Two New Pentacyclic Triterpene Saponins from the Leaves of Akebia trifoliata

Two new pentacyclic triterpene saponins, named akebiaoside K (1) and akebiaoside N (2), were isolated from the leaves of Akebia trifoliata, together with five known triterpenoids 3–7. They were all isolated from the leaves of A. trifoliata for the first time. Their structures were established by spectral and chemical means. Triterpenes 5 and 7 were found to show moderate in vitro cytotoxicity against human tumor A549, HeLa and HepG2 cell lines, with IC50 values ranging from 0.023 to 0.038 mM. Triterpenes 5–7 were further revealed to show significant in vitro α-glucosidase inhibitory activity with IC50 values from 0.040 to 0.220 mM, making them more potent than the reference compound acarbose (IC50 0.409 mM). Meanwhile, no obvious inhibitory effects were observed for the isolated triterpene saponins 1–4 in both bioactivity assays.


Introduction
Akebia trifoliata (Thunb.)Koidz. is a perennial, woody liana mainly distributed in the eastern part of Asia [1].Its fresh fruit, commonly called 'Ba-Yue-Gua' 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 traditionally been used in China for centuries as an antiphlogistic, antineoplastic and diuretic agent [3,4].To date, phytochemical studies have revealed structurally diverse triterpenes, triterpene saponins, phenolics and lignans from this plant, and some of them displayed significant biological activities [5][6][7][8][9][10].However, those studies were mainly concentrated on the stems and fruits, and seldom focused on the leaves, though our thin layer chromatography (TLC) examination of the ethanol extract showed that it was highly possible that the leaves of A. trifoliata could also be a promising source of functional natural products.To examine those potential uncharacterized chemicals in the leaves of A. trifoliata, a phytochemical investigation on this plant part was carried out, whereby seven pentacyclic triterpenoids 1-7, including two new triterpene saponins (compounds 1 and 2) were obtained (Figure 1).Herein, the isolation and structure elucidation of these compounds are described.Besides, compounds 1-7 were also tested for their α-glucosidase inhibitory activity and their cytotoxic activity against three human tumor cell lines.also tested for their α-glucosidase inhibitory activity and their cytotoxic activity against three human tumor cell lines.
Compounds 1-7 were evaluated for their in vitro cytotoxicity against human cancer cell lines A549, HeLa and HepG2, using a MTT method as described.The resulting IC 50 values are displayed in Table 3, compared to adriamycin as positive control.Compounds 5 and 7 showed moderate cytotoxicity against all the three cancer cell lines, with IC 50 values ranging from 22.7 to 38.1 µM.Meanwhile, no obvious activity was detected for the four saponins 1-4.This result reveals an obvious negative effect on the cytotoxicity when the triterpenes were linked with the linear tetrasaccharide chain at C-28.Comparison of the chemical structures and their cytotoxicity of 5 versus 6 indicated that the replacement of the structure fragment C-20(Me) 2 by the exocyclic double bond of C-20(29) also had a negative effect on the cytotoxicity.These compounds were further tested for their α-glucosidase inhibitory activity with acarbose used as a reference compound.Compounds 5-7 were revealed to show strong α-glucosidase inhibitory activity, with IC 50 values of 0.047, 0.220 and 0.040 mM, respectively, which were about two to ten-fold stronger than acarbose (IC 50 0.409 mM).These results suggested that triterpenes 5-7 from the leaves of A. trifoliata are effective α-glucosidase inhibitors with potential value for development as effective hypoglycemic agents for diabetes chemotherapy [16].Like in the cytotoxicity bioassay, no obvious α-glucosidase inhibitory activity was detected for the four triterpenoid saponins 1-4, indicating that a negative effect of the tetrasaccharide chain linked at C-28 on the α-glucosidase inhibitory activity was also evident.
A. trifoliata is a medicinal plant naturally widely distributed in the eastern part of Asia.Its air-dried stems and fruits have long been used in China as an antiphlogistic, antineoplastic and diuretic agent.To date phytochemical investigations have revealed a series of triterpenes and triterpene saponins from this plant species.However, those studies were mainly focused on the stems and fruits, and seldom concentrated on the leaves.Our present study revealed that the leaves of A. trifoliata is also rich in triterpenes and triterpene saponins.At the same time, however, it is also demonstrated that much future work is needed to unravel the complexity of the chemical constituents in the leaves of A. trifoliata.

Plant Materials
The leaves of Akebia trifoliata were collected in August 2014, at Sangzhi, Hunan Province, China, were identified by Prof. Fu-Wu Xing at the South China Botanical Garden, the Chinese Academy of Sciences (CAS).A voucher specimen (No. 20140815) was deposited at the Laboratory of Bioorganic Chemistry of the South China Botanical Garden, Chinese Academy of Sciences.

Acid Hydrolysis of 1 and 2
Each of compounds of 1 and 2 (3 mg) was heated in 2 M HCl (4 mL) at 90 ˝C for 2 h.The reaction mixture was extracted with EtOAc (3 ˆ4 mL).The EtOAc extract was purified by passing through a Sephadex LH-20 column (1500 mm ˆ25 mm i.d.) eluted with MeOH.By TLC comparison, the free aglycone of 1 was determined to be identical as 5, while that of 2 was identified as the same as 6, respectively.The aqueous layer was concentrated under reduced pressure to dryness to give a sugar-containing residue, which was reacted with L-cysteine methyl ester hydrochloride in pyridine at 60 ˝C for 2 h, then added with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and stirred under reflux at 60 ˝C for 10 h.The supernatant was then analyzed by GC-MS using a GCMS-QP2010 PLUS instrument, HP-5ms capillary column (30 m, 0.25 mm ID), Helium at constant rate of 46.5 cm/s, 1 µL injection volume, injector temperature at 230 ˝C, temperature program as 2 ˝C/min to 180 ˝C, then 20 ˝C/min to 280 ˝C.Electron ionization mode was used at 70 eV.In the acid hydrolysate of 1 and 2, D-glucose, L-rhamnose and D-xylose were confirmed by comparison of their retention times of their derivatives with those of authentic D-glucose (t R 11.852 min), L-rhamnose (t R 5.292 min) and D-xylose (t R 6.842 min) derivatives prepared in the same way, respectively.

Cytotoxic Assay
Compounds 1-7 were testeded for their cytotoxity against A549 (human lung adenocarcinoma), HeLa (human cervical carcinoma) and HepG2 (human liver hepatocellular carcinoma) cell lines.The three tumor cell lines were obtained from Kunming Institute of Zoology, Chinese Academy of Sciences.The cytotoxic activity of tested chemicals were assayed according to the MTT method using 96 well plates [17].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. 100 µL adherent cells at the density of 5 ˆ10 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, the supernatant per well was removed and subsequently added with 100 µL fresh medium and 100 µL 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 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 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 ˆ100%.IC 50 values were calculated by SPSS 16.0 statistic software.The values were based on three individual experiments and expressed as means ˘standard deviation (SD).
a Recorded in C 5 D 5 N at 500 MHz; b Recorded in C 5 D 5 N at 600 MHz.