Cytotoxic and Radical Scavenging Nor-Dammarane Triterpenoids from Viburnum mongolicum

The ethanol extract of the whole plants of Viburnum mongolicum afforded six new nor-dammarane triterpenoids: 3β,12β-dihydroxy-25,26,27-trinordammara-22-en-24,20-olide (1), 3β,12β-dihydroxy-24α-methoxy-25,26,27-trinordammara-20,24-epoxy (2), 3β-O-acetyl-12β-hydroxy-23,24,25,26,27-hexanordammarane-20-one (3), 12β-O-acetyl-15α-hydroxy-17β-methoxy-3-oxo-20,21,22-23,24,25,26,27-octanordammanrane (4), 12β-O-acetyl-15α,17β-dihydroxy-3-oxo-20,21,22-23,24,25,26,27-octanordammanrane (5), and 12β,15α-dihydroxy-3-oxo-17-en-20,21,22-23,24,25,26,27-octanordammanrane (6), together with two known nor-dammarane triterpenoids, 12β-hydroxy-3-oxo-24α-methoxy-25,26,27-trinordammara-20,24-epoxy (7) and 3β,12β-dihydroxy-23,24,25,26,27-hexanordammarane-20-one (8). The structures of the isolated compounds were established based on 1D and 2D (1H-1H COSY, HMQC, and HMBC) NMR spectroscopy, in addition to high resolution mass spectrometry. The isolated compounds were tested in vitro for cytotoxic potential against seven tumor cell lines and radical scavenging activities. Compounds 4–6 exhibited significant cytotoxic activities against all tested tumor cell lines and radical scavenging activities against ABTS·+ radicals comparable with the standard drug Trolox.


Cytotoxic Activity
All these compounds were evaluated for their in vitro cytotoxic potential against seven tumor cell lines by using the revised MTT method as described in the Experimental part. The results are summarized in Table 3 Hexanordammarane triterpenoids 3 and 8 possessed weak cytotoxic activities (50 μM < IC 50 ≤ 80 μM), while no cytotoxicity was exhibited for compound 1 (IC 50 ≥ 80 μM). The nor-dammarane triterpenoids 1-8 isolated from V. mongolicum shared the same basic skeletal structure with a wide variety of side chains. Due to their intrinsic structural variety and impressive biological activities, we suggest the viewpoint that the introduction of appropriate side chains into a planar polycyclic pharmacophore might strengthen the cytotoxic action.

Radical Scavenging Activity
The compounds 1-8 were tested in vitro for their radical scavenging activities by both the DPPH and ABTS assays. The results (Table 4) showed that compounds 1-8 had no activities towards the tested DPPH radical. Comparing the IC 50 values with that shown by Trolox in the two assays, compounds 4-6 seem to possess higher antiradical activities than other compounds on ABTS ·+ radicals. The differences of antiradical activities of compounds 4-6 between DPPH and ABTS ·+ could be explained by the different mechanisms of the reactions for the two radicals, as has been reported for some compounds which possessed ABTS ·+ scavenging activity, but did not exhibit DPPH scavenging activity [27].

Plant Material
The whole plants of V. mongolicum were collected in Hanzhong Shanxi Province, China, in April 2010. The sample was identified by one of the authors (W. Wang). A specimen (VM201004001) was deposited in the Herbarium of Shengyang Medicine College, Shengyang, China.

Cytotoxicity Assay in Vitro
The isolated compounds 1-8 were subjected to cytotoxic evaluation against A-549 cells (lung cancer), BGC-823 cells (human gastric carcinoma), HepG2 cells (human hepatocellular carcinoma), HL-60 (human myeloid leukemia), MCF-7 cells (human breast cancer), SMMC-7721 (hepatocellular carcinoma), and W480 (colon cancer) by employing the revised MTT method as described in the literature [28,29]. Doxorubicin was used as the positive control. All tumor cell lines were cultured on RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U mL −1 penicillin and 100 μg/mL streptomycin in 25 cm 2 culture flasks at 37 °C in humidified atmosphere with 5% CO 2 . For the cytotoxicity tests, cells in exponential growth stage were harvested from culture by trypsin digestion and centrifuging at 180 ×g for 3 min, then resuspended in fresh medium at a cell density of 5 × 10 4 cells per mL. The cell suspension was dispensed into a 96-well microplate at 100 μL per well, and incubated in humidified atmosphere with 5% CO 2 at 37 °C for 24 h, and then treated with the compounds at various concentrations (0, 1, 10, 100 μM). After 48 h of treatment, 50 μL of 1 mg/mL MTT solution was added to each well, and further incubated for 4 h. The cells in each well were then solubilized with DMSO (100 μL for each well) and the optical density (OD) was recorded at 570 nm. All drug doses were tested in triplicate and the IC 50 values were derived from the mean OD values of the triplicate tests versus drug concentration curves. The 50% inhibition concentration (IC 50 value) was determined by curve fitting and was used as criteria to judge the cytotoxicity (active: IC 50 ≤ 20 μM; moderately active: 20 μM < IC 50 ≤ 80 μM; not active: IC 50 > 80 μM). All cell lines were purchased from Cell Bank of Shanghai Institute of Biochemistry & Cell Biology, Chinese Academy of Sciences. Other reagents were purchased from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China).

Microplate Assay for Radical Scavenging Activity DPPH
Microplate DPPH assay was performed as described by [30]. Briefly, in a 96-well plate, successively sample dilutions (standard stocks of different samples 5 mM), in triplicate, received DPPH solution (40 μM in methanol) in a total volume of 0.2 mL and absorbance was measured at 550 nm with a microplate reader. Results were determined each 5 min until 60 min in order to evaluate kinetic behavior of the reaction. The percentage of remaining DPPH was calculated as follows: %DPPH rem = 100 × ([DPPH] sample /[DPPH] blank ). A calibrated Trolox (6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid, 3.9 mM initial concentration) standard curve was also made. The percentage of remaining DPPH against the standard concentration was then plotted in an exponential regression, to obtain the amount of antioxidant necessary to decrease the initial DPPH concentration by 50% (IC 50 ).

2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) Radical Cation Decolorization Assay
ABTS ·+ radical scavenging activity was determined according to Re [31]. The ABTS ·+ cation radical was produced by the reaction between 7 mM ABTS in H 2 O and 2.45 mM potassium persulfate, stored in the dark at room temperature for 16 h. Before usage, the ABTS ·+ solution was diluted with phosphate buffer (0.05 M, pH 7.4) to get an absorbance of 0.800 ± 0.035 at 734 nm. The solution is stable for 2 days. Different concentrations of extracts or pure compounds in methanol solution were added to 1 mL of ABTS ·+ solution. The mixture was incubated at 37 °C in the dark. After 30 min of incubation, the percentage inhibition of absorbance at 734 nm was calculated for each concentration relative to a blank absorbance (methanol). All determinations were carried out at least three times, and in triplicate. The capability to scavenge the ABTS ·+ radical was calculated using the following equation: where in A Control is the initial concentration of the ABTS ·+ and A Sample is absorbance of the remaining concentration of ABTS ·+ in the presence of different compounds. Trolox was used as reference. The stock concentrations of Trolox and of different compounds tested are the same as reported in DPPH assay.  (6), together with two known compounds, 12β-hydroxy-3-oxo-24α-methoxy-25,26,27-trinordammara-20,24-epoxy (7) and 3β,12β-dihydroxy-23,24,25,26,27-hexanordammarane-20-one (8). Previous studies have indicated that significant antioxidant property of some natural products might be responsible for their antitumor property [27], suggesting that a triterpenoid possessing both antioxidant and antiproliferative activities could offer a broad spectrum of bioactivities. In our present study, all the isolated compounds were evaluated in vitro for their cytotoxic activities against seven tumor cell lines and radical scavenging properties. Octanordammarane tertripenoids 4-6 showed particular cytotoxic activities against all tested tumor cell lines, with low IC 50 values of less than 20 μM, and exhibited radical scavenging activities against ABTS ·+ radicals with IC 50 values comparable with that of the standard drug Trolox.