Optical rotation were measured on a Perkin-Elmer 341 polarimeter (Perkin- Elmer, Waltham, MA, USA) with MeOH as solvent at the wavelength of 589 nm and 20 °C to gain their specific optical rotation [α] values after calculation. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Advance 600 NMR spectrometer (Bruker Biospin corporation, Billerica, MA, USA), Bruker advance 500M NMR spectrometer (Bruker) and a Bruker DRX-400 NMR spectrometer (Bruker Biospin, Rheistetten, Germany) with the solvent residual peaks of δ_H 7.22, 7.58, 8.74 and δ_C 123.4, 135.4, 149.7 for pyridine-d₅, and δ_H 2.50 and δ_C 39.5 for DMSO-d₆. HR-ESI-MS mass spectra were obtained on a Bruker maXis instrument (Bruker Daltonik GmbH, Bremen, Germany) in a positive ion mode after direct injection of the test solutions. ESI-MS data were obtained using a MDS SCIEX API 2000 LC/MS/MS system (Applied Biosystems, Foster City, CA, USA) in both positive and negative ion modes in the range of m/z 50–1000 after the test solutions were directly injected into the ESI source by a syringe pump. Preparative HPLC was carried out on a CXTH P3000 HPLC pump and a UV 3000 UV-Vis Detector with a Fuji-C18 column (10 um–100 A, ChuangXinTongHeng Science And Technology Co., Ltd, Beijing, China); the Performance of MPLC (medium pressure liquid chromatography) is a CXTH P3000 HPLC pump, a UV 3000 UV-Vis Detector and a C18 column (400 × 25 mM i.d, 50 μM, YMC Co. Ltd., Kyoto, Japan).

Column chromatography (CC) was performed with silica gel (80–100 and 200–300 mesh, Qingdao Haiyang Chemical Co., Qingdao, China), YMC ODS-A (50 μm, YMC Co. Ltd., Kyoto, Japan), Sephadex LH-20 (Pharmacia Fine Chemical Co. Ltd., Uppsala, Sweden), MCI gel CHP 20P (75–150 μM, Mitsubishi Chemical Corp., Tokyo, Japan). Analytical grade petroleum ether (b.p. 60–90 °C), methanol, ethyl acetate, chloroform, n-butanol, acetone were purchased from Tianjin Fuyu Fine Chemical Industry Co. (Tianjin, China); HPLC grade methanol was purchased from J&K Chemical Ltd. (Beijing, China); Fraction were monitored by precoated HSGF₂₅₄ TLC (Yantai Jiangyou Silica Gel Co. Ltd, Yantai, China), and spot detection was performed under fluorescent light (λ = 254 nm), and then spraying 10% H₂SO₄ in ethanol, followed by heating. Kanamycin sulfate, resazurin, pyridine-d₅, DMSO-d₆, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and α-glucosidase were purchased from Sigma Chemical Co. (Sigma-Aldrich, St. Louis, MO, USA). Roswell Park Memorial Institute (RPMI)-1640 medium and fetal calf serum were purchased from Gibco BRL (Gaithersburg, MD, USA). Adriamycin was obtained from Pfizer Italia SRL (Roma, Italy). p-Nitrophenyl-α-d-glucopyranoside (PNPG) and acarbose were obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan).

The pericarps of Akebia trifoliata were collected in September 2009, at Liye of Longshan, Hunan Province, China, identified by Prof. Fu-Wu Xing at South China Botanical Garden, the Chinese Academy of Sciences (CAS). A voucher specimen (No. 20090920) was deposited at the Laboratory of Bioorganic Chemistry of the South China Botanical Garden, Chinese Academy of Sciences.

Air-dried pericarps of Akebia trifoliata (3 kg) were powdered and extracted three times (3 days each) with 95% EtOH (9 L × 3) at room temperature (25–32 °C). The concentration of the solution under vacuum gave a reddish solid. Then, the resulting residue was suspended in H₂O (1.5 L) and successively partitioned with petroleum ether (1.5 L × 3) and ethyl acetate (1.5 L × 3) to afford petroleum ether-soluble (40.0 g) and EtOAc-soluble (180 g) fractions after condensation to dryness in vacuo. The petroleum ether-soluble fraction was subjected to silica gel column chromatography (1000 × 105 mM i.d.) using a gradient system of petroleum ether-acetone (100:0, 20:1, 10:1, 5:1, 2:1, 1:1, 0:100, v/v, each 1.5 L) to give nine fractions (E₁-E₉) after pooled according to their TLC profiles. E₃ (0.45 g) was repeatedly chromatographied on Sephadex LH-20 column (1550 × 13.4 mM i.d.) eluted with acetone to yield compound 5 (6.5 mg). E₄ (1.7 g) was purified on silica gel column chromatography (1000 × 105 mM i.d.) using petroleum ether-acetone (10:1, 9:1, 8:1, 7:1, 6:1, v/v, each 250 mL) as elution system, and then further purified by using MPLC eluted with MeOH-H₂O (95:5, v/v) to yield compound 3 (2.2 mg). E₇ (1.8 g) and E₈ (2 g) were passed through a MCI gel column (200 × 40 mM i.d.) for depigmentation. The resultant methanolic eluate (0.9 g) of E₇ was sequentially separated by MPLC eluted with a gradient of methanol in water (75:25, 80:20, 85:15, 90:10, 100:0, v/v, each 70 mL), and by Sephadex LH-20 column (1550 × 13.4 mM i.d.) chromatography eluted with MeOH, and silica gel CC using CHCl₃-MeOH (98:2, v/v) elution to obtain compounds 2 (4 mg) and compound 4 (2.0 mg). The resultant methanolic eluate (1.1 g) of E₈ was sequentially separated by MPLC eluted with a gradient of methanol in water (60:0, 70:30, 80:20, 90:10, 100:0, v/v, each 100 mL) to give eight fractions (E_8-1-E_8-8). The EtOAc-soluble fraction was subjected to silica gel CC (1000 × 105 mM i.d.) using a gradient of CHCl₃-MeOH (97:3, 90:10, 85:15, 70:30, 60:40, 0:100, v/v, each 3 L) to give ten fractions (F₁-F₆). Fraction F₃ (6.8 g), obtained by elution CHCl₃-MeOH (85:15, v/v), was further subjected to silica gel CC (800 × 50 mM i.d.) and successively eluted with CHCl₃-MeOH (98:2, 95:5, 90:10, v/v, each 0.5 L) to yield six sub-fractions (F_5-1–F_5-6), Sub-fraction F_5-3 (1.6 g) was separated by MPLC using MeOH-H₂O (60:40, 70:30, 80:20, 90:10, 100:0, v/v, each 350 mL) at a flow rate of 10 mL/min, and further purified by Sephadex LH-20 column (1550 × 13.4 mM i.d.) chromatography eluted with MeOH to obtain compound 6 (9 mg). Fraction F₅ (20.5 g), obtained by elution CHCl₃-MeOH (60:40, v/v), was further subjected to silica gel CC (1000 × 105 mM i.d.), and successively eluted with CHCl₃-MeOH (90:10, 80:20, 70:30, 60:40, v/v, each 1.5 L) to obtain six sub-fractions (F_5-1-F_5-6). F_5-3 (10 g) was separated by MPLC, and eluted with MeOH-H₂O (70:30, v/v) to yield compound 1 (5 mg).

2α,3β,20α-Trihydroxy-30-norolean-12-en-28-oic acid (1). White amorphous powder. +106.4 (c 0.70, MeOH). IR (KBr) ν_max 3428, 2939, 2873, 1699, 1459, 1380, 1286, 1450, 1116, 1047 cm⁻¹. ESI-MS (+) m/z: 497 [M+Na]+, 971 [2M+Na]+; ESI-MS (−) m/z: 473 [M−H]^–, 947 [2M−H]^–;. HR-ESI-MS m/z: 497.3235 [M+Na]+ (calcd for C29H46NaO5, 497.3237). For ¹H-NMR (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data, see Table 1.

2α,3β-Dihydroxy-23-oxo-30-norolean-12,20(29)-dien-28-oic acid (2). White amorphous powder. +190.0 (c 0.17, MeOH); IR (KBr) ν_max 3413, 2935, 2861, 1720, 1689, 1456, 1384, 1292, 1220, 1054 cm⁻¹. ESI-MS (+) m/z: 493 [M+Na]⁺, ESI-MS (−) m/z: 469 [M−H]^–. HR-ESI-MS m/z: 493.2922 [M+Na]⁺ (calcd for C₂₉H₄₂NaO₅, 493.2924). For ¹H-NMR (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data, see Table 1.

The bacteriostatic activity of compounds 1–6 were monitored according to the method of Rahman with slight modifications [19]. Briefly, indicator solution (100 μL, resazurin in sterile water, 100 μg/mL) was first placed into each of the sterility control wells (11th column) on the 96 well plates, and indicator solution (about 7.5 mL, 100 μg/mL) was mixed with test organism (5 mL, 10⁶ cfu/mL, OD₆₀₀ = 0.07) followed by transferring (100 μL, each) to growth control wells (12th column) and all test wells (1–10th column). Then, each of 100 μL of the test samples in beef extract peptone medium, the positive control solution were prepared by adding kanamycin sulfate and cefradine instead of the samples and the negative control solution (3% DMSO of beef extract peptone medium) were applied to the wells in the 1st column of the plates. Once all samples and controls were properly applied to the 1st column of wells on the plate, half of the homogenized content (100 μL) from these wells was then parallel transferred to the 2nd column of wells, and each subsequent well was treated similarly (doubling dilution) up to the 10th column, followed by discarding the last 100 μL aliquot. Finally, the plates were incubated at 37 °C for 5–6 h until the color of growth control change to pink. The lowest concentration for each test compound at which color change occurred was recorded as the MIC value of the test compound. The Gram-(+) bacteria, Staphyloccocus aureus (CMCC26003), Bacillus cereus (CMCC63302) and Bacillus subtilis (CMCC63501), and Gram-(‒) bacteria Escherichia coli (CMCC44102), Salmonella typhimurium (CMCC44102) and Shigella dysenteriae (CMCC51252) were obtained from the Guangdong Institute of Microbiology (Guangzhou, China). The multi-drug resistant Staphyloccocus aureus (MRSA) was kindly provided by Dr. Wei, X.Y. (South China Botanical Garden).

Compounds 1−6 were evaluated for their cytotoxicity against three human cancer cell lines, human lung adenocarcinoma (A549), human cervical carcinoma (HeLa) and human liver hepatocellular carcinoma (HepG2). The three tumor cell lines were generously provided by Kunming Institute of Zoology, Chinese Academy of Sciences. The cytotoxic activities of the tested compounds were assayed according to the MTT method by using 96 well plates [20]. Briefly, the cells were cultured in RPMI-1640 medium, supplemented with 10% fetal bovine serum in a humidified atmosphere with 5% CO₂ at 37 °C. One hundred μL of adherent cells at the density of 5 × 10⁴ cell/mL was seeded into each well of 96-well cell culture plates and incubated in 5% CO₂ at 37 °C for 24 h to form a monolayer on the flat bottoms. Then, removed the supernatant per well and subsequently added with 100 μL of fresh medium and 100 μL of medium containing a test compound. The plate was then incubated in 5% CO₂at 37 °C. After 72 h, 20 μL of 5 mg/mL MTT in DMSO was added into each well and incubated for 4 h. The supernatant per well was carefully removed and 150 μL of DMSO was added. The plate was then vortex shaken for 15 min to dissolve blue formazan crystals. The optical density (OD) of each well was measured on a Genois microplate reader (Tecan GENios, Männedorf, Switzerland) at the wavelength of 570 nm. All experiments were performed in triplicate and adriamycin was used as a positive control. In each experiment, each of the tumor cell lines was exposed to the test compound at concentrations of 50, 25, 12.5, 6.25, 3.125, 1.5625 µg/mL. The inhibitory rate of cell growth was calculated according to the following formula: Inhibition rate (%) = (OD_control− OD_treated)/OD_control× 100%. IC₅₀ values were calculated by SPSS 16.0 statistic software. The values were based on three individual experiments and expressed as means ± standard deviation (SD).

The a-glucosidase inhibitory activity of 1, 2, 5 and 6 were determined spectrophotometrically in a 96-well microtiter plate based on p-nitrophenyl-α-d-glucopyranoside (PNPG) as a substrate following the method described in the literature with slight modifications [21,22]. In brief, α-glucosidase (20 μL, 0.8 U/mL) and various concentrations (500, 250, 125, 62.5, 31.25, 15.625 µg/mL) of tested compounds (120 μL) in 67 mM phosphate buffer (pH 6.8) were mixed at room temperature for 10 min. Reactions were initiated by addition of 5.0 mM PNPG (20 μL). The reaction mixture was incubated for 15 min at 37 °C in a final volume of 160 μL. Then, 0.2 M Na₂CO₃ (80 μL) was added to the incubation solution to stop the reaction. The activities were detected in a 96-well plate, and the absorbance was determined at 405 nm (for p-nitrophenol). The negative blank was set by adding phosphate buffer instead of the sample via the same way as the test. Acarbose was utilized as positive control. The blank was set by adding phosphate buffer instead of the α-glucosidase using the same method. Inhibition rate (%) = [(OD_{negative control}− OD_blank) − (OD_test − OD_{test blank})]/(OD_{negative blank}− OD_blank) × 100%. IC₅₀ values of the samples were calculated using the Microsoft Office Excel soft.