A new anthraquinone glycoside from Rhamnus nakaharai and anti-tyrosinase effect of 6-methoxysorigenin

Abstract In continual study on the heartwood of Rhamnus nakaharai, a new alaternin-8-O-glucoside, namely 1,2,6,8-tetrahydroxy-3-methylanthraquinone-8-O-β-glucopyranoside (1), together with some known compounds were further isolated and characterised by 1-D, 2-D NMR and other spectral evidences. The free radical scavenging and antityrosinase activities of the isolates, including alaternin (1a), emodin (2a), emodin-8-O-β-glucopyranoside (2), 6-methoxysorigenin-8-O-β-glucopyranoside (3) and 6-methoxysorigenin (3a) were tested. Alaternin (1a) exhibited to be mild DPPH radical scavenger with half as potent as vitamin C, while both alaternin (1a) and emodin-8-O-β-glucopyranoside (2) exhibited stronger SOD-like activity than that of BHA. 6-Methoxysorigenin (3a), a reported potential antioxidant, and its 8-O-glucoside (3) both performed significant inhibitory effect on mushroom tyrosinase with about twice as potent as kojic acid, the positive control. Graphical abstract

Inhibition assays for blank, each concentration of sample and positive control n ≥ 3; ns: no significance; sc 50 represents the concentration needed for 50% of radical scavenged.

Mushroom tyrosinase inhibitory assay
Mushroom tyrosinase (eC 1.14.18.1) inhibitory assay is the common method for preliminary screening of skin-whitening agent. Cosmetic ingredients that have dual antioxidant and antimelanogenesis effects will be beneficial in skin protection as well as in economic value. The result of antityrosinase assay displayed that anthraquinones were not suitable substrates for tyrosinase, only emodin (2a) exhibited mild inhibitory effect on this enzyme (Table 1). However, 6-methoxysorigenin (3a) and its 8-O-glucopyranoside (3) performed significant IC 50 values compared with kojic acid, a usual commercial whitening ingredient used as positive control. 6-Methoxysorigenin (3a) showed almost twofold of inhibitory effect than kojic acid (IC 50 = 82.1 μM) with IC 50 = 42.2 μM on tyrosinase at first 30 min of incubation though the IC 50 value faded to 277.3 μM after 60 min of further incubation. However, 6-methoxysorigenin-8-O-glucopyranoside (3) performed a stable inhibition on mushroom tyrosinase in two incubation intervals (IC 50 97.4 and 98.6 μM) with almost twice as potent as kojic acid (182.8 μM) on the final incubation. Apparently, the C-1 OH and γ-lactonic carbonyl of 6-methoxysorigenin (3a) played a key role in anti-tyrosinase activity. In contrary, the C-8 OH seemed to be an unstable factor in tyrosinase inhibition, which was cured by glycosylation of this phenol. However, the C-8 O-glucosyl might somewhat too bulky to bind the enzyme that IC 50 value of 3 displayed only less than half as potent as that of 3a at first 30 min (Table 1).

Plant material
The heartwood of R. nakaharai was collected from Yang-Ming Shan, Taipei, Taiwan in January 2001. After confirming the authenticity of the species, a voucher specimen was deposited in the herbarium (T89-Rh-001) of Tajen university, Pingtung, Taiwan.

Extraction and isolation
Five kilograms of dried heartwood of R. nakaharai was chipped and extracted with 10 L of methanol at room temperature for one week. The methanolic extract was condensed in vacuum to obtain a residue of 186 g. This residue was column chromatographed (silica gel 900 g, 70-230#) and eluted with a solvent system of CH 2 Cl 2 /MeOH (v/v) from 10:0-3:1 in 2000 mL per fraction, then washed by 1500 mL of MeOH. The methanolic wash presented in a suspension and readily dissolved in water. After condensed, the residue weighed 28 g were dissolved in 30% MeOH/H 2 O (v/v) and chromatographed on an Rp-18 Si-gel (100 g, 60 μM) column by gradient elucidation with 30-100% of MeOH/H 2 O (v/v) solvent system (5%/500 mL). The MeOH/H 2 O fraction with 50% obtained 4 (30 mg) and 5 (35 mg) (Lin et al. 1991), 65% yielded 3 (28 mg, Ng et al. 2007), 70% yielded 1 (12 mg) and 2 (65 mg, Coskun et al. 1990), and 90% yielded 1a (10 mg, Kitanaka et al. 1985), 2a (24 mg) and 6 (10 mg), respectively. each of the above fractions was further purified by repeated CC with proper solvent system if needed. Known compounds were identified all by spectral evidences compared with reported or authentic samples.

ABTS· + radical scavenging assay
The ABTS· + scavenging capacity was measured according to the methods reported previously (Ng et al. 2009;Lu et al. 2015). Briefly, a mixture containing 180 μL of ABTS and 40 μL of tested compounds or negative control (PBS; phosphate buffered saline) or positive control (vitamin C) were thoroughly mixed and incubated for 7 and 24 min at room temperature, then measured the absorbance at 734 nm.

DPPH· radical-scavenging assay
The method of DPPH· scavenging assay had also been reported previously (Ko et al. 2008;Ng et al. 2009;Lu et al. 2015). In brief, 1 mL (0.1 mM) of DPPH· solution was mixed with 3 mL of various concentrations of anthraquinones or positive control (vitamin C) dissolving in methanol. The mixture was then vortexed vigorously and left at 40 °C for 30 min in the dark. The absorbance was measured at 517 nm.

Mushroom tyrosinase inhibition
The tyrosinase inhibitory assay was performed according to the method reported previously (Ko et al. 2008;Ng et al. 2009;Lu et al. 2015). Briefly, tested compounds or kojic acid (positive control) were dissolved in DMSO/methanol before diluted by phosphate buffer (pH 6.8).
The assay was performed in a 96-well plate containing 20 μL of tested compounds or kojic acid, 20 μL of mushroom tyrosinase (1000 u/mL), and 80 μL of L-tyrosine (2.0 mM) that were stocked to 200 μL with buffer. The absorbance of the mixture was measured at 490 nm after 30 and 90 min of incubation.

Conclusions
Buckthorns have a long history of being used in constipation treatment since ancient time.
Modern chromatographic methods extended the research on constituents that not only contribute on the understanding of many folklore usages but also make possible in expanding the application of these medicinal plants (Locatelli et al. 2013). Alaternin (1a) performed more active DPPH scavenging effect than emodin (2a) and better SOD-like effect than BHT that could be due to the existence of additional C-2 OH (Pietta 2000). 6-Methoxysorigenin (3a) and its 8-O-glucoside (3) are valuable candidates for further development in application of cosmetic ingredients not only because of potential tyrosinase inhibitors but also of reported potential antioxidants.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was partially funded by Ministry of Science and Technology [grant number NSC89-2320-B-127-010], Taiwan ROC.