Antitumor Triterpenoid Saponin from the Fruits of Avicennia marina

The fruits of Avicennia marina are widely used for both medicine and food in Guangxi of China. As a part of our continuous effort to search for bioactive molecules from the plant, the fruits of A. marina were investigated, which has led to one new triterpenoid saponin (1) and 29 known compounds been isolated and their structures were established by using spectroscopic methods and comparing with literature data. The new triterpenoid saponin showed cytotoxicity against GSC-3# and GSC-18# with the IC50 values were 12.21 and 5.53 μg/mL respectively, and most of the known compounds had significant antioxidant capacity with the IC50 values ranging from 0.36 to 13.07 μg/mL. Electronic supplementary material The online version of this article (10.1007/s13659-018-0167-9) contains supplementary material, which is available to authorized users.


Introduction
Functional foods not only provide variously essential nutrition to body, but also be benefit for preventing diseases and maintaining health [1,2]. These foods especially have multiple bioactivities, such as antioxidant, anti-inflammatory, antibacterial and anticancer activities [3,4], so people can benefit from daily consumption of them to keep from or relieve the occurrence of chronic age-related diseases or life-style diseases [5,6]. With the benefits, people have paid more and more attention to these foods.
Avicennia marina is one of mangrove plants and widely distribute in southeastern coast in China and its fruits are often picked and eaten by local residents [7]. Water extract of the fruits has traditionally been used for the treatments of colds, larynx and dysentery [8]. And the fruits have the effects of releasing inflammatory and dieresis as a vegetable directly [9]. Thus the fruits of A. marina reasonably meet the interests of modern people's healthy food habits. Continuation of our study on searching for more bioactive molecules from the plant had led to the isolation of one new triterpenoid saponin (1) and 29 known compounds from the fruits of A. marina. Pharmacological experiments showed that the new triterpenoid saponin had antitumor activity and most of the known compounds had potential antioxidant activity.

Anticancer Activity
All compounds were evaluated for their bioactivities against two human glioma stem cell lines (GSC-3# and GSC-18#) by the cell viability assay and phenotypic screening. The results showed that compound 1 exhibited the moderate cytotoxicity against GSC-3# and GSC-18# at the concentration of 20 lg/mL (Fig. 4), and the IC 50 values were 12.21 and 5.53 lg/mL, respectively (Fig. 5).

Antioxidant Activity
DPPH is a widely-used stable free radical to evaluate antioxidant activity of bioactive. In this present assay, the free radical scavenging activities of the polyphenols and phenylethanoid glycosides were evaluated and compounds 4, 5, 8, 18-23, 27, 28 showed potent scavenging activities with IC 50 values from 0.36 to 13.07 lg/mL. Especially compounds 4, 8, 18, and 21 exhibited well scavenging capacities than vitamin C in the experiment ( Table 2).

General Experimental Procedures
1D and 2D NMR spectra were recorded on Bruker DRX-500 spectrometers using TMS as an internal standard. Chemical shifts (d) were expressed in ppm with reference to the solvent signals. Optical rotations were measured on a JASCO P-1020 polarimeter. IR spectra were determined on a Bruker FT-IR Tensor-27 infrared spectrophotometer with KBr disks. UV spectra were detected on a SHMADZU UV-2401PC spectrometer. MS and HRMS analysis were carried out on Waters Xevo TQS and Waters AutoSpec Premier P776 mass spectrometers, respectively. Semipreparative HPLC was performed on a Waters 600 with a COSMOSIL C18 (10 9 250 mm) column. Silica gel (100-200 and 200-300 mesh, Qingdao Marine Chemical Co., Ltd., People's Republic of China), and MCI gel (75-150 lm, Mitsubishi Chemical Corporation, Tokyo, Japan) were used for column chromatography. Fractions were monitored by thin-layer chromatography (TLC) (GF254, Qingdao Marine Chemical Co., Ltd.), and spots were visualized by 10% sulfuric acid ethanol solution.

Plant Materials
The fresh fruits of A. marina were collected in Beibu Gulf of Guangxi province, China, and identified by Dr. Ya-Ping Liu, Kunming Institute of Botany, CAS. A voucher specimen (No. 20170402) has been deposited in the Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China.

Extraction and Isolation
The fresh fruits of A. marina (14.2 kg) were extracted with 95% EtOH (40 L) three times under ambient temperature.

Anticancer Activity
GSC-3# and GSC-18# were human glioma stem cell lines that were established by Kunming institute of zoology from two human glioblastoma multiform samples. The glioma stem cell was cultured in serum-free medium DMEM F12 supplied with 1xB27 and 50 ng/mL EGF, BFGF and 1% penicillin/streptomycin. The glioma stem cells were seeded in the laminin pre-coating dishes and cultured in 37°C, 5% CO 2 incubator. Cell viability assay was performed by the MTS method as previously described. GSCs were digested and counted, seeded in laminin pre-coating 96-well-plate with 20000 cells/well. The compounds were added with a serial gradient concentration (20, 10, 5, 2.5, 1.25, 0.625 lg/ mL) and cultured in cell incubator for 72 h. MTS reagent was diluted 1:5 with fresh medium and mixed well. The old medium was removed and subsequently the fresh medium was added with 100 lL/well. The cells were incubated for 1.5 h. Absorbance was measured by Hybrid Reader (Bio-Tek synergy H1) at 490 nm. The cell viability was evaluated by percentage compared with DMSO control group. The half-maximal inhibitory concentration (IC 50 ) was measured and calculated by Graph Pad Prism 5 software.

DPPH Free Radical Scavenging Activity
The antioxidant activity of compounds was evaluated using the DPPH free radical scavenging assay. The procedure used is an adaptation of those previously [38]. Samples were dissolved to various concentrations (25, 15, 5, 2, 0.5 lg/mL) with methanol. In this assay, reaction mixtures containing a methanolic solution of 0.1 mmol/L DPPH (160 lL) and serial dilutions of test samples (40 lL) were placed in a 96-well-plate. MeOH was used as a negative control and vitamin C was used as a positive control. The reactive mixtures were incubated at 37°C for 30 min in darkness. The absorbance of the mixtures at 517 nm was measured by microplate reader. The scavenging activity (%) was determined by the following equation: DPPH scavenging activity % ð Þ ¼ 1 À A x =A o ð ÞÂ100%: Percent inhibition was plotted against concentration, and the equation for the line was used to obtain the IC 50 value.