to give many fractions with antimicrobial, antimalarial and cytotoxic activities. Particularly, fractions C7, C8 and C9 displayed the most potent antimicrobial activity against many bacterial strains with MICs of 64 μg/mL. Extensive chromatographic separations afforded two azafluorenone alkaloids; onychine (1) and 7-methoxyonychine (2) together with a mixture of β-sitosterol and stigmasterol. In this study, the two alkaloids 1 and 2 were isolated from P. debilis for the first time. Compound 1 when isolated from other Annonaceae plants exhibited antibacterial and antifungal actions. The findings suggest the potential use of the P. debilis in medicinal applications and as a guide to synthetic chemistry in order to find new bioactive lead compounds.

General
Melting points were determined on an Electrothermal melting point apparatus (Electrothermal 9100) and are uncorrected. 1 H-and 13 C-NMR spectra were recorded on Bruker AVANCE 300 and 600 NMR spectrometers (operating at 300 and 600 MHz for 1 H and 75 and 125 MHz for 13 C, respectively).

Plant material
Roots of P. debilis were collected from Ubonratchatanee Province, Thailand. It has been identified (BKF 135063) by The Forest Herbarium, Royal Forestry Department, Bangkok. A voucher specimen has been deposited at Department of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok, Thailand.

Antimicrobial assay
Antimicrobial activity of the tested compounds was carried out using the agar dilution method [14]. Briefly, the tested compounds dissolved in DMSO were individually mixed with 1 mL Müller Hinton (MH) broth while the negative control was the MH broth with omission of the tested compounds. The solution was then transferred to the MH agar solution to yield the final concentrations of 32-256 g/mL. Twenty seven strains of microorganisms as listed below, cultured in MH broth at 37 C for 24 h, were diluted with 0.9 % normal saline solution to adjust the cell density of 3 × 10 9 cell/mL. The organisms were inoculated onto each plate and further incubated at 37 °C for 18-48 h. Compounds which possessed high efficacy to inhibit bacterial cell growth were analyzed. Tested microorganisms were gram-negative bacteria:

Antimalarial assay
Antimalarial activity of the tested compounds was evaluated against chloroquine resistant Plasmodium falciparum (T9.94) using the literature method [19]. Human erythrocytes (type O) infected with chloroquine resistant P. falciparum (T9.94) were maintained in continuous culture, according to the method described previously [17]. RPMI 1640 culture medium supplemented with 25 mM of HEPES, 40 mg/L gentamicin sulfate and 10 mL of human serum was used in continuous culture. Before performing the experiment, P. falciparum culture was synchronized by using sorbitol induced hemolysis according to the method of Lambros and Vanderberg [20] to obtain only ring stage-infected red blood cells and then incubated for 48 h prior to the drug testing to avoid effect of sorbitol.
The experiments were started with synchronized suspension of 0.5% to 1% infected red blood cell during ring stage. Parasites were suspended with culture medium supplemented with 15% human serum to obtain 10% cell suspension. The parasite suspension was put into 96-well microculture plate; 50 L in each well and then add 50 L of various tested drug concentrations. These parasite suspensions were incubated for 48 h in the atmosphere of 5% CO 2 at 37 C. The percents parasitemia of control and drug-treated groups were examined by microscopic technique using methanol-fixed Giemsa stained of thin smear blood preparation. The efficacy of the drugs were evaluated by determining the drug concentration that reduced parasite growth by 50% (IC 50 ).

Cytotoxic assay
Cells were grown in Ham's/F12 medium containing 2 mM L-glutamine supplemented with 100 U/mL penicillin, streptomycin and 10% fetal bovine serum. Except HepG2 cell was grown in DMEM. Cytotoxic assay was performed using the modified method as previously described [18]. In brief, cell lines suspended in RPMI-1640 containing 10% FBS were seeded at 1  10 4 cells (100 L) per well in 96-well plate and incubated in humidified atmosphere, 95% air, 5%CO 2 at 37 C. After 24 h, additional medium (100 L) containing the test compound and vehicle was added to a final concentration of 50 g/mL, 0.2% DMSO, and further incubated for 3 days. Cells were subsequently fixed with 95% EtOH, stained with crystal violet solution, and lysed with a solution of 0.1 N HCl in MeOH, after which absorbance was measured at 550 nm. Whereas A549 and HepG2 cells were stained by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). IC 50 values were determined as the drug and sample concentrations at 50% inhibition of the cell growth.