The ethanolic extract of the dried fruits of R. chingii was partitioned into n-hexane, ethyl acetate, n-butanol, and water fractions. The ethyl acetate and n-butanol fractions were subjected to silica gel chromatography, prep-Lobar RP-8, and preparative high-pressure liquid chromatography (prep-HPLC) to isolate one new alkaloid compound, rubusine (1), together with eight known compounds (Figure 1). Their structures were established on the basis of spectral evidence (Figure 2). The compounds, which comprised three alkaloids: methyl (3-hydroxy-2-oxo-2,3-dihydroindol-3-yl)-acetate (2) [19], methyl dioxindole-3-acetate (3) [20], 2-oxo-1,2-dihydroquinoline-4-carboxylic acid (4) [21]; two phenolics: vanillic acid (5) [18] and p-hydroxybenzoic acid (6); three flavonoids—kaempferol (7) [15], nicotiflorin (8) [17], and tiliroside (9) [14] were identified by comparing the spectroscopic data with those in reported literature.

Rubusine (1) was an amorphous powder in methanol. HR-EIMS revealed an [M⁺] at m/z 189.0420, corresponding to the molecular formula C₁₀H₇NO₃. The IR spectrum of 1 revealed absorption bands at ν_max 3450–3200 (OH), 2978, 2895 (NH), and 1650 (C = O) cm⁻¹ indicating hydroxyl, imide, and ketone groups, respectively. The ¹H NMR spectrum of 1 exhibited an aromatic ring at δ 7.22 (t, J = 7.7 Hz), 7.36 (d, J = 7.7 Hz), 7.54 (t, J = 7.7 Hz), and 8.13 (d, J = 7.7 Hz). The ¹³C NMR spectrum was δ 115.8, 122.3, 126.1, 130.9, 139.4, and 141.2. The ¹³C NMR chemical shifts for two ketone groups at δ 161.1 were in the B ring. The B ring has a double bond as indicated by the ¹H NMR peak of δ 6.86. The ¹³C NMR peaks of δ 123.4 and 166.8 suggest that the double bond possesses one hydroxyl group. The results of correlation spectroscopy (COSY), heteronuclear multiple quantum coherence (HMQC), heteronuclear multiple bond connectivity (HMBC), and NOESY of the ¹H and ¹³C NMR signals are shown in Table 1. These data agreed with the structure of rubusine (1) (Figure 2).

Methyl (3-hydroxy-2-oxo-2,3-dihydroindol-3-yl)-acetate (2) was a pale tinted powder and exhibited [α]_D +10° (MeOH). HR-EIMS revealed an [M⁺] at m/z 221.0683, corresponding to the molecular formula C₁₀H₇NO₃. The UV, IR, ¹H and ¹³C NMR spectral data of 2 were similar to those of methyl dioxindole-3-acetate (3), except that the methylene proton peaks were recorded at δ 3.05 and 3.08 (each d, J = 15.6 Hz, H-8), and shifted downfield. In addition, C-2 (δ 180.9) and C-7a (δ 143.7) were shifted downfield by δ 6.1 and δ 5.3, respectively. Comparing with the ¹H and ¹³C NMR data of compound 3, indicated that compound 2 may be an enantiomer of compound 3. The ¹H NMR spectrum of 2 also exhibited an ABCD system of aromatic rings at δ 6.88 (dd, J = 7.5, 1.0 Hz), 7.01 (td, J = 7.5, 1.0 Hz), 7.25 (td, J = 7.5, 1.0 Hz), and 7.35 (dd, J = 7.5, 1.0 Hz) and a methoxyl group of esters at δ 3.46. HMBC spectrum of compound 2 showed H-8 (δ 3.05 and 3.08; each d, J = 15.6 Hz) correlation to C-2 (δ 180.9), C-3 (δ 42.3), C-3a (δ 131.8), and C-9 (δ 171.1). H-4 (δ 7.35, dd, J = 7.5, 1.0 Hz) was correlated with C-3 (δ 42.3), C-3a (δ 131.8), and C-7a (δ 143.7). Taken together, the structural data point to 2 being methyl (3-hydroxy-2-oxo-2,3-dihydroindol-3-yl)-acetate (2). The ¹H- and ¹³C-NMR signals of 2 were completely assigned using contemporary 2-D NMR techniques (COSY-45, HMQC, HMBC and NOESY) and are listed in Table 2.

Antioxidants are extensively used in cosmetics, food, and medicine because they can counteract cellular free radicals and reduce metal ions, thereby, interrupting the oxidizing chain reaction before any damage occurs. Antioxidants, therefore, can combat many health problems [22]. The DPPH free radical scavenging activity of the fupenzi fruit extracts and partitions were in the order: ethyl acetate layer, n-butanol layer, water layer, ethanol extract, ascorbic acid, n-hexane layer, and ascorbic acid (Table 3). The compounds methyl (3-hydroxy-2-oxo-2,3-dihydroindol-3-yl)-acetate (2), vanillic acid (5), kaempferol (7), and tiliroside (9) showed significant free radical scavenging activity with IC₅₀ values of 45.2, 34.9, 78.5, and 13.7 μM, respectively. Compound 9 appeared to 9.62-fold as potent as ascorbic acid, whereas 2, 5, and 7 were about 2.92, 3.78, and 1.68-fold, respectively, as potent. Compound 8 had the same antioxidant activity as that of ascorbic acid (Table 4).

In the present study, the radical scavenging activity of the compounds tested was influenced by the number and the location of the hydroxyl groups, glycosylation, and other substitutions [23]. Compared with nicotiflorin (8, IC₅₀ = 130.6 μM, 23), the DPPH scavenging activity of kaempferol (7, IC₅₀ = 78.5 μM) was 1.66-fold higher. However, tiliroside (9, IC₅₀ = 13.7 μM) exhibited 5.73-fold higher activity than that of kaempferol (7). Many of the benefits associated with consumption of phenolic-rich foods are correlated with their antioxidant activities [24]. Research has suggested that phenol may prevent lipid peroxidation via hydrogen atom donation from the hydroxyl group attached to the benzene ring. The present study further demonstrated that the phenolic compounds possessing hydroxyl groups exhibited much higher DPPH free radical scavenging activity. Vanillic acid (5, IC₅₀ = 34.9 μM) exhibited higher activity when compared with p-hydroxybenzoic acid (6, IC₅₀ = 311.5 μM) [25]. An adjacent substituted methoxyl group in the aromatic ring was the most significant difference between the two phenolics. A possible explanation for the difference in DPPH free radical scavenging activity is the difficulty in forming the reaction complex between the phenolics and the free radicals. The alkaloid compound 2-oxo-1, 2-dihydroquinoline-4-carboxyloic acid (4, IC₅₀ = 1216.1 μM) has no hydroxyl group, and its DPPH free radical scavenging activity was not significant.

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.

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. ¹H and ¹³C NMR spectra were determined with a Bruker AM-500 spectrometer using DMSO-d₆ and MeOH-d₄. 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 F₂₅₄ (Merck, Darmstadt, Germany) was used for the thin-layer chromatography.

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 H₂O 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, 56.0 mg), methyl (3-hydroxy-2-oxo-2,3-dihydroindol-3-yl)-acetate (2, 71.0 mg), methyl dioxindole-3-acetate (3, 22.0 mg), and 2-oxo-1,2-dihydroquinoline-4-carboxylic acid (4, 16.0 mg). Similarly, the second fraction produced tiliroside (9, 16.0 mg), and the third fraction produced nicotiflorin (8, 60.0 mg).

Rubusine (1): Colorless powder; mp 291–292 °C; UV λ_max nm: 210.0, 230.0, 278.0, 289.0; IR ν_max cm⁻¹: 3450–3200, 2978, 2895, 1650; HREI-MS: The calculated molecular mass of C₁₀H₇NO₃, m/z: 189.0426 [M]⁺, Measured: 189.0420; EI-MS m/z (posit., rel. int. %): 189 ([M]⁺, 100), 161 (12), 144 (31), 117 (34); ¹H and ¹³C NMR data are presented in Table 1.

Methyl (3-hydroxy-2-oxo-2,3-dihydroindol-3-yl)-acetate (2): white powder; mp 131–132 °C; [α]_D +10° (c = 1.0, MeOH); UV λ_max nm: 254.3, 285.5; IR ν_max cm⁻¹: 3550–3280, 2982, 2894, 1646; HREIMS m/z 221.0688 [M]⁺ (calculated for C₁₁H₁₁NO₄, 221.0683); EI-MS m/z (posit., rel. int. %): 221 ([M]⁺, 48), 161 (81), 148 (100), 133 (20), 120 (38); ¹H and ¹³C NMR data are presented in Table 2.

Methyl dioxindole-3-acetate (3): Pale yellow oil; [α]_D −3.1°(c = 1.0, MeOH); UV λ_max (MeOH) nm: 252.8; IR v_max (KBr) cm⁻¹: 3590–3125, 2975, 1649; EI-MS m/z (posit., rel. int. %): 221 ([M]⁺, 22), 204 (100), 162 (90); ¹H-NMR(CD₃OD, 500 MHz): δ 2.81, 3.07 (each 1H, d, J = 16.0 Hz, H-8), 3.73 (3H, s, OCH₃), 6.92 (1H, dd, J = 7.8, 1.0 Hz, H-4), 7.05 (1H, td, J = 7 .8, 1.0 Hz, H-6), 7.28 (1H, dd, J = 7.8, 1.0 Hz, H-5),7.37(1H, dd, J = 7.8, 1.0 Hz, H-7); ¹³C NMR (CD₃OD, 125 MHz): δ 43.1 (C-8), 53.5 (OCH3), 75.2 (C-3), 117.2 (C-7), 124.5 (C-5), 125.9 (C-6), 127.3 (C-4), 131.1 (C-3a), 138.4 (C-7a), 170.8 (C-9), 174.8 (C-2).

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 IC₅₀ value was determined as the concentration of each sample required to give 50% of the absorbance shown by the blank test.