New Chemical Constituents from the Bark of Dendropanax morbifera Leveille and Their Evaluation of Antioxidant Activities

Four new constituents, as cis-6-oxogeran-4-enyl-10-oxy-O-β-arabinopyranosyl-4′-O-β-arabinopyranosyl-2″-octadec-9‴,12‴,15‴-trienoate (1), geran-3(10)-enyl-1-oxy-O-β-arabinopyranosyl-4′-O-β-arabinopyranosyl-2″-octadec-9‴,12‴,15‴-trienoate (2), geranilan-8-oxy-O-α-d-xylopyranosyl-2′-n-octadec-9″,12″,15″-trienoate (3), 1-cyclohex-2′, 5′-dienyl 1-cyclohexylethanol-O-β-d-xylopyranoside (4), along with six known constituents, guaiacol-O-β-d-arabinopyaranoside (5), n-tetradecanyl oleate (6), oleyl-O-β-d-xyloside (7), n-octadec-9,12-dienoyl-O-β-d-arabinopyranoside (8), linolenyl-O-β-d-arabinofuranoside (9) andglyceryl-1,3-dipalmito-2-olein (10), were isolated and identified from the Dendropanax morbifera bark. The new structures were established by one-and two-dimensional NMR (and in combination with IR, FAB-MSand HR-ESI-FTMS. The comparative evaluation of antioxidant potential by phosphomolybdenum, DPPH, FRAP and the NO assay of four different compounds (1–4), we have found that the compounds 1 and 2 have power as a natural antioxidant, whereas the compound 3 and 4 exhibited mild activity in comparison to compounds 1 and 2.


Introduction
The genus Dendropanax belongs to the family Araliaceae, can be found distributed in East Asia, Korea, Japan, the Malay Peninsula, and central South America. Dendropanax morbifera Leveille (Araliaceae) is an endemic species in Korea, and distributes within the southern part of Korea [1]. The roots and stems of this plant are used in folk medicine for the treatment of migraine headaches, dysmenorrhea and to remove wind dampness [2,3]. The plant is commonly cultivated in gardens, and it is sometimes used for flower arrangements. The stem is erect and, can grow to a height of 5 m. The leaves are 3-lobed and glossy green in color.
Compound 3 was obtained as a white semi-solid. The IR absorption band at 3315 cm −1 indicated the presence of a hydroxyl group. Its molecular ion peak at m/z 551 [M + H] + was determined on the basis of FAB mass and 13 C NMR spectra consistent with the molecular ion peak of a monoterpene sugar derivatives, indicating five degrees of unsaturation. High resolution of ESIFTMS provided the exact mass of the protonated molecular ion m/z (551.4312), from which the molecular composition C 33 H 59 O 6 was calculated. The fragmentation patterns of compound 3 are shown in Figure 3.

Free Radical Scavenging Activity
The free radical scavenging activities of the mono-and disaccharide with monoterpene along with aliphatic chain-isolated compounds from Dendropanax were tested using the DPPH method [18]. Table 1 presents the antioxidant activity of four compounds at the concentration of 1.0 mg/mL as systematic by the DPPH scavenging assay. The IC 50 values of the entire four constituents were 1-4 (10 µg/mL), (25 µg/mL), (50 µg/mL) and (100 µg/mL), respectively. Of the different compounds isolated from the acetone extract from the Dendropanax bark, compounds 1 and 2 exhibited the highest activity, which was more than about 65% and 50% at 100 µg/mL concentration, respectively, when compared with the compounds 3 and 4 ( Table 1). The compounds 3 and 4 demonstrated moderate antioxidant activity. The DPPH activity of tocopherol showed a higher degree of free radical-scavenging activity (91%) than that of the compounds at a very low concentration point (100 µg/mL). Our finding for antioxidant activities in four different sugar containing compounds support our previous results obtained in fruits of Lycium barbarum and Lycium chinense [19,20]. This is similar to other studies, wherein they have reported that only 300 µg/mL tocopherol, 230 µg/mL BHT (Butylated hydroxytoluene) and 100 µg BHA (Butylated hydroxyanisole) exhibited a free radical scavenging activity equivalent to 390 µg/mL of red bean and 1000 µg/mL of sesame coat extract [20,21]. Table 1. Antioxidant activities of the compounds (C-1 to C-4) as measured by Nitric Oxide scavenging power, Ferric reducing antioxidant power assay (FRAP) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay. Compound 1 and 2 exhibited significant activities at all the concentrations.

Reducing Power
The antioxidant effect exponentially increases as a function of the development of the reducing power, indicating that the antioxidant properties concomitant with the development of reducing power have been reported [22,23], that the reducing power of such type of compounds from medicinal plants prevents liver injury by inhibiting formation of lipid peroxides. As seen in Table 1, the reducing power of the constituents 1-4 from the Dendropanax bark isolated compounds enhanced with escalating concentration from 10 to 100 µg/mL. Reducing power of the compounds 1-4 followed in the order 1 > 2 > 3 > 4. The antioxidant potential of tocopherol was markedly greater than the test samples at a very low concentration point. This finding also supports the outcome of other researchers, where the reducing power of BHT and tocopherol [20,24] was higher than the isolated compounds.

Nitric Oxide Scavenging Activity
Nitric oxide free radicals were improved and scavenged by compounds 1 and 2 in comparison with compounds 3 and 4 at low concentrations of 10-100 µg/mL. Scavenging of NO was well interacted with the existence of sugars in compounds, and the sequence was found to be 1 > 2> 3 >4 (Table 1).

Antioxidant Capacity by Phosphomolybdenum Method
Antioxidant potential of compounds 1-4 was measured spectrophotometrically by the phosphomolybdenum method, which is based on the reduction of Mo (IV) to Mo (V) by the sample analyte, ensuing the formation of greenish phosphate/Mo (V) compounds with a maximum absorption at 695 nm.
The antioxidant potential of compounds 1-4 was established as 15.31, 13.20, 6.70 and 5.63 µg/mL, respectively. The antioxidant capacities of the compounds were found to be in the order of 1 > 2 > 3 > 4 ( Table 2). Table 2. Antioxidant capacity of compounds C-1 to C-4 by the phosphomolybdenum method.

Chemical and Instruments
Melting points of the compounds were determined using a model IA9100 melting point apparatus (Electrochemical Engineering, Seoul, Korea). Optical rotations were measured with a model AA-10 polarimeter (Instrument Ltd., Seoul, Korea). Infrared (IR) spectra were recorded after compound mixing with potassium bromide (KBr) on a Thermo Scientific FT-IR model Nicolet 6700 (Thermo Fisher Scientific, Waltham, MA, USA) spectrophotometer at the Korea Institute of Science and Technology (KIST), Seoul, Korea. Both nuclear magnetic resonance (NMR) spectra 1 H (600 MHz) and 13 C NMR (150 MHz) were measured with a Bruker Avance-600 spectrometer (Bruker Corporation, Billerica, MA, USA) using deuterated solvents, and the machine was available at the National Instrumentation Centre for Environmental Management (NICEM), College of Agriculture and Life Science, Seoul National University (SNU), Seoul, Korea. NMR spectra were recorded in deuterated chloroform, and methanol-d 4 using tetramethylsilane (TMS) as an internal standard, with chemical shifts expressed in parts per million (d) and coupling constants (J) in Hertz. High-resolution electrospray ionization Fourier transform (ESI/FT) mass spectra were recorded on a Thermo-Finnigan LTQ-Orbitrap instrument (Thermo Scientific, Bartlesville, OK, USA) equipped with a Dionex U 3000 HPLC system with UV-VIS detector (SPD-10A) (Dionex Corporation, Sunnyvale, CA, USA). A C 18 (octadecylsilyl, or ODS) column was used with a mobile phase of 0.1% TFA (Trifluoroacetic acid) in acetonitrile: water (80:20), and a flow rate of 4 mL −1 . All chemicals were of analytical grade. n-Hexane, ethyl acetate, methanol, ethanol, sulfuric acid (H 2 SO 4 ) and vanillin were purchased from Daejung Chemicals and Metals (Seoul, Korea). Thin-layer chromatography (TLC) was performed on pre-coated silica gel 60 F 254 plates (Merck, Darmstadt, Germany). Visualization of TLC plates was performed in a developing glass chamber, and after drying, they were dipped in solution of 5% vanillin and H 2 SO 4 in an ethanol spray reagent (5:5:90). Column chromatography was performed using silica gel (70-230 mesh) and LiChroprep RP-18 (40-63 µm; Octadecyl silica (ODS) gel) from Merck. Standards were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Plant Material
Dried bark (558 g) of D. morbifera was collected from Konkuk University Farm, Seoul, South Korea. A voucher specimen (No. DML-KU-2013) has been deposited at the Department of Applied Bioscience, Konkuk University, Seoul, South Korea.

Extraction and Isolation
The powdered bark of D. morbifera (558 g) was immersed in acetone (3 × 2.5 L) for three days at room temperature, and then the supernatant was concentrated under vacuum to yield 24.5 g of an extract.

Chemicals and Instruments in Antioxidant Activity
All the chemicals, reagents and the solvents used in the assay protocols were of analytical grade. Ascorbic acid, tocopherol, BHT, BHA, water, phosphate buffer, potassium ferricyanide, trichloroacetic acid, ferric chloride DPPH, DMSO, sulfanilamide and naphthylethylenediamine for Griess reagent, sodium hydroxideand sodium citrate were obtained from Sigma Aldrich, (St. Louis, MO, USA). Also, all other chemicals were purchased from Sigma Aldrich, USA. Spectrophotometer was used from Thermo Scientific, Multiskan GO Spectrophotometer (Sl. No. 1510033456).

Free Radical Scavenging Activity
Antioxidant activity of the different constituents (1)(2)(3)(4), based on the scavenging activity of the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical, was examined by the method described by Katerere and Eloff, [18]. A wide range of concentrations (10, 25, 50 and 100 µg/mL) of the compounds to be tested (0.2 mL; tocopherol) were taken in different test tubes with 4 mL of a 0.006% MeOH solution of DPPH. Water (0.2 mL) was taken as a standard. Absorbance at 517 nm was determined after 30 min. Radical scavenging activity was measured in terms of the inhibition percentage, and was calculated using Equation (1).
where A 0 is the absorbance (control) and A 1 is the absorbance (compounds/standard).

Reducing Power
The reducing power of the ginseng compounds was determined according to the method of Oyaizu [31]. Different compounds of concentrations 10, 25, 50 and 100 µg were dissolved in 1 mL of distilled water and mixed with phosphate buffer (2.5 mL, 0.2 M/L, pH 6.6) and potassium ferricyanide [K3Fe (CN)6] (2.5 mL, 1%). The mixture was kept for incubation at 50 • C for 20 min. Trichloroacetic acid (10%) was added (2.5 mL) to the mixture, which was then centrifuged at 1000 rpm for 10 min. The upper layer of the centrifuged solution (2.5 mL) was mixed with distilled water (2.5 mL) and FeCl 3 (0.5 mL, 0.1%) and the absorbance was optimized at 700 nm. The enhanced absorbance of the reaction mixture indicated improved reducing power. All analyses were done in triplicate, and averaged. The reducing power was calculated using Equation (2).

Nitric Oxide Scavenging Activity
Sodium nitroprusside in an aqueous solution at a physiological pH generates nitric oxide, which interacts with oxygen to produce nitric ions that can be estimated using Griess reagent. The complex formed during the diazotization of nitrite with sulphanilamide and the subsequent coupling with naphthylethylenediamine (Griess reagent) was read at 546 nm and the methods of Marcocci [32]. Different concentrations of samples (10,25,50 and 100 µg/mL) were prepared and added in sodium nitroprusside with a phosphate buffer. The above prepared reaction mixture was incubated at room temperature for 30 min. Then 50 µL of incubated reaction mixture was transferred to another microplate followed by the addition of Griess reagent, and absorbance was recorded at 546 nm. The percentage nitrite radical scavenging activity of the ethanol extracts and gallic acid were calculated using Equation (3).
Percentage nitrite radical scavenging activity: Where = absorbance of control sample and = absorbance in the presence of the samples of extracts or standards.

Evaluation of Antioxidant Capacity by Phosphomolybdenum Method
The total antioxidant capacity of the compounds (1-4) was evaluated by the method of Prieto [33] based on reduction of Mo (VI) to Mo (V) in acidic conditions resulting into development of a greenish complex of phosphate and Mo (V). The details of sample, reagents and standard were given in the literature [34].

Conclusions
Dendrodropanax morbifera is an endemic species in Korea, and is found in the southern parts of Korea. The D. morbifera have been reported as several classes of compounds, and many biological activities were also found in this plant's constituents and extracts. Four new compounds (1-4) have been isolated in this study, and we evaluated its antioxidant exercise as a radical scavenging effect, reducing power, phosphomolybdenum and the nitric oxide activity. The results showed the compounds (1 and 2) have good antioxidant activity in comparison with compounds (3 and 4). The developed path has been verified, and found to be useful in the investigation of active constituents in natural medicines. Further studies are needed to investigate more isolation work of novel constituents from other Dendropanax species that show strong activities as above, and also other activities.