Bioactive compounds from the bark of beilschmiedia palembanica (MIQ.) kosterm

A dienamide, named (-) kunstleramide (1), were isolated from the bark of Beilschmiedia Palembanica (Miq.) Kosterm together with one flavanone: pinocembrin (2), one phenanthrenol: perakensol (3) and 12 known alkaloids: sebiferine (4), reticuline (5), norisoboldine (6), norboldine (7), norisodomesticine (8), crychine (9), cassythicine (10), argentinine (11), isoboldine (12), o-methylnorflavinantine (13), nornantenine (14), and milonine (15). Their structures were established from spectroscopic techniques, most notably 1D- and 2D-NMR, UV, IR, OR, circular dichroism (CD) spectra and LCMS-IT-TOF. (-)-Kunstleramide (1) exhibited very poor dose-dependent inhibition of DPPH activity, with an IC50 value of 179.5 ± 4.4 μg/mL, but showed a moderate cytotoxic effect on MTT assays of A375, A549, HT-29, PC-3 and WRL-68 with EC50 values of 64.65, 44.74, 55.94, 73.87 and 70.95 µg/mL, respectively.

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Introduction

In continuation of our research on the medicinal plants from Malaysian flora [1,3] , we have performed a phytochemical investigation on the bark of a Malaysian Lauraceae, Beilschmiedia Palembanica (Miq.) Kosterm, which has led to the isolation of a dienamide, named (-)-kunstleramide [1] , were isolated from the bark of Beilschmiedia Palembanica (Miq.) Kosterm together with one flavanone: pinocembrin [2] , one phenanthrenol: perakensol [3] and 12 known alkaloids: sebiferine [4] , reticuline [5] , norisoboldine [6] , norboldine [7] , norisodomesticine [8] , crychine [9] , cassythicine [10] , argentinine [11] , isoboldine [12] , o-methylnorflavinantine [13] , nornantenine [14] , and milonine [15] were also isolated (Figure 1). This paper describes the structural elucidation, the DPPH activity with an IC 50 value and cytotoxic effect of (-)-kunstleramide [1] . spectrometer 70 eV. NMR spectra were recorded in deuterated chloroform (CDCl 3 ) and deuterated methanol CD 3 OD) on JEOL LA400 FT-NMR and JEOL ECA 400 FT-NMR as a JEOL JNM-FX400 (400 MHz for 1 H and 100 MHz for 13 C; unless stated otherwise) and signal of spectra calibrated using TMS. Chemical shifts were reported in ppm on δ scale, and the coupling constants were measured in Hertz (Hz). Silica gel 60, 70-230 mesh ASTM (Merck 7734) was used for various column chromatography methods such as CLC and FLC.TLC aluminum sheets and PTLC (20 × 20 cm Silica gel 60 F 254 ) were used in the thin layer chromatography analysis. The TLC and PTLC spots were visualized under UV light (254 and 366 nm) followed by spraying with Dragendorff's reagent for detection of alkaloids. Dragendorff's reagent and Mayer's reagent were used for alkaloid screening. All solvents used were of AR grade except those used for bulk extraction.

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Chemical structures of compounds 1–15

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Plant Materials

The bark of Beilschmiedia palembanica (Miq.) Kosterm (KL 5373) was collected from Hutan Simpan Sungai Temau, Kuala Lipis, Pahang by L.E. Teo & Din. at the Herbarium of the Department of Chemistry, University of Malaya, Kuala Lumpur, Malaysia and at the Herbarium of the Forest Research Institute, Kepong, Malaysia.

Extraction and Isolation of Chemical Constituents

The air-dried bark (4.00kg) of Beilschmiedia palembanica (Miq.) Kosterm was extracted comprehensively and defatted with hexane (12.0L) for 72 hours then hexane extract was filtered. The residual plant material was dried and left to soak for 6.30h after moistening and rinsed with 10% NH 4 OH. It was then macerated with CH 2 Cl 2 (13.0L) for 7 days. After filtration, the supernatant was concentrated to 500 mL at room temperature (30°C) followed by acidic extraction with 5% HCl until a negative Mayer's test result was obtained. This procedure was repeated three times and the hydrochloric acid portion was kept and washed with CH 2 Cl 2 . The aqueous solution was made alkaline to pH=11 with NH 4 OH and re-extracted with CH 2 Cl 2 . This was followed by washing with distilled H 2 O, dried over anhydrous sodium sulphate, and evaporation to give an alkaloid fraction (3.07g). The extraction of alkaloids was repeated by using MeOH solvent and after acid base extraction obtained another (11.07g) of crude alkaloid. The CH 2 Cl 2 extracts were dried with Na 2 SO 4 and evaporated to dryness to give crude alkaloid. Finally the yields of the crude alkaloid extracts from this plant. The crude alkaloid (3.07g of CH 2 Cl 2 ) was submitted to exhaustive column chromatography over silica gel (column dimension =3 cm, length =100 cm, silica gel 60, 70-230 mesh ASTM; Merck 7734) using CH 2 Cl 2 gradually enriched and increasing the polarity with methanol (1% until 90% MeOH) to give 5 fractions. Fractions 2, afforded five alkaloids as identified as (-)-kunstleramide [1] (2.88%), norboldine [7] (1.08%), norisodomesticine [8] (1.38%), argentinine [11] (1.63%), cassythicine [10] (1.79%) and nornantenine [14] (1.64%) (PTLC Merck KGaA silica gel 60 F254; CH 2 Cl 2 -MeOH; 90:10). Fraction3 also produced one phenanthrenol identified as perakensol [3] (1.49%), and five alkaloids as recognized as sebiferine [4] (0.54%), reticuline [5] (0.91%), crychine [9] (1.28%), o-methylnorflavinantine [13] (0.82%) and isoboldine [12] (2.38%) (PTLC Merck KGaA silica gel 60 F254; CH 2 Cl 2 -MeOH; 97:3).

Fraction 4 afforded one flavanone as pinocembrin [2] (0.80%) (PTLC Merck KGaA silica gel 60 F 254 ; CH 2 Cl 2 -MeOH; 95:5). Fraction 5 produced two alkaloids as norisoboldine [6] (0.66%) and milonine [15] (2.15%) using PTLC (Merck KGaA silica gel 60 F 254 ; CH 2 Cl 2 -MeOH; 85:15).

Antioxidant Assay

The DPPH assay was performed according to the method reported by Orhan et al.

[4] and Brem et al. [5] , with modifications. Briefly, 0.02% stable DPPH free radical (50 µL) in methanol (100 mL) was added to standard/sample/control (20 µL) and methanol (130 µL, total assay volume 200 µL) in a 96-well plate. Ascorbic Acid (vitamin C) was used as the standard and blank solvent methanol as the negative control. The absorbance was read at 517 nm using SUNRISE Microplate Absorbance Reader after 30 min of incubation at room temperature. The percentage of DPPH free radical inhibition activity was determined according to the formula:

Where a (Blank) refers to the absorbance of the blank solvent and DPPH at 517 nm while A (Standard/Sample) refers to the absorbance of Ascorbic Acid and the samples at 517 nm. This formula was also used to determine the concentration of each sample required to quench 50% of the DPPH free radical activity (IC 50 value) [6] .

Statistical Analyses

Each experiment was performed at least twice. Results are expressed as the means value ± standard deviation (SD). Log IC 50 calculations were performed using the built-in algorithms for dose-response curves with variable slope using GraphPad Prism software (version 4.0; Graph Pad Software Inc., San Diego, CA, USA). A fixed maximum value of the dose-response curve was set to the maximum obtained value for each drug.

Cytotoxic Activity Studies 2.6.1. Cell Culture

All the cells used in this study were obtained from American Type Cell Collection (ATCC) and maintained in a 37 °C incubator with 5% CO 2 saturation. A375 human melanoma, HT-29 human colon adenocarcinoma cells and WRL-68 normal hepatic cells were maintained in Dulbecco's modified Eagle's medium (DMEM), whereas A549 non-small cell lung cancer cells and PC-3 prostate adenocarcinoma cells were maintained in RPMI medium. Both medium were supplemented with 10% fetus calf serum (FCS), 100 units/mL penicillin, and 0.1 mg/mL streptomycin.

Cellular Viability

Different cell types mentioned above were used to evaluate the inhibitory effect of (-) kunstleramide [1] on cell growth using the MTT assay. The MTT assay was modified as described by Cheah et al. and Mosmann [7,8] . Briefly, cells were seeded at a density of 1 × 10 5 cells/mL in a 96-well plate and incubated for 24 h at 37 °C, 5% CO 2 . The next day, cells were treated with the compounds respectively and incubated for another 24 h. After 24 h, MTT solution at 2 mg/mL was added and incubatefor 1 h. Absorbance at 570 nm was measured and recorded using a Plate Chameleon V microplate reader (Hidex, Turku, Finland). Results were expressed as a percentage of control giving percentage cell viability after 24 h exposure to test agent. The potency of cell growth inhibition for each test agent was expressed as an EC 50 value, defined as the concentration that caused a 50% loss of cell growth. Viability was defined as the ratio (expressed as a percentage) of absorbance of treated cells to untreated cells [9] .

Results and Discussion

Results and Discussion

Antioxidant Activity

The antioxidant activity of (-)-kunstleramide (1) was tested using a DPPH assay. Antioxidants are substances that may protect cells from the damage caused by unstable molecules known as free radicals [10]. Free radicals from oxidative stress are involved in many disorders like neurodegenerative diseases and cancer [11]. The new dienamide, (-)-kunstleramide (1) exhibited very poor DPPH activity, with an IC 50 value of 179.5 ± 4.4 μg/mL compared to ascorbic acid (Figure 2). Factors such as growth conditions, stability of the specific antioxidant components, including variations in the process of extraction can influence the variations in the antioxidant activity [12] .

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IC50 values of (−)-kunstleramide (1). AA: ascorbic acid as

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Cytotoxic Activity

To evaluate the cytotoxic activity, the new compound (-)kunstleramide (1) was tested with a series of different doses on A549, PC-3, A375, HT-29 and WRL-68, respectively (Figure 3). After 24 h, cell viability was determined by the MTT assay. Test agents induced cell cytotoxicity in a concentration dependent manner. These dose titration curves allowed determining EC 50 for the test agents towards different cell lines (Table 1).

Cell line	(-)-kunstleramide (1)	Doxorubicin
A375	64.65	1.364
A549	44.74	1.550
HT-29	55.94	1.957
PC-3	73.87	2.125
WRL-68	70.95	1.731

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Effect of compounds (−)-kunstleramide (1) and doxorubicin

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Dose-response curves (using GraphPad Prism) tested with (−)-

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Conclusions

(-)-kunstleramide (1) as a dienamide, were isolated from the bark of Beilschmiedia Palembanica (Miq.) Kosterm together with one flavanone named pinocembrin (2), one phenanthrenol named perakensol (3) and 12 known alkaloids: sebiferine (4), reticuline (5), norisoboldine (6), norboldine (7), norisodomesticine (8), crychine (9), cassythicine (10), argentinine (11), isoboldine (12), o-methylnorflavinantine (13), nornantenine (14), and milonine (15). (-)-Kunstleramide (1) exhibited very poor DPPH, activity with an IC 50 value of 179.5 ± 4.4 μg/mL compared with the DPPH inhibitor ascorbic acid. Kunstleramide (1) showed moderate cytotoxic effect with EC 50 values. This study revealed that this plant showed promising cytotoxic activity but poor DPPH activity. Further investigation should be carried out to evaluate the cytotoxicity of compound 1 at lower concentrations.

Acknowledgement

AcknowledgementsThe authors acknowledge the financial support provided by University of Malaya Research Grant (UMRG 045/11BIO), Centre of Natural Products and Drugs Development (CENAR), Postgraduate Research Grant of University of Malaya (PS366/2010B) and High Impact Research (HIR) Grant of University of Malaya (F000009-21001). We also acknowledge the support provided by Faulty of science, Qom University of Technology, and Din Mat nor and Rafly Syamsir for the plant samples and also Hairin Taha for editorial assistance.

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Dose-response curves (using GraphPad Prism) tested with

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