Phenolic Constituents with Antioxidative, Tyrosinase Inhibitory and Anti-aging Activities from Dendrobium loddigesii Rolfe

Abstract Aqueous ethanol extracts of powdered stems of Dendrobium loddigesii afforded three new phenolics including threo-7-O-ethyl-9-O-(4-hydroxyphenyl)propionyl-guaiacylglycerol (1), (R)-4,5,4ʹ-trihydroxy-3,3ʹ,α-trimethoxybibenzyl (2) and (S)-5,5′,7-trihydroxy-3′,4′-dimethoxyflavanone (3), together with eleven known analogues. Their structures were determined by extensive spectroscopic analysis. To identify natural antioxidants, whitening, and anti-aging agents, the abilities of these phenolics were assessed to scavenge the 1,2-diphenyl-2-picrylhydrazyl (DPPH) radical, their abilities to inhibit tyrosinase production, and their abilities to stimulate collagen production by human dermal fibroblasts-adult (HDFa) assay. It was found that compounds 1, 4–8, 13 and 14 exhibited significant DPPH radical scavenging activities, compound 10 exhibited tyrosinase inhibitory activity (IC50 37.904 μg/mL), and compound 9 showed significant collagen production with an EC50 value of 3.182 μg/mL. These results suggest that phenolic constituents from D. loddigesii may be candidate antioxidants, skin-whitening and/or anti-aging agents. Graphic Abstract Electronic supplementary material The online version of this article (10.1007/s13659-019-00219-y) contains supplementary material, which is available to authorized users.


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are known as "Shi-Hu", which have been used for thousands of years as both traditional Chinese medicine and folk remedies for the treatment chronic atrophic gastritis, skin aging, fever, cardiovascular disease and a tonic for promoting the production of body fluid [2]. Previous studies on this genus led to the isolation of a series of polysaccharides, phenolic compounds, alkaloids, and sesquiterpenoids [1,[3][4][5], some of which possess various bioactivities including anti-inflammatory [6], antimicrobial [2], antioxidant [7], antitumor [8], antiplatelet aggregation [9], immunomodulatory [10], and against influenza A activities [11].
Phenolic compounds, are an essential part of the human diet and are known as powerful antioxidants due to their Fig. 2 The key HMBC and COSY correlations of compounds 1-3 potent chain breaking action and they may contribute directly to the anti-oxidative activity [33]. The DPPH radical scavenging assay is one of the most common and relatively quick methods used for evaluating antioxidant activity. Compounds that can donate a hydrogen atom to the DPPH radical, and then gives rise to the reduced form of DPPH which will be considered as potential antioxidant agents. All compounds were evaluated for their DPPH radical scavenging activities. The present results (Table 2) exhibited that the majority of the phenolic compounds (1, 4-8, 13 and 14) showed significant activities with scavenging capacities ranging from 89.411 to 94.278% at 100 μg/ mL.
On the other hand, tyrosinase is the copper containing enzyme and plays a critical role in controlling melanin biosynthesis pathway in melanocytes [34]. Therefore, tyrosinase inhibitors became important constituents of cosmetics or as medicinal products for hyperpigmentation and developing skin whitening agents. In the present study, all the isolates were evaluated for their tyrosinase inhibitory activity (Table 2). Kojic acid, a purported skin-lightening agent, was used as a positive control. 3,5,3′-hydroxybibenzyl (10) revealed a significant inhibitory activity with an IC 50 value of 37.904 μg/mL. Aphyllals C (11) showed moderate inhibition (IC 50 , 152.56 μg/mL). All remaining compounds were inactive at concentrations up to 200 μg/mL. In this study, it can be concluded that compounds 10 and 11 can be potential candidate for the treatment of melanin biosynthesis related skin diseases.
Considering that this species medicinally used for skin aging, since, collagen is critical for skin strength and elasticity, and its degradation leads to wrinkles that accompany aging [35]. Hence, all the compounds were also purposely evaluated for their effects on collagen production in HDFa. The results ( Table 2) showed that compound 9 significant stimulation HDFa collagen production activity (EC 50 3.182 μg/mL). Compounds 6 and 7 showed weaker activities, with collagen production of 33.062% and 29.157% at 10 μg/mL, respectively. The present results not only supported the ethnopharmacological usage of D. loddigesii but also provided a reliable structure template

General Experimental Procedures
Optical rotation was obtained on a JASCO P-1020 digital polarimeter (Horiba, Tokyo, Japan). UV spectra were measured using a Shimadzu UV-2401 PC spectrophotometer (Shimadzu, Kyoto, Japan). IR spectra were obtained on a Bruker Tensor 27 infrared spectrophotometer (Bruker Optics GmbH, Ettlingen, Germany) with KBr pellets. Mass spectra were performed on an API QSTAR time-of-flight spectrometer (MDS Sciqaszex, Concord, Ontario, Canada) and LCMS-IT-TOF (Shimadzu, Kyoto, Japan) spectrometer. NMR spectra were recorded on DRX-500 and Av III-600 instruments with TMS as the internal standard (Bruker, Bremerhaven, Germany). The chemical shifts were given in δ (ppm) with reference to the solvent signal.

Extraction and Isolation
The dried and powdered stems (10.2 kg) of D. loddigesii were extracted three times with 80% ethanol under room temperature and concentrated under reduced pressure. Then, the residue was suspended in H 2 O and partitioned with EtOAc to obtain the EtOAc fraction (220 g), which was subjected to silica gel column chromatography eluted with a gradient of petroleum ether/acetone (15:1 to 0:1) to afford 22 fractions (Fr.1-22

DPPH Radical Scavenging Activity Assay
The free radical scavenging activity assay was carried out according to previous method [36] with some modifications. Briefly, 30 μL samples (1000 μg/mL, dissolved in ethanol) and Trolox (1 mM) were added to 270 μL DPPH solution (100 μM, dissolved in methanol), respectively. The reaction proceeded for 1 h at 37 °C on a 96-well microplate. The absorbance was then read at 515 nm and percentage of total radical scavenging activity was calculated using the following formula: inhibition % = [(A 0 − A 1 )/A 0 ] × 100%, where A 0 is the absorbance of the DPPH without samples (control reaction) and A 1 is the absorbance of DPPH incubated with the samples. All the tests were conducted in triplicate and Trolox was used as a positive control agent.

Mushroom Tyrosinase Inhibitory Assay
Tyrosinase activity inhibition was determined spectrophotometrically according to the method described previously [36] with some modifications. Briefly, different concentrations of test compounds were prepared in 10% DMSO. Each of the sample solution (20 μM) were mixed with L-Dopa (1.25 mM), and diluted with 970 μL of 0.05 mM sodium phosphate buffer (PBS, pH 6.8) in the test tubes. The reaction was initiated by adding mushroom tyrosinase (25 U/mL). The reaction mixture was incubated for 5 min at room temperature. The amount of Dopachrome in the mixture was determined by the measurement of the absorbance of each well at 490 nm. Kojic acid was used as positive control. The inhibitory percentage of tyrosinase was calculated according to the following equation: Percent inhibition = [(A 0 − A 1 )/A 0 ] × 100%, where A 0 is the absorbance of the Dopachrome without test compounds (control reaction) and A 1 is the absorbance of Dopachrome incubated with the test compounds.

Collagen Production by HDFa Assay
The HDFa cell line was obtained from Cascade Biologics. HDFa cells were seeded in 96-well plates containing DMEM with 10% FBS under a humidified atmosphere of 5% CO 2 at 37 °C. After 24 h of incubation, the cells were treated with the test samples for 72 h (37 °C, 5% CO 2 ). TGF-β was used as the positive control. Media (50 µL) was collected from each well, and froze at − 80 °C until it was assayed with procollagen peptide ELISA kit. The concentration of pro-collagen was obtained by measuring the absorbance at 450 nm on the microplate reader. Remove all media from cells and add 100 µL diluted MTS reagent to each well. The reaction incubated for 40 min at 37 °C. The absorbance was measured at 490 nm with a microplate reader. The increase percentage of collagen I production was calculated according to the following equation: cell viability (%) = (Mean OD 490 sample/Mean OD 490 control); increase of collagen production % = (A 1 /B/A 0 − 1) × 100%. Where A 1 is the absorbance with the samples, A 0 is the absorbance without samples (control reaction), and B is cell viability.