(+)- Kunstlerone, a New Antioxidant Neolignan from the Leaves of Beilschmiedia kunstleri Gamble

A new neolignan, 3,4-dimethoxy-3′,4′-methylenedioxy-2,9-epoxy-6,7-cyclo-1,8-neolign-11-en-5(5H)-one, which has been named (+)-kunstlerone (1), together with six known alkaloids: (+)-norboldine (2), (+)-N-methylisococlaurine (3), (+)-cassythicine (4), (+)-laurotetanine (5), (+)-boldine (6) and (-)-pallidine (7), were isolated from the leaves of Beilschmiedia kunstleri. The structures were established through various spectroscopic methods notably 1D- and 2D-NMR, UV, IR and LCMS-IT-TOF. (+)- Kunstlerone (1) showed a strong antioxidant activity, with an SC50 of 20.0 µg/mL.


Introduction
The Lauraceae family normally, with 30 genera and over 2,000 species, occurs throughout Southeast Asia and tropical America [1,2]. In Malaysia, its contribution is about 213 species, from 16 OPEN ACCESS genera [2]. Beilschmiedia species are known to produce many types of phytochemicals [3][4][5][6][7][8][9][10][11][12] with varied biological activities such as O,O-dimethylcoclaurine isolated from Malaysian B. brevipes, which exhibited significant cytotoxicity against P-388 murine leukemia cells with an IC 50 value of 6.5 μg/mL [13]. Besides the alkaloids, epoxyfuranoid lignans were also reported in the leaves of B. tsangii [10]. Neolignans, whose precursors are the di-and trioxygenated cinnamic acids, have never been reported so far in the species of Beilschmiedia; however they were detected in parts of other species of Lauraceae such as in the fruits of Aniba riparia and in the leaves of Ocotea catharinensis [14][15][16].

General
The optical rotations were recorded on a JASCO (Japan) P1020 Polarimeter equipped with a tungsten lamp; MeOH as solvent. The mass spectra were obtained from LCMS-IT-TOF, Shimadzu. The ultraviolet spectra were obtained in MeOH on a Shimadzu UV-310 ultraviolet-visible spectrometer. The Fourier Transform Infrared (FTIR) spectra were obtained with CHCl 3 (NaCl window technique) on a Perkin Elmer 2000 instrument. The 1 H-NMR and 13 C-NMR spectra were recorded in deuterated chloroform on a JEOL 400 MHz spectrometer; chemical shifts are reported in ppm on δ scale, and the coupling constants are given in Hz. Mayer's reagent was used for alkaloid screening. Aluminum TLC sheets and PTLC (20 × 20 cm Silica gel 60 F 254 ) were used in the TLC analysis. The TLC and PTLC spots were visualized under UV light (254 and 366 nm) followed by spraying with Dragendorff's reagent for an alkaloid detection. All solvents, except those used for bulk extraction, were AR grade.

Plant materials
The leaves of Beilschmiedia kunstleri (Lauraceae) was collected from Hutan Simpan Sungai Tekam, Jerantut, Pahang, Malaysia. The plant was identified by Mr. Teo Leong Eng. A voucher specimen (KL5627) was deposited 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.

Antioxidant assay
The free radical scavenging activity was determined using DPPH as described by Shimada et al. [26]. The DPPH radical scavenging activity assay is a decolorization assay that determines the activity of antioxidants to directly react with DPPH stable free radical by observing its absorbance at 517 nm with a spectrophotometer. A purple colored 1,1-diphenyl-2-picryl hydrazyl (DPPH), a stable free radical which is reduced to α,α-diphenyl-β-picryl hydrazine and give yellow color when reacts with antioxidant. The decolorization of purple color indicates the potential of antioxidants of the samples in which increased decolorization shows the higher scavenging activity of the samples.
Briefly, 0.1 mM DPPH (1 mL) dissolved in ethanol was added to an ethanol solution (3 mL) of the tested compound at different concentrations (25, 50, 100, 150, 200 µg/mL). An equal volume of ethanol was added in the control test. The mixture was shaken vigorously and allowed to stand at room temperature for 30 min. Then the absorbance at 517 nm was measured with a UV-VIS spectrophotometer. Lower absorbance of the reaction mixture indicated higher free radical scavenging activity. The percentage of scavenging of DPPH was calculated using the following equation: where A o is the absorbance of the control reaction and A1 is the absorbance in the presence of the sample.

Conclusions
This is the first communication on neolignans from the Beilschmiedia Kunstleri. To the knowledge of the authors, kunstlerone (1) is the first neolignan reported in the family of Lauraceae bearing a propenyl group at C-1 and the ether ring that is attached to C-2 and C-9. (+)-Kunstlerone (1) showed a strong antioxidant activity with an SC 50 of 20.0 µg/mL. This compound was also detected in neutral fractions, showing that it could be isolated using an acid and base extraction. A proposed plausible biogenetic pathway for 1 as shown in Scheme 1. Apparently, it results from the oxidative coupling between the two precursors coniferyl alcohol and allyl-3,4,5-trihydroxybenzene. The primary hydroxyl would give a Michael-type addition to α,β-carbonyl function and then, an enol addition to the quinone methide (attack of C-6 to C-7) would give the cyclobutane ring. Finally, the dimer was O-methylated at C-3 and C-4 and the methylenedioxy group was formed between the hydroxyl and methoxyl groups of the coniferyl alcohol moiety. Alkaloids 2-7, which were identified in this study, belong to the aporphine, benzylisoquinoline and morphinandienone type of alkaloids. Scheme 1. Biogenetic pathway for kunstlerone (1).