Antimicrobial and cytotoxic acetogenin from Polyalthia debilis

Somchai Boonpangrak, Rungrot Cherdtrakulkiat, Ratchanok Pingaew, Patumporn Manam, Supaluk Prachayasittikul, Somsak Ruchirawat, Virapong Prachayasittikul Center for Innovation Development and Technology Transfer, Faculty of Medical Technology,Mahidol University, Bangkok 10700, Thailand. Department of Clinical Microbiology and Applied Technology, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand. Department of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok 10110, Thailand. Center of Data Mining and Biomedical Informatics, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand. Laboratory of Medicinal Chemistry, Chulabhorn Research Institute and Program in Chemical Biology, Chulabhorn Graduate Institute, Bangkok 10210, Thailand. Center of Excellence on Environmental Health and Toxicology, Commission on Higher Education (CHE), Ministry of Education, Thailand.


INTRODUCTION
Polyalthia debilis (Annonaceae), a Thai medicinal plant, has been used as folk remedy for treatment of abdominal pain and tuberculosis and as a febrigue and a galactogogue (Prachayasittikul et al., 2009).A variety of compounds were found in the P. debilis i.e., diterpenes, triterpenes, polyacetylene and alkaloids (Panthama et al., 2010;Kanokmedhakul et al., 2003).Previously, bioactive azafluorenone alkaloids from roots of the plant species were reported (Prachayasittikul et al., 2009).To continue our study on the Thai medicinal plant, herein, antimicrobial and cytotoxic compounds as well as antioxidant activity of the P. debilis have been investigated. .

Antimicrobial Assay
Antimicrobial activity of the tested compound was assayed using the agar dilution method (Srisung et al., 2013).
Briefly, the tested compound dissolved in DMSO was mixed with 1 mL Müller Hinton (MH) broth while the MH broth was used as the negative control.The two-fold dilution solution was transferred to the MH agar solution to yield the final concentrations of 32-256 µg/mL.The DMSO was tested in parallel with the compound and showed no effect on the tested organisms.Twenty -seven strains of microorganisms, cultured in the 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.Efficacy of the compound to inhibit bacterial cell growth was analyzed.

Antimalarial assay
Antimalarial activity of the tested compound was performed against chloroquine resistant Plasmodium falciparum (T9.94) using the literature method (Targer and Jensen, 1976;Lambros and Vanderberg, 1979).Human erythrocytes (type O) infected with chloroquine resistant P. falciparum (T9.94) were maintained in continuous culture as described previously (Targer and Jensen, 1976).RPMI 1640 culture medium supplemented with HEPES (25 mM), gentamicin sulfate (40 mg/mL) and human serum (10 mL) was used in continuous culture.Before performing the experiment, P. falciparum culture was synchronized (Lambros and Vanderberg, 1979) by using sorbitol induced hemolysis 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 carried out using synchronized suspension (0.5% to 1%) of infected red blood cell during ring stage.Parasites were suspended in culture medium supplemented with human serum (15%) to obtain 10% cell suspension.The parasite suspension was added into 96-well microculture plate; 50 µL in each well and then add 50 µL of various tested drug concentrations.The parasite suspensions were incubated for 48 h in the atmosphere of 5% CO2 at 37 °C.Percents parasitemia of the control and tested compound were examined by microscopic technique using methanol-fixed Giemsa stained of thin smear blood preparation.Efficacy of the compound was determined by compound concentration that reduced parasite growth by 50% (IC 50 ).

Antioxidant assay
Antioxidant activity of compound was tested using 2,2diphenyl-1-picrylhydrazyl (DPPH) assay.The DPPH (a stable purple color radical) reacts with an antioxidant to form a lightyellow colored of diphenylpicrylhydrazine, the reduced product that can be spectrophotometrically detected.The assay (Prachayasittikul et al., 2012) was performed by adding 1 mL solution of DPPH in methanol (0.1 mM) to a sample solution (0.45 mL, 1 mg/mL dissolved in DMSO).The reaction mixture was incubated for 30 min in a dark room and the absorbance at 517 nm was measured using UV-visible spectrophotometer (UV-1610, Shimadzu).Percentage of radical scavenging activity (RSA) was deduced from the following equation: where Abs.control is the absorbance of the control reaction and Abs.sample is the absorbance of the tested compound.
Cells were subsequently fixed with 95% EtOH, stained with crystal violet solution, then lysed with a solution of 0.1 N HCl in MeOH, and an absorbance was measured at 550 nm.Whreas HuCCA-1, A549 and HepG2 cells were stained by MTT and for MOLT-3 cell was stained by XTT.IC 50 values were determined as the drug and sample concentration at 50% inhibition of the cell growth.The tested cancer cell lines were human cholangio carcinoma cell line (HuCCA-1), human epidermoid carcinoma of the mouth (KB), human promyelocytic leukemia cell line (HL-60), murine leukemia cell line (P388), cervical adenocarcinoma cell line (HeLa), hormone-independent breast cancer cell line (MDA-MB231), hormone-dependent breast cancer cell line (T47D), multidrug-resistance small cell lung cancer cell line (H69AR), human hepatocellular carcinoma cell line (HepG2), human lung carcinoma cell line (A549), and hepatocellular carcinoma cell line (HCC-S102).Cells were grown in Ham's/F12 medium containing L-glutamine (2 mM) supplemented with 100 U/mL penicillin-streptomycin and FBS (10%), except for, HepG2 cell was grown in DMEM medium.

Isolation
The fraction C6 of chloroform extract (PC) was extensively isolated by the silica gel column to give acetogenin

Biological activities Antioxidant activity
The plant extracts and isolated fractions were tested for their radical scavenging activity using DPPH assay.Results (Table 1) showed that the extracts (PC,PE and PM) displayed antioxidant activity with IC 50 values of 457.0, 154.9 and 128.8 µg/mL, respectively.The isolated fractions (C9, E4 and E5) exhibited the activity with IC 50 range of 100.0 -354.8 µg/mL.The fractions (C2,C4,C6,C7 and E3.6) and the extract (PH) were shown to be inactive antioxidants.

DISCUSSION
Previously, two bioactive azafluorenone alkaloids, onychine and 7-methoxyonychine together with a mixture of βsitosterol and stigmasterol were isolated from roots chloroform extract (PC) of the P.debilis (Prachayasittikul et al., 2009).This study, the fraction C6 of PC extract was isolated by silica gel column to afford a linear acetylenic and olefinic C25 acetogenin (1) known as debilisone E (Panthama et al., 2010).Its structure was confirmed by spectral data (H 1 and C 13 NMR, MS, IR and UV).The acetogenin 1 was reported to be found in methanol extract of roots P. debilis (Panthama et al., 2010).Antioxidant potency of the palnt extracts and isolated fractions was investigated (DPPH assay).
The results showed that most of the extracts, except for PH, and some isolated fractions displayed relatively weak radical scarvenging activity.However, antioxidant activity of the P. debilis has never been reported elsewhere.The antioxidant activity of the plant extracts and isolated fractions could possibly be derived from the origin of plant triterpenoids i.e., stigmasterol (Prachayasittikul et al., 2009;Prachayasittikul et al., 2009).Stigmasterol is a phytosterol showing antioxidant effect, determined by the thiocyanate mehod (Hung and Yen, 2001), and lipid peroxdation (Ramadan et al., 2007).
In conclusion, the P. debilis extracts and isolated fractions exhibited antioxidant activity and cytotoxicity toward a panel of cancer cells.The isolated acetogenin 1 (debilisone E) displayed novel bioactivities as antibacterial and anticancer agents.The finding demonstrates compound 1 as potential lead to be further structurally modified for new bioactive derivatives.

CONFLICT OF INTEREST STATEMENT
Authors declare that we have no conflict of interest.

compound 1 (
Fig 1).Its structure was determined by comparison of 1 H and 13 C NMR,MS, IR and UV spectra with the reported debilisone E (Panthama et al., 2010).

Table 5 :
Cytotoxic activity (IC50) of compound 1.Etoposide was used as the reference drug. b