Extracts and Constituents of Rubus chingii with 1,1-Diphenyl-2-picrylhydrazyl (DPPH) Free Radical Scavenging Activity

The 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity of the fruits of Rubus chingii was studied in vitro. Ethanolic extract, ethyl acetate and n-butanol fractions from dried R. chingii fruits revealed strong DPPH free radical scavenging activity with IC50 values of 17.9, 3.4 and 4.0 μg/mL, respectively. The ethyl acetate and n-butanol fractions were further purified by a combination of silica gel chromatography, Lobar RP-8 chromatography, and high-pressure liquid chromatography (HPLC). Nine compounds were isolated, where methyl (3-hydroxy-2-oxo-2,3-dihydroindol-3-yl)-acetate (2), vanillic acid (5), kaempferol (7), and tiliroside (9) showed stronger DPPH free radical scavenging activity than that of ascorbic acid (131.8 μM) with IC50 values of 45.2, 34.9, 78.5, and 13.7 μM, respectively. In addition, rubusine (1) is a new compound discovered in the present study and methyl (3-hydroxy-2-oxo-2,3-dihydroindol-3-yl)-acetate (2), methyl dioxindole-3-acetate (3), and 2-oxo-1,2-dihydroquinoline-4-carboxylic acid (4) were isolated from the fruits for the first time.


Introduction
Fruits and vegetables are known to contain different antioxidant compounds, and high consumption of these products has long been associated with a lower incidence of degenerative diseases. This beneficial association is considered to be partially due to the various antioxidant compounds present in OPEN ACCESS these foods; these antioxidants scavenge free radicals and, thereby, reduce the manifestation of degenerative pathologies [1][2][3]. Colorful fruits and green leafy vegetables are rich sources of phenolic and flavonoid compounds, which exhibit a range of antioxidant, antibacterial, anti-inflammatory, antiallergic, hepatoprotective, vasodilatory, and neuroprotective activities [4][5][6][7].
For many years, dried fruits of R. chingii Hu (Rosaceae), referred to as ''fu-pen-zi'' in Chinese, have been used as a food and a tonic in traditional Chinese medicine [8]. Fupenzi is used to improve the functioning of the kidney and to treat seminal discharge and excessive polyuria [9]. In recent decades, modern pharmacological experiments have revealed that R. chingii has immunomodulatory effects [10] on bacterial infection, anxiety, pain, inflammation [11], and the hypothalamus-pituitary-sex gland axis [12]. Previous phytochemical studies of this plant have led to the isolation of triterpene acids, flavonoids, phenolics, and steroids [13][14][15][16][17][18]. In the present study, the DPPH free radical scavenging activity of the extracts and constituents of Chinese medicine was investigated.

Plant Material
The dried fruits of R. chingii were purchased from Chung-Yuan Company, Kaoshiung, Taiwan. Prof. Hang Chang Lin at the National Defense Medical Center, Taipei, Taiwan, identified the plant. The R. chingii specimen (No. 960801) has been deposited at the herbarium of the Chia Nan University of Pharmacy and Science, Tainan, Taiwan.

General Experimental Procedures and Apparatus
We used a Yanagimoto micromelting point apparatus to determine the melting points, which are reported as uncorrected values. Optical rotations were measured in a GENESYS 20 polarimeter (Rochester, New York, NY, USA) The IR spectra were recorded on KBr disks with a Perkin-Elmer 983 G spectrophotometer, and the UV spectra were obtained on a Shimadzu UV-160 spectrometer. 1 H and 13 C NMR spectra were determined with a Bruker AM-500 spectrometer using DMSO-d 6 and MeOH-d 4 . The FABMS measurements were measured using a JEOL JMX-HX110 mass spectrometer. The EIMS were recorded at 70 eV using a Finnigan MAT TSQ 46C GC/MS/MS/D spectrometer.
The column chromatography was carried out on silica gel (70-230 mesh, Merck). The preparative liquid chromatography was performed by a Shimadzu LC-8A chromatograph on a reverse C-18 column (Nacalai Teaqe shim-pack, pre-CODSL, 15 um, 50 × 250 mm, flow rate 15 mL/min). Two Shimadzu LC-8A pumps and a Shimadzu SCL-8A system controller were used for preparative HPLC (Shimadu, Tokyo, Japan). The chromatography peaks were detected by UV (254 nm) using a Shimadzu SPD6VA UV detector. A FMI RP-SY-ICSC Lobar pump was used for the low pressure liquid chromatography (Lobar). Silica gel 60 (Merck 70-230 mesh, 230-400 mesh; ASTM) was used for the column chromatography, and silica gel 60 F 254 (Merck, Darmstadt, Germany) was used for the thin-layer chromatography.

Extraction, Partition, and Isolation of R. chingii Compounds
The detailed purification procedure involved in the extraction and separation of the nine compounds from R. chingii is shown in Figure 1. The dried fruits (5.0 kg) were ground in a power grinder and soaked in 95% ethanol (×4) at room temperature for 3 days. The extracts were decanted, filtered under vacuum, and concentrated in a rotating evaporator to produce dark brown syrup (extraction yield 5.63% (w/w) dry weight) of 281.4 g. The crude extract was partitioned between n-hexane and 95% methanol. The 95% methanol fraction was concentrated and partitioned between ethyl acetate and water. The aqueous extract was again partitioned between n-butanol and water. The resulting portion generated fractions in the n-hexane layer (41.7 g, 14.8% (w/w) yield), ethyl acetate layer (31.9 g, 11.3% (w/w) yield), n-butanol layer (101.8 g, 36.2% (w/w) yield), and H 2 O layer (104.8 g, 37.2% (w/w) yield). The ethyl acetate layer was introduced into the silica gel chromatography column and eluted with an n-hexane-ethyl acetate mix (3:1, 1:1), an ethyl acetate-ethyl acetate-methanol mix (3:1, 1:9), and methanol to obtain vanillic acid (5,8.0 mg), p-hydroxybenzoic acid (6, 67.0 mg), and kaempferol (7, 6.5 mg). The n-butanol layer was introduced to the chromatography column on silica gel with ethyl acetate, the ethyl acetate-methanol mix (19:1, 9:1, 4:1, 1:1), and methanol. Re-chromatography of the first fraction on prep-Lobar RP-8 was conducted, followed by HPLC separation, which yielded rubusine (1,56 Table 1.

DPPH (1,1-Diphenyl-2-picryhydrazyl) Free Radical Scavenging Assay
To measure the antioxidant activity, the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity was determined by the spectro-photometric method previously described [26]. A methanol solution of a sample (100 µL) was mixed with a 0.5 mM DPPH methanol solution (800 µL) and 0.1 M acetate buffer (pH 5.5; 100 µL). The absorbance of the mixture at 517 nm was measured after standing for 30 min. The IC 50 value was determined as the concentration of each sample required to give 50% of the absorbance shown by the blank test.