Bioactive 30-Noroleanane Triterpenes from the Pericarps of Akebia trifoliata

Two new 30-noroleanane triterpenes, 2α,3β,20α-trihydroxy-30-norolean-12-en-28-oic acid (1), 2α,3β-dihydroxy-23-oxo-30-norolean-12,20(29)-dien-28-oic acid (2), were isolated from the pericarps of Akebia trifoliata, together with four known ones, 3β-akebonoic acid (3), 2α,3β-dihydroxy-30-noroleana-12,20(29)-dien-28-oic acid (4), 3α-akebonoic acid (5) and quinatic acid (6). Their structures were established on the basis of detailed spectroscopic analysis, and they were all isolated from the pericarps of A. trifoliata for the first time. Compounds 3−6 showed in vitro bacteriostatic activity against four assayed Gram-positive bacterial strains. In particular 3 showed antibacterial activity toward MRSA with a MIC value 25 μg/mL, which was more potent than kanamycin (MIC 125 μg/mL). No compounds showed antibacterial activity toward the three Gram-negative bacteria tested. Compounds 4 and 5 showed interesting in vitro growth inhibitory activity against human tumor A549 and HeLa cell lines, with IC50 values ranging from 8.8 and 5.6 μM, respectively. Compounds 1, 2, 5 and 6 were further revealed to show significant in vitro α-glucosidase inhibitory activity with IC50 values from 0.035 to 0.367 mM, which were more potent than the reference compound acarbose (IC50 0.409 mM).


Introduction
Akebia trifoliata (Thunb.) Koidz., belonging to the family Lardizabalaceae, is a perennial, woody liana mainly distributed in the eastern part of Asia [1]. The fruit of A. trifoliata, commonly called 'Bayuezha' in China, has long been consumed by the local people as a delicious food [2]. The air-dried stems and fruits of A. trifoliata have also traditionally been used in China as an antiphlogistic, antineoplastic and diuretic agent for hundreds of years [3,4]. Previously, phytochemical studies have revealed many triterpenes and triterpene saponins from A. trifoliata [5][6][7], and phenolics and lignans were also reported from this species [8,9]. However, those studies were mainly concentrated on the stems and seldom focused on the pericarps, although it is highly possible that the pericarps of A. trifoliata would be a promising source of functional bioactive natural products [10][11][12]. Very recently, a phytochemical study revealed fifteen compounds, including eleven noroleanane triterpenoids from a methanol extract of pericarps of A. trifoliata [13], suggesting noroleanane triterpenoids to be characteristic in the pericarps of this species. Noroleanane triterpenoids are a group of novel natural products bearing skeletons with one or two carbons missing from the basic oleanane skeleton, which are so far only discovered in a small group of plants and some of them have been revealed to show significant bioactivities [14]. With the aim of clarifying the uncharacterized bioactive compounds in the pericarps of A. trifoliata, a phytochemical study on the pericarps of this species was carried out, whereby two new (compounds 1-2) and four known 30-noroleanane triterpenes (compounds 3-6) were obtained ( Figure 1). Herein reported are the isolation and structure elucidation of these compounds, as well as their bacteriostatic, cytotoxic and α-glucosidase inhibitory activities.
These compounds were also tested for their in vitro cytotoxicity against three human tumor cell lines, A549 (human lung adenocarcinoma), HeLa (human cervical carcinoma) and HepG2 (human liver hepatocellular carcinoma), using the MTT method as described. The resulting IC 50 values are displayed in Table 3. Compounds 4 and 5 showed interesting cytotoxicity against A549 and HeLa cell lines, with IC 50 values 8.8 and 5.6 μM, respectively. Compound 3 also showed cytotoxic activity (IC 50 49.48, 28.63 and 52.89 μM) against the three tested tumor cell lines, but it was weaker than 4 and 5. New compounds 1-2 and compound 6 did not exhibit cytotoxic activity (IC 50 > 100 μM) in this bioassay. It could be deduced that the exocyclic double bond at C-20(29) might be an important active center for this type of nortriterpenoids to "maintain" their potential cytotoxicity, based on comparison of the structures and activities of 1 to 4. Comparison of the chemical structures of 3 and 4 versus 5 indicated that the α-orientation of the hydroxyl group at C-3 could strengthen the cytotoxic activity of this type of nortriterpenoids, while the α-hydroxyl group at C-2 seems not necessary. Moreover, a negative effect on the cytotoxicity was evident when Me-23 was oxidized (i.e., replaced by an -CHO group), as supported by analyzing the structure-active relationship of 2 and 4. Compounds 1, 2, 5 and 6 were further tested for their α-glucosidase inhibitory activity. The results are listed in Table 4, with acarbose used as a reference compound. Compound 5 showed the best α-glucosidase inhibitory activity with IC 50 value 0.035 mM, which was about twelve-fold stronger than acarbose (IC 50 0.409 mM). Compound 6 showed significant α-glucosidase inhibitory activity with IC 50 value (0.10 mM) about four-fold stronger than the reference compound. Though the α-glucosidase inhibitory activities of 1 and 2 (IC 50 0.367 and 0.220 mM, respectively) were inferior to 5 and 6, they were still more potent than acarbose. These results indicated that 30-noroleanane triterpenes in the pericarps of A. trifoliata were effective α-glucosidase inhibitors promising to be developed as effective and safe hypoglycemic agents for diabetes chemotherapy [18].

General Information
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.

Plant Materials
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.

Antibacterial Assay
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, 10 6 cfu/mL, OD 600 = 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).

Cytotoxic Assay
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% CO 2 at 37 °C. One hundred μL of adherent cells at the density of 5 × 10 4 cell/mL was seeded into each well of 96-well cell culture plates and incubated in 5% CO 2 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% CO 2 at 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