Flavonoids from the leaves and heartwoods of Artocarpus lowii King and their bioactivities

Abstract A new dihydrochalcone, 2′,4′-dihydroxy-3,4-(2″,2″-dimethylchromeno)-3′-prenyldihydrochalcone (1) together with 4-hydroxyonchocarpin (2), isobavachalcone (3), 4′,5-dihydroxy-6,7-(2,2-dimethylpyrano)-2′-methoxy-8-γ,γ-dimethylallyflavone (4), artocarpin (5) and cycloheterophyllin (6) were successfully isolated from the leaves and heartwoods of Artocarpus lowii King (Moraceae). The structures of these compounds were fully characterised using spectroscopic methods and by direct comparison with published data. These compounds were tested for their antioxidant and tyrosinase inhibitory activities. Compound (1) displayed moderate antioxidant activity towards DPPH and tyrosinase inhibitory activities with SC50 value of 223.8 μM and IC50 value of 722.5 μM, respectively. Among the isolated compounds, cycloheterophyllin (6) showed the most potential antioxidant activity with SC50 value of 320.0 and 102.8 μM for ABTS and DPPH radicals scavenging activities, respectively, and also exhibited highest FRAP equivalent value of 4.7 ± 0.09 mM. Compound (6) showed tyrosinase inhibitory activity with the IC50 value of 104.6 μM.

Compounds (1-6) were tested for their antioxidant and tyrosinase inhibitory activities. The results for antioxidant and tyrosinase inhibitory activities are showed in Table 1. The antioxidant activity was evaluated using DPPH, ABTS and FRAP assays while the tyrosinase inhibitory activity (TIA) of all compounds was tested against mushroom tyrosinase. Butylated hydroxyanisole (BHA) and Trolox were used as the standard antioxidant while kojic acid was used as standard TIA. Compound (1) exhibited moderate antioxidant activity towards DPPH radicals and tyrosinase inhibitory activities with SC 50 value of 223.8 μM and IC 50 value of 722.5 μM, respectively. Compound (6) showed the most significant antioxidant activity by inhibiting DPPH and ABTS free radicals with SC 50 values of 102.8 and 320.0 μM, respectively. Compound (6) also exhibited higher FRAP value (4.7 ± 0.09 mM) compared to other tested compounds. Compound (6) displayed significant tyrosinase inhibitory activity (IC 50 = 104.6 μM) against mushroom tyrosinase compared to the standard kojic acid (IC 50 = 219.6 μM). The presence of hydroxyl groups in the structure is responsible for antioxidant potentials and strongest radical absorption. The presence of 2,3-double bond in conjunction with 4-oxo function is also responsible for electron delocalisation (Lee et al. 2007). This study revealed that flavonoids isolated from A. lowii might be beneficial in the development of antioxidant and antityrosinase agents.

General experimental procedures
Melting points were measured using melting point apparatus equipped with microscope, Leica Gallen III and were uncorrected. The IR spectra were recorded on Shimadzu Fourier Transform Infrared Spectrophotometer as thin film (NaCl windows) for liquid samples or KBr disc for solid samples. The ultraviolet (uV) spectra were obtained on Shimadzu uV 1601PC spectrophotometer. The mass spectra were obtained from Mass Spectrometry Services, university College London, united Kingdom and National university of Singapore. The 1D and 2D NMR spectra were recorded in either deuterated chloroform or acetone on Bruker Avance 400 MHz Spectrometer, chemical shifts (δ) are reported in ppm on δ scale, and the coupling constants (J) are given in Hz. Vacuum liquid chromatography (VLC) was carried out on Merck silica gel 60 (230-400 mesh). Gravity column chromatography (CC) was performed on Merck silica gel 60 (70-230 mesh). Silica gel 60 F254 precoated aluminium sheets (0.20 mm, Merck) were used for TLC analysis of extracts, fractions and pure compounds. The TLC spots were visualised under uV light (254 and 365 nm) followed by spraying with 5% H 2 SO 4 and 1% vanillin in MeOH and heating at 120°C for 5 min. All solvents were AR grade.

Plant material
The leaves and heartwoods of A. lowii were collected from Paka, Terengganu, Malaysia in September, 2010. A voucher specimen, AZ7094 was deposited at the Herbarium of universiti Kebangsaan Malaysia, Bangi, Selangor.

DPPH free radical scavenging assay
The free radical scavenging assay was conducted based on method described by Najihah et al. (2012) with minor modification. Detail of the protocol is provided as supplementary materials.

Ferric reducing antioxidant potential (FRAP) assay
The ferric reducing antioxidant potential (FRAP) assay was carried out according to Channarong et al. (2012) and Shahwar et al. (2012) with minor modification. Detail of the protocol is provided as supplementary materials.

Tyrosinase inhibitory activity
This assay was performed using method described by Kamkaen et al. (2007) and Likhitwitayawuid and Sritularak (2001) with minor modification. Detail of the protocol is provided as supplementary materials.